Differential immune responses elicited by two tetravalent epitope-based DNA vaccines differently designed for human hand-foot-and-mouth disease

Hand-foot-and-mouth disease (HFMD) remains a significant public health concern for children. Enterovirus A71 (EV71) and coxsackievirus A16 (CVA16) have been the main causes of HFMD; however, CVA10 and CVA6 have become more prevalent in recent years. A universally effective vaccine that covers all four major enteroviruses is of importance. In this study, we developed and assessed the immunogenicity of two differently designed tetravalent DNA-based HFMD vaccines in a murine model. The new immunogen, EVCVAme, was designed with a string of reported B-cell epitopes from the four enteroviruses, while VP1EVCVAme was designed with the full-length EV71 VP1 fused with a string of reported B-cell epitopes from the four enteroviruses. In silico analysis indicated favorable structural and functional features of both immunogens. The immunization of mice with DNA vaccines expressing those two immunogens elicited HFMD-specific antibody responses, but the IgG levels were lower compared to the immunization with our previous tetravalent vaccine pVAX1.VP1me. Interestingly, only the vaccines containing full-length VP1 elicited detectable enterovirus-specific CD8 T cell response. Intracellular cytokine staining revealed polyfunctional vaccine-activated CD8 T cells upon restimulation with mixtures of the B-cell epitope peptides, indicating a dual role of these B-cell epitopes. Notably, co-administration of two vaccines neither improved the breadth nor magnitude of the immune responses. B-cell epitope mapping revealed different patterns of responding epitopes across the vaccine groups. Importantly, pVAX1.EVCVAme elicited antibodies that cover all four enteroviruses. This study offers valuable insights and important information for the improved design of a next-generation tetravalent vaccine for HFMD.