Chapter 4-Prophylactic Immunization.docx

**Chapter 4: Examining the efficacy of MPT63 derived lipopeptides in enhancing immune responses and reducing bacterial burden in H37Ra infected mice when administered prophylactically** **4.1 Introduction** ** Ever since the discovery of *Mycobacterium tuberculosis* (*Mtb*) as the causative agent...

**Chapter 4: Examining the efficacy of MPT63 derived lipopeptides in enhancing immune responses and reducing bacterial burden in H37Ra infected mice when administered prophylactically** **4.1 Introduction** ** Ever since the discovery of *Mycobacterium tuberculosis* (*Mtb*) as the causative agent of tuberculosis (TB) disease by Robert Koch in 1882, research aimed at developing vaccines and therapeutics has accelerated (**Cambau and Drancourt, 2014)**. However, the efficacy of these strategies soon became a matter of concern as the cases seemed to rise regardless of the widely used Bacillus Calmette--Guérin (BCG) vaccine and first and second-line anti-TB drugs. This is evident by the Global Tuberculosis Report published by the World Health Organization (WHO), which identified TB as a significant contributor to deaths associated with antimicrobial resistance. The rise in TB cases worldwide is due to both inefficiencies of the current treatment regimens as well as socio-economic factors such as living conditions and limited access to healthcare (**World Health Organization, 2023) **       As an individual inhales the *Mtb* particles suspended as aerosol droplets, the *Mtb* infection is established by interacting with the host mucosal tissue of the respiratory tract. The respiratory mucosa serves two functions: one as a physical barrier against the invasion of pathogens and the second as a first line of defense for the host by initiating mucosal immune responses (Li et al., 2012). The role of mucosal immunity in conferring protection against mycobacterial infection is evident by various studies suggesting its essential role in stimulating a cascade of events such as enhanced recognition of pathogen-associated molecular patterns (PAMPs) by immune cells such as macrophages, dendritic cells, and leukocytes and subsequent activation of antimycobacterial immune responses such as activation of specific T-cell and antibody synthesis (Li et al., 2012). However, the currently available vaccine, BCG, is administered through an intradermal route. It poses a significant challenge to our fight against TB as it induces inefficient CD4+ and CD8+ T cell-based immune responses (Qu et al., 2021). Therefore, we need to look for alternative vaccination routes, such as the intranasal route of administration, that stimulate the production of effective mucosal immune responses by interacting with the mucous membranes and nasal passages.** **    While one area of tuberculosis vaccination research focuses on improving the efficacy of the BCG vaccine, the other branch aims to explore innovative vaccine designs and formulations such as live attenuated, inactivated, protein subunit, recombinant, and adjuvanted (Whitlow et al., 2020). Our research focuses on investigating the role of MPT63 derived lipopeptides as a potential prophylactic and therapeutic subunit vaccine to enhance host immunity and eradicate *Mtb* infection. MPT63 is among the most highly abundant proteins found in the culture filtrate of *M. tuberculosis* \[MT-CF\] as demonstrated by protein fractionation methods. MPT63 has a molecular mass of 18 kDa and is essential in *Mtb* virulence (Mustafa, 2009). Furthermore, the investigation of the DNA region encoding for the MPT63 protein through nucleotide sequence analysis revealed that it possesses an open reading frame (ORF) that encodes for 159 amino acids (aa), which comprises a 130-aa mature MPT63 protein and 29-aa secretion signal peptide. In addition, T-cell epitope mapping illustrates that MPT63 has a highly immunodominant region within the first 30 residues of the amino-terminal of the mature protein (Mustafa, 2009). According to the immunological studies conducted to characterize the function of MPT63, it has been shown to induce robust humoral immune responses in various animal species. MPT63-derived peptides have also been shown to generate protective T helper 1 (Th1) cellular immune responses in peripheral blood mononuclear cells (PBMCs) of healthy individuals vaccinated with BCG. Furthermore, MPT63 promiscuously binds to Human Leukocyte Antigen-DR isotype (HLA-DR), which is an MHC class II cell surface receptor and functions to present peptide fragments derived from pathogens to T-helper cells to trigger an adaptive immune response. In addition, the Th1 cell epitopes are dispersed throughout the protein sequence, making MPT63 a potential vaccine candidate against TB (Mustafa, 2009).** **The effective stimulation of adaptive immune responses is essential to generate protective immunity against *Mtb* and to control bacterial replication. The adaptive immune response against *Mtb* is characterized by cytokine secretion and direct antimicrobial actions of antigen-specific T cells. In addition, the enduring quality of memory T cells specific to antigens forms the groundwork for creating vaccines that stimulate antimycobacterial defense (Sia and Rengarajan, 2019). During both latent and active forms of *Mtb* infection, CD4+ T cells play an essential role in controlling the infection via secretion of cytokines such as IFN-γ, TNF-α, and IL-2macrophage activation through dendritic cell-T cell axis and aiding in the development of cytotoxic CD8+ T cell population (Sia and Rengarajan, 2019). On the other hand, CD8+ T cells also produce vital cytokines such as IFN-γ, TNF-α, and IL-2. However, in addition to that, they also perform cytolytic activities such as the release of perforin, granzymes, and induction of apoptosis (Sia & Rengarajan, 2019).