A MULTI-EPITOPE VACCINE CONSTRUCT FOR TUBERCULOSIS

Abstract

The present disclosure provides a multi-epitope vaccine construct against Mycobacterium tuberculosis. Particularly, the present disclosure provides a multi-epitope vaccine construct by targeting signature proteins of Mycobacterium tuberculosis.

Specification

Description:FIELD OF THE INVENTION
The present disclosure in general relates to immunology. Particularly, the present disclosure relates to vaccine constructs and compositions for infectious diseases. More particularly, the present disclosure provides a multi-epitope vaccine construct by targeting signature sequences of Mycobacterium tuberculosis.

BACKGROUND OF THE INVENTION
Mycobacterium tuberculosis bacteria is responsible for the spread of Tuberculosis (TB), the most lethal bacterial infection. According to the World Health Organization (WHO), TB is extremely infectious and has been projected to cause 10.4 million new cases and 1.3 million deaths. The vast majority of antimicrobial medication combinations are being employed in place of the traditional TB treatment. As second-line injectables for tuberculosis therapy, fluoroquinolones are combined with the first-line drugs isoniazid, rifampicin, ethambutol, and pyrazinamide.

Drug-resistant mutations in M. tb are more likely to develop as a result of the prolonged treatment cycle, which usually lasts nine to twelve months or longer. The emergence and rising prevalence of M. tb, a condition that is highly resistant to drugs, has recently resulted in a decline in the efficacy of therapy. By 2035, the END-TB initiative of the WHO seeks to eradicate tuberculosis worldwide. With significant governmental support, India has likewise set an ambitious target to eradicate tuberculosis by 2025. But eliminating tuberculosis by the year 2035 is a big task in itself. Vaccination is the most efficient means of controlling deadly infections including TB. Bacille Calmette Guerin (BCG), an attenuated vaccine derived from a subculture of Mycobacterium bovis, is the only vaccine available for combating TB. BCG protects against disseminated TB, such as miliary TB, and pediatric tuberculosis. However, with time, its effectiveness diminishes, and it offers very little protection against adult pulmonary TB, which causes TB to spread among people who are at risk. Enhanced vaccines are needed given that TB infection persists even after the BCG vaccination. Global TB transmission can be considerably reduced by developing a new TB vaccination for people of all ages.
However, till date, no successful vaccines have been developed that can confer long-lasting immunity against the infection caused by Mycobacterium tuberculosis, thereby preventing or treating latent bacterial infections. Many vaccine candidates have been developed and are undergoing varying phases of clinical trials. These vaccinations fall into various categories, including protein subunit, viral vector, and whole cell live attenuated vaccines like BCG. With whole cell attenuated vaccines, the vaccine's main drawbacks include a decreased immune response and the possibility of reversion to the wild-type pathogenic strain; with viral vector vaccines, these drawbacks include an incorrect fusion between the viral coat protein and the target membrane. Utilizing protein subunit vaccines is the most promising strategy which are made only from the immunodominant epitopes of the chosen protein candidates and ensure a strong immune response.
Thus, the present disclosure offers a comprehensive approach for developing a multi- epitope vaccine construct employing mycobacterium's hallmark proteins in order to cater to the unfulfilled demand for an appropriate vaccine against Mycobacterium tuberculosis. The vaccine construct developed in the present disclosure exhibits a stronger humoral and cell-mediated immune response.
SUMMARY OF THE INVENTION
This summary is provided to introduce a selection of concepts in a simplified format that are further described in the detailed description of the invention.
In an aspect, the present disclosure relates to a multi-epitope vaccine construct, said construct comprising epitopes from signature proteins of Mycobacterium tuberculosis and a Toll-like receptor (TLR) agonist.
In another aspect, the present disclosure relates to a multi-epitope vaccine construct, wherein the proteins are selected from the group consisting of Rv1507a, Rv1509, Rv1954a, and Rv2231a.
In another aspect, the present disclosure relates to a multi-epitope vaccine construct, wherein the signature proteins are having an amino acid sequence of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4.
In yet another aspect, the present disclosure relates to a multi-epitope vaccine construct, wherein the Toll-like receptor (TLR) agonist is TLR-4 agonist.
In still another aspect, the present disclosure relates to a multi-epitope vaccine construct, wherein the TLR-4 agonist is laterosporulin agonist of an amino acid sequence of SEQ ID NO. 5, and wherein the TLR-4 agonist function as an adjuvant.
In another aspect, the present disclosure relates to a multi-epitope vaccine construct, wherein the epitopes are connected through linkers selected from GPGPG, EAAK, AAY and KK, and wherein the linkers are arranged to ensure translation of all epitopes of the construct.
In yet another aspect, the present disclosure relates to a multi-epitope vaccine construct, wherein the linker GPGPG is present: between a B-cell epitope and HLA class I epitope, and between two HLA class II epitopes, wherein the linker EAAK is present between Toll-like receptor (TLR) agonist and HLA class II epitope, wherein the linker AAY is present between two HLA- class I epitopes; and wherein the linker KK is present between two B-cell epitopes.
In still another aspect, the present disclosure relates to a multi-epitope vaccine construct, wherein the epitopes of the construct are capable of functioning as B-cell epitope, human leukocyte antigen (HLA) class I epitope and human leukocyte antigen (HLA) class II epitope and thereby simultaneously generating a B-cell response, CTL response and HTL response.
In yet another aspect, the present disclosure relates to a multi-epitope vaccine construct, wherein the construct comprises of three human leukocyte antigen (HLA) class I epitopes; four human leukocyte antigen (HLA) class II epitopes; and two B-cell epitopes.
In another aspect, the present disclosure relates to a multi-epitope vaccine construct, wherein the epitopes in construct are arranged in a consecutive sequence of HLA class II epitopes- HLA class I epitopes- B cell epitopes, and wherein the adjuvant TLR-4 agonist is positioned at N-terminal to the epitopes.
In yet another aspect, the present disclosure relates to a multi-epitope vaccine construct, wherein the construct is of an amino acid sequence of SEQ ID NO. 6.
In still another aspect, the present disclosure relates to a multi- epitope vaccine construct, wherein the construct is having a molecular weight of 21 kDa, a theoretical isoelectric point (pI) of 9.49, aliphatic index of 66.52, solubility of 0.63 and instability index of 37.61.
In one of the aspects, the present disclosure relates to a vaccine composition against Mycobacterium tuberculosis, said composition characterized in comprising a multi-epitope vaccine construct as defined in the present disclosure.
In one of the aspects, the present disclosure relates to a genetic construct characterized in comprising a nucleic acid sequence encoding a vaccine construct as defined in the present disclosure.
In one of the aspects, the present disclosure relates to a vector construct characterized in comprising a genetic construct as defined in the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, the inventions of which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
Figure 1 illustrates the workflow of study employed to develop multi-epitope vaccine construct using signature proteins of Mycobacterium tuberculosis.
Figure 2 illustrates the schematic representation of the final multi-epitope vaccine construct components. The multi-epitope vaccine construct (MEV) with TLR4 adjuvant Laterosporulin. HTL epitopes are represented in blue color, CTL epitopes are represented in pink color, and B-cell epitopes are represented in green color. EAAAK linkers are used to link adjuvants with epitopes. GPGPG and AAY linkers connect two CTL and HTL epitopes, KK linkers are used to connect two B cells.
Figure 3 illustrates the predicted solubility score of the multi-epitope vaccine construct using protein sol webserver.
Figure 4 illustrates (a) the secondary structure prediction of the multi-epitope vaccine construct obtained using the Psipred web server; (b) Ramachandran plot analysis of secondary structure of vaccine construct; (c) Tertiary structure modelled using Robetta server and refined using Galaxy refine server; and (d) Quality index of tertiary structure of vaccine construct using vadar server.
Figure 5 illustrates the docked structures between the vaccine construct and human TLR4 receptor obtained via ClusPro server. The multicolor chain indicates TLR4 receptor, and the red chain indicates the vaccine construct.
Figure 6 illustrates protein-protein interaction data of the vaccine construct and TLR4 receptor generated using PDBsum server. The figure shows interacting residues between the two protein chains.
Figure 7 illustrates (A) RMSD (B) RMSF, (C) SASA and (D) Rg of multi-epitope vaccine construct with TLR-4 human receptor was analyzed after 200 ns of MD simulation using Gromacs software.
Figure 8 illustrates in-silico immune simulation results of the vaccine construct using C-ImmSim represented as follows: (A) Active B-cell population (B) Helper-T cells (C) Cytotoxic-T cells; (D) Cytokine levels induced by the vaccine construct, D for IL-2, which measures diversity (E) Antibodies produced; (F) Dendritic cell population; and (G) state NK-cell population
Figure 9 illustrates sequence of the vaccine construct optimized and inserted into E. coli vector PET28a using (XhoI and NdeI) restriction sites. The red color in the map represents inserted DNA sequence of vaccine construct (SNAPGENE TOOL).

