Description:FIELD OF THE INVENTION
The present invention in general relates to immunology and infection biology. Particularly, the present invention provides antigenic proteins and compositions for infectious diseases. More particularly, the present invention provides an antigenic protein subunit vaccine composition against Mycobacterium tuberculosis. The present invention also provides a process for the preparation of antigenic protein variant, a key component of the vaccine composition.

BACKGROUND OF THE INVENTION
Tuberculosis (TB) remains one of the leading causes of morbidity and mortality worldwide, with Mycobacterium tuberculosis (Mtb) infecting approximately one-quarter of the global population. The Bacillus Calmette-Guérin (BCG) vaccine, the only licensed TB vaccine, provides limited protection in the case of infants and fails to prevent latent infections or reactivation in adults. Apart from BCG, there is no approved vaccine against TB till now. However, several potential TB vaccine candidates are at various clinical trial stages. Out of which according to WHO, (M72/AS01E) was shown to be strongly protective against TB disease in people with signs of latent TB infection in a Phase IIb trial that was carried out in Kenya, South Africa, and Zambia. During almost three years of follow-up, the point estimate of vaccination effectiveness was 50%. M72:AS01E is a recombinant fusion protein (m72) of Rv1196 (PPE protein) and Rv0125 (Serine proteinase) in AS01E adjuvant, a liposomal formulation of Toll-like receptor (TLR)-4 ligand (MPL-A) and QS21. Significant protection can be obtained, according to the findings of the M72:AS01E trial in humans and a recent investigation of lung BCG immunization in NHPs6,7. In addition to lung cells' expression of IL-10 and mycobacterium-specific IgA antibodies, the NHP study indicates that mycobacterium-specific TH1 cells in the lung that co-express IL-17 (referred to as Th1/Th17 cells) can offer protection against infection and illness6. While the significance of the immunogens is unclear from the M72:AS01E results, the overall efficacy of the test indicates that the adjuvant may be crucial. However, more research is required to see whether adding more protective antigens to M72 will boost its effectiveness.

Further, some of the other TB vaccine candidates for 2019 include live-attenuated mycobacterial vaccine candidates (VPM1002, BCG revaccination, and MTBVAC); mycobacterial killed, whole-cell, or extract vaccine candidates (Vaccae, MIP, DAR-901, and RUTI); and recombinant live-attenuated or replication-deficient virus-vectored candidates that express an M. tuberculosis protein(s) (TB/FLU-04L, Ad5Ag85A, and some of the other mycobacterial fusion protein or proteins in an adjuvant formulation (H56:IC31, ID93:GLA-SE, and GamTBvac); and ChAdOx1.85A/MVA85A 1,2.

Although several existing vaccine strategies are available, however, they only make use of a few numbers of concepts and vaccine classes are one of their limitations. Most feature a restricted repertoire of immune-dominant target antigens, primarily from the Ag85 and ESAT-6 family of secreted proteins and concentrate on eliciting "conventional" TH1 response. Challenges in developing vaccines employing these proteins are highlighted by the finding in mice that functional exhaustion restricts immunity by ESAT-6-specific T cells 3, while antigen availability decreases the protective immunity conferred by Ag85B-specific T cells during chronic infection. There appears to be little variety in the functional characteristics of memory T-cell responses elicited by six-subunit vaccine candidates in clinical trials4. Unnatural antigens, also known as poorly recognized antigens during natural infection, may simply not be recognized by the immune system during infection, and it is unclear how they contribute to protection 5.

Therefore, there is a need in the art to investigate vaccine approaches that make use of immunological and antigenic diversity. Particularly, strategies are required which could involve the use of donor-unrestricted T cells that can identify antigens other than traditional peptides and/or antibodies to induce unusual immunity8–10. Investigation is also required into the function of rare immune responses brought on by unique post-translationally modified antigens in defense against disease11–14. Thus, it is important to investigate vaccination ideas that stimulate memory T cells that can proliferate sustainably when they come into contact with M. tuberculosis-infected cells in the lung15. Characterization of such memory precursor effectors is crucial because long-lived memory T cells may develop from a subpopulation of effector cells16.
Accordingly, the present invention addresses the above problems and provides an antigenic protein and a vaccine composition against Mycobacterium tuberculosis (Mtb) comprising the antigenic protein, an adjuvant, and an immunologically- active co-adjuvant. The present invention also provides a process for preparing the antigenic protein.
SUMMARY OF THE INVENTION
These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description. This summary is provided to introduce a selection of concepts in a simplified form.

