A broad and potent IgM antibody against tetra-EV-As induced by EVA71 and CVA16 co-immunization

Dual blockages of a broad and potent neutralizing IgM antibody targeting GH loop of EV‐As

“…The EVA71‐VP1′ GH loop, is a well‐known target for broad and potent neutralizing antiviral design. Our study indicated that M20 [21] and engineered antibodies 20‐IgM and 20‐IgG1 bind to the peptides 43, 44 and 45 of EVA71, which are located at the GH loop (Figure S1A). To better understand the 20‐IgM and 20‐IgG1′ mechanism of the antiviral activity, the corresponding peptides 43, 44 and 45 of CVA16, CVA10 and CVA6 were synthesized (Table S2).…”

“…Human diploid cell line KMB17 cells (IMBCAMS), Vero and RD cells (ATCC) were cultured as described previously [21]. CHO‐S cells (Invitrogen, catalogue no: R800‐07, USA) were cultured in FreeStyle CHO expression medium containing l ‐glutamine at a final concentration of 8 mM at 37°C in 8% CO 2 .…”

“…The molecular weight and purity of the antibodies were analysed by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and native‐PAGE [21]. The antibodies' binding specificity was analysed by enzyme‐linked immunosorbent assay (ELISA).…”

“…The standard neutralization assay was performed by (w/v) 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based method (Meilunbio, catalogue no: MB4698, China) [21,26,27]. Briefly, 200 TCID 50 EV-As were mixed with different concentrations of antibody, then incubated with 15 000 RD cells at 37 C for 72 h. Finally, the cells' viability was analysed by the MTTbased method.…”

“…In addition, some IgM antibodies have been proved to possess broad and cross-neutralizing efficacy against emerging variants of HIV, influenza B virus and ASRS-CoV2 [18][19][20]. In a previous study, we generate an IgM antibody M20 by classic hybridoma technology, directed to much more conserved components of GH Loop of VP1, broadly neutralize a greater fraction of circulating EV-As (EVA71, CVA16, CVA10 and CVA6) than the mAbs development [21]. Indeed, M20 is the first antibody that possessed broad and potent antivirus activity against EV-As.…”