Analysis of the horse V(H) repertoire and comparison with the human IGHV germline genes, and sheep, cattle and pig V(H) sequences.

We have constructed a chimeric antibody single-chain Fv (scFv) fragments phage-displayed library that combines an invariant human V(L) chain with the repertoire of V(H) domains amplified from a horse immunized against scorpion venom. To gain insight into the equine V(H) repertoire, the V(H) sequences of 46 unique clones randomly chosen from the library prior to antigenic selection were analyzed. Comparisons with previously reported equine V(H) sequences, as well as with the repertoire of human IGHV germline genes and known V(H) sequences of sheep, cattle and pig, suggest that the equine IGH locus harbors at least three IGHV gene families. Two families belong to clan II while the other was classified into clan I. The horse sequences were also found to encode a diverse repertoire of canonical structures. The most populated equine IGHV gene family, named IGHV1, and another family termed IGHV3, encode two out of the three canonical structures so far described for CDR1. The IGHV2 gene family has the third canonical structure at CDR1. In CDR2, nine loop lengths were found, with four of them matching the pattern of typical canonical structures. The remaining five CDR2 loop lengths are shorter or longer than those reported for human IGHV germline genes and known sequences of sheep, cattle and pig. The analysis of CDR3 loops indicates a length distribution broader than previous reports for horses; being similar to that of humans, sheep and pigs. Moreover, equine CDR3 loops were found to have a combination of lower content of cysteine and higher proportion of glycine not seen in the other species. This implies less constrained loops and therefore more apt for searching the conformational space of antigen-binding sites. Altogether, these findings reveal a more diverse perspective of the horse V(H) repertoire than previous estimations and lay foundations for future studies of the equine IGH locus.