Identification of novel VH1/J558 immunoglobulin germline genes of C57BL/6 (Igh b) allotype

Although a rich database of Igh a allotype mouse immunoglobulin germline genes exists, current information on Igh b allotype immunoglobulin germline genes is limited. Among the immunoglobulin VH genes, single-cell amplified from six Igh b (C57BL/6 background) spleens in this study, 602 clonally independent immunoglobulin VH sequences belonging to the VH1/J558 family were identified. Whereas 335 of these sequences could be traced to have originated from 29 different VH1/J558 germline genes deposited in the NCBI Igblast database, the remaining 267 sequences appeared to have originated from 21 novel germline genes. Of the 50 VH1/J558 germline genes utilized in the peripheral repertoire of these Igh b allotype mice, the most frequently used genes included 45.21.2, V165.1, J558.6, J558.18A, and V23. Whereas the majority of the novel genes uncovered represented allelic counterparts of previously described Balb/c (Igh a allotype) genes, some appeared to represent truly novel germline genes. Collectively, the VH1/J558 germline genes exhibited high amino acid residue usage variability at the CDR1 positions, H31, H33, and H35, and the CDR2 positions, H50, H52, H53, H54, H56, and H58. The 50 VH1/J558 germline genes expressed in the peripheral Igh b repertoire also varied widely in the net charge of their CDR regions, raising the possibility that they may be differentially utilized to encode anti-nuclear autoantibodies.     

Autoimmune VH gene family: PCR-generated murine germline VH10 genes.

A relatively large number of variable region genes (V) contribute, via gene rearrangements with smaller numbers of additional gene elements (D and J), to generate diversity in the immune response. While some VH gene families are thought to contain 100- 1000 members, the VH10 family has only two known functioning members with 99% sequence homology. Both members (monoclonal antibodies) are capable of binding DNA, and since they were derived from inbred mice afflicted with the lupus syndrome they are considered autoimmune antibodies. Relative uniqueness of the VH10 primary nucleotide sequence presents a model system with which to examine unrearranged VH genes and attempt to identify germline genes eventually expressed as autoantibodies. PCR amplified germline sequences of the VH10 family are highly conserved, with few base substitutions evenly distributed between both framework and CDR regions. It was determined that the PCR amplified germline sequences are highly similar to the DNA sequences of the two monoclonal VH10 antibodies, and a non-functional psuedo-germline gene was found that is identical to a non-functional cDNA derived from a hybridoma cell line. These findings indicate that the use of unique CDR DNA sequences for the identification and amplification of specific germline V genes via PCR can yield vital information that may answer fundamental questions about the origins of autoimmune anti-DNA antibodies in afflicted individuals. The nature of the germline gene populations and the possible microheterogeniety of these genes may prove to be important in understanding the role of autoimmune antibodies in normal and diseased individuals.     

Fundamental characteristics of the expressed immunoglobulin VH and VL repertoire in different canine breeds in comparison with those of humans and mice

Complementarity determining regions (CDR) are responsible for binding antigen and provide substantial diversity to the antibody repertoire, with VH CDR3 of the immunoglobulin variable heavy (VH) domain playing a dominant role. In this study, we examined 1200 unique canine VH and 500 unique variable light (VL) sequences of large and small canine breeds derived from peripheral B cells. Unlike the human and murine repertoire, the canine repertoire is heavily dominated by the Canis lupus familiaris IGHV1 subgroup, evolutionarily closest to the human IGHV3 subgroup. Our studies clearly show that the productive canine repertoire of all analyzed breeds shows similarities to both human and mouse; however, there are distinct differences in terms of VH CDR3 length and amino acid paratope composition. In comparison with the human and murine antibody repertoire, canine VH CDR3 regions are shorter in length than the human counterparts, but longer than the murine VH CDR3. Similar to corresponding human and mouse VH CDR3, the amino acids at the base of the VH CDR3 loop are strictly conserved. For identical CDR positions, there were significant changes in chemical paratope composition. Similar to human and mouse repertoires, the neutral amino acids tyrosine, glycine and serine dominate the canine VH CDR3 interval (comprising 35%) although the interval is nonetheless relatively depleted of tyrosine when compared to human and mouse. Furthermore, canine VH CDR3 displays an overrepresentation of the neutral amino acid threonine and the negatively charged aspartic acid while proline content is similar to that in the human repertoire. In general, the canine repertoire shows a bias towards small, negatively charged amino acids. Overall, this analysis suggests that functional canine therapeutic antibodies can be obtained from human and mouse sequences by methods of speciation and affinity maturation.     

