Amino acid substitutions in hemagglutinin of the 2009 pandemic influenza A(H1N1) viruses that might affect the viral antigenicity

BACKGROUND: During 2009 to 2012, Thailand had encountered 4 distinctive waves of the 2009 pandemic influenza A(H1N1) (H1N1pdm) outbreaks. Considering the RNA nature of the influenza viral genome, a mutation in hemagglutinin (HA) gene which led to change in antigenicity of the strains circulating during those epidemic periods is anticipated. It is also uncertain whether the A/California/07/2009 (H1N1) (CA/07) vaccine strain still confers protective immunity against those evolved viruses, the causative agents of the later epidemic waves. METHODS: HA gene segments of 10 H1N1pdm isolates obtained during 2009 to 2012 were sequenced and phylogenetically analysed using ClustalW and MEGA5 programs. A total of 124 convalescent serum samples collected from patients naturally infected during 3 epidemic waves were employed as tools to investigate for antigenic change in HA of these 10 circulating H1N1pdm viruses by hemagglutination inhibition (HI) assay. RESULTS: A phylogenetic analysis showed that the 10 virus isolates were grouped into 4 clusters corresponding to the time of 4 consecutive outbreaks. An accumulation of amino acid substitutions in HA was observed in viruses derived from the late epidemic waves. Significantly lower antibody titers were observed when CA/07 was tested against convalescent sera collected from the 3 waves (p < 0.05) compared to most of Thai isolates; and significantly lower antibody titers were also obtained when virus isolates, retrieved from the third epidemic wave were tested against convalescent sera collected during the first and second wave. These results were suggestive of change in antigenicity of the evolved viruses. Our results also showed some mutation position residing outside the previously reported antigenic site that may involve in an alteration of the viral antigenicity. CONCLUSIONS: Our study demonstrated that convalescent sera collected from individuals naturally infected with H1N1pdm virus were successfully used to reveal a statistically significant change in antibody titers against the currently evolved H1N1pdm viruses as determined by HI assay. Nevertheless, the antibody titers of individual serum against various viruses were less than 4-folded difference as compared to that against the CA/07 vaccine strain. Therefore, CA/07 is still a potent vaccine strain for those evolved H1N1pdm viruses.