Investigation of the human antibody response to influenza virus infection has been largely limited to serology, with relatively little analysis at the molecular level. The 1918 H1N1 influenza virus pandemic was the most severe of the modern era 1 . Recent work has recovered the gene sequences of this unusual strain 2 , so that the 1918 pandemic virus could be reconstituted to display its unique virulence phenotypes 3,4 . However, little is known about adaptive immunity to this virus. We took advantage of the 1918 virus sequencing and the resultant production of recombinant 1918 hemagglutinin (HA) protein antigen to characterize at the clonal level neutralizing antibodies induced by natural exposure of survivors to the 1918 pandemic virus. In our study, each of 32 individuals tested that were born in or before 1915 exhibited seroreactivity with 1918 virus, nearly 90 years after the pandemic. Seven of 8 donor samples tested had circulating B cells that secreted antibodies that bound 1918 HA. We isolated B cells from subjects and generated five monoclonal antibodies that exhibited potent neutralizing activity against 1918 virus from three separate donors. These antibodies also cross-reacted with the genetically similar HA of a 1930 swine H1N1 influenza strain, but not with HAs of more contemporary human influenza viruses. The antibody genes exhibited an unusually Correspondence should be addressed to JEC (James.Crowe@vanderbilt.edu), CFB (Chris.Basler@mssm.edu) or ELA (Eric.Altschuler@umdnj.edu). Supplementary Information is linked to the online version of the paper at www.nature.com/nature.Author Contributions XY and TT contributed equally to this work. XY, PAM, MDH and FSH made and cloned the mAbs, sequenced antibody genes, and performed IF experiments, CJK performed biosensor studies, TMT, CP, and LAP designed and performed in vivo studies, OM sequenced the HA genes of the H1N1 viruses used in this study and performed ELISA assays with these viruses. PVA assisted with HAI and neutralization assays and with cloning of recombinant HA molecules. JS and IAW provided recombinant HA; ELA led the clinical recruitment, ELA, CFB and JEC conceived of the experimental plan. CFB and JEC wrote the manuscript. All authors discussed the results and commented on the manuscript.Antibody nucleotide sequences are deposited in GenBank, accession numbers EU169674 through EU169679 and EU825947 through EU825950.Reprints and permissions information is available at npg.nature.com/reprints and permissions.The authors declare no competing financial interests. NIH Public Access Author ManuscriptNature. Author manuscript; available in PMC 2010 April 3. . We collected transformed cells from the wells corresponding to supernates exhibiting the highest levels of specific binding to the 1918 HA (derived from five donors) and fused them to the HMMA2.5 nonsecreting myeloma partner 7 using an electrofusion technique 8 . We isolated 17 unique hybridoma cell lines that secreted antibodies reactive with the 1918 HA from cell lines derived fro...

The conserved influenza virus hemagglutinin (HA) stem domain elicits cross-reactive antibodies, but epitopes in the globular head typically elicit strain-specific responses because of the hypervariability of this region. We isolated human monoclonal antibody 5J8, which neutralized a broad spectrum of 20th century H1N1 viruses and the 2009 pandemic H1N1 virus. Fine mapping of the interaction unexpectedly revealed a novel epitope between the receptor-binding pocket and the Ca 2 antigenic site on HA. This antibody exposes a new mechanism underlying broad immunity against H1N1 influenza viruses and identifies a conserved epitope that might be incorporated into engineered H1 virus vaccines.

The recent 2009 pandemic H1N1 virus infection in humans has resulted in nearly 5,000 deaths worldwide. Early epidemiological findings indicated a low level of infection in the older population (>65 years) with the pandemic virus, and a greater susceptibility in people younger than 35 years of age, a phenomenon correlated with the presence of cross-reactive immunity in the older population. It is unclear what virus(es) might be responsible for this apparent cross-protection against the 2009 pandemic H1N1 virus. We describe a mouse lethal challenge model for the 2009 pandemic H1N1 strain, used together with a panel of inactivated H1N1 virus vaccines and hemagglutinin (HA) monoclonal antibodies to dissect the possible humoral antigenic determinants of pre-existing immunity against this virus in the human population. By hemagglutinination inhibition (HI) assays and vaccination/challenge studies, we demonstrate that the 2009 pandemic H1N1 virus is antigenically similar to human H1N1 viruses that circulated from 1918–1943 and to classical swine H1N1 viruses. Antibodies elicited against 1918-like or classical swine H1N1 vaccines completely protect C57B/6 mice from lethal challenge with the influenza A/Netherlands/602/2009 virus isolate. In contrast, contemporary H1N1 vaccines afforded only partial protection. Passive immunization with cross-reactive monoclonal antibodies (mAbs) raised against either 1918 or A/California/04/2009 HA proteins offered full protection from death. Analysis of mAb antibody escape mutants, generated by selection of 2009 H1N1 virus with these mAbs, indicate that antigenic site Sa is one of the conserved cross-protective epitopes. Our findings in mice agree with serological data showing high prevalence of 2009 H1N1 cross-reactive antibodies only in the older population, indicating that prior infection with 1918-like viruses or vaccination against the 1976 swine H1N1 virus in the USA are likely to provide protection against the 2009 pandemic H1N1 virus. This data provides a mechanistic basis for the protection seen in the older population, and emphasizes a rationale for including vaccination of the younger, naïve population. Our results also support the notion that pigs can act as an animal reservoir where influenza virus HAs become antigenically frozen for long periods of time, facilitating the generation of human pandemic viruses.

The 2009 pandemic influenza A (H1N1) virus exhibits hemagglutinin protein sequence homology with the 1918 pandemic influenza virus. We found that human monoclonal antibodies recognized the Sa antigenic site on the head domains of both 1918 and 2009 hemagglutinins, a site that is hypervariable due to immune selection. These antibodies exhibited high potency against the 2009 virus in vitro, and one exerted a marked therapeutic effect in vivo.

We generated from a single blood sample five independent human monoclonal antibodies that recognized the Sa antigenic site on the head of influenza HA and exhibited inhibitory activity against a broad panel of H1N1 strains. All five Abs used the VH3-7 and JH6 gene segments, but at least four independent clones were identified by junctional analysis. High throughput sequence analysis of circulating B cells revealed that each of the independent clones were members of complex phylogenetic lineages that had diversified widely using a pattern of progressive diversification through somatic mutation. Unexpectedly, B cells encoding multiple diverging lineages of these clones, including many containing very few mutations in the antibody genes, persisted in the circulation. Conversely, we noted frequent instances of amino acid sequence convergence in the antigen combining sites exhibited by members of independent clones, suggesting a strong selection for optimal binding sites. We suggest that maintenance in circulation of a wide diversity of somatic variants of dominant clones may facilitate recognition of drift variant virus epitopes that occur in rapidly mutating virus antigens, such as influenza HA. In fact, these Ab clones recognize an epitope that acquired three glycosylation sites mediating escape from previously isolated human antibodies.