The repertoire of human antibody to the Haemophilus influenzae type b capsular polysaccharide.

Human antibody to the Haemophilus influenzae capsular polysaccharide (Hib CP) is restricted in diversity in the individual and the population with a limited number of variable region genes encoding antibody. Antibody to the Hib CP shows restricted isoelectric focusing gel patterns and light chain usage with frequent restriction to use of only kappa light chains. Shared cross-reactive idiotypes are expressed on antibody. The heavy chain of antibody to the Hib CP is predominantly encoded by two members of the VH3 family--LSG 6.1/M85-like and VH26/30P1-like. In VH the CDR1, based on complete identity in LSG 6.1/M85-like antibodies, CDR2, based on the suggestion of mutation in this region, and CDR3, based on conserved CDR3 usage in unrelated individuals, may be important for antigen binding. Six or more different VL gene families encode antibody. The predominant antibody of the majority of individuals uses the A2-V kappa II gene in germline or near germline configuration, which encodes an idiotype designated HibId-1. Antibody can also be encoded by V kappa I, non-A2 V kappa II, V kappa III, V kappa IV, V lambda II, and V lambda VII genes. Although different VL genes can be used, unrelated individuals appear to use the same V kappa III (A27), V lambda II (V lambda 2.1 and V lambda VII (4A) genes. The VL diversity accounts for differences in fine binding specificity, with A2-V kappa II genes not encoding E. coli K100 CP cross-reactive antibodies and V lambda VII genes and some of the non-A2 V kappa genes encoding cross-reactive antibodies. The arginine in CDR3 of both antibody kappa and lambda light chains and the asparagine in CDR2 of VL sequences and in CDR1 of LSG6.1-M85 VH sequences of antibody appear to be important residues for antigen binding. A relatively limited degree of somatic mutation has occurred in the non-A2 VL genes, V lambda VII, and the VH genes. Further studies comparing the polymorphism of germline V genes to antibody-encoding V genes are needed to clarify this issue. Research comparing this repertoire to repertoires directed to other bacterial CP and to self antigens and defining structure-antigen binding relationships is in progress.     

Somatic diversity of the immunoglobulin repertoire is controlled in an isotype-specific manner

We have studied two aspects of the IgE immune response. First, we have compared the kinetics of the IgE response to the T cell-dependent antigen ph-Ox coupled to ovalbumin with that of the IgG1 response and we have assessed the quality of the IgE response. Second, we have studied the generation of somatic diversity, understood as the combined effect of somatic mutation and the selection of D(iversity) and J(oining) elements, in germinal center B cells at the molecular level, using the germ-line sequence of the prototype anti-ph-Ox heavy chain variable element V(H)Ox1 as reference. We evaluated sequences derived from mu-, gamma 1- and epsilon-variable elements and showed that somatic diversification was different for all isotypes studied. We further compared the IgE responses of wild-type mice with those of mice expressing a truncated cytoplasmic IgE tail (IgE(KVK Delta tail)). IgE(KVK Delta tail) mice showed a more diverse sequence pattern. We corroborated previous results suggesting that short CDR3 regions are indicative for high-affinity antibodies by measuring relative affinities of phage-expressed Fab fragments with prototype long and short CDR3 regions. Therefore, the composition of the antigen-receptor is responsible for the selection process and the expansion of antigen-specific cells, leading to an isotype-specific antibody repertoire.     

