A/PR/8/34 (H1N1) HA was amplified as previously described [19, 20] using the following PCR-oligonucleotides: APR834_s: 59-CACCATGAAGGCAAACCTACTGGTCCTGTTATGTG-39; APR834_as: 59-TCAGATGCATATTCTGCACTGCAAAGAT-CCATTAGA-39. A/Aichi/2/1968 (H3N2) HA was amplified using the following PCR-oligonucleotides: Aichi/2/68H3N2f: 59-CACCATGAAGACCATCATTGCTTTG-39; Aichi/2/68H3-N2r: 59-TCAAATGCAAATGTTGCACCTAATG-39. The PCR products were cloned into the pcDNA 3.1D/V5-His-TOPO vector (Invitrogen, Carlsbad, CA, USA). Subsequently, alanine mutants for H1N1 and H3N2 HA were generated using Gene Tailor Site-Directed Mutagenesis System (Invitrogen, Carlsbad, CA, USA). A total of 20 HA mutants were generated for H1N1 and 7 for H3N2 (Table S1 ).