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PubMed2002

Epitope mapping of human monoclonal antibodies recognizing conformational epitopes within HTLV type 1 gp46, employing HTLV type 1/2 envelope chimeras.

The majority of the antibody response to HTLV-1 surface glycoprotein, gp46, is directed at conformational epitopes. However, the regions of HTLV-1 gp46 that contain conformational epitopes are poorly defined. We previously reported on human monoclonal antibodies (hMAbs) to conformational epitopes within the HTLV-1 surface glycoprotein (gp46) that inhibit HTLV-1-mediated syncytium formation (Hadlock KG, Rowe J, Perkins S, et al.: J Virol 1997;71:5828-5840). To localize the conformational epitopes recognized by these antibodies, chimeric envelope proteins were constructed in which selected regions of the HTLV-1 envelope were replaced with the corresponding sequences from other members of the HTLV family of retroviruses. The chimeras were tested for reactivity with three hMAbs to conformational epitopes in HTLV-1 gp46, PRH-7A, PRH-3, and PRH-4, and one hMAb to a linear epitope, 0.5alpha. hMAb PRH-3 was specifically nonreactive with a chimera that replaced amino acids 32-36 of HTLV-1 gp46 and exhibited sharply reduced reactivity with a chimera that replaced amino acids 224-251 of HTLV-1 with the corresponding HTLV-2 sequence. hMAb PRH-4 was specifically nonreactive with a construct replacing amino acids 1-162 of HTLV-1 gp46 with the corresponding HTLV-2 sequence. Thus, HTLV-1 gp46 contains multiple conformational epitopes located in the amino-terminal portion of the protein.

Amino Acid Sequence↗
PubMed1990

Distribution and specific identification of papillomavirus major capsid protein epitopes by immunocytochemistry and epitope scanning of synthetic peptides.

Monoclonal (MAbs) and polyclonal antibodies were produced against the major capsid protein of detergent-disrupted, purified bovine papillomavirus type 1 (BPV-1). The precise locations of the corresponding epitopes were identified by the reactivity of MAbs and selected polyclonal antibodies with synthetic, overlapping, hexameric peptides corresponding with 95% of the BPV-1 major capsid protein. The topography of these epitopes was determined by reactivity of antibodies with intact (conformational and nonconformational surface epitopes) and disrupted (external or internal nonconformational epitopes) BPV-1 virions. The distribution of epitopes in various papillomaviruses of 13 different species was determined by reactivity of the MAbs and polyclonal sera with productively infected, formalin-fixed papillomas, fibropapillomas, and fibromas. Epitope scanning, using MAbs and polyclonal antisera, resulted in the precise location of BPV-1 hexameric epitopes that could be correlated with their topography on the capsid and distribution in papillomatous lesions of various species.

Animals↗
PubMed1993

Mapping of linear B cell epitopes on capsid proteins of bovine papillomavirus: identification of three external type-restricted epitopes.

Immunodominant, conserved and type-restricted external epitopes of bovine papillomavirus (BPV) major (L1) capsid protein have been identified using BPV particles and synthetic peptides. Antisera to disrupted BPV-1 recognized BPV-1 and BPV-2 particles in immune electron microscopy (IEM) studies and inhibited BPV-2-induced focus formation of NIH/3T3 cells. Thus BPV-1/BPV-2 cross-reactive epitopes occur on the surface of virions. The L1 protein appeared to be immunodominant as the antisera reacted with three dominant BPV-1/BPV-2 conserved B cell epitopes (amino acids 111 to 125, 131 to 145 and 191 to 205) in Pepscan assays of BPV-1 L1, whereas no common epitopes and less frequent antibody binding to peptides were detected in Pepscans of the L2 protein of BPV-1. Four discrete variable regions were identified in the sequences of L1 proteins of BPV-1 and BPV-2. Antisera against synthetic peptides corresponding to three of the four variable regions (amino acids 42 to 56, 435 to 449 and 485 to 499) of BPV-2 L1 caused clumping of BPV-2, but not of BPV-1, particles as examined by IEM, and antisera to one peptide (amino acids 485 to 499) inhibited BPV-2-induced focus formation of NIH/3T3 cells. These data suggest that these regions are type-specific BPV-2 L1 epitopes and that they occur on the virion surface. Although conformation-dependent epitopes remain to be identified on papillomaviruses, the linear epitopes identified in this study may be worthy of further study as constituents of experimental prophylactic vaccines.