** **       Therefore, due to the immunogenic properties previously demonstrated by the MPT63 peptides, we custom-synthesized lipopeptides spanning the full-length sequence of the MPT63 protein for this study. In this study, we aimed to examine (i) the role of lipopeptides in inducing effective CD4+ and CD8+ T cell responses in the absence of active TB infection and (ii) the protective efficacy of MPT63 lipopeptides in stimulating effective T cell responses while reducing bacterial loads in an active TB BALB/c mouse model infected with H37Ra strain of *Mtb*. The lipopeptides spanning the full-length sequence of MPT63 protein were divided into Pool 1 and Pool 2 based on the shared overlapping sequences.** **We intentionally grouped the lipopeptides to help us study the antimycobacterial, effector/immunosuppressive, and regulatory properties of the MPT63 lipopeptides. We aimed to identify any differences in immune responses elicited by Pool 1 and Pool 2 lipopeptides and reductions in bacterial burden in the lungs, spleen, and liver. For the lipopeptide design, the peptides used by Mustafa (2009) were conjugated with a lysine-palmitoyl-glycine chain at the carboxy terminus, which conferred enhanced immunogenicity to our lipopeptide construct.** **In this study, we demonstrated that Pool 2 immunizations lead to enhanced splenocyte proliferation and increased production of effector and antimicrobial CD4+ and CD8+ T cell populations compared to Pool 1 lipopeptides, which stimulated more robust production of T cell populations secreting immunosuppressive/regulatory cytokines. This phenomenon was further highlighted by the infection experiments, which showed similar differences between the Pool 1 and Pool 2 immunization groups in terms of eliciting T cell responses and a trend towards reduced bacterial loads in the Pool 2 immunized group in lungs, spleen and liver of BALB/c immunized mice.** **4.2 Results** **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** Before testing the efficacy of MPT63 lipopeptides in counteracting the *Mtb* infection, we wanted to examine if the lipopeptides can induce robust antigen-specific splenocyte proliferation within a non-active TB infection context. Splenocyte proliferation and increased percentages of CD4+ and CD8+ T cells in the spleen indicate more robust immune responses against *Mtb.* Splenocyte proliferation is often associated with and is used as a hallmark of immune activation during vaccination experiments (Ning et al., 2021). To determine that the MPT63-derived synthetic lipopeptides lead to enhanced splenocyte proliferation, we divided the lipopeptide overlapping sequences into Pool 1 and Pool 2 groups. We immunized the control group with Phosphate-buffered saline (PBS). The immunizations were carried out through an intranasal route to stimulate mucosal and systemic immune responses. The antigen-specific splenocyte proliferation was measured using the amount of BrdU incorporation. The splenocytes obtained from Pool 1 and 2 immunized groups were stimulated ex vivo with the MPT63 lipopeptides. The ex vivo stimulation of Pool 1 and Pool 2 spleen cells with Pool 1 lipopeptides resulted in significantly higher antigen-specific splenocyte proliferation compared to the PBS immunized group (**Figure 4.1**). The *ex vivo* stimulation of Pool 1 and Pool 2 spleen cells with Pool 2 lipopeptides resulted in significantly higher antigen-specific T-cell proliferation compared to the PBS immunized group **(Figure 4.1).** The data was statistically analyzed using two-way ANOVA, followed by Tukey's multiple comparison test**. Note: The steps for the splenocyte proliferation assay have been described in Chapter 3: Materials and Methods.**   **Figure 4.1:** **Splenocyte proliferation upon restimulation with MPT63 lipopeptides.** The antigen specific proliferation of splenocytes was measured by incorporation of BrDu and *ex vivo* stimulation of **(A)** control (PBS-immunized), Pool 1 and Pool 2 immunized groups with Pool 1 lipopeptides and **(B)** control (PBS-immunized), Pool 1 and Pool 2 immunized groups with Pool 2 lipopeptides. The data is represented as mean ±SEM values from triplicate wells and statistically analyzed using two-way ANOVA which defined the significance as (i) Control (PBS-immunized) vs Pool 1 immunized \*p≤ 0.05 (ii) control (PBS-immunized) vs Pool 2 immunized \*p≤ 0.05 (iii) Pool 1 Vs Pool 2 immunized ^σ^p≤ 0.05 as shown on the bar graphs. In comparison to unimmunized group, both Pool 1 and Pool 2 immunizations resulted in significantly higher splenocyte proliferation when stimulated with Pool 1 lipopeptides. Similarly, when stimulated with Pool 2 lipopeptides, significantly greater splenocyte proliferation was observed for Pool 1 and Pool 2 immunized groups compared to the control. **4.2.2 The immunization with Pool 1 and Pool 2 lipopeptides lead to differential upregulation of effector/antimicrobial