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Studies using the European rabbit Oryctolagus cuniculus contributed to elucidating numerous fundamental aspects of antibody structure and diversification mechanisms and continue to be valuable for the development and testing of therapeutic humanized polyclonal and monoclonal antibodies. Additionally, during the last two decades, the use of the European rabbit as an animal model has been increasingly extended to many human diseases. This review documents the continuing wide utility of the rabbit as a reliable disease model for development of therapeutics and vaccines and studies of the cellular and molecular mechanisms underlying many human diseases. Examples include syphilis, tuberculosis, HIV-AIDS, acute hepatic failure and diseases caused by noroviruses, ocular herpes, and papillomaviruses. The use of rabbits for vaccine development studies, which began with Louis Pasteur’s rabies vaccine in 1881, continues today with targets that include the potentially blinding HSV-1 virus infection and HIV-AIDS. Additionally, two highly fatal viral diseases, rabbit hemorrhagic disease and myxomatosis, affect the European rabbit and provide unique models to understand co-evolution between a vertebrate host and viral pathogens., Infectious disease: A leap forward for disease models Rabbits offer a powerful complement to rodents as a model for studying human immunology, disease pathology, and responses to infectious disease. A review from Pedro Esteves at the University of Porto, Portugal, Rose Mage of the National Institute of Allergy and Infectious Disease, Bethesda, USA and colleagues highlights some of the areas of research where rabbits offer an edge over rats and mice. Rabbits have a particularly sophisticated adaptive immune system, which could provide useful insights into human biology and produce valuable research and clinical reagents. They are also excellent models for studying - infectious diseases such as syphilis and tuberculosis, which produce pathology that closely resembles that of human patients. Rabbit-specific infections such as myxomatosis are giving researchers insights into how pathogens and hosts can shape each other’s evolution.

Leporid VH genes used in the generation of their primary antibody repertoire exhibit highly divergent lineages. For the European rabbit (Oryctolagus cuniculus) four VHa lineages have been described, the a1, a2, a3 and a4. Hares (Lepus spp.) and cottontail (Sylvilagus floridanus) express one VHa lineage each, the a2L and the a5, respectively, along with a more ancient lineage, the Lepus spp. sL and S. floridanus sS. Both the European rabbit and the Lepus europaeus use a third lineage, VHn, in a low proportion of their VDJ rearrangements. The VHn genes are a conserved ancestral polymorphism that is being maintained in the leporid genome.Their usage in a low proportion of VDJ rearrangements by both European rabbit and L. europaeus but not S. floridanus has been argued to be a remnant of an ancient European leporid immunologic response to pathogens. To address this hypothesis, in this study we sequenced VDJ rearranged genes for another North American leporid, L. americanus. Our results show that L. americanus expressed these genes less frequently and in a highly modified fashion compared to the European Lepus species. Our results suggest that the American leporid species use a different VH repertoire than the European species which may be related with an immune adaptation to different environmental conditions, such as different pathogenic agents.

Microbial and host cell interactions stimulate rabbit B cells to diversify the primary Ab repertoire in GALT. B cells at the base of appendix follicles begin proliferating and diversifying their V-(D)-J genes around 1 wk of age, ∼5 d after B cells first begin entering appendix follicles. To gain insight into the microbial and host cell interactions that stimulate B cells to diversify the primary Ab repertoire, we analyzed B cell trafficking within follicles during the first week of life. We visualized B cells, as well as chemokines that mediate B cell homing in lymphoid tissues, by in situ hybridization, and we examined B cell chemokine receptor expression by flow cytometry. We found that B cells were activated and began downregulating their BCRs well before a detectable B cell proliferative region appeared at the follicle base. The proliferative region was similar to germinal center dark zones, in that it exhibited elevated CXCL12 mRNA expression, and B cells that upregulated CXCR4 mRNA in response to signals acquired from selected intestinal commensals localized in this region. Our results suggest that after entering appendix follicles, B cells home sequentially to the follicle-associated epithelium, the follicular dendritic cell network, the B cell/T cell boundary, and, ultimately, the base of the follicle, where they enter a proliferative program and diversify the primary Ab repertoire.