In an aspect of the present invention, there is provided an antigenic protein of SEQ ID NO.1 comprising 88 residues long characteristic alpha crystallin domain (residues 41-128) flanked by a stretch of 40 residues of non-conserved N-terminal region (residues 1-40) and a 12 residues long truncated C-terminal region (129-140).

In another embodiment of the present invention, there is provided an antigenic protein as claimed in the present invention, wherein the antigenic protein is a C-terminal truncated Hsp16.3 protein of Mycobacterium tuberculosis (Hsp16.3ΔC4).

In yet another embodiment of the present invention, there is provided an antigenic protein as claimed in the present invention, wherein the antigenic protein is a Hsp16.3 subunit vaccine (Hsp16.3ΔC4).

In still another embodiment of the present invention, there is provided an antigenic protein as claimed in the present invention, wherein the antigenic protein is administered alone or is co-administered with BCG vaccine.

In an embodiment of the present invention, there is provided an antigenic protein as claimed in the present invention, wherein the antigenic protein enhances the effector and memory T cell response.

In another aspect of the present invention, there is provided a vaccine composition against Mycobacterium tuberculosis, said composition comprising:
(i) an antigenic protein of SEQ ID NO.1, wherein the antigenic protein is a C-terminal truncated Hsp16.3 protein of Mycobacterium tuberculosis (Hsp16.3ΔC4);
(ii) an adjuvant; and
(iii) an immunologically- active co-adjuvant.

In an embodiment of the present invention, there is provided a vaccine composition as claimed in the present invention, wherein the adjuvant is N, N’-dimethyl-N, N’-dioctadecylammonium bromide (DDA).
In another embodiment of the present invention, there is provided a vaccine composition as claimed in the present invention, wherein the immunologically- active co-adjuvant is polyionosinic - polycytidylic acid (PIC).
In yet another embodiment of the present invention, there is provided a vaccine composition as claimed in the present invention, wherein the composition is formulated in a solution form and suitable for subcutaneous administration.
In another aspect of the present invention, there is provided a use of a vaccine composition as defined in the present invention for treatment of infection caused by Mycobacterium tuberculosis.
In yet another aspect of the present invention, there is provided a process for preparing the antigenic protein as defined in the present invention, the process comprising:
- generating a truncated clone encoding antigenic protein Hsp16.3ΔC4 by polymerase chain reaction (PCR) using a pQE8-Hsp16.3 expression construct harbouring wild-type Hsp16.3 (SEQ ID NO. 2) as DNA template and primers of SEQ ID NO. 3- 6;
- subjecting purified PCR product and pET28b vector to double digestion with NdeI and XhoI restriction enzymes separately for a duration of 1 hour at 37°C;
- ligating the digested PCR product and the vector to generate expression plasmid pET28b-Hsp16.3ΔC4 plasmid and transforming to E. coli DH5α cells;
- transforming E. coli BL21 (DE3) cells with pET28b-Hsp16.3ΔC4 plasmid for the expression of the antigenic protein Hsp16.3ΔC4; and
- obtaining the purified antigenic protein Hsp16.3ΔC4 from the cell lysate after sonication and centrifugation.
In an embodiment of the present invention, there is provided a process for preparing the antigenic protein, wherein the ligation of the digested PCR product and the vector was carried out for a duration of 18 hours at 16°C using rapid DNA ligation kit to generate expression plasmid pET28b-Hsp16.3ΔC4 plasmid.
These and other features, aspects, and advantages of the present disclosure will become better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

The following figures form part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the figures in combination with the detailed description of the specific embodiments presented herein.

Figure 1 is a graphical representation of wild-type Hsp16.3 protein and C-terminal truncated Hsp16.3 (Hsp16.3ΔC4).
Figure 2 illustrates mycobacterial burden in the lung of different subunit vaccinated mice groups.
Figure 3 illustrates results showing the impact of different vaccine candidates on the viability of THP-1 monocyte-derived macrophages using the MTT assay.
Figure 4 illustrates the kidney function test by assessing the serum creatinine and urea levels in various mice groups immunized with different vaccine candidates (Serum collected on day 42 i.e. 14 days after the last vaccination).
Figure 5 illustrates the lipid profile by estimating the cholesterol and triglyceride levels in various mice groups immunized with different vaccine candidates (serum collected on day 42 i.e. 14 days after the last vaccination).
Figure 6 illustrates the blood glucose level of various mice groups immunized with different vaccine candidates (serum collected on day 42 i.e. 14 days after the last vaccination).
Figure 7 illustrates the proliferation of CD4 & CD8 T cells as Median Fluorescence Intensity (MFI) in various mice groups immunized with different vaccine candidates as measured by flow cytometer.
Figure 8 illustrates the MFI of CD4 & CD8 effector T cells in various mice groups immunized with different vaccine candidates as measured by flow cytometer.
Figure 9 illustrates percentages of CD4 & CD8 effector T cells in various mice groups immunized with different vaccine candidates as measured by flow cytometer.
Figure 10 illustrates percentages of CD4 & CD8 central memory T cells in various mice groups immunized with different vaccine candidates as measured by flow cytometer.
Figure 11 illustrates percentages of CD4 & CD8 activated T cells in various mice groups immunized with different vaccine candidates as measured by flow cytometer.
Figure 12 illustrates percentages of IFNγ secreting CD4 & CD8 T cells in various mice groups immunized with different vaccine candidates as measured by flow cytometer.
Figure 13 illustrates percentages of IL2 & IL17 secreting CD4 T cells in various mice groups immunized with different vaccine candidates as measured by flow cytometer.
Figure 14 illustrates total IgG & IgG1 induced by different vaccinate candidate vaccines as a representative of the induced humoral immune response. The antibody titres in the serum were measured using the ELISA method.

