Determination of seroprevalence and kinetics of humoral response using mpox virus A29 protein

BACKGROUND: Mpox virus (MPXV), previously known as monkeypox virus, has spread globally in 2022. An accurate and convenient antibody test is essential for the determination of seroprevalence and for studying immune response after natural infection or vaccination. Most seroprevalence or vaccine studies used either live MPXV (or vaccinia virus [VACV]) or inactivated MPXV (or VACV) culture lysate for serological assays, but MPXV culture can only be performed in biosafety level 3 (BSL-3) facilities. Here, we developed and evaluated an enzyme immunoassay (EIA) based on the MPXV A29 surface envelope protein. METHODS: We compared the specificity of the MPXV A29, VACV A27, and VACV lysate EIA using serum specimens collected prior to the global spread of MPXV. Next, we performed these EIAs for serum specimens collected from two mpox patients and an MVA-BN vaccine recipient. We also assessed the kinetics of plasmblast and MPXV A29-specific B-cell response. RESULTS: Using sera collected from different age groups in Hong Kong, we found that most individuals, including those born before 1981 who have received the smallpox vaccine, tested negative using the MPXV A29 protein. MPXV A29-specific antibody could be detected in the serum of mpox patients and an MVA-BN recipient. In a mpox patient, the frequency of plasmablast and MPXV A29-specific B cell peaked on day 8 post-symptom onset and gradually decreased. Finally, we demonstrated that antibodies against the A29 protein can be used for immunofluorescence staining of MPXV-infected cells. CONCLUSIONS: MPXV A29 protein is suitable for studying the immune response against MPXV infection.