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 ).