Reverse Vaccinology Approach To Design A Multi-epitope Subunit Vaccine And Immune Detection Of M-Ag85B Against Tuberculosis

Tuberculosis(TB),mainly caused by Mycobacterium tuberculosis(M.tuberculosis),is a severe infectious disease with devastating effects on global public health.The only used vaccine BCG could provide protection against severe TB in the childhood,but the protective efficacy waned along growing up and has limited protection against TB in adult.Novel vaccines against TB including subunit vaccines are being developed.The traditional tuberculosis subunit vaccine mainly induces cellular immunity.However,in recent years,the researchers have paid attention to reverse vaccinology and also to mucosal immunity and antibody-mediated protective immune response.Therefore,it is urgently warranted to develop a novel safe and effective TB subunit vaccine as a supplement to the traditional vaccine.Reverse vaccinology represents a major advance in the strategies to identify vaccine candidates.The evaluation of vaccine antigens discovered by Reverse vaccinology offers the possibility to explore the protective capabilities of proteins that cannot be identified with traditional techniques.In this study the highly immunogenic,antigenic and secreted fibronectin-binding proteins(Fbps),which are essential for maintaining the integrity of the mycobacterial cell envelope Ag85 B antigen was used for the development of novel TB candidate subunit vaccine.The immunological characteristics of tuberculosis novel subunit vaccine were analyzed through intranasal immunization.The thesis can be divided into two parts.In the first part we applied advanced computational techniques to develop a novel multi-epitope subunit vaccine against tuberculosis.In the second part,we constructed a single protein mutant Ag85B(m Ag85B)emulsified with different adjuvants to induce mucosal immunity against M.tuberculosis.1.In silico analysis of epitope-based vaccine candidate against tuberculosis using reverse vaccinologyWe applied advanced computational techniques to develop a universal TB vaccine.In the current study,we apply the Reverse vaccinology approach to design a multi-epitope(B-cell and T-cell)subunit vaccine against tuberculosis from very conserved,experimentally confirmed Mycobacterium tuberculosis antigens,including Rv2608,Rv2684,Rv3804c(Ag85A),and Rv0125(Mtb32A).The epitopes were linked with each other by linker.Different servers were used to predict various physicochemical properties,protein solubility and secondary structures of the protein.Bioinformatics tools were used to predict,refined,and to validate the 3D structure and then docked with toll-like-receptor(TLR-3)and did in silico cloning,codon optimization and immune stimulation.Four B cell epitopes,four HTL epitopes,and ten CTL epitopes were selected to design a novel vaccine,fulfilling the criteria of binding affinity,antigenicity,non-toxicity,and non-allergenicity.The vaccine sequence on different servers was estimated to be antigenic and non-allergenic.The final protein’s molecular weight was estimated to be 31.9 k Da.The instability index value was 25.51,suggesting the protein is extremely stable.The protein is highly soluble upon expression.Secondary structure prediction of vaccine having(20.0%)alpha-helices,(21.0%)beta-strands,and(58.0%)coils.The codon optimization index(CAI)value was predicted 1.0,and the average GC content was 59.2%,which indicates high expression in the Escherichia coli(E.coli)host.The in silico immune simulation results showed significant response for immune cells.This research also sets out a good sign for the design of a peptide-based tuberculosis vaccine.Our findings show that the known multi-epitope vaccine may activate humoral and cellular immune responses and maybe a possible tuberculosis vaccine candidate.Therefore,more experimental validations should be exposed to it.2.The construction of mutant Ag85B and evaluation of immune responses by intranasal vaccination in C57BL/6 mice modelWe introduced point mutation in Ag85B to get the Ag85B protein in soluble state.Prokaryotic expression plasmid p ET30a-m Ag85 B was constructed and induced by isopropyl-β-D thiogalactopyranoside(IPTG)and purified from the inclusion bodies by dialysis method.The m Ag85 B was emulsified in an adjuvant composed of adjuvant aluminum hydroxide/polyinosinic-polycytidylic acid [poly(I: C)](AP)and N,N’-Dimethyl-N,N’-dioctadecyl ammonium bromide(DDA)/poly(I: C)(DP).C57BL/6 mice were immunized intranasally three times with a 2-week interval.Mice immunized with BCG and PBS were used as a control.The level of specific Ig G,Ig G1 and Ig G2 c in serum and secretory Ig A(s Ig A)antibodies were detected by ELISA after five weeks from last immunization.The levels of IFN-γ secreted by spleen lymphocyte were evaluated in vitro with stimulation by specific antigen m Ag85 B and Mycobacterium.tuberculosis purified protein derivative(PPD).The immunological characteristics of different subunit vaccines with different compound adjuvants were analyzed.We have cloned,expressed,purified,and evaluate the immune responses of the mutant mycobacterial antigen 85 B in mice.The m Ag85 B protein was highly expressed in the E.coli BL21 and purified on dialysis method.The m Ag85 B showed strong immune-stimulating properties by inducing high cytokine levels interferon-gamma(IFN-γ),potent antibodies immunoglobulin G(Ig G)subtypes i.e.,Ig G1,Ig G2 a,Ig G2 b and secretory Ig A(s Ig A)responses in immunized mice.The immunogenicity of m Ag85 B with the stimulation of either m Ag85 B or PPD,the levels of IFN-γ,Ig G subtypes and s Ig A from mice immunized with m Ag85 B were higher than that from BCG or PBS controls(p < 0.05).The mAg85 B protein mixed with adjuvants aluminum hydroxide and poly(I:C)(AP)and DP by intranasal vaccination can induce antigen-specific cell immunity.therefore,it can effectively be applied in TB vaccine researches.