Quantitative Analysis of Repertoire-Scale Immunoglobulin Properties in Vaccine-Induced B-Cell Responses

Abstract

Recent advances in the next-generation sequencing of B-cell receptors (BCRs) enable the characterization of humoral responses at a repertoire-wide scale and provide the capability for identifying unique features of immune repertoires in response to disease, vaccination, or infection. Immunosequencing now readily generates 103–105 sequences per sample; however, statistical analysis of these repertoires is challenging because of the high genetic diversity of BCRs and the elaborate clonal relationships among them. To date, most immunosequencing analyses have focused on reporting qualitative trends in immunoglobulin (Ig) properties, such as usage or somatic hypermutation (SHM) percentage of the Ig heavy chain variable (IGHV) gene segment family, and on reducing complex Ig property distributions to simple summary statistics. However, because Ig properties are typically not normally distributed, any approach that fails to assess the distribution as a whole may be inadequate in (1) properly assessing the statistical significance of repertoire differences, (2) identifying how two repertoires differ, and (3) determining appropriate confidence intervals for assessing the size of the differences and their potential biological relevance. To address these issues, we have developed a technique that uses Wilcox’ robust statistics toolbox to identify statistically significant vaccine-specific differences between Ig repertoire properties. The advantage of this technique is that it can determine not only whether but also where the distributions differ, even when the Ig repertoire properties are non-normally distributed. We used this technique to characterize murine germinal center (GC) B-cell repertoires in response to a complex Ebola virus-like particle (eVLP) vaccine candidate with known protective efficacy. The eVLP-mediated GC B-cell responses were highly diverse, consisting of thousands of clonotypes. Despite this staggering diversity, we identified statistically significant differences between non-immunized, vaccine only, and vaccine-plus-adjuvant groups in terms of Ig properties, including IGHV-family usage, SHM percentage, and characteristics of the BCR complementarity-determining region. Most notably, our analyses identified a robust eVLP-specific feature—enhanced IGHV8-family usage in B-cell repertoires. These findings demonstrate the utility of our technique in identifying statistically significant BCR repertoire differences following vaccination. More generally, our approach is potentially applicable to a wide range of studies in infection, vaccination, auto-immunity, and cancer.

Analyzing Immunoglobulin Repertoires

“…Like JSD, these tests compare probability distributions and hence there is no limitation to number of sequences. A mouse study used SK and KMS tests to compare the V family usage within GC B cell repertoire of animals vaccinated with complex Ebola virus-like particle and unvaccinated controls ( 125 ). Enhanced use of IGHV8 was observed in the vaccinated group.…”

Isolation of monoclonal antibodies from anti-synthetase syndrome patients and affinity maturation by recombination of independent somatic variants

“…The number of VH-gene residues that had undergone somatic mutation ranged up to 28% with a mean of 10.9% (supplementary Table 2 and Table 1 ), consistent with reports of the degree of somatic mutation in responses to infection. 34 , 35 By subdivision of antibodies by V-gene, J-gene, and CDRH3 or CDRL3 length, 91 heavy chain clusters and 63 light chain clusters are formed; these are arranged into 130 clonal families schematically represented by their heavy/light chain pairing, and antigen specificity (Fig. S1).…”

“…Previous studies looking at responses to vaccination with complex antigens in humans have demonstrated that in some cases, clonal diversity can increase during maturation rather than decrease as a result of clonal competition. 34 , 35 , 45 To utilize this diversity, we recovered a number of related clones from high-throughput single-cell isolation and complementary repertoire sequencing. Initial identification of related clones will, by necessity, be centered on CDR3 homology.…”

Yclon: Ultrafast clustering of B cell clones from high-throughput immunoglobulin repertoire sequencing data

“…A B-cell clone or clonotype is defined as a group of antibody sequences derived from a common progenitor B cell (Hershberg et al, 2015) that often bind to the same epitope. Clone size reflects on repertoire diversity, which is related to age (Davydov et al, 2018), response to vaccination or infection (Khavrutskii et al, 2017), allergy (Wu et al, 2014), cancer (Zhang et al, 2019), among several other conditions. B-cell clone identification is crucial, and different approaches attempt to do it.…”