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PubMed1985

The idiotypic characterization of the immune response to a defined epitope of a protein antigen and the specific in vivo suppression of the immune response to this epitope by anti-idiotypic antibodies.

C10, a monoclonal antibody of C3H.SW (CSW) origin, binds a decapeptide epitope of the tobacco mosaic virus protein (TMVP) representing residues 103-112 of the protein. In vivo administration of syngeneic anti-idiotypic antibodies to C10 (anti-C10) prior to immunization with TMVP suppressed the expression of antibodies to this decapeptide determinant in CSW mice without a significant reduction of the total anti-TMVP titer. The suppression could not be overcome with repeated challenges by antigen even 6 months after administration of anti-C10. Analysis of anti-C10 showed that it contains antibodies to at least two idiotopes found on C10. One of these idiotopes, C10-Idm, is found on a very small fraction of CSW anti-TMVP antibodies capable of binding the decapeptide epitope. The other idiotope, C10-IdX, is found on most of the anti-TMVP antibodies which bind the decapeptide determinant. With synthetic analogues of the decapeptide determinant, a correlation was established between the presence of the C10-IdX and the fine specificity of the decapeptide-binding antibodies. The studies reported herein demonstrate that anti-idiotypic antibodies are potent modulators of the immune response and that the C10-IdX is important in the determination of the fine specificity of antibodies to this decapeptide epitope of TMVP.

Animals↗
PubMed1984

Recognition of two Dermatophagoides pteronyssinus-specific epitopes on antigen P1 by using monoclonal antibodies: binding to each epitope can be inhibited by serum from dust mite-allergic patients.

Monoclonal antibodies were raised against Antigen P1, the major allergen of the house dust mite (Dermatophagoides pteronyssinus). The majority were Antigen P1 specific, isotype IgG1, and did not react with a comparable D. farinae allergen. These antibodies bound 38 to 50% of 125I Antigen P1 in antigen-binding assays (titer greater than or equal to 1/1,000,000), and the quantities of IgG antibody in ascites were 2 to 4 logs greater than those in polyclonal mouse antiserum or in serum from a mite-allergic patient. Two IgM antibodies showed weak binding to Antigen P1 but reacted strongly with D. pteronyssinus in enzyme immunoassay (titer greater than or equal to 1/100,000). Assessments of the specificity of the IgG antibodies by using two inhibition radioimmunoassays suggested that they were directed against two different epitopes. Antibodies 10B9 F6 and 5H8 C12 were purified by preparative isoelectric focusing (isoelectric points of pI 6.25 and 7.4, respectively) and radiolabeled with 125I. Cross-inhibition experiments, using ascites dilutions to inhibit binding of each radiolabeled antibody to Antigen P1, confirmed that these antibodies recognized two distinct epitopes. Analysis of antibodies from 39 clones/hybrids showed that the majority were directed against the same epitopes as either 10B9 F6 or 5H8 C12 (3 out of 39 [8%] and 29 out of 39 [74%], respectively). None of the monoclonal antibodies significantly inhibited (greater than 10%) human IgE binding to Antigen P1 in the radioallergosorbent test. However, 12 of 14 sera from mite allergic patients inhibited binding by the monoclonal antibodies. One serum from a mite-allergic patient inhibited binding of both 10B9 F6 and 5H8 C12 by greater than 85% and showed parallel inhibition curves. The results suggest that these monoclonal antibodies could be used to assay Antigen P1 in both D. pteronyssinus and house dust extracts. It should also be possible to use monoclonal antibodies in inhibition assays to define the antigenic/allergenic determinants recognized by human IgG and IgE antibodies on this mite allergen.

Allergens↗
PubMed1993

A peptide variant of an arthritis-related T cell epitope induces T cells that recognize this epitope as a synthetic peptide but not in its naturally processed form.

