The Lung Mucosa and Its Impact on Mycobacterium Tuberculosis Pathogenesis and Bacillus Calmette-Guérin Vaccine Efficacy

The Lung Mucosa and its Impact on Mycobacterium tuberculosis Pathogenesis and Bacillus Calmette-Guérin Vaccine Efficacy

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Juan Ignacio Moliva

Biomedical Sciences Graduate Program

The Ohio State University

2017

Dissertation Committee:

Jordi B. Torrelles, PhD, Advisor

Stefan Niewiesk, DVM, PhD

Susheela Tridandapani, PhD

Daniel J. Wozniak, PhD

Juan Ignacio Moliva

2017

Abstract

Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis (TB), is the current leading cause of death due to a single infectious organism. Although curable, the broad emergence of drug resistant M.tb strains has hindered eradication efforts.

Furthermore, computational models predict a quarter of the world’s population is infected with M.tb in a latent state, effectively serving as the largest reservoir of any human pathogen with the ability to cause significant global morbidity and mortality.

Mycobacterium bovis Bacillus Calmette-Guérin (BCG) is the only vaccine approved for use to prevent TB. In humans, BCG is highly efficacious against disseminated forms of

TB, but fails to fully protect against the development of pulmonary TB. Thus, new vaccination strategies are urgently needed if we are to eradicate this pathogen. The World

Health Organization has prioritized research and development of novel TB vaccines, however our incomplete understanding on the requirements for protective immunity to

M.tb has made it difficult to develop new successful vaccines. In this collective work, we explore how manipulation of the mycobacterial cell wall can further advance our knowledge of M.tb pathogenesis and the development of effective protective immunity.

The microenvironment of the lung has emerged as an important contributor to antimicrobial immunity, especially in the context of M.tb. Homeostatic components within the alveolar lining fluid (ALF) of the lung mucosa have been shown to play a ii

significant role in the pathogenesis of M.tb via modulation of host immunity. Previous studies demonstrated that exposure of M.tb to human ALF can modify the M.tb cell wall,

stripping virulent lipids, glycolipids, and lipoglycans used by M.tb to subvert host

immunity. This interaction renders M.tb more susceptible to killing by host immune cells.

We demonstrate that human ALF can affect the development of mycobacterial-specific

immunity by BCG in the lung, increasing its efficacy against M.tb pathogenesis and

virulence. These results led us to hypothesize that effective pulmonary immunity against

M.tb brought forth by BCG can be improved by taking into consideration the influence of

cell wall lipids, glycolipids, and lipoglycans on the development of immunological

responses. We uncovered that chemical treatment of BCG with the aliphatic hydrocarbon

solvent petroleum ether accomplishes similar effects as human ALF; it reduces the

presence of virulent lipids and glycolipids (but not lipoglycans) from the cell wall of

BCG with high reproducibility. Pulmonary vaccination with ‘delipidated’ BCG was

superior to conventional BCG against M.tb morbidity in mice. Lastly, we evaluated

whether the co-morbidity of aging had any effects on human ALF physiology and if ‘age-

related’ changes to ALF could impact M.tb pathogenesis. We show that increases in age are associated with decreases in the innate antimicrobial properties of ALF, and that M.tb

may benefit from the declining host defense functions of the human lung mucosa in the

elderly. Overall, this collective work expands upon our understanding on how the human

lung mucosa can have a significant impact on the pathogenesis of M.tb, and how

understanding the mycobacterial cell wall can lead to new efficacious vaccination

strategies.

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Dedication

To all my teachers, counselors, and mentors who have been part of my scientific career.

To my family who have shaped me into the person I am. And to Becky for always

standing by my side. Thank you.

Acknowledgments

I would first like to extend my most sincere and grateful appreciation to my advisor, Dr.

Jordi B. Torrelles for dedicating countless hours helping me design experiments, review

documents, and perform experiments. Thank you for allowing me to be part of your

laboratory. Thank you for allowing me to develop and pursue my own ideas. And thank

you for always supporting me through my education. Your help was instrumental

throughout my PhD.

I would also like extend my most profound gratitude to Dr. Joanne Turner for being the

best collaborator/ad-hoc mentor anyone could ask for. Thank you for taking the time and

patience to teach me all of the mouse techniques. Thank you for the continued advice,

guidance, and support throughout the past five years which enabled my success as a

graduate student.

A big thank you to current and former lab members: Jesús Arcos, Smitha Sasindran, MS,

Poornima Venigalla, Sayeed Silwani, Cynthia Canan, PhD, Varun Dwivedi, PhD, Julia

Scordo, Youngmin Lee, Holden Kelley, Nicole Reinhold-Larson, Michael Duncan,

Colwyn Headley, Russell Ault, Tucker Piergallini, and Shalini Gautam, PhD. You are all terrific individuals and it was the opportunity of a lifetime to get to work with you. I wish you only the best of luck in your future endeavors! v

A big thank you to the BSL3 program and staff, Lena Lynch and Austin Hossfeld. I could

not have accomplished any of this without your continued support. Also a big thank you

to The Ohio State University’s laboratory animal resource, the comparative pathology and mouse phenotyping shared resource, the campus chemical instrument center, and the campus microscopy and imaging facility.

A heartfelt thank you to my committee members, Daren Knoell (former), PharmD, Stefan

Niewiesk, DVM, PhD, Susheela Tridandapani, PhD, and Daniel Wozniak, PhD for

dedicating the time to support and guide me throughout my PhD.

A big thank you to all of our collaborators: Joan-Miquel Balada-Llasat, PharmD, PhD,

D(ABMM), Gillian Beamer, VMD, PhD, DACVP, Evelyn Guirado, PhD, William

Lafuse, PhD, Robert Merrit, MD, Xueliang Pan, PhD, Murugesan Rajaram, PhD, Patrick

Ross Jr., MD, Larry Schlesinger, MD, Arpad Symomogyi, PhD, Shu-Hua Wang, MD,

MPH&TM, Mark Wewers, MD, and Liwen Zhang, PhD. I would also like to thank all the

individuals within the Department of Microbial Infection and Immunity and the Center

for Microbial Interface Biology for advice, guidance, support, and contributions that have

helped make me a better scientist.

Lastly, but most certainly not least, thank you Becky for your unconditional support over the past five years. This journey would not have been the same without you.

Vita

November 1989 ...... Born, Río Cuarto, Córdoba, Argentina

2004-2008 ...... Diploma, William Mason High School

2008-2012 ...... B.S. Biochemistry and Molecular Biology, ...... The Pennsylvania State University

2012-2017 ...... Graduate Research Associate, Department ...... of Microbial Infection and Immunity, The Ohio State University

Publications

Moliva, J.I.*, M.V.S. Rajaram*, S. Sidiki, S.J. Sasindran, E. Guirado, S.H. Wang, P. Ross Jr., L. Lafuse, L.S. Schlesinger, J. Turner, and J.B. Torrelles (2014). “Molecular Composition of the Alveolar Lining Fluid in the Aging Lung.” AGE, 36(3): 1187-1199. Arcos, J., L.E. Diangelo, J.M. Scordo, S.J. Sasindran, J.I. Moliva, J. Turner, and J.B. Torrelles (2015). “Lung mucosa lining fluid modifies Mycobacterium tuberculosis to reprogram human neutrophil killing mechanisms.” J Infect Dis, 212(6): 948-958. Moliva, J.I., J. Turner, and J.B. Torrelles (2015). “Prospects in Mycobacterium bovis Bacille Calmette et Guérin (BCG) Vaccine Diversity and Delivery: Why does BCG fail to protect against Mycobacterium tuberculosis Infection?” Vaccine, 33(39): 5035- 5041.

Arcos, J., S.J. Sasindran, J.I. Moliva*, J.M. Scordo*, S. Sidiki*, H. Guo, P. Venigalla, H. Kelley, G. Lin, L.E. Diangelo, N.S. Silwani, J. Zhang, J. Turner, and J.B. Torrelles (2016). “Mycobacterium tuberculosis Cell Wall released Fragments by the Action of the Human Lung Mucosa modulate Macrophages to Control the Infection in a partially IL-10 Dependent Manner.” Mucosal Immunol. doi: 10.1038/mi.2016.115 Moliva, J.I., J. Turner, and J.B. Torrelles (2017). “Immune Responses to Bacillus Calmette-Guérin Vaccination: Why Do They Fail to Protect against Mycobacterium tuberculosis? Front Immunol, 8: 407.

*Authors contributed equally

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Awards

Travel Award to attend the Midwest Microbial Pathogenesis Conference ...... 2013

Recipient of the College of Medicine Systems and Integrative Biology Training Grant Fellowship (NIH/NIGMS T32-GM068412) ...... 2013

Trainee Abstract Award to participate at the American Association of Immunologist Annual Meeting ...... 2014

Keystone Symposia Trainee Scholarship to participate at the meeting on ‘Host Response in Tuberculosis ...... 2014

Recipient of a Diversity Supplement Fellowship (NIH/NIAID R01-AI093570-S1) ....2014

Travel award for best poster presented at the 14th Trainee Research Day at the Ohio State University College of Medicine ...... 2015

Accepted into the NIAID Research Opportunities (INRO) program at the National Institutes of Allergy and Infectious Disease (NIH/NIAID) ...... 2015

Keystone Symposia Trainee Scholarship to participate at the meeting on ‘Tuberculosis Co-Morbidities and Immunopathogenesis’...... 2015

Procter & Gamble best student speaker award at the College of Medicine Biomedical Science Graduate Program annual retreat ...... 2015

Trainee Abstract Award to participate at the American Association of Immunologist Annual Meeting ...... 2016

Poster presentation award (3rd place) in the graduate student category presented at the 2016 Center for Microbial Interface Biology Symposium ...... 2016

Hodges Family Legacy Trainee Travel Award for Infectious Diseases from the Department of Microbial Infection and Immunity...... 2016

Oral presentation award (2nd place) under the Biological Sciences category presented at the 31st Edward F. Hayes Graduate Research Forum ...... 2017

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Fields of Study

Major Field: Biomedical Sciences

Emphasis: Immunology

Table of Contents

Abstract ...... ii Dedication ...... iiv Acknowledgments ...... v Vita ...... vii Fields of Study ...... ix List of Tables ...... xv List of Figures ...... xvi List of Abbreviations ...... xix

Chapter 1: The pathogen Mycobacterium tuberculosis and its vaccine, Mycobacterium bovis BCG ...... 1 A brief history of tuberculosis ...... 1 The pathogen Mycobacterium tuberculosis ...... 3 The M. tuberculosis cell wall structure ...... 4 Immune Responses to the peripheral lipids on the M. tuberculosis cell wall ...... 6 Immune responses to the peripheral lipoglycans on the M. tuberculosis cell wall ... 10 The immune response to Mycobacterium tuberculosis ...... 12 The infection cycle of M. tuberculosis ...... 12 Immune response to M. tuberculosis in the mouse model ...... 14 Immune response to M. tuberculosis in humans ...... 16 Other animal models of tuberculosis research ...... 18 Mycobacterium tuberculosis in the lung mucosa interface ...... 20 Physiology of the lung alveolus...... 20 Alveolar lining fluid and pulmonary surfactant ...... 21 The lung mucosa as an active player in the control of M. tuberculosis ...... 23 The tuberculosis vaccine: Mycobacterium bovis Bacillus Calmette-Guérin...... 25 The BCG vaccine ...... 25 x

Innate immune responses to M. bovis BCG vaccination ...... 27 Macrophages ...... 27 Dendritic Cells ...... 32 Neutrophils ...... 36 Adaptive immune responses to BCG ...... 40 T Lymphocytes ...... 40 CD4+ and CD8+ T cells ...... 40 + CD4 Th17 cells ...... 44 Treg cells ...... 46 CD1-restricted T cells ...... 47 Mucosal associated invariant T cells ...... 48 Multifunctional T cells ...... 48 B lymphocytes ...... 50 Antibody responses to BCG vaccination ...... 51 Cell-mediated responses to BCG vaccination...... 53 The quest for a better animal model to evaluate TB vaccines...... 54 Influence of the route of immunization on BCG efficacy ...... 57 Oral immunization ...... 57 Cutaneous and intradermal immunization ...... 58 Intranasal immunization ...... 59 Influence of BCG substrain on efficacy against M. tuberculosis ...... 60 Novel approaches to enhance BCG efficacy against M. tuberculosis ...... 63 Impact of the lung mucosa on BCG vaccine efficacy ...... 68

Chapter 2: Lung alveolar lining fluid enhances M. bovis BCG vaccine efficacy against M. tuberculosis in a CD8+ T cell dependent manner ...... 78 Abstract ...... 78 Introduction ...... 79 Materials and Methods ...... 81 Ethics statement ...... 81 Mice ...... 82 Growth conditions of M. tuberculosis and M. bovis BCG ...... 82 Collection of human ALF and exposure to bacteria ...... 83 Vaccination ...... 84 M. tuberculosis aerosol infection and colony forming unit enumeration ...... 84 Lung cell isolation ...... 84 Immunophenotyping by flow cytometry ...... 85 xi

Cell stimulation and ELISA ...... 86 Cell depletion ...... 87 Histopathology...... 87 Statistical analysis...... 88 Results ...... 88 Vaccination with ALF-exposed BCG preferentially stimulates CD8+ T cells in the lung ...... 88 Vaccination with ALF-exposed BCG reduces bacterial burden in the lung and spleen of C57BL/6J and C3HeB/FeJ mice ...... 89 Vaccination with ALF-exposed BCG reduces pulmonary inflammation in the lung and extends survival ...... 92 Vaccination with ALF-exposed BCG enhances T cell responses in the lung post M.tb challenge ...... 94 CD8+ T cells in the lungs of ALF-exposed BCG vaccinated mice are required for enhanced protection against M.tb ...... 95 Discussion ...... 98

Chapter 3: Selective delipidation of Mycobacterium bovis BCG enhances its vaccine potential against Mycobacterium tuberculosis infection by accelerating IL-17A responses in the lung ...... 119 Abstract ...... 119 Introduction ...... 120 Materials and Methods ...... 123 Ethics statement ...... 123 Mice ...... 123 Growth conditions of mycobacteria and delipidation of M. bovis BCG ...... 123 Analysis of extracted lipids ...... 124 Intranasal vaccination with BCG and M. tuberculosis aerosol infection ...... 125 Histopathology...... 126 SDS-PAGE and whole-cell ELISA for ManLAM ...... 127 Isolation, preparation, and in vitro infection of human macrophages ...... 127 Lung cell isolation ...... 128 Analysis of immune cells by flow cytometry ...... 129 Cytokine/LDH quantification by ELISA ...... 130 Statistical analysis...... 131 Results ...... 131 Petroleum ether treatment extracts non-polar lipids from BCG without affecting viability of the bacteria ...... 131

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Delipidated BCG is significantly attenuated in vitro and in vivo and reduces inflammation in the lung...... 133 Pulmonary vaccination with dBCG is superior to conventional BCG against M.tb challenge and attenuates pulmonary immunopathology ...... 136 Pulmonary vaccination with dBCG alters innate immune cell kinetics and increases memory T cell population of the lung ...... 137 Pulmonary vaccination with dBCG accelerates effector T cell responses in the lung upon challenge with M.tb ...... 139 Discussion ...... 140 Chapter 4: Natural aging increases the oxidation and inflammatory status of alveolar lining fluid leading to decreases in innate immune protein function, driving susceptibility to Mycobacterium tuberculosis ...... 164 Abstract ...... 164 Introduction ...... 166 Evidence for increased susceptibility to tuberculosis in the elderly ...... 166 Increasing age comes with decreases in innate and adaptive immune function ...... 167 Inflammaging, or increases in chronic inflammation due to advancing age ...... 168 Pulmonary immunity and chronic inflammation ...... 170 Elucidating changes to ALF due to aging that may impact M.tb pathogenesis ...... 171 Material and Methods ...... 173 Ethics statement ...... 173 Proteomic analysis of human ALF by Liquid Chromatography-Tandem Mass Spectrometry ...... 173 Ingenuity Pathway Analysis ...... 174 Mice ...... 175 Human subjects...... 175 Collection of bronchoalveolar lavage fluid and concentration to ALF ...... 176 Enzyme-linked immunosorbent assay of cytokines and myeloperoxidase assay .... 177 Western blotting ...... 178 Hydrolytic enzyme activity in ALF ...... 179 Alveolar lining fluid lipid content ...... 180 Complement and surfactant protein binding assays ...... 181 In vitro infections using human monocyte derived macrophages ...... 181 Immunocytochemistry and confocal microscopy ...... 182 In vivo M.tb infections of C57BL/6J mice ...... 183 Histopathology...... 183 Statistical analysis...... 184 Results ...... 184 Signatures of inflammaging are evident in human ALF ...... 184 Pro-inflammatory cytokines are higher in the ALF of aged humans and mice ...... 185 xiii

Levels of SP-A and SP-D in ALF increase in old age...... 185 Complement proteins within ALF are altered in old age ...... 186 Enzymatic activity of ALF hydrolases decreases with age ...... 187 Myeloperoxidase levels and protein oxidation within ALF increases with age ...... 187 POPC, a marker of inflammation, is elevated in ALF in old age ...... 188 SP-A, C3 from elderly human ALF are less capable of binding to M.tb ...... 189 Exposure of M.tb to elderly ALF provides a growth advantage in human macrophages ...... 190 Exposure to elderly-ALF reduces phagosomal biogenesis in M.tb infected macrophages ...... 190 Exposure of M.tb to elderly ALF increases its pathogenicity in vivo and accelerates immunopathology of the lung...... 191 Discussion ...... 192

Chapter 5: Significance of findings ...... 220 Using the mouse as a model for TB vaccine research ...... 222 Exploiting the mycobacterial cell wall to enhance TB vaccine efficacy ...... 226 Manipulating CD8+ T cell responses to improve TB vaccines ...... 230 Future directions ...... 232 Concluding remarks ...... 236

References ...... 240

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List of Tables

Table 1.1. Mycobacterial cell wall components and their described host interactions ... 76

Table 1.2. BCG substrains used for production, distribution and administration ...... 77

Table 4.1. Top 32 disease and function categories revealed by Ingenuity Pathway Analysis of the global proteome of adult and elderly human ALF...... 218

Table 4.2. Top 20 upstream regulators categories revealed by Ingenuity Pathway Analysis of the global proteome of adult and elderly human ALF...... 219

List of Figures

Figure 1.1. A cartoon representation of the cell wall structure of Mycobacterium tuberculosis and Mycobacterium bovis BCG ...... 71

Figure 1.2. Sequence of events following M.tb infection ...... 72

Figure 1.3. Innate immune cell responses to BCG vaccination ...... 73

Figure 1.4. Adaptive immune cell responses to BCG vaccination ...... 74

Figure 1.5. Influence of route of vaccination on BCG protective efficacy and novel vaccines against M.tb ...... 75

Figure 2.1. Vaccination with ALF-exposed BCG preferentially stimulates CD8+ T cells in the lung ...... 110

Figure 2.2. Gating scheme of lymphocytes ...... 111

Figure 2.3. Vaccination with ALF-exposed BCG preferentially stimulates Th1 responses and does not affect proliferation of T cells ...... 112

Figure 2.4. Vaccination with ALF-exposed BCG reduces M.tb bacterial burden in the lung and spleen of C57BL/6J and C3HeB/FeJ mouse strains ...... 113

Figure 2.5. Vaccination with ALF-exposed BCG reduces pulmonary inflammation in the M.tb infected lung of vaccinated mice ...... 114

Figure 2.6. Vaccination with ALF-exposed BCG extends survival of C57BL/6J mice, but not C3HeB/FeJ mice ...... 115

Figure 2.7. Vaccination with ALF-exposed BCG enhances CD8+ T cell responses in lung of M.tb infected mice ...... 116

Figure 2.8. Vaccination with ALF-exposed BCG preferentially stimulates Th1 responses in the M.tb infected lung ...... 117

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Figure 2.9. Reduction in bacterial burden associated with ALF-exposed BCG is dependent on CD8+ T cell responses ...... 118

Figure 3.1. Petroleum ether extracts TDM, PGL-Tb1, MycB, PDIMs, and TAGs without affecting viability of BCG ...... 152

Figure 3.2. ManLAM is not extracted by petroleum ether ...... 153

Figure 3.3. Delipidation of BCG significantly reduces its survival and attenuates inflammatory responses in human macrophages ...... 154

Figure 3.4. Delipidated BCG is eliminated from the mouse lung, while BCG is not .....155

Figure 3.5. Vaccination with delipidated BCG is associated with reduced inflammation in the lung ...... 156

Figure 3.6. Vaccination with delipidated BCG is associated with reduced formation of pulmonary immune aggregates ...... 157

Figure 3.7. Pulmonary vaccination with dBCG reduces M.tb bacterial burden in the lung and peripheral organs of infected mice ...... 158

Figure 3.8. Pulmonary vaccination with dBCG is associated with decreased M.tb lung pathology across time ...... 159

Figure 3.9. Gating scheme of innate and adaptive immune cells ...... 160

Figure 3.10. Pulmonary vaccination with delipidated BCG is associated with decreased monocyte and neutrophil influx to the lung early post inoculation ...... 161

Figure 3.11. Pulmonary vaccination with delipidated BCG increases memory and effector T cell responses in the lung ...... 162

Figure 3.12. Pulmonary vaccination with dBCG boosts CD69 and IL-17A, but not IFNγ, responses in the lung of M.tb infected mice ...... 163

Figure 4.1. Basal levels of pro-inflammatory cytokines in ALF increase with age ...... 207

Figure 4.2. Basal levels of SP-A and SP-D in ALF increase with age ...... 208

Figure 4.3. Basal levels of some complement proteins in ALF increase with age ...... 209

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Figure 4.4. ALF hydrolase activity in the mouse lung decreases with age...... 210

Figure 4.5. Nitrosative and oxidative stress to ALF proteins in the mouse lung increases with age ...... 211

Figure 4.6. Increasing age is associated with increases in POPC in mouse ALF ...... 212

Figure 4.7. C3 and SP-A from elderly human ALF are less capable of binding to the M.tb cell wall ...... 213

Figure 4.8. Exposure of M.tb to elderly-ALF favors survival within human macrophages ...... 214

Figure 4.9. Reduced phagosomal-lysosomal fusion in macrophages infected with elderly- ALF exposed M.tb ...... 215

Figure 4.10. Exposure of M.tb to elderly-ALF favors growth in mouse organs, but does not affect survival ...... 216

Figure 4.11. Exposure of M.tb to elderly-ALF increases lung inflammation early post infection ...... 217

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List of Abbreviations

ALF alveolar lining fluid ANOVA analysis of variance APC antigen presenting cell AR arabinogalactan ATCC American Type Culture Collection BAL bronchoalveolar lavage BALF bronchoalveolar lavage fluid BCG Mycobacterium bovis Bacillus Calmette-Guérin BSL-3 biosafety levels three CFP culture filtrate protein CFSE carboxyfluorescein succinimidyl ester CFU colony forming unit ConA concacavalin A CRP c-reactive protein C:M chloroform:methanol DAP diaminopimelic acid DAT diacyltrehalose DC dendritic cell DC-SIGN dendritic cell-specific intercellular adhesion molecule-3-grabbing non- integrin DLN draining lymph node DMEM Dulbecco’s modiﬁed Eagle's medium xix

DPI days post infection DPPC dipalmitoylphosphatidylcholine DPV days post vaccination DTH delayed type hypersensitivity ESAT-6 6 kDa early secretory antigenic target GFP green fluorescent protein HAM human alveolar macrophage H&E hematoxylin and eosin HIV human immunodeficiency virus IFNγ interferon-gamma ICAM-3 intercellular adhesion molecule 3 IL interleukin IP intraperitoneal IRB Institutional Review Board KO knockout LAM lipoarabinomannan LDH lactose dehydrogenase LM lipomannan LPS lipopolysaccharide LTBI latent tuberculosis infection MA mycolic acid MAIT mucosal associated invariant T cells ManLAM mannose-capped lipoarabinomannan MDM monocyte derived macrophage MHC major histocompatibility complex MINCLE macrophage inducible Ca2+-dependent lectin

MLN mediastinal lymph node MOI multiplicity of infection MR mannose receptor mRNA messenger ribonucleic acid M.tb Mycobacterium tuberculosis MVA modified vaccinia virus Ankara NBF neutral buffered formalin NIAID National Institutes of Allergy and Infectious Disease NF-κB nuclear factor kappa-light-chain-enhancer of activated B cells NHP non-human primate NO nitric oxide OADC oleoyl-albumin-dextrose-catalase OVA ovalbumin PBMC peripheral blood mononuclear cells PBS phosphate buffered saline PC phosphatidylcholine PDIM phthiocerol dimycocerosate PE petroleum ether PEtha phosphatidylethanolamine PG peptidoglycan PGly phosphatidyglycerol PI phosphatidyl-myo-inositol PILAM phosphatidyl-myo-inositol capped lipoarabinomannan PIM phosphatidyl-myo-inositol mannoside PGL phenolic glycolipid PL phagolysosome

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POPC 1-palmitoyl-2-oleoylphosphatidylcholine PPD purified protein derivative PRR pattern recognition receptor PTB pulmonary tuberculosis QTF quantiferon r recombinant ROS reactive oxygen species RPIM Roswell Park Memorial Institute medium RU relative unit SEM standard error of the mean SL sulfolipid SP surfactant protein TAT triacyltrehalose TB tuberculosis

+ Tc CD8 cytotoxic T cell TCR T cell receptor TDM trehalose dimycolate

TFH follicular helper T cell TGFβ transforming growth factor β

+ Th CD4 T helper cell TCH 2-thiophencarboxylic acid hydrazide TMM trehalose monomycolate TL total lipid TLC thin layer chromatography TLR Toll-like receptor TNF tumor necrosis factor

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Treg regulatory T cell UK United Kingdom WHO World Health Organization

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Chapter 1: The pathogen Mycobacterium tuberculosis and its vaccine,

Mycobacterium bovis BCG

A brief history of tuberculosis

Tuberculosis (TB) has been a part of humanity since antiquity (1). In some sense, it has

evolved with us, and we have evolved with it. The primary causative agent of TB,

Mycobacterium tuberculosis (M.tb), has likely killed more people than any other

pathogen throughout the course of human history (2). Estimates indicate that an early

progenitor of M.tb was present in East Africa over three million years ago (3) However,

as humans evolved, mycobacteria evolved with us and modern strains of M.tb likely

originated from a common ancestor 20,000-15,000 years ago (4). The oldest report of TB originates from a Mediterranean settlement more than 9,000 years ago when humans began abandoning their hunter-gatherer lifestyle and instead began establishing small settlements (5). Despite written mentions of TB in India, China, classic Greece, and in the

Roman Empire, it was the French physician René Laennec in 1819 who published on the

pathogenesis of TB and unified the concept of the disease (6). At the time, 0.8-1% of the

population of Europe succumbed to TB (2). It wasn’t for many years later that TB was

suspected to be transmitted from infected individuals. In 1865 the French surgeon Jean-

Antoine Villemin proved that TB was an infectious ‘substance’ by inoculating a rabbit 1

with pus from a lung lesion of an individual who had died of TB (2). However, it was

Robert Koch in 1882 that identified the tubercle bacillus and presented it along with his

famous postulates initiating the foundation for infectious disease research for many years

to come (7).

An important development in the diagnosis of TB came from Charles Mantoux in 1908.

He pioneered the use of purified protein derivative (PPD), a mixture of proteins secreted

by M.tb during growth, as a potential diagnostic tool for TB (8). When PPD was injected intradermally (in between the layers of the skin) to TB patients, less than 1% failed to

develop a delayed type hypersensitivity response (9). The potential use of the Mantoux

test, as it later came to be called, did not come from testing whether individuals had TB,

but rather developed to determine the prevalence of TB within populations. When the

Mantoux test was used in school children from endemic countries, it was discovered that

many responded as equally strong as individuals with TB, despite the lack of clinical

symptoms. It was the pediatrician Clemens von Pirquet who noted that these children had

developed latent tuberculosis infection (LTBI), a hallmark of TB (2). Thus, M.tb was not

only directly capable of causing disease, but could also infect individuals and remain in a

latent state. The advanced understanding of TB and improvements in health care through

the course of the 20th century led to drastic decreases in the incidence of TB in Europe and in North America (10;11). However, despite global decreases, TB continues to kill millions in underdeveloped countries in Asia, Africa, and Central and South America.

Inspired by the work of Edward Jenner on his discovery and development of the smallpox

vaccine, the French scientists Albert Calmette and Camille Guérin sought to use

Mycobacterium bovis, the causative agent of bovine TB, as a potential vaccine against

TB in humans (7;12). In 1908, they began by growing M. bovis on potato slices coated with glycerol and sub-passaging onto a new slice when the culture became too dense

(13). After thirteen years of continued passage they observed that the M. bovis they had been culturing failed to cause disease when injected into guinea pigs. This new strain came to be known as Mycobacterium bovis Bacillus Calmette-Guérin (BCG). In 1921 they inoculated the first individual with BCG, an infant whose mother had died of TB and whom had been placed under the care of his tuberculous grandmother. The child never developed TB. BCG was deemed a success and soon children all across Europe began receiving the vaccine (14). Today, BCG is one of the most administered vaccines in the world (15).

The pathogen Mycobacterium tuberculosis

M.tb is an obligate pathogenic bacillus in the family Mycobactericeae (16) and belongs in the Mycobacterium tuberculosis complex. In addition to M.tb, this complex contains nine other species including M. tuberculosis sensu strictu, M. africanum, M. canetti, M. bovis,

M. caprae, M. microti, M. pinnipedii, M. mungi, and M. orgis (17). M.tb is an obligate aerobe incapable of forming spores and lacks mechanisms for motility (18). The replication cycle for M.tb requires approximately 15-20 hours, making it one the slowest

replicating bacterial species (19). It differs from Gram positive and Gram negative bacteria in the sense that it possesses an unusual, waxy coating on its cell surface primarily due to the presence of mycolic acids (18). Mycolic acids form a tight, semi- impermeable layer that provides M.tb with resistance to desiccation (20). Due to the high abundance of mycolic acids, M.tb cannot be properly stained by Gram staining (false gram positive), instead the mycolic acid-specific stain, Ziehl-Neelsen, is used (21). M.tb can be grown in vitro in the laboratory, but requires caution and must be cultured in a biosafety levels three (BSL3) environment.

The M. tuberculosis cell wall structure

The M.tb cell wall is similar in structure and composition to the M. bovis BCG cell wall

(22) (Fig. 1.1). It is primarily divided into two sections, an inner layer and an outer layer, that surround the plasma membrane (23). Anchored to the plasma membrane is a short layer of peptidoglycan (PG) which in turn is covalently linked to a layer of arabinogalactan (AG). Mycolic acids are covalently linked in a perpendicularly fashion to the AG and form the bulk of the cell wall. This entire structure, the inner layer, is termed the mycolyl-arabinogalactan-peptidoglycan complex (mAGP). The mAGP forms the essential core of the mycobacterial cell wall (23). The PG on the mycobacterial cell wall, as in most eubacteria, serves to maintain shape and protect from osmotic pressure.

Mycobacteria have a chemotype IV cell wall, meaning their PG is composed of L- alanine-D-iso-glutaminyl-meso-diaminopimelic acid (DAP) and AG and contains both N-

acetyl and N-glycolyl modifications (24;25). Additionally, approximately 25% of cross-

links in the PG of mycobacteria are DAP-DAP linkages, with the remaining 75% being

DAP-alanine (26). AG, composed of arabinan and galactan, is the dominant

polysaccharide on the mycobacterial cell wall. Like PG, it is important for integrity of the

bacillus, but also serves as an anchor for the mycolic acids (23). AG is covalently

connected to PG through a phophoryl-N-acetylglucosaminosyl-rhamnosyl linkage (27).

The last major component in the mAGP complex are the mycolic acids (MA). They are made up of a short α-alkyl- and a long β-hydroxy-fatty acid ranging from 60 to 90 carbons per chain (28). The large chains of carbon atoms in MA make the mycobacterial cell wall extremely impermeable. MA are covalently linked to the AG via ester linkages and exist as tetramycolyl-pentaarabinofuranosyl clusters on the AG (27;29).

Atop the mycolic acids sits the outer layer. It is composed of non-covalently linked proteins, lipids, and lipoglycans, which include mannose-capped lipoarabinomannan

(ManLAM), lipomannan (LM), phosphatidyl-myo-inositol mannosides (PIMs), phthiocerol containing lipids such as phthiocerol dymicocerosate (PDIM), acyl trehaloses

[trehalose mono- and di-mycolate (TMM/TDM), sulfolipids (SL), and di- and triacyltrehaloses (DAT and TAT)], and oligosaccharide containing lipids

[lipooligosaccharides (LOSs), and phenolic glycolipids (PGLs )]. ManLAMs are capped at the non-reducing terminal β-Ara residue with α-(1→2) mannose residues (30;31). The outermost portion of the M.tb surface is dominated by polysaccharides. However, this appears to be directly dependent on the composition of the growth medium and whether 5

the bacteria is grown in vitro or in vivo (32-34). The major components on the outermost layer are the polysaccharides arabinomannan, mannan, α-glucan, and xylan (35;36).

Some evidence indicates that this polysaccharide layer may be required for virulence of

M.tb (37). Though the mAGP is important in the context of mycobacterial virulence, the peripheral lipids are the mediators of entry into host cells and initial modulators of the host response to infection.

Immune Responses to the peripheral lipids on the M. tuberculosis cell wall

The most abundant and toxic of the glycolipids is TDM, also known as cord factor. It is one of the primary M.tb lipids that elicit immunological responses (38;39), and was first purified by extracting mycobacteria using the solvent petroleum ether. The purified extract was highly toxic when injected into mice causing intense peritonitis and acute pulmonary hemorrhage (40). Additionally, injection of purified TDM resulted in foreign- body lung granuloma formation that lasted for up to two weeks (41). Biochemically,

TDM toxicity is attributed to its stimulation of NADase activity, lowering host NAD in the lung, liver, and spleen. This in turn reduces electron flow across mitochondrial membranes ultimately disrupting the process of oxidative phosphorylation (42). In vitro

studies of mycobacteria-infected macrophages showed that TDM actively blocks the

process of phagosome maturation (43), and thus allows for mycobacteria to survive

within host cells. TDM can also be used exclusively by mycobacteria for entry into

macrophages by utilizing the macrophage-inducible C-type lectin (MINCLE) receptor

(44). Once inside macrophages, it has been suggested that TDM may impair the

development of an effective immune response by decreasing the expression of MHC II,

CD40, CD80, and CD1d, all important requirements for effective antigen presentation

and stimulation of T cells (45). Additionally, TDM helps inhibit communication to the

adaptive immune system by inhibiting macrophage-induced IL-12p40, a key cytokine required for IFNγ secretion by T cells (46-48).

Another major trehalose containing lipid on the mycobacterial cell wall, sulfolipid-1 (SL-

1), can exert similar effects as TDM. In cell culture models, SL-1 can inhibit phagosome maturation in macrophages, induce cellular cytotoxicity, disrupt mitochondrial oxidative phosphorylation, and activate or suppress the production of cytokines and reactive oxygen species (ROS) in human leukocytes (49-53). However, injection of pure SL-1

was non-toxic and non-granulomatogenic. Conversely, SL-1 may offset some of the

effects of TDM. Co-administration of TDM and SL-1 prevented excessive lung

inflammation, and SL-1 inhibited TDM-induced release of TNF from alveolar

macrophages (54). Thus, the effects of SL-1 on the immune system remain unclear. The

other two major trehalose containing lipids on the mycobacterial cell wall,

diacyltrehalose (DAT) and triacyltrehalose (TAT) may also be important for virulence of

mycobacteria, but remain poorly described. M.tb mutants lacking DAT failed to inhibit

phagosome maturation suggesting that DAT is also important in for M.tb survival within macrophages (53). DAT was also shown to inhibit the proliferation of mouse lymphocytes (55).

Lipooligosaccharides (LOSs) also contain a trehalose in a polyacylated form with long chain fatty acids and an oligosaccharide chain (31). Though heavily disputed, LOSs are not abundant on the M. tuberculosis cell wall (56). They are considered immunogenic

(57), but immunological responses of M.tb LOSs remains unknown. More recently it has been suggested that the absence of LOSs may be associated with virulence, and the success of M.tb as a human pathogen may be attributed to its loss of LOSs (58). A strain of M. canetti (closest relative to M.tb) deficient in the pks5 locus was shown to be deficient in LOSs. This loss of LOSs resulted in M. cannetii becoming more virulent in cellular and animal models of infection. One other oligosaccharide containing lipid on the mycobacterial cell wall are phenolic glycolipids (PGLs). Most M.tb clinical isolates and all the laboratory strains do not produce PGLs (59). However, some M.tb clinical isolates are able to synthesize a specific form of PGL named PGL-Tb1. A clinical isolate of M.tb belonging to the East-Asian lineage (i.e. HN878) was found to display a hypervirulent phenotype in animal models due to the presence of PGL-Tb1 on its cell wall (60). The trisaccharide domain of M.tb PGL was found to be able to inhibit Toll-like receptor 2

(TLR2)-induced NF-κB activation, and thus production of TNF, IL-6, and CCL2, suggesting PGL-Tb1 may enhance mycobacterial subversion of the immune system

(61;62).

Of all the non-polar lipids present on the cell wall of M.tb, phthiocerol dimycocerosate

(PDIM) has received the most attention. PDIM is classified as a wax, and in M.tb, is

composed of a mixture of long-chain β-diols esterified by multimethyl-branched fatty acids named mycocerosic acids (63). Similar to other lipids on the outer surface of M.tb,

PDIMs appear to play a role in the virulence of mycobacteria. M.tb strains lacking PDIM were shown to be less capable of causing disease in mice (64;65). Genetic M.tb mutants lacking PDIM were less capable of binding to the plasma membrane of host macrophages leading researchers to conclude that PDIMs directly contribute to the initial step of macrophage infection and participated in preventing phagosomal acidification (66). In

support, other studies have shown that lack of PDIM on M.tb reduces the survival of the bacteria within macrophages, suggesting PDIMs protect M.tb from early innate host

responses (67). Thus, the literature suggests an important role for PDIMs on the virulence

of M.tb.

Another important lipid class on the cell wall of mycobacteria is phosphatidyl-myo-

inositol (PI) and its mannosylated derivatives, phosphatidyl-myo-inositol mannosides

(PIMs) (31). Aside from phosphatidylethanolamine (PEtha), PIMs are the major

phospholipid species present on the M.tb cell wall (68). PIMs can be distinguished based

on the number and position of mannose residues and fatty acids present on the molecule.

The basic PIM consists of a mannose subunit attached to position C-2 of the myo-inositol

PI anchor. Further addition of mannose to positions C-6 of the myo-inositol via a α–D- mannose linkage or a linked trimannose unit gives rise to PIM2 and PIM4, respectively.

PIM4 can be further mannosylated at position C-2 giving rise to PIM5, which can in turn

can also be mannosylated giving rise to PIM6 (31). PIMs are sub-grouped into low-order 9

(PIM1-PIM4) and high order (PIM5 & PIM6) and their degree of mannosylation appears to

affect immune responses. PIM2 and PIM6 have been shown to interact with TLR2 and induce strong inflammatory responses (69), whereas other studies have associated PIM2 and PIM6 with inhibition of TLR4 responses (70). Furthermore, higher-order PIMs have been shown to interact with the mannose receptor (MR) present on some phagocytes and promote trafficking events that inhibiting phagosome biogenesis (71). Table 1.1 summarizes the major mycobacterial cell wall components, the receptors with which they interact, and their effects on host immunity.

Immune responses to the peripheral lipoglycans on the M. tuberculosis cell wall

Major constituents of the mycobacterial cell are the lipoglycans, LM and ManLAM. In contrast to the PIMs, LM and LAM contain a α-1,6-linked mannan backbone (21-34 mannose residues) branched with α-1,2-mannose residues (72;73). ManLAM is distinguishable from LM by the presence of arabinan, which consist of α-1,5-linked arabinose backbone with α-1,3 branching (74). ManLAM, present in pathogenic mycobacteria (i.e. M.tb, M. bovis, M. leprae), differs from non-pathogenic mycobacteria

LAM by the presence of mono-, di- and tri-mannose-caps on its non-reducing end. In non-pathogenic mycobacteria such as M. smegmatis, LAM is terminally capped with phosphatidyl-myo-inositol (PI) giving rise to PILAM (75). The last described form of

LAM is AraLAM. Found in M. chelonae, AraLAM lacks mannose or PI caps, and instead arabinose is exposed at the terminal ends (76). 10

The different lipoglycans on mycobacteria have been described to stimulate immune

responses in different manners. Whole LM extracts have been described as agonists of

TLR1/TLR2. However, the number of fatty acids present on LM can influence the

interaction with host receptors. In this context, tri-acylated forms have been shown to

bind to TLR2/TLR1, while tetra-acylated preferentially bind to TLR4 (77). While LM from M.tb was shown to induce strong inflammatory response by stimulating the secretion of TNF and IL-12 by macrophages in a TLR2 dependent manner, a recent study reported that LM is capable of blocking TNF mRNA synthesis suggesting that LM may exert a dual role during M.tb infection (75;78-80).

Total LAM [independent of being AraLAM, PILAM or ManLAM] can signal through

TLR1/TLR2 and/or TLR4 depending on the number of fatty acids present, and drive a pro-inflammatory response (81-83). However, there is a large body of literature suggesting that ManLAM serves primarily to subvert the host immune response against

M.tb allowing it to survive by undermining acquired immune responses (84). Among the plethora of inhibitory effects of ManLAM, its inhibition of the fusion between phagosomes and lysosomes is regarded as the ultimate survival strategy by intracellular bacteria (85-88). Phagocytosis of M.tb via the MR has been shown to drive this arrest

(89;90) and inhibit macrophage antigen presentation (91) allowing M.tb to survive within infected macrophages. ManLAM can also inhibit M.tb-induced apoptosis by inhibiting

Ca2+-dependent signaling (92;93). Downstream, ManLAM can inhibit T cell proliferation 11

and T cell derived IFNγ by downregulating macrophage innate immune responses (94).

Furthermore, LAM also serves as a free radical scavenging agent that can neutralize oxidative burst responses by macrophages (95). Phagocytosis of M.tb via ManLAM-MR

mediated mechanisms induces the secretion of IL-10 by infected macrophages (96). In

turn, IL-10 downregulates innate and adaptive immune responses (97). Thus, ManLAM

is a powerful virulence factor associated with the M.tb cell wall that is utilized by the

bacteria to survive within infected hosts.

The immune response to Mycobacterium tuberculosis

The infection cycle of M. tuberculosis

M.tb is an airborne pathogen (98). Infection is passed from one infected individual to

another by coughing (99;100). The infection cycle can by subdivided into four main

stages. First, M.tb is inhaled and deposited in the alveoli of the lung (101). Here, M.tb is

bathed in alveolar lining fluid (ALF) prior to interaction with alveolar macrophages

(AMs). AMs phagocytose M.tb and initiate innate immune response (102). Clearance of

the bacteria at this stage hangs in the balance of host factors designed to destroy

pathogens as well as mycobacterial virulence factors designed to inhibit host immunity.

The second stage is initiated soon after by the infected macrophages. Secretion of

chemokines and cytokines by infected macrophages attract resident macrophages and

also monocytes/neutrophils from the circulation into the infected tissues (103). These

monocytes differentiate into macrophages and dendritic cells, but remain incapable of

clearing the infection. M.tb continues replicating uncontrolled and immune cells continue

infiltrating the lung. The third stage is initiated when infected cells, typically dendritic

cells, exit the lung and migrate to lymph nodes. In the lymph nodes, dendritic cells

present antigen to naïve T cells and initiate the adaptive immune response. Antigen-

specific T cells exit the lymph nodes and migrate to the lung where they provide

necessary IFNγ to macrophages that enable them to kill the intracellular bacteria through the production reactive nitrogen species (RNSs) (102). This process occurs 2-3 weeks or

4-8 weeks post infection in mouse and humans, respectively (104). T cell responses

inhibit the logarithmic growth of M.tb in the lung. As a result, the infection may become

stationary, at which point there is no net change in bacterial numbers, or latent, in which

bacterial numbers are reduced to negligible levels but never cleared (105). The cellular

immune system, if unable to clear M.tb, forms the granuloma, a well-structured collection

of host cells that surrounds infected cells to contain the infection and a hallmark of

human TB. An individual may live their entire life with M.tb infection and never

experience symptoms of the disease (106). In the fourth stage, under conditions of

declining immunity, granulomas may rupture causing necrotic cavities allowing M.tb to

initiate its replication cycle once again (107). The immune response to M.tb is

summarized in Fig. 1.2.

