Evaluation of a multiplexed immunoassay for assessing long-term humoral immunity Orthopoxviruses

Abstract

Background: The 2022 Monkeypox virus (MPXV) global outbreak boosted development of multiple serological assays to aid understanding of Mpox immunology.

Objectives: The study aimed to assess a multiplexed solid-phase electrochemiluminescence immunoassay (Meso Scale Discovery (MSD)) for simultaneous detection of antibodies against MPXV, including A35, E8 and M1 antigens, along with corresponding Vaccina Virus (VACV) homologues and demonstrate its accuracy in assessing antibody titres post-vaccination and infection.

Methods: Assay performance was assessed for simultaneous detection of antibodies against MPXV and corresponding VACV antigens. Sensitivity and specificity were evaluated with paediatric negatives (n = 215), pre- and post-IMVANEX vaccinated (n = 80), and MPXV (Clade IIb, n = 39) infected serum samples.

Results: The assay demonstrated high specificity (75.68 % (CI: 69.01-81.29) - 95.98 % (CI:92.54-97.87)) and sensitivity (62.11 % (CI:52.06-71.21) - 98.59 % (CI:92.44 %-99.93 %)) depending on the Orthopoxvirus antigen. Preferential binding was observed between MPXV-infected individuals and MPXV antigens, while vaccinated individuals exhibited increased binding to VACV antigens. These results highlight differential binding patterns between antigen homologues in related viruses.

Conclusion: Overall, this assay demonstrates high sensitivities in detecting antibodies for multiple relevant MPXV and VACV antigens post-infection and post-vaccination, indicating its utility in understanding immune responses to Orthopoxviruses in current and future outbreaks and evaluating the immunogenicity of new-generation Mpox-specific vaccinations.

Keywords: Immunoassay; Immunology; Mpox; Orthopox.