The immune system has the potential to utilize a diverse T cell repertoire for the recognition of Ag in the context of MHC molecules. Here we describe the analysis of two rat T cell clones, both of which recognize a synthetic peptide comprised of the arthritis-associated 180-188 amino acid sequence of the mycobacterial 65-kDa heatshock protein (hsp65 180-188), but which differ in the recognition of the naturally processed hsp65. The arthritogenic T cell clone A2b, generated by immunization with whole Mycobacterium tuberculosis, recognized the hsp65 180-188 synthetic peptide as well as the processed hsp65, whereas T cell clone ATL11, generated after immunization with a single amino acid substituted peptide analog of hsp65 180-188, recognized peptide hsp65 180-188 but not the processed hsp65. For both T cell clones the minimal stimulatory sequence was hsp65 180-186. However, within this minimal stimulatory sequence marked differences between the clones were found with regard to peptide residues interacting with the TCR. Furthermore, addition of extra residues at the N terminus of the hsp65 180-186 sequence abrogated the recognition by clone ATL11, but not by A2b. These findings demonstrate that, upon in vivo immunization with a synthetic peptide comprised of a single amino acid variant of a T cell epitope, T cells can be triggered that recognize a peptide comprised of the original epitope sequence, but that do not recognize this epitope in its naturally processed protein fragment. The possibility of triggering such T cells by immunization with synthetic peptides, may well have consequences for the design of peptide vaccines or peptide immunomodulatory agents.

Amino Acid Sequence↗
PubMed1998

Quinine-dependent antibodies bind a restricted set of epitopes on the glycoprotein Ib-IX complex: characterization of the epitopes.

Severe immune thrombocytopenia is an idiosyncratic complication of quinine therapy. Although in most cases the responsible antibody is directed against platelet membrane glycoprotein (GP) Ib-IX, specificity for GPIIb-IIIa or both epitopes has also been reported. The objective of this study was to characterize the binding site of GPIb-IX-specific quinine-dependent antibodies. Antibody binding to Chinese hamster ovary cells or mouse L cells stably transfected with various combinations of the three genes (Ibalpha, Ibbeta, or IX) that encode this complex was detected using flow cytometry, monoclonal antibody-specific immobilization of platelet antigens assay, and differential adsorption studies. IgG in sera from 15 patients with quinine-induced thrombocytopenia binding to the cells, in the presence of quinine, showed three distinct patterns. Group 1 sera contained at least two antibody populations, one which binds to GPIbalpha and another which recognizes GPIX. Group 2 sera contained an antibody which binds drug dependently to GPIX, and Group 3 sera contained an antibody which recognizes a quinine-dependent epitope on GPIbalpha. Thus, the quinine-dependent antibodies fall into two distinct populations that bind to GPIbalpha and GPIX independently. Using proteases which cleave GPIbalpha at specific sites, we have shown that the GPIbalpha-specific antibody binds to an 11-amino acid (283 to 293) region. Peptide inhibition studies provide confirmatory evidence that this region contains the epitope for the GPIbalpha-specific quinine-dependent antibody.

Amino Acid Sequence↗
PubMed1998

Epitope blocking: positive and negative effects on the biodistribution of 125I-labeled anti-Tac disulfide-stabilized Fv fragment of two antibodies against different epitopes of the circulating antigen.

Prior in vivo studies using the 125I-labeled anti-Tac disulfide-stabilized variable region fragment (125I-anti-Tac dsFv) of monoclonal antibody in the presence of the circulating soluble alpha subunit of the interleukin-2 receptor (sIL-2R alpha) have shown formation of complexes which interfere with biodistribution. In this study we evaluated the effects of preinjecting HuTac and 7G7/B6, two immunoglobulin Gs (IgGs) that recognize different epitopes of sIL-2R alpha, on the biodistribution of 125I-anti-Tac dsFv in mice bearing SP2/Tac tumor xenografts, which produce sIL-2R alpha, or on nude mice injected with 500 ng of sIL-2R alpha. We also evaluated the biodistribution in mice of 125I-labeled sIL-2R alpha injected alone or with HuTac and 7G7/B6. Injection of either HuTac or 7G7/B6 resulted in complexes with the sIL-2R alpha in serum. Injection of HuTac before 125I-anti-Tac dsFv, in SP2/Tac tumor-bearing mice, resulted in faster clearance of the dsFv from the blood (7.6% ID/g at 30 min), compared to 23.2% ID/g for the no-antibody control; preinjection of 7G7/B6 prolonged the retention of 125I-anti-Tac dsFv to 35.3% ID/g, with more complexes in serum. In mice pre-injected with 7G7/B6 the concentration of 125I-anti-Tac dsFv in tumor was lower (5.2 +/- 0.3% ID/g) than in mice preinjected with HuTac (7.9 +/- 1.2% ID/g) or in the control group (5.6 +/- 0.7% ID/g). In conclusion, while both IgGs formed complexes with sIL-2R alpha and prolonged its retention, preinjection of 7G7/B6 was detrimental, because the increased circulating sIL-2R alpha still had the epitope recognized by the dsFv available for binding and neutralized the anti-Tac dsFv upon injection, whereas preinjection of HuTac blocked the epitope.