Immune response to M. tuberculosis in the mouse model

Most of the immunological requirements for mycobacterial immunity have been

discovered in the mouse model. Of all the mouse strains available, the C57BL/6 strain

has dominated the field due to the wide availability of genetic mutants. Though previous

models utilized intravenous and intratracheal infection routes, the low dose aerosol model

has risen to be the most akin to the natural route of infection in humans (108). Aerosol infection is calculated to deliver 40-100 viable bacteria into the lower respiratory tract

(109). Once in the lung, M.tb enters AMs via host receptors including the MR, the class

A scavenger receptor, MINCLE, and complement receptors 3 and 4 (CR3/4). However, it remains unclear whether interactions with different receptors influence disease severity in vivo (110). It has been suggested that through the course of evolution M.tb has evolved to engage a wide variety of receptors to maximize its ability to initiate a successful infection. As AMs are considered to be one of the first cells infected by M.tb when delivered via aerosol, they contribute to the first line of defense. AMs exert their antimycobacterial response through phagolysosome fusion, by generating ROS through the oxidative burst response and RNSs via NOS2-dependent mechanisms, and through the catabolic process of autophagy (102;111). Ingestion of M.tb by AMs activates the

NF-κB pathway and triggers the production of pro-inflammatory cytokines and chemokines (112;113). Thus, AMs respond by initiating the inflammatory cascade required for the extravasation of leukocytes from the circulation into infected tissues, an essential event required for the initiation of adaptive immunity. In the mouse model, AMs

appear to be incapable of controlling the infection as M.tb grows logarithmically for three

weeks. Bacterial numbers peak at approximately one million bacteria in the lung and only

stabilize due to entry of antigen-specific T cell.

In the mouse, T cells are incapable of clearing the infection, and thus bacterial numbers plateau and enter a stationary phase at approximately three weeks post infection. The three week delay in the generation of adaptive T cell responses following M.tb infection is due in part to the slow replication nature of M.tb. The stationary phase is maintained by antigen-specific αβ T cells that secrete IFNγ. In resistant mouse strains, including

C57BL/6 and BALB/c, the stationary phase lasts for 200-300 days post infection until the mouse succumbs to the disease (114;115). In susceptible mouse strains such as

C3HeB/Fe, the stationary phase only lasts for 100-150 days (116;117). The cause of death is typically due to exacerbated inflammation resulting in pulmonary failure. The primary cells responsible for the containment of M.tb are CD4+ T cells (102). Their main

function, and most importantly in the context of M.tb, is the secretion of IFNγ, which is

required for resistance to fatal infection. This was demonstrated by the extreme lethality

of M.tb in class II MHC- and IFNγ-deficient mice (118-122). IFNγ exerts its function by

inducing the production of nitric oxide (NO) by macrophages, thus providing

macrophages with an enhanced antimicrobial defense mechanism. However, despite the

absolute requirement of CD4+ T cells, they remain incapable of eliminating the infection

or preventing fatal disease. Despite their inability to clear the infection, recruitment of

CD4+ T cells to the lung remains the hallmark of protection against mycobacterial 15

disease. While CD4+ T cells are absolutely essential for the control of M.tb, CD8+ T cells

appear to play a lesser role (102;123;124). In fact, the role of CD8+ T cells appears to be

largely dependent on the experimental method used to infect mice. In the intravenous

route of infection, the absence of CD8+ T cells results in a rapid disease onset and the

mice rapidly succumb to the disease (125). In contrast, infection of CD8+ T cell deficient

mice with a low dose aerosol of M.tb had little impact on disease progression (115;126).

However, CD8+ T cells were better suited for protecting mice against M.tb compared to

CD4+ T cells using the high dose aerosol infection where mice are infected with 500-

1000 bacteria instead of the usual 40-100 (127). Thus, while CD4+ T cells are critical in

the control of M.tb, the role of CD8+ T cells may be as equally important depending on

the context of the infection.

Immune response to M. tuberculosis in humans

The cellular immune response to M.tb in mice described above also applies to humans.

However, stark differences exist in the immunopathology and outcome of the disease between humans and mice. The most significant differences are: i) in humans only a small fraction of individuals infected with M.tb develop clinical signs of the disease, and ii) infection with M.tb can result in different outcomes of disease that range from spontaneous eradication, latent non-symptomatic, mild chronic, rapidly progressing, and reactivation. These stages of the disease do not occur in conventional laboratory mice

(128). In contrast to mice, humans develop central necrosis in lung granulomas (129), the

spatial organization of macrophages and T cells in the granuloma are not identical between the two species (130;131), and granulomas in the lung of mice remain aerobic, whereas in humans they do not, and develop hypoxic centers (130;132).

The process of generating a human granuloma is initiated by the influx of neutrophils to

the lung. The neutrophils surround the infected tissues and recruit monocytes from

circulation. After the initiation of adaptive immunity, CD4+ and CD8+ T cells enter the

lung and surround the layer of neutrophils and macrophages to form a tightly packed

structure that restricts the spread of M.tb (133). Established granulomas can form a

necrotic central core that supports the survival of M.tb (101). In immunocompetent

humans, M.tb-containing granulomas are small, compact, and characterized by the

presence of IFNγ+CD4+ T cells, whereas immunocompromised humans develop large

granulomas that are typically disorganized and predominantly composed of activated

macrophages with few lymphocytes (128;134). Although human granulomas are

primarily composed of blood-derived macrophages and lymphocytes, epitheliod cells and

multinucleated giant cells (also known as Langerhans cells) are present (135;136). In

humans the most typical granulomas are caseous, meaning these structures are formed by

macrophages surrounding a central necrotic core with a rim of B and T lymphocytes.

Other types of granulomas also exists. Non-necrotizing granulomas lack the necrotic

center, but retain the macrophage and lymphocyte layers. It has been suggested that these

may be transitioning toward caseus granulomas or be in the process of healing if the

infection is successfully cleared (137;138).

Thus, the two biggest differences in the mouse model compared to humans are: i) the

outcome of the disease following infection (clearance, latency, or active disease), and ii)

the differences in pathology due to infection (granulomagenesis). Some of the

disadvantages of the mouse model have been remedied by the use of the non-human

primate model, however its use remains controversial and costly (139). Furthermore, the

basic immunological requirements for the containment of mycobacteria are present in

mice (140) and they remain an invaluable tool for the continued quest of identifying

immunological mechanisms that protect against mycobacterial disease.

Other animal models of tuberculosis research

Aside from the mouse, the three best studied animal models of TB research are the guinea

pig, the rabbit, and the non-human primate (NHP). They each offer their own individual

advantages and disadvantages compared to using the mouse model. Guinea pigs are significantly more susceptible to TB than mice, often succumbing to the disease more rapidly (141). They have been extensively used for testing antibiotics (142;143), new

diagnostic tools (144;145), to discern immunological responses to M.tb infection (146),

and to test new vaccine formulations (147). One of the most notorious advantages of the

guinea pig model is the similarity in pathology to human TB, wherein guinea pigs

develop necrotic granulomas. However, the rabbit model is much more suitable for

assessing immunopathology as their granulomas progress in a manner very similar to

those observed in humans (i.e. cavitation) (148). However, the higher we jump on the vertebrate ladder, the more costly and more challenging experiments become.

Additionally, the lack of reagents have left the guinea pig and rabbit model as a last resort and today are only used to screen chemical compounds (i.e. antibiotics) and vaccines.

Despite the lack of reproducibility between humans and small vertebrate animals in terms of M.tb infection, most of the current knowledge has been acquired using these models.

Due to the inability to infect humans with M.tb in a controlled setting, the best available model that faithfully recapitulates human TB is the NHP. The two commonly used NHP models of TB research are the cynomolgus and rhesus macaques (139;149;150). Rhesus and cynomolgus macaques faithfully recapitulate human TB. They can develop asymptomatic TB, develop latent infection, progress to active TB, and even reactivate

(151-154). An important advantage of the NHP model of TB is our ability to monitor clinical correlates of infection (e.g. complete blood counts, toxicology reports, X-rays, diagnostics) to the same degrees as in humans (155). This allows us to directly translate discoveries with clinical trial design and execution. Although their use is associated with a plethora of difficulties (e.g. costs, regulations, reagent availability, handling), to date no animal model can reproduce human TB like the NHP model. For this reason many scientists have argued that the development of an effective vaccine for TB will require the extensive use of the NHP model.

Mycobacterium tuberculosis in the lung mucosa interface

Physiology of the lung alveolus

The mammalian lung has evolved to optimize exposure of blood to oxygen. During deep inhalation, the lungs can be inflated with up to six liters of air. In humans, similar to mice, the lung is subdivided into three lobes on the right and two lobes on the left. Air travels through the nose and mouth and passes from the larynx to the trachea and flows through 16 generations of conductive bronchi and bronchioles before reaching the alveoli. The lung is composed of 50 different types of cells (156). Mucus, secreted by

submucous glands and Goblet cells, serves to maintain a moist environment, whereas

ciliated columnar cells help prevent dust particles, pollutants, and infectious organisms

from reaching the lower airways. Together they form part of the mucociliary escalator, an

important process for the maintenance of a healthy lung. Alveoli are located after the 17th

generation of bronchioles, called the respiratory bronchioles. At the 20th generation, the entire wall of the airway is composed of alveoli and are called alveolar ducts. Alveolar ducts end at the 23rd generation in blind sacs, which are lined with alveoli and referred to as alveolar sacs (156). Alveoli are hollow cavities found in the lung parenchyma and

serve as the primary site of gas exchange. There are over 300 million alveoli in the lung,

occupying a total surface area of approximately seventy square meters (156-158).

Anatomically, the alveoli are formed of an epithelial layer and an extracellular matrix

surrounded by capillaries (159). There are three major types of cells on the alveolar wall:

type I alveolar epithelial cells (ATs), type II ATs, and AMs. Type I ATs are thin and flat

and provide the alveolus with its structural support. Type II ATs are cuboidal and secret a

homeostatic solution called surfactant, a lipid-rich solution used to lower the surface

tension at the air-liquid interface (160). The lipid free portion of surfactant, the hypophase, is called alveolar lining fluid.

Alveolar lining fluid and pulmonary surfactant

Alveolar lining fluid (ALF) is a homeostatic solution the lines the surface of the alveoli

(161;162). ALF can be further subdivided into pulmonary surfactant and a hypophase.

The development of a surfactant layer at the surface of the alveolus was essential in the

transition of animals from sea-dwelling to land-dwelling creatures (163). According to

the Young-LaPlace law, the larger the vessel radius, the larger the wall tension required

to withstand a given internal fluid pressure. In other words, in a sufficiently narrow tube

of circular cross-section, changes in pressure are due to two times the surface tension

divided by the radius (Δp=2γ/R; where p is pressure, γ is surface tension, and R is the

radius) (164). As the radius increases, the pressure decreases and when the radius

decreases, the change in pressure increases. Given that individual alveoli have a very

small radiuses (0.05 mm - 0.1 mm), the absence of components that reduce surface

tension would exert extreme forces on the alveolus and cause it to implode (165).

Pulmonary surfactant is an essential lipid-protein rich solution that stabilizes the alveoli-

air interface permitting gas exchange to occur in the lung. It forms a thin film at the air-

water interphase and dramatically decreases the surface tension of water, thereby

preventing collapse of the alveolus during the normal breathing process. Pulmonary

surfactant lipids are produced by type II ATs in the endoplasmic reticulum, deposited in

lamellar bodies, the surfactant storage organelle, and secreted via exocytosis (166;167).

Pulmonary surfactant is approximately 90% lipid by weight. The lipid species include:

phosphatidylcholine (PC), phosphatidylglycerol, and smaller amounts of PEtha, phosphatidylinositol, phosphatidylserine, the plasmalogen analog of phosphatidylcholine,

and cholesterol (168). The dominant lipid in pulmonary surfactant is

dipalmitoylphosphatidylcholine (DPPC), comprising of 41-70% of total PC (169;170).

Two essential hydrophobic proteins directly associate with pulmonary surfactant,

surfactant protein (SP)-B (8.7 kDa), and SP-C (4.2 kDa) (171). SP-B and SP-C stabilize the surfactant monolayer that forms at the air-water interface, a process that cannot occur in their absence (172;173).

The remaining fraction of ALF, the aqueous portion, among its many functions serves primarily to maintain the alveoli in a moist environment to prevent desiccation (174).

This facilitates blood circulation through the alveoli and allows for efficient gas exchange. Another important function of ALF is in host defense. The hydrophilic glycoproteins SP-A (35 kDa) and SP-D (43 kDa) play important roles in homeostasis of the lung and also serve as innate immune defenses (172). They belong to the Ca2+-

dependent carbohydrate-binding collectin family and contain a short N-terminal region, a

collagen-like domain, a coiled-coil ‘neck’, and a C-terminal carbohydrate recognition

domain (172). They are predominantly located in the aqueous phase of ALF and serve as important opsonizing factors of carbohydrates present in bacteria, fungi, and viruses. SP-

A in ALF outnumbers SP-D 10:1. SP-A has been suggested to act in a negative feedback loop during surfactant recycling by inhibiting its secretion from type II ATs (175;176).

Instead, SP-A mediates recycling surfactant by promoting uptake by type II ATs via the receptor P63/CKAP4 (177). ALF is also rich in homeostatic alveolar hydrolases that are produced and secreted by type II ATs (162). The diversity of alveolar hydrolases present in ALF indicates they may be an evolutionarily conserved antimicrobial mechanism against pathogens, including mycobacteria (discussed below). ALF is also rich in antimicrobial peptides (178-180) and complement components (181-183) which can facilitate clearance of pollutants, debris, and infectious particles. Thus, the importance of

ALF is highlighted not only by its physiological role, but also in its ability to protect the lung from foreign particles.

The lung mucosa as an active player in the control of M. tuberculosis

More recently, pulmonary ALF has been found to possess additional antimicrobial properties than previously thought. As mentioned, the protein fraction of ALF also includes a highly variable amount of serum proteins and a wide variety of specific hydrolases in addition to surfactant proteins. Alveolar compartment cells secrete this array of hydrolases into the ALF (162). The three most common hydrolases in ALF are alkaline phosphatase (AlkP), acid phosphatase (AcP), and a non-specific esterase (Est),

but other hydrolases including α-mannosidase, α-galactosidase, β-galactosidase, α-

glucosidase, β-glucosidase, α-xylosidase, α-fucosidase, arylsulfatase, fatty acid esterase-

I, alkaline phosphodiesterase, phospholipase C, peroxidase, α-rhamnosidase, and fatty acid esterase-II are also active. It was shown that at physiological concentrations, these enzymes are capable of stripping lipids and carbohydrates from the cell wall of M.tb, specifically incubation of M.tb with ALF decreased the amount of ManLAM and TDM present on the cell surface by 60% and 30%, respectively (162). Furthermore, the M.tb

cell wall fragments generated by exposing M.tb to ALF have biological activity,

demonstrated by the ability of phagocytes stimulated with these supernatants to produce

TNF, IL-6, CCL5, IL-10, and G-CSF (184;185).

The consequences of incubating M.tb with ALF become more impressive when ALF-

exposed M.tb is used to infect human phagocytes in vitro. Macrophages infected with

ALF-exposed M.tb were more capable of killing the bacteria across time compared to

bacteria that were not exposed with ALF (exposed to 0.9% NaCl as a control).

Macrophages infected with ALF-exposed M.tb had increased phagosomal-lysosomal (P-

L) fusion events compared to 0.9% NaCl-exposed bacteria (162;184). The authors

attributed the increase in P-L fusion to decreases in the amount of ManLAM on the

bacteria cell surface as ManLAM has been shown to inhibit processes associated with

phagosome biogenesis (186). Not only were macrophages more capable of containing

M.tb after it was exposed to ALF, human neutrophils were also more effective at killing

M.tb after it had been exposed to human ALF (187). Thus, ALF appears to play a critical 24

antimicrobial function in the context of infection. Given the constant exposure of the lung

to environmental stimuli (e.g. pollutants, toxins, smoke, infectious organisms), the role of

ALF in maintaining homeostasis of the lung merits further consideration.

The tuberculosis vaccine: Mycobacterium bovis Bacillus Calmette-Guérin

The BCG vaccine

Mycobacterium bovis Bacillus Calmette-Guérin is a live, attenuated strain of

Mycobacterium bovis. BCG was developed for its potential to prevent TB, not M.tb

infection, an important distinction. TB disease, defined by symptoms cause by infection

with M.tb, is the leading cause of death from an infectious agent (101;188). M.tb causes a

highly persistent infection that continues to devastate communities due to the existence of

a large reservoir of latently infected individuals that could reactivate at any time (189).

BCG vaccination is highly effective at preventing TB-meningitis and extra-pulmonary

disseminated TB; however, its efficacy against pulmonary TB (PTB) in different human

populations (children, youth, adult and elderly) varies; while some studies have shown

80% efficacy, other have shown complete lack of protection (190-192). Nonetheless,

BCG is the most widely administered vaccine around the world for the prevention of

M.tb-associated diseases (193).

The poor efficacy conveyed by BCG at preventing TB has been attributed to many

factors, including human and mycobacterial genetics, exposure to environmental mycobacteria, co-infections with viruses and/or parasites, geographical location, and importantly, socioeconomic and nutritional factors (194-198). However, the fundamental question remains as to what constitutes sterilizing immunity to TB, and why BCG fails to confer this state. BCG is primarily believed to mediate immunity through the development of antigen specific memory T cells (199-201), which act quickly following a subsequent infection with M.tb. Indeed, this is the fundamental reason why BCG works against disseminated TB and TB meningitis (202;203). However, why the same mechanism fails to prevent PTB remains poorly understood. Despite this, researchers across the world have continuously sought to modify and/or improve BCG. This began with genetic manipulations through deletions and insertions of genes from virulent mycobacteria that has today evolved into generating sophisticated multifunctional vaccine strains (204). Albeit conceptually and experimentally promising, the majority of these recombinant BCG vaccines fail at fully preventing the development of PTB

(197;198).

It is still unclear why BCG fails to prevent primary infection, reactivation and PTB, but it is widely accepted that generating long term protective immunity is essential. Thus, developing a successful vaccine against M.tb infection or TB requires an understanding of how immunity develops following BCG vaccination, and the roadblocks behind why protective immunity is not sustained. This section discusses the host innate and adaptive

immune responses to BCG, and how these further influence the host response to M.tb

infection and progression to TB. An important and commonly overlooked factor in BCG

vaccine design, the influence of the human lung environment and its consequences in

directing the M.tb pathway of infection, is also discussed.

Innate immune responses to M. bovis BCG vaccination

Macrophages

Macrophages play a critical role during innate immune responses to pathogens by

inducing inflammation early after encounter of foreign molecules (205;206). Their main

functions are phagocytosis, secretion of cytokines and chemokines, generation of

oxidative/nitrogen mediators, degradation of ingested microbes, and antigen presentation

to stimulate the adaptive immune response (207-209). Following BCG intradermal inoculation, resident epidermal macrophages interact with BCG via several pattern recognition receptors (PRRs), including CR3 (207) and TLRs 2 and 4 (206). C-type lectin

family receptors such as the MR and MINCLE receptor are expressed on macrophages

(207-209), but direct interaction between them and BCG, and its subsequent outcome,

has not yet been described. Because the peripheral lipid portion of the cell wall is very

similar between BCG and M.tb (22), it is predicted that their ability to infect tissue

macrophages will be similar. However, BCG’s first contact occurs with resident

epidermal macrophages whereas M.tb contact, in the majority of cases, occurs with

resident AMs. Differences in the mechanisms of antigen recognition, antigen uptake, antigen processing, and antigen presentation between these two types of resident tissue macrophages remains unclear, and may contribute to the reasons behind why BCG is not fully protective. Thus, resolving this discrepancy will be important to identify if epidermal vaccination is sufficient to protect against lung disease.

In fact, the effect of serum opsonization is often overlooked during BCG vaccination

(210-212). This process is thought to be crucial in initiating immune responses to the

BCG vaccine. As an example, the host opsonin factor H, a regulatory protein of the complement system that downregulates the alternative complement cascade, can bind to the BCG cell surface (213) and partially inhibit its uptake by epidermal macrophages.

Macrophages infected by factor H opsonized-BCG can secrete elevated amounts of pro- inflammatory cytokines, potentially driving an acute response (high IL-6/TNF) (213).

Thus, factor H opsonization of BCG could be detrimental to the primary goal of BCG vaccination (to generate a strong T cell memory response). This is probably because opsonized BCG gets killed too quickly, reducing the amount of time antigen is available for presentation, and thus negatively impacting T cell proliferation and recruitment to the site of infection. Like factor H, there are other serum opsonins in the epidermal tissue and thus, further studies are necessary to assess their effects in the generation of immunity to

BCG. Innate immune responses to BCG are summarized in Fig. 1.3.

From the mycobacterial perspective, the TB field assumes that BCG cell wall

components will interact in a similar fashion with macrophage receptors as M.tb cell wall

components do (31). However, differences exist, such as in the case of the ManLAM of

M.tb vs. BCG. The degree and pattern of mannose-capping and fatty acid content in M.tb

ManLAM vs. BCG ManLAM differs (31) and thus, it may dictate how this molecule is differentially recognized by macrophage receptors such as the MR, TLR2 (dimerized with TLR1 or TLR6) or TLR4, generating different immune responses that subsequently may affect antigen-presentation. In fact, BCG macrophage stimulation via TLR2 or

TLR4 drives differences in pro-inflammatory responses, T cell proliferation, and

IFNγ secretion in vitro, and results in differences in bacterial burden in the lung in vivo

(206). Indeed, prolonged antigen stimulation of TLR2 in macrophages downregulates the expression of major histocompatibility complex (MHC) class II and affects MHC I antigen cross processing; thus reducing antigen-presentation to T cells (214). It is still not clear to which degree suppression of MHC I and II via TLR2 negatively impacts the protective immune response generated by BCG vaccination and/or against M.tb infection.

In this regard, studies have shown the potential of introducing an adjuvant during BCG vaccination targeting TLR-7/9 signaling, which restores and/or increases the expression of MHC II in macrophages, thus enhancing their ability to present antigen (215).

From the host perspective, differences in tissue resident macrophages (epidermal vs. lung) expressing different PRR types and levels (216;217); as well as human polymorphisms in these receptors, could influence immune responses to BCG and

subsequently its effectiveness. Studies performed using BCG and M.tb indicate that the nature of their cell wall components engaging a specific macrophage PRR determines the host immune response generated. For example in macrophages, engagement of ManLAM or higher-order PIMs to the MR drives an anti-inflammatory response; however, engagement of TDM to MINCLE or engagement of lower-order PIMs to CR3 and/or

TLRs results in pro-inflammation (31). Thus, the balance between pro- and anti- inflammatory lipids present on the BCG and M.tb cell wall is a factor to consider in the protective ability of BCG. Reducing pro-inflammation at the site of vaccination may be beneficial as macrophages will be exposed to the antigen for a longer period of time, and thus provide additional time for the generation of T cell immunological memory. If we consider that epidermal macrophages and AMs in the lung have and/or use different

PRRs for BCG recognition which leads towards site-distinct mechanisms of antigen- presentation, then a safe delivery of BCG into the lungs could be a way to optimize BCG efficacy.

Differences in intracellular trafficking and antigen processing could also explain the failure of BCG to confer long term immunity. In fact, BCG, as well as M.tb, can block phagosome maturation (218-220) by engaging specific receptors, i.e. the MR (88), and although MHC antigen presentation and activation of the adaptive immune system is unaffected, it may be less than optimal (221;222). In this context, exogenous induction of phagosome-lysosome (P-L) fusion (223), autophagy (197), and/or increasing phagosome

leakage of Ag across the phagosome membrane (224), could enhance the antigen-

presentation process and thus, BCG efficacy.

An important step forward in the development of a new, effective TB vaccine, focused on optimizing antigen-presentation, came from the development of the rBCG ΔureC::hly+

vaccine (204). This vaccine, when administered percutaneously, has superior efficacy

than BCG by further reducing the bacterial burden in the lung of M.tb infected mice.

Recent studies also indicate that vaccination with rBCG ΔureC::hly+ increases

macrophage apoptotic vesicle formation, thereby inducing more robust CD4+ and CD8+ T

cell responses (204), as well as increasing the gene expression of ‘Absent In Melanoma

2’ (AIM2). This increases the autophagy pathway and inflammasome activation, which in

turn improves control of M.tb infection (225). Thus, although P-L fusion is an important

mechanism, modulation of apoptosis and autophagy can also offer novel avenues for

vaccines against TB. Mechanistic studies such as these, focusing on the underlying

factors behind enhanced immunity offer valuable insight into the requirements for

efficient mycobacterial clearance.

Extending on the above findings, ex vivo M.tb infected lung macrophages from BCG

vaccinated guinea pigs are shown to secrete larger amounts of IFNγ, TNF and IL-12p40,

highlighting their appropriate anti-mycobacterial response (226). Other supporting ex

vivo studies indicate that alveolar macrophages from BCG vaccinated guinea pigs

challenged with M.tb express significantly less IL-10 and more IL-12p40, compared to

unvaccinated controls (227). This same study reported an elevated expression of MHC II on peritoneal macrophages from BCG vaccinated guinea pigs (227). Thus, based on our current understanding of the requirements for mycobacterial immunity; immunity induced by the BCG vaccine results in responses to M.tb at the early phase of infection that directly impact macrophage function.

Altogether, there is a significantly large body of knowledge on the interactions between

BCG and macrophages and how these responses can influence protective immune responses against M.tb. Receptor interaction between the macrophages and bacteria are critical in initiating the response, where interactions with different macrophage receptors can differentially modulate trafficking pathways and processing and presentation of antigen. However, additional studies determining the exact role of epidermal macrophages vs. AMs following BCG vaccination are needed before macrophage functions can be exploited to further improve vaccine development strategies.

Dendritic Cells

DCs are classified as one of the most potent APCs (228) and classically described as the modulators of crosstalk between innate and adaptive immunity (229). Phenotypic and functional differences exist between DCs and macrophages; however the primary difference is that DCs, in contrast to macrophages, migrate from tissues via the lymphatic system and enter the draining lymph nodes, where they present antigen to naïve T cells

(230) leading to the induction of effector T cell responses (231;232). As DCs migrate,

they mature by up-regulating MHC II and CD80, CD86, CD40 and CD54 co-stimulatory

molecules, all involved in the activation of adaptive immune cells (230;233). Several

phagocytic receptors on DCs recognize BCG, including CR3 (CD11b/CD18), CR4

(CD11c/CD18), Dendritic Cell-Specific ICAM-3 Grabbing Non-Integrin (DC-SIGN,

CD209), and DEC-205 (CD205) (234-236). Of all these receptors, DC-SIGN is possibly one of the most important as neutralization of DC-SIGN with antibodies inhibits the interaction of BCG with DCs by 80% (233). Signaling receptors such as TLR2 and TLR4 are also shown to be involved in DC activation and maturation by their interaction with

BCG cell wall components (i.e. mAGP complex) (233;237;238).

Following BCG vaccination epidermal dendritic cells initiate adaptive immune responses in vivo by trafficking from the site of inoculation to draining lymph nodes where they present antigen to adaptive immune cells. In this context, migratory DCs display an

EpCAMlowCD11bhigh phenotype and are capable of priming CD4+ T cells via interleukin-

1 receptor (IL-1R) and MyD88 (myeloid differentiation primary response gene 88) signaling pathways (239). However, most of our understanding of DCs and BCG immunology is from studies conducted in vitro. Stimulation of DCs with BCG in vitro

induces homotypic aggregation, up-regulation of surface antigen presenting molecules,

down-regulation of endocytic activity, and release of TNF (240) implying DCs initially

respond to BCG. However, it is difficult to discern whether these responses are adequate

for optimal immunity. BCG can be engineered to enhance DC activation by the addition

of M.tb genes encoding specific mycobacterial proteins into BCG (241), suggesting that

DC responsiveness to BCG may not be optimal. Though immunity can be improved by

augmenting DC activation, we still lack a fundamental understanding of the exact

interactions or processes that result in optimal immunological protection. From the

perspective of the host, mice lacking specific genes (e.g. IFNγ, IL-12, and TNF) have been valuable in deciphering some of the requirements for effective immunity to BCG and M.tb; however they offer little insight as to the concentration and spatial organization required for their optimal efficacy. Transgenic mouse models allowing manipulation of specific gene products or conditional knockout mice allowing for spatial regulation could be useful in elucidating the necessary elements for optimal BCG efficacy in terms of space and time.

Stimulation of human DCs with BCG can increase surface expression of MHC-II, CD40,

CD44, CD54, CD80 and CD86, all markers involved in DC activation, maturation, migration, and antigen presentation to T cells (242). DCs infected with BCG also secrete

TNF, IL-1β, IL-6, IL-4 and IL-10, but not IL-12 (205). Strikingly, IL-4 is secreted in large quantities indicating that its presence could shift immunity from a Th1 polarized

response towards a Th2 response, potentially affecting BCG efficacy (243). However,

recent studies disproved this hypothesis by showing that BCG-infected DCs co-cultured

with T cells are capable of inducing T cell proliferation and IFNγ secretion in vitro, the defined Th1 cytokine critical for the control of M.tb (244). However, IFNγ correlates

poorly with protection against mycobacteria infection and disease (245;246). Thus, more 34

refined markers such as abundance of memory lymphocytes in the lung and

multifunctional T cell diversity have arisen as better indicators of functional immunity

(discussed below) (203;247). Furthermore, reports indicate that M.tb hinders the

efficiency and effectiveness of antigen presentation by macrophages and DCs (91;248),

whereas BCG is much more efficient at stimulating CD4+ T lymphocytes. Thus, although

BCG may effectively stimulate the process of antigen presentation, the capacity of BCG- infected DCs to present antigen may be at a saturation point, and any further stimulation of the adaptive immune system may not be achievable (249). In this context, attempts such as BCG expressing Fms-like tyrosine kinase 3 ligand [Flt3L, a hematopoietic growth factor that stimulates DC proliferation, and whose deletion reduces T cell responses by 50% (250)], did not show further enhancement of BCG efficacy against

M.tb challenge. This finding is independent of early expansion of DCs and increased stimulation of BCG-reactive IFNγ-secreting T cells (251). As BCG may be less efficient in inducing DC maturation than M.tb, understanding the differences between them could lead to greater approaches in improving the interaction between BCG and DCs and thus, host immunity (252).

Several groups have exploited DC maturation to improve BCG immunity, with studies centered in modulating TNF and IL-10 dominating the field. TNF neutralization inhibits

DC maturation post-BCG inoculation suggesting an important role for TNF in immunity generated by BCG (242). As a result, an adequate concentration of TNF is required for optimal DC antigen presenting efficacy. BCG infected DCs also secrete large quantities

of IL-10 but not IL-12 (205). IL-12 is, however, highly expressed in IL-10-/- mice

following inoculation with BCG indicating the existence of an important balance between

IL-10 and IL-12 which could activate/accelerate maturation of DCs (220) and serve as a

potential host-directed therapy. These findings may explain why DCs from IL-10-/- mice are more efficient at activating T cells in the draining lymph nodes (DLN) (253). Thus,

IL-10 may not only down-regulate the migration of infected DCs to the DLN, but also regulate IL-12 production and subsequent DC capacity to mature, diminishing T cell activation and proliferation, repressing the adaptive immune response generated by BCG

(254;255), and possibly affecting the development of immunological memory. Hence down-regulation of IL-10 and up-regulation of IL-12 could to be important for generating an optimal immune response to BCG.

Apart from being powerful mediators of the immune response to BCG by initiating innate responses, DCs also translate information to the adaptive branch of the immune system and initiate the first steps that subsequently give rise to immunological memory. With this in mind, it is logical to explore mechanisms that can enhance DC-BCG interactions with intentions of amplifying BCG efficacy. Although the DC-BCG interaction appears to be highly efficient, it remains unclear why they fail in the lung. One could speculate that too much focus has been placed on DC responses to BCG in vitro and thus, it has misconstrued our notion of DC responses.

Neutrophils

Circulating human neutrophils comprise approximate 60% of the blood cells, have a short

half-life of 6 to 10 hours, and are one of the first cells to respond to foreign molecules

(255). Neutrophils secrete large amounts of chemokines and cytokines, priming long lived phagocytes (256). Interaction of neutrophils with BCG increases their expression of adhesion markers CD11b and CD18, FcγRs II and III, and stimulates their up-regulation of cytokines (e.g. IL-1α, IL-1β and TGFβ) and chemokines (e.g. IL-8, CCL2 and CCL3)

(257). How changes in neutrophil phenotype upon contact with BCG can influence the generation of protective immunity against M.tb is still unclear.

Neutrophils are capable of shuttling live BCG via the lymphatic system into DLNs and into the vicinity of DCs and T cells (258). The cross-talk between BCG-infected

neutrophils and DCs in this location delivers maturation signals to immature DCs, and

also assists DCs in their presentation of BCG antigens to prime CD4+ and CD8+ T cells

(258-261). The delay in apoptosis observed in BCG-infected neutrophils supports the

concept that BCG may use them as a vehicle to disseminate from the site of inoculation

to DLNs (261). However, whether the host benefits from the neutrophil-BCG interaction remains unanswered. Additionally, the longer BCG remains within neutrophils increases the probability the bacteria will be killed by neutrophil intracellular mechanisms, possibly enhancing pathways involved in immune activation via antigen presentation (262).

Studies conducted in the C3HeB/FeJ mice, which develop necrotic and hypoxic tubercle granulomas, found that BCG vaccination in these mice was associated with long lasting 37

immunity and reduced bacterial burden. The association was attributed to a reduction in

the numbers of neutrophils (263). Thus, neutrophils may behave as a double-edged

sword; on one hand they seem to facilitate antigen presentation by shuttling live bacilli to

the vicinity of DCs, but on the other hand they may be responsible for preventing the

development of long-lasting immunity. The balance between BCG’s intracellular survival

and/or digestion, processing, and presentation by the neutrophil may impact DC

maturation, the subsequent adaptive immune response (limiting the clonal expansion of

antigen specific effector lymphocytes), and the long term establishment of a state of

protective immunity.

Conversely, neutrophils can also direct activation of T cells in lymph nodes, and thus

participate in the generation of adaptive immune responses (260;264). A recent study

indicates that neutrophils expressing CCR7 migrate to the lymph nodes in response to

CCR7 ligands, CCL19 and CCL21. This migration seems enhanced during in vivo

injection of complete Freund’s adjuvant (containing inactivated M.tb cell wall) in wild-

type mice but not CCR7-/- mice (260). To further illustrate a role for neutrophils, studies

using mice inoculated with BCG via the intranasal route show neutrophils entering the

lungs in two waves. The first wave arrives between one to three days post inoculation and

could kill M.tb. The second wave of neutrophils enters the lung three weeks post

inoculation together with IFNγ- and IL-17A-producing T cells. This second wave of neutrophils is not associated with the ability to kill M.tb, and their movement into the lungs is dependent on the expression of IL-17RA (265). Subsequent studies show that

M.tb-infected neutrophils are a prominent population in the lungs early during infection

(266), and that M.tb-infected neutrophils promote adaptive immune responses to M.tb infection. Recent studies using BCG also demonstrate that neutrophils regulate inflammation via the secretion of IL-10, impairing the control of M.tb growth during chronic infection (267). This further establishes the dual role for neutrophils during infection, being active in controlling M.tb infection, but also regulating the inflammatory response generated during infection. Since neutrophils enter tissues much sooner relative to other host cells, they could be a critical mediator in clearance or persistence of mycobacteria. However, how the regulatory function(s) of neutrophils influence adaptive memory responses generated by BCG vaccination remains unanswered.

Altogether, neutrophils can have a wide range of effects in the context of BCG vaccination. On one hand they may help facilitate adaptive immune responses by aiding in antigen presentation and shuttling live bacteria into the vicinity of professional APCs, but on the other hand their presence and propensity to induce strong inflammatory responses may be detrimental to the tissue in which they reside, propagating the disease and potentially inhibiting the development of long lasting immunity. Indeed, it remains unclear whether neutrophil shuttling of BCG is beneficial or detrimental as they may ultimately be used as a vehicle from which to disseminate to other organs. Further studies as to the role of neutrophils and mechanism in which they may be involved post-BCG vaccination could shed some light on the complex behavior of neutrophils.

Adaptive immune responses to BCG

T Lymphocytes

T cell responses arise in parallel through engagement of the T receptor with foreign

antigen presented by antigen presenting cells (268). With few exceptions, all vaccines

+ + stimulate the proliferation of CD4 helper T cells (Th) and CD8 cytotoxic T cells (Tc)

(200). In the context of BCG immunity, helper T cells primarily differentiate into two

distinct classes of effector cells during an immune response to vaccination: Th1 cells identified by the production of IFNγ, and Th17 cells identified by the production of IL-

17A, though IL-4 producing Th2 cells can also be generated (269). Other cell subsets

such as T regulatory cells (Treg) and CD1-restricted T cells also arise following BCG

vaccination, albeit to a lesser extent. BCG also induces cytotoxic T cells, whose main

function is to lyse infected cells through osmotic disruption (270). It is clear that both

CD4+, and to a lesser extent CD8+, T cells are critical for protection against M.tb

infection; experimentally highlighted using mouse knockout models (120;125;271;272)

supporting a dominant role for T cells as the main effector cells following immunization

with BCG. The adaptive immune responses to BCG are highlighted in Fig. 1.4.

CD4+ and CD8+ T cells

Mouse models have confirmed that α/β T cell receptor (TCR) expressed by CD4+ and

CD8+ T cells and MHC I and II are necessary for control of mycobacterial infections

(119;273), highlighting a dominant T cell response to BCG. Adoptive transfer studies

provide the best evidence that protective T cell mediate immunity is generated by BCG.

The transfer of CD4+ or CD8+ T cells from BCG-vaccinated mice into rag1-/- mice, an in

vivo model deficient for both B and T cells, show that CD4+ T cells are necessary to

reduce bacterial burden in the lung and spleen, while CD8+ T cells control bacterial

burden only in the spleen. These results implicate CD4+ T cells as the main effector cell

generated by BCG in the lung, and also highlight the importance of CD8+ T cells in

preventing dissemination (273-276), potentially making them the main effector cell responsible for preventing miliary TB and TB meningitis. However, despite the fact that

CD4+ T cells reduce BCG burden in the lung, BCG is not eliminated, indicating that their

effector functions in the lung may be limited. BCG vaccination followed by M.tb

challenge confirms that in the absence of CD4+ T cells, CD8+ specific T cells are able to

reduce M.tb bacterial burden in the lung at later time-points post-infection, supporting the

importance of CD8+ T cells during the later phases of the disease (273;277). These data

demonstrate that BCG can stimulate protective CD4+ and CD8+ T cells but also suggest

that CD4+ T cells are not as efficient as their CD8+ T cell counterparts when it comes to

clearing a mycobacterial infection in a tissue-specific context, or perhaps the unique

environment of the lung makes it difficult for CD4+ T cells to assert their functions. The

answer could also lie in the pathogen itself as M.tb possesses many virulence factors that

are not present in BCG that may be used to inhibit CD4+, but not CD8+, T cell responses.

The effector function of CD8+ T cells following BCG vaccination has been characterized

to a lesser extent than CD4+ T cells (246). Whereas CD4+ T cells mediate their function

by primarily activating cells of the innate immune system through engagement of co-

stimulatory molecules on innate cells, CD8+ T cell responses, though similar, are not as

capable of this function (273). Although there is a widely held view that BCG induces

poor CD8+ T cell responses in vivo, human studies line up with in vitro studies showing

that BCG vaccination induces robust antigen specific CD8+ T cell responses

characterized by increased IFNγ production, degranulation, proliferation and elevated

levels of cytotoxic proteins (278). However, the mechanism by which CD8+ T cells

contribute to the efficacy of BCG, such as cytotoxic function, stimulation of other cells

via cytokines, chemokines, or microbicidal molecules, remains elusive. One mechanism

that has received little attention is the ability of CD8+ T cells to induce apoptosis of cells

via the FasL-Fas pathways whereby CD8+ T cells lyse target cells expressing Fas (279).

By inducing apoptosis of infected cells, other healthy innate immune cells can phagocytose the produced apoptotic bodies and further stimulate cells of the adaptive immune system. Thus, another explanation for the superior ability of CD8+ T cell to

control dissemination of M.tb could lie in their ability to induce the FasL-Fas pathway in

the spleen or liver, whereas apoptosis of cells in the lung is highly restricted to prevent

excessive tissue damage that can lead to pulmonary failure. In this context, one could

envision an engineered BCG vaccine that induces apoptosis of infected cells allowing for

a more efficacious vaccine against M.tb infection.

One argument frequently linked to the poor efficacy of BCG is the age at which BCG is

administered, usually at the time of birth. As neonates are exposed to a large variety of

antigens when born, it is believed that T cells are biased toward a Th2 response to prevent excessive inflammation. As a Th2 response can be detrimental to mycobacterial immunity, BCG vaccination of neonates may not fulfill its potential (280-284). However, studies in human newborns and infants reported a Th1 biased response following BCG

immunization similar to immunized adults (285;286). These studies observed a high

number of IFNγ+CD4+ T cells (287;288), and also a significant population of CD4+ T

cells negative for IFNγ but instead positive for TNF and IL-2 (289) indicating that BCG

generates diverse and adequate immune responses required to contain M.tb in infants.

Thus, it seems that poor efficacy of BCG against M.tb may not be due to the failure of

generating Th1 cellular immunity at the time of vaccination. On the other hand, it is reported that cytokine and chemokine production differs in children vaccinated with BCG

in Malawi and the United Kingdom (UK) (290;291). These studies pointed out that

Malawian infants produce more cytokines associated with Th17 and Th2 immunity

compared to infants from the UK. An explanation for this could be the environment

surrounding the BCG vaccinated infants [e.g. parasitic co-infections that dampen

protective immune responses (292); or environmental mycobacteria (293)]. Indeed, oral

tolerance to environmental mycobacteria is a detrimental factor following intradermal

BCG vaccination, an effect that could be overcome by vaccinating via the pulmonary

route due to lack of tolerance to environmental mycobacteria in the lung (294). However, 43

virulent lipids on the mycobacterial cell wall, (including M.tb and M. bovis BCG)

particularly TDM, have prevented the use of pulmonary vaccination. TDM is a highly

toxic glycolipid that induces the formation of granulomas and a significant contributor of

immunopathological damage to the lung during mycobacterial infections (295). Thus,

another approach would be to focus on the bacteria itself and the biochemical properties

associated with its cell wall that enable it to subvert host immunity. Despite whether

BCG is efficacious or not at generating a strong Th1 response at birth, immunity appears

to wane as we age. Thus the problem of sustaining anti-mycobacterial immunity arises.

Careful analysis of the mechanisms that protect against M.tb in the first decades of life post-BCG immunization could yield valuable clues to extend the duration of immunity generated by this vaccine.

+ CD4 Th17 T cells

Th17 responses in the lung are associated with both increased protection against M.tb

infection (296) and/or exacerbated pathology (297). On one hand, repeated BCG vaccination exacerbates the influx of granulocytes into the lung in an IL-17A-dependent

manner leading to extensive immunopathology. On the other hand, IL-17A is required to

+ sustain IFNγ responses by CD4 T cells in the lung. In mice, BCG stimulates Th17

responses within the lung (298). Thus, the presence of IL-17A in the lung may be important in generating an effective immune response that benefits the host (bacterial control with limited inflammation) or an immune response that ultimately damages the

host (initial bacterial control with too much inflammation that subsequently leads to

uncontrolled bacterial growth). Studies point out the role of IL-17A in the immune response to M.tb (299), particularly during initial granuloma formation (300).

