Fundamental Characteristics of the Immunoglobulin VH Repertoire of Chickens in Comparison with Those of Humans, Mice, and Camelids

Abstract

Examination of 1269 unique naive chicken VH sequences showed that the majority of positions in the framework (FW) regions were maintained as germline, with high mutation rates observed in the CDRs. Many FW mutations could be clearly related to the modulation of CDR structure or the VH–VL interface. CDRs 1 and 2 of the VH exhibited frequent mutation in solvent-exposed positions, but conservation of common structural residues also found in human CDRs at the same positions. In comparison with humans and mice, the chicken CDR3 repertoire was skewed toward longer sequences, was dominated by small amino acids (G/S/A/C/T), and had higher cysteine (chicken, 9.4%; human, 1.6%; and mouse, 0.25%) but lower tyrosine content (chicken, 9.2%; human, 16.8%; and mouse 26.4%). A strong correlation (R2 = 0.97) was observed between increasing CDR3 length and higher cysteine content. This suggests that noncanonical disulfides are strongly favored in chickens, potentially increasing CDR stability and complexity in the topology of the combining site. The probable formation of disulfide bonds between CDR3 and CDR1, FW2, or CDR2 was also observed, as described in camelids. All features of the naive repertoire were fully replicated in the target-selected, phage-displayed repertoire. The isolation of a chicken Fab with four noncanonical cysteines in the VH that exhibits 64 nM (KD) binding affinity for its target proved these constituents to be part of the humoral response, not artifacts. This study supports the hypothesis that disulfide bond-constrained CDR3s are a structural diversification strategy in the restricted germline v-gene repertoire of chickens.

An Ultra-specific Avian Antibody to Phosphorylated Tau Protein Reveals a Unique Mechanism for Phosphoepitope Recognition

“…A recent bioinformatic study has shown that the chicken V H domain repertoire is highly homologous to that of humans and mice in both the frameworks and CDR1 and CDR2 (32). In contrast, chickens use a much longer V H CDR3 repertoire and frequently stabilize these long binding loops by introducing non-canonical disulfide bonds (32).…”

“…S1. Each of these clones was found to have the high framework uniformity that is typical of chicken antibodies (32). They are highly divergent in their V H CDR3 sequences, however, with the exception of pT231/pS235_1 and pT231/ pS235_2, which have identical heavy chain sequences but exhibit promiscuity in light chain usage.…”

“…They are highly divergent in their V H CDR3 sequences, however, with the exception of pT231/pS235_1 and pT231/ pS235_2, which have identical heavy chain sequences but exhibit promiscuity in light chain usage. Clones pT212/ pS214_1, pT212/pS214_2, and pS396/pS404_1 are type 2 chicken V H , whereas clones pT231/pS235_1, pT231/pS235_2, and pS396/pS404_2 all contain a pair of non-canonical cysteine residues in CDR-H3 and are representatives of the major structural type 1 chicken V H (32).…”

“…Disulfide bonds are often seen in chicken CDR-H3 loops and are proposed to play important roles in creating chicken paratopes of great structural complexity in the context of a single framework repertoire (32). A large insertion within CDR-H3 (18 amino acids in length) forms a surface that makes numerous interactions with the non-phosphorylated part of the epitope (Fig.…”

“…These D-like sequences are donated by pseudogenes and may replace the entire D segment or only a small section, leading to the creation of "mosaic CDRs: (28,29). The frequent use of cysteine in V H CDR3 of chickens coupled with double D segment insertions leads to Ͼ50% of all B-cell clones in the chicken repertoire containing intra-CDR disulfide bonds (32). The high frequency of these probable disulfides suggests that they play an important functional role in CDR structural diversification.…”