Construction Of A Chimeric Neisseria Meningitides Epitopes Vaccine Combined With Hepatitis B Virus Core Protein And Evaluation Of Its Immune Effects

Objective: Bioinformatics software was used to screen the dominant epitope peptides of Neisseria meningitidis serogroup B surface protein A(NspA)of MC58 strain.The selected epitope peptide was inserted into the HBc-N144 to construct the recombinant epitope vaccine HBc-N144-epitope(1+2),investigate both cellular and humoral immune response,especially the level of the mucosal immune response induced by HBc-N144-epitope(1+2),and evaluate the immuno-protective effect of the epitope vaccine in mice immunized by intramuscular or intranasal route,so as to search and screen more functional epitopes of NspA and provide the relevant experimental basis for the development of efficient vaccine candidate against Neisseria meningitides serogroup B.Methods:1.Construction of a recombinant HBc-N144-epitope(1+2)prokaryotic expression plasmid.The NspA gene sequence of MC58 strain was obtained in GenBank.Bioinformatics websites and tools were used to predict the dominant epitopes of NspA,and two fragments selected from NspA were inserted into the major immunodominant region(MIR)between 78-79 of HBc-N144 DNA sequences to create recombinant plasmid pET28a-HBc-N144-epitope(1+2).2.Preparation of a HBc-N144-epitope(1+2)vaccine.E.coli BL21 strain containing the fusion plasmid pET-28a-HBc-N144-epitope(1+2)was cultured,the induced and expressed recombinant protein was purified by nickel column affinity chromatography and then identified by Western Blot.The formed virus-like particles of purified protein was detected and observed by the negative staining transmission electron microscopy(TEM).3.Evaluation of the immune effect of a HBc-N144-epitope(1+2)vaccine.Age of 3 weeks BALB/c mice were injected intramuscularly or intranasally,the levels of serum specific IgG,IgG1,IgG2 a and secretory IgA were determined by ELISA,Th1(IFN-?),the Th2(IL-4)and Th17(IL-17A)cytokines in the supernatant of immuno-splenocytes were detected by ELISA.Meanwhile,the subsets of Th1 and Th2 cells in the immuno-splenocytes were analyzed by flow cytometry.Two weeks after the final immunization,a lethal dose of MC58 bacterial solution of Neisseria Meningitidis serogroup B was prepared and an attack experiment was conducted to record the protection rate of the HBc-N144-epitope(1+2)vaccine.The bactericidal activity of serum antibody in vitro was carried by SBA(serum bactericidal activity)using human serum as complement sources.4.Observation of the inflammatory cells infiltration at the site of vaccination.Muscle tissues at the inoculation site of intramuscularly immunized mice and lung tissues from the intranasally immunized mice were collected for pathological sections to assess the inflammatory cell infiltration.Result:1.Epitope vaccine of HBc-N144-epitope(1+2)virus-like particle was constructed successfully.TEM results showed that HBc-N144-epitope(1+2)was formed a virus-like particles structure with diameter about 30 nm.2.HBc-N144-epitope(1+2)vaccine could elicit specific anti-NspA antibodies levels in immunized mice intramuscularly or intranasally,and the antibody level showed a rising trend with the increase of the immunization times.(1)The serum specific IgG level in both intramuscular and intranasal immunized mice injected with HBc-N144-epitope(1+2)was significantly higher than that of the NspA,HBc-N144 and PBS groups(p <0.05),while there was no significant difference between the HBc-N144-epitope(1+2)and the HBc-N144-NspA group(p >0.05).The specific IgG1/IgG2 a ratios in the HBc-N144-epitope(1+2),HBc-N144-NspA and NspA group were all greater than 1.(2)The level of specific sIgA in the genital secretions and alveolar lavage fluid of both intramuscular and intranasal immunized mice injected with HBc-N144-epitope(1+2)was significantly higher than that of the HBc-N144 and PBS groups(p <0.01),of which the HBc-N144-epitope(1+2)group was significantly higher than that of the NspA group(p <0.05),while there was no significant difference between the HBc-N144-epitope(1+2)and the HBc-N144-NspA group(p >0.05).(3)The flow cytometry result indicated that both intramuscular or intranasal injection with HBc-N144-epitope(1+2),HBc-N144-NspA and NspA could induce Th2 tendentious immune response.The cytokines secreted in the splenocytes supernatant were detected by ELISA,the result showed that IL-4,IFN-?and IL-17 A cytokines produced by the HBc-N144-epitope(1+2)and HBc-N144-NspA group were higher than those by the NspA group(p <0.05),and there was no significant difference between the two groups(p >0.05).3.Immune protection effects :(1)Survival rate.Intramuscular injection : The survival rate of HBc-N144-epitope-(1+2),HBc-N144-NspA and NspA group within 72 h was 90%,90% and 80% respectively;the survival rate of the HBc-N144 and PBS group within 48 h was 0.Intranasal injection : The survival rate of HBc-N144-epitope-(1+2),HBc-N144-NspA and NspA group within 72 h was 85%,85% and 70% respectively;the survival rate of the HBc-N144 and PBS group within 48 h was 0.(2)Complement mediated bactericidal activity of immune serum in vitro.Intramuscular injection : The SBA(serum bactericidal activity)of HBc-N144-epitope(1+2),HBc-N144-NspA and NspA groups at 42 d was 1:16,1:16 and 1:8 respectively;one month after the last immunization,giving a boost immunization,the SBA of HBc-N144-epitope(1+2)group was up to 1:32;Intranasal injection: The SBA of HBc-N144-epitope(1+2),HBc-N144-NspA and NspA group at 42 d was 1:8,1:8 and 1:4 respectively;after giving a boost immunization,the SBA of HBc-N144-epitope(1+2)group was up to 1:16.In addition,the results of pathological sections showed that no inflammatory cell infiltration at the injection site was observed in immunized mice intramuscularly with HBc-N144-epitope(1+2),HBc-N144-NspA,NspA and HBc-N144 and that scattered and small quantities of inflammatory cell infiltration in the lung tissue was observed in immunized mice intranasally with HBc-N144-epitope(1+2),HBc-N144-NspA,NspA and HBc-N144.Conclusion:1.The constructed HBc-N144-epitope(1+2)vaccine has better immunogenicity,which can elicit a robust specific cellular and humoral immune response,including mucosal immunity.2.The chimeric epitope vaccine HBc-N144-epitope(1+2)has a strong protective effect on experimental mice infected with Neisseria MC58 and SBA and survival rate of experimental mice in HBc-N144-epitope(1+2)group were obviously higher than that in NspA group.