DETAILED DESCRIPTION OF THE INVENTION

Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps of the process, features of the product, referred to or indicated in this specification, individually or collectively, and any combinations of any or more of such steps or features.

Definitions
For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are collected here. These definitions should be read in the light of the remainder of the disclosure and understood by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

The articles “a”, “an” and “the” are used to refer to one or more than one (i.e., to at least one) of the grammatical object of the article.

The terms “comprise” and “comprising” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as “consists of only”.

Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated element or step or group of elements or steps but not the exclusion of any other element or step or group of element or steps.
The term “including” is used to mean “including but not limited to”. “Including” and “including but not limited to” are used interchangeably.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of the ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.

The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally equivalent products and methods are clearly within the scope of the disclosure, as described herein.

The present invention provides an antigenic protein variant against Mycobacterium tuberculosis. Particularly, the antigenic protein is a C-terminal truncated Hsp16.3 protein of Mycobacterium tuberculosis (Hsp16.3ΔC4). Hsp16.3 is a small heat shock protein belonging to a family of molecular chaperones. This class of proteins has a conserved alpha-crystallin domain. Mtb, the etiological agent that causes tuberculosis, harbors two alpha-crystallin-related (Acr) small heat shock proteins Acr1 (Hsp16.3) and Acr2 (HrpA). Hsp16.3 often plays an important role in Mtb proliferation, pathogenicity, and cell wall thickening. This mycobacterial small heat shock protein has also been demonstrated to be essential for Mtb survival during extended periods of dormancy. Furthermore, Hsp16.3 has advantages for the host as well. Since Hsp16.3 consistently appears in patient sera and has potent immunogenic qualities, it has been widely linked to vaccination. Additionally, Hsp16.3 is induced inside bacteria during stress conditions like ROS, and NOS imposed by the host. Hence, the immunity developed against this antigenic protein provides better protection. Furthermore, Hsp16.3 has specific B cell and T cell epitopes 17,18.

