Mycobacterium tuberculosis Surface-Binding Antibodies ...

MycobacteriumtuberculosisSurface‐BindingAntibodiesInfluenceEarlyInfectionEvents

by

Casey Perley

Department of Molecular Genetics and Microbiology

Duke University

Date:_______________________

Approved:

___________________________

Richard Frothingham, Supervisor

___________________________

Jörn Coers

___________________________

Jen‐Tsan Ashley Chi

___________________________

Micah Luftig

___________________________

Gregory Taylor

Dissertation submitted in partial fulfillment of

the requirements for the degree of Doctor

of Philosophy in the Department of

Molecular Genetics and Microbiology in the Graduate School

of Duke University

2015

ABSTRACT

MycobacteriumtuberculosisSurface‐BindingAntibodiesInfluenceEarlyInfectionEvents

Casey Perley

Department of Molecular Genetics and Microbiology

Duke University

Date:_______________________

Approved:

___________________________

Richard Frothingham, Supervisor

___________________________

Jörn Coers

___________________________

Jen‐Tsan Ashley Chi

___________________________

Micah Luftig

___________________________

Gregory Taylor

An abstract of a dissertation submitted in partial fulfillment of

the requirements for the degree of Doctor

of Philosophy in the Department of

Molecular Genetics and Microbiology in the Graduate School

of Duke University.

2015

Copyright by

Casey Perley

2015

iv

Abstract

Mycobacterium tuberculosis, the etiologic agent of tuberculosis (TB), is among the

leading causes of death from infectious disease world‐wide. An intracellular pathogen,

M. tuberculosis infects phagocytic cells, and subverts the host immune response,

preventing eradication once infection has been established. Even after successful

chemotherapy, exogenous re‐infection occurs, indicating that sterilizing immune

responses are not generated during natural infection. While a TB vaccine exists, it does

not alter M. tuberculosis infection rate, rather it prevents the progression from latent TB

infection to active TB disease. Vaccines against Haemophilus influenzae and Streptococcus

pneumonia protect from bacterial colonization and infection through the induction of

antibodies to capsular surface components. This dissertation explores if antibodies to

the surface of M. tuberculosis can alter the initial interaction between a bacterium and

host cell, leading to a reduction in infection rate.

When pre‐mixed with M. tuberculosis prior to in vitro infection of macrophages,

or retropharyngeal instillation of mice, monoclonal surface‐binding, but not non‐surface‐

binding antibodies, decrease bacterial burden and the number of infected cells within

the first twenty‐four hour of infection. If administered retropharyngeally prior to aerosol

exposure, surface‐binding antibodies decreased pulmonary bacterial burden at twenty‐

four hours post infection in an FcγR independent manner. Despite decreasing early

v

bacterial burden, pre‐administration of surface‐binding antibodies prior to ultra‐low

dose aerosol infection did not alter infection rate compared to mice instilled with PBS

(Chapters 4 and 5).

To evaluate the surface‐binding antibody response in humans, plasma from

uninfected controls, individuals with latent TB infection, and active TB disease was

assayed by ELISA to determine the titer, avidity and IgG/IgM ratio for antibodies to the

surface, whole cell lysate, and additional subcellular bacterial fractions . In contrast to

antibodies to antigenic fractions, individuals with active TB disease had decreased

avidity, and no augmentation of the IgG/IgM ratio for antibodies to the live M.

tuberculosis surface, as compared to uninfected controls (Chapter 3).

Overall these findings demonstrate that surface‐binding monoclonal antibodies

alter early infection events, both in vivo and in vitro, though the magnitude of protection

was not sufficient to decrease M. tuberculosis infection rate. Given the failure of patients

with active TB disease to produce highly avid IgG surface‐binding antibodies, they may

represent the target of a protective vaccine.

vi

Dedication

To my grandmother: You have taught me two of the most valuable lessons in life. Always

be yourself, and never let anyone tell you that something is not possible, especially just

because you are a woman. You marched to the beat of your own drummer, and lived life

to the fullest. I inspire to live by your example. You also taught me the secret ingredient

to make the best chocolate chip cookies. I am honored to share your name.

To my parents: Thank you for your unwavering love for all these years. You have always

supported me unconditionally in whatever I did, be it science, or music, and I wouldn’t

have made it to this point without you both.

To Neil: You have sacrificed so much in letting me pursue my doctorate, and hardest

part of writing this dissertation is knowing you won’t be here to see me defend it. I am

so proud of you, and what you are doing. Not all girls are lucky enough to marry their

hero. I am. I love you. I can’t wait until you return.

vii

Contents

Abstract ........................................................................................................................... iv

List of Tables ................................................................................................................ xiv

List of Figures .............................................................................................................. xvi

Acknowledgements ..................................................................................................... xix

1. Tuberculosis ................................................................................................................. 1

1.1 Tuberculosis disease......................................................................................................... 1

1.1.1 Mycobacterium tuberculosis complex .......................................................................... 1

1.1.2 Global disease burden ................................................................................................ 1

1.1.3 Transmission and early infection events .................................................................. 3

1.1.4 The onset of the adaptive immune response and progression to active disease 4

1.2 Vaccines ............................................................................................................................. 6

1.2.1 M. bovis Bacille Calmette‐Guérin (BCG) vaccine .................................................... 6

1.2.2 Vaccines in clinical trials ............................................................................................ 7

1.2.3 Tuberculosis vaccines that induce sterilizing immunity ..................................... 11

1.3 Animal models of tuberculosis ..................................................................................... 13

1.3.1 Murine model of active TB disease ......................................................................... 13

1.3.2 The ultra‐low dose murine infection model .......................................................... 15

1.3.3 Alternative animal models ....................................................................................... 17

1.4 Antibodies and tuberculosis ......................................................................................... 19

1.4.1 Serum therapy as treatment for tuberculosis ........................................................ 19

viii

1.4.2 Antibodies as a principal component of diagnostic testing and as a biomarker

for disease progression ...................................................................................................... 21

1.4.3 Antibodies alter disease course in mice ................................................................. 22

1.4.4 Disease progression in mice lacking components of the humoral immune

response ............................................................................................................................... 24

2. Differences in baseline lung characteristics of BALB/c and C57BL/6J may influence differences in infection rate ........................................................................ 26

2.1 Introduction ..................................................................................................................... 26

2.2 Methods ........................................................................................................................... 27

2.2.1 Animals ....................................................................................................................... 27

2.2.2 Retropharyngeal M. tuberculosis infection ............................................................. 27

2.2.3 Bacterial CFU determinations .................................................................................. 28

2.2.4 Sample collection and processing for Luminex multiplex assay ........................ 28

2.2.5 Luminex multiplex cytokine assay ......................................................................... 28

2.2.6 Single cell suspension of mouse lung ..................................................................... 29

2.2.7 Cell staining and flow cytometry ............................................................................ 30

2.2.8 Flow cytometry gating .............................................................................................. 30

2.2.9 Statistics ...................................................................................................................... 32

2.3 Results .............................................................................................................................. 32

2.3.1 Early CFU differences in BALB/c in and C57BL/6J mice are observed after

aerosol exposure but not retropharyngeal instillation .................................................. 32

2.3.2 Cytokines and chemokines are differentially expressed in the BAL fluid and

lung homogenate of BALB/c and C57BL/6 mice ............................................................ 33

2.3.3 Differences in baseline cell populations in BALB/c and C57BL/6 mice ............. 36

ix

2.4 Discussion ........................................................................................................................ 37

2.5 Future Directions ............................................................................................................ 40

3. Human antibodies to the surface of M. tuberculosis ................................................. 43

3.1 Introduction ..................................................................................................................... 43

3.2 Methods ........................................................................................................................... 44

3.2.1 Bacterial cultures and lysates ................................................................................... 44

3.2.2 Additional ELISA antigens ...................................................................................... 45

3.2.3 ELISA assay ................................................................................................................ 45

3.2.4 Avidity ELISA assay ................................................................................................. 47

3.2.5 Statistical Analysis ..................................................................................................... 48

3.2.6 Human subjects ......................................................................................................... 48

3.2.7 Cytokine profiling ..................................................................................................... 49

3.3 Results .............................................................................................................................. 50

3.3.1 Demographic information on human population ................................................ 50

3.3.2 Human antibody titers to the surface of live M. tuberculosis and to inactivated

antigenic fractions .............................................................................................................. 52

3.3.3 Relative IgG avidity to the surface of live M. tuberculosis and to inactivated

antigenic fractions .............................................................................................................. 55

3.3.4 Ratio of IgG and IgM antibodies to the surface of live M. tuberculosis and to

inactivated antigenic fractions .......................................................................................... 55

3.3.5 Correlations between antibody titers and clinical characteristics ...................... 56

3.3.6 Correlations between relative IgG avidity and clinical characteristics .............. 60

x

3.3.7. Association of cytokine production with surface‐binding and secreted protein

antibody titers and IgG avidity scores ............................................................................ 61

3.3.8 Antibody titers and avidity scores to environmental mycobacteria .................. 62

3.4 Discussion ........................................................................................................................ 64

3.5 Future directions ............................................................................................................. 72

4. Surface-binding antibodies decrease bacterial burden in the murine lung by blocking M. tuberculosis uptake ................................................................................. 75

4.1 Introduction ..................................................................................................................... 75

4.2 Methods ........................................................................................................................... 76

4.2.1 Hybridoma generation ............................................................................................. 76

4.2.2 Hybridoma growth and antibody purification ..................................................... 77

4.2.3 Antibodies .................................................................................................................. 78

4.2.4 ELISA assay ................................................................................................................ 80

4.2.5 Avidity ELISA assay ................................................................................................. 80

4.2.6 Murine immune sera ................................................................................................. 80

4.2.7 Western Blot ............................................................................................................... 81

4.2.8 Bacterial strains and growth conditions ................................................................. 81

4.2.9 Biotin fixation to M. tuberculosis .............................................................................. 82

4.2.10 Macrophage uptake and survival assay ............................................................... 84

4.2.11 Bacterial clumping assay ........................................................................................ 85

4.2.12 Mice ........................................................................................................................... 85

4.2.13 Isolation of dsRed M. tuberculosis from peritoneal lavage ................................. 85

xi

4.2.14 Murine administration of antibody pre‐mixed with M. tuberuclosis ................ 86

4.2.15 Single cell suspension of infected mouse lung and pneumocyte staining ...... 87

4.2.16 Flow cytometry analysis ......................................................................................... 87

4.2.17 Statistics .................................................................................................................... 90

4.3 Results .............................................................................................................................. 90

4.3.1 Screening of M. tuberculosis antibodies from a repository ................................... 90

4.3.2 Generation and screening of surface‐binding antibodies against LAM, HSP‐X

and Apa ................................................................................................................................ 92

4.3.3 Purified antibody reactivity to M. tuberculosis fractions and the live cell surface

............................................................................................................................................... 93

4.3.4 Determination of whole cell lysate avidity for purified antibodies ................... 96

4.3.5 Purified M. tuberculosis antibodies bind to linear epitopes and are polyreactive

............................................................................................................................................... 97

4.3.6 Pre‐mixing of biotin coated M. tuberculosis with antibody, prior to macrophage

infection, decreases bacterial burden ............................................................................... 98

4.3.7 Pre‐mixing of M. tuberculosis with surface‐binding antibodies prior to

macrophage infection decreases bacterial burden ......................................................... 99

4.3.8 Surface‐binding antibodies do not induce bacterial agglutination .................. 103

4.3.9 BTN.4 does not decrease pulmonary bacterial load at three days post infection

when mixed with biotin coated M. tuberculosis prior to retropharyngeal instillation

............................................................................................................................................. 104

4.3.10 Surface‐binding, but not non‐surface‐binding antibodies, decrease bacterial

burden at three days post infection when mixed with M. tuberculosis prior to

retropharyngeal instillation ............................................................................................ 105

xii

4.3.11 Surface‐binding antibodies decrease bacterial burden at three, but not six,

days post infection when mixed with M. tuberculosis prior to retropharyngeal

instillation .......................................................................................................................... 108

4.3.12 Pre‐incubation with surface‐binding antibodies reduces pulmonary bacterial

load three days after infection with mammalian adapted bacteria........................... 110

4.3.13 Pre‐incubation of M. tuberculosis with surface‐binding antibodies reduces the

number of infected cells by one day post retropharyngeal instillation .................... 112

4.3.14 Incubation of M. tuberculosis with surface‐binding antibodies alters the

infected cell profile during the first three days post infection ................................... 114

4.3.15 Total lung cell profile after M. tuberculosis infection ........................................ 121

4.4 Discussion ...................................................................................................................... 124

4.5 Future Direction ............................................................................................................ 131

5. Surface-binding antibodies decrease pulmonary bacterial burden after aerosol infection ....................................................................................................................... 136

5.1 Introduction ................................................................................................................... 136

5.2 Methods ......................................................................................................................... 137

5.2.1 Animals ..................................................................................................................... 137

5.2.2 Retropharyngeal instillation of purified antibody .............................................. 137

5.2.3 Pharmacokinetics of monoclonal antibodies in BAL fluid ................................ 138

5.2.4 Aerosol innocula preparation ................................................................................ 138

5.2.5 Aerosol challenge .................................................................................................... 138

5.2.6 Determination of aerosol dose presented ............................................................ 139

5.2.7 Bacterial CFU determination ................................................................................. 140

5.2.8 Lung culture ............................................................................................................. 140

xiii

5.2.9 Statistics .................................................................................................................... 140

5.3 Results ............................................................................................................................ 141

5.3.1 Antibodies persist in BAL fluid for at least twenty‐four hours after

retropharyngeal instillation ............................................................................................ 141

5.3.2 Surface‐binding antibodies decrease bacterial CFU in a concentration

dependent manner when administered prior to a standard dose M. tuberculosis

aerosol ................................................................................................................................ 142

5.3.3 Retropharyngeal administration of surface‐binding antibodies leads to

variable decreases in bacterial burden three days post aerosol infection ................ 143

5.3.4 Retropharyngeal administration of surface‐binding antibodies one day prior to

standard dose aerosol infection decreased bacterial burden in the lungs in a time

dependent manner ........................................................................................................... 146

5.3.5 Decreased bacterial burden is FCγR independent ............................................. 148

5.3.6 Retropharyngeal administration of surface‐binding antibodies prior to ultra‐

low dose aerosol does not reduce murine infection rate ............................................ 149

5.3.7 The presence of 1mg of antibodies prior to ultra‐low dose M. tuberculosis

exposure leads to decreased bacterial load one month post infection in infected

mice .................................................................................................................................... 151

5.4 Discussion ...................................................................................................................... 153

5.5 Future Directions .......................................................................................................... 157

Appendix A .................................................................................................................. 160

Appendix B .................................................................................................................. 163

Appendix C .................................................................................................................. 172

References ................................................................................................................... 178

Biography .................................................................................................................... 209

xiv

List of Tables

Table 1: M. tuberculosis vaccines in clinical trials ...................................................................... 9

Table 2: Infection rates after ultra‐low dose exposure to M. tuberculosis ............................ 16

Table 3: Cytokine values for C57BL/6 and BALB/c mice in BAL and lung homogenate.. 35

Table 4: Demographics and clinical data ................................................................................. 51

Table 5: Univariate correlation between clinical variables and total antibody titers to the

live M. tuberculosis surface or whole cell lysate in patients with active TB disease........... 58

Table 6: Univariate correlation between clinical variables and avidity of antibodies to the

live M. tuberculosis surface or whole cell lysate in patients with active TB disease........... 59

Table 7: Experimental Antibodies............................................................................................. 79

Table 8: Components of surface‐binding antibody pools ..................................................... 79

Table 9: Screening of anti‐M. tuberculosis monoclonal antibodies from repository ........... 91

Table 10: Peak OD values for monoclonal antibodies to M. tuberculosis fractions and the

live cell surface ............................................................................................................................ 95

Table 11: Peak OD values for monoclonal antibodies to their cognate antigens ............... 95

Table 12: Avidity scores to whole cell lysate for purified antibodies .................................. 96

Table 13: M. tuberculosis antibodies do not induce agglutination ...................................... 104

Table 14: Infection rates after ultra‐low dose M. tuberculosis aerosol exposure ............... 151

Table 15: BAL, lung tissue and sera cytokine data for C57BL/6 and BALB/c mice ......... 160

Table 16: Univariate correlation between clinical variables and total antibody titers to

LAM, cell wall, and secreted proteins in patients with active TB disease ........................ 164

Table 17: Univariate correlation between clinical variables and total antibody titers to the

live M. tuberculosis surface and whole cell lysate in patients with latent TB infection ... 165

xv

Table 18: Univariate correlation between clinical variable and total antibody titers to

LAM, cell wall, and secreted proteins for patients with latent TB infection .................... 166

Table 19: Univariate correlation between clinical variables and relative IgG avidity of

antibodies to LAM, cell wall, and secreted proteins in patients with active TB disease 167


antibodies to the live M. tuberculosis surface and to whole cell lysate in patients with

latent TB infection ..................................................................................................................... 168

Table 21: Univariate correlation between clinical factors and relative IgG avidity of

antibodies to LAM, secreted proteins, and cell wall in patients with latent TB infection

..................................................................................................................................................... 169

Table 22: Univariate correlation between cytokine levels in whole blood after

Quantiferon‐Gold peptide stimulation and total antibody titers ....................................... 170


Quantiferon‐Gold peptide stimulation and relative IgG avidity ....................................... 171

Table 24: Baseline characteristics and infection status for mice receiving ultra‐low dose

aerosol exposure ........................................................................................................................ 172

Table 25: Baseline characteristics of each exposure group in infection rate studies ........ 177

xvi

List of Figures

Figure 2: Early lung burden in C57BL/6 and BALB/c mice after standard dose aerosol

infection ........................................................................................................................................ 17

Figure 3: Gating scheme for flow cytometry analysis:........................................................... 31

Figure 4: Early lung CFU in C57BL/6 and BALB/c mice after retropharyngeal instillation

....................................................................................................................................................... 33

Figure 5: Pnemocyte populations in C57BL/6 and BALB/c lungs ........................................ 37

Figure 6: Reproducibility of human sera on different ELISA antigens. .............................. 47

Figure 7: Antibody responses to M. tuberculosis live cell surface or whole cell lysate ...... 53

Figure 8: Antibody responses to M. tuberculosis lipoarabinomannan, cell wall, and

secreted protein fractions ........................................................................................................... 54

Figure 9: Reactivity of human plasma to surface of environmental mycobacteria ........... 64

Figure 10: Optimization of biotin fixation to M. tuberculosis ................................................ 83

Figure 11: Gating scheme for dsRed isolation from peritoneal lavage ............................... 86

Figure 12: Gating scheme for infected cell flow cytometry analysis ................................... 89

Figure 13: Comparison of dsRed positive cells in infected vs. uninfected mice ................ 90

Figure 14: M. tuberculosis monoclonal antibodies are polyreactive ..................................... 97

Figure 15: BTM.4 decreases biotin‐coated M. tuberculosis survival in macrophages ......... 99

Figure 16: Surface‐binding but not control antibodies decrease bacterial survival in

macrophages in a concentration dependent manner ........................................................... 100

Figure 17: Surface‐binding, but not non‐surface‐binding antibodies decrease three hour

M. tuberculosis survival in murine macrophages .................................................................. 102

Figure 18: Pre‐incubation with BTN.4 does not decrease biotin coated M. tuberculosis

bacterial burden at three days post infection ........................................................................ 105

xvii

Figure 19: Surface‐binding antibodies decrease pulmonary bacterial load in mice ........ 107

Figure 20: Surface‐binding antibodies decrease bacterial burden at three, but not six,

days post retropharyngeal instillation ................................................................................... 109

Figure 21: Surface‐binding antibodies decrease pulmonary bacterial load three days after

infection with mammalian adapted bacteria ........................................................................ 111

Figure 22: Pre‐incubation of M. tuberculosis with surface‐binding antibodies prior to

retropharyngeal instillation decreases the number of infected cells by flow cytometry 113


retropharyngeal instillation decreases the number of infected cells by plate count ....... 114

Figure 24: Effect of surface‐binding antibodies on the type of infected cell after pre‐

mixing with M. tuberculosis infection prior to retropharyngeal instillation ..................... 118

Figure 25: Comparison of the number of infected neutrophils and recruited

macrophages between flow cytometry and plate counts .................................................... 120

Figure 26: Proportion of total cells in the lung after M. tuberculosis infection with and

without surface‐binding antibodies ....................................................................................... 122

Figure 27: retropharyngeal instillation of PBS does not alter the total lung profile within

twenty‐four hours post infection ............................................................................................ 123

Figure 28: Levels of antibody in BAL fluid within first twenty‐four hours after

retropharyngeal administration .............................................................................................. 142

Figure 29: Surface‐binding antibodies reduce pulmonary bacterial burden at 3 days post

infection in a concentration dependent manner. .................................................................. 143

Figure 30: Surface‐binding antibodies provide inconsistent protection at three days post

M. tuberculosis aerosol infection .............................................................................................. 145

Figure 31: The ability of surface‐binding antibodies to decrease pulmonary bacterial

burden wanes over time........................................................................................................... 147

Figure 32: Surface‐binding antibodies decrease bacterial burden at one day post infection

..................................................................................................................................................... 148

xviii

Figure 33: The effect of surface‐binding antibodies is FCγR independent ....................... 149

Figure 34: Pulmonary bacterial burden one month post ultra‐low dose aerosol infection

..................................................................................................................................................... 152

Figure 35: Reactivity of human plasma to protein lysates from M. tuberculosis and

environmental mycobacteria ................................................................................................... 163

xix

Acknowledgements

I want to thank the members of the Frothingham Lab for their invaluable

assistance with these studies: Richard Frothingham for his mentorship and guidance,

Sarah Seay for her assistance with animal work, Eva Click, for the development of the C‐

ELISA assay, Rose Asrican for keeping our lab running, and Ning Zhao, our talented

undergraduate, for antibody purification and a portion of the ELISA data presented in

Chapter 4. Aerobiology would not have been possible without the Aerobiology Core

Facility at the Duke Regional Biocontianment Lab (RBL), and the assistance of Divey

Saini, Chris Sample, and Jim Burch in delivering the aerosol exposures.

Outside of our lab I would like to thank our numerous collaborators. Jason Stout,

of the Department of Medicine, for his gift of human plasma from patients with latent

TB infection and active TB disease, Marc Frahm and Guido Ferrari for their cytokine

profiling data of these same patients, and the patients themselves for donating blood

samples for study. Ian Cumming of the DHVI flow core was invaluable for both his flow

cytometry expertise and assistance in running our samples, especially at BSL‐3.

Biomarker profiling was performed under the direction of Dr. Gregory D. Sempowski in

the Immunology Unit of the Duke RBL, with assistance from Kristina Riebe and Heather

Lynch. Monoclonal antibody generation was conducted with help from the Haynes Lab,

specifically Barton Haynes, Richard Scearce, Daria Pause and Bradley Lockwood. Wes

xx

Rountree provided invaluable statistics assistance. Karen Dobos at Colorado State

University, and Barton Haynes at Duke University provided the hybridoma cell lines for

anti‐M. tuberculosis antibodies, and isotype controls. Gifts of strains were made by

Sunhee Lee (Mycobacterium bovis Bacille‐Calmette‐Guerin Danish, Mycbacterum

tuberculosis H37Rv, and Mycobacterium fortuitum) and the Duke Clinical Microbiology

Lab (Mycobacterum avium). I would especially like to thank my ccommittee, of Jorn

Coers, Jen‐Tsan Ashely Chi, Micah Luftig and Gregory Taylor, for their assistance,

critical review, and support over the past four years.

Working at biosafety level 3 (BSL‐3), where the majority of these studies were

conducted, requires a team of people. Thank you to Scott Alderman and Jim Burch for

the BSL‐3 safety training and assistance. Also, thank you to Doug Elliot for constantly

fixing my autoclave (I swear it wasn’t my fault!), Rose Asrican and Ching‐ju Chen for

keeping the BSL‐3 facility running, and our security guards for the around the clock

monitoring.

This work was supported by NIH grants U54 AI057157 (Southeast Regional

Center of Excellence for Emerging Infections and Biodefense), P30 AI051445 (Duke

Center for Translational Research), and UC6 AI058607 (Regional Biocontainment

Laboratory at Duke), and by National Science Foundation Graduate Research

Fellowship 2011083676 (C.C.P.). Biomarker profiling was conducted by the Immunology

xxi

Unit of the Duke RBL, which received partial support for construction from the National

Institutes of Health, National Institute of Allergy and Infectious Diseases (UC6‐

AI058607). Mycobacterial antigen fractions from Colorado State University were

produced under NIH Contract HHSN266200400091C (Tuberculosis Vaccine Testing and

Research Materials) and distributed by the Biodefense and Emerging Infections Research

Resources Repository. Specimen collection and storage for the TB‐infected subjects was

supported by the Duke NIH grants N01 AI040082 and P30 AI064518 (Center for AIDS

Research)

1

1. Tuberculosis

1.1 Tuberculosis disease

1.1.1 Mycobacterium tuberculosis complex

Mycobacterium tuberculosis is one of the eight members of M. tuberculosis complex

(MBTC) [1]. All members of the complex share >99% nucleotide similarity, identical 16s

RNA sequences, replicate slowly in vitro and in vivo, and possess waxy outer coats of

mycolic acid which make them impervious to gram staining. Despite these similarities,

the members exhibit radically different host tropisms ranging from sea lions to banded

mongooses [2‐4]. M. tuberculosis is the primary cause of tuberculosis (TB) in humans.

Infection with Mycobacterium africanum and Mycobacterium canetti also cause human TB

disease, though these cases are primarily limited to Africa [5,6]. While other members of

the MBTC can infect humans, they contribute relatively little to disease burden.

Mycobacterium bovis and Mycobacterium caprae, account for only seven cases of TB per

one‐hundred thousand, while Mycobacterium microti and Mycobacterium pinnipedi have

resulted in only few documented cases of human infection world‐wide [7‐9].

1.1.2 Global disease burden

Tuberculosis is among the leading causes of death from infectious disease world‐

wide. Approximately one‐third of the global population is infected with the causative

agent, though 90‐95% of these infections are latent and will never progress to active TB

2

disease. 8.8 million cases of active disease and 1.4 million deaths occurred in 2011 [10].

TB most commonly manifests as a pulmonary infection, though it can also present as a

severe disseminated infection, known as miliary TB [11]. Other, far less frequent, sites of

disease include the joints, central nervous system, gastrointestinal tract, pleural space,

and lymph nodes [12‐16]. Effective antibiotic treatment for TB exists, though the course

of therapy requires multiple drugs and takes a minimum of six months to complete [17].

Failure of some patients to adhere to the course of therapy has led to the rise of multi‐

drug and extremely‐drug resistant strains of the bacteria, which further lengthen and

complicate treatment [18].

Though global in its distribution, TB disease burden is most heavily concentrated

in sub‐Saharan Africa, and southeast Asia [10]. While disease incidence is stable or

decreasing across most of the globe, it continues to rise in southern Africa, largely due to

high rates of human immunodeficiency virus (HIV) infection [19]. Of the numerous risk

factors for TB, including substance abuse, malnutrition, a weakened immune system,

and poor living and working conditions, HIV co‐infection is the most notable [20,21].

Individuals with HIV are twenty to thirty‐seven times more likely to contract and

develop active TB disease than their uninfected counterparts, and one‐hundred times

more likely to reactivate a latent infection [19,22].

3

1.1.3 Transmission and early infection events

Tuberculosis is an airborne infection, transmitted when an individual with

pulmonary disease coughs, aerosolizing bacteria. During aerosolization, droplet nuclei,

five micron particles that contain between one and three bacteria, are formed and

remain airborne for a few hours after generation [23]. The particle’s small size facilitates

its travel to the alveoli of the lungs where it infects and colonize mononuclear

phagocytes by preventing phagosomal maturation [24]. A single M. tuberculosis

bacterium is sufficient to cause infection [25,26].

The precise interactions between M. tuberculosis and its host cell, which lead to

productive infection, are not well known. The bacterium is believed to primarily infect

alveolar macrophages, dendritic cells (DCs), and neutrophils; however, the precise

identity of infected cells prior to fourteen days post infection is unknown [27]. M.

tuberculosis is able to interact with at least thirteen host receptors, with receptor

interactions helping to shape each bacterium’s intracellular environment [28,29]. Binding

to some, such as the mannose receptor, leads to impairment of the host cell’s bactericidal

response by blocking phagosomal maturation, suppressing radical species generation,

and damping pro‐inflammatory cytokine production [30,31]. In contrast, M. tuberculosis

binding to toll‐like receptor 2, or the FCγ receptor (FCγR), enhances cellular anti‐

mycobacterial responses through phagosomal maturation and acidification [32‐34].

4

Typically, after internalization of a bacterial pathogen, the nascent phagosome

rapidly acidifies and fuses with lysosomes, leading to bacterial killing. M. tuberculosis

modifies the protein composition of the phagosome surface, inhibiting this fusion

process by retaining Rab22a, a protein critical for the Rab5 to Rab7 conversion that leads

to lysosomal fusion [35,36]. Selective retention and exclusion of additional proteins

impairs the recruitment of inducible nitric oxide synthase (iNOS), lysosomal hydrolases

such as Cathepsin D, and the V‐type ATPase necessary for phagosomal acidification

[37‐39]. The resulting endosomal compartment remains at a close to neutral pH, and is

accessible to fusion with early endosomes, allowing for nutrient trafficking to the

bacterium, creating an environment highly conducive to bacterial replication [37,40‐42].

1.1.4 The onset of the adaptive immune response and progression to

active disease

By nine days post infection M. tuberculosis has migrated to the lymph nodes

inside of mononuclear phagocytes, presumably dendritic cells [27,43]. Once there, the

bacteria trigger the onset of the adaptive immune response by stimulating the

production of CD4+ specific T‐cells, which appear two to three days thereafter [44].

Fifteen to eighteen days post infection, antigen specific T‐cells arrive in the lungs [45].

The presence of these CD4+ T‐cells is correlated with the stabilization of pulmonary

bacillary burden in mice; however, the infection is too established to be eradicated [46].

CD4+ T‐cells produce three cytokines, interferon‐γ (IFN‐γ), tumor necrosis factor‐α

5

(TNF‐α), and interleukin 2 (IL‐2), the former two of which activate infected host cells, a

mechanism crucial for controlling M. tuberculosis infection. The loss of these cytokines in

mice, through germ‐line knock‐outs, or in humans, through somatic mutations or TNF‐α

blocking rheumatoid arthritis drugs, leads to enhanced disease severity and susceptibly

[47‐50].

In addition to secreting cytokines, CD4+ and CD8+ T‐cells, in conjunction with

uninfected macrophages, epitheloid cells, multinucleated giant cells, and B‐cells form

granulomas, walling off infected host cells [51]. These structures can drive bacteria into a

dormant state, triggering latent infection. In individuals susceptible to progression to

active disease, the interior of the granuloma becomes necrotic, causing tissue damage

and allowing bacteria access to the airways, facilitating their spread. The precise cellular

factors that lead to the formation of necrosis and progression to active disease are

unknown [52].

In patients with either active disease or latent infection, the immune response

generated is insufficient to protect from concurrent infection with multiple strains, or

exogenous re‐infection after curative treatment [53,54]. The generation of protective

vaccines, therefore, must simulate immune responses that are not triggered during

natural infection.

6

1.2 Vaccines

1.2.1 M. bovis Bacille Calmette‐Guérin (BCG) vaccine

M. bovis Bacille Calmette‐Guerin (BCG) was developed in the early 20th century

by serially passaging on glycerine‐bile agar [55]. Since its initial tests in humans in 1921,

over four billion doses of the vaccine have been given [56]. BCG is highly efficacious

against miliary TB; however, its efficacy against the pulmonary form of the disease is

variable, ranging from 0‐80% in numerous studies [57,58]. While the variability has been

attributed to numerous causes including environmental mycobacterial exposure,

different strains and growth conditions of the vaccine, poor cold chain management, and

concurrent helminth infection, the true cause is most likely a combination of these and

additional factors [59‐62].

Aside from incomplete protection, BCG has additional limitations. As a live‐

attenuated bacterial vaccine, it cannot be given to HIV+ individuals, a critical flaw given

the high degree of geographic co‐localization of the two infections [63]. Additionally,

protection wanes over time, with re‐vaccination of adolescents and adults affording little

increased effectivity [64,65]. Lastly, the BCG vaccine interferes with the tuberculin skin

test, the easiest and most widely used TB diagnostic test, triggering a positive result in

uninfected individuals within eight to twelve weeks of vaccination [66].

7

1.2.2 Vaccines in clinical trials

As of 2014, twelve novel vaccines have undergone Phase I or Phase II clinical

trials (Table 1) [67]. All but two are designed to boost prior BCG vaccinations. In

countries childhood mortality is high, BCG is given at birth and displays protection from

miliary TB, the most severe childhood form of the disease, and a non‐specific beneficial

effect on childhood survival [58] . Building off BCG harness these protections and

sidesteps the ethical issues of withholding a potentially beneficial vaccine to conduct

clinical trial with a placebo arm [67‐69].

The eight vaccines that function as the boost component of BCG prime‐boost

regimens primarily use two strategies: recombinant viral vectors or adjuvanted fusion

proteins [68,70]. Both strategies seek to augment BCG by improving CD4+/CD8+ T‐cells

responses to immunodominant antigens that are either present in M. tuberculosis but not

the BCG, or are highly expressed by bacteria during latent infection. Ultimately, these

vaccines aim to better control latent infections and prevent the progression to active

disease (Table 1).The two vaccines designed to replace BCG work in a similar manner.

Both MTBVAC and VMP1002 enhance T‐Cell responses, either through induction of M.

tuberculosis specific CD4+/CD8+ T‐cells, or by increasing antigen‐specific central memory

T‐cells [71,72].

8

Two vaccines are currently in clinical trials as post‐exposure therapeutics.

Mycobacterium vaccae is a rapid growing, non‐pathogenic, soil‐dwelling saprophyte [73].

Randomized controlled clinical trials have demonstrated variable efficacy of heat‐killed

M. vaccae preparations in treating active TB when administered after M. tuberculosis

infection. While some studies demonstrate decreased time to sputum conversion and

improved chest radiographic findings, especially in individuals with multidrug resistant

TB, others have shown no benefit [74‐77]. RUTI is a therapeutic vaccine comprised M.

tuberculosis, grown under conditions of progressive starvation and stress, prior to

fragmentation [78]. Designed to augment short‐term chemotherapy in latently infected

individuals, it has a promising safety profile in both HIV‐ and HIV+ individuals [79].

