Gene-edited vero cells as rotavirus vaccine substrates
Nichole Orr-Burks,
Jackelyn Murray,
Weilin Wu,
Carl D. Kirkwood,
Kyle V. Todd,
Les Jones,
Abhijeet Bakre,
Houping Wang,
Baoming Jiang,
Ralph A. Tripp
Affiliations
Nichole Orr-Burks
Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA, USA
Jackelyn Murray
Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA, USA
Weilin Wu
Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA, USA
Carl D. Kirkwood
Enteric and Diarrheal Diseases, Bill & Melinda Gates Foundation, Seattle, WA, USA
Kyle V. Todd
Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA, USA
Les Jones
Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA, USA
Abhijeet Bakre
Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA, USA
Houping Wang
Division of Viral Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA
Baoming Jiang
Division of Viral Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA
Ralph A. Tripp
Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA, USA; Corresponding author at: 201 Animal Health Research Center, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA.
Background: Rotavirus (RV) is a leading cause of severe gastroenteritis globally and can cause substantial morbidity associated with gastroenteritis in children <5 years of age. Orally administered live-attenuated RV vaccines offer protection against disease but vaccination efforts have been hampered by high manufacturing costs and the need to maintain a cold chain. Methods: A subset of Vero cell host genes was identified by siRNA that when knocked down increased RV replication and these anti-viral host genes were individually deleted using CRISPR-Cas9. Results: Fully-sequenced gene knockout Vero cell substrates were assessed for increased RV replication and RV vaccine antigen expression compared to wild type Vero cells. The results showed that RV replication and antigen production were logs higher in Vero cells having an EMX2 gene deletion compared to other Vero cell substrates tested. Conclusions: We used siRNAs to screen for host genes that negatively affected RV replication, then CRISPR-Cas9 gene editing to delete select genes. The gene editing led to the development of enhanced RV vaccine substrates supporting a potential path forward for improving RV vaccine production. Keywords: Rotavirus, Vaccine substrates, CRISPR-Cas9, Enhanced vaccine cell line