Thus, in accordance with the present invention, there is provided an antigenic protein of SEQ ID NO.1 comprising 88 residues long characteristic alpha crystallin domain (residues 41-128) flanked by a stretch of 40 residues of non-conserved N-terminal region (residues 1-40) and a 12 residues long truncated C-terminal region (129-140).
In an embodiment of the present invention, there is provided an antigenic protein, wherein the antigenic protein is a C-terminal truncated Hsp16.3 protein of Mycobacterium tuberculosis (Hsp16.3ΔC4). Due to the modification in C-terminal end, the antigenic protein shows enhanced cellular and humoral immune response which is helpful for effective clearance of pathogen during infection. Further, apart from cellular and humoral response, (Hsp16.3ΔC4) shows strong T cell memory response. It produces central memory T cells, effector memory T cells and antigen specifically activated memory T cells upon restimulation.
In another embodiment of the present invention, there is provided an antigenic protein as claimed in the present invention, wherein the antigenic protein is a Hsp16.3 subunit vaccine (Hsp16.3ΔC4).
In yet another embodiment of the present invention, there is provided an antigenic protein, wherein the antigenic protein is administered alone or is co-administered with BCG vaccine.
In yet another embodiment of the present invention, there is provided an antigenic protein, wherein the antigenic protein enhances the effector and memory T cell response.
The present invention also provides a vaccine composition comprising the antigenic protein, adjuvant and an immunogenically active co-adjuvant. Particularly, the vaccine composition disclosed in the present invention against Mtb, comprises a single immune-dominant Mtb antigen along with an advanced adjuvant system. The vaccine composition comprises of Hsp16.31-140, a C-terminal region-truncated Hsp16.3/Hsp16.31-144 (referred here onwards as Hsp16.3ΔC4) selected for its ability to stimulate both robust T-cell responses (Th1 & Th17) and durable humoral immunity (Total IgG and IgG1). The composition comprises an adjuvant DDA (N, N’-dimethyl-N, N’-dioctadecylammonium bromide) and a co-adjuvant i.e., PIC (polyionosinic- polycytidylic acid), enhancing antigen presentation and promoting long-lasting immune memory.
The preclinical studies in murine models demonstrate that the vaccine composition induces strong interferon-gamma (IFN-γ) production, CD4+ and CD8+ T-cell activation, and high titers of antigen specific antibodies, all critical for Mtb clearance. Further, vaccinated animals challenged with a virulent Mtb strain (H37Rv) exhibit significantly reduced bacterial burdens in the lungs and spleen compared to controls, indicating superior protective efficacy. Furthermore, the preliminary safety assessments reveal no adverse effects, highlighting the vaccine’s potential for clinical translation.
In another aspect of the present invention, there is provided a vaccine composition against Mycobacterium tuberculosis, said composition comprising:
(i) an antigenic protein of SEQ ID NO.1, wherein the antigenic protein is a C-terminal truncated Hsp16.3 protein of Mycobacterium tuberculosis (Hsp16.3ΔC4);
(ii) an adjuvant; and
(iii) an immunologically- active co-adjuvant.
The adjuvant used in the present invention is N, N’-dimethyl-N, N’-dioctadecylammonium bromide (DDA), which is a synthetic cationic lipid with a positive charge that facilitates interaction with negatively charged antigens, enhancing delivery and uptake by antigen-presenting cells (APCs). DDA forms a depot at the injection site, allowing for slow release of the antigen. This sustained antigen exposure enhances immune response duration and strength. Further, the immunologically- active co-adjuvant used in the present invention is polyionosinic - polycytidylic acid (PIC), which is a synthetic analog of double-stranded RNA, mimicking viral components. It is a potent agonist for Toll-like receptor 3 (TLR3) and RIG-I-like receptors, activating innate immune pathways. In order to fight intracellular infections like Mtb, DDA promotes a robust Th1 immune response, which is typified by elevated interferon-gamma (IFN-γ) production. Together, the two adjuvants strengthen Th1 and Th17 immune responses, which are essential for containing Mtb infection. Long-lasting protection and a strong, well-balanced immunological response against Mtb are ensured by this combination, which activates both innate and adaptive immunity. Further, DDA is well-tolerated and has shown good safety profiles in preclinical and clinical studies. PIC has been shown to enhance the immunogenicity of subunit vaccines against various infectious diseases, including tuberculosis.
In an embodiment of the present invention, there is provided a vaccine composition against Mycobacterium tuberculosis, wherein the adjuvant is N, N’-dimethyl-N, N’-dioctadecylammonium bromide (DDA).
In another embodiment of the present invention, there is provided a vaccine composition against Mycobacterium tuberculosis, wherein the immunologically- active co-adjuvant is polyionosinic - polycytidylic acid (PIC).
In still another embodiment of the present invention, there is provided a vaccine composition against Mycobacterium tuberculosis, wherein the composition is formulated in a solution form and suitable for subcutaneous administration.
In another aspect of the present invention, there is provided a use of a vaccine composition as defined in the present invention, for the treatment of infection caused by Mycobacterium tuberculosis.
In still another aspect of the present invention, there is provided a process for preparing the antigenic protein as defined in the present invention, wherein the process comprises:
- generating a truncated clone encoding antigenic protein Hsp16.3ΔC4 by polymerase chain reaction (PCR) using a pQE8-Hsp16.3 expression construct harbouring wild-type Hsp16.3 (SEQ ID NO. 2) as DNA template and primers of SEQ ID NO.: 3- 6;
- subjecting purified PCR product and pET28b vector to double digestion with NdeI and XhoI restriction enzymes separately for a duration of 1 hour at 37°C;
- ligating the digested PCR product and the vector to generate expression plasmid pET28b-Hsp16.3ΔC4 plasmid and transforming to E. coli DH5α cells;
- transforming E. coli BL21 (DE3) cells with pET28b-Hsp16.3ΔC4 plasmid for the expression of the antigenic protein Hsp16.3ΔC4; and
- obtaining the purified antigenic protein Hsp16.3ΔC4 from the cell lysate after sonication and centrifugation.
In an embodiment of the present invention, there is provided a process for preparing the antigenic protein, wherein the ligation of the digested PCR product and the vector was carried out for a duration of 18 hours at 16°C using rapid DNA ligation kit to generate expression plasmid pET28b-Hsp16.3ΔC4 plasmid.
Advantages of the present invention:
• The antigenic protein (Hsp16.3ΔC4) disclosed in the present invention is a C-terminal truncated Hsp16.3 and is found to induce a strong immune response due to its high chaperone type activity and immunogenicity. Hsp16.3ΔC4 shows robust cell-mediated and humoral immunity which is even better than the wild-type counterpart.
• The vaccine composition comprising the antigenic protein is found to be non-toxic in acute toxicity tests in mice and appears to be safe.
• Hsp16.3 is secreted during host cell infection by the Mtb pathogen and the antigenic protein is not present in humans. Hence, the subunit vaccine would be highly specific to the Mtb pathogen.
• Most existing vaccines focus on active TB, while Hsp16.3-based subunit vaccines specifically target proteins expressed during latency, addressing the reservoir of latent Mtb.
• The vaccine composition disclosed in the present invention is prepared by recombinant biotechnology which reduces the production costs and facilitates large-scale vaccine manufacturing.
• The vaccine composition is designed for conventional routes of administration i.e. subcutaneous and hence formulations are easy to use and administer.
• The developed vaccine composition shows better protection in all aspects in comparison to BCG. Particularly, the Hsp16.3 subunit vaccine does not face interference issues from prior BCG vaccination, allowing them to be administered as boosters. Coadministration of BCG along with our vaccine candidate enhances cell-based immunity specifically effector and memory T cell responses.
• The flexibility of subunit vaccines allows for optimized formulations, including slow-release systems and thermostable designs for low-resource settings.

