Isolation, preparation, and assessment of the immunogenicity effectiveness of a novel inactivated rotavirus vaccine strain: ZTR-68, rotavirus A genotype G1P[8]

Abstract Background Rotavirus is one of the leading causes of diarrhoea in children in both developed and developing countries, resulting in an estimated 600 000 deaths worldwide each year. In China, roughly 13 400 children younger than 5 years die from rotavirus infection each year. We aimed to isolate and characterise a human virus for use as a candidate human inactivated rotavirus vaccine strain. We studied the preparation of the vaccine and assessed its immunogenicity effectiveness. Methods From September, 2009, to November, 2012, we collected 206 stool specimens from children in Yunnan, China, and tested for the presence of rotaviruses by use of enzyme immunoassay and double-stranded RNA-PAGE. G and P typing of the positive samples were done by one-step RT-PCR and sequencing. A new human rotavirus was isolated from a child aged 3 months with acute gastritis from the First People's Hospital of Zhaotong City, Zhaotong, China, and was designated ZTR-68. We did a full genomic analysis of ZTR-68 and tested for identifiable characteristics of the virus. A scalable inactivated rotavirus vaccine was cultured by rolling bottles, purified using low-velocity centrifuge, microfiltration clarification, ultrafiltration concentration, and size-exclusion chromatography, and inactivated by the addition of formaldehyde. Its immunogenicity was studied in Balb/c mice by intramuscular injection. Findings Strain ZTR-68 was isolated and plaque purified, and its complete genome was sequenced. ZTR-68 is a rotavirus A of genotype G1P[8] and has a close phylogenetic relation to Wa. We confirmed that our rotavirus vaccine was completely inactivated, and rotavirus antigen can be measured by ELISA. After intramuscular injection of the vaccine into BalB/c mice aged 6 weeks, high titres of rotavirus-specific IgG (>20 000) and neutralising activity (roughly 3000) were recovered in the sera of these mice. Interpretation This newly isolated rotavirus, ZTR-68, is being used to establish a new strain of inactivated rotavirus vaccine. Our findings show that the inactivated rotavirus vaccine has good immunogenicity and provide evidence for further clinical development as an alternative candidate vaccine. Funding This research was financially supported by the State Project for Essential Drug Research and Development, the Chinese national "Twelfth Five-Year" plan (project number: 2014ZX09102041004); the National High Technology Research and Development Program ("863" Program) of China (project number: 2012AA02A404); and the Yunnan Research Program of Application Foundation and Advanced Technology, self-raised funds (project number: 2013FZ140).