The Receptor Binding Specificity of the Live Attenuated Influenza H2 and H6 Vaccine Viruses Contributes to Vaccine Immunogenicity and Protection in Ferrets

To prepare for influenza pandemics that may be caused by the H2 and H6 subtype influenza viruses, live attenuated influenza virus (LAIV) H2 and H6 vaccines are being developed and evaluated. The H2 and H6 vaccine candidates with different receptor binding preferences specified by amino acid substitutions at residues 226 and 228 were generated and evaluated for their growth in embryonated chicken eggs and their immunogenicity and protection against wild-type virus challenge in the ferret model. The viruses containing Q226 and G228 in the hemagglutinin (HA) protein bound to the avian-like alpha 2,3-sialic acid (SA) receptor and replicated efficiently in chicken eggs. The viruses with L226 and G228 bound preferentially to the human-like alpha 2,6-SA receptor. The viruses containing L226 and S228 displayed dual binding to both alpha 2,3-SA and alpha 2,6-SA receptors and replicated efficiently in eggs. The strains containing L226/G228 or L226/S228 that preferentially bound to alpha 2,6-SA receptors replicated efficiently in the upper respiratory tract of ferrets, induced high levels of neutralizing antibody, and conferred a high level of protection against wild-type virus challenge infection compared to the strain with the Q226/G228 residues. Our data suggest that pandemic vaccines with receptor binding preference to both avian- and human-like receptors might be desired for efficient viral replication in eggs and for inducing protective immune responses in humans.