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 and all 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 as 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 to 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 elements 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 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.
As used in the present disclosure, "epitope" refers to an antigenic determinant, which is the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells.
As used in the present disclosure, "agonist" refers to a chemical that activates a receptor to produce a biological response.
As used in the present disclosure, "adjuvant" refers to a substance that increases or modulates the immune response to a vaccine. It is defined as any substance that acts to accelerate, prolong, or enhance antigen-specific immune responses when used in combination with specific vaccine antigens.
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 disclosure relates to a multi-epitope vaccine construct against Mycobacterium tuberculosis. The vaccine construct comprising epitopes from signature proteins of Mycobacterium tuberculosis and a Toll-like receptor (TLR) agonist. In the development of the vaccine construct, thirteen mycobacterial species strictly pathogenic, opportunistic, and nonpathogenic were submitted to a comparative proteome analysis and twenty-five sequences unique to the M. tuberculosis complex were identified. Potential hits and genes selected by the workflow process do not have more than 20% sequence identity in proteins, more than 35% sequence identity in nucleotides, or a shared protein domain with the bacteria on the list. Thus, using the best selection criterion, four sequences (SS1, SS2, SS3, and SS4) were selected as possible candidates: Rv1507a, Rv1509, Rv1954a, and Rv2231a. The four M. tuberculosis hallmark proteins, SS1, SS2, SS3, and SS4, interact with cell surface receptors and start downstream signaling cascades, which play a role in host-pathogen interaction. These proteins are termed as Mycobacterium's hallmark proteins, designated SS1, SS2, SS3, and SS4, which demonstrates the ability to modulate the immune system in mice, promoting T and B cell responses and stimulating the production of pro-inflammatory cytokines. Additionally, studies also revealed increased levels of reactive nitrogen species (RNS), reactive oxygen species (ROS), and IgG antibodies following protein administration. In silico analysis suggests that this vaccine construct elicits a superior humoral and cell-mediated immune response compared to the existing options. The stability, solubility, antigenicity, and immunodominance of the vaccine construct developed from these signature proteins is a promising candidate with the potential to target a significant portion of the world's population.
Thus, with accordance with the present disclosure, there is provided a multi-epitope vaccine construct comprising epitopes from signature proteins of Mycobacterium tuberculosis and a Toll-like receptor (TLR).
In one of the aspects, the present disclosure relates to a multi-epitope vaccine construct, wherein the signature proteins are selected from the group consisting of Rv1507a, Rv1509, Rv1954a, and Rv2231a. The proteins are expressed in H37Rv strain of M. tb, wherein R stands for rough morphology and "V" stands for virulent. The nucleotide sequences of the selected proteins were retrieved from the NCBI database and Mycobrowser database. The FASTA sequence of the four signature proteins (Rv1507a, Rv1509, Rv1954a, and Rv2231a) are as follows:
SEQUENCE ID NO. 1
Rv1507a
MQSGQNILAKVCNLIEQSRLSSTRCLQFRITNTSRPRQLWSEFKRFCDIFNMVLGKARMGRDPGRPVRDERRIVSCEIIASDHIGLAAARLLAKRYRGRSVSGFVLMIKSASVHEIDSWSSPSVAMSIGVALCSYPHYAAARTSPPNRDWGEDTTRSRPVTGLLAG
SEQUENCE ID NO. 2
Rv1509
VFALSNNLNRVNACMDGFLARIRSHVDAHAPELRSLFDTMAAEARFARDWLSEDLARLPVGAALLEVGGGVLLLSCQLAAEGFDITAIEPTGEGFGKFRQLGDIVLELAAARPTIAPCKAEDFISEKRFDFAFSLNVMEHIDLPDEAVRRVSEVLKPGASYHFLCPNYVFPYEPHFNIPTFFTKELTCRVMRHRIEGNTGMDDPKGVWRSLNWITVPKVKRFAAKDATLTLRFHRAMLVWMLERALTDKEFAGRRAQWMVAAIRSAVKLRVHHLAGYVPATLQPIMDVRLTKR
SEQUENCE ID NO. 3
Rv1954a
MARGRVVCIGDAGCDCTPGVFRATAGGMPVLVVIESGTGGDQMARKATSPGKPAPTSGQYRPVGGGNEVTVPKGHRLPPSPKPGQKWVNVDPTKNKSGR
SEQUENCE ID N0. 4
Rv2231a
LTMACTACPTIWTLRCQTTCSNAFTGEALPHRHPRLAADAVNETRAIVQDVRNSILLSAASAWEIAINYRLGKLPPPEPSASYVPDRMRRCGTSPLSVDHAHTAHRRASGSPSTSIRPCAHRPGTAAWPDDHHRRRPVSCL
In another aspect, the present disclosure relates to a multi-epitope vaccine construct, wherein the Toll-like receptor (TLR) agonist is TLR-4 agonist. The TLR-4 agonist functions as an adjuvant. The adjuvant used in the present disclosure stimulates immune cells, to induce, and to boost protective immune responses, linking the innate and adaptive immunity.
In a preferred aspect, the present disclosure relates to a multi-epitope vaccine construct, wherein the TLR-4 agonist is Laterosporulin of an amino acid sequence of SEQ ID NO.5. Laterosporulin is a bacteriocin, or antimicrobial peptide, produced by the Brevibacillus sp. strain GI-9. It's a heat-stable peptide that is effective against a wide range of Gram-positive and Gram-negative bacteria.
SEQUENCE ID NO. 5 (Adjuvant)
ACVNQCPDAIDRFIVKDKGCHGVEKKYYKQVYVACMNGQHLYCRTEWGGPCQL


