Recombinant Influenza viruses as a vaccine vector for HIV

Recombinant Influenza viruses as a vaccine vector for

HIV

By

Mohammed Jasim Mohammed Shallal

Submitted in fulfillment of the requirements for the degree of

Doctor of Philosophy

Deakin University

March, 2015

sfol

Retracted Stamp

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Preface The work presented in this thesis represents of my original laboratory work performed at

CSIRO (AAHL) under an agreement with Deakin University.

The pseudo-challenge experiments in Chapter 5 were performed in the laboratory of Dr.

Charani Ranasinghe, Head of the Mucosal Immunology group, at the John Curtin School of

Medical Research, Australian National University.

Publications Shallal, M. J. M., Ye, S., Izzard, L., Ranasinghe, C., Trivedi, S., Wijesandara, D., Khanna,

M., Bean, A., Ward, A. and Stambas, J."Prime boost vaccination with recombinant

influenza virus vectors stimulates mucosal HIV specific CD8+ T cell immune responses in

BALB/c mice". (In preparation)

Shallal, M. J. M., Ye, S., Izzard, L., Ranasinghe, C., Trivedi, S., Wijesandara, D., Khanna,

M., Bean, A., Ward, A. and Stambas, J. "Mucosal HIV-specific CD8+ T cells protect mice

challenged with recombinant vaccinia virus expressing whole HIV-Gag protein". (In

preparation)

Conference presentations AIDS Vaccine Conference, October 2013 (Barcelona, Spain).

"Prime boost HIV vaccination with recombinant influenza virus vectors stimulates specific

and mucosal CD8+ T cell immune response in BALB/c mice".

Australian Society of Immunology (ASI), Annual Conference, December 2012

(Melbourne, Australia). "Highly expressed α4β7+CD8+ T cells induced following intranasal

prime boost vaccination by recombinant influenza-HIV vaccine in a mouse model".

Immunology Group of Victoria (IGV) Meeting, September 2012 (Melbourne, Australia).

"Recombinant influenza viruses as a vaccine vector for HIV".

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Acknowledgments I would like to acknowledge and thank the School of Medicine at Deakin University, for

providing the opportunity to undertake this PhD project. Further appreciation is acknowledged

to CSIRO/AAHL for providing facilities to support this project.

Sincere thanks to my supervisors, A/Prof. John Stambas, Prof. Alister Ward and Dr. Andrew

Bean for their encouragement and kind attention throughout this research. I am grateful

especially to John Stambas for his guidance and support throughout all stages of my project.

Also, I would like express grateful thanks to Dr. Charani Ranasinghe and her group at JCSMR

for their support. Special thanks to my colleagues at CSIRO/AAHL and Deakin University

who have helped and supported the successful completion of this project.

To Siying Ye and Leonard Izzard, thanks for the kind help you provided throughout my

research in our laboratory in the CSIRO/AAHL.

To Mathew Bruce, I would like to thank you for training me in the area of flow cytometry.

Also, I’d like to thank all staff in the DSR Sequencing Laboratory, CSIRO/AAHL, for their

efforts for sequencing my samples.

To Susanne Wilson, I would like to express my deep thanks and appreciation for your kind

help and support during all stages of my experiments.

To Adam Karpala, thank you so much for all your efforts to support me during the period of

my PhD research.

I would like also to thank and acknowledge the Iraqi Ministry of Higher Education and

Scientific Research, particularly the Iraqi Cultural Attaché and all staff who worked honesty

and kindly to provide help and support for all Iraqi students in Australia, in particular

Mohammed Al-Mayyahi. I also thank my dearest friends Saleem Al-Jebbori, Gazi Al-Sumaily,

Saad Al-Saadi and Dhia Al-Shimmary for their kind attention during my stay in Australia.

Finally, I’d like to thank Terri Grote from Academic Expressions for proofreading my thesis.

V

To my family and friends Mother and Father, I would like to thank you for the patience and love you have provided me

throughout my life and I promise you that I will not forget the sacrifices you have made to

support my studies.

To my brothers, sisters and extended family, thank you very much for your support throughout

my time away from my home.

Also, to my wonderful brother, Abed Ali, I would like to say thank you for your gracious help

and support of my family during my PhD.

Finally, to my lovely wife Khitam and my flowers (my daughters) Kawther and Fatima, a

sincere and grateful thank you for your patience throughout my PhD studies and my time away

from our home. You gave me the strength and hope to finish my PhD, so I would like to express

my appreciation and endless love to you.

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List of abbreviations AAHL Australian Animal Health Laboratory AIDS Acquired Immunodeficiency Syndrome ADH Alcohol dehydrogenase ALVAC Recombinant Canary Pox Vector vaccine expressing HIV Gag APC Antigen Presenting Cell CD8α/β CD8+ T cell Alpha/Beta CSIRO Commonwealth Scientific Industrial Research Organization CTL Cytotoxic T lymphocyte cell DMEM Dulbecco’s Modified Eagle Medium DNA Deoxyribonucleic Acid Env HIV envelope glycoprotein FCS Fetal Calf Serum Gag HIV group-specific antigen Gag197 AMQMLKETI peptide spanning residues 197-205 of Gag HBSS Hank’s buffered salt solution HA Haemagglutinin HEVs High Endothelial Venules HIV Human Immunodeficiency Virus HLA Human Leukocyte Antigen HNE Human Neutrophil Eastase inhibitor ICS Intracellular Cytokine Staining IFN-γ Interferon gamma IL-2 Interleukin 2 IL-4 Interleukin 4 IL-5 Interleukin 5 IL-6 Interleukin 6 IL-10 Interleukin 10 IL-12 Interleukin 12 IL-15 Interleukin 15 i.n Intranasal i.v Intravaginal i.n-i.n Intranasal-intranasal i.n-i.v Intranasal-intravaginal i.v-i.n Intravaginal-intranasal i.v-i.v Intravaginal-intravaginal LPAM Lymphocyte Peyer’s Patches Adhesion Molecule MAdCAM Mucosal Adhesion Molecules MBL Mannose Binding Lectin MDCK Madin–Darby Canine Kidney MDCs Myeloid Dendritic cells MHC-1 Major Histocompatibilty Complex class I N or NA Neuraminidase NP Viral Nucleoprotein NP147 TYQRTALV peptide spanning residues 147–155 of NP

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PBS Phosphate Buffer Saline PCR Polymerase Chain Reaction PDCs Plasmacytoid Dendritic Cells pfu Plaque Forming Unit PR8 A/PR8/34 (H1N1) PR8NA-Gag197 Recombinant PR8 influenza virus expressing Gag197 peptide PR8NA-Tat17 Recombinant PR8 influenza virus expressing Tat17 peptide rIL-2 Recombinant interleukin 2 RALDH Retinal Aldehyde dehydrogenase RNA Ribonucleic acid RT-PCR Reverse Transcriptase-Polymerase Chain Reaction RV144 Thailand clinical trial of HIV vaccine 2009 SD Standard Deviation of the mean SHIV Simian-Human Immunodeficiency Virus STD Sexually Transmitted Disease (s) SEM Standard Error of the Mean Tat HIV Transcriptional transactivator Tat17-25 QPKTACTNC peptide spanning residues 17–25 of Tat TB Tuberculosis TGF-β Transforming Growth Factors-β Th1 T helper 1 Th2 T helper 2 TNF-α Tumor Necrosis Factor alpha UNAIDS United Nations Program on HIV/AIDS VCAM-1 Vascular Adhesion Molecules VLA VRC

Very Late Antigens The Vaccine Research Center

WHO World Health Organization X31 A/HK/X31 (H3N2) X31NA-Gag197 Recombinant X31 influenza virus expressing Gag197 peptide X31NA-Tat17 Recombinant X31 influenza virus expressing Tat17 peptide

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Abstract Human immunodeficiency virus/Acquired immunodeficiency syndrome, HIV/AIDS is a

significant medical problem worldwide with nearly 60 million infected people. Therefore, an

effective and safe vaccine remains a high priority. To date, most HIV vaccine candidates have

failed to elicit effective humoral and cellular immune responses that are necessary to control

HIV infection. A promising phase III trial was conducted in Thailand in 2009 using a

recombinant canary pox vector vaccine (ALVAC) expressing HIV Gag protein in combination

with recombinant HIV Env glycoprotein gp120 (AIDSVAX), and was the first trial to show

significant (31.2%) efficacy in humans.

This project aimed to use influenza viruses as a mucosal live vaccine vector to stimulate an

effective CD8+ T cell immunity. Reverse genetics technology was used to generate

recombinant influenza A viruses H3N2 (HK-X31) and H1N1 (A/PR8/8/34) expressing a

defined mouse HIV-CD8+ T cell epitope (H-2Kd Gag197) in the neuraminidase (NA) stalk (X31-

NA-Gag197 and PR8-NA-Gag197). This epitope was cloned into a pHW2000 reverse genetics

plasmid encoding the appropriate influenza virus NA segment using recombinant PCR.

Following co-transfection with the other seven influenza gene segments in a co-culture of 293T

and MDCK cells, facilitated the generation of live influenza-HIV vaccine vectors.

Live recombinant influenza viruses were successfully used as HIV vaccine vectors, utilising

various mucosal prime-boost vaccination routes in a mouse model, as evidenced by the

induction of robust CD8+ T cell responses. This was shown through intracellular cytokine and

tetramer staining, which detected HIV and influenza-specific CD8+ T cell immunity. Mucosal

CD8+ T cells were also characterized through expression of specific mucosal homing integrins.

These HIV-specific CD8+ T cells migrated to mucosal regions following intranasal or

intravaginal prime boost administration of the recombinant vaccine.

Following intranasal prime-boost vaccination, tetramer and intracellular cytokine staining

showed comparable HIV and endogenous influenza-specific CD8+ T cell responses in the

spleen, and broncho-alveolar lavage, the mediastinal and inguinal lymph nodes. These mucosal

HIV-specific CD8+ T cells expressed the integrins α4β7, CD103 and CCR9. The mucosal

α4β7+CD8+ T cells expressed higher levels of the cytokine IL-15 cytokine and the IL-15Rα

receptor compared to α4β7- controls.

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Finally, challenge with a rVac-Gag virus revealed that Gag197+α4β7+CD8+ T cells contributed

to protection following vaccination. This research highlights the importance of mucosal CD8+

T cells in viral immunity and emphasizes the need for additional studies to provide key insights

to underpin future vaccine development.

X

List of Contents

Declaration I

Preface III

Publications III

Conference presentations III

Acknowledgments IV

Abstract VIII

List of Contents X

List of Figures XV

List of Tables XVII

1. Chapter 1: Literature review 1

1.1. General introduction 1

1.2. Retroviridae 2 1.2.1. Lentiviruses 5 1.2.2. Discovery of HIV 6 1.2.3. HIV-1 genes 7

1.2.3.1. Envelope glycoprotein (Env) 7 1.2.3.2. Group specific or associated antigen (Gag) 8 1.2.3.3. Transcriptional transactivator (Tat) 9

1.2.4. HIV-1 pathogenesis 10

1.3. HIV immunity 11 1.3.1. Innate immunity 11 1.3.2. Adaptive Immunity 13

1.3.2.1. CD4+ T lymphocytes and HIV immunity 13 1.3.2.2. Cytotoxic CD8+ T lymphocytes immune response 14 1.3.2.3. HIV-1 specific binding neutralizing antibodies 17

1.3.3. Mucosal immunity 17 1.3.3.1. Mucosal CD8+ T cells and homingintegrin receptors 19 1.3.3.2. Basic structure of integrins 20

1.3.3.2.1. LPAM-1 (α4/β7) integrin 22 1.3.3.2.2. CD103 (αEβ7) integrin 24

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1.3.3.2.3. CD199 (CCR9) integrin 25 1.3.3.2.4. Interleukin 15 (IL-15) 26 1.3.3.2.5. CD62L and CD44 molecules 29

1.4. HIV-1 Vaccines 30 1.4.1. Traditional vaccines strategies 30

1.4.1.1. Live attenuated vaccines 31 1.4.1.2. Whole killed and subunit vaccines 32

1.4.2. Novel vaccine strategies 33 1.4.2.1. DNA vaccines 33 1.4.2.2. Recombinant viral vaccine vectors 34

1.4.2.2.1. Adenovirus vectors 34 1.4.2.2.2. Modified vaccinia virus vectors 35 1.4.2.2.3. Canarypox virus vectors 36 1.4.2.2.4. Rabies virus vectors 36

1.5. Clinical trials of HIV vaccines 38 1.5.1. Phase 1 and phase 2 clinical trials 38 1.5.2. Phase 3 clinical trials 40

1.6. Influenza virus 41 1.6.1. Pathogenesis of the influenza virus 42 1.6.2. Influenza immunity 44

1.6.2.1. Innate immune response 44 1.6.2.2. Adaptive immune response 46

1.6.2.2.1. Humoral immune response 46 1.6.2.2.2. Cellular immune response 48

1.6.3. Influenza virus as a vaccine vector 53 1.6.3.1. Influenza virus vectors in HIV vaccine candidates 54

1.7. Project hypotheses 56

1.8. Project aims 56

2. Chapter 2: Materials and Methods 57

2.1. Materials 57

2.2. Methods 57 2.2.1. Cloning strategy to generate X31-NA and PR8-NA with HIV-CD8+ T cell epitopes 57 2.2.2. Gel electrophoresis 60 2.2.3. DNA gel extraction and purification 60 2.2.4. Ligation 60 2.2.5. Transformation 60 2.2.6. Plasmid purification 61 2.2.7. Restriction analysis 61 2.2.8. Sequencing 61 2.2.9. Rescue of recombinant influenza virus by reverse genetics 61

2.2.9.1. MDCK and HEK-293 T co-culture 61

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2.2.9.2. Transfection 62 2.2.9.3. Hemagglutination assay 62 2.2.9.4. Amplification of modified viruses in 10 day old embryonated chicken eggs 63 2.2.9.5. RNA extraction of harvested allantoic fluids for sequencing 63 2.2.9.6. Reverse transcription of RNA to cDNA for NA sequencing 63 2.2.9.7. Plaque assay 64

2.2.10. Vaccination of mice 64 2.2.10.1. Procedures for intravaginal and intranasal vaccination of mice 65

2.2.10.1.1. Primary vaccination 65 2.2.10.1.2. Prime boost vaccination 65 2.2.10.1.3. Lymphocyte isolation and preparation 65

2.2.10.2. Phenotype and surface marker staining 66 2.2.10.3. Intracellular cytokine staining 66 2.2.10.4. Tetramer staining 68 2.2.10.5. Cell sorting of HIV Gag+LPAM-1+CD8+ T cell populations 68 2.2.10.6. Real time PCR (RT-PCR) for IL-15 and IL-15Rα expression 69 2.2.10.7. Adoptive transfer and challenge of BALB/c mice 69 2.2.10.8. Pseudo-challenge of vaccinated mice with a recombinant Vaccinia virus expressing whole Gag protein. 70 2.2.10.9. Statistical analysis 70

3. Chapter 3: Generation of influenza viruses expressing HIV-CD8+ T cell epitopes 71

3.1. Overview 71 3.1.1. Generation of a recombinant X31(H3N2) or PR8 (H1N1) influenza viruses expressing HIV-CD8+ T cell epitopes 72 3.1.2. Cloning of modified NA plasmids into the pHW2000 reverse genetic plasmid system 76 3.1.3. Rescue of recombinant influenza viruses using influenza reverse genetics 84 3.1.4. Amplification of generated recombinant influenza viruses 84 3.1.5. Sequencing of recombinant viruses amplified in allantoic fluid 84

3.2. Discussion 88

4. Chapter 4: Induction of CD8+ T cell responses using recombinant influenza viruses. 93

4.1. Overview 93 4.1.1. Characterization of CD8+ T cell responses following primary infection with recombinant X31-NA-Gag197 influenza virus 94 4.1.2. Comparison of CD8+ T cell responses following various prime-boost vaccination regimes detected by ICS. 102 4.1.3. Induction of HIV-specific CD8+ T cells detected by tetramer staining assay 111 4.1.4. Detection of mucosal surface markers (LPAM-1; α4β7 and CD103; αEβ7) and other markers, CD44, CD62L, CCR9 and CD69 on antigen specific CD8+ T cells using flow cytometry 114

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4.1.5. Detection of IL-15 and IL-15Rα expression on mucosal HIV-Gag+CD8+ T cells by RT-PCR 129

4.2. Discussion 133

4.3. Conclusion 136

5. Chapter 5 Pseudo-Challenge study 137

5.1. Overview 137

5.2. Results 138 5.2.1. Adoptive transfer of mucosal CD8+ T cells into naive mice 138 5.2.2. Pseudo-challenge of BALB/c mice with recombinant vaccinia virus expressing HIV-Gag (rVac633-gag) 138 5.2.3. CD8+ T cell immune responses following the r-Vac-gag challenge mice trial 142

5.2.3.1. CD8+ T cell immune response detected by tetramer staining 142 5.2.3.2. Functional analysis of CD8+ T cell immune response detected by ICS following the pseudo-challenge 145 5.2.3.3. Analysis of α4β7+ and CD103+ surface markers on CD8+ T cells following a pseudo-challenge 147

5.3. Discussion 150

6. Chapter 6: General discussion 153

6.1. Overall discussion 153

6.2. Research outcomes 158

6.3. Future directions 158

7. References 160

A. Appendix A 211

A.1. Solutions, buffers and reagents 211 A.1.1. Agarose solution (0.8%) for gel electrophoresis 211 A.1.2. Agarose solution (1.8%) for plaque assay 211 A.1.3. FACS buffer (1%BSA; 0.02% sodium azide) 211 A.1.4. TAE Buffer (40x) (Sambrook et al., 1989) 211 A.1.5. Red cell lysis buffer (Ammonium-Tris Chloride buffer) 211 A.1.6. RNeasy Mini Kit for RNA extraction (Qiagen) 212 A.1.7. Plasmids 212 A.1.8. Competent cells 212 A.1.8.1. MAX Efficiency® DH5α™ Competent Cells (Invitrogen) 212 A.1.8.2. High efficiency JM109 Competent cells (Promega) 212 A.1.9. Reverse transcription kit for conversion RNA to cDNA 212 A.1.10. Permeabilizing solution 212

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A.1.11. Restriction endonuclease enzymes 212 A.1.12. QIAquick PCR Purification Kit protocol 213 A.1.13. QIAquick Gel Extraction Kit protocol 213 A.1.14. PureYield™ Plasmid Miniprep protocol (Promega, USA) 213 A.1.15. Cell lines 213 A.1.16. PureYield™ Maxiprep System protocol (Promega, USA) 214 A.1.17. OPTI-MEM 1 medium (Gibco) 214 A.1.18. Easy-flu medium (Invitrogen) 214 A.1.19. Fugene® 6 Transfection Reagent (Roche) 214 A.1.20. RPMI-antimedia 214 A.1.21. 2x L15 (Leibovitz’s) medium 214 A.1.22. LB agar culture media 215 A.1.23. 1% TPCK-trypsin Worthington (1 μg/ μl) 215 A.1.24. Trizol kit for RNA extraction 215 A.1.25. Plaque assay medium 215 A.1.26. Hanks' Balanced Salt Solution (HBSS) 215 A.1.27. DNA size ladder 215 A.1.28. C-RPMI medium 215 A.1.29. 4 % Paraformaldehyde 216 A.1.30. 6x DNA Loading Dye 216

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List of Figures Figure 1.1. Phylogeny of Reteroviruses 4 Figure 1.2. Phylogenic relationships among lentiviruses 5 Figure 1.3. Structure of the HIV-1 virion 6 Figure 1.4. Organization of the HIV-1 genome 10 Figure 1.5. Stages of HIV infection 11 Figure 1.6. The basic structure of mucosal homing CD8+ T cell receptors 20 Figure 1.7. The multi-steps model of CD8+ T cell migration to mucosal surfaces 22 Figure 3.1. Amino acid sequences of the X31-NA (A) and PR8-NA (B) genes used for the

generation of recombinant HIV influenza vaccine vectors 73 Figure 3.2. Generation of a recombinant neuraminidase (NA) gene segment containing HIV-

H2d Gag197 (AMQMLKETI) and Tat17 (QPKTACTNC) CD8+ T cell epitopes 74 Figure 3.3. Generation of a recombinant influenza virus expressing CD8+T cell epitopes 75 Figure 3.4. Gel electrophoresis of PCR products used a 0.8% of agarose gel 77 Figure 3.5. Gel electrophoresis of ligated PCR products into the pHW2000 reverse genetics

plasmid used a 0.8% agarose gel 78 Figure 3.6. Gel electrophoresis of restriction enzyme digestion of the pHW2000-X31-NA-

Gag197 plasmid using endonucleasePvuI and PstI 79 Figure 3.7. Nucleotide sequence of X31-NA-Gag197 80 Figure 3.8. Nucleotide sequence of pHW2000-PR8-NA-Tat17 81 Figure 3.9. Nucleotide sequence of pHW2000-X31-NA-Tat17 82

Figure 3.10. Nucleotide sequence of pHW2000-PR8-NA-Gag197. 83

Figure 3.11. Reverse genetics strategy for the rescue of recombinant influenza viruses expressing HIV-CD8+ T cell epitopes. 85

Figure 3.12. Hemagglutination assay of three rescued recombinant influenza viruses. 86

Figure 4.1. Weight loss measured in BALB/c mice following primary infection with recombinant X31-NA-Gag197 influenza virus 96

Figure 4.2. Cytokine staining profile for CD8+ T cells duringa primary response in BALB/c mice following intranasal influenza infectionError! Bookmark not defined. 99

Figure 4.3. Cytokine staining assay used for comparison of the primary functional functional IFN-γ+ CD8+ T cells response to the influenza Kd NP147 and Gag197 100

Figure 4.4. CD8+ T cell subsets detected by cytokine staining assay (ICS) 101 Figure 4.5. Dot plots depicting cytokine production ICS for CD8+ T cells specific for HIV-

H2Kd Gag197 following prime boost vaccination by various mucosal routes in BALB/c mice 104

Figure 4.6. Number of IFN-γ+CD8+ T cells specific for HIV-H2Kd Gag197 in different organs following different routes of vaccination in BALB/c mice 106

Figure 4.7. Number of IFN-γ+CD8+ T cells specific for influenza H2-Kd NP147 in different organs following different routes of vaccination in BALB/c mice 107

Figure 4.8. Cytokine profiles of poly-functional CD8+ T cells 108 Figure 4.9. Percentage of CD8+ T cell subsets including TNF-α+IFN-γ+CD8+ T cells in the

spleen following different routes of vaccination in BALB/c mice 109

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Figure 4.10. A smaller subset of CD8+ T cells including IL-2+TNF-α+IFN-γ+CD8+ T cells detected by cytokine staining assay in the spleen 110

Figure 4.11. Number of tetramer+CD8+ T cells induced for HIV-Gag197 in different organs following different routes of vaccination in BALB/C mice 112

Figure 4.12. Number of tetramer+CD8+ T cells induced for influenza NP147 epitope in different organs following different routes of vaccination in BALB/c mice 113

Figure 4.13. Gating strategy used to analyze mucosal α4β7+Gag+tetramer+CD8+ T cells 115 Figure 4.14. Percentage of HIV-Gag197+α4β7+CD8+T cells detected in different organs

following different routes of mucosal vaccination in BALB/c mice 116 Figure 4.15. Percentage of influenza NP147

+α4β7+CD8+T cells detected in different organs following different routes of mucosal vaccination in BALB/c mice 117

Figure 4.16. Percentage of CD103+HIV-H-2Kd Gag197+CD8+ T cells detected in different

organs following different routes of mucosal vaccination in BALB/c mice 118 Figure 4.17. Percentage of CD103+NP147

+CD8+ T cells detected in different organs following different routes of mucosal vaccination in BALB/c mice 119

Figure 4.18. Percentage of CD44+Gag197+CD8+ T cells detected in different organs following

different routes of mucosal vaccination in BALB/c mice 121 Figure 4.19. Percentage of CD44+NP147


Figure 4.20. Percentage of CD62L+Gag197+CD8+ T cells detected in different organs

following different routes of mucosal vaccination in BALB/c mice 123 Figure 4.21. Percentage of CD62L+NP147


Figure 4.22. Percentage of CD69+Gag197+CD8+ T cells detected in different organs following

different routes of mucosal vaccination in BALB/c mice. 125 Figure 4.23. Percentage of CD69+NP147


Figure 4.24. Percentage of CCR9+Gag197+CD8+ T cells detected in different organs following

different routes of mucosal vaccination in BALB/c mice 127 Figure 4.25. Percentage of CCR9+NP147


Figure 4.26. Gating strategy used to sort and analyze both HIV-Gag+α4β7+ and HIV-Gag+α4β7-CD8+ T cell populations 130

Figure 4.27. IL-15Rα gene expression in α4β7+Gag197+CD8+ T cells following intranasal-

intranasal prime boost vaccination quantified in the spleen, MedLN and BAL by quantitative real time PCR (RT-PCR) 131

Figure 4.28. IL-15 cytokine expression in α4β7+Gag197+CD8+ T cells following intranasal-

intranasal prime boost vaccination quantified in the spleen, MedLN and BAL by quantitative real time PCR (RT-PCR) 132

Figure 5.1. Plaque assay on infected lungs when challenged with the rVac-gag challenge virus 139

Figure 5.2. Plaque assay on ovaries of challenged mice 140 Figure 5.3. Graph of a virus titer in the lungs of r-Vac-gag-challenged BALB/c mice 141 Figure 5.4. Tetramer staining of spleen cells from intranasal r-Vac-gag-challenged BALB/c

mice for tetramer+CD8+ T cells 143 Figure 5.5. The number of tetramer+HIV-H2d Gag197

+CD8+ T cells following a pseudo-challenge 144

Figure 5.6. Number of IFN-γ+HIVKd Gag197+CD8+ T cells following pseudo-challenge 146

146

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Figure 5.7. Percentage of HIV-H2Kd Gag197+4β7+CD8+ T cells following

pseudo-challenge 148 Figure 5.8. Percentage of CD103+HIV-H2Kd Gag197

+CD8+ T cells detected by MHC-1 tetramer staining following a challenge to BALB/c mice 149

List of Tables Table 2.1. Nucleotide sequences of primers used for PCR amplification 58 Table 2.2. Conditions for PCR reactions 59 Table 2.3. Conditions of preparation DNA samples for sequencing reaction 64 Table 2.4. Stimulation mixture solutions used in intracellular cytokine staining (ICS) 67 Table 3.1. Titration of rescued recombinant influenza viruses by plaque assay 87 Table A.1. Fluorochrome conjugated Anti-mouse monoclonal antibodies 217

Chapter 1: Literature Review

1

1. Chapter 1: Literature review 1.1. General introduction Since its identification in 1983, Human immunodeficiency virus/Acquired immunodeficiency

syndrome (HIV/AIDS) has become a serious threat to humanity. More than 60 million

individuals have been infected in this epidemic, with 34 million of those currently living with

HIV/AIDS. Moreover, more than 2.6 million new cases of infection are reported every year

(UNAIDS 2013; UNAIDS 2012). Most cases of infection have occurred in the developing

world, with nearly half of those infected succumbing to the disease (Duerr, Wasserheit and

Corey, 2006; Mathers and Loncar, 2006; UNAIDS 2010).

The first cases of HIV/AIDS were identified among homosexual men with complicated

respiratory distress from unusual pneumonia in mid-1981 in the United States of America

(USA). It was later discovered that the disease had been transmitted from central Africa to Haiti

and then onto the USA. Several different modes of transmission were identified, and included

blood transfusions, mother to-child and sexual transmission (Piot, 1989; Quinn et al., 1986).

Many approaches have been used to reduce the risk of HIV transmission from infected persons

to healthy individuals. For example, testing of blood and blood products of donors before

transfusion, use of condoms during sexual intercourse and immediate use of antiretroviral

therapy as a prophylactic treatment in neonates or in adults after exposure. However, none of

these interventions have completely prevented the spread of HIV infection (Carvalho et al.,

2011; Haile-Selassie, Townsend and Tookey, 2011; Shevkani et al., 2011).

The generation of a safe and effective HIV vaccine remains the most likely approach to

eliminate infection (Johnston and Fauci, 2008). Several clinical trials have been conducted to

test new HIV vaccine candidates in the last 25 years. Many of these vaccine candidates are still

under study. Since 1987, more 200 Phase I/II trials have been used for testing 60 HIV-1

candidate vaccines. These include DNA vaccines and recombinant vector and subunit vaccines.

However, only a few candidate vaccines have been tested for efficacy in humans at the phase

III level (Esparza and Van Regenmortel, 2014; Esparaza, 2013; Richert et al., 2014). These

vaccine candidates were designed to induce HIV-specific T cells alone or both T cell and

antibody responses (Chanzu and Ondondo, 2014). All have resulted in poor efficacy with the

exception of one vaccine (RV144: phase III trial performed in Thailand 2009) using a

heterologous prime-boost vaccine strategy that elicited a moderate level of protection against


2

HIV (31%) (Nath, 2010; Vaccari, Poonam and Franchini, 2010; Rerks-Ngarm et al., 2009).

The need for an alternative, more robust vaccine is the motivating factor for this body of work.

The following literature review firstly introduces the Retroviridae family, while the second part

discusses influenza virus and its use as a vector in HIV vaccine research.

1.2. Retroviridae Retroviral diseases have been known for more than two centuries, with the first reported cases

being ovine pulmonary adenocarcinoma and bovine leucosis identified in 1825 and 1870,

respectively. In the decades following, the causative agent of these two diseases was reported

as a retrovirus (Johnson and Kannene, 1992; Voisset, Weiss and Griffiths, 2008). Retroviruses

were first isolated from mice and birds between 1908-1910 in the form of oncogenic viruses

(cancer-causing viruses), such as the mouse mammary tumor virus (MMTV) and the avian

leukosis virus (ALV) (Mathews, 1982). However, other retroviruses were subsequently

identified that led to non-malignant diseases characterized by immunological and neurological

symptoms, such as HIV-1 and HIV-2 (Burmeister et al., 2007; Voisset, Weiss and Griffiths,

2008).

All Retroviridae family viruses have the ability to invade host cells and incorporate into their

genome. Indeed, about 5-10% of the human genome is composed of sequences resulting from

reverse transcription of viral-derived genes called retro-sequences (Lander et al., 2001; Levy,

1993). The current classification of retroviruses depends on the genetic variation of the reverse

transcriptase protein to differentiate between seven major genera within this family (Coffin,

Hughes and Varmus, 1997) (Figure 1.1).

These seven major genera of the retroviridae family included the following:

1) Alpha-retroviruses: simple retroviruses characterized by C-type morphology. These

viruses are widespread in avian species, such as an avian leukemia virus (ALV) (Coffin,

Hughes and Varmus, 1997).

2) Beta-retroviruses: simple retroviruses characterized by a round eccentric core and B-

type morphology. These viruses infect primates, sheep and mice causing tumor

diseases, such as the mouse mammary tumor virus (MMTV) (Hardy, 2009).

3) Gamma-retroviruses: simple retroviruses characterized by C-type morphology that

cause leukemia in different animals, such as the feline leukemia virus (FLV) (Cohen

and Fauci, 2001).


3

4) Delta-retroviruses: complex retroviruses characterized by C-type morphology. These

infect humans and other animals causing leukemia, for example human T-lymphotropic

virus (HTLV) (Burmeister, Schwartz and Thiel, 2001).

5) Epsilon-retroviruses: simple retroviruses characterized by C-type morphology. These

are found in fish and reptiles, for example, Walleye dermal sarcoma virus (WDSV)

(Hardy, 2009).

6) Spumaviruses: complex retroviruses characterized by unique virion morphology with

an uncondensed core and foamy pathogenic changes in infected cells, for example

human foamy virus (HFV). These viruses originated in chimpanzees and were

subsequently transmitted to humans (Buchschacher, 2001; Delelis, Saïb and Sonigo,

2003).

7) Lentiviruses: complex retroviruses distinguished from other retroviruses by unique

virion morphology with cylindrical cores. These viruses infect humans, cattle, horses,

sheep, primates and rabbits causing malignancies as well as lymphatic and neurological

disorders, such as HIV/AIDS (Baig et al., 2008; Gilbert et al., 2009; Katzourakis et al.,

2007).


4

Figure 1.1. Phylogeny of Reteroviruses. Phylogenetic tree showing the seven genera of endogenous retroviruses, including simple and complex genome viruses, with HIV-1 grouped with the Lentiviruses (Weiss, 2006).

ALV: Avian Leukemia Virus; RSV: Rous Sarcoma Virus; JRSV: Jaagsiekte Sheep Retrovirus; MMTV: Mouse

Mammary Tumor Virus; SRV: Squirrel Retrovirus; HERV-K: Human Endogenous Retrovirus-K; HERV-W:

Human Endogenous Retrovirus-W; PERV: Porcine Endogenous Retrovirus; GALV: Gibbon Ape Leukemia

Virus; MLV: Murine Leukemia Virus; FeLV: Feline Leukemia Virus; HTLV: Human T-Lymphotropic Virus;

BLV: Bovine Leukemia Virus; WDSV: Walleye Dermal Sarcoma Virus; SnRV: Snakehead Retro-Virus; FFV:

Feline Foamy Virus; BFV: Bovine Foamy Virus; SFVagm: Simian Foamy Virus (African green monkey);

SFVcpz: Simian Foamy Virus (in Chimpanzees); EIAV: Equine Infectious Anemia Virus; FIV: Feline

Immunodeficiency Virus; MVV: Maedi-Visna Virus; SIVmac: Simian Immunodeficiency Virus (in Macaques);

HIV: Human Immunodeficiency Virus.


5

1.2.1. Lentiviruses Lentiviruses are a genus of viruses that represent one of the largest subgroups of the

Retroviridae family (Şenocak et al., 2009). The name of the virus originates from the Latin

words, lent, lenta, lento and lenti, to indicate the slow nature of disease progression. They are

found endemically worldwide (Ross and Craigo, 2010). These viruses are characterized by

continuous genome evolution in the host cell due to high mutation rates (Hirsch et al., 1995).

Lentiviruses include large and ubiquitous viruses that cause highly infectious diseases in

humans, such as HIV-1 and HIV-2, as well as other primate lentiviruses, such as simian

immunodeficiency virus (SIV), which shares genetic similarity with HIV-1 and 2 (Peeters,

Courgnaud and Abela, 2001). In addition to the major structural genes that are represented in

all retroviruses (pol, gag and env), lentiviruses encode accessory genes, such as rev and tat,

(Buchschacher, 2001; Kerbs, Goldstein and Kilpatrick, 2010; Liu et al., 2009) (Figure 1.2).

HIV is a member of the lentivirinae subfamily within the lentivirus genus that causes

significant disease in humans (Chiu et al., 1985).

Figure 1.2. Phylogenic relationships among lentiviruses. Classification of the lentiviruses on the basis of accessory genes, such as nef, vif, vpr and vpu, with HIV-1 showing similarity to other lentiviruses within the primate group (Gifford et al., 2008).


6

Figure 1.3. Structure of the HIV-1 virion. The genome of HIV-1 is composed of 2 copies of single stranded RNA covered by a double lipid envelope in which two surface glycoproteins (Envgp120 and gp41) are embedded (Roberts, 2009).

1.2.2. Discovery of HIV Between 1970 and 1980, several cases of unknown infection with symptoms of immunologic

dysfunction were recognized as an unusual syndrome among homosexual men in the USA and

Europe (Cohen and Fauci, 2001). This syndrome was characterized by several distinctive

symptoms, such as generalized lymphadenopathy cytomegalovirus-associated retinitis, and

cryptococcal meningitis (Cohen and Fauci, 2001; Mayer and Bell, 2007).

By June 1981, HIV was proposed as the causative agent of AIDS reported mainly in

homosexual males, intravenous drug users and hemophiliacs. This designation was officially

confirmed in 1983 (Barré-Sinoussi et al., 1983; Luft, Seme and Poljak, 2004). Indeed,

HIV/AIDS has now become one of the largest causes of death around the world, particularly

in Sub-Saharan Africa and within the African American population in the USA (Carter and

Saunders, 2009; Crampin et al., 2002).


7

1.2.3. HIV-1 genes The genome of HIV-1 is 9749 nucleotides in size, consisting of nine genes, which are flanked

by two long terminal repeats (Gallo et al., 1988; Varmus, 1988) (Figure 1.4). These genes are

classified into three types: i) structural genes, such as gag, pol, and env; ii) regulatory genes

including tat and rev; and iii) accessory genes, such as vpu, vpr, vif and nef (Berger et al.,

1991). Various HIV genes have been expressed in delivery vectors for induction of HIV-

specific immune responses (Yusim et al., 2002).

1.2.3.1. Envelope glycoprotein (Env) Env is an envelope glycoprotein (Env gp160) trimer that forms the viral envelope and

surrounds HIV particles with 72 little spikes. It mediates virus entry into host cells using the

trimeric envelope glycoprotein spike in order to attach to the cellular receptors and then fuses

to the viral and target cell membrane. It also represents a direct target for antiviral antibodies.

The Env glycoprotein trimer is encoded by a 2.5 kilo-base (Kb) gene and contains 850 amino

acids in total (Sattentau, 2013; Mao et al., 2013). It is made up of two glycoproteins subunits:

(i) surface gp120 (SU), located on the outside of the HIV-1 virus, which plays an important

role in initiating attachment to target cells by binding to the CD4 receptor; and (ii)

transmembrane gp41 (TM), a hydrophobic protein normally embedded within the viral

envelope but exposed after conjugation of gp 120 with the CD4 receptor molecule, which leads

to activation of gp 41 for enhancement of viral-cell membrane fusion (Capon and Ward, 2003).

Some regions of the Env protein are conserved between different strains of HIV-1, but this

protein is generally characterized by alternative glycosylation and divergent sequences. Each

conserved region of the Env protein (C1-C5) is separated by highly variable regions (V1-V5),

leading to altered immunogenicity of Env glycoproteins that play an important role in the

evasion of the immune response. This is because of the high diversity of the HIV-1 Env amino

acid sequence among the nine clades of the HIV-1 virus, A, B, C, D, F, G, H, J and K, and also

because there is more than 35 circulating recombinant forms that can be mutated to 20% within

a clade and to 35% between different clades (Walker and Korber, 2001; Gaschen et al., 2002).

Accordingly, the HIV-1 clades are distinguishable on the basis of their Env glycoprotein

sequences (Dorn et al., 2000; Go et al., 2009; Ho et al., 2008; Huang et al., 1997). Despite this,

Env has also been considered as a potential candidate for HIV-1 vaccines (Ahmad et al., 1994;

Betts, Yusim and Koup, 2002).


8

As a consequence of high variable regions of Env gene sequences of the same HIV-1 virus

strain or between different clades that contributes to inducing a less protective CD8+ T cell

response (Go et al., 2009; Ho et al., 2008), it is highly recommended that multiple Env peptides

or the highly conserved positions of other HIV-1 peptides like Gag and Tat be used in HIV

vaccine candidates.

1.2.3.2. Group specific or associated antigen (Gag) The gag, or group-associated antigen, encodes capsid proteins that influences the physical

structure of the virus. Gag is directly associated with the membrane of the host cell and

enhances the assembly of new immature virions (Bryant and Ratner, 1990). After virus

maturation, gag is cleaved into 4 major specific proteins. The first of these is the matrix protein

(MA or P17) that is embedded into the envelope of the virus where it initiates binding of the

precursor polyprotein (Pr55 gag) to the host cellular envelope. This is an important step in

regulating and controlling assembly and maturation of the virion (Hayakawa et al., 1992). The

capsid protein (CA or P24) forms the main shell structure surrounding the viral genome, located

in the central core of the viral ribonucleoprotein (Göttlinger, Sodroski and Haseltine, 1989).

The nucleocapsid protein (NC or P7) is localized in the virion and has an important role in

assembly of RNA proteins into the HIV-1 virion. A fragment of NC (P6) also has an important

function in facilitating the release of complete viruses from infected cell membranes (Clavel

and Mammano, 2010; Patil, Gautam and Bhattacharya, 2010). Most previous HIV vaccine

candidates failed to control HIV infection because a high diversity of the HIV-1 protein

sequences between different clades of virus enabled the virus to escape the immune response

as the majority of induced CD8+ T cell responses were directed against variable HIV epitopes

(Kunwar et al., 2013; Yoshimura et al., 1996). Therefore, the main aim in the development of

an effective vaccine against HIV is to induce a strong immune response targeting conserved

regions of the HIV-1 virus. This would elicit a breadth of CD8+ T cell responses that recognize

the most conserved positions of HIV epitopes that have an important role in controlling HIV

viremia (Kunwar et al., 2013; Kulkarni et al., 2013). There is a high degree of functional

conservation in the sequence of the whole Gag protein among the 8 major clades of HIV-1,

especially in the capsid subunit. This was evident by a low diversity of amino acid sequences

throughout the full length of the Gag protein subunit, with 17% between different clades of the

HIV-1 virus, and only 9% within the same virus clade (Li et al., 2013). Also, Gag is commonly

targeted by CD8+ T cell responses across various HIV-1 clades as a result of a high cross

reactivity of Gag peptide sequences among these clades. Gag-specific CD8+ T cells obtained


9

from HIV-1 clade C infected individuals were able to recognize at least one Gag peptide from

different HIV-1 clades. For instance, these CD8+ T cells recognized the Gag peptide of 92%

from African clade CDu422, 90% for the Chinese clade CCH, 77% for clade A, 69% for clade B,

and 87% for clade D (Zembe et al., 2011). This was demonstrated by a higher magnitude of

functional HIV-1 CD8+ T cells detected in HIV-1 infected case with 96% against the Gag pool

compared to 86% for Pol and Nef. Moreover, the level of Gag-specific CD8+ T cells were

inversely correlated with the HIV-1 virus load in those infected individuals (Edwards et al.,

2002). Indeed, highly conserved regions of the HIV-1 Gag protein encode the main HIV-1

cytotoxic CD8+ T cell epitopes that are used as vaccine targets and induced CD8+ T lymphocyte

responses (Goonetilleke et al., 2009; Nchinda et al., 2010; Tritel et al., 2003). As a result of the

highly conserved regions of the HIV-1 Gag, this protein is regarded as one of the best

candidates for use in HIV vaccines (Addo et al., 2001; Currier et al., 2006; Yusim et al., 2002).

Therefore, the HIV-1 Gag antigen is widely considered as one of the most conserved viral

proteins used in CD8+ T cell based vaccines, and has also induced a breadth of Gag specific

CD8+ T cell responses. For instance, there was a significant level of IFN-γ+CD8+ T cell

response detected in BALB/c mice vaccinated subcutaneously with multiple doses of the HIV-

1 Gag protein alone or when combined with the Tat protein. Broad CD8+ T cell responses were

directed to the limited nine amino acids of 197 to 205 of the Gag compared to the other Gag

peptides (Cellini et al., 2008).

1.2.3.3. Transcriptional transactivator (Tat) The HIV-1 Tat gene encodes two specific proteins; the 72 residue “Tat-1 protein” and the 86

residue “Tat-2 protein” (Howoroft et al., 1993; Ma and Nath, 1997). These proteins serve to

regulate transcription and translation of polymerase (pol) and transcription of double stranded

DNA after incorporation into the host genome. They do so by binding to specific

transactivation elements called the Tat activating region (TAR), which stimulate the activity of

RNA polymerase II and enhance transcription of viral RNA (Braddock et al., 1993; Jones and

Peterlin, 1994; Liang and Wainberg, 2002; Raha et al., 2005). Tat proteins are also released by

cells infected by HIV-1 and transmitted to non-infected cells which interfere with normal

immune responses. This is achieved through the inhibition of MHC-1 presentation of HIV-1

antigens by dendritic cells and other antigen presenting cells (APCs) (Gavioli et al., 2004;

Petersen, Morris and Solheim, 2003; Raha et al., 2005). Tat is also considered one of the main

regulatory proteins that is frequently targeted by cytotoxic CD8+ T cells and is induced


10

following natural HIV-1 infection; therefore it may be considered an important target for CD8+

T cell based vaccines to control HIV (Addo et al., 2001).

Figure 1.4. Organization of the HIV-1 genome. Specific gene architecture distinguishing HIV-1, including structural genes (gag, pol and env), and each of the regulatory (tat and rev) and accessory genes (vpu, vpr, vif and nef), arranged between the two long terminal repeats (LTR) (Frankel and Young, 1998).

1.2.4. HIV-1 pathogenesis HIV-1 enters the human body through mucosal surfaces and is dependent on the high affinity

binding of surface envelope glycoprotein (Env gp120) with the host cell CD4+ receptor. HIV-

1 also needs to bind to specific major co-receptors such as the chemokine receptors CCR5 and

CXCR4, which are located on the surface of target cells (Kramer, 2007; Lee and Montaner,

1999; Nguyen and Taub, 2002), in order to facilitate the effective entry of the HIV virus into

CD4+ cells. Indeed, the mechanism of interaction between HIV-1 and these co-receptors is a

critical step in the HIV-1 life cycle that influencesthe progression of the disease (Gorry and

Ancuta, 2011). Acute HIV-1 infection generates a high level of viremia, leading to accelerated

depletion of CD4+ cells accompanied by severe acute symptoms. This is followed by a partial

recovery of CD4+ T cell numbers with management of the virus infection, followed by clinical

latency that lasts for 3-10 years (Weiss, 2001; Mayer and Bell, 2007) (Figure 1.5). HIV-1

emerges from latency through alteration of the host’s immune responses through ongoing

exhaustion of immune cells, high depletion of CD4+ T cells (<200 cells/μl) and decreased


11

cytokine activity (Banda et al., 1999; Berger et al., 1998; Gorry and Ancuta 2011). Most cases

of HIV-1 infected individuals are known as chronic progressors, characterized with a high level

of virus replication which leads to extreme decline of CD4+ T cells (Blankson, 2010). As a

result, opportunistic diseases occur that ultimately lead to death 10-15 years following initial

infection (Lü and Jacobson, 2007; Mayer and Bell, 2007; Rodrı´guez et al., 2006; Weiss, 2001)

(Figure 1.5).

Figure 1.5. Stages of HIV infection. Delineation of the different stages of HIV-1 infection with respect to CD4+ T cell count and number of HIV RNA copies. The primary infection, which extends from the initial infection for several months, is accompanied with acute symptoms followed by a long clinical latency period and complete destruction of host immunity (Coffin, Hughes and Varmus, 1997).

1.3. HIV immunity The immune system defends the human body against a wide range of different pathogenic

agents. The recognition of these microorganisms by the immune system is a necessary step in

initiating the battle against pathogens. The host immune system includes two major arms:

innate and adaptive immunity (Levy, Scott and Mackewicz, 2003; Mogensen et al., 2010; Sui

et al., 2010).

1.3.1. Innate immunity The innate immune response is the first line of immune defense following acute HIV-1

infection. The immune response starts with recognition of the HIV-1 antigen and includes


12

various parts, such as the epithelial barrier, phagocytic and antigen presenting cells (Mogensen

et al., 2010). The innate immune response is characterized by a fast onset and a short period of

response (Guatelli, 2010; Lehner et al., 2008). The main aim of the innate immune response

against HIV infection is to reduce virus replication by inducing the cascade of inflammatory

cytokines like interferons (IFNs) and natural killer (NK) cells, which play a critical role in

controlling of infection. The natural killer cells contain specific receptors and killer

immunoglobulin-like receptors (KIRs) that recognized the HIV-1 infected cells and then

destroy these infected cells (Carrington and Alter, 2012). Activation of innate immunity that is

induced directly following early HIV-1 infection includes a contribution by recruited cells,

such as granulocytes, macrophages and lymphocytes. Both lymphocytes and macrophages

target the HIV-1 virus (Mogensen et al., 2010). There are other antigen-specific immune cells

that contain surface Toll-like receptors that recognize molecules from the HIV-1 virus (Bafica

et al., 2004; Datta and Raz, 2005; Klotman and Chang, 2006). The dendritic cell (DC) also acts

as an early innate immune effecter cell that responds rapidly following HIV infection and arms

the immune system by recognizing of the HIV-1 antigen and stimulating naïve T lymphocytes

(Howie, Ramage and Hewson, 2000). There are two main types of dendritic cells: myeloid

dendritic cells (MDCs), which secrete IL-2 to stimulate a proliferation of T lymphocytes, and

plasmacytoid dendritic cells (PDCs), which produce interferons (mostly IFN-α) (Colonna,

Trinchieri and Liu, 2004; Fitzgerald-Bocarsly and Jacobs, 2010; Hardy et al., 2007). Innate

immune response to HIV-1 infection includes trigerring specific Toll-like receptors, such as

TLR7 and TLR8, and activating the protective role of dendritic cells (DCs) (Meier et al., 2009).

As a result, PDCs are able to directly inhibit replication of HIV in the infected cells, particularly

in the spleen and lymph nodes, by releasing both type 1 IFNs and tumor necrosis factor α (TNF-

α) (McKenna, Beignon and Bhardwaj, 2005; Nascimbeni et al., 2009). Furthermore, dendritic

cells in women induce a higher level of IFN-α compared tothe same age group of men, which

is influenced by their response to the HIV RNA triggering of TLR7/8. This would lead to a

lower level of virus replication, thus enhancing control of a HIV-1 infection, compared to HIV-

1 infected individuals (Meier et al., 2009).

Another important component of the innate immune response are neutrophils, which secrete

cytokines such as IL-12 into the circulation to stimulate mucosal immune responses,

particularly in the genital mucosa (Fahey et al., 2005; Gaudreault and Gosselin, 2009).

Additionally, neutrophils release specific proteinase inhibitors such as human neutrophil


13

elastase inhibitor (HNE). HNE is one of the serine proteinase released into the mucosa that

targets invading microorganisms. HNE also plays an important role in the migration of

neutrophils through infected tissues and up-regulates the expression of several types of

cytokines, such as IL-6, IL-8 and transforming growth factors (TGF-β). This further stimulates

the innate immune response (Fitch et al., 2006; Wiedow and Meyer-Hoffert, 2005).

Innate immunity functions also include serum specific proteins called mannose-binding lectin

(MBL), which have the ability to attach to HIV and evoke a host cell response to destroy the

invading virus (Alfano and Poli, 2005; Bouwman, Roep and Roos, 2006; Ezekowitz et al.,

1989). Depletion of MBL enables a high titer of the virus, leading to a rapid progression of

HIV infection (Martin et al., 2003; Rantala et al., 2008; Spear, 1993).

1.3.2. Adaptive Immunity The adaptive immune response involves contributions from different immune components, and

are referred to as the humoral and cellular immune responses (Vieillard et al., 2006; Yamamoto

and Matano, 2008). The humoral immune response includes B lymphocytes, responsible for

the production of antibodies, while the cellular immune response involves T lymphocytes,

including cytotoxic CD8+ T lymphocytes and CD4+ T lymphocytes. Both the humoral and cell

mediated immune responses play critical roles in the defense against HIV-1 (Clerici et al.,

1993; Weber et al., 2001; Zhu et al., 2001).

1.3.2.1. CD4+ T lymphocytes and HIV immunity CD4+ T lymphocytes are characterized by a specific receptor located on their surface, CD4.

This molecule is the primary target of the HIV-1 envelope glycoprotein, Env (gp120/41) and

the HIV-1 infection (Bourgeois et al., 2006; Selig et al., 2001). As such, the CD4+ T cells are

specifically targeted by HIV-1 during acute infection, leading to an abrupt depletion of CD4+

cells (Bentwich, 2005; López-Balderas et al., 2007; McKinnon et al., 2010). This is highly

significant as CD4+ T lymphocytes play a critical role in the control of HIV/AIDS. Activating

B lymphocytes to produce antibodies assists helper T cells to secrete cytokines necessary for

the regulation of a productive immune response (Ostrowski et al., 2000). Two types of helper

T cells perform this function. The first T helper 1 (TH1) cells, which release the cytokines

interferon-γ (IFN-γ) and interleukin-2 (IL-2) necessary for activation of macrophages and

cytotoxic CD8+ T lymphocytes (Bandera et al., 2010; Fortna et al., 2004). The second type of

helper T (TH2) cells, which produce other interleukins, such as IL-4, IL-5, IL-6 and IL-10,


14

which are involved in antibody production. These cells collectively act to reduce the spread of

HIV by stimulating antibodies and enhancing the immune response, through the recruitment of

neutrophils, eosinophils and lymphocytes (Bandera et al., 2010; Fortna et al., 2004; Plana et

al., 2004).

1.3.2.2. Cytotoxic CD8+ T lymphocytes immune response Cytotoxic CD8+ T cells also play a key role in controlling HIV infection (Gómez, Smaill and

Rosenthal, 1994; Landay et al., 1993; Liu et al., 1998). The protective role of HIV-1 specific

CD8+ T cells can be identified by a rise in the initial HIV-1-specific CD8+ T cell responses

during the first weeks of infection, which are directed against a limited number of HIV-1

epitopes, like Env, Pol and Gag (Streeck and Nixon, 2010). The level of these initial CD8+ T

cells is positively related to the slow progression of the virus and alow viremia rate and there

is a direct link between the rapid progression of HIV infection and the reduction of HIV-1

specific CD8+ T cells (Betts et al., 2001; Streeck et al., 2009). CD8+ T cells are normally

stimulated during primary viral infections in humans to support the host immune response and

inhibit HIV-1 virus replication (Callan et al., 1998; Reddehase and Koszinowski, 1984; Reusser

et al., 1999; Rickinson and Moss, 2003). During acute HIV-1 infection, CD8+ T cells are

involved in the reduction of the viral load through the destruction of HIV-1 infected cells by

secreting a high amount of HIV-1 specific-IFN-γ (Cao et al., 2003; Borrow et al., 1994).

Despite the low number of CD8+ T cells induced following acute HIV infection, they do inhibit

the virus titer after an early period of HIV-1 viremia. These CD8+ T cells induced for chronic

HIV infection are also less effective in inhibiting virus replication. In addition, following

persistent viremia caused by the rapid progression of the HIV-1 infection, the level and

functional activity of specific CD8+ T cells is impaired and exhausted as a result of certain

death factors expressed on HIV tetramer+CD8+ T cells, such as inhibitory receptor programmed

death 1 (PD-1 or PDCD1), which down-regulates the activated CD8+ T cells (Day et al., 2006;

Miguele et al., 2002). This evident in the observation that first CD8+ T cells stimulated in an

acute infection has a highly functional role in the suppression of virus replication compared to

the impaired suppressive role of equivalent cells that are induced later. This suggests that early

CD8+ T cells directly targeta limited number of HIV-1 epitopes (Streeck and Nixon, 2010). For

example, there was a high detection level of Env-specific CD8+ T cells responses in acute HIV-

1 infected individuals showing a rapid reduction of viremia compared to a high viremic stage

of infection characterized by a low level of CD8+ T cell response (Borrow et al., 1994). Also,

CD8+ T cell responses directed for HIV-1 antigens like Gag, Pol and Env, detected in most


15

primary infected individuals showlow levels of virus replication. These cells were detected

within a period of a few weeks to a few months while no neutralizing antibodies were detected

during the same period of infection. This can be regarded as evidence there is a critical role of

CD8+ T cell response to the primary HIV-1 infection (Koup et al., 1994).

Furthermore, highly active CD8+ T cells are also involved in the immune response against HIV

infection. These poly-functional CD8+ T cells are characterized by high proliferative capacity

and the ability to secrete a range of cytokines, including IFNγ, IL-2 and TNF-α, thus resulting

in a high level of cytotoxicity (Betts et al., 2006; Harari et al., 2007; Kiepiela et al., 2004).

HIV-specific CD8+ T cells are highly stimulated by specific HIV antigens, such as Gag, Pol

and Env. These cells were detected in HIV-1 infected individuals after 3-6 months following

presentation of the HIV-1 virus antigen (Koup et al., 1994; Duvall et al., 2008; Holmgren and

Czerwinski, 2005). Human leukocyte antigen-restricted CTLs (HLA-class 1) were also

detected, which induced highly protective immune responsesthat controlled HIV infection

through the reduction of viremia (Koup et al., 1994). These cytotoxic CD8+ T cells have the

ability to eliminate HIV-1 infected cells by secreting soluble factors, such as β-chemokines and

specific CD8+ T cell antiviral factor (CAF), to combat HIV-1 infection by suppressingthe

binding and transcription of the virus (Gulzar and Copeland, 2004). For example, HIV-1

specific CD8+ T cells have been shown to play an effective role in preventing replication of the

virus at mucosal surfaces, such as the genital mucosa, thus resulting in control of HIV infection.

Also, sero-negative, highly exposed but resistant Kenyan sex workers have been shown to

possess a high level of mucosal cytotoxic CD8+ T cells when compared to HIV-1 seropositive

sex workers (Kaul et al., 2000). The protective role of HIV-1 specific CD8+ T cells includes

their ability to clear the low level of infection and prevent HIV-1 virus dissemination (Card,

Ball and Fowke 2013). In general, 5-10% of commercial female sex workers in the Pumwani

district in Kenya were identified as HIV-exposed seronegative (HESN). These individuals were

resistant to HIV infection, characterized by a higher level of HIV-CD8+ T cells detected in the

genital mucosa compared to the HIV sero-positive female sex workers (Fowke et al., 1996).

Furthermore, less than 1% of HIV-1 infected individuals, known as Elite controllers or

suppressors (ES), have shown a clinically undetectable infection in addition to a low level of

virus replication (<50 RNA copies/ml) without using antiretroviral treatment (ART). These

individuals contain a higher significant level of polyfunctional CD8+ T cells which enhance

suppression of HIV-1 replication compared to chronic progressive HIV-1 infected individuals


16

(Blankson, 2011; Buckheit et al., 2012). All of these studies indicate the critical role of highly

functional subsets of CD8+ T cells in control of HIV-1 infection in non-progressive HIV-1

chronic cases (Betts et al., 2006).

A high response of polyfunctional CD8+ T cells was also detected in uninfected volunteers in

the United Kingdom. These were administered subcutaneously with a prime-boost vectored

vaccine expressing highly conserved amino acid positions of Gag, Pol, Vif and Env proteins

obtained from the most conserved regions of the HIV-1 clades. This induced CD8+ T cells to

target both Gag and Pol conserved epitopes which led to inhibited virus replication (Borthwick

et al., 2014). Moreover, HIV-1 resistant individuals were similarly characterized by high levels

of granzyme and perforin compared to progressive HIV-1 infected cases. This led to high

cytolytic activity of cytotoxic CD8+ T cells. Although both non-progressive and progressive

HIV-1 infected individuals were included in HIV-specific CD8+ T cells, only non-progressor

individuals characterised with a higher significant proliferation were up-regulated by the

highest perforin expression compared to HIV progressors (Migueles et al., 2002). This was

also evident by measuring the level of granzymes and perforin expressed on human CD8+ T

cells obtained from four groups of chronic-HIV-infected individuals in relation to their stage

of progression and viremia. According to the results, the highest level of granzymes and

perforin was detected in the elite controllers compared to viremic controllers, chronic

progressive and viremic non-progressive individuals. Also, a negative correlation was detected

between the HIV-1 RNA copy number and the level of granzymes and perforin expressed by

CD8+ T cells. This was due to the highly proliferative capacity of induced CD8+ T cells up-

regulated by the highest level of expressed perforin and also because of the higher lytic function

of granzyme B granules destroying the HIV-1infected cells (Migueles et al., 2008; Hersperger

et al., 2010).

Furthermore, the protective role of SIV-CD8+ T cells was detected in macaques (macaca

mulatta) injected subcutaneously with the recombinant RhCMV/SIV virus expressing multiple

SIV proteins, like Gag, Rev, Tat, Nef and Env. Polyfunctional CD8+ T cells were induced that

enabled control of the intrarectal challenge of the SIVmac239 virus (Hansen et al., 2009).

This protective role was also confirmed in SIVmac-infected macaques (Macaca mulatta) which

were administered intravenously for three days with anti-CD8+ T cell monoclonal antibodies

(OKT8F). The effectiveness of these anti-CD8+ T cell monoclonal antibodies was firstly

apparent with the early depletion of CD8+ T cells following the first hour of first dose, with


17

99.9% depletion CD8+ T cells, thus leading to an increase in SIV virus replication. Following

a complete reduction of these monoclonal antibodies, a reduced SIV virus titer had been

detected (Jin et al., 1999). Similar results have also been identified in African green monkeys

injected intravenously with anti-CD8+ T cell antibodies (cM-T807) following infection with

the SIVagm virus (Gaufin et al., 2010).

1.3.2.3. HIV-1 specific binding neutralizing antibodies HIV-1 specific neutralizing antibodies are directed mainly against the HIV-1 Env proteins and

control the primary HIV infection by preventing the viral envelope from binding to CD4+

receptor molecules (Mascola and Montefiori, 2010). In general, the effectiveness of

neutralizing antibodies is mainly restricted to halting the progression of chronic HIV infection

(Morris, 2002). Their effectiveness has been shown in macaque trials where animals have been

vaccinated with recombinant HIV Env proteins and have acquired a high level of anti-Env

neutralizing antibodies that protected them against a homologous highly virulent simian-

human immunodeficiency virus (SHIV) (Someya et al., 2005).

Moreover, heterologous multi-clade HIV-1 epitopes of HIV Env gp140, were identified from

six patients with HIV infection. The antibodies were neutralizing and poly-reactive when tested

against HIV-1 virus in vitro (Mouquet et al., 2010). High levels of neutralizing antibodies, such

as HIV specific mucosal IgA, have been detected in the genital tract and the plasma of resistant

groups of female sex workers (Broliden et al., 2001; Devito et al., 2002; Kaul et al., 1999).

However, one limitation of HIV specific antibodies induced for HIV-1 Env (gp120) is the lack

of cross clade protection. This is due to the high diversity of HIV envelope glycoprotein

sequences between HIV-1 clades (Letvin, 2009; Letvin, 2009a), which results in the escape of

virus recognition of the antibody (Anzinger, Olinger and Spear, 2008; McGrath et al., 2001;

Montpetit, 1995; Morris, 2002). However, this has been changed in recent years after the 2009

isolation of broadly neutralizing antibodies (Nabs) from chronic HIV-1 infected patients that

can bind to different regions of HIV-Env gp120 i.eV1/V2 loop and V3 glycan region to control

HIV-1 virus titers in long-term non-progressors (Benjelloun, et al., 2012; Mascola and Haynes,

2013).

1.3.3. Mucosal immunity The first line of defense for the human body against the external environment involves skin

and the mucosal system (Jakasa et al., 2010; Proksch et al., 2008). The mucosal system includes

nasal, oral, gastrointestinal, respiratory, conjunctival and urogenital mucosal surfaces, and


18

these are important for mucosal transmitted pathogens, such as influenza virus, Herpes simplex

virus and HIV (Kaetzel and Bruno, 2007; MacDonald, 2003). Mucosal defense is characterized

by physical barriers (such as epithelial cells) to protect against the invasion of pathogens and

immune cells, such as dendritic cells and macrophages stimulated as a result of a bacterial or

viral infection. This mode of defense also leads to the induction of both mucosal IgA and

cytotoxic T lymphocytes (Kunisawa and Kiyono, 2005). Mucosal CD8+ T lymphocytes are

important for providing long term protection against mucosal transmitted viruses, such as HIV-

1 (Belyakov and Berzofsky, 2004; Belyakov etal., 2004; Gallichan and Rosenthal, 1996; Suvas

et al., 2007). For instance, HIV-resistant female sex workers in Kenya are characterized by

persistent mucosal CD8+ T cells in the cervix compared to those at risk of HIV infection (Kaul

et al., 2000). Additionally, the humoral immune response also plays an important role in

mucosal immunity by stimulation of specific mucosal antibodies, such as IgA, blocking the

main portal of HIV virus entry through mucosal surfaces like urogenital mucosa (Mascola et

al., 2000).

The main aim of developing HIV vaccines is to induce a strong and stable level of mucosal

immune response in addition to systemic immunity. This may be achieved by using the mucosal

route for the delivery of HIV vaccines rather than the systematic route. This is not to say that

systemic vaccination cannot induce mucosal immunity. For example, Gag-specific CD8+ T

cells were shown to migrate to different effector mucosal sites after a prime boost intramuscular

vaccination with recombinant adenovirus vectors expressing Gag protein in mice and macaques

(Kaufman et al., 2008). It should be noted there is also an important role for HIV-specific

neutralizing antibodies in immunity against human immunodeficiency virus type 1 (HIV-1)

infection. This antibody immune response may include a local induction of IgA but this role is

very limited when compared to more potent role of IgG for controlling HIV-1 at different

mucosal sites. This role was highlighted in maccaque studies where administered IgG induced

protection following vaginal challenge using SHIV virus compared to control macaques (Klein

et al., 2013). Mucosal immune responses induced by mucosal HIV vaccines aim to decrease

virus transmission and progression (Gardner and Luciw, 2008; Gherardi and Esteban, 2005;

Im and Hanke, 2004), by stimulating mucosal IgA and IgG responses, in addition to a cytotoxic

CD8+ T cell responses (Bretscher, 1999; Caley et al., 1997). There are several routes of mucosal

immunization; however, HIV mucosal vaccines are the most effective when administrated

intrarectally or intragastriclly (Belyakov et al., 1998; Duerr, 2010). Vaccines administered


19

intranasally, leading to high mucosal and systemic CD8+ T cell immune responses that have

protected BALB/c mice against the challenge virus, and the recombinant vaccinia virus

expressing HIV- 1IIIB gp160 when compared to the subcutaneous route of vaccination

(Belyakov et al., 1998). Also, in macaques there was no significant difference between various

mucosal route of vaccination, such as intrarectal, intragastric, oral and intranasal vaccination,

that induced robust CD8+ T cells in different mucosal regions following vaccination (Duerr,

2010).

1.3.3.1.Mucosal CD8+ T cells and homingintegrin receptors Integrins are a large family of heterodimeric glycoprotein receptors first identified over 25

years ago (Tamkun et al., 1986). In addition to their direct role in cell adhesion, integrins play

also important role in cell migration which is essential in many vital activities, such as

embryonic development, tissues repair and regeneration, and immune responses (Ridley et al.,

2003). Lymphocytes are continually migrating between the peripheral blood circulation and

different tissues. These cells are trafficked from circulation to various organs via lymph nodes

and pass through venues high endothelial venules (HEV) (Girard and Nerem, 1995).

In order to detect mucosal CD8+ T cell responses, research groups have been studying various

homing receptors, such as LPAM-1, CD103 and CCR9 expressed on antigen-specific CD8+ T

cells migrating to different lymphoid organs.These homing CD8+ T cell integrins include α4β7

(LPAM-1), αEβ7 (CD103) and CCR9 (CD199) integrins that are mainly expressed following

HIV-1 infection or vaccination to prevent dissemination of the HIV-1 virus (Hogg et al., 2003).

