Hemagglutinin stalk antibodies elicited by the 2009 pandemic influenza virus as a mechanism for the extinction of seasonal H1N1 viruses

Abstract

After the emergence of pandemic influenza viruses in 1957, 1968, and 2009, existing seasonal viruses were observed to be replaced in the human population by the novel pandemic strains. We have previously hypothesized that the replacement of seasonal strains was mediated, in part, by a population-scale boost in antibodies specific for conserved regions of the hemagglutinin stalk and the viral neuraminidase. Numerous recent studies have shown the role of stalk-specific antibodies in neutralization of influenza viruses; the finding that stalk antibodies can effectively neutralize virus alters the existing dogma that influenza virus neutralization is mediated solely by antibodies that react with the globular head of the viral hemagglutinin. The present study explores the possibility that stalk-specific antibodies were boosted by infection with the 2009 H1N1 pandemic virus and that those antibodies could have contributed to the disappearance of existing seasonal H1N1 influenza virus strains. To study stalk-specific antibodies, we have developed chimeric hemagglutinin constructs that enable the measurement of antibodies that bind the hemagglutinin protein and neutralize virus but do not have hemagglutination inhibition activity. Using these chimeric hemagglutinin reagents, we show that infection with the 2009 pandemic H1N1 virus elicited a boost in titer of virus-neutralizing antibodies directed against the hemagglutinin stalk. In addition, we describe assays that can be used to measure influenza virus-neutralizing antibodies that are not detected in the traditional hemagglutination inhibition assay.

Fig. 1.

Chimeric HA (cHA) proteins and recombinant chimeric virus. (A) Schematic representation of cHAs. The globular head domain is defined as the intervening amino acid sequence between residues C52 and C277 (H3 numbering). Using this disulfide bond as the demarcation between head and stalk, exotic HA heads were introduced atop heterologous stalks. The stalk domain is defined as the remaining portions of HA1 and HA2 subunits. CT, cytoplasmic tail; SP, signal peptide; TM, transmembrane domain. The full-length HA structures were downloaded from the Protein Database (PDB): PR8 (H1) HA (PDB ID 1RU7) and A/guinea fowl/Hong Kong/WF10/99 HA [represented by A/swine/Hong Kong/9/98 (H9; PDB ID 1JSD)]. Final images were generated with PyMol (Delano Scientific). Because no structure of an H6 HA has been published, the image of the head-folding of the PR8 HA is used for the cH6/1 construct. (B) Immunofluorescence to confirm expression of cHA. MDCK cells were infected with either WT PR8 or cH9/1 N3 virus, or they were mock-infected. Antibodies specific for the head and stalk of PR8 virus as well as an antibody with H9 reactivity were used to confirm cHA expression. (Magnification bar: 40×.)

Fig. 2.

Adult patients infected with pandemic H1N1 virus have high titers of neutralizing antibodies that are reactive with the HA stalk. Reactivity of sera of pH1N1-infected adults (n = 9), children not infected with pH1N1 (n = 5), and adults not infected with pH1N1 virus (n = 11) with cH6/1 protein (A), cH9/1 protein (B), the LAH of the HA2 protein (anti-LAH antibody was used as a positive control; C), H5 HA protein (mouse polyclonal serum raised against H5 HA was used as a positive control and a pan-H3 antibody, 12D1, was used as negative control; D) (13), or H3 HA protein (12D1 was used as a positive control and mouse polyclonal serum raised against H5 HA was used as a negative control; E). All were assessed by ELISA; data points represent average titers with SE or reactivity of pooled samples.

Fig. 3.

Adult patients infected with pandemic H1N1 virus have high titers of neutralizing antibodies that are specific for the HA stalk. (A and B) Sera from pH1N1-infected (n = 14) and adults not infected with pH1N1 (n = 5) were pooled separately, and total IgG from both pools was purified. Neutralizing capability of stalk antibodies was assessed by plaque reduction assay using cH9/1 N3 virus. Data points represent the mean and SE of two experiments. Plaques were immunostained with anti-H9 antibody G1-26. B shows plaque reduction of the four dilutions of sera shown along the top. (C) Pseudotype particle neutralization assay measures neutralizing antibody activity of the human-purified IgG preparations (sera from pH1N1-infected adults and adults not infected with pH1N1). Total IgG concentrations were 50, 10, and 2 μg/mL. As a positive control, the stalk-specific monoclonal antibody 6F12 was used.