Animals↗
PubMed1998

Localisation of a protein core-specific epitope from gastrointestinal mucin (MUC2). The effect of epitope immobilisation on antibody recognition.

Human intestinal mucins are high molecular weight glycoproteins which protect and lubricate the epithelium of the gastrointestinal tract. In cases of malignant disease, mucins are abnormally expressed, overproduced or underglycosylated. This feature may enable the mucins to serve as tumour markers. The MUC2 mucin largely consists of a variable number of tandem repeats of a 23 amino acid sequence, 1PTTTPITTTTTVTPTPTPTGTQT23. In this study we have localised the minimal and the optimal epitope within this region by the previously developed protein core specific 996 monoclonal antibody using synthetic peptides. Several overlapping and truncated peptides related to the tandem repeat unit have been prepared by solid-phase methodology. Other mucin peptides were synthesised on the tips of polyethylene pins, and these remained C-terminally attached to the pins for comparative investigations. The interaction of the 996 monoclonal antibody with the synthetic peptides was studied either in solution by competition RIA or on immobilised peptides by indirect ELISA experiments. These experiments show that the minimal epitope recognised by the 996 antibody is the Ac-19TGTQ22 (IC50=3100 microM in solution). For the optimal 996 antibody binding in solution the 16PTPTGTQ22 heptapeptide (IC50 = 3 microM) is required.

Antigen-Antibody Reactions↗
PubMed2001

Natural T-helper immunity against human papillomavirus type 16 (HPV16) E7-derived peptide epitopes in patients with HPV16-positive cervical lesions: identification of 3 human leukocyte antigen class II-restricted epitopes.

Tumor-specific T-helper (Th) immunity was found to play a pivotal role in the natural and vaccine-induced immune defense against tumors. Since the majority of cervical cancers express human papillomavirus type 16 (HPV16) E7 oncoprotein, it is important to investigate the Th response against this target antigen in detail. By means of PBMC cultures from HLA-typed healthy donors, we identified the central part of HPV16 E7 (E7(41-72)) as the major immunogenic region within this antigen. Furthermore, we mapped 3 distinct Th epitopes within this region (DR15/E7(50-62), DR3/E7(43-77), DQ2/E7(35-50)). In a parallel approach, employing IFN-gamma ELISPOT analysis, we detected Th immunity against HPV16 E7 in subjects with HPV16+ lesions. Several of these responses matched with the 3 Th epitopes defined in our study. A number of other HPV16+ subjects did not display any E7-specific type 1 cytokine-producing T-cell immunity, indicating failure of the immune response. Our combined data argue for more extensive as well as longitudinal analysis of HPV16-specific T-cell immunity using the ELISPOT assay described, as well as for HPV-specific vaccination of individuals with HPV+ lesions.

Cancer Vaccines↗
PubMed2006

Association between protein tyrosine phosphatase 22 variant R620W in conjunction with the HLA-DRB1 shared epitope and humoral autoimmunity to an immunodominant epitope of cartilage-specific type II collagen in early rheumatoid arthritis.