Mucosal homing integrins are heterodimeric transmembrane protein receptors that work as

bridges for cell-cell and cell-extracellular matrix (ECM) interactions. They participate in many

important biological functions, such as migration, differentiation and apoptosis (Gupta et al.,

2005; Tang and Rosenthal, 2010). These homing integrins up-regulated trafficking of antigen-

specific CD8+ T cells to mucosal compartments. For instance, polyfunctional HIVGag-specific

CD8+ T cells obtained from prime boost intramuscular vaccination of BALB/c mice using the

recombinant rAd26-Gag and Ad5HVR48-Gag virus, transferred intravenously into naïve mice,

led to the migration of these cells to the gastro-intestinal tract with higher expression of homing

integrins, such as β7 integrin, CCR9, and CD103 compared to non-transferred mice (Kaufman

et al., 2008). Furthermore, transfer of mucosal CD8+ T cells expressing these homing integrin

receptors has been shown to be critical for protection against the intranasal A/PR/8/34 influenza

infection in BALB/c mice. This included an intravenous adoptive transfer of 107 effector CD8+


20

T cells after one hour of intranasal influenza infection.The influenza virus titer was reduced by

the fourth day following infection compared to the negative control (Cerwenka et al., 1999).

These mucosal CD8+ T cells were functionally important in the clearance of the rotavirus from

intraperitoneal infected mice following the intravenous adoptive transfer of α4β7+ expressed

CD8+ T cells (Rose et al., 1998).

1.3.3.2.Basic structure of integrins Homing T cell integrins include α (alpha) and β (beta) subunits that form the main chain

subunits of the homing integrin. Both subunits differ in their nature of expression by

lymphocytes and endothelial cells. There are 8β subunits (β1-β8) and 18α chain subunits. Both

α and β subunits combine for 24 different integrins with distinct αβ integrin subunit pairs (Hogg

et al., 2003). The main structure of integrins consists of two parts. The first part is the

extracellular part, which is elongated from stalk and ends with a globular head region. This part

includes approximately 700 amino acids in the length of α subunit and 1000 amino acids in the

length of the β subunit. The second part is the cytoplasmic tail of less than 75 amino acid in

length (Xiong et al., 2001) (Figure 1.6).

Figure 1.6. The basic structure of mucosal homing CD8+ T cell receptors. These integrins are transmembrane receptors consisting of a heterodimer of alpha and beta subunits. They are expressed on mucosal homing T cells and mediate the attachment specific to endothelial cells (Eslami, 2005).


21

These integrins also mediate cell adhesion through attachment of their globular head region

(ligand binding extracellular part) with the extracellular matrix glycoproteins, such as laminins

and collagens located on the cellular plasma membrane. The β subunit is shared between

different integrin molecules located in different animals. For example, the β1 subunit works as

the main active subunit of integrins in chickens, the human fibronectin receptor, the human

laminin receptor and VLA proteins (Stroeken, 2002). T cells mainly express the two β7 subunit

integrins, α4β7 and αEβ7, that are highly involved in the migration of effector T cells to

peripheral lymph nodes (Hogg et al., 2003). About 12 of these integrins are expressed by T

cells and are highly involved in T cell mediated immunity. The ability of T cells to express

these integrin heterodimers depends on their specific subsets and maturation (Pribila et al.,

2004). For example, low levels of these integrins, such as αLβ2 (leukocyte function associated

antigen-1: LFA-1), α4β1 (very late antigen-4: VLA-4) and α4β7 (lymphocyte Peyer's patch

adhesion molecule integrin: LPAM) are expressed by naïve CD8+T cells compared to effector

or memory CD8+ T cells. In order to enhance migration of T cells to lymphoid effector sites,

these integrins are binding specific ligands like intracellular adhesion molecule-1 (ICAM-1),

vascular cellular adhesion molecule-1 (VCAM-1), and mucosal addressin cell adhesion

molecule-1 (MAdCAM-1) (Egger et al., 2002; Pribila et al., 2004). This facilitates the adhesion

of CD8+ T cells to the endothelial layer and their migration into different lymphoid organs like

the peripheral lymph nodes (DeNucci, Mitchell and Shimizu, 2009; Hogg et al., 2003;

Stroeken, 2002) (Figure 1.7).


22

Figure 1.7. The multi-steps model of CD8+ T cell migration to mucosal surfaces. Migration of specific CD8+ T cells from the effector sites of infection to mucosal regions includes expression of specific mucosal integrins, such as α4β7 (LPAM-1), αEβ7 (CD103) and CCR9 (CD199) on epithelial cells transmit through perivascular membranes on their way to mucosal surfaces of different lymphoid tissue (Hogg et al., 2003).

In addition, a migration strategy includes a contribution of these integrins with specific secreted

chemokines, such as CD44, CD62L and CCR9, which combine with these expressed integrins

to facilitate the migration of antigen-specific T cells to the effector sites of infection (Fousteri

et al., 2009; Wermers et al., 2011). In general, high expression of beta 1 or 7 integrin subunits

facilitates the attachment of high endothelial venules of Peyer’s patches and T cells with

adhesion molecules, such as LPAM-2 (α4β1) which only binds the very late antigen 4 (VLA-

4). While the beta 7 integrin subunit molecule has a high affinity of adhesion property by

binding to specific legand molecules, such as (MAdCAM-1) for LPAM-1(α4β7) and vascular

adhesion molecules (VCAM-1) for CD103 (αEβ7) (Strauch et al., 1994; Yang et al., 1998).

1.3.3.2.1. LPAM-1 (α4/β7) integrin The lymphocytepeyer's patch adhesion molecule (LPAM-1 or α4β7 integrin heterodimer) is

one of the homing integrin receptors expressed on T cells characterized by adhesive properties

responsible for the recognition of migrated lymphocytes into lymphoid compartments, such as

the gut-associated lymphoid tissues (Rosé et al., 1998). LPAM-1 is binding to its ligand called

the mucosal addressin cell adhesion molecule (MAdCAM), which is located mainly on high


23

endothelial venules (HEVs) of mucosal lymphoid organs (Gorfu, Rivera-Nieves and Ley 2009;

Petrovic et al., 2004). This enables the antigen-specific T cells to migrate from the main

circulatory system to mucosal areas, particularly the endothelium of the gut or other related

lymphoid tissues (such as Peyer’s patches and other lymph nodes except the peripheral lymph

nodes) (Gorfu, Rivera-Nieves and Ley, 2009). Indeed, the homing integrins are differentially

expressed on migrated T cells depending on the status of these cells (Petrovic et al., 2004). For

instance, the low level of α4β7 integrin is expressed on naïve T and B cells while it increases

following inflammation or infection. The α4β7 integrin is also expressed on different types of

cells, such as natural killer (NK) cells, and stimulated monocytes, macrophages and eosinophils

(Erle et al., 1994). However, the α4 subunit has the main role to mediate migration of activated

CD8+ T cells to the intestinal mucosa. Therefore, α4β7 has been considered the more dependent

T cell homing marker which has been highly expressed in the intestinal mucosa following the

intravenous injection of C57BL/6J mice using the recombinant vesicular stomatitis virus

expressing ovalbumin (VSV-ova) peptide, SIINFEKL. Mucosal CD8+ T cells expressing α4β7

were transferred intravenously into naïve mice. These mice were infected intravenously with a

wild type of vesicular stomatitis virus which led to high trafficking of the transferred cells to

effector mucosal regions to control virus infection (Lefrançois et al., 1999). Some pathogenic

microorganisms that invade mucosal regions, such as rotavirus, herpes simplex virus type-2,

HIV and Chlamydia, generate a mucosal CD8+ T cell response that provides protection against

the invading pathogens. The overall response is affected by the migration of CD8+ T cells to

infected mucosal tissues and is mediated by the α4β7 integrin homing receptor which facilitates

attachment to specific lymph nodes in the intestine, respiratory and vaginal mucosal surfaces

(Gupta et al., 2005; Tang and Rosenthal, 2010).

There are some differences in the structure and affinity of binding between LPAM-1 and

LAPAM-2. For instance, LPAM-2 (α4β1) integrin contains an α subunit which is recognize

the specific analogous to α chain of the human specific integrins heterodimer (VLA-4) while

LPAM-1 works analogous to other T cell homing heterodimer integrin molecules in humans

(Holzmann, McIntyre and Weissman, 1989). Therefore, LPAM-1 also binds other ligands, such

as VCAM-1 or the CS-1 subdomain of human fibronectin, but with lower affinity compared to

MAdCAM (Rott et al., 1996). The LPAM-1 integrin and other groups of integrins work as the

CD8+ T cell homing receptors detect mucosal CD8+ T cell immune responses. These can also

be used to distinguish memory and effector T cells in response to a rotavirus infection in mice.

For example, functional CD8+ T cells obtained from rotavirus infected mice induced a high


24

percentage of the α4/β7+ subset of CD8+ T cells compared to naïve T cells that included a

subset α4/β7-CD8+ T cell. Therefore, LPAM-1 is mainly expressed on antigen-specific CD8+

T cells and is used for detecting mucosal CD8+ T cell immune responses that are induced

following an intraperitoneal (i.p) rotavirus infection in mice.These mucosal cells are highly

effective in clearing infection compared to other mucosal homing CD8+ T cell integrin

receptors (Rosé et al., 1998).

1.3.3.2.2. CD103 (αEβ7) integrin CD103 or αEβ7 is a mucosal integrin receptor and is composed of a heterodimer of two

subunits, αE and β7. This integrin works mainly as a homing receptor in various cell types,

such as epithelial cells that are localized in various mucosal compartments including the gastro-

intestinal and female reproductive tract, and the important entry sites of mucosal infections,

such as herpes simplex and HIV (Gebhardt et al., 2009; Tang and Rosenthal, 2010; Kiravu et

al., 2011). It is characterized as one of the T cell homing receptors that have an important role

in trafficking of the activated CD8+ T cells from the blood to the genital and gastrointestinal

tract mucosa (Kiravu et al., 2011). Also, CD103 mediates an interaction or adhesion of mucosal

T cells and it surrounds the epithelial layers through its binding to a specific ligand called E-

cadherin or L-CAM, which has a combined function for determining the shape and motility of

lymphocyte cells (Kiravu et al., 2011; Schlickum et al., 2008). A high percentage of mucosal

HSV-2 specific CD8+ T cells found in genital tract mucosa have been shown to stain positive

for the homing integrins CD103 (αEβ7 integrin) and CD49a (α1β7 integrin or VLA-1) one

week after HSV-2 infection in a mouse model (Tang and Rosenthal, 2010), thus suggesting an

important role for these integrin expressing cells in anti-viral immunity and in the control of

these viruses.

The ability of these integrins to be expressed on migrating effector T cells depends not only on

the integrin but also on the area of localization. For instance, CD103 (αEβ7) was expressed on

90% of gut intraepithelial lymphocytes (IEL) localized on Peyer’s patches and other villi to

mediate their attachment to epithelial cells (Koseki et al., 2001). This was not available for

other integrins like L-Selectin. Indeed, it has been confirmed that CD103 is directly involved

in trafficking HIV-specific CD8+ T cells to the cervical mucosa, which are mainly included in

specific Gag+CD8+ T cells (Kiravu et al., 2011). The highly effective role of the αEβ7 integrin

heterodimer is represented by the induction of CD8+ Tcells in both peripheral and intestinal

epithelium as a response to an infection or tumor. For instance, there is a highly effective role

for CD103 chemokine receptors in regulatory functions to stimulate the migration of effector


25

T cells to the infected foci, such as mesenteric lymph nodes and Peyer’s patches, which are

represented by a CD103+CD8+ subset of T cells (Wermers et al., 2011; Apostolaki et al., 2008).

1.3.3.2.3. CD199 (CCR9) integrin CCR9 or CD199 is a mucosal homing integrin mainly expressed on lymphoid cells following

its binding to the specific legand (CCL25) that is presented on epithelial cells in the gut with

enhanced migration of T cells from the periphery and secondary lymphoid organs to the gut

mucosa (Johansson-Lindbom and Agace, 2007). Indeed, the CCR9 integrin heterodimer has an

important regulatory role in the migration of T cells to the infected regions during a specific

inflammatory status, particularly in the intestine, which was represented by an increase in the

subset of CCR9+CD8+T cells found in the spleen, MLN, the lamina propria and the mesenteric

lymph nodes in infected mice. This regulatory role of CCR9 has also been been confirmed by

intraperitoneal administration of the anti-mouse CCR9 monoclonal antibody which led to an

increase in the severity of ileitis in B6 mice (Wermers et al., 2011).

It has also been confirmed that mucosal CD8+ T cells are differentiated by expressing CCR9,

which mediates the fast recovery of BALB/c mice from acute colitis. This was achieved by the

migration of effector CD8+ T cells from blood circulation to Peyer’s patches and the small

intestine, compared to a low level of expressed CCR9 in the large intestine (Wurbel et al.,

2011). As a result of induced mucosal CD8+ T cells expressing CCR9, wild mice were shown

with normal weight after 17 days of infection compared to CCR9 deficient mice who suffered

severe lose weight and a high mortality rate (Wurbel et al., 2011). Furthermore, CCR9 was also

highly required to control ileitis in mice through its expression on lymphocytes at the last stage

of infection that were localised in intestinal lamina propria and mesenteric lymph nodes

(Wermer et al., 2011). In addition, a high level of SIV-specific CD8+ T cells expressing both

CCR9 and αEβ7 were detected especially in mesentric lymph nodes of intranasally infected

monkeys using recombinant modified vaccinia virus expressing SIV-Gag, Pol and Env proteins

(rMVTTSIVgpe). The monkeys were then boosted with an intramuscular infection with

recombinant adenovIrus expressing SIV-Gag, Pol and Env proteins (rAd5SIVgpe). This

mucosal T cell response was able to protect mokeys against mucosal infection from the

SIVmac239 challenge virus (Tang et al., 2012).

Indeed, there are many factors regulating expression of mucosal homing integrins and

stimulating the migration of CD8+ T cells to various mucosal compartments. For instance,


26

retinoic acid is up-regulating both α4β7 and CCR9 expressed on CD8+ T cells. Also, retinoic

acid works through enhancing the signaling receptors of retinoic acid in CD8+ T cells in the

spleen and mesenteric lymph nodes by expressing CD103+CCR9+ subset of CD8+ T cells

(Svensson et al., 2008). Furthermore, 90% of T cells in the intestinal mucosa express α4β7 and

CCR9 as they are trafficked from the peripheral to the gut and this would be impaired in α4β7-

and CCR9- deficient mice (Berlin et al., 1993; Wurbel et al., 2001). There are several other

factors affecting the migration of effector CD8+T cells to mucosal surfaces.

1.3.3.2.4. Interleukin 15 (IL-15) IL-15 was first discovered in 1994 and identified as one of the T cell growth factors.

Interleukin-15 (IL-15) is a glycoprotein and is a member of the 4 α-helix bundle cytokine

family of approximately 15-kDa (Waldmann and Tagaya, 1999). There is a high similarity of

IL-15 sequences among different species. For instance, there is about a 97% of identical

residues between human and simian IL-15, and 82% identical residues between human and

porcine IL-15 (Canals et al., 1997; Anderson et al., 1995). IL-15 is mainly produced by

dendritic cells (Kuniyoshi et al., 1999), monocytes, macrophages (Sandau et al., 2004) and T

cells (Grabstein et al., 1994; Bamford et al., 1996).

IL-15 is a pleiotropic cytokines highly involved in an innate and adaptive immune response

and is mainly active on natural killer (NK) cells (Ma, Koka, and Burkett, 2006). IL-15 utilizes

the same common receptors chains as IL-2, β (CD122) and γ (CD132) chain of their trimeric

receptors. Both cytokines also share similar biological properties and particular function

enhancements of T cell proliferation, generation of cytotoxic T cells and survival of NK cells

(Bodnar et al., 2008; DiSanto et al., 1997). However, IL-15 has little sequence homology with

IL-2. Indeed, IL-15 has an important role in the development and function of NK/NKT cells

and also in the maintenance of naive and memory CD8+ T cells (Gill and Ashkar, 2009).

Furthermore, IL-15 is highly required for the differentiation and persistence of both effector

and memory CD8+ T cells (Munger et al., 1995). Also, the regulatory role of cytokine IL-15

for the development and maintenance of different types of immune cells is confirmed by a high

decrease of CD8+ T cells, natural killer cells, NKT cells and intraepithelial T cells detected in

defective cytokine IL-15 mice (Yajima et al., 2006). IL-15 also stimulates the immunological

function of CD8+ T cells by enhancing their cytolytic activity and inducing long lived memory

T cells that controlling malaria re-infection in mammalian host as IL-15 gives rise the level of


27

IFN-γ producing CD8+ T cells in addition to its role in increasing CD44hiCD62Llo CD8+T cell

subset necessary to control malaria re-infection (Krzych et al., 2012).

The main role of cytokine IL-15 is to maintain, survive, proliferate and turnover CD8+ T cells

detected with high availablibility during periods of inflammation in mice. This is necessary to

improve the immune response in the infected mice (Rubinstein et al., 2008), with less antigen-

specific CD8+ T cells maintained in IL-15-deficient mice. Also, the cytokine IL-15 is

considered one of the key cytokines generated in response to mucosal infection. IL-15 enhances

the proliferation and migration of effector CD8+ T cells to the infected areas such as the lung

following intranasal influenza infection (Verbist et al., 2011), the gut in mice after infection

with the lymphocytic choriomeningitis virus (LCMV) (Masopust et al., 2006) and the skin

following epicutaneous infection using the vaccinia virus (Jiang et al., 2012). For example,

high levels of IL-15 have been identified in the lung airways at day 6 of intranasal influenza

infection in mice. This promotes specific CD8+ T cells to migrate throughout the mucosal area

of the lung (Rubinstein et al., 2008).

IL-15 also works as an adjuvant used to increase the ability of the antigen to be stimulated for

a longer time in order to induce higher immune response and enhance the migration of specific

natural killers or CD8+ T cells to effector sites following intranasal influenza infection of mice

(Verbist et al., 2011; Verbist et al., 2012). Moreover, the removal of specific dendritic cells

that produce cytokine IL-15, or the administration of a specific IL-15 antagonist, inhibits the

proportion of influenza specific CD8+T cells in the lungs and increases the virus load (McGill,

Van Rooijen and Legge 2010; McInnes et. al., 1996).

It is also recognized that cytokine IL-15 has a responsibility to stimulate effector influenza

specific CD8+ T cells to migrate to the site of infection (lungs airways) in the intranasal infected

IL-15-deficient B6 mice using either A/HK-X31 (X31, H3N2) or A/Puerto Rico 8 (PR8, H1N1)

influenza viruses and intranasally administered with IL-15 plus IL-15Rα soluble complex

(Verbist et al., 2011). Furthermore, IL-15 is necessary to stimulate a protective CD8+ T cell

response in HIV infected chimpanzees as a result of the regulatory role of interleukin IL-15 to

maintain the level of interferon-gamma (IFN-γ). For instance, a highly significant reduction of

both IL-15 and tumor necrosis factor alpha (TNF-α) interferon-gamma (IFN-γ) were detected

in progressive HIV-infected chimpanzees compared to a robust maintained level of IL-15 and


28

interferon-gamma (IFN-γ) in those resistant to infection (Rodriguez et al., 2007). The role of

IL-15 is directly controlled by the specific receptor, IL-15Rα, which is mainly expressed

mainly on various types of cells, such as dendritic cells, monocytes and macrophages (Steel,

Waldmann and Morris 2012; Anderson et al., 1995) and T cells (Rowley et al., 2009). The IL-

15Rα receptor ischaracterized by a high affinity binding to soluble IL-15, and IL-15 acts

through its binding to IL-15Rα receptors on CD8+ T cells to enhance the survival and

proliferation of CD8+ T cells (Mortier et al., 2006; Mortier et al., 2008). For example, higher

levels of expressed IL-15 mRNA on CD8+ T cells were detected in BAL, mediastinal draining

lymph nodes and the spleen of intranasally infected BL/6 mice with A/Hong Kong-

X31influenza virus. There was a four fold increase in its expression at day 3 of infection

compared to naïve mice, and expression of IL-15 mRNA continued to increase in the lung

airways until the day 7 of infection (Verist et al., 2011). Furthermore, IL-15 had an important

role for the expansion of primary antigen-specific CD8+ T cells detected indifferent organs,

such as the spleen, peripheral lymph nodes, mesenteric lymph nodes, lung, liver, and small

intestinal lamina propria (LP) of the intravenous infected C57BL/6J mice with vesicular

stomatitis virus (VSV). However, VSV specific CD8+ T cells were reduced significantly (less

than 50%) in IL-15−/− and IL-15Rα−/− mice. Also, the level of IL-15Rα expression was

correlated to the status of a CD8+ T cell as it was fairly low on naive CD8+ T cells while up-

regulated on Ag-specific effector cells. As a result, IL-15 is necessary for the generation and

maintenance of specific antiviral CD8+ T cells (Kimberly et al., 2002). Therefore, both IL-15

cytokine and IL-15Rα receptor expression by specific immune cells, such as dendritic and

CD8+ T cells are considered as an indicator of highly migrated T cells to mucosal regions to

control various mucosal infections, such as an influenza infection and or other inflammatory

diseases like rheumatoid arthritis (McGill, Van Rooijen and Legge 2010; McInnes et. al.,

1996). In addition, other cytokines have a role in the migration of T cells to mucosal effector

sites following mucosal or parenteral vaccination. One of these cytokines is IL-12, which was

highly expressed in draining lymphoid tissues, such as mesenteric lymph nodes and Peyer’s

patches following oral or intraperitoneal reovirus infection in BALB/c mice (Mathers and Cuff,

2004).

On the other hand, high expression of IL-4 and IL-13 down-regulates HIV-1 specific CD8+ T

cells. This was identified in IL-14-/- and IL-13-/- mice primed intranasally with recombinant

Fowl Pox Virus expressing the HIV-Gag, Pol and Env proteins.The mice were then inoculated


29

with an intramuscular dose of recombinant vaccinia virus expressing the Gag and Pol protein.

As a result, polyfunctional CD8+ T cells specific for H-2Kd Gag197-205 were only detected in the

IL-14-/- and IL-13-/- compared to the wild type of infected mice. Accordingly, it is recognized

that higher protective HIV-specific CD8+ T cells induced following mucosal vaccination can

be enhanced perfectly by down-regulating expression of IL-4 and IL-13 at mucosal lymphoid

regions (Wijesundara et al., 2013). Furthermore, other cytokines were generally suppressed

following vaccination. For example, IL-10 is one of the inhibited cytokines and suppressed in

lymphoid tissues like Peyer's patches and mesenteric lymph nodes. Also, IL-4 is highly

inhibited and suppressed in the mesenteric lymph nodesin oral or intraperitoneal vaccinated

miceusing reovirus compared to unvaccinated mice (Mathers and Cuff, 2004; Jackson et al.,

2014

1.3.3.2.5. CD62L and CD44 molecules CD62L or L-Selectin is a cell adhesion molecule or homing receptor expressed on lymphocytes

as a member of the Selectin family of protein. It enhances lymphocytes moving to secondary

lymphoid tissues and regulates migrationof T cells to the peripheral lymph nodes via their

rolling through the endothelium of blood vessels (Robbins et al., 1998). CD62L, in addition to

IFN-γ and TNF-α, was highly produced by both CD8+ and CD4+ T cells induced in lymph

nodes isolated from vaccinia virus naturally infected individuals after a zoonotic outbreak that

occurred in Brazil in 2005 (Medeiros-Silva et al., 2013). It was also detected in 40% of CD4+

and 50% of CD8+ T cells located in cervical draining lymph nodes, or spleen and distal lymph

nodes after intranasal immunization of C57BL/6 mice using ovalbumin (OVA) plus CpG oligo-

deoxynucleotide adjuvant (Ciabattini et al., 2011). CD62L also differentiates effecter T cells

from naïve T cells. For instance, T cells can be recognized by subsets of CD62Lhi CD8+ T

lymphocytes in naïve mice comparedto CD62Llo specific subset of CD8+ T cells in influenza

infected mice, and C57BL/6J using recombinant influenza virus (HKx31-OVA) expressing the

ovalbumin (SIINFEKL) (Kedzierska et al., 2007).

CD44 is a polymorphic group of proteins controlled by highly conserved gene, the CD44 gene

and binds to various legands, such as osteopontin, collagens, and matrix metalloproteinases

(MMP) (Spring et al., 1988). The CD44 is within the size range of 80–200 KDa, which was

firstly identified as an antigen recognized by a monoclonal antibody raised against human

leukocytes (Screaton et al., 1992). The CD44 is a transmembrane cell surface glycoprotein


30

molecule working as a homing cell adhesion molecule, which regulates certain changes like

differential glycosylation in extracellular matrix composition. This leads tothe control of

different cellular functions, such as growth, survival and differentiation (Ponta, Sherman and

Herrlich, 2003). The CD44 is also involved in several cellular functions, such as cell–cell

interactions, and the migration of lymphocytes to effector sites (Screaton et al., 1992).

Furthermore, CD44 is the most dominant subset of CD8+ T cells as lymph nodes homing

receptors recognize the specific CD8+ T cell induced during primary intranasal influenza

infection in mice (Kedzierska et al., 2007).

1.4. HIV-1 Vaccines Vaccines are still regarded as the most effective way to control the majority of infectious

diseases, particularly those diseases that are caused by highly pathogenic viruses (Johnston and

Fauci 2008). Since the first identification of HIV/AIDS, more than 50 vaccine candidates have

been tested to control HIV infection (Barouch, 2008; Girard, 2006).

All of these trials have failed due to the following key challenges:

(i) Highly variable antigensof HIV-1;

(ii) The high mutation rate of HIV-1 permitting the virus to escape host immunity;

(iii) The lack of efficient immune responses against latently infected cells;

(iv) The absence of synergistic activity between humoral and cellular immunity to control HIV;

and

(v) The limitations of standard laboratory animal models used for HIV vaccine trials (Barouch,

2008; Girard, 2006).

The most important function of any HIV-1 vaccine is to produce an immune response that will

prevent transmission and replication of the virus at the entry portal, such as the vaginal, rectal,

urethral and oral mucosa (Haase, 2010; Haynes and Shattock, 2008; Veazey, 2005). To achieve

this, HIV vaccines must be safe, immuno-efficient, highly prophylactic and capable of reducing

viral loads to less than 1500 RNA copies per ml in the HIV infected persons, at which point

HIV progression can be controlled (Newman et al., 2010; Quinn et al., 2000).

1.4.1. Traditional vaccines strategies Traditional strategies for HIV vaccines include live-attenuated virus vaccines, whole killed

virus vaccines and subunit vaccines (Barouch 2008).


31

1.4.1.1.Live attenuated vaccines Live attenuated vaccines have been used to control highly infectious diseases such as small

pox, poliomyelitis and herpes viruses, and are still regarded as potentially the most protective

vaccines (Chakrabarti et al., 1996; Plotkin, 1994; Riedel, 2005). Use of live attenuated SIV

vaccines in monkeys provided significant protection to macaques against a virulent

homologous SIV (Koff et al., 2006; Wyand et al., 1996). Also, there is an inverse correlation

between the level of virus attenuation and the level of protective immune response in macaque

studies (Wodarz, 2008). The use of attenuated viruses with deleted or truncated genes, such as

nef or deletion of both nef and vpr resulted in a strong level of protective immune responses in

vaccinated macaques (Johnson and Desrosiers, 1998). Moreover, intravenous vaccination of

rhesus monkeys using attenuated SIV with deleted nef and vpr sequences (SIVmac239), or the

deleted nef and vpx sequences, or deleted nef, vpr, vpx sequences, then intravenously or

intravaginally infected with the SIVmac251 challenge virus. The less attenuated SIV vaccine

induced robust SIV specific T cell responses leading to higher protection in the intravaginal

challenged group than protection against those intravenously challenged (Johnson et al., 1999).

Furthermore, other trials with macaques used attenuated simian immunodeficiency virus

(SIVmac239) constructed with the six deleted accessory genes, nef, vpr, vpx, vif, tat, and rev

genes. These were the given within multiple doses of intravenously to macaques and then

challenged intrarectally using pathogenic type of homologous SIV challenge virus, produced

protection in 75% of vaccinated macaques with decreasing viral load to less than 1,000

copies/ml for 6 months, and with a stable count of CD4+ T cell for 2 years following challenge

(Guan et al., 2001). However, there was no complete protection for the rhesus monkeys who

were vaccinated intravenously with SIVmac316-D-nef (deleted nef), or with SIVmac239-D3

(deleted nef and vpr), and then challenged intravenously using the wild type of SIV

(SIVmac251). The result was the most vaccinated monkeys were infected after 18 to 44 weeks

of challenge (Wyand et al., 1996). Also, there was a concern with the safety of the SIV

attenuated vaccine represented by a persistent rise of viremia in vaccinated neonate macaques

because of inability of immune response induced for nef or vpr to decrease the pathogenicity

of SIV in neonate macaques (Baba et al., 1999). In another study in macaques vaccinated with

highly attenuated SIVmac239-Delta 3 which included large deletions of nef and vpr, and then

challenged with a wild type of the SIV virus, saw the level of immune response decline,

accompanied with serious adverse reactions. These reactions included recurrent viremia,


32

thrombocytopenia, wasting, and opportunistic infections aftera few years of vaccination in

vaccinated macaques compared to non-vaccinated macaques (Baba et al., 1999). Accordingly,

a live attenuated vaccine is unlikely to be used in humans, this is because of the risk of reversion

to the virulence of virus and the attenuated virus strain could cause HIV infection as a result of

a virus mutation which can lead to immune dysregulation in the vaccinated individuals (Baba

et al., 1999; Baba et al., 1995; Foster and Garcia, 2008; Gorry et al., 2007).

1.4.1.2.Whole killed and subunit vaccines Whole killed and subunit vaccines have also been used for immunization against HIV/SIV

infection. Preclinical trials of formalin-killed potentiated with respective adjuvants or subunit

SIV vaccines have resulted in protection against experimental homologous infection

ofpathogenic SIV virus in rhesus macaques (Macaca mulatta) (Mascola and Montefiori, 2010;

Patterson, Robey and Robert-Guroff, 2000). However, whole killed viruses and protein

subunits are recommended in vaccination programs because of their inability to induce broad

antibody or CD8+ T cell responses multiple doses potentiated adjuvants (Murphey-Corb et al.,

1989; Flynn et al., 2005). The mucosal adjuvants used included combinations of cytokines,

such as IL-1α, IL-12, and IL-18 to potentiate an antibody immune response in BALB/c mice

intranasally vaccinated with the polyvalent C4/V3 HIV-1 Env peptide as the HIV vaccine

candidate (T1SP10 MN) (Bradney et al., 2002). When used to deliver the HIV-1 Env peptide

with multiple doses, it resulted in a high level of antibodies involving serum IgG1 and secretory

IgA in different sites of mucosal surfaces, such as saliva, and the vaginal lavage in vaccinated

mice (Bradney et al., 2002; Staats and Ennism, 1999; Staats et al., 2001). One of the first

generations of subunit vaccines represented by the Phase I HIV vaccine trial was conducted in

50 healthy adult participants who were at risk of HIV infection but were sero-negative for HIV-

1. This group was given the vaccine composed of recombinant HIV-1 Env proteins (gp 120 or

gp160) conjugated with alum adjuvant, which led to the induction of a specific HIV-1 Env

antibody response in sera. There were samples of sera obtained from 16 HIV-1 infected persons

used to evaluate neutralizing antibody activity against the 5 primary viruses (US 1-US5) that

were isolated from the HIV-1 infected volunteers in addition to the heterologous laboratory-

adapted HIV strains or (MN, IlIB, and SF2) (Frey, 1999; Mascola et. al., 1996; VanCott et al.,

1997). However, there was no long term protection against virus infection due to the lack of

long active HIV neutralizing antibodies (nAbs) (Goebel et al., 1999; Mascola et. al., 1996).


33

1.4.2. Novel vaccine strategies Traditional HIV vaccine strategies have failed to provide significant protection (Foster and

Garcia, 2008; Das, Jeeninga and Berkhout, 2010). Accordingly, there is currently no

commercially available vaccine to prevent HIV infection (Koup and Douek, 2011; Wahren et

al., 2010). Due to safety concerns and the ineffectiveness of traditional HIV candidate vaccines,

alternative strategies involving various genetic approaches are being used to develop novel

vaccines (Excler, 2007; Letvin, 2004; Voronin, Manrique and Bernstein, 2010). These novel

HIV vaccine strategies include DNA vaccines and live recombinant vectors (Dubensky, Liu

and Ulmer, 2000; Letvin, 2005).

1.4.2.1. DNA vaccines DNA vaccines utilize plasmids that encode specific viral proteins as immunogens for the

induction of strong immune responses (Casimiro et al., 2003; Graham et al., 2006; Vasan et

al., 2010). In general, most DNA candidate vaccines for HIV-1 have been shown to be safe

when injected in mice or macaques, although they have failed to induce a protective immune

response (Johnston and Flores 2001). The immunogenicity of plasmid DNA vaccines has been

improved with adjuvants (Barouch et al., 2000a; Chong et al., 2007; Kutzler et al., 2009; Xu

et al., 2008). For example, DNA vaccines have been conjugated with conventional adjuvants,

such as aluminum salts, which increase antibody responses 100-fold in mice and 10-fold in

macaques compared to non-conjugated DNA vaccines. They also enhance cellular immunity

as represented by Th1 and Th2 cytokine responses (Petrovsky and Aguilar, 2004; Ulmer et al.,

1999). Recent preclinical trials of DNA plasmid vaccine candidates have expressed genes

within the highly conserved regions, such as gag, pol and nef. These have been isolated from

multiple HIV-1 clades, and then followed by a recombinant adenovirus vector expressing

SIVmac Gag, Pol and Nef proteins alone or combined with the Gag protein. Following these

trials, all vaccinated Indian-origin rhesus monkeys were challenged using the simian-human

immunodeficiency virus (SHIV-89.6P) with no protection against the virus. These monkeys

expriences a rapid loss of CD4 T cells, despite of robust IFN-γ ELISPOT responses to the

Gag/Pol/Nef and Env plus the high-titer antibodies, especially in the Env vaccinated group

(Letvin et al., 2004). The same strategy was also conducted in Russia was used in BALB/c

mice who were vaccinated intramuscularly with recombinant plasmids expressing the HIV-1

genes, env, gag, pol and nef, the induced robust cytotoxic T cell and IFN-γ+CD8+ T cell

responses for HIV-1 peptides mainly for Env (Murashev et al., 2007). Another trial of a DNA

vaccine, expressing recombinant SIV-Gag and HIV-1 Env was used in macaques induced with


34

a low level of cytotoxic T lymphocytes and CD8+ T cell responses that resulted in high viremia

and rapid loss of CD4+ T cells following an intravenous challenge infection using the

pathogenic SHIV-89.6P virus (Barouch et al., 2000).

1.4.2.2. Recombinant viral vaccine vectors Recombinant live vaccine vectors express HIV antigens or epitopes from a recombinant virus.

The most common viral vectors used as HIV vaccine candidates are the adenoviruses, modified

vaccinia viruses, canarypox and rabies viruses. It is important that there is a difference in the

ability of various HIV genes to induce immune responses (Liu, 2010). For example, gag, vif

and nef expressed in Hela-CD4 cells leads to the stimulation of an immune response sufficient

to reduce the viral titers, while the use of other HIV genes, such as tat, rev and vpu increased

the rate of infection compared to no inhibitory effect of HIV env, vpr and pol expressed genes

to control the homologous T-cell-line-adapted HIV-1 strain (D'Aloja et al., 1998). Indeed, viral

vector vaccines were considered the most likely used vaccine candidates to induce both a

humoral and T cell immune response against infectious diseases in humans and animals

(Draper and Heeney, 2010). Recently, viral vector vaccines have been regarded as the most

likely vaccines to be used to stimulate both a humoral and cellular immune response (Draper

and Heeney, 2010).

1.4.2.2.1. Adenovirus vectors The adenovirus serotype 5 (Ad5) has been used as a vector in many preclinical and clinical

trials of recombinant HIV vaccines. For example, one clinical trial of a randomized, double

blind phase 1 trial conducted by the Vaccine Research Center (VRC), utilized 4 rAd5 vectors

to express the recombinant HIV-1 Gag Pol fusion protein (subtype B) and also the Env protein

(subtypes A, B, and C) to evaluate safety. The immunogenicity of this recombinant vaccine

was administered intramuscularly to 30 healthy non-infected volunteers, with induced T cell

responses included in 93.3% of vaccine recipients, IFN-γ+CD8+ T cells in 60% of vaccine

recipients, and Env specific antibody responsesin only 50% with moderate adverse reactions,

such as headache, malaise, myalgia, chills or mild fever persisting for 1 day after vaccination

(Catanzaro et al., 2006). A trivalent MRK Adenovirus type 5 has also been used in a Merck

trial for the expression of HIV-1 antigens, such as Gag, Pol and Nef (MRKAd5-Gag MRKAd5-

Pol, and MRKAd5-Nef of clade B), in 259 intramuscular vaccinated recipients. This trivalent

vaccine induced cell mediated T cell included CD8+ T cells immune responses without serious

adverse reactions, however this trial was discontinued due to lack of efficacy (Priddy et al.,

2008).


35

Recombinant adenovirus serotype 5 vectors were also used to express whole SIV Gag/Pol and

Env proteins in a prime-boost vaccine given to rhesus macaques primed with a plasmid DNA

vaccine, including SIVmac239 gag-pol-nef genes, that stimulated both cytotoxic CD8+ T cells

and antibodies (Santra et al., 2005). Another preclinical trial of the SIV vaccination used

replication-deficient Ad5 expressing HIV-1 Tat, Env (gp140), recombinant Env (SHIV-89.6P

gp140) and a combination of SIV Gag and Env (gp140) as one of the SHIV challenge studies

administered intramuscularly in macaques (Macaca mulatta), and then challenged

intravenously using SHIV-89.6P. Each of the vaccines induced significant protective cytotoxic

CD8+ T cells targeting Env gp140, and Gag compared to lower induced immune response to

Tat (Liang et al., 2005). However, rAd5 vectors used in the phase 2 STEP study to express

HIV-1 Gag, Pol, and Nef antigens, induced insufficient T cell immune responses and no env-

specific antibody responses which failed to protect vaccinated individuals against HIV-1

infection (Barouch, 2010). However, serious adverse reactions after vaccination including

viremia were shown in participants. This was due in part to a high mutation of the virus

escaping from the host immune response (Flynn et al., 2005; Patterson and Robert-Gouroff,

2008). Furthermore, the recombinant adenovirus 5 expressing HIV-1 Gag protein (rAd5-gag)

was used in BALB/c mice as an intramuscular or intravaginal prime dose of prime boost

vaccine following the recombinant Listeria monocytogenes expressing HIV-Gag protein (rLm-

Gag). This was administered via different mucosal routes, such as orally, intrarectally or

intravaginally, and resulted in a robust Gag-specific CD8+ T cell response in the spleen and the

vaginal secretion and lamina propria. The best CD8+ T cell response detected in the vagina was

induced after the intravaginal route of vaccination, particularly in intravaginal-intravaginal

prime boost vaccinated mice that produced higher protection against intravaginal challenge

using vaccinia virus expressing the whole HIV-Gag protein compared to other routes of

vaccination (Li et al., 2008).

1.4.2.2.2. Modified vaccinia virus vectors Most HIV vaccine studies in humans or animals have utilized modified vaccinia virus as a safe

and effective vector for expressing viral proteins and for delivering them to mucosal surfaces

to induce a mucosal protective mucosal immune response against respiratory pathogens, such

as the influenza virus, respiratory syncytial virus and mycobacterium tuberculosis (Artenstein,

2008; Gherardi and Esteban, 2005; McCurdy et al., 2004). In addition, the recombinant

modified vaccinia virus vector (rMVA) has been used to deliver HIV or SIV antigens, induced

HIV specific immune responses not only in genital and rectal mucosa in mice or macaques but


36

also controlled HIV or SIV progression and dissemination of the virus (Gherardi and Esteban,

2005). For example, a modified vaccinia virus vector expressing the HIV-1 Env IIIB protein

(MVA-Env) conjugated to cholera toxin was given to BALB/c mice intranasally and

intravaginally, which induced mucosal CD8+ T cells with both IgA and IgG1 specific

antibodies (Gherardi et al., 2004). Another trial in the UK, using recombinant modified

vaccinia viruses expressing HIV-Gag proteins (p24 and p17) from HIV-1 clade A administered

intradermally in healthy males and females between 18-60 years of age after an intramuscular

priming dose of HIV DNA plasmid (expressing Gag protein of HIV-1 clade A). This vaccine

induced specific CD4+ and CD8+ T cell immune responses in 89% of volunteers (Mwau et al.,

2004). In a similar clinical trial, DNA containing HIV-Env gp160 (subtypes A, B, and C), Rev

(subtype B) and the p17/p24 Gag (subtype A) was injected intradermally or intramuscularly

into 40 healthy volunteers as a priming, followed by a recombinant HIV Env, Gag and Pol-

vaccinia virus boost. This vaccine trial induced IFN-γ+ from CD8+ and CD4+ T cells in 30% of

volunteers following the prime dose of vaccine, which increased to 92 % after boosting

(Sandström et al., 2008). Also, SIV proteins, such as Gag, Pol and Env, expressed by the

modified vaccinia virus Ankara (MVA) were injected intramuscularly into rhesus macaques

and then challenged intravenously using pathogenic SIVsm-E660, thus resulting in a reduction

of viremia in the vaccinated macaques (Ourmanov et al., 2000).

1.4.2.2.3. Canarypox virus vectors Canarypox is a strain of the avian pox virus strain from the Orthopoxvirus family. It is isolated

from the foci of the pox infection in canaries and attenuated by multiple passaging (200 serial

passages) in chicken embryo fibroblasts to produce recombinant virus vectored vaccines

(ALVAC) that express specific genes of pathogenic viruses, such as HIV, influenza, and

African horse sickness (Bruyn et al., 2004; Bublot et al., 2006; Guthrie et al., 2009).

Recombinant canarypox has also been used as a vector to express retroviral proteins, such as

the feline leukemia virus (FeLV) Gag and Env, and when given intranasally induced Gag and

Env specific cytotoxic T cell immune responses (Poulet et al., 2003; Tartaglia et al., 1993).

1.4.2.2.4. Rabies virus vectors The rabies virus is a non-segmented RNA virus of the Rhabdoviridae family. It is used as a

safe and immunogenic vectored vaccine inoculated in animals as a result of complete

inactivation of the virus (Faber, Dietzschold and Li, 2009). The recombinant rabies virus vector

(RV) has the ability to express a large viral genes, sequence of up to 4 Kb, such as the HIV-1

Gag-Pol precursor (Pr160) or 2.2 Kb of the Env protein which is necessary to elicit a broad


37

host immune response in mice and monkeys (Gomme et al., 2010; McGettigan et al., 2003;

Addo et al., 2001). For example, the recombinant rabies virus expressed HIV-1 Gag (SPBN-

ΔG-Gag) was given intramuscularly in female BALB/c mice as a priming dose and then

intraperintoneally administered with the vaccinia virus expressed HIV-1 Gag (VV-Gag). This

induced a significant increase of Gag-specific CD8+ T cell responses (Gomme et al., 2010).

Recombinant rabies expressing the HIV-1 Gag-Pol precursor protein was given

intramuscularly in BALB/c mice, and after 6 weeks they were challenged using an

intraperitoneal dose of the vaccinia virus expressing the whole HIV-1 Gag. This induced about

12-14% of CD8+ T cells specific for a Gag p24 epitope detected after 5 days of challenge

(McGettigan et al., 2003). Recombinant rabies viruses expressing the HIV-1 Env (gp 160)

protein were able to induce a robust humoral and T cell immune responsetargeting Env in

BALB/c mice vaccinated with a intramuscular-intraperitoneal prime boost regime (Schnell,

2000). Furthermore, BALB/c mice primed intramuscularly with the recombinant rabies virus

expressing the HIV-1 Gag protein induced a significant increase of cell response targeting the

HIV-1 Gag. This is in addition to 26.8% of Gag+IFN-γ+ producing CD8+ T cells in the

vaccinated mice after an intraperitoneal challenge with a recombinant vaccinia virus expressing

a HIV-1 Gag (McGettigan et al., 2001). Also, six adult male macaques (Macaca mulatta) were

injected intramuscularly using the rabies virus vector expressed SHIV-89.6P Env and

SIVmac239 Gag proteins as a priming dose, which induced seroconversion in vaccinated

macaques following their initial vaccination. After 45 weeks, they were given an intranasal or

intramuscular boost dose of the same vaccine but there was no immune response induced

following the boosting dose. This was as a result of preexisting antibodies for the G protein of

the rabies virus. Therefore, when this boosting vaccine was replaced by the G protein of the

vesicular stomatitis virus (VSV) expressing SHIV-89.6P Env protein and SIVmac239 Gag

protein, strong humoral immune responses and low T cell responses were induced. After four

months of the boost dose, all vaccinated macaques were intravenously challenged using the

pathogenic SHIV-89.6Pvirus. Induced robust neutralizing antibodies and T cell responses

resulted in a reduction of the viral load and maintained a level of CD4+ T cells in vaccinated

macaques compared to high viremia and rapid loss of CD4+ T cells in the non-vaccinated group

(McKenna et al., 2007).


38

1.5. Clinical trials of HIV vaccines 1.5.1. Phase 1 and phase 2 clinical trials Many HIV-1 candidate vaccines have been tested in Phase I and II clinical trials and found to

be safe and immunogenic. Some key examples include:

1) The ALVAC vaccine trial: This was the first clinical trial of HIV-1 vaccines involving 150

high and low behavioral risk HIV-1 uninfected volunteers (18-60 years old), using

recombinant canarypox vaccine candidates expressing whole Gag and Pol proteins (ALVAC-

HIV vCP205) alone or combined with the recombinant subunit Env protein boosting dose

(Envgp120). All volunteers were divided into 8 treated groups and then vaccinated

intramuscularly using various schedules of multiple doses of vaccines in addition to four

control groups followed for a period of 2 years. At the end of the vaccination period, a

significant cytotoxic T cell response (64-89%) was detected in all vaccinated groups depending

on the number of doses compared to control groups. A significant response of neutralizing

antibodies was also detected in all vaccinated groups compared to controls with no serious

diverse reactions which confirmed the vaccine was safe and immunogenic (Gupta et al., 2002).

A similar trial was also followed in phase II that involved 330 healthy HIV seronegative

volunteers using intramuscular administrating canarypox vaccine candidates (vCP1452)

expressing Gag and Pol proteins alone or combined with a Env gp 120 subunit vaccine boosting

dose. This result was strong neutralizing antibody response in the range of 57-95%, while

cytotoxic CD8+ T cell response was only 13-16% in all vaccinated groups (Russel et al., 2007).

2) The HIV Envgp120/Nef-Tat/AS02A vaccine trial: This was a randomized phase 1 trial of

the subunit protein vaccine conducted in 20 HIV-1 chronically infected volunteers who were

well controlled by highly active antiretroviral therapy (HAART). This vaccine included a

plasmid DNA encoding HIV-1 proteins, Nef, Tat and Env gp120 (clade B), and was conjugated

with AS02 adjuvant. It also included strong and highly significant HIV Env specific CD4+ cells

that produced IL-2 cytokine, with proliferation of both CD4+ and CD8+ T cells following

multiple doses of intramuscular immunization compared to controls (Lichterfeld et al., 2012;

de Rosa and McElrath, 2008).

3) The EUROVAC 02 trial: This was a randomized phase 1 trial using recombinant attenuated

poxvirus (vaccinia virus: NYVAC) expressing genes isolated from the HIV-1 clade C. These

genes included gag, env, pol and nef. This clinical trial involved 50 healthy participants (low

behavioral risk of HIV infection) who were injected intramuscularly by the recombinant


39

NYVAC vaccine alone or primed first by intramuscular administration of the recombinant

DNA expressing the same HIV-1 proteins. The prime boost vaccine induced a higher T cell

response, with 90% of participants compared to only 33% following the NYVAC vaccine only.

Also, this vaccine induced polyfunctional CD4+ and CD8+ T cells as IL-2+IFN-γ+TNF-α+

secreting cells. Furthermore, the highest and most frequent T cell responses targeted Env gp120

(91% of vaccinees) compared to only 48% for Gag-Pol-Nef. However, this vaccine has induced

lower antibody response, and was only IgG specific for the Env protein, which was detected in

75% of participants who received the DNA/NYVAC prime boost vaccine compared to only

25% induced for the NYVAC vaccine alone (Harari et al., 2008; Midgley et al., 2008).

4) The RV 172 trial: This was a randomized phase I/II clinical trial that included a mixture of

three plasmid DNA expressing HIV Env glycoprotein (isolated from HIV-1 clades A, B, and

C) and three plasmids encoding Gag, Pol, and Nef proteins (clade B), in addition to a boosting

dose of replication-defective recombinant adenovirus 5(rAd5) expressing the HIV-1 Gag/Pol

polyprotein (clade B) and Env glycoprotein (clades A, B, and C). This vaccine was conducted

in 324 of HIV-1 non-ninfected African participants from Kenya, Tanzania, and Uganda who

were injected intramuscularly with a single dose of rAd5 or intramuscular prime boost vaccine

(DNA/rAd5) of multiple dosesover 24 weeks. Induced HIV-specific T cells responses were

represented by IFN-γ+ ELISPOT in 63% of the total participants, with no significant difference

between T cell responses specific for Env, Gag and Pol induced in groups vaccinated by rAd5

alone (63%) or prime boost DNA/rAd5 (62%). Also, there was a low level of HIV specific

antibody responses in all groups and no effect of preexisting Ad5 neutralizing antibodies on T

cell responses in prime-boost vaccinated individuals (Kibuuka et al., 2010).

5) The trial (HVTN 041): This was a randomized phase 1 trial conducted in 84 HIV-1

uninfected volunteers who were injected intramuscularly with multiple doses of HIV-1

recombinant proteins included Env gp120, Nef and Tat (HIV-W61D). These were expressed

by recombinant Pichia pastoris yeast cells, and formulated with AS02A adjuvant. This vaccine

induced robust neutralizing antibodies in all vaccine recipients following the second week with

third dose of the vaccination. There was also a strong titer of binding antibody responses

induced specifically for Env gp120, which was detected by antibody-dependent cellular

cytotoxicity assay (ADCC), also with a significant increase of HIV-specific CD4+ T cells.

However, no CD8+ T cell response was detected in vaccinated individuals (Goepfert et al.,

2007).


40

1.5.2. Phase 3 clinical trials 1) The VAX004 trial: This was a double-blind and randomized trial held in Canada and the

Netherlands in 1998-1999. This included two recombinant HIV-1 Env gp120 proteins, MN-

rgp120/HIV-1 and GNE8-rgp120/HIV-1 clade B administered intramuscularly within multiple

doses in 5403 healthy volunteers, such as men who had sex with men and women at risk of

heterosexual transmission of HIV-1. This vaccine was used as a subunit vaccine with multiple

doses to induce specific Env gp120 neutralizing antibodies. This clinical trial of HIV vaccine

resulted in low specific binding neutralizing antibodies (nAbs) that led to a low level of

immuno-efficacy (6%) but with no significant overall protection in vaccinated individuals

(Flynn et al., 2005). A similar trial called the VAX003 trial was also conducted in 2546 healthy

volunteers among heavy intravenous drug users at high behavioral risk of HIV-1 infection.

This trial also used also two recombinant Env glycoproteins, MN-rgp120/HIV-1 and A244-

rgp120/HIV-1 (clade B and E). Volunteers were injected intramuscularly within multiple doses

held in Thailand in 1999-2000. However, this trial only resulted only 0.1% efficacy in all

vaccinated participants without a significant difference between vaccinated and control

individuals following 36 months of vaccination (Pitisuttithu et al., 2006).

2) The STEP trial: This trial started in 2004 and concluded in 2007 with approximately 1500

healthy participants who were at high risk of HIV infection, including homosexual men and

female sex workers. This trial included the development of recombinant adenovirus serotype 5

(rAd5) by Merk, which was used as a vector for expressing the HIV-1 whole proteins, Gag,

Pol and Nef (MRKAd5-gag/pol/nef). To begin with results were promising as this vaccine

induced a CD8+ T cell response in intramuscular vaccinated volunteers without a serious

adverse reaction. However, the stimulated immune response could not prevent the transmission

and progression of a HIV-1 infection and the trial was terminated because of an increased risk

of HIV-1 infection represented by a high titer of the virus at the last stage of the trial. 40,000

copies/ml were detected in the vaccinated group and 37,000 copies/ml in the control group.

This was as a result of the high prevalence of preexisting Ad5-specific neutralizing antibodies

that led to no suitable efficacy after vaccination (O’Brien et al., 2009; Takahashi, Rolling and

Owen, 2010; Zak et al., 2012).

3) The RV144 phase 3 trial: This trial used a combination of the recombinant Canarypox live

virus vector (ALVAC-HIV,vCP1521) that was genetically modified to express the HIV-1 Gag

and Pro protein obtained from the HIV-1 subtype B combined with Env gp120 protein (subtype


41

E) boost with (AIDSVAX B/E HIV-subtypes) the vaccine containing bivalent Env

glycoproteins. This was undertaking during two trials. The first was conducted in Thailand in

1999-2000 with a high risk group including 2546 intravenous drug users using the AIDSVAX

B/E HIV (VaxGen) vaccine for 36 months. No protection was induced against HIV-1 infection

in vaccinated subjects (Pitisuttithum et al., 2006). The second was also held in Thailand,

involved 16395 subjects divided into three groups: female sex workers, drug users and

homosexual men, and the results of this trial were concluded in 2009 (Rerks-Ngarm et al.,

2009; Vaccari, Poonam and Franchini, 2010). As a result of a HIV-specific response (90.1%)

and (49.3%), a T cell proliferation induced in the vaccinated participants targeting both Env

and Gag proteinsalong with the T cells that produced an interferon-γ, were also detected in

19.7% of vaccine recipients by ELISPOT, and 11.1% and 7.6% of IFN-γ+CD8+ T cells were

detected for Env and Gag by ICS. Also, binding antibody responses induced for Env (98.6%)

and Gag (52.1%) were detected by antibody-dependent cellular cytotoxicity assay (ADCC)

(Rerks-Ngarm et al., 2009). Overall results demonstrated partial protection (31.2 %) with

modest reduction in the rate of HIV-1 infection (Pitisuttithum et al., 2010; Rerks-Ngarm et al.,

2009). This provides hope for future work to control HIV/AIDS infection rates (Pantaleo et al.,

2010; Vaccari, Poonam and Franchini, 2010).

1.6. Influenza virus Influenza virus is a single stranded, negative sense segmented RNA virus infecting a wide

range of mammals and bird species. It belongs to the Orthomyxoviridae family and causes

respiratory illness, ranging from mild respiratory symptoms to severe pneumonia causing

death, particularly in infants, elderly and immuno-compromised individuals (Barnard, 2009;

Bouvier and Lowen, 2010). Five influenza virus genera have been identified: A, B, C,

Thogotovirus and Isavirus (Kawaoka et al., 2005). Influenza virus type A and B are comprised

of a genome with eight RNA segments compared to type C viruses, which have seven segments

(Lamb and Krug, 1996; Lewis, 2006). The three types differ in their pathogenicity and ability

to infect humans (Mateo et al., 2007). The most pathogenic influenza viruses are the type A

viruses which are characterized by high virulence causing the most severe disease in humans.

For instance, the H1N1 influenza virus infected humans and led to a high mortality rate with

the Spanish flu in 1918 (Johnson and Mueller, 2002), the Asian flu of 1957 (the result of a re-

assorted H2N2 virus), the Hong Kong flu of 1968 (caused by a re-assorted H3N2 virus), and

the swine flu in 2009 (caused by re-assorted H1N1) (Garten et al., 2009; Klenk, 2005). In

addition, the avian re-assorted H5N1 influenza virus is considered one of the highest


42

pathogenic influenza viruses in humans as it has the ability to infect humans leading to severe

complications, such as multiple-organ and respiratory failure and ending with death (de Jong

et al., 2006; Uyeki et al., 2009; Tran et al, 2004; Beigel et al., 2005).

Influenza A viruses have a spherical or filamentous virion with a diameter of 80-120 nm. These

viruses have two surface glycoproteins, hemaglutinin (HA) and neuraminidase (NA),

embedded in a lipid envelope surrounding the virus in addition to other viral glycoproteins,

such as matrix proteins (M1 and M2), nucleoprotein (NP), polymerase complex proteins PB1,

PB2 and PA, and non-structural proteins NS1, NS2 (Palese and Shaw, 2007; Jagger et al.,

2012). HA plays a direct role in regulating the infectivity of the influenza virus through

attachment to the host cell membrane by binding to sialic acid on specific receptors located at

the cell surface. NA is responsible for the assembly and release of new progeny viruses from

infected cells. There are 17 subtypes of HA and 9 subtypes of NA amongst influenza A viruses

(Kaverin et al., 2000; Pushko et al., 2005; Taubenberger, 2006; Tong et al., 2012). Therefore,

antigenic drift caused by gradual accumulated mutations in highly variable regions of HA

resulting seasonal influenza epidemic outbreaks, with antigenic shift introducing new subtypes

of influenza A viruses in the human population, thus leading to pandemic outbreaks of an

influenza virus infection (Smith et al., 2004; Herfst et al., 2012). Both mutation mechanisms

enable influenza viruses to evade an immune response through escape recognition by virus

specific antibodies because of an absence of neutralizing antibodies to these viruses in the total

population (Fouchier et al., 2005; de Jong et al., 2000).

1.6.1. Pathogenesis of the influenza virus Influenza viruses have a high affinity for replicating at the mucosal surfaces of the upper and

lower respiratory tract. The first step of influenza infection is the attachment of the virus to

molecules on the surface of the target cells by the haemagglutinin glycoprotein (HA) binding

to the epithelial surface receptor (sialic acid) followed by the cleavage of HA into two linked

subunits, HA1 and HA2, by membrane-associated proteolytic enzymes. This enables the virus

to enter the cytoplasm of the target cells and then replicate in these infected target cells

(Taubenberger, 2006; Zhirnov, Ikizler and Wright, 2002). Following the first few hours of

initial infection, newly forming viruses are released to infect other cells facilitated by the

neuraminidase glycoprotein (NA) (Lamb and Krug, 1996; Murphy and Webster, 1996). The

infectivity of the influenza virus depends on the level of HA cleavage. For example, highly

pathogenic influenza viruses, such as H1, H5, and H7 subtypes, are characterized by high levels


43

of HA cleavage compared to low pathogenic or non-pathogenic influenza viruses (Senne et al.,

1996; Shortridge et al., 1998). After its replication in respiratory epithelial cells, the influenza

virus subsequently infects macrophages and monocytes located in the alveoli, leading to the

destruction of these cells and the formation of necrotic areas as a result of damaged and dying

cells accumulating in these locations and ending with pneumonia (Fesq et al., 1994;

Taubenberger and Morens, 2008).

There are different models of laboratory animals that are characterized with high sensitivity to

infectin by influenza viruses and can be used in preclinical experiments to evaluate vaccines

produced for influenza viruses. These animal models include animals naturally susceptible to

be infected by human influenza viruses like ferrets and guinea pigs, and other animals which

need adaptation to be infected by influenza viruses like mice (Bouvier and Lowen, 2010). The

susceptibility of mice to be infected by the influenza viruses depends on the strain of mice and

the laboratory adapted influenza viruses. For example, BALB/c and C57BL/6 mice are

considered the main commonly laboratory strains used in research. These mice are the highest

sensitive strain of mice infected intranasally with adapted influenza viruses, such as A/Puerto

Rico/8/1934 (H1N1; PR8), A/WSN/1933 (H1N1; WSN) or A/Aichi/2/68 (H3N2; X31). These

viruses replicate in the respiratory tract and lead to weight loss or death depending on the

pathogenic strain and lethal doses of the influenza viruses used in the two lab strains of mice,

with wild mice mainly resistant to these influenza viruses (Staeheli et al., 1988; Haller, 1981).

Indeed, the ability of these mice adapt to the influenza viruses that induce the pathogenic effects

following primary intranasal infection depends on the dose of infective viruses. For example,

BALB/c mice start losing weight on day 2 of intranasal infection of a highly lethal dose (10x

the 50% lethal dose, LD50) of the PR8 (H1N1: A/PR/8/34) influenza virus strain (2 × 103 pfu),

and died between days 6 to 8 of infection as a result of rapid replication of the virus in the

lungs. The X31 (H3N2: A/Aichi/2/68) influenza virus strain resulted in less pathogenic effects

detected in BALB/c mice who were infected intranasally using (10× LD50) 3.5 × 106 pfu of

X31, with gradual weight loss beginning with day 2 of infection and then death on day 10

(Quan et al., 2008; Yetter et al., 1980; Tamura et al., 1992). This is compared to a sub-lethal

dose of the X31 influenza virus of 1× 104 pfu which showed a peak of viremia within days 3 to

5 of primary intranasal infection accompanied with gradual weight loss in BALB/c mice. This

declined within days 6 to 7 to a complete recovery and clearance of the virus on day 10 of

infection (Cukalac et al., 2009).


44

A similar course of infection was also detected after the intranasal influenza infection in

BALB/c mice using a sub-lethal dose of 50 pfu of the PR8 influenza virus but with higher

weight loss (Edenborough, Gilbertson and Brown, 2012; Tamura and Karuta, 2004; Tamura et

al., 1998). Similar results were also detected in BALB/c mice infected by a sub-lethal dose of

the PR8 influenza virus, 10 TCID50. The infected mice showed a short period of mild weight

loss and then began to recover on day 7 post-infection, with complete recovery on day 9 post-

infection. This was compared to a moderate pathogenic effect of a higher dose (100 TCID50)

of the PR8 (A/PR8/34) influenza virus represented by higher weight loss that started on day 3

of infection, and reached 25% weight loss. These infected mice began to recover on day 10 and

then reached their starting weight within 10-14 days post-infection. While rapid viremia and

severe weight loss were detected in mice after intranasal infection using a higher lethal dose,

1000 TCID50 of the PR8(A/PR8/34) influenza virus saw mice reaching 30% weight loss on day

8 and then dying on day 15 post- infection (Verhoeven, Teijaro and Farber, 2009).

1.6.2. Influenza immunity Mucosal surfaces represent not only the main portal of entry for influenza, but also the first

line of non-specific defense. This involves the epithelium, mucin layer, non-specific cytokine

responses and secretory IgA released by the epithelial cells. Following the infection of

epithelial cells, the innate and adaptive immune system play the major role in defense (Lamb

and Krug, 1996; Murphy and Webster, 1996; Tamura and Kurata, 2004).

1.6.2.1. Innate immune response Innate immune response is the first line of defense mechanisms against an influenza virus

infection characterized by a fast onset and a short period of effectivity following primary

influenza infection. Innate immunity includes several components facilitated by recognition of

influenza antigens that also induce other parts of the immune response to control infection

(Banchereau and Steinman, 1998; Guermonprez et al., 2002). Dendritic cells (DCs) are

considered the main professional antigen presenting cells (APCs) during early influenza

infection by degrading viral proteins into small peptides by proteasomes or phagocytosis.

Dendritic cells transport these small peptides to the endoplasmic reticulum and then load to

MHC class I molecules to be recognized by the CD8+ T cells or present them to the MHC class

II peptide complexes and then recognized by CD4+ T helper cells (Bhardwaj et al., 1994;

Yewdell et al., 2003). As a result of early recognition of influenza viral antigens, naïve T and

B lymphocytes are activated to control infection. Dendritic cells also work as a link between


45

an innate and adaptive immune response, induced activation and proliferation of cytotoxic

CD8+ T cells against influenza viral antigens, and also migrate to various draining lymph nodes,

such as the mediastinal lymph node (Braciale, Sun and Kim, 2012; Kim and Braciale, 2009).

Influenza viruses are first detected after a few hours of primary infection in an infected human

or in animals by the innate immunity.Viruses are then destroyed as a result of non-antigen

specific mechanisms involving macrophages, natural killer cells, and cytokines, such as IFN-

α and β interferons. These are not active for a long time during induction, thus giving influenza

viruses the opportunity to escape this early mechanism of defense (Murphy and Webster, 1996;

Ada and Jones, 1986). High amounts of IFN-α and β, secreted by influenza infected cells, are

rapidly detected following influenza infection in both nasal and pulmonary regions and their

level is directly related to reducing the replication of influenza viruses in humans and animals

(Husseini, Sweet, Collie and Smith, 1982; Wyde, Wilson, and Cate, 1982). Natural killer cells

are cytotoxic lymphocytes activated in an innate immune response that israpidly detected in

upper respiratory mucosa and the lungs within 24 hours following primary influenza infection.

These cells produce IFN-γ in addition to their direct role in reducing the spread of the virus by

the lysis of influenza infected cells enhanced by perforin activity (Hicks et al., 1978; Kopf et

al., 2002). IFN-γ acts as a linking factor between the innate and adaptive immune response

against influenza infection as it induces the cytotoxic CD8+ T cell response and regulates

homeostasis and proliferation of these cells in secondary lymph nodes and their trafficking to

the site of infection necessary to control the influenza infection (Turner et al., 2007; Bot, Bot,

and Bona, 1998). The early innate immune response for the influenza virus includes some

inhibitory factors located in the respiratory tract mucosa, which have an identical structure to

acetyl-neuraminic acid biding to the HA glycoprotein and highly reduces its infectivity and

spread of influenza viruses inthe host cells of BALB/c mice infected nasally with mouse

adapted influenza viruses such as the PR8 influenza virus (A/PR/8/34; H1N1) (Yoshikawa et

al., 2004; Shugars, 1999). Also, macrophages localized in the alveoli have an important

inhibitory role against influenza virus infection by direct lysis of infected cells or influenza

virus particles by phagocytosis. Activated macrophages in alveoli also induce a pro-

inflammatory response, including IL-6 and TNF-α (van Riel et al., 2011; Becker, Quay and

Soukup, 1991), and they regulate an adaptive immune response as there is a direct correlation

between the depletion of alveolar macrophages leading to low levels of antibodies and a

reduction of CD8+ T cells induced following influenza infection (Kim et al., 2008). There is


46

also an indirect immune role of macrophages during early influenza infection by activating

natural killer cells through their secreting specific cytokines, such as IL-1, IL-6, IL-12 and

TNF-α, which are also necessary for decreasing virus shedding following primary influenza

infection (Monteiro, Harvey and Trinchieri, 1998).

However, influenza viruses easily escape the early innate immunity defense mechanism but

these viruses can be detected and eliminated by the main active components of the adaptive

immune mechanism, such as influenza specific antibodies and cytotoxic T cells targeted

influenza virus and the infected cells (Tamura and Kurata, 2004). There was a high level of

both T and B cells accumulated in the upper respiratory mucosa of BALB/c mice intranasally

infected with the A/PR/8/34 influenza virus. The infected mice reached their peak on day 7

post-infection. The first detected antibody response was on day 5 post-infection and reached

its peak on day 7 of infection, with IgM being the first antibody detected for the primary

influenza infection, followed by IgA and IgG (Tamura et al., 1998). IgM was first detected in

a few days of primary influenza infection and then diminished quickly within a few days (Qiu

et al., 2011). This is in comparison to IgA and IgG immunoglobulins, which are the only

immunoglobulins that detected following secondary influenza infection (Tamura et al., 1998;

van de Sandt, Kreijtz and Rimmelzwaan, 2012).

