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 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).