Although the subject matter has been described in considerable detail with reference to certain preferred embodiments thereof, other embodiments are possible.

EXAMPLES
The disclosure will now be illustrated with working examples, which are intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of the ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.

Example 1: Development of Antigenic Protein
Selection of protein: Small heat shock proteins are ubiquitous and are a family of molecular chaperones. This class of proteins has a conserved alpha-crystallin domain. Mtb, the etiological agent that causes tuberculosis, harbours two alpha-crystallin-related (Acr) small heat shock proteins Acr1 (Hsp16.3) and Acr2 (HrpA). Hsp16.3 often plays an important role in Mtb proliferation, pathogenicity, and cell wall thickening. This mycobacterial small heat shock protein has also been demonstrated to be essential for Mtb survival during extended periods of dormancy. Furthermore, Hsp16.3 has advantages for the host as well. Since Hsp16.3 consistently appears in patient sera and has potent immunogenic qualities, it has been widely linked to vaccination and was selected.
Truncation of C-terminal region of Hsp16.3
The wild-type (Hsp16.31-144/Hsp16.3WT) and C-terminal truncated protein (Hsp16.31-140/Hsp16.3ΔC4) of Mycobacterium tuberculosis Hsp16.3 were generated by the polymerase chain reaction (PCR) using a pQE8-Hsp16.3 expression construct harboring wild-type Hsp16.3 as a DNA template. To construct this truncated clone, forward primers flanking the NdeI restriction site and reverse primers flanking the XhoI restriction site were designed. The following forward and reverse primers were used.
Primers for Hsp16.3WT:
SEQ ID NO. 3: Forward primer
5′GGG AAT TCC ATA TGG CCA CCA CCC TTC 3′
SEQ ID NO. 4: Reverse primers
5′-CCG CTC GAG TCA GTT GGT GGA CCG GAT C-3′;
Primers for Hsp16.3ΔC4:
SEQ ID NO. 5: Forward primer
5′GGG AAT TCC ATA TGG CCA CCA CCC TTC 3′
SEQ ID NO. 6: Reverse primer
5′- CCG CTC GAG TCA GAT CTG AAT GTG CTT TTC GG-3′.
The PCR reaction was carried out in 50μl reaction volume containing dNTP, 10× Taq polymerase buffer, respective primers, template DNA, and Taq DNA polymerase (Fermentas, Glen Burnie, MD, USA). Amplifications were performed for 30 cycles with conditions for denaturation at 95°C for 45s, annealing at 60°C for 30 s, extension at 72°C for 45 s, and finally extended at 72°C for 10 min. The PCR products were analyzed on 0.8% agarose gel and were purified by a QIAquick gel extraction kit (Qiagen). Purified PCR products and pET28b vector were double digested with NdeI and XhoI (New England Biolabs) restriction enzymes separately for 1 hour at 37°C. The digested product was purified from 0.8% agarose gel by QIAquick gel extraction kit (Qiagen). The digested PCR product and vector were then ligated by using a rapid DNA ligation kit (Fermentas) for ~18 hours at 16°C to generate pET28b-Hsp16.3WT and pET28b-Hsp16.3ΔC4 plasmids. Afterwards, these plasmids were transformed into E. coli DH5α cells. The plasmids were then purified from the E. coli DH5α cells with the aid of a QIAprep spin miniprep kit (Qiagen). The presence of the desired truncation was confirmed by DNA sequencing carried out at the sequencing facility of Chromous Biotech Pvt Ltd, Bangalore, India.
Expression and purification of wild-type and C-terminal-truncated Hsp16.3
As mentioned above, Hsp16.3ΔC4 and wild-type Hsp16.3 genes were cloned, expressed in bacterial systems, and purified before the experiment. The expression plasmids pET28b-Hsp16.3WT, and pET28b-Hsp16.3ΔC4 were transformed into E. coli BL21 (DE3) cells for the protein expression. These proteins were induced in bacterial systems using 0.4 M IPTG. The cell lysates were obtained in the soluble fraction (after sonication and centrifugation) and mixed with Ni-NTA resins to purify the proteins. The purified fractions (250mM) were dialyzed against 50mM phosphate buffer containing 10 mM imidazole (pH 7.5). After thrombin treatment, the eluents were passed through the p-benzamidine agarose column followed by mixing with Ni-NTA resins. The eluents were collected accordingly which contained the thrombin cut proteins (devoid of 6X His-Tag). Purity was accessed using 15% SDS-PAGE.
Example 2: Evaluating toxicity and potential effects
Protection against Mycobacterium tuberculosis