3T3 Cells↗
PubMed1989

Human antibodies react with an epitope of the human papillomavirus type 6b L1 open reading frame which is distinct from the type-common epitope.

Recombinant proteins encoded by the human papillomavirus type 6b (HPV6b) L1 open reading frame react with sera from patients with condylomata acuminata and also react with rabbit antiserum raised against sodium dodecyl sulfate-disrupted bovine papillomavirus type 1 (BPV1) virions. To map the immunoreactive epitopes, a series of procaryotic expression plasmids was made which contained a nested set of 3' to 5' deletions in the HPV6b L1 open reading frame. The deleted plasmids expressed a set of carboxy to amino terminus truncated fusion proteins. Regions containing the immunoreactive epitopes were mapped by determining which of the deleted fusion proteins retained reactivity with sera in Western immunoblot assays. The coding sequence for a human antibody-reactive linear epitope mapped between HPV6b nucleotide coordinates 7045 and 7087, and the rabbit anti-BPV1-reactive epitope coding sequence mapped between coordinates 6377 and 6454. Synthetic peptides derived from the epitope mapping were reacted with sera in enzyme-linked immunosorbent assay. Human sera reacted with synthetic peptide QSQAITCQKPTPEKEKPDPYK (HPV6b L1 amino acids 417 through 437). Rabbit anti-BPV1 and rabbit antisera raised against HPV16 L1 recombinant proteins reacted with the synthetic peptide DGDMVDTGFGAMNFADLQTNKSDVPIDI (HPV6b L1 amino acids 193 through 220). Human sera which reacted with HPV6b L1 fusion proteins cross-reacted with an HPV11 L1 fusion protein but did not react with fusion proteins encoded by HPV1a, HPV16, or HPV18. Rabbit anti-BPV1 reacted with L1 fusion proteins encoded by all of these HPV types. In contrast to the type-common (rabbit anti-BPV1-reactive) epitope, the human antibody-reactive epitope appears to be relatively HPV type specific.

Amino Acid Sequence↗
PubMed1996

Cloning of monoclonal autoantibodies to epitopes of oxidized lipoproteins from apolipoprotein E-deficient mice. Demonstration of epitopes of oxidized low density lipoprotein in human plasma.

Many reactive products may be formed when LDL undergoes lipid peroxidation, which in turn can react with lipids, apoproteins, and proteins, generating immunogenic neoepitopes. Autoantibodies recognizing model epitopes of oxidized low density lipoprotein, such as malondialdehydelysine, occur in plasma and in atherosclerotic lesions of humans and animals. Because apo E-deficient mice develop particularly high titers of such autoantibodies, we used their spleens to clone 13 monoclonal antibodies to various epitopes of oxidized LDL ("E0 antibodies"). Binding and competitive RIAs demonstrated significant differences in fine specificity even between E0 antibodies initially selected for binding to the same screening antigen. For example, some E0 antibodies selected for binding to malondialdehyde-LDL also recognized copper oxidized LDL, acrolein-LDL, or LDL modified by arachidonic or linoleic acid oxidation products. Circulating IgG and IgM autoantibodies binding to copper-oxidized LDL, 4-hydroxynonenal-LDL, acrolein-LDL, and LDL modified with arachidonic or linoleic acid oxidation products were found in apo E-deficient mice, suggesting that the respective antigens are formed in vivo. Epitopes recognized by some of the E0 monoclonal antibodies were also found on human circulating LDL. Each of the E0 monoclonal antibodies immunostained rabbit and human atherosclerotic lesions, and some of them yielded distinct staining patterns in advanced lesions. Together, this suggests that the natural monoclonal antibodies recognize different epitopes of complex structures formed during oxidation of lipoproteins, or epitopes formed independently at different lesion sites. Our data demonstrate that a profound immunological response to a large number of different epitopes of oxidized lipoproteins occurs in vivo. The availability of "natural" monoclonal autoantibodies should facilitate the identification of specific epitopes inducing this response.

Acrolein↗
PubMed2006

Ii-Key/MHC class II epitope hybrids: a strategy that enhances MHC class II epitope loading to create more potent peptide vaccines.