In yet another aspect, the present disclosure relates to a multi-epitope vaccine construct, wherein the epitopes are connected through linkers selected from GPGPG, EAAK, AAY and KK, and wherein the linkers are arranged to ensure translation of all epitopes of the construct. For the effective development of a vaccine, three essential components are required i.e., adjuvants, epitopes and linkers. Linkers are short amino acid segments that enhance immune responses, prevent epitope folding, and connect adjuvants and neighboring epitopes. Adjuvants are linked to B cell and T cell epitopes via a variety of immunogenic linkers, including KK, GPGPG, AAY, and EAAAK. The integrity of the multi-epitope vaccine construct and the immune system's capacity to target antigenic epitopes are both enhanced by the frequent usage of the AAY linker, which joins two proteins. Another popular linker that aids in the correct folding of vaccine antigens, minimizing antigen aggregation, and maintaining immunogenicity and stability is the GPGPG linker, which is high in glycine-proline amino acids. Adjuvants with predicted epitopes are linked using the a-helical EAAAK linker to stabilize their interaction. Adjuvants and CTL epitopes were connected using the EAAAK linker, CTL epitopes with AAY linkers, HTL epitopes with GPGPG linkers, and B cell epitopes with flexible KK linkers.
For TLR4, Laterosporulin is used as an adjuvant (Dinata and Baindara, 2023) in designing the vaccine construct, which is an anti-TB peptide (https://aps.unmc.edu/database/peptide).
For the development of the vaccine construct, a careful selection of non-toxic and non-allergic antigenic epitopes is crucial. The selection of non-toxic and non-allergenic antigenic epitopes was done for the development of the vaccine construct. Only those epitopes that successfully passed rigorous screening for antigenicity, non-allergenicity, and non-toxicity, were incorporated into the vaccine construct. Particularly, HTL and CTL epitopes were assessed for antigenicity using vaxijen server (https://www.ddgpharmfac.net/vaxijen/VaxiJen/VaxiJen.html). Other properties like allergenicity and toxicity of the selected epitopes were analyzed with the help of AllerTOPv.2.0 server (https://www.ddg-pharmfac.net/AllerTOP/) and ToxinPred server (http://crdd.osdd.net/raghava/toxinpred/).
T-cell epitopes were predicted using the IEDB server, and CTL/MHC-I prediction was performed using a reference set of HLA alleles from (http://tools.iedb.org/mhci/). After ranking the predicted epitopes from lowest to highest values, top 10 non-overlapping peptides were selected with the lowest percentile rank, indicating the highest binding affinity for MHC-I cells.
For the prediction of HTL/MHCII cell epitopes (http://tools.iedb.org/mhcii/), while many different alleles exist, Human/HLA-DR allele and a 7-allele human leukocyte antigen (HLA) reference set was focused due to their extensive study in immunology. These alleles are well-characterized and have been extensively researched, making them reliable choices for our analysis. Based on their percentile score, which indicates the likelihood of binding to MHCII molecules, epitopes that were 15 amino acids long were selected. A lower percentile score suggests a stronger binding affinity, meaning these epitopes are more likely to interact effectively with MHC II molecules and trigger.
To predict B-cell epitopes within the target proteins, an antibody epitope prediction tool was utilized, available on the Immune Epitope Database (IEDB) analysis website (http://tools.iedb.org/main/bcell/). The retrieved protein sequences were then screened for linear B-cell epitopes. Through this analysis, 16-mer epitopes were identified using a neural network with a 0.51 threshold for overlapping filter values. The epitope with the highest score was subsequently selected for the development of the vaccine construct.
In yet another aspect, the present disclosure relates to a multi-epitope vaccine construct, wherein the linker GPGPG is present: between a B-cell epitope and HLA class I epitope, and between two HLA class II epitopes, wherein the linker EAAK is present between Toll-like receptor (TLR) agonist and HLA class II epitope, wherein the linker AAY is present between two HLA- class I epitopes; and wherein the linker KK is present between two B-cell epitopes.
In still another aspect, the present disclosure relates to a multi-epitope vaccine construct, wherein the epitopes of the construct are capable of functioning as B-cell epitope, human leukocyte antigen (HLA) class I epitope and human leukocyte antigen (HLA) class II epitope and thereby simultaneously generating a B-cell response, CTL response and HTL response.
In an aspect, the present disclosure relates to a multi-epitope vaccine construct, wherein the construct comprises of three human leukocyte antigen (HLA) class I epitopes; four human leukocyte antigen (HLA) class II epitopes; and two B-cell epitopes.
In another aspect, the present disclosure relates to a multi-epitope vaccine construct, wherein the epitopes in construct are arranged in a consecutive sequence of HLA class II epitopes- HLA class I epitopes- B cell epitopes, and wherein the adjuvant TLR-4 agonist is positioned at N-terminal to the epitopes.
In a preferred aspect, the present disclosure relates to a multi-epitope vaccine construct, wherein the construct is of an amino acid sequence of SEQ ID NO. 6.
In another aspect, the present disclosure relates to a multi-epitope vaccine construct, wherein the construct is having a molecular weight of 21 kDa, a theoretical isoelectric point (pI) of 9.49, aliphatic index of 66.52, solubility score of 0.63 and instability index of 37.61.
In one of the aspects, the present disclosure relates to a vaccine composition against Mycobacterium tuberculosis, said composition characterized in comprising a multi-epitope vaccine construct as defined in the present disclosure.
In one of the aspects, the present disclosure relates to a genetic construct characterized in comprising a nucleic acid sequence encoding a vaccine construct as defined in the present disclosure.
In one of the aspects, the present disclosure relates to a vector construct characterized in comprising a genetic construct as defined in the present disclosure.
In one of the aspects, in the present disclosure, the developed vaccine construct was compared against the human proteome, a comprehensive database of human proteins obtained from UniProt. The comparison allowed for the identification of any regions in the vaccine that might resemble human proteins, potentially increasing the risk of an autoimmune reaction. Further, the safety profile of the vaccine construct was confirmed by running pBLAST in NCBI database against proteins found in Homo sapiens and it was reported that there was no resemblance between the multi-epitope vaccine construct and any human protein. Thus, the vaccine construct is safe and efficacious to provide and that there wouldn't be any chance of an autoimmune reaction after immunization.
The multi-epitope vaccine construct developed in the present invention is antigenic, non-toxic, and targets a significant portion of the world's population. Further, the vaccine construct structure is stable at the secondary and tertiary levels and binds to the immunological receptor TLR-4 with high affinity. The construct is able to stimulate the release of interferon-gamma and promotes both helper and cytotoxic T-cell proliferation. It also stimulates the production of various cytokines, including IFN-g, TGF-b, TNF-a, IL-12, IL-6, IL-4, IL-18, IL-10, IL-28, and IFN-b and the production of targeted antibodies, including IgM, IgG, IgG1, and IgG.
EXAMPLES
Example 1: Preparation of multi-epitope vaccine construct.
For the preparation of the vaccine construct, four signature (SS) proteins were identified and selected/shortlisted based on their unique characteristics and potential to elicit a robust immune response. The sequence of four Mycobacterium tuberculosis signature proteins, identified as potential vaccine candidates, were obtained in FASTA format from the NCBI database (Table 1). Four M. tuberculosis proteins, Rv1509, Rv1507a, Rv2231a, and Rv1954a, were selected based on their unique presence in M. tuberculosis and their demonstrated high antigenic properties. The nucleotide sequences of the selected proteins were retrieved from the NCBI database (https://www.ncbi.nlm.nih.gov/) and Mycobrowser database.
Table 1: FASTA sequence of four signature proteins of M. tb used to develop vaccine were retrieved from the NCBI database.
Name of Protein FASTA Sequence of protein

