High-throughput multiplex immunoassay for the detection of mpox and MVA-BN vaccination up to 2 years after exposure in Belgium: a retrospective diagnostic accuracy study

Abstract

Background Mpox, caused by monkeypox virus (MPXV), has gained global attention following several international outbreaks. Accurate and high-throughput detection of MPXV-specific antibodies is essential for surveillance programmes, diagnosis, and vaccine trials. We therefore evaluated the long-term accuracy of an mpox multiplex immunoassay that measures IgG responses to 11 orthopoxvirus antigens. Methods The assay was validated in a diagnostic accuracy study involving a Belgian cohort of clade IIb MPXVinfected individuals (n=211) and modified vaccinia virus Ankara-Bavarian Nordic (MVA-BN) vaccine recipients (n=202), who were followed up for 2 years after exposure. We used receiver operating characteristic analysis to assess diagnostic performance at multiple timepoints after infection or vaccination compared with negative controls (n=287). To explore the assay's broader utility, we also investigated correlations between antigen-specific IgG responses and neutralising antibody titres. Findings In individuals without previous smallpox vaccination (n=149), the assay accurately detected mpox infection up to 2 years after infection, achieving 92% sensitivity (95% CI 89-97) at 95% specificity (91-98) when multiple antigens were combined. By contrast, diagnostic performance was low in previously vaccinated individuals (n=62), with sensitivities of most antigens dropping below 60% (38-77) after 6 months. Most antigen-specific IgG responses correlated strongly with neutralising antibody titres. Interpretation The assay shows high sensitivity and specificity for detecting previous mpox infection in unvaccinated individuals, with reliable performance up to 2 years after infection. Strong concordance with neutralising antibody titres supports the assay's potential for monitoring long-term infection and vaccine-induced immunity. However, previous vaccination status should be considered when interpreting results. Copyright (c) 2025 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).