OBJECTIVE: To analyze the genetic impact of allelic variants of the protein tyrosine phosphatase N22 (PTPN22) and HLA-DRB1 alleles on IgG autoantibody formation directed toward an immunodominant conformational epitope (C1(III); amino acid residues 359-369) of type II collagen (CII) in early rheumatoid arthritis (RA). METHODS: Sera obtained at study inclusion from an inception cohort of RA patients (n = 221; mean symptom duration 6 months) were analyzed for circulating anti-C1(III) IgG autoantibodies. An enzyme-linked immunosorbent assay based on solid-phase-coupled synthetic triple-helical collagen peptides was used to quantify humoral autoimmune responses. HLA-DRB1 genotypes were determined by allele-specific polymerase chain reaction amplification of genomic DNA and sequence-specific hybridization. PTPN22*620W genotyping was performed using an allelic discrimination TaqMan assay. RESULTS: Anti-C1(III) IgG autoantibody titers were significantly elevated in patients with early RA as compared with those in healthy controls (n = 70). The increased titers were more pronounced in RA patients harboring alleles of the RA-associated HLA-DRB1 shared epitope (SE) consensus sequence than in those lacking the SE. In addition, the PTPN22*620W variant was strongly associated with a vigorous humoral autoimmune response to the cartilage-specific CII determinant C1(III). CONCLUSION: Allelic variants encoding the binding pocket for peptide presentation (SE) to T cells and a functional domain of a negative regulator of T cell receptor signaling (PTPN22*620W), respectively, synergize in early RA to break self tolerance toward C1(III), an evolutionarily conserved cartilage determinant that is also frequently targeted in arthritogenic humoral autoimmunity in mice.

Arthritis, Rheumatoid↗
PubMed2004

Potent T cell response to a class I-binding 13-mer viral epitope and the influence of HLA micropolymorphism in controlling epitope length.

The BZLF1 antigen of Epstein-Barr virus includes three overlapping sequences of different lengths that conform to the binding motif of human leukocyte antigen (HLA) B*3501. These 9-mer (56LPQGQLTAY64), 11-mer (54EPLPQGQLTAY64), and 13-mer (52LPEPLPQGQLTAY64) peptides all bound well to B*3501; however, the CTL response in individuals expressing this HLA allele was directed strongly and exclusively towards the 11-mer peptide. In contrast, EBV-exposed donors expressing HLA B*3503 showed no significant CTL response to these peptides because the single amino acid difference between B*3501 and B*3503 within the F pocket inhibited HLA binding by these peptides. The extraordinarily long 13-mer peptide was the target for the CTL response in individuals expressing B*3508, which differs from B*3501 at a single position within the D pocket (B*3501, 156Leucine; B*3508, 156Arginine). This minor difference was shown to enhance binding of the 13-mer peptide, presumably through a stabilizing interaction between the negatively charged glutamate at position 3 of the peptide and the positively charged arginine at HLA position 156. The 13-mer epitope defined in this study represents the longest class I-binding viral epitope identified to date as a minimal determinant. Furthermore, the potency of the response indicates that peptides of this length do not present a major structural barrier to CTL recognition.

Antigen Presentation↗
PubMed1994

T cell epitope analysis with peptides simultaneously synthesized on cellulose membranes: fine mapping of two DQ dependent epitopes.

Several MHC class II restricted, CD4+ human T cell clones from three donors were induced with soluble matrix protein of influenza virus. The epitopes recognized by these clones were mapped using a complete set of overlapping 15-mer peptides synthesized with the newly developed SPOT method which allows the simple simultaneous synthesis of numerous peptides on cellulose membranes. Fine analysis of two clones by truncation of the stimulatory peptide by single subsequent amino acids from either the NH2- or the COOH-terminus revealed the minimal stimulatory determinants of two DQ dependent T cell epitopes.

Amino Acid Sequence↗
PubMed1997

Potent immunogenic short linear peptide constructs composed of B cell epitopes and Pan DR T helper epitopes (PADRE) for antibody responses in vivo.

Induction of humoral immune responses against protein antigen requires that two independent signals be delivered to B cells. It is currently assumed that simple monovalent synthetic peptides would not be effective immunogens for antibody responses because they would not be anticipated to effectively generate the necessary signals unless conjugated to a complex carrier system. In this study, the immunogenicity of short linear peptide constructs comprising Plasmodium vivax B cell epitopes (PVB) and non-natural Pan-DR T helper cell epitopes (PADRE) was assessed in mice and compared to other types of antigen constructs. The 33-residue PADRE-PVB linear constructs were highly immunogenic and induced responses comparable to those obtained with the multiple antigen peptides (MAP) constructs, both in terms of absolute titers and quality of antibody responses. The anti-PVB antibody responses were of long duration, composed mostly of IgG and reactive with intact sporozoites. The PADRE-PVB constructs were immunogenic when formulated in adjuvants such as Alum and Montanide ISA 51 underlining the relevance of these findings for vaccine development.

