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.     

Determination of gene organization in the human IGHV region on single chromosomes

Organization of the IGHV genes (n=108) on single human chromosomes has been determined by detecting these sequences in single sperm using multiplex PCR amplification followed by microarray detection. A total of 374 single sperm samples from five Caucasian males were studied. Three deletion/insertion polymorphisms (Del I-Del III) with deletion allele frequencies ranging from 0.1 to 0.3 were identified. Del I is a previously reported polymorphism affecting three IGHV genes (IGHV1-8, IGHV3-9, and IGHV2-10). Del II affects a region 2-18 kb containing two pseudogenes IGHV(II)-28.1 and IGHV3-29, and Del III spans approximately 21-53 kb involving genes IGHV4-39, IGHV7-40, IGHV(II)-40-1, and IGHV3-41. Deletion alleles of both Dels II and III were found in a heterozygous state, and therefore, could not be easily detected if haploid samples were not used in the study. Results of the present study indicate that deletions/insertions together with other possible chromosomal rearrangements may play an important role in forming the genetic structure of the IGHV region, and may significantly contribute to antibody diversity. Since these three polymorphisms are located within or next to the 3' half of the IGHV region, they may have an important role in the expressed IGHV gene repertoire during immune response.     

The human anti-bullous pemphigoid monoclonal autoantibody P22 is encoded by genes of the IGHV4 and IGLV4 families

We identified, cloned, and biochemically characterized the full-length cDNAs encoding the heavy and light chains of a human monoclonal antibody (mAb) from the Epstein-Barr virus (EBV)-cell line P22. The cell line P22, which originated from a patient with bullous pemphigoid (an autoimmune disease causing skin blistering) expressed immunoglobulin-G (IgG) with a lambda light chain. Although the variable heavy (IGHV) chain gene family could not be assigned by IGHV repertoire analysis, the determination of its nucleotide sequence demonstrated that the heavy chain of P22 belonged to the IGHV4 family. The limited IGHV4 gene usage by memory IgG, IGA and IgE-expressing cells supports the notion of the autoreactivity-associated IGHV4 genes and stresses the strong selection pressure within germinal centres towards IGHV4 family. Alignment of P22 IGHV4 cDNA sequence to germline sequences from gene databases, revealed a remarkable divergence, suggesting that the heavy chain of the P22 mAb encodes a distinct IGHV4 gene. The variable light chain (IGLV) encodes a IGLV4 gene and is 98% similar to a previously reported IGLV gene. Furthermore, fluorescent staining with the recombinant mAb showed the same reactivity to that of the native antibody. The data reported herein, (a) reveal an autoantibody encoding a distinct IGHV4 gene, (b) confirm the notion that autoantibodies preferentially use IGHV4 genes, and (c) hypothesize that somatic hypermutation within GC may be a mechanism by which autoreactive B lymphocytes escape negative selection.     

家族性腺瘤性息肉病一家系调查及APC基因胚系突变分析

目的 探讨家族性腺瘤性息肉病(familial adenomatous polyposis,FAP)家系调查及高危亲属基因筛查的意义,报道云南省一FAP家系发病相关基因APC基因的胚系突变结果.方法 查阅对2001年昆明医学院第一附属医院1例FAP患者病例,电话联系及登门随访进行其家系调查,绘制家系图谱.抽取该家系成员外周静脉血提取DNA,利用PCR方法扩增APC基因,应用DNA自动测序仪进行测序.结果 该家系三代共计9人,成员Ⅰ1、Ⅱ1、Ⅱ2、Ⅱ3、Ⅱ4、Ⅲ2、Ⅲ3、Ⅲ48人检出APC基因胚系突变c.3587C>A(S1196X),其中Ⅱ2、Ⅱ2、Ⅱ4、Ⅲ2、Ⅲ3经肠镜检查证实有结直肠多发息肉,Ⅲ4未检出息肉,为基因突变携带者.结论 通过家系调查对高危亲属进行基因筛查可以发现早期患者,尤其是无临床表现的FAP基因突变携带者,以早期进行医学干预及预防性手术治疗,降低FAP的癌变率、病死率;APC基因c.3587C>A(S1196X)胚系突变是引起该家系FAP患者发病的原因.     