A popular microbiological method for determining the number of live bacteria in a sample (tissue/ cells) is the CFU (Colony-Forming Unit) assay, which measures the bacteria's capacity to establish colonies on solid growth media. Studying bacterial growth, survival, and the impact of antimicrobial drugs or immunological responses requires the use of this test. The bacterial burden in lung tissues of mice infected with Mtb is measured using CFU assays. Hence, the subunit vaccine efficacy of Hsp16.3ΔC4 with and without BCG co-administration along with its control counterpart was evaluated. Immunized mice were challenged with Mtb by intranasal route and bacterial burdens were evaluated in the lungs at 3 weeks post-infection (p.i.). While WT Hsp16.3 only led to minor improvements over BCG-induced protection three weeks post-infection, Hsp16.3ΔC4 induced superior protection compared to Adjuvant, BCG, and WT Hsp16.3. BCG co-administration enhances the protection against Mtb significantly. Both BCG + Hsp16.3 and BCG + Hsp16.3ΔC4 vaccinated mice groups showed significant protection in comparison to Adjuvant, BCG, Hsp16.3, and Hsp16.3ΔC4 vaccinated counterparts. However, animals immunized with BCG + Hsp16.3ΔC4 were the most effective at eliminating the pathogen. (Figure 2).
MTT assay
Before moving on to mice experiments, an MTT assay was performed in monocyte-derived macrophage cells in vitro which is a crucial tool for assessing the cytotoxicity of vaccine candidates. The metabolic activity of living cells, which converts the yellow MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) reagent into purple formazan crystals, is the basis for this colorimetric assay's measurement of cell viability. The MTT assay aids in evaluating if the antigens cause harmful effects on host cells, including immunological or fibroblast cells while testing a vaccination candidate. Here, no toxicity was observed in either wild-type Hsp16.3 or Hsp16.3ΔC4 (Figure 3).
Kidney Function Test
In order to evaluate the safety profile of the vaccine candidate and, in particular, to identify any possible nephrotoxicity, it is crucial to monitor kidney function in mice during vaccine trials. Adjuvants, excipients, or antigenic formulations used in vaccines may unintentionally impair renal function, which is crucial for the kidney’s ability to filter waste and maintain fluid and electrolyte balance. Serum biomarkers that suggest kidney filtration efficiency, such as creatinine levels and urea levels. Figure 4 depicts the results of the kidney function test and demonstrates that no abnormality was found in kidney functioning.
Lipid Profile
During vaccine studies, a lipid profile test is a crucial evaluation to track any possible impacts of the vaccine on lipid metabolism. This test provides information on the safety and any adverse effects of the vaccine by measuring important lipid parameters such as total cholesterol, triglycerides, low-density lipoprotein (LDL), and high-density lipoprotein (HDL). Assessing the lipid profile in preclinical and clinical research helps guarantee that the vaccination won't cause side effects that could jeopardize cardiovascular health. The cholesterol and triglyceride levels were checked, and no significant changes were observed in any of the vaccinated mice groups as compared to the control groups (Figure 5).
Blood Glucose Level
An essential component of evaluating the vaccine candidate’s metabolic safety and potential effects on general health during trials is blood glucose monitoring. Immune-mediated metabolic pathways may be impacted by vaccines. This is particularly important in studies where immune stimulation or adjuvants may change insulin sensitivity or glucose homeostasis. Furthermore, tracking glucose levels can reveal information about immune-metabolic interactions, which is important for conditions like tuberculosis where immunological responses may be influenced by metabolic status. Random blood glucose was checked every 15-day intervals in control and vaccinated mice groups and no significant change was found in blood glucose level by the vaccine candidate (Figure 6).
Example 3: Evaluating the Immunogenicity and Efficacy of Vaccine Composition
Mice were subcutaneously injected with 40µg of subunit vaccine emulsified in an adjuvant composed of DDA (250µg/dose; Sigma-Aldrich, D2779) and Poly (I: C) (50µg/dose; Sigma-Aldrich, P1530). The vaccine composition was administered thrice by subcutaneous injection at three different body parts of the mice at 14-day intervals. After 3 doses of vaccination, mice were sacrificed to collect spleen, and antigen recall response was checked by re-stimulating the spleno-lymphocytes. T cell and differentiation were checked thoroughly. Along with that blood serum was collected from those vaccinated mice and total IgG antibody and its IgG1 subtype titer were checked by ELISA using respective protein subunits. After 28 days of the last vaccine administration, the mice were infected with a virulent strain of Mtb, H37Rv (100 CFU) intranasally. Body weight change monitored at regular intervals till sacrifice. Post 21 days of Mtb infection, mice were sacrificed, and bacterial load was checked in the lungs by CFU assay.
Coadministration of Hsp16.3 wildtype and Hsp16.3ΔC4 subunit vaccine along with BCG
BCG was administered along with Hsp16.3 wildtype and Hsp16.3ΔC4 subunit vaccine to check cellular responses (T cell proliferation, effector T cell formation, memory T cell formation, and cytokine responses).
