Design Of Recombinant Multi-Epitope Vaccine Of Japanese Encephalitis Virus Envelope Protein And Evaluation On The Immunogenicity In Mice Model

Japanese encephalitis virus (JEV), being a member of the family Flaviviridae, is usually infected to the human body and animal through mosquitoes and then may cause acute viral encephalitic and neurologic disease. Therefore, it is commonly regarded as a serious public health problem, with JEV being the infectious agent in many domestic and wild animals, especially with swine (viral associated abortions). Pig is considered the main vertebrate host and represents an important amplifier and reservoir for JEV. Therefore prevention of JE in swine can benefit for swine production and health of human being. Hence, it is essential to prevent swine JE, which is useful for swine production as well as health of human being.Current JE vaccines for use in swine consist of attenuated or inactivated virus, and there are concerns about the safety and cost of producing and using these products. The World Health Organization has been calling for the development of improved and/or new vaccines for JEV. The envelope (E) protein of JEV is associated with viral binding to cellular receptor(s) and membrane fusion. It is also the major antigen that elicits neutralizing antibodies and protection in hosts.Epitopes are the antigenic determinants, which are recognized by the T-cell receptors and B-cell receptors. T-cell epitopes, as presented in the context of the MHC molecule and recognized by the T-cell receptor, are the minimal essential units that stimulate cellular (T cell) immune responses. B-ell epitopes, as presented on the external surface of antigen and recognized by the B-cell receptor, stimulate humoral (B cell) immune responses. The ideal antigens include T-cell epitopes and B-cell epitopes, which can stimulate complete immune responses, and educe its effects of prevention athd treatments. The strategy of the epitope vaccine design has made greater progress up to day, so epitope vaccine became the emphasis of noval virus vaccines in recent years.In this study, we constructed a multiple-epitope gene using gene recombination technology, which comprising of six B cell epitopes,a CTL epitope and a Th epitope from E protein of Japanese encephalitis virus.Then the multiple-epitope gene was ligated into prokaryotic expression vector, and expressed in E.coli BL21. The Immunogenicity and protective efficacy of the recombinant multi-epitope protein of Japanese encephalitis virus envelope protein was observed in BALB/c mice.The results would be helpful to developed an attractive multi-epitope vaccine with high immunoprotection.The contents of the paper contain two parts as follows:1. Design of multi-epitope gene from Japanese encephalitis virus envelope protein and fusion exprssion in E.coliAccording to the reports of literatures, as well as the construction strategy of multi-epitope vaccine, in the present study, we selected six B cell epitopes from E protein which are able to induce neutralizing antibodies (amino acid residues75-92aa,149-163aa,258-285aa,356-362aa,373-399aa and397-403aa).As well as a CTL epitopes and a Th epitopes (amino acid residues60-68aa and436-445aa). A glycine and a serine residue as a spacer were introduced between two epitopes, which not only minimized junctional epitopes, but also simultaneously augmented proteosome processing. According to the frenquency of E. coli codons,we designed and synthesized ten specific primers, the artificial multiple-epitope gene (designated MEP)was paiallelled as a single chimeric gene with an unique open reading frame by using Splicing by Overlap Extension (SOEing) and polymerase chain reaction (PCR).Then the multiple-epitope gene was cloned to the pMD18-T vector and definited by sequencing analysis. In order to construct the recombinated expression plasmid pET-MEP,the MEP gene was double-digested with EcoRI and Xhol, and followed by ligation into the corresponding restriction sites of pET-28a(+) vector. Then the plasmid was transformed into host bacterium BL21(DE3).After IPTG inducing and spallation with ultrasonic waves,the soluble analysis of expression products were done with SDS-PAGE,and the fusion protein was purified on a His·Bind chelation affinity column,then the purified fusion protein (designated rMEP) was analysis by Western Blotting. The result show that recombinant fusion protein rMEP was expressed by means of inclusion.After purified on a His·Bind, its purify reach to97.2%. Western Blotting indicated that rMEP had specific antigenicity of JEV.2. Immunogenicity and protective efficacy of the recombinant multi-epitope vaccine of Japanese encephalitis virus envelope protein in mice model 4-6weeks old female BALB/c mice were randomly separated into5grouos,10mice per group, mice were intraperitoneally (i.p.) immunized with rMEP、EDⅢ protein、E protein and JEV inactivated vaccine three times at2weeks intervals, and with PBS control. Level of antibody titers,antibody isotypes,neutralizing antibody, cytokine,CTL,as well as mice challenged were detection to evaluate humoral immune and cellular immune responses of rMEP.The results showed immunization with rMEP was able to induce stronger humoral and cellular immune responses than recombinant EDIII protein or E recombinant protein,and protect against lethal JEV challenge in mice. ELISA test of antibody isotypes result indicated that rMEP induced secretion both of IgG1and IgG2, partial to IgG1.The seem result from cytokines detection showed that secretion of both of Thl(IFN-γ and IL-2) and Th2(IL-4), partial to IL-4, these results indicated that rMEP induced a more Th2immune response.In summary, the recombinant multi-epitope from E protein of Japanese encephalitis virus can induce humoral and cellular immune responses, and protect against lethal JEV challenge in mice. So it might be an attractive candidate vaccine to be tested for preventing JEV infection and the research may lead bo a new approach for the development of JE vaccine.