and regulatory T cell subsets** Along with splenocyte proliferation, we aimed to characterize if immunization with Pool 1 and Pool 2 lipopeptides of MPT63 protein resulted in upregulation or downregulation of antimicrobial cytokines such as TNF-α, IFN-γ and IL-17A, which play an essential role in controlling the *Mtb* infection or regulatory cytokines such as IL-10 and LAP which contribute to impairment of the T cell function (Domingo-Gonzalez et al., 2017). The specific gating strategies and categorization of essential cytokines into their respective functional groups examined for flow cytometry analysis have been described in **Chapter 3: Materials and Methods**. The flow cytometric analysis was specifically performed to determine the multifunctional characteristics of Cytotoxic T cells (CTLs) and helper T cells (TH) upon immunizations with MPT63-derived lipopeptides and the control group immunized with PBS. The tsNE plots described in **(Figure 4.2 and Figure 4.4)** allowed us to visualize the differences in activated cell clusters among the experimental and control groups. The corresponding heatmaps provided the information on which illuminated cluster of the tsNE plot was positive for a particular cytokine. Using the information gathered from the tsNE plots, the generated heatmaps, and phonographs, we developed bar graphs that represented the functional groups as percentage values **(Table provided in Chapter 3: Materials and Methods)**. The functional groups, such as effector/antimicrobial, pleiotropic, and regulatory, allowed us to gain insight into the cytokine expression profiles resulting from immunizations.       The analysis revealed that in spleen, intranasal immunization with Pool 2 lipopeptides led to heightened production of effector CTLs with a 98% increase compared to control (70%) and Pool 1(26%) groups. It was surprising to observe that immunization with Pool 1 lipopeptides on the other hand, lead to an increase in production of regulatory CTLs (69.9%) compared to control. (17.68%) and Pool 2 (1.62%). In terms of TH population, there was an increased production of antimicrobial TH cells (99%) in Pool 2 immunized group compared to control and Pool 1 groups (79% and 75% respectively). Pool 1 immunization resulted in a slightly higher percentage of regulatory TH cells (24%) compared to the control (20%) however the percentage of regulatory TH cells for Pool 2 immunized group was the lowest at 0.8% **(Figure 4.3).** The flow cytometric analysis of the lung samples from the immunized and control groups revealed that Pool 2 immunizations induced increased percentage of effector CTLs (82%) compared to Pool 1 (31%) and control group (59%). Contrastingly, Pool 1 immunizations gave rise to higher percentage of regulatory CTL cell populations (43%), with a slightly lower percentage in control (22%) and the lowest percentage for Pool 2 immunized group (4.7%). Furthermore, Pool 2 immunizations lead to higher production of antimicrobial TH cell populations (94%) in comparison with control (84%) and Pool 1 groups (58%). Pool 1 immunizations also induced increased production of regulatory TH cell population (42%) compared to control (16%) and Pool 2 (6%) immunized groups **(Figure 4.6).**   **Figure 4.2: The pseudo color tSNE plots, generated using FlowJo software, depict the distribution of cytotoxic T cells from spleens of the** **(A)** Control (PBS-immunized), **(B)** Pool 1 immunized group, and **(C)** Pool 2 immunized group, as well as helper T cells from spleens of the **(D)** Control (PBS-immunized) group, **(E)** Pool 1 immunized group, and **(F)** Pool 2 immunized group. These plots were utilized to identify differences in the multifunctionality of cytotoxic and helper T cells among the immunized groups. Additionally, heatmaps **(G)** and **(H)** provide detailed information on the expression levels of pro-inflammatory/effector/antimicrobial cytokines (IL-17A, TNF-α, IFN-γ) and anti-inflammatory/regulatory cytokines (IL-10 and LAP). Heatmap **(G)** represents the expression profiles for cytotoxic T cells, while heatmap **(H)** illustrates the expression profiles for helper T cells in the splenocytes of MPT63 lipopeptide-immunized and PBS-immunized groups **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. The percentage values represent changes in the effector, pleiotropic, and regulatory populations of cytotoxic T cells following immunization with MPT63-derived lipopeptides. The expression levels of various TB-associated cytokines are also shown, as described in the legend. Notably, Pool 1 immunization leads to an increased regulatory CTL population, whereas Pool 2 immunization results in enhanced effector CTL production. These differential expression patterns highlight the distinct immunological responses induced by the two different lipopeptide pools.**   **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. These profiles were analyzed to assess whether immunization with either Pool of MPT63-derived lipopeptides enhances key antimicrobial immune responses, which are crucial for controlling active TB infection. Each colored bar represents the frequency of specific cell clusters, categorized into functional groups. The detailed expression profiles of these groups are provided in the legend.