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PubMed1994

Immunization with a multiple antigen peptide containing defined B- and T-cell epitopes: production of bactericidal antibodies against group B Neisseria meningitidis.

Previous analysis of the class 1 outer-membrane (OM) protein of Neisseria meningitidis has identified discrete epitopes to be potential targets for immune attack. The conformation of these epitopes is important for inducing antibodies which can react with the native protein and promote complement-mediated lysis of the meningococcus. The multiple antigen peptide (MAP) system, which consists of an oligomeric branching lysine core to which are attached dendritic arms of defined peptide antigens, confers some conformational stability and also allows for the preparation of immunogens containing both B-cell and T helper (Th)-cell epitopes. In this study, MAPs were synthesized to contain (i) the subtype P1.16b meningococcal class 1 protein B-cell epitope (B-MAP), and (ii) the P1.16b epitope in tandem with a defined Th-cell epitope, chosen from tetanus toxin (BT-MAP). The B-MAP was nonimmunogenic in animals. In contrast, incorporation of the Th-cell epitope into BT-MAP induced a strong humoral response towards the class 1 protein B-cell epitope. Antisera from immunized mice and rabbits reacted in ELISA with synthetic peptides containing the B-cell epitope, and also cross-reacted with meningococcal OMs from strains of subtype P1.16b and P1.16a. Murine and rabbit antisera showed similar reactivity and epitope specificity, but did not react with denatured class 1 protein in Western blotting, indicating the predominance of antibodies directed towards conformational epitopes. The antisera from rabbits immunized with BT-MAP promoted complement-mediated bactericidal killing not only of the homologous meningococcal subtype P1.16b strain but also of subtype P1.16a.

Amino Acid Sequence↗
PubMed2004

B cell epitope specificity in ANCA-associated vasculitis: does it matter?

Pauci-immune idiopathic small-vessel vasculitis is strongly associated with the presence of antineutrophil cytoplasm autoantibodies (ANCA). Antibodies to PR3 predominate in patients with Wegener's granulomatosis; antibodies to myeloperoxidase (MPO) are found more frequently in patients with microscopic polyangiitis. There is increasing in vivo and in vitro evidence for a pathogenic role of ANCA in systemic vasculitis based on associations of ANCA with disease activity. If ANCA are pathogenic, why is the course of disease different from one patient to another? Antibodies can recognize different binding sites (epitopes) on their corresponding antigens. Differences in binding specificity may influence the pathogenic potential of the antibodies. Differences between epitope specificity of ANCA between patients or changes in epitope specificity of ANCA in time in an individual patient may, accordingly, result in differences in disease expression. This review will focus on epitope specificity of autoantibodies in systemic autoimmune diseases and especially on the epitope specificity of PR3- and MPO-ANCA. We will discuss whether PR3-ANCA or MPO-ANCA recognize different epitopes on PR3 and MPO, respectively, and whether the epitopes recognized by ANCA change in parallel with the disease activity of ANCA-associated vasculitis. Finally, we will speculate if the direct pathogenic role of ANCA can be ascribed to one relapse- or disease-inducing epitope. Characterization of relapse- or disease-inducing epitopes bound by PR3-ANCA and MPO-ANCA is significant for understanding initiation and reactivation of ANCA-associated vasculitis. Elucidating a disease-inducing epitope bound by ANCA may lead to the development of epitope-specific therapeutic strategies.

Amino Acid Sequence↗
PubMed1997

A multivalent minigene vaccine, containing B-cell, cytotoxic T-lymphocyte, and Th epitopes from several microbes, induces appropriate responses in vivo and confers protection against more than one pathogen.

The development of safe and effective vaccines remains a major goal in the prevention, and perhaps treatment, of infectious diseases. Ideally, a single vaccine would confer protection against several pathogens and would induce both cellular and humoral arms of the immune response. We originally demonstrated that two virus-specific cytotoxic T-lymphocyte (CTL) epitopes, from the same virus but presented by different major histocompatibility complex alleles, when linked in tandem as minigenes in a recombinant vaccinia virus, could confer complete protection against subsequent viral challenge. In the study, we extended this approach, which we termed string of beads, expanding the immunogenic scope in two ways: first, by introduction of T helper (Th) and B-cell (antibody) epitopes alongside CTL epitopes and second, by including immunogenic sequences from a variety of infectious agents, five viruses and one bacterium. The vaccine (VV-sv) comprises CTL epitopes from Sendai virus, respiratory syncytial virus, and lymphocytic choriomeningitis virus (LCMV); Th epitopes from vesicular stomatitis virus and Mycobacterium tuberculosis; and an antibody epitope from mengovirus. The construct contains a single start codon, and the epitopes are linked directly, without intervening spacer amino acids. There was some concern that the combination of several normally immunodominant epitopes might result in a new hierarchy of dominance, in which certain epitopes predominated and others exhibited reduced immunogenicity. However we show that when analyzed in tissue culture and in vivo, all six epitopes are expressed. CTL and Th cells are induced in vivo, along with neutralizing antibody. The induced immunity is biologically relevant: after VV-sv immunization, the antimengovirus antibody confers protection against mengovirus challenge. Similarly, CTL induced by the LCMV epitope protected mice against challenge with this agent. Thus, a polyvalent, minigene-based vaccine can simultaneously induce several classes of immune response and thereby can confer protection against diverse pathogens.