Although IgD first appeared, along with IgM, in the cartilaginous fishes and has been retained throughout subsequent vertebrate evolution, it has been lost in a diverse group of vertebrate species. We previously showed that, unlike vertebrates that express IgD, the rabbit lacks an IgD (Cδ) gene within 13.5 kb downstream of the IgM gene. We report here that, by conducting BLAST searches of rabbit Ig heavy chain genomic DNA with known mammalian IgD exons, we identified the remnant of the rabbit Cδ gene approximately 21 kb downstream of the IgM gene. The remnant Cδ locus lacks the δCH1 and hinge exons, but contains truncated δCH2 and δCH3 exons, as well as largely intact, but non-functional, secretory and transmembrane exons. In addition, we report that the Cδ gene probably became non-functional in leporids at least prior to the divergence of rabbits and hares ~12 million years ago.

Studies using the European rabbit Oryctolagus cuniculus contributed to elucidating numerous fundamental aspects of antibody structure and diversification mechanisms and continue to be valuable for the development and testing of therapeutic humanized polyclonal and monoclonal antibodies. Additionally, during the last two decades, the use of the European rabbit as an animal model has been increasingly extended to many human diseases. This review documents the continuing wide utility of the rabbit as a reliable disease model for development of therapeutics and vaccines and studies of the cellular and molecular mechanisms underlying many human diseases. Examples include syphilis, tuberculosis, HIV-AIDS, acute hepatic failure and diseases caused by noroviruses, ocular herpes, and papillomaviruses. The use of rabbits for vaccine development studies, which began with Louis Pasteur's rabies vaccine in 1881, continues today with targets that include the potentially blinding HSV-1 virus infection and HIV-AIDS. Additionally, two highly fatal viral diseases, rabbit hemorrhagic disease and myxomatosis, affect the European rabbit and provide unique models to understand co-evolution between a vertebrate host and viral pathogens.

The rabbit has long been a model for studies of the immune system. Work using rabbits contributed both to the battle against infectious diseases such as rabies and syphilis, and to our knowledge, of antibodies' structure, function, and regulated expression. With the description of rabbit Ig allotypes, the discovery of different gene segments encoding immunoglobulins became possible. This challenged the "one gene-one protein" dogma. The observation that rabbit allotypic specificities of the variable regions were present on IgM and IgG molecules also led to the hypothesis of Ig class switching. Rabbit allotypes contributed to the documentation of phenomena such as allelic exclusion and imbalance in production of allelic gene products. During the last 30 years, the rabbit Ig allotypes revealed a number of unique features, setting them apart from mice, humans, and other mammals. Here, we review the most relevant findings concerning the rabbit IGHV. Among these are the preferential usage of one VH gene in VDJ rearrangements, the existence of trans-species polymorphism in the IGHV locus revealed by serology and confirmed by sequencing IGHV genes in Lepus, the unusually large genetic distances between allelic lineages and the fact that the antibody repertoire is diversified in this species only after birth. The whole genome sequence of a rabbit, plus re-sequencing of additional strains and related genera, will allow further evolutionary investigations of antibody variation. Continued research will help define the roles that genetic, allelic, and population diversity at antibody loci may play in host-parasite interactions.

Gene conversion, mediated by activation-induced cytidine deaminase (AID), has been found to contribute to generation of the primary antibody repertoire in several vertebrate species. Generation of the primary antibody repertoire by gene conversion of immunoglobulin (Ig) genes occurs primarily in gut-associated lymphoid tissues (GALT) and is best described in chicken and rabbit. Here, we discuss current knowledge of the mechanism of gene conversion as well as the contribution of the microbiota in promoting gene conversion of Ig genes. Finally, we propose that the antibody diversification strategy used in GALT species, such as chicken and rabbit, is conserved in a subset of human and mouse B cells.

