The 2009 H1N1 HA diverges considerably from recent seasonal H1N1 HA and is more closely related to the NJ/76 HA (), raising doubts about the extent of protection that could be afforded by vaccination with recent seasonal influenza vaccines. Our studies show that sera from the NJ/76 swine influenza vaccine trials and contemporary sera from subjects who received recent seasonal influenza vaccines, regardless of whether they had been immunized with the NJ/76 swine influenza vaccine, frequently have cross-neutralizing activity to the 2009 H1N1. Further, these sera revealed one or more cross-neutralization epitopes that were sensitive to a conservative amino acid change in position 89 in the HA2 subunit, corresponding to a naturally-occurring amino acid variant that emerged in seasonal H1N1 influenza viruses in recent years.
Several groups have reported that prior infections or vaccinations can confer some immunity to 2009 H1N1, though findings vary. There is agreement that individuals >65 years have substantial cross-reactive antibodies to the 2009 H1N1, consistent with the epidemiology of the 2009 H1N1 pandemic showing that younger age groups were disproportionately affected 
, but the extent of cross-immunity induced by recent seasonal influenza vaccines is more ambiguous 
. Differences in methodologies and history of vaccination or infection with NCD/20/99 may have affected the outcomes. Our results involving persons aged 48–64 years (Table S5
) extend other reports showing that older persons generally have some pre-existing immunity to the 2009 H1N1, but more significantly highlight the presence of cross-neutralizing antibodies between 2009 H1N1 and NCD/20/99. Because all subjects in our contemporary cohort received yearly seasonal influenza vaccines for at least the past five years, and NCD/20/99 was repeatedly used in seasonal vaccines during the 2000/01–2006/07 influenza seasons, we cannot determine the extent to which influenza vaccinations and/or natural infections contributed to the generation of cross-neutralizing antibodies to 2009 H1N1 and NCD/20/99.
To investigate potential cross-neutralizing determinants in NCD/20/99 and 2009 H1N1, we used chimeric HA-pseudotypes involving HA1 and HA2 subunits of NCD/20/99 and Bris/59/07 and sera that lacked neutralization to Bris/59/07 (Table S2
). Both contemporary and archived sera from the NJ/76 swine influenza vaccine trials contained cross-neutralizing antibodies that depended on the HA2 subunit ( and ). Most remarkable, we found that the cross-neutralization was influenced by a single conservative amino acid change at position 89 in HA2, which differed between NCD/20/99 and Bris/59/07 (). Thus, these data reveal a new determinant in the C helix region of the HA2 stalk that modified sensitivity to cross-neutralizing antibodies present in human sera from two different cohorts separated by more than three decades.
Growing interest in the generation of broadly neutralizing influenza antibodies has led to the discovery of several new monoclonal antibodies that bind to HA2 
. The first reported heterosubtypic neutralizing antibody, C179, derived from a mouse immunized with the A/Okuda/57 H2N2 strain, was found to be directed to a conformational epitope involving the A helix in the HA2 stalk () and a region in HA1 
. More recently, several other HA2 heterosubtypic neutralizing monoclonal antibodies that are potent against strains from H1 and H5 subtype (Group 1) influenza viruses have been isolated using various methods. Some of these antibodies have been also shown to make contacts with the A helix of HA2 
(). Other HA2 monoclonal antibodies have been shown to bind to a highly conserved pocket in the stalk region containing the fusion peptide 
or undetermined regions of the HA2 stalk 
. Another potent broadly neutralizing monoclonal antibody against H3N2 (Group 2) but not H1N1 (Group 1) strains was shown to bind to a peptide corresponding to the C helix region in the HA2 stalk 
Structural implications for the L89I (L415I) mutation in HA2.
