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PubMed1996

Vaccination with peptides from MHC class II beta chain hypervariable region causes allele-specific suppression of EAE.

In our earlier studies we showed that successful immunotherapy of EAE in SJL/J mice can be achieved either by the use of antibodies to MHC class II antigens or by vaccination with synthetic peptide analogs of the beta chain of MHC class II molecules. We proposed that inhibition of EAE following vaccination with synthetic peptides derived from the beta chain of mouse I-A, was in part due to the generation of auto-anti-MHC class II antibodies that interfered with T cell sensitization. In our present study we show that suppression of EAE following vaccination results in poor sensitization of MBP reactive T cells, and that the lack of immune response is allele-specific. In F1(SJL(I-AS) x Balb/cI-Ad) mice, in which susceptibility to EAE is linked closely to the I-AS allele, vaccination with peptides from beta chain of I-AS results in inhibition of proliferative response to MBP and prevents the development of EAE. Vaccination with peptide from the beta chain of I-Ad did not affect either the development of immune response to MBP or the induction of EAE, indicating allele-specific suppression. Since global immunosuppression is not induced by vaccination with I-A peptides, we propose that this strategy can be extended to human autoimmune diseases wherein a clear association between certain MHC class II alleles and autoimmune disease is evident.

Alleles↗
PubMed1994

Acidic residues in the DR beta chain third hypervariable region are required for stimulation of a DR(alpha, beta 1*0402)-restricted T-cell clone.

We investigated the minimal requirements for stimulation of an antigen-specific HLA-DR(alpha, beta 1*0402)-restricted T-cell clone (Een217) by using transfectants expressing mutant DR beta chains as APCs. Antigen-specific stimulation of Een217 was induced with transfectants expressing DR(alpha, beta 1*0402) and DR(alpha, beta 1*0403) but not other DR4 subtypes that also bind the peptide recognized by this clone. Analysis of the effects of single amino acid substitutions in the beta chains of each of the DR4 subtypes revealed a requirement for acidic residues in the third HVR, particularly amino acid 71, in stimulation of clone Een217. Functional class II mutants were generated from nonstimulatory DR4 subtype beta chains by acidic residue substitutions within the third HVR. These data define the requirement for negatively charged residues in this region for peptide-induced stimulation of this T-cell clone. The required acidic residues can be located at either position 70, 71, or 74 in the DR beta chain. The negative charge in this segment of the DR beta chain alpha-helix may be required for direct interactions with the T-cell receptor of Een217 or may affect peptide conformation.

Amino Acid Sequence↗
PubMed2006

Identification of T-helper and linear B epitope in the hypervariable region of nucleocapsid protein of PPRV and its use in the development of specific antibodies to detect viral antigen.

Peste des petits ruminants is a highly contagious viral disease of small ruminants making its diagnosis difficult from the similar symptoms of Rinderpest. Computer based prediction algorithms was applied to identify antigenic determinants on the nucleocapsid (N) protein of PPRV. Specificity and antigenicity of each peptide was evaluated by solid phase ELISA. Six specific peptide sequences were evaluated in multiple antigenic peptide (MAP) form and immune response was evaluated by supplementing universal T-helper epitope human IL-1beta peptide (VQGEESNDK, amino acids 163-171). Out of the six peptides 19mer sequence corresponding to 454-472 region of N protein of PPRV was found to be highly immunogenic and specific to PPRV. Evaluation of overlapping peptides differing in length for this 452-472 region, showed minimum length of 14 amino acid residues were required for the stable affinity binding of antigen-antibody. The results of immunization and indirect ELISA indicated the presence of T-helper epitope at the N-terminal end and linear B epitope at the C-terminal region of 454-472 19mer of nucleocapsid peptide of PPRV-nucleocapsid protein. The antipeptide antibodies developed against this region showed specificity to PPRV antigen differentiating it from RPV when used in indirect ELISA and western blot analysis.

Amino Acid Sequence↗
PubMed2002

Critique of interpretation of high levels of heteroplasmy in the human mitochondrial DNA hypervariable region I from hair.

The phenomenon known as heteroplasmy can be operationally observed in some human mitochondrial DNA (mtDNA) samples. Typically, heteroplasmy manifests itself in an individual presenting two mtDNA species that differ at a single base. Heteroplasmy at two, and even possibly three sites, also may occur, but at very low rates. A recent report (Grzybowski, 2000, see ref. [13]) suggests that much higher levels of mtDNA (point substitution) heteroplasmy can occur in hair. This observation is contrary to the experience of the forensic mtDNA community. There are several explanations for the unusual findings of high levels of heteroplasmy. First, the template quantities of DNA are approximately three orders of magnitude higher than required for mtDNA sequencing, and an excessive number of amplification cycles were used. Thus, the protocol used did not follow routine practices by the forensic community. Second, there are misidentifications and tabular errors that call into question the reliability of the findings. Third, by comparing the natural human mtDNA variation with a reference sample population with that observed in the heteroplasmy in hair study, the data are inconsistent with population genetic expectations. The observation of high levels of heteroplasmy may be due to contamination of the samples and/or possibly the amplification of nuclear pseudogenes. The results observed in the heteroplasmy in hair study do not apply to other methods of mtDNA analysis and cannot be used to question the reliability of the current forensic mtDNA practices.