Rv1507a

MQSGQNILAKVCNLIEQSRLSSTRCLQFRITNTSRPRQLRWSEFKRFCDIFNMVLGKARMGRDPGRPVRDERRIVSCEIIASDHIGLAAARLLAKRYRGRSVSGFVLMIKSASVHEIDSWSSPSVAMSIGVALCSYPHYAAARTSPPNRDWGEDTTRSRPVTGLLAG
Rv1509 VFALSNNLNRVNACMDGFLARIRSHVDAHAPELRSLFDTMAAEARFARDWLSEDLARLPVGAALLEVGGGVLLLSCQLAAEGFDITAIEPTGEGFGKFRQLGDIVLELAAARPTIAPCKAEDFISEKRFDFAFSLNVMEHIDLPDEAVRRVSEVLKPGASYHFLCPNYVFPYEPHFNIPTFFTKELTCRVMRHRIEGNTGMDDPKGVWRSLNWITVPKVKRFAAKDATLTLRFHRAMLVWMLERALTDKEFAGRRAQWMVAAIRSAVKLRVHHLAGYVPATLQPIMDVRLTKR
Rv2231a LTMACTACPTIWTLRCQTTCSNAFTGEALPHRHPRLAADAVNETRAIVQDVRNSILLSAASAWEIAINYRLGKLPPPEPSASYVPDRMRRCGTSPLSVDHAHTAHRRASGSPSTSIRPCAHRPGTAAWPDDHHRRRPVSCL
Rv1954a MARGRVVCIGDAGCDCTPGVFRATAGGMPVLVVIESGTGGDQMARKATSPGKPAPTSGQYRPVGGGNEVTVPKGHRLPPSPKPGQKWVNVDPTKNKSGRG

