Study On The Oral Vaccine Of Enterovirus71

Hand, Foot and Mouth disease (HFMD) generally describe diseases characterized by fever, rash and herpangina, which is mainly caused by enterovirus71(EV71). HFMD typically affects infants and children. Some patients present with neurological manifestations including acute flaccid paralysis, aseptic meningitis and brainstem encephalitis, and systemic features such as shock and pulmonary edema, which usually indicate a high mortality. The Southeast-Asia region was affected heavily by HFMD. In1990s, a series of outbreaks happened in Malaysia, Taiwan, Singapore, Japan and Vietnam and so on. The latest large Asian-Pacific epidemic was in China in2008, with around490,000infections and126children died. Nowadays, the specific treatment for HFMD is still limited, and there is no effective vaccine for prevention. Therefore, to develop effective vaccine is important and urgently needed to control HFMD.EV71virus is mainly transmitted through the "fecal-oral" route or the respiratory route. Thus, it is feasible to induce mucosal antibodies and cellular immune responses by mucosal immunization to block viral infection. In addition, mucosal immunization is more secure, economical, acceptable and easier for large-scale vaccination, which makes that scientist showed much more interest and attention to the mucosal vaccination strategy in the recent years. It has been reported that, the main linear neutralizing epitopes and CD4+T cell epitopes of EV71were both located on the VP1protein. Intramuscular immunization of VP1protein can induce a protective immune response, suggesting that VP1is a potential subunit vaccine. Therefore, the oral route for immunization was used in this study to investigate the immunogenicity and protective efficacy of VP1oral vaccine.In this study, C4subtype, the EV71prevalent virus strain in China, was used as (?)accine strain. VPl protein was expressed by E. coli expression system and then (?)cntified by SDS-PAGE analysis which showed the molecular weight of recombinant VP1was around36KD, similar to that of natural virus VP1protein. Western blot was also performed with specific VP1monoclonal antibodies for further identification.(?)e formulation was prepared by mixing the purified VP1protein with chitosan solution. The immunogenicity and protective efficacy of developed oral vaccine were evaluated in rabbits and mice model, respectively.The immunogenicity of oral vaccine was evaluated in New Zealand rabbit. The results showed that the immunogenicity of the oral vaccine was satisfied. Specific IgG antibodies could be detected in all vaccine-immunized groups, among which high dose-immunized group induced IgG antibodies up to1:10240. In the adjuvanted high dose-immunized group and low dose-immunized, oral vaccine elicited neutralizing antibodies, the highest titer of which was1:16. Specific IgA antibodies were detected in the small intestine, vaginal, nasal, pulmonary mucosa, besides, in the small intestinal surface specific IgG antibodies were also detected. After stimulation of spleen cells in vitro, the cytokine IFN-y and IL-4in culture supernatants were investigated and showed significant elevation, suggesting that oral immunization of VP1oral vaccine can induce cellular immune response.The immunogenicity and protective efficacy of the oral vaccine were further evaluated in ICR mouse. The results showed that oral delivery of the prepared vaccine based on VP1protein stimulated specific IgG antibodies and neutralizing antibodies in ICR mice, and in the adjuvanted high dose-immunized group neutralizing antibodies were induced with titer of1:8. Mucosal specific IgA and IgG antibodies were detected in the small intestine, respiratory tract and vaginal washings, and feces. After stimulation of spleen cells in vitro, the cytokine IFN-y, IL-4and TGF-β in culture supernatants were investigated and showed significant elevation, indicating that the oral vaccine could induce immune response of Thl (IFN-γ), Th2(IL-4) and Th3(TGF-β) type. To evaluate the protective effect of specific mucosal antibodies induced by oral vaccine, the immunized mice were challenged with EV71virus in oral route. Although no apparent clinical manifestation was observed, shedding virus load and time in the immunized mice was significantly lower and shorter, respectively, than that in the control group, indicating that the oral vaccine could prevent the EV71infection through mucosal surfaces. In addition, the effect of maternal antibodies to protect suckling mice was also evaluated. The results showed that maternal antibodies could protect neonatal mice from103PFU virus challenge, and the suckling mice born to dams in high dose-immunized group showed a protective efficacy of30.0%.In conclusion, EV71VP1protein was successfully expressed and purified using E. coli expression system. Immunogenicity and protective efficacy of prepared oral vaccine based on VP1protein were preliminary studied in New Zealand rabbit and mouse models. The results showed that oral delivery of the vaccine induced specific humoral and cellular immune responses; the mucosal specific antibody could protect mice against EV71challenge through oral route; the maternal transmitted antibody could protect the neonatal mice against lethal challenge. Our study laid foundation for the EV71oral vaccine research, and further research could focus on the optimization of antigen component and adjuvant type to improve immunogenicity.