The HA2 monoclonal antibodies bind to regions in the HA2 stalk and interfere with conformational changes that are needed for virus entry 
, but they do not block HA attachment to receptors. These HA2 antibodies lack HI activity and were discovered using neutralization assays that sometimes involved HA-pseudotypes 
. We 
and others 
have shown that HA-pseudotypes neutralization titers are highly correlated with microneutralization titers for replicating influenza virus, but the correlate of protection using HA-pseudotype neutralization titers has not been determined. Also, glycoproteins on the surface of HIV-based retroviral particle may be less densely packed and more exposed compared to HA on the surface of influenza viral particles, perhaps making them more susceptible to HA2-directed neutralization compared to influenza virus, as suggested in some studies 
. While sensitive screening assays have allowed many groups to fish out broadly neutralizing antibodies, it is generally believed that HA2 heterosubtypic neutralizing antibodies are present at relatively low concentrations, as compared with antibodies directed to HA1 
. The need to change annual seasonal influenza vaccines to match dominant circulating strains indicates that such HA2 cross-neutralizing antibodies may not be present at high enough titers to provide robust protection. It is therefore difficult to discern the degree to which HA2 antibodies in our sera samples could contribute to protection to 2009 H1N1 virus. However, studies in animal models have provided proof of concept that induction 
or passive transfer of HA2 antibodies alone 
can provide protection. Appropriately designed vaccines may be able to induce robust immune responses to conserved neutralizing epitopes in HA2 
. Recent examples involving several approaches are showing promise 
Our finding that the conservative substitution of isoleucine for 89 L reduced sensitivity to cross-neutralizing antibodies present in our sera was surprising. The crystal structure of the A/Cal/04/2009 HA 
shows that 89 L packs tightly into a poorly exposed crevice underneath the HA1 crown (), making intimate contact with HA1 through a lysine and tyrosine at residues 310 and 308, respectively (). Substitution of 89 L with isoleucine may cause the interactions between HA1 and HA2 in this region to shift in order to accommodate the alternate side chain (Figure S2
), and in doing so, could directly alter exposure or conformation of the antibody binding site. Alternatively, residue 89 may be evolving in response to immune pressure at distant sites. For example, 89I may reflect an adaptive change in HA2 resulting from direct immune pressure on epitopes in HA1. The 89I substitution could also impose allosteric changes on nearby or more distant neutralizing epitopes in either HA1 or HA2. The observation that Bris/59/07 was less sensitive to neutralization by an HA2 antibody compared to NCD/20/99 is consistent with the notion that this residue could influence neutralization by HA2 antibodies 
. We also note that 89L is not near any of the contact residues for the recently described HA2 monoclonals specific for Group 1 HAs, although it is located on the C helix region of the HA2 stalk that has recently been suggested to contain an epitope for the 12D1 monoclonal antibody that binds H3 strains from Group 2.
Review of the database of human H1N1 HA also offers intriguing clues about the potential significance of the change of leucine to isoleucine at position 89 in HA2. We note that 89L has been maintained in seasonal H1N1 influenza viruses from at least 1918 until 2006 when it started to change to isoleucine, and 89L disappeared in 2008 (). It is tempting to speculate that this change could reflect immune escape. We also note that H3 strains from Group 2 influenza viruses generally have an isoleucine at the corresponding position in HA2. Interestingly, unlike Group 1 H1N1 HA, a carbohydrate can be seen in the H3N2 HA crystal structure extending in the vicinity of the isoleucine (coming from N285) (PDB 3HMG) 
, which could conceivably have evolved to shield it from neutralizing antibodies. These observations offer a cautionary note that antigenic drift in this region may arise under strong selection pressure. Nonetheless, the viable substitutions may be limited due to the fact that residue 89 and others in the stalk regions make important contacts in both the native and low pH structures of HA, consistent with the difficulties in generating escape mutants with some of the HA monoclonal antibodies 
. Perhaps this explains why H3N2 strains have incorporated a carbohydrate in the vicinity of this region.
In summary, our studies showed that cross-neutralizing antibodies to 2009 H1N1 influenza that involve the HA2 subunit could be detected in sera collected in 1976 from NJ/76 swine influenza vaccine trials and sera from persons aged 48–64 who received annual influenza vaccines for at least the past five years. A conservative substitution at position 89 in HA2, found in drifted seasonal influenza virus variants from the 2006/07 and 2007/08 influenza seasons, abrogated this neutralization. Future studies involving vaccines that elicit strong antibody responses to HA2 will reveal the extent to which mutations can lead to immune escape.