**    **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, analyzed across three groups: (A) Control (PBS-immunized) , (B) Pool 1-immunized, and (C) Pool 2-immunized. Additionally, cytokine expression levels in helper T cells from lung cells were examined in the (D) Control (PBS-immunized), (E) Pool 1-immunized, and (F) Pool 2-immunized groups. The pseudo-color density plots, generated using FlowJo software, highlight the differences in cytokine expression patterns between the lipopeptide-immunized and PBS-immunized groups. Red illuminated areas indicate clusters of cells/populations with significantly higher presence, enabling a clear comparison of immune responses induced by the different immunizations. Heatmap (G) represents the cytokine expression profiles for cytotoxic T cells in the lungs, while heatmap (H) depicts the cytokine expression profiles for helper T cells in the lungs of PBS-immunized and MPT63 lipopeptide-immunized groups.** **Figure 4.6: Using data derived from heatmaps, violin box plots, and pseudo-color tSNE plot analysis, the lipopeptide-stimulated expression of key TB-associated cytokines was represented in bar graphs for lung cells obtained from (A) Control (PBS-immunized), (B) Pool 1-immunized, and (C) Pool 2-immunized groups. Each colored bar indicates the frequency of distinct cell clusters, categorized into functional groups. The detailed expression profiles for these groups are outlined in the legend.** **Figure 4.7: This figure displays the expression profiles of antimicrobial and regulatory T helper cells from lung cells across three groups: (A) Control (PBS-immunized), (B) Pool 1-immunized, and (C) Pool 2-immunized. The analysis aimed to evaluate whether immunization with MPT63-derived lipopeptide pools enhances critical antimicrobial immune responses necessary for controlling active TB infection within the lungs. The colored bars indicate the frequency of specific cell clusters, which are grouped according to their function. A detailed breakdown of these expression profiles is provided in the legend.** **4.2.3 The splenocyte proliferation induced by MPT63 derived synthetic lipopeptides in H37Ra infected mice shows no significant difference** Following immunization experiments, we investigated whether MPT63 lipopeptides could boost host immune responses and splenocyte proliferation in an active tuberculosis infection induced in BALB/c mice. These infection experiments provided insights into the efficacy of MPT63-derived lipopeptides in reducing bacterial loads and enhancing cytotoxic and helper T-cell responses, which are crucial for eliminating the infection. The immunization and infection schedule for this experiment can be found in **Chapter 3: Materials and Methods.**    Splenocytes were collected from three groups: (i) PBS-immunized and infected, (ii) Pool 1-immunized and infected, and (iii) Pool 2-immunized and infected. These splenocytes were subjected to ex vivo stimulation with Pool 1 and Pool 2 lipopeptides at varying concentrations (1 µg/ml, 0.1 µg/ml, and 0.01 µg/ml). The incorporation of BrdU into the DNA of actively dividing cells was measured using an ELISA reader and reported as optical density (OD) values.     Statistical analysis of splenocyte proliferation using two-way ANOVA revealed that *ex vivo* stimulation with Pool 1 and Pool 2 lipopeptides did not significantly increase antigen-specific T cell proliferation compared to the PBS-immunized group. Additionally, no significant differences in T cell proliferation were observed among the PBS, Pool 1, and Pool 2 groups when stimulated with Pool 2 lipopeptides (**Figure 4.8**).  **Figure 4.8: The antigen-specific proliferation of splenocytes was evaluated by measuring BrdU incorporation following ex-vivo stimulation with lipopeptides (LPs). Splenocytes from three groups---Control (PBS-immunized), Pool 1 immunized, and Pool 2 immunized---were stimulated with (A) Pool 1 LPs and (B) Pool 2 LPs. The results are presented as mean ± SEM values from triplicate wells, and statistical significance was assessed using two-way ANOVA followed by Tukey's multiple comparisons test. The analysis determined significance levels as follows: (i) Control (PBS-immunized) vs. Pool 1 immunized (\*p ≤ 0.05), (ii) unimmunized vs. Pool 2 immunized (\*p ≤ 0.05), and (iii) Pool 1 vs. Pool 2 immunized (σp ≤ 0.05), as indicated on the bar graphs. In the context of an infection model, immunizations with MPT63 lipopeptides did not lead to significant splenocyte proliferation when *ex vivo* stimulation was performed with either Pool 1 or Pool 2 MPT63 lipopeptides.** **4.2.4 MPT63 lipopeptide immunizations lead to differential T cell and cytokine expression profiles in an active TB infection model** **In our previous experiments, we explored the expression profiles and regulation of cytokines, cytotoxic T cells (CTLs), and helper T cells induced by MPT63 immunizations in the absence of active TB infection. This study aims to further investigate the impact of MPT63 lipopeptide immunization on T cell-mediated immune responses during active TB infection in BALB/c mice.** **Flow cytometry was used to analyze T cell populations in the lungs and spleens of infected mice, as detailed in Chapter 3: Materials and Methods. The results, reported as percentage values, provide insights into how different immunization pools affect T cell subsets during active TB infection.