Characterization of human IgG repertoires in an acute HIV-1 infection

All known broadly neutralizing antibodies (bnAbs) are highly somatically mutated and therefore significantly differ from their germline predecessors. Thus although the mature bnAbs bind to conserved epitopes of the HIV-1 envelope glycoprotein (Env) with high affinity their germline predecessors do not or weakly bind Envs failing to initiate an effective immune response. The identification of less somatically mutated bnAbs and/or antibody maturation intermediates that are clonally related to bnAbs may be useful to circumvent the major problem of initiating immune responses leading to elicitation of bnAbs. Here, we describe the identification of IgG antibodies from an acutely HIV-1-infected patient using a combination of phage display and high-throughput sequencing. We found two antibodies with only a single point mutation in the V region of their heavy chain variable domains compared to their putative germline predecessors which bound with high affinity to several Envs. They targeted the Env gp41 and did not neutralize HIV-1. Using high-throughput sequencing, we identified several highly abundant CDR3s, germline-like as well as somatically mutated V genes in the VH/VL repertoires of the patient which may provide antibody intermediates corresponding to known bnAbs as templates for design of novel HIV-1 vaccine immunogens.     

The J558 V(H) CDR3 region contributes little to antibody avidity; however, it is the recognition element for cognate T cell control of the alpha(1-->3) dextran-specific antibody response [In Process Citation]

Analysis of the humoral immune response of BALB/c mice to alpha(1-->3) dextran (Dex) reveals novel aspects of T cell-mediated control of 'type 2 thymus-independent' responses against polysaccharide antigens. The IgM and IgG antibody response, dominated by the J558 idiotype (Id), is controlled by Id-specific T cells. These regulatory T cells, for which the T cell clone 178-4 Ts with characterized TCR alpha and beta chain sequences is the prototype, expand in all BALB/c mice upon immunization with Dex. They suppress in a cognate interaction the expansion of J558 Id-bearing B cells, committed for production of IgG antibodies. Furthermore they provide a gate which precludes variability in the VH CDR3 region of IgG antibodies appearing occasionally in the periphery. The VH CDR3 region is the recognition element of 178-4 Ts analogous T cells but contributes little to affinity for the antigen. For recognition by 178-4 Ts cells not even minimal sequence deviations of the J558 Id peptide are allowed. The tight germline programmed complementarity between J558 Id-bearing Dex-specific B and J558 Id- specific 178-4 Ts analogous T cells leaves little room on both sides for ontogenetic variability.      

Characterization of a murine immunoglobulin VH gene segment in subgroup III: a new member of the 7183 gene family.

A new gene for the variable region of the immunoglobulin heavy chain (VH gene) has been isolated from BALB/c adult liver DNA using a cDNA plasmid probe containing a mouse VH sequence. The complete nucleotide sequence of this germline gene (VH10-19), shows that it belongs to the 7183 gene family. The VH gene appears to contain an intervening 104-base-long sequence and displays the same recombination signal sequences that those observed in the germline 81X. The presence of an internal heptamer at the 3' end of the VH10-19 coding region let an alternative recombination event that could increase the representation of this gene in the immature repertoire.     

Genes coding evolutionary novel anti-carbohydrate antibodies: studies on anti-Gal production in alpha 1,3galactosyltransferase knock out mice

This study analyzes the gene repertoire coding for antibodies to an evolutionary novel immunogenic carbohydrate antigen in mice. The alpha-gal epitope (Gal alpha 1-3Gal beta 1-4GlcNAc-R) is an autoantigen, abundantly expressed in wild type mice, but absent in alpha 1,3galactosyltransferase knock-out (KO) mice, where it can induce the production of the anti-Gal antibody. Hybridoma clones secreting anti-Gal were isolated from different mice and their immunoglobulin genes were analyzed. All anti-Gal clones were found to be encoded by the heavy chain gene VH22.1 and light chain gene VK5.1. Moreover, one 'forbidden' anti-Gal clone, produced in a wild type mouse, was also encoded by VH 22.1 and VK 5.1. The genes coding for the different anti-Gal clones were found to contain somatic mutations and different CDR3 domains. These data imply that a highly restricted gene usage combined with junctional diversity and somatic mutations can generate new antibodies that have not been produced in the course of the evolution of a species.     

