Generation and characterization of mouse monoclonal antibodies against the VP4 protein of group A human rotaviruses.

Human rotaviruses (RVs) are the leading cause of severe diarrhea in infants and young children worldwide. Among the structural proteins, as a spike protein, rotavirus VP4 plays a key role in both viral attachment and penetration. Currently, studies on monoclonal antibodies (mAbs) against VP4 are limited. In this study, mice were immunized with truncated VP4* to produce murine mAbs. In total, 50 mAbs were produced and characterized. Twenty-four mAbs were genotype-specific and 20 mAbs recognized the common VP4 epitopes shared by P[8], P[4], and P[6] viruses. Thirty-five of the 50 mAbs were neutralizing mAbs, among which nine mAbs could neutralize all three P-genotype RVs, and 10 neutralizing mAbs exhibited conformational sensitivity. Ten mAbs recognized dominant neutralizing epitopes, including the broadly neutralizing mAb 9C4 recognized conformational epitope. Further investigation shows that S376 and S464 are key amino acids for 9C4 binding, however, the exact binding sites of 9C4 remain to be fully defined. Overall, this panel of mAbs has demonstrated utility as immunodiagnostic and research reagents, and could potentially serve as crucial tools for exploring the neutralizing mechanisms and quality control of VP4* protein-based RV subunit vaccines. Further evaluation of cross-neutralizing mAbs could not only improve the understanding of the heterotypic protection conferred by RV vaccines, but also facilitate the development of broadly protective RV vaccines.