A cocktail vaccine with monkeypox virus antigens confers protection without selecting mutations in potential immune evasion genes in the vaccinia WR strain challenge.

Faced with the global monkeypox outbreak, current vaccine development predominantly focuses on the mRNA platform despite its limitations in stability and long-term efficacy. Here, we engineered a recombinant vesicular stomatitis virus (rVSV)-vectored cocktail vaccine encoding four conserved monkeypox virus (MPXV) antigens (A35R, A29L, M1R, and B6R; >94% clade homology), leveraging the thermostable properties of the VSV platform validated for 4°C storage in Ebola vaccines. In BALB/c mice, this multi-antigen vaccine elicited a rapid humoral response with specific IgG detectable by day 7, effectively neutralized the virus, and induced a robust Th1/Th2 balanced cytokine response. Immunization conferred 100% survival against lethal vaccinia virus WR strain challenge, with undetectable viral loads in the lungs and serum, and sustained efficacy against secondary infection at 60 days. Histopathology confirmed minimal lung damage in vaccinated mice. Crucially, upon the successive challenges, mutations in key poxvirus immune evasion genes (E3L and B7R) emerged in the single-component vaccine groups but were absent in the cocktail vaccine group. This finding provides direct evidence that the cocktail strategy suppresses viral escape, underscoring a fundamental advantage over single-antigen approaches. Our findings demonstrate the rVSV-based cocktail vaccines as a potent, scalable, and thermostable candidate for global MPXV control, particularly in regions with limited settings. IMPORTANCE: The global emergence of the monkeypox virus (MPXV) underscores the urgent need for effective and accessible vaccines. We developed a recombinant vesicular stomatitis virus (rVSV)-vectored cocktail vaccine expressing four conserved MPXV antigens. This multivalent vaccine elicits rapid and potent immune responses in mice, conferring complete protection against lethal vaccinia virus challenge. A critical finding is that while successive viral challenges selected for mutations in key immune evasion proteins in single-antigen vaccine groups, these mutations were absent in the cocktail-vaccinated group. This suggests that the cocktail strategy may suppress viral genetic drift, potentially limiting escape pathways. Combined with the thermostability of the VSV platform, our vaccine presents a promising and scalable candidate for combating monkeypox.