Gene conversion, mediated by activation-induced cytidine deaminase (AID), has been found to contribute to generation of the primary antibody repertoire in several vertebrate species. Generation of the primary antibody repertoire by gene conversion of immunoglobulin (Ig) genes occurs primarily in gut-associated lymphoid tissues (GALT) and is best described in chicken and rabbit. Here, we discuss current knowledge of the mechanism of gene conversion as well as the contribution of the microbiota in promoting gene conversion of Ig genes. Finally, we propose that the antibody diversification strategy used in GALT species, such as chicken and rabbit, is conserved in a subset of human and mouse B cells.

Vertebrates have a large antibody repertoire with diverse antigen specificities, poised to react to invading pathogens, such as bacteria, viruses and helminths. In some species, microbes such as these are required for development of both diverse antibody and B-lymphocyte repertoires. Recent studies demonstrate that B-lymphocyte expansion, selection and somatic diversification of Ig genes are regulated in these species, in part, by the interaction between commensal intestinal bacteria and gut-associated lymphoid tissue (GALT). These findings indicate that the commensal microbiota can shape the repertoire of peripheral B lymphocytes and can potentially influence the health of the host. Here, how the interactions between commensal microbiota and lymphoid cells of GALT might affect the development of the peripheral B-lymphocyte repertoire is discussed.

Gut-associated lymphoid tissues (GALTs) interact with intestinal microflora to drive GALT development and diversify the primary antibody repertoire; however, the molecular mechanisms that link these events remain elusive. Alicia rabbits provide an excellent model to investigate the relationship between GALT, intestinal microflora, and modulation of the antibody repertoire. Most B cells in neonatal Alicia rabbits express V(H)n allotype immunoglobulin (Ig)M. Within weeks, the number of V(H)n B cells decreases, whereas V(H)a allotype B cells increase in number and become predominant. We hypothesized that the repertoire shift from V(H)n to V(H)a B cells results from interactions between GALT and intestinal microflora. To test this hypothesis, we surgically removed organized GALT from newborn Alicia pups and ligated the appendix to sequester it from intestinal microflora. Flow cytometry and nucleotide sequence analyses revealed that the V(H)n to V(H)a repertoire shift did not occur, demonstrating the requirement for interactions between GALT and intestinal microflora in the selective expansion of V(H)a B cells. By comparing amino acid sequences of V(H)n and V(H)a Ig, we identified a putative V(H) ligand binding site for a bacterial or endogenous B cell superantigen. We propose that interaction of such a superantigen with V(H)a B cells results in their selective expansion.

Rabbits generate a diversified primary antibody repertoire by somatically mutating, in gut-associated lymphoid tissue (GALT), an initial repertoire that is limited by preferential rearrangement of the 3'-most IGVH gene segment. To determine when repertoire diversification begins in GALT, we performed in situ hybridization on neonatal rabbit appendix sections with an activation-induced cytidine deaminase (AID) riboprobe, because AID is required for the mutational processes that diversify the primary antibody repertoire. We first detected AID mRNA expression around 1 week of age, in the basal region of developing follicles. By PCR-amplifying V-D-J genes from AID mRNA(+) B cells isolated by laser capture microdissection, we found evidence of somatic hypermutation, and one likely instance of somatic gene conversion. Our results suggest that V-(D)-J gene diversification begins during early postnatal appendix development, in B cells stimulated to enter a proliferative program by signals derived from select intestinal commensals.