Life-threatening diseases, such as cancer and pandemic influenza, demand new efforts towards effective vaccine design. Peptides represent a simple, safe and adaptable basis for vaccine development; however, the potency of peptide vaccines is insufficient in most cases for significant therapeutic efficacy. Several methods, such as Ligand Epitope Antigen Presentation System and ISCOMATRIX, have been developed to enhance the potency of peptide vaccines. One way of increasing the loading of MHC class II peptides occurs through the use of Ii-Key technology. Ii-Key (LRMK), a portion of the MHC class II-associated invariant chain (Ii), facilitates the direct loading of epitopes to the MHC class II molecule groove. Linking the Ii-Key moiety via a simple polymethylene bridge to an MHC class II epitope, to generate an Ii-Key/MHC class II epitope hybrid, greatly enhances the vaccine potency of the tethered epitope. The combination of such Ii-Key/MHC class II epitope hybrids with MHC class I epitope-containing peptides might generate a potent peptide vaccine for malignancies and infectious diseases. The Ii-Key hybrid technology is compared with other methods that enhance the potency of a peptide vaccine.

Animals↗
PubMed1995

Megalin (gp330) possesses an antigenic epitope capable of inducing passive Heymann nephritis independent of the nephritogenic epitope in receptor-associated protein.

The Heymann nephritis antigenic complex (HNAC) consists of two glycoproteins, megalin (gp330), and the receptor-associated protein (RAP). HNAC is expressed on the surface of the glomerular epithelium where it plays a primary role in the pathogenesis of Heymann nephritis (HN). Several models were previously proposed describing how antibody binding epitopes in HNAC may contribute to the initiation and progression of HN. Although these models suggest that nephritogenic epitopes capable of initiating HN are present in both megalin and RAP, the structural relationship between these epitopes has not been established. Previously a nephritogenic epitope was identified and characterized in RAP that initiates immune complex formation in HN. In this report, the immunologic relationship between nephritogenic epitopes in megalin and RAP were examined to determine whether these epitopes are immunologically distinct or antigenically related. To this end, a polyclonal antibody to megalin was generated that does not recognize RAP by immunoblotting or immunoprecipitation and whether this antibody is capable of inducing passive HN was determined. It was found that antimegalin antibodies devoid of RAP cross-reactivity induced the formation of subepithelial immune deposits (passive HN) when injected into rats. Antibodies eluted from glomeruli of the injected rats recognized only megalin by immunoblotting a cortical extract and did not recognize a RAP fusion protein or any other renal protein. In addition, the eluted antibodies immunoprecipitated two proteolytic fragments of megalin (140 and 75 kd) identifying a pathogenic epitope within a smaller fragment of megalin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2005

Expression and immunoreactivity of an epitope of HCV in a foreign epitope presenting system.

AIM: To construct and highly express an epitope of hepatitis C virus (HCV) in a foreign epitope presenting vector based on an insect virus, and to study the antigenicity of the epitope. METHODS: The HCV epitope sequence (amino acid residues 315 to 328: EGHRMAWDMMMNWS) of the E1 region was constructed at different positions of a foreign epitope presenting vector based on an insect virus, flock house virus (FHV) capsid protein encoding gene as a vector, and expressed in E. coli cells. Western blotting and ELISA were used to detect the immunoreactivity of these recombinant proteins. RESULTS: The gene encoding of the concerned B-cell epitope of HCV E1 envelope protein was expressed on FHV capsid carrier protein at positions I1 (aa 106), I2 (aa 153) and I3 (aa 305), respectively, on the surface of FHV capsid protein. The recombinant proteins in this system could be highly expressed in more than 40% of total cell protein of E. coli BL21. All the expressed recombinant proteins were in inclusion body form, and showed obvious immunoreactivity by Western blotting. Further purified recombinant proteins were detected by indirect ELISA as coating antigen respectively. All recombinant proteins could still show immunoreactivity. CONCLUSION: The epitope of HCV E1 envelope protein can be highly expressed in FHV carrier system as a chimeric protein with high immunoreactivity. This system has multiple entry sites conferring many possible conformations closer to the native one for a given sequence.

