MSc Thesis: Exploring MPT63-Derived Lipopeptides as Tuberculosis Vaccines (PDF)

Exploring MPT63-Derived Lipopeptides as Prophylactic and Therapeutic Vaccine Candidates against Mycobacterium tuberculosis Infection by Diksha Sharma A thesis submitted in partial fulfillment of the requirements for the degree Master of Science Department of Surgery University of Alberta © Diksha Sharma, 2024 1 Abstract Tuberculosis (TB) is an infectious disease which caused an estimated 1.30 million deaths in 2022 globally. Mycobacterium tuberculosis (Mtb) is the etiological agent of TB disease and is responsible for the global TB burden. Due to emergence of multi drug-resistance (MDR), totally drug-resistant (TDR-TB) and extensively drug-resistant (XDR-TB) tuberculosis in recent years, the toxic side effects associated with the current treatment regimen, lengthy treatment, lack of adherence and additional challenges posed by HIV-co-infections it has become increasingly challenging to treat the afflicted patients. Furthermore, the widely used vaccine Bacillus Calmette- Guérin (BCG) confers variable protection against the disease, while being ineffective at protecting immunized individuals against pulmonary TB as adults. Therefore, provided the rising the cases of TB, despite the existence of current treatments and vaccine, there is an urgent need to investigate a new and innovative vaccine candidate that can mediate infection control through enhanced stimulation of the protective host immune responses. Animal studies have shown that the culture filtrate of Mtb is rich in secretory antigens such as MPT63 which exhibit vaccine potential. For this study, we developed MPT63 derived synthetic lipopeptide construct to investigate their efficacy as both prophylactic and therapeutic subunit vaccines. The MPT63 lipopeptides (LPs) spanning the full-length sequence of MPT63 antigen were divided into Pool 1 and Pool 2 to test investigate for differences in immune mechanisms elicited by each pool. The study aimed at enhancing the functionality of effector and antimicrobial cellular immune responses to lower bacterial burden in lungs, liver and spleen of infected BALB/c mice. For the prophylactic immunization experiments, the BALB/c mice first received intranasal immunization which was followed by infection with H37Ra strain of Mtb via intravenous route. The results from prophylactic immunization provided us with an insight into differential expression of T cell subsets by revealing that Pool 1 LP immunizations promoted increase in ii regulatory cytotoxic T cell and regulatory helper T cell populations while Pool 2 LP immunizations lead to robust production of effector cytotoxic T cell population and antimicrobial helper T cell populations. Pool 2 LP immunizations also resulted in a trend towards reduced bacterial burden compared to control and Pool 1 LP immunized groups. Both Pools also lead to significant splenocyte proliferation in the absence of active TB model, but no such significance was noted in the case of presence of active TB infection. The histological studies revealed that immunization with Pool 1 and Pool 2 LPs causes a reduction in lung damage compared to the control (PBS- immunized) while the immune cell infiltration remained consistent within all groups. During the therapeutic experiments, the mice were first infected with M.Avium strain of Mtb, through intravenous route which was followed by intranasal immunizations. The results from splenocyte proliferation analysis showed that Pool 2 LP immunization led to significant splenocyte proliferation, compared to the control while Pool 1 LP immunizations did not lead to significant splenocyte proliferation in the context of a therapeutic model of the disease. Furthermore, there was a trend towards lowered bacterial burden in lungs, liver and spleen of Pool 2 immunized mice compared to Pool 1 LP immunization and control groups. Overall, our study demonstrates that the MPT63 lipopeptides belonging to Pool 2 show promise in generating effective cellular immune responses in lungs, spleen and liver of mice when administered as both prophylactic and therapeutic vaccine. This observation provides impetus for future investigations in testing efficacy of MPT63 lipopeptides as vaccine candidate against Mtb. iii Preface This thesis presents the original work done by Diksha Sharma and was submitted as partial fulfillment of the degree Master of Science (MSc) in Surgery at the University of Alberta. The animal experiments were conducted in accordance with the guidelines laid down by the Canadian Council of Animal Care (CCAC) and specific training protocols provided by the University of Alberta’s Health Sciences Laboratory Animal Services (HSLAS). The standard operating procedures for this project were ethically approved by the University of Alberta’s Animal Care and Use committee (ACUC). iv Dedications I dedicate my thesis and graduate research to my real-life hero, my father, Dr. Deepak Sharma. I watched him work tirelessly and study for his PhD growing up, while being the best father in the world. I also dedicate this thesis to my mom, Sapna Sharma, who always ensured I received abundant love and care while finishing my graduate studies. My mother pursued her graduate studies while raising two little kids and taught me patience, resilience, and perseverance. During my degree, my little brother, Ujjawal, always assured I never felt away from home and that I felt happy and cared for. I thank him for always sending me food after a long day of experiments. My little brother is the kindest and most heartwarming individual I know, and he inspires me to be the same. v Acknowledgements I am immensely thankful to Dr. Babita Agrawal for her pivotal role in my academic journey. Her constant support and encouragement have significantly contributed to my success in her lab. She has consistently pushed me to do my best, whether in experiments, research presentations, or thesis preparation. Dr. Agrawal's guidance has been invaluable in overcoming setbacks, managing time effectively, and producing high-quality work. I am deeply grateful to her for fulfilling my aspirations to pursue a master’s program at the Department of Surgery. I am thankful for the invaluable feedback I received from my committee members, Dr. Stephanie Yanow and Dr. Afsaneh Lavasanifar. Their insights and suggestions were crucial in improving my experimental design, statistical analysis, and overall research approach. I highly appreciate their contributions to my work. I want to express my gratitude to my lab members, who have been instrumental in making this project possible. I am thankful to Jane Li and Raj Patel (PhD candidate) for their assistance with the onboarding process, training me on cardiac blood sampling, bronchoalveolar lavage, and organ harvesting, and instructing me on operating various experimental equipment. Additionally, I appreciate Shanika Werellagama, a PhD candidate, and Adrien Lam, an undergraduate thesis student, for their help with extensive experiments and continuous moral support. I am tremendously grateful for the support I received from the University Flow Core and Animal Services. Their training, equipment, and facilities were essential for successfully executing my experiments. I truly appreciate their assistance and their significant role in my academic journey. As I pursued my master’s away from home, my parents and brother always ensured that I felt loved and supported in every way possible. From sending me treats to making my visits home memorable, they encouraged me to keep going despite the challenges I faced. I also want to express my heartfelt gratitude to my best friends, Harasees and Khushi, for supporting my mental well- vi being. They called me every day, despite their busy schedules, to ensure I was doing okay and constantly encouraged me to keep moving forward. Thank you to Tracey and Dr. Berry from the Department of Surgery for always making sure that I transitioned smoothly into the program and for always offering help and advice. I would also like to thank the Canadian Institutes of Health Research for funding my graduate education. vii Table of Contents Chapter 1: Introduction and Literature Review...............................................................................1 SECTION 1: Understanding Tuberculosis: Disease Overview and Pathogenesis.........................2 1.1.1 Tuberculosis Disease.................................................................................................................. 2 1.1.2 TB Pathogenesis and Clinical Presentations............................................................................ 3 SECTION II: The BCG Vaccine Induced Immunity and Limitations.........................................6 1.2.1 The BCG Vaccine....................................................................................................................... 6 1.2.3 BCG Associated Challenges.................................................................................................... 11 1.3.1 Treatment for TB..................................................................................................................... 14 1.3.2 Treatment Associated Challenges........................................................................................... 16 Section IV: Fighting the Silent Enemy: Immune Warriors Against Tuberculosis...................... 17 1.4.1 The Innate Immune Response................................................................................................ 17 1.4.2 The Adaptive Immune Responses.......................................................................................... 22 Section V: The Advent of Subunit Vaccines and MPT63.......................................................... 30 1.5.1 The Development of Subunit Vaccines................................................................................... 30 1.6 References........................................................................................................................ 40 Chapter 2: Rationale and Research Plan...................................................................................... 56 2.1 Rationale for the Research Project..................................................................................... 57 2.3 Chapter Organization and Structure................................................................................. 63 2.4 References........................................................................................................................ 64 Chapter 3: Materials and Methods............................................................................................... 67 3.1 The Mice.......................................................................................................................... 68 3.3 Mycobacterial Strains-Mycobacterium bovis BCG, Mtb H37Ra and M.avium....................... 69 3.4 MPT63 Immunization Schedule-Prophylactic and Therapeutic Experiments...................... 70 3.5 Mycobacterial Challenge and the CFU Assay..................................................................... 72 3.7 Splenocyte Proliferation Assay.......................................................................................... 73 3.9 Flow Cytometry Analysis of Immune cells.......................................................................... 