Effects Of Adjuvants On Protection Against Lethal EV71 Infection Elicited By Active Immunization In Suckling Mice And Preparation Of Novel EV71 VLPs

Hand-foot-mouth disease(HFMD)is a common illness in infants and children,which has been circulating in Asia-Pacific region and designed as a category "C" infectious disease.HFMD is mainly caused by Coxsackie virus A16(CA16)and Human enterovirus 71(EV71)infection.CA16 causes self-limited HFMD with few complication diseases,while EV71 infection can cause severe central nervous system complications and even death.The morbidity and mortality of HFMD can be reduced by anti-viral drugs and vaccines against EV71,however,there are not available anti-EV71 drugs and vaccines.Among the developing vaccines,inactivated whole-virus vaccines have been proved to protect infants and children efficiently in phase ? clinical trials.However,inactivated vaccines may cause adverse side effects and virus harmess as a result of survival virus in the inactivation process.So,it's still necessary to develop more efficient and safe vaccines.Virus-like particles(VLPs)can't replicate and reduce the side effects due to the lack of genome,thus it might be a potentially safer vaccine candidates.In terms of efficiency,the highly repetitive antigens and epitopes on the surface of VLPs can induce strong humoral responses.Chung et al reported that EV71 VLPs produced by recombinant baculovirus could induce humoral responses efficiently and confer partial protection in suckling mice by passive immunization.However,due to the lack of RNA encapsidation of EV71 VLPs,the differences in the structure between EV71 VLPs and mature virions may cause the loss of immunogenicity.Meanwhile,baculovirus can infect insect cells only,the difference of protein post-translational modification from mammalian cells may result in the structure differences between EV71 VLPs and empty particles produced in the EV71 life cycle,and lead to the further loss of immunogenicity.So,the aims of this dissertation are as follow:firstly,we compared the effects of different adjuvants on the protection efficiency elicited by VLPs in suckling mice to find a more efficient adjuvant for EV71 VLPs.Secondly,we explored a mammalian expression system to express EV71 VLPs.In the first part,we selected CpG(ODN 1826),Al(OH)3 and Poly(I:C)as different adjuvants,and their effects on the protection elicited by EV71 VLPs were studied.The results were that:1.the protection elicited by EV71 VLPs was dose-dependent,600U VLPs and 400U VLPs can provide protection at the similar level,which is better than that by 100U VLPs.2.CPG and Poly(I:C)can enhance the immunogenicity of EV71 VLPs most efficiently at 10?g and 50?g respectively.3.CPG can enhance the protection more efficiently than the other two adjuvants.These results indicated that CPG is a relatively better vaccine adjuvant for EV71 VLPs,which could guide the adjuvants selection in developing EV71 VLPs vaccines.In the second part,EV71 VLPs were expressed by semliki forest virus(SFV)expression system for the first time.In this study,we produced conditionly infectious recombinant SFV(rSFV)particles by co-transfection of replicon plasmids and helper plasmids that has three amino acid changes surrounding endoprotease cleavage site in the P62(E2-E3)spike glycoprotein.We further optimized the procdure to produce more rSFV particles,and finally expressed EV71 VLPs successfully in BHK-21 cells that were infected by rSFV.This dissertation demonstrated that CPG is a relatively better vaccine adjuvant for EV71 VLPs and EV71 VLPs can be expressed successfully by SFV expression system.These results will be of some significance to develop EV71 VLPs vaccines.