9

Table 1: M. tuberculosis vaccines in clinical trials

Vaccine Form Route Composition Mechanism Phase Clinical Trial

Results

Vaccines designed to boost BCG

Ad5Ag85A

[80]

Viral vector

boost

Intranasal Recombinant

adenovirus 5 carrying

Ag85A

Induction of Ag85A

specific CD4+ and

CD8+ T‐cells in

airway

Phase

1

Phase 1 trial halted

for unknown

reason.

Aeras‐402

[81,82]

Viral vector

boost

Intramuscular Recombinant

adenovirus 35 carrying

TB fusion protein

Induction of

polyfunctional

CD4+ and CD8+ T‐

cells

Phase

2

Safe, immunogenic

in infants. Efficacy

trials ongoing.

Aeras‐404

[83]

Protein‐

adjuvant boost

Intradermal Ag85B‐TB10.4 fusion

protein with IC31

adjuvant

Induction of

polyfunctional

CD4+ T‐cells

Phase

2

Safe in humans,

recruiting for

immunogenicity

and efficacy trials.

H1/IC31

[84,85]

Protein‐

adjuvant boost

Intramuscular ESAT6‐Ag85B fusion

protein with IC31

adjuvant

Induction of

polyfunctional

CD4+ T‐cells

Phase

2

Safe, immunogenic

in immunodeficient

adults.

H56/IC31

[86]

Protein‐

adjuvant boost

Intramuscular Ag85B‐ESAT6‐RV2660c

fusion protein with IC31

adjuvant

Induction of

polyfunctional

CD4+ T‐cells

preventing latent

reactivation

Phase

1/2a

Human safety and

immunogenicity

trials ongoing.

ID93/GLA‐

SE

[87]

Protein‐

adjuvant boost

Intramuscular 4 TB protein fusion with

GLA‐SE adjuvant

Induction of

polyfunctional

CD4+ T‐cells

Phase

1

Human safety trials

ongoing.

10

M72/AS01E

[88,89]

Protein‐

adjuvant boost

Intramuscular M. tuberculosis39a‐M.

tuberculosis32a fusion

protein with AS01E

adjuvant

Indunction of

polyfunctional

CD4+ T‐cells

Phase

2a

Safe, immunogenic

in PPD+ and PPD‐

humans.

MVA85A

[90,91]

Viral vector

boost

Intradermal Modified Vaccinia virus

carrying Ag85A

Induction of

polyfunctional

CD4+ T‐cells and

CD8+ T‐cells

Phase

2b

Safe, immunogenic

but not protective in

infants.

Vaccines designed to replace BCG

MTBVAC

[72]

Attenuated M.

tuberculosis

Intradermal M. tuberculosis with

deletions in PhoP and

fadD26 genes

Increased antigen

specific central

memory T‐cells

Phase

1

Human safety trials

ongoing

VPM1002

[71]

Recombinant

BCG

Intradermal Urease C deficient BCG

expressing listeriolysin

Stronger CD4+,

CD8+ and type‐1

cytokine responses

Phase

2

Safe, immunogenic

in adult humans.

Studies in infants

pending.

Therapeutic vaccines

M. vaccae

[92]

Attenuated

mycobacteria

Intradermal Heat‐killed M. vaccae Unknown Phase

3

Safe, immunogenic,

conflicting reports

on efficacy

RUTI

[79]

M. tuberculosis

fragments

Subcutaneous Detoxified M. tuberculosis

liposomal fragments

Induces combined

TH1/TH2/TH3

response

Phase

2

Safe and

immunogenic in

HIV‐ and HIV+

adults with LTBI

11

1.2.3 Tuberculosis vaccines that induce sterilizing immunity

The severity of the HIV/TB co‐infection epidemic has led to increased interest in

developing a TB vaccine that protects individuals from infection by inducing sterilizing

immunity. Current vaccines and vaccine candidates rely on sustained T‐Cell driven

immune responses to prevent latent TB infections from progressing to active TB disease.

The weakening of T‐Cell immunity in HIV+ individuals therefore diminishes vaccine‐

mediated protection leaving a population already at increased risk of primary

progressive and reactivation TB disease doubly vulnerable [56]. Only two TB vaccines,

BCG and MVA85A have been evaluated for their ability to prevent M. tuberculosis

infection, rather than TB disease. Neither vaccine excels in this area; a meta‐analysis of

fourteen studies demonstrated a 19% decrease in TB infection among children

vaccinated with BCG, while MVA85A vaccinated children possessed a similar infection

rate as BCG vaccinated children in a recent Phase IIb efficacy trial [90,93].

Five childhood vaccines are against bacterial pathogens, and all lead to

sterilizing immunity. Two, those against Haemophilus influenzae type b and Pneumoccocus

sp. act by preventing colonization or invasive infection by the causative microbe [94,95].

In both cases high‐titer, high‐avidity antibodies to the capsule correlate with protection

from disease [96‐98]. Of the other three vaccines, those against Corynebacterium diphtheria

and Clostridium tetani, work through antibody‐mediated toxin neutralization, and the

protective mechanism for the Bordetella pertussis vaccine remains unclear [99‐101].

12

M. tuberculosis is very different from the causative bacteria of the vaccine‐

preventable infections mentioned above. It lacks both a polysaccharide capsule, as well

as secreted toxins. However, it is hypothesized that anti‐M. tuberculosis antibodies could

function in two ways that lead to eradication, rather than control of the infection. First,

antibodies that block uptake by phagocytes could leave bacteria vulnerable to

pulmonary clearance mechanisms or to extracellular killing by myeloperoxidase or a

combination of secreted perforin and granulysin [56,102‐105]. Second, antibody‐

mediated uptake of M. tuberculosis results in enhance phagosome‐lysosome fusion, Ca2+

signaling, and reactive oxygen species generation, all of which impair mycobacterial

survival [34,106‐108]. Antibodies that shunt M. tuberculosis away from receptors that

favor bacterial subjugation of the lysosomal degredation pathway, to FCγRs, could

prevent bacterial infection. Similar strategies have been employed against other

intracellular pathogens including Toxoplasma gondii, B. pertussis, and Salmonella enterica.

In each case, antibodies, which direct bacteria away from complement receptors and

trigger uptake by FCγRs, result in decreased survival by impairing the pathogen’s

ability to alter its vacuolar environment [109‐112].

Induction of M. tuberculosis specific antibodies by vaccines has not attracted

much study. Serum IgG responses have been examined for eight of the twelve vaccines

in clinical trials; however, monitoring of the humoral immune response has been

conducted to either serve as a marker of immune induction, or to help determine the

13

TH1/TH2 polarization of the response [78,87‐89,113‐119]. Correlation between IgG titers

and disease outcome, or functional studies of the antibodies generated, have not been

undertaken.

1.3 Animal models of tuberculosis

1.3.1 Murine model of active TB disease

As human infection with M. tuberculosis is primarily a respiratory disease,

aerosol infection of experimental animals is the most physiologically relevant route of

exposure. In the laboratory setting, nebulizers generating high titer M. tuberculosis

aerosols are used to expose animals. This generation process is harsh, decreasing the

viability of numerous bacterial species, including M. tuberculosis, leading to aerosols

containing a mixture of live and dead bacteria [120‐123]. The murine standard dose

model, the most commonly used animal models of TB disease, provides a uniform,

reproducible infection of between 50‐400 culture forming units (CFU) [124‐126].

Two other commonly used murine models of M. tuberculosis infection exist.

Intravenous instillation of bacteria leads to more rapid colonization of the liver and

spleen than the aerosol model, as the bacteria are systemic and do not need to

disseminate from the lung [127]. Additionally, adaptive immune responses begin

quicker and in the spleen, rather than the lymph node, leading to greater control of the

bacteria and less severe disease [128]. The retropharyngeal model instills M. tuberculosis

into the space above the trachea in a small volume of liquid. While more physiologically

14

relevant than the intravenous route of exposure, the fluid has the ability to wash out

soluble lung factors and is less reproducible in both delivery and lung distribution than

the aerosol model [129,130]. Despite their limitations there are two critical benefits to

each model: both allow for infection with higher dose innocula than the aerosol model,

as well as the pre‐mixing of M. tuberculosis with antibodies or other biomolecules prior

to instillation.

No matter the route of exposure, the use of mice as an experimental animal has

many benefits. Inbred strains are genetically uniform and inexpensive [124]. Knock‐out,

knock‐in, and immunodeficient mouse strains allow for the probing of specific

components of the immune response to M. tuberculosis, and have been critical for

demonstrating the importance of CD4+ T‐cells, TNF‐α and IFN‐γ in controlling infection

[49,50,131]. Despite its many advantages, the murine model does not completely reflect

the spectrum of human M. tuberculosis infection. Mice do not develop the full range of

granulomas, forming only non‐necrotic lesions unless manipulated through the loss of

cytokines or injections of microbial products [50,125,132]. Murine infection also leads to

disseminated active disease, not the pulmonary form primarily seen in

immunocompetent adult humans [44]. Lastly, no true latent M. tuberculosis infection

model exists in mice. Current latency models require antibiotic treatment regimens that

lead to persistent, but unculturable bacteria, “reactivating” months after the cessation of

antibiotic treatment or upon administration of immunosuppressives [133,134].

15

1.3.2 The ultra‐low dose murine infection model

Studies in humans and in animal models have examined numerous factors that

influence M. tuberculosis disease progression; however it is not known if these factors can

influence infection rate [102,135‐137]. The standard‐dose aerosol model is an incredibly

valuable research tool, as all mice are infected at a relatively uniform dose. However, it

does not represent how M. tuberculosis spreads in the environment. There, individuals

are exposed to low aerosol concentrations of bacteria, leading to a range of infection

rates depending on numerous factors including race, proximity to the index case, and

the duration of exposure [138,139]. To better study factors affecting infection, a model

was developed in which mice are exposed to ~1‐2 M. tuberculosis bacterium and not all

mice become infected [25].

Using this ultra‐low dose aerosol model both the impact of BCG vaccination and

genetics on murine infection rate have been examined [130]. Regardless of strain

background, BCG vaccination did not impact infection rate, though it did decrease

bacterial lung burden at one month post infection. While differences in infection rate

between BCG and sham vaccinated mice were not found, infection rate differences were

seen between the C57BL/6 and BALB/c strains (Table 2 from [130]).

16

Table 2: Infection rates after ultra‐low dose exposure to M. tuberculosis

Infected/

Total

Percent

Infected

Unadjusted

Odds Ratio

(95% CI)

P‐

value*

Adjusted

Odds Ratio

(95% CI)

P‐

value

Experimental Groups

C57Bl/6J – PBS 14/36 38.9% 0.014

C57Bl/6J – BCG 15/36 41.7%

BALB/c – PBS 25/36 69.4%

BALB/c – BCG 21/35 60.0%

Mouse Strain

C57Bl/6J 29/72 40.3%

BALB/c 46/71 64.8% 2.73

(1.39, 5.37)

0.004 4.07

(1.49‐11.08)

0.006

Vaccine Status

Naïve 39/72 54.2%

Vaccinated 36/71 50.7% 0.87

(0.45, 1.68)

0.740 0.88

(0.45, 1.72)

0.699

Total 75/143 52.5%

*P‐values and unadjusted odds ratios were calculated using Fisher’s exact test, for

pairwise comparison, and the Chi‐squared test for multiple comparisons.

The main limitation of the ultra‐low dose infection model is that it requires large

numbers of mice to provide enough statistical power to detect a 20% difference between

the partially infected groups. Work comparing the ultra‐low dose and standard dose

model suggests that early lung burden can act as a surrogate marker for infection.

C57BL/6 mice, which have a lower infection rate compared with BALB/c mice, also have

consistently lower lung burdens at two hours, one, and two days post infection (Figure

1, from [130]). The ability to use early lung burden as a proxy for infection rate allows

for smaller group sizes and therefore the ability to screen numerous genotypes or

vaccines for the ability to alter early infection events.

17

Figure 1: Early lung burden in C57BL/6 and BALB/c mice after standard dose

aerosol infection

Naïve C57BL/6 (open red circles) and BALB/c (open blue square) mice were

simultaneously infected with a standard dose aerosol of M. tuberculosis H37Rv and were

sacrificed at two hours (day 0), day one through four, and day twenty‐eight post

infection. Two‐sided p‐values were calculated by Mann‐Whitney test: (*) p ≤ 0.05, (**) p

≤ 0.01, (***) p ≤ 0.001.

1.3.3 Alternative animal models

While no existing animal model perfectly recapitulates the spectrum of M.

tuberculosis infection in humans, mice are the animal model of choice due to the wide

range of immunological reagents, small size, and low cost [124]. Three other animal

models are used to study latent TB infection and active TB disease; however, unique

limitations of each model prevent more wide‐spread use.

The low‐dose aerosol guinea pig infection model is most commonly used to

evaluate novel vaccines and therapeutic regimens prior to testing in non‐human

primates [124]. The pulmonary physiology and inflammatory response of guinea pigs is

more similar to humans than mice, and after infection, guinea pigs display granulomas

18

with caseous necrotic cores, and haematogenous spread of the bacteria [140].

Additionally, the susceptibility of guinea pigs to low quantities of M. tuberculosis allows

for a better recapitulation of the human exposure environment [141]. Despite these

advantages, this experimental animal is not as widely used as the mouse due to a dearth

of immunologic reagents for evaluating cytokines and cell phenotypes, and the extreme

susceptibility of the animal to disease [124].

The initial studies on the genetic basis of M. tuberculosis susceptibility were

conducted in rabbits in the 1930s and 1940s. After exposure to M. bovis resistant rabbits

displayed a progression of pulmonary disease similar to immunocompetent adult

humans, while susceptible rabbits displayed a disseminated phenotype similar to

miliary disease [142‐144]. No other animal model displays this range of phenotypes

without inducing germline mutations that impair the host immune response.

Additionally, resistant rabbits progress to cavitary disease unlike mice and guinea pigs

[140]. Despite the similarities with human disease, the paucity of immunologic reagents,

the lack of inbred rabbit strains, special housing requirements, higher cost, and the need

to use M. bovis rather than M. tuberculosis make the rabbit model less widely used [124].

Initially used solely to evaluate vaccines and therapeutics prior to human testing,

recent advances in reagents have allowed for broader characterization of the non‐human

primate response to M. tuberculosis. After low dose aerosol exposure, macaques produce

the full spectrum of human granulomas, from non‐necrotic lesions, to those with

19

caseous necrotic cores, and rarely, cavitary, fibrotic or calcified lesions [145]. Non‐

human primates are also the only natural model of latent infection with, low‐dose

exposure triggering a mix of primary progressive and latent disease [146]. Though the

non‐human primate model is the most similar to human disease, the challenges of

animal availability, the high cost, and the housing and care of non‐human primates in

BSL‐3, have limited the use of this model [124].

1.4 Antibodies and tuberculosis

1.4.1 Serum therapy as treatment for tuberculosis

In the pre‐antibiotic era administration of immune sera, typically derived from

animals, was the primary means of treatment for numerous infectious diseases including

diphtheria, pneumonia, H. influenza meningitis, erysipelas, whooping cough, dysentery,

measles and pre‐paralytic poliomyelitis, though its efficacy varied [147]. Beginning in

the late 1800s a small number of attempts were made to find similar immune sera that

could be used to treat M. tuberculosis. Various animal species ranging from cows to fowl

were infected with live or attenuated M. tuberculosis, or vaccinated with mycobacterial

preparations. Serum from these animals was then administered to laboratory animals or

humans, through various routes and for non‐standard amounts of time [148]. Even with

the limitations in study design, inexact treatment regimens and lack of controls, two

overarching principles were observed: one, that long treatment durations were

20

necessary to see any effect, and two, that the sera worked best on localized cases that

began treatment early in disease [148].

Believing antibodies to be the critical component of serum preparations, in the

1920s Calmette systematically analyzed antibody content in numerous batches of

clinically used immune sera. Antibody levels ranged widely between sera types, as well

as between individual batches of sera. [149]. These differences are not surprising, as

variations in antigen and vaccination regimes have since been shown to lead to varying

levels of vaccine‐induced antibodies [150‐152]. The inconsistent antibody levels within

serial preparations of a particular serum may explain the varying and sometimes

contradictory results of both human and animal trials.

The failure of these studies, despite the fact that they were poorly controlled,

possibly caused animal to human transmission of M. tuberculosis, and lacked

reproducible sera preparations, lead to the belief that antibodies did not play a role in

protection from or treatment of M. tuberculosis. Recently, however, there has been

renewed interest in serum therapy to treat TB disease. Transfer of sera from infected

mice has been shown to decrease post‐chemotherapy relapse in immunocompromised

mice, and high dose human intravenous immunoglobulin (IVIG) can decrease

pulmonary bacterial burden in mice two months post administration [153‐155].

21

1.4.2 Antibodies as a principal component of diagnostic testing and as a

biomarker for disease progression

Well established methods for diagnosing latent TB infection and TB disease exist.

The tuberculin skin test, using purified‐protein derivative (PPD), the IFN‐γ release

assay, and sputum smear microscopy/culture are the most common. These methods all

have limitations, primarily interference from BCG vaccination or immunodeficiency, or

difficulty of execution in resource poor settings [156]. In order to reach the United

Nation’s millennial goals for a world‐wide reduction of TB cases, new diagnostic

methods are needed [157].

In the last decade there has been a proliferation of studies evaluating the

potential of antibodies to serve as a component of diagnostic tests. These studies have

elucidated two hallmarks of the human antibody response to M. tuberculosis, both of

which lead to difficulties for the development of serologic testing. HIV seropositivity,

infecting strain serotype, and differing disease manifestations all produce antibody

profiles that differ in titer, subtype and antigens recognized [158‐164]. Additionally,

bacillary burden correlates with peak antibody titer making smear negative TB and

latent infection difficult to detect [161,165,166].

Second, cross reactivity of antibodies against environmental mycobacteria lead to

detectable anti‐tuberculosis antibodies in uninfected controls [167,168] . Even when

using multiplex panels, and focusing on IgG rather than IgM or IgA antibodies, there is

a prohibitively large amount of overlap between uninfected controls and patients with

22

active TB disease. [159,169‐176]. While commercial tests have been tried, their

performance in the field remains inferior to sputum smear microscopy and sputum

culture [176‐181]. Based on the body of evidence, the World Health Organization

currently recommends against the use of serodiagnostic testing, citing higher costs,

fewer disability adjusted years of life, and a higher false positive rate in regions where it

is used [179,182,183] .

Research into use of serum diagnostics has elucidated the lack of a “golden

antigen”, one that triggers high‐titer, high avidity antibodies in infected, but not

uninfected controls [161]. The absence of “golden antibodies”, led to further

discouragement about the ability of antibodies to prevent M. tuberculosis infection or

alter disease progression. However, protective antibodies to a wide range of intracellular

and extracellular pathogens including C. tetani and Listeria monocytogenes have been

identified in laboratories, despite not being produced during natural infection [184‐187] .

1.4.3 Antibodies alter disease course in mice

Both M. tuberculosis specific IgA and IgG antibodies have shown protective

effects when passively administered around the time of infection. Two different anti‐

Hsp‐X IgA monoclonal antibodies have been developed. TBA61 decreases pulmonary

bacterial burden in mice if administered either before or after infection [188,189].The

same antibody, when administered with recombinant IFN‐γ and anti‐IL‐10 antibodies

decreases post chemotherapy relapse [190]. 2E9 is a human antibody, selected from a

23

phage library, that, when administered with IFN‐γ two hours prior to infection, has

been shown to decrease pulmonary bacillary burden, in CD89 transgenic mice, one

month post treatment [191].

IgG antibodies against numerous M. tuberculosis targets have been generated as

well. When administered prior to, or at the time of, exposure M. tuberculosis specific IgG

antibodies confer a survival advantage, decrease bacterial burden and reduce

pulmonary pathology [188,192‐194]. One antibody, against HBHA, works through a

different mechanism, drastically decreasing dissemination from the lung rather than

altering pulmonary burden [195].

The precise mechanism through which these monoclonal antibodies work is not

yet known. Antibody‐mediated agglutination can lead to either non‐opsonic clearance

or killing of the bacteria; however, as protection persists when monoclonal antibodies

are administered prior to M. tuberculosis infection, this mechanism appears unlikely

[188,189,194,196,197]. Broadly neutralizing antibodies impair the ability of a pathogen to

interact with host cell receptors, preventing infection in an FCγR independent manner,

while antibody opsonization leads to FCγR mediated uptake, and ultimately killing for a

wide range of pathogens [109,110,112,198,199]. Published studies suggest there is no

overarching mechanism of action for anti‐M. tuberculosis monoclonal antibodies: F(ab)’2

fragments of SMITB14 mediate protection in a FCγR indepdent manner, while murine

expression of human CD89 is necessary for 2E9 mediated protection [191,194]. Antibody

24

target, isotype, and infection route may all influence the mechanism of action for

protective antibodies.

1.4.4 Disease progression in mice lacking components of the humoral

immune response

In addition to the use of monoclonal antibodies, numerous laboratories have

studied the impact of the humoral immune response on M. tuberculosis infection through

knock‐out mice. There is contradictory evidence that mice lacking B‐cells, a key

component of humoral immunity have greater susceptibility to M. tuberculosis infection.

Two types of b‐cell knock out mice were used in these studies: both IgH 6‐/‐ and μMT

mice have a germline deletion of IGHM, the heavy chain variable region, leading to a

lack of mature B‐cells. While two studies demonstrate that B‐cell knockout mice have

decreased pulmonary pathology, decreased bacterial burden and elevated TH2 cytokine

levels post M. tuberculosis exposure, another demonstrated enhanced pulmonary

pathology and dissemination [200‐202]. Additionally, B‐cell knock‐out mice showed

similar pulmonary responses to wild‐type mice after aerogenic rechallenge [203].

Though B‐cell knock out mice have impaired humoral immunity, B‐cells are capable of

antibody independent modulation of the immune system. Through cytokine secretion

B‐cells modulate the local environment around granulomas in infected animals, further

shaping the host’s response, and complicating the interpretation of results from B‐cell

knockout mice [204‐206].

25

Studies have also examined the pathology of TB disease in mice lacking various

antibody subclasses, transporters, and receptor components. In a pair of studies wild‐

type, IgA‐/‐ and IgR‐/‐ mice were intransasally vaccinated with M. tuberculosis

lipoprotein PstS‐1 conjugated to cholera toxin. IgA was produced in wild‐type and

IgR‐/‐ mice (though its localization to mucosal surfaces was impaired in the later) and

was not produced in IgA‐/‐ mice. In both cases, knockout mice had higher pulmonary

bacterial burden at one and four weeks post infection, as well as altered pro‐

inflammatory cytokine profiles in their lungs [207,208]. In 2007 Maglione et al, also

examined the effect the loss of Fc‐receptors have on disease progression. Mice lacking

FcγRIIB, the inhibitory antibody receptor, had decreased bacterial load in the lung and

spleen, enhanced IFN‐γ and IL‐12p40 production and reduced immunopathology one

month post infection. Contrastingly, mice lacking the FCγ chain display increased

bacterial load, enhanced IL‐10 producton, and decreased survival [33]. As with B‐cell

deficient mice, the ability of the antibody receptors themselves to alter the local lung

environment, through cytokine production, makes interpretation of these results

difficult in the context of antibody‐mediated effects.

26

2. Differences in baseline lung characteristics of BALB/c and C57BL/6J may influence differences in infection rate

2.1 Introduction

Previous work in our laboratory has focused on infection rate differences

between C57BL/6 and BALB/c mice. In these studies, C57BL/6 mice possessed both a

decreased infection rate, and decreased pulmonary bacterial burden within the first

three days post infection (Figure 1, Table 2). We hypothesized that this phenotype was

due to differences in the pulmonary environment at the time of infection. To probe this,

we have compared the baseline cytokine/chemokine profiles and pneumocyte

populations in naïve mice. BALB/c mice have higher levels of chemokines in the lung

tissue and BAL fluid than C57BL/6 mice. Though past research has shown differences in

TH1/TH2 polarization between the strains in response to infection, no differences in

baseline systemic TH1 or TH2 cytokine levels were observed [209,210]. Additionally,

pneumocyte populations differ between the strains, with C57BL/6 mice possessing 2.5

times the number of myeloid dendritic cells, than BALB/c mice.

Recently, there has been increased interest in vaccines which induce sterilizing

immunity, a process most likely achieved by preventing the early stages of M.

tuberculosis infection [211]. In order to design vaccines which induce sterilizing

immunity, we need to understand factors that can influence the initial infection event.

Studies in humans and animal models of M. tuberculosis infection have examined factors

that influence M. tuberculosis disease progression; however it is not known if these

27

factors influence infection rate [102,135‐137]. By comparing the pulmonary environment

in two mouse strains, with different infection rates, we can better understand factors

that influence the initial interactions between M. tuberculosis and its host.

2.2 Methods

2.2.1 Animals

The animals in this study were handled under a protocol approved by the Duke

Institutional Animal Care and Use Committee. C57BL/6 (027) and BALB/c (028) from

Charles River Laboratories were house in the Duke RBL for at least one week prior to

aerosol exposure. The animal rooms are environmentally controlled at 21oC, 50% relative

humidity and a twelve‐hour light/dark cycle.

2.2.2 Retropharyngeal M. tuberculosis infection

M. tuberculosis H37Rv was grown to log phase in 7H9 liquid media (BD

Bioscience, 271310) supplemented with Oleic Albumin Dextrose Catalase (OADC)

growth supplement (BD Biosciences, 212351), 0.5% glycerol (Sigma, 5516), and 0.05%

tyloxapol (Sigma, T8761). Cultures were washed twice in phosphate buffered saline with

0.05% tyloxapol (PBS‐Ty) and resuspended to a final optical density (OD) of 0.002. Six

week old female C57BL/6 and BALB/c mice were instilled with 50μL of innocula

retropharyngeally either two or twenty‐four hours prior to sacrifice.

28

2.2.3 Bacterial CFU determinations

Mice were sacrificed at either two hours or one day post retropharyngeal

exposure. Murine lungs were manually homogenized in PBS‐Ty and plated on in‐house

made 7H10 agar plates. Sterility of homogenates was ascertained by plating on

Chocolate II agar with hemoglobin and IsoVitalex (BD Biosciences, 221169). CFU were

counted after three to four weeks incubation at 37oC.

2.2.4 Sample collection and processing for Luminex multiplex assay

Six week old C57BL/6 and BALB/c mice were sacrificed by isofluorane overdose

and terminally bled from the submandibular vein. Mice were then perfused with

endotoxin free PBS. To collect bronchoalveolar (BAL) fluid, 750 μL of PBS was pushed

into the lungs through a nineteen gauge‐needle inserted into the trachea. Fluid was then

drawn out and saved for analysis. Lastly, the lung was dissected and flash frozen in an

ethanol‐dry ice bath.

After flash freezing, lungs were weighed and a volume ice cold PBS was added,

prior to bead beating, so that the final concentration of the homogenate would be 20

mg/mL. Sera was isolated from blood clotted at room temperature by centrifugation at

200g for 15 minutes.

2.2.5 Luminex multiplex cytokine assay

Sera, BAL, and tissue homogenate from both C57BL/6 and BALB/c mice were

assayed on a mouse chemokine/cytokine magnetic bead panel (Millipore, MCYTMAG‐

29

70K‐PX32) according to manufacturer’s instruction. A BioPlex 200 system running

BioPlex Manager v.6.1 was used for acquisition and analysis. Using standard curves the

assay reports the following cytokine/chemokine concentrations in pg/mL: IFN‐γ, IL‐1α,

IL‐1β, IL‐2, IL‐3, IL‐4, IL‐5, IL‐6, IL‐7, IL‐9, IL‐10, IL‐12 (p40),IL‐12 (p70), IL‐13, IL‐15, IL‐

17, IP‐10, KC, leukemia inhibitory factor (LIF), LIX, monocyte chemoattractant protein

(MCP‐1), macrophage colony stimulating factor (M‐CSF), monokine induced by γ‐

interferon (MIG), macrophage inflammatory protein (MIP‐1α), MIP‐1β, MIP‐2, RANTES,

TNF‐α, vascular endothelial growth factor (VEGF), Eotaxin, granulocyte colony

stimulating factor (G‐CSF), and granulocyte macrophage stimulating factor (GM‐CSF).

The limit of detection is defined as the minimum value on the standard curve for each

cytokine. Samples whose values fell below the minimum on the standard curve are

listed at the limit of detection.

2.2.6 Single cell suspension of mouse lung

Following isofluorane overdose, lungs from eight week old C57BL/6 and BALB/c

mice were minced into a single cell suspension in R10 media (RPMI 1610 (Gibco, 11875‐

093), 10% Fetal bovine sera (FBS)(Gibco, 16000), 1% L‐glutamine (Gibco, 25030), and

0.01M HEPES (Gibco, 153600)) containing 50 μg/mL elastase (Sigma, E1250), 0.02 mg/mL

DNAse (Sigma, D4257), 0.5 μg/mL collagenase (Worthington Biochemical, CLS‐1) and 1

μg/mL streptomycin (Sigma, S6501) as previously described [212]. After vortexing the

suspension was incubated at 37oC for 30 minutes, washed in antibiotic‐free R10 media,

30

and passed through a 70 μm cell strainer. The sample was then spun at 450g to pellet,

resuspended in 1x ACK red blood cell lysis buffer (0.155M NH4Cl2, 10mM KHCO3, 1mM

EDTA∙2Na) and washed a final time. The resultant single cell suspension was then

stained for flow cytometry.

2.2.7 Cell staining and flow cytometry

Primary pneumocytes were blocked with 2.4G2, an anti‐CD16/CD32 antibody

(BD Biosciences, 553131), for 15 minutes at room temperature. Pneumocytes were then

stained with the following directly conjugated antibodies for analysis: CD11b Pac Blue

(Biolegend, 101224), CD11c APC‐Cy7 (BD Biosciences, 561241), GR‐1 Alexafluor 700

(AF700) (Biolegend, 108422), and Live/Dead fixable yellow (Invitrogen, L‐34967). All

antibodies were titrated on either RAW264.7 cells or primary pneumocytes prior to use.

APC counting beads (BD Biosciences 3404871) were added to each tube to serve as a

counting standard. Live cells were phenotyped using a FACSAria II SORP with

FACSDiva™ 8.0 (BDIS, San Jose CA).

2.2.8 Flow cytometry gating

The gating scheme for flow cytometry analysis is shown in (Figure 2).

31

Figure 2: Gating scheme for flow cytometry analysis:

Single cells were discriminated from electronic noise by plotting forward‐scatter area vs.

side scatter area. Live cells were then selected on the basis of Live/Dead fixable yellow

stain exclusion. Non‐neutrophilic cells were separated from neutrophils on the basis of

GR‐1 positivity. GR1‐ cells were then gated into four groups on the basis of CD11b and

CD11c signal: (1) myeloid DCs, (2) alveolar macrophages, (3) recruited macrophages, (4)

monocytes.

Single cells were discriminated from electronic noise by plotting forward‐scatter

area vs. side scatter area. Live cells were then selected on the basis of Live/Dead fixable

yellow stain exclusion. On the basis of published literature [27,213] cell types were

delineated on the basis of GR‐1, CD11b, and CD11c positivity. Non‐neutrophilic cells

were separated from neutrophils by GR‐1 positivity, with neutrophils ultimately being

identified as GR1+ CD11b+. GR1‐ cells were then gated into four groups based onCD11b

32

and CD11c signal. (1) myeloid DCs (CD11bhi CD11chi), (2) alveolar macrophages

(CD11blo CD11chi) (3) migratory macrophages (CD11bhi CD11clo) (4) monocyes (CD11bhi

CD11clo). Flowjo v10.0 (Treestar) was used for all flow cytometry analysis.

2.2.9 Statistics

Prism Software (GraphPad) was used for statistical analyses. For pairwise

comparisons among continuous variables a two‐sided student’s t‐test was used for

normally distributed data, and a Mann‐Whitney test was used for non‐normally

distributed data. To determine if total chemokines were upregulated in BALB/c vs.

C57BL/6 mice, aligned ranks tests were used to make the comparisons within the three

samples (sera, lung, BAL). The observations were standardized to a mean of zero within

each chemokine and then ranked. The aligned rank test statistic was computed in SAS

9.2 using PROC FREQ.

2.3 Results

2.3.1 Early CFU differences in BALB/c in and C57BL/6J mice are observed after aerosol exposure but not retropharyngeal instillation

Previous studies from our laboratory have demonstrated that C57BL/6 mice have

decreased pulmonary bacterial burden, compared to BALB/c mice, in the first three days

after aerosol M. tuberculosis exposure (Figure 1). To examine if differences in early

infection CFU were specific to the aerosol infection model, C57BL/6 and BALB/c mice

were instilled with 3.5x105 M. tuberculosis H37Rv retropharyngeally, and sacrificed at

one and twenty‐four hour post infection. In contrast to the aerosol model, no differences

33

were observed when the BALB/c strain was compared with the C57BL/6 strain at either

time point (day 0: Δ= ‐0.04 log10 CFU, p=0.37; day 1: Δ= ‐0.05 log10 CFU, p=0.13) (Figure

3).

Figure 3: Early lung CFU in C57BL/6 and BALB/c mice after retropharyngeal

instillation

Six week old female C57BL/6 (open red circles, n=4) and BALB/c (open blue squares,

n=4) mice were instilled with 3.47x105 M. tuberculosis H37Rv retropharnygeally. At two

and twenty‐four hours post instillation, mice were sacrificed and right lung CFUs

determined. The black line represents the mean.

2.3.2 Cytokines and chemokines are differentially expressed in the BAL fluid and lung homogenate of BALB/c and C57BL/6 mice

The inability to recapitulate the early CFU difference between BALB/c and

C57BL/6 mice in the retropharyngeal model lead us to hypothesize that the baseline lung

environment, which is disrupted upon retropharyngeal installation, plays a critical role

in the decreased bacterial survival observed in C57BL/6 mice within the first day after

34

aerosol infection. Differences in soluble signaling molecules could play an important

role in shaping the pulmonary environment, and dilution of their levels in the BAL fluid

by the volume of the retropharyngeal instillation, could contribute to the elimination of

the early infection CFU differences between the strains.