1.6.2.2. Adaptive immune response Adaptive immune response is the second line of the defense mechanism for influenza virus

infection characterized by slow induction against primary infection but with faster onset and

stronger effectiveness for the secondary influenza infection (van de Sandt, Kreijtz and

Rimmelzwaan, 2012). The adaptive immune response includes two parts, humoral immune

response represented by influenza-specific antibodies produced by B cells, and cellular

immune response represented by T cell response, which includes both CD4+ and CD8+ T cells

(Tamura et al., 1998; van de Sandt, Kreijtz and Rimmelzwaan, 2012; Baumgarth et al., 2000).

1.6.2.2.1. Humoral immune response The humoral immune response represents the main defense against primary influenza infection

in humans (Strugnell and Wijburg, 2010; Lamb and Krug, 1996), especially in young infants

who are mainly protected against influenza virus infection by maternal antibodies produced

following vaccination in humans and mice (Hwang et al., 2010; Zuccotti et al., 2010). The

early humoral immune response against influenza virus infection is characterized by the

production of specific IgM, IgA and IgG antibodies, while later stages involve IgA and IgG


47

only (van de Sandt, Kreijtz and Rimmelzwaan, 2012; Qiu et al., 2011). IgM antibodies are

highly active in neutralizing the influenza virus, but only for a short lived period. They also

contribute to activating the complement system necessary to inhibit virus replication

(Fernandez Gonzalez, Jayasekera and Carroll, 2008; Jayasekera, Moseman, and Carroll, 2007).

IgA works as the predominant immunoglobulin necessary for recovery from recent influenza

infection in humans (Tamura et al., 1998). On the other hand, IgG antibodies directed for

influenza antigens are characterized with a long lived protective role against influenza infection

(Koutsonanos et al., 2011; Onodera et al., 2012). Most immunoglobulins mainly target

influenza HA, NA, NP and M proteins, although the most predominant antibodies are those

induced by HA and NA, which are highly involved with protective immunity against influenza

infection. The Ig-molecules play a major role in preventing replication of the influenza virus

(Glathe, Bigl and Grosche, 1993; Lamb and Krug, 1996; Murphy and Webster, 1996). For

instance, antibodies directed to HA bind tothe receptor located on the globular head of HA, and

prevent the virus from binding to targeted host cells (de Jong et al., 2003; Knossow and Skehel,

2006), and most HA-specific antibodies are not effective for neutralizing new variants of

influenza subtypes produced by antigenic drift mutation (Yu et al., 2008; de Jong et al., 2000).

Antibodies produced targeting the NA glycoprotein act either by inhibiting the sialidase

activity of NA and preventing the release and spread of newly formed viruses from virus

infected cells, or antibodies are actively involved in destroying virus-infected cells by

facilitating the ADCC mechanism important for virus clearance (Palese and Shaw, 2007;

Hashimoto, Wright and Karzon, 1983; Bosch et al., 2010). Furthermore, antibodies induced

against influenza NP are potentially involved in protection against hetero-subtypes of influenza

A viruses as it is highly conserved glycoprotein between these viruses, and their protective role

may include ADCC mechanism of infected cells and activating T cell responses in human and

mice (Lamere et al., 2011; Sambhara et al., 2001; Carragher et al., 2008).

The protective role of influenza specific antibodies was also evident between 1991 and 1992.

Using one dose of trivalent influenza vaccine containing A/Singapore/6/86 (HIN1),

A/Beijing/353/89 (H3N2) and B/Yamagata/16/88, five different groups of people were

vaccinated. The result was an increased level of antibodies directed for HA, with 96-100%

protection in the vaccinated groups (Glathe, Bigl and Grosche, 1993). Furthermore, the

therapeutic role of antibodies to control influenza infection was proven in B-cell-deficient

muMT (-8) mice, who were highly susceptible to the intranasal infection of mouse-adapted


48

influenza type A virus A/PR/8/34 (H1N1). Around 80%-94% of these infected mice survived

by intravenous transferred spleen cells obtained from normal B6 mice and included a higher

virus-specific precursor B cell response. This was compared to muMT (-8) mice which did not

produce antibodies and were highly sensitive to influenza infection, with BCR-transgenic

spleen cells transferred, these cells contained 10 times lower virus-specific precursor B cells,

and were not protected from PR8 influenza infection (Mozdzanowska et al., 2005). Also,

specific-influenza antibodies have an important role in the control of primary influenza

infection in mice using intranasal PR8 infection. This was detected by a higher increase of the

virus titer in B cell-deficient (muMT) mice compared to less sensitive IgM-/-and wild-type

infected mice (Kopf et al., 2002).

1.6.2.2.2. Cellular immune response Cellular immune response for influenza A viruses consist of CD4+ and CD8+ T cells as they

have a regulatory role for influenza specific immune response and recovery from infection (van

de Sandt, Kreijtz and Rimmelzwaan, 2012). Indeed, the cellular immune response is more

efficient than the humoral arm in the protection against influenza virus infection because the

humoral response is induced specifically for each virus subtype and is less efficient in

recognizing newly mutant influenza A subtypes resulting from antigenic drift. This includes

altered HA or NA compared to high cross activity of cytotoxic T cells in recognition of hetero-

subtypes of influenza A viruses, thus giving the cellular immune response a higher efficient

role in the clearance of influenza virus infection in humans (Biddison, Shaw and Nelson, 1979;

Shaw and Biddison, 1979; McMichael and Askonas, 1978). For instance, CD4+ and CD8+ T

cells are mainly involved in broad cross-protection for hetero-subtypic influenza viruses. This

was recognized by impaired hetero-subtypic protection represented by no efficient control of

virus replication in acute CD4 and CD8 depleted mice infected intranasally using a prime dose

of H1N1, H2N2 or H3N2. The mice were then challenged intranasaly using H1N1 or N3N2

influenza viruses, represented by higher virus titer in the lungs and a higher mortality rate

compared to depleted Ig-/- immunoglobulin infected mice (Benton et al., 2001). This protective

role was also recognized by intranasal inoculation of the live influenza virus, A/Munich/1/79

virus (H1N1) into 63 volunteers conducted in the United Kingdom to check their levels of

Hemagglutination-inhibition titer (HAI). An anti-neuraminidase titer and cytotoxic T cell

response in infected individuals, with induced cytotoxic T-cell responses higher than 10% with

no shedding of the virus following three weeks of infection was compared to lower effective

antibodies in the clearance of the influenza virus (McMichael et al., 1983).


49

As the first component of cellular response in regulating immune responses to influenza

infection, CD4+ T cells are activated directly after recognition of influenza viral epitopes loaded

with MHC class II molecules. They are then differentiated into two functional types, T helper

1 cells (Th1) or Th2 CD4+ T cells based on the cytokine they produce (Zhu and Paul, 2010).

The T helper 1 lymphocyte secretes IFN-γ, TNF-α and IL-2 is also directly involved in CTL

responses and induction of memory CD8+ T cells (Mosmann et al., 1986; Deliyannis et al.,

2002). The CD4+ T helper 1 cell also mediates delayed types of hypersensitivity (DTH)

reactions inhibiting the replication of the influenza virus bysecreting IFN-γ (Cher and

Mosmann, 1987; Tamura et al., 1996). In addition, robust IgG and IgA antibodies are induced

following intranasal influenza infection in mice produced by the CD4 T helper 1 lymphocyte

(Topham et al., 1996; Roman et al., 2002). While the CD4+ T helper 2 lymphocyte plays an

important role in producing IL-4, IL-5 and IL-13, and in activating B cells to produce

antibodies, such as IgG1 and IgE in BALB/c mice (Gerhard et al., 1997). In addition, it also

acts as a bridge between CD4+ and CD8+ T lymphocytes that are required for a protective

immune response against hetero-subtypic influenza A viruses, which share the same internal

proteins but differ in HA and NA subtypes (Okoye and Wilson, 2011; Lamb et al., 1982;

Kamperschroer et al., 2006). Furthermore, the CD4+ T cell has another protective role in the

cell mediated immune response following intranasal PR8 influenza infection in BALB/c mice.

These mice were transferred intravenously with the CD4+ effectors harvested from TCR Tg

BALB/c mice directly with the HA126-138 peptide from influenza A/Puerto Rico/8/34 (PR8)

CD4+ T cells 18-24 hours previous to an influenza challenge. The function of these CD4+ T

cells in the transferred mice was represented by enhanced perforin and granzymes to lyse

influenza-infected cells, inducing high amounts of anti-influenza antibodies, thus leading to

clearance of the influenza virus and significant survival of the mice compared to controls

(Brown et al., 2006). Also, memory CD8+ T cells were mainly induced following primary

influenza infection and then localized in the respiratory airways compartments especially in

the lungs, to provide faster control of secondary influenza infection (Teijaro et al., 2011; Strutt

et al., 2010).

The protective role of CD4+ T cells in clearance of the influenza virus infection in mice was

also evident in CD4- depleted C57BL/6J (B6) (Ig-/- mMT) mice infected intranasally using a

prime or prime boosting dose of HK-X31 (H3N2) influenza A virus. Results obtained from this

study indicated a significant delay of influenza virus clearance from lungs harvested from CD4-


50

depleted mice for a period extending from one to three months of infection. A significantly low

percentage of survival for the CD4- depleted (-CD4mMT) challenged mice was detected and a

significantly diminished number of recruitment CD8+ T cells directed to Db NP366, which were

detected in BAL and the spleen compared to the control group of mice (Riberdy et al., 2000).

Also, there was highly significant reduction of secreted cytokines, such as IL-2, IFN-γ, IL-4

and IL-5, detected in draining lymph nodes, mediastinal LN and BAL of the CD4-deficient

mice following intranasal influenza infection compared to the wild type of mice (Tripp,

Sarawar and Doherty, 1995). This was highlighted the very important role of CD4+ T cell in

clearing influenza virus by those secreted cytokines at the site of the infection. Furthermore,

the μMT mice generated from BALB/c back-cross mice, and then maintained as homozygous

CD4- depleted mice (-CD4 μMT-/-) by treating them with anti-CD4 depleting antibodies, did

present with a high mortality rate (80%) when they were infected intranasally using a small

dose of a virulent strain of the PR8 influenza virus. This was in comparison to only 15% in

normal μMT mice (Mozdzanowska, Maiese and Gerhard, 2000).

The CD8+ T cell is the second major component of cellular immune response induced to control

primary or secondary influenza virus infection (Benton et al., 2001; Hemann, Kang and Legge,

2013). After influenza virus antigen has presented by the MHC-I molecule and loaded on

antigen presenting cells (APCs), a T cell receptor (TCR) of a naïve CD8+ T cell binds to a

specific influenza peptide for activation (Norton et al., 1992). The activated CD8+ T cells

undergo high differentiation into cytotoxic CD8+ T cells localised in respiratory lymphoid

organs, especially in draining lymph nodes. These cells then migrate to the site of the infection

and destroy infected cells through lytic activity of perforin and granzymes, and inhibit the

replication and the spread of newly formed viruses by producing pro-inflammatory cytokines,

such as IFN-γ and TNF-α (Nakanishi et al., 2009; Topham, Tripp and Doherty, 1997).

Influenza specific CD8+ T cells are characterized by high cross-reactivity against

herterosubtypes of influenza A viruses in humans when they directed to the highly conserved

internal viral proteins, such as NP, PA, PB2 and M, among various influenza virus subtypes

(Assarsson et al., 2008; Kreijtz et al., 2008).

The protective role of CD8+ T cells, response directed for a heterosubtypic immune response

was also detected in mice following influenza A virus infections (Grebe, Yewdell, and

Bennink, 2008). For instance, C57BL/6 mice primed intranasally with the influenza virus X-


51

31 (H3N2) and then challenged intraperitoneally following 4 weeks with a virulent strain of

the A/PR/8/34 (H1N1) virus, presented a higher significant level of specific CD8+ T cells

detected for the H-2Db restricted NP366–374 epitope, with fast clearance of the A/PR/8/34 virus

from the lungs and increased survival rates of mice compared to controls which were not

primed with the X31 virus. Furthermore, it was evident that there were no neutralizing

antibodies directed to the HA or NA of the PR8 challenge virus, allowed the heterosubtypic

CD8+ T cell response to be protective against the virulent challenge virus (Kreijtz et al., 2007).

The hetero-subtypic protective CD8+ T response detected in C57BL/6J mice primed

intranasally with the human influenza virus, A/Hong Kong/2/68 (H3N2), which was

characterized by the ability to replicate in mice, actually protected mice from the intranasal

challenge with the highly pathogenic influenza virus A/Indonesia/5/05 (H5N1). This robust

hetero-subtypic CD8+ T cell response was mainly directed to the highly conserved influenza

epitopes, such as NP366–374 and PA224–232, and inhibit replication of the virus in the lungs and

significantly reduce body weight loss and mortality rates compared to unvaccinated infected

mice with the highly pathogenic avian influenza virus (HPAI A/Indonesia/5/05) (Kreijtz et al.,

2009).

Indeed, the majority of cross-reactive immune response against influenza A viruses included

CD8+ cytotoxic T lymphocytes that were directed toward the highly conserved viral proteins,

such as NP and M1. These CD8+ T cells were detected in the spleen of the CBA/J strain of

mice after 5 days of intraperitoneal administration of hetero-subtypes of influenza A viruses,

such as PR8 (A/PR/8/34: H0N1), and AA (A/Ann Arbor/23/57: H2N2) (Effros et al., 1977).

Therefore, the effective vaccine used for protection against various subtypes of influenza

viruses needs to induce the heterosubtypic immunity (HIS) included specific CD8+ T cells that

recognize influenza specific epitopes located in the highly conserved six shared internal viral

proteins (Grebe, Yewdell, and Bennink, 2008; Hillaire, Osterhaus and Rimmelzwaan, 2011).

Several studies related to influenza vaccines in humans and mice have shown the most

commonly detected proportion of the CD8+ T cell response has been that directed toward the

the relatively conserved NP and M1 protein, which allows cross-protection against various

subtypes of influenza viruses (Yewdell et al., 1985; Kees and Krammer, 1984; Lee et al., 2008).

The protective role of the CD8+ T cell in controlling influenza virus infection was also

associated by a significant delay with virus clearance in CD8+ T cell-deficient mice (Thomas


52

et al., 2006). As recognised in transgenic homozygous B2-M mice (-/-), lack of MHC-I

molecules and being unable to produce mature CD8+ T cells showed a significant delay in the

clearance of the from the lungs until day 15 after intranasal administration of the mice using

influenza viruses, such as A/Port Chalmers/1/73 (H3N2) or A/Puerto Rico/8/34 (H1N1). This

was in comparison to complete clearance of the virus between day 6 and 8 in normal mice or

heterozygous B2-M (+/-) mice who were able to express MHC-I molecules (Bender et al.,

1992). The CD8+ T cell response induced following both primary and secondary influenza

virus infection in mice was detected within a highly protective role in humans and mice (Flynn

et al., 1998; McMichael et al., 1983). The CD8+ T cells were induced firstly in lungs and BAL,

and later detected in MLN of the C57BL/6J (B6) in significant numbers after seven days

following the intranasal primary infection using a sub-lethal dose of A/HKX31 (H3N2) which

was higher than that induced for primary intranasal infection using the B/HK influenza virus.

The percentage of NP366-374 specific CD8+ T cell responses induced robust influenza specific

CD8+ T cell responses in the BAL of X31 infected mice with 7.3% compared to less than 0.1%

for the CD8+ T cell induced for the primary infection the B/HK influenza virus (Flynn et al.,

1998). This was because the virus did not include the NP366-374 peptide (Allan et al., 1993).

However, influenza specific CD8+ T cells that produced IFN-γ+ were first detected after five

days of the secondary intranasal challenge using the PR8 influenza virus, and the detected

response in BAL was 5 fold higher than that detected in the primary response at the peak of

response on day 10 of infection (Flynn et al., 1998). The CD8+ T cells were also detected

expressing IFN-γ+ directed to endogenous influenza H2-Kd NP147 in BALB/c mice following

secondary intranasal infection using a prime boosting dose of recombinant X31-NA-Env311

plus PR8-NA-Env311. This was higher than the primary response of 9 fold in the spleen and 4

fold in the BAL (Cukalac et al., 2009).

Moreover, it was also detected that the highly important protective function of CD8+ T cells in

B cell–deficient mice promoted their recovery from lethal dose influenza virus infection. The

B cell deficient mice (μKO mice; H-2b) were firstly intranasally inoculated using 10x LD50

influenza A/Japan/57 (H2N2) virus and 30 minutes later they were intravenously administered

with 107 clone cells (including CD8+ T cells) obtained from spleen cells. The cells were

harvested from μKO and C57Bl/6 mice who had received a sub-lethal intranasal dose of A/

Japan /57 and stimulated in vitro with influenza A/ Japan /57 virus-infected cells. This induced

complete protection of CD8+ T cell transferred mice represented by the fast clearance of the


53

virus from the lungs compared to non-transferred mice who were more than 100-fold more

susceptible to a lethal virus infection than the wild-type C57Bl/6 mice. On the other hand, there

was only a partial protective role of CD4+ cells adoptively transferred in B cell-deficient or

antibody-deficient mice (Graham and Braciale, 1997; Epstein et al., 1998).

1.6.3. Influenza virus as a vaccine vector Influenza virus type A has been used as an efficient viral vector for the expression of

recombinant viral antigens, particularly those targeting various infectious diseases, especially

mucosally transmitted diseases (Marsh and Tannock, 2005; Satterlee, 2008; Smith, 2007). For

instance, HIV, tuberculosis and malaria antigens have been inserted into influenza virus vectors

for use for vaccination (Martínez-Sobrido and García-Sastre, 2007). This is due to the ability

of this virus to infect through mucosal portals, resulting in broad and long term humoral

immune responses (Mueller et al., 2010; Yao and Wang, 2008).

It is also relatively easy to manipulate gene segments of the influenza virus by reverse genetics

to generate novel recombinant viruses for use as vaccine vectors (Steinhauer and Skehel, 2002;

Steinhauer et al., 2009; Hoffmann et al., 2002). Furthermore, influenza virus vectors are not

integrated in the host chromosomal sequences, and so will not induce any adverse mutation in

the host DNA, and there is no ability for influenza viruses to progress into a persistent or

chronic infection in humans, which avoids neutralizing antibodies affected by immune

response induced by the virus vector (Martínez-Sobrido and García-Sastre, 2007). Most of the

recombinant influenza viruses generated by reverse genetics have involved the manipulation

of HA and NA genes (Hwang et al., 2000; Kobayashi et al., 1992; Poon et al., 1998). The

strategy of reverse genetics has been used for developing many live vaccine vectors generated

from different segmented and non-segmented viruses, such as the influenza virus (Neumann,

Whitt and Kawaoka, 2002). The first attempt to express foreign sequences into the modified

genome of the influenza virus as a negative-sense RNA virus was in 1989. At this time, it was

used a recombinant influenza virus bearing recombinant RNA containing the coding sequence

of the chloramphenicol acetyl-transferase gene that replaced the NS gene for re-assorting the

influenza helper virus but this attempt failed as the generated virus rapidly lost this gene

following few passages (Luytjes et al., 1989). However, the first successful recombinant

influenza virus was generated in 1999 by using the plasmid based technique which resulted in

12 modified plasmid recombinant influenza viruses, such as A/WSN/33 (H1N1) and

A/PR/8/34 (H1N1) that have generated from the clone cDNA (Neumann et al., 1999).


54

Subsequently, various foreign epitopes from the infectious agents have been inserted into HA,

NA and NS gene segments in order to generate a live influenza virus vector using the reverse

genetics strategy (Li et al., 2005; Nakaya et al., 2004; Takasuka et al., 2002). In this way,

influenza viruses have been used as vector expressed malaria-specific CD8+ T and B cell

epitopes which induced robust CD8+ T cell and antibody responses in intraperitonealy-

vaccinated BALB/c mice. For instance, the SYVPSAEQI CD8+ T cell epitope sequence of

Plasmodium yoelii was inserted in the NA protein of the influenza virus A/HIQ8/68 (H3N2;

HK virus), while the B cell epitope, QGPGAPQGPGAP was inserted in the HA protein of the

A/WSN/33 virus (WSN virus) (Rodrigues et al., 1994). Another attempt in BALB/c involved

intraperitoneally or intranasally infected using the recombinant HIV-1/MN-A/WSN/33

influenza virus (HK-WSN) including a modified HA protein, expressing HIV-Env gp120

peptide (IHIGPGRAFYTT), induced neutralizing antibodies and cytotoxic CD8+ T cells in the

vaccinated mice (Li et al., 1993). A similar study in BALB/c mice primed using recombinant

influenza virus with HK-WSN expressing the Env protein on the HA segment. A boosting dose

of the recombinant vaccinia virus expressing the same peptide, which induced robust CD8+ T

cells (Gonzalo et al., 1999) was then given. Furthermore, a robust CD8+ T cell response was

induced in macaques following multiple doses of a prime boosting dose of intratracheal and

intranasal administration of influenza viruses like X31, (H3N2, A/HKx31) and PR8, (H1N1,

A/Puerto Rico/8/1934). Expressed SIV-CD8+ T cell epitopes, such as SIV Gag146-172, SIV

Tat87-96 and SIVTat114-123 inserted in the manipulated NA gene segment. (Sexton et al., 2009).

Also, both X31 and PR8 influenza viruses expressed the CD8+ T cell Env311 (RGPGRAFVTI)

epitope that was also inserted in the NA segment. These were used in BALB/c mice to induce

a protective CD8+ T cell response (Cukalac et al., 2009). The methodology is discussed for the

whole procedure used in generating our influenza virus vectors in Chapter 2, Section (2.2.8),

and more details about their results are mentioned in Chapter 3, Section (3.1.1).

1.6.3.1. Influenza virus vectors in HIV vaccine candidates Live recombinant influenza viruses have been used as viable HIV vaccine vectors because of

their ability to stimulate immune responses in the respiratory and urogenital tracts to control

various mucosal infectious diseases (Holmgren and Czerkinsky, 2005; Johansson et al., 2001).

Indeed, recombinant influenza viruses manipulated in this way have been used to express HIV

or SIV-CD8+ T cell epitopes to stimulate cytotoxic T cell responses (Liu et al., 2009; Pachler,

Mayr and Vlasak, 2010; Sexton et al., 2009). For example, a recombinant influenza-HIV

vaccine (rFlu-p17) involved the PR8 (A/PR/8/34, H1N1) influenza virus expressing the HIV


55

Gag protein (P17) which was inserted into the NA segment to stimulate both the humoral and

CD8+ T cell responses in prime boost intranasal or intraperitoneal vaccinated BALB/c and

C57BL/6 mice. Both the specific p17 neutralizing antibody and the CD8+ T cell responses were

highly induced in the two groups of mice vaccinated with the rFlu-p17 vaccine compared to

those groups given the rFlu-Rev vaccine as a negative controls (de Goede et al., 2009).

Furthermore, the HIV-1 matrix Gag p17 protein is considered a good vaccine candidate. It

regulates most stages of HIV-1 virus replication and reduces HIV-1 pathogenesis by inducing

specific CD8+ T cell and neutralizing antibody responses directed to the HIV-Gag p17 protein

(Fiorentini et al., 2010).

Furthermore, both the core/matrix proteins (p24 and p17) and the Env glycoproteins (gp120

and gp41) which are considered the most frequent HIV proteins targeted by high levels of

neutralizing antibodies (Robey et al., 1985). These are regarded as the most frequent HIV

protein recognized by specific cytotoxic T cells in HIV/AIDS patients (Addo et al., 2003). The

HIV-H-2Dd Env311 epitope has been inserted into the NA stalk of recombinant influenza viruses

using reverse genetics and also used in prime boost experiments to stimulate immunity

(Cukalac et al., 2009). In this instance, vaccination involved a primary dose using mouse-

adapted (A/HKx31), X31-NA-Env311 as the intranasal primary infection in BALB/c mice. This

was compared with secondary infection in another group of BALB/c mice primed with a dose

of virulent (A/Puerto Rico/8/34, H1N1), PR8-NA-Env311 administered intraperitonealy and

then challenged with an intranasal dose of X31-NA-Env311. This vaccination regime induced a

robust polyfunctional Env311+CD8+ T cell with IFN-γ, IL-2 and a TNF-α subset of HIV-Env311

CD8+ T cells detected in the spleen and BAL following secondary infection compared to a

lower response detected in primary infected BALB/c mice (Cukalac et al., 2009). Also,

recombinant influenza viruses expressing CD8+ T cell epitopes (SIV Gag164-172), (SIV Tat87-96),

and (SIV Tat114-123), inserted separately into the NA stalks of viral vectors were generated for

using in the vaccination regimen of macaques (Rollman et al., 2008). These two lab strains of

the influenza viruses were also used to induce SIV-mucosal CD8+ T cell response in macaques.

The macaque study included an inoculation of naïve and chronic SIVmac251 infected macaques

with a prime dose of initial intranasal X31-SIV, boosted with PR8-SIV, and then challenged

intravenously using infective doses of SIVmac251. Data from this study indicated the ability of

this vaccination regimen to maintain high levels of SIV mucosal CD8+ T cells. However, there

was no control of viremia in all infected vaccinated macaques (Sexton et al., 2009) because of


56

the ability of the virus to escape the narrow immune response directed to highly variable SIV-

CD8+ T cell epitopes. Recombinant influenza virus vectors (H1N1 and H3N2) expressing

CD8+ T cell epitopes have also been used previously in mouse model trials as a vector of the

HIV vaccine because they are mouse adapted strains of influenza viruses and are serologically

distinct containing the same six internal gene segments, and differ in the expression of HA and

NA genes. This is important to avoid neutralizing antibodies that inhibit the secondary T cell

response (Cukalac et al., 2009).

1.7. Project hypotheses The project is based on two hypotheses:

1- Vaccination with recombinant influenza viruses expressing HIV epitopes will enhance

the specific and mucosal CD8+ T cell immune response in mice.

2- Adoptive transfer of induced mucosal CD8+ T cells protected naïve mice against the

challenge virus (recombinant Vaccinia virus expressing the whole HIV-Gag protein).

1.8. Project aims In order to investigate these hypotheses, the followingspecific aims will be explored:

1- To generate live influenza vaccine vectors using a reverse genetics strategy expressing

HIV-CD8+ T cell epitopes.

2- To compare the ability of different mucosal routes of vaccination inducing CD8+ T cell

immune responses.

3- To study both the quality and characteristics of induced mucosal CD8+ T cell responses.

4- To transfer induced mucosal CD8+ T cells into naïve mice for protection against the

challenge rVac expressing the whole HIV-Gag protein.

Chapter 2: Materials and Methods

57

2. Chapter 2: Materials and Methods

2.1. Materials A detailed list of materials is provided in Appendix A.

2.2. Methods 2.2.1. Cloning strategy to generate X31-NA and PR8-NA with HIV-CD8+

T cell epitopes The 8 plasmid reverse genetics system was used to generate and rescue recombinant influenza

viruses, X31 or PR8, expressing a modified NA gene segment inserted with the exogenous

CD8+ T cell epitopes, H2Kd Gag197-205 or H-2d Tat17-25 as described by Hoffmann et al., (2000).

Generation and rescue of recombinant viruses involved in all viral RNAs and mRNAs were

generated through bi-directional transcription and translation from the specific single DNA

template (Andreansky et al., 2005). In order to generate these recombinant influenza viruses,

nucleotide sequences of H-2Kd Gag197 (AMQMLKETI: 5’-GCC ATG CAA ATGTTA AAA

GAG ACC ATC-3’) or of H-2d Tat17 (QPKTACTNC: 5’-CAG CCT AAA ACT GCT TGT

ACC AAT TGC-3’) were inserted into the sequence corresponding to position 44-45 of the

NA stalk of X31 (H3N2) or PR8 (H1N1) influenza laboratory strains, using two PCR reactions.

Standard PCR was performed to generate two overlapping NA fragments inserted with Gag197

or Tat17 epitopes. The first DNA fragment with a 200 base pair (bp) size and the second DNA

fragment was with a 1.2 kilobase (Kb) size. In order to form the whole NA fragment inserted

with the HIV-CD8+ T cell epitopes, recombinant PCR was performed to combine the two NA

fragments using specific forward primers, such asBm-NA-1 and reverse primers like X31-

NA197-R. All primers used are presented in Table 2.1, and PCR reactions conditions are

provided in Table 2.2.


58

Table 2.1. Nucleotide sequences of primers used for PCR amplification. Shown are forward and reverse primer sequences used in the amplification of modified NA segments expressing HIV CD8+ T cell epitopes (Gag197-205 and Tat17-25).

HIV epitope Primer nucleotide sequences (5’- 3’) Primer name Range of Nucleotides

Gag 197-205 AMQMLKETI (5’-GCC ATG CAA ATGTTA AAA GAG ACC ATC-3’) into X31-NA.

Forward (short fragment) 5’TAT TCG TCT CAG GGA GCA AAA GCA GGA GT-3’. Reverse (short fragment) 5’-GAT GGT CTC TTT TAA CAT TTG CAT GGC GGA GTC GCA CTC ATA TTG CTT AAA ATG CAA TG-3’. Forward (large fragment) 5’-GCC ATG CAA ATG TTA AAA GAG ACC ATC CCC GCG AGC AAC CAA GTA ATG CCG TGT GAA-3’. Reverse (large fragment) 5’ATA TCG TCT CGT ATT AGT AGA AAC AAG GAG TTT TTT-3’

Bm-NA-1- F X31-NA197- R X31-NA197- F Bm-NA-1413-R

924-950

Tat17-25 QPKTACTNC (5’-CAG CCT AAA ACT GCT TGT ACC AAT TGC-3’) into X31-NA.

Forward (short fragment) 5’TAT TCG TCT CAG GGA GCA AAA GCA GGA GT-3’ Reverse (short fragment) 5’- GCA ATT GGT ACA AGC AGT TTT AGG CTG GGA GTC GCA CTC ATA TTG CTT AAA ATG CAA TG-3’ Forward (large fragment)5’-CAG CCT AAA ACT GCT TGT ACC AAT TGC CCC GCG AGC AAC CAA GTA ATG CCG TGT GAA-3’ Reverse (large fragment) Reverse (large fragment) 5’ATA TCG TCT CGT ATT AGT AGA AAC AAG GAG TTT TTT-3’

Bm-NA-1-F X31-Tat17- R X31-Tat17- F Bm-NA-1413- R

5425-5451

Gag197-205 AMQMLKETI (5’-GCC ATG CAA ATG TTA AAA GAG ACC ATC-3’) into PR8-NA.

Forward primer (short fragment) 5’-TAT TGG TCTCAG CGA GCA AAA GCA GGA GT-3’

Reverse (short fragment) 5’-GAT GGT CTC TTT TAA CAT TTG CAT GGCTTG ACT TCC AGT

TTG AAT TGA ATG GCT AAT CC-3’

Forward (large fragment)5’-GCC ATG CAA ATG TTA AAA GAG ACC ATCAAC CAT ACT GGA

ATA TGC AAC CAA AAC ATC-3’

Reverse Primer (large fragment) 5’-ATA TGG TCT CGT ATT AGT AGA AAC AAG GAG TTT TTT-3’

Ba-NA-1- F

PR8- NA197- R

PR8- NA197- F

Bm-NA-1413- R

924-950

Tat17-25 QPKTACTNC (5’-CAG CCT AAA ACT GCT TGT ACC AAT TGC-3’)into PR8-NA

Forward primer (short fragment) 5’-TAT TGG TCTCAG CGA GCA AAA GCA GGA GT-3’

Reverse primer (large fragment) 5’- GCA ATT GGT ACA AGC AGT TTT AGG CTG TTG ACT TCC AGT TTG AAT TGA ATG GCT AAT CC-3’

Forward primer (large fragment) 5’-CAG CCT AAA ACT GCT TGT ACC AAT TGCAAC CAT ACT GG ATA TGC AAC CAA AA ATC-3’

Reverse Primer (large fragment) 5’-ATA TGG TCT CGT ATT AGT AGA AAC AAG GAG TTT TTT-3’.

Ba-NA-1-F

PR8-NA-Tat 17-R

PR8-NA-Tat 17-F

Ba-NA-1413-R

5425-5451


59

Table 2.2. Conditions for PCR reactions. A) Typical PCR conditions used in cloning work, B) Conditions for the recombinant PCR reactions to generate the modified NA gene segments.

A) Standard PCR conditions.

Step of reaction Temp. Time Cycle number

1 94°C 4 min 1

2 94°C 20 sec

30 3 57°C 30 sec

4 72°C 2 min

5 95°C 1 min

1 6 57°C 1 min

7 72°C 7 min

8 4°C Hold

B) Recombinant PCR conditions.

Step of reaction Temp. Time Cycle number

1 94°C 2 min

2 2 55°C 30 sec

3 72°C 2 min

4 94°C 30 sec

33 5 59°C 30 sec

6 72°C 2 min

7 72°C 7min 1

8 4°C Hold


60

2.2.2. Gel electrophoresis PCR products and restriction digests were examined by gel electrophoresis using 0.8% (w/v)

Agarose (Analytical grade, Promega) in 1x TAE buffer (Tris-acetate EDTA, Promega)

containing SYBR safe DNA gel stain (Invitrogen). DNA samples were mixed with a 6x loading

dye (Bromophenol blue, Xylene cyanol, glycerol and water) (see Appendix A), and loaded

onto the gel using agarose solution (0.8%) which was electrophoresed at 100 V for 40 min. A

1 Kb-Plus DNA Ladder (Invitrogen) was used as a size marker for estimation of fragment sizes.

2.2.3. DNA gel extraction and purification Gel slices containing recombinant NA-H-2d Gag197 or NA-Tat17 PCR products were extracted

using a QIAquick Gel Extraction Kit (manufacturer) using the manufacturer’s instructions and

quantified using gel electrophoresis with pBR322 standards (New England Biolabs) (NEB).

2.2.4. Ligation Recombinant NA segments containing the H-2d Gag197 or Tat17 epitopes were ligated into

plasmid pHW2000 using a ligation mixture including 1 μl of T4 DNA ligase, 20 ng of the

digested and purified plasmid pHW200 treated with CIAP (Alkaline phosphatase, calf

intestinal; Promega M182A: 1 u/μl), 19 ng of recombinant NA-Gag197, 2 μl of 10x DNA ligase

buffer. The total volume of this mixture was adjusted to 20 μl by adding DNAase free H2O,

and then incubated at 16°C O/N in the PCR machine. The ligation products were then examined

by gel electrophoresis.

2.2.5. Transformation Ligation products were transformed into E. coli, JM109 or DH5α competent cells (Life

Technologies). Frozen competent cells were removed from storage at -70°C and placed on ice

for thawing. 30 μl of the competent cells were thenmixed with 10 μl of the DNA ligation

mixture. The mixture of DNA and competent cells was incubated on ice for 30 minutes prior

to heat shock for 45 seconds at 42°C without shaking. The cells were immediately returned to

ice for 2 minutes, and 900 μl of LB broth (RT) was added to each transformation reaction, prior

to incubation at 37°C for 1 hour with shaking at 225 rpm. The transformation culture was

centrifuged for 5 minutes at 2000 RPM and the supernatant poured off. The cell pellet was then

suspended in the residual LB broth medium (˜100μl) and spread onto a LB agar medium

containing 100 μg/ml Ampicillin, and incubated O/N at 37°C.


61

2.2.6. Plasmid purification Single colonies were placed into 2 ml LB broth containing Ampicillin (100 μg/ml) and

incubated at 37°C with shaking at approximately 250 RPM for 8 hours. Purification of the

recombinant plasmid DNA from these cells was achieved by using the PureYield™ Plasmid

Mini Prep-protocol (Promega, USA). For larger cultures, the PureYield™ Plasmid Maxiprep

protocol (Promega, USA) was used.

2.2.7. Restriction analysis The purified recombinant plasmid DNA was digested with the specific restriction

endonucleases enzymes, PvuI and PstI, to allow for NA cloning (Hoffmann et al., 2002; Webby

et al., 2003), using appropriate buffers and temperatures. It was then subjected to

electrophoresis.

2.2.8. Sequencing Plasmids containing the inserted HIV-1 epitopes in the NA segment, were prepared for whole

sequencing by mixing each with 1 μl template ds DNA (300 ng), 1 μl of 2.5x Big-Dye

terminator Premix (v3.1), 3.5 μl of 5x Reaction buffer, 1 μl of Primer 3.2 pmol (5 mM), with

the total volume brought up to 20 μl by nuclease free water (Table 2.3). Samples were then

sent to the DSR Sequencing laboratory at CSIRO/AAHL for sequencing using the dideoxy

chain termination method (Sanger et al., 1980).

2.2.9. Rescue of recombinant influenza virus by reverse genetics 2.2.9.1. MDCK and HEK 293 T co-culture Plasmids containing modified NA genes were combined with the 7 plasmids encoding each of

the other 7 influenza virus gene segments (pHW2000 PB1, PB2, PA, NP, M, NS and HA) from

A/HK/X31 (H3N2) as both influenza laboratory strains (X31 and PR8) contained the same 6

internal influenza viral genes, with different pHW2000 HA plasmids corresponding to the X31

or PR8 influenza lab strains. All 8 plasmids were collectively transfected into HEK 293 T cells

and then amplified in MDCK cells (as described in Chapter 3, Section 3.1.2 and shown in

Figure 3.11). Two 75 cm2 flasks containing confluent MDCK and HEK cells were maintained

in a 5% FCS-OPTI-MEM medium. Cells were harvested with 5 ml of (1x) Trypsin-Versene®

(Trypsin-EDTA) and incubated at 37°C for 5 min. They were then centrifuged at 1600 rpm for

5 min with 10 ml of DMEM medium for washing added and then re-suspended in 10 ml Easy

Flu-medium-Invitrogen (OPTI-MEM-1-Reduced-Serum-medium; GIBCO), supplemented

with hypoxanthine, thymidine, sodium pyruvate, L-glutamine, trace elements, and growth


62

factors (but no fetal calf serum, FCS). The MDCK and HEK 293T cells were then mixed in a

1:2 ratio (1 ml of MDCK cells + 2 ml of HEK 293 T cells) in a 50 ml tube to seed a co-culture

of cells. 15 ml of Easy Flu medium was then added to 3 ml of the cell mixture and aliquoted

into individual wells of a 6-well plate. This was followed by incubation at 37°C, and 10% CO2

for 18-24 hours to attain confluency.

2.2.9.2. Transfection Transfections involved mixing 1 μg of each plasmid (NP, NS, PB1, PB2, M, PA, HA and NA-

Gag197 or NA-Tat17) into a 1.5 ml tube, with 10 μl of each plasmid taken total volume of 80 μl

of the mixture of 8 plasmids, followed by incubation at room temperature for 45 min. In a

second tube, 3 μl of Fugene 6 (Roche Transfection Reagent) for each 1μg of plasmid DNA was

added (total amount = 24 μl). The total volume of the transfection reagent was brought up to

200 μl by adding 176 μl of the OPTI-MEM medium (no FCS and no antibiotics) per

transfection reaction and incubated for 5 min at room temperature. Next, the plasmid solution

was added to the (Fugene 6 reagent + Opti-MEM) and incubated at room temperature for 45

min, after which the total volume of the mixture was increased to 1000 μl by adding 720 μl of

Opti-MEM. Media was removed from each MDCK/HEK 293 T cell co-culture well and

replaced with 1 ml of transfection mixture for 6 hours at 37°C and 10% CO2. The transfection

mix was then carefully removed and replaced with 1 ml of Easy flu medium, followed by

incubation of the 6 well plates for 18-30 hours at 37°C and 10% CO2. To assist replication, 1

ml of the Easy flu medium containing 1 μg of TPCK-trypsin in PBS (Trypsin stock 1μg/μl)

was added to the co-culture to give a final volume of 2 ml in each well, with cells incubated

for a total time of 72 hours at 37°C followed by collection of virus-containing supernatants.

2.2.9.3. Hemagglutination assay The presence of the influenza virus in reverse genetics transfection supernatants was detected

by hemagglutination activity with the use of chicken red blood cells (RBCs). Firstly, chicken

red blood cells (RBCs) were washed twice with PBS and then diluted with PBS to 0.5% packed

cell volume (PCV). Two-fold dilutions of the virus were made and chicken RBCs (0.5%) were

then added to all wells followed by mixing and incubation for 30 min at room temperature after

which agglutination was scored as either positive or negative.


63

2.2.9.4. Amplification of modified viruses in 10 day old embryonated chicken eggs

Recombinant influenza viruses were amplified in 10 day old fertilized eggs. The transfection

supernatants (100 μl) were added directly to 10 day old embryonated chicken eggs and

incubated for 2 days in a 35°C incubator. Allantoic fluids were then harvested into 50 ml Falcon

tubes on ice and then at -80°C after being aliquoted into small volumes into cryogenic vials

(Nunc®CryoTubes®1.8 ml).

2.2.9.5. RNA extraction of harvested allantoic fluids for sequencing RNA was extracted using the RNeasy Mini Kit (Qiagen) as follows. First, 150 μl of harvested

allantoic fluid was mixed well with 500 μl of 70% (v/v) ethanol and then 500 μl of RLT buffer

was added. Next, 750 μl aliquots of the mixture were transferred to a purification column,

followed by centrifugation for 30 seconds at 1500×g. The eluate was discarded and the previous

step repeated with the remaining mixture. 500 μl of the RW1 buffer was then added to a wash

column with centrifugation at 1500×g for 30 seconds and the eluate discarded. The column

was washed twice using a 500 μl RPE buffer in a similar manner. The column was then dried

by centrifugation for 1 min at 1500×g, followed by elution of RNA with 20 μl of nuclease-free

water. This eluate was left at room temperature for 1 min to dissolve the extracted RNA and

followed by centrifugation for 30 seconds at 1500×g to obtain the purified RNA product.

2.2.9.6. Reverse transcription of RNA to cDNA for NA sequencing Purified RNA (1 μg) was added to an RNase-free PCR tube in a 10 μl volume. To this, 2 μl of

a 10× reverse transcriptase buffer (RT buffer), 2 μl dNTP mix (5 mM each dNTP), 2 μl of 10

μM oligo-dT primer (or BmNA-1 forward primer), 1μl of Omniscript reverse transcriptase,

and 3 μl RNA (up to 2μg per 20 μl reaction), were added and mixed well before incubation for

60 min at 37°C. An aliquot of the resultant cDNA (5 μl) was mixed with 10 μl of 1× phusion

master mix (PhusionTM Flash High-fidelity PCR master mix, Finnzymes #F-548S), 2 μl of

forward and reverse neuraminidase primers (BmNA-1 and BmNA-1413), and 1 μl of nuclease

free water. PCR conditions (Table 2.2 B) were used to amplify DNA.The PCR product was

electrophoresed on a 0.8% agarose gel, as previously described to check the accurate size of

the amplified DNA. After that, the sequencing PCR reaction was performed on the amplified

DNA samples using the reaction mixture included in the 1 μl template ds DNA (300 ng), 1 μl

of 2.5x Big-Dye terminator Premix (v3.1), 3.5 μl of 5x Reaction buffer, and 1 μl primer 3.2

pmol (5 mM). The total volume was brought up to 20 μl by nuclease free water. The sequencing


64

reaction mixture followed the reaction cycle conditions (Table 2.3). After this, all samples were

sent to the DSR Sequencing laboratory in the CSIRO/AAHL for sequencing.

Table 2.3. Conditions of preparation DNA samples for sequencing reaction. This table shows typical cycles of sequencing reaction performed on the amplified DNA samples.

Step of reaction Temperature Time Cycle

1 96 oC 1 min 1

2 96 oC 10 sec

30 3 50 oC 5 sec

4 60 oC 4 min

5 4 oC Hold

2.2.9.7. Plaque assay To measure the titer of newly generated recombinant influenza viruses, a plaque assay was

performed on Madin-Darby Canine Kidney (MDCK) cell monolayers, using the viral titer of

pfu/ml for each virus (Tannock, Paul and Barry, 1984; Thomas et al., 2006). A monolayer of

MDCK cells was grown on 6 well tissue culture plates. The MDCK cell growth media was

removed and duplicate wells were inoculated with 150 μl of ten-fold serially diluted virus stock

(10-1 -10-7). The plates were then incubated at 37°C, 5% CO2 for 45 min with shaking every 15

minutes. A 200 μl aliquot of 0.1% Trypsin Worthington TW (TPCK Trypsin Worthington from

Scima-R; Cat. No. #LS003740; (stock 1 g/ml)) was added to 2 × L15 medium (Invitrogen)

and mixed with 1.8% agarose solution (A-6013 Agarose, Sigma-Aldrich) to generate the

agarose overlay. 3 ml volume of the agarose overlay was added to the infected cell monolayer,

followed by incubation at 37°C, with 5% CO2 for 3 days. Plaques in each well were counted

and plaque forming units per ml calculated (Cukalac et al., 2009).

2.2.10. Vaccination of mice Six week old BALB/c mice were purchased from the Animal Resources Centre (ARC) and

inoculated with the recombinant influenza vaccine strains following environmental adaptation

for 2 weeks. All animal experiments were completed according to protocols approved by the

CSIRO/AAHL Animal Ethic Committee.


65

2.2.10.1. Procedures for intravaginal and intranasal vaccination of mice The vaccination program followed six different vaccine strategies involving primary and

secondary vaccination to stimulate CD8+ T cell responses.

2.2.10.1.1. Primary vaccination In order to recognize the ability of each of the generated HIV-CD8+ T cell epitopes (H-2Kd

Gag197 and H-2d Tat17) to induce a specific HIV-CD8+ T cell response, two groups of BALB/c

mice were used for primary infection using influenza virus vectors expressing Gag197 or Tat17

epitopes. The first group of BALB/c mice were primed intranasally with 30 μl (1×104 pfu) of

the recombinant influenza vaccine (X31-NA-Gag197). Also, there is another group of mice were

vaccinated with a primary intranasal infection of 30 μl (1×104 pfu) recombinant influenza

vaccine (X31-NA-Tat17). Both groups were rested for 10 days following infection and then

euthanased to harvest their spleen to detect CD8+ T cell responses.

2.2.10.1.2. Prime boost vaccination There were four different procedures of prime boost vaccination used in BALB/c mice as

follows: i) Intranasal administration of 30 μl (1 × 104 pfu) of the recombinant influenza vaccine

(X31-NA-Gag197) as a prime dose, and then after six weeks by intranasal infection with 30μl

(50 pfu) of PR8-NA-Gag197; ii) Intravaginal administration of 30 μl (1 × 104 pfu) recombinant

influenza vaccine (X31-NA-Gag197) as a prime dose followed 6 weeks later by intravaginal

infection with 30μl (50 pfu) of PR8-NA-Gag197; iii) Intranasal administration of 30 μl (1 × 104

pfu) of the recombinant influenza vaccine (X31-NA-Gag197) as a prime dose followed 6 weeks

later by intravaginal infection with 30 μl (50 pfu) of PR8-NA-Gag197; and iv) Intravaginal

administration of 30 μl (50 pfu) of PR8-NA-Gag197 as a prime dose followed 6 weeks later by

intranasal immunization with 30 μl (1 × 104 pfu) of the recombinant influenza vaccine (X31-

NA-Gag197). To obtain CD8+ T cell population, all gropus of infected mice were euthanased

after 7 days of the boost dose of the infection for harvesting different organs, such as the spleen,

brochoalveolar lavage (BAL), mediastinal (MedLN) and inguinal (ILN) lymph nodes.

2.2.10.1.3. Lymphocyte isolation and preparation After 10 days of primary infection or 7 days of prime boost infection, groups of infected mice

were euthanased by CO2 asphyxiation. After that, spleens, inguinal (ILN) and mediastinal

(MLN) lymph nodes were harvested from the infected mice. These organs were added to 10

ml of Hank’s buffered salt solution (HBSS) for the preparation of single cell suspensions of

lymphocytes. Two frosted slides were used to gently disrupt solid organs (spleen and lymph


66

nodes) before filtration of cell suspensions through 70 μm cell strainer. Enriched T cells were

obtained from the spleen by the panning of B cells using 15 ml anti-mouse IgM + IgG

immunoglobulins (5 vials + 200 mL of PBS, H + L; Jackson Immuno-Research Laboratories)

in a petri dish for 45 min. The non-adherent cell suspension was harvested and then centrifuged

for 6 min at 1600 RPM and re-suspended in 3 ml of HANKS solution. The supernatant was

then gently discarded before adding 3 ml of Ammonium Tris-Chloride buffer (see in Appendix

A) to each tube and incubating for 5 min at room temperature to lyse RBCs. The reaction was

stopped by adding 7 ml of HBSS to each tube before centrifugation for another 6 min.

Following removal of the supernatant cells, they were washed twice using10 ml of HBSS and

re-suspended in 3 ml of complete RPMI for intracellular cytokine staining assay preparation

and tetramer staining. To obtain T cells from broncho-alveolar lavage (BAL), 3 ml (1x) of

harvested BAL samples (included PBS as a diluent) were plated in 6 well plates and incubated

for 1 hour at 37ºC to remove adherent cells (macrophages). The non-adherent cells were

collected into 10 ml tubes and centrifuged at 1600 rpm for 6 min at 4ºC. The cell pellet was re-

suspended in 2 ml of complete RPMI for intracellular cytokine staining assay preparation and

tetramer staining. Lymphocytes from all samples were counted using a hemocytometer.

2.2.10.2. Phenotype and surface marker staining T cells were phenotyped using rat-anti-mouse antibodies CD4-FITC (BD-Pharmingen, Clone

RM-4.4) and CD8-α-PerCP5.5 (BD-Pharmingen, Clone 53-6.7). A 100 μl aliquot of

lymphocyte suspension was added into a 96 well culture plate (BD Falcon) followed by 50μl

of an antibody cocktail of rat anti-mouse CD4-FITC (1/400) and CD8-α-PerCP5.5 (1/200)

antibodies (Appendix A). This was then incubated on ice for 30 min and the cells washed twice

with FACS buffer (Reagent section, Appendix A). Samples were then analyzed on FACS BD-

FACS LSR II using BD DIVA software.

2.2.10.3. Intracellular cytokine staining For intracellular cytokine staining, 100 μl of enriched CD8+ T cells was added to a 96 well

round bottom plate (Corning® Costar®), followed by the addition of a 100 μl stimulation

mixture (Table 2.4). This stimulation mixture of 1 ml included 50 μl of IL-2 (50 U/ml), 2 μl of

Golgi-plug (Brefeldin) (10 μg/ml) and 20 μl of the H-2Kd Gag197 or H-2Kd NP147 peptides (2

μg/ml). It was then made up for 1000 μl by adding c-RPMI. After that, mixture of 100 μl CD8+

T cells and 100 μl of stimulation mixture (including Gag197 or NP147 peptide or no-peptide

stimulation mix) were incubated for 5 hours at 37ºC with 5% CO2. The plate was then

centrifuged at 1600 rpm for 4 min at 4ºC. The supernatant was discarded and cells were washed


67

twice with a 150 μl FACS buffer. Cells were then stained in a 50 μl volume of FACS buffer

that included anti-CD8-α-PerCP5.5 (1/200).

These samples were then incubated on ice for 20 minutes, washed and centrifugation performed

at 1600 rpm for 4 min at 4 C. Cells were then re-suspended in 100 μl of Golgi-plug solution to

keep the stimulated intracellular cytokines inside cells and not escaping out of cells (see

Appendix A). One hundred μl of 0.5% paraformaldehyde was added before incubation for 15

min at room temperature to fix the treated cells without undergoing any changes. The plate was

then covered in foil and incubated overnight at 4oC. Following centrifugation, cells were re-

suspended in 150 μl of Perm wash solution (10%) and incubated for 20 min at 4 C prior to the

addition of a cytokine antibody cocktail (anti-IFNγ-FITC, anti-IL2-PE and anti TNF-α-APC,

1/100, in a 50 μl volume.The plate was incubated on ice for 30 min and the cells washed twice

with 150 μl of a 10% Perm Wash buffer. Finally, cells were re-suspended in a final volume of

200 μl FACS buffer for analysis on the BD-FACS LSR II.

Table 2.4. Stimulation mixture solutions used in intracellular cytokine staining (ICS). The three types of stimulation mixture are shown, including the H-2Kd NP147, H-2KdGag197 peptide. No peptide stimulation mixture used as a negative control.

Item used NP147 peptide Gag197 peptide No peptide

Peptide

20 μl 20 μl -

rIL-2

50 μl 50 μl 50 μl

Golgi

(Brefeldin)

2 μl 2 μl 2 μl

C-RPMI

928 μl 928 μl 948μl

Total volume

1000 μl 1000 μl 1000 μl


68

2.2.10.4. Tetramer staining Tetramers were prepared by complexing MHC-1 molecules with the specific CD8+ T cell

receptor, conjugated with specific peptides (glycoprotein H-2Kd NP147 or H-2Kd Gag197) and

then treated with Streptavidin-Phycoerythrin. The Allophycocyanin conjugated H-2Kd NP147

or H-2Kd Gag197 tetramers were kindly synthesized by the Biomolecular Resource Facility,

John Curtin School of Medical Research. Aliquots (100 μl) of enriched CD8+ T cells were

added to a 96 well round bottom plate, followed by centrifugation at 1600 rpm for 4 min at

4°C. The cell pellet was re-suspended in 50 μl of appropriate tetramer, H-2Kd NP147-APC

(TYQRTRALV) or H-2Kd Gag197-APC (AMQMLKETI) at 1/100 dilution. The 96 well plate

was then covered with foil and incubated at room temperature for 1 hr, followed by washing

(two times) with 150 μl of FACS buffer. An antibody cocktail (anti-CD8-PerCP (1/200), anti-

CD62L-FITC (1/400), anti-CD44-PE (1/400), anti-LPAM-1-PE (1/400), anti-CD103-FITC

(1/400), anti-CD69-PE (1/1400) and anti-CCR9-FITC (1/400) was added in a 50 μl volume

using appropriate combinations. The plate of stained cells was incubated for 30 min on ice

samples washed with 150 μl of FACS buffer and cells re-suspended in a final volume of 200

μl FACS buffer before FACS analysis (BD-FACS-LSR II).

2.2.10.5. Cell sorting of HIV Gag+LPAM-1+CD8+ T cell populations The total HIV Gag197

+CD8+ T cells were harvested from the spleen, mediastinal and broncho-

alveolar lavage from vaccinated BALB/C mice. CD8+ T cells (from the spleen and MLN) were

enriched as per section (2.2.14.2) and then stained with a H-2Kd Gag197 tetramer for 1 hr at

room temperature as described above. Two populations of CD8+ T cells were stained with a

specific purified H-2Kd Gag197 tetramer. Both were HIV-H-2Kd Gag+CD8+ T cells but with

differentially expressed the mucosal homing integrin, α4β7+ (LPAM-1+) or α4β7- (LPAM-1-).

The HIV H-2Kd Gag+CD8+ T cells were then stained with anti-mouse LPAM-1(α4β7) (1/400)

and anti-mouse CD8α PerCP (1/200 dilution) in 2 ml of a sort buffer (0.1% BSA) on ice for 30

min to sort using the sorter (BDFACS AriaTM II). RNAs were extracted from the HIV-

Gag197+α4β7+CD8+ T cell or HIV-Gag197

+α4β7-CD8+ T cell populations using an RNeasy Mini

Kit (Qiagen), as described in the previous Section (2.2.11). RNA was then reverse transcribed

to cDNA using a SuperScript kit as previously described (Section 2.2.12). (Superscript III First-

Strand Synthesis Supermix).


69

2.2.10.6. Real time PCR (RT-PCR) for IL-15 and IL-15Rα expression Real time PCR (RT-PCR) was performedon the corresponding cDNA synthesized from the

Gag197+α4β7+ and Gag197

+α4β7-CD8+ T cells using the clear optical 96-well plate. This assay

used ABI-Taq-Mn MGB primer/probes specific for IL-15 or IL-15Rα supplied with ABI

Taqman gene expression assays (Applied Biosystem), the Taqman gene expression assay mix

(including probe and primers), (mouse IL-15-FAM (#Mm00434210-ml) (20×) or

(#Mm04336046-ml). The mouse IL-15Rα was then combined with the ABI Universal PCR

Master Mixof Taqman gene expression (2×) for standard PCR amplification. The Applied

Biosystems-StepOneTM/Real-Time PCR protocol of duplicate wells for each cDNA sample

was used. Firstly, 5 l of template cDNA synthesized from RNAs were extracted from

Gag197+α4β7+ or Gag197

+α4β7-CD8+ T cells, and added to each well of a clear optical 96-well

plate to 20 l of the PCR Master-Mix added and mixed gently. After that, a clear film was used

to cover the plate and then spin down the plate at 1600 rpm for 1 min to be ready for turning

on the iCycler machine. The cycle conditions used were 2 min at 50ºC and 10 min at 95ºC,

followed by a denaturation step of 40 cycles, 15 sec at 95ºC and an annealing step with 60 sec

at 60ºC.

All samples were normalized to the house-keeping gene, GAPDH (#Mm99999915-gl) as an

internal standard control of genes used in each run to act as a normalizer, and the experiment

was repeated three times. Data from the IL-15 cytokine or IL-15Rα expression detected by

real-time PCR used StepOneTM-Software v2.0. The average CT was calculated for both α4β7+

or α4β7- samples and GAPDH and then the ΔCT (CT, α4β7+ - CT, GAPDH) and ΔCT (CT,

α4β7- - CT, GAPDH) were determined. After that, the absolute value of the slope was made

up to zero and efficiencies of relative quantification for expressed IL-15 cytokine or IL-15Rα

were detected by calculating the average ΔΔCT (ΔC, α4β7+- ΔCT, α4β7-).

2.2.10.7. Adoptive transfer and challenge of BALB/c mice Gag197

+α4β7+ and Gag197+α4β7- CD8+ T cells were sorted (as described in Section 2.2.17.5)

from intranasal-intranasal prime-boosted BALB/c mice. Gag197+α4β7+or Gag197

+α4β7-CD8+ T

cells, 5 × 104 cells/200 μl were transferred intravenously (tail vein) into two groups of naïve

BALB/c mice, in addition to a negative group of mice given PBS only (Mock group) and then

rested for 6 days. These mice were then challenged with a pseudo-virus, a recombinant vaccinia


70

virus expressing the whole HIV Gag protein (rVac-Gag) (see Section 2.2.17.8) to test the

protective capacity of these populations.

2.2.10.8. Pseudo-challenge of vaccinated mice with a recombinant Vaccinia virus expressing whole Gag protein.

Six days following the adoptive transfer, BALB/c mice were infected intranasally with 1 × 107

pfu recombinant vaccinia 633-Gag (rVac-gag) virus expressing the whole HIV-Gag protein.

Five days after the pseudo-challenge, lungs and ovaries were harvested from individual mice

and placed on ice before complete homogenization with a clean probe for 1 min. Lung

homogenates were placed into a 10 ml sealed screw cup tube and centrifuged at 2000 rpm for

7 minutes, with supernatants tested by a plaque assay to determine the titer of rVac-gag in all

three groups of infected mice.Both the spleen and mediastinal lymph nodes were harvested

from the three infected groups of mice at this time point in order to determine HIV-H-2Kd

Gag197+CD8+ T cell responses using an ICS and tetramer staining assay.

2.2.10.9. Statistical analysis Means and standard deviation (SD) were used to calculate weight change and CD8+ T cell

immune responses amongst the different groups of mice infected with recombinant influenza-

HIV viruses. ANOVA with Tukey's test were used to make statistical comparisons of immune

responses induced in different routes of infection. P Values were considered statistically

significant when levels were P<0.05. In addition, the two sided t-test was used as the statistical

analysis to compare the two groups of vaccinated animals in some laboratory experiments.

Chapter 3: Generation of influenza viruses expressing HIV-CD8+ T cell epitopes

71

3. Chapter 3: Generation of influenza viruses expressing HIV-CD8+ T cell epitopes

3.1. Overview This Chapter describes the generation of recombinant influenza viruses that express the H-2Kd

HIV Gag197 or H-2d Tat17 CD8+ T cell epitopes. Two laboratory strains of mouse-adapted

influenza viruses, X31 (H3N2) and PR8 (H1N1), were generated using established reverse

genetics systems. Reverse genetics systems is the novel molecular strategy discovered in 1978

to generate newly modified live viruses that belong to various families like Rhabdoviridae,

Paramyxoviridae, Filoviridae, Bunyaviridae and Orthomyxoviridae through a modification of

their genome resulting from the cloned cDNA (Neumann, Whitt, and Kawaoka, 2002). Reverse

genetics systems were first used to modify non-segmented positive stranded RNA viruses by

transfecting a full length of the RNA genome into appropriate cells that permitted expression

of viral proteins and generation of attenuated vaccine viruses able to induce an immune

response to control infectious diseases (Taniguchi, Palmieri and Weissmann, 1978; Racaniello

and Baltimore, 1981). In 1994, modified rabies virus was the first example of a non-segmented

negative stranded RNA virus that was successfully generated by a reverse genetics strategy

(Neumann, Whitt, and Kawaoka, 2002; Schnell, Mebatsion and Conzelmann, 1994). However,

this was more difficult with segmented negative stranded RNA viruses as there was a big

challenge to generate the segmented negative-sense RNA viruses because of a complication

related to treating the segmented RNA genome. In addition, there was also the need to co-

express the viral polymerase complex and vRNAs to initiate transfection of viral RNA

segments and generate modified viruses (Neumann, Whitt, and Kawaoka, 2002). Fortunately,

all of these challenges have been overcome using a plasmid based technique. The first

segmented negative stranded RNA virus, generated in 1996, was a member of the Bunyviridae

family and included three RNA segments (Bridgen and Elliott, 1996). This was followed by

the successful generation of an eight segmented negative stranded RNA virus, the influenza A

virus (Neumann, Whitt, and Kawaoka, 2002; Neumann et al., 1999; Fodor et al., 1999).

The plasmid-based technique used to generate the genetically modified recombinant influenza

virus contained a total of 12 plasmids, including eight plasmids transfected into eukaryotic

cells. The virus also included Vero cells to encode precise copies of eight viral RNA gene

segments (PB2, PB1, PA, NP, HA, NA, M and NS) (Zobel, Neumann and Hobom, 1993)

combined to the other four expressing plasmids to encode the four viral polymerase complex


72

proteins, PB1, PB2, PA and NP necessary for the transcription and replication of newly forming

virus (Hoffmann et al., 2000; Neumann et al., 1999). After two years, a more developed reverse

genetics technique has been used to generate recombinant genetically engineered influenza

viruses entirely from cloned cDNA by the eight plasmids reverse genetics system. This system

uses only eight plasmids encoding the eight viral protein segments transfected into eukaryotic

cells, such as HEK-293T cells, thus resulting in expression of influenza viral proteins and

newly formed virus replication that is mainly used as attenuated viral vaccines (Hoffmann et

al., 2000; Neumann et al., 2005). In this Chapter, live influenza virus vectors were generated

using a cloned viral genome recombining the eight segments of the virus including modified

NA segment in which was inserted with HIV-H-2Kd Gag197 or H-2d Tat17 CD8+ T cell epitopes.

This step included the generation of plasmids containing NA segments from the two influenza

viruses, X31 (H3N2) and PR8 (H1N1) into HIV-epitopes, H-2Kd Gag197 and H-2d Tat17. HIV-

CD8+ T cell epitopes were amplified by providing all negative sense of the RNA converted

from the cloned cDNA, and utilizing specific enzymes, such as RNA polymerase 1. The

recombinant plasmid combined with the additional seven influenza plasmids were transfected

into HEK-293T cells and further amplification of the virus was carried out via MDCK cells to

generate the recombinant influenza viruses. Following this, Hemagglutination (HA) assay and

sequencing were used to confirm the generated viruses.

3.1.1. Generation of a recombinant X31(H3N2) or PR8 (H1N1) influenza viruses expressing HIV-CD8+ T cell epitopes

HIV-H-2Kd Gag197 (AMQMLKETI) and H-2d Tat17 (QPKTACTNC) CD8+ T cell epitopes

were cloned into the NA segments of X31 and PR8 viruses after amino acid residue 44 (Figure

3.1). A recombinant PCR based strategy was used to introduce these epitopes. The first set of

PCR reactions generated 2 NA fragments (200 bp and 1200 bp) that were then recombined to

generate a modified NA gene segment expressing the introduced epitopes as depicted in the

cloning strategy (Figure 3.2). The two amplified fragments totaling 1.4 Kb were then

recombined using PCR to generate recombinant X31-NA-Gag197-205, X31-NA-Tat17 and PR8-

NA-Tat17 gene segments. These would then be cloned into an established influenza reverse

genetics backbone plasmid pHW2000 (Figure 3.2) for use in virus rescue experiments to

generate a modified virus expressing exogenous HIV-CD8+ T cell epitopes (Figure 3.3).


73

Figure 3.1. Amino acid sequences of the X31-NA (A) and PR8-NA (B) genes used for the generation of recombinant HIV-influenza vaccine vectors. The position into which the H-2Kd Gag197 (AMQMLKETI) or H-2d Tat17 (QPKTACTNC) epitopes were inserted are shown with an asterisk (*).


74

Figure 3.2. Generation of a recombinant neuraminidase (NA) gene segment containing HIV-H-2Kd Gag197 (AMQMLKETI) and H-2d Tat17 (QPKTACTNC) CD8+ T cell epitopes. The schematic diagram depicts the stepwise PCR strategy used to generate recombinant reverse genetics plasmids.


75

Figure 3.3. Generation of a recombinant influenza virus expressing HIV-CD8+ T cell epitopes. This schematic structure shows the full length modified neuraminidase segment of PR8-NA and X31-NA, where HIV-CD8+ T cell epitopes, H-2Kd Gag197 and H-2d Tat17 were inserted.

NH2

X31 or PR8-NA Stalk

45 458COOH

35 61

Tat17-25 (QPKTACTNC)*Gag197-205 (AMQMLKETI)*

Membrane anchor

Neuraminidase (NA) segment

Recombinant influenza NA

Virus Membrane surface


76

PCR reactions were performed to generate the two fragments noted in Figure 3.2 which

expressed the HIV-H-2Kd Gag197 CD8+ T cell epitope. A DNA fragment of band size 200 bp

and a second DNA fragment (1.2 Kb) were amplified as predicted with BsmB1 restriction sites

(Figure 3.4 A). A second recombinant PCR reaction generated a single recombined modified

NA gene segment (1.4 Kb) with BsmB1 restriction sites at both the 5’ and 3’ends (Figure 3.4

B). Data shown for the generation of a modified X31-NA-Gag197 gene segment was

representative of two other manipulated gene NA segments, X31-NA-Tat17 and PR8-NA-Tat17.

The PR8-NA-Gag197 recombinant virus was generated by the Stambas group at the University

of Melbourne and was not cloned by the candidate.

