Comprehensive Profiling of Monkeypox Virus Antigens Identifies Potent Targets for Next-Generation mRNA Vaccine Development

The 2022 Monkeypox virus (MPXV) outbreak renewed interest in vaccines for orthopoxviruses. Initial development efforts focused on well-established antigen targets, especially A35, B6, and M1. However, orthopoxvirus surfaces are complex, displaying many antigens across two infectious forms, mature virions (MV) and extracellular virions (EV) and targets relevant to protection remain to be comprehensively defined. We leveraged advances in orthopoxvirus protein biology and mRNA vaccine technology to compare immunity to all feasible targets. Mice were immunized with mRNAs encoding each antigen, or antigen complex, and neutralizing antibody responses were measured prior to heterologous challenge with vaccinia virus. Among MV antigens, A28 induced potent complement-mediated neutralizing antibodies, and the A17:G10 complex induced neutralizing antibodies and protected from challenge. For EV antigens, A36 induced neutralizing antibodies and protected from challenge. Our results affirm the consensus strategy focusing on key antigens while highlighting additional targets that could enhance updated MPXV mRNA vaccines. SIGNIFICANCEMonkeypox virus, a member of the Orthopoxvirus genus along with variola virus, has been associated with two recent outbreaks of mpox disease leading to a renewed focus on orthopoxvirus vaccine development. We report an agnostic screen of all monkeypox virus surface antigens where we combined recent advances in structural biology and mRNA technology to evaluate these potential new vaccine targets. We confirmed that historically prioritized antigens M1, A35 and B6 were protective but also discovered new antigens of interest including A28, the A17:G10 complex and A36 that can be the targets of protective immune responses. These findings are critical to inform next-generation vaccine designs should novel orthopoxviruses emerge as human pathogens.