** **Immunization with Pool 1 led to an increased proportion of pleiotropic CTLs in both the spleen (48%) and lungs (48%) compared to the control group (40% in both cases) and Pool 2 (20% in the spleen; not specified for the lungs). On the other hand, Pool 2 immunization resulted in upregulation of the effector CTL population, which was higher in the spleen (67%) and lungs (67%) compared to the control (43% in both cases) and Pool 1 (43% in both cases).** **Regarding regulatory helper T cells, Pool 1 immunization resulted in a slight increase (22%) compared to the control (24%), while Pool 2 produced a more pronounced elevation (40%). For antimicrobial helper T cells, Pool 1 immunization showed the highest proportion (78%) in the spleen, exceeding the control (76%), whereas Pool 2 immunization resulted in the lowest proportion (60%). In the lungs, all immunization groups exhibited elevated levels of antimicrobial** **helper T cells, with Pool 1 (95%) and Pool 2 (97%) slightly higher than the control (93%).** **Figure 4.9: Using data from heatmaps, violin box plots, and pseudo-color tSNE plot analyses, we represented the expression of key TB-associated cytokines stimulated by lipopeptides in bar graphs. These graphs depict the cytokine expression profiles for splenocytes obtained from three different groups: (A) Control (PBS-immunized), (B) Pool 1-immunized, and (C) Pool 2-immunized. Each bar, color-coded to denote distinct cell clusters, reflects the frequency of these clusters categorized into functional groups. Detailed expression profiles for each group are provided in the legend.** **Figure 4.10: We analyzed the expression profiles of antimicrobial and regulatory T helper cells from splenocytes in three different groups: (A) Control (PBS-immunized), (B) Pool 1-immunized, and (C) Pool 2-immunized. This analysis aimed to determine whether immunization with MPT63-derived lipopeptide pool enhances key antimicrobial immune responses essential for managing active TB infection. Each colored bar in the graphs represents the frequency of specific cell clusters, which are categorized into functional groups. Detailed expression profiles for these groups are provided in the legend.**   **Figure 4.11: The expression profiles of cytotoxic T cells from splenocytes were analyzed in three groups of BALB/c mice: (A) Control (PBS-immunized) , (B) Pool 1 lipopeptides, and (C) immunized with Pool 2 lipopeptides. The percentages reflect the changes observed in the effector, pleiotropic, and regulatory populations of cytotoxic T cells following immunization with MPT63-derived lipopeptides. The expression levels of various TB-associated cytokines are also provided, as detailed in the legend. Notably, Pool 1 immunization results in an increased regulatory CTL population, whereas Pool 2 immunization enhances effector CTL production. These differential expression patterns underscore the distinct immunological responses elicited by the two lipopeptide pools.** **Figure 4.12: We examined 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. The goal of this analysis was to assess whether immunization with MPT63-derived lipopeptide pools could boost critical antimicrobial immune responses necessary for controlling active TB infection. In the graphs, each colored bar indicates the frequency of distinct cell clusters, which are grouped based on their functional roles. A detailed breakdown of these expression profiles is available in the legend.** **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** **Analyzing bacterial loads is a crucial component in evaluating the efficacy of a vaccine for tuberculosis. Bacterial load serves as a direct indicator of infection severity and provides insight into the immune system\'s capacity to control and eliminate the pathogen following vaccination (Rodriguez et al., 2018). Therefore, assessing bacterial loads in infection experiments is essential for understanding the effectiveness of MPT63 lipopeptides in reducing *Mycobacterium tuberculosis* (*Mtb*) burden within the host, specifically in BALB/c mice for our experiments. Given that the progressive form of the disease leads to *Mtb* dissemination to organs such as the spleen, lungs, and liver, evaluating bacterial load in these organs can reveal whether the vaccine is effective in controlling disseminated and extrapulmonary form of TB infections.** **We conducted a colony-forming unit (CFU) assay using spleen, liver, and lung samples from three groups: control (PBS-immunized), Pool 1 lipopeptide-immunized, and Pool 2 lipopeptide-immunized. The data was statistically analyzed using two-way ANOVA, which revealed no significant differences in bacterial load reduction among the spleen, lungs, and liver across the control and experimental groups. However, we noticed a trend in reduction of bacterial burden in lungs, spleen and liver of Pool 2 immunized mice.**   **Figure 4.13:** **For the CFU assay, the harvested spleen, lungs, and liver were homogenized and serially diluted to concentrations of 10¹ and 10². A volume of 100 µl from each dilution was plated and incubated for three weeks until colonies appeared. (B-D) In the spleen, lungs, and liver, we observed a relatively lower formation of H37Ra colonies in the group immunized with Pool 2 lipopeptides compared to both the control and Pool 1 immunized groups. The results were analyzed using two-way ANOVA and, although not statistically significant (p ≥ 0.05), they suggest a potential effectiveness of Pool 2 lipopeptides.