DNA, Mitochondrial↗
PubMed1996

Affinity improvement of single antibody VH domains: residues in all three hypervariable regions affect antigen binding.

BACKGROUND: Through antibody engineering, immunoglobulins can be tailored for their particular application. In this respect, small recognition units are desired for the targeting of antigens in obstructed locations like solid tumors. OBJECTIVES: To design efficient, minimum size recognition units, heavy chain variable regions (VH) had previously been modified for the use as antigen specific, single domain antibody fragments. To develop a rational approach to improve affinity, antigen binding is investigated here by analysing the effect of randomisations of CDR1 and 2 residues in VH domains specific for hapten and protein ligands. STUDY DESIGN: Randomised repertoires were displayed on phage and affinity selected to improve and analyse antigen binding. Affinities of newly selected VH domains were determined in their soluble format to assess the role of modified residues in binding. RESULTS: In four of five randomisation experiments, a new VH with an improved antigen affinity compared to the primary VH was selected. Dissociation constants decreased from 160 nM to 25 nM or 47 nM (CDR1 or CDR2 randomisation of an anti-Ox VH), from 300 nM to 31 nM (CDR2 randomisation of an anti-NIP VH) and from 3.1 microM to 1.6 microM (CDR2 randomisation of an anti-lysozyme VH). CONCLUSIONS: Thus the affinity of VH domains can be improved after site specific, secondary randomisations in CDR1 and CDR2, phage display and antigen selection. As differences in the CDR3 sequences had formed the only difference between the primary VH domains used in this study, the effect of CDR1 and CDR2 mutations of affinity is consistent with a participation of all three CDRs in antigen binding by single VH domains.

Amino Acid Sequence↗
PubMed1985

Human cellular fibronectin: comparison of the carboxyl-terminal portion with rat identifies primary structural domains separated by hypervariable regions.

We report the isolation and characterization of four overlapping cDNA clones coding for human cellular fibronectin which continuously cover more than 3 kilobases in length. The nucleotide sequence of these cDNAs has been determined, thus elucidating the amino acid sequence of the C-terminal 794 residues of human fibronectin, which cover the edge of cellular-, heparin-, and fibrin-binding domains of this protein. Comparisons of the nucleotide sequences and the deduced amino acid sequences with those of rat [Schwarzbauer, J. E., Tamkun, J. W., Lemischka, I. R., & Hynes, R. O. (1983) Cell (Cambridge, Mass.) 35, 421] indicate a high degree of conservation at both nucleotide and amino acid levels. Comparison with previously published data on amino acid sequences of bovine fibronectin made it possible to identify structurally important features of the protein during the evolution of human, calf, and rat. The deduced human amino acid sequences contain five type III and three type I repeats of internal homologies. The interspecies conservation in amino acids is more pronounced in regions containing the internal repeats and within each functional domain. The implications of these interspecies conservation and divergence are discussed.

Amino Acid Sequence↗
PubMed1987

cDNA clones encoding rabbit immunoglobulin lambda chains. Evidence for length variation of the third hypervariable region and for a novel constant region.

Five cDNA clones designated pDH2, pDH8, pDH9, pDH31 and pDH101 encoding rabbit immunoglobulin lambda light chain sequences have been characterized. Comparison of the V lambda sequences suggests that, in addition to an increased divergence in all of the complementarity-determining regions (CDRs), variable-region diversity is amplified by the length heterogeneity of the CDR3, at the V lambda-J lambda junction. An insertion of four codons at positions 48a-d has been noted in three cDNA sequences. This insert, not found in lambda nor kappa light chains of other species, has a variable sequence, suggesting its possible implication in expanding variability of the CDR2. One of the cDNA clones was shown to encode a novel C lambda region which differs by four amino acid substitutions from the C lambda region common to all the other clones. Thus, the rabbit can use two different C lambda genes, which might correlate with the expression of the two known allotypes of lambda chains, C7 and C21. Southern blotting experiments indicate a small number of germ-line V lambda genes and the cDNA nucleotide sequence data reported here suggest that several of these genes can be expressed. The possibility of at least two V-J-C gene clusters is discussed.

Alleles↗
PubMed1998

Co-existence of somatic hypermutation and gene conversion in hypervariable regions of single Igkappa clones.

