Abstract
Single-round infectious rotavirus, which lacks a gene essential for virion assembly, serves not only as a safe and effective rotavirus vaccine but also as an orally-administrable viral vector vaccine that induces mucosal immunity. Previously, we generated a single-round infectious rotavirus by partially deleting the viral VP6 gene, and demonstrated its potential as a promising vaccine platform. However, this system has several limitations; namely, low viral protein expression levels and safety concerns. Here, we addressed these challenges by introducing large deletions into the VP7 or VP4 genes, which are dispensable for viral protein expression but essential for virion assembly. These VP7- or VP4-defective viruses exhibited markedly higher protein expression in wild-type MA104 cells than the previously developed VP6-defective virus. In addition, the large deletions reduce the risk of viral reversion, thereby increasing both efficacy and safety. In a mouse model, these viruses induced neutralizing antibodies at levels comparable with those elicited by wild-type rotavirus, indicating their potential as rotavirus vaccines. Moreover, a VP4-defective rotavirus harboring a heterologous gene achieved high expression of heterologous proteins, warranting its application as a viral vector vaccine. To further increase safety, we established a reverse genetics system for the bovine rotavirus WC3 strain, a parental strain of the licensed live attenuated rotavirus vaccine, and successfully generated a single-round VP4-defective rotavirus based on the WC3 backbone. Taken together, these optimizations facilitate development of safe and effective single-round infectious rotavirus platforms suitable for human use.