The toxicity, allergenicity, and antigenicity of these identified proteins were examined using the ToxinPred server, AllerTop v2.0 server, and VaxiJen v2.0 server, respectively (Table 2). The theoretical pI, aliphatic index, molecular weight, instability index, and GRAVY score were calculated using the Expasy ProtParam software (Table 3). A few epitopes from the selected candidate signature proteins were found to be antigenic, and non-allergenic and exhibited a negative GRAVY value. Illustration of study workflow is summarized in (Figure 1).
Table 2: Antigenicity, allergenicity, and toxicity of four signature proteins used for the development of multi-epitope vaccine using IEDB database.
Protein
Antigenicity score Allergenicity Toxicity
Rv1507a 0.37 Non-allergenic Non-toxic
Rv1509 0.46 Non-allergenic Non-toxic
Rv2231a 0.53 Non-allergenic Non-toxic
Rv1954a 0.80 Non-allergenic Non-toxic

Table 3: Physiochemical properties of signature proteins used for multi-epitope vaccine development (PDBsum server)
Protein pI Aliphatic index Molecular weight Gravy score Instability index
Rv1507a 10.59 81.80 18kDa -0.280 71.58
Rv1509 8.83 92.29 33kDa -0.007 45.94
Rv2231a 9.48 70.07 15kDa -0.408 57.83
Rv1954a 10.04 54.50 10 kDa -0.501 47.75

This was followed by the selection of antigenic, non-allergen, and non-toxic epitope regions from each of the shortlisted proteins. The epitopes obtained from the proteins were aligned in a customized order to achieve a novel construct of a vaccine. The epitopes were selected based on their ability to function as B-cell epitope, human leukocyte antigen (HLA) class I epitope, and human leukocyte antigen (HLA) class II epitope thereby simultaneously generating a B-cell response, CTL response, and HTL response.
HTL and CTL epitopes were assessed for antigenicity using vaxijen server (https://www.ddgpharmfac.net/vaxijen/VaxiJen/VaxiJen.html). Other properties like allergenicity and toxicity of the selected epitopes were analyzed with the help of AllerTOPv.2.0 server (https://www.ddg-pharmfac.net/AllerTOP/) and ToxinPred server (http://crdd.osdd.net/raghava/toxinpred/).
To predict highly antigenic CTL epitopes, an IEDB server was used. For every protein, top 10 non-overlapping epitopes were selected with a low percentile rank score. After that, assessments were conducted for the allergenicity, antigenicity and toxicity of the selected epitopes. From the expected epitope collection, 4 CTL epitopes and 11 HTL epitopes were chosen from four M. tb signature sequence proteins based on the docking of each epitope with the TLR-4 receptor using the HPEPDOCK (Zhou et al., 2018) and MDOCKPEP (Xu, Yan and Zou, 2018) server. Ultimately, 3 CTL epitopes, and 4 HTL epitopes were selected based on their docking score, high antigenic score, lack of allergenicity, and toxicity. Seven T-cell epitopes that met all criteria were used to create the final multi-epitope vaccine (MEV) construct.
For the prediction of B-cell epitopes within the target proteins, antibody epitope prediction tool was utilized available on the Immune Epitope Database (IEDB) analysis website (http://tools.iedb.org/main/bcell/). The retrieved protein sequences were then screened for linear B-cell epitopes. Through the analysis,16-mer epitopes were identified using a neural network with a 0.51 threshold for overlapping filter values. The epitope with the highest score was subsequently selected for the development of the vaccine. Particularly, a total of 4 B-cell epitopes with lengths ranging up to 40 amino acids from the output collection of epitopes were chosen. The number of epitopes was then further reduced to two from four by performing individual docking of epitopes with TLRs, predicting antigenic and allergenicity properties of the selected epitopes. This was done based on high docking scores of epitopes, antigenicity, and non-allergenicity (Table 4).
Table 4: Docking score, antigenicity, allergenicity, and toxicity of selected HTL, CTL, and B-cell epitopes using Cluspro and IEDB webtool.
Epitopes
Docking score with TLR
(kj/mol) Antigenicity Allergenicity Toxicity
IEQSRLSSTRC
B-CELL -209.6 ?G 0.91 Non allergen Non-toxic
RALTDKEFAGRRAQW
B-CELL -236.4 ?G 0.5 Non allergen Non-toxic
RSVSGFVLM
CTL/MHC-I -171.1 ?G 0.52 Non allergen Non-toxic
SAWEIAINY
CTL/MHC-I -178.3 ?G 1.64 Non allergen Non-toxic
VPATLQPIM
CTL/MHC-I -151.2 ?G 0.60 Non allergen Non-toxic
SVSGFVLMIKSASVH
HTL/MHC-II -202.7 ?G 0.62 Non allergen Non-toxic
LARIRSHVDAHAPEL
HTL/MHC-II -215.9 ?G 0.60 Non allergen Non-toxic
PGQKWVNVDPTKNKS
HTL/MHC-II -208.1 ?G 0.58 Non allergen Non-toxic
TSPLSVDHAHTAHRR
HTL/MHC-II -217.6 ?G 0.85 Non allergen Non-toxic




