Human immunoglobulin gene allelic variation impacts germline-targeting vaccine priming

Abstract

Vaccine priming immunogens that activate germline precursors for broadly neutralizing antibodies (bnAbs) have promise for development of precision vaccines against major human pathogens. In a clinical trial of the eOD-GT8 60mer germline-targeting immunogen, higher frequencies of vaccine-induced VRC01-class bnAb-precursor B cells were observed in the high dose compared to the low dose group. Through immunoglobulin heavy chain variable (IGHV) genotyping, statistical modeling, quantification of IGHV1-2 allele usage and B cell frequencies in the naive repertoire for each trial participant, and antibody affinity analyses, we found that the difference between dose groups in VRC01-class response frequency was best explained by IGHV1-2 genotype rather than dose and was most likely due to differences in IGHV1-2 B cell frequencies for different genotypes. The results demonstrate the need to define population-level immunoglobulin allelic variations when designing germline-targeting immunogens and evaluating them in clinical trials.

Fig. 1. IGHV1-2 genotype, allele, and pre-vaccination IgM repertoire distributions for IAVI G001 trial participants.

a The IGHV1-2 allele content in each study participant was determined by sequencing bulk IgM libraries and inferring the IGHV allele content in each case with IgDiscover. Quantitative analyses of mRNA expression levels and HCDR3 frequencies followed. b Number of each genotype and c number of each allele, for all trial participants (n = 48). d Number of vaccine recipients per group, out of 18, with each allele (*02, *04, *05, and *06), with the *02 variant *02_S4953 classified as *02. P-values are based on a Fisher’s exact test with values >0.05 marked as not significant (NS). For all 48 participants, pre-vaccination e mRNA expression frequencies and f unique HCDR3 frequencies for each IGHV1-2 allele are shown as points color-coded by allele and grouped by homozygous and heterozygous genotype. Each point represents a trial participant, with heterozygous participants represented by two points. Thick lines are median values and boxes are the 25% and 75% quantiles. g Correlation between mRNA count and unique HCDR3 count; h Ratio of unique HCDR3 count to mRNA count versus mRNA count; i Correlation between frequency of mRNA expression and frequency of unique HCDR3s; and j Ratio of the unique HCDR3 frequency to the mRNA frequency versus the mRNA frequency, are shown for pre-vaccination repertoires. Points are shape- and color-coded by IGHV1-2 allele as shown in the legend of (g–j). Pearson correlation coefficients (r) for counts and frequencies are shown in (g and i), respectively. In panels h and j, the solid line is the median ratio, and the shaded region shows the inter-quartile range.

Fig. 2. Model estimates and 95% confidence intervals (CIs) from the Allele model for each genotype and time point.

Estimates and CIs for the frequency of VRC01-class IgG B cells at each time point by genotype are shown as open diamonds and vertical lines, respectively. Thick lines are median values and boxes are the 25% and 75% quantiles. Experimentally measured frequencies for each participant are shown as color- and shape-coded points by dose group as indicated by the legend. Genotypes containing the *05 and *06 alleles are grouped together (e.g., *02/*05 or *02/*06), because the estimated mean response from the Allele model depends only on the count of *02 and *04 alleles. Week 11 germinal center (GC) results are not shown since the Null model ranked higher than the Allele model for that sample time point.

Fig. 3. Relative contribution of *02 versus *04 alleles to Allele-model-derived post-vaccination VRC01-class frequency, pre-vaccination IGHV1-2 mRNA expression level, and post-vaccination BCR assignments.

a Allele model estimates for the relative contribution of *02 versus *04 (as a ratio) to the post-vaccination VRC01-class frequency are shown with 95% confidence intervals (CIs) for germinal center (GC) B cell, memory B cell (MBC), and plasmablast (PB) samples taken at the indicated week (Wk) after first vaccination. b Experimentally measured pre-vaccination ratios of *02 to *04 mRNA expression levels for homozygous or heterozygous genotypes. For homozygotes, the ratio of means and CI for *02 and *04 individuals is shown. For heterozygotes, ratios for each individual are shape- and color-coded (N = 4 for low dose; N = 4 for high dose), and the overall mean ratio and CI are shown in black. c Experimentally measured pre-vaccination ratios of *02 to *04 unique HCDR3 frequencies for homozygous or heterozygous genotypes. Homo- and heterozygote data are displayed as in (b). d Ratio of *02 to *04 usage for germline allele assignments among post-vaccination BCRs recovered from eight vaccine recipients known to be heterozygous for *02 and *04 by pre-vaccination genotyping are shape- and color-coded (N = 4 for low dose; N = 4 for high dose), and the overall mean ratio and CI are shown in black.

Fig. 4. Correlations between pre-vaccination IgM unique HCDR3 frequency (IGHV1-2*02 or *04) and the percent VRC01-class B cell response by visit.

Points are shape- and color-coded as shown in the legend. Spearman correlation coefficient (r) and P-values are displayed for each time point.

Fig. 5. Affinity analyses of VRC01-class IGHV1-2 allele variants.

a Affinities of VRC01-class precursors from two bnAb iGLs, four human naive precursors, and four iGLs from week 3 post-vaccination BCRs from IAVI G001, with original *02 or *04 Abs on the left, and W50R variants of those Abs on the right. b Affinities of *02 (N = 71) and *04 (N = 47) iGLs from post-vaccination BCRs from IAVI G001. c Affinities of *02 VRC01-class iGL antibodies from IAVI G001 and for *04 (R66W) variants of the same antibodies (N = 28 each). Lines connect matched Ab variants. In (a, b), horizontal lines indicate median and interquartile range. In (a–c), all affinities were measured for eOD-GT8 monomer analyte, and all iGLs from IAVI G001 are from the low dose group.