74 3.10 Categorizing the Cytokine Expression Profiles................................................................. 76 3.11 Statistical Analysis.......................................................................................................... 76 3.12 References...................................................................................................................... 77 Chapter 4: Examining the efficacy of MPT63 derived lipopeptides in enhancing immune responses and reducing bacterial burden in H37Ra infected mice when administered prophylactically.................... 78 viii 4.1 Introduction..................................................................................................................... 79 4.2 Results.............................................................................................................................. 82 4.2.1 Intranasal immunization of BALB/c mice with MPT63 derived lipopeptides lead to significant antigen specific splenocyte proliferation in the absence of infection......................... 82 4.2.2 The immunization with Pool 1 and Pool 2 lipopeptides lead to differential upregulation of effector/antimicrobial and regulatory T cell subsets..................................................................... 85 4.2.3 The splenocyte proliferation induced by MPT63 derived synthetic lipopeptides in H37Ra infected mice shows no significant difference................................................................................. 92 4.2.4 MPT63 lipopeptide immunizations lead to differential T cell and cytokine expression profiles in an active TB infection model.......................................................................................... 95 4.2.6 Immunization with Pool 2 MPT63 lipopeptides is associated with a trend of reduced bacterial loads in the spleen, lungs, and liver of H37Ra-infected BALB/c mice....................... 100 4.2.7 Immunization with Pool 1 and Pool 2 MPT63 derived lipopeptides lead to reduction in lung lesion evident by the histological study of the hematoxylin and eosin (H&E) slides of the lung tissue........................................................................................................................................ 102 4.3 Discussion....................................................................................................................... 103 4.4 References...................................................................................................................... 111 Chapter 5: Therapeutic administration of MPT63 derived lipopeptides to investigate the impact of immunization in lowering bacterial burden in M.Avium infected mice.......................................... 113 5.1 Introduction................................................................................................................... 114 5.2 Results............................................................................................................................ 116 5.2.1 The ex vivo stimulation of splenocytes derived from BALB/c mice immunized with Pool 2 MPT63 lipopeptides resulted in a significant increase in splenocyte proliferation compared to the control group............................................................................................................................. 116 5.2.2 Therapeutic immunization with Pool 2 MPT63 lipopeptides showed a trend toward lowered bacterial burden in M. avium-infected mice................................................................... 118 5.2.3 Qualitative examination of the hematoxylin and eosin (H&E) slides of lungs tissues indicate no major differences amongst MPT63 immunized and control groups...................... 119 5.3 Discussion....................................................................................................................... 120 5.4 References...................................................................................................................... 124 Chapter 6: General Discussions and Future Directions................................................................ 126 6.1 Summary of Findings...................................................................................................... 127 6.2 Limitations..................................................................................................................... 131 6.3 Future Directions............................................................................................................ 132 6.3.1 Examining the antibody responses to gain a full insight into MPT63 lipopeptide induced adaptive immune responses............................................................................................................ 132 6.3.2 Testing the efficacy of individual MPT63 lipopeptide in eliciting a robust immune response within the context of an active TB model...................................................................... 132 ix 6.3.3 Studying the function of MPT63 lipopeptides in inducing immune responses when combined with existing vaccines and therapies............................................................................ 133 6.5 References...................................................................................................................... 134 x List of Tables Table 1.1 Current Subunit Vaccines in clinical trials and their Adjuvants…………………….32 Table 3.1 The Synthetic lipopeptides spanning the full-length sequence of MPT63 antigen……69 Table 3.2 Pool 1 and Pool 2 MPT63 Lipopeptides……………………………………………...70 Table 3.3 The extracellular and intracellular markers used to stain the spleen and lung cells for flow cytometry analysis ………………………………………………………………………....75 Table 3.4 The following categories were used to characterize the multifunctional nature of T Lymphocytes expressing both pro-inflammatory and anti-inflammatory cytokines………….…76 xi List of Figures Figure 1.1 The transmission and pathogenesis of Mycobacterium tuberculosis……………….4 Figure1.2 The Structure of a granuloma……………………………………………………….5 Figure 1.3 BCG induced cellular, humoral and memory immune responses in immunized individuals………………………………………………………………………………………11 Figure 1.4 The Innate and Adaptive Immune responses involved in Mtb infection…………….30 Figure 1.5 The Structure of MPT63 protein……………………………………………………...38 Figure 3.1 The Immunization and infection schedules for prophylactic and therapeutic experiments……………………………………………………………………………………..71 Figure 4.1: Splenocyte proliferation upon restimulation with MPT63 lipopeptides……………………………………………………………………………….…....84 Figure 4.2: The pseudo color tSNE plots and heatmaps generated using FlowJo software…………………………………………………………………………………………87 Figure 4.3: The expression profiles of cytotoxic T cells derived from splenocytes in three groups of BALB/c mice: (A) Control (PBS-immunized), (B) Pool 1 immunized, and (C) Pool 2 immunized……………………………………………………………………………………...88 Figure 4.4 The expression profiles of antimicrobial and regulatory T helper cells derived from splenocytes in three groups: (A) Control (PBS-immunized), (B) Pool 1-immunized, and (C) Pool 2-immunized……………………………………………………………………..…………….89 Figure 4.5: This figure illustrates the expression profiles of various effector/antimicrobial (TNF- α, IFN-γ, IL-17A) and regulatory (LAP, IL-10) cytokines in cytotoxic T cells from lung cells………………………………………………………………………………………..…..90 Figure 4.6: Lung Cytotoxic T cell analysis…………………………………………..……….91 Figure 4.7: Lung Helper T cell analysis…………………………………..…………………..92 Figure 4.8: The antigen-specific proliferation of splenocytes was evaluated by measuring BrdU incorporation following ex-vivo stimulation with lipopeptides (LPs)………………..……….94 Figure 4.9: Spleen Cytotoxic T cell analysis…………………………………………………96 Figure 4.10: Splenocyte Helper T cell analysis……………………….……………………..97 Figure 4.11: Lung Cytotoxic T cell analysis………………………………………………….98 Figure 4.12: Lung Helper T cell analysis……………………………………………………..99 Figure 4.13: Colony forming units’ assay of Spleen, Lungs and Liver………………………101 Figure 4.13: Histological analysis of the infected lung tissue……………………..…………102 Figure 5.1: Splenocyte proliferation upon ex vivo stimulation with MPT63 lipopeptides…...117 xii Figure 5.2: Bacterial burden in lungs, liver spleen of M.avium infected mice treated with MPT63 derived synthetic lipopeptides………………………………………………………………….119 Figure 5.3: Histological analysis of the infected lung tissue…………………………………..120 xiii List of Abbreviations AA: Amino Acid Ab: Antibodies ABL2: Animal Biosafety 2 ACUC: Animal care and use committee AECs: Airway epithelial cells Ag: Antigen APCs: Antigen presenting cells BCG: Bacillus Calmette-Guerin Be1: B Effector 1 Be2: B Effector 2 Breg: B Regulatory Cells CAF: Cationic adjuvant formulation cART: Combined antiretroviral therapy CCAC: Canadian council of animal care CLRs: C-type lectin receptors CR3: Complement receptor 3 CTLs: Cytotoxic T Lymphocytes DCs: Dendritic cells DHT: Delayed-Type Hypersensitivity DLNs: Draining Lymph nodes DR-TB: Drug-resistant TB EM: Environmental mycobacteria EPI: Expanded programme on immunization EPTB: Extrapulmonary Tuberculosis ESAT-6: Early secreted antigenic target 6 KDa protein Fab: Antigen-binding fragments Fc: Crystallizable fragment xiv GLA: Glycopyranosyl lipid adjuvant GSH: Glutahione HIV: Human Immunodeficiency virus HLA-DR: Human Leukocyte antigen HSLAS: Health Science Laboratory Animal Services IFN-γ: Interferon-γ IgM : Immunoglobulin M IL-2: Interleukin-2 LTBI: Latent Tuberculosis Infection MAIT: Mucosal-associated invariant T cells MDR-TB: Multi drug -resistant tuberculosis MHC I/II: Major Histocompatibility Complex MPL: Monophosphoryl lipid A MTBC: Mycobacterium tuberculosis complex MT-CF: Mycobacteria culture filtrate NCR: Natural cytotoxicity receptor NHPs: Non-human primates NK: Natural Killer cells NOD2: Nucleotide-bindig oligomerization domain-containing protein 2 pAgs: Phosphoantigens PAMPs: Pathogen-associated molecular patterns PBMCs: Peripheral blood mononuclear cells PPD: Purified progestin derivative preXDR: Pre-extensviely drug-resistant PRRs: Pattern recognition receptors PTB: Pulmonary Tuberculosis RCTs: Randomized control trials RNIs: Reactive nitrogen Species ROS: Reactive Oxygen Species RR: Rifampicin-Resistant TB xv SE: Squalene emulsion SOPs: Standard operating procedures TB: Tuberculosis Tc: Cytotoxic T cells TCR: T cell receptor Th: Helper T cells Th1: T helper 1 Th17: T helper 17 TLR2: Toll-like receptors 2 TLR4: Toll-like receptors 4 TNF-α: Tumor necrosis factor-α VADS: Adjuvant-delivery systems WHO: World Health Organization XDR-TB: Extensively drug-resistant tuberculosis xvi Chapter 1: Introduction and Literature Review 1 SECTION 1: Understanding Tuberculosis: Disease Overview and Pathogenesis 1.1.1 Tuberculosis Disease In 1882, German bacteriologist Robert Koch made a significant discovery, identifying Mycobacterium tuberculosis (Mtb) as the etiological agent of Tuberculosis (TB) disease in humans (Cambau and Drancourt, 2014). This discovery marked a crucial point in the long history of TB, a disease that has impacted humankind over thousands of years. His discovery of Mtb, a bacillus- shaped, obligate intracellular pathogen that belongs to the Mycobacterium tuberculosis complex (MTBC), marked a significant milestone in the understanding and fight against TB (Wang et al., 2022). Despite the development of innovative therapeutic and diagnostic tools over the years, the disease continues to thrive and acts as a severe threat to public health security. According to the World Health Organization (WHO) TB holds the status of being the second leading infectious killer after COVID-19. The 2023 global tuberculosis report suggested that TB caused a total of 1.3 million deaths in 2022. Most of the people residing in WHO regions of Southeast Asia (46%), Africa (23%), and Western Pacific (18%) contracted TB, with a small portion of individuals infected in Eastern Mediterranean (8.1%), the Americas (3.1%) and Europe (2.2%) (World Health Organization, 2023). In Canada, it was reported that 1,971 people were diagnosed with active tuberculosis in 2022 with an incidence rate of 5.1 active TB cases per 100,000 population. Although the overall TB rate in Canada remained stable from 2012 to 2022, the First Nations, Inuit populations and immigrants made up most of the active TB cases (Public Health Agency of Canada, 2024). Multiple factors contributing to the increase in global TB burden include drug resistance, malnutrition, diabetes, and HIV co-infection (Luies and du Preez, 2020). These collectively lead to a slow decline in the incidence rate. Therefore, proposing an urgent therapeutic strategy is essential to overcome the challenges in treating TB and ending the global epidemic. 