Use of family specific leader region primers for PCR amplification of the human heavy chain variable region gene repertoire.

We have designed a set of six, non-degenerate oligonucleotide primers, corresponding to the 5' leader regions of each of the six human VH gene families. A general strategy for family specific polymerase chain reaction amplification is described using these primers and a conserved 3' primer corresponding to frame work 3, JH, or constant region. This strategy was used to isolate and sequence novel human germline VH genes belonging to the VH2 and VH4 families. Under certain conditions, chimeric VH sequences were created by a "jumping polymerase chain reaction", combining DNA segments from different germline genes, but this could be avoided by limiting the number of amplification cycles. PCR amplification with these family specific primers will facilitate studies of the repertoire of germline VH genes as well as studies on VH gene usage in normal and aberrant (B cell malignancies, autoimmune diseases, etc.) B cell populations.     

Structural basis of stimulatory anti-idiotypic antibodies

In order to design and produce effective vaccines based upon the idiotype network hypothesis of Jerne, a thorough understanding of the biological and structural aspects underlying the stimulating activities of anti-idiotypic antibodies is needed. Here we determined the nucleotide sequence of the variable heavy and light chain regions of two monoclonal anti-idiotypic antibodies which induce different anti-phosphorylcholine responses. The nucleotide sequences of the variable domains of two monoclonal anti-TEPC 15 (T15) antibodies (F6-3 and 4C11) were determined by the primer extension and Maxam-Gilbert techniques. The nucleotide sequence data show that 4C11 and F6-3 have homologous VH segments and JH segments, but different D regions. The VH segments of both clones belongs to the J558 VH family. Most of the differences among the VH segments are located in CDR2. The VK segments of 4C11 and F6-3 are homologous to the VK gene group 4 and group 8, respectively. Comparison of the sequences of 4C11 and F6-3 with other published anti-idiotype antibodies shows that there is no preferential utilization of immunoglobulin genes. An analysis of the distribution of charged residues and hydropathic comparison studies were used to interpret the sequence of 4C11 in terms of the biological mimicry of antigenic stimulation.     

Sequences of variable regions of a monoclonal antibody specific to the thyroid hormone, triiodo-L-thyronine.

We have determined the nucleotide sequences of the variable regions of H and L chains of a monoclonal antibody 98QQ that interacts with the thyroid hormone triiodo-L- thyronine with high affinity. Analysis of the nucleotide sequence of the light chain V region of 98QQ revealed that the VL sequence is 99% identical to Balb/c germline Vk 21-E sequence. That is an interesting finding with this high affinity anti-T3 antibody, since occurence of predominantly germline variable region sequences is observed in some autoantibodies to self antigens but not usually in high affinity IgG antibodies. The sequence analysis also revealed that the heavy chain variable region sequence of 98QQ is similar to a V region of an anti-DNA antibody (MRL DNA 22). Thus the sequence analysis of our anti-T3 mAb 98QQ has revealed some features of autoantibodies to self antigens.     

