Antibody Heavy Chain Variable Domains of Different Germline Gene Origins Diversify through Different Paths

Abstract

B cells produce antibodies, key effector molecules in health and disease. They mature their properties, including their affinity for antigen, through hypermutation events; processes that involve, e.g., base substitution, codon insertion and deletion, often in association with an isotype switch. Investigations of antibody evolution define modes whereby particular antibody responses are able to form, and such studies provide insight important for instance for development of efficient vaccines. Antibody evolution is also used in vitro for the design of antibodies with improved properties. To better understand the basic concepts of antibody evolution, we analyzed the mutational paths, both in terms of amino acid substitution and insertions and deletions, taken by antibodies of the IgG isotype. The analysis focused on the evolution of the heavy chain variable domain of sets of antibodies, each with an origin in 1 of 11 different germline genes representing six human heavy chain germline gene subgroups. Investigated genes were isolated from cells of human bone marrow, a major site of antibody production, and characterized by next-generation sequencing and an in-house bioinformatics pipeline. Apart from substitutions within the complementarity determining regions, multiple framework residues including those in protein cores were targets of extensive diversification. Diversity, both in terms of substitutions, and insertions and deletions, in antibodies is focused to different positions in the sequence in a germline gene-unique manner. Altogether, our findings create a framework for understanding patterns of evolution of antibodies from defined germline genes.

A novel framework for characterizing genomic haplotype diversity in the human immunoglobulin heavy chain locus

“…An added advantage of our approach is the ability to capture variation outside of IG coding segments and more fully characterize SVs. Although several studies have begun to demonstrate the extent of SV haplotype variation 4,16,17,63 , information on polymorphisms within these SVs, and within IGH regulatory and intergenic space remains sparse 21 . It is worth noting that, in the few samples analyzed here, the majority of variants were detected in non-coding regions, including SNVs within RS, leader, intronic, and intergenic sequences.…”

“…The numbers of putative indel errors were 276 (NA19240) and 188 (NA12878) (Supplementary Table 5). Of these indels, 254/276 and 180/188 were 1-2 bp indels, 71.26% (181/254) and 63.33% (114/180) of which were within homopolymers ( Supplementary Table 6). Finally, we additionally assessed assembly accuracy using publicly available 30x PCR-free paired-end Illumina NovaSeq data from NA19240 and NA12878 51 .…”

Hydrophobicity and the Physico-Chemical Basis of Immunotolerance

“…AVYYC is endowed with a hydrophobicity level equal to +1.180 [9]. In the mature antibody, AVYYC is allocated at the juncture with the third complementarity-determining region of the Ig heavy chain, which has a predominant role in recognizing and binding antigens and also influences antibody specificity, affinity, and polyreactivity [35].…”

IgEvolution: clonal analysis of antibody repertoires

“…The first 1-8 and the last 1-7 positions of FR3 also correspond to both binding sites (extending CDR2 and CDR3) and contact sites. Also, many FR3s contain non-conventional binding sites centered at distance 16 from the beginning of FR3 (this region named CDR4 in Kirik et al, 2017). The last position of FR4 corresponds to contact sites that are extended in constant regions.…”