2 1.1.2 TB Pathogenesis and Clinical Presentations The transmission of TB occurs when a person with active pulmonary TB infection coughs and sneezes, releasing and aerosolizing the tubercle bacilli into the air, forming aerosol droplets (Alsayed and Gunosewoyo, 2023). These infectious particles may vary in size, ranging from 0.65 to over 7.0 µm, allowing the bacteria to spread and cause infection at different sites of the respiratory tract (Shiloh, 2016). For instance, studies reveal that the larger particles initiate infection at the oropharynx or the cervical lymph nodes, and the smaller particles can travel across the tracheobronchial region to adhere to the distal airways (Shiloh, 2016). Upon the arrival of Mtb in the aerosolized form at the alveoli, the activation of the host innate immune system serves as the first line of defense, causing phagocytosis and destruction of the Mtb by the professional alveolar macrophages and resident dendritic cells (Agyeman and Ofori-Asenso, 2017; Alsayed and Gunosewoyo, 2023). However, if some tubercle bacilli can withstand the attack by alveolar macrophages, they can actively replicate within the macrophages and be released upon lysis of the macrophages (Figure 1.1). These released bacilli can rapidly replicate exponentially, causing a high bacterial burden within a few weeks (Agyeman and Ofori-Asenso, 2017). The live and active Mtb can travel through the host bloodstream or lymphatic channels to reach the lymph nodes, larynx, spine, bone, or kidneys which are the primary sites of extrapulmonary infection (Agyeman and Ofori-Asenso, 2017). The adaptive immune system is activated approximately 2 to 8 weeks after the initial infection, leading to the migration of neutrophils, lymphocytes, and other immune cells to the primary infection sites. The primary function of these immune cells is to prevent further replication of the bacilli by sequestering the bacteria within a granuloma (Delogu et al., 2013). 3 Figure 2.1 The transmission and pathogenesis of Mycobacterium tuberculosis (Adapted from: Alsayed and Gunosewoyo, 2023) The formation of granulomas is considered one of the most significant hallmarks of pulmonary TB (Figure 1.2). Granulomas are aggregate structures composed of various immune cells, such as macrophages and lymphocytes. They act as barriers, encapsulating the tubercle bacilli and preventing them from interacting with the host immune system (Cronan, 2022). If the bacilli are successfully contained within the granuloma, they cannot spread further, and this mechanism results in the state of latent tuberculosis infection (LTBI). During LTBI, the granuloma formed at the site of the primary lesion is called the Ghon focus, whereby the bacteria reside in a dormant and non-metabolically active state from decades to an individual's lifetime (Delogu et al., 2013). Individuals with LTBI are asymptomatic, cannot transmit TB, and demonstrate no evidence of TB on their chest imaging, and sputum test results are negative. In 90% of the patients, the bacteria exist in a state of latency (Gong and Wu, 2021). 4 Figure1.2 The Structure of a granuloma (Adapted from: Cronan, 2022) However, if an individual with LTBI develops an HIV infection, cancer, diabetes, sepsis, chronic renal failure, malignancy, undergoes immunosuppressive therapy, or experiences a decline in components of the host immune system like the granuloma, CD4 T cells, and cytokines such as TNF-α and IFN-γ, then TB reactivation can occur (Luies and du Preez, 2020; Chai et al., 2018; Alsayed and Gunosewoyo, 2023). The host's immunocompromised state contributes to the granuloma's liquefaction and cavitation, which releases the bacteria that can reactivate and replicate, resulting in active TB infection. Active TB is highly infectious and symptomatic as Mtb can cause infection of the lungs, travel through the blood capillaries, and disseminate to other organs while also being transmissible to other individuals. Common clinical presentations of the disease include flu-like symptoms such as cough, anorexia, fever, weight loss, or night sweats (Ocepek et al., 2005; Cerezo-Cortés et al., 2019). 5 Mtb infections primarily cause lung infections, referred to as pulmonary TB (PTB), but dissemination of the bacteria to other parts of the body results in extrapulmonary tuberculosis (EPTB). EPTB commonly manifests as meningitis, lymphadenitis, pericarditis, cutaneous, musculoskeletal, ocular, oral, pleuritis, abdominal, genitourinary, and other miliary forms of TB. EPTB can be divided based on the site of infection. If the infection occurs at the initial site, it is called primary EPTB. If it occurs because of the spread of bacteria from the primary organ through the blood or lymphatic system, it is referred to as secondary EPTB (Kim et al., 2018 ; Roberto Eduardo Guerra Estrada et al., 2022). SECTION II: The BCG Vaccine Induced Immunity and Limitations 1.2.1 The BCG Vaccine Albert Calmette and Camille Guerin discovered the Bacillus Calmette-Guerin (BCG) vaccine at the Institut Pasteur, France, between 1908 and 1921 (Tran et al., 2014). The vaccine, derived from a live-attenuated strain of Mycobacterium bovis, a related subspecies of M. tuberculosis (>90%) homology (Behr et al., 1999), is notably safe for humans due to the deletion of the region of difference (RD1) locus in the genome, which encodes for ESX-1 (Tran et al., 2014). The ESX- 1 genetic locus encodes for the secretion of proteins ESAT-6 (early secreted antigenic target of 6 kDa) and CFP-10 (culture filtrate protein of 10 kDa), which are essential and dominant antigens that contribute to the development of the disease by aiding Mtb pathogenesis (Wong, 2017). This safety feature has led to its widespread implementation worldwide. The BCG vaccine is administered via the intradermal route or percutaneously, both of which have been proven to be equally protective (Dockrell and Smith, 2017). In many countries, the BCG vaccine is administered as part of the routine immunization programs. The World Health Organization (WHO) Expanded Programme on Immunization (EPI) has included BCG vaccination since 1974 and is aimed at infant vaccination worldwide. In 2020, 154 countries 6 implemented BCG vaccination as a standard part of childhood immunization programs, and 53 countries had more than 95% coverage (Qu et al., 2021). 1.2.2 How does the BCG vaccine protect against TB? BCG provides protection against TB by generating effective innate, adaptive, and trained memory responses in immunized individuals. During intradermal immunization, resident epidermal macrophages initiate the initial innate immune responses (Singh et al., 2022). These macrophages interact with live BCG using pattern-recognition receptors (PRRs) such as complement receptor 3 (CR3) and toll-like receptors 2 and 4 (TLR2/4). They efficiently recognize various components of the mycobacterial cell wall, such as peptidoglycans, arabinogalactan, and mycolic acids, which act as pathogen-associated molecular patterns (PAMPS) of BCG (Hossain and Norazmi, 2013;Maitra et al., 2019; Moliva et al., 2017). This communication helps establish a connection with the host. The dendritic cells (DCs) play a crucial role in initiating and directing antigen-specific immune responses and modulating the activity of the innate and adaptive immune systems (Moliva et al., 2017). DCs can induce effector T cell responses by migrating from tissues to draining lymph nodes (DLNs) via the lymphatic system. As they migrate, DCs upregulate MHC class II and CD80, CD86, CD40, and CD54 costimulatory molecules, which helps in their maturation and enables them to activate the adaptive immune cells (Demangel et al., 1999; Lagranderie et al., 2003) The costimulatory molecules CD80 and CD86 have two primary functions: (i) priming the T cells in lymphoid tissues and (ii) presenting the antigenic peptides on major histocompatibility (MHC) class II molecules (Moliva et al., 2017 ; Singh et al., 2022). The CD4 T cell adaptive immune response is induced through the presentation and recognition of BCG antigens by MHC class II molecules, while the cross-presentation of BCG antigens by MHC class I molecules leads 7 to CD8 T cell responses (Singh et al., 2022). Additionally, neutrophils can help transfer the BCG using lymphatic systems closer to the dendritic cells (DCs) and T cells in draining lymph nodes (DLNs). In the DLNs, the interaction between DCs and the BCG-infected neutrophils aids in the maturation of the DCs and in the presentation of BCG antigens, which is essential to priming the CD4 and CD8 T cells (Moliva et al., 2017 ; Singh et al., 2022). Overall, the innate immune cells— macrophages, DCs, and neutrophils—mediate the phagocytosis of live BCG and the activation of appropriate adaptive immune responses. The Function of T Lymphocytes BCG triggers the activation and growth of CD8+ cytotoxic T cells (Tc) and CD4+ helper T cells (Th) as a result of interaction between T cell receptors with the BCG antigens which is mediated through APCs (Siegrist, 2012). The CD4+ responses induced by BCG are essential in reducing bacterial load in the lung and spleen, as evidenced by mouse knockout models. Contrastingly, BCG vaccination-induced activation of CD8+ T cells control the prevention of disseminated forms of TB, such as miliary TB and TB meningitis (Wang et al., 2004; Derrick et al., 2004). The vaccination also causes helper T cells to differentiate into two distinct effector cells: Th17 cells, which produce IL-17A, and Th1 cells, which produce IFN-γ. Both cytokines act as protective effector molecules that collaborate to produce effective immune responses, helping to control further dissemination and bacterial loads in the lungs (Umemura et al., 2007; Moliva et al., 2017). The Function of B Lymphocytes and Antibodies The studies on TB vaccine development have primarily focused on cell-mediated immunity due to the intracellular nature of the Mtb infection. However, newer studies demonstrate the significant contribution of humoral responses in generating long-lasting immunity by producing 8 protective