Derivation and characterization of a live attenuated equine influenza vaccine virus

Objective-To develop and characterize a cold-adapted live attenuated equine -2 influenza virus effective as an intranasal vaccine. Animals-8 ponies approximately 18 months of age. Procedures-A wild-type equine-2 virus, A/Equine/Kentucky/1/91 (H3N8), was s erially passaged in embryonated chicken eggs at temperatures gradually redu ced in a stepwise manner from 34 C to 30 C to 28 C to 26 C. At different pa ssages, infected allantoic fluids were tested for the ability of progeny vi rus to replicate in Madin-Darby canine kidney (MDCK) cells at 34 C and 39.5 C. Virus clones that replicated at 26 C in eggs and at 34 C in MDCK cells, but not at 39.5 C in MDCK cells, were tested for stability of the cold-ada pted, temperature-sensitive (ts), and protein synthesis phenotypes. A stabl e clone, P821, was evaluated for safety, ability to replicate, and immunoge nicity after intranasal administration in ponies. Results-Randomly selected clones from the 49th passage were all ts with pla quing efficiencies of < 10(-6) (ratio of 39.5 C:34 C) and retained this phe notype after 5 serial passages at 34 C in either embryonated eggs or MDCK c ells. The clone selected as the vaccine candidate (P821) had the desired de gree of attenuation. Administered intranasally to seronegative ponies, the virus caused no adverse reactions or overt signs of clinical disease, repli cated in the upper portion of the respiratory tract, and induced a strong s erum antibody response. Conclusion and Clinical Relevance-A candidate live attenuated influenza vac cine virus was derived by cold-adaptation of a wild-type equine-2 influenza virus.