Animals↗
PubMed2004

The myelin-associated oligodendrocytic basic protein region MOBP15-36 encompasses the immunodominant major encephalitogenic epitope(s) for SJL/J mice and predicted epitope(s) for multiple sclerosis-associated HLA-DRB1*1501.

Autoimmune response to the myelin-associated oligodendrocytic basic protein (MOBP), a CNS-specific myelin constituent, was recently suggested to play a role in the pathogenesis of multiple sclerosis (MS). The pathogenic autoimmune response to MOBP and the associated pathology in the CNS have not yet been fully investigated. In this study, we have characterized the clinical manifestations, pathology, T cell epitope-specificity, and TCRs associated with experimental autoimmune encephalomyelitis (EAE) induced in SJL/J mice with recombinant mouse MOBP (long isoform, 170 aa). Analysis of encephalitogenic MOBP-reactive T cells for reactivity to overlapping MOBP peptides defined MOBP15-36 as their major immunodominant epitope. Accordingly, MOBP15-36 was demonstrated to be the major encephalitogenic MOBP epitope for SJL/J mice, inducing severe/chronic clinical EAE associated with intense perivascular and parenchymal infiltrations, widespread demyelination, axonal loss, and remarkable optic neuritis. Molecular modeling of the interaction of I-A(s) with MOBP15-36, together with analysis of the MOBP15-36-specific T cell response to truncated peptides, suggests MOBP20-28 as the core sequence for I-A(s)-restricted recognition of the encephalitogenic region MOBP15-36. Although highly focused in their epitope specificity, the encephalitogenic MOBP-reactive T cells displayed a widespread usage of TCR Vbeta genes. These results would therefore favor epitope-directed, rather than TCR-targeted, approaches to therapy of MOBP-associated pathogenic autoimmunity. Localization by molecular modeling of a potential HLA-DRB1*1501-associated MOBP epitope within the encephalitogenic MOBP15-36 sequence suggests the potential relevance of T cell reactivity against MOBP15-36 to MS. The reactivity to MOBP15-36 detected in MS shown here and in another study further emphasizes the potential significance of this epitope for MS.

Animals↗
PubMedDate not supplied

Sequential order of T and B cell epitopes affects immunogenicity but not antibody recognition of the B cell epitope.

Synthetic peptide constructs, co-linearly linking a MUC1 mucin B cell epitope peptide to a known murine T cell epitope, both in T-B and B-T orientations, show that induction of high murine anti-MUC1 antibody titers is dependent on the presence and orientation of the T cell determinant. However, the sequential order of the epitopes does not affect binding of anti-B cell epitope antibodies to the constructs. Haplotype mismatching leads to a significant lowering of the anti-MUC1 antibody responses, implicating a central role for the T cell epitope in eliciting anti-B cell epitope responses. Secondary structure analysis by circular dichroism spectroscopy reveals the T-B construct to be partially ordered, while the B-T peptide adopts a highly ordered conformation in trifluoroethanol. These studies suggest that the sequential order of epitopes may significantly alter the immunogenicity of the peptide but may not necessarily affect its antigenicity. Immunogenicity of the peptide constructs may be governed by subtle differences in secondary structure, leading to variation in the way peptides are presented or processed within cells governing immune responses. These findings have relevance for the construction of peptides to be utilized as potential synthetic vaccines and for the design of peptide immunogens.

Amino Acid Sequence↗
PubMed1997

Analysis of epitope structure of PSP94 (prostate secretory protein of 94 amino acids): (II). Epitope mapping by monoclonal antibodies.

PSP94 has shown potential to be a serum biomarker for evaluating prostate cancer. Studies of the epitope structure is crucial for this endeavour. In this article, we have used 15 different monoclonal antibodies (MAb) to analyse the epitope structure of PSP94 and to compare with the results obtained from our previous work using polyclonal antibody and recombinant PSP94. Firstly, we determined the relative activities of the 15 MAb population by direct and competitive ELISA. The two predominant MAbs (MAb PSP-6 and -19) in 15 MAbs were selected for further studies of the epitope structure. By comparing the binding activities of recombinant GST-PSP94 and natural PSP94 with MAbs, and by comparing their affinity with MAbs in an in vitro denaturing experiment, PSP94 was shown to have a similar, prevalently linear epitope structure as we demonstrated by polyclonal antibody. Using recombinant GST fusion protein with PSP94 and with each half of the N- and C-terminal 47 amino acids (GST-PSP-N47/C47) in E. coli cells, the different epitopes recognized by 15 monoclonal antibodies were delineated and the polar distribution of the epitope structure of PSP94 was characterized. Results of direct ELISA of recombinant N47 and C47 and their competitive binding against natural PSP94 (competitive ELISA) showed that the N- and C-termini represent the immuno-dominant and immuno-recessive area separately. A majority of the monoclonal antibodies (12/15) showed preferential binding of the N-terminal sequence of the PSP94 protein. Using GST-PSP-N47 as a standard protein, an epitope map of the 15 monoclonal antibodies was obtained. The results of this study will help to define the clinical utility of PSP94.

