The Distinctive Immune Systems of Rabbits and Chickens Made them good Choices for Antibody Development

All jawed vertebrates use immunoglobulins (Ig) produced by B-lymphocytes for acquired immunity. Immunoglobulins are glycoproteins, consisting of two heavy and two light chains, bonded together by disulfide bonds, and often drawn in a cartoon as a “Y”. Immunity presents a genetic challenge. How do you encode a gene whose products are flexible enough to bind to unpredictable invaders? The complex genetic mechanisms of vertebrate immunity involve diversification of Igs by joining together multiple V (for variable), D (for diversity), and J (for joining) gene segments. This VDJ Rearrangement takes place through somatic mutagenesis upon subsequent B-cell encounter with foreign antigens (Somatic Hypermutation).

Both Chicken and Rabbit immune systems have been extensively studied for over 60 years. Despite many similarities among all vertebrate immune systems, B cell development and repertoire diversification does vary among species. Both Rabbits and Chickens are among the most commonly used animals for commercial antibody production. Obviously, both are larger than lab rats and mice, and this alone provides for a larger quantity of available serum. But it turns out that both Rabbit and Chicken immune systems have unique properties that make them ideal for generating commercial antibodies.

Rabbits

Rabbits are not technically rodents, but are closely related. Rabbits, hares, and pikas make up the order Lagomorpha, which is the sister group to rodents. This group combined (Glires), is a member of Euarchontoglires (synonymous with Supraprimates), which includes primates (and us). We have thus benefited from being so closely related to our commonly used lab animals, including rats, mice, and rabbits.

Compared to humans and mice, which use a diverse assortment of germline VH gene segments during VDJ rearrangement of the heavy chain, the rabbit IgH repertoire displays highly restricted VH gene segment usage. While VH genes in humans can be divided into groups A, B, and C, rabbit VH genes are all in the C group, but this C group has undergone an ancient duplication (Su et al.,1999). Rabbits use both gene conversion (Surridge et al., 2008) and somatic hypermutation to generate new arrangements of heavy and light chain genes, likely due to limited VH genes diversity. This pattern is similar to that seem in chickens, which likely converged on a similar gene conversion mechanism. Rabbit antibodies are unusually stable. Rose Mage, an authority of rabbit immune systems, speculated that the stability of rabbit antibodies could be from the stabilization of kappa 1 light chain structures by an unusual intrachain disulfide bridge between cysteine 80 in VK and cysteine 171 in CK (Mage et al., 2016). Rabbits are also unusual in having only one gamma gene, but 13 alpha genes (Burnett et al., 1989). According to Mage and colleagues, Rabbits produce “some of the most highly specific high affinity antibodies” (2016). Rabbits lack the IgD gene, and have single IgG and IgE genes (Gertz et al., 2013). Although equally distant in evolutionary terms, rabbit tissues are more similar to human tissues with respect the activity and development of the appendix, which is involved in immunity. Different from rodents, rabbits share characteristics of the chicken’s Bursa of Fabricius with humans (Mage et al. 2016). The Bursa of Fabricius is a specialized organ in birds involved with B cell development. Which brings us to Chickens.

Chickens

While mice have been favored for making monoclonal antibodies the rabbit is typically used for making polyclonal antibodies.  However, some mammalian antigens fail to induce an immune response in either rodents or rabbits especially when the proteins are highly conserved. In these cases, investigators have turned to the chicken. Monoclonal antibodies had been harder to produce with chickens in the past, because hybridoma technology has been developed for mice. However, this limitation has been overcome in recent years for both rabbit and chicken. Phage Display technology and Single Plasma cell Interrogation  technology (SPIN®) have been utilized in the field and permit the production of chicken monoclonal antibodies.

Despite diverging from the common ancestor of mammals hundreds of millions of years ago, the chicken immune system is remarkably similar to those of mammals, with some exceptions. Birds don’t have lymph nodes, but have a unique organ called the Bursa of Fabricius. Birds have three classes of molecules, homologous to mammalian IgM, IgA, and IgG, while lacking homologs to IgD, IgG, and IgE. The bird homolog to IgG is called IgY, and IgY is the predominant agent of secondary immune responses. IgY has a longer heavy chain than IgG. It lacks a hinge region, and is made of 4 rather than 3 constant domains  (Kaspers and Göbel, 2016). Unlike mammals, who possess large numbers of VL genes that rearrange with JL genes, chickens have only one of each, and VDJ rearrangements do not significantly contribute to heavy chain diversity, as they do in mammals (Kaspers and Göbel, 2016). Limited variation is formed by unique VH and JH genes, but is then diversified through a process of gene conversion in the bursa. Thus gene conversion is a significant mechanism for Ig diversity in both chickens and rabbits.

Benefits

Thus rabbit and chicken immune systems generate antibody diversity and optimize affinity by mechanisms that are more efficient than those of mice and other rodents. Other than the common Somatic Hypermutation, B cells from both rabbits and chickens also employ a mechanism called Gene Conversion. This involves the non-reciprocal homologous recombination of upstream pseudo V gene loci into the recombined VDJ (and VJ) locus. The gene conversion with the approximate of 300 pseudo V-genes dramatically enhanced the diversity of its antibody repertoire and increased the possibility of generating functional antibody clones.

The SPIN® technology based custom rabbit monoclonal antibody service and chicken monoclonal antibody custom service harnessed these unique properties of the immune systems of Rabbit and Chicken.

References:

Burnett, R. C., Hanly, W. C., Zhai, S. K., and Knight, K. L.: The IgA heavy-chain gene family in rabbit: cloning and sequence analysis of 13 Cα genes. EMBO J 8: 4041–4047, 1989

Gertz et al., 2013 Accuracy and coverage assessment of Oryctolagus cuniculus (Rabbit) Genes Encoding Immunoglobulins in the Whole Genome Sequence Assembly (OryCun2.0) and Localization of the IGH Locus to Chromosome 20 Immunogenetics 65(10)

Kaspers, B, and Göbel, TWF. (2016) The Avian Immune system. In: Encyclopedia of Immunology.  Michael J.H. Ratcliffe (Ed.) Academic Press.

Mage, R.G., Pinheiro, A., Lemos de Matos, A., and Esteves, P.J. (2016) The immune system of Lagomorphs. In: Encyclopedia of Immunology.  Michael J.H. Ratcliffe (Ed.) Academic Press. 2016. ISBN0080921523, 9780080921525

Pinheiro, A., Neves, F., Lemos de Matos, A. et al. (2016) Immunogenetics 68: 83. doi:10.1007/s00251-015-0868-8

Su, C., And Nei, M. (1999). Fifty-million-year-old polymorphism at an immunoglobulin variable region gene locus in the rabbit evolutionary lineage Proc. Natl. Acad. Sci. USA 96:9710-9715

Surridge, A.K., van der Loo, W., Abrantes, J., Carneiro, M., Hewitt, G.M., and Esteves, P.J.  (2008) Immunogenetics 60(9)515–525.