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 serially passaged in embryonated chicken eggs at temperatures gradually reduced in a stepwise manner from 34 C to 30 C to 28 C to 26 C. At different passages, infected allantoic fluids were tested for the ability of progeny virus 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-adapted, temperature-sensitive (ts), and protein synthesis phenotypes. A stable clone, P821, was evaluated for safety, ability to replicate, and immunogenicity after intranasal administration in ponies. Results - Randomly selected clones from the 49th passage were all ts with plaquing efficiencies of < 10^-6 (ratio of 39.5 C:34 C) and retained this phenotype after 5 serial passages at 34 C in either embryonated eggs or MDCK cells. The clone selected as the vaccine candidate (P821) had the desired degree of attenuation. Administered intranasally to seronegative ponies, the virus caused no adverse reactions or overt signs of clinical disease, replicated in the upper portion of the respiratory tract, and induced a strong serum antibody response. Conclusion and Clinical Relevance - A candidate live attenuated influenza vaccine virus was derived by cold-adaptation of a wild-type equine-2 influenza virus, A/Equine/Kentucky/1/91, in embryonated eggs.