Molecular characterization of human antibodies to bacterial antigens: utilization of the less frequently expressed VH2 and VH6 heavy chain variable region gene families.

Structural analysis of the human immunoglobulin repertoire holds promise for determining the basis of variable region gene usage in response to a variety of auto and exogenous antigens. Here we report the nucleotide sequences of the heavy and light chain variable regions expressed by three human monoclonal antibodies specific for two clinically relevant bacterial pathogens, Bordetella pertussis and Haemophilus influenzae type b. The cell lines were derived by in vitro stimulation of lymphocytes from spleen or tonsillar tissue, respectively, and bind to different antigens from the two organisms. The single B. pertussis antibody is of the IgM lambda isotype and utilizes the single VH6 gene segment in combination with a V lambda 2 gene and demonstrates limited somatic mutation, yet is highly indicative of an antigen-driven immune response. One H. influenzae antibody is of the IgG2 lambda isotype and expresses a VH3 gene segment with a V lambda 1 gene, while the second cell line produces an IgG3 lambda antibody expressing a combination of VH2/V lambda 3. Both molecules show evidence of somatic mutation. The D gene segments of the heavy chains vary in length and display limited sequence homology with known germline D segments. As demonstrated previously, JH4 predominates (two JH4 and one JH3) and all three utilize the J lambda 3 gene segment. In addition, we have isolated and sequenced a number of germline VH2 gene segments in an attempt to better understand the nature of the VH2 germline repertoire. In addition to contributing to the understanding of the human antibody repertoire, such clinically relevant molecules may prove to be a source of passive immunotherapy for those at risk to developing disease.

Variable region genes of anti-HIV human monoclonal antibodies: non-restricted use of the V gene repertoire and extensive somatic mutation.

The extent of the expressed human V gene repertoire for the most part has been derived from fetal cDNA libraries, autoantibodies, and myeloma proteins. In order to continue to explore the utilization of the VH and VL gene repertoire in response to exogenous viral antigens, the heavy and light chain cDNAs from four human anti-HIV monoclonal antibodies were PCR amplified from human-mouse heterohybridomas, cloned, and nucleotide sequence analysis performed. Of the monoclonals analyzed, three were directed against gp120 and one reacted with gp41. Three of the antibodies were of the IgG1 lambda isotype and one was an IgG1 kappa. Three of the four heavy chains were derived from VHI gene segments and one VHII was observed. D segments showed evidence of D-D joining and three JH4 and one JH5 gene were utilized. Two V lambda II lambda chains and one from the V lambda III gene family were observed and the single kappa chain sequenced was from the V kappa III family. DNA sequence comparison with known germline gene segments identified putative precursor V gene segments for one of the heavy chains and two light chains. Comparison of the expressed amino acid sequences with the predicted germline sequences indicated that changes were clustered in the CDRs and FR3 regions of the V gene segments. We reported previously the nucleotide sequences of five human monoclonal antibodies from HIV-infected individuals, three of which utilized VHIV, one VHV and one a VHI gene segment and also found extensive evidence of somatic mutation. Collectively, our results indicate that an antigen driven response is functioning following HIV infection and, surprisingly, to date we have not encountered a VHIII gene segment. Since VHIII is the largest human VH gene family, it may well be that this under-representation has both functional and clinical implications.

Variable region gene segment utilization in rhesus monkey hybridomas producing human red blood cell-specific antibodies: predominance of the VH4 family but not VH4-21 (V4-34).

Structural analyses of human immunoglobulin gene segments from monoclonal cell lines provide valuable information regarding the antibody repertoire. This information, in conjunction with a nearly complete knowledge of the human immunoglobulin germline repertoire, now allows further investigation into the underlying molecular basis responsible for some of the observed biases found in the expressed repertoire. One human heavy chain variable region gene segment, V4-34 (VH4-21), is one of the most prevalent gene segments in the expressed repertoire. The overwhelming presence of the V4-34 gene segment suggests that it may play an important role in immune responses. However, there is currently little information regarding its presence and potential importance in nonhuman primates. In order to determine if this gene segment is used by lower primates in a similar manner we determined the molecular structure of the variable region gene segments that are expressed by macaque monoclonal heterohybridomas that are specific for human red blood cell antigens. Eleven of the 12 hybridomas are derived from Rhesus monkeys (Macaca mulatta) and one is from a cynomologous monkey (Macaca fascicularis), all of which have been immunized with human red blood cells. The predominance of a VH4-like family and the specific absence of a VH4-21 equivalent led us to further characterize the macaque VH4 gene family at the germline level. Therefore, germline gene segments from the macaque equivalent to the human VH4 gene family are also described.

The human antibody repertoire: heavy and light chain variable region gene usage in six alloantibodies specific for human HLA class I and class II alloantigens.