To test this hypothesis, baseline cytokine and chemokine levels in sera, BAL

fluid, and lung tissue homogenate from naïve BALB/c and C57BL/6 mice were examined

to determine if differences existed between the strains (Table 3; Appendix A, Table 15).

The median cytokine values for IFN‐γ, TNF‐α, IL‐2, IL‐12(p40), IL‐12(p70), IL‐4, IL‐5, IL‐

6, G‐CSF, IL‐1α, IL‐3, IL‐7, IL‐15, IL‐17, LIF fell below the limit of detection for both

C57BL/6 and BALB/c mice in BAL and lung homogenate, and are only displayed in

Table 15 (Appendix A). No differences between TH1 and TH2 cytokines were observed

between the strains in either lung homogenate or BAL fluid, with most cytokines falling

below the limit of detection. Differences were noted for three other cytokines. M‐CSF

(Δ=2.04, p=0.008) was more highly expressed in tissue homogenates of BALB/c mice as

compared to C57BL/6 mice. Conversely, VEGF (Δ=0.36, p=0.008) and IL‐9 (Δ=0.65,

p=0.008) were more highly expressed in lung homogenates of C57BL/6 mice (Table 3).

Several chemokines were upregulated in BALB/c mice. RANTES (Δ=3.26,

p=0.016), and KC (Δ=1.64, p=0.008) were elevated in the lung tissue, while, KC (Δ=2.76,

p=0.016) and MIG (Δ=3.04, p=0.008) were elevated in the BAL fluid. Additonally, Eotaxin

and MIP‐1β had elevated levels in lung tissue and MIP‐2 had elevelated levels in BAL

35

Table 3: Cytokine values for C57BL/6 and BALB/c mice in BAL and lung homogenate

Median and range are displayed. P‐values were determined by Mann‐Whitney test. Cytokine’s whose

median values were at the limit of detection in BAL and lung homogenate for both strains are not shown

here, but can be found in Table 15.

Tissue BAL

B6

(n=5)

BALB/c

(n=5)

Bc/

B6

P‐

value

B6

(n=5)

BALB/c

(n=4)

Bc/

B6

P‐

value

Cytokines associated with a Th2 response

IL‐10 <3.6 <3.6 N/A N/A 7.2

(4.0‐10.2)

5.6

(<1.7‐11.4) 0.78 0.73

IL‐13 755.4

(<360.4‐769.6) <360.6 0.48 0.17 <180.3 <180.3 N/A N/A

Additional cytokines

GM‐CSF 35.4

(4.5‐62.4)

28.1

(22.3‐71.08) 0.79 0.73

2.3

(<1.1‐2.3)

14.1

(6.7‐17.7) 6.19 0.08

M‐CSF 55.4

(43.6‐69.4)

112.8

(80.9‐171.0) 2.04 0.008 <1.9 <1.9 N/A N/A

VEGF 670.6

(574.5‐883.0)

243.1

(208.0‐511.4) 0.36 0.008

7.4

(3.3‐9.8)

3.6

(<1.6‐6.0) 0.49 0.064

IL‐1β <4.1

(<4.1‐9.1)

<4.1

(<4.1‐25.1) N/A 0.44

<2.1

(<2.1‐5.0)

5.8

(<2.1‐8.3) 2.83 0.30

IL‐9 595.8

(489.3‐1179.8)

393.12

(302.0‐445.9) 0.65 0.008

116.0

(21.2‐175.2)

99.1

(37.4‐373.3) 0.85 1.00

IP‐10 306.7

(181.9‐419.4)

334.4

(204.6‐404.2) 1.09 0.55

6.8

(<1.7‐9.0)

7.7

(5.7‐14.1) 1.12 0.56

CCL chemokines

Eotaxin 670.5

(551.3‐1540.5)

879.8

(538.0‐2158.3) 1.31 0.84

<1.6

(<1.6‐6.9)

9.5

(6.2‐23.3) 5.82 0.056

MCP‐1 57.3

(54.9‐80.4)

65.3

(51.0‐73.6) 1.13 0.50 <2.0

<2.0

(<2.0‐4.08) N/A 0.44

MIP‐1α 84.56

(46.3‐118.9)

70.1

(57.5‐93.5) 0.83 1.00

37.5

(24.7‐61.9)

38.7

(31.5‐52.3) 1.03 0.78

MIP‐1β 18.5

(17.6‐49.5)

36.2

(22.5‐43.6) 1.95 0.15 <1.6

<1.6

(<1.6‐3.4) N/A 0.44

RANTES 27.5

(25.0‐52.9)

89.6

(44.0‐107.5) 3.26 0.016 <1.7 <1.7 N/A N/A

CXCL chemokines

KC 166.1

(129.7‐191.1)

274.7

(207.7‐388.0) 1.64 0.008

5.9

(4.4‐8.6)

16.4

(10.5‐18.4) 2.76 0.016

LIX 38.4

(<17.7‐244.7)

<17.7

(<17.7‐80.6) 0.48 0.52 <8.8 <8.8 N/A N/A

MIG 456.1

(393.5‐665.9)

583.0

(398.6‐811.3) 1.27 0.42 <1.8

5.3

(4.0‐9.9) 3.04 0.008

MIP‐2 32.6

(<11.8‐42.5)

24.7

(<11.8‐45.3) 0.75 1.00

<5.9

(<5.9‐12.4)

13.4

(<5.9‐16.3) 2.26 0.13

36

fluid, though the differences were not statistically significant (Δ=5.82, p=0.056; Δ=1.95,

p=0.15, and Δ=2.26, p=0.13 respectively) (Table 3). Multivariant analysis, considering all

cytokines together, demonstrated higher levels of total chemokines in the BAL fluid and

lung tissue homogenate of BALB/c mice (p<0.001 and p=0.038 respectively), but not in

sera (p=0.17). Interestingly, the cytokine and chemokine profile in sera differed

significantly from that observed in BAL and tissue (Appendix A, Table 15).

2.3.3 Differences in baseline cell populations in BALB/c and C57BL/6 mice

In addition to soluble signaling molecules, differences in the percent composition

of pulmonary phagocytes could also play a role in M. tuberculosis survival early in

infection. A bacterium is capable of infecting numerous phagocytic cell types; however,

its ability to survive in each varies [24,27,214,215]. To test this hypothesis primary

pneumocytes were isolated from the lungs of naïve mice, and stained with surface

markers to identify neutrophils, monocytes, myeloid DCs, recruited macrophages, and

alveolar macrophages. While no differences between the strains were observed for

neutrophils, migratory macrophages, alveolar macrophages or monocytes, the

proportion of myeloid DCs were elevated in C57BL/6 mice (0.45% vs. 0.19% of total

pneumocytes, p=0.028) (Figure 4).

37

Figure 4: Pnemocyte populations in C57BL/6 and BALB/c lungs

Primary pneumocytes were isolated from lungs of naïve C57BL/6 (n=4) and BALB/c

(n=4) mice and stained with antibodies against extracellular markers. Mean ± SEM is

displayed. Statistical significance was calculated using the Mann‐Whitney test: (*)

p<0.05, (**) p<0.01, (***) p<0.001.

2.4 Discussion

As compared with C57BL/6 mice, BALB/c mice are more susceptible to disease

from intracellular pathogens, both mycobacterial and non‐mycobacterial [216‐220]. The

increased bacterial burden in the lungs and spleen of BALB/c mice, within the first few

weeks after infection with mycobacterial pathogens, is generally attributed to broad

differences in the immune response: the C57BL/6 strain has a TH1 skewed immune

response, while the BALB/c strain is TH2 skewed [216,217,221]. In recent years there has

been increased interest in the development of vaccines that prevent M. tuberculosis

infection and human TB vaccine trials have used infection rate as a secondary endpoint

[56,90,211]. While past studies have systematically identified factors affecting disease

38

susceptibility, none have examined if these factors correlate with differences in infection

rate [102,135‐137].

Previous work in our laboratory has demonstrated early infection bacterial

burden, and infection rate differences between C57BL/6 and BALB/c mice (Figure

1,Table 2). The loss of this phenotype upon retropharyngeal instillation lead to the

hypothesis that soluble factors, diluted out due to the volume of the retropharyngeal

instillation, were critical for the decreased bacterial burden in C57BL/6 mice after aerosol

exposure (Figure 3). To test this hypothesis we characterized the lung environment in

naïve mice from both strains.

Chemokines regulate cell migration into the lungs, and contribute to the TH1/

TH2polarization of the immune response. Differences in chemokine and cytokine levels

between the strains have been documented both systemically, and within the lung in

response to infection [217,219,222‐225]. Given, however, that bacterial load differs by as

soon as two hours post infection, in the standard dose aerosol model, if differential

chemokine/cytokine expression plays a role, it is most likely due to baseline differences

between the strains, rather than their response to M. tuberculosis. In this study we noted

that BALB/c mice have elevated levels of both CCL and CXCL chemokines compared to

C57BL/6 mice (Table 3; Appendix A, Table 15). Further studies exploring if differences

in chemokine levels between the strains contribute either to differences in cellular

39

activation, or differences in mycobactericidal activity of pulmonary phagocytes need to

be undertaken.

After noting differences in baseline chemokine levels, we hypothesized that the

higher levels of chemokines in the lungs of BALB/c mice would lead to higher

proportions of myeloid DCs and recruited macrophages in the naïve lung, providing a

larger number of cellular targets for M. tuberculosis to infect. Surprisingly, we noted the

opposite with myeloid DC’s being elevated in the lungs of C57BL/6 mice (Figure 4). M.

tuberculosis infects numerous cell types including macrophages, DCs, neutrophils, and

alveolar epithelial cells, both in vivo and in vitro [27,143,214,215,226]. M. tuberculosis

survival in DCs is constrained, as compared with alveolar macrophages, due to

impairment of nutrient trafficking to the infected phagosome [214,227]. Further studies,

examining if early CFU differences between the strains are attributable to a higher

proportion of infected DCs in C57BL/6 mice are warranted.

An alternate hypothesis is that differences in airway architecture, leading to

decreased M. tuberculosis deposition in the lung, are responsible for the strain differences

in early infection bacterial burden. Several studies have compared respiratory

parameters among inbred strains, and while statistically significant differences have

been found, the magnitude of the differences is small [228‐230]. In addition, detailed

measurements of ventilatory volumes show no differences between C57BL/6 and

BALB/c mice [230]. To exclude airway architecture as a contributing factor in early lung

40

burden differences between the strains, both C57BL/6 and BALB/c mice were infected

retropharyngeally. In this model, the difference between the strains is lost, at both two

and twenty‐four hours post infection: there is less than a 2% difference in CFU between

the two strains at each time point (Figure 3). While this method of infection bypasses the

upper airway, it also introduces 50 μL PBS into the lung, potentially washing out critical

soluble factors, preventing us from distinguishing between hypotheses.

The ability to examine factors which affect M. tuberculosis infection rate, rather

than disease, is critical for a both a better understanding of the M. tuberculosis infection

event, and the development of anti‐infective vaccines. Taken together, this study

demonstrates that genetic factors altering the pulmonary environment in naïve mice

may play a role in influencing the initial interaction between the host and M. tuberculosis.

Further studies with this experimental system have the ability to elucidate a mechanism

by which baseline differences in pneumocyte populations and cytokine levels influence

early infection events, and to identify novel factors influencing M. tuberculosis infection

rate.

2.5 Future Directions

Although this study has generated the interesting hypothesis that the pulmonary

environment at the time of infection can influence infection rate, the precise mechanism

by which this occurs is unknown. Further studies are needed to elucidate the precise

role that differences in the baseline chemokine profile and pneumocyte populations play

41

in altering early infection events. Additionally, examining early bacterial burden in

alternate strains of mice, has the potential to identity genetic factors that play a role in

infection rate differences.

One hypothesis is that the differences in baseline cytokine and chemokine levels

affect the activation state of phagocytes in the lungs. Chemokines and cytokines play a

role in altering macrophage activation status, and in inducing naïve dendritic cell and

monocyte maturation [231,232]. To test this hypothesis, single cell pneumocyte

preparations from naïve mice could be stained with markers for classical (CD40, CD80,

CD86) and alternative (Mannose Receptor (CD 206), transferrin receptor (CD71) and

resistin‐like molecule alpha (RELMα)) macrophage activation, as well as DC activation

(CD40, CD80, CD83, pDL1), prior to interrogation by flow cytometry [233,234]. Even if

no differences are observed in classical activation markers, the cytokine environment in

C57BL/6 mice could predispose lung pneumocytes to M. tuberculosis control or killing.

Ex vivo infection of primary myeloid DCs, neutrophils, alveolar and migratory

macrophages with M. tuberculosis, followed by lysis at early time points and plating for

bacterial CFUs, could elucidate if C57BL/6 phagocytes are more primed for bacterial

killing.

An additional hypothesis is C57BL6 mice, due to differences in the baseline

phagocyte composition in the lung, have a greater proportion of infected myeloid DCs

than BALB/c mice. Myloid DCs are the predominantly infected cell by fourteen days

42

post infection, and constrain M. tuberculosis survival better than macrophages [27,214].

To test this, primary pneumocytes isolated from mice recently exposed to aerosolized M.

tuberculosis can be sorted on the basis of cell surface markers. Alveolar macrophages,

recruited macrophages, neutrophils and myeloid DCs can then be plated on 7H10 agar

plates, with the resultant number of infected cells determined by the number of CFUs.

The studies undertaken in this chapter have attempted to understand the

mechanism underlying the infection rate difference between C57BL/6 and BALB/c mice.

Factors influencing infection rate are most likely complex, influenced by numerous

genetic factors. Cross breeding of C57BL/6 and BALB/c mice, in an attempt to map the

location of these factors, is unlikely to succeed as the magnitude of the early CFU

difference between the strains is small. The collaborative cross project has used cross

breeding of eight genetically diverse strains of mice, to derive hundreds of inbred mouse

strains, with high degrees of genetic variation [235,236]. Screening mice from a

collaborative cross project could identify strains with a more pronounced early infection

CFU differences that are amenable to interrogation through cross breeding.

Identification of genetic loci that contribute to the host response early in M. tuberculosis

infection will not only further our understanding of factors that influence M. tuberculosis

infection rate, it may even identify targets that can be harnessed in an anti‐infective

vaccine.

43

3. Human antibodies to the surface of M. tuberculosis

3.1 Introduction

Studies on antibody production against specific M. tuberculosis proteins abound,

largely focused on their use in the diagnosis of either TB infection or TB disease.

Antibodies to the surface of other bacterial species correlate with protection from

infection, and colonization [95,237,238]. Identifying the types of antibodies produced

during M. tuberculosis infection, especially those against surface components, is

important as these antibodies could potentially modify the course of infection.

To test this, we have developed a novel whole‐cell ELISA assay to detect

antibodies to the live M. tuberculosis surface, and have compared these antibodies to

those produced against a variety of inactivated antigenic fractions. Antibodies to the

surface of live M. tuberculosis are present at low levels in uninfected individuals. Their

titers increase in those with either latent TB infection or active TB disease. Paradoxically,

IgG antibodies to the live M. tuberculosis surface have lower relative avidity in persons

with active TB disease than in uninfected individuals. Antibodies to the M. tuberculosis

surface are comprised of similar amounts of IgG and IgM, while antibodies to antigenic

fractions are predominately IgG. Among patients with active disease, M. tuberculosis

surface‐binding antibodies have higher titers and higher relative IgG avidity scores in

those were BCG‐vaccinated or foreign‐born. These correlates were not seen for

antibodies to the inactivated antigenic fractions.

44

Humoral immunity is increasingly recognized as a component of the protective

immune response to M. tuberculosis, and as a target for TB vaccines [107]. Antibodies

that bind to the surface of live M. tuberculosis are a potentially protective class, and they

have different properties from antibodies produced against other mycobacterial targets.

In particular, most patients with TB infection and disease fail to produce highly‐avid

IgG antibodies to the surface of the live bacterium. These results have important

implications for TB vaccine development.

3.2 Methods

3.2.1 Bacterial cultures and lysates

Mycobacterium fortuitum (ATCC 6841), Mycobacterium bovis BCG Danish (ATCC

35733), Mycobacterium avium subsp. avium Chester 1901 (ATCC 25291), Mycobacterium

tuberculosis H37Rv (ATCC 25681), and Mycobacterium intracellulare (ATCC 13950) were

grown in 7H9 media supplemented with 10% OADC, 0.5% glycerol, 0.05% tyloxapol, at

37oC, in a shaking incubator at 100 ‐ 130 rpm, to an OD between 1.0‐1.5 as measured by a

Cell Density Meter (Biowave, CO8000). For preparation of protein lysates, cultures were

centrifuged at 600g for 5 minutes and pellets were washed twice with PBS, pH7.4,

containing 0.05% tyloxapol. Pellets were lysed by vortexing with 0.1 micron glass beads

in the presence of 50mM tris hydrochloride, 0.5mM EDTA, 60 mM sodium phosphate

0.67% SDS, and protease inhibitors (Roche 11836170001). Lysates were clarified by

45

centrifugation at 600g, then again at 16,000g. Protein concentrations were determined

using BCA protein assay (Pierce 23227).

3.2.2 Additional ELISA antigens

M. tuberculosis H37Rv fractions including culture filtrate (NR‐14825), cell wall

(NR‐14828), whole cell lysate (NR‐14822), and lipoarabinomannan (LAM) (NR‐14848)

were obtained from Colorado State University through BEI resources.

3.2.3 ELISA assay

Antigen fractions and lysates were diluted in CBC buffer (7.5 mM sodium

carbonate, 17.4 mM sodium bicarbonate, pH 9.0) and plated at a concentration of 200

ng/well in 96 well high‐binding plates (Costar 3590). For enzyme‐linked immunosorbant

assays (ELISAs) to the live bacterial surface, growing cultures of mycobacteria at OD of

0.7‐1.5 were pelleted at 200g for 10 minutes. Live bacterial pellets were resuspended in

CBC buffer to an OD of 1.5‐2.5 and approximately 5x107 bacteria were plated per well.

After overnight incubation of plates at 4OC, wells were washed with PBS, pH 7.4

containing 0.05% Tween‐20 (PBST), then blocked with 5% nonfat dry milk in CBC buffer.

PBST‐washed plates were incubated overnight at 4OC with immune and control plasma

diluted in PBST containing 5% nonfat dry milk. A 1:128 dilution of human plasma from

a patient with active TB disease was used as a positive control. PBST with 5% nonfat dry

milk was used as a negative control. PBST washed plates were then incubated for 1

hour with a 1:3000 dilution of either alkaline phosphatase conjugated goat‐anti‐human

46

Ig (whole molecule) (Sigma A15431), IgG (Fc‐specific) (Sigma A9544), or IgM (Fc‐

specific) (Sigma A5937). After the addition of 1 μg/ml para‐nitrophenyl phosphate

(PNP) solution in CBC buffer containing 1.1 mM magnesium chloride (CBC‐Mg), color

development was recorded when positive control (POS) reached an OD of 2.0 as

measured at 405nm by spectrophotometer. Titers were defined as the reciprocal dilution

which produced an OD of twice background, and were determined by exponential

interpolation.

The whole cell ELISA assay is intrinsically more variable due to the nature of its

antigen. Bacteria clump together, and can wash off, during early wash steps, and the

assay takes longer to develop leading to higher backgrounds. All whole cell ELISAs

used in this study possess backgrounds of less than twice seen for whole cell lysate (for

Ig whole molecule and Ig (Fc‐specific) background <0.125, for IgM (Fc‐specific)

background <0.160). The reproducibility of the assay is robust (r2=0.80) and is

comparable to whole cell lysate (r2=0.86) (Figure 5).

47

Figure 5: Reproducibility of human sera on different ELISA antigens.

Human plasma was randomly selected from our cohort of 71 individuals and run in

duplicate on (A) live M. tuberculosis cell surface (n=20) (B) whole cell lysate (n=20) and

(C) lipoarabinomannan (n=10).

3.2.4 Avidity ELISA assay

Antigenic fractions, protein lysates and live mycobacteria were plated as

described above. After 4oC incubation overnight, plates were washed with PBST and

blocked with 3% bovine serum albumin (BSA) in PBST. Plates were sequentially washed

in PBST, incubated with immune and control plasma diluted at a 1:64 in 0.5% BSA in

PBST for two hours at room temperature, re‐washed in PBST and incubated with

between 0.01 and 6M sodium thiocyanate (Sigma) diluted in 0.5% BSA in PBST for 15

minutes at room temperature. PBST‐washed plates were then incubated overnight at

4OC in 0.5% BSA in PBST with a 1:3000 dilution of alkaline‐phosphate conjugated goat‐

anti‐human IgG (Fc specific) (Sigma9544). The same 1:128 dilution of plasma from a

48

patient with active TB disease was used as a positive control. PBST with 0.5% BSA was

used as a negative control. After a final PBST wash plates were incubated in 1μg/mL

PNP in CBC‐Mg solution. Color development was recorded when POS control reached

OD 2.0 as measured at 405nm by spectrophotometer. The avidity scores are defined as

the concentration of sodium thiocynate where the OD is equal to 50% of the no sodium

thiocynate control value.

3.2.5 Statistical Analysis

For comparison between groups with normal distributions either a two‐tailed

student’s t‐test, for comparisons between two variables, or ANOVA, for comparisons of

more than two variables, was used. A Mann‐Whitney test was used to compare two

groups with non‐normal distributions. For comparison of groups containing categorical

variables, a Chi‐squared or Fisher’s exact test was used as appropriate. Correlations

between two continuous variables were probed through linear regression with r and p‐

values reported. All statistical calculations were conducted using PRISM (Graphpad

Software).

3.2.6 Human subjects

Patients with latent TB infection and active TB disease were enrolled in Durham

and Wake Counties, North Carolina, as part of the “TB Epitope study.” Latent TB

infection was defined by a positive interferon‐gamma release assay (Quantiferon® Gold

In‐Tube, Cellestis Inc, Valencia, CA) with no radiographic or clinical findings suggestive

49

of active TB. Active TB disease was defined by culture‐proven TB disease or a diagnosis

of clinical TB. This group included patients with a recent TB diagnosis, patients who

were in the midst of therapy and patients who had completed therapy in the past.

Uninfected patients were HIV‐seronegative, tuberculin skin test‐negative, healthy

volunteers with no history of BCG vaccination. Informed written consent was obtained

from participants, and the study was approved by the Duke University Medical Center

Institutional Review Board.

3.2.7 Cytokine profiling

The cytokine profiling data used in this study was first reported in Frahm et al.

2011[239]. In brief, whole blood was collected from subjects and assayed by

Quantiferon® Gold In‐Tube (Cellestis Inc, Valencia, CA) testing. Each sample was

incubated with either (a) a mixture of three TB specific antigens (ESAT‐6, CFP‐10, and

TB7.7), (b) a mitogen positive control, or (c) a negative control tube containing no

antigens. Following stimulation supernatant was collected and assayed for 25 cytokines

and chemokines (IL‐1β, IL‐1Ra, IL‐2, IL‐2R, IL‐4, IL‐5, IL‐6, IL‐7, IL‐8, IL‐10, IL‐12

p40/70, IL‐13, IL‐15, IL‐17, TNF‐α, IFN‐α, IFN‐γ, GM‐CSF, MIP‐1α, MIP‐1β, IP‐10, MIG,

Eotaxin, RANTES, MCP‐1) through a Human Cytokine 25‐plex assay (Biosource,

Camarillo, CA).

50

3.3 Results

3.3.1 Demographic information on human population

The 71 patients in our study included healthy uninfected volunteers (n=9), patients with

latent TB infection (n=23), and patients with active TB disease (n=40). Demographic and

clinical data are shown in Table 4. As expected, several clinical characteristics

differenced among the groups. Male gender was less common in uninfected and latent

TB infection groups as compared to the active TB infection group (33% and 48% vs 73%;

p = 0.05 and 0.06 respectively). BCG vaccination was less common in the uninfected

group than in the latent TB infection or the active TB disease group (0% vs. 39% and

43%; p= 0.04 and 0.02 respectively). Similarly, birth in the US was more

common in the uninfected group than in the latent TB infection or the active TB disease

group (78% vs. 44% and 50%; p= 0.05 and 0.06 respectively).

51

Table 4: Demographics and clinical data

Variable Uninfected

(n=9)

Latent TB

infection (n=23)

Active TB

disease (n=40)

N

Percent*/

mean ± SD N

Percent/

mean ± SD N

Percent/

mean ± SD

Age (yrs) 9 38 ± 14 23 42 ± 17 40 43 ± 15

Gender

Male 3 33% 11 48% 29 73%

Female 6 67% 12 52% 11 28%

Race/ethnicity

White 6 67% 8 35% 7 18%

White Hispanic 1 11% 4 17% 11 28%

Black 2 22% 9 39% 17 43%

Black Hispanic 0 0% 0 0% 1 3%

Asian 0 0% 2 9% 4 10%

HIV seropositivity

HIV + 0 0% 2 8% 7 18%

HIV – 9 100% 19 83% 33 83%

Unknown 2 8%

Diabetes

Yes 2 9% 6 15%

No 21 91% 34 85%

Tobacco usage

Yes 7 30% 11 28%

No 16 70% 29 73%

Alcohol consumption (days)

None consumed 14 61% 26 65%

1‐3 drinks 5 22% 6 15%

>3 drinks or binge 4 17% 8 20%

BCG vaccination history

Yes 0 100% 9 39% 17 43%

No 9 0% 14 61% 22 55%

Unknown 0 0% 0 0% 1 3%

US‐born

Yes 7 78% 10 44% 20 50%

No 1 11% 13 57% 20 50%

Unknown 1 11% 0 0% 0 0%

PPD induration (mm) n/a n/a 19 24 ± 19 37 20 ± 16

Disease state n/a n/a n/a n/a

Pulmonary 22 55%

Extra‐pulmonary 14 35%

Both 4 10%

*Percentages may not add to 100% due to rounding.

52

3.3.2 Human antibody titers to the surface of live M. tuberculosis and to

inactivated antigenic fractions

Plasma was assayed by ELISA to determine total antibody titers against the

surface of live M. tuberculosis H37Rv and against four inactivated M. tuberculosis

antigenic fractions (Figure 6A‐B, Figure 7A‐C). Plasma from the uninfected group

reacted to the live M. tuberculosis surface (mean titer 2.0 log10) and to all four antigenic

fractions (mean titers, 2.2 to 3.1 log10). Plasma from the active disease group had higher

antibody titers to the surface of live M. tuberculosis (Δ=0.72 log10), and to two of the

antigenic fractions, whole cell lysate (Δ=0.82 log10) and secreted proteins (Δ=0.62 log10),

when compared to uninfected controls, while cell wall (Δ=0.46 log10) had elevated

antibody titers when compared to individuals with latent infection. The latent TB

infection group had intermediate titers, falling between the uninfected controls and the

active TB disease groups for the surface of live M. tuberculosis, whole cell lysate and

secreted proteins. Antibody titers to lipoarabinomannan were similar among the three

patient groups.

53

Figure 6: Antibody responses to M. tuberculosis live cell surface or whole cell lysate

Plasma from PPD‐negative volunteers (Uninfected), patients with latent TB infection

(Latent), or patients with active TB disease (Active) were assayed by ELISA against the

surface of live M. tuberculosis H37Rv or H37Rv whole cell lysate. (A‐B) Log10 total Ig

titers. (C‐D) Relative IgG avidity. (D‐E) Log10 ratio of IgG to IgM titers. Two‐sided p‐

values by student’s t‐test or Mann‐Whitney test: (*) p ≤ 0.05, (**) p ≤ 0.01, (***) p ≤ 0.001.

54

Figure 7: Antibody responses to M. tuberculosis lipoarabinomannan, cell wall, and

secreted protein fractions



surface of live M. tuberculosis H37Rv or H37Rv whole cell lysate. (A‐C) Log10 total Ig

titers. (D‐F) Relative IgG avidity. (G‐I) Log10 ratio of IgG to IgM titers. Two‐sided p‐

values by student’s t‐test or Mann‐Whitney test: (*) p ≤ 0.05, (**) p ≤ 0.01, (***) p ≤ 0.001.

Persons with active TB disease had variable responses to the live M. tuberculosis

surface (Figure 6A) with a range of 1.6 to 3.8 log10 and an interquartile range from 2.3 to

3.3 log10. Variability was generally less in the uninfected and latent infection groups.

55

This variability resulted in overlap among the patient groups in total antibody titers in

spite of statistically significant differences between groups (Figure 6A). The same trend

was observed for all the antigenic fractions as well (Figure 6B, Figure 7A‐C). Antibody

titers across antigens were correlated for each individual (data not shown).

3.3.3 Relative IgG avidity to the surface of live M. tuberculosis and to

inactivated antigenic fractions

ELISA was used to determine the relative IgG avidity to the live M. tuberculosis

surface and to the four inactivated antigenic fractions. Repeated exposure to antigens, as

in prime/boost vaccinations, normally leads to increased relative avidity [97]. As

expected, persons with active disease had higher relative IgG avidity than the

uninfected controls for all four inactivated fractions (Δ=1.4 to 2.6, Figure 6D, Figure 7D‐

F), and these differences were statistically significant (p = 0.004 for whole cell lysate, and

p < 0.001 for the other fractions). Patients with latent TB infections had avidities

intermediate between uninfected controls and active TB disease for all four fractions.

Surprisingly, the relative IgG avidity to the live M. tuberculosis surface was actually

lower in those with active TB disease (Δ=‐1.53, p=0.004), and in those with latent TB

disease (Δ=‐1.39, p=0.011), when compared to uninfected controls (Figure 6C).

3.3.4 Ratio of IgG and IgM antibodies to the surface of live M.

tuberculosis and to inactivated antigenic fractions

ELISA was used to measure the titers of IgG and IgM‐specific antibodies to the

live M. tuberculosis surface and to four inactivated antigenic fractions (Figure 6E‐F,

56

Figure 7G‐I). Infectious agents typically generate an initial IgM response that is

replaced by an IgG response [240]. Humans in all groups showed a mixed pattern of

response with both IgG and IgM antibodies to the live M. tuberculosis surface, and to all

four fractions. Patients in the uninfected and latent infection groups had roughly equal

quantities of IgG and IgM to the M. tuberculosis surface, whole cell lysate,

lipoarabinomannan, and cell wall (mean IgG/IgM ratios, ‐0.24 to 0.32 log10; Figure 6E‐F,

Figure 7G‐H), and modest IgG predominance against secreted proteins (mean IgG/IgM

ratios, 0.53 and 0.72 log10; Figure 6I). Patients with active TB disease had higher IgG than

IgM titers to all antigenic fractions (mean IgG/IgM ratios, 0.45 to 1.05 log10; Figure 6F,

Figure 7G‐I). In contrast, patients with active disease had equal amounts of IgG and IgM

to the live M. tuberculosis surface (mean IgG/IgM ratio, 0.03 log10; Figure 6E).

3.3.5 Correlations between antibody titers and clinical characteristics

We explored potential correlations between antibody responses and clinical

characteristics. Table 5 displays correlations between clinical characteristics and total

antibody titers to the live M. tuberculosis surface and to whole cell lysate for patients

with active TB disease. For discrete clinical variables the log10 titer is reported (mean ±

SEM). For continuous variables (age, PPD induration), the R‐value is reported based on

linear regression. P‐values are not adjusted for multiple comparisons. Higher titers of M.

tuberculosis surface‐binding antibodies were observed in active TB disease patients who

were BCG vaccinated versus unvaccinated (Δ=0.55 log10, p=0.008) or foreign born versus

57

US born (Δ=0.61 log10, p=0.004). Race/ethnicity was also correlated with surface‐binding

antibody titers (p=0.02), with white Hispanics and Asians possessing higher titers than

other racial/ethnic groups. There was a high degree of overlap in these three variables,

with many patients falling into one of two groups: BCG negative/US born/black or BCG

positive/foreign born/non‐black. HIV seropositivity was associated with lower surface‐

binding antibody titers (Δ=‐0.60 log10, p=0.039), as was increased age (r=‐0.35, p=0.027).

Antibody titers to whole cell lysate showed weak trends in the same direction for several

of these clinical characteristics (age, race/ethnicity, BCG vaccination, and foreign birth),

but there were no statistically significant correlations (Table 5). Similar analyses are

shown in Table 16 (Appendix B) for antibody titers to lipoarabinomannan, cell wall,

and secreted protein fractions. Increased age was associated with lower antibody titers

to lipoarabinomannan (r=‐0.35, p=0.027). No other statistically significant correlations

were observed. Patients with latent TB infection showed no statistically significant

correlations between antibody titers and clinical characteristics (Appendix B Table 17,

Table 18).

58


the live M. tuberculosis surface or whole cell lysate in patients with active TB disease

Log10 total antibody titer by ELISA

Variables M. tuberculosis surface Whole cell lysate

R value or

mean ± SEM

P

value*

R value or

mean ± SEM

P value

Age R=‐0.35 0.027 R=‐0.21 0.19

Gender 0.89 0.67

Male (n=29) 3.1 ± 0.1 3.1 ± 0.1

Female (n=11) 3.1 ± 0.3 3.2 ± 0.2

Race/ethnicity 0.018 0.11

White (n=7) 3.1 ± 0.3 2.9 ± 0.2

White Hispanic (n=11) 3.6 ± 0.2 3.5 ± 0.2

Black (n=17) 2.8 ± 0.2 2.9 ± 0.2

Black Hispanic (n=1) 2.7 2.0

Asian (n=4) 3.5 ± 0.2 3.2 ± 0.3

HIV seropositivity 0.039 0.84

HIV + (n=7) 2.6 ± 0.2 3.1 ± 0.2

HIV – (n=33) 3.2 ± 0.1 3.0 ± 0.1

Diabetes 0.65 0.051

Yes (n=6) 3.1 ± 0.1 3.5 ± 0.3

No (n=34) 3.2 ± 0.3 3.0 ± 0.1

Tobacco usage 0.15 0.48

Yes(n=11) 2.9 ± 0.2 2.9 ± 0.1

No (n=29) 3.3 ± 0.1 3.1 ± 0.1

Alcohol consumption (days) 0.87 0.76

None consumed (n=26) 3.1 ± 0.2 3.1 ± 0.1

1‐3 drinks (n=6) 3.2 ± 0.3 2.9 ± 0.3

>3 drinks or binge (n=8) 3.2 ± 0.2 3.1 ± 0.1

BCG vaccination history 0.008 0.087

Yes (n=17) 3.4 ± 0.1 3.2 ± 0.1

No (n=22) 2.9 ± 0.2 2.9 ± 0.2

US‐Born 0.004 0.18

Yes (n=20) 2.8 ± 0.2 2.9 ± 0.2

No (n=20) 3.4 ± 0.1 3.2 ± 0.2

PPD induration (mm) R=‐0.18 0.28 R=‐0.23 0.17

Disease site 0.96 0.20

Pulmonary (n=22) 3.1 ± 0.2 3.1 ± 0.1

Extra‐pulmonary (n=14) 3.1 ± 0.2 2.8 ± 0.2

Both (n=4) 3.1 ± 0.4 3.4 ± 0.2

*P‐value calculated using one‐way ANOVA or student’s t‐test for categorical variables

or F‐statistic for continuous variables. P‐values are unadjusted for multiple comparisons.

