Immunogenicity of poxvirus A16/G9 entry-fusion subcomplex and its restriction by A56/K2 protein informs vaccine...

Poxviruses rely on a conserved multiprotein entry-fusion complex, providing numerous potential antibody targets, although the immunogenicities of only a few have been analysed. Here we tested the ectodomains of ten orthopoxvirus entry-fusion complex proteins in rabbit and mouse immunization experiments and determined that six induce neutralizing antibodies. Focusing on the apical A16/G9 heterodimer, we show that it induces antibodies that prevent vaccinia virus (VACV) entry, cross-neutralize cowpox virus (CPXV) and monkeypox virus (MPXV), and protect mice against lethal challenge infections with VACV and CPXV. However, antibodies to A16/G9 had limited detection following infections with attenuated or virulent VACV, CPXV or MPXV, but were robustly induced upon immunization with recombinant VACVs secreting G9 or that have deletions of the genes encoding the viral fusion suppressor A56/K2 complex, which specifically binds A16/G9. Our work identifies previously unrecognized immunogens for incorporation into recombinant vaccines and characterizes a mode of immune evasion via the A56/K2 complex.