Peripheral blood B lymphocytes have been isolated from healthy individuals who were immunized with lymphocytes from HLA-incompatible donors and transformed with Epstein-Barr virus to produce human monoclonal cell lines specific for human HLA molecules. The cell lines have been previously characterized and are known to bind to various class I and class II alloantigens. In this report we describe the molecular characterization of the heavy and light chain variable region gene segments that are utilized by these monoclonal antibodies. Using the polymerase chain reaction and primer pairs specific for the respective constant region and VH or VL family, rearranged variable region gene segments were amplified from cDNA from individual cell lines. Products were then subcloned, sequenced and analysed for gene usage and apparent somatic mutation. The results show that the VH3 gene family predominates in a group of six heavy chains (four out of six) with one VH1 and one VH4 gene segment. The light chain variable region gene family usage is more diverse with 2 V kappa 3, 1 V kappa 1, 2 V lambda 2 and 1 V lambda 3. The extent of apparent somatic mutation is minimal, relative to our previous observations in a group of high affinity human monoclonal antibodies specific for pathogenic organisms.

The molecular structure of human antibodies specific for the human immunodeficiency virus.

The molecular structure of human antibodies that are specific for human immunodeficiency virus-1 (HIV-1) are of increasing interest as AIDS research progresses toward passive immunotherapeutics in the maintenance and prevention of infection. In recent years a number of human, HIV-specific hybridomas and EBV-transformed B cell lines, as well as a combinatorial library, have been developed and characterized at the molecular level. These sources have provided valuable information on the immunoglobulin heavy- and light-chain variable-region gene usage and the extent and appearance of somatic mutation in a disease where the immune system is under constant stimulation over a long period of time. In this article we review the current data available on the molecular structure of these antibodies.

Probing the human antibody repertoire to exogenous antigens. Characterization of the H and L chain V region gene segments from anti-hepatitis B virus antibodies.

Structural studies of human antibody V regions have been largely limited to those involving the fetal repertoire, autoantibodies, and malignant cell rearrangements, leaving the "normal" repertoire relatively unexplored. In this study we describe the nucleotide sequences of the H and L chain V regions of four antibodies specific for the surface Ag of the hepatitis B virus. Monoclonal cell lines were derived from healthy individuals who received standard immunizations with the serum-derived or recombinant hepatitis B virus vaccines by fusion of PBL to a heterohybridoma cell line, SPAZ-4. We utilized the polymerase chain reaction to amplify the H and L chain V regions for cloning and sequencing. The four antibodies express the following V region combinations: VHIII/V lambda V, VHIII/V kappa II, VHIV/V kappa I, VHV/V lambda III. When compared to germline genes with the closest sequence homology, all of the V regions appear to have undergone somatic mutation, ranging from 3.4 to 11.3% for the H chain, and 5.1 to 9.2% for the L chain. Analysis of the mutations shows them to be typical for an Ag-driven immune response.

Molecular characterization of five human anti-human immunodeficiency virus type 1 antibody heavy chains reveals extensive somatic mutation typical of an antigen-driven immune response.

We report the heavy chain variable region sequences from the cDNAs of five previously described monoclonal cell lines producing human antibodies specific for the human immunodeficiency virus type 1 and detail the molecular characteristics, germ-line origins, and extent of somatic mutation among these antibodies. Three of the five heavy chain variable regions derive from the VHIV gene family, but each has arisen from a different heavy chain variable region (VH) gene segment within the VHIV family. In addition, one is derived from a VHI gene segment, and one is derived from a VHV gene segment. Since four of the five antibodies arise from known germ-line VH elements, a precise determination of the extent of somatic variation is possible. The amount of variation from the closest germ-line sequence ranges from 4.5% to 14.8% among these antibodies, most of which is concentrated in the complementarity-determining regions. In general, the diversity (D) segments are long, characteristic of D-D fusions and/or extensive terminal deoxynucleotidyltransferase activity; however, definitive homologies cannot be found with the known germ-line D segments. Joining (JH) gene segment utilization appears random. The use of five different germ-line VH gene segments and extensive somatic mutation provides evidence that a polyclonal, antigen-driven immune response occurs during the natural infection with human immunodeficiency virus.

Analysis of D2d: a D-region class I gene.

The mouse major histocompatibility complex is composed of several genes arranged into the K, D, Qa, and Tla regions. The D region of the BALB/c mouse includes genes D2d, D3d, and D4d, in addition to H-2Dd and H-2Ld. We have determined the DNA sequence of the D2d gene and compared it with the known sequences of several class I genes. The exon/intron structure of the D2d gene is similar to other class I genes. It also contains similar 5' regulatory elements. A frameshift occurs in exon seven, resulting in a gene product with a truncated cytoplasmic tail. To examine the surface expression of the D2d molecule, we generated an exon-shuffled construct containing the promoter and exons 1-3, encoding the signal peptide, alpha 1, and alpha 2 external domains of the D2d gene linked to exons 4-8, encoding the alpha 3, transmembrane and cytoplasmic domains, of the H-2Dd gene. The construct was transfected into mouse L cells, and a protein was detected at the cell surface by a monoclonal antibody (mAb) specific for the alpha 3 domain of H-2Dd, as well as by other class I-specific mAbs. Although D2d is expressed at low levels, it may be a functional class I gene that most probably evolved from a Qa region gene.