IC-ELISAs for the quantitative detection of monkeypox virus cross-immunogenic modified vaccinia virus Ankara antigens L1 and A33.

Vaccination is always the most effective approach to control and prevent monkeypox epidemics. The efficacy testing of vaccines is an important component of vaccine quality control. Based on the 3Rs principles, measuring the content of effective proteins in vaccines to evaluate vaccine efficacy is an excellent alternative to traditional in vivo assays. In this study, we reported two indirect competitive ELISA (IC-ELISA) methods based on the use of M1- and A35-specific antibodies that recognize the L1 and A33 proteins of the modified vaccinia virus Ankara (MVA) vaccine, which are cross-immunogenic to monkeypox virus (MPXV), respectively. M1-IC-ELISA was shown to be linear over the range of 31.25-2000 ng/mL with an LOD value of 48.36 ng/mL. A35-IC-ELISA was shown to be linear over the range of 3.90-1000 ng/mL with an LOD value of 3.74 ng/mL. These two methods were found to be specific, precise, accurate and robust in the quantification of cross-immunogenic L1 and A33 in final MVA vaccine and showed a strong correlation with viral titer, MPXV cross-antibody titer and MVA neutralizing antibody titer. The developed methods may be an alternative to the in vivo assay in quality control testing of MVA vaccine.