A recombinant fusion protein consisting of West Nile virus envelope domain III fused in-frame with equine CD40 ligand induces antiviral immune responses in horses

•A subunit vaccine consisting of the E glycoprotein domain III fused in-frame to CD40L generates robust immune responses against West Nile virus (WNV).•The CD40L-WNV-DIII protein generated high anti-DII specific antibody and strong neutralization titers against WNV.•The CD40L-WNV-DII subunit vaccine stimulated CD4 + T amd CD8 + T cellular proliferation.•CD40L could be utilized as a non-toxic, alternative adjuvant to boost the immunogenicity of subunit vaccines in horses.

West Nile Virus (WNV) is endemic in the US and causes severe neurologic disease in horses since its introduction in 1999. There is no effective pharmaceutical treatment for WNV infection rendering vaccination as the only approach to prevention and control of disease. The purpose of this study was to evaluate a recombinant vaccine containing domain III (DIII) of the WNV envelope glycoprotein with and without a natural adjuvant equine (CD40L) in producing virus neutralizing antibodies in horses. Serum IgG1 concentration in the groups of horses vaccinated with the DIII-CD40L + TiterMax and DIII-CD40L proteins were significantly increased (p < 0.05) after the second booster vaccination compared to other groups. Serum IgG4 and IgG7, IgG3 and IgG5 concentrations were not significantly increased among all groups. Western blot results showed that animals immunized with the DIII-CD40L protein (with or without TiterMax) exhibited the highest specific anti-DIII antibody activities after vaccinations. Moreover, animals immunized with the DIII-CD40L protein (with or without TiterMax) exhibited significantly stronger neutralization activity (p < 0.05) compared to other groups starting at week eight. The DIII-CD40L protein (with or without TiterMax) stimulated more CD8+ T cells, but not CD4+ T cells in equine PMBCs. The results demonstrated that vaccination with recombinant WNV E DIII-CD40L protein induced superior humoral and cellular immune response in healthy horses that may be protective against WNV-associated disease in infected animals. CD40L could be utilized as a non-toxic, alternative adjuvant to boost the immunogenicity of subunit vaccines in horses.