The analysis of VH and VL genes repertoires of Fab library built from peripheral B cells of human rabies virus vaccinated donors

A human combinatorial Fab antibody library was generated from immune repertoire based on peripheral B cells of ten rabies virus vaccinated donors. The analysis of random Fab fragments from the unselected library presented some bias of V gene usage towards IGHV-genes and IGLV-gen families. The screening of the Fab library on rabies virus allowed specific human Fab antibody fragments characterized for their gene encoding sequences, binding and specificities to RV. Genetic analysis of selected Fabs indicated that the IGHV and IGLV differ from the germ-line sequence. At the level of nucleotide sequences, the IGHV and IGLV domains were found to share 74–92% and 90–96% homology with sequences encoded by the corresponding human germ-line genes respectively. IGHV domains are characterized most frequently by IGHV3 genes, and large proportions of the anti-RV heavy chain IGHV domains are obtained following a VDJ recombination process that uses IGHD3, IGHD2, IGHD1 and IGHD6 genes. IGHJ3 and IGHJ4 genes are predominantly used in RV-Fab. The IGLV domains are dominated by IGKV1, IGLV1 and IGLV3 genes. Numerous somatic hypermutations in the RV-specific IGHV are detected, but only limited amino acid replacement in most of the RV-specific IGLV particularly in those encoded by J proximal IGLV or IGKV genes are found. Furthermore, IGHV3–IGKV1, IGHV3–IGVL1, and IGHV3–IGLV3 germ-line family pairings are preferentially enriched after the screening on rabies virus.     

Germline diversity of the expressed BALB/c VhJ558 gene family

Although the mouse immunoglobulin heavy chain (Igh) locus contains 15 heavy chain V (Vh) gene families, at least half of the Vh gene segments are members of the VhJ558 family. This large Vh gene family represents the least characterized germline coding regions of any of the mouse antigen receptor loci and the contribution of individual VhJ558 genes to the preimmune repertoire is poorly understood. In fact, relatively few germline VhJ558 sequences have been reported for BALB/c, the foundation strain for mouse immunoglobulin genetics and the prototypic strain of the Igh(a) haplotype. Here we present a database consisting of 66 sequences estimated to represent one-half of the total number of functional BALB/c VhJ558 genes. Our results indicate that a subset of the VhJ558 genes is highly expressed in the preimmune repertoire, with just nine Vh sequences accounting for nearly 50% of the VhJ558 heavy chains expressed by splenic B cells. We show that this disparity in the expressed Vh gene repertoire is not due to the position of the Vh genes relative to the Dh cluster or to multiple germline copies of the highly expressed VhJ558 genes. Together, these data constitute the first detailed analysis of functional BALB/c VhJ558 genes, demonstrate a striking bias in the use of particular VhJ558 genes in the preimmune repertoire, and provide sufficient information to study the regulation of the Dh-distal region of the Igh-V locus at the level of individual genes.     

The immunoglobulin heavy chain locus: genetic variation, missing data, and implications for human disease

The immunoglobulin (IG) loci consist of repeated and highly homologous sets of genes of different types, variable (V), diversity (D) and junction (J), that rearrange in developing B cells to produce an individual's highly variable repertoire of expressed antibodies, designed to bind to a vast array of pathogens. This repeated structure makes these loci susceptible to a high frequency of insertion and deletion events through evolutionary time, and also makes them difficult to characterize at the genomic level or assay with high-throughput techniques. Given the central role of antibodies in the adaptive immune system, it is not surprising that early candidate gene approaches showed that germline polymorphisms in these regions correlated with susceptibility to both infectious and autoimmune diseases. However, more recent studies, particularly those using high-throughput genome-wide arrays, have failed to implicate these loci in disease. In this review of the IG heavy chain variable gene cluster (IGHV), we examine how poorly we understand the distribution of haplotype variation in this genomic region, and we argue that this lack of information may mask candidate loci in the IGHV gene cluster as causative factors for infectious and autoimmune diseases.     

Crystal structure of human germline antibody 3-23/B3

The human antibody repertoire is dominated by a few combinations of germline sequences, with the genes 3-23 for the heavy chain and B3 for the light chain being among the most frequently used. Despite this fact, there was no experimental structure of the antibody composed of 3-23 and B3. The crystal structure of the Fab fragment of the synthetic antibody composed of the 3-23 and B3 germline sequences was determined to provide a template for antibody modeling. The antigen-binding loops were found in the canonical conformations. Comparison to the other structures where either 3-23 or B3 is paired with a different chain reveals a significant deviation in the orientation of the variable domains.     

A new germline VH36-60 gene is used in the neonatal primary and adult memory response to (T,G)-A--L.