Interestingly, high concentrations of IL-17A that limit lung pathology are correlated to the presence of IL-10 (300). Although, the relationship between IL-17A and IL-10 remains unclear, IL-10 appears to have an immunosuppressive role during the generation of BCG immunity (301), and like IL-17A (296), IL-10 plays an important role during the initial stages of M.tb infection in vivo (302;303). The role of Th17 cellular responses in

the context of BCG immunity remains uncertain, as indicated by recent studies

demonstrating that the M.tb glycoprotein Rv1860/mpt32 (Apa or 45 KDa, present also in

BCG) can downregulate Th17 and Th1 immune responses, abrogating BCG immunity

against M.tb (304). However, this is in direct contrast to other studies where the same

protein is shown to stimulate IFNγ-secreting CD4+ and CD8+ T cells (305) preventing the

waning of BCG immunity, thus decreasing M.tb burden post challenge (306). An

explanation for these opposing results could lie in how the Rv1860 protein is utilized; in

the former study a recombinant BCG expressing Rv1860 was used to vaccinate mice,

whereas in the latter, Rv1860 was administered as a booster in BCG-primed mice. This

example highlights the complexity of the immune system and how certain mycobacterial

proteins may be detrimental or beneficial in the development of the initial immune

response depending on time and place, and may or may not be required for optimal long-

term immunity. In further support of Th17 cells as critical mediators of immunity to BCG,

it has been shown that accelerated Th1 memory responses in the lung of BCG vaccinated

mice are dependent on IL-17A and IL-23 derived from antigen-specific memory Th17 cells, and that these lung resident memory Th17 cells quickly respond to M.tb infection

(296). With this in mind, BCG vaccination in unison with host directed therapies that

augment Th17 responses in the lung could be a powerful strategy to increase the efficacy

of BCG.

Treg cells

T regulatory cells (Treg) have also emerged as important players following immunization

with BCG. BCG vaccination trials have partially associated BCG efficacy with the

geographical location where the trial is conducted. Low BCG protective efficacy is

reported in regions of the world closer to the equator, where environmental mycobacteria

are common. Thus, BCG protective efficacy could be affected by prior host Treg cell development due to pre-exposure to environmental mycobacteria (294). The importance

+ of CD4 Treg cells mainly relies on studies showing that their depletion decreases M.tb

burden in BCG-vaccinated mice (307). Similarly, BCG boosted with an Ag85 M.tb-

protein construct (Ag85-Mpt64190-198-Mtb8.4) significantly decreases the number of Treg

cells and that their depletion correlates with reduced bacterial burden in the lung of M.tb

infected mice (308). Furthermore, human studies in BCG vaccinated adults, who

responded with strong immunization-induced local skin inflammation, showed

significantly increased levels of protective multifunctional CD4+ T cells (309). However,

BCG vaccinated adults who developed mild local skin inflammation showed increased

levels of regulatory-like CD8+ T cells (309). Thus, factors that influence vaccination- induced inflammation could affect the development of Treg cells, for example, due to genetic polymorphisms within populations or their exposure to certain environments. If certain populations have a predisposition toward generating a strong Treg cell response,

BCG vaccination may be less effective. Thus, understanding genetic differences between human populations, and in particular changes brought forth by their living environment, could yield useful clues as to why BCG fails to protect them against M.tb. In fact, an important factor to consider in this matter is that many studies on environmental mycobacteria are carried out by first exposing animals to environmental mycobacteria followed by BCG vaccination. Since BCG is given at birth, exposure to environmental mycobacteria likely occurs after BCG administration. Hence, the above studies highlight that a vaccine booster (BCG or other) after exposure to environmental mycobacteria may not aid in the development of any further immunity where Treg cells may play a role

(310). Overall, Treg cells may be detrimental to BCG vaccine efficacy against TB if there are pre-exposures to environmental mycobacteria, thus blocking their function during

BCG vaccination could be critical in inducing optimal immunity to M.tb.

CD1-restricted T cells

As the mycobacterial cell wall is approximately 80% lipid (68), studies have also focused on elucidating the role of CD1-restricted antigen presentation during BCG vaccination

(311). BCG immunized humans harbor a pool of CD1-restricted CD8+ T cells

recognizing BCG infected DCs (312). Studies in guinea pigs support this finding, showing that BCG vaccination induces humoral and CD1-restricted cytotoxic T cell-

mediated immune responses stimulated by mycobacterial lipids and lipoglycans (313).

Perhaps these lipid-restricted CD8+ T cells mediate immune responses in peripheral

organs such as the spleen and liver and are thus responsible for maintaining the chronic

stage of the disease and/or help establish latent infection. This CD1-restricted CD8+ T

cell population could also be the mediator that prevents disseminated TB and TB

meningitis. Unfortunately, the absence of group one CD1 molecules (CD1a, CD1b and

CD1c) in mice (314) has made it difficult to elucidate the exact role of the CD1-restricted

CD8+ T cell population to BCG immunity and during M.tb infection.

Mucosal associated invariant T cells

Innate-like MR1-(non-classical MHC-1b)-restricted CD8+ T cells, called mucosal

associated invariant T (MAIT) cells, could also play an important role in mycobacterial

immunity by potentially acting as early sentinels to M.tb infection (315). Despite individuals with TB having low levels of circulating MAIT cells (316), these cells

respond to BCG stimulation by producing higher levels of TNF and IFNγ (317). Thus,

further studies are necessary to clarify the positive or negative impact of MAIT cells in

the context of BCG immunity.

Multifunctional T cells

Evidence for other correlates of protective immunity rose with the discovery of multifunctional CD4+ T cells, which simultaneously produce multiple cytokines (usually

TNF, IFNγ, IL-17A, and IL-2). The contribution of these cells to mycobacterial immunity have been thoroughly discussed elsewhere (318), but it is important to stress that multifunctional T cells do not always correlate with protective immunity (319), and their importance should be carefully evaluated. As an example, the MVA85A vaccine

(modified vaccinia virus Ankara made to express Ag85A from M.tb) is developed as an intranasal booster to BCG. BCG-vaccinated, MVA85A-boosted individuals have significant increases in multifunctional T cells compared to BCG alone leading many to speculate that MVA85A would enhance the efficacy of BCG. However, further studies revealed that with or without the MVA85A booster, BCG-vaccinated individuals are equally susceptible to TB (319). Thus, multifunctional T cells may be a part of the puzzle

(320), but may not contribute so significantly to protective immunity against TB as initially thought.

Overall these findings reveal the complex nature of vaccinating with a live multifaceted organism such as BCG, where it is difficult to target only one branch of the immune system and generate effective, long-lasting immunity that prevents M.tb infection and the development of TB. Although the necessity of CD4+ T cells is without question, perhaps the reason why immunity fails in the lung is not due to poor CD4+ T cell responses per se, but rather due to inhibitory mechanisms in the lung preventing immunopathology. As

highlighted above, the major role for CD8+ T cells appears to be in maintaining the chronic phase of the disease, and seem to be particularly important in peripheral organs.

Thus, it may not necessarily be that BCG is an ineffective vaccine; it may be that the lung inhibits BCG from living up to its full potential. Defining the contributions, whether good or bad, of other adaptive immune cell subsets (Th2, Th17, Treg, CD1-restricted, multifunctional, etc.) is also important. Additionally, variability in human genetics and the geographical environment in which we reside may have consequences on our ability to mount immune responses to vaccines, and should be considered when designing future trials to test new TB vaccines. The problem of understanding the adaptive immune response to M.tb is further complicated by animal models that do not exactly recapitulate

TB in humans. M.tb has evolved to infect humans and thus, humans may be the only species possessing the key to elucidating the mechanisms responsible for clearance of mycobacteria.

B lymphocytes

The generation of long-lasting immunity to most pathogens by current effective vaccines relies on long-lived humoral immune responses that mediate protection via antibodies

(200). The BCG vaccine is a strong inducer of humoral immunity; however, due to the intracellular nature of M.tb infection, the potential of antibodies as significant contributors to protective immunity has been largely disregarded. However, new evidence has emerged identifying a potential role for antibodies and B cells in the

immune response generated by BCG vaccination (321;322). Researchers have begun to

unravel a more significant role for B cells than solely antibody production in the context

of mycobacterial infections (323;324).

Antibody responses to BCG vaccination

Although M.tb intracellular modus vivendi within host cells limits antibody function, rapid and effective antibody opsonization of M.tb prior to entry into phagocytes could be a mechanism resulting in clearance by innate immunity. Studies have demonstrated that

BCG vaccination induces long-lived mycobacteria-specific memory B cells in healthy individuals (325), but details about their role in establishing immunity to BCG remain unclear. Early studies looking at antibodies following BCG vaccination show agglutination in serum from BCG vaccinated patients when incubated with M.tb antigens

(326). Following this discovery, the humoral immune response following BCG vaccination (327-330) is associated with high levels of antibody production (331;332), specifically linked to a progressive increase in the levels of immunoglobulin (Ig) M and

IgG Ab isotypes IgG1, IgG2, and IgG3, the latter being induced by Th1 cytokines

(333;334). Overall, these studies identified robust humoral immune responses following

BCG immunization, indicating that the BCG vaccine itself can reliably induce antibody

responses. In the context of M.tb infection, low IgM and IgG levels are also partially

linked to TB susceptibility (335;336); an indication that perhaps a rapid humoral immune

response in the lung may prevent M.tb infections by mitigating M.tb entry via portals that

favor its establishment [i.e. M.tb ManLAM/host the MR (337)].

IgA is the most abundant antibody in the lung mucosa comprising approximately 30% of

the total (338). Studies of IgA during BCG vaccination showed that IgA deficient (IgA-/-)

mice have increased susceptibility to BCG infection and reduced production of both IFNγ

and TNF in their lungs (339). IgA is shown to have a dual role, increasing phagocytosis

of microbes and synergize with IgG to enhance cell-mediated cytotoxicity by effector T cells, but also blocking IgG pathogen opsonization (338), potentially preventing

interactions with Fcγ receptors. Thus, proper levels of IgA in the lung mucosa may

determine the initial establishment of infection with M.tb. This finding is supported by

other studies using polymeric IgR knockout (pIgR-/-) mice, where pIgR mediates active

transport of dimeric IgA (340). As in the case of IgA-/- during BCG infection, pIgR-/-

mice are also more susceptible to M.tb within the first three weeks of infection, mainly due to an increased influx of neutrophils to the site of the infection. Thus, the development of a new recombinant BCG strain capable of inducing the production of

IgA-committed memory B cells may provide a good strategy to consider for the development of a mucosal vaccine. Given that we do not yet know how long M.tb

remains in an acellular state following initial encounter with a host, antibodies could

mediate the very first interaction between the host cells and M.tb. As an example,

opsonization of M.tb with antibodies and subsequent entry via Fcγ receptors could result

in increased bacterial killing compared to entry of M.tb via the MR (337). Given the large 52

number of individuals exposed to M.tb, it is surprising that few develop the disease and

instead remain healthy (PPD and/or QTF negative). BCG could be engineered to increase

the presence of protective neutralizing/opsonizing antibodies in the lung mucosa, so when

M.tb is encountered it is quickly neutralized. Further research on antibodies in the context

of M.tb vaccine design could answer some of these pressing questions.

Cell-mediated responses to BCG vaccination

The specific role of B cells has also been studied in the context of BCG vaccination (323) to a certain extent. In this regard, studies using BCG vaccinated µMT mice [lacking one of the IgM μ-chain transmembrane regions and thus cannot produce mature B cells or secrete antibodies of any isotype (341)] showed that these can retain the typical1.0-log10

reduction in bacterial burden in their lungs following M.tb infection (342). This finding

directly questions the role of B cells in the control of M.tb infection, and suggests that B

cells may not play a measurable protective role during BCG vaccination in mice.

However, these studies do not completely rule out that BCG induced antibody responses

or B-cell dependent co-stimulation could be targeted to prevent M.tb infection in the first

place. In fact, a non- antibody mediated role of B cells has recently emerged with the

+ study of Follicular B helper T cells (THF). TFH are antigen-experienced CD4 T cells found within secondary lymph node organs (e.g. spleen, lymph nodes) in the vicinity of B cell follicles (343). These cells are important in mediating the selection and survival of B cells and stimulate their transition into plasma cells and memory B cells (344). Recently,

TFH cells have emerged as important mediators in the development of BCG immunity. 53

While studying the BCG ΔureC::hly+ vaccine, it was discovered that the superior efficacy behind this vaccine correlates with higher levels of central memory T cells and

TFH cells (345;346). Though promising, further research will reveal the importance of TFH in the context of mycobacterial immunity.

There are still many unknowns regarding B cell immunity against M.tb, in particular in the context of BCG immunization. The role of antibodies, though previously largely dismissed, could be targeted to prevent infection with M.tb. For example, by generating a long lasting pool of M.tb-specific antibodies within the lung mucosa, M.tb could be neutralized before it has the opportunity to encounter alveolar compartmental cells where it is shielded from opsonins. The crosstalk between B and T cells could also play an important role in host defense against M.tb, including priming effective memory T cell responses. An important antibody-independent function for B cells is also plausible, as reported for other intracellular pathogens (347). Hence whether B cells can be targeted by new vaccines to contribute via antibody-mediated processes or by interaction with other cells of the adaptive immune system to prevent M.tb infection requires further investigation.

The quest for a better animal model to evaluate TB vaccines

The lack of a validated animal model for TB vaccine development is a current critical issue in the field. There are several animal models routinely being used for TB

vaccinology studies; mice, guinea pigs, and non-human primates are the most common

ones.

Infection with M.tb in humans typically results in one of three outcomes. First, on rare

occasions, M.tb can be quickly contained and the infection cleared with or without the

assistance of the adaptive immune system. Although there is little evidence for this

scenario, some individuals remain PPD negative despite exposure to M.tb (348). Second,

in 90-95% of cases the infection progresses into latency. During latent tuberculosis

infection (LTBI) adaptive immune cells surround infected macrophages and form an

enclosed structure called a granuloma. Individuals with LTBI can live their entire life without any symptoms of the disease. However, they have a 10% lifetime risk of developing active TB. Third, the remaining 5-10% of individuals progress directly to active TB and become contagious (106). In this context, the mouse model fails to replicate the natural progression of infection in humans. Although several groups are now using very low dose aerosol infections, the majority of studies still deliver 50-100 viable

M.tb bacilli into the lung to reproduce natural infection in humans. In most mouse laboratory strains, M.tb replicates in the lung until antigen-specific T cells develop. At approximately three weeks post infection, T cells enter the lung and stunt M.tb growth

(147). M.tb bacterial burden peaks at approximately one million bacteria (6 log10) in

standard mouse strains and remains at this level for an extended period of time before

increasing at the end of life (349).

Outcome of BCG vaccination in mice and humans is also quite distinct. BCG-vaccinated mice are able to contain the M.tb infection sooner than naïve mice. Where it requires three weeks for mice to establish stable M.tb CFU, BCG-vaccination shifts this pattern from three to two weeks. This allows the mouse to contain the infection more rapidly, establishing the bacterial burden at approximately 100,000 bacteria in the lung (5 log10)

(350). This mycobacterial ‘immunity’ has been attributed to immunological memory

(199-201). However, immunity wanes across time. M.tb bacterial burden gradually

increases back up to one million somewhere between 3-5 months post infection. In

humans, no studies have been conducted to evaluate the direct effects of BCG on the

control of M.tb at early stages of infection mostly because it is extremely challenging to

predict when infection will occur in a non-controlled setting. Thus, the current data on the

efficacy of BCG comes from clinical trials assessing whether individuals developed TB

or not. Human clinical trial data on the effects of BCG vaccination indicate that BCG

efficacy against PTB in humans is predicted to be 60% and wanes with increasing age

(197;198;351).

The best relevant model for vaccine development that faithfully recapitulates human TB

is the non-human primate (NHP). The two commonly used NHP models of TB research

are the cynomolgus and rhesus macaques (139;149;150). Most research with BCG,

however, has utilized the rhesus model. Rhesus macaques faithfully recapitulate human

TB. They can develop asymptomatic TB, LTBI, progress to active TB, and even

reactivate (151-154). The evidence clearly indicates that BCG can reduce M.tb bacterial

burden in the lung, but does not eliminate it (352). Thus, data obtained using animal

models suggests that BCG may in itself not prevent primary infection, but rather may

exert its protective effects by containing M.tb and preventing progression to disease.

Thus, the NHP is the ideal to model to screen TB vaccines. However, high costs, ethical issues, and challenges of animal handling have left the NHP as a last resort model for screening vaccine efficacy, and further, it is non-feasible as a high throughput model to test TB vaccine candidates.

Influence of the route of immunization on BCG efficacy

Oral immunization

Although this route of immunization is not widely used today, the first dose of BCG administered in 1921 was given orally. Oral BCG remained the chosen route of immunization until 1924 when it was replaced with more immunogenic routes (353).

Several advantages place oral vaccination above other forms of vaccination; it eliminates the need for needles, the requirement for trained clinical staff, and it is more feasible to implement on a large scale (354). In 1993, it was first to demonstrated that systemic and mucosal immunity could be generated through oral vaccination by producing a recombinant BCG (rBCG) strain expressing the immunogen LacZ, encoding a β- galactosidase from the lac operon (355). Currently, alternative formulations are being developed to improve oral BCG, mainly focused on lipid-based BCG preparations.

Formulas such as lipid microencapsulation of BCG administered via the oral route to

mice and guinea pigs can establish specific systemic cell-mediated immune responses

(356-358), and induce long-lived, diverse memory CD4 T cell populations (358)

associated with reduced bacterial burden and pathological scores in the lung when

compared to unformulated oral vaccines (359-361). However, similar studies in humans failed to generate systemic IFNγ responses to oral BCG vaccination (362). Hence it

seems clear that oral BCG vaccination may modestly reduce TB morbidity, and further

exploration of novel delivery systems is needed to enhance its potential. The influence of

immunization route on BCG vaccine efficacy is summarized in Fig. 1.5A.

Cutaneous and intradermal immunization

Although BCG was first administered orally, the vaccine later became cutaneously

administered due to enhanced induction of delayed type hypersensitivity responses

(DTH) to the PPD diagnostic test (363). Currently, WHO recommends intradermal injection of BCG in the deltoid region (364), although cutaneous injections are performed

in some cases. Several studies comparing cutaneous vs. intradermal immunization

concluded that both stimulation of a DTH response and production of T helper (Th1)

cytokines were more prominent when BCG was intradermally delivered (287;365;366).

These findings were supported by studies where intradermal vaccination reduced the

incidence of childhood TB meningitis (367-369). Conversely, a randomized trial in South

Africa evaluating the efficacy of these two vaccination routes found no significant

differences among documented TB cases (370). These conflicting outcomes were further addressed using animal models, where no immunological differences were found between cutaneous and intradermal BCG delivery (371-373). Such discrepancies could be attributed to many different factors, including but not limited to the genetics of the subject population, environmental factors, and the origin of the BCG substrain used for vaccination. Referring to the latter, it has been documented that different BCG substrains can induce diverse immune responses and degrees of protection in humans (374;375) and animal models (376;377), although differences were minimal and not significant (378).

Thus, a direct association between BCG substrain immunogenicity and protection cannot

be inferred, mainly because correlates of protection against the development of active TB

in infected individuals have yet to be identified.

Intranasal immunization

Using different animal models, aerosolized BCG immunization was shown to limit M.tb

growth in the lung and enhance the immunogenic control of TB development potentially

due to the combination of mucosal and systemic immune activation (379-383). However, the disadvantage associated with this vaccination route is the immunopathologic damage

generated in the lung, as demonstrated by larger and more numerous perivascular and

peribronchiolar granulomatous lesions (382). Thus, by defining bacterial and host

components that elicit such inflammatory damage in the lung during intranasal

immunization without affecting its protective efficacy, a candidate vaccine could be

engineered to induce systemic and mucosal immunity while limiting undesired tissue

damage. This could potentially be achieved through limiting the expression/production of

some BCG cell wall components such as phosphatidyl-myo-inositol dimannosides, phosphatidyl-myo-inositol, trehalose mono- and di-mycolates, phenolic glycolipid mycoside B and/or other waxes (22); all abundantly present on the mycobacterial cell wall and known to cause substantial damaging inflammation in tissue (31;295). However, this needs to be carefully balanced, as pathology produced by aerosol vaccination may also be the driving force for the increased protection observed using this route of vaccination. Overcoming intranasal vaccination driven pathologies may allow for the emergence of highly effective tissue specific TB vaccines, where the manipulation of the lung mucosa components (discussed below) may play a significant role in defining the protective immune response to M.tb infection and subsequent development of TB.

Influence of BCG substrain on efficacy against M. tuberculosis

The efficacy of different BCG substrains has been thoroughly discussed (376;377), and reviewed (384); however it is necessary to describe some of their aspects in the context of vaccination studies. BCG vaccination currently covers 80% of the countries where TB is considered endemic. Although one strain of BCG was originally produced, subsequent passages by many laboratories has generated 21 BCG substrains (194;384;385) (Table

1.2). Denmark/Copenhagen strain 1331, Russian/Moscow, and Japanese/Tokyo 172 BCG are the predominant substrains currently used for vaccine production, distribution, and

administration worldwide (364). Though unclear why, Japanese/Tokyo 172 appears to provide the lowest efficacy at reducing M.tb growth in animal models (384).

Russia/Moscow and Denmark/Copenhagen substrains, although biochemically and genetically different, are equally protective against the development of TB (331;386).

The presence of different Regions of Difference (RD) with different open reading frames has been suggested as the potential source of variability observed among several BCG substrains in their ability to prevent TB morbidity (194;385-387). Although genetic and phenotypic analyses have revealed substantial information on the requirements for virulence of M. bovis and the derived BCG strain, they have failed to clarify our understanding of the ability of the original BCG and daughter substrains to stimulate a durable immune response.

An additional concern that should not be overlooked is the way BCG substrain vaccines are selected, prepared, and administered by different laboratories and countries (388).

There remains no consensus or proper regulations in place to control this process. For example, China has developed four BCG substrains; where the protective effects of BCG

Shanghai and BCG Beijing are comparable to the Danish BCG Copenhagen-1331 strain

(strain from which these were originated); however, BCG Lanzhou is slightly less potent, and BCG Chanchun is completely devoid of protection as measured by the recovery of

M.tb colony forming units (CFUs) in the spleen of vaccinated guinea pigs (both were originally derived from BCG Tokyo-172) (384). These four BCG substrains are still widely used in China. Moreover, the Russia BCG substrain is predominantly used in

countries with high TB burdens (389). These discrepancies highlight problems

associated with lack of standardization. In addition, it underlines that vaccination success

may directly depend on the country’s social, political, and economic status.

An additional element to consider is the human manipulation of the original BCG vaccine

strain, when compared to the natural selection of M.tb strains. Recent studies showed that

188 T cell epitopes essential to the human immune response to M.tb infection had been

lost, to varying degrees, in all BCG substrains (390). BCG Tokyo-172 substrain had the highest number of T cell epitopes in relation to M.tb strains; however this BCG substrain has consistently induced poor immunity against TB in animal models and in clinical trials

(384;391). Thus, the number of expressed epitopes is not necessarily a good indicator for vaccine design. In fact, some BCG substrains with fewer epitopes may prevent TB more efficiently compared to substrains with greater number of epitopes (390).

Overall, bacterial genetics may play an important role in determining the ability of BCG substrains to prevent TB morbidity, but this is not the only factor. Other factors, including global standardization of BCG vaccination, minimizing adverse reactions to

BCG vaccination, and the potential variable susceptibility to mycobacterial chemotherapeutics by different BCG substrains are critical to develop an efficient TB vaccine program. In this regard, it is considered costly to standardize the global use of a given BCG vaccine substrain, which is likely one of the reasons why we still use poor- protecting substrains for vaccination programs. In the end, the question of substrain

diversity and use lingers, and whether this diversity is harmful or beneficial remains

unanswered. Directing the use of a specific BCG substrain for vaccination while keeping

in mind M.tb strain diversity and environmental/co-morbidity factors in the targeted

region could reveal that a certain BCG substrain may be ideal for one region but not for

another. This may also be an approach to bypass the potential interference of other

endemic co-infections (i.e. helminthic, HIV, non-tuberculous mycobacteria) and co-

morbidities (i.e. smoking, diabetes, aging) that could interfere with the protection induced by BCG substrains; though no evidence has yet been linked between this potential masking effect and the BCG substrain efficacy in protecting against the development of

TB (392).

Novel approaches to enhance BCG efficacy against M. tuberculosis

Past and current TB vaccines under development have been extensively discussed in the

literature [reviewed in (393-395)]. Here we provide examples of successes and failures

and what we believe are experimentally promising solutions to improve the BCG

vaccine. Published results are summarized in Fig. 1.5B. Despite an incomplete

understanding of the basic mechanisms of immunity conferred by BCG against the

development of TB, researchers have continuously sought to improve it. As a result,

many rBCG strains have been generated throughout the years. One of the pioneering studies tested the protective efficacy of a rBCG vaccine expressing the outer surface protein A (OspA) antigen of Borrelia burgdorferi in mice and found antibody responses

against M.tb (396). Following this initial study, many rBCG vaccines to protect against

TB have been developed.

Researchers have aimed to improve BCG by having it expressing single and/or multiple

M.tb molecules. One of the first successful attempts to improve BCG was conducted by

Horwitz et al. (397). They constructed a rBCG strain expressing and secreting the M.tb

30-kDa major secretory protein (or Ag85B). rBCG30 vaccinated guinea pigs challenged

with M.tb via aerosol had fewer lung lesions and 0.5 log10 fewer CFUs in the lung when

compared to conventional BCG vaccinated animals, while spleen CFU decreased ten-fold

(397). This study highlighted Ag85B antigenicity and linked it to protective immunity

against TB. In this regard, rBCG30 can cross-protect against Mycobacterium leprae

challenge, which is further enhanced by M.tb 30-kDa Ag85B boosting (398). In order to address if M.tb secreted antigens are also capable of inducing a protective response, Pym et al.. constructed a rBCG strain (BCG:RD1-2F9) containing the complete region of deletion-1 (RD1) locus, which contains 11 genes including the ones encoding the potent, secreted T cell antigens ESAT-6 (6-kDa early secretory antigenic target) and CFP-10 (10- kDa culture filtrate protein) (399). Contrary to rBCG30, vaccination of guinea pigs with rBCG:RD1-2F9 did not significantly reduce M.tb CFUs in the lung when compared to

BCG alone; however, dissemination to the spleen was reduced by 10% (399). The failure to reduce M.tb CFUs in lung by rBCG:RD1-2F9 was considered to be tissue specific as this rBCG vaccine was able to reduce dissemination and prolong survival, thus fueling the idea that a vaccine that inhibits M.tb dissemination from the lungs may have a major

positive impact on TB outcome (399). However, the majority of subsequent studies that

are focused on improving TB vaccination emphasize controlling M.tb infection and active

TB development in the lung.

Many other rBCG vaccines have been constructed, including rBCG strains expressing a

fusion protein of Ag85A-ESAT-6 (400;401), the M.tb hspX, perfringolysin O from

Clostridium perfringens, human IL-12p70 and Ag85A individually or jointly, Mtb72f

(Mtb39 + Mtb32), and many more, all with variable levels of success in reducing M.tb

CFUs after challenge. However, similar to BCG, none fully prevent the development of

TB (399;402-409). Nevertheless, a promising development in the TB vaccine field may be the vaccine engineered by Grode et al. based on improving access of mycobacterial antigens to the MHC class I pathway to boost CD8+ T cell responses (410). Their strain,

rBCG ΔUre::CHly+ (a BCG strain that secretes listeriolysin (Hly) from Listeria

monocytogenes), is highly protective against M.tb challenge via the aerosol route with

almost 3 log10 reduction in bacterial load in the lung at day 200 post infection. This

enhanced efficacy was attributed to an efficient perforation of the phagocyte phagosomal

membrane by Hly (411), promoting antigen translocation into the phagocyte cytoplasm

and enhancing cross-priming to both helper and cytotoxic T cells (204). The protection

afforded by rBCG ΔUre::CHly+ supports an important role for CD8+ T cells in reducing

M.tb CFUs and potentially establishing protection against the development of active TB.

Furthermore, the ability of rBCG ΔUre::CHly+ to induce greater numbers of central

memory CD4+ T cells was also considered an important step in this process (412). Due to

its increased potential at protecting against TB, rBCG ΔUre::CHly+ is now in clinical

trials (393-395). Similarly, autophagy, a catabolic process by which the cell digests

cellular components, has been shown to assist DCs in processing extracellular antigens

for MHC class I presentation (413). Jagannath et al. hypothesized that by inducing

autophagy, immunity to BCG could be enhanced by increasing MHC class I antigen

presentation. Indeed, induction of autophagy in BCG infected DCs which were

subsequently transferred to mice reduced M.tb CFU in the lungs by an additional 2 log10

compared to conventional BCG (414). Thus, in combination with the enhanced protection

+ + shown by rBCG ΔUre::CHly , these data indicate that CD8 T cells may play an

underestimated role in reducing the burden of M.tb in the lung and perhaps protection

against TB.

Researchers have sought to improve BCG in other ways, by vaccinating with cytokines

(415), chemokines (416), lipid mediators (417), nucleic acids (418), and antigenic

components from M.tb (419); all with variable levels of success. Other studies have

focused on the role of IL-10 as an immunomodulator (420). The absence of IL-10

enhances the ability of the immune system to clear M.tb infection in the lung and spleen

of mice (421). Pitt et al. reasoned that by inhibiting IL-10 and simultaneously vaccinating

with BCG, a more robust immune response could be mounted against M.tb challenge.

Their results showed enhanced cellular activation and 10-fold decrease in bacterial

burden in the lung compared to conventional BCG vaccination (422). This study

highlights the importance of understanding the innate immune responses to BCG and the

mechanisms that can lead to effective immunity to mycobacteria. As outlined by the

many studies above, a basic understanding of the cellular responses generated after BCG

vaccination can elucidate bacterial and/or host components required for optimal

protection against the development of active TB.

Unfortunately, the failure of the MVA85 vaccine trial was a significant step back for the

TB vaccine development field (423). MVA is an attenuated strain of the vaccinia virus

lacking all virulence factors including its ability to replicate within human cells (424).

MVA was turned into MVA85A when it was made to express Ag85A from M.tb. When

mice were vaccinated with BCG and boosted with MVA85A, this combination generated

higher levels of antigen specific CD4+ and CD8+ T cells (425). In phase I human clinical trials, MVA85A was found to induce high levels of antigen-specific IFNγ secreting T cells (425) and showed enhanced IFNγ activity in volunteers who were previously vaccinated with BCG (range of 0.5-38 years) (424). However, despite its success in

animal models and early phase human clinical trials, MVA85A (delivered to a BCG

vaccinated cohort of infants) was 17.3% effective against the development of TB as

detected by microbiological, radiological, and clinical criteria, and had worse efficacy

against controlling M.tb infection measured by the QuantiFERON TB Gold In-tube

conversion test (423). No additional protection against the development of TB, on top of

that afforded by BCG, was reported in this study (423). This significant setback

highlights the required need for a better understanding of the host immune responses

generated by BCG during vaccination, and the need to identify irrefutable correlates of

protection against pulmonary and disseminated TB.

Other vaccines designed against TB relate to therapeutic vaccines, useful for TB patients

that can no longer be cured by chemotherapy (i.e. patients infected with multi-, or extensively-drug resistant M.tb). These vaccines are mainly based on mycobacterial products (i.e. heat killed whole or fragmented (426) M.tb or its lysates) thought to generate a strong immune response; however, there are concerns about their safety and immunogenicity (427). To bypass these issues, the concept of using auxotroph attenuated

M.tb strains as vaccine candidates appeared more than 20 years ago (428), where recent advances in the development of attenuated M.tb auxotrophs containing mutations that enhance the adaptive immune response are reported (429).

Impact of the lung mucosa on BCG vaccine efficacy

Little is known about the role of the lung environment in determining the quality of immune responses generated during M.tb infection and the outcome of active or latent TB disease. The primary function of the lung is gas exchange and thus, the immune response generated within the lung is orchestrated to minimize inflammation. There is a delicate balance between generating a massive immune response that will be initially detrimental for the host and M.tb (i.e. cavities in active TB, destroying lung tissue and forcing the bacillus to accelerate replication and abandon the dying host), or an immune response

that will favor both (i.e. granuloma formation in latent M.tb infection, where both host

and M.tb live in harmony). The lung environment has an impact on this balance. In this

regard, the lung mucosa contains an array of homeostatic components (hydrolytic

enzymes, complement proteins, surfactant proteins, antimicrobial enzymes,

immunoglobulins, and many others) whose function is to maintain homeostasis of the

lung (430;431). All of these components are associated with the lung alveolar lining fluid

(ALF) (432;433). Studies from our laboratory have shown that some ALF components

(i.e. hydrolytic enzymes or hydrolases) are capable of altering the cell wall of M.tb with two distinct outcomes, modifications on the M.tb cell wall exposing ‘de novo’ motifs on the bacterium cell surface and the release of M.tb cell wall fragments to the lung milieu

(162;184;187). The interaction of M.tb with human ALF reduces the amount of

ManLAM and TDM by approximately 60% and 30%, respectively, from the M.tb cell wall surface (162). It is likely that as M.tb is deposited in the alveolar space it will encounter ALF hydrolases that will modify its cell wall prior to encountering host cells.

These M.tb cell wall alterations consequently alter M.tb recognition by human phagocytes (162;184;187) with subsequent impact on antigen processing and presentation. One question that remains is whether these human lung mucosa-induced alterations to the M.tb cell wall during its natural path of infection could explain the reason why the protective immune response generated by BCG vaccination is inadequate against PTB. For example, dominant cell wall motifs that drive antigen-specific B and T cell responses to intradermal administered BCG may be absent on ALF-exposed M.tb in the lung. Alternatively, BCG receptor mediated uptake by epidermal resident APCs may

differ from those of M.tb in the lung due to newly exposed motifs on the M.tb cell surface by the action of human ALF.

To understand the role of the lung environment in the context of BCG vaccination, a valuable approach could be to determine how M.tb is modified in the lung prior to encountering alveolar compartment cells, how this affects its metabolism, and how it differs from the ones that BCG undergoes within the epidermis. Indeed, M.tb and BCG cell walls have few biochemical differences when grown on agar plates, but their cell wall and metabolism may differ as they are exposed to different microenvironments during infection (M.tb/lung) or vaccination (BCG/epidermis), with potential consequences in establishing effective or non-effective BCG immunity. Since the lung mucosa may play a significant role in how BCG may protect us against TB, the direct delivery of BCG into the lungs of humans may prove to be superior to the conventional systemic intradermal route of BCG administration. Whether it is by modulation of innate immune cell activity, activation of T cells, development of a rapid and robust antibody response, or by targeting specific components within the bacteria itself, BCG has potential to be further manipulated to enhance its efficacy against TB.

Figure 1.1. A cartoon representation of the cell wall structure of Mycobacterium tuberculosis and Mycobacterium bovis BCG. All mycobacteria species have a layer of peptidoglycan covalently anchored to the plasma membrane. The peptidoglycan is in turn covalently linked to a layer of arabinogalactan which is covalently linked to a layer of mycolic acids. On top of the mycolic acids sits a layer of non-covalently linked lipids, glycolipids, and lipoglycans. These include: TDM, trehalose dimycolate; PIMs, phosphatidyl-myo-inositol mannosides; PDIM, phthiocerol dimycocerosates; LM, lipomannan; LAM, lipoarabinomannan; ManLAM, mannose-capped lipoarabinomannan; *PGL-Tb1, phenolic glycolipid Tb-1; *LOS, lipooligosaccharide, DAT/TAT, diacyltrehalose/triacyltrehalose; TMM, trehalose monomycolate; SL, sulfolipid. *Designates they are not universally expressed by all M.tb strains. On top of the peripheral lipids sits a capsule-like layer made up primarily of the polysaccharides α- glucan, xylan, arabinomanna, and mannan (not depicted).

Figure 1.2. Sequence of events following M.tb infection. (1) Upon inhalation of M.tb lung resident macrophages phagocytose bacteria and initiate the innate immune response. (2) Soluble immunomodulators, including cytokines and chemokines, produced by macrophages diffuse into the vasculature. (3) Circulating monocytes and neutrophils, attracted by cytokines and chemokines, extravasate into the lung. (4) Monocytes differentiate into macrophages and dendritic cells, while activated neutrophils degranulate and secrete chemokines to attract more cells to the site of infection. (5) Dendritic cells phagocytose M.tb and traffic to the mediastinal lymph nodes. (6) Dendritic cells present antigens to naïve CD4+ and CD8+ T cells in the mediastinal. (7) Antigen-specific CD4+ and CD8+ T cells exit the MLNs and traffic to the lung where they initiate the production of IFNγ. The IFNγ augments the macrophage’s antimycobacterial activity, but they remain incapable of clearing the infection. (8) Antigen-specific T cells surround infected macrophages, neutrophils, and monocytes, and form an enclosed structure called a granuloma. Most conventional laboratory mice do not develop ‘classic/well-structured’ granulomas like humans do. Instead, inflammatory cells continue to infiltrate the lung until the animal succumbs to the disease. Humans develop well- structured granulomas with hypoxic and necrotic centers. (9) Declining immunity can cause granulomas to rupture allowing bacteria to enter the airways rendering its host infectious. Abbreviations: MФ, macrophage; DC, dendritic cell; NФ, neutrophil; Mon, monocyte; MLN, mediastinal lymph nodes; CD4+, CD4+ T cell; CD8+, CD8+ T cell.

Figure 1.3. Innate immune cell responses to BCG vaccination. The initial immune response to BCG occurs at the site of inoculation (usually the dermal layer of the skin) where resident macrophages and dendritic cells interact with the bacillus via different receptors expressed on their surface. Macrophages and dendritic cells phagocytose the bacteria initiating the innate immune response through the secretion of immunomodulatory components such as cytokines and chemokines. Bacteria are degraded via intracellular killing mechanisms and their peptides are trafficked to the plasma membrane along with MHC class I and II where they are presented to cells of the adaptive immune system. Neutrophils also enter the site of inoculation and participate in the response. Finally, dendritic cells, loaded with bacteria, and expressing antigen on their surface, home to draining lymph nodes. Abbreviations: P-L, phagosome-lysosome; ROS, reactive oxygen species; RNS, reactive nitrogen species; CRs, complement receptors; TLR, Toll Like Receptors; GPCRs, G-protein coupled receptors; CLRs, C-type lectin receptors; MR, mannose receptor; MINCLE, Macrophage inducible Ca2+-dependent lectin; NLRs, Nod-like receptors; FcγRs, Fcγ receptors; SRs, scavenger receptors; DLN, draining lymph nodes, DC-SIGN, Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin.

Figure 1.4. Adaptive immune cell responses to BCG vaccination. Upon entering the + + + lymph nodes, DCs stimulate CD4 , CD8 , CD1 -restricted T cells, TFH, Treg, and B cells. CD4+ and CD8+ T cells migrate out of the lymph nodes toward the site of inoculation and + provide the necessary stimulation to innate cells. CD4 T cells differentiate into Th1, Th17 or Th2 cells depending on the stimuli present in their microenvironment and aid in the activation of macrophages, whereas CD8+ T cells mediate their functions by lysing infected cells or by secreting cytokines. B cells differentiate into Ab producing plasma cells or memory B cells. Throughout the process, memory cells arise from those that responded to the infection and populate peripheral organs, such as the lung. Together, the cells of the adaptive immune systems orchestrate the immune response in an attempt to establish mycobacteria immunity. Abbreviations: EM, environmental mycobacteria; TFH, follicular helper T cell; Treg, regulatory T cell.

Figure 1.5. Influence of route of vaccination on BCG protective efficacy and novel vaccines against M.tb. (A) Different routes of vaccination with BCG vary in their reduction of M.tb CFU in the lung of vaccinated animals. Shown is a graph compiling the relative protection conferred by distinct routes of vaccination against M.tb relative to BCG-Pasteur. The protection by BCG via subcutaneous/intradermal inoculation results in approximately one-log reduction in M.tb CFU in the lung. More immunogenic routes such as oral and intraperitoneal vaccination offer a greater degree of protection mainly due to their ability to stimulate systemic and mucosal immune responses. Immunization via BCG aerosol elicited the greatest degree of protection however this route is associated with lung tissue pathology. (B) New vaccines/concepts that have shown greater reduction of M.tb CFU in the lung of vaccinated animals compared to BCG. There is a plethora of vaccines that have been investigated for enhanced protection against M.tb compared to BCG, however here only a few have been summarized. Based on published data, the graph shows the relative number of M.tb CFU in the lung of vaccinated animals challenged with M.tb. Novel strategies, such as the induction of autophagy or blocking of the IL-10R, have been shown to enhance the protective efficacy of the BCG vaccine. Interestingly, rBCG30 was shown to be more protective than BCG RD1-2F9, both of which encode highly immunogenic antigens. To date, the rBCG ΔUre::CHly+ vaccine has been shown to be the most protective vaccines in terms of reducing∷ lung CFU. Abbreviations: SC, subcutaneous; ID, intradermal; IP, intraperitoneal; AR, aerosol.

Mycobacterial Tissue Mycobacterial Cell Wall Host Cell Sero- Cell Wall Inflammation Damage Component Receptor(s) Activity Location Inducer DC-SIGN, Outer Material α-Glucan Anti-inflammatory ND ND CR3?

Diacyl- and Triacyl- ND Pro-inflammatory ND Yes trehalose (DAT & TAT)

Higher-order phosphatidyl- MR, myo-inositol mannosides Anti-inflammatory ND Yes DC-SIGN (PIMs) Lipooligosaccharides ND Pro-inflammatory ND Yes (LOSs) TLRs, Lipomannan (LM) Pro-inflammatory ND Yes DC-SIGN Lower-order phosphatidyl- CR3, TLRs, myo-inositol mannosides Pro-inflammatory ND Yes DC-SIGN (PIMs) Mannose-capped MR, Peripheral lipid lipoarabinomannan Anti-inflammatory ND Yes DC-SIGN layer (ManLAM) Phenolic glycolipid CR3? Pro-inflammatory Yes Yes (PGL, Mycoside D) Direct Phthiocerol dimycocerosate insertion into Pro-inflammatory Yes ND (PDIM) host Membranes

Sulfolipid-1 (SL-1) ND Pro-inflammatory Yes Yes

Trehalose dimycolate Mincle-FcγR Pro-inflammatory Yes Yes (TDM) TLRs Trehalose monomycolate ND Pro-inflammatory Yes Yes (TMM)

Triglycerides TLRs Pro-inflammatory Yes ND

Arabinogalactan (AG) ND ND ND Yes

CD1 (in Ag- Cell wall Core Mycolic Acids Pro-inflammatory Yes Yes presentation Peptidoglycan Nod2 Pro-inflammatory ND ND (PG)-MDP Table 1.1. Mycobacterial cell wall components and their described host interactions.

Origin BCG Strains

• Bulgarian BCG Sophia 222 • Czechoslovakian BCG Prague • Danish Denmark/Copenhagen strain 1331* • French Original Bacille Calmete et Guérin strain Europe • French Pasteur 1173P2 • Polish BCG Poland • Romanian BCG Romania 192 • Russian BCG Moscow* • Swedish BCG Gothenburg • UK Glaxo strain 1077 • Chinese BCG Beijing • Chinese BCG Chandan Asia • Chinese BCG Lanzhou • Chinese BCG Shanghai • Japanese BCG Tokyo strain 172* • American BCG Birkhaug • American BCG Phipps North America • American BCG Tice • Canadian BCG Connaught • Canadian BCG Frappier • Mexican BCG Mexico South America • Brazilian BCG Moreau

Table 1.2. BCG substrains used for production, distribution and administration. Frequently used substrains accounting for 90% of BCG vaccine administered worldwide are indicated by *.

Chapter 2: Lung alveolar lining fluid enhances M. bovis BCG vaccine

efficacy against M. tuberculosis in a CD8+ T cell dependent manner

Abstract

Mycobacterium tuberculosis (M.tb) continues to kill millions every year. Current

tuberculosis (TB) treatments include chemotherapy and preventative vaccination with

Mycobacterium bovis Bacillus Calmette-Guérin (BCG). In humans, BCG vaccination

confers protection against childhood TB; but its efficacy wanes over time. Although

several hypotheses have been generated to explain why BCG efficacy is poor, few have

considered the pulmonary environment as a contributing factor. Our previous studies

demonstrate that exposure of M.tb to human lung mucosa [alveolar lining fluid (ALF)] can modify the M.tb cell wall, revealing alternate antigenic epitopes on the bacterium surface that effect its pathogenicity. We hypothesize that ALF modification of BCG will induce better protection against aerosol infection with M.tb. To test this hypothesis we vaccinated mice with ALF-exposed BCG, mimicking the mycobacterial cell surface properties that would be present in the lung during M.tb infection. Following M.tb challenge, ALF-exposed BCG vaccinated mice were more effective at reducing M.tb bacterial burden in the lung and spleen, and had reduced lung inflammation at late stages of M.tb infection. Improved BCG efficacy was associated with increased numbers of 78

memory and IFNγ secreting CD8+ T cells in the lung in response to M.tb challenge.

