Abstract
The ongoing global monkeypox outbreak since 2022 has highlighted the urgent need for vaccine development. Current vaccination strategies rely on cross-protective immunity provided by orthopoxvirus-based live-attenuated vaccines. However, these vaccines not only exhibit suboptimal efficacy against monkeypox virus (MPXV) but also raise safety concerns, particularly given the significant global overlap between MPXV infections and HIV. Owing to their superior safety profile and accessibility, recombinant subunit vaccines represent a highly promising platform for monkeypox vaccine development. In this study, we developed a subunit vaccine comprising A29L, B6R, and M1R antigens formulated with a proprietary nanoemulsion adjuvant and evaluated its immunogenicity and protective efficacy. In mice immunized with a prime-boost regimen of the three individual antigens combined with the nanoemulsion adjuvant, comparable serum IgG levels against each antigen were elicited. Both A29 and M1 formulations induced serum antibodies with potent neutralizing activity against MPXV and Vaccinia virus Western Reserve strain (VACV-WR). Notably, M1 antiserum exhibited stronger neutralization than A29 antiserum, whereas B6R immune serum showed no significant neutralizing activity. Splenocytes from B6R-immunized mice mounted a robust IFN-γ response, which was markedly lower in those immunized with A29 or M1. All three monovalent vaccines conferred complete survival following an intranasal lethal MPXV challenge, with M1 providing the strongest protection. In a lethal VACV-WR challenge model, only M1 immunization conferred significant protection. Histopathological analysis of lung tissues on day 5 post-infection revealed more pronounced inflammatory features in B6R-immunized mice compared to the nanoemulsion adjuvant control group. Furthermore, the nanoemulsion-adjuvanted bivalent A29L + B6R formulation induced significantly higher IgG and neutralizing antibody titers and demonstrated superior protective efficacy compared to the aluminum hydroxide-adjuvanted formulation. This comparative preclinical evaluation provides important evidence to support the development of a safe and effective subunit vaccine against monkeypox.