Fatal human respiratory disease associated with the 1918 pandemic influenza virus and potentially pandemic H5N1 viruses is characterized by severe lung pathology, including pulmonary edema and extensive inflammatory infiltrate. Here, we quantified the cellular immune response to infection in the mouse lung by flow cytometry and demonstrate that mice infected with highly pathogenic (HP) H1N1 and H5N1 influenza viruses exhibit significantly high numbers of macrophages and neutrophils in the lungs compared to mice infected with low pathogenic (LP) viruses. Mice infected with the 1918 pandemic virus and a recent H5N1 human isolate show considerable similarities in overall lung cellularity, lung immune cell sub-population composition and cellular immune temporal dynamics. Interestingly, while these similarities were observed, the HP H5N1 virus consistently elicited significantly higher levels of pro-inflammatory cytokines in whole lungs and primary human macrophages, revealing a potentially critical difference in the pathogenesis of H5N1 infections. These results together show that infection with HP influenza viruses such as H5N1 and the 1918 pandemic virus leads to a rapid cell recruitment of macrophages and neutrophils into the lungs, suggesting that these cells play a role in acute lung inflammation associated with HP influenza virus infection. In addition, primary macrophages and dendritic cells were also susceptible to 1918 and H5N1 influenza virus infection in vitro and in infected mouse lung tissue.

The proapoptotic PB1-F2 protein of influenza A viruses has been shown to contribute to pathogenesis in the mouse model. Expression of full-length PB1-F2 increases the pathogenesis of the influenza A virus, causing weight loss, slower viral clearance, and increased viral titers in the lungs. After comparing viruses from the Hong Kong 1997 H5N1 outbreak, one amino acid change (N66S) was found in the PB1-F2 sequence at position 66 that correlated with pathogenicity. This same amino acid change (N66S) was also found in the PB1-F2 protein of the 1918 pandemic A/Brevig Mission/18 virus. Two isogenic recombinant chimeric viruses were created with an influenza A/WSN/33 virus background containing the PB1 segment from the HK/156/97: WH and WH N66S. In mice infected with WH N66S virus there was increased pathogenicity as measured by weight loss and decreased survival, and a 100-fold increase in virus replication when compared to mice infected with the WH virus. The 1918 pandemic strain A/Brevig Mission/18 was reconstructed with a pathogenicity-reducing mutation in PB1-F2 (S66N). The resultant 1918 S66N virus was attenuated in mice having a 3-log lower 50% lethal dose and caused less morbidity and mortality in mice than the wild-type virus. Viral lung titers were also decreased in 1918 S66N–infected mice compared with wild-type 1918 virus–infected mice. In addition, both viruses with an S at position 66 (WH N66S and wt 1918) induced elevated levels of cytokines in the lungs of infected mice. Together, these data show that a single amino acid substitution in PB1-F2 can result in increased viral pathogenicity and could be one of the factors contributing to the high lethality seen with the 1918 pandemic virus.

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...