Mouse Antibody Repertoires Research Articles

Mice possess a more limited natural antihuman factor VIII antibody repertoire than humans that is produced disproportionately by marginal zone B cells

BackgroundOne-third of patients with severe hemophilia A develop neutralizing antibodies to the factor VIII (FVIII) protein in response to intravenous replacement therapy. Patients may also generate natural, nonneutralizing antibodies to FVIII before FVIII exposure. These patients are at increased risk of developing neutralizing antibodies to FVIII. However, natural anti-FVIII antibodies are also present in healthy human donors. ObjectivesTo further characterize the natural antihuman (h) FVIII antibody repertoire in mice and humans. MethodsAn in-house ELISA was developed using a purified polyclonal immunoglobulin (Ig) standard to quantify anti-hFVIII Ig in cell culture supernatant or plasma from mice (wild-type and FVIII-/-) and adult human donors. ResultsAll naïve wild-type and FVIII-/- mice, as well as healthy human donors, possess natural anti-hFVIII antibodies. Mice only have natural anti-hFVIII IgM, which is present in germ-free mice, suggesting that they are germline encoded. Although murine marginal zone B cells (MZBs) contribute 44% to all circulating natural IgM, they contribute disproportionately to the anti-hFVIII IgM repertoire (82%). This naturally occurring murine MZB-derived IgM is not B-domain specific and is reduced by intravenously administered hFVIII, suggesting that it may form immune complexes immediately upon hFVIII administration. Natural anti-hFVIII antibodies of IgG, IgM, and IgA isotypes can be detected in adult human donors. There were increased levels of B-domain-favoring anti-hFVIII IgG in 14% of healthy donors, which were markedly different from the rest of the “low-titer” population. ConclusionsThere is a preponderance of natural anti-hFVIII antibodies in both mice and healthy adult human donors.

Antibody Repertoires Identify β-Tubulin as a Host Protective Parasite Antigen in Mice Infected With Trypanosoma cruzi.

Few studies investigate the major protein antigens targeted by the antibody diversity of infected mice with Trypanosoma cruzi. To detect global IgG antibody specificities, sera from infected mice were immunoblotted against whole T. cruzi extracts. By proteomic analysis, we were able to identify the most immunogenic T. cruzi proteins. We identified three major antigens as pyruvate phosphate dikinase, Hsp-85, and β-tubulin. The major protein band recognized by host IgG was T. cruzi β-tubulin. The T. cruzi β-tubulin gene was cloned, expressed in E. coli, and recombinant T. cruzi β-tubulin was obtained. Infection increased IgG reactivity against recombinant T. cruzi β-tubulin. A single immunization of mice with recombinant T. cruzi β-tubulin increased specific IgG reactivity and induced protection against T. cruzi infection. These results indicate that repertoire analysis is a valid approach to identify antigens for vaccines against Chagas disease.

The mouse antibody heavy chain repertoire is germline-focused and highly variable between inbred strains.

The human and mouse antibody repertoires are formed by identical processes, but like all small animals, mice only have sufficient lymphocytes to express a small part of the potential antibody repertoire. In this study, we determined how the heavy chain repertoires of two mouse strains are generated. Analysis of IgM- and IgG-associated VDJ rearrangements generated by high-throughput sequencing confirmed the presence of 99 functional immunoglobulin heavy chain variable (IGHV) genes in the C57BL/6 genome, and inferred the presence of 164 IGHV genes in the BALB/c genome. Remarkably, only five IGHV sequences were common to both strains. Compared with humans, little N nucleotide addition was seen in the junctions of mouse VDJ genes. Germline human IgG-associated IGHV genes are rare, but many murine IgG-associated IGHV genes were unmutated. Together these results suggest that the expressed mouse repertoire is more germline-focused than the human repertoire. The apparently divergent germline repertoires of the mouse strains are discussed with reference to reports that inbred mouse strains carry blocks of genes derived from each of the three subspecies of the house mouse. We hypothesize that the germline genes of BALB/c and C57BL/6 mice may originally have evolved to generate distinct germline-focused antibody repertoires in the different mouse subspecies.

Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing

High-throughput sequencing of the antibody repertoire is enabling a thorough analysis of B cell diversity and clonal selection, which may improve the novel antibody discovery process. Theoretically, an adequate bioinformatic analysis could allow identification of candidate antigen-specific antibodies, requiring their recombinant production for experimental validation of their specificity. Gene synthesis is commonly used for the generation of recombinant antibodies identified in silico. Novel strategies that bypass gene synthesis could offer more accessible antibody identification and validation alternatives. We developed a hybridization-based recovery strategy that targets the complementarity-determining region 3 (CDRH3) for the enrichment of cDNA of candidate antigen-specific antibody sequences. Ten clonal groups of interest were identified through bioinformatic analysis of the heavy chain antibody repertoire of mice immunized with hen egg white lysozyme (HEL). cDNA from eight of the targeted clonal groups was recovered efficiently, leading to the generation of recombinant antibodies. One representative heavy chain sequence from each clonal group recovered was paired with previously reported anti-HEL light chains to generate full antibodies, later tested for HEL-binding capacity. The recovery process proposed represents a simple and scalable molecular strategy that could enhance antibody identification and specificity assessment, enabling a more cost-efficient generation of recombinant antibodies.