59

Table 6: Univariate correlation between clinical variables and avidity of antibodies to

the live M. tuberculosis surface or whole cell lysate in patients with active TB disease

Relative IgG avidity

Variable M. tuberculosis surface Whole cell lysate

R value or mean ± SEM

P value*

R value or mean ± SEM

P value

Age R=‐0.13 0.46 R=‐0.04 0.78

Gender 0.10 0.34

Male (n=29) 2.7 ± 0.2 3.6 ± 0.3

Female (n=11) 2.1 ± 0.4 3.1 ± 0.3


White (n=7) 1.8 ± 0.4 4.0 ± 0.4 White Hispanic (n=11) 3.0 ± 0.3 3.2 ± 0.5

Black (n=17) 2.3 ± 0.2 3.3 ± 0.4

Black Hispanic (n=1) 2.5 6.0

Asian (n=4) 3.9 ± 0.4 3.4 ± 0.4

HIV seropositivity 0.30 0.15

HIV + (n=7) 2.1 ± 0.3 2.7 ± 0.4

HIV – (n=33) 2.7 ± 0.2 3.6 ± 0.3

Diabetes 0.30 0.27

Yes (n=6) 2.1 ± 0.4 2.8 ± 1.0

No (n=34) 2.7 ± 0.2 3.6 ± 0.2

Tobacco usage 0.64 0.31 Yes(n=11) 2.7 ± 0.4 3.9 ± 0.4

No (n=29) 2.5 ± 0.2 3.3 ± 0.3


None consumed (n=26) 2.5 ± 0.2 3.1 ± 0.2

1‐3 drinks (n=6) 3.0 ± 0.6 4.2 ± 0.7

>3 drinks or binge (n=8) 2.5 ± 0.2 4.1 ± 0.7

BCG vaccination history <0.001 0.40

Yes (n=17) 3.2 ± 0.2 3.2 ± 0.3

No (n=22) 2.1 ± 0.2 3.8 ± 0.4

US‐Born 0.010 0.80 Yes (n=20) 2.1 ± 0.2 3.5 ± 0.4

No (n=20) 3.0 ± 0.2 3.4 ± 0.3


Disease site 0.090 0.018

Pulmonary (n=22) 2.5 ± 0.3 3.0 ± 0.3

Extra‐pulmonary (n=14) 3.0 ± 0.3 4.4 ± 0.3 Both (n=4) 1.7 ± 0.3 3.2 ± 0.8



60

3.3.6 Correlations between relative IgG avidity and clinical

characteristics

We conducted similar analyses for relative IgG avidity. Table 6 displays

correlations between clinical characteristics and relative IgG avidity to the live M.

tuberculosis surface and to whole cell lysate for patients with active TB disease. The three

clinical factors that correlated most strongly with surface‐binding antibody titers, were

also correlated with relative IgG avidity to the M. tuberculosis surface. Individuals with a

history of BCG vaccination possessed higher relative IgG avidity to the surface of M.

tuberculosis, than their non‐vaccinated counterparts (Δ=1.12, p<0.001), as did foreign born

individuals versus those born in the US (Δ=0.87, p=0.01). Race/ethnicity was also

correlated with surface‐binding antibody titers (p=0.008), with white Hispanics and

Asians possessing higher relative IgG avidity than other racial/ethnic groups. Taken

together, the group of BCG‐vaccinated/foreign born/non‐black patients had higher

antibody titers and higher relative IgG avidity to the surface of live M. tuberculosis.

Relative IgG avidity to whole cell lysate was correlated with disease site (p=0.018) with

higher avidity in patients with extra‐pulmonary disease. A similar trend was noted for

higher relative IgG avidity to the live M. tuberculosis surface in patients with extra‐

pulmonary disease. No statistically significant correlations were observed for relative

IgG avidity to lipoarabinomannan, cell wall, or secreted protein fractions (Appendix B,

Table 19,). Higher relative IgG avidity to the surface of live M. tuberculosis was observed

in patients with latent TB infection who consumed higher levels of alcohol (p=0.045) and

61

in those born in the US (Δ=0.8, p=0.033) (Appendix B, Table 20). No other statistically

significant correlations were observed between relative IgG avidity and clinical

characteristics in patients with latent TB infection (Appendix B, Table 20 , Table 21).

3.3.7. Association of cytokine production with surface‐binding and

secreted protein antibody titers and IgG avidity scores

All subjects with latent TB infection (n=23) and a subset of subject with active TB

disease (n=10) were included in a previously published M. tuberculosis biomarker study

[239]. Whole blood was incubated with M. tuberculosis‐specific peptides and the

concentrations of cytokines and chemokines determined. These values were plotted

against surface‐binding and whole cell lysate antibody titers (Appendix B, Table 22)

and surface‐binding and whole cell lysate IgG avidity scores (Appendix B, Table 23),

with r and p‐values reported. In the ten patients with active TB disease, there was a

positive correlation between IL‐1β and antibody titers to the surface of live M.

tuberculosis (r=0.70, p=0.02) and to whole cell lysate (r=0.69, p=0.03). The significance of

this finding is unclear: since 96 pairwise comparisons were made, this rate of p‐values

below 0.05 would be expected by chance alone. When cytokines were grouped into those

associated with either TH1 or TH2 responses, there was no pattern of associations.

A greater number of cytokines were correlated with surface‐binding antibody

and whole cell lysate antibody IgG avidity. There was a positive association between

IFN‐γ (r=0.73, p=0.02), IL‐6 (r=0.65, p=0.04), IL‐8 (r=0.89, p=0.001), MIP‐1α (r=0.77, p=0.01)

and MIP‐1β (r=0.85, p=0.002) and surface‐binding antibody IgG in patients with active

62

disease. Associations were also observed between IL‐10 (r=0.77, p=0.01), IL‐13 (r=0.65,

p=0.04), Eotaxin (r=‐0.71, p=0.02), and RANTES (r=‐0.71, p=0.02) and whole cell lysate

antibodies in patients with active disease. As with antibody titers, there was no pattern

of association seen with TH1or TH2 cytokines, and despite the large number of

correlations with IgG avidity scores, no overall pattern was observed.

3.3.8 Antibody titers and avidity scores to environmental mycobacteria

As shown in Figure 6 and Figure 7, subjects with no evidence of TB infection had

detectable antibody to the surface of live M. tuberculosis and to all four antigenic

fractions derived from M. tuberculosis. Humans are exposed to environmental

mycobacteria with significant antigenic similarity to M. tuberculosis. We generated

protein lysates from M. tuberculosis H37Rv, and the type strains of M. intracellulare, M.

avium, and M. fortuitum. We compared antibody titers to these lysates in our nine

uninfected and a randomly selected group of ten patients with active TB disease

(Appendix B, Figure 34A‐D). Uninfected patients had modest antibodies to lysates from

all four mycobacterial species, and these titers were increased to all four species in all

patients with active TB (Δ=0.65 to 0.76 log10). Among those with active TB disease,

antibody titers to the M. tuberculosis protein lysates were correlated to antibody titers to

M. avium (r2=0.92, p<0.001), M. intracellulare (r2=0.94, p<0.001), and M. fortuitum (r2=0.58,

p=0.01) as shown in Figure 34E‐G (Appendix B).

63

As shown in Figure 6C, relative IgG avidity to the surface of live M. tuberculosis

was lower in persons with active TB disease than in uninfected patients. We measured

antibody titers and relative IgG avidity to the surface of environmental mycobacteria to

determine whether this reduced avidity is specific to M. tuberculosis. Antibody titers to

the surface of M. avium, M. intracellulare, and M. fortuitum did not differ significantly

between uninfected patients and patients with active TB disease (Figure 8A‐C). Relative

IgG avidity did not differ between uninfected volunteers and individuals with active TB

disease for the surface of live M. intracellulare (Δ=0.37, p=0.37) or the surface of live M.

avium (Δ=0.62, p=0.33) (Figure 8D‐E). Patients with active TB disease had increased IgG

avidity to the surface of live M. fortuitum (Δ=1.54, p=0.01).

64

Figure 8: Reactivity of human plasma to surface of environmental mycobacteria



surface of live M. intracellulare (A,D) M. avium (B,E), and M. fortuitum (C,F). Log10 total Ig

titers and relative IgG avidity are displayed. Two‐sided p‐values by student’s t‐test or

Mann‐Whitney test: (*) p ≤ 0.05, (**) p ≤ 0.01, (***) p ≤ 0.001.

3.4 Discussion

Previous work has characterized the human humoral response against M.

tuberculosis by examining selected antigens to determine their utility for inclusion in

diagnostic tests for tuberculosis, or as potential biomarkers [164,241‐251]. Our results are

65

in agreement with an overarching principle from these studies. Antibody titers in

persons with latent TB infection or active TB disease are highly variable, and overlap

with the titers found in uninfected individuals [249‐252]. We observed a wide range of

antibody titers among patients with active TB disease to the surface of live M.

tuberculosis and to all four antigenic fractions (Figure 6, Figure 7). In each case, titers

from patients with active TB disease overlapped with titers from uninfected subjects.

In addition our work has identified that antibodies to the surface of M.

tuberculosis do not follow the same trends as antibodies against other M. tuberculosis

antigens. While patients with active disease have slightly elevated titers compared to

uninfected controls, there is a decrease in avidity, and no increase in the IgG/IgM ratio

(Figure 6), suggesting that there is a prolonged IgM response to the live surface of M.

tuberculosis. This contrasts with antibodies to whole cell lysate, secreted proteins, and

two components of the cell wall: LAM, and the cell wall fraction. The decrease of avidity

to cell surface antigens may represent a lowering of overall avidity, conversion of

antibodies from IgM to low‐avidity IgG, or exhaustion of B‐cells producing higher‐

avidity antibodies. The absence of increased avidity is notable none the less, as surface‐

binding antibodies are a potentially protective class, and humans lack high titer‐high

avidity antibodies against the surface. We identified three clinical factors (BCG

vaccination, race/ethnicity, and country of origin) in persons with active TB disease that

66

were strongly correlated with antibody titer (Table 5) and relative IgG avidity (Table 6)

to the live M. tuberculosis surface. These factors were not correlated with antibody titers

or relative IgG avidity to the inactivated antigenic fractions.

Numerous studies have examined the correlation of a variety of clinical factors

with antibody production in tuberculosis and other diseases, though none have

evaluated antibodies to the surface of live M. tuberculosis. While these studies have

examined numerous factors found in this study including age [253], country of origin,

BCG vaccination status and gender [254], HIV status [255], , disease site [246], , and PPD

size [256], their results have varied depending on the antigen or cellular fraction used to

quantitate antibody levels. Among persons with active TB disease, higher surface‐

binding antibody titers and higher relative IgG avidity were strongly correlated with

BCG vaccination, foreign birth, and with race/ ethnicity of either white Hispanic or

Asian. Lower surface‐binding antibody titers were associated with increasing age and

with HIV seropositivity (Table 6).

There was high overlap among the clinical variables of BCG vaccination, country

of birth, and race/ethnicity among patient with active TB disease: 100% of BCG

vaccinated patients were foreign born, 91% of BCG unvaccinated patients were US born,

and 77% of patients identifying themselves as black were US born and BCG

unvaccinated. This overlap was too high to permit meaningful multivariate analysis.

67

The association with higher surface‐binding antibodies may be primarily driven by one

of these three factors, but the present data set does not define this factor. Other studies

have examined race, BCG vaccination status, and country of origin in relation to

antibody responses to antigenic fractions from M. tuberculosis [254,257,258]. Associations

were noted with country of origin, but not with race or BCG vaccination. None of these

studies evaluated antibodies to the surface of live M. tuberculosis.

Antibody titer and antibody avidity have been identified as correlates of

protection for other bacterial vaccines including vaccines against Haemophilus influenzae,

Streptococcus pneumoniae, and Bordetella pertussis [97,98,259,260]. BCG vaccines may

induce increased antibody titers and increased relative avidity to the surface of M.

tuberculosis, and surface‐binding antibodies may serve as a correlate of vaccine‐induced

protection. These questions could be tested using samples from BCG vaccine trials.

Among active TB disease patients, lower surface‐binding antibody titers were

associated with increasing age (r=‐0.35, p=0.027) (Table 5). A similar association was

seen for antibodies to lipoarabinomannan (r=‐0.35, p=0.027) (Appendix B, Table 16), and

trends were seen with other antigenic fractions. Elderly patients also have reduced

antibody responses to vaccination [253]. Previous work focusing on TB has shown either

a negative correlation between antibody titer and age, or no correlation at all depending

on the specific antigens studied [246,254].

68

Among patients with active TB disease, HIV seropositivity was associated with

lower surface‐binding antibody titers (Δ=0.5 log10) (Table 5). Other studies have shown

that HIV infection induces dysregulation in antibody responses to LAM, altering IgG

subtype profiles, and decreasing the levels of IgG2 anti‐M. tuberculosis antibodies as

compared to HIV negative controls [255]. In both aging and HIV infection, T‐cells are

also affected. Increased age is also known to lead to a collapse in T‐Cell diversity [261]

and function [262], while HIV infection leads to a decrease in CD4+ T‐Cell count [263].

Older age and HIV infection are both associated with reactivation TB disease. While T‐

cells are primarily responsible for controlling M. tuberculosis infection [264‐266], our

results raise the possibility of a role for surface‐binding antibodies as well.

We also examined cytokine levels in whole blood after TB‐specific antigen

stimulation for all patients with latent TB infection and ten active TB disease patients

(Appendix B, Table 22). No meaningful correlations were found for either antibody titer

or antibody avidity. Elevated levels of Th2 cytokines, promote B‐cell proliferation and

differentiation to antibody‐producing plasma cells [267] and humans with active TB

disease have also been demonstrated to generate Th2‐like profiles in response to

mycobacterial antigens [268]. However, studies in mice have demonstrated altered

cytokine profiles over the course of infection, starting with a pro‐inflammatory TH1

response, and switching to a TH0 balance (equal amounts of IL‐2 and IL‐4 production)

69

during chronic infection [269]. Having a cross‐sectional study design with patients at

varying stages of disease may have hidden disease‐state dependent cytokine differences

We examined the antibody response to the surface of other live environmental

mycobacteria to determine if the decreased avidity score observed to the surface of live

M. tuberculosis was species‐specific. As expected, antibodies from humans with active

disease cross‐reacted with protein lysates as well as the live surface of M. avium, M.

intracellulare, and M. fortuitum. (Figure 8; Appendix B, Figure 34). This is unsurprising

as previous studies have shown monoclonal antibodies produced against M. tuberculosis

cross react with numerous other mycobacterial species including M. kansasii, M. gastri,

M. fortuitum and M. marinum [167]. The cross‐reactive nature of mycobacterial antibodies

may also explain the presence of M. tuberculosis specific antibodies in the uninfected

control population. Notably, however, avidity scores did not decrease in individuals

with active TB disease to the surface of live environmental microbes, indicating that the

decreased avidity to the surface of M. tuberculosis is species‐specific.

The cohort used for this study has several limitations which affect the scope of

our conclusions. Due to the cross‐sectional nature of this study, we cannot determine

whether antibodies to the surface of M. tuberculosis reduce progression to disease in

patients with latent TB infection, and we are unable to track changes in antibody levels

associated with successful treatment. Prospective studies of patients with latent TB

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infection and active TB disease would provide additional insights. We also did not

collect data on parasitic infection, in any of our patient groups. The presence of parasites

can induce Th2‐polarize adaptive immune responses, which could mask correlations

between biomarkers and antibody titers/IgG avidity scores [270]. Our uninfected cohort

is also small. Despite its low number, antibody titers and avidity scores had a similar

range and standard deviation as latently infected and actively diseased individuals. In

addition our uninfected cohort contained no BCG vaccinated individuals, making us

unable to comment on the ability of the BCG vaccine to generate high titer/high avidity

surface‐binding antibodies in the absence of M. tuberculosis infection. Expansion of this

cohort to include individuals with BCG vaccination, HIV seropositivity and a higher

percentage of foreign born individuals would provide important information on the

baseline status of surface‐binding antibodies across a broad population.

Our ELISA assay also utilizes bacteria grown in liquid media in the presence of

tyloxapol to reduce bacterial clumping. As tyloxapol has the potential to alter the

bacterial surface, the surface of these culture‐grown bacteria may differ from the surface

of M. tuberculosis that is present in human tissues or sputum [271]. Future studies

detailing changes to the surface of the bacteria when grown in vivo versus in vitro will

enable us to better characterize the human surface‐binding antibody response to the

most physiologically relevant form of the bacterium.

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Natural TB infection in humans results in incomplete protection from re‐

infection [53]; therefore, a fully effective vaccine may need to generate responses not

found in infected humans. Antibodies to many microbes have the capacity to alter

infection rate when they are present at the time of exposure, and novel TB vaccines with

the goal of preventing infection may need to rely on antibodies, a they can be present in

the lung at the time of infection [272]. Our work demonstrates that most humans with

active TB infection lack high avidity surface‐binding antibodies. Vaccines targeting

specific antigenic components or epitopes that are accessible on the surface of M.

tuberculosis may induce high‐avidity surface‐binding antibodies. Further research is

warranted into the potential protective role of high‐avidity antibodies to the M.

tuberculosis surface.

In summary, these data demonstrate the difference between antibody responses

to the surface of live M. tuberculosis and the responses to inactivated cellular fractions.

When compared to uninfected humans, patients with active TB disease had increased

avidity to cellular fractions, but decreased avidity to the M. tuberculosis surface. Patients

with active TB disease also had increased IgG/IgM ratios to cellular fractions but not to

the M. tuberculosis surface. Lastly, higher surface‐binding antibody titers were correlated

with clinical factors associated with reduced rates of active TB disease: BCG vaccination,

younger age, and HIV seronegativity. As highly‐avid antibodies to surface components

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of other bacteria are associated with protection, the lack of high‐avidity antibodies to the

surface of M. tuberculosis in humans is notable.

3.5 Future directions

Although this study has generated interesting hypotheses regarding the use of

surface‐binding antibodies as clinical correlates and the failure of humans with active

disease to produce high‐avidity, high titer surface‐binding antibodies, its cross sectional

nature limits its power. Future studies need longitudinal samples, preferably pre‐ and

post‐BCG vaccination, with sufficient clinical tracking to diagnose both latent TB

infection and active TB disease years after enrollment. Large cohorts, with these

characteristics, and uniform countries of origin, exist and plasma from enrollees could

be used to expand on our findings [273,274].

In our study, BCG vaccinated individuals with active disease possess higher‐

titer, higher‐avidity surface‐binding antibodies, than their non‐vaccinated counterparts

(Table 5, Table 6). While factors such as antigen‐specific CD4+ T‐cells, and pro‐

inflammatory cytokine production are critical for disease control, currently, no true

correlates for BCG mediated protection are known [90,275]. Antibodies are correlates of

protection for numerous other vaccines against both viral and bacterial pathogens [276].

However, due to a high degree of confounding between race, country of origin, and

BCG vaccination status, it was impossible to determine if BCG vaccination was the

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driving factor behind the higher‐titer/higher‐avidity surface‐binding antibodies. To test

the hypothesis that BCG vaccination drove the difference, future studies should

compare surface‐binding antibody titers and relative IgG avidity in BCG vaccinated and

unvaccinated individuals, while controlling for race and country of origin. In addition, it

would be interesting to compare baseline surface‐binding antibody levels in BCG

vaccinated individuals who become infected after exposure to M. tuberculosis, to those

who do not, to determine if surface‐binding antibody levels are a predictor of the failure

of BCG vaccination to protect.

Another intriguing conclusion from our study is that surface‐binding antibodies

have decreased avidity in individuals with active TB disease as compared to uninfected

controls (Figure 6). Examination of relative IgG avidity in longitudinal samples from the

same individual prior to, and after the contraction of TB infection, would elucidate if this

finding is an artifact of the cohorts used. Decreasing IgG avidity over the course of

infection is not unique to M. tuberculosis, as infection of mice with the tapeworm parasite

Echinococcus granulosus leads to decreasing avidity, and corresponding IgG subclass

change over the course of infection[277]. If avidity is observed to decrease in the same

individual over the course of disease, two pronged additional studies should follow.

Standard and avidity ELISA assays of sequential plasma samples can determine if the

decreased avidity is due to a shifting of IgG subclasses from one with higher relative

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avidity to one with lower relative avidity. Additionally, isolation and immortalization of

plasma and memory B‐cells, followed by sequencing of individual B‐cell clones at

multiple time points, can monitor if there is constant conversion of antibodies from IgM

to low‐avidity IgG, or if there is exhaustion of B‐cells producing high avidity antibodies.

Lastly, in this study, HIV + individuals with active TB disease had decreased

antibody titers to the surface of live M. tuberculosis, but not antigenic fractions, as

compared with HIV ‐ patients (Table 5; Appendix B, Table 16). However, the number

of seropositive individuals examined was small (n=7). HIV seropositivity is known to

impair the formation of some, but not all, antibodies post vaccination, as well as

decrease pre‐existing antibody levels, and trigger shifts in IgG subclass [255,278‐280].

Obtaining plasma samples from a larger cohort of HIV patients to validate this finding is

essential. If HIV seropositivity is confirmed to lead to decreased surface‐binding

antibody titers, the correlation between antibody titers and T‐Cell levels should be

determined. Demonstrating a CD4+ T‐Cell dependence for surface‐binding, but not

other M. tuberculosis antibodies, would provide valuable information on the formation

and maintenance of these potentially critical antibodies during infection.

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4. Surface-binding antibodies decrease bacterial burden in the murine lung by blocking M. tuberculosis uptake

4.1 Introduction

Antibodies that bind to the surface of bacteria are capable of preventing both

colonization and infection [94,95] . However, humoral immunity has been disregarded

as a potential protective mechanism against M. tuberculosis, largely due to the failure of

passive protection studies at the turn of the 20th century [148]. Recently, there has been

renewed interest in antibody‐mediated immunity to M. tuberculosis, especially as it

related to vaccine design [107]. Monoclonal antibodies have been shown to be protective

in mice, and serum antibody levels correlate with reduced disease severity and impaired

dissemination in humans [188,189,191‐195,281,282]. On the basis of these studies, we

hypothesized that antibodies, specifically those against the surface of the bacteria, could

alter interactions between M. tuberculosis and host immune cells, changing the course of

early infection.

In this study we have identified murine monoclonal antibodies that bind to the

surface of live M. tuberculosis. Surface‐binding, but not non‐surface‐binding antibodies

decrease early pulmonary burden in mice, through an agglutination independent

mechanism, when mixed with M. tuberculosis prior to infection. These changes in early

CFU are accompanied in vivo by a decrease in the number of infected cells, and a change

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in the cell types that are infected. The ability of surface‐binding antibodies to alter early

infection events makes them a promising component of anti‐infective vaccines.

4.2 Methods

4.2.1 Hybridoma generation

Female BALB/c mice were vaccinated five times intraperitoneally with 50μg of

either purified H47rV heat‐shock protein‐X (HSP‐X) (BEI, NR‐14860), LAM or Apa (BEI

Resources, NR‐14862) diluted 1:1 in SAS adjuvant (Sigma, S6322). Vaccinations occurred

at two week intervals, with a final boost of antigen alone coming three days prior to

fusion. Splenocytes and NS0 Bcl2 myeloma cells were fused using polyethylene glycol

(Roche, 10783641001) according to manufacturer’s instructions. After fusion, cells were

grown in selection media (RPMI 1640, 10% Fetal Calf Serum (FCS) (GE Healthcare,

SH3008002), 1% non‐essential amino acids (Gibco, 11140‐050), 2 mM glutamine, 1 mM

sodium pyruvate (Gibco, 11360‐070), 2% HAT‐media supplement (Duke Cell Culture

Facility, H0262‐VL), and 10% J774A.1 cell supernatant).

Ten to fourteen days after fusion hybridoma supernatants were screened by

ELISA against H37Rv whole cell lysate and their cognate antigens. LAM and Apa

vaccinated mice were screened against the purified antigen used for vaccination, while

HSP‐X vaccinated mice were screened against a recombinant HSP‐X reference standard

(BEI Resources, NR‐31384). Hybridoma lines with high reactivity to both their cognate

77

antigen and whole cell lysate were cloned by limiting dilution. A cell line was

considered clonal when after repeated limiting dilutions, 20/20 clones demonstrated

reactivity to their cognate antigen. Once clonal, cell lines were adapted to hybridoma

growth media (DMEM/F‐12+ glutamax (Gibco, 10565‐018), 1% antibiotic/antimycotic

(GE Healthcare, SV3007901), 10% J774A.1 cell supernatant, 10% FCS, 0.7% β‐

mercaptoethanol (Sigma, M2650)) for propagation and freezing.

4.2.2 Hybridoma growth and antibody purification

Hybridoma lines were next adapted to serum free hybridoma media (SFM

Hybridoma media (Gibco, 1245‐076) with 1% antibiotic/antimycotic). After adaptation,

cells were washed and incubated in serum free hybridoma media in either 1000mL or

300mL cell‐line flasks (VWR WCL1000 and WCL0350). Media in flasks was changed

weekly.

Supernatant from cell line flasks was spun at 200g for six minutes, and then

filtered through a 0.45 μM filter prior to antibody purification. Supernatants were

purified on HiTrap protein G columns (GE Healthcare, 17‐040‐01) according to

manufacturer’s instructions. In brief, columns were washed with binding buffer (20mM

Na2HPO4, pH 7.0), followed by application of hybridoma supernatants diluted 1:1 in

binding buffer. The column was then washed in binding buffer, followed by antibody

elution from the column in 0.1 M glycine‐HCl, pH 2.7. Eluant was immediately restored

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to neutral pH with 1M Tris‐HCL, pH 9.0. All samples were passed over the column at

1mL/min flow rate, and purified antibody concentrations were determined via the

Nanodrop 2000 (Thermo Scientific).

All samples with over a 0.250mg/mL concentration were buffered exchanged by

centrifugation (Millipore, 4307) into PBS. The isotypes of purified antibodies were

determined using IsoStrip mouse monoclonal antibody isotyping kit (Roche,

11493027001).

4.2.3 Antibodies

In addition to the antibodies generated in this study, hybridoma lines for eight

additional M. tuberculosis‐specific antibodies (gift of Karen Dobos) along with three

control antibodies (gift of Barton Haynes) were obtain. A list of all antibodies used, their

isotypes, sources, and targets can be found in Table 7. Surface‐binding antibody pools

were also tested. A list of the components and ratios for these antibody pools can be

found in Table 8.

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Table 7: Experimental Antibodies

Antibody Isotype Source Target

Control antibodies

15H10 IgG2a Barton Haynes [283] Ebola Env

6D11 IgG1 Barton Haynes[284] Ebola Env

13H11 IgG2a Barton Haynes [285] HIV gp41

Experimental antibodies

CS‐40 IgG1 BEI Resources NR‐13812

[286]

LAM

IT‐70 IgG1 BEI Resources NR‐13657

[287]

GroEL2


[288]

GroEL2

18D11 IgG1 this study HSP‐X

15A4 IgG1 this study HSP‐X

16G2 IgG1 this study HSP‐X

CS‐49 IgG1 BEI Resources NR‐13814 HSP‐X


[288]

HSP‐X

1H11 IgG1 BEI Resources NR‐13779 GLcB


[288]

PhoS1

Table 8: Components of surface‐binding antibody pools

Surface‐binding

antibody pool

Ratio Antibodies

surface‐binding

pool A

1:1 IT‐70 : 18D11

surface‐binding

pool B

1:1:1:1 IT‐70 : IT‐13 : CS‐40 : 18D11

surface‐binding

pool C

1:1:1 IT‐70 : CS‐40 : 18D11

surface‐binding

pool D

2:2:1 IT‐70 : CS‐40 : 18D11

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4.2.4 ELISA assay

The experimental protocol for both antigenic and C‐ELISA assays can be found

in Chapter 3, with a few minor alterations. First, in addition to whole cell lysate and

LAM, the HSP‐X recombinant reference standard and the soluble cell wall protein

fraction (BEI Resources, NR‐ 14840) were used as antigens. Second, since the samples

assayed were mouse, not human, a 1:32 dilution of sera from a mice with chronic M.

tuberculosis was used as a positive control, and a 1:3000 dilution of alkaline‐phosphatase

conjugated goat‐anti‐mouse‐IgG (Sigma, A7434) was used as the secondary antibody.

Third, assay results are displayed as the OD value at a 0.25mg/mL concentration for

purified antibodies and at a 1:64 dilution for murine sera.

4.2.5 Avidity ELISA assay

The experimental protocol for the avidity ELISA assay can be found in Chapter 3,

and was performed using the same murine‐specific secondary antibody and positive

control described above.

4.2.6 Murine immune sera

Twelve week old female BALB/c mice were infected with a viable aerosol

concentration of 0.38 CFU/mL of M. tuberculosis as described in Chapter 5. Blood was

harvested from the submandibular vein every two weeks for one year post infection.

Sera were isolated as described in Chapter 2.

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4.2.7 Western Blot

15μg of either M. tuberculosis whole cell lysate, secreted proteins, cell wall, or

cytosolic (BEI Resources, NR‐14834) fractions were mixed with Laemmli sample buffer

and run at 130V for 45 minutes on an SDS‐PAGE gel (BioRad, 567‐1098) in 25mM Tris,

192mM glycine, 0.1% SDS, pH8.3 (BioRad, 161‐0772). Proteins were transferred to PDVF

membrane at 30V for 1 hour in 25mM Tris, 192mM glycine, 20% methanol, pH8.3

(BioRad, 161‐0771). Membranes were blocked in 5% non‐fat dry milk in Tris‐Buffered

Saline + 0.1% Tween 20 (TBST) for one hour at room temperature, and incubated

overnight at 4oC with primary antibody. Primary antibodies were diluted as follows in

5% non‐fat dry milk in TBST: IT‐70 (0.6 μg/mL), 18D11 (0.4 μg/mL), 15A4 (1.1 μg/mL),

CS‐49 (1.2 μg/mL), hybridoma supernatants (1:100 to 1:500). Membranes were then

washed 3 times in TBST, and incubated for one hour at room temperature with 0.2

μg/mL goat‐anti‐mouse IgG‐HRP (Genscript, A00160). Membranes were washed a final

three times and developed via chemiluminescence (Roche, 12015796001) and x‐ray film.

4.2.8 Bacterial strains and growth conditions

M. tuberculosis H37Rv and M. tuberculosis H37Rv dsRed were grown in 7H9

media supplemented with 10% OADC, 0.5% glycerol, 0.05% tyloxapol, at 37oC, in a

shaking incubator at 100 ‐ 130 rpm. Media for H37Rv dsRed was supplemented with

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1μg/mL kanamycin (Sigma, K0129). For CFU determination, M. tuberculosis H37Rv was

grown on the in house 7H10 agar plates described in Chapter 2.

4.2.9 Biotin fixation to M. tuberculosis

M. tuberculosis biotin fixation was adapted from Jhingran et al [289]. To optimize

the ratio of biotin to M. tuberculosis H37Rv dsRed, log phase bacteria were washed in

50mM carbonate buffer. Varying concentrations of bacteria were then rotated with 0.5

mg/mL Biotin‐XX, SSE (Invitrogen, B‐6352) for two hours at 4oC, in 1mL of 50mM

carbonate buffer. Bacteria were washed in 0.1M Tris‐HCL pH 8.0, and stained with

0.02mg/mL FITC‐streptavidin (Life technologies, SNN1008) at room temperature for 30

minutes. FITC signal intensity served as a proxy for the amount of biotin affixed to the

surface. Live bacteria were phenotyped using a FACSAria II SORP and FACSDiva™ 8.0.

Single bacteria were identified on the basis of forward side scatter profiles, and dsRed

positivity (Figure 9). Mean FITC‐intensity increased in a concentration dependent

manner. 0.5 mg/mL Biotin‐XX,SSE per OD 1.0 M. tuberculosis was selected as the

optimum ratio for future studies.

For cell culture and animal experiments M. tuberculosis H37Rv was grown to log

phase and resuspended in 50mM carbonate buffer. 2x108 bacteria were rotated with 0.5

mg/mL of Biotin‐XX, SSE for two hours at 4oC in 1mL of 50mM carbonate buffer.

Bacteria were then washed in 0.1M Tris‐HCL pH 8.0, resuspended in either PBS or cell

83

culture media, and incubated with BTN.4, an IgG1 anti‐biotin antibody (Thermo

Scientific, MA5‐11251), in lieu of M. tuberculosis specific antibodies.

Figure 9: Optimization of biotin fixation to M. tuberculosis

Varying amounts of M. tuberculosis H37Rv dsRed were incubated with 0.5 mg/mL biotin

for two hours at 4C, prior to washing with 0.1M Tris‐HCL (pH 8). After the wash

bacteria were labeled with 0.02 mg/mL FITC –streptavidin in PBS for 30 minutes prior to

flow cytometry. (A) gating strategy for isolating single bacteria (B) Histograms of mean

FITC fluorescence (C) plot of mean FITC intensity per mg/OD∙mL of biotin.

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4.2.10 Macrophage uptake and survival assay

Raw 264.7 cells were grown to 50‐80% confluence in Raw growth media (DMEM

(Gibco, 11965‐084) with 10% FBS, 1% antibiotic/antimycotic, 1% sodium pyruvate, and

1% non‐essential amino acids). Cells were washed in Raw cell experimental media

(DMEM with 10% FBS, 1% sodium pyruvate and 1% non‐essential amino acids), plated

in 96‐well plates at ~10,000 cells per well, and left to settle at 37oC.