3.1.2. Cloning of modified NA plasmids into the pHW2000 reverse genetic plasmid system

Recombinant influenza NA gene segments (X31-NA-Gag197, X31-NA-Tat17, and PR8-NA-

Tat17) with BsmB1 restriction sites were then cloned into the pHW2000 plasmid for use in

downstream virus rescue experiments. The ligated products were transformed into competent

cells to generate new plasmids pHW2000-X31-NA-Gag197, pHW2000-X31-NA-Tat17 and

pHW2000-PR8-NA-Tat17. These were extracted and purified using the PureYield™ Plasmid

Miniprep system and run on a 0.8% agarose gel (Figure 3.5). Three products were visualised

on gel electrophoresis. The largest ligation product (4.4 Kb) contained both the pHW2000

vector (3 Kb) and the recombinant NA with inserted H-2Kd Gag197 sequence (1.4 Kb). To

confirm the insertion of the NA segment containing the H-2Kd Gag197 or H-2d Tat17 CD8+ T cell

epitopes, the recombinant pHW2000 plasmids were digested with PvuI (which cuts within the

vector), and PstI (which cuts within the insert). Clones that contained the appropriate modified

NA gene segment generated two products (1.9 Kb and 2.2 Kb), as shown in lane 3, Figure 3.6).

Lane 2 shows the undigested plasmid (including different forms of DNA) and lane 4 shows the

pHW2000-X31-NA-Gag197 cut with PvuI alone resulting in a single 4.4 Kb band (Figure 3.6).

To further confirm insertion of HIV-CD8+ T cell epitopes, sequencing of three plasmids,

pHW2000-X31-NA-Gag197, pHW2000-X31-NA-Tat17 and pHW2000-PR8-NA-Tat17 was

carried out in full (Figures 3.7, 3.8 and 3.9). We also confirmed the sequence of pHW2000-

PR8-NA-Gag197 previously generated by the Stambas laboratory (Figure 3.10).


77

Figure 3.4. Gel electrophoresis of PCR products used a 0.8% of agarose gel. Panel A shows the predicted PCR products contained the HIV-H-2Kd Gag197 CD8+ T cell epitope. Panel B shows the recombined PCR product representing the full length modified NA gene segment expressing H-2Kd Gag197. Data shown in this figure is representative of other PCR products generated for the rescue of X31 or PR8 influenza viruses expressing HIV-H-2Kd Gag197 or H-2d Tat17 CD8+ T cell epitopes.


78

Figure 3.5. Gel electrophoresis of ligated PCR products into the pHW2000 reverse genetics plasmid used a 0.8% agarose gel. Lane 1 shows the use of 1 Kb plus a DNA ladder ranging from 100 bp to 12 Kb. Lane 2 shows the ligation reaction with three products. These bands represent the non-ligated plasmid (pHW2000) at 3 Kb and the recombinant modified NA at 1.4 Kb. The ligation band size at 4.4 Kb represents the recombined pHW2000-NA-Gag197 plasmid.

500 bp

1.6Kb3 Kb4Kb

1Kb

5Kb

100 bp

Recombinant PCR 1.4 Kb

Vector 3 Kb Ligation 4.4 Kb

40 msec exposure time1 2


79

Figure 3.6. Gel electrophoresis of restriction enzyme digestion of the pHW2000-X31-NA-Gag197 plasmid using endonucleases PvuI and PstI. Lane 1 contains the 1 Kb plus DNA ladder with band sizes ranging from 100 bp to 12 Kb. Lane 2 shows undigested plasmid and lane 4 shows the pHW2000-X31-NA-Gag197 plasmid cut with PvuI alone. The pHW2000-X31-NA-Gag197 plasmid was digested with both PvuI and PstI in lane 3. Similar results were shown for the other generated plasmids, pHW2000-X31-NA-Tat17 and pHW2000-PR8-NA-Tat17.

1Kb

3kb

4.4 Kb2.2 Kb

1.9 Kb

500bp

100bp

1 2 3 4

1.6Kb2Kb


80

Figure 3.7. Nucleotide sequence of X31-NA-Gag197. The HIV-H-2Kd Gag197 epitope inserted into the NA gene segment is highlighted in bold letters (AMQMLKETI-protein and GCCATGCAAATGTTAAAAGAGACCATC-DNA sequence).

1/1 ATG M

AAT N

CCA P

AAT N

CAA Q

AAG K

ATA I

ATA I

ACA T

ATC I

CAG G

TGT C

CTG L

TAA S

GTA V

GTC V

GGA G

CTA L

ATT I

AGC S

CTA L

ATA I

CTC L

ATG M

CAG Q

ATT I

GCC A

ATC I

CTG L

GTA V

ACT T

ACT T

GTA V

ACA T

TTG L

CAT H

TTT F

AAG K

CAA Q

TAT Y

GAG E

TGC C

GAC D

TCC S

GCC A

ATG M

CAA Q

ATG M

TTA L

AAA K

GAG E

ACC T

ATC I

CCC P

GCG A

AGC S

AAC N

CAA Q

GTA V

ATG M

CCG P

TGT C

GAA E

CCA P

ATA I

ATA I

GAA E

AGG R

AAC N

ATA I

ACA T

GAG E

ATA I

GTG V

TAT Y

TTG L

AAT N

AAC N

ACC T

ACC T

ATA I

GAC D

AAA K

GAC E

AAA K

TGC C

CCC P

AAA K

GTA V

GTG V

GAA E

TAC Y

AGA R

AAT N

TGG W

TCA S

AAG K

CCG P

CAA Q

TGT C

CAA Q

ATT I

ACA T

GGA G

TTT F

GCA A

CCT P

TTT F

TCT S

AAG K

GAC D

AAT N

TCA S

ATC I

CGG R

CTT L

TCT S

GCT A

GGT G

GGG G

GAC D

ATT I

TGG W

GTG V

ACG T

AGA R

GAA E

CCT P

TAT Y

GTG V

TCA S

TGC C

GAT D

CAT H

GGC G

AAG K

TGT C

TAT Y

CAA Q

TTT F

GCA A

CTC L

GGG G

CAG Q

GGG G

ACC T

ACA T

CTA L

GAC D

AAC N

AAA K

CAT H

TCA S

AAT N

GAC D

ACA T

ATA I

CAT H

GAT D

AGA R

ATC I

CCT P

CAT H

CGA R

ACC T

CTA L

TTA L

ATG M

AAT N

GAG E

TTG L

GTG G

GTT V

CCA P

TTT F

CAT H

TTA L

GGA G

ACC T

AGG R

CAA Q

GTG V

TGT C

ATA I

GCA A

TGC W

TCC S

AGC S

TCA S

AGT S

TGT C

CAC H

GAT D

GGA G

AAA K

GCA A

TGG W

CTG L

CAT H

GTT V

TGT C

ATC I

ACT T

GGG G

GAT D

GAG D

AAA K

AAT N

GCA A

ACT T

GCT A

AGC S

TTC F

ATT I

TAT Y

GAC D

GGG G

AGG R

CTT L

GTG V

GAC D

AGT S

ATT I

GGT G

TCA S

TGG W

TCT S

CAA Q

AAT N

ATC I

CTC L

AGA R

ACC T

CAG Q

GAG E

TCG S

GAA E

TGC C

GTT V

TGT C

ATC I

AAT N

GGG G

ACT T

TGC C

ACA T

GTA V

GTA V

ATG M

ACT T

GAT D

GGA G

AGT S

GCT A

TCA S

GGA G

AGA R

GCC A

GAT D

ACT T

AGA R

ATA I

CTA L

TTC F

ATT I

GAA E

GAG E

GGG G

AAA K

ATT I

GTC V

CAT H

ATT I

AGC S

CCA P

TTG L

TCA S

GGA G

AGT S

GCT A

CAG Q

CAT H

GTA V

GAA E

GAG E

TGT C

TCC S

TGT C

TAT Y

CCT P

AGA R

TAT Y

CCT P

GGC G

GTC V

AGA R

TGT C

ATC I

TGC C

AGA R

GAC D

AAC N

TGG W

AAA K

GCC G

TCT S

AAT N

AGG R

CCC P

GTC V

GTA V

GAC D

ATA I

AAT N

ATG M

GAA E

GAT D

TAT Y

AGC S

ATT I

GAT D

TCC S

AGT S

TAT Y

GTG V

TGC C

TCA S

GGG G

CTT L

GTT V

GGC G

GAC D

ACA T

CCT P

AGA R

AAC N

GAC D

GAC D

AGA R

TCT S

AGC S

AAT N

AGC S

AAT N

TGC C

AGG R

AAT N

CCT P

AAT N

AAT N

GAG E

AGA R

GGG G

AAT N

CAA Q

GGA G

GTG V

AAA K

GGC G

TGG W

GCC S

TTT F

GAC D

AAT N

GGA G

GAT D

GAC D

GTG V

TGG W

ATG M

GGA G

AGA R

ACG T

ATC I

AGC S

AAG K

GAT D

TTA L

CGC R

TCA S

GGT G

TAT Y

GAA E

ACT T

TTC F

AAA K

GTC V

ATT I

GGT G

GGT G

TGG W

TCC S

ACA T

CCT P

AAT N

TCC S

AAA K

TCG S

CAG Q

ATC I

AAT N

AGA R

CAA Q

GTC V

ATA I

GTT V

GAC D

AGC S

GAT D

AAT N

CGG R

TCA S

GGT G

TAC Y

TCT S

GGT G

ATT I

TTC F

TCT S

GTT V

GAG E

GGC G

AAA K

AGC S

TGC C

ATC I

AAT N

AGG R

TGC C

TTT F

TAT Y

GTG V

GAG E

TTG L

ATA I

AGG R

GGA G

AGG R

AAA K

CAG Q

GAG E

ACT T

AGA R

GTG V

TGG W

ACC T

TCA S

AAC N

AGT S

ATT I

GTT V

GTG V

TTT F

TGT C

GGC G

ACT T

TCA S

GGT G

ACC T

TAT Y

GGA G

ACA T

GGC G

TCA S

TGG W

CCT P

GAT D

GGG G

GCG A

AAC N

ATC I

AAT N

TTC F

ATG M

CCT P

ATA I


81

Figure 3.8. Nucleotide sequence of pHW2000-PR8-NA-Tat17. The HIV-H-2d Tat17 epitope inserted into the NA gene segment is highlighted in bold letters (QPKTACTNC-protein and CAGCCTAAAACTGCTTGTACCAATTGC-DNA sequence).

1/1 ATG M

AAT N

CCA P

AAT N

CAA Q

AAG K

ATA I

ATA I

ACA T

ATC I

CAG G

TGT C

CTG L

TAA S

GTA V

GTC V

GGA G

CTA L

ATT I

AGC S

CTA L

ATA I

TTG L

CAA Q

ATA I

GGG G

AAT N

ATA I

ATC I

AAT I

TCA S

ATA I

TGG W

ATT I

TGG W

AGC S

CAT H

TCA S

ATT I

CAA Q

ACT T

GGA G

AGT S

CAA Q

CAG Q

CCT P

AAA K

ACT T

GCT A

TGT C

ACC T

AAT N

TGC C

AAC N

CAT H

ACT T

GGA G

ATA I

TGC C

AAC N

CAA Q

AAC N

ATC I

ATT I

ACC T

TAT Y

AAA K

AAT N

AGC S

ACC T

TGG W

GTA V

AAG K

GAC D

ACA T

ACT T

TCA S

GTG V

ATA I

TTA L

ACC T

GGC G

AAT N

TCA S

TCT S

CTT L

TGT C

CCC P

ATC I

CGT R

GGG G

TGG W

GCT A

ATA I

TAC Y

AGC S

AAA K

GAC D

AAT N

AGC S

ATA I

AGA R

ATT I

GGT G

TCC S

AAA K

GGA G

GAC D

GTT V

TTT F

GTC V

ATA I

AGA R

GAG E

CCC P

TTT F

ATT I

TCA S

TGT C

TCT S

CAC H

TTG L

GAA E

TGC C

AGG R

ACC T

TTT F

TTT F

CTG L

ACC T

CAA Q

GGT G

GCC A

TTA L

CTG L

AAT N

GAC D

AGG R

CAT H

TCA S

AAT N

GGG G

ACT T

GTA V

GAG K

GAC D

AGA R

AGC S

CCT P

TAT Y

AGG R

GCC A

TTA L

ATG M

AGC S

TGC C

CCT P

GTC V

GGT G

GAA E

GCT A

CCG P

TCC S

CCG P

TAC Y

AAT N

TCA S

AGA R

TTT F

GAA E

TCG S

GTT V

GCT A

TGG W

TCA S

GCA A

AGT S

GCA A

TGT C

CAT H

GAT D

GGC G

ATG M

GGC G

TGG W

CTA L

ACA T

ATC I

GGA G

ATT I

TCA S

GGT G

CCA P

GAT D

AAT N

GGA G

GCA A

GTG V

GCT A

GTA V

TTA L

AAA K

TAC Y

AAC N

GGC G

ATA I

ATA I

ACT T

GAA E

ACC T

ATA I

AAA K

AGT S

TGG W

AGG R

AAG K

AAA K

ATA I

TTG L

AGG R

ACA T

CAA Q

GAG E

TCT S

GAA E

TGT C

GCC A

TGT C

GTA V

AAT N

GGT G

TCA S

TGT C

TTT F

ACT T

ATA I

ATG M

ACT T

GAT D

GGC G

CCG P

AGT S

GAT D

GGG G

CTG L

GCC A

TCG S

TAC Y

AAA K

ATT I

TTC F

AAG K

ATC I

GAA E

AAG K

GGG G

AAG K

GTT V

ACT T

AAA K

TCA S

GAG E

TTG L

AAT N

GCA A

CCT P

AAT N

TCT S

CAC H

TAT Y

GAG E

GAA E

TGT C

TCC S

TGT C

TAC Y

CCT P

GAT D

ACC T

GGC G

AAA K

GTG V

ATG M

TGT C

GTG V

TGC C

AGA R

GAC D

AAT N

TGG W

CAT H

GGT G

TCG S

AAC N

CGG R

CCA P

TGG W

GTG V

TCT S

TTC F

GAT D

CAA Q

AAC N

CTG L

GAT D

TAT Y

CAA Q

ATA I

GGA G

TAC Y

ATC I

TGC C

AGT S

GGG G

GTT V

TTC F

GGT G

GAC D

AAC N

CGT R

CCC P

AAA K

GAT D

GGA G

ACA T

GGC G

AGC S

TGC C

GGT G

CCA P

GTG V

TAT Y

GTT V

GAT D

GGA G

GCA A

AAC N

GGA G

GTA V

AAG K

GGA G

TTT F

TCA S

TAT Y

AGG R

TAT Y

GGT G

AAT N

GGT G

GTT V

TGG W

ATA I

GGA G

AGG R

ACC T

AAA K

AGT S

CAC H

AGT S

TCC S

AGA R

CAT H

GGG G

TTT F

GAG E

ATG M

ATT I

TGG W

GAT D

CCT P

AAT N

GGA G

TGG W

ACA T

GAG E

ACT T

GAT D

AGT S

GAG E

TTC F

TCT S

GTG V

AGG R

CAA Q

GAT D

GTT V

GTG V

GCA A

ATG M

ACT T

GAT D

TGG W

TCA S

GGG G

TAT Y

AGC S

GGA G

AGT S

TTC F

GTT V

CAA Q

CAT H

CCT P

GAG E

CTA L

ACA T

GGG G

CTA L

GAC D

TGT C

ATA I

AGG R

CCG P

TGC C

TTC F

TGG W

GTT V

GAA E

TTA L

ATC I

AGG R

GGA G

CGA R

CCT P

AAA K

GAA E

AAA K

ACA T

TGG W

ACC T

AGT S

GCG A

AGC S

GCG A

AGC S

AGC S

ATT I

TCT S

TTT F

TGT C

GGC G

GTG V

AAT N

AGT S

GAT D

ACT T

GTA V

GAT D

TGG W

TCT S

TGG W

CCA P

GAC D

GGT G

GCT A

GAG E

TTG L

CCA P

TTG L

CCA P

TTC F

ACC T

ATT I

GAC D

AAG K


82

Figure 3.9. Nucleotide sequence of pHW2000-X31-NA-Tat17. The HIV-H-2d Tat17 epitope inserted into the NA gene segment is highlighted in bold letters (QPKTACTNC-protein and CAGCCTAAAACTGCTTGTACCAATTGC-DNA sequence).

1/1 ATG M

AAT N

CCA P

AAT N

CAA Q

AAG K

ATA I

ATA I

ACA T

ATC I

CAG G

TGT C

CTG L

TAA S

GTA V

GTC V

GGA G

CTA L

ATT I

AGC S

CTA L

ATA I

CTC L

ATG M

CAG Q

ATT I

GCC A

ATC I

CTG L

GTA V

ACT T

ACT T

GTA V

ACA T

TTG L

CAT H

TTT F

AAG K

CAA Q

TAT Y

GAG E

TGC C

GAC D

TCC S

CAG Q

CCT P

AAA K

ACT T

GCT A

TGT C

ACC T

AAT N

TGC C

CCC P

GCG A

AGC S

AAC N

CAA Q

GTA V

ATG M

CCG P

TGT C

GAA E

CCA P

ATA I

ATA I

GAA E

AGG R

AAC N

ATA I

ACA T

GAG E

ATA I

GTG V

TAT Y

TTG L

AAT N

AAC N

ACC T

ACC T

ATA I

GAC D

AAA K

GAC E

AAA K

TGC C

CCC P

AAA K

GTA V

GTG V

GAA E

TAC Y

AGA R

AAT N

TGG W

TCA S

AAG K

CCG P

CAA Q

TGT C

CAA Q

ATT I

ACA T

GGA G

TTT F

GCA A

CCT P

TTT F

TCT S

AAG K

GAC D

AAT N

TCA S

ATC I

CGG R

CTT L

TCT S

GCT A

GGT G

GGG G

GAC D

ATT I

TGG W

GTG V

ACG T

AGA R

GAA E

CCT P

TAT Y

GTG V

TCA S

TGC C

GAT D

CAT H

GGC G

AAG K

TGT C

TAT Y

CAA Q

TTT F

GCA A

CTC L

GGG G

CAG Q

GGG G

ACC T

ACA T

CTA L

GAC D

AAC N

AAA K

CAT H

TCA S

AAT N

GAC D

ACA T

ATA I

CAT H

GAT D

AGA R

ATC I

CCT P

CAT H

CGA R

ACC T

CTA L

TTA L

ATG M

AAT N

GAG E

TTG L

GTG G

GTT V

CCA P

TTT F

CAT H

TTA L

GGA G

ACC T

AGG R

CAA Q

GTG V

TGT C

ATA I

GCA A

TGC W

TCC S

AGC S

TCA S

AGT S

TGT C

CAC H

GAT D

GGA G

AAA K

GCA A

TGG W

CTG L

CAT H

GTT V

TGT C

ATC I

ACT T

GGG G

GAT D

GAG D

AAA K

AAT N

GCA A

ACT T

GCT A

AGC S

TTC F

ATT I

TAT Y

GAC D

GGG G

AGG R

CTT L

GTG V

GAC D

AGT S

ATT I

GGT G

TCA S

TGG W

TCT S

CAA Q

AAT N

ATC I

CTC L

AGA R

ACC T

CAG Q

GAG E

TCG S

GAA E

TGC C

GTT V

TGT C

ATC I

AAT N

GGG G

ACT T

TGC C

ACA T

GTA V

GTA V

ATG M

ACT T

GAT D

GGA G

AGT S

GCT A

TCA S

GGA G

AGA R

GCC A

GAT D

ACT T

AGA R

ATA I

CTA L

TTC F

ATT I

GAA E

GAG E

GGG G

AAA K

ATT I

GTC V

CAT H

ATT I

AGC S

CCA P

TTG L

TCA S

GGA G

AGT S

GCT A

CAG Q

CAT H

GTA V

GAA E

GAG E

TGT C

TCC S

TGT C

TAT Y

CCT P

AGA R

TAT Y

CCT P

GGC G

GTC V

AGA R

TGT C

ATC I

TGC C

AGA R

GAC D

AAC N

TGG W

AAA K

GCC G

TCT S

AAT N

AGG R

CCC P

GTC V

GTA V

GAC D

ATA I

AAT N

ATG M

GAA E

GAT D

TAT Y

AGC S

ATT I

GAT D

TCC S

AGT S

TAT Y

GTG V

TGC C

TCA S

GGG G

CTT L

GTT V

GGC G

GAC D

ACA T

CCT P

AGA R

AAC N

GAC D

GAC D

AGA R

TCT S

AGC S

AAT N

AGC S

AAT N

TGC C

AGG R

AAT N

CCT P

AAT N

AAT N

GAG E

AGA R

GGG G

AAT N

CAA Q

GGA G

GTG V

AAA K

GGC G

TGG W

GCC S

TTT F

GAC D

AAT N

GGA G

GAT D

GAC D

GTG V

TGG W

ATG M

GGA G

AGA R

ACG T

ATC I

AGC S

AAG K

GAT D

TTA L

CGC R

TCA S

GGT G

TAT Y

GAA E

ACT T

TTC F

AAA K

GTC V

ATT I

GGT G

GGT G

TGG W

TCC S

ACA T

CCT P

AAT N

TCC S

AAA K

TCG S

CAG Q

ATC I

AAT N

AGA R

CAA Q

GTC V

ATA I

GTT V

GAC D

AGC S

GAT D

AAT N

CGG R

TCA S

GGT G

TAC Y

TCT S

GGT G

ATT I

TTC F

TCT S

GTT V

GAG E

GGC G

AAA K

AGC S

TGC C

ATC I

AAT N

AGG R

TGC C

TTT F

TAT Y

GTG V

GAG E

TTG L

ATA I

AGG R

GGA G

AGG R

AAA K

CAG Q

GAG E

ACT T

AGA R

GTG V

TGG W

ACC T

TCA S

AAC N

AGT S

ATT I

GTT V

GTG V

TTT F

TGT C

GGC G

ACT T

TCA S

GGT G

ACC T

TAT Y

GGA G

ACA T

GGC G

TCA S

TGG W

CCT P

GAT D

GGG G

GCG A

AAC N

ATC I

AAT N

TTC F

ATG M

CCT P

ATA I


83

Figure 3.10. Nucleotide sequence of pHW2000-PR8-NA-Gag197. The HIV H-2Kd Gag197 in the NA gene segment is highlighted in bold letters (AMQMLKETI-protein and nucleotides GCCATGCAAATGTTAAAAGAGACCATC-DNA sequence).

1/1 ATG M

AAT N

CCA P

AAT N

CAA Q

AAG K

ATA I

ATA I

ACA T

ATC I

CAG G

TGT C

CTG L

TAA S

GTA V

GTC V

GGA G

CTA L

ATT I

AGC S

CTA L

ATA I

TTG L

CAA Q

ATA I

GGG G

AAT N

ATA I

ATC I

AAT I

TCA S

ATA I

TGG W

ATT I

TGG W

AGC S

CAT H

TCA S

ATT I

CAA Q

ACT T

GGA G

AGT S

CAA Q

GCC A

ATG M

CAA Q

ATG M

TTA L

AAA K

GAG E

ACC T

ATC I

AAC N

CAT H

ACT T

GGA G

ATA I

TGC C

AAC N

CAA Q

AAC N

ATC I

ATT I

ACC T

TAT Y

AAA K

AAT N

AGC S

ACC T

TGG W

GTA V

AAG K

GAC D

ACA T

ACT T

TCA S

GTG V

ATA I

TTA L

ACC T

GGC G

AAT N

TCA S

TCT S

CTT L

TGT C

CCC P

ATC I

CGT R

GGG G

TGG W

GCT A

ATA I

TAC Y

AGC S

AAA K

GAC D

AAT N

AGC S

ATA I

AGA R

ATT I

GGT G

TCC S

AAA K

GGA G

GAC D

GTT V

TTT F

GTC V

ATA I

AGA R

GAG E

CCC P

TTT F

ATT I

TCA S

TGT C

TCT S

CAC H

TTG L

GAA E

TGC C

AGG R

ACC T

TTT F

TTT F

CTG L

ACC T

CAA Q

GGT G

GCC A

TTA L

CTG L

AAT N

GAC D

AGG R

CAT H

TCA S

AAT N

GGG G

ACT T

GTA V

GAG K

GAC D

AGA R

AGC S

CCT P

TAT Y

AGG R

GCC A

TTA L

ATG M

AGC S

TGC C

CCT P

GTC V

GGT G

GAA E

GCT A

CCG P

TCC S

CCG P

TAC Y

AAT N

TCA S

AGA R

TTT F

GAA E

TCG S

GTT V

GCT A

TGG W

TCA S

GCA A

AGT S

GCA A

TGT C

CAT H

GAT D

GGC G

ATG M

GGC G

TGG W

CTA L

ACA T

ATC I

GGA G

ATT I

TCA S

GGT G

CCA P

GAT D

AAT N

GGA G

GCA A

GTG V

GCT A

GTA V

TTA L

AAA K

TAC Y

AAC N

GGC G

ATA I

ATA I

ACT T

GAA E

ACC T

ATA I

AAA K

AGT S

TGG W

AGG R

AAG K

AAA K

ATA I

TTG L

AGG R

ACA T

CAA Q

GAG E

TCT S

GAA E

TGT C

GCC A

TGT C

GTA V

AAT N

GGT G

TCA S

TGT C

TTT F

ACT T

ATA I

ATG M

ACT T

GAT D

GGC G

CCG P

AGT S

GAT D

GGG G

CTG L

GCC A

TCG S

TAC Y

AAA K

ATT I

TTC F

AAG K

ATC I

GAA E

AAG K

GGG G

AAG K

GTT V

ACT T

AAA K

TCA S

GAG E

TTG L

AAT N

GCA A

CCT P

AAT N

TCT S

CAC H

TAT Y

GAG E

GAA E

TGT C

TCC S

TGT C

TAC Y

CCT P

GAT D

ACC T

GGC G

AAA K

GTG V

ATG M

TGT C

GTG V

TGC C

AGA R

GAC D

AAT N

TGG W

CAT H

GGT G

TCG S

AAC N

CGG R

CCA P

TGG W

GTG V

TCT S

TTC F

GAT D

CAA Q

AAC N

CTG L

GAT D

TAT Y

CAA Q

ATA I

GGA G

TAC Y

ATC I

TGC C

AGT S

GGG G

GTT V

TTC F

GGT G

GAC D

AAC N

CGT R

CCC P

AAA K

GAT D

GGA G

ACA T

GGC G

AGC S

TGC C

GGT G

CCA P

GTG V

TAT Y

GTT V

GAT D

GGA G

GCA A

AAC N

GGA G

GTA V

AAG K

GGA G

TTT F

TCA S

TAT Y

AGG R

TAT Y

GGT G

AAT N

GGT G

GTT V

TGG W

ATA I

GGA G

AGG R

ACC T

AAA K

AGT S

CAC H

AGT S

TCC S

AGA R

CAT H

GGG G

TTT F

GAG E

ATG M

ATT I

TGG W

GAT D

CCT P

AAT N

GGA G

TGG W

ACA T

GAG E

ACT T

GAT D

AGT S

GAG E

TTC F

TCT S

GTG V

AGG R

CAA Q

GAT D

GTT V

GTG V

GCA A

ATG M

ACT T

GAT D

TGG W

TCA S

GGG G

TAT Y

AGC S

GGA G

AGT S

TTC F

GTT V

CAA Q

CAT H

CCT P

GAG E

CTA L

ACA T

GGG G

CTA L

GAC D

TGT C

ATA I

AGG R

CCG P

TGC C

TTC F

TGG W

GTT V

GAA E

TTA L

ATC I

AGG R

GGA G

CGA R

CCT P

AAA K

GAA E

AAA K

ACA T

TGG W

ACC T

AGT S

GCG A

AGC S

GCG A

AGC S

AGC S

ATT I

TCT S

TTT F

TGT C

GGC G

GTG V

AAT N

AGT S

GAT D

ACT T

GTA V

GAT D

TGG W

TCT S

TGG W

CCA P

GAC D

GGT G

GCT A

GAG E

TTG L

CCA P

TTG L

CCA P

TTC F

ACC T

ATT I

GAC D

AAG K


84

3.1.3. Rescue of recombinant influenza viruses using influenza reverse genetics

Once the sequence of the recombinant plasmids was confirmed, the recombinant influenza

viruses containing the modified NA genes were generated using reverse genetics. This

technique is summarized in Figure 3.11 and involved the transfection of the eight pHW2000

plasmids (pHW2000-NP, pHW2000-PA, pHW2000-PB1, pHW2000-PB2, pHW2000-M,

pHW2000-NS, pHW2000-HA and the pHW2000-NA-Gag197 or pHW2000-NA-Tat17), each

containing one of the influenza gene segments into a co-culture of HEK-293T and MDCK

cells. Supernatant containing the recombinant virus was then further amplified in 10 day old

embryonated chicken eggs (Figure 3.11).

3.1.4. Amplification of generated recombinant influenza viruses Following reverse genetics, three rescued influenza viruses were further amplified by

inoculation of the 10 day old embryonated eggs (Methods, Section 2.2.12) and the supernatant

collected. The allantoic fluids containing the amplified viruses were then harvested and kept at

-80ºC. All allantoic fluid samples were subjected to a semi-quantitative HA assay to confirm

the rescued viruses. HA titers were then determined for each virus. X31-NA-Gag197 was 1/256,

PR8-NA-Tat17 was 1/256, and X31-NA-Tat17 was 1/128 (Figure 3.12). Following confirmation

of the viral titers, the rescued viruses were determined by plaque assay (see Methods Section

2.2.14). The virus titer for X31-NA-Gag197 was 6.0 × 107 pfu/ml, X31-NA-Tat17 was 6.7 × 107

pfu/ml and PR8-NA-Tat17 was 8.2 × 107 pfu/ml. The titer of the fourth virus already generated

in the Stambas laboratory for PR8-NA-Gag197 was 4.5 × 108 pfu/ml (Table 3.1).

3.1.5. Sequencing of recombinant viruses amplified in allantoic fluid RNA was extracted from the allantoic fluid and PCR was amplified using NA primers, forward

primer, Bm-NA-1-F (5’TAT TCG TCT CAG GGA GCA AAA GCA GGA GT-3’) and

reverse primer, Bm-NA-1413-R (5’ATA TCG TCT CGT ATT AGT AGA AAC AAG GAG

TTT TTT-3’). This PCR product was run on an agarose gel electrophoresis, excised, purified

and sequenced. It was at the expected 1.4 Kb site as previously described in Figure 3.4 B.

Sequencing confirmed the NA gene contained our inserted HIV-CD8+ T cell epitopes as

described previously in Figure 3.7. The confirmed construct was appropriately labelled X31-

NA-Gag197. In addition, the sequencing results confirmed the NA gene segment with inserted

H-2d epitopes, Gag197 and Tat17, had been faithfully maintained in the amplification process of

all rescued viruses as shown previously when plasmids were sequenced in Figures 3.8, 3.9 and

3.10.


85

Figure 3.11. Reverse genetics strategy for the rescue of recombinant influenza viruses expressing HIV-CD8+ T cell epitopes. This schematic diagram shows the procedure used to rescue recombinant influenza viruses. The manipulated pHW2000-X31-NA-Gag197 or PR8-NA-Tat17 or X31-NA-Tat17 plasmid was co-transfected with 7 other pHW2000 encoding influenza gene segments (pHW2000-PB1, PB2, PA, HA, NP, M and NS) in a co-culture of HEK-293T and MDCK cells (Hoffmann et al., 2000).

HA NS

NPPAPB1PB2

M

NA

7 master strain plasmids of PR8 or

X31 viruses

Rescued reverse genetics viruses were plaqued to determine titre of viruses

pHW2000–PR8 or X31-NA-Gag197-205

Transfection and amplification


86

Figure 3.12. Hemagglutination assay of three rescued recombinant influenza viruses. Supernatants were harvested from the reverse genetic cultures of three viruses, PR8-NA-Tat17, X31-NA-Gag197 and X31-NA-Tat17. They were diluted two fold in a Hemagglutination assay (HA) in combination with 0.5% chicken RBCs. This figure shows the positive results of hemagglutination applied on a round bottom plate using two serial dilution presented for three rescued viruses (A) anda graph that presents the HA titers of these rescued viruses (B).


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Table 3.1. Titration of rescued recombinant influenza viruses by plaque assay. Allantoic fluids harvested following 10 day old embryonated chicken eggs were used in a plaque assay on MDCK cell monolayers to determine viral titers.

Recombinant virus Titer by plaque forming unit (pfu)/ml

X31-NA-Gag197

6.0 × 107 pfu/ml

X31-NA-Tat17 6.7 × 107 pfu/ml

PR8-NA-Tat17 8.2 × 107 pfu/ml

PR8-NA-Gag197 4.5 × 108 pfu/ml (Stambas group)


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3.2. Discussion As a result of the failure of most traditional vaccines to induce appropriate immune responses

for protection against viral diseases, such as HIV/AIDS, new strategies including live

virus/bacterial vectors are currently being investigated to use for expressing specific antigens

or peptides of infectious viruses or bacteria as an effective vaccine control infection (Dudek,

2006; Fitzgerald et al., 2003; Izumi et al., 2003). These vectors can be generated by a number

of methodologies, one of which is reverse genetics. Reverse genetics is a powerful strategy

used in a range of different applications such as gene therapy and vaccine development. This

involves the manipulation of plasmid DNA in order to create novel modified features in viral

or bacterial agents when rescued as whole organisms (Jalilian et al., 2010; Stoff-Khalili, Dall,

and Curiel 2006; Tung et al., 2007). In order to generate these vectors, it is necessary to place

antigens, polypeptides or epitopes into genes for further manipulation. Given the availability

of a mouse model for live vaccination (influenza based strategy), well defined HIV-CD8+ T

cell epitopes derived in the H-2d BALB/c mouse model and influenza reverse genetics plasmids

can be used to generate recombinant vectors for use in vaccination studies. Influenza virus type

A has been considered a strong candidate for live viral vector delivery, given its ability to infect

via mucosal surfaces such as the respiratory tract (Marsh and Tannock, 2005; Satterlee, 2008;

Smith, 2007). Negative stranded RNA viruses, such as influenza are characterized by their

ability to infect humans and laboratory animals through mucosal portals of entry resulting in

broad and long term humoral and cellular immunity (Fodor et al., 1999; Mueller et al., 2010;

Neumann and Kawaoka, 2001; Yao and Wang, 2008). In addition, influenza viruses are

especially amenable for use as recombinant vaccine vectors due to the presence of a segmented

genome and the availability of plasmids that allow manipulation and rescue of novel

recombinant viruses (Fodor et al., 1999; Palese et al., 1996). As such, influenza viruses have

been used as vectors to express antigens from HIV, SIV, tuberculosis, malaria and lympho-

cytic-chorio-meningitis virus as a part of the HA or NA gene segments to generate novel

vaccines (Jalilian et al., 2010; Rodrigues et al., 1994; Sexton et al., 2009). Influenza A viruses

have also been recombinant vaccine vectors that express HIV antigens or CD8+ T cell epitopes

that have been inserted into the NA stalk PR8 (H1N1) and X31 (H3N2) (Pleschka et al., 1996).

Early work in particular focused on the expression of HIV envelope antigens or epitopes

(Kramer, 2007; Letvin, 2009; Zolla-Pazner, 2004).


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Various recombinant influenza viruses expressing HIV-CD8+ T cell epitopes have been

successfully generated to use as HIV or SIV vaccines. For instance, A/PR/8/34 (HIN1) and

A/Aichi/2/1968 (H3N2) were manipulated by a reverse genetics strategy in order to use in a

preclinical trial of vaccination in mice or macaques. The result was stimulation of a mucosal

CD8+ T cell response. These stimulated mucosal CD8+ T cells were critical to decrease viral

copies and control of HIV or the SIV infection depending on the breadth of stimulated CD8+

T cell responses (Casimiro et al., 2005; Hoffmann et al., 2002; Liu et al., 2009; Pachler, Mayr

and Vlasak, 2010; Rimmelzwaan et al., 2007). Some preclinical and clinical trials of HIV-1

vaccine candidates have used HIV-1 Env proteins or HIV-Env CD8+ T cell epitopes which

stimulated strong humoral and cellular immune responses (Sattentau, 2013; Cukalac et al.,

2009; Rerks-Ngarm et al., 2009).

Historically, most HIV-1 candidate vaccines using Env protein/epitopes have been unable to

elicit protective responses (Cho et al., 2001; McBurney and Ross, 2008; Plotkin, 2009). This

is because of high variability in envelope glycoprotein amino acid sequences amongst different

HIV-1 clades (Sreepian et al., 2009; Stamatatos et al., 2009; Zolla-Pazner and Cardozo, 2010).

This study has focused on the generation of strong mucosal cytotoxic CD8+ T cell responses,

and thus have revolved around the use of HIV-Gag+CD8+ T cell epitopes. Gag is a structural

protein necessary for the assembly of newly generated virions during the life cycle of HIV-1

in infected individuals and is highly conserved making it an ideal candidate for CTL based

vaccines (Bryant and Ratner, 1990; Currier et al., 2006). Tat epitopes were also incorporated

into our vaccine constructs. Tat is also a frequently targeted by cytotoxic CD8+ T cells as a

highly conserved regulatory protein with defined CD8+ T cell epitopes (Addo et al., 2001;

Gavioli et al., 2008; Gavioli et al., 2004; Scriba et al., 2005).

In this project, three recombinant live influenza viruses expressing HIV-CD8+ T cell epitopes,

H-2Kd Gag197 or H-2d Tat17 epitopes have been successfully generated. These two candidate

HIV-1 epitopes were inserted individually into the NA stalk of A/Puerto Rico/8/34 (H1N1) or

A/Aichi/2/1968 (H3N2) influenza viruses and then rescued using reverse genetics. These virus

strains were used as they were adapted in mice. Three rescued influenza viruses, X31-NA-

Gag197, X31-NA-Tat17 and PR8-NA-Tat17 were obtained with similar titers tested by HA and

plaque assay. However, they were different from the titer of PR8-NA-Gag197 generated

previously by the Stambas group. This was probably because the ability of three newly


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generated viruses to modify and replicate in allantoic fluids was similar as they were

transfected and modified into a similar batch of co-culture. The viruses were also inoculated as

supernatant into the same batch of embryonated chicken eggs, which was completely different

from the co-culture inoculated with the PR8-NA-Gag197. This may also be the result of the

different batches of embryonated eggs inoculated with supernatants of these generated viruses

compared to the fourth previously generated virus which might be adapted to grow on different

types of chicken eggs. There was also a highly significant increase in the affinity of egg-adapted

variants of influenza A and B viruses to replicate compared to the non-adapted. This occurred

by increased binding of the plasma membranes of the chorio-allantoic cells of the chicken eggs

and also by increasing their multiplication (Gambaryan, Robertson and Matrosovich, 1999). It

was anticipated that targeting two different epitopes would broaden the overall CD8+ T cell

response and decrease opportunities for the generation of CTL escape in the future if the virus

was further studied in pre-clinical trials in humans or macaques. Given the success of initial

studies with X31 and PR8-NA-Gag197 recombinant viruses, analysis of X31 and PR8-NA-Tat17

viruses was not taken further but could be explored at a later point in time.

However, there are several limitations that can affect highly efficient influenza reverse genetics

systems in HIV vaccine production. For instance, a limited number of mammalian cells are

likely to be infected to amplify the eight plasmids to generate influenza viruses (Palache et al.,

1999; Halperin et al., 2002; Brühl et al., 2000). Although MDCK and Vero cells are mainly

used to produce the recombinant influenza virus, there is no high efficiency to produce a large

amount of the virus (Fodor et al., 1999; Nicolson et al., 2005). Also, some modified influenza

viruses are unable to grow with high titer which leads to delay in virus production (Neumann

et al., 1999; Hoffmann et al., 2002). Another limitation of using HA or NA in the influenza

reverse genetics system is due to their small size. For instance, there is space for approximately

10 amino acids in a foreign amino acid sequence to be inserted into the HA molecule (Garcia-

Sastre and Palese, 1995; Palese et al., 1997; Staczek et al., 1998). Although the NA stalk

tolerates a longer foreign peptide sequence to be introduced into the stalk region, the maximum

inserted length would be less than 82 amino acids with high opportunity for poor presentation

of these peptides to the immune system. Another study using mice recognized that the

neuraminidase (NA) stalk can tolerate as many as 41 amino acid peptide insertions expressed

on the rescued influenza virus A/WSN/33 (HlNl), which was well replicated in intranasal

infected BALB/c mice (Castrucci and Kawaoka, 1993; Luo, Chung and Palese, 1993).


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Furthermore, it was clearly detected that the maximum length of a foreign peptide is 58 amino

acids can be introduced into the neuraminidase stalk without the loss of viral function. The

peptide was introduced with the H-2d LCMV nucleoprotein that expressed on the A/WSN/33

(HIN1) influenza virus and was used intranasally and intraperitonealy in BALB/c mice. This

induced CD8+ T cell protected vaccinated mice for the intracerebral challenge CA 1371 strain

of LCMV (Castrucci et al., 1994). In addition, a limited size of the NA gene segment inserted

with efficient amount of the HIV-1 protein was noted with loss of its functionality. This was

detected by the insertion of the HIV-1 p17 Gag protein (rFlu-p17) gene that included 1-132

amino acids inserted into the NA segment of the PR8 (A/PR/8/34) influenza virus. Insertion of

the amino acids was not possible until complete removal of the start codon of NA and most of

the NA coding sequence was excised by MluI and SpeI endonucleases and replaced by the

HIV-1 p17 Gag protein, or HIV-1 Rev protein for use as a HIV vaccine in C57BL/6 and

BALB/c mice (de Goede et al., 2009; van Baalen et al., 2005). Furthermore, recombinant

influenza viruses generated from this transgenic process were replication deficient viruses

because they lacked functional NA, despite the inducementof a detectable immunity response

as they were depended on the exogenous source of NA (Sigma-Aldrich) added to enhance the

replication (De Wit et al., 2004; Rimmelzwaan et al., 2007). As a result, the strategy for this

project was to use only a limited number of amino acids as specific epitopes inserted into the

NA stalk, which requires knowledge of the MHC haplotype of specific peptides or epitopes to

generate a vaccine. An alternative strategy to rescue recombinant influenza viruses expressing

full HIV polypeptide sequences is possible. For example, the residue of 137 amino acids from

the HIV-1 Nef protein that contains nucleotides 210-618 inserted into the NS1 segment of PR8

(A/PR/8/34: H1N1), or the A/Aichi/1/168 (H3N2) influenza virus that uses an intranasal

vaccine in BALB/c mice to induce robust antibodies and CD8+ T cell responses in the systemic

and mucosal compartments, especially in the urogenital tract (Ferko et al., 2001). This problem

can be avoided by using the NS gene segment with an inserted whole green fluorescent protein

(GFP) containing 125 amino acids in the NS1 only (consisted of 230 amino acids), that

maintains the fully functional non-structural proteins of the recombinant influenza viruses

(Kittel et al., 2004). This provides an opportunity to introduce large sequences of HIV-1

proteins into the NS segment (Takasuka et al., 2002; Kittel et al., 2005). Therefore, it is highly

recommended any future work involve expressing full length HIV proteins using the NS as this

would overcome the need to know the MHC haplotypes.


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Live influenza vaccines are currently in use and licensed by MedImmune in the USA. Using

seasonal vaccines as a backbone or uncommon HA and NA combination will avoid issues with

neutralizing antibodies.

Chapter 4: Induction of CD8+ T cell responses using recombinant influenza viruses

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4. Chapter 4: Induction of CD8+ T cell responses using recombinant influenza viruses.

4.1. Overview Chapter 3 described the generation of recombinant influenza viruses expressing the HIV-H-2d

CD8+ T cell epitopes, Gag197 or Tat17. This chapter tests these recombinant vaccines in vivo in

a mousee study using different mucosal routes of vaccination in order to elucidate the optimal

approach for inducing HIV-specific CD8+ T cell immune responses in various lymphoid organs

and regional lymph nodes distributed in different parts of the body, especially in the urogenital

region which would provide a good opportunity to control HIV-1 infection (Garulli, Kawaoka

and Castrucci, 2004). For this reason, different mucosal routes were used, specifically

intranasal and intravaginal routes. This is because influenza virus naturally infects various

mucosa, especially pulmonary mucosa. The intranasal route of vaccination is commonly used

to deliver recombinant influenza A viruses in order to generate antigen specific mucosal

responses in the spleen and respiratory tract mucosa that are represented by CD8+ T cells and

neutralizing antibody responses targeting endogenous influenza antigens and exogenous HIV-

1 peptides (Byron et al., 2011; Takasuka et al., 2002). A study using a modified A/WSN/33

expressing HIV-Env protein (HIV-1 IIIB gp160) in BALB/c mice resulted in a 4 fold increase

of CD8+ T cell responses in the spleen and less frequent but detectable response in the genital

lymph nodes (Gherardi et al., 2003).

In contrast the intravaginal route of vaccination is rarely used in women (Johansson et al.,

2001). It is not common for the intravaginal route to be used to induce a mucosal immune

response following influenza infection in mice given the lack of trypsin like enzymes to assist

replication. There are a couple of examples however to note. Intravaginal using the

recombinant influenza A virus vector expressing the HIV-Env protein produced strong durable

mucosal CD8+ T cells specific for the influenza H-2Kd NP147 (TYQRTRALV) and HIV- H-2

Dd Env (RGPGRAFVTI) peptide in regional vaginal draining lymph nodes, the inguinal lymph

nodes and the spleen when compared to intranasally vaccinated mice that produced robust

CD8+ T cell responses in the distant urogenital lymph nodes and the respiratory lymphoid

tissues (Garulli, Kawaoka and Castrucci, 2004). In addition recombinant adenovirus (AdC6)

expressing HIV-H-2Kd Gag197 (AMQMLKETI) viruses have been used in BALB/c mice via

intranasal and intravaginal, vaccination to induce large numbers of CD8+ T cells in the spleen,


94

iliac lymph nodes and e vaginal tract (IELs). Higher vaginal Gag197+CD8+ T cell responses

were mainly induced in the intravaginaly primed mice (de Souza et al., 2007). The responses

induced through intravaginal vaccination have also been shown to be protective following

pseudo-challenge (Li et al., 2008).

The work in this chapter extends previous studies through an analysis of HIV-H-2d CD8+ T

cell epitopes, Gag197 or Tat17. A direct comparison between intranasal and intravaginal

vaccination, and a combination of intranasal and intravaginal routes in eliciting CD8+ T cell

responses will be undertaken. To characterize induction of HIV-specific and mucosal immune

responses in a mouse model, two laboratory strains of the influenza virus, (X31, H3N2 and

PR8, H1N1) were used as vectors expressing CD8+ T cell epitopes (as described in Chapter 3).

CD8+ T cell responses from different effector sites including the broncho-alveolar lavage

(BAL), the spleen, mediastinal lymph nodes (MedLN) and inguinal lymph nodes (ILN) were

characterized using intracellular cytokine and tetramer staining in combination with staining

for specific mucosal phenotypic markers. Finally, CD8+ T cells expressing mucosal surface

markers were sorted using flow cytometry and RNA extracted to detect expression of cytokine

IL-15 and its surface receptor IL-15Rα, all of which are known to be involved in the migration

of CD8+ T cells into effector mucosal sites.

4.1.1. Characterization of CD8+ T cell responses following primary infection with recombinant X31-NA-Gag197 influenza virus

To evaluate the immunogenicity of the recombinant viruses and their ability to induce immune

responses against the exogenous HIV-H-2Kd Gag197 epitope and the endogenous influenza

virus H2-Kd NP147 epitope, various mucosal routes (i.n and i.v) of vaccination were tested in

mice to determine which route provided robust HIV specific and mucosal immune responses,

and whether these recombinant influenza viruses were safe to use in mice. Six to eight week

old BALB/c mice purchased from the Animal Resources Centre (ARC) were housed in a

conventional animal house for 7 days for environmental adaptation before inoculation with the

recombinant influenza virus. All animal experiments were conducted according to protocols

approved by the CSIRO AAHL and Deakin University’s Animal Ethics Committees.

BALB/c mice were primed intranasally or intravaginally with 1 × 104 pfu X31-NA-Gag197

influenza viruses and their body weight monitored daily for ten days following infection.


95

Following the first two days of intranasal infection, mice maintained normal weight but started

to lose weight gradually on the third day. The weight loss continued into the fourth fifth and

sixth days, dropping approximatey (7.8 ± 1.0 %) from their original starting weight. This

weight loss was significant and consistent with results in the literature (Cukalac et al.,

2009).Mice gradually began regaining weight on day 7 with complete recovery of original

weight occurring by day 10, thus indicating clearance of the influenza virus (Figure 4.1A).

However, mice in the intravaginal infected group showed no signs of weight loss throughout

the monitoring period following primary infection with the recombinant influenza virus, X31-

NA-Gag197 (Figure 4.1B). Weight loss for intranasally infected mice was expected as influenza

viruses replicate in the respiratory tract due to the presence of tryptase Clara an enzyme

necessary for cleavage of hemagglutinin (HA) (Garulli, Kawaoka and Castrucci, 2004).

In contrast, the absence of protease enzyme activity in the vaginal mucosa results in abortive

replication (Garulli, Kawaoka and Castrucci, 2004; Böttcher-Friebertshäuser et al., 2010;

Zhirnov, Ovcharenko and Bukrinskaya, 1984). This expected lack of replication in the genital

tract mucosa translated to a lack of weight loss. However, there was an immune response

induced in the inguinal lymph nodes following intravaginal influenza infection due to this

abortive infection. This is because influenza antigens were presented by the regional antigen

presenting cells to the immune system in these nodes. Further studies such as a quantitative

PCR or immuno-histochemistry could be used to determine antigen levels at these sites.

Importantly, these observations serve as evidence that there was no cross contamination

between the vagina and respiratory tract during experimental infection as there was no

influenza specific CD8+ T cell response detected in the pulmonary mucosal compartments,

such as BAL or Mediastinal lymph nodes following intravaginal vaccination.


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Figure 4.1. Weight loss measured in BALB/c mice following primary infection with recombinant X31-NA-Gag197 influenza virus. BALB/c mice (each group n = 5) were infected intranasally (A) or intravaginally (B) with 1 × 104 pfu of X31-NA-Gag197 influenza virus and were monitored for changes in body weight over a ten day period following infection. Also, change in the actual weight of individual BALB/c mice infected intranasally (C) or intravaginally (D). All data represents the mean ± SD of three experiments, using the Student t-test **p<0.01 and ***p<0.001 compares drop in weight of the infected mice with the second day following infection.


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Next, the HIV-Gag197 and influenza NP147 specific CD8+ T cell responses were analyzed to

determine the efficacy of the newly generated recombinant viruses. As mentioned above two

groups of six to eight week old BALB/c mice were infected intranasally or intravaginally with

1 × 104 pfu/30 μl recombinant influenza viruses X31-NA-Gag197. After 10 days of infection,

the spleens of infected mice were harvested to determine responses at the known peak of

adaptive immunity (Figure 4.2). A single dose of X31-NA-Gag197 given intranasally to BALB/c

mice induced a detectable IFN-γ+CD8+ T cell response against HIV-Gag197 peptide using ICS

of the harvested spleen. We also saw a co-dominat influenza NP147-specific response (2.56 ×

105 ± 2.29 ×104) IFN-γ+CD8+ T cells for influenza NP147 compared to (2.32 × 105 ± 8.77 × 103)

IFN-γ +CD8+ T cells for Gag197 (Figure 4.3).

This is unusual for exogenous CD8+ T cell responses induced using a live vaccine vector, where

responses are often directed towards the vector. An example of this is the Step/Phambili HIV

trial vaccine that used a recombinant adenovirus (rAd5) to express multiple HIV-1 proteins,

Gag/Pol/Nef or Env. These epitopes induced both CD8+ and CD4+ T cell responses in

vaccinated groups of homosexual men who were at risk of HIV infection but the majority of

the immune responses weredirected against the Ad5 vector (Koup and Douek, 2011; Koup et

al., 2010). In addition our results are similar to those observed by Cukalac and colleagues who

found co-dominance of Kd NP147 and exogenous HIV-Dd Env311 in the spleen and BAL of

BALB/c mice following a single intranasal dose of the X31-NA-Env311 influenza virus

(Cukalac et al., 2009). We were unable to detect IFN-γ+CD8+ T cell responses against HIV-H-

2d Tat17 following primary intranasal infection of mice using the X31-NA-Tat17 virus (data not

shown) and so all subsequent experiments have focused on the HIV Gag197 CD8+ T cell epitope.

We believe this may have been a processing issue but given time constraints we were unable

to analyse this further.

We further analysed the quality of the CD8+ T cell responses by analysing the proportion of

TNF-α+IFN-γ+CD8+ and IL-2+IFN-γ+CD8+ T cells for the endogenous H-2Kd NP147 and the

exogenous H-2Kd Gag197 epitopes as described previously (La Gruta, Turner and Doherty,

2004). No significant difference in TNF-α+IFN-γ+CD8+ T cells specific for H-2Kd NP147+ (74.8

± 3.0331%) and H-2Kd Gag197+ (72 ± 3.4641%), (p<0.210) responses were observed. This was


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also the case for IL-2+TNF-α+IFN-γ+CD8+T cells were the H-2Kd NP147 epitope (33.2 ±

3.4205%) and H-2Kd Gag197 epitope (30.8 ± 3.9623%) were equivalent (Figure 4.4).


99

Figure 4.2. Cytokine staining profiles for CD8+ T cells during a primary response following intranasal influenza virus infection in BALB/c mice. After panning of B cells, FACS dot plots depicting intracellular cytokine staining for CD8+ T cell responses were gated to firstly identify T lymphocytes (A), CD8+ T cells (B) and then IFN-γ+CD8+ T cells specific for HIV-H-2Kd Gag197 (C), influenza H-2Kd NP147 (D) and No peptide negative control (E). Lymphocytes were obtained from the spleens of BALB/c mice (n = 5 mice) at 10 days following primary intranasal influenza infection.


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Figure 4.3. Intracellular Cytokine staining assay used for comparison of functional IFN-γ+ CD8+ T cells responses targeting the influenza Kd NP147 and HIV-Kd Gag197 epitopes. Naïve BALB/c mice (number of mice used for each group (n = 5)) were intranasally infected with the HK-X31-NA-Gag197 influenza virus and lymphocytes obtained from spleens and stimulated with the H-2Kd Gag197 or NP147 peptide. The data shown was statistically analyzed to compare the responses between the Gag197 and NP147

+IFN-γ+CD8+T cell response using the Student t-test. All data represents the mean ± SD representative of three experiments, p > 0.05 is non-significant (ns).


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Figure 4.4. Quality of antigen-specific CD8+ T cell subsets detected by cytokine staining assay (ICS). Naïve BALB/c mice (number of mice used for each group (n = 5)) were intranasally infected with the HK-X31-NA-Gag197 influenza virus and lymphocytes obtained from spleens and stimulated with the H-2Kd Gag197 or NP147 peptide. The proportion of TNF-α+IFN-γ+CD8+ (A) and IL-2+TNF-α+IFN-γ+CD8+ T cells (B) are depicted. All data represents the mean ± SD of three experiments, using the Student t-test *p<0.05 and **p<0.01.


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4.1.2. Comparison of CD8+ T cell responses following various prime-boost vaccination regimes detected by ICS.

In order to compare different mucosal routes for prime-boost infection in BALB/c mice,

lymphocytes harvested from spleen, bronchoalveolar lavage, respiratory draining lymph nodes

(mediastinal) and genital draining lymph nodes (inguinal) lymph nodes of various mucosal

routes of immunized BALB/c mice were assayed for production of cytokines, such as IFN-γ,

TNF-α and IL-2 specific for HIV-Gag197-205 and influenza-NP147-155 peptides.

Four groups of BALB/c mice were administered different prime-boost vaccination

combinations. Mice were primed intranasally using 1 × 104 pfu/mouse of X31-NA-Gag197. This

was followed six weeks later with an intranasal boost using 50 pfu of PR8-NA-Gag197. PR8-

NA-Gag197 and X31-NA-Gag197 are serologically distinct and so neutralizing antibodies are

avoided allowing boosting of CD8+ T cell responses. The second vaccination group followed

the same regimen but viruses were administered via an intravaginal-intravaginal route. The

intent here was to prime immunity in the regional draining node at a site of known viral entry.

The third vaccination group used a intranasal-intravaginal route and the fourth vaccination

group a intravaginal-intranasal route. Seven days after boosting, lymphocytes were harvested

from the spleen, broncho-alveolar lavage, respiratory draining lymph nodes (mediastinal,

MedLN) and genital draining lymph nodes (inguinal, ILN). These were analyzed for CD8+ T

cell ex vivo responses, particularly for the production of cytokines, such as IFN-γ, TNF-α and

IL-2 in response to the HIV-Gag197 and influenza-NP147 peptides. These polyfunctional cells

are present in non-progressive HIV-infected individuals compared to those people

characterized with progressive HIV infection (Betts, et al., 2006; de Keersmaecker et al., 2012).

Our results demonstrated the ability of various mucosal routes of vaccination to induce CD8+

IFN-γ+ T cells in different lymphoid tissues that are directed against the exogenous HIV-H-2Kd

Gag197 and endogenous influenza H-2Kd NP147 peptides in BALB/c mice (Figure 4.5). In

addition, it was clearly demonstrated that an intranasal-intranasal prime boost vaccination

strategy with recombinant influenza viruses expressing HIVGag197 induced a significantly

higher proportion of IFN-γ+CD8+ T cells in the spleen, BAL and MedLN (p< 0.001) compared

to other vaccination strategies like intravaginal-intravaginal, intranasal-intravaginal or


103

intravaginal-intranasal). In contrast, in ILN the highest proportion of IFN-γ+CD8+ T cell

responses were detected in the intravaginal-intravaginal prime boost vaccinated group of mice

(Figure 4.5).


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Figure 4.5. Representative dot plots depicting cytokine production for CD8+ T cells specific for HIV-H-2KdGag197 following prime-boost vaccination by various mucosal routes in BALB/c mice. ICS was used to detect cytokine production in the spleen (A), MedLN (B), BAL (C) and ILN (D).


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Overall our data shows a significant increase in HIV-Gag197+IFN-γ+CD8+ T cells following

intranasal-intranasal prime boost vaccination in the spleen, BAL and MedLN (Figure 4.6). In

total, (9.51 × 105 ± 1.12 × 105) cells were detected following i.n/i.n prime boost infection in the

spleen, compared to (1.49 × 105 ± 1.31× 104) for i.v/i.v, (4.58 × 105 ± 4.41 × 104) for i.v/i.n

and (1.16 × 105 ± 3.87 × 104) cells for i.n/i.v (Figure 4.6A). Similar trends were observed in

the BAL (Figure 4.6B) and MedLN (Figure 4.6C). This can be explained by the fact that

influenza viruses replicate in the lung and respiratory system due to the presence of a protease

required for the cleavage of haemagglutinin. The higher levels viral antigen are likely to drive

increased CD8+ T cell responses (Garulli, Kawaoka and Castrucci, 2004; Lu et al., 2012, Lu et

al., 2013). However, results were different in the ILN as the intravaginal-intravaginal prime

boost strategy induced the largest increase in HIVGag197 specific IFN-γ+CD8+ T cells given it

is the draining lymph node of the genital tract (Figure 4.6 D). Moreover, the intravaginal-

intravaginal route induced responses in the spleen and ILN while there were no obvious antigen

specific CD8+ T cells detected in either the BAL or MedLN of the respiratory compartments.

The secondary NP147+IFN-γ+CD8+ T cell immune response following intranasal-intranasal

prime boost vaccination elicited a higher response in the spleen, BAL and MedLN compared

to the other routes of mucosal vaccination. In the spleen (9.31 × 105 ± 1.86 × 105) IFN-γ+CD8+

T cells were detected following i.n/i.n prime boost infection, compared to the (1.44 × 105 ±

2.21 × 104 ) cells for i.v/i.v, (4.17 × 105 ± 6.64 × 104) cells for i.v/i.n and (1.27 × 105 ± 4.99 ×

104) cells for i.n/i.v (Figure 4.7A). Similar trends were observed for the BAL and MedLN.

Intravaginal-intravaginal prime boost vaccination again led to the greatest increase in NP147+

CD8+ T cells in the ILN, the draining lymph node of the urogenital tract (Figure 7D). Moreover

H-2Kd Gag197 and NP147 CD8+ T cell responses were co-dominant following i.n/i.n, i.v/i.v,

iv/i.n and i.n/i.v prime boost vaccination in spleen, BAL and MedLN (Figure 4.6 and Figure

4.7).


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Figure 4.6. Number of HIV-H-2Kd Gag197 IFN-γ+CD8+ T cells in different organs following different routes of vaccination in BALB/c mice. Four groups of BALB/c mice were infected i.n/i.n, i.n/i.v, i.v/i.v and i.v/i.n with X31-NA-Gag197 and PR8-NA-Gag197 influenza viruses and ICS use to determine the number of HIV-Gag197

+CD8+ T cells in the spleen (A), BAL (B) MedLN (C) and ILN (D). Lymphocyte populations were harvested and stimulated for five hours with 1μM HIV-H-2Kd Gag197 peptide before staining with anti-mouse CD8α and anti-IFN-γ antibodies. Data were normalized using a no-peptidecontrol. Data represent mean ± SD from three repeated experiments, and statistical differences were determined using a one way of variance (ANOVA) and Tukey's multiple comparison test, ***p<0.001.


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Figure 4.7. Number of H-2KdNP147

+IFN-γ+CD8+ T cells in different organs following different routes of vaccination in BALB/c mice. Four groups of BALB/c mice were infected i.n/i.n, i.n/i.v, i.v/i.v and i.v/i.n prime boost infection with X31-NA-Gag197 and PR8-NA-Gag197 influenza viruses and ICS use to determine the number of NP147

+CD8+ T cells responses in the spleen (A), BAL (B) MedLN (C) and ILN (D). Lymphocyte populations were harvested and treated for five hours with 1μM influenza H-2Kd NP147 peptide before staining with anti-mouse CD8α and anti-IFN-γ+ antibodies. Data represents the mean ± SD from three repeated experiments, and statistical differences determined using a one way of variance (ANOVA) and Tukey's multiple comparison test, **p<0.01 and ***p<0.001.


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Further analyses focused on poly-functional subsets of the antigen specific CD8+ T cells

(Figure 4.8). There was no significant difference in the proportion of TNF-α+IFN-γ+CD8+ T

cells for NP147+ (64 ± 3.8%) and Gag197

+ (62 ± 2.8%) (p<0.373) populations induced following

intranasal-intranasal vaccination. The i.n-i.n prime boost vaccination induced the highest

proporation of TNF-α+IFN-γ+CD8+ T cell compared to other routes of vaccination (p<0.001)

(Figure 4.9). Similar results and trends were observed for the IL-2+TNF-α+IFN-γ+CD8+ T cell

subsets following intranasal-intranasal prime boost vaccination for NP147 (15.8 ± 1.9%) and

Gag197 (13.2 ± 2.7%) antigen specific CD8+ T cells in the spleen (Figure 4.10).

Figure 4.8. Cytokine profiles of poly-functional CD8+ T cells. Schematic figure shows three subsets of CD8+ T cells including IFN-γ+CD8+ T cells (A), IFN-γ+ and TNF-α+ CD8+ T cells (B), and CD8+ T cells producing IL-2, TNF-α and IFN-γ (C).


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Figure 4.9. Proportion of TNF-α+IFN-γ+CD8+ T cells in the spleen detected by ICS following different routes of vaccination in BALB/c mice. This figure compares HIV-H-2Kd Gag197 (A) and influenza H-2Kd NP147 (B) CD8+ T cell responses. Data represents the mean ± SD from three individual experiments, and statistical significance was determined using the one way of variance (ANOVA) and Tukey's multiple comparison test, ***p<0.001.


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Figure 4.10. Proportion of IL-2+TNF-α+IFN-γ+CD8+ T cells in the spleen detected by ICS following different routes of vaccination in BALB/c mice. This figure compares HIV-H-2Kd Gag197 (A) and influenza H-2Kd NP147 (B) CD8+ T cell responses. Data represents the mean ± SD from three individual experiments, and the statistical significance was determined using the one way of variance (ANOVA) and Tukey's multiple comparison test, ***p<0.001.


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4.1.3. Induction of HIV-specific CD8+ T cells detected by tetramer staining assay

Previous mice studies have demonstrated that the number of antigen specific CD8+ T cells

producing IFN-γ in an ICS assay are comparable to the number of antigen specific

tetramer+CD8+ cells detected following primary and secondary influenza A virus infection

(Flynn et al., 1998). To confirm this, the number of tetramer+CD8+ T cells induced against the

HIV-H2-Kd Gag197 epitope (Figure 4.11) and H-2Kd NP147 epitope (Figure 4.12) were analysed.

Harvested CD8+ T cells from the spleen, BAL, MedLN and ILN of prime-boost vaccinated

BALB/c mice were stained with H-2Kd Gag197 and NP147 labelled tetramers. No significant

difference was detected between the total number of IFN-γ+and tetramer+ CD8+ T cells in the

spleen following i.n/i.n, i.v/i.v, iv/i.n and i.n/i.v prime boost vaccinationfor both antigen-

specific cell populations in all tissues tested.

The highest numbers of tetramer+CD8+ T cells were induced following i.n/i.n prime boost

vaccination for HIV Gag197 (Figure 4.11), and influenza NP147 (Figure 12) in the spleen, BAL

and MedLN as per results obtained using intracellular cytokine staining. In the ILN the higheest

tetramer response was detected following intravaginal-intravaginal prime boost vaccination

(Figure 4.11D and Figure 4.12D). HIV-H-2Kd Gag197+ and NP147

+CD8+ T cell responses were

detected in the urogenital draining lymph nodes following intranasal infection. Previous studies

have shown that CD8+ T cell responses can be induced in the vaginal regional lymph nodes of

mice infected intranasally with recombinant influenza A virus vectors expressing the HIV-

Env311 peptide (Garulli, Kawaoka and Castrucci 2004; Lu et al., 2012). Moreover, an increase

of HIV-H-2Kd Nef (EWRFDSRLAFHHVAREL) and H-2Kd NP147 (TYQRTRALV) specific

CD8+ T cell responses have been detected in the urogenital draining lymph nodes, in addition

to the spleen and respiratory regional lymph nodes, of mice infected intranasally with live

recombinant influenza A viruses expressing the Nef protein (Ferko et al., 2001).


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Figure 4.11. Number of H-2Kd Gag197 tetramer+CD8+ T cells in different tissues following different routes of vaccination in BALB/C mice. Four groups of BALB/c mice (each group n=5) were infected with 1 × 104 pfu X31-NA-Gag197 influenza virus and then challenged after six weeks with 50 pfu PR8-NA-Gag197 influenza virus. Seven days following the final boost, the spleen (A), BAL (B), MedLN (C) and ILN (D) lymphocyte populations were sampled for analysis of tetramer+CD8+ T cells. Data shows a representative example of 3 expepriments and indicates the mean ± SD. Statistical significance was ascertained using the one way of variance (ANOVA), Tukey's multiple comparison test, ***p<0.001.


