IGHV1-69 polymorphism modulates anti-influenza antibody repertoires, correlates with IGHV utilization shifts and varies by ethnicity

Abstract

IGHV polymorphism provides a rich source of humoral immune system diversity. One important example is the IGHV1-69 germline gene where the biased use of alleles that encode the critical CDR-H2 Phe54 (F-alleles) to make broadly neutralizing antibodies (HV1-69-sBnAb) to the influenza A hemagglutinin stem domain has been clearly established. However, whether IGHV1-69 polymorphism can also modulate B cell function and Ab repertoire expression through promoter and copy number (CN) variations has not been reported, nor has whether IGHV1-69 allelic distribution is impacted by ethnicity. Here we studied a cohort of NIH H5N1 vaccinees and demonstrate for the first time the influence of IGHV1-69 polymorphism on V-segment usage, somatic hypermutation and B cell expansion that elucidates the dominance of F-alleles in HV1-69-sBnAbs. We provide evidence that Phe54/Leu54 (F/L) polymorphism correlates with shifted repertoire usage of other IGHV germline genes. In addition, we analyzed ethnically diverse individuals within the 1000 genomes project and discovered marked variations in F- and L- genotypes and CN among the various ethnic groups that may impact HV1-69-sBnAb responses. These results have immediate implications for understanding HV1-69-sBnAb responses at the individual and population level and for the design and implementation of "universal" influenza vaccine.

Figure 1. Correlation between IGHV1-69 polymorphism and Ab response to the H5 vaccine.

The pre-vaccinated sera of the 85 individuals were diluted 1/1250 and analyzed for binding activities against the anti-IGHV1-69 idiotype mAb G6. Binding activities were normalized by subtracting the G6 MSD signal with the MSD signal obtained from an isotype control, and by using a standard curve made with the IGHV1-69 F-allele-based IgG Ab D80. (b) Post-vaccination sera (diluted 1/125) were competed with the anti-stem Ab F10 IgG for binding to H1CA0709. Cuzick’s trend test was used to further confirm that the occurrence of F-alleles increases the ability of serum to block F10 binding (L/L = 0, F/L = 1, F/F = 2). Error bars represent standard error of mean.

Figure 2. Analyzing IGHV1-69 V-segment gene utilization among the three IGHV1-69 genotypic groups.

(a) The frequency of IGHV1-69 IgM clones defined by unmutated V-segments (b) the frequency of IGHV1-69 IgG clones. Error bars represent standard error of mean.

Figure 3. Correlating IGHV1-69 F-allele copy number with IGHV1-69 utilization.

(a) Correlating F-allele CN with the frequency of IGHV1-69 IgM clones defined by unmutated V-segments. (b) Correlating F-allele CN with the frequency of IgG clones. The insets in both panels (a) and (b) describe IGHV1-69 clone frequency in individuals that lack IGHV1-69 gene duplication (Arrows point to overly of two L/L individuals).

Figure 4. The antibody repertoire of the three IGHV1-69 genotypic groups.

V-gene frequencies were averaged for the L/L group (n = 3), F/L group (n = 11), and F/F group (n = 4) from the datasets of IgM clones characterized by unmutated V-segments (a) and IgG clones (b). The majority of the functional V-genes were tabulated according to their respective positions in the IGH locus (further detailed in Supplementary Fig. 11). Asterisks denote V-genes utilized differently among the three genotypic groups as determined by Kruskal-Wallis test (P < 0.05). Error bars represent standard error of mean. In panels (c,d) Spearman correlation coefficients are derived for the data presented in panels (a,b) with L/L = 0, F/L = 1, and F/F = 2. Asterisks indicate statistically significant correlations (P < 0.05). Red rectangles point to the location of IGHV1-69 and IGHV2-70, for which their usages were significantly different among the three genotypic groups, being the highest in the F/F group and lowest in the L/L group, in both the unmutated IgM and IgG datasets. The inset panels are enlarged cropped sections from Panel (a,b) of the IGHV4-30-4/31-to-IGHV3-23 region that is negatively correlated with F-alleles.

Figure 5. IGHV1-69 F/L polymorphism and CN variations among various ethnicities.

(a) Table including mean IGHV1-69 copy number estimates after partitioning by IGHV1-69 rs55891010 genotype, provided for the total combined population, for three broad ethnic groups and the NIH cohort samples that were analyzed by NGS. Bubble plots corresponding to IGHV1- 69 CN for each genotypic class in the total combined population (a, lower) and individual ethnic groups (b). In each plot, the area of a given circle is proportional to the number of individuals observed for that particular combination of IGHV1-69 CN and rs55891010 genotype relative to the number of samples analyzed in each group (e.g., Combined, African, Asian, or European).