Evaluation Of Recombinant Subunit ZIKV Vaccines In Non-human Primates And Investigation Of Mechanism Of Protective Immunity

Zika virus(ZIKV)is an arbovirus that belongs to the Flavivirus genus,Flaviviridae family.The worldwide spread and the damage to human health of ZIKV highlights the importance of ZIKV prevention.The fact that ZIKV can be transmitted by mosquitos,as well as mother-to-child transmission and sexual transmission suggests that a safe and effective ZIKV vaccine is much needed to prevent the virus.To date,no drugs or vaccines specific for ZIKV are available;however,several vaccines are under development,including attenuated and inactivated vaccines,DNA and mRNA vaccines,and protein subunit vaccines.The information from ZIKV studies and flavivirus vaccine studies is helpful for ZIKV vaccine development.In DENV studies,researchers found that antibodies elicited by infection and immunization can enhance viral infection by antibody-dependent enhancement response.ZIKV has similar structure,sequence and distribution with DENV,and antibodies elicited by ZIKV can enhance the DENV infection,suggesting that an ideal ZIKV vaccine should prevent ZIKV infection and should not enhance DENV infection.We are working on the development of recombinant subunit ZIKV vaccine,because this vaccine platform is high safe,and it is suitable for immunogen design/optimization study.We designed ZIKV EDⅢ and E80 proteins,the former contains the third domain of envelope protein and the latter contains the entire ectodomain domain of envelope protein.In mouse studies,we have reported that both immunogens could induce neutralizing antibody response and antigen-specific T-cell response,and inhibited the replication of ZIKV upon viral challenge in vivo.These results indicate that EDⅢ and E80 are potential candidates for recombinant subunit ZIKV vaccine.In the current project,we further evaluated the immunogenicity and protective efficacy of EDⅢ and E80 proteins in nonhuman primates,and investigated the mechanisms of vaccine-induced protective immune responses.The results demonstrated that both immunogens induced antigen-specific antibodies and cellular immune responses.At 2 weeks after the last immunization,antibodies induced by both proteins could bind to ZIKV virion and inhibit ZIKV infection in plaque reduction neutralization test,suggesting that the immune responses induced by EDⅢ and E80 should be capable of in vivo protection.Thus,we then preformed an in vivo protection experiment.Unexpectedly,results showed that the protective efficacy of EDⅢ and E80 was distinctly different,as that E80 immunization inhibit ZIKV infection in monkeys whereas EDⅢ immunization did not inhibit the infection.To investigate the reason of differential protection of EDⅢ and E80,we then compared several properties of the antibody response induced by the non-protective EDⅢ protein with the protective E80 protein.We verified that E80 immunization suppressed the replication of ZIKV in monkeys,and EDⅢ immunization significantly enhanced the viremia.The results of in vitro ADE assay indicated that ADE activities of antibodies elicited by two proteins were not significantly different,that monkey sera from both groups could enhance ZIKV infection when they were diluted to low concentration.Considering the 2-weekinterval between blood sample collection and ZIKV injection,we compared the decrease of antibodies induced by two proteins.Results showed that the durability of virion-binding antibodies induced by EDⅢ protein was less than that of E80 protein,leading to the significant lower neutralizing activity in EDⅢ monkeys when they were challenge with ZIKV.This phenomenon may explain why E80 was more potent than EDⅢ as an immunogen.The high antigen-binding titer and low virion-binding titer of EDⅢ sera indicate that the antibody response elicited by EDⅢ protein was dominated by epitopes that were not exposed on virion surface.We thus investigated the epitopes of antibodies.Results revealed that the antibodies bound to multiple non-neutralizing epitopes that cover a large area of EDⅢ protein but are not well-exposed on the virus surface.This phenomenon suggests that the low exposure on virion limit the function of EDⅢI protein.We also investigated the targets of E80-incuced antibodies.Results indicated that EDI/II region was the major target of these antibodies.In peptide-based epitope mapping,E80-induced antibodies bound to multiple exposed EDI/II epitopes and only few non-neutralizing EDⅢ epitopes.These data implies that it was EDI/II of E80 that conferred monkey protection against ZIKV.Because of the genetic and structural similarity between ZIKV and DENV,we also tested the cross-reactivities of antibodies to DENV.Results showed that only E80-induced antibodies could cross-reacted with DENV,with both neutralizing and enhancing activities.These data revealed advantages and potential risks of the E80 immunogen,suggesting further optimization of this protein to provide broadly protection.In conclusion,we evaluated ZIKV EDⅢ and E80 proteins as candidate vaccines in non-human primates.The results not only revealed that E80 was a more promising vaccine candidate,but also indicated that the quantity,quality and durability of virion-binding antibodies induced by the immunogen are all critical for a successful ZIKV vaccine,This study is of great significance to the development of ZIKV vaccine,the adequate evaluation of ZIKV vaccine,and the vaccine design of other similar virus.