Animals↗
PubMed2001

Tetramer-guided epitope mapping: rapid identification and characterization of immunodominant CD4+ T cell epitopes from complex antigens.

T cell responses to Ags involve recognition of selected peptide epitopes contained within the antigenic protein. In this report, we describe a new approach for direct identification of CD4+ T cell epitopes of complex Ags that uses human class II tetramers to identify reactive cells. With a panel of 60 overlapping peptides covering the entire sequence of the VP16 protein, a major Ag for HSV-2, we generated a panel of class II MHC tetramers loaded with peptide pools that were used to stain peripheral lymphocytes of an HSV-2 infected individual. With this approach, we identified four new DRA1*0101/DRB1*0401- and two DRA1*0101/DRB1*0404-restricted, VP16-specific epitopes. By using tetramers to sort individual cells, we easily obtained a large number of clones specific to these epitopes. Although DRA1*0101/DRB1*0401 and DRA1*0101/DRB1*0404 are structurally very similar, nonoverlapping VP16 epitopes were identified, illustrating high selectivity of individual allele polymorphisms within common MHC variants. This rapid approach to detecting CD4+ T cell epitopes from complex Ags can be applied to any known Ag that gives a T cell response.

CD4-Positive T-Lymphocytes↗
PubMed2001

A novel approach of direct ex vivo epitope mapping identifies dominant and subdominant CD4 and CD8 T cell epitopes from Listeria monocytogenes.

We used a novel approach for the direct ex vivo identification and characterization of T cell epitopes based on the screening of peptide spot libraries with freshly isolated splenocytes in a sensitive enzyme-linked immunospot (ELISPOT) assay. This technique was applied for the analysis of splenocytes from Listeria monocytogenes-infected BALB/c and C57BL/6 mice. The screening of peptide spot libraries covering the whole listeriolysin O and p60 of L. monocytogenes confirmed all known CD4 and CD8 T cell epitopes of these proteins and additionally revealed six new H-2(d) and six new H-2(b)-restricted T cell epitopes. New epitopes were categorized into CD4 and CD8 T cell epitopes by ex vivo ELISPOT analysis with separated T cell populations. The quantitative analysis of cells reactive with these CD4 and CD8 T cell epitopes revealed the existence of dominant and subdominant CD4 and CD8 T cell populations during L. monocytogenes infection. As a consequence of these data we suggest that ELISPOT-based screening of peptide spot libraries could be a general approach for the rapid identification and characterization of pathogen-specific T cell populations during various infectious diseases.

Animals↗
PubMed2005

Prototype Alzheimer's disease vaccine using the immunodominant B cell epitope from beta-amyloid and promiscuous T cell epitope pan HLA DR-binding peptide.

Immunization of amyloid precursor protein transgenic mice with fibrillar beta-amyloid (Abeta) prevents Alzheimer's disease (AD)-like neuropathology. The first immunotherapy clinical trial used fibrillar Abeta, containing the B and T cell self epitopes of Abeta, as the immunogen formulated with QS21 as the adjuvant in the vaccine. Unfortunately, the clinical trial was halted during the phase II stage when 6% of the participants developed meningoencephalitis. The cause of the meningoencephalitis in the patients that received the vaccine has not been definitively determined; however, analysis of two case reports from the AN-1792 vaccine trial suggest that the meningoencephalitis may have been caused by a T cell-mediated autoimmune response, whereas production of anti-Abeta Abs may have been therapeutic to the AD patients. Therefore, to reduce the risk of an adverse T cell-mediated immune response to Abeta immunotherapy we have designed a prototype epitope vaccine that contains the immunodominant B cell epitope of Abeta in tandem with the synthetic universal Th cell pan HLA DR epitope, pan HLA DR-binding peptide (PADRE). Importantly, the PADRE-Abeta(1-15) sequence lacks the T cell epitope of Abeta. Immunization of BALB/c mice with the PADRE-Abeta(1-15) epitope vaccine produced high titers of anti-Abeta Abs. Splenocytes from immunized mice showed robust T cell stimulation in response to peptides containing PADRE. However, splenocytes from immunized mice were not reactivated by the Abeta peptide. New preclinical trials in amyloid precursor protein transgenic mouse models may help to develop novel immunogen-adjuvant configurations with the potential to avoid the adverse events that occurred in the first clinical trial.