In the rabbit, recent investigations have provided evidence that gene conversion leads to the generation of diversity of heavy chain rearranged VH-DH-JH genes. No data have been published on a similar mechanism for rabbit light chains. In our laboratory, we initially infected rabbits with Trypanosoma brucei, which stimulates B-cell hyperplasia and hypergammaglobulinaemia. The heterozygous rabbits exhibited the Ckappa1 b4 and b9 kappa light chain allotypes. After reverse transcription of mRNA, and cloning and sequencing of cDNA, the Vkappa-Jkappa-Ckappa genes provided evidence for both somatic hypermutation and gene conversion. We saw that in each of the b4 and b9 kappa light chain cDNA, CDR1 and CDR3 carried both point mutation and provisional gene conversion traits. In the CDR2 region, point mutation and gene conversion inserts were observed in the b4 genes, with only gene conversion in two b9 genes. In the CDR regions, although some genes exhibited only somatic hypermutation or gene conversion, others showed linkage of both somatic hypermutation and gene conversion in the same sequence. This also marks the first time that somatic hypermutation and gene conversion in the same cloned CDR region has been observed in Vkappa1 genes; however, it has been seen earlier in rabbit heavy chain VH sequences. Furthermore, the addition of several codons to the CDR3 segment by gene conversion may have provided a mechanism for length variation. In addition, we demonstrated that Jkappa and framework region segments contained examples of somatic hypermutation. Confirmation of gene conversion necessitates that donor sequences be identified as providing the templated inserts. Thus after cloning two pseudogenes we found putative CDR3 donor segments for two CDR3 rearranged genes. The results offer additional mechanisms for the generation of diversity among rearranged rabbit kappa light chain genes. Whether there is a relationship or influence of gene conversion upon somatic hypermutation or vice versa is not discernable at present.

Amino Acid Sequence↗
PubMed1989

Allelic sequence variations in the hypervariable region of a T-cell receptor beta chain: correlation with restriction fragment length polymorphism in human families and populations.

Direct sequence analysis of the human T-cell antigen receptor (TCR) V beta 1 variable gene identified a single base-pair allelic variation (C/G) located within the coding region. This change results in substitution of a histidine (CAC) for a glutamine (CAG) at position 48 of the TCR beta chain, a position predicted to be in the TCR antigen binding site. The V beta 1 polymorphism was found by DNA sequence analysis of V beta 1 genes from seven unrelated individuals; V beta 1 genes were amplified by the polymerase chain reaction, the amplified fragments were cloned into M13 phage vectors, and sequences were determined. To determine the inheritance patterns of the V beta 1 substitution and to test correlation with V beta 1 restriction fragment length polymorphism detected with Pvu II and Taq I, allele-specific oligonucleotides were constructed and used to characterize amplified DNA samples. Seventy unrelated individuals and six families were tested for both restriction fragment length polymorphism and for the V beta 1 substitution. The correlation was also tested using amplified, size-selected, Pvu II- and Taq I-digested DNA samples from heterozygotes. Pvu II allele 1 (61/70) and Taq I allele 1 (66/70) were found to be correlated with the substitution giving rise to a histidine at position 48. Because there are exceptions to the correlation, the use of specific probes to characterize allelic forms of TCR variable genes will provide important tools for studies of basic TCR genetics and disease associations.

Alleles↗
PubMed1985

Characterization of human rheumatoid factors with seven antiidiotypes induced by synthetic hypervariable region peptides.

Recently, we have used synthetic peptides corresponding to the complementarity-determining regions (CDR) of Ig molecules to induce antiidiotypic antisera. Peptide PSH3, representing the third CDR of the IgM rheumatoid factor (RF) Sie heavy (H) chain, induced a private antiidiotype that reacted with only one out of five IgM-RF. Peptide PSL2, corresponding to the second CDR of Sie light (L) chain, induced an antibody against a crossreactive idiotype (CRI), expressed by 10 out of 12 human IgM-RF analyzed. Herein, we report that five additional antiidiotypic antibodies were generated by immunization with synthetic peptides identical to the third L chain CDR of IgM-RF Sie (PSL3), the second and third H chain CDR of IgM-RF Wol, and the second and third CDR of IgM-RF Pom. As analyzed by immunoblot assay, both anti-PSL3 and anti-PSL2 reacted with the majority of 16 IgM-RF. In contrast, all five antiidiotypes induced by the H chain peptides reacted only with the parent proteins, except anti-PSH3, which reacted weakly with one additional RF. These results suggest that one (or very few) VL gene(s), but a larger number of VH genes, are used to encode IgM-RF autoantibodies.

Antibodies, Anti-Idiotypic↗
PubMed1985

Synthetic peptides corresponding to third hypervariable region of human monoclonal IgM rheumatoid factor heavy chains define an immunodominant idiotype.

Synthetic peptides corresponding to eight individual heavy chain complementarity-determining regions (CDR) of three human monoclonal IgM anti-IgG (rheumatoid factor [RF]) paraproteins elicited rabbit antibodies with markedly different properties. All antisera recognized the immunizing peptide, and several reacted with the isolated IgM heavy chain on immunoblots. However, only the antisera against peptides representing the third CDR bound consistently and specifically to the intact IgM-RF molecule. These data indicate that the third CDR of human mu chains comprises an immunodominant idiotype, and suggest that the D gene segment may be especially important in creating idiotypic diversity. Synthetic peptides corresponding to the third heavy chain CDR of human paraproteins may be clinically useful for the specific induction of antiidiotypic antibodies.

Amino Acid Sequence↗