Background Since the first report of the antiretroviral restriction factor TRIM5α in primates, several orthologs in other mammals have been described. Recent studies suggest that leporid retroviruses like RELIK, the first reported endogenous lentivirus ever, may have imposed positive selection in TRIM5α orthologs of the European rabbit and European brown hare. Considering that RELIK must already have been present in a common ancestor of the leporid genera Lepus, Sylvilagus and Oryctolagus, we extended the study of evolutionary patterns of TRIM5α to other members of the Leporidae family, particularly to the genus Sylvilagus. Therefore, we obtained the TRIM5α nucleotide sequences of additional subspecies and species of the three leporid genera. We also compared lagomorph TRIM5α deduced protein sequences and established TRIM5α gene and TRIM5α protein phylogenies. Results The deduced protein sequence of Iberian hare TRIM5α was 89% identical to European rabbit TRIM5α, although high divergence was observed at the PRYSPRY v1 region between rabbit and the identified alleles from this hare species (allele 1: 50% divergence; allele 2: 53% divergence). A high identity was expected between the Sylvilagus and Oryctolagus TRIM5α proteins and, in fact, the Sylvilagus TRIM5α was 91% identical to the Oryctolagus protein. Nevertheless, the PRYSPRY v1 region was only 50% similar between these genera. Selection analysis of Lagomorpha TRIM5α proteins identified 25 positively-selected codons, 11 of which are located in the PRYSPRY v1 region, responsible for species specific differences in viral capsid recognition. Conclusions By extending Lagomorpha TRIM5α studies to an additional genus known to bear RELIK, we verified that the divergent species-specific pattern observed between the Oryctolagus and Lepus PRYSPRY-domains is also present in Sylvilagus TRIM5α. This work is one of the first known studies that compare the evolution of the antiretroviral restriction factor TRIM5α in different mammalian groups, Lagomorpha and Primates.

Extract: The function of the immune system is to protect the host from invasion by pathogenic microorganisms. Accordingly, most studies of host-microbial interactions have focused on pathogenic agents. The most common and intricate host-microbial interactions are, however, those between the host and non-pathogenic microorganisms that have taken up residence within the host, especially within the gastrointestinal tract. The lower gastrointestinal tract of mammals provides one of the best examples in nature of mutually beneficial association between host and colonizing foreign microorganisms. Between 500 and 1,000 microbial species colonize the mammalian colon to a density of approximately 1,000,000,000,000 bacteria per gram of content, comprising in total 100 times more cells than those that make up the host itself. Over the period of evolution, mammals have not only developed immunological tolerance mechanisms to accommodate this resident community, but they also have come to rely on these intestinal commensals for their own normal development. Studies of germfree and gnotobiotic animals revealed that the intestinal microbiota influences many aspects of immunity including development and function of mucosal immunity, development of oral tolerance and generation of the antibody repertoire. A recent study demonstrated that capsular polysaccharide produced by the commensal bacteria, Bacteroides fragilis, stimulates maturation of the developing immune system by contributing to the generation of CD4+ T cells and the proper T helper 1/T helper 2 cytokine balance.

Intestinal microbiota affect multiple aspects of the mucosal immune system, including promotion of mucosal organogenesis, TH cytokines balance, somatic diversification of Ig genes, and production of IgA. The mechanism by which microbiota interacts with vertebrate hosts and mediates these processes remains largely unexplored. Here we discuss the organogenesis of mucosal-associated lymphoid tissues, mechanisms by which microbiota promote MALT development, and how IgA can maintain a peaceful coexistence between commensals and their host.

Gut-associated lymphoid tissue (GALT) development requires interaction with the intestinal microbiota. Because murine secondary lymphoid tissue development is driven by positive feedback interactions between B cells and stromal cells, we used in situ hybridization to determine whether intestinal commensals influence such interactions during rabbit appendix development. The features of positive feedback interactions we examined (CXCL13 mRNA expression, B cell accumulation and FDC differentiation) increased during early follicle development, but stalled in the absence of intestinal commensals. These features were reinitiated by commensals that stimulated follicle development and intrafollicular B cell proliferation. Our results suggest that rabbit appendix follicles develop in two phases: an initial phase of B cell recruitment to nascent follicles, possibly through positive feedback interactions, and a subsequent phase of intrafollicular B cell proliferation stimulated by intestinal commensals. In addition, we found that intestinal commensals stimulate appendix CCL21 mRNA expression and T cell area formation.