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Figure 4.12. Number of H-2Kd NP147 tetramer+CD8+ T cells in different tissues following different routes of vaccination in BALB/c mice. Four groups of BALB/c mice (each group n=5) were infected with 1 × 104 pfu X31-NA-Gag197 influenza virus and then challenged after six weeks with 50 pfu PR8-NA-Gag197 influenza virus. Seven days following the final boost, the spleen (A), BAL (B), MedLN (C) and ILN (D) lymphocyte populations were sampled for analysis of tetramer+CD8+ T cells using i.n-i.n,i.v-i.v, i.v-i.n, i.n-i.v and other vaccination strategies. Data shows a representative example of 3 experiments and indicates the mean ± SD. Statistical significance was ascertained using the one way of variance (ANOVA), Tukey's multiple comparison test, ***p<0.001.


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4.1.4. Detection of mucosal surface markers (LPAM-1; α4β7 and CD103; αEβ7) and other markers, CD44, CD62L, CCR9 and CD69 on antigen specific CD8+ T cells using flow cytometry

In order to further characterize antigen-specific CD8+ T cells in vaccinated mice, cells were

examined for the expression of homing CD8+ T cell integrins, LPAM-1(α4β7) and CD103

(αEβ7) which are considered important for the trafficking of mucosal lymphocytes into

lymphoid tissues. These mucosal markers were detected using species-specific anti-mouse

mucosal integrin antibodies, and the gating used is shown in Figure 4.13. Cells were first gated

on total lymphocytes, then on the CD8+ T cell subset before identification of the the HIV-

Gag197+CD8+T cell population using tetramer staining. Cells were then analyzed for expression

of the mucosal markers LPAM-1 and CD103 in addition to other markers, such as CD44,

CD62L, CCR9 and CD69 in samples derived from the spleen, MedLN, ILN and BAL. The

intranasal-intranasal prime boost vaccination strategy produced the highest proportion of

α4β7+HIVGag+CD8+T cells following various routes of vaccination in the spleen, BAL and

MedLN (Figure 4.14A, 4.14B and 4.14C). The intravaginal-intravaginal prime boost strategy

induced the highest proportion of α4β7+HIVGag+CD8+ T cells in the ILN (Figure 4.14D). The

levels of CD103+HIVGag+CD8+ T cells were similar to those observed for α4β7+ in the spleen,

BAL and to a lesser extent the MedLN (Figure 4.16A, 4.16B and 4.16C). However, there was

little correlation between the two integrins for the ILN (Figure 4.16D). Moreover similar

patterns LPAM-1+ and CD103+ expression on CD8+ T cells were observed for influenza-

specific NP147 CD8+ T cells (Figure 4.15 and 4.17).


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Figure 4.13. Gating strategy used to analyze mucosal α4β7+Gag+tetramer+CD8+ T cells. Isolated cells were first gated for total lymphocytes (A) followed by CD8+ T cells using an anti-mouse CD8α-PercP antibody (B). HIV-specific CD8+ T cells were gated on the basis of binding to a specific H-2Kd Gag197-APC tetramer (C). Cells were then interrogated for their expression of the mucosal marker α4β7+ using anti-mouse LPAM-1 (α4β7)-PE antibody (D).


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Figure 4.14. Proportion of HIV-Gag197

+α4β7+CD8+ T cells detected in tissues following different routes of mucosal vaccination in BALB/c mice. T lymphocytes were obtained from different lymphoid organs and then stained with H-2Kd Gag197-APC tetramer, anti-mouse CD8α PerCP and anti-α4β7 (LPAM-1)-PE antibodies. α4β7+Gag197

+CD8+ T cell numbers were determined (as described in Figure 4.13) with data representative of three repeated experiments. Data is shown as mean ± SD and significance determined using a one way of variance (ANOVA) and Tukey's multiple comparison test, ***p<0.001.


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Figure 4.15. Proportion of influenza NP147

+α4β7+CD8+ T cells detected in tissues following different routes of mucosal vaccination in BALB/c mice. T lymphocytes were obtained from different lymphoid organs and then stained with H-2KdNP147-APC tetramer, anti-mouse CD8α PerCP and anti-α4β7 (LPAM-1)-PE antibodies. α4β7+NP147+CD8+ T cell numbers were determined (as described in Figure 4.13) with data representative of three repeated experiments. Data is shown as mean ± SD and significance determined using a one way of variance (ANOVA) and Tukey's multiple comparison test, *p<0.05 and ***p<0.001.


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Figure 4.16. Proportion of CD103+HIV-H-2KdGag197

+CD8+ T cells detected in tissues following different routes of mucosal vaccination in BALB/c mice. T lymphocytes were obtained from different lymphoid organs and then stained with H-2Kd Gag197-APC tetramer, anti-mouse CD8α PerCP and anti-CD103-FITC antibodies. CD103+CD8+ T cell numbers were determined (as described in Figure 4.13) with data representative of three repeated experiments. Data is shown as mean ± SD and significance determined using a one way of variance (ANOVA) and Tukey's multiple comparison test; ***p<0.001.


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Figure 4.17. Proportion of CD103+NP147+CD8+ T cells detected in tissues following different routes of mucosal vaccination in BALB/c mice. T lymphocytes were obtained from different lymphoid organs and then stained with H-2Kd NP147-APC tetramer, anti-mouse CD8α PerCP and anti-CD103 FITC antibodies. CD103+NP147

+CD8+ T cell numbers were determined (as described in Figure 4.13) with data representative of three repeated experiments. Data is shown as mean ± SD and significance determined using a one way of variance (ANOVA) and Tukey's multiple comparison test, ***p<0.001.


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Following the analysis of mucosal markers, we checked the level of expression of as the marker

of antigen experience, CD44, and the marker for effector function/memory CD62L, in the

spleen, BAL, MedLN and ILN harvested from all groups of vaccinated mice. In general, CD44

and CD62L were differentially expressed on the CD8+ T cell population in naïve, acute effector

or memory cells as has been previously described by Kedzierska and colleagues (2007) and

also by Kaufman and colleagues, (2008). CD44-CD62L+CD8+ T cells were isolated from

different lymphoid tissues, such as Peyer’s patches, inguinal, mesenteric, peripheral lymph

nodes and the spleen in naïve mice, while effector CD44+CD62L-CD8+ T cells were obtained

following vaccination of mice with recombinant adenovirus expressing the SIV-Gag+CD8+ T

cell epitope (Kaufman et al., 2008). Furthermore, in response to a novel vaccine containing

recombinant Bacillus subtilis or Listeria monocytogenes as a vector expressing HIV-Gag in

mice, vaginal CD62L+CD44+ CD8+ memory CD8+ T cells were identified five weeks after

vaccination (Li et al., 2008).

Following our vaccination regimen, we identified the highest proportion of HIV-

Gag197+CD44+CD8+ T cells and influenza NP147

+CD44+CD8+ T cells following intranasal-

intranasal prime boost vaccination in the spleen, MedLN and BAL. In the ILN the highest

proportion of antigen experienced cells was found after intravaginal-intravaginal vaccination

(Figures 4.18 and 4.19). As expected after acute infection only a small proportion of antigen

specific CD8+ T cells cells were CD62L+(Figures 4.20 and 4.21).

Two final cell surface markers where characterized following vaccination. These included the

T cell activation marker CD69 and the T cell migration marker CCR9 (Norment et al., 2000;

Kathuria et al., 2012). Previous studies have shown that expression of CCR9 and α4β7 on CD8+

T cells was a characteristic of homing/mucosal T cells (Mavigner et al., 2012). Intranasal-

intranasal vaccination induced the highest proportion of CD69+ antigen specific CD8+ T cells

(Figures 4.22 and 4.23) whilst differences in CCR9 expression on antigen specific cells were

only detected in the spleen Figures 4.24 and 4.25).


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Figure 4.18. Percentage of CD44+Gag197

+CD8+ T cells detected in different organs following mucosal vaccination in BALB/c mice. T cells obtained from different lymphoid organs were stained with the H-2Kd Gag197-APC tetramer, in addition to anti-mouse CD8α PerCP and CD44-PE antibodies. Data shows a representative example of three experiments. Results are expressed as mean ± SD and statistical significance assessed using the one way of variance (ANOVA) and Tukey's multiple comparison test; *p<0.05 and ***p<0.001.


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Figure 4.19. Percentage of CD44+NP147

+CD8+ T cells detected in different organs following mucosal vaccination in BALB/c mice. T cells obtained from different lymphoid organs were stained with the H-2Kd NP147-APC tetramer, in addition to anti-mouse CD8α PerCP and CD44-PE antibodies. Data shows a representative example of three experiments. Results are expressed as mean ± SD and statistical significance assessed using the one way of variance (ANOVA) andTukey's multiple comparison test; *p<0.05 and ***p<0.001.


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Figure 4.20. Percentage of CD62L+Gag197

+CD8+ T cells detected following mucosal vaccination in BALB/c mice. T cells obtained from different lymphoid organs were stained with the H-2Kd Gag197-APC tetramer, in addition to anti-mouse CD8α PerCP and CD62L-FITC antibodies. Data shows a representative example of three experiments. Results are expressed as mean ± SD and statistical significance assessed using the one way of variance (ANOVA) and Tukey's multiple comparison test; **p<0.01 and ***p<0.001.


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Figure 4.21. Percentage of CD62L+NP147

+CD8+ T cells detected following mucosal vaccination in BALB/c mice. T cells obtained from different lymphoid organs were stained with H-2Kd NP147-APC tetramer, in addition to anti-mouse CD8α-PerCP and CD62L-FITC antibodies. Data shows a representative example of three experiments. Results are expressed as mean ± SD and statistical significance assessed using the one way of variance (ANOVA) and Tukey's multiple comparison test; ***p<0.001.


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Figure 4.22. Percentage of CD69+Gag197

+CD8+ T cells detected following mucosal vaccination in BALB/c mice. T cells obtained from different lymphoid organs were stained with the H-2Kd Gag197-APC tetramer, in addition to anti-mouse CD8α PerCP and CD69-PE antibodies. Data shows a representative example of three experiments. Results are expressed as mean ± SD and statistical significance assessed using the one way of variance (ANOVA) and Tukey's multiple comparison test; **p<0.01 and ***p<0.001.


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Figure 4.23. Percentage of CD69+NP147

+CD8+ T cells detected following mucosal vaccination in BALB/c mice. T cells obtained from different lymphoid organs were stained with the H-2Kd

NP147-APC tetramer, in addition to anti-mouse CD8α-PerCP and CD69-PE antibodies. Data shows a representative example of three experiments. Results are expressed as mean ± SD and statistical significance assessed using the one way of variance (ANOVA) and Tukey's multiple comparison test; *p<0.05, **p<0.01 and ***p<0.001.


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Figure 4.24. Percentage of CCR9+Gag197

+CD8+ T cells detected following mucosal vaccination in BALB/c mice. T cells obtained from different lymphoid organs were stained with the H-2Kd Gag197-APC tetramer, in addition to anti-mouse CD8α PerCP and CCR9-FITC antibodies. Data shows a representative example of three experiments. Results are expressed as mean ± SD and statistical significance assessed using the one way of variance (ANOVA) and Tukey's multiple comparison test; *p<0.05 and ***p<0.001.


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Figure 4.25. Percentage of CCR9+NP147

+CD8+ T cells detected following mucosal vaccination in BALB/c mice. T cells obtained from different lymphoid organs were stained with the H-2Kd NP147-APC tetramer, in addition to anti-mouse CD8α-PerCP and CCR9-FITC antibodies. Data shows a representative example of three experiments. Results are expressed as mean ± SD and statistical significance assessed using the one way of variance (ANOVA) Tukey's multiple comparison test; *p<0.05, **p<0.01 and ***p<0.001.


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4.1.5. Detection of IL-15 and IL-15Rα expression on mucosal HIV-Gag+CD8+ T cells by RT-PCR

Interleukin 15 (IL-15) is a T cell-stimulating cytokine and has an important role in regulating

survival, proliferation and development of CD8+ T cells (Zhang et al., 1998). The IL-15Rα

receptor binds to IL-15 cytokine on T cells with high affinity (Anderson et al., 1995; Lodolce

et al., 1998). Both IL-15 and IL-15Rα have an important role in generating and maintaining

antigen-specific CD8+ T cell responses and are necessary for primary expansion of CD8+ T

cells following viral infections (Schluns et al., 2002). IL-15Rα is expressed on dendritic,

natural killer and lymphocytic cells (Anderson et al., 1995). IL-15 also regulates the

maintenance of lymphoid homeostasis by supporting lymphocyte homing and proliferation

(Lodolce et al., 1998). IL-15 has been shown to regulate the migration of effector CD8+ T cells

to pulmonary airways and as such has been used as an adjuvant to induce influenza-specific

CD8+ T cells to facilitate clearance of virus (Verbist et al., 2011). Its role in migration of

antigen-specific CD8+ T cells has been demonstrated using trans-well chemotaxis chambers

through experiments that show 10 fold increases in movement of influenz-specifc NP+CD8+ T

cell when compared to media alone controls (Verbist et al., 2011).

We set out to determine if IL-15 and/or the IL-15R could be detected in vaccinated mice. We

chose to focus our attention on HIV Gag197+CD8 T cells that that had shown higher expression

levels of the α4β7+ mucosal marker. We prepared two HIV Gag197+CD8 T cell populations in

the spleen, BAL and MedLN (α4β7+ and α4β7-). These populations were sorted following

intranasal-intranasal prime-boost infection of BALB/c mice with the X31-NA-Gag197 and PR8-

NA-Gag197 recombinant influenza viruses using the gating strategy shown in Figure 4.26. High

purity populations (98.5%) of Gag197+α4β7+ and Gag197

+α4β7-CD8+ T cells were obtained.

RNA was then extracted from HIV-Gag197+α4β7+and HIV-Gag197

+α4β7-CD8+ T cells to

determine the mRNA levels of IL-15-Rα and IL-15 by RT-PCR. The HIV-Gag197+α4β7+CD8+T

cell population expressed higher levels of IL-15-Rα when compared to α4β7-CD8+ T cells in

spleen cells (23 fold), MedLN (18 fold) and BAL (11 fold) (Figure 4.27). There was also

increased expression of the IL-15 cytokine on the mucosal α4β7+CD8+ T cell population in the

spleen (10.5 fold), MedLN (9.6 fold) and in BAL (4.8 fold) compared to Gag197+α4β7-CD8+T

cells (Figure 4.28).


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Figure 4.26. Gating strategy used to sort and analyze HIV-Gag+α4β7+ and HIV-Gag+α4β7-

CD8+ T cell populations. Total T lymphocytes were gated (A), followed by CD8+ T cells (B), H-2Kd Gag197-APC tetramer+CD8+ T cells (C), and finally α4β7+CD8+ T cells (D) to identify the Gag+α4β7+ and Gag+α4β7-CD8+ T cell populations.


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Figure 4.27. IL-15Rα gene expression in α4β7+Gag197

+CD8+ T cells following intranasal-intranasal prime boost vaccination in the spleen, MedLN and BAL by quantitative real time PCR (RT-PCR). Data is presented as the fold change expression compared to α4β7-

Gag197+CD8+ T cells, and normalized to 1 using the Δ cycle threshold method. All values are

representative of three repeated experiments with a mean ± SD, with different significance by one way of variance (ANOVA) and Tukey's multiple comparison test; *p<0.05, **p<0.01 and ***p<0.001.


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Figure 4.28. IL-15 cytokine expression in α4β7+Gag197

+CD8+ T cells following intranasal-intranasal prime boost vaccination in the spleen, MedLN and BAL by quantitative real time PCR (RT-PCR). Data is presented as the fold change expression compared to the α4β7-

Gag197+CD8+ T cells, and normalized to 1 using the Δ cycle threshold method. All values are

represented as a mean ± SD of three repeated experiments, with different significance by one way of variance (ANOVA) and Tukey's multiple comparison test; *p<0.05, **p<0.01 and ***p<0.001.


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4.2. Discussion Similar to other infectious diseases, the need for an effective vaccine to control HIV infection

is of prime importance in the efforts of global HIV programs. A number of CD8+ T cell

properties are thought to be important in a vaccine-induced response. Mucosal immune

responses stimulated by HIV/AIDS vaccines are considered highly desirable for blocking the

main portal of HIV-1 entry which is through mucosal surfaces (Shattock et al., 2008),

particularly gastrointestinal, vaginal, or oral surfaces (Haynes and Shattock, 2008;

Challacombe and Sweet, 2002; Clerici et al., 2001; Demberg and Robert-Guroff, 2009; Jespers

et al., 2010).

As the main portal of entry for most infectious agents is through the mucosal surfaces, the best

way to control their transmission is by strengthening the mucosal immune responses to ensure

that infections do not pass local draining lymph nodes and disseminate throughout the body.

Previous studies that looked at mucosal immune responses in HIV infected or high risk

individuals have noted high interferon-gamma (IFNγ) and interleukin-2 (IL-2) expression in

CD8+ T cells and IgA production in the cervix and blood of resistant HIV female sex workers

in Nairobi. There was also a direct correlation between the level of induced mucosal HIV-CD8+

T cells and control of the HIV infection (Orga, Faden and Welliver 2001; Stevceva et al., 2002).

This chapter describes the use of different routes of vaccination to determine which strategy

generates not only the highest immune response but also has the ability to stimulate a high

proporation of antigen specific cells that express mucosal markers.

Many routes of vaccination are thought to induce mucosal immune responses. Vaccines

administered intrarectally, intranasally or orally in BALB/c mice using the synthetic HIV

peptide PCLUS3–18IIIB and combined with a cholera toxin were effective in stimulating long

lived HIV-specific CD8+ T cells in regional and distant lymph nodes in addition to the spleen

when compared to subcutaneous vaccinated mice (Belyakov et al., 1998). The intranasal and

intravaginal route of vaccination is also thought to facilitate the induction of strong mucosal

CD8+ T cell immuity as a strong link has been established between tthe mucosa of the upper

respiratory tract and the urogenital tract following administration of antigen on these surfaces

(Holmgren and Czerkinsky 2005). Influenza viruses have been used as mucosal vectors to

induce mucosal CD8+ T cell immunity following both intransal and intravaginal vaccination.


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Intravaginal infection of BALB/c mice with recombinant influenza viruses expressing the H-2

Dd HIV-Env+CD8+ T cell epitope (P18IIIB) stimulated CD8+ T cell responses in both the

inguinal lymph node and the spleen (Garulli, Kawaoka and Castrucci, 2004). Recombinant

influenza viruses expressing the HIV-1 Env311 epitope have been shown to stimulate IFN-γ+

and tetramer specific CD8+ T cell responses a in the spleen following a prime boost vaccination

(Cukalac et al., 2009).

Previous studies using HIV vaccines in animal models have involved the use of numerous

vaccine vectors to deliver specific HIV/SIV epitopes or proteins, i.e., adenovirus, vaccinia virus

and fowl pox virus. All of these viral vectors were capable of inducing strong immune

responses (Gonzalo et al., 1999; Sexton et al., 2009; Ranasinghe et al., 2011). Use of these

recombinant HIV vaccines has induced mucosal CD8+ T cell immune responses that have led

to decreased RNA copies of SIV virus and controlled the viremia in non-human primate models

(Casimiro et al., 2005; Hoffmann et al., 2002; Liu et al., 2009; Pachler, Mayr and Vlasak,

2010; Rimmelzwaan et al., 2007; Sexton et al., 2009; Wit et al., 2004). Moreover recombinant

influenzaA viruses (H3N2, A/HKx31 and PR8 H1N1, A/Puerto Rico/8/1934) have been used

as candidate SIV vaccine vectors to express SIV-CD8+ T cell epitopes, SIV-Gag164-172, SIV-

Tat87-96, and SIV-Tat114-123. Importantly intranasal or intramuscular vaccination of macaques

with the HIV-1SF162 envelope protein facilitated the development of mucosal immune

response in the vaginal mucosa of macaques, and protected them against virulent SHIV

(SHIVSF162P4) challenge (Barnett et al., 2008). This result indicates the intranasal route of

vaccination can induce e mucosal immune responses at distant urogenital lymph nodes to

enable vaccinated macaques to resist SIV challenge.

Our main aim was to stimulate mucosal Gag197+CD8+ T cells however we did also assess the

magnitude and quality of the endogenous influenza NP147+specific CD8+ T cell response.

Similar levels of endogenous influenza NP147+specific CD8+ T cells were induced in our

experiment following primary intranasal vaccination to those results achieved from the study

performed by Cukalac and colleagues (Cukalac et al., 2009). The two main integrin molecules

α4 and β7, presented in as α4β7, α4β1 and αEβ7, are components regulating the migration of

lymphocytes into mucosal areas (Kang et al., 2011). Expression of β7 integrin has previously

been associated with the use of recombinant influenza viruses. SIV-specific CD8+ T cell

responses following vaccination and challenge with SIVmac251 (Sexton, et al., 2009). Use of


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other live vaccine vectors has also been associated with an increases in α4β7 mucosal marker

expression on CD8+ T cells following oral vaccination in macaques. Recombinant Salmonella

expressing the SIV-Gag protein and vaccinia virus expressing the SIV-Gag protein were both

used in these experiments to stimulate SIV-Gag181-189 specific CD8+ T cells (Evans et al., 2003).

The importance of the α4β7 mucosal homing integrin in virus control has also been observed

in other model systems. Rotavirus specific α4β7+CD44+CD8+ T cells has been very effective

in the clearance of rotavirus infection. Adoptive transfer of 2 × 104 α4β7+CD44+ CD8+ T cells

showed that mice were protected from rotavirus (Rosé et al., 1998). Moreover, the transfer of

increased numbers of α4β7+CD44+ CD8+ T cells improved protective efficacy. Finally blocking

of both CD62L and α4β7 has been shown to impair the migration of CD8+ T cells to mesenteric

lymph nodes (Ciabattini et al., 2011).

We noted high levels of mRNA expression of interleukin IL-15Rα and IL-15 in HIV-H-2Kd

Gag197+α4β7+CD8+ T cells compared to the HIV-H-2Kd Gag197

+α4β7-CD8+ T cells following

intranasal-intransal vaccination in multiple tissues (spleen, BAL and mediastinal lymph nodes)

as seen in Figures 4.19 and 4.20. Removal of dendritic cells that produce IL-15 or blocking of

IL-15R has been previously shown to inhibit the migration of influenza specific CD8+ T cells

to the site of infection (Verbist et al., 2011; Brincks and Woodland, 2010; McGill, Van Rooijen

and Legge, 2010; McInnes et. al., 1996). This may be a direct result of th use of live influenza

virus as similar results (high IL-15 Rα mRNA expression) were detected in the BAL,

mediastinal lymph nodes (MedLNs) and spleens harvested of intranasally infected (X31

(A/Hong Kong x31)) C57BL/6 mice. These mRNA levels were detected for the first time on

day 3 and continued increasing until day 7 with a 4 fold change compared to naïve mice

(Verbist et al., 2011). Therefore, increased expression of IL-15 Rα in mucosal CD8+ T cells

might provide evidence for the important role of this cytokine in regulating survival of HIV-

H-2KdGag197+CD8+ T cells at mucosal sites. This is further supported by the fact that

expression of IL-15 Rα correlated with a high level of IFN-γ+ expression in CD8+ T cells

obtained from HIV positive chimpanzees (Rodriguez et al., 2007). An increase of IL-15 Rα

was also expressed in response to intranasal influenza infection in mice which was highly

effective in facilitating the trafficking of CD8+ and natural killer cells into mucosal sites of

infection and assist in the control of virus replication (Verbist et al., 2012).


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4.3. Conclusion According to the results of our mice studies, we conclude that intranasal prime boost

vaccination in BALB/c mice has an important role in stimulating antigen specific CD8+ T cells

that express known mucosal markers such as α4β7 and CD103. In addition, these mucosal HIV-

Gag197+α4β7+CD8+ T cells stimulated following intranasal prime boost vaccination expressed

high levels of IL-15R and also produced small but detectable amounts of expressed IL-15

compared to HIVGag197+α4β7- CD8+ T cells.

The next chapter will test the ability of these cells to protect against a pseudo challenge

infection, recombinant vaccinia virus expressing the whole HIV-Gag protein.

Chapter 5: Pseudo-Challenge study

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5. Chapter 5 Pseudo-Challenge study

5.1. Overview Human immunodeficiency virus (HIV) naturally uses mucosal surfaces, such as those of the

intestinal and urogenital regions as the preferential sites for rapid replication. Therefore, these

mucosal compartments are considered the most important in which to stimulate immune

protection in order to control HIV infection and transmission. Previous studies have used

various HIV-1 proteins to induce mucosal immunity. For example, HIV-1 Gag protein

delivered orally using a recombinant Listeria monocytogenes vector given as prime-boost

vaccine induced HIV-Gag-specific cytotoxic CD8+ T cell immunity in a mouse model. HIV

Gag-specific CD8+ T cells producing both interferon gamma (IFN-γ+) and tumour necrosis

factor (TNF-α+) were induced in the spleen and mucosal-associated lymphoid tissue (MALT)

of vaccinated BALB/c mice. These CD8+ T cell responses completely protected mice against

a intravaginal or intranasal pseudo-challenge using recombinant vaccinia virus expressing the

whole Gag protein (rVac-HIVGag) (Zhao et al., 2006). An additional pseudo-challenge study

in mice also indicated a protective role of HIV-1 Env-PCLUS3–18 IIIB

(RIQRGPGRAFVTIGK) specific CD8+ T cells in BALB/c mice. These mice were initially

primed intrarectally, intranasally, intragastrically or subcutaneous with HIV-1 Env-PCLUS3–

18 IIIB. CD8 T cells were then intravenously transferred into naïve mice 5-6 days prior to

intrarectal pseudo-challenge with 2.5 × 107 pfu rVac virus expressing HIV-1 Env gp160 protein

(Belyakov et. al., 1998). Recombinant Listeria monocytogenes expressing HIV-1 Gag protein

has also been used in pseudo challenge experiments following vaccination. HIV Gag specific

CD8+ T cells protected BALB/c mice against an intraperitoneal pseudo-challenge using 2.5 ×

107 pfu recombinant vaccinia virus expressing whole HIV-1 Gag protein (vVK1) (Rayevskaya

and Frankel, 2001).

Our results from Chapter 4 indicate the presence of a large number of HIV-Gag197+α4β7+CD8+

T cells following i.n-i.n prime boost vaccination. These cells expressed higher levels of IFN-

γ+ and IL-15R when compared to HIV Gag197+α4β7-CD8+ T cells. This chapter will assess the

protective efficacy of this population using adoptive transfer experiments using a pseudo-

challenge mouse model.


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5.2. Results 5.2.1. Adoptive transfer of mucosal CD8+ T cells into naive mice To assess the effectiveness of our recombinant influenza HIV vaccine, a pseudo-challenge

experiment was performed in BALB/c mice using the recombinant vaccinia virus expressing

the whole Gag protein (Rosé et al., 1998; Kaufman et al., 2008). This involved the adoptive

transfer of mucosal HIV-1 Gag197+ CD8+ T cells induced in response to the vaccination of

BALB/c mice as described in Chapter 4. Seven days after the booster vaccination, spleens and

mediastinal lymph nodes were harvested from mice and used for the isolation of mucosal

HIVGag197+α4β7+CD8+ T cells. These HIV-Gag197

+α4β7+CD8+T cells along with

HIVGag+α4β7-CD8+T cell controls were sorted (as previously described in Chapter 4, Section

4.1.5 and Methods, Section 2.2.15.6) and enumerated. A total of 5 × 104 cells/ 200μl PBS of

HIV-Gag+α4β7+CD8+T cells, HIV-Gag+α4β7-CD8+ T cells or PBS (Mock control) were

adoptively transferred via the intravenous route into separate groups of naïve BALBC mice

and six days later used in challenge studies as per previous studies (Rose et al., 1998)

(Kaufman et al., 2008) .

5.2.2. Pseudo-challenge of BALB/c mice with recombinant vaccinia virus expressing HIV Gag (rVac633-gag)

Three groups of BALB/c mice containing adoptively transferred cells (HIV-Gag+α4β7+CD8+

T cells, HIVGag+α4β7-CD8+ T cells and Mock) were challenged intranasally with 1 × 107

plaque forming units (pfu) of recombinant vaccinia virus-633 expressing the whole Gag protein

(rVac-gag) to test the protective efficacy of these cells. Five days of challenge, the mice were

euthanized and the lungs and ovaries harvested and homogenized to ascertain the titer of the

challenge virus (rVac-Gag) as determined by plaque assay (Belyakov et. al., 1998). Analysis

of our three vaccinated groups revealed lower number of plaques in the lungs of mice following

adoptive transfer (Figure 5.1) of Gag+α4β7+CD8+ T cells (7.82×105 pfu/lung) in comparison to

those in which Gag+α4β7-CD8+ T cells were transferred (1.36×106 pfu/lung) or those Mock

treated BALB/c mice (1.80 ×106 pfu/lung) infected with the rVac-Gag challenge virus (Figure

5.3). Moreover, none of the harvested ovaries showed any plaques (Figure 5.2). The data was

analysed using a one way of variance test (ANOVA), which determined a significant difference

among the three groups of infected mice. Our results demonstrate an important role for mucosal

Gag+α4β7+CD8+ T cells in the control and dissemination of the rVac-Gag virus in this BALB/c

mouse model when compared to both Gag+α4β7-CD8+ T cells and the Mock challenged group

(Figure 5.3).


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Figure 5.1. Plaque assay on infected lungs when challenged with the rVac-gag challenge virus. Lungs were harvested from three groups of r-Vac-Gag-challenged BALB/c mice, homogenised and then diluted to 10-2, 10-3 and 10-4, as indicated. These dilutions were used to infect 143B cells and after two days of infection, the supernatant was removed and plaques visualised by staining with 0.1% crystal violet. Data shown are representative assay for Gag+α4β7+CD8+ transferred T cells (A), Gag+α4β7-CD8+ transferred T cells (B) and Mock (C).


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Figure 5.2. Plaque assay on ovaries of challenged BALB/c mice. Ovaries were harvested from the three groups of rVac-Gag-challenged BALB/c mice, homogenized and then diluted to ten fold dilutions, 10-2, 10-3 and 10-4 and used to infect 143B cells as described in Figure 1. Groups containing Gag+α4β7+CD8+ transferred T cells (A), Gag+α4β7-CD8+ transferred T cells (B) or Mock (C) as displayed.


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Figure 5.3. Graph of a virus titer in the lungs of r-Vac-Gag-challenged BALB/c mice. All data shows the mean ± SD from two repeated experiments with significance determined by a one way of variance test (ANOVA) and Tukey's multiple comparison test, *p<0.05 and ***p<0.001, which compares Gag197

+α4β7+CD8+ T cell intravenous recipient mice to both Gag197

+α4β7-CD8+ T cell transferred mice and Mock controls. It also compares reduction of the challenge virus titer between the group of mice that received the Gag197

+α4β7-CD8+ T cells and mock controls.


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5.2.3. CD8+ T cell immune responses following the r-Vac-gag challenge mice trial

Our three groups of BALB/c mice were also assayed for CD8+ T cell responses 5 days

following the intranasal r-Vac-gag challenge. Both spleens and mediastinal lymph nodes were

harvested for detection of HIVGag197+CD8+ T cell responses using ICS and tetramer staining

assays.

5.2.3.1. CD8+ T cell immune response detected by tetramer staining Spleen and MedLN samples were assayed by tetramer staining to determine the percentage of

HIV-H-2Kd Gag197+CD8+ T cells. In summary, there was a significant increase in the

percentage of tetramer HIV-H-2Kd Gag197+CD8+ T cells in both the spleen and MedLN of the

group of mice that received adoptively transferred Gag197+α4β7+CD8+ T cells when compared

to both Gag197+α4β7-CD8+ T cells and Mock as detected by flow cytometry in Figure 5.4.

Analysis of the numbers followed a similar pattern. The total number of tetramer+HIV-Kd

Gag197+CD8+ splenic T cells was (2.87 × 105 ± 5.03 × 104) in the group of mice that received

adoptively transferred Gag197+α4β7+CD8+ T cells (p<0.01) compared to (1.38 × 105 ± 2.65 ×

104) total cells for the group of mice that received adoptively transferred Gag197+α4β7-CD8+ T

cells and (2.53 × 104 ± 7.30 × 103) HIV-H-2 KdGag197+CD8+ T cells (p< 0.001) in Mock

controls (Figure 5.5 A). A similar pattern was observed in the MedLN (Figure 5.5 B)

Significant differences were also found in the Gag+α4β7-CD8+ T cell transfer group when

compared to Mock controls in both the spleen (p< 0.05) and MedLN (p< 0.01) (Figure 5.5).


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Figure 5.4. Tetramer staining of spleen cells from following r-Vac-gag-challenge. Flow cytometry of single cell spleen supsensions was used to detect the percentage of HIV-Kd Gag197+CD8+ T cells in three groups of mice: mice that received adoptively transferred Gag197

+α4β7+CD8+ T cells (A), mice that received adoptively transferred Gag197+α4β7-CD8+ T

cells (B) and PBS control only (Mock) (C). A lymphocyte CD8+ T cell and tetramer+ gated were used for analysis.


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Figure 5.5. The number of tetramer+HIV-H-2Kd Gag197+CD8+ T cells following a pseudo-

challenge. Flow cytometry of single cell spleen suspension was used to detect the percentage of HIV-H-2Kd Gag197

+CD8+ T cells in three groups of mice: mice that received adoptively transferred Gag197

+α4β7+CD8+ T cells, mice that received adoptively transferred Gag197+α4β7-

CD8+ T cells and PBS only (Mock) in spleen (A) and MedLN (B). Data are represent the mean ± SD of two repeated experiments. Statistical differences were analysed using one way of variance (ANOVA),*p<0.05, **p<0.01 and***p<0.001 comparing Gag197

+α4β7+CD8+ T cells to Gag197

+α4β7-CD8+ T cells. In addition, there was a comparison between results of Gag197

+α4β7-CD8+ T cells transfer group and Mock controls.


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5.2.3.2. Functional analysis of CD8+ T cell immune response detected by ICS following the pseudo-challenge

The spleen and MedLN samples were also assayed for functional cytokine responses using the

intracellular cytokine assay to measure the number of IFN-γ+HIVGag197+CD8+ T cells. T cells

harvested from the spleen and individual MedLN were stimulated for five hours with the

stimulation mixture containing HIV-Kd Gag197 peptide, IL-2 and Golgi-plug (Brefeldin). The

number and function of specific CD8+ T lymphocytes was then detected by flow cytometry.

This revealed a statistically higher number of specific IFN-γ+HIVKd Gag197+CD8+ T cells in

the group of mice that received adoptively transferred Gag197+α4β7+CD8+ T cells, (3.05 × 105

± 2.64 × 104) cells (p< 0.001) compared to the group of mice that received adoptively

transferred Gag197+α4β7-CD8+ T cells (1.07 × 105 ± 2.07 × 104) cells, and this was also

statistically higher in the group of mice that received adoptively transferred Gag197+α4β7+CD8+

T cells (p< 0.001) compared to Mock controls (2.42 × 104 ± 8.53 × 103) cells.

It is important to note that the group of mice that received adoptively transferred Gag197+α4β7-

CD8+ T cells had significantly higher numbers of HIVKdGag197+CD8+ T cells than Mock

controls (p< 0.001). Similar results to tetramer staining of the spleen were observed (Figure

5.6 A). The MedLN data was consistent with the observations noted via tetramer staining

(Figure 5.6 B) when compared to mice that received adoptively transferred Gag197+α4β7-CD8+

T cells. Statistical significance was also established when compared to mock controls (Figure

5.6).


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Figure 5.6. Number of IFN-γ+HIV-Kd Gag197

+CD8+ T cells following pseudo-challenge. Functional CD8+ T cell populations were analyzed for their ability to express IFN-γ+ in the spleen (A) and MedLN (B). Data is representative of the mean ± SD from two repeated experiments. Statistical significance was determined using a one way of variance (ANOVA), *p<0.05, **p<0.01 and ***p<0.001 when comparing the Gag197

+α4β7+CD8+ T cell transfer mouse group to both Gag197

+α4β7-CD8+ T cells and Mock controls. There was also a comparison of results between the Gag197

+α4β7-CD8+ T cell transfer mouse group and Mock controls.


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5.2.3.3. Analysis of α4β7+ and CD103+ surface markers on CD8+ T cells following a pseudo-challenge

Mucosal integrins, such as α4β7+ and CD103+ were assessed on HIV-Kd Gag197+tetramer+

CD8+ T cells following pseudochallenge. We detected an increase in the percentage of mucosal

markers on HIV-Kd Gag197+α4β7+ CD8+ T cells following challenge in both spleen and

mediastinal lymph nodes in the group of mice that received adoptively transferred

Gag197+α4β7+CD8+ T cells (12.95 ± 0.78%), compared to the group of mice that received

adoptively transferred Gag197+α4β7-CD8+ T cells (9.18 ± 0.13%) and Mock controls (5.1 ±

2.12%) (Figure 5.7). Similar trends were observed in the MedLN (Figure 5.7 B).

CD103 has been used in previous studies as a mucosal marker expressed on specific Gag+CD8+

T cells detected in the vaginal and gut mucosa of mice or macaques (Kiravu et al., 2011; Cerf-

Bensussan et al., 1987; Schon et al., 1999; Stevceva et al., 2002). A similar percentage of

HIVGag197+CD103+CD8+ T cells was detected in the group of mice that received adoptively

transferred Gag197+α4β7+CD8+ T cells compared to the group of mice that received adoptively

transferred Gag197+α4β7-CD8+ T cells and Mock controls suggesting there may be co-

expression of these molecules on mucosal cells (Figure 5.8).


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Figure 5.7. Percentage of HIV-H-2Kd Gag197

+4β7+CD8+ T cells following a pseudo-challenge. The percentage of HIV-Kd Gag197

+α4β7+CD8+ T cells in the spleen (A) and MedLN (B) were compared in three groups of mice that received adoptively transferred cells. Data is representative of the mean ± SD from two repeated experiments. Statistical significant difference was analyzed using a one way of variance test (ANOVA), **p<0.01 and ***p<0.001. This was then used to compare Gag197+α4β7+CD8+ T cells to Gag197

+α4β7-CD8+ T cells and Mock controls. There was also a comparison between the Gag197

+α4β7-CD8+ T cells transfer group and Mock controls.


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Figure 5.8. Percentage of CD103+HIV-H2Kd Gag197

+CD8+ T cells following challenge. The percentage of HIV-KdGag197

+CD103+CD8+ T cells in spleen (A) and MedLN (B) were compared in three groups of mice that received adoptively transferred cells.The data represents the mean ± SD from two repeated experiments. Statistically significant differences were analyzed using a one way of variance test (ANOVA), **p<0.01 and***p<0.001 to compare the Gag197

+α4β7+CD8+ T cell transfer mouse group to the Gag197+α4β7-CD8+ T cell transfer

group and Mock transfer group. A statistical analysis was also performed between the Gag197

+α4β7-CD8+ T cell transfer group and Mock controls.


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5.3. Discussion The ultimate aim of a HIV-1 vaccine is to successfully control HIV-1 infection by decreasing

HIV-1 virus progression and dissemination via the mucosal points of entry. Therefore, the aim

of this chapter was to investigate the protective capabilities of a subset of HIV-specific mucosal

CD8+ T cells (α4β7+Gag197+CD8+ T cells) induced by an intranasal prime boost strategy using

a recombinant influenza-HIV vaccine (see results from Chapter 4) to control naïve mice

challenged with the recombinant vaccinia virus expressing the whole Gag protein. This

involved the adoptive transfer of α4β7+or α4β7-Gag197+CD8+ T cells that were also shown to

express high levels of IL-15Rα and IL-15 cytokine as described in chapter 4.

The use of adoptive transfer has been utilized in experimental human trials. HIV-specific CD8+

T cells sorted from a non-infected identical twin brother of a HIV infected patient following

vaccination with recombinant vaccinia virus expressing Env glycoprotein, and then transferred

into the HIV-infected individual, led to a decrease in the titer of the HIV-1 virus (Bex et al.,

1994). This showed that adoptive transfer of HIV-specific CD8+ T cells could control virus in

a limited capacity as the cells were able to reduce virus dissemination through mucosal

compartments (Jespers et al., 2010; Shattock et al., 2008).

Indeed, mucosal CD8+ T cells expressing HIV-interferon-gamma (IFNγ), tumor necrotic factor

(TNF-α) and interleukin-2 (IL-2) have been detected at high levels in the cervix and blood of

female sex workers in Nairobi, thus indicating that mucosal CD8+ T cells represent a key

important line of defence against HIV-1 transmission and progression (Kaul et al., 2000;

Trivedi et al., 2001; Steers et al., 2009). Moreover, strong induction of a mucosal immune

responses represented by CD8+ T cells that secrete soluble proteins called alpha or beta

defensins in the oral cavity may contribute to HIV immunity(Weinberg et al., 2006; Tomescu,

Abdulhaqq and Montaner, 2011; Furci et al., 2007; Zhang et al., 2002). These protective

immune responses were also detected in rhesus macaques that were administered enteric coated

capsules containing an adenovirus vector expressing Gag protein via the oral route. The rhesus

macaques were then given an intranasal dose of adenovirus expressing Env polypeptide that

was shown to induce robust mucosal CD8+ and CD4+ T cells in the oral cavity, vaginal mucosa

and intestine in addition to a Gag and Env-specific antibody response (Mercier et al., 2007).

The main aim of this chapter was to test the protective efficacy of mucosal Gag197+α4β7+CD8+

T cells against a rVac-Gag pseudo-challenge.


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Our results clearly demonstrate that significant partial protection can be achieved with a sorted

Gag197+α4β7+CD8+ T cell population. Clearly the responses aren’t fully protective, and

therefore a combination of other mucosal subsets (CD103+ or CCR9+) Kd Gag197+CD8+ T cells

or those specific for other HIV antigens may be required to improve efficacy. Previous studies

in BALB/c mice have examined the protective role of mucosal CD8+ T cells induced against

the Env 18 IIIB (RIQRGPGRAFVTIGK) peptide. CD8+ T cell repsonses were detected in the

spleen, peyer’s patches and lamina propria following intrarectal immunisation in mice. Mice

were then challenged intrarectally with 2.5 × 107 plaque-forming units of recombinant vaccinia

virus expressing the HIV Env protein (gp160IIIB) (Belyakov et al., 1998 ) and 6 days later a

4.5 log reduction in challenge virus titer was detected in the ovaries of vaccinated mice

compared to mock controls. Furthermore studies in both rhesus macaques and C57BL/6 mice

following recombinant adenovirus (rAd5) SIVmac239 Gag protein vaccination and adoptive

transfer showed upregulation of mucosal homing integrins, (CD103 and CCR9). The transfered

mucosal CD8+ T cells facilitated protection in mice and macaques following recombinant

vaccinia-Gag challenge (Kaufman et al., 2008).

Other studies have used an alternative delivery system, such as Listeria monocytogenes as a

live recombinant vector vaccine to induce Gag-specific cytotoxic CD8+ T cell immune

responses in mice following vaccination. These Gag specific cytotoxic CD8+ T cells produced

interferon gamma (IFN-γ+) and tumour necrosis factor (TNF-α) in various mucosal lymphoid

tissues, such as the spleen and MALT and protected mice against a recombinant vaccinia virus

(rVac-Gag) vaginal challenge (Zhao et al., 2006). BALB/c mice primed intramuscularly using

either a rabies virus expressing HIV Gag protein (RABV) and boosted with either RABV-Gag,

a vesicular stomatitis virus (VSV-Gag), or a Newcastle disease virus expressing Gag protein

(NDV-Gag), induced a robust functional HIV Gag+CD8+ T cell responses (Lawrence et al.,

2013).

Our pseudo-challenge experiments demonstrated a 1/2 log reduction in the titer of challenge

virus by Gag197+α4β7+ CD8+ T cells. Complete protection was not achieved by Gag197

+α4β7+

CD8+ T cells. This is in contrast to Belyakov and colleagues, (1998) where a 4.5 log reduction

in the titer of pseudo-challenge virus (rVac-Env) was observed in the ovaries follwoing 4 doses

of HIV Env peptide IIIB in combination with cholera toxin (Belyakov et al., 1998).


152

Our experimental results are in line with a recent study in BALB/c mice where less than one

log reduction was observed following an adenovirus 5 Gag protein (Ad5-Gag) vaccination

regimen (Kanagavelu et al., 2014). In summary, the reduction in challenge virus titer in our

experiments is promising and provides opportunities to test the protective efficacy of the other

mucosal markers that could be used in combination with Gag197+α4β7+ CD8+ T cells. These

markers include CD103+, CCR9+ or a combination of both (CD103+CCR9+).

Pseudo-challenge studies assessing efficacy of HIV vaccines in mice have focused on

intraperitoneal, rectal, vaginal or intranasal administration of recombinant vaccinia virus

expressing recombinant antigens (Belyakov et al., 1998; Kaufman et al., 2008; Zhao et al.,

2006; Nakaya et al., 2004). These vaccines were able to induce protection against challenge

virus in ovaries, spleen and regional lymph nodes as rVV is capable of disseminating

systemically. (Belyakov et al., 1998; Zhao et al., 2006).

Previous studies have shown a critical role for mucosal CD8+ T lymphocytes induced following

intranasal influenza infection. Mucosal surface markers such as α4β7, have been identified in

the lymph nodes of infected mice (Schnorrer et al., 2006; Sallusto, 1999; Dieli et al., 2003).

Furthermore, transfer of mucosal influenza-specific CD8+ T cells expressing homing integrin

receptors has been shown to be critical for protection against intranasal A/PR/8/34 influenza

infection in BALB/c mice. This involved intravenous adoptive transfer of 107 CD8+ T cells

and challenge using 10 times the lethal dose of PR8 (A/PR/8/34: H1N1). Results showed a

significant reduction of influenza virus titer 4 days following infection compared to control

mice (Cerwenka et al., 1999). As such we can conclude that the ability of mucosal CD8+ T

cells to control virus infection can be associated with the expression of α4β7 integrin in our

study and may also involve CCR9 and CD103 and (Tan et al., 2011; Rudraraju et al., 2012;

Chattha et al., 2013). The cytotoxic potential of the Gag197+α4β7+ CD8+ T cells generated

following vaccination was not determined in these studies. A comparison with Gag197+α4β7-

CD8+ T cells may offer further insights.

Chapter 6: General Discussion

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6. Chapter 6: General Discussion 6.1. Overall discussion HIV/AIDS remains a serious threat to humanity with more than 1.8 million HIV-1 infected

individuals dying world-wide every year because of the global spread of the HIV epidemic.

According to World Health Organization reports, the mortality rate associated with HIV/AIDS

infection will remain at current levels until 2030 (McElrath and Haynes, 2010; Boily et al.,

2009; Brown et al., 2008; Bongaarts et al., 2008). Therefore, international efforts to uncover

effective solutions to control infection remain vital (Duerr, Wasserheit and Corey, 2006;

Mathers and Loncar, 2006). For this purpose, development of an effective HIV vaccine is still

regarded as the most appropriate way to control this epidemic (Johnston and Fauci, 2008).

Since 1983, more than 60 vaccine candidates have been generated to control HIV infection

(Barouch, 2008; Girard, 2006). A variety of approaches have been trialled, which include DNA

vaccines, recombinant vector vaccines and subunit vaccines, most of which have been

administered to non-human primates. A very limited number of candidate vaccines have been

tested in humans, with most of these unable to elicit significant efficacy. The exception,

however, was a vaccine tested in Thailand in 2009 (RV144: Phase III). For the first time, this

vaccine elicited a moderate level of protection against HIV-1 (31%). Antibody responses were

induced for HIV Env and Gag, as detected by antibody dependent cellular cytotoxicity assay

(ADCC) and also a cell mediated response represented by CD8+ T cell proliferation directed

against Gag and Env proteins (Rerks-Ngarm et al., 2009). CD8+ T lymphocytes play a major

role in controlling HIV infection and may contribute to mucosal immune responses (Gómez,

Smaill, and Rosenthal, 1994; Levy, 2003; Liu et al., 1998). CD8+ T lymphocytes are normally

stimulated during primary viral infections in humans to support the host immune response

(Callan et al., 1998; Reddehase, 1984; Reusser et al., 1999; Rickinson and Moss, 2003).

Mucosal CD8+ T lymphocytes are thought to be important for providing long-term protection

against HIV-1 virus which is transmitted by mucosally, through blood transfusion and by

mother-to-child transmission (Belyakovet al., 1998; Gallichan and Rosenthal, 1996; Suvas et

al., 2007). HIV-resistant female sex workers in Kenya possessed persistent mucosal HIV-1-

specific CD8+ T cells in the cervix (Kaul et al., 2000). In addition, but not specifically covered

in this thesis, the humoral immune response also plays an important role through the

stimulation of HIV-specific mucosal antibodies, such as IgA, which acts to directly block virus

entry through mucosal surfaces in the urogenital mucosa. This has been shown following


154

vaginal challenge with SHIV virus in macaques treated with passive IgA or in humanized mice

given anti-HIV-polymeric IgA inhibited HIV mucosal transmission. However, the role IgA

plays in controlling mucosal transmission of HIV-1 is not completely characterized in human

at this point in time (Mascola et al., 1996; Mascola et al., 2000; Hur et al., 2012; Wolbank et

al., 2003). Poly-functional CD8+ T cells are stimulated by specific HIV peptides, such as those

derived from the Gag protein (Duvall et al., 2008; Holmgren and Czerkinsky, 2005; Gillespie,

2005). During acute and chronic HIV-1 infection, these polyfunctional CD8+ T cells have been

shown to be involved in the reduction and control of the viral load. Firstly, they control virus

replication by producing high levels of multiple cytokines, such as IFNγ, TNF-α and IL-2

(Kobayashi et al., 2004; Viganò et al., 2008). Secondly, CD8+ T lymphocytes can destroy HIV-

1 infected cells through cytolytic action involving granzymes and perforin (Froelich, Dixit and

Yang, 1998), with HIV-resistant individuals shown to express high levels of both granzymes

and perforin. Moreover, there is a negative correlation between HIV-1 RNA copy number and

the level of granzymes and perforin expressed by CD8+ T cells (Kaul et al., 2000; Hersperger,

2010).

The studies described in this dissertation explored a reverse genetics strategy for generating a

vaccine that stimulates mucosal HIV-specific immunity. This strategy involved the use of

recombinant DNA technology and reverse genetics to generate influenza viruses that expressed

HIV-1 specific CD8+ T cell epitopes in order to induce mucosal CD8+ T cell immunity. Two

H-2d HIV-1 epitopes, Gag197 (AMQMLKETI) and Tat17 (QPKTACTNC), were inserted into

the NA gene segment of influenza virus strains PR8 (HIN1) and X31 (H3N2) using reverse

genetics and mice were immunized via the mucosal route using a prime-boost strategy.

Previous HIV vaccine studies in animal models have used a range of vectors (including

adenovirus, vaccinia virus and fowl pox virus) to deliver HIV epitopes or proteins. All of these

vectors were able to induce HIV-specific immune responses but many did not focus on

stimulation of mucosal immunity. The influenza virus was chosen as a live vaccine vector

candidate as it efficiently expresses viral epitopes in the respiratory and urogenital

compartments (Marsh and Tannock, 2005; Satterlee, 2008; Smith, 2007) and has the ability to

generate broad and durable long-term immunity at different mucosal surfaces (Fodor et al.,

1999; Mueller et al., 2010; Neumann and Kawaoka, 2001; Yao and Wang, 2008). Moreover,

there is a documented strong connection between the mucosa of the upper respiratory tract and

of the urogenital tract (Holmgren and Czerkinsky, 2005). This was detected using nasal Cholera


155

toxin B subunit immunization in humans to induce both IgA and IgG antibody responses in

cervicovaginal mucosa in addition to the upper respiratory mucosa and regional secretions

(saliva, nasal secretions) (Holmgren and Czerkinsky, 2005; Kozlowski et al., 1997; Johansson

et al., 2004). Another advantage of this approach include the fact that the gene segments of the

influenza virus could be easily manipulated using reverse genetics technology which allowed

the generation of recombinant viruses as vaccine vectors (Palese et al., 1996; Steinhauer and

Skehel, 2002; Steinhauer et al., 2009).

Recombinant influenza viruses manipulated in this way have been used as vaccine vectors

expressing HIV-CD8+ T cell epitopes to successfully stimulate cytotoxic CD8+ T cell responses

(Casimiro et al., 2005; Hoffmann et al., 2002; Pachler, Mayr and Vlasak, 2010; Sexton et al.,

2009). Intranasal vaccination of macaques with recombinant HIV vaccines has led to

stimulation of a strong mucosal immune response in vaginal mucosa when challenged

intravaginally with a virulent SHIV virus (Barnett et al., 2008). Other mucosal HIV-1

vaccination trials were highly effective in inducing both systematic and mucosal immune

responses against HIV-1. Examples of this include intranasal immunisation with a recombinant

influenza vector expressing HIV-1 Env (gp41) that stimulated strong humoral immune

responses, as shown by HIV-1 specific antibodies in the lung, spleen and urogenital mucosa of

mice (Ferko et al., 1998). Importantly, the two strains of laboratory influenza viruses associated

with our study had previously been used to express the HIV-H-2Dd Env311 CD8+ T cell epitope

in mice (Cukalac et al., 2009).

The H-2Kd HIV-Gag197 epitope was used as an antigen for this project for several reasons.

Firstly, this Gag epitope is highly conserved, and so should offer protection against diverse

viral strains (Mata et al., 1998; Lorgeoux, Guo and Liang, 2012). Indeed, the nine amino acids

of Gag from 197 to 205 were the most dominant epitope targeted by a breadth of HIV-CD8+ T

cell responses when compared to other Gag peptides (Cellini et al., 2008). Moreover,

polyfunctional HIV-specific CD8+ T cells obtained from HIV-elite controllers have been

shown to be Gag-specific (Ndhlovu et al., 2012; Berger et al., 2011). Secondly, HIV Gag

protein can stimulate specific HIV-CD8+ T cell immune responses with the mostly dominant

HIV-1 restricted Gag H-2Kd CD8+ T cell in mouse models (Faul et al., 2009; Lawrence et al.,

2013; McGettigan et al., 2001; McGettigan et al., 2003; McGettigan et al., 2003a). This

includes conserved regions of the HIV-Gag197 (AMQMLKETI) epitope detected in the wild


156

type virus genome which induced a robust HIV-1 CD8+ T cell immune response in vaccinated

mice (Lawrence et al., 2013; Wallace et al., 2013). Also, strong HIV-1 Gag H-2Kd

(AMQMLKETI) restricted CD8+ T cell responses have previously been identified following

vaccination of BALB/c mice using a DNA vaccine expressing the HIV-1 Gag protein (Wallace

et al., 2013). Also, robust HIV-Gag+CD8+ T cell responses were detected in intramuscularly

vaccinated BALB/c mice using recombinant virus vectors, such as the rabies virus (RABV),

vesicular stomatitisvirus (VSV) and Newcastle disease virus expressing the Gag protein

(Lawrence et al., 2013). In addition, Gag-specific CD8+ T lymphocytes have been shown to be

highly migratory following intramuscular infection with a recombinant adenovirus vector

expressing the whole Gag protein in mice and macaques (Kaufman et al., 2008). These HIV-

Gag-specific CD8+ T cells dominant in the Gag Db-restricted epitope (AAVKNWMTQTL)

were found mainly in the spleen and different mucosal sites, Peyer’s patches, the inguinal and

mesenteric lymph nodes, and vaginal tract mucosa (Kaufman et al., 2008). It is also important

to note that the dominant CD8+ T cell specificities induced in macaques following intratracheal

and intranasal recombinant influenza viruses, X31 (H3N2, A/HKx31) and PR8 (H1N1,

A/Puerto Rico/8/1934) expressing SIV-CD8+ T cell epitopes and NA stalks were derived from

the SIV-Tat87–96, SIV-Tat114–123 and SIV- Gag164–172 epitopes that were separately inserted into

the NA stalk of influenza virus vectors (Sexton et al., 2009).

Our study clearly demonstrate the ability of the influenza virus to replicate in the upper

respiratory tract of mice when infected intransally. Replication in the genital mucosa is

normally non-productive due to the lack of trypsin-like enzymes required for the cleavage of

HA in order to facilitate entry of the viral genome into the cell cytoplasm (Klenk, Rott, Orlich,

and Blödorn, 1975; Garulli, Kawaoka and Castrucci, 2004; Garulli et al., 2007). As was

detected in this project, using the recombinant X31-NA-Gag197 influenza virus, intranasally

infected BALB/c mice experience significant weight loss compared to intravaginally infected

mice in which no weight loss was detected. This can be explained by the ability of influenza

viruses to replicate in the respiratory tract mucosa as a result of appropriate protease activity in

the pulmonary mucosal area, thus increasing the pathogenicity of influenza virus infection

because this enzyme is lacking in the vaginal mucosa (Garulli, Kawaoka and Castrucci, 2004).

However, this does not mean that no immune response was induced in the urogenital mucosa

following influenza infection. Moreover, methods have been developed to improve the

replication rates of viruses in the urogenital area. For example, subcutaneous administration of


157

progesterone has been shown to improve sensitivity of mice to intravaginal infection with

viruses such as herpes simplex virus type 2 (Parr, Bozzola and Parr, 1998; Parr et al., 1994)

or influenza virus (Garulli, Kawaoka and Castrucci, 2004).

Experiments by Garulli and colleagues, (2004) demonstrated that the most highly induced

CD8+ T cells were specific for HIV-Env epitope (RGPGRAFVTI) of the gp160 envelope

protein and the immuno-dominant restricted H-2Dd NP147-155 epitope (TYQRTRALV) from the

HIV-1 IIIB isolate. Most of these CD8+ T cells were detected in the spleen, iliac lymph nodes

and in the genital mucosa of progesterone treated BALB/c mice that were then infected with a

high dose of recombinant influenza viruses either A/WSN/33, A/PR/8/34 (PR8) or X-31

viruses expressing the HIVEnv311 epitope (Flu/P18IIIB; Env gp160). In addition, HIV-specific

antibody responses were detected in both sera and vaginal secretions of these infected mice.

However, the group of mice that received a lower dose of the recombinant influenza virus

vector showed low or undetectable virus titres following intravaginal infection (Garulli,

Kawaoka and Castrucci, 2004). According to this study, influenza A viruses could induce acute

infection in the vaginal mucosa of the progesterone-treated BALB/c mice. This means vaginal

replication could be enhanced by treatments that mimic protease function thus increasing

pathogenicity of the influenza virus. This was also observed in the female genital organs of

mice during the estrous cycle on progesterone and resulted in high susceptibility of adult

BALB/c mice to vaginal infection with herpes simplex virus 2 compared to non-estrous mice

(Parr, Bozzola and Parr, 1998; Parr et al., 1994).

Mucosal immune responses are characterized by markers such as α4β7, CD103 and CCR9. An

important role for LPAM-1 (α4β7) as a mucosal homing CD8+ T cell integrin in protective

immune responses has been suggested in various virus infections in mice studies. (Holzmann,

McIntyre and Weissman, 1989; Rudraraju et al., 2012; Tan et al., 2011). When comparing

different mucosal routes of prime-boost infection in BALB/c mice, the results in this project

indicate that intranasal vaccination with recombinant influenza virus vaccine vectors induces

the best polyfunctional HIVGag197+CD8+ T cell response

The main aim of mucosal vaccines used in this project was to induce HIV specific mucosal

CD8+ T cells that enable control of a pseudovirus challenge. Therefore, we conducted

experiments to find out whether the induced mucosal HIV-Gag197+4β7+CD8+ T cells were

functionally protective. HIV-Gag197+4β7+CD8+ T cells were sorted for adoptive transfer into


158

naïve mice prior to pseudochallenge with the recombinant vaccinia virus expressing HIV-Gag

(rVac). Importantly this transferred population was partially protective.

6.2. Research outcomes This project has successfully investigated a novel approach to stimulate, detect and characterize

mucosal CD8+ T cells responses using recombinant influenza viruses as a vaccine vector. The

key research outcomes of this work were:

1. The generation of recombinant influenza viruses expressing the H-2Kd restricted HIV-

Gag197 or H-2d HIV-Tat17 CD8+ T cell epitopes using reverse genetics technology.

2. Demonstration that recombinant influenza viruses expressing the HIV-H-2KdGag197

epitope stimulated strong CD8+ T cell immunity at multiple anatomical sites as seen

through the use of tetramer staining and ICS.

3. Intranasal-intranasal prime-boost vaccination induced the highest proportion and

numbers of HIV and influenza-specific mucosal CD8+ T cells in the spleen, draining

lymph nodes and distal mucosal sites.

4. Adoptive-transfer of Gag197+α4β7+CD8+ T cells into naïve mice enabled partial control

of whole Gag protein expressing vaccinia virus challenge.

6.3. Future directions Due to time limitations, a generic adoptive transfer regime was adopted for protection

experiments. As such, the most critical experiments to perform are ones that involve fine-tuning

the adoptive transfer conditions given, since only partial protection was achieved. Experiments

would therefore involve repeating the intranasal prime-boost vaccination strategy, and

transferring a higher number of mucosal α4β7+Gag+CD8+ T cells to naïve mice to determine if

recombinant vaccinia virus challenge could be fully controlled. In addition, these studies could

be expended by transferring HIVGag197 specific CD8+ T cells that express more than one

mucosal integrin, such as Gag197+CD103+CD8+ T cells, to improve protection from a pseudo-

challenge virus. This work could also be extended to vaccination that could induce both a

mucosal and systematic CD8+ T cell immune responses necessary to control challenge virus.

If successful, the transfer experiments could be repeated in macaques. These experiments

would involve the use of SIV-Gag197+CD8+ T cell epitopes to initially prove the ability of the


159

intranasal prime boost immunisation to induce mucosal CD8+ T cells prior to commencement

of the adoptive transfer experiments. These experiments are costly and must be carefully

considered given the ethic issues around the use of non-human primates. Availability and/or

cross reactivity of mucosal markers in this non-human primate model must also be verified

prior to commencement. In conclusion, studies that enhance our understanding of mucosal HIV

immunity are of utmost importance given the portal of entry for most HIV infections in humans

is the mucosa. Development of vaccines that can stop dissemination of the virus, halt its

progress at the local draining lymph nodes and therefore avoid latency may prove the most

effective in bringing this worldwide epidemic under control.

Finally it would be important to test if influenza-specific α4β7+CD8+ T cells could behave

similarly in protection experiments against intranasal influenza virus challenge when

adoptively transferred to mice to establish this mucosal subset as a universal protective marker.

References

160

7. References Ada, G. L. and Jones, P. D. (1986)."The immune response toinfluenza infection". Curr.Top.

Microbiol. Immunol., vol. 128:1-45.

Addo, M. M., Yu, X. G., Rathod, A., Cohen, D., Eldridge, R. L., Strick, D., Johnston, M. N., Corcoran, C., Wurcel, A. G., Fitzpatrick, C. A., Feeney, M. E., Rodriguez, W. R., Basgoz, N., Draenert, R., Stone, D. R., Brander, C., Goulder, P. J., Rosenberg, E. S., Altfeld, M. and Walker, B. D. (2003)."Comprehensive epitope analysis of human immunodeficiency virus type 1 (HIV-1)-specific T-cell responses directed against the entire expressed HIV-1 genome demonstrate broadly directed responses, but no correlation to viral load".J. Virol., vol. 77 (3):2081-2092.

Addo, M. M., Altfeld, M., Rosenberg, E. S., Eldridge, R. L., Habeeb, K., Khatri, A., Brander, C., Robbins, G. K., Mazzara, G. P., Goulder, P. J. and Walker, B. D. (2001)."The HIV-1 regulatory proteins Tat and Rev are frequently targeted by cytotoxic T lymphocytes derived from HIV-1-infected individuals". Proc. Natl. Acad. Sci. USA, vol. 98 (4):1781-1786.

Ahmad, A., Yao, X. A., Tanner, J. E., Cohen, E. and Menezes, J. (1994)."Surface expression of the HIV-1 envelope proteins in env gene-transfected CD4-positive human T cell clones: characterization and killing by an antibody-dependent cellular cytotoxic mechanism". J. Acquir. Immu. Defic. Syndr., vol. 7 (8):789-798.

Alfano, M. and Poli, G. (2005)."Role of cytokines and chemokines in the regulation of innate immunity and HIV infection". Molecu. Immunol.,vol. 42 (2):161-182.

Allan, W., Carding, S. R., Eichelberger, M. and Doherty, P. C. (1993)."hsp65 mRNA+ macrophages and γδ T cells in influenza virus-infected mice depleted of the CD4+ and CD8+ lymphocyte subsets". Micro. Patho., vol. 14 (1):75-84.

Allen, T. M., O’Connor, D. H., Jing, P., Dzuris, J. L., Mothé, B. R., Vogel and T. U. (2000)."Tat-specific cytotoxic T lymphocytes select for SIV escape variants during resolution of primary viraemia". Nature, vol. 407(6802):386-390.

Anderson, D. M., Johnson, L., Glaccum, M. B., Copeland, N. G., Gilber, D. J., Jenkins, N. A., Valentine, V., Kirstein, M. N., Shapiro, D. N., Morris, S. W., Grabstein, K., Cosman, D. (1995)."Chromosomal assignment and genomic structure of IL-15". Genomics, vol. 25:701-706.

Anzinger, J. J., Olinger, G. G. and Spear, G. T. (2008)."Donor variability in HIV binding to peripheral blood mononuclear cells". Virology, vol.5:95. doi: 10.1186/1743-422X-5-95.

Apostolaki, M., Manoloukos, M., Roulis, M., Wurbel, M. A., Müller, W., Papadakis, K. A., Kontoyiannis, D. L., Malissen, B. and Kollias, G. (2008)."Role of β7 integrin and the chemokine/chemokine receptor pair CCL25/CCR9 in modeled TNF-dependent Crohn's disease". Gastroenterology, vol. 134 (7):2025-35.

Artenstein, A. W. (2008)."New generation smallpox vaccines: a review of preclinical and clinical data". Rev. Med. Virol., vol. 18 (4): 217-231.

Assarsson, E., Bui, H. H., Sidney, J., Zhang, Q., Glenn, J., Oseroff, C., Mbawuike, I. N., Alexander, J., Newman, M. J. and Grey, H. (2008)."Immunomic analysis of the

References

161

repertoire of T-cell specificities for influenza A virus in humans". J. Virol., vol. 82: 12241-12251.

Baba, T. W., Liska, V., Khimani, A. H., Ray, N. B., Dailey, P. J., Penninck, D., Bronson, R., Greene, M. F., McClure, H. M., Martin, L. N. and Ruprecht, R. M. (1999)."Live attenuated, multiply deleted simian immunodeficiency virus causes AIDS in infant and adult macaques". Nat. Med., vol. 5 (2):194-203.

Baba, T., Jeong, Y. S., Pennick, D., Bronson, R., Greene, M. F. and Ruprecht, R. M. (1995)."Pathogenicity of live, attenuated SIV after mucosal infection of neonatal macaques". Science, vol. 267 (5205):1820-1825.

Bafica, A., Scanga, C. A., Equils, O. and Sher, A. (2004)."The induction of Toll-like receptor tolerance enhances rather than suppresses HIV-1 gene expression in transgenic mice". J. Leuko. Biol., vol. 75 (3):460-466.