Furthermore, protection was abrogated when CD8+ T cells were depleted before M.tb

challenge. Therefore, ALF treatment can modify BCG specific host responses sufficient

to improve efficacy against a pulmonary challenge with M.tb, and our data demonstrate

the importance of CD8+ T cells in this process. We conclude that ALF modifications to

the M.tb cell wall in vivo are relevant in the context of vaccine design.

Introduction

Mycobacterium tuberculosis (M.tb) causes significant morbidity and mortality around the

globe every year. Tuberculosis (TB), the disease, is now the leading cause of death

attributed to a single infectious organism, surpassing the human immunodeficiency virus

(HIV) (434). Mathematical models predict approximately 1.7 billion individuals, a

quarter of the world’s population, to be infected with M.tb in a latent state serving as a

large reservoir for the disease (189). Current chemotherapy against TB, though effective,

has led to the rise of drug resistant strains making it more difficult to curtail this disease

(434;435). Thus, the best approach to contain and, potentially eradicate, TB may lie in the

development of an effective vaccine. Mycobacterium bovis Bacillus Calmette-Guérin

(BCG) is the only vaccine currently supported by the World Health Organization (WHO)

for the prevention of TB. However, the efficacy of BCG at preventing TB is highly

variable (198;391), ranging from 0%-80%. Furthermore, its protective immunity only appears to last for 10-15 years (436) and current vaccination boosters are ineffective

(423). Despite many efforts to develop new effective TB vaccines over the last few

decades, these approaches have resulted in little success (437;438).

During the natural course of infection with M.tb, bacilli are inhaled and deposited in the

alveolar sacs of the lung (439) where they are bathed in alveolar lining fluid (ALF) (440).

ALF is composed of surfactant lipids and its associated proteins, as well as an aqueous hypophase rich in innate host defense molecules such as hydrolytic enzymes (187), complement proteins (161), surfactant proteins A and D (441), and immunoglobulins

(442). We have shown that interaction between ALF attenuates M.tb pathogenicity in

vitro (162;187), likely due to the action of hydrolytic enzymes removing cell wall

peripheral lipids such as mannose-capped lipoarabinomannan (ManLAM) and trehalose dimycolate (TDM) (162). Thus, exposure to human ALF modifies M.tb, reducing virulence factors and exposing new antigenic motifs on its surface prior to encountering and infecting host cells. Such changes modify uptake and phagocytosis by phagocytes

(162;187), which initiate altered innate immune responses to M.tb that we consider to be influential in the generation of appropriate adaptive immune responses in vivo. It is therefore plausible to hypothesize that immunogenic cell wall motifs and virulence factors associated with M.tb are mediated by the ALF-M.tb contact during infection.

In this study we determined whether the underlying mechanism behind poor BCG efficacy may be due to discrepancies associated with the natural route of infection with

M.tb via the lung vs. inoculation with BCG via the skin. We hypothesized that ALF-

exposed BCG (with ALF cell surface modifications) would generate an immune response

against similar motifs generated on ALF-exposed M.tb that are accessible to the immune system during M.tb infection in the lung, resulting in improved control of M.tb during challenge. We identified differences in immune responses to ALF-exposed BCG vaccination in the lung, particularly within the CD8+ T cell subset. When challenged with

M.tb, ALF-exposed BCG vaccination led to a significant decrease in M.tb bacterial

burden, reduced pulmonary inflammation, and extended survival. The reduction in

bacterial burden was dependent on CD8+ T cell responses in the lung and was associated

with increased IFNγ. Hence, we provide proof of principle that changes on the BCG cell

wall surface, akin to the ones observed by M.tb after exposure to human ALF, have the

potential to generate superior host immune responses and induce better protection against

infection. Our studies highlight the importance of considering the properties of human

ALF when developing an effective vaccine against TB.

Materials and Methods

Ethics statement. All experimental procedures with animals were approved by The Ohio

State University Institutional Animal Care and Use Committee (IACUC protocol number:

2012A00000132-R1). For human subjects (to obtain BALF), this study was carried out in

strict accordance with US Code of Federal and Local Regulations [University Human

Subjects Institutional Review Board (IRB) protocol number: 2008H0135], and Good

Clinical Practice as approved by the National Institutes of Health/National Institute of

Allergies and Infectious Diseases/Division of Microbiology and Infectious Diseases

(NIH/NIAID/DMID protocol number: 12-0086). In this study only adult human subjects

participated, and all of them provided written informed consent.

Mice. Specific- pathogen-free, female mice aged 6-8 weeks of the C57BL/6J or

C3HeB/FeJ background were purchased from Jackson Laboratories (Bar Harbor, ME).

Upon arrival, mice were supplied with sterilized water and chow ad libitum and

acclimatized for at least one week prior to experimental manipulation. Mice were

maintained in micro-isolator cages located in either a standard vivarium for all

noninfectious studies or in a biosafety level three (BSL-3) core facilities for all studies

involving M.tb. Mice were divided into three groups: Mock-vaccinated (vehicle), NaCl- exposed BCG-vaccinated, or ALF-exposed BCG-vaccinated.

Growth conditions of M. tuberculosis strain Erdman and M. bovis BCG strain Pasteur.

GFP-M.tb strain Erdman (provided by Dr. Horwitz, University of California, Los

Angeles) and M. bovis BCG strain Pasteur [American Type Culture Collection (ATCC),

#35734] were grown as previously described (443). Briefly, bacterial stocks were plated weekly onto OADC [0.06ml/L oleic acid (Fisher Scientific), 5.0g/L bovine albumin

(Sigma-Aldrich, St. Louis, MO), 2.0g/L dextrose (Fisher Scientific), 0.0040g/L catalase powder (Spectrum Chemical MFG Co.), and 0.850g/L sodium chloride (Fisher

Scientific)] supplemented Middlebrook 7H11 agar and incubated at 37°C. Bacteria were harvested between 9-14 days of growth. Single cell suspensions were generated by

transferring a few loop-fulls of bacteria into a tube containing 0.9% NaCl. The clumps were dissociated by pressing the loop to the side in of the tube in a circular motion several times. The tubes were then pulse vortexed 5 times and allowed to sit for 30 min.

Supernatants were transferred into a new tube and allowed to sit for 5 min. Supernatant from the second tube were then transferred to a new tube and counted using a Petroff-

Hausser chamber.

Collection of human ALF and exposure to bacteria. Human alveolar lining fluid (ALF) was isolated from bronchoalveolar lavage fluid (BALF) donated by consented human donors in 80 ml sterile 0.9% NaCl. All protocols were approved by the Institutional

Review Board at The Ohio State University and by the Division of Microbiology and

Infectious Diseases (DMID) at the National Institute of Allergy and Infectious Disease

(NIAID). BALF was concentrated 20-fold to physiological concentrations of 1 milligram phospholipid per milliliter by the use of a 10 kDa molecular mass cutoff membrane

Centricon Plus (Amicon Bioseparations). The process removed surfactant lipids, while the functional components remained in the ALF (>10 kDa fraction). ALF (defined in this study by BALF >10 kDa fraction) was stored at -80°C until use. Single cell suspensions of freshly plated M.tb or M. bovis BCG were exposed to physiological concentrations of

ALF and incubated for 12 h at 37°C, 5% CO2. The bacteria were then washed extensively with 0.9% NaCl and resuspended at working concentrations for aerosol infections or subcutaneous injections with M.tb or M. bovis BCG, respectively.

Vaccination. Mice were subcutaneously injected in the in the scruff of the neck with 100

µl of 0.9% NaCl (saline, vehicle), 0.9% NaCl-exposed, or ALF-exposed M. bovis BCG

Pasteur (7.5×105 CFU), diluted in sterile 0.9% NaCl. Mice were housed without further

experimental manipulation for six weeks.

M. tuberculosis aerosol infection and colony forming unit (CFU) enumeration. Mice

were infected aerogenically with a low dose of M. tuberculosis Erdman using the Glas-

Col (Terre Haute, IN) inhalation exposure system. Prior to infection, a single cell

suspension of M.tb was exposed to the same ALF used to vaccinate mice as described

above. The nebulizer compartment was filled with a suspension of bacteria calculated to

deliver between 40-100 viable bacteria into the lung per mouse during a 40 min exposure

cycle. The bacterial burden was assessed at various time-points post infection by

culturing serial dilutions of organ homogenates onto Middlebrook 7H11 agar (Becton

Dickinson, San Jose, CA), supplemented with OADC. Colonies were enumerated

following 14-21 days incubation at 37°C. Data are expressed as the log10 value of the mean number of CFU recovered per organ (n=4–5 mice). Lung homogenate was also plated onto OADC supplemented 7H11 media containing 2 µg/ml of 2- thiophenecarboxylic acid hydrazide (TCH; Sigma-Aldrich) to exclude BCG growth

(310).

Lung cell isolation. Mice were sacrificed, and the lungs were cleared of blood via perfusion through the pulmonary artery with 10 ml PBS containing 50 U/ml heparin 84

(Sigma-Aldrich). Lungs were removed from the thoracic cavity and placed into 2 ml cold

DMEM (500 ml, Mediatech, Herndon, VA), supplemented with 10% heat-inactivated

FBS (Atlas Biologicals, Ft. Collins, CO), 1% HEPES buffer (1 M, Sigma-Aldrich), 1%

L-glutamine (200 nM, Sigma-Aldrich), 10 ml 100x nonessential amino acid solution

(Sigma-Aldrich), 5 ml penicillin/streptomycin solution (50,000 U penicillin, 50 mg streptomycin, Sigma-Aldrich), and 0.1% 2-Mercaptoethanol (50 mM, Sigma-Aldrich; complete DMEM [c-DMEM]). The lungs were then supplemented with 2 ml complete

DMEM containing collagenase XI (0.7 mg/ml, Sigma-Aldrich) and type IV bovine pancreatic DNase (30 µg/ml, Sigma-Aldrich), before being partially dissociated using the mouse lung dissociator program one on the gentleMACS tissue dissociator (Miltenyi

Biotec). The tissue was then incubated for 30 min at 37°C, 5% CO2. After incubation, the

tissue was further dissociated by using program two on the gentleMACS dissociator.

Digested lungs were dispersed gently through a 70 µm nylon screen to obtain a single-

cell suspension. Residual red blood cells were lysed using Gey’s lysis buffer (0.15 M

NH4Cl, 1 mM KHCO3), and washed with c-DMEM. Cell suspensions were counted using

trypan blue to exclude dead cells and resuspended at a working concentration of 1x107 cells/ml in c-DMEM or fixed for flow cytometry as described below.

Immunophenotyping by flow cytometry. Cell suspensions were prepared for flow

cytometry by first adjusting the concentration to 1x107 cells/ml in d-RPMI buffer and

incubated at 4°C for one hour. Cells (1×106) were labeled with 25 µg/ml of specific

fluorescent-labeled antibody for 30 min at 4°C in the dark followed by extensive 85

washing. For intracellular staining, 5x106 unfixed cells were first stimulated with 10

µg/ml CD3ε, 1 µg/ml of CD28, and 3 µM of monensin for 4 h at 37°C, 5% CO2. Cells

were first labeled with cell surface antibodies, permeabilized using BD

Cytofix/Cytoperm™ Plus and then labeled with intracellular antibodies as directed by

manufacturer for 30 min at 4°C in the dark. Samples were read on a Becton Dickinson

LSRII flow cytometer (in BSL3 facility) or FACSCanto II, and data were analyzed using

FlowJo version 10 software. Lymphocytes were gated according to their forward- and

side-scatter profiles, and CD4+ or CD8+ T cells were identified by the presence of

specific, fluorescent-labeled antibody in combination with CD3ε. Cell surface markers

analyzed were: FITC-conjugated CD8 (5H10-1-BioLegend), PE-conjugated CD69

(H1.2F3-BD Biosciences), PE-conjugated CD197/CCR7 (4B12-BD Biosciences), PerCP-

Cy5.5-conjugated CD44 (IM7-BD Biosciences), PerCP-Cy5.5-conjugated CD3 (145-

2C11-BD Biosciences), APC-conjugated IFNγ (XMG1.2-BD Biosciences), APC- conjugated CD62L (MEL-14-BD Biosciences), PE-Cy7-conjuated CD8 (53-6.7-BD

Biosciences), and APC-Cy7-conjugated CD4 (GK1.5-BD Biosciences). Appropriate isotype controls recommended by the manufacturer were included in each experiment and used to set gates for analysis.

Cell stimulation and ELISA. Lung cell suspensions at 1x106 in c-DMEM were plated per

well in a 96-well flat plate. Lung cells were incubated with 10 µg/ml ovalbumin (OVA)

as a negative control, 10 µg/ml M.tb H37Rv CFP, or 10 µg/ml of the mitogen concacavalin

A as a positive control at 37°C, 5% CO2 for 72 h. Following the incubation period, 86

supernatants were analyzed for levels of IFNγ and IL-4 by ELISA following manufacturer’s instructions (BD OptEIA). To assess proliferation, lung cells were incubated with the conditions described above in the presence of CFSE (CellTrace,

ThermoFisher Scientific) at 37°C, 5% CO2 for 72 h, fixed and then stained for flow cytometry. Concentration of IFNγ and IL-12p40 in lung homogenates was assessed by

ELISA (BD OptEIA) following the manufacturer’s protocol. ELISAs were read on a

Spectramax M2 Microplate reader (Molecular Devices LLC, Sunnyvale, California).

Cell depletion. Cell depletion was carried out by injecting 500 µg of anti-CD8+ (Clone:

53.6.72) depletion antibody (BioXCell, West Lebanon, NH) or whole rat IgG2a (Isotype-

Clone: 2A3) (BioXCell) in 100 µl into the intraperitoneal cavity four times over a period of two weeks beginning one day prior to M.tb infection. One lung lobe per mouse was digested to obtain single cell suspensions and stained with fluorophore conjugated antibodies to confirm CD8+ T cell depletion.

Histopathology. The middle right lung was isolated from each individual mouse and inflated with and stored in 10% neutral buffered formalin (NBF). Lung tissue was processed, sectioned, and stained with hematoxylin and eosin (H&E) for light microscopy with lobe orientation designed to allow for maximum surface area of each lobe to be seen. Sections were examined by a board-certified veterinary pathologist without prior knowledge of the experimental groups and evaluated for according to severity, granuloma size and number, cellular composition, lymphocytic cuffing, and 87

necrosis. Areas of cell aggregation (inflammation) were quantified using Aperio

Imagescope at 4x magnification by calculating the total area of the inflamed tissue over

the total area of the lobe for each individual mouse.

Statistical analysis. Statistical significance was determined using Prism 4 software

(GraphPad Software, San Diego, CA). The unpaired, two-tailed Student’s t-test was used for two group comparisons. Multiple comparisons were analyzed using one-way

ANOVA with Tukey’s post-hoc test. Log-Rank test was used to determine statistical significance of survival experiments. Statistical significance was reported as *p<0.05;

**p<0.01; or ***p<0.001.

Results

Vaccination with ALF-exposed BCG preferentially stimulates CD8+ T cells in the lung

To evaluate the impact of ALF-exposed BCG on host immune responses (independent of

M.tb infection) we subcutaneously vaccinated mice with BCG that had been exposed to

0.9% NaCl (control) or human ALF and assessed vaccine specific responses. Six weeks after vaccination, mice were euthanized and lung specific T cell responses assessed (Fig.

2.1, for gating scheme see Fig. 2.2). The total number of CD4+ and CD8+ T cells (Fig.

2.1A) in the lungs of mice that received NaCl-exposed or ALF-exposed BCG were

equivalent, with no enhancement in the number of CD4+ or CD8+ effector (CCR7-CD62-

CD44+) and memory (CCR7-CD62L+CD44+) T cell subsets, or T cells capable of

producing IFNγ (Fig. 2.1B-D). However, a significant increase in CD8+ T cells

expressing CD69 was observed in the lungs of mice vaccinated with ALF-exposed BCG

(Fig. 2.1E).

To assess mycobacterial antigen specific cytokine production we cultured lung cells from

NaCl- or ALF-exposed BCG vaccinated mice with M.tb H37Rv CFP and determined IFNγ or IL-4 secretion. Ovalbumin and concacavalin A were included as negative and positive controls, respectively (Fig. 2.3A). Lung cells from both treatment groups were equally capable of secreting IFNγ, although we observed an increasing trend in IFNγ secretion from the ALF-exposed BCG vaccinated group. Antigen specific IL-4 secretion was reduced in the ALF-exposed BCG group relative to the NaCl-exposed BCG group, suggesting a shift towards Th1 immunity (Fig. 2.3A). We further assessed antigen specific cell proliferation by measuring CFSE dilution in lung cells stimulated with M.tb

H37Rv CFP by flow cytometry from NaCl-exposed BCG or ALF-exposed BCG

vaccinated mice. No significant differences were observed between NaCl-exposed BCG or ALF-exposed BCG vaccinated mice suggesting that lymphocytes from both treatment groups were equally capable of proliferating in response to M.tb antigens (Fig. 2.3B).

Vaccination with ALF-exposed BCG reduces bacterial burden in the lung and spleen

of C57BL/6J and C3HeB/FeJ mice

To assess the efficacy of ALF-exposed BCG as a vaccine, C57BL/6J (resistant to TB

disease) and C3HeB/FeJ (susceptible to TB disease) mice were vaccinated with either

vehicle (mock-vaccinated; no BCG), NaCl- exposed BCG, or ALF- exposed BCG. Six

weeks after vaccination, all groups were infected with a low dose aerosol of ALF-

exposed M.tb [the same ALF used for BCG vaccination]. ALF-exposed, and not broth grown M.tb, was specifically used to model the composition of the M.tb cell wall after exposure to human ALF. Groups of C57BL/6J and C3HeB/FeJ mice were sacrificed at 14 days post infection (DPI) to assess early responses to M.tb infection (Fig. 2.4). Additional groups of mice were sacrificed at 250 DPI (C57BL/6J) or 100 DPI (C3HeB/FeJ), to assess later phases of infection where mice begin to display signs of progressive disease

(117). Differences in timing are because C3HeB/FeJ mice are significantly more susceptible to M.tb, with a median survival of 150 days (444).

We did not anticipate a major reduction in M.tb CFU in the lung and spleen of BCG vaccinated mice at 14 DPI, as the typical 1.0 Log10 reduction is not observed until 21-30

DPI (445;446). At day 14 post M.tb challenge, vaccination with ALF-exposed BCG

(black bars) resulted in a superior reduction in M.tb CFU in the lung beyond that afforded

by NaCl-exposed BCG (grey bars), in both C57BL/6J (0.56 log10) and C3HeB/FeJ (0.24

log10) mice (Fig. 2.4A, C). ALF-exposed BCG also conferred a better protection against dissemination in C57BL/6J mice (an additional 0.8 log10 protection), as indicated by a

significant decrease in the bacterial burden in the spleen (Fig. 2.4A). Although not

significant, the same trend was also observed in C3HeB/FeJ mice (Fig. 2.4C). Our data

demonstrate that modification of BCG through exposure to ALF can further boost the

protective efficacy of BCG in vivo, allowing for more rapid control of M.tb in the lung as

demonstrated by significantly less M.tb CFU at day 14 of infection.

We also determined M.tb CFU at 250 DPI to assess the ability of ALF-exposed BCG to

provide long term control of M.tb infection and its impact on development of progressive disease. NaCl-exposed BCG was ineffective at conferring a reduction of M.tb CFU in the lung or spleen at 250 DPI (Fig. 2.4B, D), similar to several studies that have shown limited long term protection by BCG (447). Vaccination with ALF-exposed BCG continued to confer superior protection in C57BL/6J mice. We observed a 0.56 log10

reduction in CFUs in ALF-exposed BCG vaccinated mice relative to vehicle, compared

to a 0.25 log10 reduction in NaCl-exposed BCG vaccinated mice relative to vehicle (Fig.

2.4B). This trend was also found to be statistically significant in the spleen of C57BL/6J

mice (Fig. 2.4B), whereas C3HeB/FeJ mice showed superior long term control in the

lung only (Fig. 2.4D). These data suggest that vaccination with ALF-exposed BCG can

enhance and extend the duration of protective immunity generated by BCG in the lung

and spleen of C57BL/6J mice, and the lungs of C3HeB/FeJ mice. Overall, exposure of

BCG to ALF prior to vaccination results in accelerated M.tb control (Fig. 2.4A, C; early

reduction in CFU) combined with the capacity to sustain control of M.tb for an extended

period of time (Fig. 2.4B, D; late reduction in CFU).

Vaccination with ALF-exposed BCG reduces pulmonary inflammation in the lung and

extends survival

To determine the capacity of ALF-exposed BCG to reduce severity of infection and progression of disease, the right middle lobe from each mouse was sectioned and stained with hematoxylin-eosin (H&E) to assess the degree of tissue inflammation through

quantification of cellular aggregation relative to the total size of the lung. At 14 DPI (A,

C), C57BL/6J mice showed no significant differences in tissue inflammation between the

three groups studied (Fig. 2.5C). However, both NaCl-exposed and ALF-exposed BCG

vaccinated mice had moderately more cellular infiltration (Fig. 2.5C, E), supportive of

accelerated immune responses in vaccinated mice. Additionally, mice vaccinated with

ALF-exposed BCG visually had more foci of cells compared to vehicle or NaCl-exposed

BCG-vaccinated mice (Fig. 2.5A, B), indicating that the response to M.tb infection is

accelerated in ALF-exposed BCG vaccinated mice.

At 250 DPI, the lungs of vehicle- and NaCl-exposed BCG-vaccinated C57BL/6J mice

contained abundant cellular infiltration and inflammation, with approximately 50% of the

entire lung being involved (Fig. 2.5A, D). Both control and NaCl-exposed BCG

vaccinated mice had macrophage dominated cellular aggregates in the lung that

consumed up to 40-50% of lung space (Fig. 2.5A, D). In contrast, ALF-exposed BCG vaccinated mice had significantly reduced lung cellular infiltrate relative to control and

NaCl-exposed BCG, with approximately 75% of the lung space remaining uninvolved

(Fig. 2.5D). Furthermore, the smaller cell aggregates within the lung were dominated by

lymphocytes (Fig. 2.5A, D). C3HeB/FeJ mice displayed similar phenotypes to C57BL/6J

mice at 14 DPI with little inflammation observed in all three groups (Fig. 2.5B, E) and at

100 DPI we again observed a significant decrease in the amount of infiltrating cells and

lung inflammation in ALF-exposed BCG vaccinated mice when compared to NaCl-

exposed BCG vaccinated mice (Fig. 2.5B, F). Importantly, when compared to vehicle

control, NaCl-exposed BCG vaccination showed extensive lung inflammation whereas

vaccination with ALF-exposed BCG did not.

We determined the effectiveness of the ALF-exposed BCG vaccine to extend survival after M.tb infection. Vaccination with ALF-exposed BCG significantly extended the survival of C57BL/6J mice (Fig. 2.6A) to a median of 71 weeks, with some mice surviving ∼80 weeks, demonstrating superior ability of ALF-exposed BCG to reduce TB severity. The median survival of vehicle-treated mice was 50 weeks, which is in line with previously published data (117) and NaCl-exposed BCG vaccinated mice had a median

survival of 64.5 weeks. Overall, exposure of BCG to ALF results in a vaccine that can

generate accelerated M.tb control (Fig. 2.4A; early reduction in CFU) combined with the

capacity to sustain control of M.tb for an extended period of time (Fig. 2.4B; late

reduction in CFUs), which ultimately extends survival by 20 weeks relative to non-

vaccinated mice (and an additional 6 weeks beyond NaCl-exposed BCG) (Fig. 2.6A).

Despite observing decreased bacterial burden and pulmonary inflammation in ALF-

exposed BCG vaccinated C3HeB/FeJ mice, we did not observe any extension in survival

in this TB susceptible mouse strain (Fig. 2.6B). This may be due to the rapid onset of TB observed in the C3HeB/FeJ strain.

Vaccination with ALF-exposed BCG enhances T cell responses in the lung post M.tb challenge

Given that ALF-exposed BCG vaccination alone induced increases in the number of

activated CD8+ T cells in the lung (Fig. 2.1), and reduced the bacterial burden (Fig. 2.4) and pulmonary inflammation (Fig. 2.5) following M.tb challenge; we next sought to characterize T cell responses in the lungs of vehicle, NaCl-, or ALF-exposed BCG vaccinated mice post M.tb challenge. C57BL/6J mice were vaccinated, challenged with

M.tb via aerosol, and sacrificed at 14 DPI to characterize early T cell responses in their lungs. The total number of CD8+ T cells in mice vaccinated with ALF-exposed BCG

significantly increased compared to vehicle (as well as a trend for more vs. NaCl-exposed

BCG), whereas the total number of CD4+ T cells did not differ between vaccination

groups (Fig. 2.7A, for gating scheme see Fig. 2.2).

We next characterized T cell phenotypic and functional responses in vaccinated and M.tb

challenged mice. Mice vaccinated with ALF-exposed BCG had significantly increased

numbers of CD8+ T cells that expressed a memory phenotype (CCR7-CD62L+CD44+)

relative to vehicle and NaCl-exposed BCG (Fig. 2.7B). Mice vaccinated with ALF-

exposed BCG also had an increased number of CD8+ T cells with the potential to secrete

IFNγ, relative to both vehicle and NaCl-exposed BCG (Fig. 2.7D). We did not observe

differences in the number of effector T cells (CCR7-CD62L-CD44+) in the lung (Fig.

2.7C) and CD69 expression was also unchanged (Fig. 2.7E).

The total number (Fig. 2.7A) of CD4+ T cells, CD4+ effector, memory, and activated

subsets, and CD4+ T cells capable of secreting IFNγ was not significantly different between mice vaccinated with ALF-exposed BCG and vehicle control (Fig. 2.7A-E).

These data indicate that, in contrast to NaCl-exposed BCG, vaccination with ALF- exposed BCG has a significant impact on CD8+ T cell numbers in the lung at day 14 DPI, and those cells expressed markers associated with a memory phenotype and had the capacity to secrete IFNγ. This phenotype was less apparent in the CD4+ T cell subset.

Furthermore, whole lung cells cultured in the presence of M.tb H37Rv CFP showed a trend

for increased secretion of IFNγ and decreased secretion of IL-4 (Fig. 2.8). This suggests

that vaccination with ALF-exposed BCG may favor Th1 immune responses more strongly

than vaccination with NaCl-exposed BCG. As a consequence, stronger Th1 immunity

could have help drive the faster immune responses to M.tb observed in ALF-exposed

BCG vaccinated mice.

CD8+ T cells in the lungs of ALF-exposed BCG vaccinated mice are required for enhanced protection against M.tb

Having observed significant increases in CD8+ T cell numbers and activity in the lung of

ALF-exposed BCG vaccinated mice, we explored whether the reduction in bacterial

burden observed in the lung of ALF-exposed BCG vaccinated mice (Fig. 2.4) was directly dependent on CD8+ T cells. Mice were vaccinated with vehicle, NaCl- or ALF-

exposed BCG. Six weeks later, CD8 neutralizing antibodies or isotype controls were

injected one day prior to M.tb infection and every four days thereafter. Mice were euthanized at 14 DPI to assess bacterial burden in the lung and spleen. Neutralizing antibodies successfully depleted CD8+ T cells from the lung without affecting the total

number of CD4+ T cells (Fig. 2.9A, B), and we observed the same trends in bacterial

burden in mice injected with isotype antibodies (Fig. 2.3, Fig. 2.9C). CD8+ T cell

depletion had no observable effect on M.tb burden in the lung and spleen of mice

vaccinated with the vehicle control (Fig. 2.9C), confirming that CD8+ T cells are not a

major contributor at this stage of primary M.tb infection (150;448). Interestingly, CD8+ T

cell depletion also had no effect on M.tb burden in the lung and spleen of mice that had

received the NaCl-exposed BCG vaccination (449), though M.tb burden was still

significantly reduced compared to vehicle-vaccinated mice (Fig. 2.9C). However, in contrast to the NaCl-exposed BCG group, CD8+ T cell depletion of mice that had been

vaccinated with ALF-exposed BCG lost the enhanced capacity to control M.tb infection

(Fig. 2.9C), restoring M.tb burden to that of NaCl-exposed BCG. These data indicate that

activation and expansion of CD8+ T cells by ALF-exposed BCG is directly responsible

for the enhanced protection induced by ALF-exposed BCG.

We also analyzed the levels of IFNγ and IL-12p40 in the lung of vaccinated and M.tb infected mice with or without CD8+ T cell depletion. Results showed significant

increases in IFNγ in the lungs of mice vaccinated with both NaCl- and ALF-exposed

BCG (Fig. 2.9D), but the highest level of IFNγ was associated with ALF-exposed BCG vaccination. Similar to M.tb CFU, when ALF-exposed BCG vaccinated mice were depleted of CD8+ T cells, IFNγ production in response to M.tb infection returned to levels

comparable to NaCl-exposed BCG. These data identify CD8+ T cells and IFNγ as

important contributors to the enhanced protection against M.tb challenge that is mediated

by ALF-exposed BCG vaccination. We did not observe any differences in IL-12p40

between the vaccinated/CD8+ T cell depleted groups (Fig. 2.9D), further supporting the

concept that CD8+ T cells were a dominant source of IFNγ (102;450) (and were not

independently modifying Th1 responses that would lead to changes in IL-12p40).

Altogether these findings reveal that exposure of BCG to ALF has the ability to

significantly alter host immunity and the ability to control M.tb infection in mice. Not

only did we observe significant differences in the modulation of the immune response,

we also observed significant decreases in bacterial burden and importantly, reduced

immuno-pathological damage to the lung. The reduction in bacterial burden was dependent on CD8+ T cell activity and was associated with increased levels of IFNγ in

the lung. Though we did not extend our neutralization studies to later stages of the

disease, we can postulate that the reduction in pulmonary inflammation observed at later

time-points of M.tb infection (Fig. 2.5) is directly linked to early (and potentially extended) CD8+ T cell activity in the lung.

Discussion

Host immune responses to M.tb and the requirements for its control have been well studied (102), yet immune mechanisms that result in efficient M.tb clearance from the host remain unclear. Here we show that vaccination with ALF-exposed BCG is more efficacious at reducing M.tb bacterial burden (compared to conventional NaCl-exposed

BCG) in the lung and spleen at early and late stages of M.tb infection. Mice vaccinated with ALF-exposed BCG had significantly lower bacterial burden, reduced pulmonary inflammation in the lung, and extended survival. Our data support the concept that vaccine formulations capable of rapidly containing M.tb following infection are the most efficacious (296;451;452), where earlier control of M.tb infection can translate into a reduction in pulmonary inflammation and M.tb bacterial burden at later stages and prolong survival.

We first observed that vaccination with ALF-exposed BCG was capable of altering basal immune cell populations within the lung. Although we did not observe changes to the total number of CD4+ and CD8+ T cells in the lung, we observed significant increases in the number of CD8+ T cells positive for the T cell activation marker CD69. CD69 is the earliest cell surface glycoprotein acquired by T cells during the activation process, but its

presence decreases in the absence of constant stimulus (453;454). The fact that CD69

remained significantly elevated on CD8+ T cells six weeks post vaccination suggests

there may have been a constant supply of activated innate immune cells such as

macrophages or dendritic cells capable of continuously stimulating the T cells.

Furthermore, the fact that we only observed increases in the CD8+, but not CD4+, T cell

population suggests that ALF-induced modifications may enhance the presentation of

cytosolic peptides (455). As mycobacteria typically enter cells via receptor-mediated

phagocytosis, the mechanism by which antigen is presented relies on the MHC-II

pathway. This pathway classically presents antigens to CD4+ T cells (456). However,

bacteria and/or bacterial components can also be processed and presented via the MHC-I

pathway, a process known as cross-presentation (248). Antigens presented along with

MHC-I are used to activate CD8+ T cells (248;456). Indeed, cross-presentation of

antigens via mechanisms such as apoptosis (39) and/or autophagy (410) have been shown to be effective methods of enhancing BCG vaccine efficacy (123;279;414). Additionally, we observed a trend for increased numbers of CD8+ T cells that expressed markers

associated with immunological memory, suggesting that vaccination with ALF-exposed

BCG may alter mechanisms that lead to the development of T cell memory. In this

context, the development of T cell memory largely relies on IL-2, IL-7, IL-15, and IL-

17A responses (457). Vaccination alone with ALF-exposed BCG could have altered immune responses, but not significantly enough in the absence of M.tb infection.

Other potential reasons for the increases in the number of activated T cells we observed

in ALF-exposed BCG vaccinated mice could be explained by some of our previous

observations with M.tb. We have shown that hydrolytic enzymes present in ALF are

capable of altering the cell wall of M.tb reducing the amount of ManLAM and TDM by

60% and 30%, respectively, and this in turn decreases intracellular survival

of M.tb within macrophages (162). Given the fact that the cell wall structure of M.tb and

BCG are biochemically similar (458), we anticipate comparable results when BCG is

exposed to ALF. It is well known that peripheral lipids on the cell wall of mycobacteria

such as ManLAM and TDM can inhibit innate immune processes such as P-L

fusion (337;459), and antigen processing and presentation (45;252). By inhibiting these

mechanisms, mycobacteria can effectively downregulate adaptive immune responses

(460;461) and potentially diminish the development of immunological responses as has

been shown for other chronic infections (462;463). For example, modulation of the cell

wall of BCG by components present in ALF could have mediated entry into macrophages

or dendritic cells via distinct pathways normally utilized by intact mycobacteria such as

the mannose receptor (MR), the dendritic cell-specific intercellular adhesion molecule-3-

grabbing non-integrin (DC-SIGN), or the macrophage inducible Ca2+-dependent lectin

(MINCLE), all of which have been implicated in the survival of mycobacteria within host cells (464). Reduction of virulence factors from the BCG cell wall (i.e. ManLAM, TDM)

by the action of hydrolytic enzymes in ALF could direct entry of BCG toward host-

beneficial mechanisms such as complement receptors or surfactant protein

receptors (464;465) and induce stronger innate immune responses that could

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subsequently boost memory responses. Indeed, adjuvants that stimulate innate immune

processes have been shown to augment adaptive immunity (466;467). Therefore, we

argue that interaction of mycobacteria with ALF is important in the context of BCG

vaccination, as ALF could be modulating important mechanisms involved in effective

immunity. Thus, our results could be explained by changes in the way ALF-exposed

BCG interacts with innate immune cells in the skin and how they communicate with cells

of the adaptive immune system. Identifying and recapitulating the ALF-induced changes on mycobacteria that lead to enhanced immunity would be a novel strategy to improve the current BCG vaccine.

Our observations that vaccination with ALF-exposed BCG was associated with increased

T cell activity in the lung led us to hypothesize that ALF-exposed BCG vaccinated mice would be more capable of controlling M.tb upon infection. Our data, and the scientific

literature, support the idea that the natural route of infection with M.tb involves it being

exposed to ALF upon entering the lung, where it undergoes modification prior to being

phagocytosed by resident alveolar cells. Thus, for this reason we opted to infect vehicle,

NaCl-, and ALF-exposed BCG vaccinated mice exclusively with ALF-exposed M.tb. We

observed that ALF-exposed BCG vaccinated mice displayed lower M.tb bacterial burden

in the lung and spleen early post infection (14 DPI); ALF-exposed BCG being superior to that of conventional (NaCl-exposed) BCG in both C57BL/6J and C3HeB/FeJ mouse strains, a resistant and a susceptible mouse model of TB research, respectively (451;468).

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Furthermore, at later stages of M.tb infection, mice vaccinated with ALF-exposed BCG

retained a significantly lower bacterial burden in the lung and spleen compared to vehicle

control, reduced pulmonary inflammation in the lung, and extended survival in the case

of C57BL/6J mice. Although we did not observe significant differences in bacterial

burden between NaCl- and ALF-exposed BCG vaccinated mice at later time points (DPI

250) in the C57BL/6J strain, trends for enhanced reduction in the ALF-exposed BCG group were evident. We did, however, observe significant difference between ALF- exposed BCG vaccinated mice and vehicle that was not significant between NaCl-BCG

and vehicle. In this context, it is well established that control of M.tb in BCG-vaccinated mice begins to wane (447). By the time animals succumb to TB, bacterial burdens in the lung are equal between unvaccinated and vaccinated groups. This has led the field to hypothesize that mycobacterial immunity conferred by BCG wanes as we age, a phenomenon that is supported by human studies (351).

In addition to altering early immune events in the lung, ALF-exposed BCG vaccination reduced manifestations of the disease (i.e. inflammation of the lung). Interestingly, M.tb infected C57BL/6J mice that received the ALF-exposed BCG vaccine experienced similar levels of inflammation early in the lung compared to non-vaccinated mice or mice vaccinated with the conventional BCG. However, at day 250 post-infection a 57% reduction in tissue damage was observed in the C57BL/6J mice vaccinated with ALF- exposed BCG when compared to vehicle. In this regard, conventional BCG vaccination only reduced inflammation by 19.5% in this mouse strain. Furthermore, in the TB

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susceptible C3HeB/FeJ mouse model, conventional BCG vaccination tended to

exacerbate lung inflammation, whereas the ALF-exposed BCG vaccine reduced

inflammation to less than 10% area of the lungs covered by inflammation. It is feasible

that the early recruitment of IFNγ+CD8+ T cells induced by ALF-exposed BCG

vaccination at day 14 post-infection may have simply reduced M.tb burden to low enough

levels to induce less inflammation, but an equally plausible mechanism is that long term

control may have been a more active process involving other cells. For example, we

observed significant increases in the number of activated (CD69+ and CD44+) CD4+ T

cells in the lung of ALF-exposed BCG, M.tb infected mice. Given that CD4+ T cells are

the primary mediators of protective immunity in the context of M.tb infection and BCG

vaccination and that their responses do not peak until 21 days post infection, it is

plausible that they could have played a larger role at later stages of the disease.

Interestingly, the reduced inflammation in ALF-exposed BCG vaccinated mice did not correlate with the levels of cytokines present in the lung during M.tb infection, as we

observed increases in the levels of IL-12p40 and IFNγ in lung homogenates of NaCl- exposed BCG and ALF-exposed BCG vaccinated mice at 14 days post-infection relative to vehicle. However, mice vaccinated with ALF-exposed BCG had significantly more

IFNγ in the lung compared to NaCl-exposed BCG vaccinated mice, indicating that exposure to ALF might shift towards generating Th1-skewed immune responses. As

discussed in chapter one, there is a strong correlation between mounting strong Th1

polarized responses and effectiveness of BCG vaccination. In addition, we also observed

that lung cells from ALF-exposed BCG vaccinated mice were more capable of secreting 103

more IFNγ and less IL-4 when stimulated with M.tb antigens (H37Rv CFP) compared to

NaCl-BCG vaccinated mice. Together, there is a strong indication that vaccination with

ALF-exposed BCG alters the immune profile of the lung to an extent that allows for

faster immune responses that mitigate pathology.

Our vaccination model also shows that exposure to ALF enhances the efficacy of BCG

not only in terms of reducing bacterial burden in target organs and reducing pulmonary

inflammation, but in its ability to increase the survival of vaccinated mice. C57BL/6J

mice vaccinated with ALF-exposed BCG had a significantly longer lifespan compared to

NaCl-exposed BCG vaccinated mice. Consistent with previously published data (263),

we observed lower bacterial burden and reduced pulmonary inflammation in BCG

vaccinated C3HeB/FeJ mice, but we did not observe the same survival trend in

C3HeB/FeJ mice as in C57BL/6J mice. In the C3HeB/FeJ mouse model, neither NaCl-

nor ALF-exposed BCG were effective at extending survival after M.tb infection, suggesting that this mouse model might present limitations for testing the efficacy of new

TB vaccines. One explanation for this discrepancy may lie in the differences in disease progression observed in C3HeB/FeJ mice, but not C57BL/6J. In contrast to the C57BL/6J strain, C3HeB/FeJ mice develop highly organized necrotic lesions and occasional cavitation in the lung after M.tb infection (116;469). The differences have been attributed to a region on chromosome 1 called the “super-susceptibility to tuberculosis-1” (sst1).

The sst1 allele appears to mediate the formation of caseous necrosis within pulmonary lesions and shift cellular death mechanisms from apoptosis to necrosis (116;470-472).

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Thus, the means by which the immune system responds to M.tb infection may play a

significant role in vaccine efficacy. Although we observed decreased pathology in

C3HeB/FeJ mice at 100 DPI, extensive necrosis of cells in the lung likely influenced the

overall survival of the animal and could explain why we did not observe differences

between NaCl- and ALF-exposed BCG vaccinations. Furthermore, though similar

experiments have been conducted to evaluate the efficacy of BCG in C3HeB/FeJ mice

(472), the route of vaccination and infection differed from ours [intravenous BCG

vaccination and intravenous M.tb infection (472) vs. subcutaneous BCG vaccination and

M.tb aerosol infection (ours)], suggesting that the route of immunization/infection is

important in the context of disease outcome as discussed in chapter one (197).

Additionally, the rapid onset of TB disease in C3HeB/FeJ mice compared to C57BL/6J

mice might explain the differences in survival (470).

The functionality of CD8+ T cell in BCG vaccination has not been extensively addressed

(473). BCG-specific cytotoxic CD8+ T cell responses in humans are reported (278); and

although it is thought that BCG induces poor CD8+ T cell responses in vivo, human

studies line up with in vitro studies in newborns showing that BCG vaccination drive

robust antigen-specific CD8+ T cell responses characterized by increased levels of IFNγ,

degranulation, proliferation, and elevated levels of cytotoxic proteins (474). We

established that the ability of ALF-exposed BCG to control M.tb infection throughout the first 14 days was mediated by CD8+ T cells, with CD8+ T cell depletion reverting ALF-

exposed BCG back to similar control as NaCl-exposed BCG. Furthermore ALF-exposed

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BCG vaccination led to increased numbers of activated (CD69+) CD8+ T cells within the lung, and an increased number of memory phenotype and IFNγ secreting CD8+ T cells in

the lungs after M.tb challenge. IFNγ levels in the lung were also reduced when CD8+ T cells were depleted. Thus, exposure of BCG to ALF resulted in a modified vaccine that could localize an increased number of activated CD8+ T cells to the lung post-

vaccination. We anticipate that these cells matured to form a reservoir of memory CD8+

T cell which could recognize and respond quickly upon encountering M.tb. This reservoir

of memory CD8+ T cell could play a key role in protection as resident memory

lymphocytes are critical for protective immune responses post BCG vaccination (119).

Furthermore, the reduction of bacterial burden in the lung and spleen of M.tb-infected

mice receiving the ALF-exposed BCG vaccine was likely mediated by CD8+ T cell

derived IFNγ as IFNγ concentration in the lung was directly dependent on the presence or absence of CD8+ T cells, despite similar numbers of CD4+ T cells in the lung. We did

not, however, perform experiments looking into whether IFNγ was directly responsible

for the decrease in M.tb bacterial burden. Although, the literature strongly supports the

fact that IFNγ is one of the primary mediators of mycobacterial immunity as discussed in

chapter one, it is still possible that our observations were not due to IFNγ, but still

dependent on CD8+ T cells. Although the secretion of IFNγ, TNF, and IL-2 are the major

effector functions of CD8+ T cells in the context of M.tb infection (269), other

mechanisms such as the cytotoxic functions of CD8+ T cells could have been amplified

by vaccinating with ALF-exposed BCG. As mentioned previously, our observations 106

could be explained by increases in cross-presentation of antigen, a process whereby

APCs present exogenous antigens via MHC class I to CD8+ T cells via mechanisms such

as apoptosis and autophagy (39;410;475). Direct lysis of host cells via the secretion of

granules containing perforin and granzyme (123) or engagement of the Fas-FasL

receptors to induce apoptosis (279) could have been affected. One such way these

mechanisms could have been engaged is if ALF-exposed BCG was superior to conventional BCG at escaping the phagosome. Translocation of BCG to the cytosol could relay “danger” signals to effector CD8+ T cells leading to the engagement of apoptosis- inducing killing mechanisms such as degranulation and Fas-mediated apoptosis as it has been shown for M.tb. As mentioned above, translocation of BCG to the cytosol could also have resulted in activation of autophagy-mediated killing mechanisms all of which could amplify cross-presentation of antigen (414). Any of these mechanisms could lead to the early M.tb control we observe, and we can also speculate that they contribute to the extended control of M.tb infection and reduced pulmonary inflammation that are observed as CD8+ T cells have typically been associated with containment of M.tb

infection (476).