Further, adjuvants and linkers are employed to improve the vaccine's efficacy, longevity, and immunogenicity when developing the vaccine construct. In this study, randomly shuffled epitopes were added, and TLR- 4 adjuvant were added to the C-terminals to create multiple MEV vaccine constructs of Sequence ID NO. 6. In this study, while developing vaccine constructs, several linkers, such as EAAAK, AAY, GPGPG, and KK, were utilized to connect adjuvants, CTL, HTL, and B-cell epitopes (Figure 2). The TLR-4 agonist is laterosporulin in designing the construct which is an anti-TB peptide (https://aps.unmc.edu/database/peptide).
The proposed constructs (MEVs) were also assessed for their allergic, antigenic, and toxicological qualities. It has been determined that vaccines are neither, toxic nor allergenic. (Sequence ID NO. 6)
SEQUENCE ID NO. 6: Vaccine construct
Final epitopes selected for vaccine construction. TLR-4 adjuvant (Laterosporulin) and epitopes are connected through EAAAK (yellow). B-cell epitopes (Green color), CTL epitopes (Pink color) and HTL epitopes (Blue color) are linked using different linkers.
ACVNQCPDAIDRFIVKDKGCHGVEKKYYKQVYVACMNGQHLYCRTEWGGPCQLEAAAKSVSGFVLMIKSASVHGPGPGLARIRSHVDAHAPELGPGPGPGQKWVNVDPTKNKSGPGPGTSPLSVDHAHTAHRRGPGPGRSVSGFVLMAAYVPATLQPIMAAYSAWEIAINYGPGPGIEQSRLSSTRCKKRALTDKEFAGRRAQW
The antigenicity of vaccine constructs was assessed using the VaxiJen v2.0 server (https://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html) with a threshold of 0.5 to. AllerTop server (https://www.ddg-pharmfac.net/AllerTOP/) was utilized to predict the allergenic nature of vaccine designs. This server is an alignment-free method that assesses allergenicity based on the chemical components of vaccines.
Worldwide, there could be small variations in the expression of HLA alleles due to geographical and ethnic variances. Specific CTL and HTL epitopes that were utilized in the development of the vaccine construct, together with the corresponding HLA alleles, were obtained from this analysis for the population coverage analysis using the IEDB server. The statistical findings indicated that the vaccine covers a worldwide population coverage is 89.3%. (Table 5).
Table 5: Information on population coverage of CTL and HTL epitopes combined using IEDB server.
Population MHC-I AND MHC-II class combined
Coverage Average hit Pc90
World 89.32 % 11.85 6.55
Standard deviation 0.0 0.0 0.0

The conformational B-cell epitopes are readily accessible to the solvent and exposed to the surface. They often result from the folding of proteins, and since B-cells and antibodies can readily reach them, they aid in the production of a strong immunological response. The vaccine construct has 5 linear continuous epitopes and 7 discontinuous epitopes. The conformational B cell linear epitopes were determined to have a total of 32, 42, 19, 7, and 8 amino acids residues in the construct with values higher than 0.5 on the ElliPro server. (Table 6).











Table 6: Confirmational B-Cell epitope prediction of MEV using IEDB server
Linear Peptides Residues Score

1. GPGPGPGQKWVNVDPTKNKSGPGPGTSPLSVD 32 0.741

2. ACVNQCPDAIDRFIVKDKGCHGVEKKYYKQVYVACMNGQHLY 42 0.681


3. YGPGPGIEQSRLSSTRCKK 19 0.644

4. ASVHGPG 7 0.559

5. TEWGGPCQ 8 0.503



Example 2: Structure validation and refinement of multi-epitope vaccine construct
The vaccine constructs were analyzed for their secondary and tertiary structures using online tools. The constructs exhibited secondary structural elements predominantly within the permissible regions and successfully passed tertiary structure validation using web-based servers. The 2D structures of the vaccine constructs were analysed using PDBsum server (https://www.ebi.ac.uk/thornton-srv/databases/pdbsum) and the PSIPRED server (http://bioinf.cs.ucl.ac.uk/psipred/). PSIPRED provides information about the type of vaccine being designed and displays charts showing the proportions of aromatic, non-polar, and polar amino acids, as well as alpha-helix, coil, and beta-strand structures. The findings demonstrated that shortlisted MEVs had distinct percentages of a-helix, random coil, and b-strand architectures without any errors. As 3D modeling significantly impacts a vaccine's ability to trigger an immune response and offer protection, it is a critical stage in the design process. The Robetta webserver (https://robetta.bakerlab.org/) was used for the prediction of tertiary structure of the designed vaccine constructs (Figure 4). The 3D models for vaccines were refined and improved in quality using the Galaxy Refine server. ERRAT values in SAVES server and Ramachandran plot evaluation in the Vadar server were used for the quality assessment and validation of selected vaccine models to choose the best models with the highest proportion of favorable areas residues. Since a higher ERRAT score corresponds to higher quality vaccine design, ERRAT values, i.e., of MEV have demonstrated that there are minimal mistakes in protein folding and structure. The Ramachandran plot results showed that our MEV (vaccine construct) had residues in their preferred area as depicted in (Table 7).
Table 7: Information about Ramachandran plot analysis of designed vaccine construct.
Ramachandran statistics Multi-epitope Vaccine
? Disallowed regions 2.5%
? Generously allowed regions 0.0%
? Most favored regions 88.1%
? Additional allowed regions 9.4%

Example 3: Evaluating physiochemical properties of multi- epitope vaccine construct
The physicochemical properties including theoretical isoelectric point (pI), aliphatic index, molecular weight, GRAVY (Grand Average of Hydropathy), instability value, and solubility (https://protein-sol.manchester.ac.uk/), are crucial for vaccine efficacy and stability. Stable vaccines should have an instability index value less than 40. For instance, a high molecular weight can influence vaccine antigenicity, while a low instability index value is indicative of a stable vaccine that can maintain its integrity under various conditions. The aliphatic index, a measure of the aliphatic amino acid content, is correlated with thermostability, ensuring the vaccine remains effective even at elevated temperatures. The GRAVY index, on the other hand, determines the hydrophobicity or hydrophilicity of the vaccine, which can impact its solubility and interaction with biological fluids. Finally, solubility ensures the vaccine can be evenly distributed and absorbed by the body, maximizing its effectiveness. The Expasy ProtPram tool (https://web.expasy.org/protparam/), utilizes the amino acid sequence and pKa values of the vaccine, which provides valuable insights into these physicochemical properties.
The theoretical pI, aliphatic index, molecular weight, aliphatic index, instability index, and GRAVY score of final multi-epitope vaccine construct were calculated using the ProtParam Expasy service (Table 8). The MEV's are of a basic nature since their theoretical pI value is about 9. It was established that our MEV was stable since their score of instability was below 40, which is the ideal number for antigen stability. The aliphatic index, which measures a vaccine's thermostability at body temperature, was 80 for MEV. The MEVs are hydrophilic, meaning they may have an interaction with the water molecules nearby as seen by their negative GRAVY values, which range from 0.15. The solubility of vaccine designs was evaluated using the protein sol server, was predicted to be 0.63 (more than the 0.5 criterion for soluble vaccines), demonstrated that the developed vaccine is soluble (Figure 3). Upon examining the antigenic qualities with VaxiJen 2.0, these epitopes stand out as a promising antigen for designing MEV. AllerTop v2.0 assessed the vaccine for non-allergen characteristics. Based on their physicochemical features, out of three candidates, one construct was chosen that appeared highly antigenic, had high binding affinity with TLR-4, stable tertiary structure, effective, soluble, thermostable, and non-allergenic.
Table 8: Physiochemical properties of the final multi-epitope vaccine construct (PDBsum server).
Pi Aliphatic index Molecular weight Gravy score Instability index Solubility
9.49 66.52 21kDa -0.388 37.61 0.636