Our previous studies of the neonatal primary response to (T,G)-A--L showed that the majority of anti-(T,G)-A--L antibodies bind the copolymer L-Glu:L-Tyr (GT), share idiotypy (Id), and use the H10 germline VH gene from the VHJ558 family and a V kappa 1 gene. We also identified two hybridomas from different neonatal donors that produced GT+, Id+ antibodies using a V kappa 1 gene with a VH gene from the VH36-60 family. In the study reported here, we show that both neonatal hybridomas use the same germline VH gene from the VH36-60 gene family. However, the VH gene sequence is different from previously identified germline genes of the VH36-60 gene family. To determine whether the expressed heavy chain gene had undergone somatic mutation, we isolated the corresponding germline gene from kidney DNA. Sequence analysis of this gene shows that it is a new member of the VH36-60 family which is not mutated in the neonatal antibodies. Furthermore, the deduced amino acid sequences of the two neonatal antibodies are identical not only in the VH region but also in the VH-D-JH joins, suggesting that there is a strong selection for CDRIII among neonatal anti-(T,G)-A--L antibodies using this germline gene (designated here as VH3A1) with a V kappa 1 gene. Also, the VH gene from the VH36-60 family that we showed previously was used by an adult memory B cell clone specific for (T,G)-A--L, can now be identified as a rearrangement of the VH3A1 germline gene. Elucidation of the germline variable region genes that are used in the antigen-specific neonatal response will help us understand the mechanisms that shape the preimmune B cell repertoire during B cell development.     

Extensive restrictions in the VH sequence usage of the human antibody response against the Rhesus D antigen

Anti-Rhesus D immunoglobulin purified from human sera is used as a prophylactic reagent in Rhesus D negative women at risk of alloimmunization during pregnancy. We are currently developing a Rhesus D antigen-specific recombinant polyclonal antibody drug lead for replacing the existing blood derived-products. By analyzing the RhD-specific antibody VH repertoires from eight alloimmunized women we found, in agreement with previous studies, a strong preference for the VH 3-33 "superspecies" gene segments which encompasses the IGHV3-30-3*01, IGHV3-30*18, and IGHV3-33*01 VH alleles. Even more extensive genetic restriction was observed among five donors, which produced antibodies of identical V-D-J usage and CDR3 loop length and joining regions of similar amino acid composition. In addition, we find a high degree of sequence relatedness to previously isolated anti-Rhesus D antibodies. Such close homology in VH domains indicates that significant structural restrictions are operating in the selection of antibodies recognizing RhD as seen for T cell receptors. Moreover, some VH domains were isolated in their germline configuration indicating that anti-RhD antibodies of relatively high affinity are present in the na?ve antibody repertoire of Rhesus negative individuals which offers an explanation for the strong and clinically significant immunogenicity of the Rhesus D.     

Identification of the V genes encoding xenoantibodies in non-immunosuppressed rhesus monkeys

The major immunological barrier that prevents the use of wild-type pig xenografts as an alternative source of organs for human xenotransplantation is antibody-mediated rejection. In this study, we identify the immunoglobulin variable region heavy (IgV(H)) chain genes encoding xenoantibodies to porcine heart and fetal porcine islet xenografts in non-immunosuppressed rhesus monkeys. We sought to compare the IgV(H) genes encoding xenoantibodies to porcine islets and solid organ xenografts. The immunoglobulin M (IgM) and IgG xenoantibody response was analysed by enzyme-linked immunosorbent assay and cDNA libraries from peripheral blood lymphocytes were prepared and sequenced. The relative frequency of IgV(H) gene usage was established by colony filter hybridization. Induced xenoantibodies were encoded by the IGHV3-11 germline progenitor, the same germline gene that encodes xenoantibodies in humans mounting active xenoantibody responses. The immune response to pig xenografts presented as solid organs or isolated cells is mediated by identical IgV(H) genes in rhesus monkeys. These animals represent a clinically relevant model to identify the immunological basis of pig-to-human xenograft rejection.     