Overall, vaccination with BCG effectively accelerated immune responses allowing mice

to contain the infection more rapidly than non-vaccinated groups thereby stunting M.tb

growth sooner. This accelerated control of M.tb translated to reduced pulmonary

inflammation and extended survival. ALF-exposed BCG was significantly better than

conventional BCG (NaCl-exposed BCG) at accelerating immune responses in the lung.

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Furthermore, our data highlight the importance of vaccine formulations that can rapidly contain M.tb following infection, (203;296;452), where earlier control of M.tb infection can translate to a reduction in pulmonary inflammation and M.tb bacterial burden at later stages, and extend survival. Thus, our data, and those of others, suggests an important parameter for vaccines that effectively prevent PTB should be how rapidly the vaccine can induce immune responses in the lung prior to and upon infection with M.tb. We extend on this concept by implementing a vaccine strategy that is directed against the phenotype of M.tb within the lung ALF, and demonstrate a clear enhancement of BCG protective efficacy. Based on our results, we speculate the mechanism likely lies within the innate immune branch. Specifically, we believe the absence of virulent glycolipids, such as ManLAM and TDM, on the BCG cell wall that inhibit innate immune responses enables macrophages to process and present BCG more effectively to adaptive immune cells.

The fact that exposure to ALF was able to increase the efficacy of BCG vaccination in terms of reducing bacterial burden and reducing pulmonary inflammation suggests that manipulation of BCG, particularly with emphasis on its cell wall composition at the time of vaccination, offers novel avenues to understand and improve on the development of protective immunity to mycobacteria. The development of vaccines against TB have largely centered around genetic manipulation of BCG, often focusing on the deletion or addition of proteins to the genome as discussed in chapter one (198). However, this “trial and error” approach has not significantly expanded our current understanding of

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mycobacterial immunity. Here we suggest an alternate approach that considered the

properties of the lung environment that M.tb resides within. Identifying and recapitulating

the ALF induced changes on mycobacteria (either by genetic manipulation or temporary

biologically induced changes) would be a novel strategy to improve the current BCG vaccine. As the cell wall is the first component recognized by innate immune cells, we argue that the first interaction between the host and the pathogen will ultimately dictate the efficacy of the vaccine. Altogether our research highlights the complexities of the first interaction of mycobacteria with the host. Understanding the changes brought forth to

BCG by the action of ALF, particularly in the context of exploiting the biochemical properties of the mycobacterial cell wall, can lead to the development of more efficacious vaccines against TB.

One large caveat associated with these studies was the fact that we were utilizing human derived ALF to induce changes to the cell wall of BCG. Although we were able to discover an important role for CD8+ T cells in the early control of M.tb infection in vaccinated individuals, the use of ALF in large vaccination programs poses significant challenges. Not only would it be controversial due to the fact that ALF is derived from humans, but it would be difficult to standardize as the composition of ALF as it varies substantially from person to person (162;187). For these reasons we sought a method that could modify the cell wall of BCG to a similar extent, but could also be standardized. In chapter three, we discuss the use of the organic solvent petroleum ether to achieve this.

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Figure 2.1. Vaccination with ALF-exposed BCG preferentially stimulates CD8+ T cells in the lung. C57BL/6J mice were vaccinated with NaCl-exposed BCG (NaCl-BCG; white bars) or ALF-exposed BCG (ALF-BCG; grey bars). Six weeks later, lungs were digested and single cell suspension of cells were labeled with fluorescent antibodies specific for CD8 or CD4 in combination with CD69, CD62L, CCR7, CD44, and intracellular IFNγ. (A) Total number of CD8+ and CD4+ T cell in the lung. (B) Total number of CD8+ or CD4+ T cells with a memory (CCR7-CD62L+CD44+) phenotype. (C) Total number of CD8+ or CD4+ T cells with an effector (CCR7-CD62L-CD44+) phenotype. (D) Total number of CD8+ or CD4+ T cells with the potential to produce IFNγ. (E) Total number of CD8+ or CD4+ T cells expressing CD69. The absolute number of T cells in the lung is shown. Representative experiments from n=2 with 5 mice per group, mean ± SEM; Student’s t-test, *p<0.05; Abs: absolute number.

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Figure 2.2. Gating scheme of lymphocytes. Lung lymphocytes were first gated based on their size (FSC) and granularity (SSC). Lymphocytes were split into CD4+ and CD8+ cells. Each lymphocyte subset (CD4+ or CD8+) was gated for CCR7- negative cells. The CCR7- gated cells were further sub-gated using CD62L and CD44. Within the gate, memory cells (CCR7-CD62L+CD44+) reside within Q2, while effector cells (CCR7- CD62L-CD44+) reside in Q3. In a second panel, each lymphocyte subset (CD4+ or CD8+) was gated for CD69. In a third panel, lung cells previously stimulated with CD3/CD28 were gated based on lymphocyte subset (CD4+ or CD8+) and gated for IFNγ.

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Figure 2.3. Vaccination with ALF-exposed BCG preferentially stimulates Th1 responses and does not affect proliferation of T cells. C57BL/6J mice were vaccinated with NaCl-exposed BCG (NaCl-BCG; white bars) or ALF-exposed BCG (ALF-BCG; grey bars). Six weeks later, lungs were digested and single cell suspensions were generated. (A) Lung cells were cultured in the presence of ovalbumin (OVA-negative control), M.tb H37Rv CFP, or concacavalin A (ConA-positive control) for 72 h. Secretion of IFNγ and IL-4 was quantified in cell culture supernatants by ELISA. (B) Lung cells were labeled with CFSE and stimulated with M.tb H37Rv CFP for 72 h. Cell were then collected and labeled with fluorescent antibodies specific for CD8+ or CD4+ T cells. Proliferation was determined by using FlowJo’s proliferation tool. Representative experiments from n=2 with 5 mice per group, mean ± SEM; Student’s t-test, *p<0.05; ns: not significant.

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Figure 2.4. Vaccination with ALF-exposed BCG reduces M.tb bacterial burden in the lung and spleen of C57BL/6J and C3HeB/FeJ mouse strains. C57BL/6J and C3HeB/FeJ mice were vaccinated with 0.9% NaCl (vehicle; white bars), NaCl-exposed BCG (NaCl-BCG; grey bars), or ALF-exposed BCG (ALF-BCG; black bars). Six weeks later, mice were infected with a low dose aerosol of ALF-exposed M.tb. (A, B) C57BL/6J mice were sacrificed at 14 and 250 days post infection and M.tb CFU determined in lung and spleen. Representative experiments from n=3 with 4-5 mice per group per time-point studied, mean ± SEM; one-way ANOVA with Tukey’s post-hoc test, *p<0.05, **p<0.01, ***p<0.001. (C, D) C3HeB/FeJ mice were sacrificed at 14 and 100 days post infection and M.tb CFU determined in lung and spleen. Data from n=1 with 5 mice per group per time-point, mean ± SEM; *p<0.05; ns: not significant.

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Figure 2.5. Vaccination with ALF-exposed BCG reduces pulmonary inflammation in the M.tb infected lung of vaccinated mice. C57BL/6J and C3HeB/FeJ mice were vaccinated with 0.9% NaCl (vehicle; white bars), NaCl-exposed BCG (NaCl-BCG; grey bars), or ALF-exposed BCG (ALF-BCG; black bars). Six weeks later, mice were infected with a low dose aerosol of M.tb. (A) At 14 and 250 DPI for C57BL/6J or (B) 14 and 100 DPI for C3HeB/FeJ, mice were sacrificed, and lungs fixed in 10% NBF, embedded in paraffin, sectioned, and stained with hematoxylin and eosin to visualize tissue morphology. Areas of cell aggregation and infiltration (inflammation) were quantified using Aperio Imagescope by calculating the area of inflammatory foci (i.e. involvement) divided by the total area of the lung; (C) C57BL/6J 14 DPI, (D) C57BL/6J 250 DPI, (E) C3HeB/FeJ 14 DPI, and (F) C3Heb/FeJ 100 DPI. For C57BL/6J mice, a representative experiment from n=3 with 4-5 mice per group per time-point, mean ± SEM; one-way ANOVA with Tukey’s post-hoc test, *p<0.05, ***p<0.001. For C3HeB/FeJ, data are from n=1 with 5 mice per group per time-point, mean ± SEM; one-way ANOVA with Tukey’s post-hoc test *p<0.05; ns: not significant. Representative images at a final magnification of 20X.

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Figure 2.6. Vaccination with ALF-exposed BCG extends survival of C57BL/6J mice, but not C3HeB/FeJ mice. (A) C57BL/6J mice were vaccinated with 0.9% NaCl (vehicle; white circles), NaCl-exposed BCG (NaCl-BCG; grey diamonds) or ALF- exposed BCG (ALF-BCG; black circles) and challenged with M.tb six weeks later. Survival was monitored across a period of 85 weeks. Mice were euthanized when they met the exclusion criteria documented in animal care and use protocols, and the date documented. Mice receiving vehicle displayed a median survival of 50.00 weeks. NaCl- BCG vaccinated mice had a median survival of 64.50 weeks. ALF-BCG vaccinated mice had a median survival of 71.00 weeks. Pooled experiment from n=3 with 4-6 mice, mean ± SEM; Log-rank test. (B) C3HeB/FeJ mice were vaccinated with 0.9% NaCl (vehicle; white circles), NaCl-exposed BCG (NaCl-BCG; grey diamonds) or ALF-exposed BCG (ALF-BCG; black circles) and challenged with M.tb six weeks post vaccination. Survival was monitored across a period of 33 weeks. Vehicle-vaccinated mice displayed a median survival of 30.50 weeks. NaCl-BCG vaccinated mice had a median survival of 32.00 weeks while ALF-BCG vaccinated mice had a median survival of 26.00 weeks. Data from n=1 with 5-8 mice per group, mean ± SEM; Log-rank test.

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Figure 2.7. Vaccination with ALF-exposed BCG enhances CD8+ T cell responses in lung of M.tb infected mice. C57BL/6J were vaccinated with 0.9% NaCl (vehicle; white bars), NaCl-exposed BCG (NaCl-BCG; grey bars), or ALF-exposed BCG (ALF-BCG; black bars). Six weeks post vaccination, mice were challenged with M.tb and euthanized at 14 DPI to characterize immune cell populations in the lung by flow cytometry. (A) Total number of CD8+ and CD4+ T cell in the lung. (B) Total number of CD8+ or CD4+ T cells with a memory (CCR7-CD62L+CD44+) phenotype. (C) Total number of CD8+ or CD4+ T cells with an effector (CCR7-CD62L-CD44+) phenotype. (D) Total number of CD8+ or CD4+ T cells with the potential to produce IFNγ. (E) Total number of CD8+ or CD4+ T cells expressing CD69. Representative experiment from n=2 with 5 mice per group, mean ± SEM; one-way ANOVA with Tukey’s post-hoc test, *p<0.05, **p<0.01; ns: not significant; Abs: absolute number.

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Figure 2.8. Vaccination with ALF-exposed BCG preferentially stimulates Th1 responses in the M.tb infected lung. C57BL/6J were vaccinated with 0.9% NaCl (vehicle; white bars), NaCl-exposed BCG (NaCl-BCG; grey bars), or ALF-exposed BCG (ALF-BCG; black bars). Six weeks post vaccination, mice were challenged with M.tb and euthanized at 14 DPI to characterize antigen responses of lung cells to different stimuli including M.tb-specific antigens. Single cell suspensions were cultured in the presence of ovalbumin (OVA-negative control), M.tb H37Rv CFP, or concacavalin A (ConA-positive control) for 72 h at 37°C, 5% CO2. Production and secretion of IFNγ and IL-4 were quantified by ELISA. Representative experiment from n=2 with 5 mice, mean ± SEM.

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Figure 2.9. Reduction in bacterial burden associated with ALF-exposed BCG is dependent on CD8+ T cell responses. C57BL/6J mice were vaccinated with 0.9% NaCl (vehicle), NaCl-exposed BCG, or ALF-exposed BCG. Six weeks after vaccination, mice were i.p. injected with 500µg of anti-CD8, or rat anti-mouse IgG2a and challenged with M.tb. Anti-CD8 or rat anti-mouse IgG2a was delivered one day prior to M.tb challenge and every 4 days thereafter. Mice were sacrificed at 14 DPI. (A) Schematic depicting effective depletion of CD8+ T cells from the lung. (B) Quantification of CD8+ and CD4+ T cells in the lung of isotype-injected (white bars) or anti-CD8-injected (black bars) mice. CD8+ T cells were depleted to less than 20 cells per 50,000 cells. Depletion of CD8+ T cells did not affect the number of CD4+ T cells in the lung. Pooled experiment from n=2 with 5 mice/per group, mean ± SEM; student’s t-test, ***p<0.001. (C) M.tb CFU were determined in the lung and spleen at 14 DPI. Pooled experiment from n=2 with 5 mice/per group, mean ± SEM; one-way ANOVA with Tukey’s post-hoc test or student’s t-test (for single comparisons), *p<0.05, **p<0.01, ***p<0.001, §p<0.05, §§§p<0.01; ns: not significant. (D) IFNγ and IL-12p40 in the lung homogenates of vehicle-treated or vaccinated mice with or without CD8+ T cells depletion at 14 DPI. Pooled experiment from n=2 with 5 mice/per group, mean ± SEM, one-way ANOVA with Tukey’s post-hoc test (for multiple groups) or student’s t-test (for single comparisons), *p<0.05, **p<0.01, ***p<0.001.

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Chapter 3: Selective delipidation of Mycobacterium bovis BCG enhances its vaccine potential against Mycobacterium tuberculosis infection by

accelerating IL-17A responses in the lung

Abstract

Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis (TB), is now the

leading cause of death due to an infectious organism. Though effective, TB treatment

requires several months to years of chemotherapy and poor outcome has led to the rise of

drug-resistant strains that threaten current eradication efforts. Mycobacterium bovis

Bacillus Calmette-Guérin (BCG) is the only vaccine approved for the prevention of TB,

however its efficacy against pulmonary TB (PTB) is poor. BCG is currently inoculated

intradermally while the natural route of infection with M.tb is via the lung. This disparity

is likely one of the underlying factors behind the poor efficacy of BCG against PTB.

Excessive lung pathology caused by direct delivery of BCG into the lungs has prevented

the use of this route of immunization. Here we show that chemical treatment of BCG

with petroleum ether removes trehalose dimycolate, phenolic glycolipid-1, mycoside B,

and other lipids from the bacterial surface without affecting the viability of BCG.

Pulmonary vaccination with delipidated BCG (dBCG) attenuated inflammatory responses

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and prevented immunopathology of the lung, abrogating the major concern for the use of

this route of immunization. Importantly, dBCG pulmonary vaccination was significantly

superior to conventional BCG at reducing bacterial burden in the lung and peripheral

organs of M.tb infected mice and reduced immunopathology of the lung caused by M.tb.

This was associated with increased numbers of effector and central memory T cells

populating the lung. dBCG pulmonary vaccination followed by M.tb challenge increased

the numbers of CD69+ and IL-17A+, but not IFNγ+, CD4+ and CD8+ T cells residing

within the lung, indicating that IL-17A responses are responsible for the improved anti-

mycobacterial immunity observed in dBCG-vaccinated mice. These results provide evidence that delipidation of BCG offers a novel, safe, and effective pulmonary vaccine against M.tb that could easily be expedited into clinical trials.

Introduction

Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis (TB), continues to cause significant morbidity and mortality around the world and the rise of extensive-,

extreme-, and total-drug resistant M.tb endanger eradication efforts (477;478). Despite

successful treatments, TB is unlikely to be eradicated without an effective vaccine. The

only currently licensed vaccine against TB, Mycobacterium bovis Bacille Calmette-

Guérin (BCG), is ineffective against pulmonary TB (PTB) despite it being efficacious

against other forms of mycobacterial disease such as TB meningitis and miliary TB

(391). One potential explanation for this discrepancy may lie in the route of

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immunization as BCG is administered intradermally whereas M.tb is a natural airborne pathogen (479). This noted disparity may not confer proper anti-mycobacterial immunity in the lung. To circumvent this problem, research has shifted toward direct pulmonary vaccination with BCG, however excessive pulmonary inflammation caused by BCG has hindered the transition to a pulmonary vaccine (382;480).

As direct immunization of the lung has been consistently shown to be more effective against M.tb infection in animal models (275;383;481-483), efforts have been made to transition BCG towards a pulmonary vaccine. However, evidence in the literature suggests that BCG is too pathogenic for direct pulmonary inoculation (382;480). For this reason no human clinical trial has been yet implemented to evaluate the efficacy of pulmonary BCG vaccination against M.tb. The primary reason behind the excessive inflammation and immunopathology caused by mycobacteria to the lung is attributed to potent toxic lipids such as trehalose dimycolate (TDM) (295;484), di- and tri- acylglycerols (DAG/TAG) (485), phthiocerol dimycocerosates (PDMIs) (66;486),

phenolic glycolipids (PGLs) (487), among others, present on the BCG cell wall. These

lipids induce rapid and robust innate immune responses that lead to tissue inflammation

and damage (60;487). Recombinant BCG strains deficient in specific lipids or

combination of these lipids were incapable of mounting sufficient immune responses to

protect mice against M.tb (488). Thus, it is suggested that absence of inflammatory lipids

is detrimental to the generation of immunity, but their presence is responsible for much of

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the excessive inflammation that can lead to lung tissue damage. Hence, a balance must be

struck to attain optimal immunity to M.tb in the lung.

In this study we show that treatment of BCG with the organic solvent petroleum ether

(PE) extracts many of the inflammatory lipids including TDM, PGL-Tb1, MycB, and

some TAGs and PDIMs without affecting the viability of the bacteria. Furthermore, using

primary human cells we demonstrated that delipidated BCG (dBCG) was significantly

attenuated compared to conventional BCG. This observation led us to hypothesize that

delipidated BCG could be used as an effective pulmonary vaccine as it would bypass the

excessive inflammatory responses associated with this route of vaccination while

retaining the efficacy associated with direct inoculation of the lung. Here we demonstrate

that pulmonary vaccination with dBCG was associated with lower levels of inflammation

in the lung compared to conventional BCG. Furthermore, dBCG was significantly

superior to conventional BCG at reducing M.tb bacterial burden in the lung and

peripheral organs of infected mice. We show that dBCG vaccination was associated with

increased numbers of effector and memory T cell populations in the lung at the time of

infection with M.tb. Furthermore, we report that vaccination with dBCG increased CD69+

and IL-17A+, but not IFNγ+, T cell responses in the lung. Thus, our data suggest that the

superior ability of dBCG to protect against M.tb may not have been due to enhanced

IFNγ responses, but rather IL-17A. Together our result provide proof of concept that dBCG can be easily adapted into a pulmonary vaccine with minimal safety concerns and enhanced efficacy against M.tb. 122

Materials and Methods

Ethics statement. All experimental procedures with animals were approved by The Ohio

State University Institutional Animal Care and Use Committee (IACUC protocol number:

2012A00000132-R1). For human subjects, this study was carried out in strict accordance

with US Code of Federal and Local Regulations [University Human Subjects Institutional

Review Board (IRB) protocol numbers: 2007H0262 and 2008H0119]. In this study only

adult human subjects participated, and all of them provided written informed consent.

Mice. Specific- pathogen-free, female mice aged 6-8 weeks of the C57BL/6J background were purchased from Jackson Laboratories (Bar Harbor, ME). Upon arrival, mice were supplied with sterilized water and chow ad libitum and acclimatized for at least one week prior to experimental manipulation. Mice were maintained in micro-isolator cages located in either a standard vivarium for all noninfectious studies or in a biosafety level three

(BSL-3) core facility for all studies involving M.tb. Mice were divided into three groups: vehicle-vaccinated (PBS), PBS-treated BCG-vaccinated (BCG), or PE-treated BCG- vaccinated (dBCG).

Growth conditions of mycobacteria and delipidation of M. bovis BCG strain Pasteur.

GFP-M.tb Erdman (provided by Dr. Horwitz, UCLA, CA) and M. bovis BCG Pasteur strains [American Type Culture Collection (ATCC), #35734] were grown as previously

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described in chapter two. Freshly plated M. bovis BCG were harvested between 9-14 days of growth into siliconated tubes (Fisher Scientific, Hampton, NH), resuspended in 1 ml of petroleum ether (PE) or phosphate buffered solution (PBS) and vortexed for two min, rested for five min, and then pelleted at 6000 g for five min. The procedure was repeated three times. The supernatants from the PE treated bacteria were collected, dried under N2, and kept at -20°C until further analysis. Treated bacteria used for in vitro or in

vivo studies were dried briefly to evaporate excess solvent, washed twice in PBS, and

resuspended in PBS prior to use. The viability of BCG and dBCG was assessed by

performing serial dilutions, plating the bacteria onto Middlebrook 7H11 agar

supplemented with OADC, and counting colonies three to four weeks later as we have

described (162).

Analysis of extracted lipids. Lipid extracts were analyzed by thin layer chromatography

(TLC). PE extracts were dried under N2 and stored at -20°C until use. To assess the

amount of certain lipids that remained on the cell wall of BCG post PE extraction, PE-

treated bacteria were resuspended in a solution of chloroform-methanol (C:M, 2:1, v/v)

and placed at 37°C for 12 h. The bacteria were then spun out and the lipids were dried

under N2 and stored at -20°C until use. Total lipids (TL) were obtained by extracting

BCG in a solution of C:M (2:1, v/v) and placed at 37°C for 12 h. All extracts were

resuspended in C:M (2:1, v/v) solution at a final concentration of 10 µg/µl. 100-200 µg of

dry-weight lipids were spotted onto aluminum-backed TLC plates and developed in the

following solvent systems; TDM, mycoside B (MycB), and PGL-Tb1

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(chloroform/methanol 95:5, v/v), phthiocerol dimycocerosates (PDMIs) and triglycerides

(TAGs) (petroleum ether/acetone 96:4, v/v) and phosphatidyl myo-inositol mannosides

(PIMs; chloroform/acetic acid/methanol/water 40:25:3:6, v/v/v/v). Semi-quantitative

analysis was performed using the NIH software ImageJ.

Intranasal vaccination with BCG and M. tuberculosis aerosol infection. C57BL/6J

mice were anesthetized with an aerosolized solution containing 2-5% isoflurane. A single

cell suspension of BCG treated with PBS (BCG) or PE (dBCG) containing approximately

5x105 viable bacilli in 50 µl was injected evenly between the two nostrils. Following

administration, mice were held in an upright position for 15 seconds to ensure the entire

inoculum was inhaled and then returned to their cage and monitored until recovery. At

the indicated time post vaccination, mice were euthanized, and the lung was removed and

processed for histological analysis, CFU enumeration, or lung cell isolation as described

below. Bacterial burden in the lung of vaccinated mice was assessed by culturing serial

dilutions of organ homogenates onto Middlebrook 7H11 agar, supplemented with OADC

as we have described (303). Colonies were enumerated after 3-4 weeks incubation at

37°C. Data are expressed as the log10 value of the mean number of CFU recovered per

organ (n=4–5 mice). A separate group of mice were immunized with BCG or dBCG, rested for 50 days, and then infected via aerosol with a low dose of M.tb using the Glas-

Col (Terre Haute, IN) inhalation exposure system as described (489). Briefly, the

nebulizer compartment was filled with a suspension of M.tb calculated to deliver 40-100

viable bacteria into the lung. Mice were sacrificed at various time-points post infection,

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the lung, spleen, liver, and mediastinal lymph node (MLN), were aseptically removed into sterile saline and the bacterial burden was assessed by culturing serial dilutions of organ homogenates onto OADC supplemented Middlebrook 7H11. Colonies were enumerated after 3-4 weeks incubation at 37°C. Data are expressed as the log10 value of the mean number of CFU recovered per organ (n=4–5 mice). For M.tb challenge studies, organ homogenates were also plated onto OADC supplemented 7H11 media containing 2

µg/ml of 2-thiophenecarboxylic acid hydrazide (TCH; Sigma-Aldrich, St. Louis, MO) to exclude BCG growth (310).

Histopathology. The middle right lung was isolated from each individual mouse and inflated with and stored in 10% neutral buffered formalin. Lung tissue was processed, sectioned at 4-5 µm, and stained with hematoxylin and eosin (H&E) for light microscopy with lobe orientation designed to allow for maximum surface area of each lobe to be visualized. Sections were examined by a board-certified veterinary pathologist without prior knowledge of the experimental groups and graded according to severity, granuloma size and number. H&E-stained slides were digitized for morphometric analysis using

Aperio ScanScope XT slide scanner (Leica, Buffalo Grove, IL) at 40X magnification.

Immune cell infiltration and granulomatous tissue was calculated by manually outlining all foci and determining the total area of inflammation as a percentage of the total area of the lung.

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SDS-PAGE and whole-cell ELISA for ManLAM. Several loop-fulls of BCG underwent

PE extraction and subsequently C:M (2:1, v/v) extraction as described above. The extracts were dried under N2 and resuspended in dimethyl sulfoxide at a final concentration of 10 µg/µl. 100 µg of each sample were separated by SDS-PAGE (12%) and stained with silver nitrate plus periodic acid (162;187). Surface-exposed ManLAM on treated M. bovis BCG bacilli was analyzed by ELISA using anti-ManLAM monoclonal antibody CS-35 (443;490). To do this, BCG bacilli were subjected to PE or

PBS treatment as described, immobilized on a medium-binding 96-well ELISA plate and dried overnight. The plates were blocked with 1% BSA in PBS with 0.05% Tween-20 for

2 h. The plates were then probed with a 1 µg/ml of rabbit anti-human CS-35 Ab overnight. The following day, the binding was assayed by standard ELISA technique using a secondary anti-rabbit HRP-conjugated antibody (162).

Isolation, preparation, and in vitro infection of human macrophages. Monocyte- derived macrophage (MDM) monolayers for cytokine and CFUs enumeration were prepared from healthy tuberculin-negative human volunteers (234). Whole blood was collected and separated using Ficoll-Paque differential centrifugation. Peripheral blood mononuclear cells (PBMCs) were isolated and differentiated over a five day period in

Teflon wells (Thermo Fisher Scientific, Waltham, MA) containing RPMI 1640 plus 20% autologous serum at 37ºC, 5% CO2. Macrophages were then collected and plated onto tissue culture plates and allowed to differentiate for an additional seven days in RPMI

1640 containing 20% autologous serum. Macrophages were infected with a single cell 127

suspension of PBS- or PE-treated bacteria at a multiplicity of infection (MOI) 1:1 or 10:1

for 2 h (162). Supernatants were collect at each time point and stored at -80°C until

further analysis. To enumerate CFUs, cells were lysed by first incubating the cells in (500

µg/ml) DNase in H2O for 10 min at 4°C followed by an addition 10 min in 0.25% sodium

dodecyl sulfate (SDS) solution. The SDS was quenched by adding a 20% solution of

human serum albumin (BSA) and serial dilutions were plated onto OADC supplemented

7H11 agar plates (162). CFUs were counted 3-4 weeks later. Images of macrophage monolayers were obtained on an Olympus CKX41SF2 microscope using an Olympus

DP71 digital camera at a final magnification of 100x.

Lung cell isolation. Lung cell were isolated as described in chapter two. The lungs were cleared of blood via perfusion through the pulmonary artery with 10 ml PBS containing

50 U/ml heparin and placed into 2 ml cold complete-DMEM (c-DMEM) [DMEM

(Mediatech, Herndon, VA), supplemented with 10% heat-inactivated FBS (Atlas

Biologicals, Ft. Collins, CO), 1% HEPES buffer (1 M, Sigma-Aldrich), 10 ml 100x nonessential amino acid solution (Sigma-Aldrich), 5 ml penicillin/streptomycin solution

(50,000 U penicillin, 50 mg streptomycin, Sigma-Aldrich), and 0.1% 2-mercaptoethanol

(50 mM, Sigma-Aldrich)]. The lungs were then supplemented with 2 ml complete

DMEM containing collagenase XI and type IV bovine pancreatic DNase, before being partially dissociated using the mouse lung dissociator program one on the gentleMACS tissue dissociator (Miltenyi Biotec). Following 30 min of incubation at 37°C, 5% CO2,

the tissue was further dissociated by using program two on the gentleMACS dissociator.

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Digested lungs were dispersed gently through a 70 µm nylon mesh to obtain a single-cell

suspension. Red blood cells were lysed using Gey’s lysis buffer, and washed with c-

DMEM. Cell suspensions were counted using trypan blue to exclude dead cells and

resuspended at a working concentration in c-DMEM or fixed for flow cytometry as

described below.

Analysis of immune cells by flow cytometry. Cells were prepared for flow cytometry as described in chapter two. Briefly, lung cell suspensions were adjusted to 1x107 cells/ml

with FACS buffer supplemented with 0.1% sodium azide and incubated at 4°C for one

hour. For intracellular staining, 2.5x106 unfixed cells were first stimulated with 10 µg/ml

CD3ε, 1 µg/ml of CD28, and 3 µM of monensin for 4 h at 37°C, 5% CO2 (303). Cells

were then labeled with cell surface markers, permeabilized using BD

Cytofix/Cytoperm™ Plus and then labeled with intracellular markers as directed by

manufacturer for 25 min at 4°C in the dark. Cells (1×106) were labeled with 25 µg/ml of specific fluorescent-labeled antibody for 30 min at 4°C in the dark followed by two washes with FACS buffer. Samples were read on a Becton Dickinson LSRII flow cytometer, and data were analyzed using FlowJo version 10 software. Lymphocytes were gated according to their forward- and side-scatter profiles, and CD4+ or CD8+ T cells were identified by the presence of specific, fluorescent-labeled antibody in combination with CD3ε. Innate immune cells were blocked with CD16/CD32 (Fc block-BD

Biosciences, East Rutherford, NJ) prior to staining. Antibodies used for phenotyping were: FITC-conjugated Gr-1 (RB6-8C5- BioLegend), FITC-conjugated CD3ε (145- 129

2C11-BD Biosciences), FITC-conjugated CD44 (IM7-BD Biosciences), FITC-

conjugated CD8a (53-6.7-BioLegend), PE-conjugated CD4 (H129.19-BD Biosciences),

PE-conjugated CD4 (RM4-5-BD Biosciences), PE-conjugated IL-17A (TC11-18H10.1-

BioLegend) PerCP-Cy5.5-conjugated CD11b (M1/70-BD Biosciences), PerCP-Cy5.5-

conjugated TCR γ/δ (GL3-BioLegend), PerCP-Cy5.5-conjugated CD69 (H1.2F3-

BioLegend), APC-conjugated CD11c (HL3-BD Biosciences), APC-conjugated CD8a

(53-6.7-BioLegend), APC-conjugated IFNγ (XMG1.2-BioLegend), PE-Cy7-conjuated

CD19 (6D5-BioLegend), PE-Cy7-conjuated NK1.1 (PK136-BioLegend), PE-Cy7- conjuated CD3ε (17A2-BD Biosciences), APC-Cy7-conjugated CD62L (MEL-14-

BioLegend), and APC-Cy7-conjugated CD4 (GK1.5-BD Biosciences). Appropriate isotype controls recommended by the manufacturer were included in each experiment and used to set gates for analysis.

Cytokine/LDH quantification by ELISA. Concentrations of IFNγ, TNF, IL-10, IL-

12p40, IL-6, and IL-1β in mouse organ homogenates or human macrophage supernatants were assessed by ELISA (BD Biosciences for mouse and R&D Systems for human) following the manufacturer’s protocol. Briefly, 96-well plates were coated with antibodies designed to detect the specific cytokine and incubated for 12-16 h. Organ homogenates or macrophage supernatants were then overlaid and incubated for 2 h at room temperature. Colorimetric analysis at OD450 along with a standard curve was used

to determine the concentration of each cytokine. Concentration of LDH was assessed

using CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Fitchburg, WI) 130

following manufacturer’s protocol. Colorimetric assays were read on a Spectramax M2

Microplate reader (Molecular Devices LLC, Sunnyvale, CA).

Statistical analysis. Statistical significance was determined using Prism 4 software

(GraphPad Software, San Diego, CA). The unpaired, two-tailed Student’s t-test was used for two group comparisons. Multiple comparisons were analyzed using one-way

ANOVA with Tukey’s post-hoc test. Statistical significance was reported as *, p<0.05;

**, p<0.01; or ***, p<0.001.

Results

Petroleum ether treatment extracts non-polar lipids from BCG without affecting viability of the bacteria

To create a modified version of BCG that has been stripped of inflammatory lipids, we treated BCG with petroleum ether (PE). First, we addressed the nature of the lipids extracted from BCG with PE treatment by thin layer chromatography (TLC). BCG total lipids (TL) were included on TLCs as a reference. Three independent extractions are shown (Fig. 3.1). As previously described, TDM was highly extractable from BCG using

PE (PE BCG Extract) (Fig. 3.1A) (43). To determine the relative amount of lipids remaining on the BCG cell wall after PE extraction, we extracted PE-treated BCG with a solution of chloroform:methanol (C:M, 2:1, v/v) (C:M BCG Extract) (Fig. 3.1A) as we

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have described (443). We used ImageJ software to calculate the relative intensity of the

bands and calculate the percent of material extracted by dividing the combined intensity

of both bands (PE BCG Extract plus C:M BCG Extract) over the intensity of each individual band (PE BCG Extract or C:M BCG Extract). On average, PE was capable of extracting 70%-90% of TDM from the BCG cell wall (Fig. 3.1D).

Due to the hydrophobic nature of PE, we reasoned that other non-polar molecules would be extractable in addition to TDM. We observed almost complete extraction of phenolic glycolipid 1 (PGL-Tb1) and MycB (Fig. 3.1A, D). We also found that some species of phthiocerol dimycocerosates (PDIMs) and triacylglycerol’s (TAGs) were extractable

(Fig. 3.1B, D). Lastly, we analyzed the amount of extracted phosphatidyl-myo-inositol

mannosides (PIMs) as these molecules are anchored to large chain carbohydrates and

their loss would indicate PE is capable of extracting polar lipids (491). As shown, PE is

incapable of extracting PIMs (Fig. 3.1E, D). We confirmed large chain carbohydrates

such as mannose-capped lipoarabinomannan (ManLAM) were not extractable with PE by

separating lipid extracts using SDS-PAGE followed by staining with silver stain plus

periodic acid (Fig. 3.2A) and bacteria whole cell ELISA (Fig. 3.2B). Lastly, to confirm

that lipid extraction was not lethal, we plated serial dilutions of PBS-treated, PE-treated,

or C:M-treated BCG onto OADC-supplemented 7H11 agar. We did not observe

significant differences in growth between PBS and PE treated bacteria, while C:M-treated

bacteria did not grow (Fig. 3.1E). Overall, the non-polar molecules on the BCG cell wall

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are highly extractable with PE, with high reproducibility, and without affecting viability

of BCG.

Delipidated BCG is significantly attenuated in vitro and in vivo and reduces

inflammation in the lung

As mentioned, the major drawback of moving BCG from a percutaneous vaccine towards

a pulmonary vaccine is the concern of pulmonary damage caused by excessive

inflammation to the lung primarily due to toxic lipids such as TDM. We reasoned that

loss of non-polar lipids would be beneficial in terms of control of the bacterial infection by cells of the innate immune system such as human macrophages. Thus, we tested the ability BCG bacilli previously treated with PBS (BCG) or PE (dBCG) to grow and replicate in vitro, using human monocyte derived macrophages (183). Despite equal

inoculums (Fig. 3.3A), we observed that dBCG was significantly less capable of growing

within human macrophages when compared to non-delipidated BCG across time (Fig.

3.3B). Non-treated BCG had an average inoculum uptake of 9.66% ± 5.51%, whereas the

average uptake for delipidated BCG was 3.00% ± 1.32%, highlighting that non-polar

lipids are important for entry and/or association with macrophages.

To further evaluate immune responses to delipidated BCG, we assessed the levels of

inflammatory cytokines secreted from macrophages following infection with BCG or

dBCG. We found that infection with dBCG significantly decreased the secretion of TNF,

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IL-1β, IL-6, and IL-10, but not IL-12p40 secreted by human macrophages across time

(Fig. 3.3C, D). Lack of differences in IL-12p40 suggests that stimulation of the adaptive immune system may not be impaired (Fig. 3.3D). Lastly, to determine cellular cytotoxicity we measured the amount of lactose dehydrogenase (LDH) secreted by infected macrophages. We observed decreased levels of LDH secreted from macrophages infected with dBCG, suggesting that loss of non-polar lipids renders macrophages more resilient against infection (Fig. 3.3E). Images of infected macrophages captured at 120 h

post infection corroborate these finding (Fig. 3.3F).

Due to our previous observations, we next sought to evaluate the potential of dBCG as a

pulmonary vaccine. We began by evaluating the survival of BCG or dBCG in the lung of

vaccinated mice. C57BL/6J mice were inoculated intranasally with 5.0x105 BCG or

dBCG bacilli. Despite equal inoculums (Fig. 3.4A), the ability of dBCG to replicate in

the lung was significantly reduced starting at 2 days post vaccination (DPV), a trend that continued for up to 150 days, with a continuous decrease in bacterial burden (Fig. 3.4B).

By 150 DPV, CFUs in the lung of dBCG-vaccinated mice were below detection levels

whereas BCG-vaccinated mice had 2-3 log10 CFUs in the lung (Fig. 3.4B). Furthermore,

to assess the levels of inflammation in the lung of vaccinated mice, lung homogenates

were assayed for levels of TNF, IL-6, IL-1β, IL-10, IL-12p40, and IFNγ by ELISA. We observed similar results as our in vitro findings in that the inflammatory response to dBCG were significantly attenuated. The levels of TNF and IL-6 were significantly decreased beginning at 7 DPV, and the levels of IL-1β, IL-10, and IFNγ displayed the

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same trend (Fig. 3.5). Similar to our in vitro findings with MDMs, we observed no changes in the levels of IL-12p40 (Fig. 3.3D, Fig. 3.5). By 50 DPV, all of the cytokines except for IL-12p40 were significantly decreased. Altogether, the data suggest that chemical removal of non-polar lipids from the BCG cell wall significantly impacts its ability to replicate in vivo and diminishes inflammatory responses within the lung.

Lastly, we sought to analyze the cellular infiltration and aggregation in the lung in BCG-

or dBCG-vaccinated mice. The middle lobe from each vaccinated mouse was sectioned

and stained with H&E to evaluate morphological changes to the composition of the lung.

Lung inflammation was determined by quantifying the size of the inflammatory foci over

the total area of the lobe. The lung of BCG-vaccinated mice had larger areas of

inflammation and perivascular cuffing compared to dBCG-vaccinated mice (Fig. 3.6A,

B). This same trend was observed at 21 and 50 DPV with larger foci clearly visible in

BCG-vaccinated mice compared to dBCG-vaccinated animals. By 150 DPV, the size and number of cellular aggregates in the lung of BCG-vaccinated mice had mostly subsided, but remained significantly higher compared to those in dBCG-vaccinated mice. Together, the data clearly demonstrates that delipidation of BCG reduces inflammatory responses and is associated with fewer cellular aggregates in the lung when it is administered directly into the lung. Thus, our data suggests that dBCG could be a safe pulmonary vaccine.

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Pulmonary vaccination with dBCG is superior to conventional BCG against M.tb

challenge and attenuates pulmonary immunopathology

In order to replace the current TB vaccine, the new candidate must be more efficacious at

reducing M.tb bacterial burden and preventing TB pathology. The best way to achieve

this is by direct inoculation of the lung. We began by testing the efficacy of conventional

BCG or dBCG against M.tb challenge. Mice were randomized into vehicle (PBS), BCG,

or dBCG groups and vaccinated with 5x105 bacteria via intranasal inoculation of the lung. Mice were housed without further manipulation for 50 days and then infected with a low dose aerosol of M.tb. Bacterial burden was assessed in the lung, spleen, liver, and mediastinal lymph node (MLN) at 21, 60, and 150 days post infection (DPI). We observed significant decreases in the bacterial burden throughout the duration of the experiment in mice vaccinated with dBCG in all organs assayed (Fig. 3.7). In the lung, we observed the typical 6 log10 bacterial burden associated with non-vaccinated mice

across all time points, while vaccination with dBCG stunted growth of M.tb in the lung

significantly better than BCG (Fig. 3.7A). We also observed a significant delay in the

colonization of the spleen (Fig. 3.7B) and liver (Fig. 3.7C) in dBCG-vaccinated mice.

We did not observe M.tb CFUs in the liver of dBCG-vaccinated mice until 150 DPI.

Furthermore, though we did not observe significant differences in bacterial burden in the

MLN at 21 and 60 DPI, bacterial burden in the MLN of dBCG-vaccinated mice was significantly decreased at 150 DPI relative to BCG-vaccinated mice (Fig. 3.7D).

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Together, the data suggest delipidation of BCG augments its efficacy as a vaccine against

M.tb challenge.

Next we assessed immunopathology of the lung at the indicated times post infection (Fig.

3.8). One lobe from each mouse was sectioned and stained with hematoxylin-eosin and

used to assess the degree of tissue inflammation by quantifying areas of cellular

aggregation relative to the total size of the lung. Vaccination with dBCG attenuated

pulmonary inflammation at every time point assessed. At 21 DPI small areas of cellular

aggregation were visible in all three groups, but foci in the lung of vehicle-treated mice were significantly larger compared to dBCG, but not BCG (Fig. 3.8A, B). Inflammatory foci doubled in size in the lung of vehicle-treated and BCG vaccinated mice at 60 DPI, whereas foci in the lung of dBCG-vaccinated mice remained small. We also observed significantly decreased pulmonary inflammation in the lung of dBCG-vaccinated mice compared to BCG-vaccinated mice at this time point. At 150 DPI, the mean area of the inflammatory foci was 20.84% ± 4.66% in vehicle-treated mice and 19.14% ± 13.54% in

BCG-vaccinated mice, compared to 8.30% ± 3.04% in dBCG-vaccinated. Together the

data suggest that not only is dBCG superior to BCG at reducing bacterial burden in the

lung, but also more effective at preventing M.tb-induced pulmonary immunopathology.

Pulmonary vaccination with dBCG alters innate immune cell kinetics and increases

memory T cell population of the lung

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Since dBCG was associated with increased control of M.tb and reduced

immunopathology in the lung, we assessed immune cell kinetics in the lung of vaccinated

mice across 50 days. A naïve group of mice was included to compare basal number of

cells present in the lung [for the gating scheme see Fig. 3.9A]. Early post vaccination (7

and 21 DPV), we observed significantly reduced percentages of monocytes and

neutrophils in the lung of mice vaccinated with dBCG (Fig. 3.10A, B), resonating with

previous findings that vaccination with dBCG was associated with lower levels of

inflammation. We did not observe any significant differences in percentages of AMs and

DCs in the lung (Fig. 3.10C, D). The number of AMs decreased, while the number of

DCs increased across time in both groups relative to naïve mice. We found that B cell

numbers increased across time in both vaccination groups, though BCG-vaccinated mice had a non-significant trend for increased numbers at later time-points (Fig. 3.10E). This increase in B cells across time suggests they may be active players in the immune response to BCG. The number of NK cells decreased across time, but significantly increased in BCG-vaccinated mice at 50 DPV (Fig. 3.10F), while the percentage of γδ+ T cells was not affected by either formulation (Fig. 3.10G).