Example 4: Evaluation of Structural and Dynamic Features of Multi-epitope vaccine construct using Molecular Dynamic Simulation
The final vaccine construct was subjected to an all-atom MD simulation with the GROMACS software suite in order to determine its structural and dynamical features. The protein structures were calibrated using the GROMOS43a1 force field. In order to reduce artifacts at the solvent-vacuum interface, the vaccine complex was positioned in the center of a cubic simulation box with periodic boundary conditions set at 1.5 nm. To simulate biological conditions, the systems were neutralized with ions, solvated with simple point charge, and water molecules. The steepest-descent approach was used to minimize energy until the maximal force on any given atom was less than 1000 kJ/mol/nm. After minimization, the systems were equilibrated twice: once at 300 K under the NVT ensemble and once at 1 bar under the NPT ensemble. Each equilibration phase ran for 1 ns followed by 200 ns MD simulation with a 2-fs time step. Using the gmx_ trjconv module, the post-simulation processing comprised aligning trajectories and carefully fixing periodicity errors. A thorough series of analyses was carried out to assess the stability and conformational alterations of the TLR-vaccine complex. Multiple studies were performed to evaluate the TLR -Vaccine complex's stability and adaptability throughout time. The gmx_ rmsd tool was utilized to measure the Root-Mean-Square Deviation (RMSD) in order to ascertain the average deviation of the complex atoms from their initial positions. This measurement yielded valuable information regarding the stability of the complex. The gmx_rmsf tool was utilized to conduct the Root-Mean-Square Fluctuation (RMSF) analysis, which reveals areas of structural variability and computes the average displacement of atoms from their starting positions. This enabled an evaluation of the flexibility of various regions within the complex. By analyzing the distribution of atoms around the complex's center of mass, the Radius of Gyration (Rg), which provides insights into folding and structural integrity, was computed using the gmx_ gyrate tool to determine how compact the protein structure was. Lastly, the gmx_ sasa tool was employed to measure the Solvent-Accessible Surface Area (SASA) in order to determine the degree of solvent exposure. (Li et al., 2023).
It was found out through the RMSD analysis, which measures the average distance between the atoms of protein structures, that the conformation of the vaccine construct was steady throughout the whole simulation (Figure 7a), with slight RMSD value fluctuation (avg. RMSD: 0.63 nm for TLR4). Using the data of RMSF, which describes the flexibility of each amino acid residue in the protein, it was quite evident that the vaccine construct was quite structurally stable for a full 200 ns of the dynamic simulation. Therefore, from the above, the results of the MD simulation proved that the vaccine construct remained stable, with some regions showing some flexibility while retaining compact and solvent accessibility of the whole structures. The average RMSD values for the TLR-4 vaccine and showed minor deviations in the initial structures in the initial duration of MD simulation, further suggesting conformational equilibration with only minor deviations during the starting period of the simulation. The RMSF analysis highlighted that region in the constructs with flexibility in many flexible loops and terminal regions portrayed a more significant atomic displacement than relatively stable regions (Figure 7b). Therefore, this difference in flexibility might infer that specific regions within TLR- 4 vaccine complexes are more dynamic and, thus, might contribute to their functional roles. Fundamentally, the Rg analysis looks at how compact the protein is, and it could, therefore, be used to examine how compact the protein structures are. The results show that TLR-4 vaccine construct is compact and relatively stable during the simulation time, for their respective Rg values remain steady, with no indication of unfolding events (Figure 7d) Then, the overall structural integrity of the TLR-4 vaccine constructs was maintained unchanged, as shown in. In addition, the SASA measurements, which assess solvent exposure into both protein complexes, confirm the conformational stability and compactness observed (Figure 7c). Overall, the MD results confirm that the TLR-4 vaccine complex is stable without further conformational changes in the structures. (Table 9)
Table 9: RMSD, RMSF, SASA, and Rg values of TLR-4 and final vaccine construct
Parameters TLR-4 receptor and vaccine complex
RMSD (nm) 0.53
RMSF (nm) 0.23
SASA (nm2) 302.75
Rg (nm) 2.92