CD4 and CD8 T cell proliferation
The proliferation of CD4 and CD8 T cells is a crucial factor in assessing the effectiveness of vaccines, especially against diseases that demand strong cellular immunity. By generating cytokines like IFN-γ and IL-2, which improve macrophage activation and antigen presentation, CD4 T cells also known as helper T cells play a crucial part in coordinating the immune response. This is especially crucial for subunit vaccinations because CD4 T cell proliferation guarantees the activation and recruitment of other immune cells that are necessary for the removal of pathogens. Conversely, cytotoxic T lymphocytes (CTLs), also known as CD8 T cells, help to produce vaccine-induced immunity by lysing infected cells directly and releasing granzyme and perforin to destroy intracellular pathogens. Since CD8 T cells can identify and eliminate Mtb-infected cells that contain dormant bacteria, their proliferation after vaccination is essential for focusing on latent reservoirs of infections. Also, CD4 and CD8 T cells indicate probable development of memory T cell development which helps in long-term protection. Co-administering BCG along with the candidate vaccine enhances CD4 and CD8 T cell proliferation. Figure 7 depicts the enhanced CD4 and CD8 T cell proliferation.
Effector T cell function
In the context of vaccine-induced immunity, CD4 and CD8 effector T cell proliferation is essential, especially against intracellular pathogens like Mtb. As discussed, earlier CD4 T cells are essential because they coordinate the immune response, encourage macrophage activation, and produce cytokines like IFNγ to improve bacterial killing. On the other hand, CD8 T cells, limit the reproduction niche of infections by directly killing infected host cells by the release of granzymes and perforins. To ensure quick and focused reactions when a pathogen is encountered, effective vaccines seek to promote the strong proliferation and differentiation of these effector T cell subsets. Co-administering BCG along with our candidate vaccine enhances CD4 and CD8 effector T cells. Figure 8 and Figure 9 depicts enhanced CD4 and CD8 effector T cells.
Memory T cell production
CD4+ and CD8+ T cells with central memory (TCM) and effector memory (TEM) are essential for the long-term immunity that vaccinations provide. Due to their high expression of CD62L and CCR7, central memory T cells are mainly found in lymphoid organs and can effectively recirculate and multiply when exposed to antigens again. TCM is essential for ongoing immune surveillance and secondary immunological responses because of its capacity to proliferate quickly and specialize into effector cells. On the other hand, activated memory T cells have been exposed to an antigen and are prepared to provide a more powerful and rapid defense against specific antigens in the future. Co-administering BCG along with vaccine composition augments both central and activated memory CD4 and CD8 T cells (Figure 10 and Figure 11).
Antigen-specific Th1 & Th17 response
Adaptive immunity relies heavily on the Th1 and Th17 responses, especially when fighting intracellular infections such as mycobacterial infections. IFNγ and IL2, which are produced by Th1 cells, activate macrophages, improve antigen presentation, and stimulate cytotoxic T-cell responses. The removal of contaminated cells depends on this. In contrast, Th17 cells release IL17, which promotes inflammation and neutrophil recruitment to limit the spread of pathogens outside of cells. CD4+ and CD8+ T cells are key mediators of this dual protective approach in infection and vaccine-induced immunity. IFNγ, IL2, and IL17 work together to produce a strong and complementary immune response, which is visible in our vaccine candidate (Figure 12 and Figure 13).
Humoral immunity
In addition to the cellular immune response, humoral protection against tuberculosis is mostly dependent on total IgG and IgG1. Pathogen detection, opsonization, and the activation of immune effector processes including complement fixation and phagocytosis are all aided by IgG antibodies, especially IgG1. IgG1 has been demonstrated to target particular Mtb antigens in the setting of tuberculosis, improving macrophages' capacity to internalize and eliminate the bacteria. An efficient humoral response, which helps to limit bacterial spread and encourage immune-mediated clearance, is frequently linked to elevated levels of total IgG and IgG1 antibodies. These antibodies can also function as indicators of disease development or protective immunity, offering information on immunological status and vaccine effectiveness. From our experiment, it is evident that Hsp16.3ΔC4 alone can produce significantly higher total IgG and IgG1 antibodies in comparison to wild-type Hsp16.3 (Figure 14).