Alzheimer Disease↗
PubMed1995

alpha-galactosyl (Galalpha1-3Galbeta1-4GlcNAc-R) epitopes on human cells: synthesis of the epitope on human red cells by recombinant primate alpha1,3galactosyltransferase expressed in E.coli.

Developing methods for in vitro synthesis of the carbohydrate structure Galalpha1-3Galbeta1-4GlcNAc-R (termed the alpha-galactosyl epitope) on human tumour cells may be of potential clinical significance in cancer immunotherapy. Tumour vaccines with this epitope would be opsonized in vivo by the natural anti-Gal antibody, which is present in large amounts in humans, and which interacts specifically with alpha-galactosyl epitopes. Binding of anti-Gal to alpha-galactosyl epitopes on tumour cell membranes is likely to increase uptake of the cell membranes by antigen-presenting cells, such as macrophages, via the adhesion of the Fc portion of anti-Gal to Fc receptors on these cells. This, in turn, may increase processing and presentation of tumour-associated antigens by antigen-presenting cells, and induce an effective immune response against tumour cells with these antigens. The present study describes a method for the synthesis of alpha-galactosyl epitopes on human cells (red cells used as a model) by recombinant alpha1,3galactosyltransferase (rec. alpha1,3GT) expressed in bacteria. Escherichia coli was transformed with cDNA of the luminal portion of New World monkey rec. alpha1,3GT linked to six histidines (His)6 at the N-terminus. The enzyme produced by the bacteria was isolated from bacterial lysates on a nickel-Sepharose column and eluted with imidazole. This recombinant enzyme displayed acceptor specificity similar to that of rec. alpha1,3GT produced in COS cells. Red cells were pre-treated with sialidase for exposure of N-acetyllactosamine acceptors, then subjected to rec. alpha1,3GT activity. This enzyme synthesized at least 4 x 10(4) alpha-galactosyl epitopes/red cell. These epitopes were found to be accessible for binding of anti-Gal, as well as Bandeiraea simplicifolia IB4 lectin. It is argued that the method presented can be used for the synthesis of alpha-galactosyl epitopes on membranes of autologous tumour vaccines in humans.

Animals↗
PubMed1996

Epitope mapping of a recombinant human TSH receptor extracellular domain: identification of a predominant epitope using animal sera.

The extracellular domain of the TSH receptor (TSHR-561, amino acids #78-389) was expressed as a hexa-histidine fusion protein in bacteria. The recombinant protein was purified to homogeneity and used to immunize porcine and ovine species. High titre antibodies were obtained from both species that recognized the recombinant protein in Western blot analysis but failed to interfere with the TSH radio receptor assay. An epitope library was constructed and screened with affinity purified ovine and porcine antisera and detected a number of positive clones. Sequence analysis revealed that all of the epitopes contained sequences derived from the carboxyl terminus of the recombinant immunogen. One clone defined an epitope covering 16 amino acids from the carboxyl terminus and was the common epitope found in all of the other clones. Western blot screening of a large panel of Graves' sera with recombinant TSH receptor protein identified one patient sera that also recognized linear epitopes in the TSHR-561 protein. Experimentation demonstrated that the linear epitope recognized by this human sera was identical to the sequence recognised by the animal antisera. This sequence is unique to the TSH receptor and will be useful in further studies to analyze the TSH receptor protein.

Amino Acid Sequence↗
PubMed2002

Rabbit anti-HMG-17 antibodies recognize similar epitopes on the HMG-17 molecule as lupus autoantibodies. Relation with histone H1 defined epitopes.

