Vaccines elicit antibodies in humans that poorly react to HA with the L194P mutation.

<p>Sera from 21 human donors were collected before <b>(A)</b> and after <b>(B)</b> vaccination with the 2015–2016 influenza vaccine. Antibody binding to virus-like particles expressing WT and L194P HA was measured using ELISA. Titer is expressed as the dilution of sera required to reach a fixed absorbance (O.D. = 0.75). The geometric mean ELISA titers calculated after testing each sera sample in triplicate are shown. Significance was determined using a paired Student’s t-test.</p

L194P is an egg-adaptive substitution in human H3N2 HA.

<p><b>(A)</b> Frequency of different amino acids observed at HA residue 194 of human H3N2 isolates. Egg: isolates that were sequenced after passaging in egg. Unpassaged: isolates that were sequenced without passaging. <b>(B)</b> Egg-passaged human H3N2 isolates are categorized based on the number of passage. The fractions of isolates in different number of passage that possess a Pro at residue 194 are shown. Error bars represent the standard error estimated from a binomial distribution. <b>(C)</b> Frequency of different amino acids observed at HA residue 194 of pandemic H1N1 (“swine influenza”) human isolates. Egg: isolates that were sequenced after passaging in egg. Unpassaged: isolates that were sequenced without passaging. Of note, no sequence information was found for unpassaged isolates in 2013. <b>(D)</b> Cα traces of the HA structures of Bris07 P194 (blue) and L194 (green) are aligned and compared.</p

Crystal structures of Bris07 P194 and L194 HAs in complex with receptor analogs.

<p>HA structures of <b>(A)</b> Bris07 P194 in complex with 3'SLNLN, <b>(B)</b> Bris07 L194 in complex with 3'SLNLN, <b>(C)</b> Bris07 P194 in complex with 6'SLNLN, and <b>(D)</b> Bris07 L194 in complex with 6'SLNLN are shown. The apo form for each structure is aligned and colored in grey. Glycan receptor analogs (3'SLNLN and 6'SLNLN) are colored in yellow and shown as stick representations. Hydrogen bonds are represented by black dashed lines.</p

<i>B</i>-value analysis of Bris07 HA.

<p><b>(A-B)</b><i>B</i>-values (Ų) of Cα atoms in <b>(A)</b> Bris07 P194 and <b>(B)</b> Bris07 L194 are projected on the HA structure. Of note, on average, the <i>B</i>-values of Cα atoms in Bris07 P194 (mean ± s.d. = 58 ± 24 Ų) are lower than that of Bris07 L194 (mean ± s.d. = 64 ± 24 Ų). <b>(C)</b> The normalized <i>B</i>-values of Cα atoms in Bris07 P194 (blue) and L194 (green) are compared. The bottom panel shows their difference in HA1: (normalized <i>B</i>-values of Cα atoms in P194)–(normalized <i>B</i>-values of Cα atoms in L194). The amino-acid position is plotted along the x-axis. Positions corresponding to the residues (Res) of interest are shaded in grey.</p