Serial Passaging of the Human Rotavirus CDC-9 Strain in Cell Culture Leads to Attenuation: Characterization from In Vitro and In Vivo Studies

Abstract

Live oral rotavirus vaccines have been developed by serial passaging in cell culture and found to be safe in infants. However, mechanisms for the adaptation and attenuation of rotavirus vaccines are not fully understood. We prepared a human rotavirus vaccine strain, CDC-9 (G1P[8]), which when grown in MA104 cells to passage 11 or 12 (P11/P12) had no nucleotide or amino acid sequence changes from the original virus in stool. Upon adaptation and passages in Vero cells, the strain underwent five amino acid changes at P28 and one additional change at P44/P45 in the VP4 gene. We performed virologic, immunological, and pathogenic characterization of wild-type CDC-9 virus at P11/P12 and its two mutants at P28 or P44/P45 using in vitro and in vivo model systems. We found that mutants CDC-9 P28 and P44 induced upregulated expression of immunomodulatory cytokines. On the other hand, the two mutant viruses induced lower STAT1 phosphorylation and grew to 2-log-higher titers than wild-type virus in human Caco-2 cells and simian Vero cells. In neonatal rats, CDC-9 P45 showed reduced rotavirus shedding in fecal specimens and did not induce diarrhea compared to wild-type virus and modulated cytokine responses comparably to Rotarix infection. These findings indicate that mutant CDC-9 is attenuated and safe. Our study is the first to provide insight into the possible mechanisms of human rotavirus adaptation and attenuation and supports ongoing efforts to develop CDC-9 as a new generation of rotavirus vaccine for live oral or parenteral administration.IMPORTANCE Mechanisms for in vitro adaptation and in vivo attenuation of human rotavirus vaccines are not known. The present study is the first to comprehensively compare the in vitro growth characteristics, virulence, and host response of a wild-type and an attenuated human rotavirus strain, CDC-9, in Caco-2 cells and neonatal rats. Our study identifies critical sequence changes in the genome that render human rotavirus adapted to growth to high levels in Vero cells and attenuated and safe in neonatal rats; thus, the study supports clinical development of CDC-9 for oral or parenteral vaccination in children.

Keywords: oral vaccines; rotavirus; vaccine; virus adaptation; virus attenuation.