Improvement of anti-Burkholderia mouse monoclonal antibody from various phage-displayed single-chain antibody libraries

To improve anti-Burkholderia monoclonal antibody (MAb) binding affinity, six single chain variable fragments (scFvs) constructed previously were used as scaffolds to construct large highly-diversified phage-displayed mouse scFv random and domain libraries. First, we employed random mutagenesis to introduce random point mutations into entire variable regions, generating six random libraries. Additionally, the oligonucleotide-directed mutagenesis was targeted on complementarity-determining region 3 (CDR3) from each variable region of heavy (VH) and light chains (VL) derived from six scFvs, and generated eighteen domain libraries including six VH CDR3, six VL CDR3, and six combined VH/VL CDR3 mutated domains, respectively. We collected high scFvs binders through panning experiment over the large (size ~1 × 10?) random and domain libraries. The quality of the libraries was validated by successful selection of high-affinity clones. Random mutagenesis generated many mutant scFv clones having more than one amino acid changes around framework regions, but not many in CDRs. Surprisingly, the resulting eight higher scFv binders were selected from CDR3 mutations, but not from random mutations. Six of them resulted from CDR3 mutations of light chain, except for two scFvs from heavy chain, showing both Burkholderia pseudomallei and Burkholderia mallei had preferentially influenced the VL CDR3. Furthermore, all eight higher scFvs converted to full format human IgG1 antibodies were expressed transiently in 293T cell line. Five chimeric MAbs showed improved higher binding activity, as much as 0.2-0.3 at O.D. 405 nm, than positive control MAbs. These libraries could be valuable sources for selection of anti-Burkholderia antibodies and discovery of the relevant epitope(s) for developing effective vaccines or therapeutics.     

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.     

Anti-idiotypic antibodies of a predefined specificity generated against CDR3VH synthetic peptides define a private anti-CD4 idiotype

A synthetic peptide corresponding to the third complementarity determining region (CDR) of the heavy chain (CDR3VH) of anti-Leu3a, a monoclonal anti-CD4 antibody which inhibits HIV gp120 binding to CD4, was used to elicit specific anti-peptide antibodies in rabbits. The anti-peptide antisera showed anti-idiotypic antibody (anti-Id) activity and recognized both the immunizing peptide and the intact cognate protein by ELISA. In addition, the antisera reacted with isolated heavy chains of anti-Leu3a by Western blot analysis. The lack of reactivity with a panel of monoclonal anti-CD4 antibodies suggested that the anti-peptide antisera recognize a private idiotype (Id) associated with the anti-Leu3a CDR3VH region. Further studies demonstrated the inability of the rabbit antisera to inhibit the binding of anti-Leu3a to the CD4 molecule. In addition, soluble recombinant CD4 was unable to inhibit the binding of the rabbit anti-peptide antisera to anti-Leu3a indicating that the CDR3VH region may not be involved in CD4 recognition. Anti-Id containing sera from mice, rabbits and nonhuman primates immunized with the intact anti-Leu3a molecule did not bind the CDR3VH synthetic peptide, suggesting that the corresponding region of anti-Leu3a may not represent an immunodominant idiotypic determinant in thes e species. These results suggest the potential use of synthetic peptides corresponding to immunoglobulin variable (V) region amino acid sequences in generating anti-Id reagents of a predefined specificity. In addition, V-region synthetic peptides may be useful in mapping the idiotopes recognized by an anti-Id response to the cognate molecule.     

Idiotypic cross-reactivity of anti-GAT and anti-alprenolol antibodies: an approach to the structural correlates of the pGAT idiotypic specificity

Most anti-GAT antibodies in the BALB/c strain express a public idiotypic specificity (pGAT), which is encoded by specific germline genes (VH10, VK5.1 and VK1A5). One or both of these germline genes, referred to as "GAT-specific genes", are also used by four anti-alprenolol antibodies. Anti-Alp and anti-GAT antibodies show no cross-reactivity for the antigens. The light chain of one anti-Alp antibody, 22C4, is encoded as the anti-GAT antibodies by a VK5.1-J2 combination and expresses part of the pGAT idiotopes, whereas the heavy chain is not "GAT"-related. Two anti-Alp using the VH10-VK5.1-J1 association do not express any of the pGAT idiotopes. Sequence comparison of the various CDR sequences points to the predominant role of the VH-CDR2 and VL-CDR3 for the constitution of the pGAT specificity. Regarding VL-CDR3, a drastic change in idiotypic determinants appears to be linked to V-J junctional diversity.     

