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 noting that none of these two specific mutations is present in any of the more than 6,000 sequences available in public influenza databases (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.