Differentiating Mpox Infection and Vaccination Using a Validated Multiplex Orthopoxvirus IgG Serology Assay

The resurgence of monkeypox virus (MPXV) outbreaks has increased demand for validated serological assays to assess exposure and immunity. Cross-reactivity among orthopoxivuses, stemming from high sequence conservation, complicates distinguishing antibody responses from natural MPXV infection versus vaccination or other orthopoxvirus exposures. We validated the Meso Scale Discovery (MSD) V-PLEX Orthopoxvirus Panel 1 (IgG) Kit, which quantifies antibody levels to five MPXV antigens and their vaccina virus (VACV) orthologs, following Good Clinical Laboratory Practice (GCLP) guidelines. We assessed assay performance using serum from 26 individuals with prior mpox, 52 JYNNEOS vaccine recipients, and 179 unexposed controls. The assay reliably detected antibody responses in all exposed cohorts with peak levels observed 2-months post-vaccination. Antibody levels to specific antigens also correlated with Modified Vaccinia Ankara (MVA) neutralization titer, particularly for MPXV B6R/VACV B5R, MPXV E8L/VACV D8L, and MPXV M1R/VACV L1. Receiver operating characteristic (ROC) analysis showed that some individual antigens achieved high sensitivity and specificity for exposure detection (AUC > 0.96 for VACV D8L, MPXV B6R, VACV B5R); however, individual antigens performed poorly in distinguishing infection from vaccination. In contrast, antibody level ratios between some MPXV and VACV orthologs effectively differentiated MPXV infection from vaccinia vaccination with high sensitivity and specificity (e.g. MPXV A35R/VACV A33R ortholog ratio, AUC = 0.97, sensitivity = 0.97, specificity = 0.96). Our findings validate the MSD assay for clinical research and serosurveillance to assess MPXV immunity and support the utility of ortholog pair ratio analysis as a strategy to discriminate vaccinated and infected individuals.