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
III
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".
IV
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
XI
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
XV
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
+CD8+ T cells detected in different organs following different routes of mucosal vaccination in BALB/c mice 122
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
+CD8+ T cells detected in different organs following different routes of mucosal vaccination in BALB/c mice 124
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
+CD8+ T cells detected in different organs following different routes of mucosal vaccination in BALB/c mice 126
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
+CD8+ T cells detected in different organs following different routes of mucosal vaccination in BALB/c mice 128
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
Chapter 1: Literature Review
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).
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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).
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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.
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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).
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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).
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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).
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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
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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
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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
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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
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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
Chapter 1: Literature Review
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,
Chapter 1: Literature Review
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
Chapter 1: Literature Review
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
Chapter 1: Literature Review
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
Chapter 1: Literature Review
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
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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
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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+
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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).
Chapter 1: Literature Review
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).
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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
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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
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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
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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,
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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
Chapter 1: Literature Review
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
Chapter 1: Literature Review
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
Chapter 1: Literature Review
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
Chapter 1: Literature Review
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).
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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,
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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).
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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
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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).
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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
Chapter 1: Literature Review
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
Chapter 1: Literature Review
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).
Chapter 1: Literature Review
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
Chapter 1: Literature Review
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).
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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
Chapter 1: Literature Review
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
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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
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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).
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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
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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
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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
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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
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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).
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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-
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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-
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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
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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
Chapter 1: Literature Review
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).
Chapter 1: Literature Review
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
Chapter 1: Literature Review
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
Chapter 1: Literature Review
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.
Chapter 2: Materials and Methods
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
Chapter 2: Materials and Methods
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
Chapter 2: Materials and Methods
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.
Chapter 2: Materials and Methods
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
Chapter 2: Materials and Methods
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.
Chapter 2: Materials and Methods
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
Chapter 2: Materials and Methods
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.
Chapter 2: Materials and Methods
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
Chapter 2: Materials and Methods
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
Chapter 2: Materials and Methods
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
Chapter 2: Materials and Methods
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).
Chapter 2: Materials and Methods
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
Chapter 2: Materials and Methods
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
Chapter 3: Generation of influenza viruses expressing HIV-CD8+ T cell epitopes
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).
Chapter 3: Generation of influenza viruses expressing HIV-CD8+ T cell epitopes
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 (*).
Chapter 3: Generation of influenza viruses expressing HIV-CD8+ T cell epitopes
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.
Chapter 3: Generation of influenza viruses expressing HIV-CD8+ T cell epitopes
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
Chapter 3: Generation of influenza viruses expressing HIV-CD8+ T cell epitopes
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).
Chapter 3: Generation of influenza viruses expressing HIV-CD8+ T cell epitopes
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.
Chapter 3: Generation of influenza viruses expressing HIV-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
Chapter 3: Generation of influenza viruses expressing HIV-CD8+ T cell epitopes
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
Chapter 3: Generation of influenza viruses expressing HIV-CD8+ T cell epitopes
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
Chapter 3: Generation of influenza viruses expressing HIV-CD8+ T cell epitopes
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
Chapter 3: Generation of influenza viruses expressing HIV-CD8+ T cell epitopes
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
Chapter 3: Generation of influenza viruses expressing HIV-CD8+ T cell epitopes
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
Chapter 3: Generation of influenza viruses expressing HIV-CD8+ T cell epitopes
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.
Chapter 3: Generation of influenza viruses expressing HIV-CD8+ T cell epitopes
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
Chapter 3: Generation of influenza viruses expressing HIV-CD8+ T cell epitopes
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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).
Chapter 3: Generation of influenza viruses expressing HIV-CD8+ T cell epitopes
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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).
Chapter 3: Generation of influenza viruses expressing HIV-CD8+ T cell epitopes
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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,
Chapter 4: Induction of CD8+ T cell responses using recombinant influenza viruses
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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.
Chapter 4: Induction of CD8+ T cell responses using recombinant influenza viruses
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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.
Chapter 4: Induction of CD8+ T cell responses using recombinant influenza viruses
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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).
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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.
Chapter 4: Induction of CD8+ T cell responses using recombinant influenza viruses
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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
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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.
Chapter 4: Induction of CD8+ T cell responses using recombinant influenza viruses
134
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
Chapter 4: Induction of CD8+ T cell responses using recombinant influenza viruses
135
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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136
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
137
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.
Chapter 5: Pseudo-Challenge study
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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).
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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.
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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
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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
Chapter 6: General Discussion
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
Chapter 6: General Discussion
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
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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
Chapter 6: General Discussion
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
Chapter 6: General Discussion
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.
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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
Appendix A
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)
Appendix A
215
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
Appendix A
216
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
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