The antibody repertoire of rabbits has interested immunologists for decades, in part because of the ease with which large quantities of high affinity antibodies can be obtained in serum, and in part because of the presence of genetic variants, allotypes, within V(H), C(H) and C(L) regions. Studies of these allotypes led to the initial descriptions of allelic exclusion, and neonatal suppression of serum Ig production (allotype suppression), and were instrumental in demonstrating that V and C regions are encoded by separate genes and are usually expressed in cis. The immune system of rabbit continues to be of interest primarily because of the use of both gene conversion and somatic hypermutation to diversify rearranged heavy and light chain genes and the role that gut-associated lymphoid tissues (GALT) and intestinal flora play in developing the primary (preimmune) antibody repertoire.

In domestic rabbit (Oryctolagus cuniculus), three serological types have been distinguished at the variable domain of the antibody H chain, the so-called V(H) a allotypes a1, a2, and a3. They correspond to highly divergent allelic lineages of the V(H) 1 gene, which is the gene rabbit utilizes in more than 80% of VDJ rearrangements. The sharing of serological V(H) a markers between rabbit and snowshoe hare (Lepus americanus) has suggested that the large genetic distances between rabbit V(H) 1 alleles (9-14% nucleotide differences) can be explained by unusually long lineage persistence times (transspecies polymorphism). Because this interpretation of the serological data is uncertain, we have determined the nucleotide sequences of V(H) genes expressed in specimens of Lepus species. Two sequence groups were distinguished, one of which occurred only in hare specimen displaying serological motifs of the rabbit V(H) a-a2 allotype. Sequences of this group are part of a monophyletic cluster containing the V(H) 1 sequences of the rabbit a2 allotype. The fact that this "transspecies a2 cluster" did not include genes of other rabbit V(H) a allotypes (a1, a3, and a4) is incompatible with the existence of a common V(H) a ancestor gene within the species, and suggests that the divergence of the V(H) a lineages preceded the Lepus vs Oryctolagus split. The sequence data are furthermore compatible with the hypothesis that the V(H)a polymorphism can be two times older than the divergence time between the Lepus and Oryctolagus lineages, which was estimated at 16-24 million years.

Intestinal bacteria are required for development of gut-associated lymphoid tissues (GALT), which mediate a variety of host immune functions, such as mucosal immunity and oral tolerance. In rabbits, the intestinal microflora are also required for developing the preimmune Ab repertoire by promoting somatic diversification of Ig genes in B cells that have migrated to GALT. We studied the mechanism of bacteria-induced GALT development. Bacteria were introduced into rabbits in which the appendix had been rendered germfree by microsurgery (we refer to these rabbits as germfree-appendix rabbits). We then identified specific members of the intestinal flora that promote GALT development. The combination of Bacteroides fragilis and Bacillus subtilis consistently promoted GALT development and led to development of the preimmune Ab repertoire, as shown by an increase in somatic diversification of VDJ-Cμ genes in appendix B cells. Neither species alone consistently induced GALT development, nor did Clostridium subterminale, Escherichia coli, or Staphylococcus epidermidis. B. fragilis, which by itself is immunogenic, did not promote GALT development; hence, GALT development in rabbits does not appear to be the result of an Ag-specific immune response. To identify bacterial pathways required for GALT development, we introduced B. fragilis along with stress-response mutants of B. subtilis into germfree-appendix rabbits. We identified two Spo0A-controlled stress responses, sporulation and secretion of the protein YqxM, which are required for GALT development. We conclude that specific members of the commensal, intestinal flora drive GALT development through a specific subset of stress responses.

This chapter focuses on the immunoglobulin (Ig) genes and development of the antibody repertoire in species that generate their primary antibody repertoires through the extensive somatic diversification of V(D)J heavy and light chain genes. In contrast, the V hand V L of mouse and human, use combinatorial joining of multiple V, (D), and J gene segments to generate the primary repertoire, comprise multiple V hand V L groups. It is possible that species in which the germline genes belong to are only a single group, and therefore have a less diverse repertoire of V gene segments have evolved to utilize somatic hypermutation, gene conversion, or both for developing a highly diversified primary antibody repertoire. Further, the species with less diverse V-gene segments utilize gut-associated lymphoid tissue (GALT) as the site for B-cell expansion and generation of the primary antibody repertoire. It is likely that the somatic diversification in GALT that leads to the primary antibody repertoire is not antigen driven, but instead occurs as a polyclonal response either to an exogenous agent, as occurred in rabbits, or possibly to an endogenous agent, such as a retroviral superantigen in species such as chicken, in which exogenous molecules are not required. Further studies are needed to elucidate the mechanism by which both GALT development and the somatic diversification of the Ig genes are initiated.

