Evaluation Of The Immune Effect Of EvV71VP1Combined With ETEC LTB Via Intranasal Route

Hand-foot-mouth disease (HFMD) is caused by enterovirus infectionresulting in multiple complications, and even death. There are two commonenteroviruses: enterovirus type71(EV71) and coxsackievirus A16(CA16).Viral protein1(VP1) as a toxic protein of EV71plays crucial roles in HFMD.VP1also is a major antigen and neutralization epitope of EV71, and becomesa candidate antigen for developing epitope vaccine and subunit vaccine ofEV71.Enterotoxigenic Escherichia coli (ETEC) secretes two kinds of toxicproteins: heat-stable enterotoxin(ST） and heat-labile enterotoxin(LT).Research indicates that B subunit of LT is an effective mucosal immuneadjuvant. It can induce strong mucosal immune response together with otherspecific antigen.Objects: To detect the immunogenicity of Enterotoxigenic Escherichiacoli heat-labile enterotoxin B subuni（tLTB）and evaluate the immune effect ofenterovirus71(EV71) capsid protein VP1together with LTB by intranasalroute. Methods:（1） The LTB gene was cloned into the prokaryoticexpression vector pET32a.The recombinant plasmid pET32a-LTB wastransformed to E．coli BL21(DE3) and induced with0.5mmol／L IPTG．（2）The expressed fusion protein6×His-LTB was purified by protein purificationkit with Ni-Agarose His-tag and refolded.(3) New Zealand white rabbits wereimmunized with the fusion protein by subcutaneous injection at several sites，at a dosage of300μg，for3times each at an interval of2weeks，of which thesera were collected7d after the last immunization and determined for titer byELISA.(3) BALB/c mice were immunized with EV71VP1alone or EV71VP1combination with LTB via intranasal drip respectively. The sera as wellas lung and intestinal mucosa washing were collected and determined for IgGand sIgA by indirect ELISA.Results:(1) PCR，restriction analysis and sequencing proved thatrecombinant plasmid pET32a-LTB was constructed successfully．(2) Theexpressed fusion protein6×His-LTB mainly existed in a form of inclusionbody with a relative molecular mass of about30kD. The product reached apurity of95％and a concentration of2.12mg／ml. The vaccination induceda serum antibody titer of1∶125000in rabbits．(3) The level of specific IgGand sIgA of mice immunized with EV71VP1plus LTB were significantlyhigher than those in EV71VP1alone and control group.Conclusion: Fusion protein6×His-LTB was successfully expressed inE.coli BL21(DE3) and showed good immunogenicity. LTB can efficiently enhance specific serum antibody response and induced mucosal immuneresponse by intranasal vaccination. The results may lay a foundation fordeveloping high efficiency and safety vaccines of EV71.