Extended Data Fig. 1: Validation of ELISA with VACV-Elstree-infected cell lysate.

(a) Sera from participants vaccinated with MVA-H5 (pre-vaccination and 4 weeks after booster vaccination) were 4-fold diluted starting at a dilution of 1:10 and added to plates coated with a VACV-infected or MVA-infected cell lysate. All sample OD450 values were normalized to the highest and lowest OD450 value on that plate and plotted as log (inhibitor) vs response curve with four parameter variable slope using GraphPad Prism 9.02. A 30% endpoint dilution was estimated by calculating the proportionate distance between two dilutions, from which a titer was determined. 30% endpoint titers are reported in the legend for both ELISAs with VACV-infected and MVA-infected cell lysate. (b) Diagnostic sera from patients with confirmed infectious diseases other than MPX (VZV: varicella zoster virus; HSV: herpes simplex virus; Ent: enterovirus; CMV: cytomegalovirus; EBV: Epstein-Barr virus; HIV: human immunodeficiency virus) were selected and added to plates coated with a VACV-infected cell lysate at a serum concentration of 1:100. Sera from MVA-H5-vaccinated participants obtained 4 weeks after booster vaccination were included as positive control. (c) Several sera from all four cohorts were selected as ‘bridging’ samples and were measured in at least two independent ELISAs with VACV-infected cell lysate. Assays were correlated by performing Spearman r analysis. (d) A reference serum (either a pool of MVA-H5-vaccinated individuals, or a pool of historically-vaccinated and Imvanex-boosted individuals [P/B]) was included on every ELISA plate.