Natural human antibodies to pneumococcus have distinctive molecular characteristics and protect against pneumococcal disease

The molecular and functional characteristics of natural antibody from the preimmune repertoire have not been explored in detail in man. We describe seven human IgM monoclonal antibodies selected on the basis of pneumococcal polysaccharide binding that share both molecular and functional characteristics with natural antibody, suggesting a common B cell lineage origin. Unlike class-switched antibodies, which are serotype-specific, the antibodies were polyreactive and bound all pneumococcal polysaccharide capsular serotypes tested. Some bound endogenous antigens, including blood group antigens and intermediate filament proteins. All the antibodies used unmutated heavy chain V (IGHV) that are expressed at an increased frequency in the elderly and in the preimmune repertoire. The CDR3 was characterized by long length (mean aa 18.4 (+/-4.2) and selective use of IGHD6 (P < 0.001) and IGHJ6 (P < 0.01) family genes. The clones expressing IGHV1-69 and IGHV 3-21 provided significant passive protection against invasive pneumococcal disease in vivo.     

Molecular characterization of the human immunoglobulin V lambda I germline gene repertoire.

To advance our understanding of the human immunoglobulin V lambda germline gene contribution to normal as well as autoimmune responses, we have isolated and sequenced six germline genes of the V lambda I subgroup. These genes can be divided into three sub-subgroups on the basis of greater than or equal to 93% nucleotide sequence homology and greater than or equal to 88% deduced amino acid sequence similarity. Examination of all cDNA and protein sequences available for expressed V lambda I genes supports the assignment of these three sub-subgroups. Sequence comparisons also suggest that germline gene members of two of these sub-subgroups, I-a and I-b, are preferentially utilized in the expressed V lambda I repertoire. This finding may be at least partially attributable to regulatory sequence abnormalities apparent in two of the other V lambda I germline genes (Humlv101 and Humlv104) which may interfere with their expression.     

A bioinformatics pipeline to build a knowledge database for in silico antibody engineering

A challenge to antibody engineering is the large number of positions and nature of variation and opposing concerns of introducing unfavorable biochemical properties. While large libraries are quite successful in identifying antibodies with improved binding or activity, still only a fraction of possibilities can be explored and that would require considerable effort. The vast array of natural antibody sequences provides a potential wealth of information on (1) selecting hotspots for variation, and (2) designing mutants to mimic natural variations seen in hotspots.The human immune system can generate an enormous diversity of immunoglobulins against an almost unlimited range of antigens by gene rearrangement of a limited number of germline variable, diversity and joining genes followed by somatic hypermutation and antigen selection. All the antibody sequences in NCBI database can be assigned to different germline genes. As a result, a position specific scoring matrix for each germline gene can be constructed by aligning all its member sequences and calculating the amino acid frequencies for each position. The position specific scoring matrix for each germline gene characterizes “hotspots” and the nature of variations, and thus reduces the sequence space of exploration in antibody engineering.We have developed a bioinformatics pipeline to conduct analysis of human antibody sequences, and generated a comprehensive knowledge database for in silico antibody engineering. The pipeline is fully automatic and the knowledge database can be refreshed anytime by re-running the pipeline. The refresh process is fast, typically taking 1 min on a Lenovo ThinkPad T60 laptop with 3G memory.Our knowledge database consists of (1) the individual germline gene usage in generation of natural antibodies; (2) the CDR length distributions; and (3) the position specific scoring matrix for each germline gene. The knowledge database provides comprehensive support for antibody engineering, including de novo library design in selection of favorable germline V gene scaffolds and CDR lengths. In addition, we have also developed a web application framework to present our knowledge database, and the web interface can help people to easily retrieve a variety of information from the knowledge database.     

The evolutionary and functional significance of germline immunoglobulin gene variation

The recombination between immunoglobulin (IG) gene segments determines an individual’s naïve antibody repertoire and, consequently, (auto)antigen recognition. Emerging evidence suggests that mammalian IG germline variation impacts humoral immune responses associated with vaccination, infection, and autoimmunity – from the molecular level of epitope specificity, up to profound changes in the architecture of antibody repertoires. These links between IG germline variants and immunophenotype raise the question on the evolutionary causes and consequences of diversity within IG loci. We discuss why the extreme diversity in IG loci remains a mystery, why resolving this is important for the design of more effective vaccines and therapeutics, and how recent evidence from multiple lines of inquiry may help us do so.     

