The molecular basis of antigenic drift was determined for the hemagglutinin (HA) of human influenza A/H3N2 virus. From 1968 to 2003, antigenic change was caused mainly by single amino acid substitutions, which occurred at only seven positions in HA immediately adjacent to the receptor binding site. Most of these substitutions were involved in antigenic change more than once. Equivalent positions were responsible for the recent antigenic changes of influenza B and A/H1N1 viruses. Substitution of a single amino acid at one of these positions substantially changed the virus-specific antibody response in infected ferrets. These findings have potentially far-reaching consequences for understanding the evolutionary mechanisms that govern influenza viruses.

The majority of currently circulating influenza A(H1N1) viruses are antigenically similar to the virus that caused the 2009 influenza pandemic. However, antigenic variants are expected to emerge as population immunity increases. Amino acid substitutions in the hemagglutinin protein can result in escape from neutralizing antibodies, affect viral fitness, and change receptor preference. In this study, we constructed mutants with substitutions in the hemagglutinin of A/Netherlands/602/09 in an attenuated backbone to explore amino acid changes that may contribute to emergence of antigenic variants in the human population. Our analysis revealed that single substitutions affecting the loop that consists of amino acid positions 151 to 159 located adjacent to the receptor binding site caused escape from ferret and human antibodies elicited after primary A(H1N1)pdm09 virus infection. The majority of these substitutions resulted in similar or increased replication efficiency in vitro compared to that of the virus carrying the wild-type hemagglutinin and did not result in a change of receptor preference. However, none of the substitutions was sufficient for escape from the antibodies in sera from individuals that experienced both seasonal and pandemic A(H1N1) virus infections. These results suggest that antibodies directed against epitopes on seasonal A(H1N1) viruses contribute to neutralization of A(H1N1)pdm09 antigenic variants, thereby limiting the number of possible substitutions that could lead to escape from population immunity. A prerequisite for the influenza virus to infect the host cell is the binding of the hemagglutinin (HA) surface protein to sialylated glycan receptors on the host cell through its receptor binding site (RBS). HA is the main target of neutralizing antibodies and is therefore a critical component of influenza vaccines (5). Influenza viruses continually escape antibody-mediated neutralization by Citation Koel BF, Mögling R, Chutinimitkul S, Fraaij PL, Burke DF, van der Vliet S, de Wit E, Bestebroer TM, Rimmelzwaan GF, Osterhaus ADME, Smith DJ, Fouchier RAM, de Graaf M. 2015. Identification of amino acid substitutions supporting antigenic change of influenza A(H1N1)pdm09 viruses.

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