antibodies (Ab) (Sebina et al., 2012; Moliva et al., 2017). B cells have been shown to modulate the immune environment in TB-infected patients' lungs and in mice's granulomas, non- human primates (NHPs) infected with Mtb, and cattle infected with M.bovis (Tanner et al., 2019). The vaccination leads to heightened production of specific Ab such as immunoglobulin (Ig) M and IgG Ab isotypes IgG1, IgG2, and IgG3 (Beyazova et al., 1995; Hoft et al., 1999). IgA is abundantly found in the lung mucosa, and studies investigating the association of BCG vaccination to IgA production demonstrate that IgA deficient (IgA-/-) mice become more susceptible to BCG infections and have lowered production of both IFN-γ and TNF-α in the lungs (Rodríguez et al., 2005). IgA plays a crucial role in promoting phagocytosis of the microbes and, along with IgG, enhances cell-mediated cytotoxicity. Furthermore, these antibody-mediated immunity functions exacerbate bacterial neutralization, enhance inflammasome activation, increase cytotoxic natural killer (NK) cell activity, and increase phagocytosis. Immunity against Mtb is further enhanced by the interaction between antibodies and T cells, which gives rise to the formation of bacterial- antibody complexes, which function to mediate enhanced T cell activation, cytotoxic responses, and more significant processing and presentation of Mtb antigens to CD4+ T cells (Moliva et al., 2017). BCG Induced Trained Innate Immunity The BCG vaccine provides immunological memory to the innate cells of vaccinated individuals, which is referred to as trained innate immunity. Trained immunity is the enduring reprogramming of innate immune cells triggered by internal or external challenges. This results in enhanced effector responses following a period of quiescence (Chen et al., 2023). The molecular mechanisms that result in trained immunity involve many epigenetic modifications, such as noncoding RNAs, histone modifications, chromatin remodeling, and DNA modifications (Zhang 9 and Cao, 2019). These epigenetic modifications work with cellular metabolic pathways that regulate and develop monocytes, macrophages, and natural killer cells to induce trained immunity (Chen et al., 2023). In human monocytes, the BCG vaccination causes histone modifications and epigenetic reprogramming at the promoter regions of genes that encode inflammatory cytokines like IL-6 and TNF-α. These monocytes and macrophages undergo a "training" process, leading to functional and epigenetic reprogramming. This reprogramming promotes heightened production of proinflammatory cytokines such as IL-6, IL-1, and TNF-α (Chen et al., 2023). Furthermore, BCG vaccination leads to enhanced production of proinflammatory cytokines such as IL-1B and IL-6 by the NK cells. The increased production of proinflammatory cytokines, including TNF-α, IL-1B, and IL-6, induces the coordination between local and systemic inflammatory responses. For example, IL-1β and IL-6 stimulate the production of acute-phase proteins by activating hepatocytes (Medzhitov, 2007;Chen et al., 2023). The production of these acute phase proteins induces phagocytosis of pathogens by macrophages and neutrophils via activation of the complement system (Medzhitov, 2007;Chen et al., 2023). Additionally, activation of the local endothelium, vasodilation, enhanced vascular permeability and recruitment of serum proteins and leukocytes to the site of the infection is sequentially achieved by the coordination between TNF- α and IL-1b. The trained immunity from BCG vaccination is beneficial as it contributes to long-term cross-protection against heterologous infections, reduces susceptibility to respiratory tract infections, and enhances innate immune responses (Ayoub, 2020; de Bree et al., 2020). In summary, the BCG vaccination impacts various parts of the innate and adaptive immune system, making it effective in shielding vaccinated individuals against Mtb infection (Figure 1.3). This is achieved by boosting immune responses, which in turn can prevent potential complications 10 like disease spread. The broad applications of BCG have been suggested in cancer therapy since the pioneering work of Old LJ. et al., who demonstrated that BCG had anti-tumor effects (OLD et al., 1959). Morales A. et al. first used BCG in 1976 to treat superficial bladder cancer, marking a significant milestone in the history of cancer treatment (Morales et al., 2017). BCG induced immune responses are illustrated in Figure 1.3. Figure 1.3 BCG induced cellular, humoral and memory immune responses in immunized individuals. (Adapted from: Qu et al., 2021). 1.2.3 BCG Associated Challenges Since its discovery, the BCG vaccine has been widely used as the primary vaccine against TB worldwide. When administered during the neonatal stage, it protects against disseminated and pulmonary TB disease in young children (Khandelia et al., 2023). However, the vaccine is 11 ineffective at protecting against pulmonary TB in adult patients. Studies have shown that the vaccine's effectiveness in controlling TB can range from 0% to 80% (Tran et al., 2014). The varying effectiveness of the BCG vaccine can be attributed to several challenges that hinder efforts to eradicate the global TB burden. Some of the limitations of the BCG vaccine include the route of administration, variability in BCG strains, and exposure to environmental mycobacteria (EM) (Moliva et al., 2017). Route of Administration For years, the BCG vaccine has been administered intradermally (ID) in TB-endemic regions. While this method triggers robust systemic immune responses, it only offers limited protection in both human and animal models (Qu et al., 2021). The limitations of this approach are clear, and it is essential to understand how the vaccine-induced immune responses fade over time. This understanding is a catalyst for change, prompting us to investigate alternative routes of administration that can induce strong cellular, humoral, memory, and mucosal responses, particularly when dealing with respiratory pathogens. Studies suggest that its effectiveness is limited while BCG induces CD4+ and CD8+ T cells. In an infection model using the guinea pig as the model organism, BCG vaccination provides initial protection by increasing the production of antigen (Ag)-specific lipopeptide-reactive CD4+ T cells in peripheral blood mononuclear cells (PBMCs) (Horvath et al., 2012; Jeyanathan et al., 2010; Kaufmann et al., 2016). However, it cannot prevent granuloma formation due to a lack of functional diversity. Vaccination with BCG leads to strong activation of Ag-specific CD8+ T cells, but it cannot efficiently deliver Ag to the sites of T cell activation. Studies also indicate that as Mtb infection progresses, there is a state of "immune system paralysis" due to the subsequent functional decline of CD4+ and CD8+ T cells (Ryan et al., 2009). Additionally, BCG is ineffective at inducing 12 T helper 1 (Th1) and Th17 cells (Orr et al., 2015;Bhattacharya et al., 2014). Although the importance and mechanisms guiding the humoral responses induced by BCG vaccination are not so well understood, the B cells and antibodies do indeed play an essential role in fighting against Mtb infection. Studies show that the antibodies induced by ID BCG vaccination is very low. For example, IgA antibodies work by targeting the surface antigens of Mtb and can block the uptake of Mtb without the need for Fc alpha receptors. However, the levels of IgA being produced are insufficient, and the BCG vaccine's inability to generate adequate humoral responses needs to be considered when developing new strategies to enhance the immune-boosting properties of the BCG vaccine (Li & Javid, 2018 ; Tanner et al., 2019). Variation in BCG Strains Since the development of the BCG vaccine in 1920, it has been distributed worldwide, leading to genetic and metabolic variability and the emergence of different BCG strains. Today, several BCG vaccine strains are used globally (Brosch et al., 2007; Abdallah et al., 2015). A particularly significant study in this field is an observational study in Kazakhstan, which compared four birth cohorts immunized with different strains of BCG vaccine (BCG-Russia, BCG-Serbia, BCG-Japan) or no BCG. The cohorts, totaling 138,059 and 168,664 children, were followed up for 29 months. The TB case notifications revealed significant differences in protective efficacy among the different strains. The protection against clinical TB was found to be 69%, 43%, and 22% and 92%, 82%, and 51% efficacy against culture-positive TB for BCG-Japan, BCG-Serbia, and BCG- Russia, respectively. However, the evidence supporting the idea that genetic variation between the different strains dictates the variability in efficacy or protective properties remains weak (Favorov et al., 2012; Ritz et al., 2008) 13 Exposure to Environmental Mycobacteria (EM) The research into how exposure to environmental mycobacteria (EM) affects the effectiveness of the BCG vaccine began after observations from clinical studies and animal models. It was found that BCG vaccination provided the greatest protection to infants and individuals who had not previously shown sensitivity to tuberculin. The mechanisms responsible for the influence of EM on the efficacy of BCG vaccination are not entirely clear, but two possible mechanisms have been suggested (McShane et al., 2012). The first theory is that the immune response triggered by BCG vaccination may be diminished due to the presence of immunity from prior EM exposure. The second theory proposes that the immunity gained from EM exposure could hinder the replication of BCG, thereby reducing its protective effects (McShane et al., 2012). Understanding these two possible mechanisms is crucial for developing new vaccine strategies and could have a significant impact on vaccine development and public health. Section III: Tuberculosis Treatment: Approaches and Limitations 1.3.1 Treatment for TB During latent TB infection (LTBI), the host immune system successfully keeps the bacteria sequestered within the granuloma. It is a persistent immune response occurring in reaction to stimulation by Mtb antigens. The absence of bacterial replication marks this stage, and there are no signs of active TB disease. However, suppose a person with latent TB is exposed to risk factors such as alcohol consumption, smoking, malignancy, diabetes, or renal failure (Gideon and Flynn, 2011). The bacteria can reactivate in that case, leading to active TB disease. Active TB disease is characterized by lung tissue damage, ongoing bacterial replication, and sometimes the development of cavities that can spread the bacteria (Dartois & Rubin, 2022). Classifying Mtb strains into fully drug-susceptible, mono-resistant, multidrug-resistant (MDR), or totally drug-resistant (XDR) determines the patient's treatment which is usually a combination 14 of three to four antibiotics. The standardized treatment schedule for drug-susceptible tuberculosis is six months long. The treatment has an induction phase during which the patient receives a combination of first-line drugs: rifampin, isoniazid, and pyrazinamide (Horsburgh et al., 2015). Additionally, ethambutol is used to protect against unrecognized resistance to one of the three core drugs. It can be discontinued once the susceptibility to isoniazid, rifampin, and pyrazinamide has been confirmed. After the initial phase, the treatment progresses to the consolidation phase. During this time, patients are given rifampin and isoniazid for an additional four months (Horsburgh et al., 2015). In the case of mono-resistant TB, an individual can become infected with strains of TB that are resistant to only first-line anti-TB drugs like isoniazid or rifampin. For example, if a patient becomes resistant to isoniazid or experiences severe toxic side effects, it can be replaced with a later-generation fluoroquinolone such as levofloxacin or moxifloxacin. Recent studies have shown that a combination of rifampin, moxifloxacin, pyrazinamide, and ethambutol administered for two months as part of a 6-month treatment regimen, followed by rifapentine and moxifloxacin for four months, is also effective (Horsburgh et al., 2015). The development of Multidrug-resistant tuberculosis (MDR-TB) occurs when an individual is infected with strains of Mtb that are resistant to both rifampin, isoniazid, and other anti-TB drugs. The course of treatment, in this case, must be determined by obtaining the results of drug susceptibility testing of Mtb isolates from the patient. According to WHO recommendations, the treatment for MDR-TB should last at least 20 months. The treatment involves a combination of four second-line anti-TB drugs such as pyrazinamide, a fluoroquinolone, and a parenteral agent (kanamycin, amikacin, capreomycin, ethionamide) and either cycloserine or PAS (Minion) (Günther et al., 2015; Horsburgh et al., 2015). 