Evaluation of a Multiplexed Immunoassay for Assessing Long-Term Humoral Immunity to Monkeypox virus infection and Orthopoxvirus Vaccination

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.