BCG is the only licensed vaccine that primarily mediates protection via activation of

CD4+, and to a lesser extent, CD8+ T cells (200;391). BCG exerts its function as a

vaccine against TB by generating a pool of memory T cells that respond rapidly upon

infection with M.tb (201;412). For these reasons, we focused on the adaptive immune

responses in the lung of BCG- or dBCG-vaccinated mice. We analyzed the proportions of

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CD4+ and CD8+ T cells expressing markers associated with immature (naïve) T cells

(CD62L+CD44-), effector T cells (CD62L-CD44+), and memory T cells (CD62L+CD44+)

[for the gating scheme see Fig. 3.9B]. We discovered that the proportion of CD4+ and

CD8+ T cells continually increased across time following vaccination. Mice vaccinated

with dBCG had significantly increased numbers of CD4+ and CD8+ T cells in the lung at

50 DPV, the time of challenge with M.tb (Fig. 3.11A). We did not observe any changes

to the proportion of naïve cells in either subset across time (Fig. 3.11B). Proportions of

effector CD4+ T cells in the lung increased across time reaching statistical significance at

50 DPV in dBCG vaccinated mice, while effector CD8+ T cells did not (Fig. 3.11C).

While effector cells are believed to possess short lived immunological memory, tissue- resident memory cells, though their numbers are significantly smaller than effector cells in tissues, are longer-lived (492). We found that dBCG induced greater proportions of

CD4+ and CD8+ memory cells in the lung compared to BCG vaccination (Fig. 3.11D).

This suggests the reason behind dBCG superior ability to control M.tb and prevent

immunopathology of the lung is due to its ability to generate a pool of memory T cells

that can respond rapidly to M.tb infection.

Pulmonary vaccination with dBCG accelerates effector T cell responses in the lung

upon challenge with M.tb

Finally, we evaluated effector T cells responses in the lung of mice that had been

vaccinated only (DPV 50) or vaccinated and then challenged with M.tb (10 and 21 DPI)

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(Fig. 3.12A). We focused on the expression of CD69 on CD4+ and CD8+ T cells, and the

ability of T cells to produce IFNγ or IL-17A [for the gating scheme see Fig. 3.9C]. In

BCG and dBCG groups, the number of CD69 expressing CD4+ and CD8+ T cells

remained unchanged at 50 DPV. However, infection with M.tb increased the number of

CD69+CD4+ and CD69+CD8+ T cells in the lung of dBCG-vaccinated mice, suggesting dBCG vaccination is associated with faster T cell responses to M.tb (Fig. 3.12B). To further assess T cell activation in the lung, we evaluated two important effector mechanisms required for M.tb control, the production of IFNγ and IL-17A by T cells

(122;296;493). We did not observe statistical significance in the number of CD4+IFNγ+

and CD8+IFNγ+ T cells in the lung of BCG- or dBCG-vaccinated mice at any time point, although dBCG-vaccinated mice displayed an increasing trend at 21 DPI (Fig. 3.12C).

This suggests that, although necessary, IFNγ responses may have a limited ability to clear

M.tb infection. We did however, observe significant increases in the number of CD4+IL-

17A+ and CD8+IL-17A+ T cells in the lung of dBCG-vaccinated mice challenged with

M.tb (Fig. 3.12D). There was a significant increase in the number of CD8+IL-17A+ T

cells at 10 DPI in dBCG-vaccinated mice, while at 21 DPI we observed significant

increases in CD4+IL-17A+ and CD8+IL-17A+ T cells in the lung of dBCG-vaccinated

mice. Overall, our data suggests that IL-17A actively participates in the control of M.tb

and is important for the development of mycobacterial immunity through immunization.

Discussion

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BCG remains the only vaccine available for the prevention of TB, yet it fails to confer

long term immunity against PTB. Despite its success against meningeal- and miliary-TB,

its efficacy against PTB must be improved if we wish to eradicate M.tb. One underlying

factor that may contribute to this phenomenon is the route of immunization with BCG

versus the route of M.tb infection. M.tb has evolved to be a pulmonary pathogen by

taking advantage of the immunoprivileged status of the lung, where inflammatory

responses are tightly regulated to prevent damage by excessive inflammation (494).

Vaccination with BCG, on the other hand, is administered into the dermal layer of the

skin (198). The immune composition of the lung and the skin has been shown to vary

significantly, and within them, immune responses to pathogens (495;496). Although

other vaccines administered via percutaneous injection such as the measles-mumps-

rubella (MMR) vaccine or the diphtheria-tetanus-pertussis (DTP) vaccines have been extremely successful, their primary target for immunological memory is through generation of long lived plasma B cells that produce high-affinity antibodies capable of

rapidly neutralizing their target pathogen (200). The BCG vaccine is unique in that it

relies primarily on CD4+ and CD8+ T cell memory for protection against M.tb. Multiple studies have suggested that BCG is more efficacious against M.tb when delivered directly into the lung (275;383;482;483). However, the presence of inflammatory lipids on the mycobacterial cell wall has inhibited the use of BCG as a pulmonary vaccine. As a result, we postulated that removal of inflammatory lipids from the cell wall of BCG will enable direct pulmonary vaccination with BCG.

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Our results show that selective delipidation of BCG with petroleum ether effectively serves as a more potent vaccine against M.tb when administered directly into the lung.

Pulmonary vaccination with dBCG was more effective than conventional BCG at reducing M.tb bacterial burden in the lung, spleen, and liver at the initial stage of infection (21, 60 DPI) and in all organs including the MLN at later stages (150 DPI). This observation is likely attributed to the removal of virulent lipids including TDM, MycB,

PGL-Tb1, TAGs, and PDIMs from the cell wall that inhibit the development of effective immunity. As discussed in chapter one, TDM can inhibit the fusion between phospholipid vesicles such as those required for fusion of phagosomes with lysosomes (497).

Phagosome-lysosome (P-L) fusion is required for the killing of intracellular pathogens and for the subsequent presentation of foreign peptides along with MHC-class II to adaptive immune cells (498). TDM can also inhibit cellular energy metabolism by stimulating NADase activity thus lowering the levels of NAD and thereby reducing the activity of NAD-dependent enzymes which can affect the generation of adaptive immune responses (499). Furthermore, the absence of TDM allows for better expression of MHC-

II, CD1d, CD40, CD80, and CD86 on macrophages, and as a result increases their ability to stimulate CD4+ T cell responses (45). TDM can also induce apoptosis of immature lymphocytes in the thymus leading to atrophy suggesting TDM may also inhibit T cell development (500). By removing TDM from BCG, we anticipate macrophages or dendritic cells will be more efficient at presenting antigen to adaptive immune cells and stimulating the development of immunological memory. Thus, our data suggests the

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absence of TDM on BCG is beneficial for the generation of protective immune responses against M.tb.

The other lipids we have removed have also been shown to downregulate immune responses. MycB is a monosaccharide PGL produced by all sub-strains of M. bovis

(including BCG), but not by M.tb (501;502). Despite being highly abundant on the BCG cell wall, its effects on the immune response remain largely unknown. One study showed that MycB was incapable of stimulating the secretion of IL-1β and IL-6 from macrophages, but could induce TNF (295). Some M.tb clinical isolates are able to synthesize the trisaccharide form of PGL, namely PGL-Tb1. A clinical isolate of M.tb belonging to the East-Asian lineage (i.e. HN878) was found to be hypervirulent in animal models due to the presence of PGL-Tb1 on its cell wall (60). The trisaccharide domain of PGL-Tb1 was found to be able to inhibit Toll-like receptor 2 (TLR2)-induced

NF-kB activation, and thus production of IL-1β, TNF, IL-6, and CCL2, suggesting PGL-

Tb1 enhances mycobacterial subversion of the immune system (61;62). Similar to PGL-

Tb1, the accumulation of TAGs on the mycobacterial cell surface can increase virulence

(503). Thus, the accumulation of PGLs and TAGs can confer an adaptive growth advantage in stressful environments. Similar to other lipids on the outer surface of M.tb,

PDIMs appear to play a role in the virulence of mycobacteria. M.tb strains lacking PDIM were shown to be less capable of causing disease in mice (64;65). Genetic M.tb mutants lacking PDIM were less capable of binding to the plasma membrane of host macrophages thus leading researchers to conclude that PDIMs directly contribute to the initial step of 143

macrophage infection and participated in preventing phagosomal acidification (66). In support, other studies have shown that lack of PDIMs on M.tb reduces the survival of the bacteria within macrophages, suggesting PDIMs protect M.tb from early innate host responses (67). In contrast, a recent study has suggested that complete absence of

PGL/PDIMs reduces the protective efficacy of BCG against M.tb (488). Thus, it is suggested by the literature that the presence of PGL/PDIM benefits mycobacterial virulence, but their complete absence is detrimental to the development of effective host immunity. In our studies, PE was incapable of removing all the PDIMs species from the cell wall of BCG and we anticipate this may be one of the reasons behind it superior efficacy compared to conventional BCG.

As mentioned above, immune responses in the lung are tightly regulated to prevent excessive tissue inflammation and damage. Despite infection, the lung must continue to oxygenate the blood or risk the death of the organism. Excessive inflammation can impair this process, and thus must be counter regulated. Several endogenous anti-inflammatory, pro-resolving molecules (e.g. lipoxins, resolvins, protectins, and maresins) are heavily involved in driving resolution of inflammation in the lung (504-507). They are secreted by most types of immune cells and act by inhibiting the production of inflammatory lipids such as leukotrienes and prostaglandins (494). In this context, lipoxin levels have been shown to increase in the lung during M.tb infection (508), although the mechanism by which M.tb induces their production remains unknown. In the absence of lipoxins, levels of IL-12, IFNγ, and NO synthase 2 (NOS2) increase suggesting that M.tb may

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specifically target lipoxins to subvert host immune responses (509). Most importantly,

mice deficient in lipoxins can control M.tb more efficiently than mice with an intact

lipoxin pathway (509). Thus, it is strongly suggested that lipoxins negatively regulate

anti-mycobacterial responses in vivo and suggests that inhibition of lipoxins could be

targeted to enhance mycobacterial immunity. Although we did not specifically measure

lipoxins levels post vaccination, we suspect that dBCG-vaccination could be associated

with low levels of pro-resolving lipids as inflammatory responses are diminished. The

absence of pro-resolving lipids could favor the development of more effective adaptive

immunity by dBCG.

In addition to our observation that pulmonary vaccination with dBCG was superior to

BCG against M.tb challenge, it was also associated with decreased immunopathology of

the lung. The mouse model has been an invaluable tool for assessing progression of M.tb

disease, despite not accurately replicating human TB pathology. We did not observe

significant differences between BCG- and dBCG-vaccinated mice at 21 DPI, although

dBCG-vaccinated mice had fewer and smaller lesions compared to vehicle-treated mice.

We did, however, observe significant decreases in in the number and size of lesions in the lung between BCG- and dBCG-vaccinated mice at 60 and 150 DPI. No significant differences were observed between vehicle and BCG-vaccination at these time points.

These observations suggest that vaccination with dBCG may accelerate immune responses to M.tb infection that quickly contain the pathogen and limit inflammation in the lung. Indeed, it has been shown that the faster immune responses develop in the lung,

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the faster M.tb can be contained (510). BCG mediates immunity through the development of antigen-specific memory T cells that respond rapidly following infection with M.tb

(199;201). As discussed in chapter one, the success of BCG against meningeal- and miliary-TB is due to the rapid response of memory T cells that allow for quick containment of the infection. The presence of resident memory T cells in the lung bypasses the delay required for T cell priming in the lymph nodes that occurs in naïve hosts (202;203). In this context, memory T cells exist within two populations: memory and effectors. Memory cells are abundant within lymphoid tissue, respond faster to stimulus, have the ability to self-renew, and are long-lived. On the other hand, effector T cells reside in larger numbers within non-lymphoid tissues, respond more slowly to stimulus, and are short-lived (203;492). Previous studies have shown that effector T cells accumulate in the lung of mice that were vaccinated with BCG via the subcutaneous route, but memory T cells did not (511). This phenomenon was also observed in humans

(512).Thus, if numbers of memory T cells could be increased in the lung it could lead to faster immune responses to pathogens. Our data show that pulmonary vaccination with dBCG amplified the number of effector CD4+ T cells, but more importantly, increased

the number of memory CD4+ and CD8+ T cells within the lung. This directly suggests

that the superior efficacy of dBCG is due to its ability to stimulate the population of the

lung with memory T cells.

In addition to increasing the number of memory and effector cells within the lung,

pulmonary vaccination with dBCG accelerated T cell activation in the lung following

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M.tb infection. We observed a significant increase in the number of CD4+ and CD8+ T

cells positive for CD69 in both BCG- and dBCG-vaccinated groups at 50 DPV or 10 DPI

relative to control groups, but no differences between vaccination groups. However,

dBCG-vaccinated mice had higher numbers of CD4+CD69+ and CD8+CD69+ T cells in

the lung 21 DPI compared to BCG-vaccinated mice, suggesting that vaccination with

dBCG does indeed accelerate T cell activation upon M.tb infection. We also assessed the

number of T cells positive for IFNγ and IL-17A in the lung following ex vivo stimulation

with CD3 and CD28 in the presence of monensin, as evidence suggests they are critical

for M.tb containment (513). To our surprise we did not observe any significant

differences in the amount of T cells positive for IFNγ prior to, or following, infection

with M.tb in the lung in vaccinated groups. It was clear that IFNγ responses develop

slowly in mice upon infection with M.tb, reaching equal proportions in vaccinated mice

at 21 DPI. The fact that we observed no significant difference in IFNγ responses in the

lung of BCG- and dBCG-vaccinated mice suggests that IFNγ may have a limited ability

to confer protective immunity (514). Since IFNγ was not mediating the additional

protection against M.tb associated with dBCG vaccination, we next assessed IL-17A

responses as they have been implicated in conferring immunity to M.tb at mucosal sites

(513;515). We observed significant increases in the number of CD8+IL-17A+ T cells in the lung of dBCG-vaccinated mice at 10 DPI, and significant increases in the number of

CD4+IL-17A+ and CD8+IL-17A+ cells in the lung at 21 DPI. This suggests that IL-17A responses are behind the increased protection observed in dBCG-vaccinated mice. Thus,

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our data suggests IFNγ has a limited ability to protect against M.tb, while IL-17A

responses can potentially be further manipulated to enhance mycobacterial immunity.

As discussed in chapter one, Th17 (IL-17A producing T cells) responses in the lung can

act as double edged swords. On one hand they are required to sustain necessary IFNγ responses, but on the other hand they can increase the influx of granulocytes into the lung leading to pulmonary damage (296;297). The fact that vaccination with dBCG augmented

IL-17A responses, but not IFNγ responses, suggests that IL-17A may be the mechanism behind the increased protection against M.tb we observed in dBCG-vaccinated mice. As a

consequence, our data suggests that lipids on the BCG cell wall may directly participate

to inhibit IL-17A responses. In fact, recent evidence suggests that accelerated Th1

memory responses in the lung of BCG vaccinated mice were dependent on IL-17A and

IL-23 derived from antigen-specific memory Th17 cells. These memory Th17 cells are

one of the first T cell subsets to respond during M.tb infection (296). We observed

significant increases in the number of effector and memory T cells prior to infection, and

although we did not observe increases in T cells with the potential to produce IL-17A at

50 DPV, their numbers, particularly in the CD8+ population, increased post infection.

This suggests that vaccination with BCG may generate a pool of rapidly responding IL-

17A cells that become activated during M.tb infection. Overall, our data suggests that

removal of lipids from BCG may enhance IL-17A responses in the lung and this strategy

could serve as a powerful approach to increase the efficacy of BCG.

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As the highest incidences of TB occur in developing nations, particularly within rural communities that may lack access to routine health care, TB vaccine design should aim to develop single dose formulations. Though some TB vaccine boosters have shown promising results, implementation of large-scale vaccination programs may be difficult

(516). Additionally, vaccines that require administration of components over a span of a few days/weeks pose significant logistical challenges. For these reasons, our design was centered on developing a single-dose vaccine. Furthermore, TB vaccines that are inoculated into the lung have been shown to be more effective than percutaneous injections. However, toxic lipids on the cell wall of BCG have prevented administration of BCG directly into the lung. We were able to overcome both of these challenges via a simple lipid extraction using the readily available solvent petroleum ether. The procedure was fast, reproducible, inexpensive, and can be easily implemented into current vaccine production. We show that pulmonary vaccination with dBCG was more effective than

BCG at reducing M.tb bacterial burden without the need for a booster or multiple vaccination regimes. More importantly, dBCG was quickly cleared from the lung after inoculation reaching undetectable levels by 150 DPV, whereas BCG was not. Lung inflammation and the development of pulmonary lesions due to dBCG vaccination were significantly lower than vaccination with conventional BCG. These findings effectively abolish the primary concern for moving BCG from an intradermal vaccine to pulmonary one. Although our vaccination was evaluated in mice and may not reflect true human pulmonary TB pathology, the immunological mechanisms that we uncovered will likely hold true.

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One caveat is that all of our experiments were conducted using the mouse model.

Although many of the immunological requirements for protection are shared between mouse and humans as described in chapter one, one is left to speculate whether dBCG could be effective in humans given some of the differences. To date, there is no data available on the efficacy of pulmonary BCG vaccination against M.tb in humans. The main challenge has been administration of the vaccine. In contrast to many other licensed vaccines (i.e. killed, attenuated, subunit), BCG is administered alive and is associated with a very strong inflammatory response at the site of injection (dermis of the skin)

(517;518). Additionally, BCG is administered to newborn infants within a few days of being born and up to six months post-partum (519). The danger of inducing strong inflammatory responses in the lung of newborn infants has prevented BCG from being used as a pulmonary vaccine. We predict that delipidation of BCG will ablate the excessive inflammation and permit direct inoculation of BCG into the lung. The next steps for moving dBCG forward would likely involve testing the vaccine in higher vertebrate mammals (e.g. guinea pigs, rabbits, and NHPs) to assess its efficacy and safety. Human trials would likely involve consenting adults prior to newborns. One benefit of testing dBCG in adults could be to assess whether dBCG can act as a booster for conventional BCG. In the end, dBCG may offer some of the answer the TB field seeks by specifically targeting mechanisms mycobacteria use to subvert host immunity.

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Our data suggest that BCG cell wall lipids directly inhibit the generation of protective

immune responses to M.tb, and that understanding of the mycobacterial cell wall can be

further exploited to improve protective immunity to mycobacteria. Whereas current

vaccines typically fall under the categories of attenuated, inactivated, or

subunit/conjugate, we have classified dBCG under a new category we call “selective

attenuation”. We have classified dBCG this way because the bacteria remain alive, and

therefore are able to replenish the extracted lipids on their cell wall. However, the removal of cell wall lipids via petroleum ether extraction significantly reduces its ability to infect cells and induce immunopathology, thus attenuating it. This allowed us to vaccinate directly into the lung, the most immunogenic route of vaccination for protection against M.tb. Given that cell wall molecules are utilized by the bacteria to engage immune cells, we argue that the initial contact between host and the bacteria will mediate the success of the vaccine. Not only was vaccination with dBCG more effective against M.tb infection, it also opened the possibility of pulmonary vaccination in humans due to the absence of excessive inflammation to the lung. Furthermore, our observations revealed that targeting IFNγ responses may not necessarily augment immunity to mycobacteria. Instead, IL-17A responses appear to contribute significantly to mycobacterial immunity in the lung in the context of vaccination. Altogether our research highlights the complexities of the initial interaction between mycobacteria and the host.

Research on mycobacterial cell wall lipid interactions with host immune cells can lead to a better understanding of the requirements for effective mycobacterial immunity, and the development of more effective vaccines.

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Figure 3.1. Petroleum ether extracts TDM, PGL-Tb1, MycB, PDIMs, and TAGs without affecting viability of BCG. To assess nature and percentage of the lipids remaining on the BCG cell wall following extraction with PE (PE BCG extract), PE treated BCG was subjected to C:M (2:1, v/v) extraction for 12 h at 37°C (C:M BCG extract) (n=3; TLC, C:M, 95:5 v/v). Representative TLC images of PE BCG extracts in triplicate. BCG total lipid (BCG TL) extracts were included as a reference. (A) TDM, MycB, and PGL-Tb1 are highly extractable by PE. (B) Some PDIMs and TAGs are extracted from BCG by PE (n=3; TLC, petroleum ether/acetone, 96:4 v/v). (C) PIMs are not extractable by PE (n=3, TLC, chloroform/acetic acid/methanol/water, 40:25:3:6 v/v/v/v). (D) Densitometry analysis of all lipids plotted as percent extracted following treatment with the corresponding solvents, (PE BCG extracts, grey bars), (C:M BCG extracts, black bars), (n=3). (E) Viability of BCG treated with PBS, PE or C:M (2:1, v/v) assessed by plating serial dilutions (n=6). Representative experiment shown, each experiment performed at least three times, mean ± SEM; Student’s t-test, ***p<0.001; ns: not significant, nd: no data.

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Figure 3.2. ManLAM is not extracted by petroleum ether. M. bovis BCG underwent PE extraction as described. (A) 100 µg of extract material was separated by SDS-PAGE. Gels were then stained with silver nitrate and periodic acid to visualize carbohydrates and proteins. Purified ManLAM was included as a control. The large chain carbohydrate ManLAM was not extractable with either condition (PE or C:M) as shown by lack of its presence in any of the lanes. Representative experiment of n=2. (B) BCG was subjected to PBS (white bar) or PE (black bar) treatment as described. Bacteria were immobilized on a medium-binding ELISA plate and dried overnight and then probed with CS-35 (ManLAM monoclonal antibody). The following day, the binding was assayed by standard ELISA technique. Representative experiment of n=2, mean ± SEM; Student’s t- test; ns: not significant.

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Figure 3.3. Delipidation of BCG significantly reduces its survival and attenuates inflammatory responses in human macrophages. MDMs monolayers (2.5x105 cells) were infected with viable BCG (grey bars) or dBCG (black bars) bacilli. Colony forming unit (CFU) assays were used to assess growth of BCG or dBCG in vitro. (A) Inoculum used to infect MDMs showed no significant differences between the two groups. A representative experiment of n=4 in triplicate is shown, mean ± SEM; Student’s t-test; ns: not significant. (B) Human macrophages were infected with BCG or dBCG at an MOI 1:1 and bacterial growth was determined at the indicated intervals (2-120 h). A representative experiment of n=4 in triplicate is shown, mean ± SEM; Student’s t-test, *p<0.05, **p<0.01, ***p<0.001. (C) MDMs were infected with BCG or dBCG at an MOI 10:1 and supernatants were probed for the inflammatory cytokines TNF, IL-6, and IL-1β. A representative experiment of n=4 in triplicate is shown, mean ± SEM; Student’s t-test, *p<0.05, **p<0.01, ***p<0.001. (D) IL-10 levels in the supernatant of dBCG infected macrophages were also significantly reduced. No significant differences were observed for IL-12p40. A representative experiment of n=4 in triplicate is shown, mean ± SEM; Student’s t-test, *p<0.05. (E) Levels of LDH as a measure of cytotoxicity in BCG or dBCG MOI 10:1 infected macrophages. A representative experiment of n=2 in triplicate is shown, mean ± SEM; Student’s t-test, **p<0.01, ***p<0.001. (F) Representative pictures of MDM monolayers at 120 h not infected or infected with BCG or dBCG at MOI 10:1; final magnification: 100x.

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Figure 3.4. Delipidated BCG is eliminated from the mouse lung, while BCG is not. C57BL/6J mice were intranasally inoculated with 5x105 viable BCG (grey bar or grey circle) or dBCG (black bar or black circle) bacilli. Mice were sacrificed at 2, 7, 21, 50, and 150 DPV to assess BCG bacterial burden in the lung. (A) Inoculum used to vaccinate mice showed no significant differences between the two groups. Representative experiment of n=2, mean ± SEM; Student’s t-test; ns: not significant. (B) BCG CFUs in the lung was assessed at 2, 7, 21, 50, and 150 DPV by plating serial dilutions onto OADC supplemented 7H11. Pooled results from n=2 with 4-5 mice/group per time-point, mean ± SEM; Student’s t-test, **p<0.01, ***p<0.001.

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Figure 3.5. Vaccination with delipidated BCG is associated with reduced inflammation in the lung. C57BL/6J mice were intranasally inoculated with 5x105 viable BCG (grey bars) or dBCG (black bars) bacilli. Mice were sacrificed at 7, 21, and 50 DPV to assess pulmonary inflammation. At 7, 21, and 50 DPV lung homogenates were probed for the inflammatory cytokines TNF, IL-6, IL-1β, IL-10, IFNγ, and IL- 12p40. Representative experiment of n=2, mean ± SEM; Student’s t-test, *p<0.05, **p<0.01, ***p<0.001.

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Figure 3.6. Vaccination with delipidated BCG is associated with reduced formation of pulmonary immune aggregates. C57BL/6J mice were intranasally inoculated with 5x105 viable BCG or dBCG bacilli. Mice were sacrificed across a period of 150 DPV to assess BCG pulmonary cellular infiltration and aggregation. (A) At 7, 21, 50, and 150 DPV, mice were sacrificed and lungs fixed in 10% NBF, embedded in paraffin, sectioned, and stained with hematoxylin and eosin to visualize tissue morphology. (B) Areas of cell aggregation and infiltration (inflammation) in BCG (grey bars) or dBCG (black bars) vaccinated mice were quantified using Aperio Imagescope by calculating the area of inflammatory foci (i.e. involvement) divided by the total area of the lung. Representative images at a final magnification of 20X. Representative experiment from n=2 with 4-5 mice/group, mean ± SEM; Student’s t-test, *p<0.05, **p<0.01, ***p<0.001.

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Figure 3.7. Pulmonary vaccination with dBCG reduces M.tb bacterial burden in the lung and peripheral organs of infected mice. C57BL/6J mice were intranasally inoculated with PBS (vehicle; white circles) or 5x105 viable BCG (grey squares) or dBCG (black circles) bacilli. Fifty days later, mice were infected with a low dose aerosol of M.tb. At 21, 60, and 150 DPI mice were euthanized to assess bacterial burden in the (A) lung, (B) spleen, (C) liver, and (D) MLN. Pooled results from n=2 with 4-5 mice/group per time-point, mean ± SEM; One way ANOVA with Tukey’s post-hoc test, *p<0.05, **p<0.01, ***p<0.001; ns: not significant.

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Figure 3.8. Pulmonary vaccination with dBCG is associated with decreased M.tb lung pathology across time. C57BL/6J mice were intranasally inoculated with PBS (vehicle) or 5x105 viable BCG or dBCG bacilli. Fifty days later, mice were infected with a low dose aerosol of M.tb. (A) At 21, 60, and 150 DPI mice were sacrificed and lungs fixed in 10% NBF, embedded in paraffin, sectioned, and stained with hematoxylin and eosin to visualize tissue morphology. (B) Areas of cell aggregation and infiltration (inflammation) in vehicle (white bars), BCG (grey bars) or dBCG (black bars) vaccinated mice were quantified using Aperio Imagescope by calculating the area of inflammatory foci (i.e. involvement) divided by the total area of the lung. Representative images at a final magnification of 20X. Pooled results from n=2 with 4-5 mice/group per time-point, mean ± SEM; One way ANOVA with Tukey’s post-hoc test, *p<0.05, **p<0.01; ns: not significant.

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Figure 3.9. Gating scheme of innate and adaptive immune cells. (A) Lung cells were subdivided based on the expression of immunological markers: B cells (CD19+), monocytes (Mon; CD11b+CD11c-), dendritic cells (DC; CD11b+CD11c+), alveolar macrophages (AM; CD11b-CD11c+), neutrophils (Neu; Gr-1+CD11b+), γδ T cells (γδ TCR+), and NK cells (NK1.1+). (B) Lung lymphocytes were first gated based on their size (FSC) and granularity (SSC) (not shown). CD3 was further used to identify the lymphocyte population. Lymphocytes were split into CD4+ and CD8+ cells. Each lymphocyte subset (CD4+ or CD8+) was identified further using CD62L and CD44. Within the gate, naïve cells (CD62L+CD44-) reside within Q1, memory cells (CD62L+CD44+) reside within Q2, and effector cells (CD62L-CD44+) reside within Q3. (C) In a separate panel, each lymphocyte subset (CD4+ or CD8+) was sub-gated for CD69. A separate group of lung cells, previously stimulated with CD3/CD28, were gated based on lymphocyte subset (CD4+ or CD8+) and sub-gated for IFNγ or IL-17A.

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Figure 3.10. Pulmonary vaccination with delipidated BCG is associated with decreased monocyte and neutrophil influx to the lung early post inoculation. C57BL/6J mice were intranasally inoculated with 5x105 viable BCG (grey squares) or dBCG (black circles) bacilli. A group of naïve mice (white circles) was included as a control. Mice were sacrificed at 7, 21, and 50 DPV to assess immune cell kinetics in the lung. (A) Monocytes (CD11b+CD11c-) in the lung of dBCG vaccinated mice were significantly decreased at 7 and 21 DPV, but returned to basal levels by 50 DPV. (B) Neutrophils (Gr-1+CD11b+) were also significantly decreased in dBCG vaccinated mice early post inoculation. (C) Alveolar macrophages (CD11b-CD11c+) decreased equally in both vaccination groups relative to naïve mice. (D) Dendritic cells (CD11b+CD11c+) increased across time in both groups. (E) B cells (CD19+) continued to increase across time for both vaccination groups. (F) Natural killer cells (NK1.1+) continued to decrease in dBCG vaccinated mice across time, but stabilized in BCG vaccinated mice at 50 DPV. (G) The number of γδ TCR+ T cells increased across time but was not significantly different between the two vaccination groups. Pooled experiment from n=2 with 4-5 mice/group, mean ± SEM; Student’s t-test, *p<0.05, **p<0.01, ***p<0.001. DPV, day post vaccination; AMs, alveolar macrophages; DC, dendritic cells; NK, natural killer; TCR, T cell receptor.

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Figure 3.11. Pulmonary vaccination with delipidated BCG increases memory and effector T cell responses in the lung. C57BL/6J mice were intranasally inoculated with 5x105 viable BCG (grey squares) or dBCG (black circles) bacilli. A group of naïve mice (white circles) was included as a control to assess changes in the immune cell population due to vaccination. Mice were sacrificed at 7, 21, and 50 DPV to characterize memory T cell responses in the lung. (A) Total number of CD4+ and CD8+ T cells in the lung across time. (B) Proportion of CD4+ or CD8+ T cells displaying a naïve phenotype (CD62L+CD44-). (C) Proportion of CD4+ or CD8+ effector T cells (CD62L-CD44+) increased across time, and reached statistical significance in dBCG vaccinated mice at 50 D P V. (D) Proportion of CD4+ or CD8+ memory T cells (CD62L+CD44+) also increased across time and peaked at 50 DPV in dBCG vaccinated mice. Pooled experiment from n=2 with 4-5 mice/group, mean ± SEM; Student’s t-test, *p<0.05. DPV, day post vaccination.

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Figure 3.12. Pulmonary vaccination with dBCG boosts CD69 and IL-17A, but not IFNγ, responses in the lung of M.tb infected mice. (A) Timeline showing experimental design. C57BL/6J mice were intranasally inoculated with vehicle (white circle) or 5x105 viable BCG (grey square) or dBCG (black diamond) bacilli. (B) T cell activation based on the expression of CD69 on CD4+ and CD8+ T cells at 50 DPV and at 10 and 21 DPI. CD69 was significantly increased in dBCG-vaccinated mice at 21 DPI. (C) Total number of CD4+ and CD8+ T cells staining positive for IFNγ after stimulation with CD3/CD28 in the presence of monensin. (D) Total number of CD4+ and CD8+ T cells staining positive for or IL-17A after stimulation with CD3/CD28 in the presence of monensin. Mice vaccinated with dBCG had significantly higher proportions of CD8+IL-17A+ cells (10 DPI) and CD4+IL-17+ and CD8+IL-17A+ (21 DPI) in the lung, while IFNγ+ cells were not significantly elevated in either vaccination group. Pooled experiment from n=2 with 4-5 mice/group, mean ± SEM; One-way ANOVA with Tukey’s post-hoc test, *p<0.05, **p <0.01, ***p<0.001.

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Chapter 4: Natural aging increases the oxidation and inflammatory status of alveolar lining fluid leading to decreases in innate immune protein function, driving susceptibility to Mycobacterium tuberculosis

Abstract

As we age, there is an increased risk for the development of pulmonary diseases,

including infections with Mycobacterium tuberculosis (M.tb). Few studies have

considered changes to lung fluid physiology and soluble components of the innate

immune system due to aging as contributing factors behind the increased susceptibility of

the elderly to infections. We and others have demonstrated that human alveolar lining

fluid (ALF) contains soluble innate factors [e.g. complement, surfactant protein (SP)-A,

SP-D, hydrolytic enzymes, and lipids] that mediate innate immune responses to microbial infections. However, there is a lack of understanding regarding the effect of increasing age on the level and function of ALF components in the lung. Here we addressed this gap in knowledge by analyzing changes in innate immune components that occur to ALF as we age. Our findings demonstrate that some pro-inflammatory cytokines, surfactant

proteins, lipids, and complement components are significantly elevated in the aged lung

in both mice and humans. We also show that the aging lung is a relatively oxidized

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environment. Furthermore, as M.tb infection results in bacterial deposition in the lung alveoli where they are bathed in ALF, we explored the impact of ALF on M.tb infection.

We hypothesized that ALF components lose their function as we age, and this deficiency favors the establishment of M.tb infection. Although the levels of SP-A and C3 were elevated in elderly ALF, their binding to M.tb was reduced, suggesting a defect in innate immune function with age. To evaluate the impact of ALF age-associated changes in

vitro, human macrophages were infected with adult- or elderly-ALF exposed M.tb.

Macrophages infected with elderly-ALF exposed M.tb were less capable of controlling

the infection and displayed fewer phagosome-lysosome fusion events, suggesting natural

aging may dampen the antimicrobial effects of ALF. Furthermore, mice infected with

elderly ALF-M.tb had significantly increased bacterial growth within the lung and spleen

compared to adult ALF-M.tb infected mice, suggesting that gradual loss of the

antimicrobial effects of ALF could have long term consequences in controlling M.tb

infection. Our studies provide new information on how the pulmonary environment in old

age can modify mucosal immune responses, thereby impacting pulmonary infections and

other pulmonary diseases in the elderly population. They also suggest that M.tb may

benefit from the declining host defense function of the lung mucosa in the elderly,

driving host susceptibility to this pathogen.

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Introduction

Evidence for increased susceptibility to tuberculosis in the elderly

Despite successful efforts by the World Health Organization to curtail the spread of M.tb,

this highly infectious disease continues to kill millions every year (434). Globally, the

number of TB rates is reported to be highest within populations over 50 years of age

(434), placing the elderly population (≥ 65 years old) in the highest risk group

for M.tb infection independent of race, ethnicity, and gender. Furthermore,

epidemiological data indicates that the highest rates of TB mortality occur within the

elderly population (520). Possible explanations of why the elderly are at such a risk

include both biological (e.g. compromised nutrition and immune status, underlying

disease, medication side effects) and socioeconomic factors (e.g. poverty, poor living

conditions, lack of access to health care) (521). However, developed countries such as the

United States where socioeconomic factors do not play as significant of a role in overall

morbidity, the elderly population is associated with the highest rates of TB, wherein the

rate of TB is 30% higher than in the adult population (522). Despite the fact that >80% of

TB cases worldwide occur in developing nations, the majority of TB cases occur in

individuals between the ages of 25-44 (434). However, increased access to health care

has led to over half of the developing nations with the highest TB rates to have life

expectancies over 65. Furthermore, the number of elderly individuals (aged greater than

65) on the planet is projected to double from 800 million to approximately 1.6 billion by

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the year 2050 and comprise of a quarter of the global population (523). With the global population of elderly individuals, especially those in developing nations where TB rates are the highest, expected to continue growing exponentially, it is imperative that we understand the role of aging in the context of lung physiology and immunology and how may affect susceptibility to M.tb.

Increasing age comes with decreases in innate and adaptive immune function

Advancing age is accompanied with failing pulmonary health and an increased susceptibility to numerous pulmonary infections including influenza, pneumococcal pneumonia, and TB (524-528). Increased susceptibility to infection is attributed to immunosenescence, the natural gradual deterioration of the immune system brought on by advancement in age (529). In this context, early observations suggested that T and B cells become less functional with increases in age. It was shown that B cells from elderly individuals were less capable of proliferating in response to vaccination, while T cell responses develop more slowly and are less capable of proliferating in response to antigen stimulation (530-533). Although adaptive immune senescence has been thought as the dominant force behind declining immunity in old age, recent studies suggest that innate immune cells may also be important contributors. Neutrophils, macrophages, and

DCs have been shown to be affected by increasing age. Aging appears to diminish neutrophil migration and chemotaxis, and thus affect their ability to respond to infection

(534;535). Macrophages derived from elderly humans or old mice appear to lose their

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ability to phagocytose, induce the process of P-L fusion, respond to microbial infection, and present antigens to adaptive immune cells (536-539). DCs may also lose their ability to activate adaptive immune cells and translocate from tissues to lymph nodes (540-542).

Together, there is clear evidence that increases in age weaken immune responses by both branches of the immune system.

Inflammaging, or increases in chronic inflammation due to advancing age

Recently, the theory of “inflammaging”, or increases in basal inflammation that comes

with natural aging resulting in chronic inflammation, has been suggested as an important

contributor to the decline in innate and adaptive immunity observed in old age.

Inflammaging is a chronic condition attributed to increases in basal amounts of

circulating pro-inflammatory mediators including cytokines, chemokines, and acute

phase proteins. In general, elderly individuals display higher basal levels of TNF, IL-6,

IL-18, IL-15, C reactive protein (CRP), serum-amyloid A, fibrinogen, Von Willebrand

factor, resistin, and leukotrienes in their plasma (543-546). Inflammaging has been linked

to age-related disease such as atherosclerosis (547), cancer (548), rheumatoid arthritis

(549), osteoporosis (550), type-two diabetes (551), and Alzheimer’s disease (552). In

particular, IL-6 displays the strongest correlation with morbidity (frailty, sarcopenia), and

mortality in older humans (553). In this context, inhibition of IL-6 has been shown to

improve inflammatory conditions such as rheumatoid arthritis that typically afflict the

elderly (554).

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A few theories have been suggested to explain the molecular mechanisms responsible for

inflammaging. First, the ‘theory of stress’ suggests that increases in stress result in

increases in basal inflammation. This theory is supported by findings that stress increases

the basal inflammation of young individuals more than in older counterparts, suggesting

that elderly individuals already have a basal level of inflammation (555;556). Second, the

‘theory of oxidation-inflammation’ suggests that oxidation is directly linked to

inflammaging. Under normal physiological conditions, reactive oxygen species (ROS)

are generated as a byproduct of oxidative phosphorylation, the process by which the body

generates energy (in the form of adenosine triphosphate) (557). Dysfunction of ROS-

neutralizing mechanisms allows residual ROS molecules to damage nucleic acids,

proteins, lipids, and carbohydrates, which in turn can dysregulate cellular function,

potentially killing the cell and result in more inflammation (558-562). Third, the ‘theory

of cytokines’ suggests that cytokine expression is dysregulated and expression of

inflammatory cytokines becomes leaky with increasing age. Elderly individuals have

elevated levels of IL-1β, IL-6, TNF in their serum relative to younger counterparts, and

elevation of these cytokines is associated with disease, disability, and mortality

(563;564). Fourth, the ‘theory of DNA damage’ suggests that accumulation of DNA

damage, caused by both exogenous and endogenous factors, can result in cellular

senescence (565;566). In turn, senescent cells have been shown to secrete pro-

inflammatory molecules in a phenomenon called senescence-associated secretory phenotype (SASP) which contributes to inflammaging (567;568). Fifth, the ‘theory of

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autophagy’ suggests that aging impairs autophagy, a process utilized by cells to remove

cellular debris and maintain homeostasis (569). As we age, cells gradually lose their autophagic capacities leading to accumulation of dysfunctional cellular components (e.g. mitochondria, proteins). This leads to accumulation of ROS which, as discussed above, can lead to cellular senescence and perpetuate inflammaging (570). Overall, although the

exact mechanisms of inflammaging have not been thoroughly defined, it is likely that inflammaging is a consequence of all these factors, amongst others.

Pulmonary immunity and chronic inflammation

Advancing age contributes to systemic immune dysfunction. However, few studies have considered whether changes in components of pulmonary surfactant and/or the innate immune system can impact lung function sufficiently to increase the risk of developing age-associated pulmonary disorders or to increase susceptibility to infection in the elderly. We and others have previously demonstrated that some components of human surfactant [the collectins surfactant protein (SP)-A and SP-D, surfactant homeostatic hydrolytic enzymes (hydrolases), and lipids] are important elements of the innate immune system during microbial infections, including to M.tb (162;571-574). In addition, the complement system is active in the alveolar space and plays an important role in the microbe-macrophage encounter (182;575-578). As described in chapters one and two,

ALF is generated, secreted, and recycled by alveolar epithelial cells (ATs) and is essential for maintaining lung function (579). Similarly, components of the complement

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system are produced by ATs (580) and macrophages in the lung (581). ALF in elderly

individuals has a slow turnover and degrades quickly because of inefficient regeneration

by senescent ATs (582). Therefore, slow ALF turnover plus the presence of inflammatory

lung and parenchymal cells can drive baseline low-grade chronic inflammation in the

lung during aging (583). These events result in changes in alveolar surfactant and

complement production and activity, the former as a result of a change in the local

oxidation state (584). Moreover, alterations in surfactant lipid composition are predicted

to negatively affect SP-A and SP-D function (585), with the latter proteins being able to

be oxidized themselves (586). Studies of ALF from cadaveric human lungs have shown that SP-A levels decreases with increasing age (from age 22–55) (587). This may indicate that the aged human lung is particularly vulnerable to ALF dysfunction and chronic inflammation. Since ALF dysfunction correlates with the defined function of SP-A in

reducing complement activity in the lung (588), as well as in modulating inflammatory

responses (589-591), alterations in the balance of SP-A, other surfactant components,

complement, and cytokines may contribute to chronic inflammation in old age.

Moreover, SP-D deficiency has been associated with a chronic inflammatory state in

which there is an increase in apoptotic and necrotic alveolar macrophages and intra-

alveolar accumulation of surfactant lipids (592).

Elucidating changes to ALF due to aging that may impact M.tb pathogenesis

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Recent connections between inflammaging and susceptibility to infectious disease have

raised questions as to whether chronic inflammation may be a mechanism exploited by

M.tb during its natural infection cycle. We sought to explore underlying mechanisms

behind decreased pulmonary immunity in old age. In this study, we examined

components of ALF from both mice and humans focusing on cytokines, surfactant

proteins, and complement proteins found within the ALF of young (3 month) or old (≥18

month) mice, and adult (25-44 years) or elderly (≥ 65 years) humans. Analysis of ALF

revealed inflammaging signatures within the lung of healthy aged mice and humans. We

demonstrate that ALF components are significantly altered in old age and that the

pulmonary environment shifts towards a pro-inflammatory state with increasing age,

supporting the theory of inflammaging. Additionally, we observed an increase in

oxidized proteins obtained from the ALF of old mice. This led us to hypothesize that

inflammaging hinders innate immune responses, particularly within antimicrobial

proteins that serve as the first host defense to infection. Using M.tb as a model, we report

that soluble innate immune proteins from elderly humans are less capable of binding to

the cell wall of M.tb, suggesting a decline in innate immunity with age. Macrophages

infected with M.tb exposed to elderly ALF were less capable of clearing the infection, suggesting that a decline in soluble innate immune components can affect the cellular innate immune response. In support of this finding, we observed decreased P-L fusion in

macrophages infected with M.tb exposed to elderly ALF. Finally, in vivo experiments

wherein mice were infected with adult- or elderly-exposed M.tb demonstrated that exposure of M.tb to elderly ALF increases virulence and pathogenicity. These findings

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provide new insights about the baseline immunologic state within the lung alveolus in old age. Such findings are critical in enabling future studies to elucidate the direct influence of these changes on the increased susceptibility of the elderly to infection and noninfectious pulmonary diseases.

Material and Methods

Ethics statement. All experimental procedures with animals were approved by The Ohio

State University Institutional Animal Care and Use Committee (IACUC protocol number:

2012A00000132-R1). For human subjects (to obtain BALF), this study was carried out in strict accordance with US Code of Federal and Local Regulations [University Human

Subjects Institutional Review Board (IRB) protocol number: 2012H0135]. For human subjects (to obtain blood), this study was carried out on strict accordance with US Code of Federal and Local regulations [University Human Subjects Institutional Review Board

(IRB) protocol numbers: 2007H0262 and 2008H0119]. For this study blood was only collected from adult human subjects, and all of them provided written informed consent.