Bhardwaj, N., Bender, A., Gonzalez, N., Bui, L.K., Garrett, M. C. and Steinman, R. M. (1994). "Influenza virus-infected dendritic cells stimulate strong proliferative and cytolytic responses from humanCD8+ T cells". J. Clin. Investig., vol. 94:797-807.

Baig, T. T., Strong, C. L., Lodmell, J. S. and Lanchy, J. M. (2008)."Regulation of primate lentiviral RNA dimerization by structural entrapment". Retrovirology, vol. 5 (1):65.

Bamford, R. N., Battiata, A. P., Burton, J. D., Sharma, H. and Waldmann, T. A. (1996). "Interleukin (IL) 15/IL-T production by theadult T-cell lymphotrophic virus type I R region/IL-15 fusion message that lacks many upstream AUGs that normally attenuateIL-15 mRNA translation". Proc. Natl. Acad. Sci. USA, vol. 93:2897-2902.

Banchereau, J. and Steinman, R. M. (1998). "Dendritic cells and the control of immunity". Nature, vol. 392:245-252.

Banda, N. K, Simon,G. R., Sipple, J. D., Terrell, K. L., Archer, P., Shpall, E. J., Akkina, R. K., Myers, A. M. and Harrison, G. S. (1999). "Depletion of CD34+CD4+ cells in bone marrowf rom HIV-1–infected individuals". Biol. Blood Marrow Transplant, vol. 5 (3):162-72.

Bandera, A., Ferrario, G., Saresella, M., Marventano, I., Soria, A., Zanini, F., Sabbatini, F., Airoldi, M., Marchetti, G., Franzetti, F., Trabattoni, D., Clerici, M. and Gori, A. (2010)."CD4+ T cell depletion, immune activation and increased production of regulatory T cells in the thymus of HIV infected individuals."PLoS. ONE, vol. 5 (5):e10788.

Barnard, D. L. (2009)."Animal models for the study of influenza pathogenesis and therapy". Antiviral. Res., vol. 82 (2):A110-A122.

Barnett, S. W., Srivastava, I. K., Kan, E., Zhou, F., Goodsell, A., Cristillo, A. D., Ferrai, M. G., Weiss, D. E., Letvin, N. L., Montefiori, D., Pal, R. and Vajdy, M. (2008)."Protection of macaques against vaginal SHIV challenge by systemic or mucosal and systemic vaccinations with HIV-envelope". AIDS, vol. 22 (3):339-348.

Barouch, D. H. (2010)."Novel adenovirus vector-based vaccines for HIV-1". Curr. Opin. HIV AIDS, vol. 5 (5):386-390.

Barouch, D. H. (2008)."Challenges in the development of an HIV-1 vaccine". Nature, vol. 455 (7213):613-619.

References

162

Barouch, D., Santra, S., Schmitz, J. E., Kuroda, M. J., Fu, T. M., Wagner, W., Bilska, M., Craiu, A., Zheng, X. X., Krivulka, G. R., Beaudry, K., Lifton, M. A., Nickerson, C. E., Trigona, W. L., Punt, K., Freed, D. C., Guan, L., Dubey, S., Casimiro, D., Simon, A., Davies, M. E., Chastain, M., Strom, T. B., Gelman, R. S., Montefiori, D. C., Lewis, M. G., Emini, E. A., Shiver, J. W. and Letvin, N. L.(2000)."Control of viremia and prevention of clinical AIDS in rhesus monkeys by cytokine-augmented DNA vaccination". Science, vol. 290 (5491):486-492.

Barouch, D. H. and Letvin, N. L. (2000a)."Cytokine-induced augmentation of DNA vaccine-elicited SIV-specific immunity in rhesus monkies". Dev. Biol., vol. 104:85-92.

Barré-Sinoussi, F., Chermann, J. C., Rey, F., Nugeyre, M. T., Chamaret, S., Gruest, J., Dauguet, C., Axler-Blin, C., Vézinet-Brun, F., Rouzioux, C., Rozenbaum, W. and Montagnier, L. (1983)."Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS) ". Science, vol. 20 (220):868-871.

Baumgarth, N., Herman, O. C., Jager, G. C., Brown, L. E., Herzenberg, L. A. and Chen, J. (2000)."B-1 and B-2 cell-derived immunoglobulin M antibodies are nonredundant components of the protective response to influenza virus infection". J. Exp. Med., vol. 192: 271-280.

Becker, S., Quay, J. and Soukup, J. (1991). "Cytokine (tumor necrosis factor, IL-6, and IL-8) production by respiratory syncytial virus-infected human alveolar macrophages". J. Immunol., vol. 147:4307-4312.

Beigel, J. H., Farrar, J., Han, A. M., Hayden, F. G., Hyer, R., de Jong, M. D., Lochindarat, S., Nguyen, T. K., Nguyen, T. H. and Tran, T. H. (2005)."Avian influenza A (H5N1) infection in humans". N. Engl. J. Med., vol. 353:1374-1385.

Belshe, R. B., Nichol, K. L., Black, S. B., Shinefield, H., Cordova, J., Walker, R., Hessel, C., Cho, I. and Mendelman, P. M. (2004)."Safety, efficacy, and effectiveness of live, attenuated, cold-adapted influenza vaccine in an indicated population aged 5–49 years". Clin. Inf. Dis., vol. 39:920-927.

Belyakov, I. M. and Berzofsky, J. A. (2004)."Immunobiology of mucosal HIV infection and the basis for development of a new generation of mucosal AIDS vaccines". Immunity, vol. 20 (3):247-253.

Belyakov, I. M., Hammond, S. A., Ahlers, J. D., Glenn, G. M. and Berzofsky, J. A. (2004)."Transcutaneous immunization induces mucosal CTLs and protective immunity by migration of primed skin dendritic cells". J. Clin. Investig., vol. 113 (7):998-1007.

Belyakov, I. M., Derby, M. A., Ahlers, J. D., Kelsall, B. L., Earl, P., Moss, B., Strober, W. and Berzofsky, J. A. (1998)."Mucosal immunization with HIV-1 peptide vaccine induces mucosal and systemic cytotoxic T lymphocytes and protective immunity in mice against intrarectal recombinant HIV-vaccinia challenge". Proc. Natl. Acad. Sci. USA, vol. 95:1709–1714.

Bender, B. S., Croghan, T., Zhang, L. and Small, P. A. (1992). "Transgenic mice lacking class I major histocompatibility complex-restricted T cells have delayed viral clearance and increased mortality after influenza virus challenge". J. Exp. Med., vol. 175: 1143-1145.

Benjelloun, F., Lawrence, P. Verrier, B., Genin, C. and Paula, S. (2012)."Role of human immunodeficiency virus type 1 envelope structure in the induction of broadly neutralizing antibodies". J. Virol., vol. 86 (24):13152-13163.

References

163

Benton, K. A., Misplon, J. A., Lo, C.-Y., Brutkiewicz, R. R., Prasad, S. A. and Epstein, S. L. (2001). "Heterosubtypic Immunity to Influenza A Virus in Mice Lacking IgA, all Ig, NKT cells, or γδ T cells". J. Immunol., vol. 166 (12):7437-7445.

Bentwich, Z. (2005)."CD4 measurements in patients with HIV: are they feasible for poor settings?". PLoS Med., vol. 2 (7):e214.

Berger, C. T., Frahm, N., Price, D. A., Mothe, B., Ghebremichael, M., Hartman, K. L., Henry, L. M., Brenchley, J. M., Ruff, L. E., Venturi, V., Pereyra, F., Sidney, J., Sette, A., Douek, D. C., Walker, B. D., Kaufmann, D. E. and Brander, C. (2011)."High-functional-avidity cytotoxic T lymphocyte responses to HLA-B-restricted Gag-derived epitopes associated with relative HIV control". J. Virol., vol. 85 (18):9334-9345.

Berger, E. A., Doms, R. W., Fenyö, E. M., Korber, B. T. M., Littman, D. R., Moore, J. P., Sattentau, Q. J., Schuitemaker H., Sodroski, J. and Weiss, R. A. (1998)."A new classification for HIV-1". Nature, vol. 391 (6664):240-240.

Berger, J., Aepinus, C., Dobrovnik, M., Fleckenstein, B., Hauber, J. and Bohnlein, E. (1991)."Mutational analysis of functional domains in the HIV-1 rev trans-regulatory protein". Virology, vol. 183 (2):630-635.

Berlin, C., Berg, E. L., Briskin, M. J., Andrew, D. P., Kilshaw, P. J., Holzmann, B., Weissman, I. L., Hamann, A. and Butcher, E. C. (1993)."α4β7 Integrin mediates binding to the mucosal vascular addressin MAdCAM-1". Cell, vol. 74 (1):185-195.

Betts, M. R., Nason, M. C., West, S. M., de Rosa, S. C., Migueles, S. A., Abraham, J., Lederman, M. M., Benito, J. M., Goepfert, P. A., Connors, M., Roederer, M. and Koup, R. A. (2006)."HIV nonprogressors preferentially maintain highly functional HIV-specific CD8+ T cells". Blood, vol. 107 (12):4781-4789.

Betts, M. R., Yusim, K. and Koup, R. (2002)."Optimal antigens for HIV vaccines based on CD8+ T response, protein length, and sequence variability". DNA Cell. Biol., vol. 21 (9):665-670.

Betts, M. R., Ambrozak, D. R., Douek, D. C., Bonhoeffer, S., Brenchley, J. M., Casazza, J. P., Koup, R. A. and Picker, L. J. (2001)."Analysis of total human immunodeficiency virus (HIV)-specific CD4+ and CD8+ T-cell responses: Relationship to viral load in untreated HIV infection". J. Virol., vol. 75:11983-11991.

Bex, F., Hermans, P., Sprecher, S., Achour, A., Badjou, R., Desgranges, C., Coquniaux, J., Franchioli, P., Vanhulle, C. and Lachgar, A. (1994)."Syngeneic adoptive transfer of anti-human immunodeficiency virus (HIV-1)-primed lymphocytes from a vaccinated HIV-seronegative individual to his HIV-1-infected identical twin". Blood, vol. 84:317-3326.

Biddison, W. E., Shaw, S. and Nelson, D. L. (1979). "Virus specificity of human influenza virus-immune cytotoxic T cells". J. Immunol., vol. 122:660-664.

Blankson, J. N. (2011)."The study of elite controllers: a pure academic exercise or a potential pathway to an HIV-1 vaccine? ". Curr. Opin. HIV AIDS, vol. 6:147-150.

Blankson, J. (2010)."Control of HIV-1 replication in Elite supressors". Division of Infectious Diseases, Department of Medicine, Johns Hopkins University School of Medicine, Maryland, USA.

References

164

Bodnar, A., Nizsaloczki, E., Mocsar, G., Szaloki, N., Waldmann, T. A., Damjanovich, S. and Vamosi, G. (2008)."A biophysical approach to IL-2 and IL-15 receptor function: localization, conformation and interactions". Immunol. Lett., vol. 116:117-125.

Boily, M. C, Baggaley, Wang, R. F. L., Masse, B., White, R., Hayes, R. and Alary, M. (2009). Heterosexual risk of HIV-1 infection per sexual act: systematic review and metaanalysis of observational studies". Lancet Infect. Dis., vol. 9 (2):118-129.

Bongaarts, J., Buettner, T., Heilig, G. and Pelletier, F. (2008)."Has the AIDS epidemic peaked?". Popul. Develop. Rev., vol. 34(2): 199-224.

Borrow, P., Lewicki, H., Hahn, B. H., Shaw, G. M. and Oldstone, M. B. A. (1994)."Virus speci¢c CD8+ cytotoxic T lymphocyte activity associated with control of viremia in primary human immunodeciency virus type-1infection". J.Virol., vol. 68, 6103-6110.

Borthwick, N., Ahmed, T., Ondondo, B., Hayes, P., Rose, A., Ebrahimsa, U., Hayton, E. J., Black, A., Bridgeman, A., Rosario, M., Hill1, A. V.S., Berrie, E., Moyle, S., Frahm, N., Cox, J., Colloca, S., Nicosia, A., Gilmour, J., McMichael, A. J., Dorrell, Lucy and Hanke, T. (2014)."Vaccine-elicited human T cells recognizing conserved protein regions inhibit HIV-1". Mol. Ther., vol. 22 (2): 464-475.

Bosch, B. J., Bodewes, R., de Vries, R. P., Kreijtz, J. H., Bartelink, W., van Amerongen, G., Rimmelzwaan, G. F., de Haan, C.A., Osterhaus, A. D. and Rottier, P. J. (2010). "Recombinant soluble, multimeric HA and NA exhibit distinctive types of protection against pandemic swine-origin 2009 A(H1N1) influenza virus infection in ferrets". J. Virol., vol. 84: 10366-10374.

Bot, A., Bot, S. and Bona, C.A. (1998). "Protective role of gamma interferon during the recall response to influenza virus". J. Virol., vol. 72:6637-6645.

Böttcher-Friebertshäuser, E., Freuer, C., Sielaff, F., Schmidt, S., Eickmann, M., Uhlendorff, J., Steinmetzer, T., Klenk, H. D. and Garten, W. (2010)."Cleavage of influenza virus hemagglutinin by airway proteases TMPRSS2 and HAT differs in subcellular localization and susceptibility to protease inhibitors". J. Virol., vol. 84 (11):5605-5614.

Bourgeois, R., Mercier, J., Paquette-Brooks, I. and Cohen, É. A. (2006)."Association between disruption of CD4 receptor dimerization and increased human immunodeficiency virus type 1 entry". Retrovirology, vol. 3:31.

Bouvier, N. M. and Lowen, A. C. (2010)."Animal models for influenza virus pathogenesis and transmission". Viruses, vol. 2 (8):1530-1563.

Bouwman, L.H., Roep, B. O. and Roos, A. (2006)."Mannose-binding lectin: clinical implications for infection, transplantation,and autoimmunity". Human Immunol., vol. 67:247-256.

Braciale, T. J., Sun, J. and Kim, T. S. (2012). "Regulating the adaptive immune response to respiratory virus infection". Nat. Rev., vol. 12:295-305.

Braddock, M., Powel, R., Blanchard, A. D., Kingsman, A. J. and Kingsman, S. M. (1993)."HIV-1 TAR RNA-binding proteins control TAT activation of traslation in Xenopus oocytes." FASEB J., vol. 7:214-222.

Bradney, C. P., Sempowski, G. D. , Liao, H., Haynes, B. F. and Staats, H. F. (2002)."Cytokines as adjuvants for the induction of anti-human immunodeficiency virus peptide immunoglobulin G (IgG) and IgA antibodies in serum and mucosal secretions after nasal immunization". J. Virol., vol. 76 (2):517-524.

References

165

Bretscher, P. A. (1999)."A two-step, two-signal model for the primary activation of precursor helper T cells."Proc. Natl. Acad. Sci. USA, 96 (1):185-190.

Bridgen, A. and Elliott, R. M. (1996)."Rescue of a segmented negative-strand RNAvirus entirely from cloned complementary DNAs". Proc. Nat. Acad. Sci. USA, vol. 93: 15400-15404.

Brincks, E. L. and Woodland, D. L. (2010)."Novel roles for IL-15 in T cell survival". Biol. Reports, vol. 2:67.

Broliden, K., Hinkula, J., Devito, C., Kiama, P., Kimani, J., Trabbatoni, D., Bwayo, J. J., Clerici, M., Plummer, F. and Kaul, R. (2001)."Functional HIV-1 specific IgA antibodies in HIV-1 exposed, persistently IgG seronegative female sex workers". Immunol. Lett., vol. 79 (1-2):29-36.

Brown, T., Salomon, J. A., Alkema, L., Raftery, A. E. and Gouws, E. (2008)."Progress and challenges in modelling country-level HIV/AIDS epidemics: The UNAIDS estimation and projection package 2007". Sex. Transm. Infect., vol. 84 (Supplement 1): i5–i10.

Brown, D. M., Dilzer, A. M., Meents, D. L. and Swain, S. L. (2006). "CD4 T cell-mediated protection from lethal influenza: perforin and antibody-mediated mechanisms give a one-two punch". J. Immunol., vol. 177:2888-2898.

Brühl, P., Kerschbaum, A., Kistner, O., Barrett, N., Dorner, F. and Gerenčer, M. (2000). "Humoral and cell-mediated immunity to Vero cell-derived influenza vaccine". Vaccine, vol. 19 (9-10):1149-1158.

Bruyn, G., Rossini, A. J., Chiu, Y. L., Holman, D., Elizaga, M. L., Frey, S. E., Burke, D., Evans, T. G., Corey, L. and Keefer, M. C. (2004)."Safety profile of recombinant canarypox HIV vaccines." Vaccine, vol. 22 (5-6):705-714.

Bryant, M. and Ratner, L. (1990)."Myristoylation-dependent replication and assembly of human immunodeficiency virus 1". Proc. Natl. Acad. Sci. USA, vol. 87 (2):523-527.

Bublot, M., Pritchard, N., Swayne, D. E., Selleck, P., Karaca, K., Suarez, D. L., Audonnet, J.C. and Michle, T. R. (2006)."Development and use of fowlpox vectored vaccines for avian influenza." Ann. N. Y. Acad. Sci., vol. 1081 (1):193-201.

Buchschacher, G. L. (2001)."Introduction to retroviruses and retroviral vectors."Somat. Cell. Mol. Genet., vol. 26 (1-6):1-11.

Buckheit, R. W., Salgado, M., Silciano, R. F. and Blanksona, J. N. (2012)."Inhibitory Potential of Subpopulations of CD8_ T Cells in HIV-1-Infected Elite Suppressors". J. Virol., vol. 86 (24):13679.

Burmeister, T., Schwartz, S., Hummel, M., Hoelzer, D. and Thiel, E. (2007)."No genetic evidence for involvement of Deltaretroviruses in adult patients with precursor and mature T cell neoplasms."Retrovirology, vol. 4:11. doi:10.1186/1742-4690-4-11.

Burmeister, T., Schwartz, S., and Thiel, E. (2001)."A PCR primer system for detecting oncoretroviruses based on conserved DNA sequence motifs of animal retroviruses and its application to human leukaemias and lymphomas". J. Gen. Virol., vol. 82 (9):2205-2213.

Byron, E. E. M., van den D. P., Koraka, P., Amerongen, G., Spohn, G., Haagmans, B. L., Provacia, L. B. V., Osterhaus, A. D. M. E. and Rimmelzwaan, G. F. (2011). "A Recombinant Influenza A Virus Expressing Domain IIIof West Nile Virus Induces

References

166

Protective Immune Responsesagainst Influenza and West Nile Virus". PLoS ONE, vol. 6 (4 ): e18995.

Cafaro, A., Titti, F., Fracasso, C., Maggiorella. M. T., Baroncelli, S. and Caputo, A. (2001).

"Vaccination with DNA containing tat coding sequences and unmethylated CpG motifs protects cynomolgus monkeys upon infection with simian/human immunodeficiency virus (SHIV89.6P)". Vaccine, vol. 19 (20-22):2862-2867.

Caley, I. J., Betts, M. R., Irlbeck, D. M., Davis, N. L., Swanstrom, R., Frelinger, J. A. and Johnston, R. E. (1997)."Humoral, mucosal, and cellular immunity in response to a human immunodeficiency virus type 1 immunogen expressed by a Venezuelan equine encephalitis virus vaccine vector". J. Virol., vol. 71 (4):3031–3038.

Callan, M. F. C., Tan, L., Annels, N. E., Ogg, G. S., Wilson, D. K., O’Callaghan, C. A., Steven, N., McMichael, A. J. and Rickinson., A. B. (1998)."Direct visualization of antigen-specific CD8+T cells during the primary immune response to Epstein-Barr virus in vivo". J. Exp. Med., vol. 187 (9):1395-1402.

Canals, A., Grimm, D. R., Gasbarre, L. C., Lunney, J. K., Zarlenga, D. S. (1997). "Molecular cloning of cDNA encoding porcine interleukin-15". Gene, vol. 195:337-339.

Cao, H., Kaleebu, P., Hom, D., Flores, J., Agrawal, D., Jones N., Serwanga, J., Okello, M., Walker, C., Sheppard, H., El-Habib, R., Klein, M., Mbidde, E., Mugyenyi, P., Walker, B., Ellner, J., Mugerwa, R. and the HIV Network for Prevention Trials. (2003). "Immunogenicity of a recombinant human immunodeficiency virus (HIV)–canarypox vaccine in HIV-seronegative ugandan volunteers: results of the HIV network for prevention trials 007 vaccine study". J. Infect. Dis., vol. 187 (6): 887-895.

Cao, J., McNevin, J., Holte, S., Fink, L., Corey, L. and McElrath, M. J. (2003). Comprehensive analysis of human immunodeficiency virus type 1 (HIV-1)-specific gamma interferonsecreting CD8+ T cells in primary HIV-1 infection. J. Virol. vol. 77(12):6867- 6878.

Capon, D. J. and Ward, R. H. R. (2003)."The CD4-gpl20 interaction and AIDS pathogenesis". Ann. Rev. Immunol., vol. 9 (1):649-678.

Caputo, A., Gavioli, R. and Ensoli, B. (2004). Recent advances in the development of HIV-1 Tat-based vaccines. Curr. HIV Res. vol. 2:5038-5042.

Card, D. M., Ball, T. B. and Fowke, K. R. (2013). "Immune quiescence: a model of protection against HIV infection". Retrovirol., vol. 10:141.

Carragher, D. M., Kaminski, D. A., Moquin, A., Hartson, L. and Randall, T. D. (2008)."A novel role for non-neutralizing antibodies against nucleoprotein in facilitating resistance to influenza virus". J. Immunol., vol. 181:4168-4176.

Carter, J. and Saunders, V. (2009)."Virology: Priciples and applications". J. Trop. Pediatr., vol. 55 (1):66.

Carvalho, F. T., Goncalves, T. R., Faria, E. R., Shoveller, J. A., Piccinini, C. A., Ramos, M. C. and Medeiros, L. R. (2011)."Behavioral interventions to promote condom use among women living with HIV". Cochrane Database Syst. Rev., vol. 9:CD007844.

Carrington, M. and Alter, G. (2012)."Innate immune control of HIV". Cold Spring Harb. Perspect Med., vol. 2 (7):a007070.

References

167

Casimiro, D. R., Wang, F., Schleif, W. A., Liang, X., Zhang, Z. Q., Tobery, T. W., Davies, M. E., McDermott, A. B., O'Connor, D. H., Fridman, A., Bagchi, A., Tussey, L. G., Bett, A. J., Finnefrock, A. C, Fu, T., Tang, A., Wilson, K. A., Chen, M., Perry, H. C., Heidecker, G. J., Freed, D. C., Carella, A., Punt, K. S., Sykes, K. J., Huang, L., Ausensi, V. I., Bachinsky, M., Sadasivan-Nair, U., Watkins, D. I., Emini, E. A. and Shiver, J. W. (2005).'Attenuation of simian immunodeficiency virus SIVmac239 infection by prophylactic immunization with DNA and recombinant adenoviral vaccine vectors expressing Gag". J. Virol., vol. 79 (24):15547-55.

Casimiro, D. R., Chen, L., Fu, T. M., Evans, R. K., Caulfield, M. J., Davies, M. E., Tang, A., Chen, M., Huang, L., Harris, V., Freed, D.C., Wilson, K. A., Dubey, S., Zhu, D., Nawrocki, D., Mach, H., Troutman, R., Isopi, L., Williams, D., Hurni, W., Xu, Z., Smith, J. G., Wang, S., Liu, X., Guan, L., Long, R., Trigona, W., Heidecker, G. J., Perry, H. C., Persaud, N., Toner, T. J., Su, Q., Liang, X., Youil, R., Chastain, m., Bett, A. J., Volkin, D. B., Emini, E. and Shiver, J. W. (2003)."Comparative immunogenicity in rhesus monkeys of DNA plasmid, recombinant vaccinia virus, and replication-defective adenovirus vectors expressing a human immunodeficiency virus type 1 gag gene". J. Virol., vol., 77 (11):6305-6313.

Castrucci, M. R., and Kawaoka, Y. (1993). "Biologic importance of neuraminidase stalk length in influenza A virus". J. Virol., vol. 67:759-764.

Castrucci, M. R., Hou, S., Doherty, P. C. and Kawaoka Y. (1994)."Protection against lethal lymphocytic choriomeningitis virus (LCMV) infection by immunization of mice with an influenza virus containing an LCMV epitope recognized by cytotoxic T lymphocytes". J. Virol., 68 (6)3486-3490.

Catanzaro, A. T., Koup, R. A., Roederer, M., Bailer, R. T., Enama, M. E., Moodie, Z., Gu, L., Martin, J. E., Novik, L., Chakrabarti, B. K., Butman, B. T., Gall, J. G. D., King, C.R., Andrews, C. A., Sheets, R., Gomez, P. L., Mascola, J. R., Nabel, G. J. and Graham, B. (2006)."Phase 1 safety and immunogenicity evaluation of a multiclade HIV-1 candidate vaccine delivered by a replication-defective recombinant adenovirus vector". J. Infec. Dis., vol. 194 (12):1638-49.

Cellini, S., Fortini, C., Gallerani, E., Destro, F., Cofano, E. B., Caputo, A. and Gavioli, R. (2008)."Identification of new HIV-1 Gag-specific cytotoxic T lymphocyte responses in BALB/c mice". Virol. J., vol. 5:81.

Cerf-Bensussan, N., Jarry, A., Brousse, N., Lisowska-Grospierre, B., Guy-Grand, D. and Griscelli, C. (1987). "A monoclonal antibody (HML-1) defining a novel membrane molecule present on human intestinal lymphocytes". Eur. J. Immunol., vol. 17:1279-1285.

Cerwenka, A., Morgan, T. M.,Harmsen, A. G. and Dutton, R. W. (1999)."Migration kinetics and final destination of type 1 and type 2 CD8+ effector cells predict protection against pulmonary virus infection. J. Exp. Med., vol.189:423-434.

Chakrabarti, B. K., Maitra, R. K., Ma, X.-Z. and Kestler, H. W. (1996)."A candidate live inactivatable attenuated vaccine for AIDS." Proc. Natl. Acad. Sci. USA, vol. 93:9810-9815.

Challacombe, S. J. and Sweet, S. P. (2002)."Oral mucosal immunity and HIV infection: current status". Oral Dis. 8 (Suppl 2):55-62.

References

168

Chanzu, N. and Ondondo, B. (2014)."Induction of potent and long-lived antibody and cellular immune responses in the genitorectal mucosa could be the critical determinant of HIV vaccine efficacy". Review Article, Frontiers in Immunol., vol. 5 (202):1-21, Plebanski, M., Australia.

Chattha, K. S., Kandasamy, S., Vlasova, A. N. and Saif, L. J. (2013)."Vitamin A deficiency impairs adaptive B and T cell responses to a prototype monovalent attenuated human rotavirus vaccine and virulent human rotavirus challenge in a gnotobiotic piglet model". PLOS ONE, vol. 8 (12):e82966.

Cher, D. J. and Mosmann, T. R. (1987). "Two types of murine helper T cell clone. II. Delayed-type hepersensitivity is mediated by Th1 clone". J. Immunol., vol. 138:3688-3694.

Chin, J. and Mann, J. M. (1988)."The global patterns and prevalence of AIDS and HIV infection". AIDS, vol. 2 (Suppl 1):247-252.

Chiu, I. M., Yaniv, A., Dahlberg, J. E., Gazit, A., Skuntz, S. F., Tronick, S. R. and Stuart, A. A. (1985)."Nucleotide sequence evidence for relationship of AIDS retrovirus to lentiviruses". Nature, vol. 317 (6035):366-368.

Cho, M. W., Kim, Y. B., Lee, M. K., Gupta, K. C., Ross, W., Plishka, R., Buckler-White, A., Igarashi, T., Theodore, T., Byrum, R., Kemp, C., MontefIori, D. C. and Martin, M. A. (2001)."Polyvalent envelope glycoprotein vaccine elicits a broader neutralizing antibody response but is unable to provide sterilizing protection against heterologous simian/human immunodeficiency virus infection in pigtailed macaques". J.Virol., vol. 75 (5):2224-2234.

Chong, S.Y., Egan, M. A., Kutzler, M.A., Megati, S., Masood, A., Roopchard, V., Garcia-Hand, D., Montefiori, D. C., Quiroz, J., Rosati, M., Schadeck, E. B., Boyer, J. D., Pavlakis, G. N., Weiner, D. B., Sidhu, M., Eldridge, J. H. and Israel, Z. R. (2007)."Comparative ability of plasmid IL-12 and IL-15 to enhance cellular and humoral immune responses elicited by a SIVgag plasmid DNA vaccine and alter disease progression following SHIV89.6P challenge in rhesus macaques". Vaccine, vol. 25 (26):4967-4982.

Ciabattini, A., Pettini, E., Fiorino, F., Prota, G., Pozzi, G. and Medaglini, D. (2011). "Distribution of primed T cells and antigen-loaded antigen presenting cells following intranasal immunization in mice". PLoS ONE., vol. 6 (4):e19346.

Clavel, F. and Mammano, F. (2010)."Role of Gag in HIV Resistance to Protease Inhibitors". Viruses, vol. 2:1411-1426.

Clerici, M., Boasso, A., Ceroni, S., Luzzeri, D. and Maffeis, G. (2001)."HIV pathogenesis: An update". J. Intern. Med. India, vol. 4:23-31.

Clerici, M., Lucey, D., Berzofsky, J. A., Pinto, L. A., Wynn, T. A., Blatt, S. P., Dolan, M. J., Hendrix, C. W., Wolf, S. F. and Shearer, G. M. (1993)."Restoration of HIV-specific cell-mediated immune responses by interleukin-12 in vitro". Science, vol. 262 (5140):1721-1724.

Coffin, J., Hughes, S. and Varmus, H. (1997)."Historical introduction to the general properties of retrovirusess". Retroviruses. Cold Spring Harbor Laboratory Press, USA

Cohen, O. J. and Fauci, A. S. in Knipe, D.M. and Howley, P. M. (2001). "Pathogenesis and medical aspects of HIV-1 infection, Field Virology". Lippincott Williams & Wikins, Philadeliphia, USA.

References

169

Colonna, M., Trinchieri, G. and Liu, Y. (2004)."Plasmacytoid dendritic cells in immunity". Nature Immunol., vol. 5 (12):1219-1226.

Corbett, M., Bogers, W. M., Heeney, J. L., Gerber, S., Genin,, C., Didierlaurent, A., Oostermeijer, H., Dubbes, R., Braskamp, G., Lerondel, S., Gomez, C.E., Esteban, M., Wagner, R., Kondova, I., Mooij, P., Balla-Jhagihoorsingh, S., Beenhakker, N., Koopman, G., van der Burg, S., Kraehenbuhl, J. P. and Le Pape, A. (2008)."Aerosol immunization with NYVAC and MVA vectored vaccines is safe, simple, and immunogenic." Proc. Natl. Acad. Sci., vol.105 (6):2046-2051.

Crampin, A. C., Floyd, S., Glynn, J. R., Sibande, F., Mulawa, D., Nyondo, A., Broadbent, P., Bliss, L., Ngwira, B. and Fine, P. E. (2002)."Long-term follow-up of HIV-positive and HIV-negative individuals in rural Malawi". AIDS, vol.16 (11):1545-1550.

Cukalac, T., Moffat, J. M., Venturi, V., Davenport, M., Doherty, P. C., Turner, S. J. and Stambas, J. (2009)."Narrowed TCR diversity for immunised mice challenged with recombinant influenza A-HIV Env311-320 virus". Vaccine, vol. 27 (48): 6755-6761.

Currier, J., Visawapoka, U., Tovanabutra, S., Mason, C. J., Birx, D. L., McCutchan, F. E. and Cox, J. H. (2006)."CTL epitope distribution patterns in the Gag and Nef proteins of HIV-1 from subtype A infected subjects in Kenya: Use of multiple peptide sets increases the detectable breadth of the CTL response". BMC Immunology, vol. 7 (1): 8.

D'Aloja, P., Olivetta, E., Bona, R., Nappi, F., Pedacchia, D., Pugliese, K., Ferrari, G., Verani, P. and Federico, M. (1998)."Gag, vif, and nef Genes Contribute to the Homologous Viral Interference Induced by a Nonproducer Human Immunodeficiency Virus Type 1 (HIV-1) Variant: Identification of Novel HIV-1-Inhibiting Viral Protein Mutants". J. Virol., vol. 72 (5):4308-4319.

Das, A. T., Jeeninga, R. E. and Berkhout, B. (2010)."Possible applications for replicating HIV-1 vectors". HIV Ther., vol. 4 (3):361-369.

Datta, S. K. and Raz, E. (2005)."Induction of antigen cross-presentation by Toll-like receptors". Springer Semin. Immunopathol., vol. 26 (3):247-255.

Day, C. L., Kaufmann, D. E., Kiepiela, P., Brown, J. A., Moodley, E. S., Reddy, S., Mackey, E. W., Miller, J. D., Leslie, A. J., DePierres, C., Mncube, Z., Duraiswamy, J., Zhu, B., Eichbaum, Q., Altfeld, M., Wherry, E. J., Coovadia, H. M., Goulder, P. J., Klenerman, P., Ahmed, R., Freeman, G. J. and Walker, B. D. (2006)."PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression". Nature, vol. 443 (7109):350-354.

Delelis, O., Saïb, A. and Sonigo, P. (2003)."Biphasic DNA synthesis in spumaviruses". J. Virol., vol. 77 (14):8141-8146.

Deliyannis, G., Jackson, D. C., Ede, N. J., Zeng, W., Hourdakis, I., Sakabetis, E. and Brown, L. E. (2002)."Induction of long-term memory CD8 (+) T cells for recall of viral clearing responses against influenza virus". J. Virol., vol. 76:4212-4221.

DeNucci, C. C., Mitchell, J. S. and Shimizu, Y. (2009)."Integrin functionin T cell homing to lymphoid and non-lymphoid sites: getting there and staying there."Crit. Rev. Immunol., vol. 29 (2):87-109.

References

170

de Goede, A. L., Boers, P. H. M., Dekker, L. J. M., Osterhaus, A. D. M. E., Gruters, R. A. and Rimmelzwaan, G. F. (2009)."Characterization of recombinant influenza A virus as a vector for HIV-1 p17Gag". Vaccine, vol. 27 (42):5735-5739.

de Jong, M. D., Simmons, C. P., Thanh, T. T., Hien, V. M., Smith, G. J., Chau, T. N., Hoang, D. M., Chau, N. V., Khanh, T. H., Dong, V. C., Qui, P. T., Cam, B. V., Ha do, Q., Guan, Y., Peiris, J. S., Chinh, N. T., Hien, T. T. and Farrar, J. (2006)."Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia". Nat. Med., vol. 12(10):1203-1207.

de Jong, J. C., Palache, A. M., Beyer, W. E., Rimmelzwaan, G. F., Boon, A. C. and Osterhaus, A. D. (2003)."Haemagglutination-inhibiting antibody to influenza virus". Dev. Biologicals, vol. 115:63-73.

de Jong, J. C., Rimmelzwaan, G. F., Fouchier, R. A. and Osterhaus, A. D. (2000)."Influenza virus: A master of metamorphosis". J. Infect., vol. 40:218-228.

de Keersmaecker, B., Allard, S. D., Lacor, P., Schots, R., Thielemans, K. and Aerts, J. L. (2012)."Expansion of polyfunctional HIV-specific T cells upon stimulation with mRNA electroporated dendritic cells in the presence of immunomodulatory drugs". J Virol., vol. 86 (17):9351-9360.

Demberg, T. and Robert-Guroff, M. (2009)."Mucosal immunity and protection against HIV/SIV infection: strategies and challenges for vaccine design". Int Rev Immunol., vol. 28 (1):20-48.

de Rosa, S. D. and McElrath, M. J. (2008)."T cell responses generated by HIV vaccines in clinical trial"s. Curr. Opin. HIVAIDS, vol. 3 (3):375-379.

de Souza, A. P. D., Haut, L. H., Silva, R., Ferreira, S. I. A. C. P., Zanetti, C. R., Ertl, H. C. J. and Pinto, A. R. (2007)."Genital CD8+ T cell response to HIV-1 gag in mice immunized by mucosal routes with a recombinant simian adenovirus". Vaccine, vol. 25: 109-116.

Devito, C., Hinkula, J., Kaul, R., Kimani, J., Kiama, P., Lopalco, L., Barass, C., Piconi, S., Trabattoni, D., Bwayo, J. J., Plummer, F., Clerici, M. and Broliden K. (2002)."Cross-clade HIV-1-specific neutralizing IgA in mucosal and systemic compartments of HIV-1-exposed, persistently seronegative subjects." J. Acquir. Immune Defic. Syndr., vol. 30 (4):413-420.

de Wit, E., Spronken, M. I., Bestebroer, T. M., Rimmelzwaan, G. F., Osterhaus, A. D. and Fouchier, R. A. (2004). "Efficient generation and growth of influenza virus A/PR/8/34 fromeight cDNA fragments". Virus Res., vol. 103 (1–2):155-161.

Dieli, F., Poccia, F., Lipp, M., Sireci, G., Caccamo, N., Di Sano, C.and Salerno, A. (2003). "Differentiation of Effector/Memory Vδ2 T Cells and Migratory Routes in Lymph Nodes or Inflammatory Sites". J. Exp. Med., vol. 198 (3):391-397.

DiSanto, J. P. (1997)."Cytokines: Shared receptors, distinct functions". Curr. Biol., vol. 7 (7):R424-R426.

Dorn, J., Masciotra, S.,Yang, C.,Downing, R., Biryahwaho, B., Mastro, T. D., Nkengasong, J., Pieniazek, D., Rayfield, M. A., Hu,D. J. and Lal, R. B. (2000)."Analysis of Genetic Variability within the Immunodominant Epitopes of Envelope gp41 from Human Immunodeficiency Virus Type 1 (HIV-1) Group M and Its Impact on HIV-1 Antibody Detection". J. Clin. Microbiol., vol. 38 (2):773-780.

References

171

Draper, S. J. and Heeney, J. L. (2010)."Viruses as vaccine vectors for infectious diseases and cancer". Nat. Rev. Micro., vol. 8 (1):62-73.

Dubensky, T. W., Liu, M. A. and Ulmer, J. B. (2000)."Delivery systems for gene-based vaccines". Mol Med., vol. 6 (9):723-732.

Dudek, T. and Knipe, D. M. (2006). "Replication-defective viruses as vaccines and vaccine vectors". Virology, vol. 344 (1):230-239.

Duerr, A. (2010)."Update on mucosal HIV vaccine vectors."CurrOpp. HIV AIDS, vol. 5 (5):397-403.

Duerr, A., Wasserheit, J. N. and Corey, L. (2006). "HIV/AIDS: HIV Vaccines: New Frontiers in Vaccine Development". Clin. Infec. Dis., vol. 43 (4):500-511.

Duvall, M. G., Precopio, M. L., Ambrozak, D. A., Jaye, A., McMichael, A. J., Whittle, H. C., Roederer, M., Rowland-Jones, S. L. and Koup, R. A. (2008)."Polyfunctional T cell responses are a hallmark of HIV-2 infection". Eur. J. Immunol., vol 38:350-363.

Effros, R. B., Doherty, P. C., Gerhard, W. and Bennink, J. (1977). "Generation of both cross reactive and virus specific T-cell populations after immunization with serologically distinct influenza A viruses". J. Exp. Med., vol. 145 (3):557-568.

Edenborough, K. M., Gilbertson, B. P. and Brown, L. E. (2012)."A mouse model for the study of contact-dependent transmission of influenza A virus and the factors that govern transmissibility". J. Virol., vol. 86 (23):12544.

Edwards, B. H., Bansal, A., Sabbaj, S., Bakari, J., Mulligan, M. J., Goepfert, P. A. (2002). "Magnitude of functional CD8+ T-cell responses to the gag protein of human immunodeficiency virus type 1 correlates inversely with viral load in plasma". J. Virol. vol. 76 (5):2298-2305.

Eger, K.A. and Unutmaz, D. (2005).'The innate immune system and HIV pathogenesis". Curr. HIV/AIDS Rep., vol. 2 (1):10-5.

Egger, L. A., Kidambi, U., Cao, J., Van Riper, G., McCauley, E., Mumford, R. A., Amo, S., Lingham, R., Lanza, T., Lin, L. S., De Laszlo, S. E., Young, D. N., Kopka, I. E., Tong, S., Pikounis, B., Benson, E., Warwood, S., Bargatze, R. F., Hagmann, W. K., Schmidt, J. A. and Detmers, P. A. (2002)."Alpha(4)beta(7)/alpha(4)beta(1) dual integrin antagonists block alpha(4)beta(7)-dependent adhesion under shear flow". J. Pharmacol. Exp. Ther., vol. 302(1):153-162.

Ensoli, B., Cafaro, A., Caputo, A., Fiorelli, V., Ensoli, F., Gavioli, R., Ferrantelli, F., Cara, A., Titti, F. and Magnani, M. (2005). "Vaccines based on the native HIV Tat protein and on the combination of Tat and the structural HIV protein variant DeltaV2 Env". Microbes Infec., vol. 7:1392-1399.

Erle, D. J., Briskin, M. J., Butcher, E. C., Garcia-Pardo, A., Lazarovits, A. I. and Tidswell, M. (1994)."Expression and function of the MAdCAM-1 receptor, integrin α4β7, on human leukocytes". J. Immunol., vol. 153:517-528.

Esparaza, R. and Van Regenmortel, M. H. V. (2014)."More surprises in the development of an HIV vaccine". General Commentary Article, Front. Immunol., Francesca Chiodi, Karolinska Institutet, Sweden.

Esparaza, R. (2013)."An HIV vaccine: how and when?". Bull. World Health Org., vol. 79 (12): 1133-1137.

References

172

Epstein, S. L., Lo, C. Y., Misplon, J. A. and Bennink, J. R. (1998). "Mechanism of protective immunity against influenza virus infection in mice without antibodies". J. Immunol., vol. 160:322-327.

Eslami, Ameneh (2005)."The role of integrins in wound healing". S. C. Q (6). CD45 in the Stem Cell Niche. R&D systems. Verkkodokumentti.

Esquivel-Pérez, R. and Moreno-Fierros, L. (2005)."Mucosal and systemic adjuvant effects of cholera toxin and cry1Ac protoxin on the specific antibody response to HIV-1 C4/V3 peptides are different and depend on the antigen co-administered". Viral Immunol., vol. 18 (4):695-708.

Evans, D. T., Chen, L. M., Gillis, J., Lin, K. C., Harty, B., Mazzara, G. P., Donis, R. O., Mansfield, K. G., Lifson, J. D., Desrosiers, R. C., Galan, J. E. and Johnson, R. P. (2003)."Mucosal priming of simian immunodeficiency virus-specific cytotoxic T-lymphocyte responses in rhesus macaques by the Salmonella type III secretion antigen delivery system". J. Virol., vol. 77(4):2400-2409.

Excler, J. L., Rida, W., Priddy, F., Fast, P. and Koff, W. (2007)."A strategy for accelerating the development of preventive AIDS vaccines". AIDS., vol. 21 (17):2259-2263.

Ezekowitz, R. A. B., Kuhlman, M., Groopman, J. E. I. and Byrn, R. A. (1989). "A human serum mannose-binding protein inhibits in vitro infection by the human immunodeficiency virus". J. Exp. Med., vol. 169:185-96.

Faber, M, Dietzschold, B. and Li, J. (2009)."Immunogenicity and safety of recombinant rabies viruses used for oral vaccination of stray dogs and wildlife". Zoonoses Public Health., vol. 56 (6-7):262-269.

Fahey, J. V., Schaefer, T. M., Channon, J. Y. and Wira, C. R. (2005)."Secretion of cytokines and chemokines by polarized human epithelial cells from the female reproductive tract". Hum Reprod., vol. 20 (6):1439-1446.

Faul, E. J., Aye, P. P., Papaneri, A. B., Pahar, B., McGettigan, J. P., Schiro, F., Chervoneva, I., Montefiori, D. C., Lackner, A. A. and Schnell, M. J. (2009)."Rabies virus-based vaccines elicit neutralizing antibodies, poly-functional CD8+ T cell, and protect rhesus macaques from AIDS-like disease after SIV(mac251) challenge". Vaccine, vol. 28 (2):299-308.

Ferko, B., Stasakova, J., Sereinig, S., Romanova, J., Katinger, D., Niebler, B., Katinger, H. and Egorov, A. (2001)."Hyperattenuated recombinant influenza A virus nonstructural-protein-encoding vectors induce human immunodeficiency virus type 1 Nef-specific systemic and mucosal immune responses in mice". J. Virol., vol. 75 (190) 8899-8908.

Ferko, B., Katinger, D., Grassauer, A., Egorov, A., Romanova, J., Niebler, B., Katinger, H. and Muster, T. (1998)."Chimeric influenza virus replicating predominantly in the murine upper respiratory tract induces local immune responses against human immunodeficiency virus type 1 in the genital tract". J. Infec. Dis., vol. 178 (5):1359-1368.

Fernandez Gonzalez, S., Jayasekera, J. P. and Carroll, M. C. (2008). "Complement and natural antibody are required in the long-term memory response to influenza virus". Vaccine, vol. 26: I86-I93.

Fesq, H., Bacher, M., Nain, M. and Gemsa, D. (1994)."Programmed cell death (apoptosis) in human monocytes infected by influenza A virus". Immunobiol., vol.190 (1-2):175-182.

References

173

Fiorentini, S., Giagulli, C., Caccuri, F., Magiera, A. K. and Caruso, A. (2010.)."HIV-1 matrix protein p17:a candidate antigen for therapeutic vaccines against AIDS". Pharmacol. Ther., vol. 128 (3):433-444.

Fitch, P. M., Roghanian, A., Howie, S. E. M. and Sallenave, J. M. (2006)."Human neutrophil elastase inhibitors in innate and adaptive immunity". Biochem. Soci. Transactions, vol. 34 (2):279-282.

Fitzgerald-Bocarsly, P. and Jacobs, E. S. (2010)."Plasmacytoid dendritic cells in HIV infection: striking a delicate balance". J. Leuk. Biol., vol. 87 (4):609-620.

Fitzgerald, J. C., Gao, G. P., Reyes-Sandoval, A., Pavlakis, G. N., Xiang, Z. Q., Wlazlo, A. P., Giles-Davis, W., Wilson, J. M.and Ertl, H. C. J. (2003). "A Simian replication-defective adenoviral recombinant vaccine to HIV-1 gag". J. Immunol., vol. 170 (3):1416-1422.

Flynn, N. M., Forthal, D. N., Harro, C. D., Judson, F. N., Mayer, K. H. and Para, M. F. (2005)."Placebo-controlled phase 3 trial of a recombinant glycoprotein 120 vaccine to prevent HIV-1 infection". J. Infec. Dis., vol.191 (5):654-665.

Flynn, K. J., Belz, G. T., Altman, J. D., Ahmed, R., Woodland, D. L. and Doherty, P. C. (1998)."Virus-specific CD8+ T cells in primary and secondary influenza pneumonia". Immunity, vol. 8 (6):683-691.

Fodor, E., Devenish, L., Engelhardt, O. G., Palese, P., Brownlee, G. G. and García-Sastre, A. (1999)."Rescue of influenza A virus from recombinant DNA". J. Virol., vol. 73 (11):9679-9682.

Fortna, A. K. Y., MacLaren, E., Marshall, K. and Hahn, G. (2004)."Regulating the regulators: immune system regulators are highly susceptible to HIV infection". PLoS Biol., vol. 2 (7):0882.

Foster, J. and Garcia, J. V. (2008)."HIV-1 Nef: at the crossroads". Retrovirology, vol. 5 (1):84.

Fouchier, R. A., Munster, V., Wallensten, A., Bestebroer, T. M., Herfst, S., Smith, D.,Rimmelzwaan, G. F., Olsen, B. and Osterhaus, A. D. ( 2005). "Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls". J. Virol., vol. 79:2814-2822.

Fousteri, G., Dave, A., Juntti, T.and von Herrath, M. (2009). "CD103 is dispensable for anti-viral immunity and autoimmunity in a mouse model of virally-induced autoimmune diabetes". J. Autoimm., vol. 32 (1):70-77.

Fowke, K. R., Nagelkerke, N. J., Kimani, J., Simonsen, J. N., Anzala, A. O., Bwayo, J. J., MacDonald, K. S., Ngugi, E. N. and Plummer, F. A. (1996)."Resistance to HIV-1 infection among persistently seronegative prostitutes in Nairobi, Kenya". Lancet, vol. 48(9038):1347-1351.

Frankel, A. D. and Young, J. A. T. (1998)."HIV-1: fifteen proteins and an RNA." Annl. Rev. Biochemistry, vol. 67 (1):1-25.

Frey, S. E. (1999)."HIV vaccines". Infec. Dis. Clin. North America, vol. 13 (1): 95-112.

Froelich, C. J., Dixit, V. M. and Yang, X. H. (1998)."Lymphocyte granule-mediated apoptosis: matters of viral mimicry and deadly proteases". Immunology Today, vol. 19 (1):30-36.

Furci, L., Sironi, F., Tolazzi, M., Vassena, L. and Lusso, P. (2007). "α-defensins block the early steps of HIV-1 infection: interference with the binding of gp120 to CD4". Blood, vol. 109 (7): 2928-2935.

References

174

Gallichan, W.S. and Rosenthal, K. L. (1996)."Long-lived cytotoxic T lymphocyte memoryin mucosal tissues after mucosal but not systemic immunization". J. Exp. Med., vol. 184 (5):1879-1890.

Gallo, R., Wong-Staal, F., Montagnier L., Haseltine W. A., Yoshida M. (1988)."HIV/HTLV gene nomenclature". Nature, vol. 333 (6173):504-504.

Galvin, J., Spaeny-Dekking, L., Wang, B., Seth, P., Hack, C. E. and Froelich, C. (1999)."Apoptosis induced by granzyme B lycosaminoglycan complexes:implications for granule-mediated apoptosis in vivo". J. Immunol., vol. 162 (9):5345-5350.

Gambaryan, A. S., Robertson, J. S and Matrosovich, M. N. (1999)."Effects of egg-adaptation on the receptor-binding properties of human influenza A and B viruses". Virology, vol. 258:232-239.

Garcia-Sastre, A. and Palese. P. (1995). "Influenza virus vectors". Biologicals, vol. 23:171-178.

Gardner, M. B. and Luciw, P. A. (2008)."Macaque models of human infectious disease". ILAR J., vol. 49 (2):220-255.

Garten, R. J., Davis, C. T., Russell, C. A., Shu, B., Lindstrom, S., Balish, A., Sessions, W. M., Xu, X., Skepner, E. and Deyde, V. (2009)."Antigenic and genetic characteristics of swine-origin 2009 A(H1N1) influenza viruses circulating in humans". Science, vol. 325:197-201.

Garulli, B., Meola, M., Stillitano, M. G., Kawaoka, Y., Castrucci, M. R. (2007)."Efficient vagina-to-lower respiratory tract immune trafficking in a murine model of influenza A virus infection".Virology, vol. 361 (2):274-82.

Garulli, B., Kawaoka, Y. and Castrucci, M. R. (2004)."Mucosal and systemic immune responses to a human immunodeficiency virus type 1 epitope induced upon vaginal infection with a recombinant influenza A virus"'. J. Virol., vol. 78 (2):1020-1025.

Gaschen, B., Taylor, J., Yusim, K., Foley, B., Gao, F., Lang, D., Novitsky, V., Haynes, B., Hahn, B. H., Bhattacharya, T. and Korber, B. (2002)."Diversity considerations in HIV-1 vaccine selection".Science., vol. 296 (5577):2354-2360.

Gaudreault, E. and Gosselin, J. (2009)."Leukotriene B4 potentiates CpG signaling for enhanced cytokine secretion by human leukocytes". J. Immunol.,vol. 183 (4):2650-2658.

Gaufin, T., Ribeiro, R. M., Gautam, R., Dufour, J., Mandell, D., Apetrei, C. and Pandrea, I. (2010)."Experimental depletion of CD8+ cells in acutely SIVagm-infected african green monkeys results in increased viral replication".Retrovirol., vol. 7:42.

Gavioli, R., Cellini, S., Castaldello, A., Voltan, R., Gallerani, E., Gagliardoni, F., Fortini, C., Cofano, E., Triulzi, C., Cafaro, A., Srivastava, I., Barnett, S., Caputo, A. and Ensoli, B. (2008)."The Tat protein broadens T cell responses directed to the HIV-1 antigens Gag and Env: Implications for the design of new vaccination strategies against AIDS". Vaccine, vol. 30:727-737.

Gavioli, R., Gallerani, E., Fortini, C., Fabris, M., Bottoni, A., Canella, A., Bonaccorsi, A., Marastoni, M., Micheletti, F., Cafaro, A., Rimessi, P., Caputo, A. and Ensoli, B . (2004)."HIV-1 Tat protein modulates the generation of cytotoxic T cell epitopes by modifying proteasome composition and enzymatic activity". J. Immunol., vol. 173 (6):3838-3843.

References

175

Gebhardt,T.,Wakim, L. M., Eidsmo, L., Reading, P. C., Heath, W. R., and Carbone, F. R. (2009) "Memory T cells in nonlymphoid tissue that provide enhanced local immunity during infection with herpes simplexvirus". Nat. Immunol., vol. 10:524-530.

Gerhard, W., Mozdzanowska, K., Furchner, M., Washko, G. and Maiese, K. (1997)."Role of the B-cell response in recovery of mice from primary influenza virus infection". Immunol. Rev., vol. 159: 95-103.

Gherardi, M. M. and Esteban, M. (2005)."Recombinant poxviruses as mucosal vaccine vectors". J. General Virol., vol. 86 (11):2925-2936.

Gherardi, M. M., Pérez-Jiménez, E., Najera, J. L. and Esteban, M. (2004)."Induction of HIV immunity in the genital tract after intranasal delivery of a MVA vector: enhanced immunogenicity after DNA prime-modified vaccinia virus ankara boost immunization schedule". J. Immunol., vol. 172 (10):6209-6220.

Gherardi, M. M., Nájera, J. L., Pérez-Jiménez, E., Guerra, S., García-Sastre, A. and Esteban, M. (2003).'Prime-boost immunization schedules based on influenza virus and vaccinia virus vectors potentiate cellular immune responses against human immunodeficiency virus Env protein systemically and in the genitorectal draining lymph nodes". J. Virol., vol. 77 (12):7048-57.

Gifford, R. J., Katzourakis, A., Tristem, M., Pybus, O. G., Winters, M. and Shafer, R. W. (2008)."A trastional endogenous lentivirus from the genome of a basal primate and implications for lentivrus evolution". Proc. Natl. Acad. Sci., vol. 105 (51):20362-20367.

Gill, N., Paltser, G., and Ashkar, A.A. (2009)."Interleukin-15 expression affects homeostasis and function of B cells through NK cell-derived interferon-gamma". Cell Immunol., vol. 258 (1):59-64.

Gilbert, C., Maxfield, D. G., Goodman, S. M. and Feschotte, C. (2009)."Parallel germline infiltration of a lentivirus in two malagasy lemurs". PLoS Genetics, vol. 5 (3):1-12.

Gillespie, G. M., Pinheiro, S., Sayeid-Al-Jamee, M., Alabi, A., Kaye, S., Sabally, S., Sarge-Njie, R., Njai, H., Joof, K., Jaye, A., Whittle, H., Rowland-Jones, S. and Dorrell, L. (2005)."CD8(+) T cell responses to human immunodeficiency viruses type 2 (HIV-2) and type 1 (HIV-1) gag proteins are distinguishable by magnitude and breadth but not cellular phenotype". Eur. J. Immunol., vol. 35:1445-1453.

Girard, M. P. (2006)."A review of vaccine research and development: meningococcal disease". Vaccine, vol. 24:4692-4700.

Girard, P. R. and Nerem, R. M. (1995)."Shear stress modulates endothelial cell morphology and F-actin organization through the regulation of focal adhesion-associated proteins". J. Cell Physiol., vol. 163:179-193.

Glathe, H., Bigl, S. and Grosche, A. (1993)."Comparison of humoral immune responses to trivalent influenza split vaccine in young, middle-aged and elderly people". Vaccine, vol. 11 (7):702-705.

Gnjatic, S., Sawhney, N. B.and Bhardwaj, N. (2010).'Toll-like receptor agonists: are they good adjuvants?". The Cancer J., vol. 16 (4):382-391.

References

176

Go, E. P., Chang, Q., Liao, H., Sutherland, L. L., Alam, S. M., Haynes, B. F. and Desaire, H. (2009)."Glycosylation site-specific analysis of clade C HIV-1 envelope proteins". J. Proteome Res., vol. 8 (9):4231-4242.

Goebel, F. D., Mannhaltera, J. W., Belsheb, R. B., Eiblc, M. M., Grobd, P. J., Gruttolae, V., Griffithsf, P. D., Erfleg, V., Kunschaka, M. and Engla, W. (1999)."Recombinant gp160 as a therapeutic vaccine for HIV-infection: results of a large randomized, controlled trial". AIDS, vol. 13:1461-1468.

Goepfert, P. A., Tomaras, G. D., Horton, H., Montefiori, D., Ferrari, G., Deers, M., Voss, G., Koutsoukos, M., Pedneault, L., Vandepapeliere, P., McElrath, M. J., Spearman, P., Fuchs, J. D., Koblin, B. A., Blattner, W. A., Frey, S., Baden, L. R., Harro, C. and Evans, T. (2007)."Durable HIV-1 antibody and T-cell responses elicited by an adjuvanted multi-protein recombinant vaccine in uninfected human volunteers". Vaccine, vol. 25:510-518.

Gomme, E., Faul, J., Flomenberg, P., McGettigan, J. P. and Schnell, M. J. (2010). "Characterization of a single-cycle rabies virus-based vaccine vector". J. Virol., vol. 84 (6):2820-2831.

Gómez, C. E., Nájera, J. L.Jilmenez, V., Bieler, K., Wild, J., Kostic, L., Heidari, S., Chen, M., Frachette, M. J. Pantaleo, G., Wolf, H., Liljestorm, P., Wagner, R. and Esteban, M. (2007)."Generation and immunogenicity of novel HIV/AIDS vaccine candidates targeting HIV-1 Env/Gag-Pol-Nef antigens of clade C". Vaccine, vol. 25 (11):1969-1992.

Gómez, A. M., Smaill, F. M. and Rosenthal, K. L. (1994)."Inhibition of HIV replication by CD8+ T cells correlates with CD4 counts and clinical stage of disease". Clin. Exp. Immunol., vol. 97: 68-75.

Gonzalo, R. M., Rodríguez, D., García-Sastre, A., Rodríguez, J. R., Palese, P. and Esteban, M. (1999)."Enhanced CD8+ T cell response to HIV-1 env by combined immunization with influenza and vaccinia virus recombinants". Vaccine, vol. 17: 887-892.

Goonetilleke, N., Liu, M. K. P., Salazar-Gonzalez , J. F., Ferrari, G., Giorgi, E., Ganusov, V.V. Keele, B. F., Learn, G. H., Turnbull, E. L., Salazar, M. G., Weinhold, K. J. and Moore, S. (2009)."The first T cell response to transmitted/founder virus contributes to the control of acute viremia in HIV-1 infection". J. Exp. Med., vol. 206 (6):1253-1272.

Gorfu, G., Rivera-Nieves, J. and Ley, K. (2009)."Role of beta7 integrins in intestinal lymphocyte homing and retention". Curr. Mol. Med., vol. 9 (7):836-850.

Gorry, P. R. and Ancuta, P. (2011)."Coreceptors and HIV-1 pathogenesis".Curr HIV/AIDS Rep., vol. 8(1):45-53.

Gorry, P. R.,McPhee, D. A.,Verity, E., Dyer, W. B.,Wesselingh, S. L., Learmont, J., Sullivan, J. S., Roche, M., Zaunders, J. J., Gabuzda, D., Crowe, S. M., Mills, J., Lewin, S. R., Brew, B. J., Cunningham, A. L. and Churchill, M. J. (2007)."Pathogenicity and immunogenicity of attenuated, nef-deleted HIV-1 strains in vivo". Retrovirology, vol. 4 (1):66.

Gottlinger, H. G., Dorfman, T., Sodroski, J. G. and Haseltine, W. A. (1991)."Effect of mutations affecting the p6 gag protein on human immunodeficiency virus particle release". Proc. Natl. Acad. Sci. USA, vol. 88: 3195-3199.

References

177

Göttlinger, H. G., Sodroski, J. G. and Haseltine, W. A. (1989)."Role of capsid precursor processing and myristoylation in morphogenesis and infectivity of human immunodeficiency virus type 1". Proc. Natl. Acad. Sci. USA, vol. 86 (15):5781-5785.

Grabstein, K. H., Eisenman, J., Shanebeck, K., Rauch, C., Srinivasan, S., Fung, V., Beers, C., Richardson, J., Schoenborn, M. A. and Ahdieh, M. (1994)."Cloning of a T cell growth factor that interacts with the b chain of the interleukin-2 receptor". Science, vol. 264: 965-968.

Graham, B. S., Koup, R. A., Roederer, M., Bailer, R. T., Enama, M. E., Moodie, Z., Martin, J. E., McCluskey, M. M. and Bimal K. (2006)."Phase 1 safety and immunogenicity evaluation of a multiclade HIV-1 DNA candidate vaccine". J. Infect. Dis., vol. 194 (12):1650-1660.

Graham, M. B. and Braciale, T. J. ( 1997). "Resistance to and recovery from lethal influenza virus infection in B lymphocyte-deficient mice". J. Exp. Med., vol. 186:2063-2068.

Grebe, K. M.,Yewdell, J. W. and Bennink, J. R. (2008)."Heterosubtypic immunity to influenza A virus: Where do we stand?". Microb. Infect., vol. 10:1024-1029.

Greenough, T. C., Babcock, G. J., Roberts, A., Hernandez, H. J., Thomas J., Coccia, J. A., Graziano, R. F., Srinivasan, M., Lowy, I., Finberg, R. W., Subbarao, K., Vogel, L., Somasundaran, M., Luzuriaga, K., Sullivan, J. L. and Ambrosino, D. M. (2005). "'Development and characterization of a severe acute respiratory syndrome-associated coronavirus-neutralizing human monoclonal antibody that provides effective immunoprophylaxis in mice". J. Infec. Dis., vol. 191 (4):507-514.

Grossman, Z., Meier-Schellersheim, M., Sousa, A. E., Victorino, R. M. M. and Paul, W. E. (2002)."CD4+ T-cell depletion in HIV infection: Are we closer to understanding the cause?". Nat. Med., vol. 8 (4):319-323.

Guan,Y., Whitney, J. B., Diallo, K. and Wainberg, M. A. (2000)."Leader sequences downstream of the primer binding site are important for efficient replication of simian immunodeficiency virus". J. Virol., vol. 74:8854-8860.

Guan, Y., Whitney, J. B., Detorio, M. and Wainberg, M. A. (2001). "Construction and in vitro properties of a series of attenuated simian immunodeficiency viruses with all accessory genes deleted". J. Virol., vol.75 (9):4056-4067.

Guatelli, J. (2010)."How innate immunity can inhibit the release of HIV-1 from infected cells". N. Engl. J. Med., vol. 362 (6):553-554.

Gudas, L. J. (2012)."Emerging roles for retinoids in regeneration and differentiation in normal and disease states', Biochimica et Biophysica Acta (BBA)". Molec. Cell Biol. Lipids, vol. 1821 (1):213-21.

Guerbois, M., Moris, A., Combredet, C., Najburg,V., Ruffiéa, C., Févriera, M., Cayet, N., Brandler, S., Schwartz, O. and Tangy, F. (2009)."Live attenuated measles vaccine expressing HIV-1 Gag virus like particles covered with gp160[Delta]V1V2 is strongly immunogenic". Virology, vol. 388 (1):191-203.

Guermonprez, P., Valladeau, J., Zitvogel, L., Thery, C. and Amigorena, S. (2002)."Antigen presentation and T cell stimulation by dendritic cells". Annu. Rev. Immunol., vol. 20: 621-667.

Gulzar, N. and Copeland, K. F. (2004)."CD8+ T-cells: function and response to HIV infection". Curr. HIV Res., vol. 2 (1):23-37.

References

178

Gupta, S., Janani, R., Bin, Q., Luciw, P., Greer, C., Perri, S., Legg, H., Donnelly, J., Barnett, S., O'Hagan, D., Polo, J. M.and Vajdy, M. (2005)."Characterization of human immunodeficiency virus Gag-specific gamma interferon-expressing cells following protective mucosal immunization with alphavirus replicon particles". J. Virol., vol. 79 (11):7135-7145.

Gupta K., Hudgens M., Corey L., McElrath, M. J., Weinhold, K., Montefiori, D. C., Gorse, G. J., Frey, S. E., Keefer, M.C., Evans, T. G., Dolin, R., Schwartz, D. H., Harro, C., Graham, B., Spearman, P. W., Mulligan, M., Goepfert, P. and AIDS Vaccine Evaluation Group (2002)."Safety and immunogenicity of a high-titered canarypox vaccine in combination with rgp120 in a diverse population of HIV-1-uninfected adults: AIDS Vaccine Evaluation Group Protocol 022A". J. Acquir. Immun. Defic. Syndr., vol. 29:254-261.

Guthrie, A. J., Quan, M., Lourens, C. W., Audonnet, J. C., Minke, J. M., Yao, J., He, L., Nordgren, R., Gaedner, I. A. and Maclachlan, N J. (2009)."Protective immunization of horses with a recombinant canarypox virus vectored vaccine co-expressing genes encoding the outer capsid proteins of African horse sickness virus". Vaccine, vol. 27 (33):4434-4438.

Haase, A. (2010)."Targeting early infection to prevent HIV-1 mucosal transmission."Nature, vol. 464 (7286): 217-223.

Haile-Selassie, H. T., Townsend, C. L. and Tookey, P. A. (2011)."Use of neonatal post-exposure prophylaxis for prevention of mother-to-child HIV transmission in the UK and Ireland, 2001–2008". HIV Med., vol. 12 (7):422-427.

Halperin, S. A., Smith, B., Mabrouk, T., Germain, M., Trépanier, P., Hassell, T., Treanor, J., Gauthier, R. and Mills, E. L. (2002)."Safety and immunogenicity of a trivalent, inactivated, mammalian cell culture-derived influenza vaccine in healthy adults, seniors, and children". Vaccine, vol. 20:1240-1247.

Hansen, S. G., Vieville, C., Whizin, N., Coyne-Johnson, L., Siess, D. C., Drummond, D. D., Legasse, A. W., Axthelm, M. K., Oswald, K., Trubey, C. M., Piatak, M., Lifson, J. D., Nelson, J. A., Jarvis, M. A. and Picker, L. J. (2009)."Effector memory T cell responses are associated with protection of rhesus monkeys from mucosal simian immunodeficiency virus challenge". Nat. Med., vol. 15(3):293-299.

