Mutations of two amino acids in VP5 mediate the attenuation of human rotavirus vaccine: evidence from <i>in vitro</i> and <i>in vivo</i> studies

Various vaccines, like polio, measles, and rotavirus, have been developed by serial-passaging in cell culture. Live oral rotavirus vaccines have been shown to be generally safe, but mechanisms of attenuation are not known. We have used a new, entirely plasmid-based reverse genetics system to artificially generate the novel human rotavirus vaccine strain CDC-9 (G1P[8]) and analyze the effect of the mutations within the VP4 gene on adaptation <i>in vitro</i> and attenuation <i>in vivo</i>. We demonstrated that out of the 6 amino acid mutations that appeared after serial passaging in Vero cells, mutations of wild-type CDC-9 P11 at VP4 AA331 and AA385 each or in combination were associated with increased replication <i>in vitro</i> comparable to cell-culture adapted CDC-9 P45. Neonatal rats infected with the single AA331 or AA385 mutant had reduced viral shedding, comparable to cell-culture passaged CDC-9 P45. We observed additional reduced shedding in neonatal rats that were infected with combination mutants harboring mutations at position AA331_385_388, indicative of a slight additive effect. Our data indicate that mutations in the VP5* region of the VP4 gene, particularly at position AA331 and AA385, are the determining factor for <i>in vitro</i> replication adaptation and <i>in vivo</i> attenuation of a G1P[8] rotavirus vaccine. This information provides great potential for targeted mutation in rotavirus vaccine generation instead of labor-consuming serial passaging in cell culture.IMPORTANCELive oral rotavirus vaccines have been developed through serial passaging in cell culture and found to be generally safe and efficacious in children. Live vaccines are also found to be associated with rare but severe adverse events, such as intussusception, in vaccinated children. Mechanisms for vaccine attenuation and adverse effects are unknown. We have developed a novel human rotavirus vaccine strain (CDC-9) and demonstrated several amino acid mutations in the VP4 gene of cell-passaged virus. In the present study, we identified two key amino acid mutations via reverse genetics technology in VP4 that mediated enhanced growth in cell culture, including a human intestinal cell line, reduced virus shedding, and downregulated inflammatory response in neonatal rats. This study is the first to identify the molecular signatures that define attenuation of human rotavirus vaccine and should help provide guidance for developing new generations of safe and effective vaccines.