Predominant VH genes expressed in innate antibodies are associated with distinctive antigen-binding sites.

Antibodies to phosphatidylcholine (PtC), a common constituent of mammalian and bacterial cell membranes, represent a large proportion of the natural antibody repertoire in mice. Previous studies of several mouse strains (e.g., C57BL/6) have shown that anti-PtC antibodies are mainly encoded by the VH11 and VH12 immunoglobulin heavy chain variable region gene families. We show here, however, that VH11 and VH12 encode only a small proportion of the anti-PtC antibodies in BALB/c mice. Instead, VHQ52-encoded antibodies predominate in this strain. In addition, two-thirds of the cells expressing VHQ52 family genes use a single gene (which, interestingly, has been previously shown to predominate in the anti-oxazolone response). We also show here that in anti-PtC antibodies from all strains, the distinctive antigen-binding sites associated with VHQ52 differ substantially from those associated with VH11 and VH12. That is, VHQ52-containing transcripts preferentially use the joining region JH4 rather than JH1 and exhibit more diverse complementarity-determining region 3 (CDR3) junctions with more N-region nucleotide additions at the gene segment junctions. Thus, the VH gene family that predominates in the anti-PtC repertoire differs among mouse strains, whereas the distinctive VHDJH rearrangements (CDR3, JH) associated with each VH gene family are similar in all strains. We discuss these findings in the context of a recent hypothesis suggesting that CDR3 structure, independent of VH framework, is sufficient to define the specificity of an antibody.

Variable domain structure of κIV human light chain Len: High homology to the murine light chain McPC603

Antibody light chains of the κ subgroup are the predominant light chain component in human immune responses and are used almost exclusively in the antibody repertoire of mice. Human κ light chains comprise four subgroups. To date, all crystallographic studies of human κ light chains were carried out on proteins of the κI subgroup. The light chain produced by multiple myeloma patient Len, was of the κIV subgroup, it differed by only one residue from the germ-line gene encoded protein. The variable domain fragment of the light chain was crystallized from ammonium sulfate in space group C222 1. The crystal structure was determined by molecular replacement and refined at 1.95 Å resolution to an R-factor of 0.15. Protein Len has six additional residues in its CDR1 segment compared to the κI proteins previously characterized. The κIV variable domain. Len, differs in only 23 of 113 residues from murine κ light chain McPC603. The RMS deviation upon superimposing their α-carbons was 0.69 Å. The CDR1 segment of the human and murine variable domains have the same length and conformation although their amino acid sequences differ in 5 out of 17 residues. Structural features were identified that could account for the significantly higher stability of the human κIV protein relative to its murine counterpart. This human κIV light chain structure is the closest human homolog to a murine light chain and can be expected to facilitate detailed structural comparisons necessary for effective humanization of murine antibodies.

The primary antibody repertoire of normal, immunodeficient and autoimmune mice is characterized by differences in V gene expression

During the last decade, the structure and organization of the immunoglobulin heavy and light chain locis have been defined in mice and humans. Studies on VH gene expression at different stages of development, in different organs and disease states have provided useful insight into the construction of a primary antibody repertoire in mice. Clearly, 3'VH genes 7183, Q52 and Vh11, which are conserved during evolution, are preferentially expressed during early development of the B-lymphocyte repertoire. A preferential use for the V kappa 4 gene family is evident during early B-cell development. The initial development of the primary antibody repertoire is therefore influenced by a restricted set of VH and V kappa gene elements. The restricted B-cell repertoire is subsequently normalized in the periphery, as revealed by stochastic VH gene expression, as a result of exposure to environmental antigens. Obviously, the peripheral B-cell pool characterized by stochastic VH gene expression is selectively replenished by newly generated B cells in bone marrow that preferentially expresses 3'VH genes. The V kappa genes are, however, expressed in a non-random manner in the neonatal and adult B-lymphocyte repertoire that is probably related to VH and V kappa association dynamics and/or positive or negative selection. Interestingly, these characteristics of neonatal and adult primary repertoire are noted in both B1 and B2 lymphocytes. No remarkable age-related differences are evident for VH and V kappa gene expression. In healthy mice, both the mitogen responsive (available) and unstimulated (expressed) B-cell repertoire show similar VH gene expression. Interestingly, VH gene expression varies in different organs which may reflect, or occur as a result of, the specialized function of each organ. For example, J558 gene expression is higher in the peripheral LN where B cells continuously encounter exogenous antigens. The skewed VH and V kappa gene expression noted in immunodeficient and autoimmune lupus-prone mice reflects the impairment of the primary antibody repertoire associated with immunodeficiency and autoimmune disorders.