** **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** **Histological study of the hematoxylin and eosin slides of the lung tissue obtained from the control (PBS-immunized), Pool 1 lipopeptide immunized and Pool 2 lipopeptide immunized groups allowed us to qualitatively determine disease markers such as lung lesion and immune cell infiltration. The slides were examined under 5X and 40x magnification. The immune cell infiltration was similar amongst the three groups while control group exhibited exacerbated damage to the lung tissue with multiple lesions.**   **Figure 4.13: The figure represents the lung lesions and immune cell infiltration in the lungs of control (PBS-immunized), Pool 1 LP immunized, and Pool 2 LP immunized groups examined at 5X and 40X magnifications. The arrows indicate possible sites of cellular infiltration (macrophages, neutrophils and lymphocytes).** **4.3 Discussion** **TB is described as a bacterial infection caused by *Mycobacterium tuberculosis* (Mtb), a member of the *Mtb* complex, which has manifested and caused suffering amongst individuals around the globe. Provided the challenges posed by the current treatment and vaccine strategies there is an urgent need for the development of a novel vaccine approach with enhanced immunogenicity as well as increased safety profile. One such approach involves incorporating proteins/antigens secreted by *Mtb* during pathogenesis in the vaccine design and such vaccine is called a protein subunit vaccine. More specifically, lipopeptides derived from the *Mtb* proteins have been shown to be efficacious at stimulating appropriate adaptive immune responses while exhibiting self-adjuvating properties (Hamley, 2021). This vaccine design entails the attachment of lipid components to peptide sequences of the protein/antigen in question which facilitates the enhancement of the immunogenicity of the vaccine due to effective antigen presentation and T cell activation (Hamley, 2021).** **    Over the years, the immunological studies carried out to study various secretory antigens of the *Mtb* complex have identified several antigens which are produced at various stages of the *Mtb* pathogenesis and have been demonstrated to be immunogenic in nature. Examples of such antigens include the early secreted antigenic target 6 (ESAT-6) which has been demonstrated to stimulate both CD4+ and CD8+ T cell responses contributing to protective immunity against TB (Pal et al., 2022). The culture filtrate protein 10 (CFP-10), when used alongside ESAT-6, has been shown to enhance the specificity of immune responses in TB diagnostics and in eliciting CD4+ T cell responses (Pal et al., 2022). Amongst several identified proteins, MPT63 is one such emerging candidate. Previous studies have shown that the MPT63 antigen is effective for diagnosing TB. It induces immune responses in rabbits and guinea pigs, as well as Th1 responses in PBMCs of BCG immunized individuals (Kim et al., 2021). However, the role of MPT63 as a potential prophylactic vaccine is not well understood. To fill that gap in our knowledge and reveal the immunogenic properties of MPT63, we conducted several immunization experiments. The primary objective of this study was to evaluate the efficacy of MPT63-derived synthetic lipopeptides as a potential prophylactic vaccine for tuberculosis. Specifically, we aimed to assess the ability of the lipopeptides in enhancing the adaptive immune responses, as evidenced by splenocyte proliferation, upregulation of CD4+ and CD8+ responses, cytokine expression profiles in spleen and lung cells, and their impact on bacterial load in infected tissues. We achieved these objectives by first conducting intranasal immunization experiments in the absence of an active TB disease model to gain insight into the role of the MPT63 lipopeptides in enhancing T cell-based immune responses. This was followed by investigating the impact of immunization in the context of an active TB disease model by using BALB/c mice as the model organism.** **   The splenocyte proliferation experiment and analysis allowed us to gain valuable insights into the effectiveness of MPT63-derived lipopeptides in augmenting host immune responses. The importance of studying splenocyte proliferation is that splenocytes comprise various immune cells, such as T cells, B cells, and macrophages, which play a vital role in orchestrating the body's defense against *Mtb* (Fan et al., 2006*)*. More specifically, antigen-specific splenocyte proliferation provides information on the vaccine's effectiveness. Our results demonstrated that in the absence of infection, prophylactic immunization with MPT63-derived lipopeptides resulted in significantly robust antigen-specific proliferation in groups immunized with Pool 1 and Pool 2 lipopeptides. In both cases, splenocytes from mice immunized with Pool 1 and 2 were restimulated with Pool 1 and Pool 2 lipopeptides. Previous studies exploring the role of lipopeptides in stimulating cellular immune responses can help us understand the potential mechanisms behind the significant splenocyte proliferation observed after administering the lipopeptide-based vaccine. Studies have shown that lipopeptide-based vaccines exhibit self-adjuvating properties and can mediate signaling via Toll-like receptor 2 (TLR2). Therefore, the