Log phase H37Rv was washed twice in PBS‐Ty and resuspended in Raw cell

experimental media. 5x104 bacteria were incubated with varying concentrations of

antibody or media alone for 30 minutes prior to incubation with cells for two hours at

4oC to synchronize uptake. Infection occurred for two hours at 37oC. After infection cells

were washed with Raw experimental media, and incubated for 1 hour at 37oC in Raw

experimental media containing 1μg /mL streptomycin (Sigma, S6501) to remove

extracellular bacteria. Cells were then either lysed in de‐ionized water (dH2O), or

incubated overnight, in antibiotic‐free Raw experimental media, at 37oC and lysed at

twenty‐four hours post infection. Lysates were serially diluted in PBS‐Ty, and plated.

Data is displayed as percent of no antibody control. Normalized twenty‐four hour data

was calculated by dividing the percent of no antibody control at twenty‐four hours by

the average percent of no antibody control at three hours.

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4.2.11 Bacterial clumping assay

M. tuberculosis H3Rv was grown to OD 0.78, washed twice in PBS‐Ty, and

resuspended to a final OD of 0.1 in PBS‐Ty. 1x105 bacteria were incubated with either

PBS‐Ty or 60μg of antibody in a total volume of 50μL for 15 minutes at room

temperature. Samples were then serially diluted, and plated on 7H10 agar plates. Each

antibody was tested in triplicate.

4.2.12 Mice

Six week old female wild‐type BALB/c mice from National Cancer Institute

(01B05) were housed in the RBL at Duke University for at least one week prior to M.

tuberculosis exposure under environmental conditions and protocols described in

Chapter 2.

4.2.13 Isolation of dsRed M. tuberculosis from peritoneal lavage

M. tuberculosis H37Rv dsRed was washed twice in PBS‐Ty and resuspended to a

final OD of 1.0. Fifteen ten‐week old female BALB/c mice were injected intraperitoneally

with 1x107 bacteria in 100μL. One week post infection, mice were sacrificed by isoflurane

overdose, with M. tuberculosis dsRed recovered from the peritoneal cavity via peritoneal

lavage with 5 mL of PBS‐Ty. Peritoneal lavages were spun at 200g for 10 minutes, with

the resulting pellet lysed in RIPA buffer (Sigma, R0278) to free intracellular M.

tuberculosis. Samples were then resuspended in 400 μL of PBS‐Ty and dsRed positive

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bacteria isolated by using a FACSAria II SORP and FACSDiva™ 8.0. The gating scheme

for flow cytometry sorting is displayed in Figure 10. RAW 267.4 cells, grown in Raw

growth media to confluence, were lysed to serve as a negative control for gating. A

median amount of 4.4log10 ± 0.7log10 M. tuberculosis were recovered from each mouse.

Figure 10: Gating scheme for dsRed isolation from peritoneal lavage

(A) uninfected RAW 267.4 cells were lysed in dH20 to serve as a negative control (B)

bacterial isolation from peritoneal lavage one week after intraperitoneal instillation of M.

tuberculosis H37Rv dsRed.

4.2.14 Murine administration of antibody pre-mixed with M. tuberuclosis

For infection with culture‐grown bacteria, log phase M. tuberculosis H37Rv was

washed twice in PBS‐Ty and resuspended to a final OD of 0.1. 1x105 M. tuberculosis were

incubated with varying concentrations of antibody in a 50μL total volume, fifteen

minutes prior to retropharyngeal instillation. For infection with mammalian adapted

bacteria, 4x102 M. tuberculosis H37Rv dsRed isolated from peritoneal lavage fluid were

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incubated with 30μg of antibody in a 50 μL total volume for fifteen minutes prior to

retropharyngeal instillation.

Mice were sacrificed at either three of six days post retropharyngeal exposure.

Murine lungs were manually homogenized in PBS‐Ty. After homogenization lungs were

serially diluted in PBS‐Ty prior to plating. Sterility of homogenates was ascertained by

plating on Chocolate II agar with hemoglobin and IsoVitalex. CFUs were counted after

3‐4 week incubation at 37oC.

4.2.15 Single cell suspension of infected mouse lung and pneumocyte staining

Seven week old female BALB/c mice were instilled with either PBS, 2.4x105 M.

tuberculosis or 2.4x105 M. tuberculosis mixed with 60μg of surface‐binding pool C as

previously described. Mice were sacrificed at two, twenty‐four, and seventy‐two hours

post infection, and lungs were harvested for processing. The protocol for pneumocyte

isolation and cell staining is the same as found in Chapter 2. Prior to flow cytometry,

10% of the total pneumocyte preparation was removed for CFU determination by

plating.

4.2.16 Flow cytometry analysis

The gating scheme for flow cytometry analysis is shown in Figure 11. Single cells

were discriminated from electronic noise by forward‐scatter area vs. side scatter area.

Live cells were then selected on the basis of Live/Dead fixable yellow stain exclusion. On

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the basis of published literature cell types were delineated on the basis of GR‐1, CD11b,

and CD11c positivity [27,213]. Non‐neutrophilic cells were separated from neutrophils

by GR‐1 signal, with neutrophils ultimately being identified as GR1+ CD11b+. GR1‐ cells

were then gated into three groups on the basis of CD11b and CD11c signal. (1) alveolar

macrophages (CD11blo CD11chi) (2) migratory macrophages (CD11bhi CD11clo) (3)

monocyes (CD11bhi CD11cneg). Infected cells were discriminated on the basis of dsRed

positivity, with their cell type determined by applying the gates previously described

(Figure 11). The average amount of dsRed background signal was determined using six

uninstilled control mice. A representative image of an infected and an uninfected mouse

can be found in (Figure 12). All reported values for infected cells have been background

subtracted. Flowjo v10.0 was used for all flow cytometry analysis.

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Figure 11: Gating scheme for infected cell flow cytometry analysis

Single cells were discriminated from electronic noise by plotting forward‐scatter area vs.

side scatter area. Live cells were then selected on the basis of Live/Dead fixable yellow

stain exclusion. Non‐neutrophilic cells were separated from neutrophils by GR‐1

positivity. GR1‐ cells were then gated into three groups on the basis of CD11b and

CD11c signal: (1) alveolar macrophages, (2) migratory macrophages, (3) monocytes.

dsRed positive cells were isolated from the live‐cell gate, with cell‐types identified by a

combination of GR‐1, CD11b and CD11c signal.

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Figure 12: Comparison of dsRed positive cells in infected vs. uninfected mice

Pneumocytes were isolated from the lungs of M. tuberculosis infected or uninfected

control mice, and dsRed positive cells were discriminated as described in Figure 11. A

representative image of the number of dsRed positive cells in a M. tuberculosis infected

mouse and an uninfected control mouse is shown.

4.2.17 Statistics

Prism Software was used for statistical analyses. For pairwise comparisons

among continuous variables a student’s t‐test was used, and for comparisons among

non‐parametrically distributed variables the Mann‐Whitney test was used.

4.3 Results

4.3.1 Screening of M. tuberculosis antibodies from a repository

Thirty‐one hybridoma supernatants against seventeen different M. tuberculosis

targets were evaluated for their ability to bind to the surface of M. tuberculosis by C‐

ELISA assay. Antibodies against GroEL2 (IT‐56, IT‐70, IT‐13), HBHA (NR‐13804), LAM

(CS‐25, CS‐35, CS‐40), and LpqH (IT‐54), demonstrated surface‐reactivity (Table 9). Not

all antibodies to surface‐expressed antigens reacted to the live cell surface. CS‐44 (anti‐

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Table 9: Screening of anti‐M. tuberculosis monoclonal antibodies from repository

Target Clone WCL titer

(log10)

Surface titer

(log10)

Western Blot/

additional ELISA*

Ag85 CS‐90 0.00 0.00 N.D.

Ag85 IT‐44 0.00 0.00 +

Ald IT‐46 2.71 0.00 +

DNAK IT‐40 0.00 0.00 +

GlcB 1H11 0.00 0.00 N.D.

GlnA IT‐58 0.00 0.00 N.D

GroEL2 IT‐56 4.39 3.05 +

GroEL2 IT‐70 2.49 2.09 +

GroEL2 IT‐13 2.00 1.44 +

GroEL2 CS‐44 0.00 0.00 +

GroES IT‐3 4.47 0.00 +

HBHA NR‐13804 2.39 1.69 +

HSP‐X CS‐49 2.79 0.00 +

HSP‐X CS‐50 2.69 0.00 +

HSP‐X IT‐20 3.07 0.00 +

KatG IT‐57 1.78 0.00 +

KatG IT‐42 3.34 0.00 +

LAM CS‐25 2.41 2.45 +

LAM CS‐40 0.00 1.69 +

LAM CS‐35 4.49 3.53 +

LpqH IT‐19 2.04 0.00 +

LpqH IT‐12 2.86 0.00 +

LpqH IT‐54 3.30 0.54 +

Mpt32 CS‐93 0.00 0.00 +

Pg1 IT‐43 0.00 0.00 N.D.

PhoS1 IT‐21 0.00 0.00 +

PhoS1 IT‐23 1.71 0.00 +

PhoS1 IT‐47 0.00 0.00 +

PhoS1 IT‐15 4.08 0.00 +

RV1411 NR‐13806 0.00 0.00 N.D.

SodA CS‐18 0.00 0.00 N.D.

Hybridoma supernatants were evaluated by C‐ELISA to determine surface‐reactivity

and by ELISA to whole cell lysate (WCL) or Western Blot to numerous bacterial fractions

to confirm the presence of M. tuberculosis specific antibody. N.D. indicates additional

ELISAs/Western Blots were not performed. Hybridoma lines for antibodies in blue were

obtained for future studies (gift of Karen Dobos).

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GroEL2), and both IT‐19 and IT‐12 (anti‐LpqH), lacked surface‐reactivity, suggesting

they bind to non‐exposed sections of the target molecule. To confirm the presence of

antibody in supernatants from non‐surface‐binding antibodies, ELISA assays and

western blots to whole cell lysate, secreted proteins, and the cell wall and cytosolic

fractions were conducted.

4.3.2 Generation and screening of surface-binding antibodies against LAM, HSP-X and Apa

Groups of three mice were immunized five times with either LAM, HSP‐X or

Apa adjuvanted with SAS, an oil in water immersion. IgG antibody titers were

monitored after each vaccination. LAM vaccinated mice did not develop IgG antibody

titers to the immunogen, and hybridomas to the lipopolysaccharide were not generated.

One Apa vaccinated mouse, and two HSP‐X vaccinated mice with high IgG antibody‐

titers to both the surface of M. tuberculosis and their cognate antigen were sacrificed to

produce hybridomas.

The supernatants from approximately two‐thousand hybridomas, derived from

HSP‐X vaccinated mice, were screened against whole cell lysate and recombinant HSP‐X

by ELISA. Based on a positive signal of twice background for both whole cell lysate and

HSP‐X, fifty‐seven hybridomas were identified as producing HSP‐X antibodies. Of these

fifty‐seven, only four (7.0%) reacted against the cell‐surface as measured by the C‐ELISA

93

assay. One surface‐binding (18D11) and two non‐surface‐binding (15A4, 16G2)

antibodies were cloned by limiting dilution for further study.

The supernatants from approximately eleven‐hundred hybridomas, derived

from Apa vaccinated mice, were screened against whole cell lysate and purified Apa

protein by ELISA. Based on a positive signal of twice background to both whole cell

lysate and Apa, two hybridomas were identified as producing Apa antibodies. One of

these dual reactive clones demonstrated surface‐binding reactivity by C‐ELISA assay,

but was lost to contamination. Using the less stringent cut‐off of a positive signal of

twice background to Apa alone, an additional one‐hundred and twenty‐eight

hybridomas were identified for future screening. Two of these clones demonstrated

weak surface‐binding reactivity. No Apa antibodies were selected for further study.

4.3.3 Purified antibody reactivity to M. tuberculosis fractions and the live cell surface

Purified monoclonal antibodies were assayed by ELISA to determine target

abundance in whole cell lysate, the soluble cell wall protein fraction and the live M.

tuberculosis surface as measured by the OD value at 0.25mg/mL antibody concentration

(Table 10). Five antibodies (IT‐70, 18D11, 15A4, 16G2, CS‐49 had strong reactivity (OD

>1.0) to whole cell lysate and moderately strong reactivity (OD>0.5) to the soluble cell

wall protein fraction. Three antibodies, IT‐20, CS‐40 and IT‐15 possessed weak reactivity

(0.5< OD) to both preparations. 6D11, an IgG1 isotype control antibody, and 1H11

94

demonstrated no‐reactivity to either fraction. Polyclonal sera from chronically infected

mice was also evaluated for comparison at a 1:64 dilution. The average OD to whole cell

lysate was 3.15 ± 0.57, a value higher than any monoclonal antibody tested.

In agreement with the initial hybridoma supernatant screens only three

antibodies, CS‐40, IT‐70, and 18D11 reacted against the surface, though peak OD values

were low (0.18‐0.3) (Table 10). Sera from chronically infected mice had an average peak

OD of 2.10 ±1.01, a value at least seven times higher than any purified monoclonal

antibody tested.

To confirm the ability of purified antibodies to bind to their cognate antigen

antibodies were tested by ELISA for reactivity to either LAM (CS‐40), or HSP‐X (18D11,

15A4, 16G2, CS‐49, IT‐20). Cognate antigens to GroEL2 (IT‐70), PhoS1 (IT‐15), and GlcB

(1H11) were unavailable. All antibodies bound well to their cognate antigen. OD values

for all anti‐HSP‐X antibodies were greater than 1.0, and CS‐40 possessed significantly

higher reactivity to purified LAM than to whole cell lysate (2.70 vs. 0.12).

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Table 10: Peak OD values for monoclonal antibodies to M. tuberculosis fractions and

the live cell surface

Antibody Whole Cell

Lysate OD*

Soluble Cell

Wall Protein

OD*

Live M.

tuberculosis

Surface OD*

Surface

binders

CS‐40 0.12 0.23 0.28

IT‐70 1.44 0.91 0.30

18D11 1.99 0.98 0.18

Non

Surface

Binders

15A4 1.85 0.52 0.03

16G2 1.61 0.48 0.04

CS‐49 1.41 0.99 0.08

IT‐20 0.52 0.30 0.07

1H11 0.00 0.00 0.00

IT‐15 0.23 0.13 0.03

Controls 6D11 0.00 0.00 0.03

Murine Polyclonal sera 3.15 ± 0.57 2.10 ±1.01

* The OD value at 0.25mg/mL for purified antibodies and a 1:64 dilution for sera is

displayed. The OD is an average of n=3 for purified antibodies on whole cell lysate and

the soluble cell wall protein fraction, and n=3 to n=6 for live cell surface with the

exception of 6D11 (n=1). Peak OD values for murine polyclonal sera, drawn from mice

32 weeks post aerosol M. tuberculosis exposure, are displayed as mean ± SEM (n=5).

Table 11: Peak OD values for monoclonal antibodies to their cognate antigens

Purified antibodies were tested against their cognate antigens by ELISA. OD value is

average of n=3 at 0.25m/mL concentration.

Antigen Antibody

Cognate

antigen OD*

Surface‐binder LAM CS‐40 2.70

Surface‐binder

HSP‐X

18D11 1.26

Non‐surface‐binder 15A4 1.19

Non‐surface‐binder 16G2 1.50

Non‐surface‐binder CS‐49 1.07

Non‐surface‐binder IT‐20 1.58

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4.3.4 Determination of whole cell lysate avidity for purified antibodies

Purified monoclonal antibodies were assayed by sodium thiocynate ELISA to

determine their avidity to whole cell lysate (Table 12). Antibody avidity was poor,

ranging from 0.09‐0.61 for the eight antibodies tested. Polyclonal mouse sera had

similarly low avidity, with an average avidity score of 0.59 ± 0.11. 6D11 and 1H11 do not

bind to whole cell lysate by ELISA and therefore could not be evaluated. Surface avidity

for 18D11, IT‐70, and CS‐40 could not be evaluated due to low plateau OD values (Table

10).

Table 12: Avidity scores to whole cell lysate for purified antibodies

Antibody

whole cell

lysate

avidity

Surface

binders

CS‐40 0.15

IT‐70 0.09

18D11 0.45

Non

Surface

Binders

15A4 0.12

16G2 0.61

CS49 0.21

IT20 0.69

1H11 NA

IT‐15 0.58

Controls 6D11 NA

Murine polyclonal sera 0.59 ± 0.11

Whole cell lysate avidity is the average of n=2 replicates for all purified antibodies and is

displayed as mean ± SEM for polyclonal sera, drawn 32 weeks after standard dose M.

tuberculosis exposure, for n=5 mice

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4.3.5 Purified M. tuberculosis antibodies bind to linear epitopes and are polyreactive

To further confirm that antibodies recognize their cognate antigens, and to

examine if they are polyreactive, purified antibodies were assayed against whole cell

lysate by Western Blot. All anti‐HSP‐X antibodies assayed (18D11, CS‐49, 15A4),

recognized a band at approximately 15 kDa, the molecular mass of the HSP‐X protein.

IT‐70, an anti‐GroEL2 antibody, recognized a band at approximately 75 kDa, near the

known molecular weight of the GroEL2 protein (70 kDa) (Figure 13).

Figure 13: M. tuberculosis monoclonal antibodies are polyreactive

M. tuberculosis monoclonal antibodies were assayed against whole cell lysate by Western

Blot. Antibody concentrations range from 0.4 μg/mL to 1.2 μg/mL.

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All antibodies, regardless of their ability to bind to the live M. tuberculosis surface

reacted non‐specifically with M. tuberculosis components (Figure 13). The ability to bind

to non‐specific targets decreases with antibody concentration, but binding ability to the

cognate antigen remains (data not shown).

4.3.6 Pre-mixing of biotin coated M. tuberculosis with antibody, prior to macrophage infection, decreases bacterial burden

The M. tuberculosis antibodies in this study are of low avidity. To test if

antibodies with high avidity to a surface moiety can alter bacterial burden in

macrophages, biotin was affixed to the surface of live M. tuberculosis H37Rv. Biotin

coated bacteria were then incubated with BTN.4, a commercially available high‐affinity

anti‐biotin antibody, at varying concentrations prior to infection of murine

macrophages. At three hours post‐infection BTN.4 decreased the number of recoverable

bacteria in a concentration dependent manner, with a maximal decrease of 59.6%

(p=0.019) with respect to the no antibody control (Figure 14). A similar trend was seen at

twenty‐four hours post infection. Incubation with 5 μg of BTN.4 resulted in a maximal

decrease of 68.1%.When twenty‐four hour bacterial counts were normalized to three

hour bacterial counts, no statistically significant decreases in twenty‐four hour survival

were observed (Figure 14).

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Figure 14: BTM.4 decreases biotin‐coated M. tuberculosis survival in macrophages

Biotin coated M. tuberculosis was mixed with BTN.4 for thirty minutes prior to Raw

264.7 cell infection at an MOI of 5. After incubation at 4oC for two hours, to ensure

synchronization of uptake, cells were then incubated at 37oC for two hours, washed, and

incubated with media containing 1μg/mL streptomycin for an additional hour at 37oC to

kill extracellular bacteria. Cells were then either lysed with dH20 (three hour time point),

or incubated overnight at 37oC in antibiotic free media for lysis at twenty‐four hours.

Graph depicts mean ± SEM for three replicates, except for 5μg of BTN.4 at twenty‐four

hours (n=2). Normalized twenty‐four hour data was calculated by dividing the percent

of no PBS control at twenty‐four hours by the average percent of no PBS control at three

hours. Statistical significance was calculated using Student’s t‐test: (*) p<0.05, (**) p<0.01,

(***) p<0.001.

4.3.7 Pre-mixing of M. tuberculosis with surface-binding antibodies prior to macrophage infection decreases bacterial burden

We next examined if M. tuberculosis surface‐binding antibodies could decrease

bacterial survival in a similar manner. M. tuberculosis H37Rv was pre‐incubated with a

concentration series of 6D11, an IgG1 control antibody, or surface‐binding antibodies

prior to macrophage infection. At three and twenty‐four hours after inoculation,

macrophages were lysed in water, and titered to determine the number of viable

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bacteria. At three hours post infection, all surface‐binding antibodies tested decreased

bacterial burden in a concentration dependent manner, with maximal decreases

occurring after pre‐incubation with 60μg of antibody. At twenty‐four hours post

infection similar concentration dependent decreases in bacterial survival were observed

(Figure 15).

Figure 15: Surface‐binding but not control antibodies decrease bacterial survival in

macrophages in a concentration dependent manner

M. tuberculosis was mixed with various murine monoclonal antibodies in a volume of

50μL for thirty minutes prior to Raw 264.7 cell infection at an MOI of 5. After incubation

at 4oC for two hours to ensure synchronization of uptake, cells were then incubated at

37oC for two hours, washed, and incubated with media containing 1μg/mL streptomycin

for an additional hour at 37oC to kill extracellular bacteria. Cells were then either lysed

with dH20 (three hour time point), or incubated overnight at 37oC in antibiotic free

media for lysis at twenty‐four hours. Graph depicts mean ± SEM. (Black triangle) 6D11

(n=4), (Blue Square) 18D11 (n=4), (Blue circle) IT‐70 (n=3), (Blue triangle) surface‐binding

(SB) pool C (n=4), (Blue Diamond) CS‐40 (n=4).

.

101

To test if the ability to bind the surface of live M. tuberculosis was necessary for

the decreased bacterial burden after macrophage infection, bacteria were pre‐incubated

with 60μg of non‐surface‐binding antibodies prior to macrophage infection and

compared to pre‐incubation with 60μg of surface‐binding antibodies from the

concentration series in Figure 15. Pre‐mixing of M. tuberculosis with individual surface‐

binding antibodies led to significant reductions in bacterial CFU at three hours post

infection, ranging from ‐28.7% (18D11, p=0.002) to ‐86.4% (CS‐40, p<0.001), with IT‐70

falling in between (Δ= ‐43.0%, p=0.051). A pool of surface‐binding antibodies did not

perform differently than individual monoclonal antibodies, with pre‐incubation of M.

tuberculosis and surface‐binding pool C resulting in a maximal decrease of ‐62.0%

(p=0.005) (Figure 16A). None of the non‐surface‐binding antibodies, nor the control

antibody tested significantly decreased bacterial burden at 3 hours post infection.

At twenty‐four hours post infection the pattern remained the same (Figure 16B).

Pre‐incubation with both surface‐binding Pool C (Δ= ‐71.2%, p=0.015) and CS‐40 (Δ= ‐

38.0%, p=0.009) significantly decreased bacterial CFU, while pre‐incubation 18D11 (Δ= ‐

19.2%, p=0.26) and IT‐70 (Δ= ‐35.3%, p=0.13), trended towards decreased bacterial

burden though the difference was not statistically significant. Neither non‐surface‐

binding nor control antibodies exerted any effect on twenty‐four hour bacterial burden

(Figure 16B).

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Figure 16: Surface‐binding, but not non‐surface‐binding antibodies decrease three

hour M. tuberculosis survival in murine macrophages

M. tuberculosis was mixed with 60μg of various control, surface‐binding and non‐

surface‐binding antibodies in a volume of 50μL for thirty minutes prior to infection. Raw

264.7 cells were infected at an MOI 5 and incubated at 4oC for two hours to ensure

synchronization of uptake. Cells were then incubated at 37oC for two hours, washed

with PBS, and incubated with media containing 1μg/mL streptomycin for an additional

hour at 37oC to kill extracellular bacteria. Following the incubation, cells were either

lysed with dH20 (3 hour time point), or incubated overnight at 37oC in antibiotic free

media for lysis at twenty‐four hours post infection. Normalized twenty‐four hour data

was calculated by dividing the percent of no PBS control at twenty‐four hours by the

average percent of no PBS control at three hours. Graph depicts mean ± SEM for n=4

except for IT‐70 where n=3. (White) IgG1 control antibody, (Green) non‐surface‐binding

antibody (Blue) surface‐binding antibody. Statistical significance was calculated using

Student’s t‐test: (*) p<0.05, (**) p<0.01, (***) p<0.001.

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Additionally, twenty‐four hour bacterial burden was normalized to three hour

bacterial burden to determine if additional killing occurred in that time frame, or if the

differences seen at twenty‐four hours post infection were due solely to the differences

observed at the earlier time point. After normalization no antibody differed significantly

from the PBS control (Figure 16C).

4.3.8 Surface-binding antibodies do not induce bacterial agglutination

To test if surface‐binding antibodies mediated their effect by agglutination, as

measured by decreased bacterial CFU, M. tuberculosis was incubated with 60μg of

antibody in a 50μL volume for thirty minutes prior to titering. Neither surface‐binding

nor non‐surface‐binding antibodies agglutinated bacteria, with as the mean titer for all

antibodies tested fell within 15.3% of the PBS control (Table 13).

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Table 13: M. tuberculosis antibodies do not induce agglutination

The titer represents the mean of n=3 replicates, except for 16G2 (n=2) and PBS (n=6).

4.3.9 BTN.4 does not decrease pulmonary bacterial load at three days post infection when mixed with biotin coated M. tuberculosis prior to retropharyngeal instillation

To evaluate if high‐affinity antibodies to surface‐molecules on M. tuberculosis can

decrease pulmonary bacillary burden, mice were inoculated with biotin coated M.

tuberculosis pre‐incubated with BTN.4, and sacrificed at three days post infection.

Pulmonary burden in BTN.4 treated animals trended lower, though the reduction was

not statistically significant (Δ= ‐22.4%, p=0.086).

Antibody Titer x105

(Mean ± SEM) % of PBS

PBS 9.8 ± 1.0

Surface

binders

CS‐40 9.8± 1.1 100.0%

IT‐70 9.1 ± 1.3 93.0%

18D11 11.3 ± 0.7 115.3%

SB pool C 8.7 ± 3.0 88.6%

Non‐ Surface

binders

16G2 9.2 93.9%

CS49 8.9 ± 1.2 90.5%

IT20 10.5 ± 0.7 106.6%

1H11 9.0 ± 0.8 92.0%

IT‐15 9.9 ± 1.3 101.2%

105

Figure 17: Pre‐incubation with BTN.4 does not decrease biotin coated M. tuberculosis

bacterial burden at three days post infection

Eight week old female BALB/c mice were instilled retropharyngeally with 1.1x105 biotin

coated M. tuberculosis was mixed with 23 μg of BTN.4, an anti‐biotin antibody. Three

days after infection lungs were harvested for CFU determination. (Black) PBS (n=8)

(Red) BTN.4 (n=4). Mean ± SEM is displayed. Statistical significance was calculated

using Student’s t‐test: (*) p<0.05, (**) p<0.01, (***) p<0.001.

4.3.10 Surface-binding, but not non-surface-binding antibodies, decrease bacterial burden at three days post infection when mixed with M. tuberculosis prior to retropharyngeal instillation

Incubation with the high‐affinity BTN.4 antibody did not reduce pulmonary

bacterial burden at three days post‐infection; however the system is artificial. Biotin

coating of the surface may alter the initial interaction between M. tuberculosis and host

cells in a manner that makes the protective effect of antibodies less pronounced. To test

if surface‐binding antibodies, despite week avidity, could alter early bacterial burden,

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mice were instilled with M. tuberculosis H37Rv pre‐incubated with either PBS, 15H10, an

anti‐Ebola control antibody, or a variety of surface and non‐surface‐binding M.

tuberculosis antibodies. At three days post infection pulmonary bacterial burden was

evaluated.

Of the four surface‐binding antibodies tested, all decreased bacterial burden

(Figure 18). Pre‐incubation with IT‐13 lead to the largest decrease in bacterial CFU, (Δ= ‐

59.1% p<0.001), with CS‐40 (Δ= ‐44.6%, p=0.008), IT‐70 (Δ= ‐35.3%, p=0.006), and 18D11

(Δ= ‐31.8%, p=0.006, Δ= ‐31.5%, p=0.001, and Δ= ‐20.4%, p=0.15 for Figure 18A, C, D

respectively) all producing significant decreases.

We hypothesized that a pool of surface‐binding antibodies would reduce

bacterial burden to a greater degree than individual monoclonal antibodies due to the

ability to bind to multiple target sites on the bacterial surface. Interestingly the surface‐

binding antibody pool (Δ= ‐33.5%, p=0.009) did not perform better than individual

monoclonal antibodies, producing a percent reduction less than two of the four surface‐

binding antibodies alone. In contrast to surface‐binding antibodies, none of the seven

non‐surface‐binding antibodies tested decreased bacterial burden (Δ= ‐9.4% to 22.5%).

Similarly, mice treated with 15H10 did not demonstrate reduced pulmonary bacillary

burden (Δ= ‐4.9% and 0.02%).

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Figure 18: Surface‐binding antibodies decrease pulmonary bacterial load in mice

1x105 M. tuberculosis was mixed with 60μg of purified antibody, in a volume of 50 μL.

Three days post infection lungs were harvested for CFU determination. (A)‐(D)

represent different experiments. (Black) PBS (n=8), (White) Control antibodies (n=4)

(Green) non‐surface‐binding antibodies (n=4) (Blue) surface‐binding antibodies (n=4).

Mean ± SEM is displayed. Statistical significance was calculated using Student’s t‐test: (*)

p<0.05, (**) p<0.01, (***) p<0.001.

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4.3.11 Surface-binding antibodies decrease bacterial burden at three, but not six, days post infection when mixed with M. tuberculosis prior to retropharyngeal instillation

To test the duration of surface‐binding antibody mediated protection, mice were

infected with M. tuberculosis pre‐mixed with PBS, 13H11, an anti‐HIV control antibody,

15A4, a non‐surface‐binding antibody, or one of two surface‐binding antibodies and

sacrificed at three and six days post infection. Similarly to Figure 18, mice treated with

M. tuberculosis mixed with 60μg of surface‐binding antibodies had decreased pulmonary

bacterial burden at three days post infection (Δ= ‐47.9%, p=0.001 and Δ= ‐38.4%, p=0.02

for 18D11 and IT‐70 respectively). This decrease was largely independent of dose, as

30μg and 120μg of antibody yielded similar decreases (Figure 19).

The surface‐binding antibody mediated decrease in bacterial burden observed at

three days post infection was not present at six days post infection for IT‐70 (60μg Δ= ‐

6.3% , p=0.50), and was decreased in magnitude for 18D11 (30μg day 6 Δ= ‐26.5% ,

p=0.03 vs 30μg day 3 Δ= ‐34.5%, p=0.006). Interestingly, pre‐incubation with the non‐

surface‐binding antibody 15A4 decreased bacterial burden at six days post infection (Δ=

‐30.7%, p=0.03) despite not differing from the PBS control three days earlier (Δ= ‐3.1%,

p=0.86). The control antibody 13H11 did not have an effect at either time point (Figure

19).

109

Figure 19: Surface‐binding antibodies decrease bacterial burden at three, but not six,

days post retropharyngeal instillation

Between 1.3x 105 and 2.3x105 M. tuberculosis was mixed with various concentrations of

purified antibody in a volume of 50 μL. At three and six days post infection lungs were

harvested for CFU determination. (A) and (B) represent separate experiments. (Black)

PBS (n=8), (White) Control antibodies (n=4) (Green) non‐surface‐binding antibodies

(n=4) (Blue) surface‐binding antibodies (n=4). Mean ± SEM is displayed. Statistical

significance was calculated using Student’s t‐test: (*) p<0.05, (**) p<0.01, (***) p<0.001.

110

4.3.12 Pre-incubation with surface-binding antibodies reduces pulmonary bacterial load three days after infection with mammalian adapted bacteria

Cultured M. tuberculosis, grown to log phase, expresses different proteins than

bacteria grown inside of mammalian hosts [290]. To test if surface‐binding antibodies

decreased pulmonary bacterial burden when pre‐incubated with mammalian adapted

bacteria, M. tuberculosis H37Rv dsRed was grown intraperionteally in BALB/c mice for

one week. Bacteria were then isolated by flow cytometry on the basis of dsRed

fluorescence, and incubated with either PBS, control or surface‐binding antibodies.

Surface‐binding antibodies decreased three day bacterial burden, similarly to

when pre‐mixed with culture grown bacteria. 18D11 (Δ= ‐15.3%, p=0.053), and IT‐70 (Δ=

‐18.4%, p=0.045) both decreased pulmonary bacterial load, though surface‐binding pool

A decreased CFUs to a greater extent (Δ= ‐36.0%, p<0.001). This is in contrast to culture

grown bacteria, where pools of surface‐binding antibodies did not decrease bacterial

burden to a greater degree than individual monoclonal antibodies. The protection of

surface‐binding antibodies waned by day six, decreasing slightly in magnitude for both

18D11 and surface‐binding pool A. The differences for both individual monoclonal

antibodies and surface‐binding pool A were not statistically significant from the PBS

control at the later time point (IT‐70 Δ= ‐32.1%, p=0.065; 18D11 Δ= ‐11.6%, p=0.51;

surface‐binding pool A Δ= ‐32.7%, p=0.061) (Figure 20).

111

At three days post infection, pre‐incubation with non‐surface‐binding antibody

15A4 did not alter bacterial load in the lungs (Δ= 6.8%, p=0.51). By six days post

infection, however, there was a trend towards decreasing bacterial burden, though the

difference was not statistically significant (Δ= ‐29.2%, p=0.10). The 13H11 control

antibody had no effect at either time point.

Figure 20: Surface‐binding antibodies decrease pulmonary bacterial load three days

after infection with mammalian adapted bacteria

M. tuberculosis dsRed was harvested from mouse peritoneum, sorted by flow cytometry,

mixed with 30μg purified antibody and inoculated into female BALB/c mice by

retropharyngeal instillation. Lungs were harvested three days post infection and

homogenized in PBS‐Ty prior to plating. (Black) PBS (n=8), (White) Control antibodies

(n=4) (Green) non‐surface‐binding antibodies (n=4) (Blue) surface‐binding antibodies

(n=4). Mean ± SEM is displayed. Statistical significance was calculated using Student’s

test: (*) p<0.05, (**) p<0.01, (***) p<0.001.

