Abstract
The 2022 global mpox outbreak highlighted the risk of zoonotic diseases establishing sustained transmission in human populations and underscored the need for accurate serological tools to monitor orthopoxvirus exposure. However, cross-reactive antibodies induced by Modified Vaccinia Ankara (MVA) vaccination make it difficult to discriminate between monkeypox virus (MPXV) infection and vaccination-induced immunity. Here we present a machine learning (ML)-assisted bead-based serological multiplex assay that distinguishes MPXV infection from MVA vaccination and pre-immune sera by targeting antibody responses to 15 poxviral antigens. Of the six algorithms tested, the Gradient Boosting Classifier (GBC) achieves the highest performance (F1 = 0.83) in sera from the 2022 outbreak and from a follow-up epidemiological cohort of at-risk men who have sex with men (MSM; n = 1,260). In an independent validation cohort (n = 143), GBC (F1 = 0.70) robustly detects MPXV infections, including breakthrough cases, with 88% specificity and 92% sensitivity. Integrating ML with high-dimensional serology enables accurate cross-sectional classification of orthopoxvirus immune status and provides a scalable framework for mpox serosurveillance and outbreak preparedness.