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18. Verbon, A. et al. The 14,000-molecular-weight antigen of Mycobacterium tuberculosis is related to the alpha-crystallin family of low-molecular-weight heat shock proteins. J. Bacteriol. 174, 1352–1359 (1992). , Claims:1. An antigenic protein of SEQ ID NO.1 comprising 88 residues long characteristic alpha crystallin domain (residues 41-128) flanked by a stretch of 40 residues of non-conserved N-terminal region (residues 1-40) and a 12 residues long truncated C-terminal region (129-140).

2. The antigenic protein as claimed in claim 1, wherein the antigenic protein is a C-terminal truncated Hsp16.3 protein of Mycobacterium tuberculosis (Hsp16.3ΔC4).

3. The antigenic protein as claimed in claim 1, wherein the antigenic protein is a Hsp16.3 subunit vaccine (Hsp16.3ΔC4).

4. The antigenic protein as claimed in claims 1-3, wherein the antigenic protein is administered alone or is co-administered with BCG vaccine.

5. The antigenic protein as claimed in claim 1, wherein the antigenic protein enhances the effector and memory T cell response.

6. A vaccine composition against Mycobacterium tuberculosis, said composition comprising:
(i) an antigenic protein of SEQ ID NO.1, wherein the antigenic protein is a C-terminal truncated Hsp16.3 protein of Mycobacterium tuberculosis (Hsp16.3ΔC4);
(ii) an adjuvant; and
(iii) an immunologically- active co-adjuvant.

7. The vaccine composition as claimed in claim 6, wherein the adjuvant is N, N’-dimethyl-N, N’-dioctadecylammonium bromide (DDA).

8. The vaccine composition as claimed in claim 6, wherein the immunologically- active co-adjuvant is polyionosinic - polycytidylic acid (PIC).
9. The vaccine composition as claimed in claims 6-8, wherein the composition is formulated in a solution form and suitable for subcutaneous administration.

10. Use of a vaccine composition as defined in claims 6-9 for treatment of infection caused by Mycobacterium tuberculosis.

11. A process for preparing the antigenic protein as defined in claim 1-5, the process comprising:
- generating a truncated clone encoding antigenic protein Hsp16.3ΔC4 by polymerase chain reaction (PCR) using a pQE8-Hsp16.3 expression construct harbouring wild-type Hsp16.3 (SEQ ID NO. 2) as DNA template and primers of SEQ ID NO. 3- 6;
- subjecting purified PCR product and pET28b vector to double digestion with NdeI and XhoI restriction enzymes separately for a duration of 1 hour at 37°C;
- ligating the digested PCR product and the vector to generate expression plasmid pET28b-Hsp16.3ΔC4 plasmid and transforming to E. coli DH5α cells;
- transforming E. coli BL21 (DE3) cells with pET28b-Hsp16.3ΔC4 plasmid for the expression of the antigenic protein Hsp16.3ΔC4; and
- obtaining the purified antigenic protein Hsp16.3ΔC4 from the cell lysate after sonication and centrifugation.

12. The process as claimed in claim 11, wherein the ligation of the digested PCR product and the vector was carried out for a duration of 18 hours at 16°C using rapid DNA ligation kit to generate expression plasmid pET28b-Hsp16.3ΔC4 plasmid.