In the present study we describe the binding features and the neutralizing activity of a human monoclonal antibody named PN-SIA28 previously described as Fab fragment [19, 20] . IgG PN-SIA28 was tested against a large panel of influenza A viruses belonging to group 1 (H1N1, H2N2, H5N1, H9N2) and group 2 (H3N2, H7N2) subtypes showing a broader neutralizing activity compared to its monovalent molecule, possibly due to the bivalency feature of the IgG. Indeed, IgG PN-SIA28 showed a Figure 1 . PN-SIA28 Neutralizing activity. Influenza hemagglutinin unrooted phylogenetic tree of the different viral strains tested in neutralization assays with PN-SIA28. Viral isolates belonging to group 1 and group 2 are presented in the box B and box A, respectively. A green '+' indicates positive neutralizing activity, a red '2' indicates negative neutralizing activity. As reported in the text, PN-SIA28 is able to neutralize all of the group 1 strains and is also able to neutralize all of the H3N2 isolates spanning 1968 and 1975. *Recombinant HA from (H1N1) A/South Carolina/1/ 1918 pandemic strain was previously shown to be bound by PN-SIA28 [19, 20] . Analogously, recombinant HA from H5N1 A/Cygnus Olor/Italy/742/ 2005 was recognized by PN-SIA28 (data not shown). # H1N1 A/New Caledonia/20/1999 was previously shown to be neutralized by PN-SIA28 as Fab fragment [19, 20] . doi:10.1371/journal.pone.0028001.g001 robust neutralizing activity (IC 50 = 0.4-3.7 mg/ml) against all tested strains belonging to group 1, thus demonstrating that its epitope is broadly shared among group 1 viruses, including the highly divergent H9 subtype. More importantly, PN-SIA28 showed a potent neutralizing activity also against group 2 viruses demonstrating that its epitope is also present in the highly Figure 2 . Sequence conservation in hemagglutinin groups and subtypes. Boxes indicate mutated residues which decrease PN-SIA28 binding to mutated HAs. Circles on the top indicate PN-SIA28 percent binding to each HA alanine mutants compared to binding to wild-type HA: red 25% binding, yellow 50-75% binding. Sequence numbering is based on H1N1 A/PR/8/34 coding region (GenBank accession number ABO21709). Neutralizing activity of PN-SIA28 against each strain is highlighted by a green '+' or a red '2' on the left, indicating neutralizing activity and no neutralizing activity, respectively. *Recombinant HA from H1N1 A/South Carolina/1/1918 pandemic strain was previously shown to be bound by PN-SIA28 [19, 20] ; analogously, recombinant HA from H5N1 A/Cygnus Olor/Italy/742/2005 was recognized by PN-SIA28 (data not shown). # H1N1 A/New Caledonia/20/1999 was previously shown to be neutralized by PN-SIA28 as Fab fragment [19, 20] . doi:10.1371/journal.pone.0028001.g002 divergent H3N2 subtype. Indeed, IgG PN-SIA28 was able to potently neutralize H3N2 strains circulating from 1968 to 1975 (IC 50 = 0.8-2.6 mg/ml), despite the important phylogenetic distance between these viruses and those belonging to group 1 ( Figure 1 ). No neutralizing activity was observed against the dramatically divergent H7N2 isolate tested in this study, nor against the more recent H3N2 isolates.