1. Longitudinal analysis of cross-group HA stem–binding lineages.
Longitudinal analysis of cross-group HA stem–binding lineages. (A) Cells were single cell–sorted from the same donor from blood collected on the day of the H7 prime (PreVacc) and 2 weeks after the H7N9 boost (PostVacc). Each bar represents the total number of sequences from each donor amplified from cells sorted after vaccination that were H7H3+ (left) or bound H7 alone (right) by flow cytometry. The sequences found after vaccination that belonged to lineages detected before vaccination as well are in black. (B) For each of the 36 lineages found before and after vaccination, we calculated the approximate percentage of this lineage of all IgG+ cells at each time point by flow cytometry and single-cell Ig sequencing. Each line connects the proportion of a single lineage in the IgG+ B cell population for each time point. Significance was determined using Wilcoxon paired nonparametric t test. (C) Mean percent mutation level of the rearranged VH gene amplified from single cell–sorted cells that were cross-group HA stem–specific (H7H3H1+, Stem+), group 2 only–specific (H7H3+ H1−), or bound H7 HA alone as indicated. Each dot connected by lines represents the mean mutation percent in each donor for each B cell specificity group. (D) For each donor, the percentage of Ig sequences from B cells with the indicated specificity as in (C) that were clonally related to another sequence is plotted. Statistical significance for (B) to (D) was determined using a paired nonparametric Wilcoxon t test. (E, G, and I) Pie charts showing sequences from all cross-reactive H5H1+ or H7H3+ B cells generated after H5N1 or H7N9 vaccination, respectively, in the same donor that received both vaccines. Individual clones are separated by white lines. Colored slices of the pie charts represent all cross-group HA stem–binding B cells as detected by flow cytometry. Representative mAbs from some of the lineages were also expressed, and cross-group binding was confirmed (Figs. 4 and 6). All other cross-reactive B cells are in gray. The total number of cross-reactive B cells sorted and sequenced is indicated in the center of the chart. (F and H) Approximate percentage of each of the indicated lineages in the total IgG+ population 2 weeks after the H5N1 boost, the day of the H7 vaccine prime (PreVacc), and 2 weeks after the H7N9 boost.
Sarah F. Andrews et al. Sci. Immunol. 2017;2:eaan2676
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