Abstract
Enterovirus 71 (EV71) and coxsackieviruses (CV) are the most common agents of hand, foot and mouth disease (HFMD). Although many factors participating in enterovirus 71 (EV71) entry and replication have been found, the precise mechanisms associated with these events are still unclear. In this study, our team showed that heat shock protein 90 (HSP90) plays a key role associated not only with EV71 replication but also with entry into human rhabdomyosarcoma (RD) cells. Reduction of HSP90 by adding HSP90β siRNA to RD cells prior to infection or blocking HSP90 with a HSP90-specific antibody or with geldanamycin (GA; a specific inhibitor of HSP90), as well asby the addition of recombinant HSP90β, resulted in inhibition of viral entry and subsequent viral replication. A co-immunoprecipitation assay showed that HSP90β can physically associate with EV71 particles.HSP90 appeared to mediate EV71 replication by preventing proteosomal degradation of the newly synthesized capsid proteins but did not influence the transcription of viral genes. This result was confirmed by the post-treatment of host cells with GA, which did not affect the expression of viral transcripts but accelerated the degradation of viral capsid proteins and interfered with the formation of assembled virions. For in vivo experiments, we used human SCARB2-transgenic mice to evaluate the protection conferred by the HSP90 inhibitor17-AAG,which elicited a similar activity to GA but with less toxicity. The results showed that two administrations of 17-AAG can help hSCARB2 mice reduce symptoms after challenge with the C2 and B4 genotypes of EV71. These data supported the hypothesis that HSP90 plays an important role in EV71 infection and that the targeting of HSP90 with clinically available drugs may provide a feasible therapeutic approach for treating EV71 infection. In contrast, there is currently no vaccine available against HFMD. Our team has designed an adenovirus-based EV71 VLP vaccine (Ad-EVVLP) with the EV71 P1 and 3CD genes inserted into a recombinant adenoviral genome.P1, digested by the 3CD protease,and virus-like particles (VLPs),produced in HEK-293A cells, were assessed by transmission electron microscopy and western blotting. Immunogenicity studies in mice showed that the antisera against Ad-EVVLP can neutralize the EV71 B4 and C2 genotypes. Ad-EVVLP was also shown to induce the activation of VLP-specific CD4+ and CD8+/IFN-γ T cells associated with Th1/Th2-balanced IFN-ɣ, IL-17, IL-4, and IL-13; in contrast, FI-EV71 induced only Th2-mediated neutralizing antibody against EV71 and low VLP-specific CD4+ and CD8+ T cell responses. Using the hSCARB2 transgenic (hSCARB2-Tg) mice as a challenge model, the antiviral immunity against EV71 was clearly demonstrated in mice vaccinated with Ad-EVVLP. Ad-EVVLP-vaccinated mice were 100% protected and demonstrated reduced viral loads in both the CNS and muscle tissues. Interestingly, anti-CVA16 immunity was successfully induced by Ad-EVVLP. The 3C-specific CD4+and CD8+/IFN-γ T cells were identified, which could mediate protection against a CVA16 challenge, although the antisera had no neutralizing activity against CVA16.In contrast, FI-EV71 had no efficacy against the CVA16 challenge and did not induce 3C-mediated immunity. These results allowed us to conclude that Ad-EVVLP can induce neutralizing antibodies and protective cellular immune responses to prevent EV71 infection and cellular immune responses against CVA16 infection.