Fig. 4: Thermostability and antigenic effects of G186D and D190N mutations.
From: Epistasis mediates the evolution of the receptor binding mode in recent human H3N2 hemagglutinin
a Recombinant Vic20 HAs (WT and mutants) were analyzed by thermal shift assay. The differences in melting temperature (ΔTm) between WT and mutants are shown. Each datapoint represents one experimental replicate. Each bar represents the mean of six independent replicates. (b) A group of 6-week-old female BALB/c mice (n = 6) were immunized intraperitoneally with 10,000 PFU of recombinant influenza virus with HA and NA from Italy20 and the six internal segments from PR8. On day 21 post-immunization, a booster was performed with the same amount of adjuvanted virus. Antisera were collected on day 21 post-boosting. 50% microneutralization (MN50) titer of the antisera against Italy20 WT and mutants was measured by a microneutralization (MN) assay. Each color represents the antiserum from one mouse. c MN50 titer of human plasma (n = 28) against an older human H3N2 strain Sing16 WT as well as Italy20 WT and mutants were measured by MN assay. Data are presented as a boxplot. The middle horizontal line represents the median. The lower and upper hinges represent the first and third quartiles, respectively. The upper whisker extends to the highest data point within a 1.5× inter-quartile range (IQR) of the third quartile, whereas the lower whisker extends to the lowest data point within a 1.5× IQR of the first quartile. Each data point represents one plasma sample. b, c The median MN50 titers were compared between Italy20 WT and different mutants using two-sided paired Wilcoxon signed-rank test without adjustment for multiple comparisons. P-value was computed between groups, with “*” indicates statistically significant difference (P < 0.05), whereas “ns” indicates non-significant. b The P-values for WT vs D186G, WT vs N190D, and WT vs D186G/N190D are 0.14, 0.04, and 0.03, respectively. c The P-values for Italy20 WT vs Sing16 WT, Italy20 WT vs Italy20 N190D, Italy20 WT vs Italy20 D186G, and Italy20 WT vs Italy20 D186G/N190D are 8e−6, 0.22, 7e−5, and 0.002, respectively. Raw data are provided as a Source Data file.