Rearrangement of only one human IGHV gene is sufficient to generate a wide repertoire of antigen specific antibody responses in transgenic mice

In recent years, mice carrying human IG transgenes are being generated for the production of human monoclonal antibodies as an alternative approach to the conventional use of mouse or chimeric-humanized antibodies. Theoretically, the size of the repertoire of human antibodies that these mice could produce would be critically dependent on the number of human V genes introduced in the transgene. This could be the case for BABkappa and BABkappa,lambda transgenic mice, which carry several genes from the human IGK (BABkappa), and IGK and IGL (BABkappa,lambda) loci, but only five human IGHV genes and the entire IGHD-IGHJ cluster linked to two human IGHC (IGHM-IGHD) genes. We analyzed the expressed human IG genes in 30 IgM-secreting hybridomas generated from transgenic mice immunized either with soluble proteins (human IgM coupled to KLH) or with cells (human PBMC, tumour cell lines or rat cells transfected with human CD69). The results show that all hybridoma cells analyzed rearranged exclusively the IGHV1-2 gene, in contrast with naive spleen B cells that used three out of the five IGHV genes present in the transgene. The configuration of the rearranged CDR3 region revealed a much higher heterogeneity in the heavy chains. A variety of IGHJ and IGHD genes were used in hybridomas, and somatic mutations were also seen in some hybrids. Regarding the rearranged light chains genes, it was a much higher variety in the use of V and J genes in both, kappa and lambda chains, than in the heavy chain, and also in the level of mutation. The results indicate that only one IGHV gene is sufficient to generate a wide repertoire of antigen specific antibody responses. Thus, efforts aimed at the generation of new transgenic mice should focus more on the integrity of the D/J region and on the DNA regions regulating somatic hypermutation, rather than on the number of V genes present in the transgene.     

Identification of an anti-idiotypic antibody that defines a B-cell subset(s) producing xenoantibodies in primates

Synthetic anti-idiotypic antibodies represent a potentially valuable tool for the isolation and characterization of B cells that produce xenoantibodies. An anti-idiotypic antibody that binds to a subset of B cells producing antibodies encoded by the variable-region heavy chain 3 (V(H)3) germline genes DP35 [immunoglobulin variable-region heavy chain 3-11 (IGHV3-11)], DP-53 and DP-54 plus a small number of V(H)4 gene-encoded antibodies in humans has recently been identified. These germline progenitors also encode xenoantibodies in humans. We tested whether the small, clearly defined group of B cells identified with this anti-idiotypic antibody produce xenoantibodies in non-human primates mounting active immune responses to porcine xenografts. Peripheral blood B cells were sorted by flow cytometry on the basis of phenotype, and cDNA libraries were prepared from each of these sorted groups of cells. Immunoglobulin V(H) gene libraries were prepared from the sorted cells, and the V(H) genes expressed in each of the sorted groups were identified by nucleic acid sequencing. Our results indicate that xenoantibody-producing peripheral blood B cells, defined on the basis of binding to fluorescein isothiocyanate (FITC)-conjugated galactose alpha(1,3) galactose-bovine serum albumin (Gal-BSA) and the anti-idiotypic antibody 2G10, used the IGHV3-11 germline gene to encode xenoantibodies and were phenotypically CD11b+ (Mac-1+) and CD5-. This novel reagent may be used in numerous applications including definition of xenoantibody-producing B-cell subsets in humans and non-human primates and immunosuppression by depletion of B cells producing anti-Gal xenoantibodies.     

Generation of llama single-domain antibodies against methotrexate, a prototypical hapten

Single-domain antibodies specific to methotrexate (MTX) were obtained after immunization of one llama (Llama glama). Specific VHH domains (V-D-J-REGION) were selected by panning from an immune-llama library using phage display technology. The antibody fragments specific to MTX were purified from Escherichia coli (C41 strain) periplasm by immobilized metal affinity chromatography with an expression level of around 10mg/L. A single band around 16,000Da corresponding to VHH fragments was found after analysis by SDS-PAGE and Western blotting, while competition ELISA demonstrated selective binding to soluble MTX. Surface plasmon resonance (SPR) analysis showed that anti-MTX VHH domains had affinities in the nanomolar range (29-515nM) to MTX-serum albumin conjugates. The genes encoding anti-MTX VHH were found by IMGT/V-QUEST to be similar to the previously reported llama and human IGHV germline genes. The V-D and D-J junction rearrangements in the seven anti-MTX CDR3 sequences indicate that they were originated from three distinct progenitor B cells. Our results demonstrate that camelid single-domain antibodies are capable of high affinity binding to low molecular weight hydrosoluble haptens. Furthermore, these anti-MTX VHH give new insights on how the antigen binding repertoire of llama single-domain antibody can provide combining sites to haptens in the absence of a VL. This type of single-domain antibodies offers advantages compared to murine recombinant antibodies in terms of production rate and sequence similarity to the human IGHV3 subgroup genes.