HMG-17 is a nucleosomal protein which is an immune target of autoantibodies in systemic lupus erythematosus (SLE) and other autoimmune diseases. Autoantibody production in SLE is believed to result from autoantigen specific immune stimulation and subsequently, it is expected that antigenic determinants recognized by SLE autoantibodies and induced antibodies by immunization are quite similar. To examine this issue, rabbits were immunized with purified HMG-17. The produced antiserum showed cross reactivity on blots and in inhibition ELISA with histone H1, even after its affinity purification with immobilized HMG-17. Finally, purification of the antiserum over H1 absorbed on nitrocellulose membrane produced specific anti-HMG-17 antibodies in the supernatant and anti-HMG-17/H1 antibodies that were bound to H1. SLE sera positive for HMG-17 had also cross reactivity with H1, and following the same procedure as before we received HMG-17 specific SLE autoantibodies and anti-HMG-17/H1 autoantibodies. Using the multipin epitope mapping technology, 19 overlapping 15-mer HMG-17 peptides and six 15-peptides, corresponding to known epitopes of histone H1, were synthesized. Four major epitopes were identified on the HMG-17 molecule, reactive with induced anti-HMG-17 antibodies, and these were the same as major autoepitopes In SLE. The sequence 25-51 of HMG-17, part of its DNA-binding domain, was recognized by the anti-HMG-17/H1 antibodies that were bound to H1. These antibodies recognized also defined epitopes of H1. Our results show that SLE autoantibodies can be directed against the same or similar epitopes as do IgGs evoked during the active immunization of animals, and provide additional evidence that autosensitization with an autoantigen might be operative. The possibility that the same or similar epitopes are found on different molecules (in this study HMG-17 and H1) supports the fact that there are rules by which nature selects the most dominant immunodeterminant to a given protein, which often represents functional or structural sites in the autoantigen.

Amino Acid Sequence↗
PubMed1999

Identification of a highly promiscuous and an HLA allele-specific T-cell epitope in the birch major allergen Bet v 1: HLA restriction, epitope mapping and TCR sequence comparisons.

BACKGROUND: Allergen-specific CD4+ T cells play an important regulatory role in atopic allergy. OBJECTIVE: To investigate the human leucocyte antigen (HLA) restriction and T-cell receptor (TCR) usage of allergen-specific T-cell clones (TCCs) that react with defined epitopes of Bet v 1, the major birch pollen allergen. METHODS: Five Bet v 1-specific TCCs derived from two birch pollen-allergic individuals and specific for Bet v 1, were epitope-mapped with overlapping synthetic peptides. In addition, HLA-restriction and TCR CDR3 sequences were determined. RESULTS: Three TCCs reacted with a Bet v 1 peptide containing amino acid residues 21-33 (BP21), the other two TCCs reacted with a minimal peptide comprising residues 37-45 (BP37). Studies using neutralizing anti-HLA-monoclonal antibodies and HLA-typed APCs showed that the BP37-specific TCCs were restricted by a HLA-DQA1*0301/DQB1*0603 heterodimer. In contrast, BP21 was recognized in a highly promiscuous manner. TCCs recognizing this sequence were restricted by HLA-DPB1*0201, a HLA-DQA1*0201/DQB1*0201 heterodimer, or HLA-DRB3*0101. Reverse transcription-polymerase chain reaction with primers for all known TCRAV and TCRBV gene segments, followed by CDR3 region sequencing, revealed the usage of five different TCRAV and four different TCRBV gene segments by the TCCs, as well as diversity in the joining region. All BP21-specific TCCs contained a negatively charged residue in their CDR3alpha regions, the CDR3beta regions showed a high concentration of polar and OH-group bearing residues. BP37-specific TCCs shared the amino acid combination LY in the middle of their CDR3alpha regions, the CDR3beta regions showed high concentration of OH-group bearing or charged residues. CONCLUSIONS: This study shows the existence of a highly promiscuous T-cell epitope in Bet v 1. The presence of additional T-cell epitopes in Bet v 1 may, however, hamper the clinical applicability of the epitope. Likewise, the diversity in TCR usage by T cells recognizing the epitope does not support the development of TCR-directed immunotherapy for birch pollen allergy.

Alleles↗