Immunogenicity of trivalent DNA vaccine candidate encapsulated in Chitosan-TPP nanoparticles against EV-A71 and CV-A16

<b>Aim:</b> To develop a trivalent DNA vaccine candidate encapsulated in Chitosan-TPP nanoparticles against hand foot and mouth disease (HFMD) and assess its immunogenicity in mice. <b>Materials &amp; methods:</b> Trivalent plasmid carrying the VP1 and VP2 genes of EV-A71, VP1 gene of CV-A16 was encapsulated in Chitosan-TPP nanoparticles through ionic gelation. <i>In vitro</i> characterization and <i>in vivo</i> immunization studies of the CS-TPP-NPs (pIRES-VP121) were performed. <b>Results:</b> Mice administered with CS-TPP NPs (pIRES-VP121) intramuscularly were observed to have the highest IFN-γ response. Sera from mice immunized with the naked pDNA and CS-TPP-NPs (pIRES-VP121) demonstrated good viral clearance against wild-type EV-A71 and CV-A16 in RD cells. <b>Conclusion:</b> CS-TPP-NPs (pIRES-VP121) could serve as a prototype for future development of multivalent HFMD DNA vaccine candidates. Currently, there are no US-FDA approved vaccine against HFMD. China-FDA approved monovalent HFMD inactivated vaccines were unable to provide cross protection against other enterovirus serotypes. We have developed a trivalent plasmid vaccine candidate, pIRES-VP121 which encodes the VP1 and VP2 genes of EV-A71 and the VP1 gene of CV-A16. We successfully synthesized Chitosan TPP nanoparticles encapsulating DNA Encapsulation of plasmid vaccine candidate pIRES-VP121 through ionic gelation, yielding highly monodisperse nanoparticles with CS-TPP-NPs (pIRES-VP121) with &gt;70% encapsulation efficiency. Encapsulation of plasmid vaccine candidate pIRES-VP121 through ionic gelation successfully formulated sub-200 nm, spherical shaped, mildly cationic (+10 mV) and highly monodisperse CS-TPP-NPs (pIRES-VP121) with &gt;70% encapsulation efficiency. Chitosan NPs encapsulations improved T cell immunogenicity resulting in higher IFN-γ expressing CD4⁺/CD8⁺ T cells population and IFN-γ secretion compared with mice immunized with naked pIRES-VP121. Enhanced B cell neutralizing efficacy were demonstrated by mice sera (1:16 dilution) immunized with CS-TPP-NPs (pIRES-VP121) against the wild-type EV-A71 B4 and CV-A16/N132 strain enteroviruses in comparison with naked pIRES-VP121 immunization. These findings showed the potential of a trivalent DNA plasmid to serve as a prototype for future development of multivalent DNA vaccine candidate against HFMD.