Analogous humoral antigen recognition between Monkeypox-infected and Smallpox-vaccinated individuals

In early 2022, a cluster of Monkeypox (now termed MPOX) virus (MPXV) cases were identified within the UK with no prior travel history to MPXV-endemic regions, suggesting localised transmission of MPXV within the UK. Subsequently, cases were identified in several other non-endemic countries and currently exceed 80,000 worldwide, primarily affecting gay, bisexual, and other men who have sex with men. Public health agencies worldwide have offered the IMVANEX Smallpox vaccination to these individuals to provide protection and limit the spread of MPXV. We have developed a comprehensive array of ELISA assays to study poxvirus-induced antibodies, utilising 24 MPXV and 3 Vaccinia virus (VACV) recombinant antigens. Panels of serum samples from individuals with one, two, or three doses of IMVANEX or ACAM2000 (Modified Vaccinia Ankara (MVA)) vaccination, and those with prior MPOX infection were tested against these antigens, with Pearson correlation, principal component analysis and receiver operator curve statistics used to further elucidate antigenic responses to pox-virus infection. Furthermore, using our data, we demonstrated the development of a pooled antigen ELISA that can reliably detect antibody responses induced by Smallpox vaccination or MPXV infection. Using diverse poxvirus antigen ELISAs, we observe that one dose of Smallpox vaccination induces a low number of antibodies, primarily against MPXV B2, with a second dose inducing considerably higher antibody responses against B2R but also to other MPXV antigens such as B5, E8, M1, and A35. Prior MPXV infection, both Clades IIa and IIb, induce variable responses, but similarly induce antibody responses to poxvirus antigens observed in Smallpox-vaccinated individuals, and additionally responses to MPXV A27, A29 and H3. Principal component and Pearson correlation matrix identified MPXV A27 as a differential between IMVANEX and MPOX-infected individuals, whilst MPXV M1 (VACV L1) is likely a serological marker of IMVANEX-vaccination. When using recombinant MPXV/VACV protein homologues, we also observe a difference in antigen binding, with variability based on the individual's originating infection/vaccination. Using a pooled-antigen ELISA, we also demonstrate a sensitivity of 97.14% (95% CI:91.93-99.22) and specificity of 98.23% (96.67-99.07) in detecting poxvirus antibodies with applicability to measuring longitudinal antibody responses post-vaccination/post-MPXV infection. Here, we show that both MPXV-infected or Smallpox-vaccinated individuals mount antibodies able to bind a diverse but core set of poxvirus antigens, with implications for future vaccine (e.g., mRNA-based) and therapeutic (e.g., monoclonal antibodies) targets. We identify low levels of antibodies observed in those post-dose one IMVANEX, but considerably higher levels of antibodies post-dose two. We also demonstrate that homologous VACV and MPXV antigens may offer a mechanism for discriminating between vaccinated and MPXV-infected individuals through differential binding, aiding in serosurveillance and future immunology studies.