Background: The 2022 Monkeypox virus (MPXV) global outbreak boosted development of multiple serological assays to aid understanding of Mpox immunology. Objectives: The study aimed to assess a multiplexed solid-phase electrochemiluminescence immunoassay (Meso Scale Discovery (MSD)) for simultaneous detection of antibodies against MPXV, including A35, E8 and M1 antigens, along with corresponding Vaccina Virus (VACV) homologues and demonstrate its accuracy in assessing antibody titres post-vaccination and infection. Methods: Assay performance was assessed for simultaneous detection of antibodies against MPXV and corresponding VACV antigens. Sensitivity and specificity were evaluated with paediatric negatives (n = 215), pre- and post-IMVANEX vaccinated (n = 80), and MPXV (Clade IIb, n = 39) infected serum samples. Results: The assay demonstrated high specificity (75.68 % (CI: 69.01-81.29) - 95.98 % (CI:92.54-97.87)) and sensitivity (62.11 % (CI:52.06-71.21) - 98.59 % (CI:92.44 %-99.93 %)) depending on the Orthopoxvirus antigen. Preferential binding was observed between MPXV-infected individuals and MPXV antigens, while vaccinated individuals exhibited increased binding to VACV antigens. These results highlight differential binding patterns between antigen homologues in related viruses. Conclusion: Overall, this assay demonstrates high sensitivities in detecting antibodies for multiple relevant MPXV and VACV antigens post-infection and post-vaccination, indicating its utility in understanding immune responses to Orthopoxviruses in current and future outbreaks and evaluating the immunogenicity of new-generation Mpoxspecific vaccinations.

In the summer of 2022, a large outbreak of Monkeypox virus (MPXV) cases occurred globally. By December 2022, a total of 3,582 Mpox cases had been confirmed within the UK. As a result, the Modified Vaccinia Ankara-Bavarian Nordic (IMVANEX) vaccine was offered to high-risk groups to protect against the spread of the virus. This outbreak led to the development of multiple serological assays to aid the current understanding of Mpox immunology. This study assessed the performance of a multiplexed solid-phase electrochemiluminescence (Meso Scale Discovery (MSD)) immunoassay for simultaneous detection of antibodies against MPXV A29, A35, B6, E8, and M1 antigens, along with the corresponding Vaccina Virus (VACV) homologues A27, A33, B5, D8, and L1. Sensitivity and specificity were evaluated with paediatric negatives (n=215), pre- and post-IMVANEX vaccinated (n=80) and MPXV (2022 Clade IIb outbreak, n=39) infected serum samples. The overall Orthopoxvirus multiplex assay demonstrated high specificity ranging from 75.68% (CI: 69.01&-81.29) - 95.98% (CI:92.54-97.87) and sensitivity from 62.11% (CI:52.06-71.21) - 98.59% (CI:92.44% - 99.93%) depending on the Orthopoxvirus antigen, either used singularly or combined. Additionally, preferential binding was observed between Mpox-infected individuals and MPXV antigens, whilst vaccinated individuals exhibited increased binding to VACV antigens. These results highlight the differential binding patterns between antigen homologues in closely related viruses. Using this assay, we show that the Orthopoxvirus MSD assay is highly sensitive in detecting IgG titres for vaccinated sera ≥24-days post dose one and ≥14-days post dose two for all antigens within the assay except for MPXV A29 and VACV A27. A similar trend was observed with convalescent sera, although differing antigens demonstrate stronger sensitivities. Overall, this assay has the capability to accurately assess antibody titres for multiple relevant MPXV and VACV antigens post infection and post vaccination, demonstrating its utility in understanding immune responses to Orthopox viruses in current and future outbreaks, and assessing the immunogenicity of new generation Orthopox and Mpox-specific vaccinations.### Competing Interest StatementThe authors have declared no competing interest.### Funding StatementThis study did not receive any funding### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:Mpox PCR-confirmed cases were granted approval for assay performance validation by the NHS Research Ethics Committee (reference 22/HRA/3321). IMVANEX Smallpox-vaccinated samples were obtained from individuals with written consent through UKHSA Research and Ethics Committee for assay validation. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors

