Immunogenicity and immunodominant linear B-cell epitopes of a new DNA-based tetravalent vaccine against four major enteroviruses causing hand, foot, and mouth disease.

Hand, foot, and mouth disease (HFMD) poses a significant public health threat primarily caused by four major enteroviruses: enterovirus 71 (EV71), coxsackieviruses A16, A10, and A6. Broadly protective immune responses are essential for complete protection against these major enteroviruses. In this study, we designed a new tetravalent immunogen for HFMD, validated it in silico, in vivo evaluated the immunogenicity of the DNA-based tetravalent vaccine in mice, and identified immunogenic B-cell and T-cell epitopes. A new tetravalent immunogen, VP1me, was designed based on the chimeric protein and epitope-based vaccine principles. It contains a complete EV71 VP1 protein and six reported neutralizing B-cell epitopes derived from the four major enteroviruses causing HFMD. In silico validation using multiple immunoinformatic tools indicated good attributes of the VP1me immunogen suitable for vaccine development. The VP1me-based DNA vaccine efficiently induced both humoral and cellular immune responses in BALB/cAJcl mice. A combination of in silico prediction and immunoassays enabled the identification of immunogenic linear B-cell and CD8 T-cell epitopes within the VP1me immunogen. Immunodominant linear B-cell epitopes were identified in six regions of VP1me, with one epitope located at the N-terminus of the VP1 protein (aa 9-23) regarded as a novel epitope. Interestingly, some B-cell epitopes could also induce the CD8 T-cell response, suggesting their dual functions in immune stimulation. These results lay the groundwork for further development of VP1me as a new vaccine candidate.