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

Abstract

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.

Evaluation of a Multiplexed Immunoassay for Assessing Long-Term Humoral Immunity to Monkeypox virus infection and Orthopoxvirus Vaccination

“…An ROC analysis evaluating paediatric negatives to vaccinated sera reveals a higher sensitivity (91.3 % CI:82.30-95.95) compared to those infected with Mpox (81% CI:66.58-90.78) and vaccinated and Mpox-infected individuals combined (83.9% CI:76.22-89.44) ( Table 1, Supplementary Table 1 ). This demonstrates that individuals vaccinated with IMVANEX generate a higher M1 antibody response compared to those infected, consistent with findings from previous studies 28 . Additionally, it has been observed that mRNA vaccines (VGPox1 and VGPox2, both encoding a fusion protein containing A35R and M1R) induce a rapid and robust production of MPXV M1 and A35 specific antibodies seven days after vaccination, indicating a more effective immune response to MPXV M1 compared to natural infection which induced humoral immunity against other antigens with non-significant levels of M1-specific antibodies seven days post exposure 33 .…”

“…Nevertheless, a subsequent decrease in antibody titres was observed ∼84 days post-dose 2 (PD2.4, Figure 2 ), suggesting a waning of the humoral immune response to MPXV and VACV antigens. A similar trend that we previously demonstrated with individual MPXV and VACV antigen ELISAs 28 . The majority of samples remain above the designated cut-off defined in this study up to 220 days post-dose two.…”

“…In response to the global Mpox outbreak in 2022, a number of assays have been developed to assess both immune responses to MPXV infection and Smallpox vaccination 20,28 . Accurate assays for detecting MPXV and VAVC antibodies are critical for evaluating new vaccinations and for conducting serosurveillance studies to determine exposure to MPXV and those previously vaccinated, especially with ongoing Mpox infections in Europe, America and Asia, in addition to high-incidence countries such as DRC and Nigeria 29 .…”

Serological responses to smallpox A33 antigen and monkeypox A35 antigen in healthy people and people living with HIV‐1 from Guangzhou, China

“…Finally, according to the examination specificity reaching 99%, the threshold of the A35 and A33 ELISA AUC values was 0.1155 and 0.0955 (Supporting Information: Figure ). In a previous study, Otter et al showed that both MPXV‐infected or smallpox‐vaccinated individuals contain A35‐ or A33‐specific antibodies 8 …”

“…In a previous study, Otter et al showed that both MPXVinfected or smallpox-vaccinated individuals contain A35-or A33-specific antibodies. 8 To determine whether we could detect A33-binding antibodies induced by previous smallpox vaccination, we first performed an ELISA assay with A33 antigen and serial dilutions of the plasma samples (Figure 1A,B). The results indicated that 29% (23/79) of the samples from the hospital staff older than 42 years were positive for antibodies against A33.…”