112

4.3.13 Pre-incubation of M. tuberculosis with surface-binding antibodies reduces the number of infected cells by one day post retropharyngeal instillation

Data from macrophage uptake experiments (Figure 16) suggests that surface‐

binding antibodies may work by blocking uptake, as they decrease bacterial burden as

soon as three hours post infection. To determine if a similar mechanism is at work in the

whole animal, mice were instilled retropharyngeally with M. tuberculosis dsRed mixed

with either PBS, or surface‐binding antibodies. At two, twenty‐four, and seventy‐two

hours post infection, mouse lungs were digested into single cell pneumocyte

preparations, treated with streptomycin to kill extracellular bacteria, and stained with a

panel of mammalian cell markers. Cells were analyzed by flow cytometry to determine

the number of infected cells as measured by dsRed fluorescence. To account for

differences in the number of pneumocytes isolated from each lung, the number of

infected cells was expressed per million pneumocytes.

As soon as two hours post infection, mice treated with M. tuberculosis mixed with

surface‐binding antibody pool C had fewer infected cells (Δ= ‐52.1%, p=0.047). This trend

continued, at both twenty‐four (Δ= ‐62.3%, p=0.011) and seventy‐two hours (Δ= ‐49.1%,

p=0.003) post infection (Figure 21).

113


retropharyngeal instillation decreases the number of infected cells by flow cytometry

Seven week old female BALB/c mice were instilled retropharyngeally with M.

tuberculosis H37Rv dsRed mixed with either PBS (n=8 at each time point) or 60μg of

surface‐binding antibody pool C (n=4 at each time point). At the time of sacrifice, two,

twenty‐four and seventy‐two hours post infection, lungs were harvested and digested

into a single cell suspension for staining. Infected cells were identified by dsRed

positivity. The number of infected cells for each mouse is expressed as the number of

infected cells per million pneumocytes to account for differences in cell recovery for each

lung. (Black) PBS (Blue) surface‐binding pool C. Mean ± SEM is displayed. Statistical


To confirm the decrease in the number of infected cells, a portion of the

pneumocyte preparation was plated prior to flow cytometry. At both twenty‐four

(Δ= ‐22.9%, p=0.24) and seventy‐two hours (Δ= ‐32.2%, p=0.042) mice instilled with M.

tuberculosis mixed with surface‐binding antibodies had fewer infected cells than mice

instilled with M. tuberculosis mixed with PBS, though the difference was only statistically

significant at the later time point (Figure 22).

114


retropharyngeal instillation decreases the number of infected cells by plate count



surface‐binding antibody pool C (n=4 at each time point). At the time of sacrifice,

twenty‐four and seventy‐two hours post infection, lungs were harvested and digested

into a single cell suspension for staining. Approximately 10% of the preparation was

plated on 7H10 agar plates prior to flow cytometry to enumerate the number of infected

cells. Mean ± SEM is displayed. (Black) PBS, (Blue) surface‐binding pool C. Statistical


4.3.14 Incubation of M. tuberculosis with surface-binding antibodies alters the infected cell profile during the first three days post infection

Pre‐incubation of M. tuberculosis with surface‐binding antibodies decreases the

number, but does not eliminate, infected cells after retropharyngeal instillation (Figure

21). To examine if surface‐binding antibody pre‐incubation alters the infected cell

profile, dsRed positive pneumocytes were classified as either alveolar macrophages,

115

recruited macrophages, monocytes, neutrophils, or “other” on the basis of GR‐1, CD11b

and CD11c staining.

Surprisingly at two hours post infection alveolar macrophages only comprised

10.9% and 7.2% of infected pneumocytes in mice instilled with M. tuberculosis mixed

with PBS and surface‐binding antibody pool C respectively. In mice instilled with M.

tuberculosis mixed with PBS, a large mixture of cells were infected at two hours post

instillation, with 59.2% of infected cells classified as “other”, 20.7% classified as

neutrophils, and small number of infected recruited macrophages and monocytes

comprising the remaining 10% of infected cells (Figure 23). The infected cell profile for

mice infected with M. tuberculosis mixed with surface‐binding antibody pool C was also

widely varied, with a larger proportion of infected neutrophils (49.1% vs 20.7%, p=0.002)

and fewer “other” cells (29.3% vs 59.2%, p=0.015) than their PBS counterparts.

In mice instilled with M. tuberculosis mixed with PBS, alveolar macrophage

comprised a larger portion of infected pneumocytes at twenty‐four hours post infection

of as compared to two hours post infection (10.9% vs. 29.1%, p<0.001). This trend,

towards an increasing number of infected alveolar macrophages, continued between

twenty‐four and seventy‐two hours post infection as well (29.1% vs. 43.5% p=0.003).

Mice infected with M. tuberculosis mixed with surface‐binding antibodies demonstrated

different kinetics of alveolar macrophage infection. At twenty‐four hours post infection

116

7.3% of infected pneumocytes were alveolar macrophages, no change from the 7.2% at

two‐hours post infection (p=0.98). By seventy‐two hours post infection, however,

alveolar macrophages accounted for 51.0% of infected pneumocytes, a large increase

from twenty‐four hours post infection (p=0.02), and not significantly different from mice

not infected with M. tuberculosis mixed with PBS (p=0.43) (Figure 23).

The kinetics of neutrophil infection also differed between the two groups. In

mice instilled with M. tuberculosis mixed with PBS, neutrophils comprised 20.7% of

infected cells at two hours post infection, with the percentage increasing to 41.1% by

twenty‐four hours post infection (p<0.001). The percentage then held relatively steady

with neutrophils comprising 38.7% of infected cells at seventy‐two hours post infection

(p=0.51). In mice infected with M. tuberculosis mixed with surface‐binding antibody pool

C, the increase in the proportion of infected neutrophils between two and twenty‐four

hours was even more pronounced. 41.1% of infected pneumocytes were neutrophils at

two hours post infection, a proportion which increased to 85.3% at twenty‐four hours

post infection (p=0.048), and fell back to 33.9% (p=0.010) at seventy‐two hours. The

proportion of infected neutrophils differed between the two groups at both two

(p=0.002) and twenty‐four (p<0.001), but not seventy‐two (p=0.54) hours post infection

(Figure 23).

117

The kinetics of recruited macrophage infection was similar between the two

groups. In mice infected with M. tuberculosis mixed with PBS, recruited macrophages

represented 8.7% of infected cells, which fell to 1.0% at twenty‐four hours (p=0.001).

Similarly, in mice infected with M. tuberculosis mixed with surface‐binding pool C,

recruited macrophages represented 13.5% of infected cells at two hours, a proportion

which fell to 1.5% by twenty‐four hours (p=0.17). Few infected monocytes were

identified during the course of the experiment (Figure 23). No differences between the

proportion of monocytes or recruited macrophages were noted between the groups at

any time points.

118

Figure 23: Effect of surface‐binding antibodies on the type of infected cell after

pre‐mixing with M. tuberculosis infection prior to retropharyngeal instillation



surface‐binding antibody pool C (n=4 at each time point). At the time of sacrifice, two,

twenty‐four, and seventy‐two hours post infection, lungs were harvested and digested


positivity, and cell types determined on the basis of GR‐1, CD11b, and CD11c signal.

Data are presented as the percentage of total infected cells for each group. (Black)

alveolar macrophage (Blue) monocyte (Green) recruited macrophage (Grey) neutrophil

(Light Purple) other. Statistical significance between M. tuberculosis mixed with PBS and

surface‐binding pool C is shown and was calculated using Student’s t‐test: (*) p<0.05, (**)

p<0.01, (***) p<0.001.

To validate these findings all recruited macrophages and neutrophils were sorted

from the pneumocyte preparations isolated at twenty‐four and seventy‐two hours post

infection. The sorted cells were then plated on 7H10 agar to enumerate the number of

infected cells, allowing for a total of eight comparisons between flow cytometry and

plate counts. Seven of those eight comparisons showed no statistically significant

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differences: the lone exception was recruited macrophages in mice instilled with M.

tuberculosis mixed with PBS at twenty‐four hours (p<0.001) (Figure 24).

Additionally, the ratio of the total number of infected cells between mice infected with

M. tuberculosis mixed with PBS and mice infected with M. tuberculosis mixed with.

surface‐binding pool C remained remarkably consistent. At twenty‐four hours post

infection, as measured by flow cytometry, mice infected with M. tuberculosis mixed with

surface‐binding pool C mixed had 25.6% as many infected neutrophils and 44.4% as

many infected macrophages as mice instilled with M. tuberculosis mixed with PBS. By

plate count these ratios were 19.8% and 9.3% respectively. The lack of concordance with

the recruited macrophage ratio is due to the far higher number of infected recruited

macrophages as identified by plate counting (Figure 24A). At seventy‐two hours post

infection, as measured by flow cytometry, mice instilled with M. tuberculosis mixed with

surface‐binding pool C had 28.6% as many infected neutrophils and 29.9% as many

infected recruited macrophages as mice instilled with M. tuberculosis mixed with PBS. By

plate count, those ratios were 31.5% and 30.8% respectively (Figure 24B).

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Figure 24: Comparison of the number of infected neutrophils and recruited

macrophages between flow cytometry and plate counts



surface‐binding antibody pool C (n=4 at each time point). At the time of sacrifice,

twenty‐four and seventy‐two, hours post infection, lungs were harvested and digested


positivity, and cell types determined on the basis of GR‐1, CD11b, and CD11c signal. All

neutrophils and recruited macrophage were sorted and plated on 7H10 plates to

determine the number of infected cells. (Black) PBS (Blue) surface‐binding antibodies.

Graphs compare the total number of infected cells in both groups at (A) twenty‐four or

(B) seventy‐two hours post infection.

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4.3.15 Total lung cell profile after M. tuberculosis infection

Lastly we wanted to examine if pre‐incubation of M. tuberculosis with surface‐

binding antibodies altered cell recruitment to the lungs. M. tuberculosis infection alone

does not drastically alter the total cell profile. No changes were observed in the

proportion of alveolar macrophages, recruited macrophages, or monocytes at any time

point. Neutrophils were elevated beginning at twenty‐four hours post infection

(p=0.099), and had increased by 75% over uninstilled control mice by seventy‐two hours

post infection (p=0.015). At seventy‐two hours post infection the number of cells

classified as “other” were higher in control vs. M. tuberculosis infected mice (p=0.034)

(Figure 25).

Instillation of M. tuberculosis mixed with surface‐binding antibodies altered the

infected cell profile, as compared to mice infected with M. tuberculosis alone. As soon as

two hours post infection, mice infected M. tuberculosis mixed with surface‐binding

antibodies had decreased alveolar macrophages (Δ= ‐26.4%, p=0.01) and increased

neutrophils (Δ= 61.6%, p=0.001). The difference in the proportion of neutrophils

disappeared by twenty‐four hours post infection; however, the decreased proportion of

alveolar macrophages remained (Δ= ‐54.3%, p=0.022). By seventy‐two hours post

infection, the number of alveolar macrophages was equal between the two groups;

however monocytes (Δ= 29.1%, p=0.021) and recruited macrophages (Δ=60.9%, p=0.035)

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were upregulated in mice infected with M. tuberculosis mixed with surface‐binding

antibodies (Figure 25).

Figure 25: Proportion of total cells in the lung after M. tuberculosis infection with and

without surface‐binding antibodies

Seven week old female BALB/c mice were either uninfected (control) (n=6), or instilled

retropharyngeally with M. tuberculosis H37Rv dsRed mixed with either PBS (n=8 at each

time point) or 60μg of surface‐binding antibody pool C (n=4 at each time point). At the

time of sacrifice, two, twenty‐four, and seventy‐two hours post infection, lungs were

harvested and digested into a single cell suspension for staining. Cell types were

determined on the basis of GR‐1, CD11b, and CD11c signal. (Purple) control, (Black)

PBS, (Blue) surface‐binding antibodies. Mean ± SEM is displayed. Statistical significance

between M. tuberculosis mixed with PBS vs surface‐binding pool C is displayed was

calculated using Student’s t‐test: (*) p<0.05, (**) p<0.01, (***) p<0.001.

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Figure 26: Retropharyngeal instillation of PBS does not alter the total lung profile

within twenty‐four hours post infection

Seven week old female BALB/c mice were either uninstilled (control)(n=6), or instilled

with PBS retropharyngeally and sacrificed at two hours (n=4) and twenty‐four hours

(n=4) post infection. At time of sacrifice lungs were harvested and digested into a single

cell suspension for staining. Cell types were determined on the basis of GR‐1, CD11b,

and CD11c signal. Mean ± SEM is displayed. (Purple) control (Grey) PBS alone.

Statistical significance between control and instilled mice was calculated using Student’s

t‐test: (*) p<0.05, (**) p<0.01, (***) p<0.001.

To confirm that the changes observed in the total cell profile, as compared to

control mice, are the result of M. tuberculosis instillation, and not caused by

retropharyngeal instillation of 50μL of liquid, single cell pneumocyte preparations were

prepared from mice administered PBS and sacrificed at two, and twenty‐four hours post

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instillation. No differences between uninfected and PBS instilled mice were observed at

either time point (Figure 26).

4.4 Discussion

Murine monoclonal antibodies against M. tuberculosis have been shown to be

protective from TB disease: blocking uptake of bacteria by host cells, preventing

dissemination, decreasing pulmonary pathology and bacterial burden, and improving

host survival [188,189,192‐195,291] . Aside from SMITB14, an anti‐LAM antibody

capable of blocking uptake, these antibodies function on more long term time scales,

rather than immediately impacting events in the first week after infection [193,194].

Bacterial growth remains stable in the lung during the first three days post M.

tuberculosis aerosol exposure, prior to a period of exponential growth [130]. After

establishing infection, neither cell mediated nor antibody responses have been able to

eradicate the bacteria, suggesting that anti‐infective vaccines must operate in a short

time frame after infection, prior to the onset of bacterial growth [90,153].

Antibodies against surface components of numerous other bacterial and parasitic

pathogens have demonstrated the ability to alter interactions with the host cell, by

enhancing FCγR mediated killing, altering the vacuolar environment triggering

transcriptional changes in the pathogen, and interfering with the colonization and

infection of the host [110,111,237,292]. We hypothesized that antibodies to the surface of

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M. tuberculosis could alter interactions between an individual bacterium and the first

infected cells. To evaluate this, we identified surface‐binding and non‐surface‐binding

antibodies and compared their ability to alter early bacterial burden both in vivo and in

vitro. Pre‐incubation with surface‐binding antibodies led to a decreased number of

recoverable bacteria three hours after in vitro macrophage infection, and lowered the

bacterial burden and the number of infected cells in mice within the first three days post

retropharyngeal instillation (Figure 16, Figure 18, Figure 21). Non‐surface‐binding and

isotype control antibodies did not protect in any assay (Figure 16, Figure 18).

Though the surface‐binding antibodies in this study decreased bacterial burden

in the lungs of mice, they were of poor avidity to whole cell lysate. This is not surprising,

as the majority of the antibodies were isolated from M. tuberculosis infected mice, and the

whole cell lysate avidity of polyclonal sera, even during chronic M. tuberculosis infection,

is poor (Table 12). High avidity antibodies to surface‐components correlate with

enhanced neutralization or bactericidal ability for numerous pathogens, including

poliovirus, dengue virus, H. influenza, Neisseria meningitis, and Streptococcus pneumoniae,

[293‐297]. It is possible that the avidity of the surface‐binding antibodies used in these

studies to their cognate antigens, is strong, and that weak cross‐reactive binding to other

components of whole cell lysate is decreasing the total avidity score (Figure 13). Even if

true, the live cell surface is a mixture of mycolic acids and proteins, that more closely

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resembles whole cell lysate than pure preparations of HSP‐X or LAM. Antibodies with

higher‐avidity to their cognate antigens both in purified preparations in and complex

mixtures are needed [298]. Plasma isolated from humans with active TB disease

possesses a broader range of antibody avidities to whole cell lysate than mice [168].

Isolation of new monoclonal antibodies from humans, with high sera avidity to both the

bacterial surface and whole cell lysate, will maximize the probability of identifying

highly avid surface‐binding antibodies that may improve upon the bacterial reductions

already seen in the murine model.

Additionally, plateau ELISA OD values to the live M. tuberculosis surface were

low (Table 10). Polyclonal sera in mice and humans have plateau OD values over seven

fold higher, suggesting that preparations containing antibodies to multiple targets may

generate stronger reactivity [168]. We hypothesized that monoclonal antibody pools

would improve the performance of surface‐binding antibodies in vivo, and in vitro, by

allowed increased antibody binding to the surface. Surprisingly, results were mixed,

showing enhanced performance of antibody pools in some studies (Figure 20), and no

difference from individual monoclonal antibodies in others (Figure 16, Figure 18).

Rather than enhancing the overall magnitude, antibody pools can provide enhanced

protection by decreasing the effective concentration of antibodies needed [299]. Future

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studies comparing individual and pools of monoclonal antibodies at lower

concentrations may display the hypothesized enhanced protection.

In vitro, surface‐binding, but not control and non‐surface‐binding antibodies,

decreased bacterial burden in RAW macrophages at three, and twenty‐four hours post

infection (Figure 15, Figure 16). The immediacy of the decrease suggests two potential

mechanisms for surface‐binding antibody mediated‐protection: either rapid FCγR

mediated killing, or a blocking of bacterial uptake. Normalization of twenty‐four hour

bacterial load in macrophages, to the three hour bacterial load, demonstrated no further

killing between the time points. Taken together with the observations that antibody‐

mediated early CFU reduction is FCγR independent (Chapter 5), and results fewer

infected cells as soon as two hours post infection in vivo (Figure 21), antibody‐mediated

blocking of bacterial uptake appears the more plausible. SMITB14, an anti‐LAM

antibody whose F(ab’)2 fragment decreases M. tuberculosis bacterial load as soon as two

hours after intranasal infection, is believed to work through a similar mechanism [194].

Another potential mechanism, agglutination, was ruled out by the inability of surface‐

binding antibodies to clump M. tuberculosis in vitro (Table 13).

In addition to culture grown bacteria, two other bacterial models were used in

these experiments. Biotin‐coated M. tuberculosis was used to evaluate the extent to which

the avidity of surface‐binding antibodies mediates their effects [289]. Biotin fixation to

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the surface of bacteria was detected by the binding of fluorescent streptavidin (Figure 9),

and was uneven, and not abundant enough to reliably distinguish labeled and unlabeled

bacteria. Pre‐incubation with BTN.4, a high‐affinity anti‐biotin antibody, showed a

concentration dependent decrease in bacterial burden in cell culture, and trended

towards decreased pulmonary burden three days post retropharyngeal instillation in

mice, though the magnitude was similar to that of the low avidity anti‐M. tuberculosis

monoclonal antibodies (Figure 14, Figure 17). Whether these results are simply a

reflection of poor biotin fixation, or representative of a phenomenon similar to HIV

where antibody avidity does not affect neutralizing activity, provided there is a

sufficient on‐rate, is unclear [293].

Surface‐binding antibodies also decreased bacterial burden in mice infected with

mammalian adapted M. tuberculosis at three days post infection (Figure 20). Sputum‐

derived M. tuberculosis has a transcriptional profile more similar to bacteria in a non‐

replicative persistence state, than log‐phase cultures [290]. Mammalian adapted bacteria

display similar features to sputum‐isolated bacteria, including the up‐regulation of HSP‐

X, a known surface‐protein, suggesting that infection‐induced transcriptional changes

can alter the bacterial surface [300]. Therefore, the ability of surface‐binding antibodies

to alter bacterial CFU when pre‐mixed with more physiologically relevant bacteria is

noteworthy. However, the mammalian adapted M. tuberculosis did encounter PBS‐Ty

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during the experiment. Detergent disrupts the protein‐polysaccharide capsule that

forms on bacteria during anexic growth and helps to mediate early interactions between

bacteria and host cells [301,302]. Disruption of the capsule may expose otherwise buried

proteins, and eliminate the true surface‐antigens found on in vivo grown M. tuberculosis.

Future studies examining the ability of surface‐binding antibodies to decrease bacterial

burden, using sputum derived M. tuberculosis, are critical for determining the potential

of surface‐binding antibodies to alter early infection events in humans.

Interestingly, 15A4, a non‐surface‐binding antibody was protective at later time

points during infection, decreasing pulmonary bacterial load at day six but not three

days post infection. This is the opposite pattern from surface‐binding antibodies, whose

protection at day three wanes by day six (Figure 19, Figure 20). This observation

confirms previously published studies that M. tuberculosis antibodies have a variety of

mechanisms of action, with some altering bacterial burden at later time points, though

an as of yet undescribed mechanism [189,194,291]. It is tempting to speculate how non‐

surface‐binding antibodies mediate this protection. M. tuberculosis components are

released from host cells through exocytosis, where non‐surface‐binding antibodies may

find and neutralize their effects, potentially damping M. tuberculosis subversion of the

host immune response [303]. Additionally, antibodies can enter cells, even when not

bound to pathogens, and neutralize bacterial toxins [185,304]. If non‐surface‐binding

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antibodies can access the intracellular space, they could impair bacterial manipulation of

the phagosome, and ultimately impinge upon M. tuberculosis survival.

When mixed with M. tuberculosis prior to infection, surface‐binding antibodies

alter the infected cell profile at two and twenty‐four hours post infection. By seventy‐

two hours post infection, however, the infected cell profile is identical to mice infected

with M. tuberculosis mixed with PBS (Figure 23). Surface‐binding antibodies impair

uptake by alveolar macrophages, and favor uptake by neutrophils. Neutrophils have

greater mycobactericidal properties than alveolar macrophages, potentially further

contributing to the decreased number of infected cells and reduced bacterial burden in

vivo [30,34,215]. In vivo models of bacterial disease are complex, and it is possible that

while blocking cellular uptake is the primarily mechanism by which surface‐binding

antibodies act, alteration of the infected cell profile may also play a role. It is tempting to

speculate that the waning of surface‐binding antibody mediated protection between

days three and six post‐infection is driven by the change in the infected cell profile,

away from neutrophils and towards alveolar macrophages. The ability to further impair

uptake by alveolar macrophages may increase the duration of the surface‐binding

antibody protection.

In summary, these studies demonstrate the ability of surface‐binding antibodies

to alter early infection events through a mechanism that both blocks bacterial uptake by

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host cells, and alters the type of host cell infected. This highlights the need for future

studies evaluating the protective role humoral immunity plays in altering M. tuberculosis

early infection events, and its ability to function as a component of an anti‐infective

vaccine.

4.5 Future Direction

The studies described in this chapter have demonstrated the ability of surface‐

binding antibodies to influence early infection events, a key step to altering infection

rate. However, the antibodies used in this study have poor avidity (Table 12). To

evaluate if enhanced avidity can improve early CFU reduction, new surface‐binding

antibodies are needed. Mice do not generate high‐avidity antibodies to the surface of

live M. tuberculosis, even during the chronic stages of infection (Table 12). Humans,

however, have a heterogeneous immune response to the surface of M. tuberculosis with

some individuals possessing relatively high surface avidity scores. As murine

vaccination with cell surface components leads to a low yield of surface‐binding

antibodies, in addition to those found being of low avidity, humans with plasma that

contains higher‐titer, higher‐avidity surface reactivity represent a potential source of

better antibodies [168]. Isolating and immortalizing memory B‐cells from such humans,

using the protocol described in Bonsignori et al., will maximize the chances of finding

high avidity clones. Once M. tuberculosis specific antibodies have been identified, their

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efficacy in decreasing early bacterial burden can be evaluated using the methods

described in this chapter [305].

Generating a larger pool of surface‐binding antibodies has other benefits.

Multiple studies have cataloged the cell wall components of M tuberculosis using mass

spectrometry [306,307]. The cell wall is a complex, multilayered structure [298,308].

Proteins may be completely buried in the outer layer of mycolic acids, or have portions

exposed. A large collection of surface‐ and non‐surface‐binding antibodies against a

particular target will allow for epitope mapping, to elucidate the portions of the

molecule that are accessible. Even if large collections of antibodies to a specific target do

not exist, the identification of a single surface‐binding antibody against that target

indicates its exposure on the surface and helps to further characterize the portion of each

bacterium exposed to the host.

Interestingly, surface‐binding and non‐surface‐binding antibodies showed

different activity kinetics: surface‐binding antibodies decreased bacterial burden at three

days post infection, though this decrease was waning by day six, while 15A4, a non‐

surface‐binding antibody, had no effect at early time points, but decreased bacterial at

six days post infection (Figure 19, Figure 20). The sample size of the non‐surface‐

binding antibodies tested is small. Future studies evaluating the potency of additional

non‐surface‐binding antibodies, both to HSP‐X and other targets, are necessary to

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confirm the phenotype. If confirmed, functional studies, including extending the

infected‐cell flow cytometry studies past three days, and the evaluation of the antibodies

in FCγR knock‐out mice should be undertaken to understand the alternate mechanism

of action.

Better animal models to measure the effect surface‐binding antibody mediated

protection are also needed. Pre‐mixing of M. tuberculosis and antibodies pre‐disposes the

system to success: antibodies have the ability to bind to the bacteria prior to infection. To

improve the physiological relevancy, antibodies should be administered prior to an

aerosol infection, as done in Chapter 5. This model, however, is incompatible with the

first‐infected cell flow cytometry assay. Wolf et al. used a standard dose aerosol model

of M. tuberculosis, followed by isolation of single pneumocytes from the lungs to

determine the identity of infected cells, though they were unable to reliability detect

cells prior to ten days post infection [27]. While higher‐dose aerosol models have been

described in the literature, they only deliver ~2000 CFU to the mice, a dose still not

reliably sufficient to detect infected cells at early time points (data not shown) [309]. We

attempted to generate a high dose aerosol, of ~5,000‐10,000 CFU using a vibrating mesh

nebulizer, which is capable of delivering higher doses of M. tuberculosis to mice than the

standard air‐jet nebulizer system. These attempted failed, delivering ~3000 bacteria, with

few to no infected cells detectable by flow cytometry at one day post infection (data not

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shown). Improvements in aerobiology that allow for longer exposure times without

increasing bacterial death in the innocula, clogging the vibrating mesh, or increasing

humidity leading to droplet condensation, are needed in order to deliver a sufficiently

high dose.

Despite its limitations, the mixing of M. tuberculosis with surface‐binding

antibodies prior to instillation, followed by isolation of pneumocytes, demonstrated an

interesting phenotype. Fewer infected cells were detected at two, twenty‐four, and

seventy‐two hours post infection, when compared to a no‐antibody control (Figure 21).

Additionally, whole‐lung homogenate bacterial burden was decreased at three days post

infection (Figure 18‐Figure 20). Taken together, these findings suggest that bacteria are

either rapidly killed or cleared from the lung. To further understand the mechanism of

early CFU reduction two approaches can be taken. Live/Dead strains of M. tuberculosis

have been described, which use tetracycline induction of green fluorescent protein (GFP)

to distinguish transcriptionally active and inactive cells [310]. Attempts at using a

Live/Dead strain in the early‐infection flow cytometry model failed, as cells must be

exposed to tetracycline for approximately twenty‐four hours to reliably distinguish live

and dead cells (data not shown). Optimization of the strain to more rapidly produce

GFP would allow for the identification of dead bacteria within cells, provided they are

not sufficiently degraded by the lysosome, and the testing of the hypothesis that the

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decreased number of infected cells is due to rapid bacterial killing. Conversely, bacteria

can be radiolabeled prior to incubation, allowing for the tracing of the bacteria beyond

the lung. After infection, if the amount of radiolabeled bacteria in the lungs of mice

treated with both PBS and antibody‐mixed M. tuberculosis is the same, despite early

infection differences in bacterial burden, it implies that M. tuberculosis is being rapidly

killed. If the amount of radiolabeled bacteria is decreased in the lungs of mice infected

with M. tuberculosis mixed with antibodies, it implies the bacteria are being cleared.

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5. Surface-binding antibodies decrease pulmonary bacterial burden after aerosol infection

5.1 Introduction

Interest in humoral immunity as a component of a TB vaccine, especially one that

acts by preventing infection, rather than preventing the progression to active disease, is

increasing [107,211,311]. In order to design vaccines which induce sterilizing immunity,

we need to understand if vaccine‐inducible factors, such as antibodies, can alter early

infection events, if present at the time of exposure. While numerous studies have

examined the effect monoclonal antibodies administered near the time of infection have

on M. tuberculosis disease, to our knowledge none have examined their ability to alter

infection rate [107].

In Chapter 4 we demonstrated that surface‐binding antibodies decrease early

infection bacterial load in vivo and in vitro when mixed with M. tuberculosis prior to

infection. While these studies demonstrate the ability of surface‐binding antibodies to

alter early infection events, neither pre‐mixing of the antibodies with M. tuberculosis, nor

the route of administration is physiologically relevant. To evaluate if surface‐binding

antibodies are protective, when present at the time of aerosol infection, we used a more

physiologically relevant animal model; surface‐binding antibodies were administered

retropharyngeally twenty‐four hours prior to M. tuberculosis aerosol exposure. In this

model, surface‐binding antibodies decreased bacterial burden in the lungs within the

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first three days of infection in an FCγR independent, concentration dependent manner.

The effect was most pronounced at one day post infection, waning in magnitude over

time, and disappearing completely by six days after exposure. When present at the time

of an ultra‐low dose M. tuberculosis aerosol exposure, surface‐binding antibodies did not

alter infection rate. However, in infected mice both an IgG1 isotype control and surface‐

binding antibodies decreased bacterial burden at one month post infection. These

studies reveal that surface‐binding antibodies are capable of altering early infection

events, but that the antibody‐induced changes are not sufficient to reduce infection rate.

5.2 Methods

5.2.1 Animals

The animals in this study were handled under a protocol approved by the Duke

Institutional Animal Care and Use Committee. Six week old female wild‐type BALB/c

mice from Charles River Laboratories (028) and Taconic (BALB‐F) as well as BALB/c

background FCγR knock‐out mice (Taconic, 584‐F) were housed in the RBL at Duke

University for at least one week prior to M. tuberculosis exposure under environmental

conditions described in Chapter 2.

5.2.2 Retropharyngeal instillation of purified antibody

Monoclonal antibodies were purified as described in Chapter 4. After buffer

exchange antibodies were further concentrated to of >20 mg/mL using the Amicon

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Ultracel‐30k centrifugation filter (Millipore UFC803096). Antibodies were then diluted to

the desired concentration in endotoxin free‐PBS and delivered to anesthetized mice via

retropharyngeal instillation twenty‐four hours prior to aerosol exposure in a volume of

50μL.

5.2.3 Pharmacokinetics of monoclonal antibodies in BAL fluid

20 μL of hybridoma supernatant was instilled retropharyngeally into BALB/c

mice. At fifteen minutes, thirty minutes, one hour, two hours and twenty‐four hours

post instillation mice were sacrificed and perfused, with BAL fluid collected and as

described in Chapter 2. BAL fluid was then assayed by ELISA against whole cell lysate,

as described in Chapter 3, to determine the amount of antibody present.

5.2.4 Aerosol innocula preparation

For ultra‐low dose aerosol exposures, Mycobacterium tuberculosis H37Rv was

diluted from frozen aliquots to an OD of 0.00025 prior to vortexing for one minute. For

standard dose aerosol exposures, M. tuberculosis H37Rv frozen aliquots were diluted to

OD 0.01 prior to horizontal vortexing for one minute. Antifoam Y‐30 (Sigma Aldrich,

A5758) was added to the innocula at a 1:5000 dilution prior to aerosolization.

5.2.5 Aerosol challenge

Mice, at least seven weeks old, were exposed to M. tuberculosis aerosol in a

whole‐body Madison exposure chamber connected to a Class III biological safety

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cabinet, as we have previously described [25,312]. The aerosol was generated using a 6‐

jet Collison nebulizer (BGI Inc, CN25), operating at 19±1 liters per minute (Lpm), 35±1

pounds per square inch of pressure, 50% relative humidity and a flow rate of 50 Lpm.

The exposure system was controlled by Aerosol Management platform, AeroMP (Biaer

Tech, LLC). Mice were loaded into the chamber with air flowing, and exposed to the

aerosol for 20 minutes. A BioSampler (SKC Inc), drawing at a flow rate of 12.5 Lpm, was

used to sample the viable aerosol concentration through a port in the Madison chamber

door. Bacterial titers were performed to determine the viable bacterial concentration in

the innocula before aerosolization (pre‐neb), in the remaining innocula post

aerosolization (post‐neb), and in the BioSampler.

5.2.6 Determination of aerosol dose presented

The dose presented represents the number of viable M. tuberculosis bacteria

inhaled by the mouse during the aerosol exposure. It is calculated by the formula Dp= VE

x Ca, where VE represents the exposure volume and Ca the viable aerosol concentration

in the aerosol chamber. VE =VM x t, where t is the duration of the aerosol exposure and

VM is the minute ventilatory volume of each mouse as determined by Guyton’s

Forumula VM= 2.1 x (mouse body weight)0.75 [313]. Ca, is defined as Ca= (CSAMPLE x

(VSAMPLE‐(Ec x t)))/(QSAMPLER x t), where CSAMPLE is the BioSampler concentration, VSAMPLER

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is the initial BioSampler volume, Ec ia evaporation rate, t is aerosol exposure duration,

and QSAMPLER is sample airflow rate.

5.2.7 Bacterial CFU determination

Mice were sacrificed between one day and four weeks post aerosol exposure, and

their lungs were homogenized as described in Chapter 4. Mice sacrificed between one

hour and nine days post infection had lung homogenates plated neat, while

homogenates from mice with a longer duration of infection were serially diluted in PBS‐

Ty prior to plating. CFU were counted after 3‐4 week incubation at 37oC.

5.2.8 Lung culture

For mice sacrificed at one month after ultra‐low dose aerosol exposure, the ~70%

of lung homogenate remaining after CFU plating was added to 9mL of 7H9

supplemented with 10% OADC, 0.5% glycerol and 0.05% tyloxapol. Cultures were

incubated at 37oC for six to seven weeks with intermittent shaking and then evaluated

for turbidity. Thirty‐three out of one‐hundred and ninety liquid cultures were streaked

onto 7H10 agar plates to confirm either negativity (in the absence of turbidity) or M.

tuberculosis growth.

5.2.9 Statistics

Prism Software was used for statistical analyses. For pairwise comparisons

among continuous variables a two‐sided student’s t‐test was used for normally

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distributed data, and a Mann‐Whitney test was used for non‐normally distributed data.

For discrete variables, a Fisher’s exact test was used for pairwise comparisons, and a

Chi‐squared test was used for comparisons between multiple variables.

5.3 Results

5.3.1 Antibodies persist in BAL fluid for at least twenty-four hours after retropharyngeal instillation

To evaluate if antibodies administered retropharyngeally persisted in the BAL

fluid, fifteen female BALB/c mice were instilled with 20μL of hybridoma supernatant,

and sacrificed at various time points during the first twenty‐four hours after instillation.

