| Summary: | 碩士 === 國立臺灣大學 === 口腔生物科學研究所 === 94 === Enterovirus 71 has been the most important enterovirus to cause hand-foot-and-mouth disease accompanied with neurological complication. EV71, like the poliovirus, belongs to the Picornaviridae family and there are four kinds of structural proteins, VP1-VP4, to assemble the virions. It is generally considered that VP1 is the most important antigenic determinant of EV71. Therefore, previous studies on the EV71 vaccine most focused on the inactivated virus and VP1, but inactivated virus vaccine had better protection than VP1 subunit vaccine. In addition, studies on poliovirus showed that three to four neutralizing antigenic sites have been described, involving residues of all three major structural proteins, VP1-VP3.
Based on the reasons above, we want to identify neutralizing epitopes of EV71 Caspid, other than VP1, to improve vaccine. We purified other recombinant caspid proteins, other than VP1, to immunize BALB/c mice accompanied with CFA/IFA or cholera toxin as adjuvant. We demonstrated the VP0 (VP0 is the propeptide of VP2 and VP4) and VP3 can induce specific antibodies. In the neutralization test, we also found that anti-VP0 and anti-VP3 serum can protect RD cell against virus infection as well as anti-VP1 serum. Further, serum samples from EV71-immunized mice had the best neutralization effect.
About the antigen saturation study, only inactivated virus could block neutralization effect of anti-virus serum. We suggested that denatured VP1, VP3, and VP0 might lose most of conformational epitopes during the purification and had low affinity with anti-virus serum. In the other hand, we estimated the component of anti-virus serum by ELISA and we found that VP1-specific antibody was more than VP3 and VP4-specific antibody.
Then, we combined different structural proteins with different ratio to immunize BALB/c mice via nasal route for the reason that induction of mucosal immunity to protect virus infection in the first line of defense. The results showed that only inactivated virus can elicit great magnitude of immune response.
In conclusion, antibodies induced by EV71 capsid proteins, other than VP1, had neutralization effect and whole virus could induce the best neutralizing antibodies and mucosal immunity. We estimated components of anti-virus serum by ELISA and considered that VP1 might be more important neutralizing epitopes in virus infection. We considered that intact virus is the proper candidate of the EV71 vaccine development. Thus, we will design enterovirus-like particle for development of mucosal vaccine.
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