The immunoglobulin D (IgD) antibody class was, for many years, identified only in primates, rodents and teleost fish. The limited distribution of IgD among vertebrates suggested that IgD is a functionally redundant antibody class that has been lost by many vertebrate species during evolution. The recent identification of IgD in artiodactyls, however, suggests that IgD might be more widely expressed among vertebrates than previously thought, possibly serving a unique role in immunity. IgD expression has been searched for but not detected in rabbits. In order to search directly for a rabbit Cdelta locus encoding the constant region of IgD, we determined the nucleotide sequence of 13.5 kb of genomic DNA downstream of the rabbit Cmu locus. We did not find a rabbit Cdelta locus in this region, but found instead that this region is densely populated by repetitive elements, including a long interspersed DNA element repeat, six C repeats, and two processed pseudogenes. We conclude that the rabbit probably does not express IgD because there is no Cdelta locus immediately downstream of the rabbit Cmu locus.

The rabbit establishes its primary Ab repertoire by somatically diversifying an initial repertoire that is limited by restricted VH gene segment usage during VDJ gene rearrangement. Somatic diversification occurs in gut-associated lymphoid tissue (GALT), and by about 1–2 mo of age nearly all Ig VDJ genes are somatically diversified. In other species that are known to establish their primary Ab repertoire by somatic diversification, such as chicken, sheep, and cattle, diversification appears to be developmentally regulated: it begins before birth and occurs independent of exogenous factors. Because somatic diversification in rabbit occurs well after birth in GALT, the diversification process may not be developmentally regulated, but may require interaction with exogenous factors derived from the gut. To test this hypothesis, we examined Ab repertoire diversification in rabbits in which the appendix was ligated shortly after birth to prevent microbial colonization and all other organized GALT was surgically removed. We found that by 12 wk of age nearly 90% of the Ig VDJ genes in PBL were undiversified, indicating that intestinal microflora are required for somatically diversifying the Ab repertoire. We also examined repertoire diversification in sterilely derived remote colony rabbits that were hand raised away from contact with conventional rabbits and thereby acquired a different gut microflora. In these remote colony rabbits, GALT was underdeveloped, and 70% of the Ig VDJ genes in PBL were undiversified. We conclude that specific, currently unidentified intestinal microflora are required for Ab repertoire diversification.

Unlike mice and humans, rabbits generate their primary antibody repertoire by somatically diversifying V(D)J genes in gut-associated lymphoid tissues (GALT). Rabbit V(D)J genes are diversified by somatic hyper-point mutation and somatic gene conversion, both of which require activation-induced cytidine deaminase (AID). To determine when repertoire diversification begins, we examined AID expression in neonatal rabbit appendix by in situ hybridization. We first detected AID mRNA expression at one week of age, in B cells in the basolateral region of appendix follicles. We asked whether AID+ B cells were undergoing V(D)J gene diversification by using laser capture microdissection (LCM) to isolate AID+ and AID- B cells from 1-week-old appendix follicles. We RT-PCR-amplified and cloned VDJ genes from the two B cell populations, and searched for evidence of somatic diversification by comparing their nucleotide sequences to those of the appropriate germline genes. We found somatic gene conversion events in the VDJ genes from AID+ B cells, but not in those from AID- B cells. These results demonstrate that diversification of the rabbit primary antibody repertoire begins around 1 week of age, in B cells residing at the basolateral region of appendix follicles. This work was supported by NIH Grant 1 RO1 AI49458-01A1.