Proteomic analysis of human ALF by Liquid Chromatography-Tandem Mass

Spectrometry (LC-MS/MS). Human ALF was collected and concentrated as described in chapter two. Protein content was determined following the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL). ALF aliquots containing 50 µg of protein were dehydrated and proteins were precipitated using trichloroacetic acid (TCA). Briefly, proteins were

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resuspended in a solution of 20% TCA, vortexed, and incubated for 30 min on ice. The

samples were then centrifuged at 13,000 x g for 30 min at 4°C. The supernatant was

removed, and the protein pellet was resuspended in acetone. The samples were spun

again at 13,000 x g for 15 min at 4°C. The acetone was removed, the protein pellets were

allowed to air dry, and separated by sodium-dodecyl-sulfate polyacrylamide gel

electrophoresis (SDS-PAGE). Mass-spectrometry was performed using a ThermoFisher

Scientific Orbitrap Fusion HPLC-MS/MS instrument. Proteins were identified by comparing peptide fragmentation patterns to the mascot protein database (Matrix

Science; http://www.matrixscience.com/).

Ingenuity pathway analysis (IPA). Global protein profiles of adult and elderly humans

were analyzed using IPA Software (Qiagen Silicon Valley, Redwood City, CA). Genes

corresponding to each identified protein were cross-referenced using Uniprot

(www.uniprot.org) and then entered into the IPA software. In our analysis we identified

the top ‘diseases and functions’ and the top ‘upstream regulators’. The p-value is

calculated using a right-tailed Fisher’s exact test and reflects the likelihood that the

association to overlap between a set of significant molecules from the experiment and a

given process/pathway/transcription neighborhood is due to random chance. The smaller

the p-value, the less likely the association is random. The ‘diseases and functions’

analysis identifies categories associated with proteins that can be explained by the

observed molecules present in a sample. The ‘upstream regulator’ analysis identifies the

cascade of upstream transcriptional regulators that can explain the observed changes in

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the dataset, which helps reveal the biological activities occurring in the samples being

studied.

Mice. Specific-pathogen-free, female, C57BL/6J mice were purchased from Charles

River Laboratories (Wilmington, MA) at 3 months of age (young) or at 18 months of age

(old) through a contract with the National Institute on Aging. For our studies, young mice

were deemed to be 3 months of age as this is when they reach sexual maturity. Old mice

were considered 18 months or older, which is physiologically equivalent to old age in

humans (65 years or older) (593). Additionally, we used 18 month old mice to avoid

confounding factors that may appear later in life such as frailty and spontaneous tumor

generation. Upon arrival, mice were supplied with sterilized water and chow ad libitum

and acclimatized for at least one week prior to experimental manipulation. Mice were maintained in micro-isolator cages located in either a standard vivarium for all noninfectious studies or in a biosafety level three (BSL-3) core facility for all studies

involving M.tb. Mice were examined at necropsy and those with gross lesions were

excluded from the study.

Human subjects. This study was carried out in strict accordance with the United States

Code of Federal Regulations, Local Regulations (institutional review board (IRB)), and

Good Clinical Practice as approved by the National Institutes of Health (National

Institute on Aging and National Institute of Allergy and Infectious Diseases). A total of

four adults (two healthy and two lung resection patients) and five elderly (all lung

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resection patients) were enrolled. For the elderly group, the average age was 77 years

(range 71–87), 100% Caucasian, non-Latino, with two females and three males. For the adult group, the average age was 31 years (range 25–42), 100% Caucasian, non-Latino, with two females and two males. Both genders were included without discrimination of race or ethnicity. For the lung resection patients, the pre-operative diagnoses were lung nodule (three adults and two elderly), lung nodule and mass (one elderly), and abnormal parathyroid and thymus (one adult and one elderly). Two elderly and two adult subjects had pulmonary function test performed prior to surgery. One elderly had moderate to severe obstructive impairment, and the other had normal spirometry with the exception of suboptimal inspiratory flow-volume loop. One young subject had restrictive disease with low diffusion capacity, and the other had normal spirometry and lung volume with reduced diffusion capacity but normalized when corrected to volume. Donors who were smokers, injection and non-injection drug users, or excessive alcohol users or those with acute pneumonia were excluded from this study. For lung resection patients, individuals with the following comorbidities were excluded: heart disease, diabetes, asthma, chronic obstructive pulmonary disease (COPD), renal failure, liver failure, hepatitis, thyroid disease, rheumatoid arthritis, immunosuppression, HIV/AIDS, cancer requiring chemotherapy, leukemia/lymphoma, and tuberculosis. Bronchoalveolar lavage was performed on the healthy lobe of the subject’s lung.

Collection of bronchoalveolar lavage fluid and concentration to ALF. Old and young mice were euthanized by CO2 asphyxiation following The Ohio State University IACUC-

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approved protocols. Upon euthanasia, BALF was obtained by BAL in mice by washing

the lungs ten times using 0.5 ml of sterile saline (0.9% NaCl) each time. ALF protein content was measured by the BCA assay, and aliquots were quickly frozen and stored at -

80°C until use. Human ALF was obtained by BAL from healthy donors and lung resection patients (adults and elderly) under strict guidance of the OSU IRB human subject-approved protocol. For lung resection patients, BAL was performed on healthy-

appearing lung segments (as determined by the surgeon and pre-operative X-rays) of live human subjects (no samples taken from isolated tissues). Protein content was determined using the BCA assay. After protein determination, crude ALF was concentrated 20-fold by using a 10 kDa molecular mass cutoff membrane column by centrifuging at 4°C to achieve the concentration of ALF present within the lung as described in chapter two.

This process separates the ALF into two different fractions: (ALF) defined as the fraction depleted of surfactant lipids leaving functional SP-A, SP-D, complement, and hydrolases

(this is the same ALF used for our previous studies), and (SL) defined as the fraction containing the surfactant lipids. Fractions were frozen at -80°C until use.

Enzyme-linked Immunosorbent Assay of cytokines and myeloperoxidase assay. The cytokine content of ALF (mouse or human) was measured by standard ELISA technique following the manufacturer’s instructions. All samples were normalized by total protein content (10 µg) and volume by adding 100 µl/well. For mice, we used the following

ELISA kits from BD Biosciences: TNF (555268), IL-6 (555240), IL-1β (559603), IL-

12p40 (555165), IL-10 (555252), and IFNγ (555138). For humans, we used R&D

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Systems ELISA kits: TNF (DY210), IL-6 (DY206), IL-1β (DY201), IL-12p40

(DY1240), IL-10 (DY217B), and BD Biosciences IFNγ (555142). For mouse cytokine studies, the number of samples was n=5/group performed in triplicate. In our studies ‘n’ is used to denote a biological replicate, as in an individual mouse or human. For human cytokine studies, the number of samples was n=4/group performed in triplicate. MPO was detected using a colorimetric detection kit (Enzo Life Sciences, ADI-907-029) as per the manufacturer’s instructions. The number of mouse samples studied for MPO detection was n=16/group.

Western blotting. Samples of 10 µg of protein were separated by SDS-PAGE and transferred onto a nitrocellulose (for detection of complement components) or polyvinylidene difluoride (PVDF- for detection of oxidation) membrane. Western blotting to detect SP-A, SP-D, and complement components and to determine the presence of nitrotyrosine and carbonyl residues were performed following the manufacturer’s recommendations (594;595). The carbonyl and nitrotyrosine presence was determined assessing the band density of all bands in the lane. Control lanes without protein in all Western blot experiments were used to subtract the background due to non- specific binding of antibody or artifacts during the membrane development process. All densitometry results are plotted after background subtraction. For mouse SP-A/SP-D studies, the number of samples was n=17 for SP-A and n=15 for SP-D. For human SP-A and SP-D studies, the number of samples was n=4. For mouse complement studies, the number of samples was n=12 for C2 and C4β and n=9 for the rest. For human

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complement studies, the number of samples was n=4. For mouse nitrotyrosine and

carbonyl studies, the number of samples was n=5. We analyzed a different number of ‘n’

values to attain statistical significance. For mouse ALF, the following antibodies from

Santa Cruz Biotechnology, Inc. were used: SP-A (sc-166914), SP-D (sc-59695), C2 (sc-

134639), C3 (sc-70474 or sc-28294), C4b (sc-74524), donkey anti-mouse IgG-HRP (sc-

2314), factor B (sc-67141), goat anti-rabbit IgG-HRP (sc-2004). OxiSelect Nitrotyrosine

Immunoblot Kit (BioCell Labs), and OxiSelect Carbonyl Immunoblot Kit (BioCell Labs) were used to determine protein oxidation. For human ALF, the following antibodies were used: SP-A (sc-166914, Santa Cruz Biotechnology, Inc.), SP-D (sc-59695, Santa Cruz

Biotechnology, Inc.), C2 (sc-134639, Santa Cruz Biotechnology, Inc.), C3 (sc-28294/B9,

Santa Cruz Biotechnology, Inc.), C5 (A220, Complement Technology, Inc.), and C7 (sc-

160195, Santa Cruz Biotechnology, Inc.).

Hydrolytic enzyme activity in ALF. Hydrolase activities present in ALF were measured

using a colorimetric method based on the release of p-nitrophenol upon specific substrate

cleavage. To determine hydrolase activity in ALF, 25 µl substrate (2.5 mM) and 25 µl

human ALF (each containing in vivo-relevant alveolar levels of 0.5–1.5 mg/ml

phospholipids by phosphate assay) were loaded in a 96-well plate. After 1 h incubation at

37°C with gentle shaking, reactions were stopped by adding 65 µl of 1 M sodium

carbonate and the optical density was measured at OD450nm. For negative controls,

substrates were incubated with 25 µl buffer under the same conditions, and reactions

were stopped as described. As a positive control, we incubated 0.1 U available α- 179

mannosidase (Prozyme, Hayward, CA) with 10 mM p-nitrophenyl-α-O-mannoside in

0.125 M sodium acetate buffer (pH 5) as substrate. Results were normalized to negative controls. Substrates used to determine the hydrolase content in ALF and human lung tissue are indicated in parentheses: 1) Acid phosphatase (p-nitrophenyl-phosphate); 2) α- mannosidase (p-nitrophenyl-α-mannoside); 3) α-galactosidase (p-nitrophenyl-α- galactopyranoside); 4) β-galactosidase (p-nitrophenyl-β-galactopyranoside); 5) α- glucosidase (p-nitrophenyl-α-D-glucoside); 6) β-glucosidase (β-Glc; p-nitrophenyl-β-D- glucoside); 7) α-xylosidase (p-nitrophenyl-α-D-xylopyranoside); 8) α-fucosidase (p- nitrophenyl-α-L-fucoside); 9) arylsulfatase (dipotassium p-nitrocatechol sulfate); 10) fatty acid esterase-I (p-nitrophenylpalmitate ester); 11) nonspecific esterase (p- nitrophenylacetate); 12) alkaline phosphatase (AlkP; p-nitrophenyl-phosphate); 13) alkaline phosphodiesterase (bis[p-nitrophenyl]phosphate); 14) phospholipase C (p- nitrophenylphosphorylcholine); 15) peroxidase (O-phenylenediamine HCl); 16) α- rhamnosidase (p-nitrophenyl-α-L-rhamnopyranoside; and 17) fatty acid esterase-II (p- nitrophenylstearate ester). Samples were normalized by ALF phospholipid content (1 mg phospholipid/ml of ALF) mimicking the physiological conditions described within the healthy lung (162). For these studies, the number of samples was n=8/group.

Alveolar lining fluid lipid content. Total lipids were extracted from ALF samples

(normalized by protein content, 100 µg) from young and old mice by following a standard lipid extraction protocol (596). First, lipids are extracted with a solution of 2:1

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chloroform:methanol (C:M) for 2 h at 37°C. The organic phase was collected and the

lipids are then extracted with 1:2 a solution of C:M for 2 h at 37°C. The organic phase is

collected, pooled with the organic phase of the first extract, and the lipids are then

extracted with a solution of 10:10:3 C:M:water for 2 h at 37°C. The remaining organic

phase in pooled with the two others. The three pooled extracts are dried under N2 and

resuspended at working concentration in chloroform. Samples were analyzed by gas-

chromatography mass-spectrometry (GC-MS). Samples were directly analyzed by

electrospray ionization tandem mass spectrometry (ESI)-MS/MS in a positive ion mode

(596).

Complement and surfactant protein binding assays. M.tb single cell suspensions were

exposed to adult or elderly ALF for 12 h at 37°C with gentle nutation. Exposed-M.tb

bacilli were washed, suspended in 0.9% NaCl, and 1.0x106 were plated onto a medium-

binding 96-well plate (572;597). Monoclonal antibodies directed against complement

component 3 (C3) or surfactant protein A (SP-A) were used to quantify the amount of protein bound to the M.tb cell wall after exposure to ALF. As controls, purified 2 µg of

C3 or SP-A were immobilized onto the plate. HRP-conjugated secondary antibodies and

TMB substrate were used to determine the relative amount of bound protein by

colorimetric measurement at OD450nm.

In vitro infections using human monocyte derived macrophages. Peripheral blood

mononuclear cells (PBMCs) from healthy adults were collected and prepared as

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described in chapter three. Monocyte-derived macrophage (MDM) monolayers were infected at a multiplicity of infection (MOI) of 1:1 with a single cell suspension of M.tb

that had been previously exposed to adult or elderly ALF. At each time point (2h-120h)

cells were lysed as described in chapter three, and colony forming units (CFUs) were

enumerated by plating serial dilutions onto OADC supplemented 7H11 agar plates. M.tb

CFUs were counted 21 days later. Human alveolar macrophages (HAMs) were collected

from healthy adults by bronchoalveolar lavage, washed twice in an excess volume of cold

of 0.9% NaCl, and allowed to adhere for three hours to cell culture plates at 37°C, 5%

CO2 prior to infection with the same conditions. At each time point, CFUs were

enumerated as described in chapter three.

Immunocytochemistry and confocal microscopy. MDM monolayers on glass coverslips

were infected for 2 h with adult- or elderly-ALF exposed M.tb. The cells were fixed in

2% paraformaldehyde for 10 min and washed in PBS. The cells were then permeabilized with a 1% solution of triton X-100 for 5 min, washed with PBS, and incubated in blocking buffer [10% FBS plus 5 mg/ml bovine serum albumin (BSA; Sigma) in DPBS] for 1 h at 37°C. Primary rabbit anti-human IgG1 antibody against CD63 (2 µg/ml) (Santa

Cruz Biotechnology) was used as an endosomal/lysosomal marker. Goat anti-rabbit IgG conjugated to Alexa Fluor 568 (Invitrogen) was used as the secondary antibody. Normal rabbit IgG1 served as the isotype control. The nucleus was stained with 400 ng/ml 4',6- diamidino-2-phenylindole [DAPI (Invitrogen)] prior to mounting onto glass microscope slides. Coverslips were mounted on slides using ProLong Gold Antifade Reagent

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(Invitrogen). The cells were visualized by laser-scanning confocal microscopy using an

Olympus Filter/Spectral FV1000 Confocal microscope set at appropriate parameters to

minimize background fluorescence at a final magnification of 600X. At least 100

internalized bacteria were counted per experiment and analyzed for co-localization of

phagosomes containing GFP-M.tb (GFP, 488nm) with CD63 (lysosomal marker,

AlexaFluor, 568nm). Microscopy data was analyzed using ImageJ or Olympus FV1000

software.

In vivo M.tb infections of C57BL/6J mice. Adult or elderly ALF-exposed M.tb was

delivered into the lung of C57BL/6J mice via aerosol using an inhalation exposure

system calibrated to deliver 40–100 CFU to the lungs of each mouse as described in

chapters two and three. At 21, 60, and 150 days post-infection, the lung and spleen were aseptically removed, homogenized, diluted, plated onto OADC supplemented 7H11 agar plates. M.tb CFUs were counted 21 days later as described in chapters two and three.

Histopathology. Mice lung lobes were fixed in 10% formalin, embedded in paraffin,

sectioned, and stained with hematoxylin-eosin to visualize lung morphology post

infection with adult or elderly ALF-exposed M.tb as described in chapters two and three.

Sections were examined by a board-certified veterinary pathologist without prior

knowledge of the experimental groups and graded according to severity, granuloma size

and number. H&E-stained slides were digitized for morphometric analysis using Aperio

ScanScope XT slide scanner (Leica, Buffalo Grove, IL) at 40X magnification. Immune

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cell infiltration and granulomatous tissue was calculated by manually outlining all foci and determining the total area of inflammation as a percentage of the total area of the lung.

Statistical analysis. Statistical significance was determined using Prism 4 software

(GraphPad Software, San Diego, CA). The unpaired, two-tailed Student’s t-test was used for two group comparisons. Multiple comparisons were analyzed using one-way

ANOVA with Tukey’s post-hoc test. Statistical significance was reported as *, p<0.05;

**, p<0.01; or ***, p<0.001.

Results

Signatures of inflammaging are evident in human ALF

We hypothesized that the aging lung environment exists in a pro-inflammatory state similar to what has been observed in the systemic circulation (598;599). Thus, we expected proteins associated with inflammatory signatures would be elevated in the ALF of elderly humans. We began our studies by analyzing the global proteome of ALF isolated from adult or elderly humans by liquid chromatography tandem mass spectrometry (LC-MS/MS). Protein hits from the MS analysis were cross-referenced to corresponding genes using the Uniprot database and analyzed with Ingenuity Pathway

Analysis (IPA) software. Under the ‘disease and function’ category, we discovered that

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elderly humans had more signatures associated with inflammation (Table 4.1).

Furthermore, upstream analysis revealed more inflammatory signatures in the ALF of

elderly individuals (Table 4.2) (600).

Pro-inflammatory cytokines levels are higher in the ALF of aged humans and mice

To validate our proteomic and bioinformatics analyses, we measured the levels of

cytokines in ALF collected from naive young (3 months) and old (18 months) mice or

human ALF from adult (25–44 years) and elderly (65+ years) subjects collected by

bronchoalveolar lavage (BAL). ALF was normalized by protein content, which was

equivalent in young and old mice (Fig. 4.1A) and adult and elderly humans (Fig. 4.1C).

In mice, increasing age was associated with a significant increase in TNF and IL-6 in the

ALF and a trend for increased IL-1β (Fig. 4.1B). IL-12p40, IL-10, and IFNγ were equivalent to those measured in young mice. We observed a similar significant increase in the level of IL-6 in ALF samples from elderly humans and a trend for increased TNF and IL-1β (Fig. 4.1D). IL-12 and IFNγ, while not significantly altered, trended toward decreased levels in the elderly. There was also a trend toward an increase in IL-10 that did not reach statistical significance. Overall, our results demonstrate that the aging lung, in both mice and humans, contains a basal increase in pro-inflammatory cytokines.

Levels of SP-A and SP-D in ALF increase in old age

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We hypothesized that SP-A and SP-D would be increased in the lungs of old mice and elderly subjects relative to their younger counterparts. This is in part thought to be a consequence of alveolar epithelial cell dysfunction, resulting in higher amounts of serum-

derived components in the lung which contributes to a relatively pro-inflammatory lung

environment (601). Levels of SP-A and SP-D were determined in ALF. Semi-quantitative

gel electrophoresis and Western blotting of individual samples showed that old mice had

significantly higher levels of SP-A and SP-D in their ALF (Fig. 4.2A) compared to young mice. Similar trends were observed for SP-A and SP-D in humans (Fig. 4.2B), although these did not reach statistical significance. The increased levels of SP-A and SP-D that we observed within the ALF in old age are in accordance with our hypothesis and supported by the fact that recycling of pulmonary ALF may be dysfunctional in old age

(579;602).

Complement proteins within ALF are altered in old age

With the knowledge that ALF components such as SP-A and SP-D are elevated in old age, we further anticipated that complement components would also be altered.

Complement protein levels were determined in ALF from old and young mice and adult and elderly humans by Western blotting. We focused primarily on the classical complement pathway as we and others have shown that components of the alternative complement pathway are at very low levels in ALF (575;594). C2, C3, and C4 molecules

were determined. For completeness, we also measured factor B (FB) of the alternative

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pathway, and C5 and C7 molecules as they assemble to form the membrane attack

complex (594). Old mice had significantly higher levels of complement proteins C3β, and

C4β (both products of C4 cleavage), and FBα and FBβ (Fig. 4.3A). C3α showed a trend for increased amounts, but did not reach significance. C2 in the ALF of old mice was significantly lower than that measured in young mice. In human samples, we observed a trend for decreased C2 and increased C7 in the ALF of elderly subjects, but other complement components were unaltered (Fig. 4.3B).

Enzymatic activity of ALF hydrolases decreases with age

We have shown that human and mouse ALF contains abundant homeostatic and antimicrobial enzymes (hydrolases) (432;433;603;604) that are capable of altering the cell envelope of M.tb (162). Thus, alveolar hydrolase serve as additional modulators of lung function that can be altered by increasing age. We determined the hydrolase activity of ALF from old and young mice. Our results indicate that old mice have an overall decrease in lung hydrolase activity (Fig. 4.4). This decrease could be attributed to poor

ALF recycling by dysfunctional alveolar epithelial cells as well as due to lung physiological dysfunction which is associated with chronic inflammation (579;602).

Myeloperoxidase levels and protein oxidation within ALF increases with age

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Increasing age is highly associated with increased oxidative stress (584;605). Therefore, we anticipated that the aged lung would represent a relatively oxidized environment.

Although ALF from young and old mice was found to contain similar protein content

(Fig. 4.1A), we observed a 2.85-fold or 65% increase in the levels of myeloperoxidase

(MPO) (Fig. 4.5A) Additionally, carbonyl residues and nitrotyrosine residues [reliable

and stable markers of ROS and reactive nitrogen species (RNS) oxidation of proteins, respectively (606;607)] (Fig. 4.5B, C) were significantly elevated in old mice suggesting that during aging there is an increase in oxidative damage in the alveolar environment. In support, LC-MS/MS of BALF indicated that proline (P) residues of SP-A (amino acids sequence 48 DGIKGDPGPPGPMGPPGGMPGLPGRDGLPGAPGAPGEHGDKGEPGE

R 94) and SP-D [(102 GLSGPPGLPGIPGPAGK 118), (168GAPGVQGAPGNAGAAG

PAGPAGPQGAPGSR 197)] found in elderly ALF were converted to 2-hydroxyproline, its oxidized form. In addition, we observed a 1.65-fold increase in proteins with oxidized tryptophan (W) [5-hydroxytryptophan, 5-nitrotryptophan] and cysteine (C) [cysteine sulfenic acid, cysteine sulfinic acid, and cysteine sulfonic acid] residues in the elderly human and old mouse ALF (608). Thus supporting that SP-A/-D, and potentially other soluble innate immune proteins, are oxidized in the aging lung.

POPC, a marker of inflammation, is elevated in ALF in old age

1-Palmitoyl,2-oleoyl-sn-glycero-3-phosphocholine (POPC or PC16:0/18:1, mass

(M) = m/z 760, shown in Fig. 4.6 as the m/z 782 [M − H + Na]+ sodiated form) is a

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marker of inflammation. 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC or

PC16:0/16:0, M = m/z 734, shown in Fig. 4.6 as the m/z 752 [M − H + Na]+ sodiated form) is the non-oxidized form of POPC that comprised of ~40% of PC lipids in surfactant. POPC is thought to originate from necrosis and membrane shedding from activated inflammatory cells within the lung. Thus, we examined the level of POPC by extracting surfactant lipids from the ALF of old and young mice and directly analyzing the samples by electrospray ionization tandem mass spectrometry (ESI/MS/MS) in a positive ion mode (596). Our data show that DPPC is the dominant lipid present within

ALF as expected. In contrast, old mice accumulate POPC rather than DPPC (Fig. 4.6).

Our ESI/MS/MS results, together with the above data, provide evidence for the existence of a pro-inflammatory environment within the aging lung.

SP-A, C3 from elderly human ALF are less capable of binding to M.tb

To test our hypothesis that soluble innate immune proteins become less functional with

age, we assessed the binding ability of SP-A and C3 to the M.tb cell wall. Both SP-A and

C3 have been shown to be important modulators of host immunity to M.tb by binding to its cell wall and influencing the initial host-pathogen interaction. M.tb was incubated in adult- or elderly-ALF as describe in chapter two. M.tb bacilli were then washed, plated, and probed with monoclonal antibodies directed against SP-A or C3. In support of our hypothesis, we discovered that C3 derived from the lung of elderly human ALF was significantly less capable of binding to the M.tb cell wall. Furthermore, although not

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statistically significant, SP-A binding was reduced (Fig. 4.7). We attribute this non-

significant decrease to a low number of human samples. Together these results further

support our initial hypothesis that inflammaging drives decreases in innate immune

function.

Exposure of M.tb to elderly ALF provides a growth advantage in human macrophages

Having observed decreases in the binding of SP-A and C3 from elderly ALF to the cell wall of M.tb, two important proteins that mediate M.tb-macrophage interactions, we hypothesized that macrophages infected with elderly-ALF exposed M.tb would be less capable of controlling the infection. We infected 2.5x105 human MDMs or 1.0x105

human alveolar macrophages (HAMs) with a single cell suspension of adult- or elderly-

ALF exposed M.tb at an MOI 1:1. Despite equal uptake 2 h post infection, MDMs

infected with elderly-ALF exposed M.tb had significantly higher bacterial burden starting

at 48 h post infection, a trend that was observable for the remaining time points (Fig.

4.8). We observed similar trend for HAMs (Fig. 4.8). Together, the data provides

evidence that homeostatic components present in ALF can influence M.tb survival

mechanisms within human macrophages.

Exposure to elderly-ALF reduces phagosomal biogenesis in M.tb infected macrophages

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We next explored whether the increased virulence associated with M.tb exposed to

elderly ALF was due to macrophage killing mechanisms. We analyzed P-L fusion events occurring within macrophages previously infected with adult- or elderly-ALF exposed

M.tb for 2 h by staining the cells with the endosome/lysosome marker CD63. The cells were visualized under scanning laser confocal microscopy. Co-localization of CD63 puncta (red) with GFP-M.tb (green) was deemed a fusion event (yellow). Non-fused bacteria remained green. Independent experiments using different human blood and ALF donors revealed that macrophages infected with elderly-ALF exposed M.tb had significantly fewer co-localization events compared to macrophages infected with adult-

ALF exposed M.tb (Fig. 4.9). This suggests that ALF serves as an important antimicrobial solution that promotes clearance of infectious particles by innate immune cells. This phenomenon appears to become impaired with increasing age.

Exposure of M.tb to elderly ALF increases its pathogenicity in vivo and accelerates immunopathology of the lung

To determine the biological significance of ALF in the context of M.tb infection in vivo, we next assessed disease outcomes after infecting mice with adult or elderly-ALF exposed M.tb. C57BL/6J mice were infected with a low dose aerosol of M.tb that had been previously exposed to adult or elderly ALF. Despite equal inoculums (Fig. 4.10A), mice infected with elderly-ALF exposed M.tb had significantly higher M.tb bacterial burden in the lung and spleen at 21, 60, and 150 days post infection (Fig. 4.10B). This

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suggests that the antimicrobial properties of ALF also play a significant role in vivo.

Furthermore, we assessed the survival of mice infected with the two conditions. We

found a non-significant trend for increased survival of mice infected with adult-ALF exposed M.tb (Fig. 4.10C). Finally, we assessed the relative amount of inflammation caused to the lung across time. Not surprisingly, mice infected with elderly-ALF exposed

M.tb developed significantly larger inflammatory foci at 21 and 60 days post M.tb infection. At 150 days post infection we observed the same trend, but it was not statistically significant (Fig. 4.11). Thus, it appears that ALF plays a role in the early stages of infection with M.tb but differences are diminished once bacteria have successfully colonized its host.

Discussion

The relevance of ALF in the elderly during microbial infection or the development of chronic pulmonary diseases is essentially unknown. Thus, the goal of this study was 1) to the characterize baseline levels of selected components that are likely to play a major role in the local alveolar immune response, and 2) discern the mechanistic role of ALF both in vitro and in vivo. Of particular significance, we observed that several ALF components are similar in both mice and humans, making the study of long-term in vivo phenotypic and functional changes possible using a mouse model. Furthermore, the changes observed in specific-pathogen-free housed old mice are representative of a natural aging phenotype without the confounding factors that are associated with environmental

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challenges seen in humans, and therefore, the similarities we observed between mouse and human indicate that those changes we found in human samples are indeed related to increasing age. Mechanistic studies focused on elucidating the impact of ALF on M.tb pathogenesis and virulence and the consequences of exposing M.tb to ALF in terms of infection in vitro and in vivo.

In these studies, we analyzed selected components of ALF in aged mice and/or humans.

We show that ALF from old mice have elevated levels of pro-inflammatory cytokines,

SP-A and SP-D, and several complement components. We further demonstrate that the

ALF of mice contains an increase in oxidized molecules consistent with oxidative damage and an imbalance in DPPC and POPC, consistent with basal pulmonary inflammation. A comparative analysis of ALF from human samples showed that ALF in the elderly also contained increased amounts of pro-inflammatory cytokines and of SP-A and SP-D. High levels of pro-inflammatory cytokines such as IL-6 in ALF from elderly individuals were reported in a previous study (609). Furthermore, we discovered that soluble innate immune proteins from elderly ALF were less capable of binding to the cell wall of M.tb, and may have facilitated survival of M.tb within human macrophages by downregulating macrophage defenses such as P-L fusion. We also show that ALF may aid in the immune response to M.tb in vivo and that the declining function of ALF brought forth by age may be one of the underlying reasons behind the increased susceptibility to M.tb observed in the elderly. Overall, our studies demonstrate that the pulmonary environment in old age is significantly altered resulting in an enhanced

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inflammatory state, supporting the concept of inflammaging that extends beyond the

systemic circulation to the lung alveolar environment. The impact of local inflammation

on the development of common pulmonary diseases of the elderly, including the

increased susceptibility to infection, has received little attention. Our studies lay the

foundation for future mechanistic studies to establish a causal link between pulmonary

inflammaging and lung disease.

The global proteome of ALF differed quite starkly between adult and elderly humans. We observed a shift from an anti-inflammatory state in adults to a more pro-inflammatory

state in the ALF of elderly individuals. The top 32 IPA hits under ‘disease and function’

revealed elderly humans had higher associations with inflammatory states. In this

context, the lower the p-value, the lower the chance the association is due to random

chance. Using a p-value cutoff of E-20, elderly ALF was associated with 15 different inflammatory disease categories, while adult ALF only had 3 (Table 4.1). Some adult humans also had some degree of inflammation and we attribute this to our patient exclusion criteria. Due to IRB constraints, we were unable to perform bronchoalveolar lavages on healthy elderly individuals. Instead, all elderly subjects underwent lung resections due to cancerous growth in the lung. For our studies, only the healthy side of the lung, where no surgery was to take place, was lavaged. To properly control our experiments, our LC-MS/MS studies were carried out using ALF from adults who also underwent a lung resection. Not surprisingly, ‘disease and function’ analysis of both adult and elderly ALF revealed pathways associated with cancer. The fact that adult samples

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were obtained from individuals with early-stage cancer could explain the increases in

inflammatory signatures we observed in adult ALF, as cancer is associated with increases

in basal inflammation (610). Analysis of the top 20 upstream modulators suggested that

the reason behind increased inflammatory signatures in the elderly was due to pathways

associated with LPS, TNF, IFNγ, IL-6, and IL-1β (Table 4.2). While LPS and TNF were also contributing factors in adults, their p-values were lower than in the elderly suggesting these pathways are more commonly associated with increasing age. Thus, our proteomic screen indicates that aging increases the basal level of inflammation in the lung.

To validate our bioinformatics analysis we assess the basal levels of inflammatory cytokines within young or old mouse ALF or adult or elderly human ALF. We observed a very clear correlation between increases in age and increases in the pro-inflammatory cytokines TNF, IL-6, and IL-1β in the ALF of both mice and humans. This was further indication that the aging lung is an increased basal state of inflammation supporting the theory of inflammaging. We also observed cytokine disparity in IL-10 and IL-12, two interrelated cytokines (611), in mice and humans. IL-10 acts as an immunomodulator of

IL-12 secretion via the extracellular signal-regulated kinases (ERK1/2) molecular switch

(612) and it is plausible that this biological switch works differently as we age and/or its functionality varies among species. These results could also be simply explained by the lack of power in some of our analyses due to the limited human ALF sample size in both groups. 195

Enhanced inflammatory signatures are suggestive of altered inflammatory cell numbers within the lung with increasing age. It was reported that an increase in neutrophils in human ALF as a marker of acute and/or chronic inflammation, i.e., from two percent total neutrophils in ALF from healthy adults (mean age 28) to eight percent in ALF from idiopathic pulmonary fibrosis patients (mean age 58) (613). However, when looking at neutrophil numbers in healthy adults and elderly individuals, the same authors found that the adults had more neutrophils in the ALF than the elderly (1.1% vs. 0.3%, respectively)

(614). Although we were unable to acquire the cytological data from our human samples, our mouse cytological data showed a similar trend with a relatively low number of neutrophils in ALF from young and old mice (4.4% and 3.1%, respectively), indicating that the basal lung inflammation observed in aging may not be driven solely by an influx of neutrophils into the alveolar space in the absence of other stimuli.

As the lung ages, it loses alveolar complexity and surface area (615), and changes in the amount and function of surfactant and other innate immune determinants occur.

Information about the composition of the lung innate immune determinants in humans as we age is relevant from a clinical perspective to understand respiratory infections.

Following infection of the lower respiratory track, any given microbe will reside in the alveoli and be bathed in ALF. Thus, ALF components such as SP-A, SP-D, hydrolases, lipids, and complement are likely to modulate and define the interaction between the microbe and host immune cells. Determinants that promote microbe survival and an

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aberrant host response in this context will be detrimental to the host, whereas factors that

promote effective microbial clearance with an appropriately focused and localized host

response will be protective. In this context, increased levels of SP-D drive susceptibility to fungal infections (616), whereas high levels of SP-A indirectly drive susceptibility to

M.tb infection (88;617) and modulate the lung inflammatory response by regulating several macrophage activities (589-591). Our results using mouse ALF showed an increase in SP-A and SP-D in old mice. This trend was also observed in human ALF. We opted to normalize by total protein content. Other studies have normalized by urine, by albumin, or by recovered ALF volume; thus, the process of normalization generates some discrepancies in the levels of SP-A and SP-D reported. This is a challenge when comparing ALF-soluble components between age groups. Human studies comparing adults vs. middle-aged/elderly subjects that normalized by recovered ALF volume showed a slight decrease in SP-A and no differences in SP-D with increasing age (614).

However, in this study (614), ALF recovery varied among subjects, and thus may have affected the comparisons made between age groups. This caveat is minimized in our study since we normalized samples by protein amount.

Complement proteins (and their cleaved products) have been detected in human ALF obtained from healthy hosts, where complement levels differed from levels in human serum (594). Complement is a complex, highly regulated protein system with an essential role in host defense through bacterial lysis, stimulation of phagocytosis, recruitment of immune cells to infected/damaged tissue, and promotion of the inflammatory response.

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The relative changes in complement components observed in the ALF of old mice

(decreased C2, increased C3 and C4) indicate that increasing age may lead to problems

associated with MAC formation or antibody complex clearance associated with the

classical arm of the complement cascade. This is supported by the reported reduced

function of antibodies in elderly donors and may contribute to their increased

susceptibility to infections (618-620). Similarly, our data indicate that the low levels of

C2 and high levels of factor B observed in the ALF of old mice may favor the alternative pathway in lower respiratory tract secretions (621). This is in contrast to what we have seen in the healthy human adult lung where the classical pathway of the complement system appears to dominate (575). We also failed to observe similar changes in complement components between adult and elderly human donors, although the sample size precludes making any firm conclusions about these data. The significance of different complement components between mouse and human requires further investigation.

A variety of hydrolases have been related to ALF recycling and degradation (622), many of which have lysosomal-type degradative functions. Some of these hydrolases are highly active (i.e., alkaline and acid phosphatases, and non-specific esterases) while others much less so (i.e., α-mannosidase and arylsulfatase) (162). The presence of low hydrolase activity in the lungs of old mice could imply that ALF is not properly recycled by type 2

ATs (623-628), a mechanism likely due to cell dysfunction or senescence that can arise due to telomere shortening (629). This, in turn, can drive hydrolase-induced tissue

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destruction and perpetuate chronic inflammation and oxidation (630). Although little is

known about the exact role of ALF hydrolases in stabilizing the healthy lung

environment or during microbial infection, we have shown that ALF hydrolases can alter

the cell envelope of M.tb during infection promoting pro-inflammation (162).

Lung surfactant is composed of ~90% lipids. Of all the species, phosphatidylcholine (PC)

comprises ~80% of the total surfactant lipid, about half of which is DPPC (587). The rest

of the lipids are charged phospholipids (PE, PG), cholesterol, acylglycerols, and free fatty

acids (585). As discussed in chapter one, these lipids reduce the surface tension of water making the essential for normal respiratory function. In this context, surfactant lipids can modulate the release of oxidative and inflammatory mediators from inflammatory cells and help regulate inflammation (631-633). For example, DPPC can reduce the oxidative response of monocytes stimulated with zymosan or phorbol-12-myristate-13-acetate

(PMA), whereas 1-palmitoyl-2-arachidonoyl (PAPC) did not (632). In contrast, alterations in the composition of surfactant lipids can lead to lung surfactant dysfunction

(592), including a reported decrease in SP-A function (634). Alterations in surfactant phospholipid profiles have also been directly linked to the cause of chronic obstructive pulmonary disease (COPD) (635), a disease that commonly plagues the elderly (636). We show that the aged lung is a relatively oxidized environment as indicated by large amounts of MPO, carbonyl and nitrotyrosine residues in ALF proteins, and by the presence of oxidized lipids such as POPC. POPC is thought to originate from necrosis

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and membrane shedding from activated inflammatory cells within the lung and its

accumulation has been reported in the lung surfactant of cystic fibrosis patients (596).

The likely explanation for the increases in inflammation we observed in the ALF may be

alveolar epithelial cell dysfunction. In old age, AT cell senescence slows down ALF

recycling and allows for the accumulation of enzymes and proteins within the lung ALF

(582). This buildup can lead to surfactant lipid oxidation which can lead to surfactant

dysfunction, and increases in complement levels with poorly regulated activity that can

contributes to chronic inflammation. In turn, chronic inflammation could result in a

perpetual state of activated macrophages that secrete more complement locally further

propagating the inflammatory cycle (581). Surfactant lipid accumulation and oxidation

could also potentially affect antimicrobial protein functions (SP-A, SP-D). In this regard,

our results show that the aging lung contains an altered DPPC:POPC ratio. SP-A binds to

DPPC but not to POPC (637), suggesting that soluble SP-A may be particularly

susceptible to oxidation in the ALF of elderly individuals. In this context, just the mere

presence of POPC directly interferes with SP-A-DPPC complex formation (637). Non-

functional oxidized SP-A would not effectively control complement activation, thereby

further enhancing inflammation and lung injury in aging. Moreover, nitrated SP-A has decreased ability to aggregate surfactant lipids (637). Reduced SP-A and/or SP-D activity is also expected to contribute to increased lipid oxidation in the lungs of old individuals, since both have been shown to be potent inhibitors of surfactant lipid peroxidation (638).

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We observed decreased binding of both SP-A and C3 to the cell wall of M.tb after incubation with elderly ALF. These observations further support our hypothesis that soluble innate immune proteins lose functionality with increasing age. In support, mass- spectrometry analysis revealed that certain amino acids of SP-A and SP-D derived from elderly ALF are oxidized. Oxidized amino acids were primarily located within non-active regions of the proteins (i.e. collagenous domain) and not on their binding domains (i.e. carbohydrate recognition domain) (639). However, oxidation of amino acids can induce conformational changes of the protein, resulting in the formation of protein-protein cross- linkages leading to fragmentation of the protein, which can inhibit their function

(640;641). In this context, binding of SP-A to microbes has been shown to enhance macrophage phagocytosis. SP-A was shown to directly bind the flagella of Pseudomonas aeruginosa thereby enhancing macrophage phagocytosis and increasing intracellular killing mechanisms (642). Similar observations were reported with Streptococcus pneumoniae (643). It was later shown that SP-A was capable of enhancing endosomal trafficking in alveolar macrophages through direct regulation of Rab7, a marker of late phagosome maturation (644). In contrast, SP-A can induce inflammatory responses and drive inflammaging. SP-A can stimulate the release of neutrophil chemotactic factors by type II alveolar epithelial cells (645;646) in the absence of infection. Thus, accumulation of SP-A due to failure of alveolar epithelial cells to recycle surfactant can propagate chronic inflammation. Lastly, although we were unable to measure differences in binding of SP-D from adult or elderly ALF due to sample limitations, we anticipate that oxidation of SP-D would also have resulted in reduced binding to M.tb. Binding of SP-D to M.tb

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was shown to promote phagosome-lysosome fusion in macrophages upon phagocytosis

of M.tb (573). In contrast, binding of SP-D to M.tb reduces phagocytosis of the bacteria

(572). Thus, oxidation of SP-D may benefit M.tb by allowing it to escape macrophage

phagocytosis and intracellular trafficking mechanisms.

The ultimate goal of our studies was to explore the consequences and relevance of

exposure to ALF in vitro and in vivo. To do this, we incubated M.tb with ALF from adult

or elderly humans and used the modified bacteria to infect macrophages and assess

bacterial survival and intracellular trafficking patterns. Macrophages infected with

elderly-ALF exposed M.tb were less capable of controlling the infection than

macrophages infected with adult-ALF exposed M.tb. This phenomenon was reproducible

using MDMs from several different human donors and in primary HAMs, suggesting

ALF plays an important antimicrobial role in the context of infection. These observations

could be attributed to the oxidation of innate immune proteins in the lung. As described

above, SP-A and SP-D are important host proteins that help mediate macrophage

responses to pathogens including M.tb. SP-A promotes the phagocytosis of pathogens, and in the case of other organisms, leads to effective clearance of the pathogen. Whereas

SP-D can enhance phagolysosomal fusion in macrophages leading to increased killing of

M.tb (573), the outcome of SP-A-mediated phagocytosis remains unclear. Although SP-A enhances uptake and killing of M. bovis BCG, it does not affect intracellular killing of M. avium (647;648). SP-A may indirectly promote the survival of M.tb within non-acidified phagosomes. SP-A was shown to increase the expression of the macrophage mannose

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receptor (MR) (649) which may promote entry of M.tb via this receptor. The MR has

been linked with survival of M.tb within non-acidified phagosomes (234;337).

Furthermore, SP-A-mediated recognition of M.tb by alveolar macrophages suppressed

the production of nitric oxide, suggesting that M.tb may utilize SP-A as an additional

mechanism to subvert host immunity (650). Thus, the literature suggests a host-

detrimental role for SP-A in the context of M.tb infection. Our results revealed that SP-A

from elderly ALF is less capable of binding to the cell wall of M.tb. Thus, our findings

appear to contradict previous reports on SP-A. However, lack of SP-A on the surface of

M.tb could direct entry towards mechanisms associated with mycobacterial survival such

as the MR.

Furthermore, our association studies did reveal significant decreases in the binding of C3

from elderly ALF to M.tb. Similar to SP-A, the outcome of deposition of C3 on M.tb in the context of whether its beneficial for the bacteria or for the host remains unclear.

Complement receptors (CRs) recognize different ligands; CR1 binds complement component C1q, C4b, and C3b, whereas CR3 and CR4 preferentially bind to C3bi

(575;651). However, no study has been performed to assess the effects of C3 deposition on M.tb and the impact it has on intracellular trafficking. Thus, it is possible decreased deposition of C3 onto the mycobacterial cell will have no effect on intracellular killing mechanisms. One important difference we did observe, however, was a significant decrease in the enzymatic activity of lung hydrolases in the ALF of aged mice. Although we were unable to measure their enzymatic activity in ALF of elderly humans, our

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combined results predict a similar phenotype. As discussed in previous chapters, we have shown that these homeostatic and antimicrobial hydrolases can dramatically modify the cell wall of M.tb, significantly decreasing the abundance of lipid virulence factors. These cell wall-modifying effects of the hydrolases altered M.tb intracellular trafficking, and favored intracellular killing of the bacilli via mechanisms related to phagosome biogenesis (162;187). Our intracellular trafficking studies revealed that macrophages infected with elderly-ALF exposed M.tb displayed fewer M.tb-CD63 co-localization events compared to macrophages infected with adult-ALF exposed M.tb. This directly suggests that macrophages infected with elderly-ALF exposed M.tb are less capable of killing the bacteria. Furthermore, it implies that proper function of ALF hydrolases is an important anti-mycobacterial mechanism of the lung. This process may be impaired in old age and may help contribute to the increased susceptibility to infection observed in elderly populations.