Immunoglobulin repertoire of B lymphocytes infiltrating breast medullary carcinoma

Tumor specific peptides recognized by T lymphocytes infiltrating solid tumors, as well as the corresponding T cell receptor (TcR) repertoire usage, have been extensively investigated. By contrast, tumor infiltrating B cells and their immunoglobulin (Ig) repertoire have been studied only in a limited number of tumors. The objective of the present study was to determine, whether DNA sequence analysis of the expressed immunoglobulin variable regions of B cells that infiltrate breast cancer, could be used to reveal a potential specific tumor binding capacity of the antibodies. To answer this question, about 200 expressed Ig heavy (VH) and light chain variable gene (VL) regions were cloned, sequenced and comparatively analysed from a typical medullary beast carcinoma (MBC), where the massive B and plasma cell infiltration correlates with favourable prognosis despite of its high grade. The tumor infiltrating B cell Ig heavy and light chain sequences could be classified into clusters, families and subgroups, based on the identity level to germline, showing a pattern of oligoclonality. Some overrepresented clusters could be determined. In the course of a detailed analysis and search in Blastn database, a number of VH and VL sequences showed more than 99% homology to DNA sequences of Ig VH region, with proved tumor antigen binding capacity. Our data suggest, that potential tumor binder Ig VH and VL sequences might be selected using a detailed immunoglobulin variable region analysis. This new approach might have a benefit for further antibody engineering, as difficulties in search for tumor binders by phage library selection might be reduced and the time for selection shortened.     

Comparison of the Frequencies of Arginines in Heavy Chain CDR3 of Antibodies Expressed in the Primary B-Cell Repertoires of Autoimmune-Prone and Normal Mice

Because the pathogenesis of anti-DNA Ab in SLE is correlated to Ab specificity for native DNA (dsDNA), understanding how such specificity is generated is important. The VH structures of most autoimmune anti-DNA antibodies include at least one arginine in VH–CDR3; moreover, antibody specificity for dsDNA can be correlated to the relative position of arginines in VH–CDR3. The coding sequences for most VH–CDR3 arginines among the anti-DNA MoAbs we have studied to date appeared to have been encoded by sequences generated during V–D–J recombination and would have been expressed in the primary B-cell repertoire. The frequency at which arginine codons are generated during V–D–J recombination therefore could potentially influence the frequency at which DNA-specific B cells are generated in the primary B-cell repertoire. The present study was undertaken to determine whether a higher percentage of B cells in the primary, preautoimmune repertoire of autoimmune-prone (NZB × NZW)F₁ mice have immunoglobulin heavy chains with at least one VH–CDR3 arginine compared to B cells in the primary, preimmune repertoire of non-autoimmune-prone BALB/c mice. The present results indicate that mature B cells in preautoimmune (NZB × NZW)F₁ mice, whether specific for DNA or not, are no more likely to have heavy chains with VH–CDR3 arginines than are B cells in BALB/c mice. The high frequency of recurrence of VH–CDR3 arginines among autoimmune anti-DNA in (NZB × NZW)F₁ mice would appear to derive from the selective oligoclonal expansion of selected B cells that express such structures.     

Monoclonal BALB/c anti-progesterone antibodies use family IX variable region heavy chain genes

Variable region nucleotide sequences and respective translated amino acid sequences for three heavy chains (DB3, 11/32 and 10/8) and two light chains (DB3 and 11/32) of monoclonal mouse IgG1 anti-progesterone antibodies have been determined by primer extension mRNA sequencing. The three VH regions exhibit the same rarely observed VH IX gene family and have greater than 88% homology between them. Two associated light chain sequences are 95% homologous and belong to the V kappa I group. The N-terminal twenty two amino acids of the kappa light chain of the third antibody 10/8 have been determined by automated protein sequencing and are identical to those of 11/32. Thus, these three monoclonal anti-progesterones derived from separate fusions all use VHIX-V kappa 1 gene combinations.