BAL fluid from each mouse was evaluated by ELISA (Figure 27). Antibody levels in the

BAL fluid remained relatively stable for the first hour, and fell sharply thereafter.

Despite the decrease, antibody was still detected at twenty‐four hours post instillation,

though it represented only 28% of peak levels.

Aerosol delivery of M. tuberculosis, to mice, two hours post retropharyngeal

instillation, led to large variability in the pulmonary dose retained. This problem was

not noted when the aerosol exposure occurred twenty‐four hours after instillation (data

not shown). The continued persistence of antibody in the BAL fluid at one day post

administration allowed for aerosol exposures to be conducted at that time point for all

future studies.

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Figure 27: Levels of antibody in BAL fluid within first twenty‐four hours after

retropharyngeal administration

20uL of hybridoma supernatant (anti‐HSPX) was instilled retropharyngeally into female

BALB/c mice. At fifteen minutes (n=3), thirty minutes (n=3), one hour (n=3), two hours

(n=3), and twenty‐four hours (n=3) post infection mice underwent terminal

bronchoalveolar lavage (BAL). BAL fluid was assayed by ELISA against M. tuberculosis

H37Rv whole cell lysate. Mean ± SEM shown for each time point.

5.3.2 Surface-binding antibodies decrease bacterial CFU in a concentration dependent manner when administered prior to a standard dose M. tuberculosis aerosol

Female BALB/c mice were given either PBS, 1mg of an IgG1 control (6D11)

antibody, or varying concentrations of surface‐binding antibody pool D one day prior to

aerosol infection with an M. tuberculosis at a concentration of 0.59 CFU/mL. At three

days post infection mice instilled with surface‐binding antibodies had a concentration

dependent decrease in pulmonary bacterial burden (Figure 28). At the maximal dose of

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1mg, mice administered surface‐binding antibodies had a 34.0% decrease in pulmonary

bacterial burden (p=0.03), while mice instilled with control antibodies did not differ from

mice instilled with PBS (Δ= ‐2.5%, p= 0.86).

Figure 28: Surface‐binding antibodies reduce pulmonary bacterial burden at 3 days

post infection in a concentration dependent manner.

BALB/c mice were given PBS (n=8), a control antibody (6D11) (n=4) or varying

concentrations of surface‐binding pool D (n=4) retropharyngeally twenty‐four hours

prior to a standard dose M. tuberculosis aerosol infection. At three days post infection

mice were sacrificed, and lungs plated to determine bacterial burden. (Black) PBS,

(White) control antibodies, (Blue) surface‐binding antibodies. Mean ± SEM is displayed.

Statistical significance was calculated using the student’s t‐test: (*) p<0.05, (**) p<0.01,

(***) p<0.001.

5.3.3 Retropharyngeal administration of surface-binding antibodies leads to variable decreases in bacterial burden three days post aerosol infection

To confirm our finding, that 1mg of surface‐binding antibodies, administered prior

to aerosol infection, decreased lung bacterial burden, we conducted three similar

studies. In each study, mice were instilled with either 1mg of individual surface‐binding

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antibodies, 1mg of a surface‐binding antibody pool, 1mg of an isotype control antibody,

or PBS one day prior to M. tuberculosis aerosol exposure with viable aerosol

concentrations of 0.24‐0.26 CFU/mL, and sacrificed at three days post infection. Overall,

the general trend in these studies confirms our previous findings (Figure 29). However,

large standard deviations within groups, as well as high degrees of variation between

experiments, highlight the inconsistency of this model.

The first study confirmed our previous findings: mice administered 1mg of IT‐70,

a surface‐binding antibody, had decreased bacterial burden at three days post infection

by as compared to a PBS control (Δ= ‐40.1%, p=0.02) (Figure 29A). In the second study,

instillation of 1mg of two different surface‐binding antibodies, 18D11 and IT‐70

decreased lung burden by ~30%, however, due to large standard deviations within the

groups, the p‐values were not significant (Δ= ‐31.3%, p=0.07 and Δ= ‐30.4%, p=0.08

respectively). Mice instilled with 1mg of either CS‐40, or a surface‐binding antibody pool

prior to infection, only had decreased lung burdens of between 10‐20% (Δ= ‐10.7%,

p=0.57 and Δ=‐17.2%, p=0.34 respectively) (Figure 29B). In the third experiment, CS‐40,

IT‐70, and a pool of surface‐binding antibodies all decreased lung CFU by over 35%,

with the pool reducing CFUs by 56%, though none of these decreases were statistically

significant due to large standard errors (CS40: Δ= ‐37.7%, p=0.23, IT‐70: Δ= ‐52.85,

p=0.26, and surface‐binding pool D Δ= ‐56.1%, p=0.16 respectively). 18D11 only reduced

145

Figure 29: Surface‐binding antibodies provide inconsistent protection at three days

post M. tuberculosis aerosol infection

Female BALB/c mice were given PBS (n=8), 1mg of a control antibody (6D11) (n=4), a

variety of surface‐binding antibodies (n=4), 15A4, a non‐surface‐binding antibody (n=4),

or a surface‐binding antibody pool (n=4) retropharyngeally twenty‐four hours prior to a

standard dose M. tuberculosis aerosol infection. At three days post infection mice were

sacrificed, and lungs plated to determine bacterial burden. (Black) PBS, (White) Control

antibodies (Green) non‐surface‐binding antibodies (Blue) surface‐binding antibodies.

Mean ± SEM is displayed. Statistical significance was calculated using the student’s t‐

test: (*) p<0.05, (**) p<0.01, (***) p<0.001.

146

lung CFU by 13.1% (p=0.68) (Figure 29C). Two of these three experiments had either

isotype or non‐surface‐binding antibody controls. The isotype control antibody, 6D11,

decreased bacterial burden by between 3.6% and 20% (p=0.52 and p=0.85), while the

non‐surface‐binding 15A4 decreased bacterial burden by 10.5% (p=0.74) (Figure 29B and

Figure 29C).

5.3.4 Retropharyngeal administration of surface-binding antibodies one day prior to standard dose aerosol infection decreased bacterial burden in the lungs in a time dependent manner

Due to the variability observed in surface‐binding antibody mediated protection

at three days post infection, we hypothesized that the antibody’s optimal effect occurred

earlier. To test this, mice were administered either PBS, 1mg of isotype control antibody,

or 1mg of a surface‐binding antibody pool, retropharyngeally one day prior to a

standard dose M. tuberculosis aerosol infection at a concentration of 0.42 mg/mL. Surface‐

binding antibodies decreased lung bacterial CFU by 41.1% at one day post infection,

compared to PBS control (p=0.006), but did not decrease CFU at three (Δ= ‐2.5%, p=0.81),

six (Δ= ‐32.3%, p=0.16), or nine (Δ= ‐3.7%, p=0.86) days post infection (Figure 30). The

6D11 isotype control antibody did not alter bacterial CFU at any time point (Figure 30).

To confirm the results of this study, a modified time course consisting of murine

sacrifice at one and three days post infection was conducted. As with Figure 30, surface‐

binding antibody pool C decreased bacterial burden in the lungs at one (Δ= ‐47.0%,

p=0.007), but not three (Δ= ‐4.5%, p=0.86) days post infection (Figure 31). At one day

147

post‐infection 18D11 also decreased bacterial burden (Δ=‐35.0%, p=0.031), while IT‐70

and CS‐40 trended towards decreased bacterial burden, though it was not statistically

significant (Δ= ‐12.0%, p=0.53, and Δ= ‐22.0%, p=0.17 respectively).

Figure 30: The ability of surface‐binding antibodies to decrease pulmonary bacterial

burden wanes over time

Mice were given either PBS, 1mg of a control antibody (6D11) or 1mg of surface‐binding

pool D retropharyngeally twenty‐four hours prior to a standard dose M. tuberculosis

aerosol infection. At varying time points post infection mice were sacrificed, and lungs

plated to determine bacterial burden. (Black) PBS, (White) Control antibodies (Green)

non‐surface‐binding antibodies (Blue) surface‐binding antibodies. Mean ± SEM is

displayed. Statistical significance was calculated using the student’s t‐test: (*) p<0.05, (**)

p<0.01, (***) p<0.001.

148

Figure 31: Surface‐binding antibodies decrease bacterial burden at one day post

infection

Six week old female BALB/c mice were given either PBS (n=8), 1mg of a surface‐binding

antibody (n=4), or 1mg of a surface‐binding pool (n=4), intratracheally twenty‐four hours

prior to a standard dose M. tuberculosis aerosol infection. At varying time points post

infection mice were sacrificed, and lungs plated to determine bacterial burden. (Black)

PBS, (Blue) surface‐binding antibodies. Mean ± SEM is displayed. Statistical significance

was calculated using the student’s t‐test: (*) p<0.05, (**) p<0.01, (***) p<0.001.

5.3.5 Decreased bacterial burden is FCγR independent

To determine if the decreased lung burden after surface‐binding antibody

treatment was due to bacterial killing mediated by the FCγR, wild‐type and FCγR ‐/‐

mice were instilled either with PBS, or 1mg of a surface‐binding antibody pool, one day

prior to a M. tuberculosis aerosol exposure at a concentration of 0.58 CFU/mL, and were

sacrificed at twenty‐four hours post infection. Wild‐type mice administered surface‐

binding antibodies had a 67.5% decrease in lung burden compared to mice administered

PBS (p<0.001). The same phenotype was observed in the FCγR ‐/‐ mice, with mice given

149

surface‐binding antibodies having a 61.7% decrease lung burden compared to mice

administered PBS (p=0.003).

Figure 32: The effect of surface‐binding antibodies is FCγR independent

Eight week old female wild‐type of FCγR knockout BALB/c mice were instilled with

either PBS, or 1mg surface‐binding pool C in 50μL, twenty‐four hours prior to aerosol

exposure. At one day post infection the mice were sacrificed, their lungs manually

homogenized, and plated to determine lung bacterial burden. Student’s t‐tests were

used to determine statistical significance: (*) p<0.05, (**) p<0.01, (***) p<0.001.

5.3.6 Retropharyngeal administration of surface-binding antibodies prior to ultra-low dose aerosol does not reduce murine infection rate

Female BALB/c mice were divided into three groups and administered with

either PBS, 1mg of an isotype control antibody, or 1mg of a surface‐binding antibody

pool one day prior to ultra‐low dose aerosol M. tuberculosis infection with aerosol

concentrations between 0.007‐0.033 CFU/mL. Aerosol concentrations in this range lead

to infection of ~60% of all animals. The experiment was repeated three times, for a total

of seventy‐two mice in the PBS and surface‐binding antibody groups, and forty‐six mice

150

in the 6D11 group. The large number of mice was necessary to detect a 20% percent

difference in infection rate between the surface‐binding antibody and PBS treated

groups with 80% power. Information on the strain, age, and weight for every mouse can

be found in Table 24 (Appendix C), while the baseline characteristics for each exposure

group can be found in Table 25 (Appendix C). Mouse weight and age did not differ

significantly between treatment or exposure groups.

The entire mouse lung was assayed to determine if a mouse was infected. One

month post infection mice were sacrificed and each lung was homogenized with ~30%

plated on 7H10 agar plates to determine bacterial CFU, and the remaining ~70%

cultured for 7 weeks in 7H9 liquid media. A mouse was determined to be infected if the

liquid culture was positive. If the liquid culture was contaminated, infection status was

determined by the presence or absence of bacterial CFU on 7H10 agar plates.

No infection rate differences were seen among the three groups Table 14. Mice

administered PBS prior to exposure had a 63.8% infection rate, and mice administered

surface‐binding antibodies had a 56.9% infection rate (OR 0.75, p=0.50). The infection

status of each mouse can be found in Table 24.

151

Table 14: Infection rates after ultra‐low dose M. tuberculosis aerosol exposure

Infected/

Total

Percent

Infected

(%)

Odds Ratio

(95% CI)* P‐value*

Experimental group A

PBS 9/24 37.5%

6D11 10/24 41.7% 1.2(0.37‐3.80) 1.0

SB pool C 7/24 29.2% 0.69(0.21‐2.30) 0.76

Experimental Group B

PBS 19/24 79.2%

6D11 17/22 77.3% 0.89(0.22‐3.64) 1.00

SB pool C 15/24 62.5% 0.44(0.12‐1.59) 0.34

Experimental Group C

PBS 18/24 75.0%

SB Pool C 19/24 79.2% 1.27(0.33‐4.89) 1.00

Total

PBS 46/72 63.8%

SB Pool C 41/72 56.9% 0.75(0.38‐1.46) 0.50

* P‐value and odds ratio were calculated using Fisher’s exact test

5.3.7 The presence of 1mg of antibodies prior to ultra-low dose M. tuberculosis exposure leads to decreased bacterial load one month post infection in infected mice

Lung burden was evaluated in all infected mice at one month post ultra‐low dose

aerosol exposure. The log10 CFU for all infected mice, as determined by plate counting,

are displayed in Figure 32, and the value for each individual mouse can be found in

Table 24 (Appendix C). Mice with positive cultures, but no plate counts, are not shown

152

in the figure. One month median CFU counts for mice administered with 1mg of

surface‐binding antibodies prior to ultra‐low dose infection (Δ=0.63 log10 CFU, p<0.001)

were significantly lower than mice administered PBS. The affect appears non‐specific, as

same reduction was observed for mice instilled with 1mg of isotype control antibody

(Δ=0.58 log10 CFU, p=0.004).

Figure 33: Pulmonary bacterial burden one month post ultra‐low dose aerosol

infection

Six week‐old female BALB/c mice were instilled with either PBS, 1mg 6D11 or 1mg

surface‐binding pool C in 50μL, twenty‐four hours prior to ultra‐low dose aerosol.

Lungs were manually homogenized, with~30% of the lung homogenate was plated to

determine lung bacterial burden. The red line represents the median value. (Black) PBS,

(White) control antibody, (Blue) surface‐binding pool C. Mann‐Whitney tests were used

to determine statistical significance. For comparison with 6D11 only Group A and

Group B CFUs for PBS mice were included, as Group C had no mice treated with 6D11:

(*) p<0.05, (**) p<0.01, (***) p<0.001.

153

5.4 Discussion

The cell‐mediated immune response is critical for the control of M. tuberculosis;

however, it does not reach the lung until between two to three weeks post infection, too

late to prevent the logarithmic growth of the bacteria [314]. The BCG vaccine induces

antigen‐specific CD4+ T‐cells, which are present at the time of infection in vaccinated

individuals, but remains unable to prevent infection [90,130,315]. In order design a

vaccine that generates sterilizing immunity, other factors need to be considered. To test

if passive administration of antibodies can alter the initial interactions between the host

and M. tuberculosis, mice were administered monoclonal surface‐binding IgG antibodies

prior to aerosol infection. High concentrations of surface‐binding, but not control,

antibodies were able to reduce early infection CFU; however, these antibodies were

unable to alter infection rate (Table 14, Figure 28, Figure 30).

Previous work has primarily focused on the ability of antibodies to exert long

term effects on M. tuberculosis infection [188,189,191‐195]. These antibodies work in a

variety of ways: altering interactions with epithelial cells to prevent dissemination,

enhancing cellular immune responses, blocking interactions between bacterial ligands

and host‐receptors, and neutralizing the pathogenic effects of secreted polysaccharides

[194,195,291]. Antibodies mediate protection against other bacterial pathogens through a

variety of additional mechanisms including recruitment of complement prior to

phagocytosis, toxin neutralization, agglutination, FCγR mediated killing and alteration

154

of cell entry mechanisms [304,316]. Administration of surface‐binding antibodies one

day prior to standard‐dose M. tuberculosis aerosol exposure reduced bacterial CFU at

twenty‐four hours post infection in both wild‐type and FCγR deficient mice (Figure 32).

When combined with the data from Chapter 4 that showed pre‐mixing of M. tuberculosis

with surface‐binding antibodies leading to fewer infected cells, as soon as two hours

post infection, (Figure 21), it is tempting to speculate that surface‐binding antibodies

mediate protection by blocking uptake by phagocytic cells in an FCγR independent

manner. This would leave the bacteria vulnerable to extracellular killing or mucocilliary

clearance from the lung into the gastrointestinal tract. Mucocilliary clearance is an

important host defense mechanism, rapidly clearing particles from the lung in as little as

three hours in humans, and twelve hours in mice [317,318]. Future studies with

radiolabeled bacteria to determine if surface‐binding antibodies result in enhanced

clearance from the lung will be able to distinguish between the two mechanisms.

In contrast to C57BL/6 and BALB/c mice, where differences in early infection

CFU corresponded to differences in infection rate, we did not see the same correlation

with surface‐binding antibodies [130]. Despite surface‐binding antibody dependent CFU

decreases at one day post infection, the same treatment did not alter infection rate

(Table 14). Two potential reasons for this discrepancy exist. First, peak differences

between the C57BL/6 and BALB/c mice were larger than in this study: at one day post

infection C57BL/6 mice had on average 66% fewer M. tuberculosis CFU, while pre‐

155

administration of surface‐binding antibodies decreased CFUs at the same time point by

only 44% [130] (Figure 30 , Figure 31). Additionally, the infect rate study described in

this chapter was powered to detect a 20% infection rate difference, assuming the peak

bacterial burden decrease by surface‐binding antibodies was 50%. Given the lower

maximal decrease in early infection bacterial reduction, as well variability in antibody

delivery after retropharyngeal instillation, infection rate differences may exist that our

study was not sufficiently powered to detect [319]. Improvement in both antibody

avidity and delivery may reveal surface‐binding antibody dependent infection‐rate

differences. Second, different mechanisms may underlie the early bacillary reduction

seen in the C57BL/6 mice, and antibody treated mice. A better understanding of the

mechanisms that drive both phenotypes is critical for the efforts to design an anti‐

infective M. tuberculosis vaccine [211].

Despite not altering infection rate, treatment with both surface‐binding and

control antibodies decreased pulmonary bacterial load at one month post ultra‐low dose

infection. The ability of the high dose control antibody to decrease bacterial burden is

not surprising, as IvIg, when administered intraperitoneally within twenty‐four hours of

intravenous M. tuberculosis infection, decreases bacterial by 2 log10 at one month post

infection [154]. High dose immunoglobulins have a profound impact on the immune

system, suppressing TH1 cytokine production, decreasing T‐Cell proliferation, and

interfering with complement deposition [167,320,321]. Given the high concentration of

156

surface‐binding antibodies needed to observed early infection CFU decreases, the long

term non‐specific effects of this high a dose may mask the specific effects of surface‐

binding antibodies. Higher‐avidity antibodies which exert protective effects at lower

doses are critical for determining if surface‐binding antibodies, and not non‐specific

effects, can decrease bacterial burden one month.

The animal model used in this study also represents an improvement on the

retropharyngeal mixing studies performed in Chapter 4 and the majority of the

published literature. [192‐195]. While the model is more physiologically relevant, as

antibodies are present at the time of an aerosol infection, retropharyngeal instillation of

antibodies is invasive, depositing 50 μL of liquid into the lungs. Delivery of vehicle can

alter the baseline lung environment, leading to loss of certain strain‐specific

mycobactericidal properties (Chapter 2). Additionally, retropharyngeal instillation is

variable, does not produce uniform delivery to all lobes of the lung, and in some animal

models does not result in reliable in alveolar delivery, a critical limitation, as that is

where initial interactions between host cells and M. tuberculosis occur [319]. To overcome

these limitations, induction of surface‐binding antibodies through vaccination should be

undertaken in future studies. Furthermore, the antibodies used in these studies bind

poorly, requiring high dosages to demonstrate effects (Figure 28). Identification of

antibodies with higher avidity, and more abundant or easily accessible targets could

157

produce a more reproducible phenotype, require a lower dosage of antibody, and

decrease early infection CFUs to a greater degree.

In summary these data demonstrate that surface‐binding antibodies are capable

of influencing early infection events, though not in a manner that is able to alter M.

tuberculosis infection rate. The results of this study further demonstrate that surface‐

binding antibodies can be protective and affirm the need for further research into

humoral immunity both as a component of the immune response to M. tuberculosis, and

as a vaccine component.

5.5 Future Directions

As with the results in Chapter 4, the avidity of the surface‐binding antibodies

used in these studies is poor. Isolating additional surface‐binding antibodies to a variety

of targets has the potential to identify high‐avidity antibodies, which may increase the

magnitude of protection from aerosol infection, and require less antibody to be

administered. Further decreases in early bacterial burden may also lead to reduction in

infection rate, the ultimate goal for surface‐binding antibodies.

The experimental system used in this chapter represents a significant

improvement from Chapter 4: here antibodies are instilled in the lung twenty‐four hours

prior to aerosol infection, rather than being mixed with M. tuberculosis prior to

retropharyngeal instillation. However, it still has significant limitations.

Retropharyngeal instillation is non‐physiologic. The method is invasive, distribution of

158

antibodies into the lung is not uniform, the liquid has the potential to dilute host factors

[322‐324]. Additionally, instilled substances do not always reach the alveolus, a critical

limitation as that is where initial M. tuberculosis‐host cell interactions occur [319].

To overcome these limitations, surface‐binding antibodies must be produced by

the mouse and be present in the lung at the time of infection. There are two predominant

strategies. The first strategy is the induction of mucosal immune responses, or spill‐over

of serum antibodies into the mucosal tract after systemic vaccination. This strategy is

employed by both the H. influenza type B and Pneumoccal sp. vaccines: the mucosal IgG

and IgA antibodies, generated after intramuscular injection are critical for protection

against colonization and infection of the upper airways [237,238,325]. The second

strategy is local production of antibodies in the lungs themselves. Inducible bronchus

associated lymphoid tissue (iBALT) is a tertiary lymphoid organ located in the

perivascular and interstitial areas of the lung, capable of supporting germinal centers

[326,327]. Antibody producing cells generated in the lung are long lived, lasting at least

11 months post induction, and produce pulmonary antibodies directly, rather than

relying on sera antibodies [328]. Regardless of the method used, murine production of

high‐titer/high‐avidity surface‐binding antibodies eliminates the need for their

instillation retropharyngeally, improving the physiologic relevancy of the model, and

potentially decreasing its variability.

159

Lastly, mechanistic studies undertaken in Chapter 4 demonstrate that pre‐mixing

of M. tuberculosis and surface‐binding antibodies results in fewer infected cells within

the first day after infection. To confirm that the FCγR independent mechanism, for the

one day CFU reductions in the aerosol model, is due to blocking of bacterial uptake, and

not FCγR‐independent cellular killing, flow cytometry studies quantifying the number

and type of infected cells, in both wild type and FCγR‐/‐ mice, should be undertaken. In

order to visualize infected cells by flow cytometry at early time points, a dose presented

of ~10,000 bacteria is needed. Due to technical limitations with aerosol delivery systems,

we have not been able to achieve this dose. Optimization of the aerobiology, followed by

isolation and staining of lung pneumocytes, will allow us to further probe the

mechanism behind surface‐binding antibody mediated protection from aerosol M.

tuberculosis infection.

160

Appendix A

Table 15: BAL, lung tissue and sera cytokine data for C57BL/6 and BALB/c mice

Tissue BAL Sera

B6

(n=5)

BALB/c

(n=5)

DiffBc/B6

P‐

value

B6

(n=5)

BALB/c

(n=4)

Diff Bc/B6

P‐

value

B6

(n=5)

BALB/c

(n=5)

Diff Bc/B6

P‐

value


IFN‐γ <3.5 <3.5 N/A N/A <1.7 <1.7 N/A N/A <1.6 <1.6 N/A N/A

TNF‐α <3.4 <3.4 N/A N/A <1.7 <1.7 N/A N/A 4.2

(<1.6‐6.4)

3.7

(<1.6‐6.4) 0.88 1.00

IL‐2 <3.3

(<3.3‐7.6) <3.3 N/A 1.00

<1.7

(<1.7‐5.0) <1.7 N/A 0.44 <1.6 <1.6 N/A N/A

IL‐12

(p40)

<3.4

(<3.4‐8.1)

<3.4

(<3.4‐11.8) N/A 1.00 <1.7 <1.7 N/A N/A

4.4

(<1.6‐8.3)

<1.6

(<1.6‐9.1) 0.38 0.69

IL‐12

(p70) <3.9 <3.9 N/A N/A <1.9 <1.9 N/A N/A

6.1

(<1.3‐9.4)

<1.3

(<1.3‐44.3) 0.21 0.83


IL‐4 <3.3 <3.3 N/A N/A <1.6 <1.6 N/A N/A <1.6 <1.6

(<1.6‐4.0) N/A 1.00

IL‐5 <3.4 <3.4 N/A N/A <1.7 <1.7 N/A N/A 6.5

(5.5‐24.1)

5.1

(<1.6‐9.4) 0.78 0.38

IL‐6 <3.7 <3.7 N/A N/A <1.8 <1.8 N/A N/A <1.7 <1.7

(<1.7‐15.6) N/A 0.44

IL‐10 <3.6 <3.6 N/A N/A 7.2

(4.0‐10.2)

5.6

(<1.7‐11.4) 0.78 0.73

<1.5

(<1.5‐3.8)

<1.5

(<1.5‐12.4) N/A 1.00

IL‐13 755.4

(<360.4‐769.6) <360.6 0.48 0.17 <180.3 <180.3 N/A N/A

79.2

(64.4‐138.1)

84.1

(69.4‐152.9) 1.06 0.90

161

Tissue BAL Sera

BAL Sera

B6

(n=5)

BALB/c

(n=5)

DiffBc/B6

P‐

value

B6

(n=5)

BALB/c

(n=4)

Diff Bc/B6

P‐

value

B6

(n=5)

BALB/c

(n=5)

Diff Bc/B6

P‐

value


G‐CSF <12.7 <12.7 N/A N/A <6.4 <6.4 N/A N/A 90.7

(34.6‐172.0)

115.8

(79.4‐176.7) 1.28 0.55

GM‐CSF 35.4

(4.5‐62.4)

28.1

(22.3‐71.08) 0.79 0.93

2.3

(<1.1‐2.3)

14.1

(6.7‐17.7) 6.19 0.008

<7.2

(<7.2‐23.4)

<7.2

(<7.2‐15.6) N/A 0.72

M‐CSF 55.4

(43.6‐69.4)

112.8

(80.9‐171.0) 2.04 0.008 <1.9 <1.9 N/A N/A

10.0

(3.2‐13.0)

6.7

(4.4‐8.9) 0.67 0.54

VEGF 670.6

(574.5‐883.0)

243.1

(208.0‐511.4) 0.36 0.008

7.4

(3.3‐9.8)

3.6

(<1.6‐6.0) 0.49 0.064 <1.6 <1.6 N/A N/A

IL‐1α <340.7 <340.7 N/A N/A <170.4 <170.4 N/A N/A 482.7

(431.4‐1009.8)

616.4

(353.2‐1094.2) 1.28 0.84

IL‐1β <4.1

(<4.1‐9.1)

<4.1

(<4.1‐25.1) N/A 0.44

<2.1

(<2.1‐5.0)

5.8

(<2.1‐8.3) 2.83 0.30

8.7

(5.8‐12.4)

3.9

(2.9‐6.8) 0.45 0.040

IL‐3 <3.3 <3.3 N/A N/A <1.6 <1.6 N/A N/A <1.6 <1.6 N/A N/A

IL‐7 <3.6 <3.6

(<3.6‐24.0) N/A 1.00 <1.8 <1.8 N/A N/A <1.6

<1.6

(1.6‐4.2) N/A 1.00

IL‐9 595.8

(489.3‐1179.8)

393.12

(302.0‐445.9) 0.65 0.008

116.0

(21.2‐175.2)

99.1

(37.4‐373.3) 0.85 1.00

138.6

(<47.1‐789.7)

109.4

(<47.1‐129.4) 0.79 0.46

IL‐15 <15.5

(<15.5‐34.0)

<15.5

(<15.5‐37.6) N/A 1.00 <7.8 <7.8 N/A N/A

<9.1

(<9.1‐25.9)

<9.1

(<9.1‐39.3) N/A 0.84

IL‐17 <3.4 <3.4 N/A N/A <1.7 <1.7 N/A N/A 8.2

(5.2‐12.6)

7.6

(4.2‐15.6) 0.93 1.00

LIF <3.3 <3.3 N/A N/A <1.6 <1.6 N/A N/A <1.6 <1.6

(<1.6‐3.7) N/A 1.00

IP‐10 306.7

(181.9‐419.4)

334.4

(204.6‐404.2) 1.09 0.55

6.8

(<1.7‐9.0)

7.7

(5.7‐14.1) 1.12 0.56

96.9

(80.6‐140.7)

176.7

(139.5‐216.5) 1.83 0.016

162

P‐values were calculated using a Mann‐Whitney test.

Tissue BAL Sera

BAL Sera

B6

(n=5)

BALB/c

(n=5)

DiffBc/B6

P‐

value

B6

(n=5)

BALB/c

(n=4)

Diff Bc/B6

P‐

value

B6

(n=5)

BALB/c

(n=5)

Diff Bc/B6

P‐

value

CCL chemokines

Eotaxin 670.5

(551.3‐1540.5)

879.8

(538.0‐2158.3) 1.31 0.85

<1.6

(<1.6‐6.9)

9.5

(6.2‐23.3) 5.82 0.056

609.5

(323.2‐789.1)

430.2

(410.0‐657.9) 0.71 0.55

MCP‐1 57.3

(54.9‐80.4)

65.3

(51.0‐73.6) 1.13 0.50 <2.0

<2.0

(<2.0‐4.08) N/A N/A

37.5

(26.3‐50.8)

35.7

(24.3‐40.9) 0.95 0.82

MIP‐1α 84.56

(46.3‐118.9)

70.1

(57.5‐93.5) 0.83 1.00

37.5

(24.7‐61.9)

38.7

(31.5‐52.3) 1.03 0.78

25.9

(18.2‐42.5)

24.1

(19.1‐32.5) 0.93 0.38

MIP‐1β 18.5

(17.6‐49.5)

36.2

(22.5‐43.6) 1.95 0.15 <1.6

<1.6

(<1.6‐3.4) N/A 0.44

9.7

(3.8‐27.3)

35.9

(34.3‐50.9) 3.70 0.008

RANTES 27.5

(25.0‐52.9)

89.6

(44.0‐107.5) 3.26 0.016 <1.7 <1.7 N/A N/A <921.7 <921.7 N/A N/A

CXCL chemokines

KC 166.1

(129.7‐191.1)

274.7

(207.7‐388.0) 1.64 0.008

5.9

(4.4‐8.6)

16.4

(10.5‐18.4) 2.76 0.016

75.8

(63.3‐101.1)

36.4

(15.8‐49.4) 0.48 0.008

LIX 38.4

(<17.7‐244.7)

<17.7

(<17.7‐80.6) 0.48 0.52 <8.8 <8.8 N/A N/A Values above range, unreadable

MIG 456.1

(393.5‐665.9)

583.0

(398.6‐811.3) 1.27 0.42 <1.8

5.3

(4.0‐9.9) 3.04 0.008

33.7

(21.5‐49.0)

70.3

(52.8‐151.4) 2.08 0.008

MIP‐2 32.6

(<11.8‐42.5)

24.7

(<11.8‐45.3) 0.75 1.00

<5.9

(<5.9‐12.4)

13.4

(<5.9‐16.3) 2.26 0.13

115.7

(85.7‐139.2)

101.6

(97‐129.7) 0.88 0.42

163

Appendix B

Figure 34: Reactivity of human plasma to protein lysates from M. tuberculosis and

environmental mycobacteria

Plasma from PPD‐negative volunteers (Uninfected) and a randomly selected subset of

patients with active TB disease (Active) were assayed against whole cell protein lysates

from M. tuberculosis H37Rv (A), M. intracellulare (B), M. avium (C), and M. fortuitum (D).

Two‐sided p‐values by student’s t‐test: (*) p ≤ 0.05, (**) p ≤ 0.01, (***) p ≤ 0.001. (E‐G)

Correlations between log10 antibody titers for M tuberculosis and environmental

mycobacteria are displayed for individuals with active disease.

164


LAM, cell wall, and secreted proteins in patients with active TB disease


Variable LAM Cell wall Secreted proteins

R value/

mean ±

SEM

P

value

*

R value/

mean ±

SEM

P

value

R value/

mean ±

SEM

P

value

Age R=‐0.35 0.027 R=‐0.23 0.15 R=‐0.14 0.39

Gender 0.93 0.37 0.55

Male (n=29) 3.2 ± 0.2 3.3 ± 0.1 3.3 ± 0.2

Female (n=11) 3.2 ± 0.2 3.6 ± 0.3 3.1 ± 0.2

Race/ethnicity 0.059 0.48 0.31

White (n=7) 2.9 ± 0.4 3.4 ± 0.3 2.9 ± 0.2

White Hispanic (n=11) 3.8 ± 0.2 3.5 ± 0.1 3.6 ± 0.2

Black (n=17) 3.0 ± 0.2 3.2 ± 0.2 3.2 ± 0.3

Black Hispanic (n=1) 2.3 3.8 2.9

Asian (n=4) 3.2 ± 0.3 3.8 ± 0.4 3.3 ± 0.4

HIV seropositivity 0.10 0.60 0.16

HIV + (n=7) 2.7 ± 0.3 3.5 ± 0.3 2.9 ± 0.2

HIV – (n=33) 3.3 ± 0.3 3.4 ± 0.1 3.3 ± 0.1

Diabetes 0.35 0.70 0.12

Yes (n=6) 3.5 ± 0.5 3.5 ± 0.4 3.7 ± 0.3

No (n=34) 3.0 ± 0.4 3.4 ± 0.1 3.2 ± 0.1

Tobacco usage 0.61 0.67 0.33

Yes(n=11) 3.2 ± 0.2 3.5 ± 0.3 3.0 ± 0.2

No (n=29) 3.1 ± 0.2 3.4 ± 0.1 3.3 ± 0.1

Alcohol consumption (day) 0.62 0.68 0.80

None consumed (n=26) 3.2 ± 0.2 3.4 ± 0.2 3.2 ± 0.1

1‐3 drinks (n=6) 3.0 ± 0.5 3.1 ± 0.2 3.2 ± 0.4

>3 drinks or binge (n=8) 3.4 ± 0.2 3.6 ± 0.2 3.4 ± 0.3

BCG vaccination history 0.057 0.18 0.24

Yes (n=17) 3.0 ± 0.2 3.6 ± 0.1 3.4 ± 0.2

No (n=22) 3.5 ± 0.2 3.2 ± 0.2 3.1 ± 0.2

US‐Born 0.10 0.11 0.32

Yes (n=20) 3.4 ± 0.1 3.2 ± 0.2 3.1 ± 0.2

No (n=20) 3.0 ± 0.2 3.6 ± 0.1 3.4 ± 0.1

PPD induration (mm) R=‐0.19 0.26 R=‐0.06 0.75 R=‐0.13 0.44

Disease Site 0.20 0.43 0.31

Pulmonary (n=22) 3.3 ± 0.2 3.4 ± 0.2 3.4 ± 0.2

Extra‐pulmonary (n=14) 2.9 ± 0.2 3.2 ± 0.2 3.0 ± 0.2

Both (n=4) 3.6 ± 0.3 3.8 ± 0.5 3.5 ± 0.3



165


the live M. tuberculosis surface and whole cell lysate in patients with latent TB

infection



‡Not done. Pairwise comparisons were limited to clinical characteristics with at least

three subjects in each group.