15 1.3.2 Treatment Associated Challenges Tuberculosis presents itself in various forms and, as such, calls for specific treatment suited to each patient. The treatment involves a combination of several antibiotics, which are given for several months. The lengthy duration of the treatment poses two significant challenges that hinder its overall success. Firstly, patients often struggle to complete the entire treatment course, with reports indicating that 16 to 49% do not finish the regimen (Volmink and Garner, 2007). The lack of adherence to the treatment can be attributed to a multitude of factors, such as the cost of the treatment, stigma, the patient’s beliefs and values, and the adverse effects caused by the drugs (Munro et al., 2007). Furthermore, the medications may cause significant toxic side effects, including liver damage, gastrointestinal issues, allergies, and joint pain (Horsburgh et al., 2015). The study results revealed that 15% of the participants experienced adverse drug reactions, leading to the interruption or discontinuation of one or more drugs. Among those affected, 7.7% experienced adverse reactions that resulted in hospitalization, disability, or death (Lv et al., 2013). Another challenge is that patients co-infected with Human Immunodeficiency Virus (HIV) require treatment with both antituberculosis and antiretroviral drugs. As a result, these patients suffer from the ill effects of the treatment, such as pill burden, compliance issues, drug interactions, overlapping toxic side effects, and immune reconstitution inflammatory syndrome (Swaminathan et al., 2011). Observational studies have shown that combined antiretroviral therapy (cART) during treatment for tuberculosis reduces mortality in ART-naïve patients, despite the increased risk of immune reconstitution syndrome (Girardi et al., 2012). 16 Section IV: Fighting the Silent Enemy: Immune Warriors Against Tuberculosis 1.4.1 The Innate Immune Response The host's innate immune response serves as the first line of defense against several pathogens, including Mtb. The innate immune system generates immediate defense mechanisms against the invading pathogen and long-lasting adaptive immunity (Iwasaki and Medzhitov, 2015). The early interaction of Mtb with host innate immune cells, such as macrophages (Mϕ), dendritic cells (DCs), neutrophils, and natural killer (NK) cells, determines the establishment of Mtb infection (Figure 1.4.) Furthermore, unconventional T cells like mucosal-associated invariant T cells (MAIT) and airway epithelial cells (AECs) also trigger effective immune responses against Mtb.The host's immune cells recognize pathogen-associated molecules (PAMPS) present on the surface of Mtb through pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs), Nod-like receptors (NLRs), and C-type lectin receptors (CLRs). These receptors also initiate immune defense-associated cellular functions such as phagocytosis, autophagy, apoptosis, and inflammasome activation (Liu et al., 2017; Iwasaki and Medzhitov, 2015; Gold et al., 2015) Unconventional Innate Immune Cells Airway Epithelial Cells (AECs) During Mtb transmission, the bacteria enter the recipient's respiratory tract through the nose and mouth. It then travels to the trachea, bronchus, bronchioles, and the alveoli. The respiratory mucosa along the airway acts as the first line of defense against Mtb (Middleton et al., 2002). For example, the respiratory mucosa consists of epithelium that contains a layer of airway epithelial cells (AECs), which serves as a barrier that prevents the invasion of the bacteria. The AECs actively express pattern recognition receptors such as Toll-like receptors, Dectin-1, C-type lectin receptors (CLRs), nucleotide-binding oligomerization domain-containing protein 2 (NOD2), dendritic cell (DC)-specific intercellular adhesion molecule-3-grabbing non-integrin and mannose receptor (Li 17 et al., 2012). These PRRs mediate the recognition of PAMPs on the Mtb surfaces. After exposure to Mtb, AECs produce cytokines and effector molecules to initiate an effective immune response. Additionally, AECs present antigens to mucosal-associated invariant T cells (MAITs) while stimulating the production of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and granzyme. These collectively contribute to the clearance of Mtb (Harriff et al., 2014; Lerner et al., 2015). Conventional Innate Immune Cells Macrophages (Mϕ) Macrophages play a crucial role in the body’s defense against Mtb infection. Granuloma formation is a significant hallmark of TB disease from a histopathological point of view. The granuloma comprises various transformed macrophages, including multinucleated giant cells, epithelioid cells, and foam cells (Feng et al., 2014; Liu et al., 2017). The recognition of Mtb PAMPs such as glycolipids, lipoproteins, and carbohydrates by macrophage-specific PRRs such as TLRs, NLRs, and CLRs mediates the initiation of a network of coordinated signaling pathways, which results in unique gene expression profiles in macrophages at different stages of infection along with an antimicrobial effector function (Killick et al., 2013;Liu et al., 2017). The development and function of macrophages is influenced by various micro-environmental signals, leading to their differentiation into M1 and M2 populations. The M1 macrophages serve as the primary effectors, and their activation in response to intracellular bacteria leads to the production of immune- stimulatory cytokines such as IFN-γ, TNF-α, and GM-CSF. Contrastingly, M2 macrophages play a crucial role in maintaining a balance between exacerbated pathology and controlling bacterial growth. The M2 population of macrophages helps establish balance by suppressing Th1 responses and is induced by cytokines such as IL-4, IL-13, IL-10, and TGF B. In addition to their effector functionality, macrophages can trigger various mechanisms to fight off Mtb infection, including 18 producing oxygen and nitrogen components and cytokines, phagosome acidification, and autophagy of intracellular Mtb (Murray, 2017; Ginhoux et al., 2015; Sica et al., 2015). Dendritic Cells (DCs) DCs are critical immune cells that bridge the innate and adaptive immune system. DCs play a crucial role in Mtb antigen presentation, and due to their ability to secrete T-helper polarizing cytokines and their costimulatory capacity, they can initiate adaptive immune responses (Prendergast & Kirman, 2013; Liu et al., 2017). Human DCs generated from monocytes express DCSIGN, CD11b, CD11c, and mannose receptors, all of which can recognize Mtb ligands. DC- SIGN facilitates the entry of Mtb into DCs by recognizing mannose-capped lipoarabinomannan (ManLAM). This interaction triggers the production of the anti-inflammatory cytokine IL-10, associated with impeding DC maturation and suppressing the generation of costimulatory molecules (Nigou et al., 2001). Mtb is a notorious pathogen that manipulates the function of DC to prevent optimal conditions for promoting adaptive immunity by producing a negative signal through ManLAMs, which inhibits IL-12 production (Nigou et al., 2001; Liu et al., 2017). The kinetics, nature, and magnitude of the T cell response are influenced by changes in DC maturation, cytokine production, and antigen presentation. If impaired, this can allow bacteria to reside in and evade the host lungs. This phenomenon has been demonstrated through studies investigating the ability of Mtb-infected monocyte-derived DC to stimulate lymphoproliferation of naive and memory CD4s and CD8s. Recent studies have shown that during Mtb infection, the expression of CD13 is high on DCs, which may be responsible for impairing T cell responses in humans. This suggests that targeting the CD13 receptor with anti-CD13 antibody treatment could reduce the impact of Mtb on inhibiting T-cell activation (Kuo et al., 2016 ; Liu et al., 2017). 19 Neutrophils Active pulmonary TB patients' bronchoalveolar lavage and sputum contain abundant neutrophils, as these cells are the first to infiltrate the lungs after Mtb infection. Neutrophils are a robust population of effector cells that can drive both antimycobacterial activity and immunopathology in humans (Nouailles et al., 2014; Niazi et al., 2015). Neutrophils engulf bacteria and release antimicrobial substances from their granules when an infection occurs. Furthermore, during the neutrophils' respiratory burst, they release factors such as elastase, collagenase, and myeloperoxidase, which cause indiscriminate damage to bacterial and host cells. During Mtb infection, neutrophils also play an essential role in the induction of adaptive immunity and granuloma cavitation (Dallenga and Schaible, 2016; Blomgran and Ernst, 2011; Ong et al., 2015). Furthermore, neutrophils can release enzymes that damage lung tissue, such as arginase and matrix metalloproteinase-9 (MMP-9 and gelatinase B). These enzymes are also released by other innate immune cells and epithelial cells, all of which are affected by Mtb infection (Elkington et al., 2007; Hesse et al., 2001). Recent transcriptional studies have shown neutrophils have an additional immunoregulatory function beyond their degranulation ability in Mtb infection. The interaction between programmed death ligand 1 (PD-L1) on myeloid cells and programmed death receptor (PD-1) on lymphocytes is thought to play a role in developing weakened or exhausted lymphocyte responses during persistent infections (McNab et al., 2011). Neutrophils, like other innate immune cells, can be manipulated by Mtb, inducing neutrophil necrosis and preventing apoptosis. This process depends on the region of difference 1 (RD-1)-encoded virulence factors (Corleis et al., 2012; Francis et al., 2014). It is worth noting that future studies focused on determining whether pharmacologic intervention targeting neutrophil necrosis can alter 20 uncontrolled inflammation and immunopathology during Mtb infection and any extra mycobacterial effectors of neutrophil necrosis. Natural Killer Cells (NK) NK cells are a subset of innate lymphocytes characterized by their granular nature and remarkable cytolytic ability (Allen et al., 2015). These cells play a critical role in the early stages of infection. Different cell wall components of Mtb, including mycolic acids, serve as direct ligands for the natural cytotoxicity receptor (NCR) NKp44 found on the NK cells. The NK cells utilize various mechanisms to control mycobacterial growth. This is achieved indirectly by stimulating the immune system through macrophage activation and directly by cytotoxic functionality, such as the release of cytoplasmic granules comprising perforin, granulysin, and granzyme (Allen et al., 2015; Liu et al., 2017). Furthermore, NK cells produce IFN-γ and IL-22, which can inhibit the intracellular growth of Mtb by enhancing phagolysosomal fusion. Additionally, they secrete CD54, TNF-α, GM-CSF, and IL-12, promoting γδ T-cell proliferation. It's important to note that NK cells can be found in fully developed granulomatous lesions in the lungs of patients infected with Mtb. This indicates that NK cells may be functionally impaired during the course of TB. One way to address this issue is to develop anti-TB treatment plans that reduce bacterial levels, partially restoring the cytolytic capabilities of natural killer (NK) cells. Glutathione (GSH) is one such substance that uses bacteriostatic mechanisms to hinder Mtb replication, thereby boosting NK cell function (Allen et al., 2015; Esin et al., 2013; Feng et al., 2006). 