Here, we have evaluated the cross-neutralization of pandemic and seasonal H1N1 influenza viruses. Despite more than 90 years of separation between these viruses that both caused human pandemics, the 1918 SC and 2009 CA viruses raised immune responses in mice that demonstrated cross-neutralization, whereas they were both resistant to antisera directed to a relatively recent seasonal influenza virus of the same subtype. To understand the molecular basis for cross-neutralization, we examined the specificity of antibody recognition by protein competition studies, as well as by site-directed mutagenesis and protein structural modeling. The RBD-A region was defined as the target of neutralization, and we demonstrated that glycosylation sites in this region are important in allowing evasion of antibody neutralization in seasonal strains. Specifically, introduction of glycosylation sites into these strains eliminated their ability to bind neutralizing antibodies, suggesting that glycan shielding from antibodies is a mechanism by which seasonal influenza viruses evolve after the emergence of a viral pandemic strain. Similar cross-neutralization of these viruses has been reported recently by others and mapped to a similar region of the RBD (15
); however, the role of glycans in modulating immune recognition and its influence on viral evolution were not defined in those studies.
The findings of the present study have implications for the evolution of pandemics and the development of herd immunity that may prevent viral spread. As a pandemic H1N1 strain evolves into a seasonal form in humans, it acquires glycosylation sites on the RBD () that can effectively mask antigenic regions from recognition by antibodies. A related process likely occurred with H2N2, H3N2, and H5N1 viruses, where glycosylation of HA has been shown to inhibit recognition by antibodies (17
) and is associated with their antigenic drift (21
). Here, we have defined a role of specific glycans in the evolution of H1N1 strains and described the structural basis for antibody resistance. In addition to the changes in glycosylation, we also find that amino acid changes in the RBD-A region contribute to neutralization resistance and viral evolution in humans, and in some cases, the glycosylation is also required for the generation of functional viral spike. For example, the 1999 NC HA lacking both 142 and 177 glycans did not give rise to a functional spike that mediated entry in the context of a pseudotyped lentiviral vector, suggesting that antigenic drift in the RBD-A region has resulted in an HA that now requires these glycans for entry into cells.
Neutralizing antibodies to the pandemic viruses of 1918 and 2009 are not elicited by either vaccines or infections from seasonal influenza viruses. Therefore, a segment of the population that had not been exposed to either pandemic virus would fail to develop protective herd immunity to the 2009 virus and remain susceptible to infection by this otherwise sensitive virus. The presence of H1N1 strains without glycosylation on the top of the RBD and relatively high sequence conservation with the 1918 SC HA in the RBD-A region in the earlier decades of the 20th century is a likely explanation for the relative degree of protection seen in the elderly against the present 2009 pandemic. This is especially true for those born around 1910, who were presumably exposed to the 1918 virus and have antibodies that neutralize A (H1N1) 2009 (22
The data reported here define the molecular basis of the recognition of chronologically distant pandemic influenza viruses by antibodies raised to other pandemic viruses and have significant implications for vaccine preparedness, both in preventing future pandemics and for the evolution of the current swine-related pandemic influenza virus. The sensitivity of 1918 SC and 2009 CA to RBD-A neutralization suggests that childhood immunization with such prototypic viruses might protect against future pandemic outbreaks with viruses of this type. Knowledge of the structures and mechanisms of cross-neutralization therefore facilitates rational vaccine design and may help to contain such outbreaks. The current A (H1N1) 2009 pandemic virus will likely develop resistance to the present vaccine through the evolution of escape mutants. The data collected in this study suggest that an effective mode of escape would be conferred by acquisition of glycosylation sites in the RBD-A region. In addition, based on analysis of H1N1 sequence variation in RBD-A and elsewhere over time, changes in primary amino acid sequence may further promote immune evasion. Surveillance for their presence will facilitate the identification of next-wave viruses. This information can be used preemptively to develop vaccine strains that could prevent the emergence of RBD-A–glycosylated viruses, thus constraining its evolution into a seasonal influenza and limiting its further spread.
Note added in proof:
Recently, four new pandemic isolates of the 2009 H1N1 virus have been added to the Influenza Virus Resource database (http://www.ncbi.nlm.nih.gov/genomes/FLU/Database/select.cgi?go=1
) that show glycosylation in the RBD-A region of HA defined in this report. These strains include A/Beijing/SE2649/ 2009, A/Russia/178/2009, A/Russia/180/2009, and A/Salekhard/01/ 2009, all of which contain amino acid mutations predicted to add a glycosylation site at position 179 (1918 numbering).