isolated in humans and in particular H1, H2 and H3 subtypes have been responsible of the reported influenza pandemic outbreaks. In this study, we describe a human mAb, named PN- SIA28, that is capable of neutralizing all tested group 1 isolates, as well as isolates belonging to H3N2, the only group 2 subtype capable, so far, of causing a pandemic. Results Neutralizing activity of PN-SIA28 The binding and neutralizing features of PN-SIA28 were initially studied using the Fab fragment molecule produced in E. coli, demonstrating that Fab PN-SIA28 recognizes an epitope on the stem region of HA and is able to strongly neutralize all tested H1N1 strains [19,20]. It is well documented in the literature that bivalency of a whole IgG molecule may be an essential features for the biological activity of a mAb [21,22,23,24]. For this reason, in this work, to evaluate PN-SIA28 features as whole IgG molecule, IgG PN- SIA28 was generated and tested in different neutralization assays against human, swine and avian influenza A viruses belonging to both HA based phylogenetic groups and encompassing all subtypes responsible of described pandemic events. The results obtained showed that IgG PN-SIA28 strongly neutralizes viruses belonging to the group 1 as well as those of the group 2. More in details, IgG PN-SIA28 neutralized all the H1N1 tested viruses with an half maximal inhibitory concentration (IC50) ranging between 0.4–3.7 mg/ml, the H5N1 viruses with IC50 ranging between 0.9–2.8 mg/ml, the H2N2 subtype isolate with an IC50 of 0.8 mg/ml and the H9N2 subtype strain with an IC50 of 0.9 mg/ml (Table 1, Figure S1). More interestingly, IgG PN-SIA28, also potently neutralizes H3N2 viruses circulating in the years from Table 1. PN-SIA28 neutralizing activity against all tested isolates. Subtype Strain IC50a mg/ml (95% CIb) A/Vietnam/1203/2004 clade 1 A/duck/Vietnam/NCVD98/2007 clade 2.3.4 2.8 (1.3–5.9) LAIV A/chicken/Hong Kong/G9/97 0.4 (0.3–0.5) 0.8 (0.2–2.9) 0.7 (0.3–1.4) 1.5 (1.3–1.6) 3.7 (2.8–4.9) 0.8 (0.1–4.8) 0.9 (0.1–8.1) 0.9 (0.2–3.4) 1.3 (0.7–2.5) 0.8 (0.5–1.4) 2.6 (1.8–3.7) 1 (0.4–2.3) .10 .10 .10 .30 .30 .10 Cross-Neutralizing mAb to Influenza Viruses 1968 to 1975 with IC50 ranging between 0.8–2.6 mg/ml (Table 1, Figure S2) despite the great phylogenetic distance of these viruses compared to those belonging to group 1 (Figure 1). No detectable neutralizing activity was observed against more recent H3N2 tested viruses (Table 1, Figure 1 and S3) as well as for the H7N2 tested isolate. Epitope mapping Screening of a random 12-mer peptide phage displayed library. In order to select peptides able to bind antibody PN- SIA28 that could possibly indicate the region that the antibody recognizes on the HA, a phagemidic random 12-mer peptide library was screened. After three rounds of panning, PN-SIA28 binding phages were eluted and used to infect E. coli XL1-Blue for an ELISA screening on PN-SIA28. Twenty-five positive clones were sequenced. An in silico analysis was performed, using Mimox and Pepitope servers, comparing the selected peptides to the available crystal structure of H1N1 A/South Carolina/1918 (A/ SC/1918) (ID code 1RD8) and A/Puerto Rico/8/34 (A/PR/8/ 34) hemagglutinins (ID code 1RU7). This study allowed the identification of several possible residues located on the stem region of HA potentially involved in the binding of PN-SIA28: Asn336, Ile337, Pro338, Trp357 and Thr358 (sequence number- ing refers to A/PR/8/34, GenBank accession number ABO21709) (Figure 2). In vitro selection of escape mutants. To further define the region bound by PN-SIA28, an assay aiming at the evaluation of PN-SIA28 capability to induce escape mutant was performed. A/ PR/8/34 H1N1 virus was cultured under constant selective pressure of PN-SIA28, and after several rounds of cell infections, two escape mutants were generated. Sequencing analysis of the generated neutralization escape variants revealed two different mutants carrying each a single amino acidic mutation in position 361 (Ile361Thr) and in position 362 (Asp362Gly) in the HA2 subunit compared to wild type. Alanine scanning mutagenesis study. On the basis of these results, HA mutants carrying an alanine substitution in position 361 (Ile361Ala) or in position 362 (Asp362Ala) were generated and the PN-SIA28 binding to the mutants was evaluated by FACS analysis evidencing that PN-SIA28 was not longer able to bind to the mutated HAs. In order to identify other amino acidic residues involved in the binding of PN-SIA28 to HA, a large panel of HA- alanine mutants was generated (Table S1). FACS analysis showed that binding of PN-SIA28 to HA was decreased by His25Ala, His45Ala mutants on HA1 and Thr358Ala, Met360Ala, Ile361Ala, Asp362Ala, Gly363Ala, Trp364Ala, Thr384Ala and Val395Ala mutants on HA2. (Figure 2). All other mutations listed in the Table S1 did not have any effect on PN-SIA28 binding. An extra HA-mutant in position 361 (Ile361Val) was generated. Indeed, in position 361 either an Isoleucine or a Valine residue can be present in different isolates belonging to either to group 1 either to group 2 (Figure 2). No binding reduction of PN-SIA28 to this mutant was observed. Based on the results obtained with the HA mutants, an in silico analysis on the HA crystal structure of A/SC/1918 and A/PR/8/ 34 was carried out for the amino acid residues that the alanine scanning study showed to influence the binding of PN-SIA28 to HA. The analysis confirmed that the residues identified lie on the stem region of HA, that they belong to the HA1 and HA2 subunits and that they are exposed on the surface of the HA molecule (Figure 3). A similar approach was followed also for the H3 subtype. Based on the results obtained for the H1N1 study, four H3 alanine mutants were initially generated on the A/Aichi/2/68 (H3N2) HA: His34Ala, Asn54Ala on HA1 and Ile363Ala and Asp364Ala H1N1 H2N2 H5N1 H9N2 H3N2 A/Wilson Smith/33 A/Puerto Rico/8/34 A/Malaya/302/54 A/Milan/UHSR1/2009 A/swine/Parma/1/97 A/Ann Arbor/6/60 A/Aichi/2/68 A/Hong Kong/8/68 A/Port Chalmers/1/73 A/Victoria/3/75 A/Philippines/01/82 A/England/648/89 A/Fukui/20/2004 A/Washington/01/2007 A/New Hampshire/01/2009 H7N2 A/New York/107/2003 aIC50: Half maximal inhibitory concentration expressed in mg/ml. bCI: confidence interval. doi:10.1371/journal.pone.0028001.t001 PLoS ONE | www.plosone.org 2 December 2011 | Volume 6 | Issue 12 | e28001