Immunoglobulin genes and generation of antibdoy repertoires in camelids

The humoral immune response of camelids is unique as these animals are the only known mammals that possess functional homodimeric heavy chain antibodies (HCAbs) besides the classical hetero-tetrameric antibodies (Abs) composed of heavy (H) and light (L) chains. Biochemical and cDNA sequence analyses have shown that the H-chains in tetrameric and homodimeric IgGs are different. Firstly, the HCAbs lack the first constant domain, possibly due to a point mutation on the donor-splicing site present in the first C exon/intron boundary. Secondly, the sequences of the hinge region are totally different. Thirdly, HCAbs differ in their V regions as compared to classical Abs (so-called VHH and VH regions). These VHHs are encoded by a distinct set of V genes where codons for strongly conserved hydrophobic amino acids in FR2 region are substituted by codons for hydrophilic amino acids. At present the generation and the structural properties of IgG-HCAbs have been well investigated, although the occurrence of other HCAb isotypes in camelids remains unknown. In camelids the C? gene, the joining (JH) and possibly the diversity (D) genes are shared for the generation of HCAbs and classical Abs. The development of a B-cell bearing HCAb IgG is thus presumed to pass through an IgM stage. In this study we generated an IgM derived V library encompassing both, the VH and VHH repertoire. Panning of this library did not retrieve antigen-specific binders. The failure to identify antigen-specific binders from the cloned VHH-D-J-C? transcripts and the presence of the C?1 exon in these transcripts imply that B-cells do not express VHH-IgM. Probably during B-cell development, the B-cell fails to produce VHH-IgM and undergoes an immediate class switch to avoid B-cell apoptosis. This suggests that for HCAbs, the IgM stage is a transient stage. The actual mechanism and the time point, during B-cell development, of the C? class switch to a dedicated C? isotype remains elusive. Analysis of camelid cDNA shows that four different isotypes (C?, C?, C?, C?) are present as classical antibodies and two as HCAbs (C?, C?). In contrast to the C? genes, which are clearly divided in C? genes for classical antibodies and dedicated C? genes for HCAbs, the camelid genomic IgH locus displays only one C? gene, suggesting that this single C? gene is capable of generating classical IgA and HCAb IgA. Also, C? does not harbor a point mutation on the donor-splicing site as found for the functional IgG-HCAbs. Nevertheless, the IgA-HCAbs were shown to be functional through antigen-binding of their VHHs. Furthermore, analyses of cDNA of the different camelid immunoglobulin isotypes revealed that VHHs are scarce in classical antibodies, whereas VHs readily contribute to the repertoire of HCAbs. This was shown by generation of such antigen-binding VHs, whose sequences were identical to the human VH4 family. Additionally, analysis of these VH4 sequences revealed that some of these genes encode for antigen-binding elements both in classical antibodies and in HCAbs. This questions the importance and the contribution of the light chain to the antigen-binding site in camelids. Thus, the origin of the variable domains of the HCAbs is not necessarily restricted to the specific VHH genes, since they can also be derived from the same or different families of classical VHs through somatic diversification processes. Consequently the HCAb Ag-binding repertoire in camelids appears to be more diverse than previously assumed as it is not exclusively dependent on the employment of dedicated C? genes and dedicated VHH genes.