Adjuvants, Immunologic↗
PubMed2003

Epitope-based vaccines: an update on epitope identification, vaccine design and delivery.

The basic premise of the epitope-based approach to vaccine development is that, in certain cases, the responses induced by the natural immunogen are not optimal, and can be improved upon by isolation or optimization of specific components of the response. For example, immunodominance is a key factor limiting the type and breadth of adaptive immunity. Recent advances in understanding the mechanisms of immunodominance thus represent an opportunity to further develop the epitope-based approach.

Cancer Vaccines↗
PubMed2001

Epitope mapping porcine reproductive and respiratory syndrome virus by phage display: the nsp2 fragment of the replicase polyprotein contains a cluster of B-cell epitopes.

We screened phage display libraries of porcine reproductive and respiratory syndrome virus (PRRSV) protein fragments with sera from experimentally infected pigs to identify linear B-cell epitopes that are commonly recognized during infection in vivo. We identified 10 linear epitope sites (ES) 11 to 53 amino acids in length. In the replicase polyprotein, a total of eight ES were identified, six of which localized to the Nsp2 replicase polyprotein processing end product. In the structural proteins, a total of two ES were identified, in the ORF3 and ORF4 minor envelope glycoproteins. The ORF4 ES was previously identified by monoclonal antibody mapping (J. J. M. Meulenberg, A. P. van Nieuwstadt, A. van Essen-Zandenbergen, and J. P. M. Langeveld, J. Virol. 71:6061-6067, 1997), but its immunogenicity had not been examined in pigs. We found that six experimentally PRRSV-infected pigs consistently had very high antibody titers against the ORF4 ES. In some animals, sera diluted 1:62,500 still gave weak positive enzyme immunoassay reactivity against the ORF4 ES. This hitherto unrecognized immunodominance likely caused phages displaying the ORF4 ES to outcompete phages displaying other ES during library screening with porcine sera and accounted for our failure to identify more than two ES in the structural genes of PRRSV. Genetic analysis showed that variable ES were also the most immunogenic in vivo. Serological analysis indicated differences in the immunoglobulin A responses between short-term and longer-term viremic pigs towards some ES. The implications of these findings for PRRSV diagnostics and immunopathogenesis are discussed.

Amino Acid Sequence↗
PubMed2026

Distinct HLA Associations for Antibody Multireactivity With Citrulline-Containing Type II Collagen Epitopes Versus More Limited Antibody Reactivity With Citrulline-Containing IgG Epitopes in Rheumatoid Arthritis.

OBJECTIVE: Anticitrullinated protein antibodies (ACPAs) in rheumatoid arthritis (RA) can be promiscuous, with cross-reactive binding to many antigens containing short motifs, or private with little cross-reactivity. Also, ACPA reactivity patterns differ among patients with RA, including for motif-containing epitopes in important self-antigens like collagen and IgG (bound by RA-associated rheumatoid factors [RFs]), with limited understanding of the underlying mechanism. The objective of this study was to determine if HLA alleles associate with ACPA reactivity patterns. METHODS: For 100 ACPA+RF+ participants with RA, serum IgG binding was quantified by enzyme-linked immunosorbent assay to 10 citrulline-containing peptides derived from Type II collagen and IgG1 (nine with motifs), and HLA loci were genotyped. Also, antibody and serum multireactivity were evaluated. HLA alleles present differentially in RA participants with high versus low IgG binding to specific peptides, as well as with multireactivity versus limited reactivity were identified by Fisher's exact test. RESULTS: Serum IgG multireactivity for citrulline-glycine motif-containing collagen peptides was high, at least partially due to promiscuous antibodies. HLA-DQA1*01:02 was present in more participants with anticitrullinated collagen antibodies and multireactive sera. In contrast, serum multireactivity was low for IgG1-derived peptides due at least in part to more private antibodies. Shared epitope-containing HLA-DRB1*04:01 was present more frequently in participants with RA-associated RFs irrespective of the citrulline-serine motif and less frequently in participants with anticitrullinated collagen antibodies. Several HLA alleles associated with specific antibody reactivities. CONCLUSION: Different HLA alleles may contribute to the different reactivity patterns of promiscuous anticitrullinated collagen antibodies and more private RA-associated RFs.