Example 5: Evaluating immunogenicity and immune response profile of multi-epitope vaccine construct
The immune response elicited by the selected vaccine construct within a host organism was analyzed using specialized online servers such as utilizing the C-ImmSim tool (https://kraken.iac.rm.cnr.it/C-IMMSIM/index.php) to assess the potential immune response elicited by the designed multi-epitope vaccine construct. This computational tool is capable of simulating the immune system's response to a vaccine, providing valuable insights into the vaccine's efficacy and safety. The C-ImmSim analysis provided valuable information on the potential immune response triggered by the synthetic vaccine. Interestingly, the vaccination stimulated the production of cytokines, including IFN-g, TGF-b, TNF-a, IL-12, IL-6, IL-4, IL-18, IL-10, IL-28 and IFN-b. Targeted antibodies produced by the designed vaccine construct included IgM, IgG, IgG1, and IgG2. Numerous activated B cells were present following vaccination, and these cells formed plasma cells, which are responsible for producing specific antibodies. A discernible rise in B-cell duplication is seen following vaccination. Furthermore, after receiving the vaccine, there was a noticeable increase in the number of activated T-cells and a corresponding decrease in inactive cells, indicating that the cells became active post-vaccination (Figure 8).
Further, the safety profile of the designed vaccine was confirmed by running pBLAST in NCBI database against proteins found in Homo sapiens. It was discovered that there was no resemblance between our multi-epitope vaccine and any human protein. This suggested that the designed vaccine construct would be safe and efficacious to provide and that there wouldn't be any chance of an autoimmune reaction after immunization.
Example 6: In-silico evaluation of the structural and dynamic behavior of multi-epitope vaccine construct with immune receptor (TLR 4)/ TLR-4 vaccine complex
A critical aspect of vaccine development which is its ability to engage with human receptors, such as Toll-like receptors (TLRs). This interaction is pivotal for triggering the production of immunological molecules that stimulate and recruit immune cells, leading to a robust immune response. By initiating a focused immune response against specific pathogens, vaccines can provide long-term protection against recurrent infections. To investigate the interactions between the designed vaccine construct and TLR-4 receptors on human immune cells, the ClusPro 2.0 software was employed (https://cluspro.org/help.php). The interaction of the designed vaccine construct with TLR-4 using the ClusPro server is depicted in (Figure 5). The protein-protein interaction of the vaccine construct with the TLR-4 receptor demonstrated a strong interaction of -17.9?G. (Figure 6).
Example 7: Expression of vaccine construct in Escherichia coli (E. coli)
The protein sequence of the designed vaccine construct was reverse translated, and its codons were optimized using Codon Optimization server (http://www.prodoric.de/JCat). The vaccine construct was expressed in the k12 strain of E. coli bacteria due to its reliability, easy handling, and high yield. The nucleotide sequence of the designed subunit vaccine was used as the input for the expression. During the optimization process, rho-independent transcription terminators, prokaryotic ribosome binding sites and restriction enzyme cleavage sites were avoided. JCat server improves the GC content, codon optimization index (CAI score), and removes incorrect characters of the designed vaccine.
The optimal GC content and CAI value were determined using the Java codon adaptation tool to provide a high production of proteins. Following sequence optimization in this study, the GC content percentage for MEV was 58.01% and the resultant CAI value was 0.75. The absolute GC content ranges from 30% to 70%, and a high score is often defined as a CAI value of 0.8. At last, an in-silico clone of the optimized MEV sequence was created using the SnapGene software. This was accomplished by inserting sequences in the pET28a (+) vector at the XhoI and NdeI restriction sites (Figure 9), which led to a final genome size of 5908 base pairs. , Claims:1. A multi-epitope vaccine construct, said construct comprising epitopes from signature proteins of Mycobacterium tuberculosis and a Toll-like receptor (TLR) agonist.

2. The multi-epitope vaccine construct as claimed in claim 1, wherein the proteins are selected from the group consisting of Rv1507a, Rv1509, Rv1954a, and Rv2231a.

3. The multi-epitope vaccine construct as claimed in claims 1-2, wherein the signature proteins are having an amino acid sequence of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4.

4. The multi-epitope vaccine construct as claimed in claims 1-3, wherein the Toll-like receptor (TLR) agonist is TLR-4 agonist.

5. The multi-epitope vaccine construct as claimed in claim 4, wherein the TLR-4 agonist is laterosporulin agonist of an amino acid sequence of SEQ ID NO.5, and wherein the TLR-4 agonist function as an adjuvant.

6. The multi-epitope vaccine construct as claimed in claims 1-5, wherein the epitopes are connected through linkers selected from GPGPG, EAAK, AAY and KK, and wherein the linkers are arranged to ensure translation of all epitopes of the construct.

7. The multi-epitope vaccine construct as claimed in claim 6, wherein the linker GPGPG is present: between a B-cell epitope and HLA class I epitope, and between two HLA class II epitopes, wherein the linker EAAK is present between Toll-like receptor (TLR) agonist and HLA class II epitope, wherein the linker AAY is present between two HLA- class I epitopes; and wherein the linker KK is present between two B-cell epitopes.


8. The multi-epitope vaccine construct as claimed in claims 1-7, wherein the epitopes of the construct are capable of functioning as B-cell epitope, human leukocyte antigen (HLA) class I epitope and human leukocyte antigen (HLA) class II epitope and thereby simultaneously generating a B-cell response, CTL response and HTL response.

9. The multi-epitope vaccine construct as claimed in claims 1-8, wherein the construct comprises of three human leukocyte antigen (HLA) class I epitopes; four human leukocyte antigen (HLA) class II epitopes; and two B-cell epitopes.

10. The multi-epitope vaccine construct as claimed in claims 1-9, wherein the epitopes in construct are arranged in a consecutive sequence of HLA class II epitopes- HLA class I epitopes- B cell epitopes, and wherein the adjuvant TLR-4 agonist is positioned at N-terminal to the epitopes.

11. The multi-epitope vaccine construct as claimed in claims 1-10, wherein the construct is of an amino acid sequence of SEQ ID NO. 6.

12. The multi-epitope vaccine construct as claimed in claims 1-11, wherein the construct is having a molecular weight of 21 kDa, a theoretical isoelectric point (pI) of 9.49, aliphatic index of 66.52, solubility of 0.63, and instability index of 37.61.

13. A vaccine composition against Mycobacterium tuberculosis, said composition characterized in comprising a multi-epitope vaccine construct as defined in claims 1 to 12.

14. A genetic construct characterized in comprising a nucleic acid sequence encoding a vaccine construct as defined in claims 1 to 12.

15. A vector construct characterized in comprising a genetic construct as defined in claim 14.

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