21 1.4.2 The Adaptive Immune Responses Due to the classification of Mtb as an intracellular pathogen, the host's immune responses to Mtb depend on specific immunity. The two subdivisions of the specific immunity are cellular and humoral immunity. Cellular immune responses are mediated by T cells, while humoral immunity is conferred by B cells and antibodies (Jasenosky et al., 2015;Zhuang et al., 2024). The initiation of cellular immune responses occurs approximately 2-6 weeks after Mtb infection, as evidenced by the development of a delayed hypersensitivity response to intradermally injected tuberculin (DHT) purified protein derivative (PPD) (de Martino et al., 2019). T-cell immunity is an immune response elicited by the host immune system in response to stimulation by Mtb antigens. The activation of T lymphocytes is triggered by DCs, which are part of the innate immune system. Subsequent activation occurs as the Mtb infection spreads inside the draining lymph nodes. In addition to T lymphocyte activation in the lymph nodes, specific lymphocyte populations also expand in response to Mtb antigens (Matucci et al., 2014). The T cells specific to Mtb start moving towards the blood and travel to the primary infection sites in the lungs, actively working to control the spread of the infection (Chackerian et al., 2002; Wolf et al., 2007). Essentially, the functional role of T lymphocytes involves the presentation and recognition of Mtb antigens by antigen- presenting cells (APCs). Subsequently, a cascade of immune responses occurs, including the activation, proliferation, and differentiation of the T-cells (Matucci et al., 2014). Ultimately, these processes lead to eradicating the Mtb antigens from the host's body in ideal scenarios In the presence of an Mtb infection, various T cell subsets, both conventional and unconventional, develop through T cell differentiation. These subsets can be identified based on the surface markers they express. The primary conventional T cells are CD4+ T cells and CD8+ T cells, while the unconventional T cells consist of γδ and CD1-restricted cells (Zhuang et al., 2024). 22 CD4+ T Lymphocytes The presentation of major histocompatibility complex class II (MHC II) molecules on antigen- presenting cells (APCs) can stimulate the differentiation of initial CD4+ T cells (Th0) into various subtypes of helper T cells, including Th1, Th2, Th17, and Treg cells (Sekiya & Yoshimura, 2016). These cells are responsible for secreting key cytokines such as IFN-γ, IL-2, and TNF-α, activating the infected macrophages to induce antibacterial activity. These cytokines help eliminate Mtb by recruiting monocytes and neutrophils, regulating macrophage cytotoxicity, inducing inflammatory mediators, and reactive oxygen and nitrogen species (Cowley & Elkins, 2003; Yahagi et al., 2010;Geginat et al., 2014). Contrastingly, Th17 cells secrete cytokines such as IL-17A, IL-21, IL- 22, and IL-26, leading to the recruitment of neutrophils to the infection site, further enhancing the inflammatory response. The essential function of Th17 cells is to combat Mtb infection by boosting the host's immune response by stimulating chemokine production in the lungs (Ouyang et al., 2008; Knochelmann et al., 2018). These cytokines attract CD4 T cells that produce IFN-γ during Mtb infection (de Martino et al., 2019). IFN-γ plays a crucial role in facilitating and enhancing the maturation of macrophage phagosomes. The significance of IFN-γ is demonstrated in knockout animal models, which shows that these knockouts experience a severe course of Mtb infection. Furthermore, patients with mutations in the genes that encode for IFN-γ or its receptors are widely recognized to experience disseminated infections resulting from BCG or other non-tuberculous members of the mycobacteria genus (Rosain et al., 2018). However, the control of infection by IFN-γ alone is limited, and association with other molecules such as IL-6, IL-1, and TNF-α is required. It has been shown that TNF-α plays a crucial role in protecting against infection and preventing reactivation in humans, non-human primates, and mice (Flynn et al., 1995; Domingo- Gonzalez et al., 2017). Additionally, CD4 T cells help activate B cells and CD8 T cell responses 23 and provide migration signals for other cells. This collective effort helps prevent the progression of Mtb (Flynn & Chan, 2022). IL-17 has been implicated in regulating chronic Mtb infection in murine models, with its production primarily attributed to γδ T cells. Furthermore, during acute infection, γδ T cells were identified as the primary source of IL-17 production, as demonstrated by Lockhart et al. in 2006. Additionally, evidence supports that IL-17 can help expand γδ T cells in non-human primates (Lockhart et al., 2006; Shen et al., 2019). What we know about the role of CD4+ T cells comes from the results obtained from studies conducted in human immunodeficiency virus-positive patients who experienced CD4+ T cell depletion. The findings indicate that these patients are at higher risk of both initial and latent infection reactivation. Studies using mice without specific immune system genes (IFN-γ, TNF, or IL-12), as well as research involving the neutralization of TNF in non-human primates and humans, along with genetic studies on susceptibility to mycobacterial infection in humans, have shown the crucial role of Th1 cytokines in resisting tuberculosis (Cooper, 2009; de Martino et al., 2019). CD8+ Lymphocytes Another group of T lymphocytes, CD8+ T cells, also fight intracellular pathogens such as Mtb.Earlier, it was believed that CD8+ T lymphocytes did not contribute to the immune response against Mtb infection. However, their specific function related to Mtb became more apparent due to the observation that they are present in the airway lumen as the infection establishes. The most evidence we have today regarding the role of CD8+ T lymphocytes comes from human models exhibiting CD8+ T lymphocyte defects. There is limited evidence compared to the vast amount of human models available for studying HIV infection, which has contributed significantly to our understanding of the function of CD4+ T lymphocytes (Canaday et al., 2001; Lin & Flynn, 2015).CD8+ T cells can recognize Mtb antigen peptides presented by MHC class I molecules. As 24 a result of this recognition, the cells undergo proliferation and differentiation into cytotoxic T lymphocytes (CTLs) (Jenkins & Griffiths, 2010). CTLs achieve the destruction of intracellularly infected Mtb cells by releasing cytokines in addition to two main mechanisms. The first mechanism by which CTLs disrupt infected Mtb cells involves the binding of CTLs to antigen complexes on the surface of these cells. This binding leads to the release of perforin and granulysin from the CTLs into the immunological synapse via exocytosis. As a result, the normal osmotic gradient of the infected cells is disrupted, leading to the lysis of the bacteria. Subsequently, the Mtb can be phagocytosed and eliminated by nearby macrophages. The activated CTLs express Fas ligand (FasL), which triggers apoptotic signals by binding to Fas receptors on infected Mtb cells, initiating the caspase8-mediated apoptotic signaling pathway. Furthermore, the secretion of IFN-γ enhances cytotoxicity by positively regulating the expression of reactive oxygen species (ROS) and reactive nitrogen intermediates (RNIs) in monocytes and macrophages (Nathan et al., 1983; Ashkenazi & Dixit, 1998; Lord et al., 2003; Jenkins and Griffiths, 2010). CD8+ T cells exhibit additional direct effector functions, such as releasing granules containing cytotoxic molecules like perforin, granzymes, and granulysin. These molecules can destroy host cells or directly eliminate Mtb and other bacteria. IFN-γ, TNF-α, and IL-2 are some cytokines produced by CD8+ T cells and are well known for their role in controlling Mtb infection. On the contrary, CD8+ lymphocytes also produce cytokines like IL-10 and TGF-β, which favor the growth of Mtb instead of controlling it (Oddo et al., 1998 ; Canaday et al., 2001; Lin and Flynn, 2015) γδ T Cells γδ T cells play a crucial role in providing anti-mycobacterial immunity by integrating innate and adaptive immune properties during Mtb infection. It was in 1989 when the significance of γδ T cells in combating Mtb infection was acknowledged (Vorkas et al., 2018; Cheng et al., 2018). It 25 has been shown that in people who suffer from active tuberculosis (TB) and acute bacterial and parasitic infections, the frequencies of γδ T cells in the peripheral blood can rise significantly. While healthy individuals typically have less than 5% of these cells, some patients can exhibit levels exceeding 45% (Chen & Letvin, 2003). The bacteria and parasites produce small phosphorylated nonpeptide antigens called phosphoantigens (pAgs), which can be detected by Vγ9Vδ2 T cell receptors (TCRs). Upon activation, the Vγ9Vδ2 T cells employ mechanisms such as TCR-dependent degranulation and antibody-dependent phagocytosis, which mediates the killing of the infected cells in vitro (Junqueira et al., 2021; Roy Chowdhury et al., 2023). In general, the production of TNF-α and IFN-γ due to the phosphoantigen-mediated activation of γδ T cells helps strengthen the protective responses to Mtb (Casetti & Martino, 2008). In children with acute bacterial meningitis, researchers have found an increase in the frequency of circulating Vγ9Vδ2 T cells that produce interleukin-17 (IL-17). This finding is consistent with studies using mouse infection models, which show that γδ T cells are essential in producing IL-17 during the early stages of acute infection, thereby triggering the inflammatory response (Caccamo et al., 2011; Papotto et al., 2017). Aside from their ability to fight against tuberculosis, γδ T cells also help in the maturation of dendritic cells. This process influences other cell types such as CD4 T helper cells and B cells, ultimately boosting the immune response against tuberculosis. The differentiation of Vγ9Vδ2 T cells affects the function of αβ T cells by stimulating CD4+ and CD8+ αβ T cells to produce TNF-α and IFN-γ, which helps in eliminating Mtb. In summary, the effectiveness of Vγ9Vδ2 T cells in combating Mtb depends on three key factors: cytokine release, cytolytic effector function, and DC maturation (Zhao et al., 2018). 