CpG/Poly (I:C) mixed adjuvant priming enhances the immunogenicity of a DNA vaccine against eastern equine encephalitis virus in mice

• The recombinant plasmids (pcDNA3.1(+)-C-E) were constructed.
• The EEEV VLPs were generated by pcDNA3.1(+)-C-E-transfected 293 T cells.
• CpG/poly(I:C) mixed adjuvant more strongly enhanced the immunogenicity of pcDNA3.1(+)-C-E than CpG or poly(I:C) alone.

Eastern equine encephalitis virus (EEEV) poses a serious public health threat in many countries. Therefore, developing efficient vaccine against EEEV remains an important challenge in the field of disease control. To identify immunogenic proteins in EEEV, we constructed an expression vector containing the protein coding genes C, E3, E2, 6k, and E1 (pcDNA3.1-C-E). After verifying the target gene expression in 293 T cells, we immunized BALB/c mice with the pcDNA3.1-C-E vector as a DNA vaccine in conjunction with either CpG or poly (I:C) or a mixture of both adjuvants and monitored various aspects of the immune response. After two immunizations, the mice vaccinated with antigen plus mixed CpG/poly (I:C) adjuvant exhibited significantly stronger IFN-gamma responses and generated high-level CD4+ cell responses for the cytokines IL-2, IL-4, and IFN-γ and CD8+ T cell responses for the cytokines IL-2 and IFN-γ compared to the mice vaccinated with the corresponding antigen plus CpG or poly(I:C) alone. In addition, the higher antibody titers against EEEV effectively neutralized the EEEV pseudoviruses in the group immunized with antigen plus mixed CpG/poly (I:C) adjuvant after tertiary immunization. This study demonstrates that the pcDNA3.1-C-E plasmids in conjunction with mixed CpG/poly (I:C) adjuvant priming maximize the cellular immune response and specific antibody generation in mice. Moreover, this mixed adjuvant priming provides a promising strategy for enhancing the immune effectiveness of a DNA vaccine against EEEV.