MPT63-derived lipopeptides can be recognized by TLR2 on the dendritic cells and other immune cells. The recognition of the lipopeptides by these receptors leads to the activation and maturation of dendritic cells (Apte et al., 2012; Wu et al., 2010; Hamley, 2021). The activated dendritic cells can also enhance T cell proliferation, specifically CD4+ and CD8+ T cells, essential for generating effective immune responses against *Mtb* infection. Moreover, the lipopeptide structure of the MPT63 antigen-based vaccine appears to facilitate antigen presentation by major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (APCs). This improved antigen presentation likely promotes the activation and clonal expansion of naïve T cells, which then differentiate into effector cells capable of combating *Mtb*. (Patel & Agrawal, 2023; Hamley, 2021). The improved antigen presentation modulates the activation of naïve T cells, leading to proliferation and differentiation into effector cells capable of combating *Mtb*. Furthermore, the proliferation of the splenocytes in response to MPT63 lipopeptides leads to the formation of memory T cells. The antigen-induced memory cells' elevation and production are essential for long-term immunity and rapid responses upon re-exposure to *Mtb* (Patel & Agrawal, 2023). However, to our surprise, in the presence of an active infection, there was no significant splenocyte proliferation observed between the control, Pool 1, and Pool 2 groups. A plausible explanation for this could be that in an infection model, upon entry into the host, the presence of *Mtb* induces a cascade of innate and adaptive immune responses, leading to clonal expansion of T and B cells and an overall enhancement of the immune response (de Martino et al., 2019). There is a possibility that the dosage of the lipopeptides being administered during the infection experiments is insufficient to induce presentation by MHC molecules and exceed the splenocyte proliferation induced in the control (PBS-immunized) group.** **To fully understand the role of MPT63 lipopeptides in inducing effective adaptive immune responses, we needed to investigate the upregulation/downregulation of the various T cell subsets, such as helper T cells and cytotoxic T cells. The goal of the flow cytometry analysis was to identify the populations of helper T cells and cytotoxic T cells using heatmaps and tsNE plots. These representations revealed specific clusters indicating the upregulation of cytokines (Patel & Agrawal, 2023). Our results revealed that in the absence of an infection, immunization with Pool 1 lipopeptides leads to upregulation of regulatory cytotoxic and helper T cell populations in both lungs and spleens. On the other hand, Pool 2 lipopeptides lead to more robust production of effector cytotoxic and helper T cell populations in both lungs and spleen. This differential activation of T cell subsets influenced by MPT63 immunizations sheds light on the distinct properties of the amino acid (AA), which comprise the Pool 1 and Pool 2 lipopeptides sequence. The generation of regulatory T cell (Tregs) populations with Pool 1 immunization is marked by the production of cytokines such as latency-associate peptide (LAP) which is a component of the inactive form of TGF-β and Interleukin-10 (IL-10). Since LAP is a part of the complex that forms the inactive precursor of TGF-β, including LAP in the panel design provided insights into the impact of MPT63 lipopeptide immunization on TGF-β modulation (Rodriguez et al., 2018).Previous studies have shown that TGF-B plays a role in inhibiting the activity and function of Th1 cells, which produces IFN-γ and further activates macrophages vital to killing intracellular pathogens like *Mtb*. Furthermore, TGF-β promotes the differentiation of Tregs, preventing excessive immune responses and suppressing the immune response against *Mtb* infection (Rodriguez et al., 2018). Tregs also produce IL-10, further limiting potentially pathogenic immune responses. Studies have shown that an increased production of Tregs expressing IL-10 has been associated with exacerbated bacterial burden and more severe TB in an Indian population. Furthermore, IL-10 can inhibit phagosome maturation and macrophage activation within the macrophages in a STAT3-dependent manner, allowing *Mtb* to replicate and survive within the phagosome. In essence, Pool 1 immunization tends to favor immune mechanisms, which favored bacterial growth more than controlling the infection (Rodriguez et al., 2018).** **On the contrary, the increased production of effector and antimicrobial T cell subsets by Pool 2 immunizations indicates the involvement of mechanisms that induce the production of cytokines such as TNF-α, IFN-γ, and IL-17A, which function to lower bacterial burden by inhibiting further bacterial replication within the host. TNF-α is a central proinflammatory mediator and plays essential functions such as proliferation and differentiation of immune cells along with multiple inflammatory processes such as migration and optimal macrophage activation. Studies have also revealed that neutralizing TNF-α following BCG infection leads to losing granulomas, the hallmarks of the *Mtb* disease (Rodriguez et al., 2018). IFN-γ is essential for the host's survival post *Mtb* infection and is often expressed by antigen-specific T cells. IFN-γ is produced