The summer of 2022 saw the first global outbreak of Mpox disease (formerly monkeypox), primarily within gay, bisexual, and other men who have sex with men (GBMSM). In response, public health agencies in the UK have offered smallpox vaccines to those individuals deemed at highest risk of infection. With Mpox cases still being detected globally, novel tools are required to aid with diagnosis, serosurveillance and the evaluation of immune responses following infection and vaccination. Here, we describe the development of a multiplexed immunoassay that is able to measure IgG responses to twelve immunogenic Orthopoxvirus proteins concurrently and distinguish between responses to infection and vaccination. Using the Luminex platform, antibody responses to vaccinia virus (VACV) proteins B5, A27, A33 and Monkeypox virus (MPXV) proteins E8, B6, B2, M1, A27, A35, H3, A29, A5 were assessed in serum from individuals post-MPXV infection (n=24) and post-vaccination (n=75) with modified vaccinia virus Ankara-Bavarian Nordic (MVA-BN, IMVANEX). Negative sera (n=435) were run alongside to assess appropriate assay cut-offs and characteristics. Using the results from a combination of eight of the twelve proteins within the immunoassay we were able to classify samples as either post-vaccination or infection, from negative samples with a sensitivity of 98.39% (9.72-99.22%) and specificity of 95.24% (86.91-98.70%). IgG responses to VACV A27, MPXV A29 and MPXV A5 provided little diagnostic advantage. IgG responses to the MPXV protein A27 were able to distinguish post-MPXV infection from negative and post-vaccination samples with a sensitivity of 87.5% (69.00-95.66%) and specificity of 96.84% (94.84-98.07%). There is an ongoing need to utilise Mpox serology to conduct disease surveillance, assess the efficacy of current and new vaccine candidates, and further understand immune responses to Mpox infection. We believe this assay will provide substantial insight into the current global outbreak of Mpox, with additional benefits over current serological assays.### Competing Interest StatementThe authors have declared no competing interest.### Funding StatementThis study did not receive any funding### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:The research ethics committee (Research and Public Health Practice Ethics and Governance Group, REGG) of the UK Health Security Agency (UKHA) granted ethical approval for sampling of individuals with written and informed consent. In addition, the NHS Research Ethics Committees (REC) granted approval for sampling from previous MPXV-infected individuals under reference 22/HRA/3321.I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors

In early 2022, a cluster of monkeypox virus (MPXV) infection (mpox) cases were identified within the UK with no prior travel history to MPXV-endemic regions. Subsequently, case numbers exceeding 80,000 were reported worldwide, primarily affecting gay, bisexual, and other men who have sex with men (GBMSM). Public health agencies worldwide have offered the IMVANEX Smallpox vaccination to these individuals at high-risk to provide protection and limit the spread of MPXV. We have developed a comprehensive array of ELISAs to study poxvirus-induced antibodies, utilising 24 MPXV and 3 Vaccinia virus (VACV) recombinant antigens. Panels of serum samples from individuals with differing Smallpox-vaccine doses and those with prior MPXV infection were tested on these assays, where we observed that one dose of Smallpox vaccination induces a low number of antibodies to a limited number of MPXV antigens but increasing with further vaccination doses. MPXV infection induced similar antibody responses to diverse poxvirus antigens observed in Smallpox-vaccinated individuals. We identify MPXV A27 as a serological marker of MPXV-infection, whilst MPXV M1 (VACV L1) is likely IMVANEX-specific. Here, we demonstrate analogous humoral antigen recognition between both MPXV-infected or Smallpox-vaccinated individuals, with binding to diverse yet core set of poxvirus antigens, providing opportunities for future vaccine (e.g., mRNA) and therapeutic (e.g., mAbs) design.

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.

1.AbstractIn March 2020, the Rare and Imported Pathogens Laboratory at Public Health England, Porton Down, was tasked by the Department of Health and Social Care with setting up a national surveillance laboratory facility to study SARS-CoV-2 antibody responses and population-level sero-surveillance in response to the growing SARS-CoV-2 outbreak. In the following 12 months, the laboratory tested more than 160,000 samples, facilitating a wide range of research and informing PHE, DHSC and UK government policy. Here we describe the implementation and use of the Euroimmun anti-SARS-CoV-2 IgG assay and provide an extended evaluation of its performance. We present a markedly improved sensitivity of 91.39% (≥14 days 92.74%, ≥21 days 93.59%) compared to our small-scale early study, and a specificity of 98.56%. In addition, we detail extended characteristics of the Euroimmun assay: intra- and inter-assay precision, correlation to neutralisation and assay linearity.