Humans↗
PubMedDate not supplied

The development of multi-epitope vaccines: epitope identification, vaccine design and clinical evaluation.

We have developed efficient methods for epitope identification and vaccine design. Our process for epitope selection based on the combined use of motif analyses, binding assays and immunogenicity evaluations is described. We also describe how the projected population coverage and vaccine design can be optimized. Finally, it is discussed how vaccine potency is evaluated by immunogenicity and antigenicity assays.

Epitopes↗
PubMed2001

A population of autoantibodies against a centromere-associated protein A major epitope motif cross-reacts with related cryptic epitopes on other nuclear autoantigens and on the Epstein-Barr nuclear antigen 1.

Autoimmune diseases arise from a host's immune response against self-antigens. The triggering events ultimately resulting in such a break of tolerance are largely unknown. It is also not known why certain molecular structures become autoantigenic. The hypothesis has long been proposed that autoimmune diseases arise from molecular mimicry followed by an epitope spreading mechanism. Recently we have shown that the anti-centromere-associated protein A (CENP-A) immune response is directed against an autoantigenic motif, G/A-P-R/S-R-R, that occurs three times in the N-terminal amino acids of CENP-A. In the present study we used mutational analyses with immobilized oligopeptide arrays to identify the amino acids in this motif that are responsible for antibody binding. In particular, we found that surprisingly mimotopes of this motif are present in a vast number of autoantigens and in the Epstein-Barr nuclear antigen 1. With affinity-purified antibodies we show that the antibodies against this motif are polyclonal and cross-react with several autoantigens. However, in these autoantigens this motif often represents a cryptic epitope explaining the obvious conflict between our results and the known high specificity of autoantibodies. The presence of such an ubiquitous structure on autoantigens suggests a novel peptide-driven mechanism for the evolution of autoantibodies.

Amino Acid Motifs↗
PubMed2004

Epitope analysis of monoclonal antibody E5/G6, which binds to a linear epitope in the nucleocapsid protein of hantaviruses.

Monoclonal antibody E5/G6 recognized a linear epitope common to hantavirus nucleocapsid proteins. Using synthetic peptides, we identified epitope E5/G6 as the 9 mer YEDVNGIRK (NP 165-173), in which D167, G170, I171, and R172 are indispensable. Furthermore, all the peptides synthesized using various hantavirus sequences bound MAb E5/G6 consistently, despite the existence of several amino acid variations in this region. These results indicate that MAb E5/G6 is a useful tool for detecting hantavirus antigen in rodent or patient tissues using Western blotting or other immunohistochemical assays.

Amino Acid Sequence↗
PubMed2005

Epitope mapping of Burkholderia pseudomallei serine metalloprotease: identification of serine protease epitope mimics.

Filamentous phage random peptide libraries were used to identify the epitopes of Burkholderia pseudomallei protease by panning against IgG polyclonal sera that exhibited protease neutralizing properties. The isolated fusion peptides presented a consensus peptide sequence, TKSMALSG, which closely resembles part of the active site sequence, 435GTSMATPHVAG445, of B. pseudomallei serine metalloprotease. By comparing the consensus sequence, TKSMALSG, with the predicted three-dimensional molecular model of B. pseudomallei serine metalloprotease, it appears that the potential antibody binding epitope was buried within the molecule. This active site was conformational whereby one continuous sub-region (SMA) was located between two discontinuous sub-regions, supplied by the flanking residues in the same polypeptide. All phages selected from the biopanning with IgG polyclonal sera showed good binding towards the polyclonal antibodies when compared to the negative control. In addition, these peptide-bearing phages showed competitive inhibition of B. pseudomallei serine metalloprotease binding to the polyclonal IgG.

Amino Acid Sequence↗