26 CD-1 Restricted T Cells The identification of nonclassical T cells that can recognize non-peptidic antigens associated with non-MHC antigen-presenting molecules is a more recent discovery compared to the well- established function of classical T cells (De Libero et al., 2014). Classical T cells mediate the recognition of peptidic antigens associated with MHC-encoded molecules during Mtb infection (Cooper, 2009). These non-MHC-restricted T cells can be classified as lipid and glycolipid-specific CD1-restricted T cells, mucosal-associated invariant T (MAIT) MRI-restricted cells, and TCR γδ BTN3A1-restricted T cells depending on the restriction molecule, antigen specificity, and the T- cell receptor (TCR) structure (De Libero et al., 2014). CD1 molecules represent a category of MHC class I-like molecules that are involved in presenting lipid antigens to T cells. In humans, three primary categories of CD1 isoforms have been recognized. These include group 1 CD1, which consists of CD1a, CD1b, and CD1c, and group 2 CD1, which includes CD1d, and these molecules are expressed in a range of cell types (Morgun et al., 2021). The studies conducted on the presentation of mycobacterial lipids in humans and other animal species reveal that group 1 CD1- restricted T cells possess effector functions similar to T helper 1 (Th1), T helper 2 (Th2) and T helper 17 (Th17) cells. An increase in the effector function allows for mounting a protective response against Mtb (Cohen et al., 2009). Furthermore, evidence of their role in protecting against Mtb can be seen in the detection of group 1 CD1-restricted T cells in people infected with Mtb. Busch and colleagues (2016) further concluded that a significant factor in limiting Mtb growth in BAL of latent and active TB infections is CD1b-dependent T cell activation. They also found that the primary lipid recognized by T cells from healthy human PBMC was LAM, a glycolipid in the Mtb cell wall and a major virulence factor (Busch et al., 2016). Additionally, researchers discovered that latently infected patients possessed CD1b-restricted LAM-specific T 27 cells with polycytotoxic functionality, including CD8+, granulysin+, granzyme B+, and perforin+, whereas actively infected patients did not.This research strongly suggests that group 1 CD1- restricted T cells play a protective role in human tuberculosis, as latently infected patients are better able to suppress disease progression than actively infected patients (Busch et al., 2016; Morgun et al., 2021).The inhibition of CD1-restricted responses by Mtb is an evolved strategy to escape the host immune system. Mtb achieves this by inducing the production of CD1a-, CD1b- and CD1c- DCs in the presence of IFN-α while inhibiting the expression of CD1 molecules due to the infection of human monocytes (Hiromatsu et al., 2002; Watanabe et al., 2006). As a result of their differentiation into host cells that resemble macrophages and their diminished ability to activate CD1-restricted T cells, Mtb-infected monocytes provide an immunoprivileged environment for the persistence of mycobacteria. In more recent years, the potential of including group 1 CD-1 presented Mtb lipid antigens in vaccine development has been explored. This is because the CD1 proteins are largely non-polymorphic which implies that similar antigen presenting capacity and similar vaccine efficacy profiles will be shared amongst genetically diverse populations (De Libero et al., 2014). 1.4.3 Humoral Immune Responses The humoral response to Mtb infection comprises the B cells, which mediate the destruction of the infected cells by producing antibodies and releasing toxins. B cells are specialized antigen- presenting cells (APCs) that become activated when they internalize antigens through surface receptors.B cells present these antigens to activate CD4+ T cells (Lund and Randall, 2010). Once activated, CD4+ T cells can produce different cytokines and differentiate into Th1 and Th2 helper T cells, which they then regulate.The modulation of B lymphocyte antibody response by Th1 and Th2 cells helps to facilitate the apoptosis of cells infected with Mtb bacteria (Hong et al., 2018; 28 Hua & Hou, 2020). Moreover, B cells can transform into various subsets such as B effector 1 (Be1), B effector 2 (Be2) and B regulatory cells (Breg). Be1 and Be2 cells can secrete diverse cytokines that promote the maturation of naïve CD4+ T cells into effector Th1 and Th2 T cells that play a crucial role in eliminating Mtb from the body of the infected host (Mosmann, 2000; Harris et al., 2000). Antibodies (Ab) impose their antimicrobial effects through antigen-binding fragments (Fab) and the interaction between the crystallizable fragment (Fc) and Fc receptors (FcRs) on the effector cells. This interaction results in the regulation of neutralization, opsonization, opsonophagocytosis, and complement fixation, as well as a wide range of effector functions of innate immune cells, which includes antigen presentation and cytokine production. FcR is a vital molecule that is involved in the regulation of the host immune system (Chan et al., 2014; Bournazos et al., 2015). It comprises three types: FcRI (CD64), FcRII (CD32), and FcRIII (CD16), each possessing distinct intracellular motifs (ITAM or ITIM). These motifs determine whether the receptors display an inhibitory or activating function (Nimmerjahn & Ravetch, 2006; Nimmerjahn & Ravetch, 2008). The engagement of these receptors plays a crucial role in the intricate stimulation of T cells either by impeding or facilitating the development and exhibition of antigens by DCs (Kalergis and Ravetch, 2002). Overall, the evidence indicates that the B cells and antibodies play an essential role in shaping immune responses to Mtb, which are also crucial in establishing effective anti- tuberculosis immunity. B cells and antibodies exert their functions via cytokine production, neutrophilic response, and granulomatous inflammation, influencing vaccine-induced immunity in mice (Chan et al., 2014). The investigation into the role of B cells and antibodies in controlling Mtb infection has recently garnered increased interest among researchers. They are seeking to understand the immune responses against tuberculosis in individuals with varying levels of 29 infection and disease progression. This is being done by using non-human primate models of the disease (Phuah et al., 2016). Figure 1.4 The Innate and Adaptive Immune responses involved in Mtb infection. (Adapted from Zhuang et al., 2024). Section V: The Advent of Subunit Vaccines and MPT63 1.5.1 The Development of Subunit Vaccines Currently, the only authorized vaccine for TB is BCG, one of the most widely used vaccines globally. The BCG vaccine helps prevent the most severe forms of childhood TB (Singh et al., 2016). Vaccination with BCG effectively protects children from severe forms of tuberculosis, reducing the risk of contracting disseminating types of TB such as meningeal and miliary TB by 85% and decreasing TB-related deaths by 66% (Roy et al., 2019). The BCG vaccine is administered to around 100 million children worldwide annually and protects against TB for up to 10 years (Mangtani et al., 2013). Despite the advantages of the BCG vaccination, several persisting problems exist, including waning protective efficacy over time and limited protection against pulmonary TB in adults (Abubakar et al., 2013). In addition, there are also safety concerns 30 associated with BCG as it is a live attenuated vaccine that comes with the risk of becoming virulent (Fatima et al., 2020). Furthermore, it possesses a disadvantageous risk profile in individuals with compromised immune systems, such as those with congenital immunodeficiency and HIV infections, due to the subsequent occurrence of BCG bacteremia post-vaccination. For example, BCG vaccination in infants exposed to HIV but not infected resulted in reduced BCG-specific T- cell proliferation, as well as lower levels of protective cytokines such as IFN-γ and TNF-α production compared to healthy infants (Mazzola et al., 2011; Hatherill et al., 2020; Scriba & Mizrahi, 2020). Therefore, given the current challenges, devising a new TB vaccine approach is necessary. This strategy would address issues concerning safety, long-term immunity, and vaccination of immunodeficient patients. Myriad approaches can be taken when implementing new vaccine strategies, such as preventive pre-exposure for uninfected individuals or infants, preventive post- exposure for latent TB in adolescents and adults, and therapeutic vaccines for active TB patients (Table 1.1) (Pipeline of vaccines, 2024). The currently used BCG vaccine uses the preventative pre-exposure approach (Whitlow et al., 2020). Another method of classifying vaccines in preclinical and clinical trials involves categorizing vaccines based on their biochemical forms. This includes live attenuated, inactivated, adjuvanted protein subunit and recombinant vaccines. Another method of classifying vaccines in preclinical and clinical trials involves categorizing vaccines based on their biochemical forms. This includes live attenuated, inactivated, adjuvanted protein subunit and recombinant vaccines (Whitlow et al., 2020). 31 Table 1.1 Current Subunit Vaccines in clinical trials and their Adjuvants (Adapted from: (Mustafa, 2021) 1.5.2 What is a Subunit Vaccine? Subunit vaccines consist of specific pathogen components compared to the whole pathogen vaccines. Subunit vaccines, primarily consisting of proteins/antigens or peptides, may be limited in inducing an immune response (Vetter et al., 2017). Furthermore, they also lack “pathogen- associated microbial patterns” which function to elicit activation of the innate immune responses. Consequently, they necessitate immunostimulatory adjuvants or delivery systems to augment the immune response (Duong et al., 2023). The most integral part when designing the subunit vaccine is to choose the suitable antigen, as most subunit TB vaccines target specific proteins from Mtb. Therefore, the scope of the immune reaction tends to be more limited. It is best to select antigens that induce protective immune responses in most immunized people. The subunit vaccine approach offers several advantages, one of the most significant ones being that due to their lack of viability, subunit vaccines possess an enhanced safety profile and can be administered to immunocompromised individuals without the risk of infection (Lai et al., 2023). In addition to that, subunit vaccines also offer an opportunity to have greater control over the dose and 32 vaccination regimen (Lai et al., 2023). The development of subunit vaccines, especially those based on peptides, can potentially overcome some of the limitations associated with the BCG vaccine. This is accomplished by directly binding to major histocompatibility complex (MHC) molecules in antigen-presenting cells while avoiding cross-reactivity with non-tuberculous mycobacteria or latent Mtb antigens (Bellini & Horváti, 2020). 