Figure 3. Position of the residues on the surface of (H1N1) A/ PR/8/34 influenza hemagglutinin trimer decreasing the bind- ing of PN-SIA28. White areas indicate the HA1 domains of the HA trimer, whereas cyan areas indicate the HA2 domains. Blue regions depict the mutated residues inhibiting PN-SIA28 binding to HA according to Figure 2. doi:10.1371/journal.pone.0028001.g003 divergent H3N2 subtype. Indeed, IgG PN-SIA28 was able to potently neutralize H3N2 strains circulating from 1968 to 1975 (IC50 = 0.8–2.6 mg/ml), despite the important phylogenetic dis- tance 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. The broad neutralizing activity of PN-SIA28 against highly divergent influenza viruses, suggests that the epitope recognized is extremely conserved. The lack of hemagglutination inhibitory activity and the competition with a mouse mAb (C179) directed against the stem region of HA, had already suggested that PN- SIA28 epitope is not localized on the globular head and that it is shared between HA1 and HA2 domains [19,20]. Several approaches have been used in this study to better define the region recognized by PN-SIA28. The screening of a phagemidic random peptide library suggested the binding of PN-SIA28 to the stem region in the proximity of the viral membrane. Interestingly, the sequence analysis of A/PR/8/34 (H1N1) escape mutants, generated under PN-SIA28 selective pressure, identified two residues (Ile361 and Asp362) localized in close proximity to the residues identified with the peptide library approach. It is worth Cross-Neutralizing mAb to Influenza Viruses noting that none of these two specific mutations is present in any of the more than 6,000 sequences available in public influenza data- bases (http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html), suggesting that the immune pressure on these residues is poor and that the region is functionally highly conserved in all subtypes (rate of non-conservative substitutions: 0.17% for residue 361, and 8.7% for residue 362). Based on these data, A/PR/8/34 (H1N1) HA mutants carrying an alanine substitution in position 361 (Ile361Ala) or in position 362 (Asp362Ala) were generated and the FACS analysis showed that PN-SIA28 lost most of its capability to bind the mutated HA, in concordance with the data obtained from the escape mutants. The residue in position 361 was also mutated in Valine (Ile361Val), an aminoacid largely present in most of the neutralized isolates (Figure 2), indeed confirming that the presence of this residue did not affect binding. With the help of available HA crystal structures, a larger panel of HA-A/PR/8/34 (H1N1) alanine mutants was analogously generated and several residues (His25Ala, His45Ala on HA1; Thr358Ala, Met360Ala, Ile361Ala, Asp362Ala, Gly363Ala, Trp364Ala, Thr384Ala and Val395Ala on HA2), close to those identified with the escape mutants approach, influenced the interaction of PN-SIA28 with HA (Figure 2 and 3). The more restricted panel of alanine mutants (His25Ala, Asn45Ala, Ile361Ala and Asp362Ala) generated for A/Aichi/2/68 (H3N2) HA, confirmed the key role of some of the residues already identified as implicated in the interaction of PN-SIA28 with HA (H1) (Figure 4). Three more mutants were generated considering the differences among solvent exposed residues close to PN-SIA28 epitope-core on HA stem region between A/Victoria/3/1975 (the most recent neutralized isolate) and A/Philippines/2/1982 (the earliest non neutralized strain) isolates. The only mutant partially affecting PN-SIA28 binding was the one on Lys57, a residue present among all H3N2 neutralized strains and substituted by several different residues among H3N2 not neutralized strains. This could partially explaining the lack of neutralizing activity against most recent strains (Figure 5 and Table S1). Overall, these results indicate that the region bound by PN- SIA28 encompasses residues on the HA stem, and that it is partially overlapping from the binding region of other human neutralizing antibodies with heterosubtipic neutralizing activity limited to influenza A group 1 viruses [25,26,27,28,29] or group 2 viruses [30,31]. In addition, the region bound is also partially shared with FI6v3, an optimized human mAb able to cross- neutralize influenza A group 1 and group 2 viruses [32]. Importantly, both the heavy chains of PN-SIA28 and FI6v3 derive from rearrangement of the same VH gene germline (VH3- 30). As already hypothesized for the VH1-69 subfamily, widely shared among human mAbs with heterosubtypic neutralizing activity limited to group 1 viruses, the VH3-30 gene structure may be important in conferring the broader heterosubtypic activity observed for PN-SIA28 and FI6v3. The future definition of HA/ PN-SIA28 crystal structure and its comparison with HA/FI6v3 complex may help confirm the possible role of conserved VH3-30 residues in conferring such unusual neutralization properties. Although the resolution of the crystal structure will be necessary for the fine definition of the PN-SIA28 epitope, several important considerations may already be made. Firstly, it was previously speculated that the presence of an additional glycosilation site (Asn54 corresponding to His45 present on group 1 viruses) on the HA1 portion of the stem region in H3N2 viruses is the reason for the lack of anti-H3N2 activity of other mAbs with heterosubtypic activity limited to group 1 subtypes [25,26,30]. The present study demonstrates that this extra glycosilation site on H3N2 does not preclude PN-SIA28 binding and neutralizing activity against PLoS ONE | www.plosone.org 5 December 2011 | Volume 6 | Issue 12 | e28001