Cross-Neutralizing mAb to Influenza Viruses 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 on HA2 (corresponding to His25, His45, Ile361 and Asp362 in H1N1 sequencing numbering) (Figure 2, Figure 4 and Table S1). None of these alanine mutated HAs was bound by PN-SIA28. Furthermore, the Asn54His mutant (corresponding to position 45 in H1N1 numbering) on A/Aichi/2/1968 HA was also generated due to the presence of a Histidine in the corresponding position in H1N1 viruses. An increased binding of PN-SIA28 to this mutant was observed compared to wild type H3 HA. In order to deeper investigate on the absence of neutralizing activity of PN-SIA28 against most recent H3N2 strains, three more alanine-mutants (Asp18, Lys57 and Ile70) (see Table S1) were generated considering the differences in the stem region between H3N2 A/Victoria/3/1975 (the most recent neutralized isolate) and H3N2 A/Philippines/2/1982 (the earliest non neutralized strain). A 30% binding decrease was observed for Lys57Ala HA mutant, while the Asp18Ala and Ile70Ala did not have any effect on PN- SIA28 binding to H3N2 HA (Figure 5 and Table S1). Discussion 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 PLoS ONE | www.plosone.org 3 December 2011 | Volume 6 | Issue 12 | e28001