1.5.3 The Need for An Adjuvant As previously mentioned, protein/peptide antigen-based subunit vaccines require a suitably formulated adjuvant to enhance their immunogenicity significantly. An adjuvant is a two-part system consisting of an immunomodulator and a delivery system designed to boost the effectiveness of the vaccines (Maisonneuve et al., 2014). One of the key roles of adjuvants is to activate the innate immune response by engaging with antigen-presenting cells (APCs), thereby guiding the adaptive immune system to generate targeted and efficient immune reactions toward the antigen. Immunodulators target pathogen recognition receptors (PRRs) and their signaling pathways, regulating the production of humoral and cellular immune responses. This system is engineered to activate specific signaling pathways that destroy Mtb while inhibiting pathways that decrease bacterial phagocytosis. Various PRRs identify Mtb pathogen-associated molecular patterns (PAMPs), such as toll-like receptors (TLRs) and C-type lectin receptors (Duong et al., 2023). The adaptive immune responses vary depending on the adjuvant's activation of a specific innate APC immune receptor. For instance, following the activation of the innate receptor, APCs can trigger T-helper (Th)1, Th17, and Th2, which constitute the T-helper cell responses. Subsequently, the T-cell response determines the nature and the extent of B-cell responses and the overall outcome of adaptive immunity (Duong et al., 2023). 33 While aluminum-based adjuvants are widely used as primary adjuvants to stimulate humoral responses, they are ineffective in producing optimal Th1-cellular immunity and CD8+ cytotoxic responses due to the intracellular nature of Mtb. This limitation underscores the need for alternative adjuvants (Skwarczynski and Toth, 2016). Liposomes and emulsions, as vaccine adjuvant-delivery systems (VADS) such as AS01, CAF01, and GLA-SE, can help enhance the Th1 responses (Bellini and Horváti, 2020). For instance, IC31 is currently being investigated and clinically evaluated with the fusion protein H56. The results indicate that the vaccine is safe to use with an acceptable tolerability profile in Mtb-infected as well as Mtb-uninfected adults and produces durable antigen- specific CD4+ T-cell responses ( Geldenhuys et al., 2015; Agger, 2016; Suliman et al., 2019). Furthermore, Cationic Adjuvant Formulation (CAF01) has demonstrated the ability to enhance long-lasting immunity with a Th1 profile in animal models while triggering a Th17 response through TDB signaling via the C-type lectin receptor Mincle. A study examining the effect of subcutaneous immunization with H56:CAF01 following intranasal boost on Th1/17 immune responses did not seem to have a great impact on the Th1/17 immune response profile established by the parenteral vaccine. Additionally, the vaccine did not mediate enhanced control of pulmonary TB post aerosol Mtb challenge. Nevertheless, the results from a phase I clinical trial conducted to assess the safety profile of CAF01 showed no significant issues related to the administration of the CAF01 adjuvanted vaccine in healthy adults (Woodworth et al., 2019; Van Dissel et al., 2014). Glucopyranosyl Lipid Adjuvant (GLA) exhibits a TLR4 agonistic action and is a synthetic derivative if Monophosphoryl Lipid A (MPL) and is formulated using a stable oil-in-water squalene emulsion (SE) ultimately leading to production of GLA-SE. Ongoing Phase IIa clinical trials are using a combination of GLA-SE adjuvant with fusion protein ID93 to evaluate its safety, immunogenicity, and efficacy in previously BCG-vaccinated healthy healthcare workers. The 34 Phase I clinical trials showed an increased magnitude of T-cell responses and significantly higher proportion of polyfunctional CD4+ T-cells producing TNF+, IL-2+, IFN-y cytokines which lead to improved quality of the overall T-cell response. Furthermore, ID93: GLA-SE vaccination resulted in increased antibody production, predominantly that of IgG1 and IgG3 subclasses. (Coler et al., 2011; Penn-Nicholson et al., 2018; Coler et al., 2018). Polysaccharides have also been receiving increased focus as adjuvants due to their biocompatibility and biodegradability. For example, dextran is an extensively studied α-glucan that is commonly used in drug and antigen delivery and has a well-established track record of medical safety. One example of a subunit vaccine utilizing a polysaccharide adjuvant currently in clinical trials is GamTBVac whereby a fusion protein with a dextran-binding domain is combined to a novel dextran/CpG adjuvant. The vaccine showed success in Phase I trials in Russia and has shown safety and immunogenicity in recently completed Phase IIa trials in healthy BCG-vaccinated adults (Vasina et al., 2019). 1.5.4 Mtb Secretory Antigens as a potential vaccine candidate In order to develop a successful vaccine against a pathogen, it is significant to identify and include antigens that can trigger appropriate protective and suppressive immune responses. Mtb produces around 4000 antigens, which makes it challenging to determine which antigen can be utilized as a potential subunit vaccine (Stylianou et al., 2018; Schrager et al., 2019). Firstly, there remains limited knowledge about the function of these antigens; secondly, they are often expressed differentially during various stages of the pathogen's life cycle (Stylianou et al., 2018; Schrager et al., 2019).For example, while Ag85B is expressed early during the infection, ESAT-6 is always expressed (Moguche et al., 2017). Antigens implicated in the active replication of bacteria, including Ag85, ESAT6, and CFP10, are widely utilized in tuberculosis vaccination due to their demonstrated high immunogenicity and efficacy in protecting various animal models (Voss et al., 35 2018). One example of vaccine design that stimulates immune responses to antigens expressed at different stages of Mtb infection is H56. H56 is a protein fusion vaccine that is synthesized with the combination of Ag85B, ESAT-6, and Rv660c. The results from a recent preclinical trial shed light on the efficacy of H56:CAF01 and indicate that the vaccine can activate innate and adaptive immunity in mice models (Thakur et al., 2020; Marasini & Kaminskas, 2019). When developing a vaccine candidate, it is crucial to select antigens focusing on enhancing the function of CD8+ T- cell mediated responses, CD4+ T-cells and increased production of interferon-gamma producing T-cells. A study investigating the subunit vaccine M72/AS01E suggested that it effectively prevents pulmonary TB in adults who were already infected with Mtb with an efficacy of 54%. Another study comparing the six TB vaccine candidates such as MVA85A, AERAS-402, H1:IC31, M72/AS01E, ID93 + GLA-SE and BCG, showed that M72/AS01E leads to induction of high memory Th1-cytokine expressing Cd4+ T-cell memory responses. This is a very remarkable observation as CD4+ T-cells are immune correlates of protective immunity, making M72/AS01E as one of the most promising candidates (Rodo et al., 2018;White et al., 2019). 1.5.5 MPT63 As Mtb evades the host's respiratory system and establishes the infection, it secretes various proteins that drive the pathogenesis process and enable the bacteria to survive in the presence of the hostile host immune system. In addition to that, as an intracellular pathogen, the secretion of these proteins modulates crucial pathways involved in protective immunity, such as antigen presentation and recognition by the effector T-cells (Zhai et al., 2019). In animal models, Mtb antigens such as Ag85B, ESAT-6, and CFP-10 have been demonstrated to induce protective immune responses (Chugh et al., 2024). The MPT63 gene is a member of the Mtb complex, such as the virulent M. tuberculosis, M.africanum, M.bovis, and BCG. The MPT63 gene functionally 36 encodes for a protein comprising 159 amino acids, including a 29 amino acid secretion signal peptide and a 130 amino acid mature MPT63 protein (Manca et al., 1997). MPT63 has emerged as a potential vaccine candidate and a diagnostic reagent in recent years due to its specificity for the Mtb complex. Additionally, the absence of serological cross-reactivity between the MPT63 antigen and proteins from other atypical mycobacterial species, particularly M.avium, contributes to its candidacy as a vaccine (Manca et al., 1997; Duan et al., 2015). 1.5.6 The Structure of MPT63 It has been confirmed that MPT63 is a secreted protein of the Mtb complex using the Tn552 phoA in vitro transposition system (Braunstein et al., 2000). It is considered to be a protein associated with the cell envelope and is implicated in Mtb-specific virulence. The immunogenic epitopes of MPT63 have been shown to consist of the first 30 residues of the fully developed protein (Lee and Horwitz, 1999). These residues are located within the first three β-strands of the structure (β1, β2, and β3) (Figure 1.5). MPT63 has a sandwich-like structure consisting of two antiparallel β-sheets, resembling an immunoglobulin-like fold, along with an additional small, antiparallel β-sheet (β5 and β8). In contrast, the longer-stranded β-sheet is composed of four antiparallel β-strands (β2, β3, β4 and β9). The fold of MPT63 can be classified as a member of the immunoglobulin superfamily due to its structural similarity with immunoglobulin structures (Halaby and Mornon 1998). Furthermore, cell surface-binding proteins indicate that MPT63 facilitates endocytosis and phagocytosis through interactions with host cells. MPT 63 also shows similarities to eukaryotic fibronectin-binding proteins, major histocompatibility domains, and T- cell receptors (Goulding et al., 2002). Another exciting feature of the MPT63 protein is the more pronounced curvature of the smaller β-sheets compared to cellular proteins such as adaptin, arrestin, and invasin. Given that MPT63 shares structural similarities with proteins with a common 37 functional theme, we can speculate that MPT63 may play a role in host-bacterial interactions significant during bacterial internalization, such as phagocytosis and endocytosis. However, it is challenging to determine the specific functions of MPT63 due to its structural features, as it has an immunoglobulin-like fold, which is present in about 24% of the structures in the Protein Data Bank (Goulding et al., 2002). Figure 1.5 The structure of MPT63 protein. (Adapted from: Goulding et al., 2002) 1.5.7 MPT63 in Modulating Immune Responses Over years, MPT63 has been investigated as a potential vaccine candidate and as a diagnostic tool for human and bovine TB with the use of antibody assays due to its specificity to Mtb complex organisms (Mustafa, 2009). Studies aiming to understand the effect of MPT63 immunization in reducing bacterial loads in guinea pigs infected with virulent form of Mtb show that MPT63 plays an essential role in inducing humoral responses when administered through aerosol route. Furthermore, MPT63 has been demonstrated to elicit antibody production and is immunogenic in rabbits. Mustafa (2009) showed that MPT63 is vital in recognition by Th1 cell and defining the 38 pathway for antigen presentation. Furthermore, MPT63 induces moderate Th1 cell reactivity as its epitope binds promiscuously to HLA-DR, which is a MHC class II cell surface receptor (Kim et al., 2021).

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