innately by phagocytes, which are stimulated via their pattern recognition receptors and contribute to early proinflammatory responses to infection. The role of IL-17A in the context of an *Mtb* infection has been established as a down regulator of IL-10 production and enhancer of IL-12 production, which leads to enhanced production of IFN-γ. Additionally, IFN-γ is important for the induction and maintenance of chemokine gradients for T cell migration (Rodriguez et al., 2018). Overall, the Pool 2 immunizations lead to induction of immune responses which aid in sequestering and controlling the bacterial infection. In the presence of an active infection, we saw a similar pattern in upregulation of effector function induced by Pool 2 immunizations in the spleen and lungs of the H37Ra infected mice. However, to our surprise, Pool 1 immunizations also lead to increase in production of antimicrobial helper T cell populations in lungs and spleens of infected and immunized mice. Since the mechanisms underlying infection and immune responses during an active infection are so much more complex than an immunization experiment, it will be important to repeat the experiments to truly confirm if the differences between the Pool 1 and Pool 2 sequences is as apparent as highlighted by the immunization experiments in the absence of an infection.** **The flow cytometry results obtained from our infection experiments aid in explaining the trends observed for bacterial burden in MPT63 lipopeptide immunized and PBS immunized control. The results reveal that immunization with Pool 1 lipopeptides leads to relatively higher bacterial loads in the lungs, liver and spleen compared to Pool 2 lipopeptide immunized groups. The flow cytometry results can rationalize these trends in reduced bacterial burden in Pool 2 immunized group, which revealed an upregulation of effector and antimicrobial helper and cytotoxic T-cell populations within immunized groups. The underlying mechanism behind this observation can be explained by previous studies that have investigated the role of other antigens, such as ESAT-6, in immunomodulation upon administration. Essentially, upon interaction with the MPT63 lipopeptides, the activation of TLRs can lead to activation of downstream signaling pathways. For instance, in MyD88-dependent pathways, the activation of TLR can lead to the production of pro-inflammatory cytokines and chemokines, immune cell recruitment, and upregulation of co-stimulatory molecules, which have a combined effect of facilitating antigen presentation to T cells as well as promoting adaptive immune responses against *Mtb* (Passos et al., 2024). In addition to that, it has been shown that vaccination with *Mtb* antigens such as ESAT-6 can activate CD4+ and CD8+ T-cells, which further produce IFN-γ and TNF-α which are essential cytokines that control bacterial dissemination, promote granuloma formation and reduce replication (Passos et al., 2024).We observed the same when it came to Pool 2 lipopeptide immunizations, which implies that MPT63 uses similar pathways to lower the bacterial burden. However, we also observed that Pool 1 immunizations lead to heightened production of antimicrobial helper T cell populations, while the bacterial loads remained high in the lungs, spleen, and liver. It is unclear if clear distinctions exist between Pool 1 and Pool 2 in stimulating and activating specific populations or subsets of T cells. Further investigation is necessary to clarify this distinction.** **      The histological analysis of the H&E slides of lung tissues obtained from the control and experimental groups revealed that control mice experience extensive lung damage and the presence of lesions in the absence of the lipopeptides. On the other hand, Pool 1 and Pool 2 MPT63 immunizations led to a decrease in lung lesions. The amount of immune cellular infiltration remained the same amongst the three groups. Studies have shown that the rapid influx of immune cells, such as macrophages and neutrophils, to the site of the active infection mediates the cessation of the infection and formation of a granuloma, which contributes to controlling the infection (Moreira-Teixeira et al., 2020). Furthermore, TB-induced lung damage clinically presents as cavitation, fibrosis, nodular infiltrates, or a mix of pulmonary pathologies (Ravimohan et al., 2018). Studying lung lesions and immune cell infiltration allows us to understand better potential vaccine candidates' role in reducing TB-associated pathologies.** **We conclude from the prophylactic immunization experiments that Pool 1 lipopeptides are associated with the induction of regulatory T cells and anti-inflammatory cytokines, which favor bacterial growth, and this is evident by the colony-forming units assay. However, it remains indefinite why there was also the production of antimicrobial helper T cell populations in the lungs and spleen of Pool 1 immunized and infected mice. On the contrary, Pool 2 lipopeptide immunizations showed promising results regarding splenocyte proliferation, production of effector and antimicrobial T cell populations, and a trend towards reduction in bacterial loads in lungs, spleen, and liver.** **References** Apte, S. H., Groves, P. L., Skwarczynski, M., Fujita, Y., Chang, C., Toth, I., & Doolan, D. L. (2012). 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