Finally, our results support the notion that ALF contributes to the control of M.tb infections in vivo. We found that despite equal inoculums, C57BL/6J mice infected with elderly-ALF exposed M.tb displayed higher bacterial burden in the lung and spleen at 21,

60, and 150 days post infection. We did not, however, observe significant differences in survival; adult-M.tb infected mice had a median survival of 55.50 weeks while mice infected with elderly-M.tb had a median survival of 51.00 weeks. Although we observed statistical significance in bacterial burden at 150 days post infection, it was clear that bacterial burden began to normalize as the infection progressed. We observed a similar

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trend in the pulmonary immunopathology. At 21 and 60 days post infection, mice infected with elderly-ALF M.tb had significantly more widespread inflammation in the lung. By 150 days post infection, inflammation was approximately equal in both groups.

Thus, the data suggest that the antimicrobial properties of ALF play a role early during in vivo infection, but does not prevent establishment of the infection or continue to inhibit

M.tb growth in the long term in the mouse model. Although little in vivo evidence exists of the antimicrobial effects of ALF, one study found that Pseudomonas aeruginosa infection in rats increased alveolar epithelial fluid (i.e. ALF) clearance by 48% and that the mechanism was dependent on TNF (652). In contrast, one report indicated that SP-A and SP-D were dispensable for the control of M.tb delivered via the aerosol route (653).

The mouse, however, is not a natural host for M.tb and does not faithfully recapitulate

M.tb pathology and outcome of infection (i.e. no granulomagenesis, no natural latency or clearance) (654). Thus, although the mouse model is a powerful tool for studying TB, the influence of ALF on the infection cycle of M.tb will have to be evaluated in the context of a more relevant animal model (i.e. non-human primate or humans).

Our studies further support the theory of inflammaging. Not only were we able to detect markers associated with inflammaging in the lung via proteomic analysis, we confirmed that basal levels of pro-inflammatory cytokines are elevated in the aging lung. As a result, loss of homeostasis may lead to increases in basal levels of oxidation and contribute to protein dysfunction. In turn, loss of function in innate immune proteins may be an important contributing factor behind the increased susceptibility of elderly individuals to

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infectious disease including M.tb. Here, we provide evidence that components present in

ALF can significantly impact the control of M.tb by human macrophages. Macrophages infected with elderly-ALF exposed M.tb were less capable of controlling the infection, a mechanism that may have been related to inhibition of intracellular killing mechanisms.

Additionally, we provide proof of principle that modifications brought forth by ALF to the cell wall of M.tb may be biologically important in the context of infection in vivo.

Altogether our data reveal an important antimicrobial and homeostatic effect of ALF that

may gradually expire as we age rendering us susceptible to respiratory infections.

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Figure 4.1. Basal levels of pro-inflammatory cytokines in ALF increase with age. ALF from young (Y, white bars) and old (O, black bars) mice or ALF from adult (A, white bars) and elderly (E, black bars) humans were obtained by BAL using 0.9% NaCl. (A) Total protein content in mouse ALF. Pooled experiments from n=16, mean ± SEM. (B) Equal quantities of mouse ALF proteins (10 µg in 100 µl) were loaded onto 96-well plate. Cytokines were measured by standard ELISA. Pooled experiments from n=5/group, mean ± SEM. (C) Total protein content in human ALF. Pooled experiments from n=4, mean ± SEM. (D) Equal quantities of human ALF proteins (10 µg in 100 µl) were loaded onto 96-well plate. Cytokines were measured by standard ELISA. Pooled experiments from n=4/group, mean ± SEM. Student’s t-test of old mouse vs. young mouse ALF or elderly vs. adult human ALF. *p<0.05; **p<0.01.

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Figure 4.2. Basal levels of SP-A and SP-D in ALF increase with age. ALF from young (Y, white bars) and old (O, black bars) mice or ALF from adult (A, white bars) and elderly (E, black bars) humans were obtained by BAL using 0.9% NaCl. (A) Equal quantities of mouse ALF proteins (10 µg) were separated by SDS-PAGE and probed for SP-A or SP-D. Western blot of SP-A and SP-D (representative blots shown) in mouse ALF and densitometry analysis of the bands on the mouse ALF Western blot with their respective backgrounds subtracted, n=17 for SP-A and n=15 for SP-D, mean ± SEM. (B) Equal quantities of human ALF proteins (10 µg) were separated by SDS-PAGE and probed for SP-A or SP-D. Western blot of SP-A and SP-D (representative blots shown) in human ALF and densitometry analysis of the bands on the human ALF Western blot with their respective backgrounds subtracted, n=4 for SP-A and n=4 for SP-D, mean ± SEM. RU, relative units. Student’s t-test of old mouse vs. young mouse ALF or elderly vs. adult ALF. *p<0.05; ***p<0.001.

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Figure 4.3. Basal levels of some complement proteins in ALF increase with age. ALF from young (Y, white bars) and old (O, black bars) mice or adult (A, white bars) and elderly (E, black bars) humans were obtained by BAL using 0.9% NaCl, normalized by protein content and separated by SDS-PAGE (10 µg). (A) Representative Western blots of mouse ALF with densitometry analysis (lower panel), n=12 for C2 and C4β, and n=9 for FB-β, FB-α, C5, C7, C3α and C3β, mean ± SEM (B) Densitometry analysis of Western blot for C2, C5, C7, and C3-β in human ALF, n=4, mean ± SEM. A representative experiment is shown, mean ± SEM; Student’s t-test of old mouse vs. young mouse ALF or elderly vs. adult ALF. *p<0.05; **p<0.01; ***p<0.001. RU, relative units.

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Figure 4.4. ALF hydrolase activity in the mouse lung decreases with age. Hydrolase activity present in concentrated ALF of young (Y, white bars) or old (O, black bars) mice were quantified using a colorimetric method based on the cleavage of substrates containing p-nitrophenol. Enzymes analyzed: 1: acid phosphatase, 2: α-mannosidase, 3: α-galactosidase, 4: β-galactosidase, 5: α-glucosidase, 6: β-glucosidase, 7: α-xylosidase, 8: α-fucosidase, 9: arylsulfatase, 10: fatty acid esterase-I, 11: non-specific esterase, 12: alkaline phosphatase, 13: alkaline phosphodiesterase, 14: phospholipase C, 15: peroxidase, 16: α-rhamnosidase, 17: fatty acid esterase-II. A representative experiment is shown, n=8, mean ± SEM; Student’s t-tests of young mouse vs. old mouse ALF. *p<0.05; **p<0.01; ***p<0.001.

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Figure 4.5. Nitrosative and oxidative stress to ALF proteins in the mouse lung increases with age. ALF from young (Y, white bars) and old (O, black bars) mice were obtained by BAL using 0.9% NaCl, normalized by protein content (10 µg) and separated by SDS-PAGE for Western blotting or incubated in a 96-well for ELISA. (A) Myeloperoxidase (MPO) was directly detected in ALF by ELISA. Representative experiment, n=16, mean ± SEM. (B) Western blot of nitrotyrosine residues present on ALF proteins (indicator of RNS oxidation). Representative experiment, n=5/group, mean ± SEM. Densitometry analysis revealed a significant increase in nitrosylation of proteins from old ALF. (C) Western blot of carbonyl residues present in ALF proteins (indicator of ROS oxidation). Representative experiment, n=5, mean ± SEM. Densitometry analysis revealed a significant increase in carboxylation of proteins from old ALF. Student’s t-test of old mouse vs. young mouse ALF. *p<0.05; ***p<0.001. RU, relative units.

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Figure 4.6. Increasing age is associated with increases in POPC in mouse ALF. Electrospray ionization-Mass Spectrometry (ESI-MS) of mouse surfactant phospholipids in a positive mode. ESI+ spectra shown mainly contain sodium adducts for DPPC [M − H + Na]+ at m/z 756 and for POPC [M − H + Na]+ at m/z 782. An increase in POPC in the aging lung surfactant is indicative of an oxidative environment. A representative experiment of n=3 is shown (top panel old mice, lower panel young mice). PC, phosphatidylcholine; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3 phosphocholine; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine.

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Figure 4.7. C3 and SP-A proteins from elderly human ALF are less capable of binding to the M.tb cell wall. M.tb was incubated in physiological concentrations of adult (A, white bars) or elderly (E, black bars) ALF at physiological conditions. The exposed bacteria were immobilized onto a cell culture plate and probed with monoclonal antibodies directed against SP-A and C3. Purified SP-A or C3 served as positive controls (C, dashed bars). Relative quantities of bound protein were measured by standard ELISA by measuring the absorbance at OD450nm. Pooled experiment of n=4 is shown, mean ± SEM; Student’s t-test of adult vs. elderly human ALF. *p < 0.05.

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Figure 4.8. Exposure of M.tb to elderly-ALF favors survival within human macrophages. Human monocyte derived macrophages or human alveolar macrophages (HAMs) were collected, plated, and infected with adult- (white bars) or elderly- (black bars) ALF exposed M.tb at an MOI 1:1. Macrophages were lysed every 24 h to assess bacterial burden by plating serial dilutions. Despite equal numbers of bacteria at 2 h post infection, elderly-ALF exposed M.tb had significant growth advantage in MDMs and HAMs at later time points. A representative experiment of n=3 performed in triplicate is shown, mean ± SEM; Student’s t-test of adult human vs. elderly human ALF. *p<0.05; **p<0.01.

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Figure 4.9. Reduced phagosomal-lysosomal fusion in macrophages infected with elderly-ALF exposed M.tb. Confocal microscopy images of P-L fusion events in M.tb- infected human macrophages. Human macrophages were adhered to glass coverslips and infected with adult- or elderly-ALF exposed GFP-M.tb (MOI 10:1) for 2 h. Cell monolayers were fixed, permeabilized, and stained with anti-human CD63. Pictures depict CD63 positive compartments in red, GFP-M.tb in unfused phagosomes in green (white arrows), and those co-localized with CD63 in yellow (orange arrows). P-L fusion events were visualized with confocal microscopy and enumerated by counting at least fifty independent bacteria. Original magnification at 600X. Pooled experiment from n=4 (blood donors) with 3 different human ALFs performed in duplicate or triplicate, mean ± SEM; Student t-test adult vs. elderly, *p<0.05.

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Figure 4.10. Exposure of M.tb to elderly-ALF favors growth in mouse organs, but does not affect survival. C57BL/6J mice were infected with a low dose aerosol of M.tb strain Erdman previously exposed to adult- (white bars or white circles) or elderly- (black bars or black circles) ALF. At indicated time points post infection (21, 60, and 150 days), mice were euthanized to assess bacterial burden in target organs. Individual organs were homogenized in 0.9% NaCl, and serial dilutions were plated. (A) Mice were infected with equal inoculums at day 0. (B) Mice infected with elderly-ALF exposed M.tb had higher bacterial burden in the lung and spleen. Pooled results from n=2 with 4-5 mice/group, mean ± SEM; Student’s t-test, *p<0.05, **p<0.01, ***p<0.001. (C) Survival was monitored across a period of 80 weeks. Mice were euthanized when they met the exclusion criteria documented in animal care and use protocols, and the date documented. Mice infected with adult-ALF exposed M.tb had a median survival of 55.50 weeks, while mice infected with elderly-ALF exposed M.tb had a median survival of 51.00 weeks. Pooled experiment from n=2 with 10-20 mice; Log-rank test; ns, not significant.

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Figure 4.11. Exposure of M.tb to elderly-ALF increases lung inflammation early post infection. C57BL/6J mice were infected with a low dose aerosol of M.tb strain Erdman previously exposed to adult- (white bars) or elderly- (black bars) ALF. (A) At 21, 60, and 150 DPI, mice were sacrificed and lungs fixed in 10% NBF, embedded in paraffin, sectioned, and stained with hematoxylin and eosin to visualize tissue morphology. (B) Areas of cell aggregation and infiltration (inflammation) were quantified using Aperio Imagescope by calculating the area of inflammatory foci (i.e. involvement) divided by the total area of the lung. Quantification for 21, 60, and 150 DPI are shown. Representative images at a final magnification of 20X. Pooled results from n=2 with 4-5 mice/group, mean ± SEM; Student’s t-test, **p<0.01.

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Disease and Function Analysis Adult Elderly Category p-value Category p-value psoriasis 4.39E-29 cell movement 5.22E-43 inflammation of organ 5.41E-28 migration of cells 3.67E-39 cell death 4.07E-26 psoriasis 9.10E-38 benign thyroid nodule 3.98E-25 cell death 1.30E-35 allergy 3.40E-23 inflammation of organ 1.00E-34 breast or ovarian cancer 7.12E-22 allergy 2.20E-29 proliferation of cells 1.44E-21 leukocyte migration 1.11E-28 neuromuscular disease 6.40E-21 proliferation of cells 1.51E-28 necrosis 1.72E-20 hypersensitivity reaction 1.78E-28 cell movement 4.30E-19 necrosis 1.75E-27 disorder of basal ganglia 5.02E-19 benign thyroid nodule 7.45E-25 hypersensitivity reaction 1.33E-18 breast cancer 2.08E-24 breast cancer 1.90E-18 cell movement of leukocytes 2.27E-24 malignant solid tumor 2.78E-18 atopic dermatitis 2.29E-24 atopic dermatitis 3.17E-18 apoptosis 1.32E-23 mammary tumor 4.24E-18 inflammatory response 3.92E-23 Dermatitis 7.82E-18 Dermatitis 4.52E-23 nodule 1.16E-17 mammary tumor 5.59E-23 metabolism of reactive oxygen species 1.22E-17 immediate hypersensitivity 5.61E-23 apoptosis 2.27E-17 invasion of cells 5.84E-23 taupathy 2.53E-17 cell movement of myeloid cells 2.74E-22 Movement Disorder 2.54E-17 viral respiratory infection 3.91E-22 chronic skin disorder 3.04E-17 breast or ovarian cancer 8.87E-22 amyloidosis 3.46E-17 cell movement of phagocytes 1.56E-21 cancer 5.23E-17 Viral Infection 2.22E-21 autosomal dominant disease 2.30E-16 severe acute respiratory syndrome 3.05E-21 Dementia 4.55E-16 Rheumatic Disease 3.86E-21 synthesis of reactive oxygen species 5.28E-16 inflammation of body cavity 8.65E-21 Alzheimer’s disease 5.63E-16 chronic inflammation disorder 1.72E-20 immediate hypersensitivity 7.27E-16 cellular infiltration 4.28E-20 migration of cells 7.76E-16 systemic autoimmune syndrome 4.34E-20 chronic psoriasis 8.38E-16 amyloidosis 9.35E-20

Table. 4.1. Top 32 disease and function categories revealed by Ingenuity Pathway Analysis of the global proteome of adult and elderly human ALF. Categories directly related to the inflammatory response have been highlighted. The p-value indicates the likelihood associations are due to chance. This type of analysis identifies categories associated with proteins that can be explained by the observed molecules present within a sample. 218

Upstream Analysis Adult Elderly Upstream Regulator p-value Upstream Regulator p-value APP 3.55E-29 Lipopolysaccharide 1.09E-34 MAPT 4.51E-28 Beta-estradiol 8.33E-27 1,2-dithiol-3-thione 1.05E-26 Nitrofurantoin 8.11E-25 Sirolimus 1.74E-26 TNF 1.25E-24 TP53 9.24E-25 Tretinoin 6.08E-24 MYC 2.18E-24 APP 4.31E-23 Nitrofurantoin 3.52E-24 MYC 1.90E-22 NFE2L2 2.09E-23 IFNG 3.31E-21 TGFB1 9.95E-23 TP53 3.98E-21 Beta-estradiol 3.27E-22 TGFB1 5.78E-20 Lipopolysaccharide 7.44E-22 OSM 5.78E-20 PSEN1 8.34E-22 IL6 2.52E-19 PLN 1.60E-21 Dexamethasone 5.22E-19 Tretinoin 1.76E-21 Sirolimus 3.98E-18 Pln 2.55E-21 MLK1 1.02E-17 Pirinixic acid 1.52E-18 CEBPA 1.54E-17 5-fluorouracil 1.08E-17 SB203580 5.53E-17 Methylprednisolone 4.83E-17 IL1B 1.47E-16 Dexamethasone 3.29E-16 1,2-dithiol-3-thione 1.82E-17 TNF 6.19E-16 FOS 1.87E-16

Table. 4.2. Top 20 upstream regulators categories revealed by Ingenuity Pathway Analysis of the global proteome of adult and elderly human ALF. Categories directly related to the inflammatory response have been highlighted. The p-value indicates the likelihood associations are due to chance. This type of analysis identifies the cascade of upstream transcriptional regulators that can explain the observed changes in the dataset, which can help reveal the biological activities occurring in the samples studied.

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Chapter 5: Significance of findings

Tuberculosis continues to devastate communities across the globe, especially in underdeveloped nations. From 2011-2015, World Health Organization estimated there were 55.4 million infections and 7.15 million deaths directly attributed to the pathogen

Mycobacterium tuberculosis. That is over 67 and 8 times greater than the population of

Columbus, Ohio, respectively at the time of writing. Despite effective chemotherapy, lack of accessibility to antibiotics due to economic factors in endemic countries has prevented successful eradication of M.tb. Furthermore, misuse and failure to complete antibiotic treatment has led to the rise of multi- extensive, extreme-, and total-drug resistant strains of M.tb. Thus, it is unlikely that current chemotherapy will effectively eliminate M.tb. For these reasons, the development of an effective vaccine will be critical for the successful eradication of M.tb. The current vaccine, Mycobacterium bovis Bacillus

Calmette-Guérin (BCG), was developed at the beginning of the 20th century without a

fundamental understanding of mycobacterial immunity. It succeeded at preventing

meningeal- and miliary-TB, but its efficacy against pulmonary TB, the most common

form of TB, is extremely variable. Clinical trials conducted across the world have

reported efficacies ranging from 0%-80%, with higher efficacies associated in countries farther from the equator. Despite its questionable success, BCG is administered to more than 100 million children annually, making it one of the most administered vaccines in 220

the world (655). In order to end TB, it will be absolutely critical for scientists to discover

new and successful vaccination strategies against M.tb.

In this work, we have demonstrated novel approaches for the development of TB vaccines by focusing on the mycobacterial cell wall. We showed that the human lung mucosa [alveolar lining fluid (ALF)] has the ability to impact the efficacy of BCG vaccination against M.tb. We discovered that vaccination with ALF-exposed BCG augmented CD8+ T cell responses in the lung prior to and after M.tb infection. This

enhanced CD8+ T cell response in the lung led to the superior efficacy of ALF-exposed

BCG vaccination at reducing M.tb bacterial burden in the lung. Secondly, we sought to

develop a more reliable method of inducing cell wall modifications to M.tb that did not

involve human-derived compounds. We provided evidence that selective delipidation

(removal of mycobacterial lipids) of BCG using petroleum ether was an effective way to

achieve similar results to that of exposing BCG to ALF. Vaccination with delipidated

BCG (dBCG) was vastly superior to conventional BCG at protecting mice against M.tb.

Importantly, delipidation of BCG can be easily implemented into current vaccine

production, and thus dBCG could be expedited into clinical trials. Lastly, we sought to

evaluate changes to the physiology and composition of ALF with increasing age (a TB

co-morbidity) and its impact on M.tb pathogenesis. We discovered that the ALF of

elderly humans and old mice is in a state of heightened inflammation and oxidation. This

loss of functionality in the antimicrobial properties of ALF may predispose the elderly to infection with M.tb.

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Using the mouse as a model for TB vaccine research

No other medical development in human history has had as large of an impact on society

than vaccines. To date, vaccines have saved more lives than any other therapeutic

intervention. Vaccines have historically been the most effective means to prevent and

eradicate infectious diseases (e.g. smallpox, polio, measles, mumps, rubella, etc.). Their

main objective is to elicit an immune response that rapidly activates following subsequent

encounters with the given infectious organism the vaccine is designed to target. This

bypasses the required time for immunity to develop and protects individuals from

developing the disease. Though simple to explain, the generation of protective immune

response is an intrinsically complex process. Our current technology limits the use of in

vitro models to test vaccines, instead the use of animal models is necessary. Animals are

used throughout processes of vaccine research, development, production, and quality

control. Throughout the vaccine research and development phase, animal models are used

for screening and selecting adjuvants, assessing immunogenicity and safety, testing

routes of administration and dosages, and developing optimal formulations. The mouse

has proven to be an invaluable tool for screening vaccine candidates ranging from

anthrax to Zika (656;657).

Although the mouse model is not without limitations for studying TB, the many benefits

of an in vivo model have made it an important laboratory tool. Mice share similar

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physiology to humans (658), and similar immunological responses to pathogens (659).

Importantly, the mouse model successfully replicates some vital elements of M.tb pathogenesis as discussed in chapter one (654). A major drawback with the use of the mouse model, however, is the relatively low protection conferred by BCG. Compared to the 2-3 log10 reduction observed in guinea pigs, mice only display a 0.5-1 log10 reduction in M.tb bacterial burden in the lung (660). In addition, the mouse model does not faithfully recapitulate human TB pathology. In contrast to humans, which develop caseous necrotic granulomas with hypoxic centers, most laboratory mouse strains develop regions of diffused cellular infiltrates (661) that are not hypoxic (132). Despite the drawbacks, TB vaccine candidates are almost always first screened in mice. Any candidate with increased protection over 0.5-1 log10 reduction in bacterial burden is typically considered an effective vaccine. Combinations of parameters including reduction in bacterial burden, reduction in immunopathology, and extension in survival have been the classic readouts for moving vaccines forward. Whether these parameters can accurately predict the success of a TB vaccine in humans remains debatable.

To discern whether protection afforded by ALF-exposed BCG vaccination was a strain dependent phenotype, we validated our findings using two distinct strains of mice. Using both resistant (C57BL/6J) and susceptible (C3HeB/FeJ) mouse models of TB allowed us to strengthen our findings. We demonstrated that the capacity of ALF-exposed BCG to confer better protection against M.tb infection, associated with reduced pulmonary cellular infiltration and inflammation, was common across mice of different genetic

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backgrounds and M.tb susceptibilities. Our data indicated that not only was vaccination

with ALF-exposed BCG superior to NaCl-exposed BCG at reducing the bacterial burden in targeted organs, but it could also significantly reduce pulmonary inflammation and pathology that is typically associated with morbidity and mortality in M.tb infected mice.

Furthermore, in C57BL/6J mice, exposure of BCG to ALF resulted in a vaccine that could generate accelerated M.tb control combined with the capacity to sustain control of

M.tb for an extended period of time, which ultimately extended survival by 20 weeks relative to non-vaccinated mice and an additional 6 weeks beyond NaCl-exposed BCG.

An extension of 6 weeks in the mouse is equivalent to approximately 3-5 years of

extended life in humans, while an extension of 20 weeks is equivalent to 10-15 years

(662). Although these numbers may not extrapolate directly, it suggests that BCG

vaccination in the mouse model is effective at protecting against M.tb. In the C3HeB/FeJ

mouse model, however, we did not observe the same trends in survival suggesting they

may lack some of the immunological mechanisms required for protection to M.tb. Indeed,

C3HeB/FeJ mice appear to be defective in some aspects of the innate immune response

that integrate signals generated by intracellular pathogens (470). This evidence further

supports the importance of the innate immune system in the early phase of M.tb infection.

Reductions in M.tb bacterial burden and immunopathology, despite the differences

relative to humans, are reliable indicators of long term survival in mice (663;664).

Although we did not perform survival experiments in our PE-delipidation studies with

BCG, we can extrapolate and predict the outcome using the survival data from our ALF-

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exposed BCG vaccination studies. At 14 DPI, vaccination with ALF-exposed BCG

resulted in a 0.56-log10 reduction in CFUs beyond that afforded by NaCl-exposed BCG

(vaccination control). ALF-exposed BCG also reduced bacterial burden by 0.8-log10 in

the spleen, indicating its superiority to NaCl-exposed BCG against dissemination.

We observed similar or even greater reductions in bacterial burden in mice vaccinated

with dBCG. At 21 DPI, vaccination with dBCG resulted in a 0.5-log10 reduction in CFUs

beyond that afforded by BCG (1.8-log10 compared to vehicle control) in the lung, while

in the spleen we observed almost a 2.0-log10 reduction in CFUs beyond that afforded by

BCG (3.2-log10 compared to vehicle control). Similar results were observed in the liver

and MLN of dBCG vaccinated mice. More strikingly, we continued to observe significant

reductions in bacterial burden at later time-points (60 and 150 DPI). In terms of

pulmonary inflammation (immunopathology), at 250 DPI both vehicle control and NaCl-

exposed BCG vaccinated mice had macrophage dominated cellular aggregates in the lung

that consumed up to 40-50% of lung space, whereas ALF-exposed BCG vaccinated mice

only had approximately 25% involvement. Although our closest time-point in our PE-

delipidation studies was 150 DPI, both vehicle and BCG vaccinated mice had

approximately 15-20% involvement compared to ~5% in dBCG vaccinated mice. Thus,

given that vaccination with dBCG was superior at reducing M.tb bacterial burden and

attenuating immunopathology of the lung caused by M.tb infection we expect that dBCG

would significantly extend survival similar to our results with ALF-exposed BCG.

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While the correlation between reduced bacterial burden leads to increased survival is a

classic readout in the context of vaccination with BCG, it does not appear to hold true in

the absence of vaccination. Despite observing reductions in bacterial burden and

pulmonary inflammation in mice infected with adult-ALF exposed M.tb or elderly-ALF

exposed M.tb, differences in survival were not apparent. In fact, bacterial burden and

immunopathology began to normalize between the two groups as time progressed. This

suggests, at least in the context of M.tb, that the development of pathogen-specific

adaptive immune responses via vaccination is superior to the development of pathogen-

specific immune responses due to M.tb infection. We found that vaccination with ALF-

exposed BCG and vaccination with dBCG accelerated adaptive immune responses (CD4+

and/or CD8+) in the lung of mice prior to M.tb infection. Thus, the development of

antigen-specific CD4+/CD8+ T cells prior to M.tb infection, even though they may not be

able to successfully clear the infection, may be more beneficial for the survival of the

host. In contrast, in the absence of prior immunity (naïve host), immune responses that

develop in response to M.tb infection alone may not be sustainable or robust to enable

containment of the pathogen resulting in inefficient protection of the organism. It would

be interesting to see if this observation holds true in superior animal models (i.e. NHP) and/or humans as differences exist in the progression of this disease compared to mice.

Thus, our data support that vaccination with BCG is still warranted despite it not being exceptionally efficacious against PTB.

Exploiting the mycobacterial cell wall to enhance TB vaccine efficacy

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The peripheral lipid layer of the mycobacterial cell wall has evolved to subvert host immune responses that intend to destroy it. This has enabled the success of M.tb at colonizing one fourth of the world’s population, thus leading our group to center our research on understanding the mycobacterial cell wall and its role in virulence and pathogenesis. Our unique approach, however, has been to merge the field of mycobacterial biochemistry with the field of mucosal immunology in an attempt to elucidate the critical, initial innate immune response to M.tb by the host. As discussed in chapter one, previous research from our laboratory has revealed the existence of hydrolytic enzymes and antimicrobial proteins present within the ALF of the lung. These powerful antimicrobial enzymes are capable of stripping immunomodulatory lipids, glycolipids and lipoglycans from the M.tb cell wall such as TDM and ManLAM. Given

the size-to-area ratio of the lung alveolus, when M.tb is inhaled, the likelihood of M.tb

coming into contact with ALF in the lung prior to coming into contact with host cells is

relatively high. In fact, fifteen minutes in ALF is sufficient to induce significant changes

to the M.tb cell wall. Thus, bacilli that interact with host cells have undergone extensive

biochemical changes to the composition of their cell wall. As a result, ALF-exposed M.tb

was more susceptible to killing by host phagocytes (i.e. macrophages and neutrophils)

(162;187). This indicates that ALF has an important antimicrobial function against M.tb

and it can be reasonably assumed that this likely holds true for other pulmonary

pathogens.

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In the studies presented here we extended beyond M.tb. Given that the cell wall structure

and peripheral lipid layer composition of M.tb and M. bovis BCG are similar (22), we

sought to evaluate whether ALF induced modifications to BCG played a role in its

efficacy against M.tb. Subcutaneous vaccination with ALF-exposed BCG was superior to

NaCl-exposed BCG (conventional BCG) at reducing M.tb bacterial burden within the lung of infected mice. This decrease in M.tb bacterial burden likely translated to reduced pulmonary inflammation at later stages of the disease, and subsequently extended survival. The superior efficacy of ALF-exposed BCG vaccination was revealed to be dependent on CD8+ T cell responses, a subset that is not commonly thought of as critical

for the containment of M.tb in humans. Hence, the composition of the M.tb cell wall

appeared to play a significant role in the development of protective immunity. Similarly, we used petroleum ether (PE) to standardize a protocol of delipidation of the BCG cell wall. Removal of virulent lipids including TDM, PGL, MycB, PDIMs, and TAGs attenuated BCG, but did not kill it. We believe the true strength of our approach was the

decision to use an organic solvent to remove lipids from the cell wall of BCG as opposed

to generating lipid-specific gene knockouts strains. Using PE provided two crucial advantages. First, PE was able to reproducibly strip a significant amount of some cell wall lipidic components, but it was incapable of stripping one-hundred percent of any lipid species. As discussed in chapter one, the lipids extracted by PE are significant inducers of inflammation. In their complete absence, innate immune responses may not be sufficient to properly stimulate adaptive immune cells, and thus result in poor protective immunity. Second, although lipid-specific knockouts would offer the

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advantage of consistency, it would be extremely challenging (if possible) to generate a mutant that lacks all of the lipids targeted by PE. Thus, we believe this ‘selective attenuation’ of BCG enabled our success. Collectively, in two independent projects we

showed that the mycobacterial cell wall can be exploited to improve vaccine efficacy.

A pressing question that remains unresolved in the TB field is: What are the key immune

elements that dictate the progression to clearance, active disease, or latency associated

with M.tb infection in humans? Does the innate immune system actively participate in the

process of eliminating M.tb, or is the host entirely reliant on the adaptive immune

system? As our data suggest that the initial interaction with ALF influences the

consequence of M.tb survival within host phagocytes, we speculate that ALF aids the

innate immune system in its attempt to clear M.tb. By stripping off and modifying

virulent lipids, glycolipids and lipoglycans on the cell wall of M.tb, the action of alveolar

hydrolases and soluble innate immune proteins present in ALF may prevent M.tb from

down-modulating macrophage killing mechanism (such as P-L fusion). This would

indicate that dysfunction of ALF favors progression towards latency or active disease.

Indeed, when elderly-ALF exposed M.tb, which contains dysfunctional ALF components

was compared to adult-ALF exposed M.tb, we found that dysfunction of ALF

components favors the establishment of M.tb infection both in vitro and in vivo. In the

context of our aging studies, we can conclude that our observations are due to ALF

intrinsic factors because our infections were carried out using healthy adult human (20-40

years) macrophages and/or young mice (2-6 months), and lack many of the morbidities

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associated with increases in age (i.e. chronic inflammation, oxidation). Additionally, the

elderly are not the only group with increased risk of developing TB. Groups that have

risk factors associated with loss of pulmonary function, including diabetes, cigarette

smoking, excessive alcohol consumption, and lack of proper nutrition, are at a higher risk

for developing TB (665-668). Although these risk factors can also affect the adaptive

immune system, an encompassing theme is that decreased pulmonary function, probably

starting at the level of ALF, may drive susceptibility to M.tb infection.

Manipulating CD8+ T cell responses to improve TB vaccines

The literature supports a critical role for T cells in immunity to M.tb. Experimental and

clinical evidence suggest that the generation of effective T cell responses will be an

essential requirement of a successful vaccine against TB. Mechanisms of improving

CD4+ T cell responses have dominated the TB field mostly due to the observations that

individuals infected with the human immunodeficiency virus 1 (HIV-1), a virus that

targets and kills CD4+ T cells, are associated with the highest risk of developing TB more

than any other group (434). Thus, mechanisms that target CD8+ T cell activation have been less commonly studied. However, recent evidence, including our own, suggest that

CD8+ T cells are important contributors to mycobacterial immunity. We previously

demonstrated that vaccination with ALF-exposed BCG enhances CD8+ T cell responses,

and in their absence, vaccination with ALF-exposed BCG is not as effective.

Furthermore, we also observed significant increases in CD8+ T cell responses in mice

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vaccinated with dBCG. Although we did not specifically assess their contribution, recent

studies to ours have reported that pulmonary vaccination with BCG is associated with

protective CD8+ T cell responses (275). Thus, CD8+ T cells are emerging as important

contributors to mycobacterial immunity in the context of vaccination.

The preferential processing of intracellular bacteria, including M.tb and BCG, via the

MHC class II pathway means that eliciting CD4+ T cells by vaccination is relatively

straightforward. However, the enhancement of antigen-specific CD8+ T cell responses

has proven to be significantly more challenging. CD8+ T cells recognize antigens that are typically derived from the cytosol, such as viral proteins that are found in the cytosol during viral assembly (669;670). Thus, the best approach to induce CD8+ T cell

responses is to target the antigen(s) of interest to the cytosol. In this context, new

approaches have been implemented to target proteins of intracellular bacterial pathogens

to the cytosol of macrophages or dendritic cells to elicit stronger CD8+ T cell responses.

One of these approaches uses in vitro osmotic shock to force a pathogen to enter the cytosol of DCs. These DCs can then be injected into the host to target CD8+ T cells.

Another approach, which can bypass the process of antigen processing and presentation,

is the discovery and/or production of M.tb antigens with high affinity for MHC class I.

By binding directly to extracellular MHC I, these “adjuvants” could favor the activation

of CD8+ T cells. Lastly, one could exploit the natural properties of pathogens that directly target or enter the cell’s cytosol. For example, Listeria monocytogenes uses virulence

factors such as listeriolysin O to escape from the phagosome and enter the cytosol. Thus,

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recombinant strains that incorporate these virulence factors could be used to enhance the

efficacy of BCG. In fact, as discussed in chapter one, the rBCG ΔUre::CHly+

incorporates a mechanism for escaping from the phagosome present in L. monocytogenes

and is currently considered one of the most promising vaccines to replace BCG. With this

in mind, it is plausible that removal of virulent lipids, either through exposure to ALF or

PE-delipidation, from BCG altered the mechanisms of phagocytosis and/or intracellular trafficking that favored CD8+ T cell activation. Alternatively, as discussed in chapters

two and three, mechanisms that result in antigen cross-presentation such as apoptosis and

autophagy could also have contributed.

Future directions

Although progress has been made in elucidating the immunological requirements for effective immunity to M.tb, the quest for a more effective TB vaccine continues. In this work we have demonstrated that manipulation of the mycobacterial cell wall can be a useful approach to improving the BCG vaccine. However, many questions remain. From our ALF-exposed BCG studies we were only able to conclude that CD8+ T cells mediated

the reduction in M.tb bacterial burden at 14 DPI. In the future, we would like to extend

CD8+ T cell depletion to 250 DPI and evaluate whether our observed decreases in

immunopathology and extension in survival were specifically due to CD8+ T cell activity.

In addition, our CD8+ T cell depletion study was designed to prevent the development of

CD8+ T cell responses during M.tb infection. It would be valuable to deplete the CD8+ T

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cells during the vaccination period (45 days prior to M.tb infection) to evaluate whether our observations were specifically due to a memory CD8+ T cell response as opposed to

effector responses that arise due to M.tb infection. Furthermore, although we observed a

direct association between IFNγ and CD8+ T cells in terms of bacterial burden in the

lung, we would like to assess if the control of M.tb was directly dependent on IFNγ

derived from CD8+ T cells. It would also be valuable to assess how vaccination with

ALF-exposed BCG augmented CD8+ T cell priming and activation in the lung. In this

context, we could evaluate mechanisms of antigen processing and presentation by

epidermal-resident macrophages and DCs and how differences translate to changes in the

host adaptive immune response. Exploring how ALF-exposed BCG interacts with the

host could also reveal novel mechanistic pathways that could be targeted to improve TB

vaccine efficacy. Similarly, some of our data suggested that vaccination with ALF-

exposed BCG was a stronger inducer of Th1-polarized immune responses compared to vaccination with NaCl-exposed BCG. Exploring how to accelerate and enhance Th1- mediated immune responses could also offer novel strategies for improving BCG. Lastly, as our data suggest an important role for CD8+ T cell responses in the control of M.tb, it

would be valuable to evaluate whether enhancement of CD8+ T cell activity via

vaccination correlates with protective immunity to TB in humans.

The work with ALF-exposed BCG led us to explore a more reliable method of stripping

lipids from the cell wall of BCG. Although previous studies have utilized petroleum ether

to evaluate the impact of TDM on M.tb pathogenesis, we were the first to assess whether 233

selectively delipidated BCG could serve as an effective vaccine. In this work, we

presented evidence that dBCG is superior to conventional BCG at protecting against M.tb in the mouse model all while bypassing the concern of direct pulmonary vaccination. In the future, we would like to extend our findings and confirm that the mechanism behind the increased protection associated with dBCG vaccination is indeed due to IL-17A by depleting IL-17A or by vaccinating IL-17A-deficient mice. In contrast to our studies with

ALF-exposed BCG, we found that vaccination with dBCG increased the number of both

CD4+ and CD8+ memory T cells. Via depletion studies, we would like to determine the specific immunological contribution of each lymphocyte subset in the control of M.tb

bacterial burden and immunopathology. We would also like to explore the mechanism of

how dBCG augmented memory T cell responses and apply that knowledge for the development of more effective TB vaccines. We hypothesize the reason for dBCG’s

superior ability to induce memory T cell population of the lung may be due to slow

priming of the adaptive immune system, which may enhance memory T cell development

in the lung . Furthermore, we would like to test the efficacy of dBCG against M.tb in

complex vertebrate animal models (i.e. guinea pig, rabbit, NHP), and/or further assess its

safety in advanced mouse models (i.e. humanized mice, immunocompromised mice). We

predict dBCG has the potential to be evaluated for safety and efficacy in human clinical

trials. In this context, dBCG could either be adapted as the primary vaccine for TB or

further developed as a booster for a later stage in life when mycobacterial immunity has

been suggested to wane. Furthermore, vaccination with dBCG could also be repurposed

for agricultural use and for the control of M. bovis in the ecosystem. Currently, M. bovis

234

infections in cattle cause significant economic losses, with estimates placing an annual

loss of $3 billion to the economy of the United States (671). Thus, dBCG would not only

be beneficial to global health, but also for economic development.

Finally, our studies comparing the effects of adult- or elderly-ALF exposed M.tb have raised many important questions as to the requirements for effective anti-mycobacterial immunity. We showed that ALF possesses antimicrobial properties that appear to wane as we age possibly due to increases in basal levels of inflammation and oxidation. In this context, ALF could play a role in dictating whether M.tb infections are rapidly cleared from the host or whether the infected individual progresses to active TB or latent infection. This is an important question that we would like to address in the future by continuing to study the properties of ALF from individuals in different risk groups (e.g. smoking, alcohol, diabetes, HIV-1+, old age, etc.). Specifically, we will explore whether a

connection exists between susceptibility to TB and decreased innate immune function,

particularly the function of soluble innate immune proteins. Similarly, we would like to

characterizing other soluble innate immune proteins (i.e. SP-D, C1q, mannose binding

lectin, antimicrobial peptides, etc.), all of which have been shown to bind to M.tb. To

evaluate the specific contribution of soluble innate immune proteins such as SP-A and

C3, we could deplete individual proteins from ALF using antibodies prior to exposing

M.tb. We could then assess changes in the survival of M.tb within host cells in the

presence or absence of specific ALF proteins. This could reveal the specific contribution

of each protein in the innate immune response to M.tb. Extending on those experiments,

235

we would also like to continue characterizing intracellular trafficking of M.tb after

exposure to ALF. Although we observed decreases in P-L fusion after exposure to elderly-ALF, we do not know whether these phagosomes are acidified as M.tb can inhibit this process, as well as if autophagy is a valid mechanisms for clearing M.tb as it is

deficient in the elderly. Another important question is whether exposure to adult- or

elderly-ALF alters M.tb-macrophage receptor engagement. We have shown in the past

that hydrolases in ALF can remove mannose from the cell wall of M.tb. This

phenomenon, in combination with the fact that hydrolase activity decreases with age,

could favor M.tb phagocytosis through the mannose receptor, a mechanism that is known

to promote M.tb survival. The absence of mannose could shift entry toward more host-

beneficial mechanisms. We could evaluate the specific contribution of phagocytic

receptors on phagocytes with the use of small interfering RNA (siRNA) technology that

would allow us to knockdown specific receptors. Overall, we have uncovered a novel

role for ALF in the context of mycobacterial innate immunity in the lung, but further

studies are needed to fully unravel and understand its specific contribution.

Concluding remarks

BCG remains the only World Health Organization supported vaccine we have for the

prevention of TB, yet lacks the ability to protect against the primary form of TB. In this

collective work, we have highlighted the current knowledge in the field regarding innate

and adaptive immune responses to BCG in the hope of stressing the importance of

236

understanding immunological mechanisms that give rise to effective mycobacterial

immunity. The importance of crosstalk between the innate and adaptive branches of the

immune system cannot be overstressed, yet the fundamental question of why BCG fails

to fully protect against PTB remains unanswered. It could be due to virulent lipids

present on the mycobacterial cell wall preventing the full development of immune

responses in the lung, or discrepancies between immune responses at the site of vaccine

administration vs. the natural route of M.tb infection through the lungs. Perhaps it is not because BCG is poor at generating effective immune responses, but that the immunosuppressive status of the lung prevents it from doing so. Similarly, BCG could be at its saturation point, and thus further stimulation of the immune system would yield no added immunity. The answer could simply lie in shifting research efforts towards a more immunogenic route of vaccination. The literature suggests that intranasal vaccination with BCG is a more effective method to develop immunity against M.tb, yet no study involving humans has been published on this matter, mainly due to increases in pathology observed in the lungs using this delivery method. Thus, research into mechanisms that can decrease this inflammation in the lungs may open new avenues for direct mucosal vaccine delivery into the lungs as we have shown with PE-delipidated BCG. Efforts directed at exploring immunological events that occur following pulmonary vaccination with BCG and the status of immune cells within the lung could yield valuable answers.

We are now beginning to fully understand that in vitro studies do not always translate to in vivo results. The development of more suitable animal models and implementation of –

237

omics research could aid in the quest of finding a suitable replacement for BCG.

Unfortunately, no effective vaccine yet exists for intracellular bacterial pathogens. A

major question still remains: Is BCG poor at stimulating mycobacterial immunity or is

M.tb simply extremely well adapted at avoiding immunological responses against it?

Furthermore, the majority of TB vaccine development research focuses on using generic

laboratory M.tb strains and thus, does not assess vaccine efficacy against M.tb clinical

isolates, with different degrees of virulence, heavily present in high TB burden regions.

Studies directed at uncovering the mechanisms behind how BCG successfully primes,

enhances, accelerates, and maximizes host immune recognition of M.tb should be

prioritized. Furthermore, we must revaluate correlates of protection vs. correlates of risk

and their implications in vaccine design. The TB field has placed much emphasis on the

study of IFNγ responses and how they can be augmented, yet new evidence suggests that

IFNγ is a better correlate of risk than of protection. Similarly, we must begin to explore the contribution of other immune cells and factors and how they can be targeted to develop a more effective vaccine. We have shown that CD8+ T cells and IL-17A responses may be imperative for effective mycobacterial immunity. We must also begin to design vaccine clinical trials that ask more refined questions about mycobacterial immunity and protection. Until we refine our understanding of immunity to mycobacteria, the development of a successful TB vaccine will remain a difficult task.

Whether by accelerating CD8+ T cell response, removing particular lipids from the BCG

cell wall, or by understanding the antimicrobial properties of ALF, we hope that this

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work can serve as groundwork for the development of more efficacious vaccines against one of the most successful pathogen in human history.

239

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