Harari, A., Bart, P.A., Stöhr, W., Tapia, G., Garcia, M., Medjitna-Rais, E., Burnet, S., Cellerai, C., Erlwein, O., Barber, T., Moog, C., Liljestrom, P., Wagner, R., Wolf, H., Kraehenbuhl, J. P., Esteban, M., Heeney, J., Frachette, M. J., Tartaglia, J., McCormack, S., Babiker, A., Weber, J. and Pantaleo, G. (2008)."An HIV-1 clade C DNA prime, NYVAC boost vaccine regimen induces reliable, polyfunctional, and long-lasting T cell responses". J. Exp. Med., vol. 205 (1):63-77.

Harari, A., Cellerai, C., Enders, F. B., Köstler, J., Codarri, L., Tapia, G., Boyman, O., Castro, E., Gaudieri, S., James, I., John, M. Wagner, R., Mallal, S. and Pantaleo, G. (2007). "Skewed association of polyfunctional antigen-specific CD8 T cell populations with HLA-B genotype". Proc. Nat. Acad. Sci. USA, vol. 104 (41):16233-16238.

Hardy, W. D. (2009)."The feline immunodeficiency virus (FIV), the FIV test patent has expired, History of retroviruses", Zuckerman, E. E., Natl. Vet. Lab., Newsletter 8 (2), Franklin Lakes, USA.

References

179

Hardy, A. W., Graham, D. R. Shearer, G. M. and Herbeuval, J. P. (2007)."HIV turns plasmacytoid dendritic cells (pDC) into TRAIL-expressing killer pDC and down-regulates HIV coreceptors by Toll-like receptor 7-induced IFN-α". Proc. Natl. Acad. Sci., vol. 104 (44):17453-17458.

Hashimoto, G., Wright, P. F. and Karzon, D. T. (1983). "Antibody-dependent cell-mediated cytotoxicity against influenza virus-infected cells". J. Infect. Dis., vol. 148:785-794.

Hayakawa, T., Miyazaki, T., Misumi, Y., Kobayashi, M. and Fujisawa, Y. (1992). "Myristoylation-dependent membrane targeting and release of the HTLV-I Gag precursor, Pr53gag, in yeast". Gene, vol. 119 (2):273-277.

Haynes, B. F.and Shattock, R. J. (2008)."Critical issues in mucosal immunity for HIV-1 vaccine development". Official J. Acad. Pediateric Assoc., vol. 122 (1):3-9.

Hemann, E. A., Kang, S.-M. and Legge, K. L. (2013)."Protective CD8 T cell-mediated immunity against influenza A virus infection following Influenza Virus -like Particle Vaccination". J. Immunol., vol. 191:2486-2494.

Herfst, S., Schrauwen, E. J. A., Linster, M., Chutinimitkul, S., de Wit, E., Munster, V. J., Sorrell, E. M., Bestebroer, T. M., Burke, D. F. and Smith, D. J. (2012)."Airborne transmission of influenza A/H5N1 virus between ferrets". Science, vol. 336:1534-1541.

Hersperger, A. R., Pereyra, F.,Nason, M., Demers, K., Sheth, P., Shin, L. Y., Kovacs, C. M., Rodriguez, B., Sieg, S. F., Teixeira-Johnson, L., Gudonis, D., Goepfert, P. A., Lederman, M. M., Frank, I., Makedonas, G., Kaul, R., Walker, B. D. and Bett, M. R. (2010)."Perforin expression directly ex vivo by HIV-specific CD8+ T cells is a correlate of HIV elite control". PLoS Pathog., vol 6 (5):e1000917.

Hicks, J. T., Ennis, F. A., Kim, E. and Verbontiz, M. (1978)."The importance of an intact complement pathway in recovery from a primary viral infection: influenza in decomplemented and in C5-deficient mice". J. Immunol., vol. 121:1437-1445.

Hillaire, M. L., Osterhaus, A. D. and Rimmelzwaan, G. F. (2011)."Induction of virus-specific cytotoxic T lymphocytes as a basis for the development of broadly protective influenza vaccines". J Biomed Biotechnol., vol. 2011:939860.

Hirsch, V., Dapolito, G., Goeken, R. and Campbell, B. J. (1995)."Phylogeny and natural history of the primate lentiviruses, SIV and HIV". Curr Opin Genet Dev., vol. 5 (6):798-806.

Ho, Y. S., Abecasis, A. B., Theys, K., Deforche, K., Dwyer, D. E., Charleston, M., Vandamme,A. M. and Saksena, N. K. (2008)."HIV-1 gp120 N-linked glycosylation differs between plasma and leukocyte compartments". Virol. J., vol. 5:14.

Hoffmann, E., Mahmood, K., Yang, C. F., Webster, R. G., Greenberg, H. B. and Kemble, G. (2002)."Rescue of influenza B virus from eight plasmids". PNAS, USA, vol. 99 (17):11411-11416.

Hoffmann, E., Neumann, G., Kawaoka,Y., Hobom, G. and Webster, R. G. (2000). A DNA transfection system for generation of influenza A virus from eight plasmids. Proc. Natl. Acad. Sci., vol. 7 (11): 6108–6113.

Hogg, N., Laschinger, M., Giles K. and McDowall, A. (2003)."T-cell integrins: more than just sticking points". J. Cell Sci., vol. 116:4695-4705.

Holmgren, J. and Czerkinsky, C. (2005)."Mucosal immunity and vaccines." Nat. Med., vol. 11 (4 Suppl):S45-53.

References

180

Holzmann, B., McIntyre, B. W. and Weissman, I. L. (1989). "Identification of a murine Peyer's patch specific lymphocyte homing receptor as an integrin molecule with an α chain homologous to human VLA-4α". Cell, vol. 56 (1):37-46.

Howie, S., Ramage, R. and Hewson, T. (2000)."Innate immune system damage in human immunodeficiency virus type 1 infection. implications for acquired immunity and vaccine design". Am. J. Respir. Crit. Care Med., vol. 162 (4):S141-145.

Howoroft, T. K., Strebel, K., Martin, M. and Singer, D. S. (1993)."Repression of MHC class I gene promoter activity by two-exon Tat of HIV". Science, vol. 260:1320-1322.

Huang, X., Barchi, J. J., Lung, F. D. T., Roller, P. P., Nara, P. L., Muschik, J. and Garrity, R. R. (1997). "Glycosylation affects both the three-dimensional structure and antibody binding properties of the HIV-1IIIB GP120 peptide RP135". Biochemistry, vol. 36 (36):10846-56.

Hur, E. M., Patel, S. N., Shimizu, S., Rao, D. S., Gnanapragasam, P. N. P., An, D. S., Yang, L. and Baltimore, D. (2012)."Inhibitory effect of HIV-specific neutralizing IgA on mucosal transmission of HIV in humanized mice". Blood, vol. 120 (23).

Husseini, R. H., Sweet, C., Collie, M. H. and Smith, H. (1982)."The relation of interferon and nonspecific inhibitors to virus levels in nasal washes of ferrets infected with influenza viruses of differing virulence". Br. J. Exp. Pathol., vol. 62:87-93.

Hwang, S. D., Shin, J. S., Ku, K. B.; Kim, H. S., Cho, S. W. and Seo, S. H. (2010). "Protection of pregnant mice, fetuses and neonates from lethality of H5N1 influenza viruses by maternal vaccination". Vaccine, vol. 28: 2957-2964.

Hwang, J. S., Yamada, K., Honda, A., Nakade, K. and Ishihama, A. (2000)."Expression of functional influenza virus RNA polymerase in the methylotrophic yeast pichia pastoris". J. Virol., vol. 74 (9):4074-4084.

Im, E. J. and Hanke, T. (2004)."MVA as a vector for vaccines against HIV-1". Expert. Rev. Vaccines, vol. 3 (4s1):S89-S97.

Izumi, Y., Ami, Y., Matsuo, K., Someya, K., Sata, T., Yamamoto, N. and Honda, M. (2003). "Intravenous inoculation of replication-deficient recombinant vaccinia virus DIs expressing simian immunodeficiency virus Gag controls highly pathogenic simian-human immunodeficiency virus in monkeys". J. Virol., vol. 77 (24):13248-13256.

Jagger, B.W., Wise, H.M., Kash, J. C., Walters, K. A., Wills, N.M., Xiao, Y. L., Dunfee, R. L., Schwartzman, L. M., Ozinsky, A. and Bell, G. L. (2012)."An overlapping protein-coding region in influenza a virus segment 3 modulates the host response". Science, vol. 337:199-204.

Jakasa, I., Koster, E. S., Calkoen, F., McLean, W. H. I., Campbell, L. E., Bos, J. D., Verberk, M. M. and Kezic, S. (2010)."Skin barrier function in healthy subjects and patients with atopic dermatitis in relation to filaggrin loss-of-function mutations". J. Invest. Dermatol., vol. 131:540-542.

Jackson, R. J., Worley, M., Trivedi, S. and Ranasinghe, C. (2014)."Novel HIV IL-4R antagonist vaccine strategy can induce both high avidity CD8 T and B cell immunity with greater protective efficacy". Vaccine, vol. 32 (43):5703-5714.

Jalilian, B., Omar, A., Bejo, M., Alitheen, N., Rasoli, M.and Matsumoto, S. (2010). "Development of avian influenza virus H5 DNA vaccine and MDP-1 gene of Mycobacterium bovis as genetic adjuvant". Genetic Vaccines Therapy, vol. 8 (1):4.

References

181

Jayasekera, J. P., Moseman, E. A. and Carroll, M. C. (2007). "Natural antibody and complement mediate neutralization of influenza virus in the absence of prior immunity". J. Virol., vol. 81: 3487-3494.

Jespers, V., Harandi, A. M., Hinkula, J., Medaglini, D., LeGrand, R., Stahl-Hennig, C., Bogers, W, El Habib, R., Wegmann, F., Fraser, C., Cranage, M., Shattock, R. J. and Spetz, A. L. (2010)."Assessment of mucosal immunity to HIV-1". Expert. Rev. Vaccines, vol. 9 (4):381-394.

Jin, X., Bauer, D. E., Tuttleton, S. E., Lewin, S., Gettie, A., Blanchard, J., Irwin, C. E., Safrit, J. T., Mittler, J., Weinberger, L., Kostrikis, L. G., Zhang, L., Perelson, A. S. and Ho, D. D. (1999)."Dramatic rise in plasma viremia after CD8(+) T cell depletion in simian immunodeficiency virus-infected macaques". J. Exp. Med., vol. 189(6):991-998.

Jiang, X., Clark, R. A., Liu, L. and Wagers, A. J. (2012)."Fuhlbrigge RC, Kupper TS. Skin infection generates non-migratory memory CD8+ T(RM) cells providing global skin immunity". Nature, vol. 483:227-231.

Johansson-Lindbom, B. and Agace, W. W. (2007)."Generation of gut-homing T cells and their localization to the small intestinal mucosa". Immunol. Rev., vol. 215:226-242.

Johansson, E.L., Wassen, L.,Holmgren, J., Jertborn, M. and Rudin, A. (2001)."Nasal and vaginal vaccinations have differential effects on antibody responses in vaginal and cervical secretions in humans."Infect. Immun., vol. 69 (12): 7481-7486.

Jones, K. A. and Peterlin, M. B. (1994)."Control of RNA initiation and elongation at the HIV-1 promoter". Annl. Rev. Biochem., vol. 63 (1):717-743.

Johansson, E.L., Bergquist, C., Edebo, A., Johansson, C. and Svennerholm, A. M. (2004). Comparison of different routes of vaccination for eliciting antibody responses in the human stomach. Vaccine, vol. 22, 984-990.

Johnson , R. P., Lifson , J. D., Czajak, S. C., Cole , K. S., Manson , K. H. and Glickman, R. (1999)."Highly attenuated vaccine strains of simian immunodeficiencyvirus protect against vaginal challenge: inverse relationship of degree of protection with level of attenuation". J. Virol., vol. 73(6):4952-4961.

Johnson, R. .P. and Desrosiers, R. C. (1998). "Protective immunity induced by live attenuated simian immunodeficiency virus". Curr. Opin. Immunol., vol. 10:436-443.

Johnson, R. and Kannene, J. (1992)."Bovine leukemia virus and enzootic bovine leukosis". Vet. Bull., vol. 62:287-312.

Johnson, N. P. and Mueller, J. (2002). "Updating the accounts: Global mortality of the 1918–1920 "Spanish"influenza pandemic". Bull. Hist. Med., vol. 76:105-115.

Johnston, M. I. and Fauci, A. S. (2008). "An HIV Vaccine Challenges and Prospects". N Engl. J. Med., vol. 359 (9):888-890.

Johnston, M. I. and Flores, J. (2001)."Progress in HIV vaccine development". Curr.t Opin. Pharmacol., vol. 1 (5):504-510.

Kaetzel, C. S. and Bruno, M. E. C. (2007)."Epithelial transport of IgA by the polymeric immunoglobulin receptor". Mucosal Immune Defense: Immunoglobulin A. C. S. Kaetzel, Springer US: 43-89.

References

182

Kamperschroer, C., Dibble, J. P., Meents, D. L., Schwartzberg, P. L. and Swain, S. L. (2006). "SAP is required for Th cell function and for immunity to influenza". J. Immunol., vol. 177:5317-5327.

Kanagavelu, S., Termini1, J. M., Gupta, S., Raffa, Francesca N., Fuller, K. A., Rivas, Y., Philip, S., Kornbluth, R. S. and Stone, G. W. (2014)."HIV-1 Adenoviral Vector Vaccines Expressing Multi-Trimeric BAFF and 4-1BBL Enhance T Cell Mediated Anti-Viral Immunity". PLOS ONE, vol. 9 (2):e90100.

Kang, S. G., Park., Cho, J. Y., Ulrich, B. and Kim, C. H. (2011)."Complementary roles of retinoic acid and TGF-β1 in coordinated expression of mucosal integrins by T cells". Mucosal Immunol., vol. 4 (1):66-82.

Kantele, A., Zivny, J., Häkkinen, M., Elson, C. O.and Mestecky, J.(1999)."'Differential Homing Commitments of Antigen-Specific T Cells After Oral or Parenteral Immunization in Humans". J. Immunol., vol. 162 (9):5173-5177.

Kathuria, N., Kraynyak, K. A., Carnathan, D., Betts, M., Weiner, D. B. and Kutzler, M. A. (2012)."Generation of antigen-specific immunity following systemic immunization with DNA vaccine encoding CCL25 chemokine immunoadjuvant". Hum. Vaccin. Immunother., vol. 8 (11):1607-1619.

Katzourakis, A., Tristem, M., Pybus, O. G. and Gifford, R. J. (2007)."Discovery and analysis of the first endogenous lentivirus". Proc. Natl. Acad. Sci., vol. 104 (15):6261-6265.

Kaufman, D. R., Liu, J., Carville, A., Mansfield, K. G., Havenga, M. J. E., Goudsmit, J. and Barouch, D. H. (2008)."Trafficking of antigen-specific CD8+ T lymphocytes to mucosal surfaces following intramuscular vaccination". J. Immunol., vol. 181 (6):4188-98.

Kaul, R., Plummer, F. A., Kimani, J., Dong, T., Kiama, P., Rostron, T., Njagi, E., MacDonald, K. S., Bwayo, J. J., McMichael, A. J. and Rowland-Jones, S. L. (2000). "HIV-1-specific mucosal CD8+ lymphocyte responses in the cervix of HIV-1-resistant prostitutes in nairobi". J. Immunol., vol. 164 (3):1602-1611.

Kaul, R., Trabattoni, D., Bwayo, J. J., Arienti, D., Zagliani, A., Mwangi, F. M., Kariuki, C., Ngugi, E. N., MacDonald, K. S., Ball, B. T., Clerici, M., and Plummer, F. (1999)."HIV-1-specific mucosal IgA in a cohort of HIV-1-resistant Kenyan sex workers". AIDS, vol. 13 (1):23-29.

Kaverin, N. V., Matrosovich, M. N., Gambaryan, A. S., Rudnevaa, I. A., Shilova, A. A. Varicha, N. L.,Makarovaa, N. V., Kropotkinaa, E. A. and Sinitsina, B. V. (2000)."Intergenic HA-NA interactions in influenza A virus: postreassortment substitutions of charged amino acid in the hemagglutinin of different subtypes". Virus Res., vol. 66 (2):123-129.

Kawaoka, Y., Cox, N. J., Haller, O., Hongo, S., Kaverin, N., Klenk, H. D., Lamb, R. A., McCauley, J., Palese, P., Rimstad, E. and Webster, R. G. (2005)."Family Orthomyxoviridae. Virus taxonomy. Classification and nomenclature of virus'. Fauquet, C. M., Mayo, M. A., Maniloff, .J, Desselberger, U. and Ball, L. A., eds Eighth Report of the International Committee on the Taxonomy of Viruses. San Diego, CA: Elsevier Academic Press; 681-693.

Kedzierska, K., Stambas, J., Jenkins, M. R., Keating, R., Turner, S. J. and Doherty, P. C. (2007)."Location rather than CD62L phenotype is critical in the early establishment of

References

183

influenza-specific CD8+ T cell memory". Proc. Nati. Acad. Sci., vol. 104 (23): 9782-9787.

Kees, U. and Krammer, P. H. (1984). "Most influenza A virus-specific memory cytotoxic T lymphocytes react with antigenic epitopes associated with internal virus determinants". J. Exp. Med., vol. 159 (2)365-377.

Kerbs, J. E., Goldstein, E. S. and Kilpatrick, S. T. (2010)."Transposons, retroviruses, and retrotransposons". Lewin's essential genes, Second Edition, Jones and Bartlett Publishers, Sudbury, Massachusetts.

Kibuuka, H.,Kimutai, R., Maboko, L.,Sawe, F., Schunk, M. S., Kroidl, A., Shaffer, D., Eller, L. A., Kibaya, R., Eller, M. A., Schindler, K. B., Schuetz, A., Millard, M., Kroll, J., Dally, L., Hoelscher, M., Bailer, R., Cox, J. H., Marovich, M., Birx, D. L., Graham, B. S., Michael, N. L., de Souza, M. S. and Robb, M. L. (2010). "A Phase 1/2 Study of a Multiclade HIV-1 DNA Plasmid Prime and Recombinant AdenovirusSerotype 5 Boost Vaccine in HIV-Uninfected East Africans (RV 172)". JID, vol. 201:600-607.

Kiepiela, P., Leslie, A. J., Honeyborne, I., Ramduth, D., Thobakgale, C., Chetty, S., Rathnavalu, P., Moore, C., Pfafferott, K. J., Hilton, L., Zimbwa, P., Moore, S., Allen, T., Brander, C., Addo, M. M., Altfeld, M., James, I., Mallal, S., Bunce, M., Barber, L. D., Szinger, J., Day, C., Klenerman, P., Mullins, J., Korber, B., Coovadia, H. M., Walker, B. D. and Goulder, P. J. R. (2004)."Dominant influence of HLA-B in mediating the potential co-evolution of HIV and HLA". Nature, vol. 432 (7018):769-75.

Kim, T. S. and Braciale, T. J. (2009)."Respiratory dendritic cell subsets differ in their capacity to support the induction of virus-specific cytotoxic CD8+ T cell responses". PLoS One, vol. 4 (1): e4204.

Kim, H. M., Lee, Y. W., Lee, K. J., Kim, H. S., Cho, S. W., van Rooijen, N., Guan, Y. and Seo, S. H. (2008)."Alveolar macrophages are indispensable for controlling influenza viruses in lungs of pigs". J. Virol., vol. 82:4265-4274.

Kimberly, S. S., Williams, K., Ma, A., Zheng, X. X. and Lefranc, L. (2002)."Cutting edge: requirement for IL-15 in the generation of primary and memory antigen-specific CD8 T cells". J. Immunol., vol. 168:4827-4831.

Kiravu, A. 1., Gumbi, P., Mkhize, N. N., Olivier, A., Denny, L. and Passmore, J. A. (2011). "Evaluation of CD103 (αEβ7) integrin expression by CD8 T cells in blood as a surrogate marker to predict cervical T cell responses in the female genital tract during HIV infection". Clin. Immunol. 141(2):143-151.

Kirchhoff, F. (2010)."Immune evasion and counteraction of restriction factors by HIV-1 and other primate lentiviruses". J. Immunol., 154 (5): 2448-2459.

Kittel, C., Ferko, B., Kurz, M., Voglauer, R., Sereinig, S., Romanova, J., Stiegler, G., Katinger, H. and Egorov, A. (2005)."Generation of an influenza A virus vector expressing biologically active human interleukin-2 from the NS gene segment". J. Virol.,vol. 79 (16):10672-10677.

Kittel, C., Sereinig, S., Ferko, B., Stasakova, J., Romanova, J., Wolkerstorfer, A., Katinger, H. and Egorov, A. (2004)."Rescue of influenza virus expressing GFP from the NS1 reading frame".Virology, vol. 324: 67-73.

Klein, K., Veazey, R. S., Warrier, R., Hraber, P., Doyle-Meyers, L. A., Buffa, V., Liao, H., Haynes, B. F., Shaw, G. M. and Shattock, R. J. (2013)."Neutralizing IgG at the portal

References

184

of infection mediates protection against vaginal simian/human immunodeficiency virus challenge". J. Virol., vol. 87(21):11604-11616.

Klein, M., van Baalen, C. A., Holwerda, A. M., Kerkhof, G. S. R., Bende, R. J., Keet, I. P., Eeftinck-Schattenkerk, J. K., Osterhaus, A. D., Schuitemaker, H. and Miedema, F. (1995)."Kinetics of Gag-specific cytotoxic T lymphocyte responses during the clinical course of HIV-1 infection: A longitudinal analysis of rapid progressors and long-term asymptomatics". J. Exp. Med., vol. 181:1365-1372.

Klenk, H. D., Rott, R., Orlich, M. and Blödorn, J. (1975)."Activation of influenza A viruses by trypsin treatment". Virology, vol. 68 (2):426-439.

Klotman, M. E. and Chang, T. L. (2006)."Defensins in innate antiviral immunity". Nat. Rev. Immunol, vol. 6 (6):447-456.

Knossow, M. and Skehel, J. J. (2006)."Variation and infectivity neutralization in influenza. Immunology, vol. 119:1-7.

Kobayashi, A., Greenblatt, R. M., Anastos, K., Minkoff, H., Massad, L. S., Young, M., Darragh, T., Weinberg, V., Smith-McCune, K. (2004)."Functional attributes of mucosal immunity in cervical intraepithelial neoplasia and effects of HIV infection". Cancer Res., vol. 64 (18): 6766-6774.

Kobayashi, M., Tuchiya, K., Nagata, K.and Ishihama, A. (1992)."Reconstitution of influenza virus RNA polymerase from three subunits expressed using recombinant baculovirus system". Virus Res., vol. 22 (3):235-245.

Koff, W. C., Johnson, P. R.,Watkins, D. I., Burton, D. R., Lifson, J. D., Hasenkrug, K. J., McDermott, A. B., Schultz, A., Zamb, T. J., Boyle, R. and Desrosiers, R. C. (2006)."HIV vaccine design: insights from live attenuated SIV vaccines". Nat. Immunol., vol. 7 (1): 19-23.

Kopf, M., Abel, B., Gallimore, A., Carroll, M. and Bachmann, M. F. (2002)."Complement component C3 promotes T cell priming and lung migration to control acute influenza virus infection'. Nat. Med., vo. 8:373-378.

Koseki, S., Miura, S., Fujimori, H., Hokari, R., Komoto, S., Hara, Y., Ogino, T., Nagata, H., Goto, M., Hachimura, S., Kaminogawa, S.and Ishii, H. (2001)."In situ demonstration of intraepithelial lymphocyte adhesion to villus microvessels of the small intestine". Int. Immunol., vol. 13(9):1165-74.

Koup, A. and Douek, D. C. (2011)."Vaccine design for CD8 T lymphocyte responses". Cold Spring Harb Perspect Med., vol. 1 (1):a 007252.

Koup, R. A., Roederer, M., Lamoreaux, L., Fischer, J., Novik, L., Nason, M. C., Larkin, B. D., Enama, M. E., Ledgerwood, J. E., Bailer, R. T., Mascola, J. R., Nabel, G. J. and Graham, B. (2010)."Priming immunization with DNA augments immunogenicity of recombinant adenoviral vectors for both HIV-1 specific antibody and T-cell responses". PLoS ONE, vol. 5 (2):e9015.

Koup, R. A., Safrit. J. T., Cao, Y., Andrews, C. A., McLeod, G., Borkowsky, W., Farthing, C. and Ho, D. D. (1994)."Temporal association of cellular immune responses with the initial control of viremia in primary human immunodeficiency virus type 1 syndrome". J. Virol., vol. 68 (7):4650-4655.

References

185

Koutsonanos, D. G., del Pilar M., M., Zarnitsyn, V. G., Jacob, J., Prausnitz, M. R., Compans, R.W. and Skountzou, I. (2011)."Serological memory and long-term protection to novel H1N1 influenza virus after skin vaccination". J. Infect. Dis., vol. 204:582-591.

Kozlowski, P. A., Cu-Uvin, S., Neutra, M. R. and Flanigan, T. P. (1997). Comparison of the oral, rectal, and vaginal immunization routes for induction of antibodies in rectal and genital tract secretions of women. Infect. Immun., vol. 65:1387-1394.

Kramer, I. (2007)."What triggers transient AIDS in the acute phase of HIV infection and chronic AIDS at the end of the incubation period?, A model analysis of HIV infection from the acute phase to the chronic AIDS stage". Comput. Math. Meth. Med., 8 (2):125-151.

Kreijtz, J. H.; Bodewes, R., van den Brand, J. M., de Mutsert, G., Baas, C., van Amerongen, G., Fouchier, R. A., Osterhaus, A. D. and Rimmelzwaan, G. F. (2009)."Infection of mice with a human influenzaA/H3N2 virus induces protective immunity against lethal infection with influenza A/H5N1 virus".Vaccine, vol. 27:4983-4989.

Kreijtz, J. H. C. M., de Mutsert, G., van Baalen, C. A. , Fouchier, R. A. M., Osterhaus, A. D. M. E. and Rimmelzwaan, G. F. (2008)."Cross-recognition of avian H5N1 influenza virus by human cytotoxic T-lymphocyte populations directed to human influenza A virus". J. Virol., vol. 82:5161-5166.

Kreijtz, J. H., Bodewes, R., van Amerongen, G., Kuiken, T., Fouchier, R. A., Osterhaus, A. D. and Rimmelzwaan, G. F. (2007)."Primary influenza A virus infection induces cross-protective immunityagainst a lethal infection with a heterosubtypic virus strain in mice". Vaccine, vol. 25:612-620.

Krzych, U., Dalai, S., Zarling, S. and Pichugin, A. (2012)."Memory CD8 T cells specific for Plasmodia liver-stage antigens maintain protracted protection against malaria". Front. Immunol., vol. 3:370.

Kulkarni, V., Rosati, M., Valentin, A., Ganneru, B., Singh, A. K., Yan, J., Rolland, M., Alicea, C., Beach, R. K., Zhang, G. M., Gall, S. L., Broderick, K. E., Sardesai, N. Y. and Felber, B. K. (2013)."HIV-1 p24gag Derived Conserved Element DNA Vaccine Increases the Breadth of Immune Response in Mice". PLoS ONE, vol. 8 (3):e60245.

Kunisawa, J. and Kiyono, H. (2005)."A marvel of mucosal T cells and secretory antibodies for the creation of first lines of defense". Cell. Mol. Life Sci., vol. 62:1308-1321.

Kuniyoshi, J. S., Kuniyoshi, C. J., Lim, A. M., Wang, F. Y., Bade, E. R., Lau, R., Thomas, E. K. and Weber, J. S. (1999)."Dendritic cell secretion of IL-15 is induced by recombinant huCD40LT and augments the stimulation of antigen-specific cytolytic T cells". Cell Immunol., vol. 193:48-58.

Kunwar, P., Hawkins, N., Dinges, W. L., Liu, Y., Gabriel, E. E., Swan, D. A., Stevens, C. E., Maenza, J., Collier, A. C., Mullins, J. I., Hertz, T., Yu, X. and Horton, H. (2013). "Superior Control of HIV-1 Replication by CD8+ T Cells Targeting Conserved Epitopes: Implications for HIV Vaccine Design". PLoS ONE, vol. 8 (5):e64405.

Kutzler, M. A., Kraynyak, K. A., Nagle, S. J., Parkinson, R. M., Zharikova, D., Chattergoon, M., Maguire, H., Muthumani, K., Ugen, K. andWeiner, D. B. (2009)."Plasmids encoding the mucosal chemokines CCL27 and CCL28 are effective adjuvants in eliciting antigen-specific immunity in vivo". Gene Ther., vol. 17 (1): 72-82.

References

186

La Gruta, N. L., Turner, S. J. and Doherty, P. C. (2004). "Hierarchies in cytokine expression profiles for acute and resolving influenza virus-specific CD8+ T cell responses: correlation of cytokine profile and TCR avidity". J. Immunol., vol. 172 (9):5553-55560.

Lamb, R. and Krug, R. M. (1996)."Orthomyxoviridae: the viruses and their replication". P.1353-1395. In Field, Knipe, B. N. and Howely, D. N. (eds), Virology, Lippincott Raven Publishers, Philadelphia, USA.

Lamb, J. R., Woody, J. N.; Hartzman, R. J. and Eckels, D. D. (1982)."In vitro influenza virus-specific antibody production in man: Antigen-specific and HLA-restricted induction of helper activity mediated by cloned human T lymphocytes". J. Immunol., vol. 129:1465-1470.

Lambe, T., Carey, J. B., Li, Y., Spencer, A. J., van Laarhoven, A., Mullarkey, C. E., Vrdoljak, A., Moore, A. C. and Gilbert, S. C. (2013). "Immunity against heterosubtypic influenza virus induced by adenovirus and MVA expressing nucleoprotein and matrix protein-1". Scientific Reports, 3 : 1443.

Lamere, M. W., Moquin, A., Lee, F. E., Misra, R. S., Blair, P. J., Haynes, L., Randall, T. D., Lund, F. E. and Kaminski, D. A. (2011)."Regulation of ant-inucleoprotein IgG by systemic vaccination and its effect on influenza virus clearance". J. Virol., vol. 85:5027-5035.

Landay, A. L., Mackewicz, C. E. and Levy, J. A. (1993)."An activated CD8+ T cell phenotype correlates with anti-HIV activity and asymptomatic clinical status." Clin. Immunol. Immunopathol., 69 (1):106-116.

Lander, E. S., Linton, L. M., Birren, B., Nusbaum, C., Zody, M. C., Baldwin, J. K., Devon, K., Dewar, K., Doyle, M., FitzHugh, W. and Funke, R. (2001)."Initial sequencing and analysis of the human genome". Nature, vol. 409 (6822): 860-921.

Lawrence, T. M., Wanjalla, C. N., Gomme, E. A., Wirblich, C., Gatt, A., Carnero, E., García-Sastre, A., Lyles, D. S., McGettigan, J. P.and Schnell, M. J. (2013)."Comparison of heterologous prime-boost strategies against human immunodeficiency virus type 1 Gag using negative stranded RNA viruses". PLoS ONE, vol. 8 (6):e67123.

Lee, L. Y., Ha Do, L. A. and Simmons, C. (2008)."Memory T cells established by seasonal human influenza A infection cross-react with avian influenza A (H5N1) in healthy individuals". J. Clin. Inv., vol. 118 (10)3478-3490.

Lee, B. and Montaner, L. (1999)."Chemokine immunobiology in HIV-1 pathogenesis". J. Leuk. Biol., vol. 65 (5): 552-565.

Lefrançois, L., Parker, C. M., Olson, S. Muller, W., Wagner, N. and Puddington, L. (1999). "The role of β7 integrins in CD8 T cell trafficking during an antiviral immune response". J. Exp. Med., vol. 189 (10) 1631-1638.

Lehner, T., Wang, Y., Pido-Lopez, J., Whittall, T., Bergmeier, L. A. and Babaahmady, K. (2008)."The emerging role of innate immunity in protection against HIV-1 infection". Vaccine, vol. 26 (24): 2997-3001.

Letvin, N. L. (2009)."A vaccine that delivers rather than induces antibodies". Gene Ther., vol. 16 (11):1283-1284.

Letvin, N. L. (2009a)."Thai HIV vaccine study, moving forward in HIV vaccine development - perspective". Science, vol. 326 (5957):1196-1198.

References

187

Letvin, N. L. (2005)."Progress toward an HIV vaccine."Annl. Rev. Med., vol. 56 (1): 213-223.

Letvin, N. L., Y. Huang, Chakrabarti, B. K., Xu, L., Seaman, M. S., Beaudry, K., Korioth-Schmitz, B., Yu, F., Rohne, D., Martin, K. L., Miura, A., Kong, W. P., Yang, Z. Y., Gelman, R. S., Golubeva, O. G., Montefiori, D. C., Mascola, J. R. and Nabel, G. J. (2004)."Heterologous envelope immunogens contribute to AIDS vaccine protection in rhesus monkeys". J. Virol., vol. 78 (14):7490-7497.

Levy, J. A. (2003)."The search for the CD8+ cell anti-HIV factor (CAF)". Trends Immunol., vol 24:628-632.

Levy, J. A., Scott, I. and Mackewicz, C. (2003)."Protection from HIV/AIDS: the importance of innate immunity". Clin. Immunol., vol. 108 (3):167-74.

Levy, J. A.(1993)."Pathogenesis of human immunodeficiency virus infection". Microbiol. Molec. Biol. Rev., vol. 57 (1): 183-289.

Lewis, D. B. (2006)."Avian flu to human influenza". Annl. Rev. Med., vol. 57 (1): 139-154.

Li, G., Verheyen, J., Rhee, S. Y. Voet, A., Vandamme, A. M. and Theys, K. (2013). "Functional conservation of HIV-1 Gag: implications for rational drug design". Retrovirology, vol. 10:126.

Li, R., Fang, H., Li, Y., Liu, Y., Pellegrini, M. and Podda, A. (2008)."Safety and immunogenicity of an MF59TM-adjuvanted subunit influenza vaccine in elderly Chinese subjects". Immun. Ageing, vol. 5 (1): 2.

Li, Z., Zhang, M., Zhou, C., Zhao, X., Iijima, N. and Frankel, F. R. (2008)."'Novel vaccination protocol with two live mucosal vectors elicits strong cell-mediated immunity in the vagina and protects against vaginal virus challenge". J. Immunol., vol. 180 (4):2504-2513.

Li, Z. N., Mueller, S. N., Ye, L., Bu, Z., Yang, C., Ahmed, R. and Steinhauer, D. A. (2005). "Chimeric influenza virus hemagglutinin proteins containing large domains of the Bacillus anthracis protective antigen: protein characterization, incorporation into infectious influenza viruses, and antigenicity". J. Virol., vol. 79:10003-10012.

Li, S., Polonis, V., Isobe, H., Zaghouani, H., Guinea, R., Moran, T., Bona, C. and Palese P. (1993)."Chimeric influenza virus induces neutralizing antibodies and cytotoxic T cells against human immunodeficiency virus type 1". J. Virol., vol. 67, 6659-6666.

Liang, X., Casimiro, D. R., Schleif, W. A., Wang, F., Davies, M. E., Zhang, Z. Q., Fu, T. M., Finnefrock, A. C., Handt, L., Citron, M. P., Heidecker, G., Tang, A., Chen, M., Wilson, K. A., Gabryelski, L., McElhaugh, M., Carella, A., Moyer, C., Huang, L., Vitelli, S., Patel, D., Lin, J., Emini, E. A. and Shiver, J. W. (2005)."Vectored Gag and Env but not Tat show efficacy against simian-human immunodeficiency virus 89.6P challenge in Mamu-A*01-negative rhesus monkeys". J. Virol., vol. 79 (19):12321-12331.

Liang, C. and Wainberg, M. A. (2002)."The Role of Tat in HIV-1 replication: an activator and/or a suppressor? ". AIDS Rev., vol. 4: 41-49.

Lichterfeld, M., Gandhi, R. T., Simmons, R. P., Flynn, T., Sbrolla, A., Yu, X. G., Basgoz, N., Mui, S., Williams, K., Streeck, H., Burgett-Yandow, N., Roy, G., Janssens, M., Pedneault, L., Vandepapelière, P., Koutsoukos, M., Demoitié, M.-A., Bourguignon, P., McNally, L., Voss, G. and Altfeld, M. (2012)."Induction of strong HIV-1-specific CD4+ T Cell Responses using an HIV-1 gp120/NefTat vaccine adjuvanted with AS02A

References

188

in ARV treated HIV-1-infected individuals". J. Acquir. Immun. Def. Syndr., vol. 59(1): 1-9.

Liu, M. A. (2010)."Immunologic basis of vaccine vectors". Immunity, vol. 33(4):504-515.

Liu, J., O'Brien, K. L., Lynch, D. M., Simmons, N. L., La Porte, A. , Riggs, A. M. , Abbink, P., Coffey, R. T., Grandpre, L. E., Seaman, M. S., Landucci, G., Forthal, D. N., Montefiori, D. C., Carville, A., Mansfield, K. G., Havenga, M. J., Pau, M. G., Goudsmit, J. and Barouch, D. H. (2009)."Immune control of an SIV challenge by a T-cell-based vaccine in rhesus monkeys". Nature, vol. 457 (7225):87-91.

Liu, Z., Cumberland, W. G., Hultin, L. E., Kaplan, A. H., Detels, R. and Giorgi, J. V. (1998). "CD8+ T-lymphocyte activation in HIV-1 disease reflects an aspect of pathogenesis distinct from viral burden and immunodeficiency". Acquir. Immun. Def. Syndr. Hum. Retrovirol., vol. 18 (4):332-340.

Lodolce, J. P., Boone, D. L., Chai, S., Swain, R. E., Dassopoulos, T., Trettin, S. and Ma, A. (1998)."IL-15 receptor maintains lymphoid homeostasis by supporting lymphocyte homing and proliferation". Immunity, vol. 9: 669.

López-Balderas, N., Huerta, L., Villarreal, C., Rivera-Toledo, E., Sandoval, G., Larralde, C.and Lamoyi, E. (2007)."In vitro cell fusion between CD4+ and HIV-1 Env+ T cells generates a diversity of syncytia varying in total number, size and cellular content". Virus Res., vol. 123 (2):138-146.

Lorgeoux, R. P. Guo, F. and Liang, C. (2012)."From promoting to inhibiting: diverse roles of helicases in HIV-1 Replication". Retrovirol., vol. 9:79.

Lü, F. X. and Jacobson, R. S. (2007)."Oral mucosal immunity and HIV/SIV infection". J. Dental Res., vol. 86 (3):216-226.

Lu, X., Shi, Y., Zhang, W., Zhang, Y., Qi, J. and Gao, G. F. (2013)."Structure and receptor-binding properties of an airborne transmissible avian influenza A virus hemagglutinin H5 (VN1203mut)". Protein Cell, vol. 4:502-511.

Lu, X., Shi, Y., Gao, F., Xiao, H., Wang, M., Qi, J. and Gao, G. F. (2012)."Insights into avian influenza virus pathogenicity: the hemagglutinin precursor HA0 of subtype H16 has an alpha-helix structure in its cleavage site with inefficient HA1/HA2 cleavage". J. Virol., vol. 86:12861-12870.

Luft, S., Seme, K. and Poljak, M. (2004)."Laboratory diagnosis of human immunodeficiency virus infection". Acta. Dermatoven., vol.13(2): 43-49.

Luo, G., Chung, J. and P. Palese. (1993)."Alterations of the stalk ofthe influenza virus neuraminidase: deletions and insertions". Virus Res., vol. 29:141-153.

Luytjes, W., Krystal, M., Enami, M., Pavin, J. D. and Palese, P. (1989)."Amplification, expression, and packaging of foreign gene by influenza virus". Cell, vol. 59: 1107-1113.

Ma, A., Koka, R. and Burkett, P. (2006)."Diverse functions of IL-2, IL-15, and IL-7 in lymphoid homeostasis". Annu. Rev. Immunol., vol. 24:657-679.

Ma, M. and Nath, A. (1997)."Molecular determinants for cellular uptake of Tat protein of human immunodeficiency virus type 1 in brain cells". J. Virol., vol. 71 (3):2495-2499.

References

189

MacDonald, T. T. (2003)."The mucosal immune system". Parasite Immunol., vol. 25 (5):235-246.

Maggiorella, M. T., Baroncelli, S., Michelini, Z., Fanales-Belasio, E., Moretti, S. and Sernicola, L. (2004). Long-term protection against SHIV89.6P replication in HIV-1 Tat vaccinated cynomolgus monkeys. Vaccine, vol. 22 (25–26):3258-3269.

Mao, Y., Wang, L., Gu, C., Herschhorn, A., Désormeaux, A., Finzi, A., Xiang, S. H. and Sodroski, J. G. (2013)."Molecular architecture of the uncleaved HIV-1envelope glycoprotein trimer". Proc. Natl. Acad. Sci. USA, vol. 110 (30):12438-12443.

Marsh, G. and Tannock, G. (2005)."The role of reverse genetics in the development of vaccines against respiratory viruses." Exp. Opin. Biol.Therapy 5 (3):369-380.

Martin, P., Lerner, A., Johnson, L., Lerner, D. L., Haraguchi, S., Good, R. A. and Day, N. K. (2003)."Inherited mannose-binding lectin deficiency as evidenced by genetic and immunologic analyses: association with severe recurrent infections". Ann. Allergy Asthma Immunol., vol. 91(4):386-92.

Martínez-Sobrido, L. and García-Sastre, A. (2007)."Recombinant influenza virus vectors". Future Virol., vol. 2 (4):401-416.

Mascola, J. R. and Haynes, B. F. (2013)."HIV-1 neutralizing antibodies: understanding nature’s pathways". Immunol. Rev., vol. 254 (1): 225-244.

Mascola, J. R. and Montefiori, D. C. (2010)."The role of antibodies in HIV vaccines". Annl. Rev. Immunol., vol. 28 (1):413-444.

Mascola, J., G. Stiegler, VanCott, T. C., Katinger, H., Carpenter, C. B., Hanson, C. E., Beary, H., Hayes, D., Frankel, S. S., Birx, D. L. and Lewis, M. G. (2000)."Protection of macaques against vaginal transmission of a pathogenic HIV-1/SIV chimeric virus by passive infusion of neutralizing antibodies". Nat. Med., vol. 6 (2):207-210.

Mascola, J. R., Snyder, S. W., Weislow, O. S., Belay, S. M., Belshe, R. B., Schwartz, D. H., Clements, M. L., Dolin, R., Graham, B. S., Gorse, G. J., Keefer, M. C., McElrath, M. J., Walker, M. C., Wagner, K. F., McNeil, J. G., McCutchan, F. E. and Burke, D. S. (1996)."Immunization with envelope subunit vaccine products elicits neutralizing antibodies against laboratory-adapted but not primary isolates of human immunodeficiency virus type 1". J. Infect. Dis., vol. 173 (2):340-348.

Masopust, D., Vezys, V., Wherry, E. J., Barber, D. L. and Ahmed, R. (2006)."Cutting edge: gut microenvironment promotes differentiation of a unique memory CD8 T cell population". J. Immunol., vol. 176:2079-2083.

Mata, M., Travers, P. J., Liu, Q., Frankel, F. R. and Paterson, Y. (1998)."The MHC class I-restricted immune response to HIV-gag in BALB/c mice selects a single epitope that does not have a predictable MHC-binding motif and binds to Kd through interactions between a glutamine at P3 and pocket D". J. Immunol., vol. 161:2985-2993.

Mateo, R. I., Xiao, S.Y., Lei, H., Aelia, P. A., Travassosda, R. and Tesh, R. B. (2007)."DHORI virus (Orthomyxoviridae: Thogotovirus) infection in mice: a model of the pathogenesis of severe orthomyxovirusinfection". Am. J. Trop. Med. Hyg., vol. 76 (4):785-790.

Mathers, A. R. and Cuff, C. F. (2004)."Role of Interleukin-4 (IL-4) and IL-10 in serum immunoglobulin G antibody responses following mucosal or systemic reovirus infection". J. Virol., vol. 78 (7):3352-3360.

References

190

Mathers, C. and Loncar, D. (2006)."'Projections of global mortality and burden of disease from 2002 to 2030". PLoS Med, vol. 3 (11):e442.

Mathews, R. E. F. (1982)."Classification and nomenclature of viruses. In Fourth Report of the International Committee on Taxonomy of Viruses", pp. 234-238 , Karger, Basel.

Mavigner, M., Cazabat, M., ubois, M.,’Faqihi, F. E. L., Requena, M., Pasquier, C., Klopp, P., Amar, J., Alric, L., Barange, K., Vinel, J. P., Marchou, B., Massip, P. Izopet, J. and Delobel, P. (2012)."Altered CD4+ T cell homing to the gut impairs mucosal immune reconstitution in treated HIV-infected individuals". J. Clin. Invest., vol. 122 (1):62-69.

Mayer, K. H. and Bell, S.K., cited by Libman, H. and Makadon, H. (2007)."Transmission, pathogenesis, and natural history." HIV, Third edition, American College of Physicians, Philadelphia, USA.

McBurney, S. P. and Ross, T. M. (2008)."Viral sequence diversity: challenges for AIDS vaccine designs". Exp. Rev. Vaccines, vol.7 (9):1405-1417.

McCurdy, L. H., Larkin, B. D., Martin, J. E. and Graham, B. S. (2004)."Modified vaccinia ankara: potential as an alternative smallpox vaccine". Clin. Infect. Dis., vol. 38 (12): 1749-1753.

McElrath, M. J. and Haynes, B. F. (2010)."Induction of immunity to human immunodeficiency virus type-1 by vaccination". vol. 33 (4): 542-554.

McGettigan, J. P., Naper, K.,Orenstein, J.,Koser, M., McKenna, P. M. and Schnell, M. J. (2003)."Functional human immunodeficiency virus type 1 (HIV-1) Gag-Pol or HIV-1 Gag-Pol and Env expressed from a single rhabdovirus-based vaccine vector genome". J. Virol., 77 (20):10889-10899.

McGettigan, J. P., Pomerantz, R. J., Siler, C. A., McKenna, P. M., Foley, H. D., Dietzschold, B. , and Schnell, M. J. (2003a)."Second-generation rabies virus-based vaccine vectors expressing human immunodeficiency virus type 1 Gag have greatly reduced pathogenicity but are highly immunogenic". J. Virol., vol. 77 (1): 237-244.

McGettigan, J. P., Sarma, S., Orenstein, J. M., Pomerantz, R. J. and Schnell, M. J. (2001). "Expression and immunogenicity of human immunodeficiency virus type 1 Gag expressed by a replication-competent rhabdovirus-based vaccine vector". J. Virol., vol. 75 (18):8724-8732.

McGill, J., Van Rooijen, N. and Legge K. L. (2010)."IL-15 trans-presentation by pulmonary dendritic cells promotes effector CD8 T cell survival during influenza virus infection". J. Exp. Med., vol. 207:521-534.

McGrath, K. M., Hoffman, N. G., Resch, W., Nelson, J. A.E. and Swanstrom, R. (2001). "Using HIV-1 sequence variability to explore virus biology". Virus Res., vol. 76 (2): 137-160.

McInnes, I. B., al-Mughales, J., Field, M., Leung, B. P., Huang, F. P., Dixon, R., Sturrock, R. D., Wilkinson, P. C. and Liew, F. Y. (1996)."The role of interleukin-15 in T-cell migration and activation in rheumatoid arthritis". Nat. Med., vol. 2:175-182

McKenna, K., Beignon, A. S. and Bhardwaj, N. (2005)."Plasmacytoid dendritic cells: linking innate and adaptive immunity". J. Virol., vol. 79: (1):17-27.

McKenna, P. M., Koser, M. L., Carlson, K. R., Montefiori, D. C., Letvin, N. L., Papaneri, A. B., Pomerantz, R. J., Dietzschold, B., Silvera, P., McGettigan, J. P. and Schnell, M. J. (2007)."Highly attenuated rabies virus-based vaccine vectors expressing simian-

References

191

Human immunodeficiency virus 89.6P Env and simian immunodeficiency virus mac239 Gag are safe in rhesus macaques and protect from an AIDS-like disease". J. Infect Dis., vol. 195 (7): 980-988.

McKinnon, L. R., Kimani, M., McKinnon, L. R., Kimani, M., Wachihi, C., Nagelkerke, N. J., Muriuki, F. K., Kariri, A., Lester, R. T., Gelmon, L. T., Ball, B., Plummer, F. A., Kaul, R. and Kimani, J. (2010)."Effect of baseline HIV disease parameters on CD4+ T cell recovery after antiretroviral therapy initiation in kenyan women". PLoS ONE, vol. 5 (7): e11434.

McMichael, A. J., Gotch, F. M., Noble, G. R. and Beare, P. A. S. (1983)."Cytotoxic T-cell immunity to influenza". N. Eng. J. Med., vol. 309 (1):13-17.

McMichael, A. J. and Askonas, B. A. (1978)."Influenza virus–specific cytotoxic T cells in man: induction and properties of the cytotoxic cells". Eur. J. Immunol., vol. 8:705-711.

Medeiros-Silva, D. C., Moreira-Silva, E. A., Gomes, J. de A. S., da Fonseca, F. G. and Correa-Oliveira, R. (2013)."CD4 and CD8 T cells participate in the immune memory response against Vaccinia virus after a previous natural infection". Results Immunol., vol. 3:104-113.

Meier, A., Chang, J. J., Chan, E. S., Pollard, R. B., Sidhu, H. K., Kulkarni, S., Wen, T. F, Lindsay, R. J., Orellana, L., Mildvan, D., Bazner, S., Streeck, H., Alter, G., Lifson, J. D., Carrington, M., Bosch, R. J., Robbins, G. K. and Altfeld, M. (2009)."Sex differences in the Toll-like receptor-mediated response of plasmacytoid dendritic cells to HIV-1". Nat. Med., vol. 15 (8):955-9.

Mercier, G. T., Nehete, P. N., Passeri, M. F., Nehete, B. N., Weaver, E. A., Templeton, N. S., Schluns, K., Buchl, S. S., Sastry, K. J. and Barry, M. A. (2007)."Oral immunization of rhesus macaques with adenoviral HIV vaccines using enteric-coated capsules". Vaccine, vol. 25 (52): 8687-8701.

Midgley, C. M., Putz,M. M., Weber, J. N. and Smith, G. L. (2008)."Vaccinia virus strain NYVAC induces substantially lower and qualitatively different human antibody responses compared with strains Lister and Dryvax". J. Gen.Virol., vol. 89 (12):2992-2997.

Migueles, S. A., Osborne, C. M., Royce, C., Compton, A. A., Joshi, R. P., Weeks, K. A., Rood, J. E., Berkley, A. M., Sacha, J. B., Cogliano-Shutta, N. A., Lloyd, M., Roby, G., Kwan, R., McLaughlin, M., Stallings, S., Rehm, C., O’Shea, M. A., Mican, J. A., Packard, B. Z., Komoriya, A., Palmer, S., Wiegand, A. P., Maldarelli, F. J., Coffin, M., Mellors, J. W., Hallahan, C. W., Follman, D. A. and Connors, M. (2008)."Lytic Granule Loading of CD8+ T Cells Is Required for HIV-Infected Cell Elimination Associated with Immune Control". Immunity, vol. 29 (6): 1009-1021.

Migueles, S. A., Laborico, A. C., Shupert, W. L., Sabbaghian, M. S., Rabin, R., Hallahan, C. W., Van Baarle, D., Kostense, S., Miedema, F., McLaughlin, M., Ehler, L., Metcalf, J., Liu, S. and Connors, M. (2002)."HIV-specific CD8+ T cell proliferation is coupled to perforin expression and is maintained in nonprogressors". Nat. Immunol., vol. 3(11):1061-1068.

Mogensen, T., Melchjorsen, J., Larsen, C. S. and Paludan, S. R. (2010)."Innate immune recognition and activation during HIV infection". Retrovirology, vol. 7 (1):54.

Monteiro, J. M., Harvey, C. and Trinchieri, G. (1998)."Role of interleukin-12 in primary influenza virus infection". J. Virol., vol 72 (6):4825-4831.

References

192

Montpetit, M. (1995)."Sequence Note:HIV-1 subtype A in Canada". AIDS Res. Hum. Retroviruses, vol. 11 (11):1421-1422.

Morris, L. (2002)."Neutralizing antibody responses to HIV-1 infection". IUBMB Life, vol. 53:197-199.

Mortier, E., Woo, T., Advincula, R., Gozalo, S. and Ma, A. (2008)."IL-15Ra chaperones IL-15 to stable dendritic cell membrane complexes that activate NK cells via trans presentation". J. Exp. Med., vol. 205: 1213-1225.

Mortier, E., Quemener, A., Vusio, P., Lorenzen, I., Boublik, Y., Grotzinger, J. Plet, A. and Jacques,Y. (2006)."Soluble interleukin-15 receptor alpha (IL-15Ra)-sushi as a selective and potent agonist of IL-15 action through IL-15Rbeta/gamma: hyperagonist IL-15- IL-15Ra fusion proteins". J. Biol. Chem., vol. 281:1612-1619.

Mosmann, T. R., Cherwinski, H., Bond, M. W., Giedlin, M. A. and Coffman, R. L. (1986). "Two types of murine helper T cell clone I. definition according to profiles of lymphokine activities and secreted proteins". J. Immunol., vol.136: 2348-2357.

Mouquet, H., Scheid, J. F., Zoller, M. J., Krogsgaard, M., Ott, R. G., Shukair, S., Artyomov, M. N., Pietzsch, J., Connors, M., Pereyra, F., Walker, B. D., Ho, D. D., Wilson, P. C., Seaman, M. S., Eisen, H., Chakraborty, A. K., Hope, T. J., Ravetch, J. V., Wardemann, H. and Nussenzweig, M. (2010)."Polyreactivity increases the apparent affinity of anti-HIV antibodies by heteroligation". Nature, vol. 467 (7315):591-595.

Mozdzanowska, K., Furchner, M., Zharikova, D., Feng, J. Q. and Gerhard, W. (2005). "Roles of CD4 T-cell-independent and -dependent antibody responses in the control of influenza virus infection: evidence for noncognate CD4 T-cell activities that enhance the therapeutic activity of antiviral antibodies. J. Virol., vol. 79(10):5943-5951.

Mozdzanowska, K., Maiese, K. and Gerhard, W. (2000)."Th cell-deficient mice control influenza virus infection more effectively than Th- and B cell-deficient mice: evidence for a Th-independent contribution by B cells to virus clearance". J. Immunol., vol. 164:2635-2643.

Mueller, S., Coleman, J. R., Papamichail, D., Ward, C. B., Nimnual, A., Futcher, B., Skiena, S.and Wimmer, E. (2010)."Live attenuated influenza virus vaccines by computer-aided rational design". Nat. Biotech., vol. 28 (7):723-726.

Munger, W., DeJoy, S. Q., Jeyaseelan, R., Torley, L. W., Grabstein, K. H., Eisenmann, J., Paxton, R., Cox, T., Wick, M. M. and Kerwar, S. S. (1995)."Studies evaluating the antitumor activity and toxicity of interleukin-15, a new T cell growth factor: comparison with interleukin-2". Cell Immunol., vol. 165:289-293.

Murashev, B., Kazennova, E., Kozlov, A., Murasheva, I. Dukhovlinova, E., Galachyants, Y., Dorofeeva, E., Dukhovlinov, I., Smirnova, G., Masharsky, A., Klimov, N. and Kozlov, A. P. (2007)."Immunogenicity of candidate DNA vaccine based on subtype A of human immunodeficiency virus type 1 predominant in Russia". Biotechnol. J., vol. 2: 871-878.

Murphy, B. R. and Coelingh, K. (2002)."Principles underlying the development and use of live attenuated cold-adapted influenza A and B virus vaccines. Viral Immunol. vol. 15:295-323.

References

193

Murphy, B. R. and Webster, R. G. (1996)."Orthomyxoviruses:the viruses and their replication. P.1353-1395". In Field, Knipe,B. N. and Howely, D. N. (eds), Virology, Lippincott Raven Publishers, Philadelphia, USA.

Murphy, B. R. (1993). "Use of live attenuated cold-adapted influenza reassortant virus vaccines in infants, children, young adults and elderly adults. Infect. Dis. Clin. Pract. vol. 2:174-81.

Murphey-Corb, M., Martin, L. N., Davison-Fairburn, B., Montelaro, R. C., Miller, M., West, M., Ohkawa, S., Baskin, G. B., Zhang, J. Y., Putney, S. D., Allison, A. C. and Eppstein, D. A. (1989)."A formalin-inactivated whole SIV vaccine confers protection in macaques". Science, vol. 246 (4935):1293-1297.

Mwau, M., Cebere, I.,Sutton, J., Chikoti, P., Winstone, N., Wee, E. G.-T., Beattie, T., Chen, Y. H., Dorrell, L., McShane, H., Schmidt, C., Brooks, M., Patel, S., Roberts, J., Conlon, C., Rowland-Jones, S. L., Bwayo, J. J., McMichael, A. J. and Hanke, T. (2004)."A human immunodeficiency virus 1 (HIV-1) clade A vaccine in clinical trials: stimulation of HIV-specific T-cell responses by DNA and recombinant modified vaccinia virus Ankara (MVA) vaccines in humans". J. Gen. Virol., vol. 85 (4):911-919.

Nakanishi, Y., Lu, B., Gerard, C. and Iwasaki, A. (2009)."CD8 (+) T lymphocyte mobilization to virus infected tissue requires CD4 (+) T-cell help". Nature, vol. 462:510-513.

Nakaya, Y., Nakaya, T., Park, M. S., Cros, J., Imanishi, J., Palese, P. and García-Sastre, A. (2004)."Induction of cellular immune responses to simian immunodeficiency virus gag by two recombinant negative-strand RNA virus vectors". J. Virol., vol. 78: 9366-9375.

Nascimbeni, M., Perie, L., Chorro, L., Diocou, S., Kreitmann, L., Louis, S., Garderet, L., Fabiani, B., Berger, A., Schmitz, J., Marie, J-P., Molina, T. J., Pacanowski, J., Viard, J. P., Oksenhendler, E., Beq, S., Abehsira-Amar, O., Cheynier, R. and Hosmalin, A. (2009)."Plasmacytoid dendritic cells accumulate in spleens from chronically HIV-infected patients but barely participate in interferon-{alpha} expression". Blood, vol. 113 (24):6112-6119.

Nath, A. (2010)."HIV/AIDS Vaccine: An Update". Indian J. Community Med., vol. 35 (2): 222-225.

Nchinda, G., Amadu, D., Trumpfheller, C., Mizenina, O.,Überla, K. and Steinman, R. M. (2010)."Dendritic cell targeted HIV gag protein vaccine provides help to a DNA vaccine including mobilization of protective CD8+ T cells". Proc. Nati. Acad. Sci. UAS, vol. 107 (9):4281-4286.

Ndhlovu, Z. M., Proudfoot J., Cesa K., Alvino D. M., McMullen A., Vine S., Stampouloglou, E., Piechocka-Trocha, A., Walker, B. D. and Pereyra, F. (2012). Elite controllers with low to absent effector CD8+ T cell responses maintain highly functional, broadly directed central memory responses". J. Virol., vol. 86 (12):6959-6969.

Neumann, G., Fujii, K., Kino, Y. and Kawaoka, Y. (2005). "An improved reverse genetics system for influenza A virus generation and its implications for vaccine production". Proc. Natl. Acad. Sci. USA, vol. 102 (46):16825-16829

Neumann, G., Whitt, M. A. and Kawaoka, Y. (2002)."A decade after the generation of a negative-sense RNA virus from cloned cDNA- what have we learned?". J. Gen.Virol., vol. 83 (11):2635-62.

References

194

Neumann, G. and Kawaoka, Y. (2001)."Reverse genetics of influenza virus". Virology, vol. 287 (2):243-250.

Neumann, G., Watanabe, T., Ito, H., Watanabe, S., Goto, H., Gao, P., Hughes, M., Perez, D. R., Donis, R., Hoffmann, E., Hobom, G. and Kawaoka, Y. (1999)."Generation of influenza A viruses entirely from cloned cDNAs". Proc. Natl. Acad. Sci. U S A., vol. 96 (16):9345-9350.

Newman, P. A., Roungprakhon, S.,Tepjan, S. and Yim, S. (2010)."Preventive HIV vaccine acceptability and behavioral risk compensation among high-risk men who have sex with men and transgenders in Thailand". Vaccine, vol. 28 (4):958-964.

Nguyen, D. H. and Taub, D. (2002)."CXCR4 function requires membrane cholesterol: implications for HIV infection". J. Immunol., vol. 168 (8): 4121-4126.

Nicolson, C., Major, D., Wood, J. M. and Robertson, J. S. (2005)."Generation of influenza vaccine viruses on Vero cells by reverse genetics: an H5N1 candidate vaccine strain produced under a quality system". Vaccine, vol. 23 (22):2943-2952.

Norment, A. M., Bogatzki, L.Y., Gantner, B. N. and Bevan, M. J. (2000)."Murine CCR9, a chemokine receptor for thymus-expressed chemokine that is up-regulated following pre-TCR signaling". J. Immunol., vol. 164:639-648.

Norton, S. D., Zuckerman, L., Urdahl, K. B., Shefner, R., Miller, J. and Jenkins, M. K. (1992)."The CD28 ligand, B7, enhances IL-2 production by providing a costimulatory signal to T cells". J. Immunol., vol. 149 (5):1556-1561.

O’Brien, K. L., Liu, J., King, S. L., Sun, Y. H., Schmitz, J. E., Lifton, M., Hutnick, N., Betts, M. R., Dubey, S. A., Goudsmit, J., Shiver, J. W., Robertson, M. N., Casimiro, D. R. and Barouch, D. H. (2009)."Adenovirus-specific immunity following immunization with an Ad5 HIV-1 vaccine candidate in humans". Nat. Med., vol. 15 (8): 873-875.

Okoye, I. S. and Wilson, M. S. (2011)."CD4+ T helper 2 cells-Microbial triggers, differentiation requirements and effector functions". Immunology, vol. 134: 368-377.

Onodera, T., Takahashi, Y., Yokoi, Y., Ato, M., Kodama, Y., Hachimura, S., Kurosaki, T. and Kobayashi, K. (2012)."Memory B cells in the lung participate in protective humoral immune responses to pulmonary influenza virus reinfection". Proc. Natl. Acad. Sci. U S A, vol. 109:2485-2490.

Ourmanov, I., Bilska, M. and Hirsch, V. M. (2000)."Recombinant modified vaccinia virus ankara expressing the surface gp120 of simian immunodeficiency virus (SIV) primes for a rapid neutralizing antibody response to SIV infection in macaques". J. Virol., vol. 74 (6):2960-2965.

Ostrowski, M. A., Justement, S. J., Ehler, L., Mizell, S. B., Lui, S., Mican, J., Walker, B. D., Thomas, E. K., Seder, R. and Fauci, A. S. (2000). "The Role of CD4+ T cell help and CD40 ligand in the in vitro expansion of HIV-1-specific memory cytotoxic CD8+ T cell responses". J. Immunol, vol. 165 (11):6133-6141.

Pachler, K., Mayr, J. and Vlasak, R. (2010)."A seven plasmid-based system for the rescue of influenza C virus". J. Mol. Genet. Med., vol. 4:239-246.

Pal, R., Venzon, D., Letvin, N. L., Santra, S., Montefiori, D. C., Miller, N. R., Tryniszewska, E., Lewis, M. G., VanCott, T. C., Hirsch, V., Woodward, R., Gibson, A., Grace, M., Dobratz, E., Markham, P., Hel, Z., Nacsa, J., Klein, M., Tartaglia, J. and Franchini, G. (2002)."ALVAC-SIV-gag-pol-env-based vaccination and macaque major histo-

References

195

compatibility complex class I delay simian immunodeficiency virus SIVmac-induced immunodeficiency". J. Virol., vol. 76 (1):292-302.

Palache, A. M., Scheepers, H. S., de Regt, V., van Ewijk, P., Baljet, M., Brands, R. and van Scharrenburg, G. J. (1999)."Safety, reactogenicity and immunogenicity of Madin Darby Canine Kidney cell-derived inactivated influenza subunit vaccine. A meta-analysis of clinical studies". Dev. Biol. Stand., vol. 98:115-125.

Palese, P., Zheng, H., Engelardt, O. G., Pleschka, S. and Garcia-Sastre, A. (1996)."Negative-strand RNA viruses: Genetic engineering and applications". Proc. Natl. Acad. Sci. USA, vol. 93:11354-11358.

Palese, P., Zavala, F., Muster, T., Nussenzweig, R. S. and Garcia-Sastre, A. (1997). "Development of novel influenza virus vaccines and vectors". J. Infect. Dis., vol. 176 (Suppl. 1):S45–S49.

Palese, P. and Shaw, M. L. (2007) "Orthomyxoviridae: The viruses and their replication". In Fields Virology, 5th ed.; Lippincott Williams & Wilkins, Philadelphia, USA, vol 2:1647-1689.

Pantaleo, G., Esteban, M., Jacobs, B. and Tartaglia, J. (2010)."Poxvirus vector-based HIV vaccines." Curr. Opin. HIV AIDS, vol. 5 (5):391-396.

Parr, E. L., Bozzola, J. J. and Parr, M. B. (1998)."Immunity to vaginal infection by herpes simplex virus type 2 in adult mice: characterization of the immunoglobulins in vaginal mucus". J. Reprod. Immunol., vol. 38:15-30.

Parr, M. B., Kepple, L., McDermott, M. R., Drew, M. D., Bozzola, J. J. and Parr, E. L. (1994). "A mouse model for studies of mucosal immunity to vaginal infection by herpes simplex virus type 2". Lab. Investig., vol. 70:369-379.

Patil, A., Gautam, A. and Bhattacharya, J. (2010)."Evidence that Gag facilitates HIV-1 envelope association both in GPI-enriched plasma membrane and detergent resistant membranes and facilitates envelope incorporation onto virions in primary CD4+ T cells". Virol. J., vol. 7 (1):3.

Patterson, L. J. and Robert-Guroff, M. (2008)."Replicating adenovirus vector prime/protein boost strategies for HIV vaccine development". Expert. Opin. Biol Ther., vol. 8 (9): 1347-1363.

Patterson, J., Robey, F. and Robert-Guroff, M. (2000)."An SIV C4 peptide subunit vaccine may enhance viral burden in rhesus macaques after intrarectal challenge with SIVmac251". 7th Conf. Retrovir. Oppor. Infect., vol. 7: 227.

Peeters, M., Courgnaud, V. and Abela, B. (2001)."genetic diversity of lentiviruses in non-human primates". AIDS Rev., vol. 3:3-10.

Petersen, J. L., Morris, C. R. and Solheim, J. C. (2003)."Virus evasion of MHC class I molecule presentation". J. Immunol, vol. 171 (9):4473-4478.

Petrovic, A., Alpdogan, O., Willis, L. M., Eng, J. M., Greenberg, A. S., Kappel, B. J., Liu, C., Murphy, G. J., Heller, G.and van den Brink, M. R. M. (2004)."LPAM (α4β7 integrin) is an important homing integrin on alloreactive T cells in the development of intestinal graft-versus-host disease". Blood, vol. 103 (4):1542-7.

Petrovsky, N. and Aguilar, J. C. (2004)."Vaccine adjuvants: Current state and future trends". Immunol. Cell Biol., vol. 82 (5):488-496.