R value/

mean ± SEM

P

value*

R value/

mean ± SEM

P

value

Age R=0.06 0.78 R=0.25 0.24

Gender 0.66 0.12

Male (n=11) 3.0 ± 0.1 2.9 ± 0.1

Female (n=12) 2.9 ± 0.2 2.6 ± 0.1


White (n=8) 2.9 ± 0.1 2.8 ± 0.1

White Hispanic (n=4) 3.1 ± 0.2 2.0 ± 0.4

Black (n=9) 3.0 ± 0.1 3.0 ± 0.1

Asian (n=2) 3.1 3.3

HIV seropositivity ND‡ ND

HIV + (n=2) 3.0 2.9

HIV – (n=19) 3.0 ± 0.1 3.0 ± 0.1

Diabetes ND ND

Yes (n=2) 3.1 3.0

No (n=21) 3.0 ± 0.1 2,7 ± 0.1


Yes(n=7) 3.0 ± 0.2 2.8 ± 0.1

No (n=16) 3.0 ± 0.1 2.7 ± 0.1


None consumed (n=14) 2.9 ± 0.1 2.6 ± 0.1

1‐3 drinks (n=5) 2.9 ± 0.1 2.7 ± 0.1

>3 drinks or binge (n=4) 3.2 ± 0.2 3.0 ± 0.1


Yes (n=9) 3.1 ± 0.2 2.7 ± 0.2

No (n=14) 2.9 ± 0.1 2.7 ± 0.1

US‐Born 0.86 0.76

Yes (n=13) 3.0 ± 0.1 2.7 ± 0.1

No (n=10) 2.9 ± 0.1 2.7 ± 0.2


166

Table 18: Univariate correlation between clinical variable and total antibody titers to

LAM, cell wall, and secreted proteins for patients with latent TB infection


Variables LAM Cell wall Secreted Proteins

R value/

mean ±

SEM

P

value*

R value/

mean ±

SEM

P

value

R value/

mean ±

SEM

P

value

Age R=‐0.22 0.32 0.18 0.42 R=0.27 0.23

Gender 0.89 0.32 0.35

Male (n=11) 2.8 ± 0.2 3.3 ± 0.2 2.9 ± 0.2

Female (n=12) 2.8 ± 0.2 3.7 ± 0.3 2.7 ± 0.2


White (n=8) 2.5 ± 0.2 3.4 ± 0.2 2.7 ± 0.1

White Hispanic (n=4) 3.1 ± 0.2 3.4 ± 0.5 3.0 ± 0.6

Black (n=9) 2.9 ± 0.1 3.7 ± 0.4 2.8 ± 0.1

Asian (n=2) 3.0 3.0 ± 0.4 2.9

HIV seropositivity ND‡ ND ND

HIV + (n=2) 3.0 5.2 ± 0.7 2.5

HIV – (n=19) 2.8 ± 0.1 3.3 ± 0.2 2.9 ± 0.1

Diabetes ND ND ND

Yes (n=2) 3.1 3.0 ± 0.9 2.8

No (n=21) 2.8 ± 0.2 3.5 ± 0.2 2.9 ± 0.1


Yes(n=7) 2.8 ± 0.2 3.8 ± 0.5 2.8 ± 0.2

No (n=16) 2.8 ± 0.2 3.3 ± 0.2 2.8 ± 0.1


None consumed (n=14) 2.8 ± 0.1 3.2 ± 0.2 2.9 ± 0.1

1‐3 drinks(n=5) 2.7 ± 0.4 3.6 ± 0.2 2.8 ± 0.2

>3 drinks/or binge (n=4) 2.9 ± 0.1 4.1 ± 0.8 2.8 ± 0.2


Yes (n=9) 2.9 ± 0.2 3.4 ± 0.3 2.9 ± 0.3

No (n=14) 2.8 ± 0.2 3.5 ± 0.3 2.8 ± 0.1

US‐Born 0.84 0.26 0.96

Yes (n=13) 2.8 ± 0.2 3.7 ± 0.3 2.8 ± 0.3

No (n=10) 2.8 ± 0.2 3.2 ± 0.3 2.8 ± 0.1

PPD induration (mm) R=0.06 0.80 0.07 0.78 R=0.05 0.83





167


antibodies to LAM, cell wall, and secreted proteins in patients with active TB disease


Variable LAM Cell wall Secreted protein

R value/

mean ±

SEM

P

value*

R value/

mean ±

SEM

P

value

R value/

mean ±

SEM

P

value

Age R=‐0.01 0.59 R=0.28 0.085 R=0.04 0.80

Gender 0.36 0.75 0.40

Male (n=29) 5.6 ± 0.1 4.2 ± 0.2 4.5 ± 0.1

Female (n=11) 5.9 ± 0.2 4.1 ± 0.2 4.3 ± 0.3


White (n=7) 6.0 4.2 ± 0.3 4.3 ± 0.1

White Hispanic (n=11) 5.4 ± 0.3 4.0 ± 0.2 4.4 ± 0.1

Black (n=17) 5.6 ± 0.2 4.2 ± 0.3 4.6 ± 0.2

Black Hispanic (n=1) 6.0 4.4 4.5

Asian (n=4) 6.0 4.1 ± 0.4 4.3 ± 0.3

HIV seropositivity 0.57 0.70 0.62

HIV + (n=7) 5.8 ± 0.2 4.0 ± 0.4 4.3 ± 0.1

HIV – (n=33) 5.6 ± 0.1 4.2 ± 0.2 4.5 ± 0.1

Diabetes 0.18 0.22 0.90

Yes (n=6) 5.8 ± 0.1 4.6 ± 0.4 4.5 ± 0.2

No (n=34) 5.4 ± 0.3 4.1 ± 0.2 4.4 ± 0.1


Yes(n=11) 5.6 ± 0.3 4.4 ± 0.4 4.4 ± 0.2

No (n=29) 5.7 ± 0.1 4.1 ± 0.1 4.5 ± 0.1


None consumed (n=26) 5.7 ± 0.1 4.1 ± 0.2 4.4 ± 0.1

1‐3 drinks (n=6) 6.0 4.1 ± 0.5 4.4 ± 0.2

>3 drinks or binge (n=8) 5.3 ± 0.4 4.4 ± 0.4 4.5 ± 0.1


Yes (n=17) 5.6 ± 0.2 4.2 ± 0.2 4.4 ± 0.1

No (n=22) 5.7 ± 0.1 4.2 ± 0.2 4.5 ± 0.1

US‐Born 0.84 0.79 0.50

Yes (n=20) 5.7 ± 0.1 4.2 ± 0.2 4.5 ± 0.2

No (n=20) 5.7 ± 0.2 4.1 ± 0.2 4.4 ± 0.1

PPD induration (mm) R=‐0.004 0.98 R=0.18 0.30 R=‐0.18 0.30

Disease Site 0.55 0.21 0.75

Pulmonary (n=22) 5.7 ± 0.2 4.0 ± 0.2 4.4 ± 0.1

Extra‐pulmonary (n=14) 5.6 ± 0.2 4.2 ± 0.2 4.4 ± 0.2

Both (n=4) 6.0 4.9 ± 0.2 4.7 ± 0.2



168


antibodies to the live M. tuberculosis surface and to whole cell lysate in patients with

latent TB infection



R value/

mean ± SEM

P

value*

R value/

mean ± SEM

P value

Age R=0.33 0.12 R=0.10 0.64

Gender 0.49 0.060

Male (n=11) 2.9 ± 0.2 3.2 ± 0.5

Female (n=12) 2.6 ± 0.3 2.8 ± 0.5


White (n=8) 2.8 ± 0.4 2.5 ± 0.5

White Hispanic (n=4) 2.0 ± 0.5 2.6 ± 0.7

Black (n=9) 2.9 ± 0.3 3.0 ± 0.6

Asian (n=2) 3.0 ± 0.8 6.0

HIV seropositivity ND‡ ND

HIV + (n=2) 3.4 ± 0.8 3.2 ± 2.2

HIV – (n=19) 2.6 ± 0.2 3.0 ± 0.4

Diabetes ND ND

Yes (n=2) 2.5 ± 0.8 2.9 ± 0.1

No (n=21) 2.7 ± 0.2 3.0 ± 0.4


Yes(n=7) 3.2 ± 0.2 2.8 ± 0.7

No (n=16) 2.5 ± 0.3 3.0 ± 0.4

Alcohol consumption (day) 0.045 0.56

None consumed (n=14) 2.3 ± 0.2 2.6 ± 0.5

1‐3 drinks (n=5) 3.4 ± 0.5 3.5 ± 0.2

>3 drinks or binge (n=4) 3.2 ± 0.3 3.4 ± 0.9


Yes (n=9) 2.5 ± 0.3 2.5 ± 0.6

No (n=14) 2.8 ± 0.3 3.3 ± 0.5

US‐Born 0.033 0.26

Yes (n=13) 3.0 ± 0.3 3.4 ± 0.5

No (n=10) 2.2 ± 0.3 2.5 ± 0.5

PPD induration (mm) R=0.004 0.99 R=0.14 0.56





169

Table 21: Univariate correlation between clinical factors and relative IgG avidity of

antibodies to LAM, secreted proteins, and cell wall in patients with latent TB

infection






Variable LAM Cell wall Secreted proteins

R value/

mean ±

SEM

P

value

*

R value/

mean ±

SEM

P

value

R value/

mean ±

SEM

P

value

Age R=‐0.33 0.13 R=0.18 0.42 R=0.35 0.10

Gender 0.81 0.32 0.31

Male (n=11) 4.2 ± 0.3 3.3 ± 0.2 4.1 ± 0.2

Female (n=12) 4.1 ± 0.2 3.7 ± 0.3 4.4 ± 0.3


White (n=8) 4.0 ± 0.4 3.4 ± 0.2 4.3 ± 0.3

White Hispanic (n=4) 4.3 ± 0.6 3.4 ± 0.5 3.8 ± 0.5

Black (n=9) 4.1 ± 0.3 3.7 ± 0.4 4.5 ± 0.3

Asian (n=2) 4.8 ± 0.1 3.0 ± 0.4 3.7 ± 0.3

HIV seropositivity ND‡ ND ND

HIV + (n=2) 4.8 ± 0.2 5.2 ± 0.7 5.3 ± 1.1

HIV – (n=19) 4.0 ± 0.2 3.3 ± 0.2 4.2 ± 0.2

Diabetes ND ND ND

Yes (n=2) 3.9 ± 1.1 3.0 ± 0.9 4.0 ± 0.0

No (n=21) 4.2 ± 0.2 3.5 ± 0.2 4.3 ± 0.2


Yes(n=7) 4.4 ± 0.4 3.8 ± 0.5 4.4 ± 0.2

No (n=16) 4.1 ± 0.2 3.3 ± 0.2 4.2 ± 0.2

Alcohol consumption days 0.61 0.24 0.11

None consumed (n=14) 4.3 ± 0.2 3.2 ± 0.2 4.0 ± 0.2

1‐3 drinks (n=5) 3.8 ± 0.5 3.6 ± 0.2 4.7 ± 0.3

>3 drinks or binge (n=4) 4.0 ± 0.5 4.1 ± 0.8 4.7 ± 0.4


Yes (n=9) 4.2 ± 0.3 3.4 ± 0.3 4.2 ± 0.3

No (n=14) 4.1 ± 0.3 3.5 ± 0.3 4.3 ± 0.2

US‐Born 0.95 0.26 0.32

Yes (n=13) 4.1 ± 0.3 3.7 ± 0.3 4.4 ± 0.2

No (n=10) 4.1 ± 0.3 3.2 ± 0.3 4.1 ± 0.3

PPD induration (mm) R=0.05 0.85 R=0.07 0.78 R=0.15 0.55

170


Quantiferon‐Gold peptide stimulation and total antibody titers

Latent TB infection (n=23) Active TB Disease (n=10)

M. tuberculosis

surface

Whole cell

lysate

M. tuberculosis

surface

Whole cell

lysate

R* P‐

value‡ R

P‐

value R

P‐

value R

P‐

value


IL‐2 ‐0.02 0.48 ‐0.20 0.37 0.08 0.83 ‐0.17 0.64

IL‐12p40/p70 0.004 0.78 ‐0.22 0.32 ‐0.06 0.86 ‐0.55 0.10

IFN‐γ ‐0.01 0.68 ‐0.13 0.54 ‐0.12 0.74 ‐0.55 0.10

TNF‐α ‐0.01 0.62 ‐0.31 0.15 0.33 0.35 0.18 0.62


IL‐4 ‐0.02 0.50 ‐0.10 0.65 0.26 0.47 0.12 0.75

IL‐5 0.01 0.73 ‐0.20 0.36 0.35 0.32 0.09 0.80

IL‐6 0.000 0.92 ‐0.34 0.11 0.52 0.12 0.40 0.26

IL‐10 ‐0.04 0.37 ‐0.11 0.61 0.28 0.43 0.01 0.99

IL‐13 ‐0.002 0.85 ‐0.03 0.87 0.00 0.99 ‐0.32 0.37


IL‐1β 0.002 0.84 ‐0.34 0.11 0.70 0.02 0.69 0.03

IL‐1Rα ‐0.02 0.56 ‐0.16 0.45 0.31 0.39 ‐0.06 0.87

IL‐2R ‐0.005 0.76 ‐0.20 0.37 0.03 0.93 0.09 0.80

IL‐7 0.000 0.95 ‐0.08 0.74 0.10 0.79 ‐0.15 0.67

IL‐8 ‐0.16 0.06 ‐0.30 0.17 0.45 0.20 0.00 1.00

IL‐15 ‐0.01 0.72 ‐0.01 0.96 ‐0.37 0.30 ‐0.44 0.21

IL‐17 ‐0.004 0.77 ‐0.14 0.52 0.42 0.22 0.00 1.00

IFN‐α ‐0.02 0.47 ‐0.09 0.68 0.48 0.16 0.11 0.76

GM‐CSF ‐0.001 0.87 ‐0.26 0.23 0.58 0.08 0.15 0.69

Chemokines

MIP‐1α ‐0.01 0.67 ‐0.23 0.30 0.51 0.14 0.29 0.41

MIP‐1β ‐0.06 0.26 ‐0.16 0.47 0.47 0.18 0.07 0.85

IP‐10 ‐0.03 0.42 ‐0.14 0.51 0.08 0.82 ‐0.22 0.54

MIG ‐0.03 0.44 ‐0.04 0.87 0.24 0.51 ‐0.27 0.44

Eotaxin 0.02 0.52 ‐0.20 0.36 0.07 0.85 0.34 0.33

RANTES ‐0.13 0.09 ‐0.31 0.15 ‐0.47 0.17 ‐0.25 0.49

MCP‐1 0.02 0.48 ‐0.06 0.77 0.57 0.09 0.07 0.84

* R values determined by linear regression of the log‐transformed antibody titers plotted

against log‐transformed cytokine concentrations

‡ P‐values determined by F‐statistic, and are not adjusted for multiple comparisons.

171


Quantiferon‐Gold peptide stimulation and relative IgG avidity

Latent TB infection (n=23) Active TB Disease (n=10)

M. tuberculosis

surface

Whole cell

lysate

M. tuberculosis

surface

Whole cell

lysate

R* P‐

value‡ R

P‐

value R

P‐

value R

P‐

value


IL‐2 0.13 0.57 0.13 0.56 0.32 0.36 0.39 0.27

IL‐12p40/p70 ‐0.25 0.24 ‐0.07 0.75 0.45 0.19 0.10 0.79

IFN‐γ ‐0.29 0.17 ‐0.24 0.28 0.73 0.02 0.38 0.28

TNF‐α 0.22 0.31 ‐0.04 0.84 0.49 0.15 0.35 0.33


IL‐4 0.06 0.77 ‐0.04 0.87 0.31 0.38 0.01 0.98

IL‐5 0.12 0.58 0.29 0.17 0.22 0.55 ‐0.09 0.80

IL‐6 ‐0.33 0.12 ‐0.23 0.29 0.65 0.04 0.03 0.94

IL‐10 0.00 1.00 ‐0.12 0.59 0.40 0.26 0.77 0.01

IL‐13 0.19 0.40 ‐0.09 0.67 0.56 0.09 0.65 0.04


IL‐1β ‐0.34 0.12 ‐0.12 0.58 0.32 0.37 0.50 0.14

IL‐1Rα ‐0.19 0.40 ‐0.02 0.95 0.39 0.27 0.13 0.72

IL‐2R ‐0.32 0.14 ‐0.20 0.36 ‐0.35 0.32 ‐0.07 0.85

IL‐7 ‐0.02 0.93 0.32 0.17 0.50 0.14 0.13 0.73

IL‐8 0.15 0.50 0.06 0.78 0.89 0.001 0.13 0.73

IL‐15 ‐0.14 0.51 0.20 0.37 ‐0.07 0.85 ‐0.49 0.15

IL‐17 ‐0.02 0.93 0.39 0.07 0.32 0.37 0.06 0.86

IFN‐α 0.01 0.98 ‐0.07 0.76 0.34 0.34 ‐0.35 0.33

GM‐CSF 0.40 0.06 0.61 0.00 0.54 0.10 0.06 0.87

Chemokines

MIP‐1α ‐0.27 0.21 ‐0.04 0.86 0.77 0.01 0.49 0.16

MIP‐1β ‐0.10 0.65 ‐0.01 0.96 0.85 0.002 0.44 0.20

IP‐10 ‐0.06 0.78 0.09 0.68 0.27 0.45 0.09 0.81

MIG 0.19 0.38 ‐0.05 0.82 0.64 0.05 0.53 0.12

Eotaxin ‐0.04 0.86 0.27 0.21 ‐0.55 0.10 ‐0.71 0.02

RANTES 0.05 0.83 0.06 0.79 ‐0.27 0.44 ‐0.71 0.02

MCP‐1 0.08 0.73 ‐0.02 0.93 0.67 0.03 0.04 0.91

* R values determined by linear regression of the log‐transformed antibody titers plotted

against log‐transformed cytokine concentrations

‡ P‐values determined by F‐statistic, and are not adjusted for multiple comparisons.

172

Appendix C

Table 24: Baseline characteristics and infection status for mice receiving ultra‐low

dose aerosol exposure

Exposure Group

Mouse ID

Treatment Age (days)

Weight(grams)

Lung Burden

Log10 CFU*

Culture Positive

Infected

A 1 PBS 44 17.3 <0.61 N N

A 2 PBS 44 21.4 <0.61 N N

A 3 PBS 44 17.1 4.56 Y Y

A 4 PBS 44 20.4 <0.61 N N

A 5 6D11 44 18.6 <0.61 Y Y

A 6 6D11 44 17.9 4.57 Y Y

A 7 6D11 44 15.6 <0.61 Y Y

A 8 6D11 44 19.5 4.48 Y Y

A 9 SB Pool C 44 18.2 <0.61 N N

A 10 SB Pool C 44 16.5 <0.61 N N

A 11 SB Pool C 44 16.9 <0.61 N N

A 12 SB Pool C 44 17.5 <0.61 N N

A 13 PBS 44 16.4 <0.61 N N

A 14 PBS 44 16.3 <0.61 N N

A 15 PBS 44 17.6 <0.61 N N

A 16 PBS 44 17 2.51 Y Y

A 17 6D11 44 19.4 <0.61 N N

A 18 6D11 44 17.4 <0.61 N N

A 19 6D11 44 17.1 <0.61 N N

A 20 6D11 44 18.5 <0.61 N N

A 21 SB Pool C 44 16.7 <0.61 N N

A 22 SB Pool C 44 16.9 4.13 Y Y

A 23 SB Pool C 44 15.6 <0.61 N N

A 24 SB Pool C 44 17.7 <0.61 N N

A 25 PBS 44 18.3 <0.61 N N

A 26 PBS 44 16 <0.61 N N

A 27 PBS 44 17 4.34 Y Y

A 28 PBS 44 18.4 4.30 CONT Y

A 29 6D11 44 18.6 <0.61 N N

A 30 6D11 44 17.2 <0.61 N N

A 31 6D11 44 16.5 3.96 Y Y

A 32 6D11 44 16.3 <0.61 Y Y

A 33 SB Pool C 44 18.3 <0.61 N N

A 34 SB Pool C 44 18.8 <0.61 N N

173

Exposure Group

Mouse ID


Weight(grams)

Lung Burden

Log10 CFU*

Culture Positive

Infected

A 35 SB Pool C 44 17.3 <0.61 Y Y

A 36 SB Pool C 44 17 <0.61 Y Y

A 37 PBS 44 20.1 4.34 Y Y

A 38 PBS 44 17.6 <0.61 N N

A 39 PBS 44 17.6 <0.61 N N

A 40 PBS 44 18.4 4.94 Y Y

A 41 6D11 44 17.3 1.08 Y Y

A 42 6D11 44 15.5 2.83 Y Y

A 43 6D11 44 17.3 4.21 Y Y

A 44 6D11 44 17.5 3.99 Y Y

A 45 SB Pool C 44 17.8 4.06 Y Y

A 46 SB Pool C 44 16 <0.61 N N

A 47 SB Pool C 44 18.2 <0.61 N N

A 48 SB Pool C 44 19 <0.61 N N

A 49 PBS 44 15.5 4.93 Y Y

A 50 PBS 44 16.8 4.88 Y Y

A 51 PBS 44 18.2 <0.61 N N

A 52 PBS 44 20.4 <0.61 N N

A 53 6D11 44 16.1 <0.61 N N

A 54 6D11 44 17.3 <0.61 N N

A 55 6D11 44 18 <0.61 N N

A 56 6D11 44 17.3 <0.61 N N

A 57 SB Pool C 44 19.7 <0.61 N N

A 58 SB Pool C 44 15.8 <0.61 N N

A 59 SB Pool C 44 18.2 <0.61 N N

A 60 SB Pool C 44 17.1 5.10 Y Y

A 61 PBS 44 16.9 <0.61 N N

A 62 PBS 44 18.3 3.97 Y Y

A 63 PBS 44 18.1 <0.61 N N

A 64 PBS 44 14.8 <0.61 N N

A 65 6D11 44 18.6 <0.61 N N

A 66 6D11 44 15.4 <0.61 N N

A 67 6D11 44 19.5 <0.61 N N

A 68 6D11 44 16.5 <0.61 N N

A 69 SB Pool C 44 15.4 4.02 Y Y

A 70 SB Pool C 44 17.2 <0.61 N N

A 71 SB Pool C 44 15.6 <0.61 N N

A 72 SB Pool C 44 17.3 4.30 Y Y

B 73 6D11 43 17.9 <0.61 N N

B 74 6D11 43 16.6 <0.61 N N

174

Exposure Group

Mouse ID


Weight(grams)

Lung Burden

Log10 CFU*

Culture Positive

Infected

B 75 6D11 43 17.6 4.26 Y Y

B 76 PBS 43 15.6 4.97 Y Y

B 77 PBS 43 16.0 <0.61 N N

B 78 PBS 43 17.0 5.30 Y Y

B 79 PBS 43 18.1 4.96 Y Y

B 80 SB Pool C 43 18.6 4.44 Y Y

B 81 SB Pool C 43 14.7 <0.61 N N

B 82 SB Pool C 43 15.4 3.91 CONT Y

B 83 SB Pool C 43 16.4 <0.61 N N

B 84 PBS 43 16.3 4.33 Y Y

B 85 PBS 43 16.0 4.52 Y Y

B 86 PBS 43 16.2 4.94 Y Y

B 87 PBS 43 17.6 4.20 Y Y

B 88 6D11 43 18.3 4.10 Y Y

B 89 6D11 43 17.0 4.97 Y Y

B 90 6D11 43 15.1 <0.61 CONT N

B 91 6D11 43 15.9 <0.61 CONt N

B 92 SB Pool C 43 16.0 4.91 CONT Y

B 93 SB Pool C 43 16.7 2.41 Y Y

B 94 SB Pool C 43 15.8 <0.61 N N

B 95 SB Pool C 43 15.8 <0.61 N N

B 96 PBS 43 18.0 4.60 Y Y

B 97 PBS 43 18.2 4.90 Y Y

B 98 PBS 43 14.8 3.94 Y Y

B 99 PBS 43 18.3 <0.61 N N

B 100 6D11 43 19.5 4.63 Y Y

B 101 6D11 43 16.9 3.72 Y Y

B 102 6D11 43 17.7 4.51 Y Y

B 103 6D11 43 15.5 <0.61 Y Y

B 104 SB Pool C 43 16.0 4.79 Y Y

B 105 SB Pool C 43 16.5 4.00 Y Y

B 106 SB Pool C 43 17.1 4.19 Y Y

B 107 SB Pool C 43 15.5 4.79 Y Y

B 108 PBS 43 16.1 4.35 Y Y

B 109 PBS 43 17.5 4.49 Y Y

B 110 PBS 43 17.3 5.21 Y Y

B 111 PBS 43 17.7 5.24 Y Y

B 112 6D11 43 15.5 4.33 Y Y

B 113 6D11 43 16.2 5.04 Y Y

B 114 6D11 43 16.3 2.26 Y Y

175

Exposure Group

Mouse ID


Weight(grams)

Lung Burden

Log10 CFU*

Culture Positive

Infected

B 115 6D11 43 15.4 3.76 Y Y

B 116 SB Pool C 43 16.1 <0.61 N N

B 117 SB Pool C 43 16.6 <0.61 CONT N

B 118 SB Pool C 43 17.7 4.27 Y Y

B 119 SB Pool C 43 18.4 <0.61 N N

B 120 PBS 43 15.3 5.32 Y Y

B 121 PBS 43 16.7 5.00 Y Y

B 122 PBS 43 14.8 4.81 Y Y

B 123 PBS 43 18.2 4.46 Y Y

B 124 6D11 43 16.6 4.62 Y Y

B 125 6D11 43 15.0 4.59 Y Y

B 126 6D11 43 17.5 4.30 Y Y

B 127 SB Pool C 43 14.5 <0.61 N N

B 128 SB Pool C 43 17.2 4.92 Y Y

B 129 SB Pool C 43 15.2 4.23 Y Y

B 130 SB Pool C 43 15.9 4.87 Y Y

B 131 PBS 43 17.6 5.25 CONT Y

B 132 PBS 43 20.5 <0.61 CONT N

B 133 PBS 43 18.7 <0.61 CONT N

B 134 PBS 43 15.7 <0.61 N N

B 135 6D11 43 14.1 <0.61 CONT N

B 136 6D11 43 17.0 4.93 Y Y

B 137 6D11 43 18.2 3.70 Y Y

B 138 6D11 43 16.5 4.23 CONT Y

B 139 SB Pool C 43 16.3 4.11 Y Y

B 140 SB Pool C 43 17.1 <0.61 N N

B 141 SB Pool C 43 15.8 2.21 Y Y

B 142 SB Pool C 43 16.2 4.41 Y Y

C 143 PBS 43 15.8 <0.61 N N

C 144 PBS 43 16.3 4.11 Y Y

C 145 PBS 43 15.2 4.00 Y Y

C 146 PBS 43 18.0 <0.61 N N

C 147 SB Pool C 43 18.3 4.85 Y Y

C 148 SB Pool C 43 15.4 2.83 Y Y

C 149 SB Pool C 43 16.6 4.07 Y Y

C 150 SB Pool C 43 15.7 3.84 Y Y

C 151 PBS 43 14.7 <0.61 Y Y

C 152 PBS 43 17.5 <0.61 N N

C 153 PBS 43 15.9 4.35 Y Y

C 154 PBS 43 16.2 <0.61 N N

176

For mice with no colonies on titer plates, Log10 CFU are marked as <0.61 log10 CFU, the limit of

detection for this study.

Exposure Group

Mouse ID


Weight(grams)

Lung Burden

Log10 CFU*

Culture Positive

Infected

C 155 SB Pool C 43 15.2 4.25 Y Y

C 156 SB Pool C 43 16.3 2.19 Y Y

C 157 SB Pool C 43 16.0 <0.61 CONT N

C 158 SB Pool C 43 15.4 3.04 Y Y

C 159 PBS 43 19.0 4.94 CONT Y

C 160 PBS 43 15.4 4.40 Y Y

C 161 PBS 43 15.9 4.48 Y Y

C 162 PBS 43 18.4 5.03 Y Y

C 163 SB Pool C 43 17.2 2.67 CONT Y

C 164 SB Pool C 43 17.5 <0.61 N N

C 165 SB Pool C 43 16.9 4.46 Y Y

C 166 SB Pool C 43 15.0 4.14 Y Y

C 167 PBS 43 16.0 5.08 Y Y

C 168 PBS 43 16.9 4.33 Y Y

C 169 PBS 43 14.8 4.96 Y Y

C 170 PBS 43 19.2 <0.61 Y Y

C 171 SB Pool C 43 14.5 4.67 CONT Y

C 172 SB Pool C 43 17.4 2.80 CONT Y

C 173 SB Pool C 43 17.0 4.88 CONT Y

C 174 SB Pool C 43 15.8 3.24 Y Y

C 175 PBS 43 15.6 4.52 CONT Y

C 176 PBS 43 16.0 2.92 CONT Y

C 177 PBS 43 16.0 4.82 CONT Y

C 178 PBS 43 14.5 <0.61 N N

C 179 SB Pool C 43 16.0 <0.61 N N

C 180 SB Pool C 43 18.4 <0.61 N N

C 181 SB Pool C 43 15.0 2.56 CONT Y

C 182 SB Pool C 43 16.2 3.89 CONT Y

C 183 PBS 43 16.4 3.89 Y Y

C 184 PBS 43 15.5 <0.61 N N

C 185 PBS 43 14.0 4.76 Y Y

C 186 PBS 43 16.3 4.10 Y Y

C 187 SB Pool C 43 16.6 4.50 Y Y

C 188 SB Pool C 43 16.1 <0.61 N N

C 189 SB Pool C 43 17.6 4.54 Y Y

C 190 SB Pool C 43 14.7 4.35 Y Y

177

Table 25: Baseline characteristics of each exposure group in infection rate studies

Group

(number)

Mean age

(days)

Mean weight

(grams) +/‐ SD

P‐value for

(weight)*

Group 1 (72) 44 17.5 ± 1.3

PBS (24) 44 17.7 ± 1.6 0.48

6D11 (24) 44 17.5 ± 1.2

SB Pool C (24) 44 17.3 ± 1.1

Group 2 (70) 43 16.7 ± 1.3

PBS (24) 43 17.0 ± 1.4 0.16

6D11 (22) 43 16.7 ± 1.3

SB Pool C (24) 43 16.3 ± 1.0

Group 3 (71) 43 16.3 ± 1.2

PBS (24) 43 16.2 ± 1.4 0.96

SB Pool C (24) 43 16.3 ± 1.1

Total (213) 43 ± 0.3 16.8 ± 1.4

PBS (72) 43 ± 0.3 17.0 ± 1.6 0.85

SB Pool C (72) 43 ± 0.3 16.6 ± 1.2

* P‐value was calculated using a one‐way ANOVA for groups with three variables and a student’s t‐test for groups with two variables

178

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Biography

Casey Crowell Perley was born in Buffalo, NY on April 30, 1986. She lived in the

small town of Boston, NY, twenty‐five miles south of Buffalo until the age of eighteen.

She matriculated from the Buffalo Seminary, an all girl’s high school, in 2000, graduating

cum laude. In 2008, Casey graduated from Yale University with a B.S in Molecular

Biophysics and Biochemistry. While at Yale, she spent two years as an undergraduate

researcher in the Lombroso Lab, receiving the Dean’s Office Fellowship to support her

research in the summer 2007. Her work, on the localization and function of Major Vault

Protein in the rodent brain, resulted in a second author paper; Major Vault Protein is

Expressed along the Nucleus‐Neurite Axis and Associates with mRNAs in Cortical

Neurons, published in Cerebral Cortex in 2008. Her fluorescent microscopy image of a

cortical neuron was selected for the journal cover.

After graduation Casey spent a year as a research technician in the Lindquist Lab

at the Whitehead Institute of MIT. Her work on understanding the role of heat‐shock

proteins in cancer, led to co‐authorship on two papers. Using the Heat‐Shock Response

to Discover Anticancer Compounds that Target Protein Homeostasis, was published in

ACS Chemical Biology in 2012, and Tight coordination of protein translation and heat

shock factor 1 activation supports the anabolic malignant state, was published in

Science in 2013.

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In 2009 Casey moved to Durham, NC, and entered the PhD program in

Molecular Genetics and Microbiology at Duke University. She received both Duke

University’s Chancellor’s Fellowship, and the National Science Foundation Graduate

Research Fellowship to support her graduate work. She was also named a Duke Scholar

in Infectious Disease in 2012.

In 2010 Casey joined the Frothingham Lab where she has studied the role M.

tuberculosis surface‐binding antibodies play in altering early infection events. These

studies have led to three published papers. Ultra‐low dose of aerosolized Mycobacterium

tuberculosis creates partial infection in mice, was published in Tuberculosis in 2012.

Human antibody response to the surface of Mycobacterium tuberculosis, a first‐author

paper, was published in PLoS One in 2014, and Rhesus immune responses to SIV

Gag expressed by recombinant BCG vectors are independent from pre‐existing

mycobacterial immunity, a second‐author paper, is current in press at Vaccine. She

has three more first/co‐first author papers that are in preparation.

Outside of lab, Casey is an avid harpist and regularly performs with symphony

orchestras in the Raleigh‐Durham area.

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