Seasonal epidemics caused by antigenic variations in influenza A virus remain a public health concern and an economic burden. The isolation and characterization of broadly neutralizing anti-hemagglutinin monoclonal antibodies (MAb) have highlighted the presence of highly conserved epitopes in divergent influenza A viruses. Here, we describe the generation and characterization of a mouse monoclonal antibody designed to target the conserved regions of the hemagglutinin of influenza A H1 viruses, a subtype that has caused pandemics in the human population in both the 20th and 21st centuries. By sequentially immunizing mice with plasmid DNA encoding the hemagglutinin of antigenically different H1 influenza A viruses (A/South Carolina/1/1918, A/USSR/92/1977, and A/California/4/2009), we isolated and identified MAb 6F12. Similar to other broadly neutralizing MAb previously described, MAb 6F12 has no hemagglutination inhibition activity against influenza A viruses and targets the stalk region of hemagglutinins. As designed, it has neutralizing activity against a divergent panel of H1 viruses in vitro , representing 79 years of antigenic drift. Most notably, MAb 6F12 prevented gross weight loss against divergent H1 viruses in passive transfer experiments in mice, both in pre- and postexposure prophylaxis regimens. The broad but specific activity of MAb 6F12 highlights the potent efficacy of monoclonal antibodies directed against a single subtype of influenza A virus.

Apoptosis may play a crucial role in the pathogenesis of pneumonia and lymphopenia caused by this virus in humans.

Avian influenza viruses preferentially recognize sialosugar chains terminating in sialic acid-␣2,3-galactose (SA␣2,3Gal), whereas human influenza viruses preferentially recognize SA␣2,6Gal. A conversion to SA␣2,6Gal specificity is believed to be one of the changes required for the introduction of new hemagglutinin (HA) subtypes to the human population, which can lead to pandemics. Avian influenza H5N1 virus is a major threat for the emergence of a pandemic virus. As of 12 June 2007, the virus has been reported in 45 countries, and 312 human cases with 190 deaths have been confirmed. We describe here substitutions at position 129 and 134 identified in a virus isolated from a fatal human case that could change the receptor-binding preference of HA of H5N1 virus from SA␣2,3Gal to both SA␣2,3Gal and SA␣2,6Gal. Molecular modeling demonstrated that the mutation may stabilize SA␣2,6Gal in its optimal cis conformation in the binding pocket. The mutation was found in approximately half of the viral sequences directly amplified from a respiratory specimen of the patient. Our data confirm the presence of H5N1 virus with the ability to bind to a human-type receptor in this patient and suggest the selection and expansion of the mutant with human-type receptor specificity in the human host environment.

Highly pathogenic H5N1 avian influenza virus has spread through at least 45 countries in three continents. Despite the ability to infect and cause severe disease in humans, the virus cannot transmit efficiently from human to human. The lack of efficient transmission indicates the incompletion of the adaptation of the avian virus to the new host species. The required mutations for the complete adaptation and the emergence of a potential pandemic virus are likely to originate and be selected within infected human tissues. Differential receptor preference plays an important role in the species-tropism of avian influenza. We have analysed quasispecies of sequences covering the receptor-binding domain of the haemagglutinin gene of H5N1 viruses derived from fatal human cases. We employed a likelihood ratio test to identify positive-selection sites within the quasispecies. Nine of seventeen positive-selection sites identified in our analyses were found to be located within or flanking the receptor-binding domain. Some of these mutations are known to alter receptor-binding specificity. This suggests that our approach could be used to screen for mutations with significant functional impact. Our data provide new candidate mutations for the viral adaptation to a human host, and a new approach to search for new genetic markers of potential pandemic viruses.

Microarray analysis of gene expression profile of lungs from two fatal H5N1 influenza cases identified 3,435 genes with higher than twofold changes in mRNA levels as compared to those of normal lung. One thousand nineteen genes and 2,416 genes were up-regulated and down-regulated commonly, respectively. Gene ontology analysis identified several ontology terms with significant association with these genes, most of which are related to cellular metabolism and regulation of cellular process including apoptosis and chemotaxis. Pulmonary surfactant protein D (SP-D) was found to be down-regulated. Quantitative RT-PCR confirmed the levels of SP-D mRNA in the lungs infected with H5N1 to be lower than those of normal lungs and lungs from patients with acute respiratory distress syndrome. SP-D plays multiple roles in respiratory innate defense against various pathogens, regulation of inflammatory responses, and maintenance of alveolar integrity. Reduction of SP-D in H5N1 influenza may play important roles in the pathogenesis of the disease.

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Codon volatility is a method recently developed to estimate selective pressures on proteins on the basis of their synonymous codon usage. Volatility of a codon was defined as the fraction of single nucleotide substitutions that would be nonsynonymous. Higher volatility may indicate that the gene has been under more positive selection in the recent past. We analyzed volatility of hemagglutinin genes of H5N1 viruses in the recent outbreaks and observed differences in the volatility among viruses of different clades. The codon volatility of subclade 2.1 viruses from Indonesia was the lowest among all H5N1 clades and subclades. Time series analyses since the beginning of the epidemic in 2004 showed that codon volatility of subclade 2.1 has gradually decreased, while those of other major clades have been increasing. This may reflect differences in the recent evolution of these viruses.

A recombinant vaccinia virus harboring the full length hemagglutinin (HA) gene derived from a highly pathogenic avian influenza A/Thailand/1(KAN-1)/2004 (H5N1) virus (rVac-H5 HA virus) was constructed. The immunogenicity of the expressed HA protein was characterized using goat antiserum, mouse monoclonal antibody, and human sera. The expressed HA protein localized both in the cytoplasm and on the cytoplasmic membrane of the thymidine kinase negative cells infected with the rVac-H5 HA virus, as determined by immunofluorescence assay. Western blot analysis demonstrated that the rVac-H5 HA protein was post-translationally processed by proteolytic cleavage of the HA0 precursor into HA1 and HA2 domains; and all of these HA forms were immunogenic in BALB/c mice. The molecular weight (MW) of each HA domain was the same as the wild-type H5 HA produced in Madin-Darby canine kidney cells infected with the H5N1 virus, but was higher than that expressed by a baculovirus-insect cell system. Sera from all H5N1 survivors reacted to HA0, HA1, and HA2 domains; whereas sera from H5N1-uninfected subjects reacted to the HA2 domain only, but not to HA0 or HA1, indicating that some cross-subtypic immunity exists in the general population. There was a lot-to-lot variation of the recombinant HA produced in the baculovirus-insect cell system that might affect the detection rate of antibody directed against certain HA domains.