References

196

Piot, P. (1989)."Genital ulcers, other sexually transmitted diseases, and the sexual transmission of HIV". Br. Med. J., vol. 298 (6674):623-624.

Pitisuttithum, P., Choopanya, K. and Rerk-Ngnam, S. (2010)."HIV-vaccine research and development in Thailand: evolution and challenges". Vaccine, vol. 28 (Supplement 2): B45-B49.

Pitisuttithum, P., Gilbert, P., Gurwith, M., Heyward, W., Martin, M., van Griensven, F., Hu, D., Tappero, J. W. and Bangkok Vaccine Evaluation Group. (2006). "Randomized, double blind, placebo controlled efficacy trial of a bivalent recombinant glycoprotein 120 HIV 1 vaccine among injection drug users in Bangkok, Thailand". J. Infect. Dis., vol. 194 (12):1661-1671.

Plana, M., Garcia, F., Oxenius A., Ortiz, G. M., Lopez, A., Cruceta, A., Mestre, G., Fumero, E., Fagard, C., Sambeat, M. A., Segura, F., Miró, J. M., Arnedo, M., Lopalcos, L., Pumarola, T., Hirschel, B., Phillips, R. E., Nixon, D. F., Gallart, T. and Gatell, J. M . (2004)."Relevance of HIV-1-specific CD4+ helper T-cell responses during structured treatment interruptions in patients with CD4+ T-cell nadir above 400/mm3". J. AIDS, vol. 36 (3):791-799.

Pleschka, S., Jaskunas, R., Engelhardt, O. G., Zürcher, T., Palese, P. and Garcı ́a-Sastre, A. (1996)."A plasmid-based reverse genetics system for influenza A virus". J. Virol., vol. 70: 4188-4192.

Plotkin, S. A. (2009)."Sang froid in a time of trouble: is a vaccine against HIV possible?". J. Int. AIDS Soc., vol 12:2.

Plotkin, S. (1994)."Vaccines for varicella-zoster virus and cytomegalovirus: recent progress". Science, vol. 265 (5177):1383-1385.

Ponta, H., Sherman, L. and Herrlich, P. A. (2003)."CD44:from adhesion molecules to signalling regulators". Nat. Rev. Mol. Cell Biol., vol. 4:33-45.

Poon, L. L. M., Pritlove, D. C., Sharps, J. and Brownlee, G. G. (1998)."The RNA polymerase of influenza virus, bound to the 5' end of virion RNA, acts in cis to polyadenylate mRNA". J. Virol., vol. 72 (10):8214-8219.

Poulet, H., Brunet, S., Boularand, C., Guiot, A.L., Leroy, V., Tartaglia, J., Minke, J., Audonnet, J. C. and Desmettre, P. (2003)."Efficacy of a canarypox virus-vectored vaccine against feline leukaemia". Vet. Rec., vol. 153 (5):141-145.

Pribila, J. T., Quale, A. C., Mueller, K. L. and Shimizu, Y. (2004)."Integrins and T cell-mediated immunity". Annu. Rev. Immunol., vol. 22:157-180.

Priddy, F. H., Brown, D., Kublin, J., Monahan, K., Wright, D. P., Lalezari, J., Santiago, S., Marmor, M., Lally, M., Novak, R. M., Brown, S. J., Kulkarni, P., Dubey, S. A., Kierstead, L. S., Casimiro, D. R., Mogg, R., DiNubile, M. J., Shiver, J. W., Leavitt, R. Y., Robertson, M. N., Mehrotra, D. V., Quirk, E. and Merck V520-016 Study Group. (2008)."Safety and immunogenicity of a replication incompetent adenovirus type 5 HIV 1 clade B gag/pol/nef vaccine in healthy adults". Clin. Infect. Dis., vol. 46 (11):1769-1781.

Proksch, E., Brandner, J. M. and Jensen, J. M. (2008)."The skin: an indispensable barrier". Exp. Dermatol., vol. 17(12):1063-1072.

Pushko, P., Tumpey, T. M., Bu, F., Knell, J., Robinson, R. and Smith, G. (2005)."Influenza virus-like particles comprised of the HA, NA, and M1 proteins of H9N2 influenza virus

References

197

induce protective immune responses in BALB/c mice". Vaccine, vol. 23 (50): 5751-5759.

Qiu, C., Tian, D., Wan, Y., Zhang, W., Qiu, C., Zhu, Z., Ye, R., Song, Z., Zhou, M., Yuan, S., Shi, B., Wu, M., Liu,Y., Gu, S., Wei, J., Zhou, Z., Zhang, X., Zhang, Z., Hu, Y., Yuan, Z. and Xu, J. (2011)."Early adaptive humoral immune responses and virus clearance in humans recently infected with pandemic2009 H1N1 influenza virus". PLoS ONE, vol. 6 (8):e22603.

Quan, F. S., Steinhauer, D., Huang, C., Ross, T. M., Compans, R. W. and Kang, S. M. (2008)."A bivalent influenza VLP vaccine confers complete inhibition of virus replication in lungs". Vaccine, vol. 26:3352-3361.

Quinn, T. C., Wawer, M. J., Sewankambo, N., Serwadda, D., Li, C., Wabwire-Mangen, F., Meehan, M. O., Lutalo, T. and Ronald H. Gray for the Rakai Project Study Group. (2000)."Viral load and heterosexual transmission of human immunodeficiency virus type 1". N. Eng. J. Med., vol. 342 (13):921-929.

Quinn, T., Mann, J., Curran, J. and Piot, P. (1986)."AIDS in Africa: An epidemiologic paradigm". Science, 234:955-963.

Racaniello, V. R. and Baltimore, D. (1981)."Cloned poliovirus complementary DNA is infectious in mammalian cells". Science, vol. 214:916-919.

Raha, T., Cheng, G. S. W. and Green, M. R. (2005)."HIV-1 Tat stimulates transcription complex assembly through recruitment of TBP in the absence of TAFs". PLoS Biol., vol. 3 (2):e44.

Ranasinghe, C., Eyersb, F., Stambas, J., Boyled, D. B., Ramshawa, I. A. and Ramsaye, A. J. (2011)."A comparative analysis of HIV-specific mucosal/systemic T cell immunity and avidity following rDNA/rFPV and poxvirus-poxvirus prime boost immunisations". Vaccine, vol. 29 (16):3008-3020.

Rantala, A., Lajunen, T., Juvonen, R., Bloigu, A., Silvennoinen-Kassinen, S., Peitso, A., Saikku, P., Vainio, O. and Leinonen, M. (2008)."Mannose-binding lectin concentrations, MBL2 polymorphisms, and susceptibility to respiratory tract infections in young men". J. Infect. Dis., vol. 198 (8):1247-1253.

Rayevskaya, M. V and Frankel, F. R. (2001)."Systemic immunity and mucosal immunity are induced against human immunodeficiency virus Gag protein in mice by a new hyperattenuated strain of listeria monocytogenes". J. Virol., vol. 2001:2786-2791.

Riberdy, J. M., Christensen, J. P., Branum, K. and Doherty, P, C. (2000)."Influenza virus-specific CD8 + T-cell diminished primary and secondary responses in CD4-depleted Ig-/- mice". J. Virol., vol. 74 (20):9762.

Reddehase, M. J. and Koszinowski, U. H. (1984)."Significance of herpesvirus immediate early gene expression in cellular immunity to cytomegalovirus infection". Nature., vol. 312 (5992):369-371.

Rerks-Ngarm, S., Pitisuttithum, P., Nitayaphan, S., Kaewkungwal, J., Chiu, J., Paris, R., Premsri, N., Namwat, C., de Souza, M., Adams, E., Benenson, M., Gurunathan, S., Tartaglia, J., McNeil, J. G., Francis, D. P., Stablein, D., Birx, De. L., Chunsuttiwat, S., Khamboonruang, C., Thongcharoen, P., Robb, M. L., Michael, N. L., Kunasol, P. and Kim, J. H. for the MOPH–TAVEG Investigators. (2009)."Vaccination with ALVAC

References

198

and AIDSVAX to Prevent HIV-1 Infection in Thailand". N. Eng. J. Med., vol. 361 (23):2209-2220.

Reusser, P., Cathomas, G., Attenhofer, R., Tamm, M. and Thiel, G. (1999)."Cytomegalovirus (CMV)-specific T cell immunity after renal transplantation mediates protection from CMV disease by limiting the systemic virus load". J. Infect. Dis., vol. 180(2):247-253.

Richert, L., Doussau, A., Lelièvre, J. D., Arnold, V., Rieux, V., Bouakane, A., Lévy, Y., Chêne, G. and Thiébaut, R. for the Vaccine Research Institute. (2014)."Accelerating clinical development of HIV vaccine strategies: methodological challenges and considerations in constructing an optimised multi-arm phase I/II trial design".Trials, 15:68, Centre INSERM U897-Epidemiologie-Biostatistique, Bordeaux, France.

Rickinson, A. B. and Moss, D. J. (2003)."Human cytotoxic T lymphocytes responses to epstein-barr virus infection". Annl. Rev. Immunol., vol. 15 (1):405-431.

Ridley, A. J., Schwartz, M. A., Burridge, K., Firtel, R. A., Ginsberg, M. H., Borisy, G., Parsons, J. T. and Horwitz, A. R. (2003)."Cell migration: integrating signals from front to back". Science, vol. 302:1704-1709.

Riedel, S. (2005)."Edward jenner and the history of smallpox and vaccination". Proc. (Bayl. Univ. Med. Cent.). 18 (1): 21-25.

Rimmelzwaan, G. F., Nieuwkoop, N. J., de Mutsert, G., Boon, A. C. M., Kuiken, T., Fouchier, R. A. M. and Osterhaus, A. D. M. E. (2007)."Attachment of infectious influenza A viruses of various subtypes to live mammalian and avian cells as measured by flow cytometry", Virus Res., vol. 129 (2):175-181.

Robbins, S. L., Cotran, R. S., Kumar, V. and Collins, T. (1998)."Robbins Pathologic Basis of Disease". W. B. Saunders Company, Philadelphia, USA.

Roberts, J. P. (2009)."Fear of the invisible : an investigation of viruses and vaccines HIV and AIDS". Impact Investigative Media Productions, 2nd ed., Bristol, England.

Robey, W. G., Safai, B., Oroszlan, S., Arthur, L. O., Gonda, M. A., Gallo, R. C. and Fischinger, P. J. (1985)."Characterization of envelope and core structural gene products of HTLV-III with sera from AIDS patients". Science., vol. 228 (4699):593-595.

Rodriguez, A. R., Arulanandam, B. P., Hodara, V. L., McClure, H. M., Cobb, E. K., Salas, M. T., White, R. and Murthy, K. K. (2007)."Influence of interleukin-15 on CD8+ natural killer cells in human immunodeficiency virus type 1-infected chimpanzees". J. Gen. Virol., vol. vol. 88(2) 641-651.

Rodrı´guez, B., Sethi, A. K., Cheruvu, V. K., Mackay, W., Bosch, R. J., Kitahata,M., Boswell, S. L. , Mathews, W. C., Bangsberg, D. R., Martin,J., Whalen, C. C., Sieg, S., Yadavalli, S., Deeks, S. G. and Lederman, M. M. (2006)."Predictive value of plasma HIV RNA level on rate of CD4 T-cell decline in untreated HIV infection". JAMA, vol. 296 (12): 1498-1506.

Rodrigues, M., Li, S., Murata, K., Rodriguez, D., Rodriguez, J. R., Bacik, I., Bennink, J. R., Yewdell, J. W., Garcia-Sastre, A. and Nussenzweig, R. S., Esteban, M., Palese, P. and Zavala, F. (1994)."Influenza and vaccinia viruses expressing malaria CD8+ T and B cell epitopes. Comparison of their immunogenicity and capacity to induce protective immunity". J. Immunol., vol. 153 (10):4636-4648.

References

199

Rollman, E., Mason, R. D., Lin, J., Brooks, A. G. and Kent, S. J. (2008)."Protection afforded by live attenuated SIV is associated with rapid killing kinetics of CTLs". J. Med. Primatol., vol. 37 (Suppl 2):24-32.

Roman, E., Miller, E., Harmsen, A., Wiley, J., Von Andrian, U. H., Huston, G. and Swain, S. L. (2002)."CD4 effector T cell subsets in the response to influenza: heterogeneity, migration, and function". J. Exp. Med., vol. 196: 957-968.

Rosé, J. R., Williams, M. B., Rott, L. S., Butcher, E. C. and Greenberg, H. B. (1998). "Expression of the mucosal homing receptor α4β7 correlates with the ability of CD8+ memory T cells to clear rotavirus infection', J. Virol, vol. 72 (1):726-730.

Ross, T. M. and Craigo, J. K. (2010)."Editorial: Hot topic: Animal Lentiviruses". Curr. HIV Res., vol. 8 (1):1.

Rott, L. S., Briskin, M. J., Andrew, D. P., Berg, E. L. and Butcher, E. C. (1996)."A fundamental subdivision of circulating lymphocytes defined by adhesion to mucosal addressin cell adhesion molecule-1. Comparison with vascular cell adhesion molecule-1 and correlation with β7 integrins and memory differentiation". J. Immunol., vol. 156: 3727-3736.

Roumeliotis, G. (2006)."MedImmune cleared to offer Flumist the reverse genetics treatment". Breaking News on Global Pharmaceutical Technology and Manufacturing.

Rowley, J., Monie, A., Hung, C. F. and Wu, T. C. (2009)."Expression of IL-15RA or an IL-15/IL-15RA fusion on CD8+ T cells modifies adoptively transferred T-cell function in cis". Eur. J. Immunol., vol. 39 (2):491-506.

Rubinstein, M. P., Lind, N. A., Filippou, P., Best, J. A., McGhee, P. A. and Goldrath, Ananda W. (2008)."Augmenting CD8+ T cell responses following infection with IL-15/sIL-15Rá complexes. Fed. Am. Soci. Exp. Biol. J., vol. 1070:7.

Rudraraju, R., Surman, S. L., Jones, B. G., Sealy, R., Woodland, D. L. and Hurwitz, J. L. (2012)."Reduced frequencies and heightened CD103 expression among virus-induced CD8(+) T cells in the respiratory tract airways of vitamin A-deficient mice". Clin. Vaccine Immunol., vol. 19: 757-765.

Sallusto, F., Lenig, D., Forster, R., Lipp, M. and Lanzavecchia, A. (1999)."Two subsets of memory T lymphocytes with distinct homing potentials and effector functions". Nature, vol. 401(6754):708-712.

Sambhara, S., Kurichh, A., Miranda, R., Tumpey, T., Rowe, T., Renshaw, M., Arpino, R.,Tamane, A., Kandil, A. and James, O. (2001)."Heterosubtypic immunity against human influenza A viruses, including recently emerged avian H5 and H9 viruses, induced by FLU-ISCOM vaccine in mice requires both cytotoxic T-lymphocyte and macrophage function". Cell. Immunol., vol. 211:143-153.

Sandau, M. M., Schluns, K. S., Lefrancois, L. and Jameson, S. C. (2004)."Cutting edge: transpresentation of IL-15 by bone marrow-derived cells necessitates expression of IL-15 and IL-15R alpha by the same cells". J. Immunol., vol. 173 (11):6537-6541.

Sandström, E., Nilsson, C., Hejdeman, B., Bråve, A., Bratt, G., Robb, M., Cox, J., Vancott, T., Marovich, M., Stout, R., Aboud, S., Bakari, M., Pallangyo, K., Ljungberg, K., Moss, B., Earl, P., Michael, N., Birx, D., Mhalu, F., Wahren, B. and Biberfeld, G. (2008)."Broad immunogenicity of a multigene, multiclade HIV-1 DNA vaccine

References

200

boosted with heterologous HIV-1 recombinant modified vaccinia virus Ankara". J. Infect. Dis., vol. 198 (10):1482-1490.

Sanger, F., Coulson, A. R., Barrell, B. G., Smith, A. J. and Roe, B. A. (1980)."Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing". J. Mol. Biol., vol. 143 (2):161-178.

Santra, S., Seaman, M. S., Xu, L., Barouch, D. H., Lord, C. I., Lifton, M. A., Gorgone, D. A., Beaudry, K. R., Svehla, K., Welcher, B., Chakrabarti, B., Huang, Y., Yang, Z. Y., Mascola, J., Nabel, G. J. and Letvin, N. L. (2005)."Replication-defective adenovirus serotype 5 vectors elicit durable cellular and humoral immune responses in non-human primates". J. Virol., vol. 79(10):6516-6522.

Sattentau, Q. J. (2013)."Envelope glycoprotein trimers as HIV-1 vaccine immunogens". Vaccines, vol. 1 (4): 497-512.

Satterlee, B. (2008)."Production of H5N1 avian influenza virus vaccine by plasmid-based reverse genetics technology". Basic Biotech. eJournal., vol. 4:93-98.

Schlickum, S., Sennefelder, H., Friedrich, M., Harms, G., Lohse, M. J., Kilshaw, P. and Schön, M. P. (2008)."Integrin alpha E (CD103) beta 7 influences cellular shape and motility in a ligand-dependent fashion". Blood, vol. 112 (3):619-625.

Schnell, M. J., Foley, H. D., Siler, C. A., McGettigan, J. P., Dietzschold, B. and Pomerantz, R. J. (2000)."Recombinant rabies virus as potential live-viral vaccines for HIV-1". Proc. Natl. Acad. Sci. U S A., vol. , vol. 97 (7):3544-3549.

Schnell, M. J., Mebatsion, T. and Conzelmann, K. K. (1994)."Infectious rabies viruses from cloned cDNA". EMBO J., vol. 13:4195-4203.

Schnorrer, P., Behrens, G. M. N., Wilson, N. S., Pooley, J. L., Smith, C. M., El-Sukkari, D., Davey, G., Kupresanin, F., Li, M., Maraskovsky, E., Belz, G. T., Carbone, F. R., Shortman, K., Heath, W. R.and Villadangos, J. A. (2006)."The dominant role of CD8+ dendritic cells in cross-presentation is not dictated by antigen capture". Proc. Nat. Acad. Sci. USA, vol. 103 (28):10729-10734.

Schon, M. P., Arya, A., Murphy, E. A., Adams, C. M., Strauch, U. G., Agace, W. W., Marsal, J., Donohue, J. P., Her, H., Beier, D. R., Olson, S., Lefrancois, L., Brenner, M. B., Grusby, M. J., Parker, C. M. (1999). "Mucosal T lymphocyte numbers are selectively reduced in integrin alpha E (CD103)-deficient mice". J. Immunol., vol. 162:6641-6649.

Schluns, K. S., Williams, K., Ma, A., Zheng, X. X. and Lefrancois, L. (2002)."Cutting edge: requirement for IL-15 in the generation of primary and memory antigen-specific CD8 T cells". J. Immunol., vol. 168:4827-4831.

Screaton, G. R., Bell, M. V., Jackson, D. G., Cornelis, F. B., Gerth, U. and Bell, J. I. (1992)."Genomic structure of DNA encoding the lymphocyte homing receptor CD44 reveals at least 12 alternatively spliced exons". Proc. Natl. Acad. Sci. USA, vol. 89:12160-12164.

Scriba, T. J., zur Megede, J., Glashoff, R. H., Treurnicht, F. K., Barnett, S. W. and van Rensburg, E. J. (2005)."Functionally-inactive and immunogenic Tat, Rev and Nef DNA vaccines derived from sub-Saharan subtype C human immunodeficiency virus type 1 consensus sequences". Vaccine, vol. 23 (9):1158.

Selig, S. M., Arthos, J., Cicala, C., White, A. A., Ravindranath, H., Van, R. D., Steenbeke, T. D., Machado, E., Hanback, M., Hanback, D., Rabin, R. and Fauci, A. S. (2001)."The

References

201

role of the CD4 receptor versus HIV coreceptors in envelope-mediated apoptosis in peripheral blood mononuclear cells". Conf. Retrovir. Oppor. Infect. 8th Chic Ill, vol. (8):75.

Senne, D. A., Panigrahy, B., Kawaoka, Y., Pearson, J. E., Süss, J., Lipkind, M., Kida, H. and Webster, R. G. (1996)."Survey of the hemagglutinin (HA) cleavage site sequence of H5 and H7 avian influenza viruses: amino acid sequence at the HA cleavage site as a marker of pathogenicity potential". Avian Dis., vol. 40 (2):425-437.

Şenocak, E., Oğuz, K. K., Özgen, B., Kurne, A., Özkaya, G., Ünal, S. and Cila, A. (2009)."Imaging features of CNS involvement in AIDS". Diagn. Interv Radiol., vol. 16 (3):193-200.

Sexton, A., De Rose, R., Reece, J. C., Alcantara, S., Loh, L.,Moffat, J. M., Laurie, K., Hurt, A., Doherty, P. C., Turner, S. J.,Kent, S. J. and Stambas, J. (2009)."Evaluation of recombinant influenza virus-simian immunodeficiency virus vaccines in macaques". J. Virol., vol. 83 (15):7619-7628.

Shattock, R. J., Haynes, B. F., Pulendran, B., Flores, J. and Esparza, J. (2008)."Improving defences at the portal of HIV entry: mucosal and innate immunity". PLoS Med., vol. 5 (4):e81.

Shaw, S. and Biddison, W. E. (1979)."HLA-linked genetic control of the specificity of human cytotoxic T-cell responses to influenza virus". J. Exp. Med., vol. 149:565-75.

Shevkani, M. B. K., Kumar, P., Purohit, H., Nihalani, U. and Shah, A. (2011)."An overview of post exposure prophylaxis for HIV in health care personals: Gujarat scenario". Indian J. Sex Transm. Dis., vol. 32 (1):9-13.

Shortridge, K. F., Zhou, N. N., Guan, Y., Gao, P., Ito, T., Kawaoka, Y., Kodihalli, S., Krauss, S., Markwell, D., Murti, K. G., Norwood, M., Senne, D., Sims, L.,Takada, A., Webster, R. (1998)."Characterization of avian H5N1 influenza viruses from poultry in Hong Kon"g. Virology, vol. 252 (2):331-342.

Shugars, D. C. (1999)."Endogenous mucosal antiviral factors of the oral cavity". J. Infect. Dis., vol. 179 (3):S431-S435.

Smith, K. (2007)."The use of plasmid-based reverse genetics to generate influenza virus strains for improved vaccine production". Basic Biotechnol. eJournal, vol. 3:123-130.

Smith, D. J., Lapedes, A. S., de Jong, J. C., Bestebroer, T. M., Rimmelzwaan, G. F. Osterhaus, A. D. and Fouchier, R. A. (2004)."Mapping the antigenic and genetic evolution of influenza virus". Science, vol. 305: 371-376.

Someya, K., Cecilia, D., Ami, Y., Nakasone, T., Matsuo,K., Burda, S., Yamamoto, H., Yoshino, N., Kaizu, M., Ando, S., Okuda, K., Zolla-Pazner, S., Yamazaki, S.,Yamamoto, N. and Honda, M. (2005)."Vaccination of rhesus macaques with recombinant mycobacterium bovis bacillus calmette-guérin Env V3 elicits neutralizing antibody-mediated protection against simian-human immunodeficiency virus with a aomologous but not a heterologous V3 motif". J. Virol., vol. 79 (3):1452-1462.

Spear, G. T. (1993)."Interaction of non-antibody factors with HIV in plasma". AIDS, vol. 7 (9):1149-1158.

Sreepian, A., Permmongkol, J., Kantakamalakul, W., Siritantikorn, S., Tanlieng, N. and Sutthent, R. (2009)."HIV-1 neutralization by monoclonal antibody against conserved

References

202

region 2 and patterns of epitope exposure on the surface of native viruses". J. Imm. Based Therap. Vacc., vol. 7:5.

Spring, F. A., Dalchau, R., Daniels, G. L., Mallinson, G., Judson, P. A., Parsons, S. F., Fabre, J. W. and Anstee, D. J. (1988)."The Ina and Inb blood group antigens are located on a glycoprotein of 80,000 MW (the CDw44 glycoprotein) whose expression is influenced by the In (Lu) gene". Immunology, vol. 64 (1):37-43.

Staats, H. F., and Ennism F. A. (1999)."IL-1 is an effective adjuvant for mucosal and systemic immune responses when coadministered with protein immunogens". J. Immunol., vol. 162:6141-6147.

Staats, H. F., Bradney, C. P., Gwinn, W. M., Jackson, S. S., Sempowski, G. D., Liao, H. X., Letvin, N. and Haynes, B. F. (2001)."Cytokine requirements for induction of systemic and mucosal CTL after nasal immunization". J. Immunol., vol. 167 (9):5386-5394.

Staczek, J., Gilleland, H. E., Gilleland, L. B., Harty, R. N., Garcia-Sastre, A., Engelhardt, O. G. and Palese, P. (1998)."A chimeric influenza virusexpressing an epitope of outer membrane protein F of Pseudomonas aeruginosa affords protection against challenge with P. aeruginosa in a murine-model of chronic pulmonary infection". Infect. Immun., vol. 66:3990-3994.

Staeheli, P., Grob, R., Meier, E., Sutcliffe, J. G. and Haller, O. (1988)."Influenza virus-susceptible mice carry Mx genes with a large deletion or a nonsense mutation". Mol. Cell Biol., vol. 8 (10):4518-4523.

Steel, J. C., Waldmann, T. A. and Morris, J. C. (2012)."Interleukin-15 biology and its therapeutic implications in cancer". Trends Pharmacol. Sci., vol. 33 (1):35-41.

Steers, N. J., Peachman, K. K., McClain, S., Alving, C. R. and Rao, M. (2009)."Liposome-encapsulated HIV-1 Gag p24 containing lipid A induces effector CD4+ T-cells, memory CD8+ T-cells, and pro-inflammatory cytokines". Vaccine, vol. 27(49):6939-6949.

Steinhauer, D. A., Langley, W.A.,Talekar, G. and Li, Z. (2009)."P12-07. novel HIV vaccines using chimeric influenza HA vectors". Retrovirology,vol. 6 (Suppl 3):173.

Steinhauer, D. A. and Skehel, J. J. (2002)."Genetics of influenza viruses". Annu. Rev. Genet. 36:305-332.

Stevceva, L., Kelsall, B., Nacsa, J., Moniuszko, M., Hel, Z., Tryniszewska, E. and Franchini, G. (2002). "Cervicovaginal lamina propria lymphocytes: phenotypic characterization and their importance in cytotoxic T-lymphocyte responses to simian immunodeficiency virus SIVmac251". J. Virol., vol. 76:(9-18).

Stoff-Khalili, M. A., Dall, P. and Curiel, D.T. (2006)."Gene therapy for carcinoma of the breast". Cancer Gene Therap., vol. 13 (7):633-647.

Stamatatos, L., Morris, L., Burton, D. R., and Mascola, J. R. (2009)."Neutralizing antibodies generated during natural HIV-1 infection: good news for an HIV-1 vaccine?". Nat. Med., vol. 15:866-870.

Strauch, U. G., Lifka, A., Gosslar, U., Kilshaw, P. J., Clements, J. and Holzmann, B. (1994). "Distinct binding specificities of integrins alpha 4 beta 7 (LPAM-1), alpha 4 beta 1 (VLA-4), and alpha IEL beta 7". Int. Immunol., vol. 6 (2):263-275.

References

203

Streeck, H. and Nixon, D. F. (2010)."T cell immunity in acute HIV-1 infection". J. Infect. Dis., vol. 202 (Suppl 2):S302-8.

Streeck, H., Jolin, J. S., Qi, Y., Yassine-Diab, B., Johnson, R. C., Kwon, D. S., Addo, M. M., Brumme, C., Routy, J. P., Little, S., Jessen, H. K., Kelleher, A. D., Hecht, F. M., Sekaly, R. P., Rosenberg, E. S., Walker, B. D., Carrington, M. and Altfeld, M. (2009)."Human immunodeficiency virus type 1-specific CD8+ T-cell responses during primary infection are major determinants of the viral set point and loss of CD4+ T cells". J. Virol., vol. 83 (15):7641-7648.

Stroeken, P. J. M. (2002)."Adhesion molecules in lymphoma cell migration and metastasis. The role of the beta1 integrin cytoplastic domain". Ponsen & Looijen, the library of the University ofAmsterdam, Faculty of Medicine,Amsterdam.

Strugnell, R. A. and Wijburg, O. L. (2010)."The role of secretory antibodies in infection immunity". Nat. Rev. Microbiol., vol. 8: 656-667.

Strutt, T. M., McKinstry, K. K., Dibble, J. P., Winchell, C., Kuang, Y., Curtis, J. D., Huston, G., Dutton, R. W. and Swain, S. L. (2010)."Memory CD4+ T cells induce innate responses independently of pathogen". Nat. Med., vol. 16: 558-564.

Sui, Y., Zhu, Q., Gagnon, S., Dzutsev, A., Terabe, M., Vaccari, M., Venzon, D., Klinman, D., Strober, W., Kelsall, B., Franchini, G., Belyakov, I. M. and Berzofsky, J. A. (2010). "Innate and adaptive immune correlates of vaccine and adjuvant-induced control of mucosal transmission of SIV in macaques". Proc. Natl. Acad. Sci. USA, vol. 107 (21):9843-9848.

Suvas, P. K., Dech, H. M., Sambira, F., Zeng, J., and Onami, T. M. (2007)."Systemic and mucosal infection program protective memory CD8 T cells in the vaginal mucosa1". J. Immunol., vol. 179:8122-8127.

Svensson, M., Johansson-Lindbom, B., Zapata, F., Jaensson, E., Austenaa, L.M., Blomhoff, R. and Agace, W.W. (2008)."Retinoic acid receptor signaling levels and antigen dose regulate gut homing receptor expression on CD8+ T cells". Mucosal Immunol., vol. 1 (1):38-48.

Takahashi, M. N., Rolling, J. A. and Owen, K. E. (2010)."Characterization of transgene expression in adenoviral vector-based HIV-1 vaccine candidates". Virol. J., vol. 7 (1):39.

Takasuka, N., Enami, M., Itamura, S. and Takemori, T. (2002). "Intranasal inoculation of a recombinant influenza virus containing exogenous nucleotides in the NS segment induces mucosal immune response against the exogenous gene product in mice". Vaccine, vol. 20: 1579-1585.

Tamkun, J. W., DeSimone, D. W., Fonda, D., Patel, R. S., Buck, C., Horwitz, A. F. and Hynes, R. O. (1986)."Structure of integrin, a glycoprotein involved in the transmembrane linkage between fibronectin and actin". Cell, vol. 46 (2):271-282.

Tamura, S. and Kurata, T. (2004)."Defense mechanisms against influenza virus infection in the respiratory tract mucosa"'. Jpn. J. Infect. Dis., vol. 57:236-247.

Tamura, S., Iwasaki, T., Thompson, A. H., Asanuma, H., Chen, Z., Suzuki, Y., Aizawa, C. and Kurata, T. (1998)."Antibody-forming cells in the nasal-associated lymphoid tissue during primary influenza virus infection". J. Gen. Virol., vol. 79 (2):291-299.

References

204

Tamura, S. I., Miyata, K., Matsuo, K., Asanuma, H., Takahashi, H., Nakajima, K., Suzuki, Y., Aizawa, C. and Kurata, T. (1996)."Acceleration of influenza virus clearance by Th1 cells in the nasal site of mice immunized intranasally with ajduvant-combined recombinant nucleoprotein". J. Immunol., vol. 156:3892-3900.

Tamura, S. I. , Ito, Y., Asanuma, H., Hirabayashi, Y., Suzuki, Y., Nagamine, T., Aizawa, C. and Kurata, T. (1992)."Cross-protection against influenza virus infection afforded by trivalent inactivated vaccines inoculated intranasally with cholera toxin B subunit". J. Immunol., vol. 149:981-988.

Tan, X., Sande, J. L., Pufnock, J. S., Blattman, J. N. and Greenberg P. D. (2011)."Retinoic acid as a vaccine adjuvant enhances CD8+ T cell response and mucosal protection from viral challenge". J. Virol., vol. 85 (16):8316-8327.

Tang, X., Du, Y., Sun, C., Chen, L., Zhang, L. and Chen, Z. (2012)."Mucosal prime with a replicating vaccinia-based vaccine promotes mucosal immunity against SIV". Retrovirol., vol. 9 (Suppl 2):P189.

Tang, V. A. and Rosenthal, K. L. (2010)."Intravaginal infection with herpes simplex virus type-2 (HSV-2) generates a functional effector memory T cell population that persists in the murine genital tract". J. Reprod. Immunol., vol. 87 (1-2):39-44.

Taniguchi, T., Palmieri, M. and Weissmann, C. (1978)."QB DNA-containing hybrid plasmids giving rise to QB phage formation in the bacterial host". Nature, vol. 247:223-228.

Tannock, G. A., Paul, J. A. and Barry, R. D. (1984)."Relative immunogenicity of the cold-adapted influenza virus A/Ann Arbor/6/60 (A/AA/6/60-ca), recombinants of A/AA/6/60-ca, and parental strains with similar surface antigens". Infect. Immun., vol. 43 (2):457.

Tartaglia, J., Jarrett, O., Neil, J. C., Desmettre, P. and Paoletti, E. (1993)."Protection of cats against feline leukemia virus by vaccination with a canarypox virus recombinant, ALVAC-FL". J. Virol., vol. 67 (4):2370-2375.

Taubenberger, J. K. (2006)."Influenza hemagglutinin attachment to target cells: ‘birds do it, we do it". Future Virol., vol. 1 (4):415-418.

Taubenberger, J. K. and Morens, D. M. (2008). "The pathology of influenza virus infections". Ann. Rev. Pathol., vol. 3:499-522.

Teijaro, J. R., Turner, D., Pham, Q., Wherry, E. J., Lefrancois, L. and Farber, D. L. (2011). "Cutting edge:Tissue-retentive lung memory CD4 T cells mediate optimal protection to respiratory virusinfection". J. Immunol., vol. 187:5510-5514.

Thomas, P. G., Keating, R., Hulse-Post, D. J. and Doherty, P. C. (2006)."Cell-mediated protection in influenza infection". Emerg Infect Dis., vol. 12 (1):48-54.

Tomescu, C., Abdulhaqq, S. and Montaner, L. J. (2011)."Evidence for the innate immune response as a correlate of protection in human immunodeficiency virus (HIV)-1 highly exposed seronegative subjects (HESN)". Clin. Exp. Immunol., vol. 164:158-169.

Tong, S., Li, Y., Rivailler, P., Conrardy, C., Castillo, D. A., Chen, L. M., Recuenco, S., Ellison, J. A., Davis, C. T. and York, I. A. (2012)."A distinct lineage of influenza A virus from bats". Proc. Natl. Acad. Sci. U. S. A., vol.,109:4269-4274.

References

205

Topham, D. J., Tripp, R. A., Hamilton-Easton, A. M., Sarawar, S. R. and Doherty, P. C. (1996)."Quantitative analysis of the influenza virus-specific CD4T cell memory in the absence of B cells and Ig". J. Immunol., vol. 157:2947-2952.

Topham, D. J., Tripp, R. A. and Doherty, P. C. (1997)."CD8+ T cells clear influenza virus by perforin or Fas-dependent processes". J. Immunol., vol. 159:5197-5200.

Tran, T. H., Nguyen, T. L., Nguyen, T.D., Luong, T. S., Pham, P. M., Nguyen, V., Pham, T. S., Vo, C. D., Le, T. Q., Ngo, T. T., Dao, B. K., Le, P. P., Nguyen, T. T., Hoang, T. L., Cao, V. T., Le, T. G., Nguyen, D. T., Le, H. N., Nguyen, K. T., Le, H. S., Le, V. T., Christiane, D., Tran, T. T., Menno, de J., Schultsz, C., Cheng, P., Lim, W., Horby, P., Farrar, J. and World Health Organization International Avian Influenza Investigative Team. (2004)."World Health Organization International Avian Influenza Investigative Team. Avian influenza A (H5N1) in 10 patients in Vietnam". N. Engl. J. Med., vol. 350:1179-1188.

Tripp, R. A., Sarawar, S. R. and Doherty, P. C. (1995)."Characteristics of the influenza virus-specific CD8+ T cell response in mice homozygous for disruption of the H-2lAb gene". J. Immunol., vol.155: 2955-2959.

Tritel, M., Stoddard, A. M., Flynn, B. J., Darrah, P. A., Wu, C. Y., Wille, U., Shah, J. A., Huang, Y., Xu, L., Betts, M.R, Nabel, G. J. and Seder, R. A. (2003)."Prime-Boost vaccination with HIV-1 Gag protein and cytosine phosphate guanosine oligo-deoxy-nucleotide, followed by adenovirus, induces sustained and robust humoral and cellular immune responses". J. Immunol., vol. 171 (5):2538-2547.

Trivedi, H. N., Plummer, F. A., Anzala, A. O., Njagi, E., Bwayo, J. J., Ngugi, E. N., Embree, J. E. and Hayglass, K. T. (2001)."Resistance to HIV-1 infection among African sex workers is associated with global hyporesponsiveness in interleukin 4 production". FASEB J., vol. 15: 1795-1797.

Tung, W., Bakar, S., Sekawi, Z. and Rosli, R. (2007)."DNA vaccine constructs against enterovirus 71 elicit immune response in mice". Genetic Vaccines Ther., vol. 5 (1):6.

Turner, S. J., Olivas, E., Gutierrez, A., Diaz, G. and Doherty, P.C. (2007)."Disregulated influenza A virus-specific CD8+ T cell homeostasis in the absence of IFN-gamma signaling". J. Immunol., vol. 178:7616-7622.

Ulmer, J. B., DeWitt, C. M., Chastain, M., Friedman, A., Donnelly, J. J., McClements, W. L., Caulfield, M. J., Bohannon, K. E., Volkin, D. B. and Evans, R. (1999)."Enhancement of DNA vaccine potency using conventional aluminum adjuvants". Vaccine, vol. 18 (1-2):18-28.

UNAIDS (2013)."Global AIDS response progress reporting 2013:Construction of core indicators for monitoring,the 2011 UN Political Declaration on HIV/AIDS". Joint United Nations Programme on HIV/AIDS (UNAIDS).

UNAIDS (2012)."Global report: UNAIDS report on the global AIDS epidemic". Joint United Nations Programme on HIV/AIDS (UNAIDS).

UNAIDS (2010)."UNAIDS Report on the global AIDS epidemic". Joint United Nations Programme on HIV/AIDS(UNAIDS).

Uyeki, T. M. (2009)."Human Infection with Highly Pathogenic Avian Influenza A (H5N1) Virus: Review of Clinical Issues". Clin. Infect. Dis., vol. (2009) 49 (2):279-290.

References

206

Vaccari, M., Poonam, P. and Franchini, G. (2010)."Phase III HIV vaccine trial in Thailand: a step toward a protective vaccine for HIV". Expert. Rev.Vaccines, vol. 9 (9):997-1005.

Van Baalen, C. A., Kwa, D., Verschuren, E. J., Reedijk, M. L., Boon, A. C. and De Mutsert, G. (2005)."Fluorescent antigen-transfected target cell cytotoxic T lymphocyte assay for ex vivo detection of antigen-specific cell-mediated cytotoxicity". J. Infect. Dis., vol. 92 (7):1183-1190.

van de Sandt, C. E., Kreijtz, J. H. C. M. and Rimmelzwaan, G. F. (2012)."Evasion of influenza A viruses from innate and adaptive immune responses". Viruses 2, vol. 4:1438-1476.

VanCott, T. C., Mascola, J. R., Kaminski, R. W., Kalyanaraman, V., Hallberg, P. L., Burnett, P. R., Ulrich, J. T., Rechtman, D. and Birx, D. L. (1997)."Antibodies with specificity to native gp120 and neutralization activity against primary human immunodeficiency virus type 1 isolates elicited by immunization with oligomeric gp160". J. Virol., vol. 71 (6):4319-4330.

van Riel, D., Leijten, L. M., van der Eerden, M., Hoogsteden, H. C., Boven, L.A., Lambrecht, B. N., Osterhaus, A. D. and Kuiken, T. (2011)."Highly pathogenic avian influenza virus H5N1 infects alveolar macrophages without virus production or excessive TNF-alpha induction". PLoS Pathog., vol. 7 (6) e1002099.

Varmus, H. (1988)."Regulation of HIV and HTLV gene expression". Genes Dev., vol. 2:1055-1062.

Vasan, S., Schlesinger, S. J., Huang, Y., Hurley, A., Lombardo, A., Chen, Z., Than, S., Adesanya P., Bunce C., Boaz M., Boyle R., Sayeed E., Clark L., Dugin D., Schmidt C., Song Y., Seamons, L., Dally, L., Ho, M., Smith, C., Markowitz, M., Cox, J., Gill, D. K., Gilmour, J., Keefer, M. C., Fast, P. and Ho, D. D. (2010)."Phase 1 safety and immunogenicity evaluation of ADVAX, a multigenic, DNA-based clade C/B' HIV-1 candidate vaccine". PLoS ONE, vol. 5 (1):e8617.

Veazey, R. S. (2005)."Protection of macaques from vaginal SHIV challenge by vaginally delivered inhibitors of virus-cell fusion". Nature, vol. 438:99-102.

Verbist, K. C., Rose, D. L., Cole, C. J., Field, M. B. and Klonowski, K. D. (2012)."IL-15 participates in the respiratory innate immune response to influenza virus infection". PLoS ONE, vol. 7 (5):e37539.

Verbist, K. C., Cole, C. J., Field, M. B.and Klonowski, K. D. (2011)."A role for IL-15 in the migration of effector CD8 T cells to the lung airways following influenza infection". J. Immunol., vol. 186 (1):174-182.

Verhoeven, D., Teijaro, J. R. and Farber, D. L. (2009)."Pulse-oximetry accurately predicts lung pathology and the immune response during influenza infection". Virology, vol. 390:151-156.

Vieillard, V., Costagliola, D., Simo Donna L.n, A., Debré, P. and French Asymptomatiques à Long Terme (ALT) Study Group. (2006)."Specific adaptive humoral response against a gp41 motif inhibits CD4 T-cell sensitivity to NK lysis during HIV-1 infection". AIDS, vol. 20 (14):1795-1804.

Viganò, A., Zuccotti, G., Pacei, M., Erba, P., Castelletti, E., Giacomet, V., Amendola, A., Pariani, E., Tanzi, E. and Clerici, M. (2008)."Humoral and cellular response to influenza vaccine in HIV-infected children with full viroimmunologic response to antiretroviral therapy". J. AIDS, vol. 48 (3):289-296.

References

207

Voisset, C., Weiss, R. A. and Griffiths, D. J. (2008)."Human RNA "Rumor" viruses: the search for novel human retroviruses in chronic disease". Microbiol. Molec. Biol. Rev., vol. 72 (1):157-196.

Voronin, Y., Manrique, A. and Bernstein, A. (2010)."The future of HIV vaccine research and the role of the Global HIV Vaccine Enterprise". Curr. Opin. HIV AIDS, vol. 5 (5):414-420.

Wahren, B., Biswas, P., Borggren, M., Coleman, A., Da Costa, K., De Haes, W., Dieltjens, T., Dispinseri, S., Grupping, K., Hallengard, D., Hornig, J., Klein, K., Mainetti, L., Palma, P., Reudelsterz, M., Seifried, J., Selhorst, P., Skold, A., van Gils, M., Weber, C., Shattock, R.and Scarlatti, G. (2010)."Rational design of HIV vaccine and microbicides: report of the EUROPRISE annual conference". J. Transl. Med., vol. 8 (1):72.

Waldmann, T. A. and Tagaya, Y. (1999)."The multifaceted regulation of interleukin-15 expression and the role of this cytokine in NK cell differentiation and host response to intracellular pathogens". Annu. Rev. Immunol., vol. 17:19-49.

Walker, B. D., and Korber, B. T. (2001)."Immune control of HIV: the obstacles of HLA and viral diversity". Nat. Immunol. vol. 2:473-475.

Wallace, A., West, K., Rothman, A. L., Ennis, F. A., Lu, S. and Wang, S. (2013)."Post-translational intracellular trafficking determines the type of immune response elicited by DNA vaccines expressing Gag antigen of Human Immunodeficiency Virus Type 1 (HIV-1)". Hum. Vacc. Immuno.ther., vol. 9 (10):2095-2102.

Webby, R. J., Andreansky, S., Stambas, J., Rehg, J. E., Webster, R. G., Doherty, P. C. and Turner, S. J. (2003)."Protection and compensation in the influenza virus-specific CD8+ T cell response". Proc. Natl. Acad. Sci. USA, vol. 100 (12):7235–7240.

Weber, F., Goldmann, C., Krämer, M., Kaup, F. J., Pickhardt, M., Young , P., Petry, H., Weber, T. and Lüke, W. (2001)."Cellular and humoral immune response in progressive multifocal leukoencephalopathy". Ann. Neurol., vol. 49 (5):636-642.

Weinberg A., Quinones-Mateu, M. E., Lederman, M. M. (2006)."Role of human beta-defensins in HIV infection". Adv. Dent. Res., vol. 19 (1):42-48.

Weiss, R. A. (2006)."The discovery of endogenous retroviruses". Retrovirology, vol. 3 (1):67.

Weiss, R. A. (2001)."Gulliver's travels in HIV land". Nature, vol. 410 (6831):963-967.

Wermers, J. D., McNamee, E. N., Wurbel, M. A., Jedlicka, P.and Rivera–Nieves, J. (2011). "The Chemokine receptor CCR9 is required for the T-cell-mediated regulation of chronic ileitis in mice"'. Gastroenterology, vol. 140 (5):1526-35.

Wiedow, O. and Meyer-Hoffert, U. (2005)."Neutrophil serine proteases:potential key regulators of cell signalling during inflammation". J. Int. Med., vol. 257:319-328.

Wijesundara, D., Jackson, R., Tscharke, D. and Ranasinghe, C. (2013)."IL-4 and IL-13 mediated down-regulation of CD8 expression levels can dampen anti-viral CD8+ T cell avidity following HIV-1 recombinant pox viral vaccination". Vaccine, vol. 31 (41): 4548-4555.

Wit, E., Spronken, M. I. J., Bestebroer, T. M., Rimmelzwaan, G. F., Osterhaus, A. D. M. E. and Fouchier, R. A. M. (2004)."Efficient generation and growth of influenza virus A/PR/8/34 from eight cDNA fragments", Virus Res., vol. 103 (1-2):155-161.

References

208

Wodarz, D. (2008)."Immunity and protection by live attenuated HIV/SIV vaccines". Virology, vol.378(2):299-305.

Wolbank, S., Kunert, R., Stiegler, G. and Katinger, H. (2003). "Characterization of human class-witched polymeric (immunoglobulin M [IgM] and IgA) anti-human immunodeficiency virus type 1 antibodies 2F5 and 2G12". J. Virol., vol. 77:4095-4103.

Wright, P. E. and Webster, R. G. (2001)."Orthomyxoviruses". In Knipe, D. M., Howley, P.M.,Griffin, D.E., Lamb, R. A., Martin, M. A., Roizman, B. and Straus,S. E. (ed), Field's Virology, 4th edition, PA: Lippincott Williams & Wilkins, Philadelphia.

Wurbel, M. A., McIntire, M. G., Dwyer, P. and Fiebiger, E. (2011). CCL25/CCR9 interactions regulate large intestinal inflammation in a murine model of acute colitis. PLoS ONE, vol. 6 (1):e16442.

Wurbel, M. A., Malissen, M., Guy-Grand, D., Meffre, E., Nussenzweig, M. C.,Richelme, M., Carrier, A. and Malissen, B. (2001)."Mice lacking the CCR9 CC-chemokine receptor show a mild impairment of early T- and B-cell development and a reduction in T-cell receptor γδ(+) gut intraepithelial lymphocytes". Blood, vol. 98:2626-2632.

Wyand, M. S., Manson, K. H., Garcia-Moll, M., Montefiori, D. and Desrosiers, R. C. (1996). "Vaccine protection by a triple deletion mutant of simian immunodeficiency virus". J. Virol., vol. 70:3724-33.

Wyde, P. R., Wilson, M. R. and Cate, T. R. (1982)."Interferon production by leukocytes infiltrating the lungs of mice during primary influenza virus infection". Infect. Immunol., vol. 38:1249-1255.

Xiong, J. P., Stehle, T., Diefenbach, B., Zhang, R., Dunker, R., Scott, D. L., Joachimiak, A., Goodman, S. L. and Arnaout, M. A. (2001)."Crystal Structure of the Extracellular Segment of Integrin αVβ3". Science, vol. 294 (5541):339-345.

Xu, R., Megati, S., Roopchand, V., Luckay, A., Masood, A., Garcia-Hand, D., Rosati, M., Weiner, D. B., Felber, B. K., Pavlakis, G. N., Sidhu, M. K., Eldridge, J. H. and Egan, M. A. (2008)."Comparative ability of various plasmid-based cytokines and chemokines to adjuvant the activity of HIV plasmid DNA vaccines". Vaccine, vol. 26 (37):4819-4829.

Yajima, T., Yoshihara, K., Nakazato, K., Kumabe, S., Koyasu, S., Sad, S., Shen, H., Kuwano, H., Yoshikai, Y. (2006)."IL-15 regulates CD8+ T cell contraction during primary infection". J. Immunol., vol. 176:507-515.

Yamamoto, H. and Matano T. (2008)."Anti-HIV adaptive immunity: determinants for viral persistence". Rev. Med. Virol., vol. 18:293-303.

Yang, Y., Cardarelli, P. M., Lehnert, K., Rowland, S.and Krissansen, G. W. (1998)."LPAM-1(integrin α4β7)-ligand binding: overlapping binding sites recognizing VCAM-1, MAdCAM-1 and CS-1 are blocked by fibrinogen, a fibronectin-like polymer and RGD-like cyclic peptides". Eur. J. Immunol., vol. 28 (3):995-1004.

Yao, K. and Wang, Z. (2008)."Reverse genetics for live attenuated virus vaccine development". Trends Bio. Pharmaceuti. Industry, vol. 2:57-62.

Yetter, R. A., Lehrer, S., Ramphal, R. and Small, P. A. (1980)."Outcome of influenza infection: effect of site of initial infection and heterotypic immunity". Infec. Immun., vol. 29:654-662.

References

209

Yewdell, J. W., Bennink, J. R., Smith, G. L. and Moss, B. (1985)."Influenza A virus nucleoprotein is a major target antigen for cross-reactive anti-influenza A virus cytotoxic T lymphocytes". Proc. Nat. Acad. Sci. U. S. A., vol. 82 (6):1785-1789.

Yewdell, J. W., Reits, E. and Neefjes, J. (2003)."Making sense of mass destruction: Quantitating MHC class I antigen presentation". Nat. Rev., vol. 3:952-961.

Yoshikawa, T., Matssuo, Ke., Matsuo, Ka., Suzuki, Y., Nomoto, A., Tamura, S. I., Kurata, T., and Sata, T. (2004)."Total viral genome copies and virus-Ig complexes after infection with influenza virus in the nasal secretions of immunized mice". J. Gen. Virol., vol. 85 (8): 2339-2346.

Yoshimura, K., Matsushita, S., Hayashi, Azusa and Takatsuki, K. (1996)."Relationship of HIV-1 Envelope V2 and V3 Sequences of the Primary Isolates to the Viral Phenotype". Microbiol. Immunol., vol. 40(4):277-287.

Yu, X., Tsibane, T., McGraw, P. A., House, F. S., Keefer, C. J., Hicar, M. D., Tumpey, T. M., Pappas, C., Perrone, L. A. and Martinez, O. (2008)."Neutralizing antibodies derived from the B cells of 1918 influenza pandemic survivors". Nature, vol. 455:532-536.

Yusim, K., Kesmir, C., Gaschen, B., Addo, M. M., Altfeld, M., Brunak, S., Chigaev, A., Detours, V. and Korber, B. T. (2002)."Clustering patterns of cytotoxic T-lymphocyte epitopes in human immunodeficiency virus type 1 (HIV-1) proteins reveal imprints of immune evasion on HIV-1 global variation". J. Virol. vol. 76 (17):8757-8768.

Zak, D. E., Andersen-Nissen, E., Peterson, E. R., Sato, A., Kristina Hamilton, M., Borgerding, J., Krishnamurty, A. T., Chang, J. T., Adams, D. J., Hensley, T. R., Salter, A. I., Morganb, C. A., Duerr, A. C., de Rosa, S. C., Aderem, A. and McElrath, M. J. (2012)."Merck Ad5/HIV induces broad innate immune activation that predicts CD8+ T-cell responses but is attenuated by preexisting Ad5 immunity". Proc. Natl. Acad. Sci. U S A., vol. 109 (50):E3503-12.

Zembe, L. Burgers, W. A., Jaspan, H. B., Bekker, L. G., Bredell, H., Stevens, G., Gilmour, J., Cox, J. H., Fast, P., Hayes, P., Vardas, E., Williamson, C. and Gray, C. M. (2011). "Intra- and Inter-clade Cross-reactivity by HIV-1 Gag Specific T-Cells Reveals Exclusive and Commonly Targeted Regions: Implications for Current Vaccine Trials". PLoS ONE, vol. 6 (10):e26096.

Zhang, L., Yu, W., He, T., Yu, J., Caffrey, R. E., Dalmasso, E. A., Fu, S., Pham, T., Mei, J., Ho, J. J., Zhang, W., Lopez, P. and Ho1, David D. (2002). "Contribution of Human α-Defensin 1, 2, and 3 to the Anti-HIV-1 Activity of CD8 Antiviral Factor". Science, vol. 298 (5595):995-1000.

Zhang, X., Sun, S., Hwang, I., Tough, D. F. and Sprent, J. (1998). "Potent and selective stimulation of memory-phenotype CD8+ T cell in vivo by IL-15". Immunity, vol. 8: 591.

Zhao, X., Zhang, M., Li, Z.and Frankel, F. (2006)."Vaginal protection and immunity after oral immunization of mice with a novel vaccine strain of listeria monocytogenes expressing human immunodeficiency virus". J Virol., vol. 80(18):8880-8890.

Zhirnov, O. P., Ikizler, M. R. and Wright, P. F. (2002)."Cleavage of influenza A virus hemagglutinin in human respiratory epithelium is cell associated and sensitive to exogenous antiproteases". J. Virol., vol. 76 (17):8682-8689.

References

210

Zhirnov, O. P., Ovcharenko, A. V. and Bukrinskaya, A. G. (1984)."Suppression of influenza virus replication in infected mice by protease inhibitors". J. Gen. Virol., vol. 65:191-196.

Zhu, J. and Paul, W. E. (2010)."Peripheral CD4+ T-cell differentiation regulated by networks of cytokines and transcription factors". Immunol. Rev., vol. 238:247-262.

Zhu, J., Shearer, G. M., Marincola, F. M., Norman, J. E., Rott, D., Zou, J. P. and Epstein, S. E. (2001)."Discordant cellular and humoral immune responses to cytomegalovirus infection in healthy blood donors:existence of a Th1-type dominant response". Int. Immunol., vol. 13 (6):785-790.

Zobel, A., Neumann, G. and Hobom, G. (1993)."RNA polymerase I catalysed transcription of insert viral cDNA". Nucleic Acids Res., vol. 21:3607-3614.

Zolla-Pazner, S. and Cardozo, T. (2010)."Structure-function relationships of HIV-1 envelope sequence-variable regions refocus vaccine design". Nat. Rev. Immunol., vol. 10 (7):527-535.

Zolla-Pazner, S. (2004)."Identifying epitopes of HIV-1 that induce protective antibodies". Nature Rev. Immunol., vol. 4 (3):199-210.

Zuccotti, G., Pogliani, L., Pariani, E., Amendola, A. and Zanetti, A. (2010)."Transplacental antibody transfer following maternal immunization with a pandemic 2009 influenza (H1N1) MF59-adjuvanted vaccine". JAMA, vol. 304:2360-2361.

Appendix A

211

A.Appendix A A.1. Solutions, buffers and reagents A.1.1. Agarose solution (0.8%) for gel electrophoresis 0.8 g agarose (Sigma-Aldrich)

Add 1x TAE buffer (100 ml)

Dissolve by microwave

Add 3 μl SYBR safe DNA gel stain (Invitrogen)

A.1.2. Agarose solution (1.8%) for plaque assay 0.9 g Agarose (Analytical grade, Promega)

Dissolve in Baxter water (50 ml)

Autoclave for 20 min at 121 oC

Keep in a 56 oC water bath for 2 hours

Transfer to a 45 oC water bath to be ready for the assay

A.1.3. FACS buffer (1%BSA; 0.02% sodium azide) 500 ml phosphate-buffered solution (PBS)

50 ml 10% (v/v) bovine serum albumin (BSA)

500 μl 20% (v/v) sodium azide A.1.4. TAE Buffer (40x) (Sambrook et al., 1989) 1.6 M Tris (Tris: hydroxymethyl methane (Merk)

40 mM EDTA (Ethylene Diamine Tetra Acetic acid) (Merk)

Dissolve in 800 ml of deionized water

pH (8.2-8.4) in a 25oC with 1 M Acetic acid (Merk)

Bring volume to 1000 ml

Store at 22-25 oC and use in 1-2 weeks

A.1.5. Red cell lysis buffer (Ammonium-Tris Chloride buffer) 0.14 M NH4CL (7.47 g) (BDH, Australia)

0.017 M Tris hydroxymethyl methane (2.06 g) (BDH, Australia)

980 ml Baxter water

Adjust pH to 7.2 with 1 M HCL

Bring volume to 1 L with Baxter water

Store at 22-25 oC and use in 1-2 weeks

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212

A.1.6. RNeasy Mini Kit for RNA extraction (Qiagen) Including the following steps: 500 μl 70% Ethanol

500 μl RLT buffer

500 μl RW1 buffer

500 μl RPE buffer

Elution buffer (Nuclease free water) dH2O

A.1.7. Plasmids pHW2000

pBR322 plasmid cloning vector (New England Biolabs (NEB)

pUC19 DNA (0.01 μg/ml)

A.1.8. Competent cells A.1.8.1. MAX Efficiency® DH5α™ Competent Cells (Invitrogen) DH5α™ Competent Cells

A.1.8.2. High efficiency JM109 Competent cells (Promega) E.coli JM109 competent cells >108 cfu/μg

A.1.9. Reverse transcription kit for conversion RNA to cDNA 2μl of 10× Reverse transcriptase buffer (RT buffer)

2μl dNTP mix buffer

2μl 10 μM oligo-dT primer

1 μl Omniscript reverse transcriptase

A.1.10. Permeabilizing solution One part Perm/washTM buffer (Becton Dickinson, UAS)

Nine part deionized distilled water

A.1.11. Restriction endonuclease enzymes Specific restriction endonuclease enzyme, PstI (Cat# R6325)

Specific restriction endonuclease enzyme, PvuI (Cat# R611A)

Appendix A

213

A.1.12. QIAquick PCR Purification Kit protocol For one volume of the PCR sample:

Add 5 volumes Buffer PB

Apply the sample to the QIAquick column

Centrifugation for 30–60 seconds and Discard flow through

Add 0.75 ml Buffer PE and centrifuge for 30–60 seconds

Discard flow-through and Place QIAquick column in 1.5 ml microcentrifuge tube

Add 30 μl elution buffer and then centrifuge

A.1.13. QIAquick Gel Extraction Kit protocol Add 3 volumes Buffer QG to 1 volume of gel

Incubate at 50°C for 10 min

Mix by vortexing the tube every 2–3 min to dissolve the gel

Add 1 volume of isopropanol

Apply the sample to the QIAquick column

Discard flow-through.

Add 0.5 ml Buffer QG and centrifuge for 1 min

Add 0.75 ml Buffer PE and centrifuge for 1 min

Discard the flow-through and Place QIAquick column into microcentrifuge tube

Add 20 μl Buffer EB (10 mM Tris·Cl, pH 8.5) and centrifuge for 1 min

A.1.14. PureYield™ Plasmid Miniprep protocol (Promega, USA) Add 600 μl transformed bacterial culture to 1.5 ml centrifuge tube

Add 100 μl cell lysis buffer and mix well

Add 350 μl cold neutralization solution and mix well

Centrifugation at 13000 rpm for 3 minutes

Transfer the supernatant to the PureYield™ Minicolumn into collection tube

Centrifuge at 13000 rpm for 15 seconds and discard the flow through

Wash by add 200 μl of Endotoxin removal wash (ERB)

Centrifugation at 13000 rpm at 15 seconds

Add 400 μl of column wash solution (CWS) and centrifuge for 30 seconds

Transfer the Minicolumn to 1.5 ml centrifuge tube

Elute the plasmid DNA by adding 30 μl of elution buffer and centrifuge for 15 seconds

A.1.15. Cell lines Madin-Darby Canine Kidney cells (MDCK cells)

Human embryonic kidney cells (HEK 293T cells)

Appendix A

214

A.1.16. PureYield™ Maxiprep System protocol (Promega, USA) Grow up 100–250 ml of transformed E. coli bacterial cell culture for overnight

Pellet the cells by centrifugation at 5,000 × g for 10 minutes at room temperature

Discard supernatant and mix the pellet with 12 ml Cell Resuspension Solution

Add 12 ml Cell Lysis Solution, mix well and incubation for 3 minutes at room temperature

Add 12 ml Neutralization Solution and mix well

Centrifuge the lysate at 14,000 × g for 20 minutes at room temperature

Pour the lysate into the blue PureYield™ Clearing Column

Apply maximum vacuum for passing the lysate debris through the PureYield™ Clearing Column

Wash the DNA by add 5 ml of Endotoxin Removal Wash and apply the vacuum

Add 20 ml of Column Wash and apply the vacuum

Dry the membrane by applying a vacuum for 5 minutes

Place a 1.5 ml microcentrifuge tube in the base of the Eluator™ Vacuum

Elute the DNA by 1 ml of Nuclease-Free Water and apply maximum vacuum for 1 minute

A.1.17. OPTI-MEM 1 medium (Gibco) OPTI-MEM® I modification of MEM (Eagle's)

A.1.18. Easy-flu medium (Invitrogen) 500 ml OPTI-MEM 1 medium (Gibco)

5 ml Penicillin (10,000 U/ml) and Streptomycin (10,000 μg/ml)

A.1.19. Fugene® 6 Transfection Reagent (Roche) Lipid blends in 80% ethanol, sterile-filtered, packaged in glass vials

A.1.20. RPMI-antimedia 500 ml Gibco RPMI 1640

300 μl Gentamycin (40 mg/ml)

5 ml Penicillin 10,000 U/ml (final concentration 100 U/ml)

Streptomycin 40 mg/ml (final concentration 24 μg/ml)

A.1.21. 2x L15 (Leibovitz’s) medium 2 packets of L15 powder media (2x 13.7 g) (Invitrogen)

Add 900 ml Baxter water

Stir for 1 hour

Adjust pH to 6.8 (1 M Hydrochloric acid)

Add 8 ml of 7% NaHCO3

Add 800 μl of Hepes buffer (pH 6.8, 1M)

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Add 20 ml of Pen/Strep (10,000 U/ml)

Add 1.2 ml Gentamycin (40 mg/ml)

A.1.22. LB agar culture media 200 ml LB agar

Dissolve by microwave for 1-2 min

Add 0.2 ml ampicillin 100 μg/ml (Sigma-Aldrich)

Pour 15 ml agar in petri-dish plates

Wait for 5 min and cover with parafilm

Keep at 4 oC

A.1.23. 1% TPCK-trypsin Worthington (1 μg/ μl) 10 mg of TPCK-trypsin Worthington (ScimaR)

Dissolve in 10 ml of PBS

Filtering by 0.45 μm Minisart filter

Aliquot into 250 μl volume

Keep at -80 oC

A.1.24. Trizol kit for RNA extraction 1 ml Trizol reagent (GibcoBRL) per 5-10x106 cells

200 μl chloroform (AnalR)

0.5 ml Propan-2-ol (isopropanol: AnalR)

1 ml 75% ethanol (AnalR)

30 μl HPLC water

Re-dissolve RNA for 10 minutes at 60oC (heat block)

A.1.25. Plaque assay medium 500 ml Gibco RPMI 1640 (Invitrogen)

300 μl Gentamycin (40 mg/ml)

5 ml Penicillin /Streptomycin (10,000 U/ml)

5 ml L-glutamine 2 mM (200 mM)

5 ml Sodium pyruvate (100 mM).

45 ml Heat inactivated FCS

A.1.26. Hanks' Balanced Salt Solution (HBSS) Gibco Hanks' Balanced Salt Solution (HBSS) (1X) (Invitrogen)

A.1.27. DNA size ladder 1 Kb plus DNA ladder (Invitrogen 1μg/μl)

A.1.28. C-RPMI medium

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500 ml RPMI media (JRH)

2 ml Penicillin/Streptomycin (10,000 U/ml)

40 ml Heat inactivated FCS

25 ml Hepes buffer

A.1.29. 4 % Paraformaldehyde 100 ml Phosphate Buffered Saline (PBS)

Pour into the conical flask containing 4g of paraformaldehyde (AnalR)

Stir inside the fume hood

Aliquot in 1 ml and store at 4oC

A.1.30. 6x DNA Loading Dye 0.25% (w/v) Bromophenol blue

0.25% (w/v) Xylene cyanol FF

30% (v/v) glycerol in distilled water

Appendix A

217

Table A.1. Fluorochrome conjugated Anti-mouse monoclonal antibodies

Anti-mouse monoclonal antibodies Fluorochrome Clone Supplier

Anti-mouse CD8-α PerCP 5.5 53-6.7 BD Pharmingen

Anti-mouse CD8-α PE 53-6.7 BD Pharmingen

Anti-mouse CD8-α APC 53-6.7 BD Pharmingen

Anti-mouse CD8-α FITC 53-6.7 BD Pharmingen

Anti-mouse CD4 FITC RM4-4 BD Pharmingen

Anti-mouse CD62L FITC MEL-14 BD Pharmingen

Anti-mouse CD62L APC MEL-14 BD Pharmingen

Anti-mouse CD44 APC 1M7 BD Pharmingen

Anti-mouse CD44 PE 1M7 BD Pharmingen

Anti-mouse CD69 PE H1-2F3 BD Pharmingen

Anti-mouse CD69 APC H1-2F3 Biolegend

Anti-mouse LPAM-1 (α4β7) PE DATK32 BD Pharmingen

Anti-mouse LPAM-1 (α4β7) APC DATK32 Biolegend

Anti-mouse CD103 (αEβ7) FITC M290 BD Pharmingen

Anti-mouse CD199 (CCR9) FITC 9B1 BD Pharmingen

Anti-mouse CD199 (CCR9) PE 9B1 Biolegend

Anti-mouse B1 integrin FITC HMB1-1 Biolegend

Anti-mouse B7 integrin (CD) FITC FIB27 Biolegend

Anti-mouse interferon (IFN-γ) FITC XMG1.2 BD Pharmingen

Anti-mouse interferon (IFN-γ) APC XMG1.2 BD Pharmingen

Anti-mouse (TNF-α) APC MP6-XT22 BD Pharmingen

Anti-mouse interleukine 2 (IL-2) PE JES6-5H4 BD Pharmingen

Recombinant Influenza viruses as a vaccine vector for HIV

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