Wa-VP4* as a candidate rotavirus vaccine induced homologous and heterologous virus neutralizing antibody responses in mice, pigs, and cynomolgus monkeys.

Group A rotavirus (RVA) is the primary etiological agent of acute gastroenteritis (AGE) in children under 5 years of age. Despite the global implementation of vaccines, rotavirus infections continue to cause over 120,000 deaths annually, with a majority occurring in developing nations. Among infants, the P[8] rotavirus strain is the most prevalent and can be categorized into four distinct lineages. In this investigation, we expressed five VP4(aa26-476) proteins from different P[8] lineages of human rotavirus in E. coli and assessed their immunogenicity in rabbits. Among the different P[8] strains, the Wa-VP4 protein, derived from the MT025868.1 strain of the P[8]-1 lineage, exhibited successful purification in a highly homogeneous form and significantly elicited higher levels of neutralizing antibodies (nAbs) against both homologous and heterologous rotaviruses compared to other VP4 proteins derived from different P[8] lineages in rabbits. Furthermore, we assessed the immunogenicity of the Wa-VP4 protein in mice, pigs, and cynomolgus monkeys, observing that it induced robust production of nAbs in all animals. Interestingly, there was no significant difference between in nAb titers against homologous and heterologous rotaviruses in pigs and mankeys. Collectively, these findings suggest that the Wa-VP4* protein may serve as a potential candidate for a rotavirus vaccine.

Bivalent rotavirus VP4∗ stimulates protective antibodies against common genotypes of human rotaviruses.

Non-replicating rotavirus vaccines are an alternative strategy to improve the efficacy and safety of rotavirus vaccines. The spike protein VP4, which could be enzymatically cleaved into VP8∗ and VP5∗, is an ideal target for the development of recombinant rotavirus vaccine. In our previous studies, we demonstrated that the truncated VP4 (aa26-476, VP4∗) could be a more viable vaccine candidate compared to VP8∗ and VP5∗. Here, to develop a human rotavirus vaccine, the VP4∗ proteins of P[4], P[6], and P[8] genotype rotaviruses were expressed. All VP4∗ proteins can stimulate high levels of neutralizing antibodies in both guinea pigs and rabbits when formulated in aluminum adjuvant. Furthermore, bivalent VP4∗-based vaccine (P[8] + P[6]-VP4∗) can stimulate high levels of neutralizing antibodies against various genotypes of rotavirus with no significant difference as compared to the trivalent vaccines. Therefore, bivalent VP4∗ has the potential to be a viable rotavirus vaccine candidate for further development.

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.

Establishment of Sandwich ELISA for Quality Control in Rotavirus Vaccine Production.

Non-replicating rotavirus vaccines are alternative strategies that may improve the protective efficacy of rotavirus vaccines in low- and middle-income countries. The truncated spike protein VP4 (aa26-476, VP4*)was a candidate antigen for the development of recombinant rotavirus vaccines, with higher immunogenicity and protective efficacy compared to VP8* and VP5* alone. This article describes the development of three genotype-specific sandwich ELISAs for P[4], P[6], and P[8]-VP4*, which are important for quality control in rotavirus vaccine production. Our results showed that the detection systems had good specificity for the different genotype VP4* and were not influenced by the E. coli host proteins. Moreover, the detection systems play an important role in determining whether the target protein was contaminated by VP4* proteins of other genotypes. They can also detect the adsorption rate of the adjuvant to the P[4], P[6], P[8]-VP4* protein during the process development. The three detection systems will play an important role in the quality control and process development of VP4* based rotavirus vaccines and facilitate the development of recombinant rotavirus vaccines.