MpoxPlex: a high-throughput and versatile multiplexed immunoassay for assessing and discriminating between IgG responses to Mpox infection and vaccination

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.