Amino Acid Sequence↗
PubMed2002

Clustering patterns of cytotoxic T-lymphocyte epitopes in human immunodeficiency virus type 1 (HIV-1) proteins reveal imprints of immune evasion on HIV-1 global variation.

The human cytotoxic T-lymphocyte (CTL) response to human immunodeficiency virus type 1 (HIV-1) has been intensely studied, and hundreds of CTL epitopes have been experimentally defined, published, and compiled in the HIV Molecular Immunology Database. Maps of CTL epitopes on HIV-1 protein sequences reveal that defined epitopes tend to cluster. Here we integrate the global sequence and immunology databases to systematically explore the relationship between HIV-1 amino acid sequences and CTL epitope distributions. CTL responses to five HIV-1 proteins, Gag p17, Gag p24, reverse transcriptase (RT), Env, and Nef, have been particularly well characterized in the literature to date. Through comparing CTL epitope distributions in these five proteins to global protein sequence alignments, we identified distinct characteristics of HIV amino acid sequences that correlate with CTL epitope localization. First, experimentally defined HIV CTL epitopes are concentrated in relatively conserved regions. Second, the highly variable regions that lack epitopes bear cumulative evidence of past immune escape that may make them relatively refractive to CTLs: a paucity of predicted proteasome processing sites and an enrichment for amino acids that do not serve as C-terminal anchor residues. Finally, CTL epitopes are more highly concentrated in alpha-helical regions of proteins. Based on amino acid sequence characteristics, in a blinded fashion, we predicted regions in HIV regulatory and accessory proteins that would be likely to contain CTL epitopes; these predictions were then validated by comparison to new sets of experimentally defined epitopes in HIV-1 Rev, Tat, Vif, and Vpr.

Amino Acid Sequence↗
PubMed2002

Identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus GP5 ectodomain.

After infection of swine with porcine reproductive and respiratory syndrome virus (PRRSV), there is a rapid rise of PRRSV-specific nonneutralizing antibodies (NNA), while neutralizing antibodies (NA) are detectable not sooner than 3 weeks later. To characterize neutralizing epitopes, we selected phages from a 12-mer phage display library using anti-PRRSV neutralizing monoclonal antibody (MAb) ISU25-C1. In addition, phages carrying peptides recognized by swine antibodies with high seroneutralizing titer were isolated after subtracting from the library those clones binding to swine anti-PRRSV serum with no neutralizing activity. Two epitopes located in the ectodomain of PRRSV GP5 were identified. One of these epitopes, which we named epitope B, was recognized both by neutralizing MAb ISU25-C1 and swine neutralizing serum (NS) but not by swine nonneutralizing serum (NNS), indicating that it is a neutralizing epitope. Epitope B is sequential, conserved among isolates, and not immunodominant. Antibodies directed against it are detected in serum late after infection. In contrast, the other epitope, which we named epitope A, is hypervariable and immunodominant. Antibodies against it appear early after infection with PRRSV. This epitope is recognized by swine NNA but is not recognized by either neutralizing MAb ISU25-C1 or swine NA, indicating that it is not involved in PRRSV neutralization. During infection with PRRSV, epitope A may act as a decoy, eliciting most of the antibodies directed to GP5 and delaying the induction of NA against epitope B for at least 3 weeks. These results are relevant to the design of vaccines against PRRSV.

Amino Acid Sequence↗
PubMed2004

Novel and promiscuous CTL epitopes in conserved regions of Gag targeted by individuals with early subtype C HIV type 1 infection from southern Africa.

Characterization of optimal CTL epitopes in Gag can provide crucial information for evaluation of candidate vaccines in populations at the epicenter of the HIV-1 epidemic. We screened 38 individuals with recent subtype C HIV-1 infection using overlapping consensus C Gag peptides and hypothesized that unique HLA-restricting alleles in the southern African population would determine novel epitope identity. Seventy-four percent of individuals recognized at least one Gag peptide pool. Ten epitopic regions were identified across p17, p24, and p2p7p1p6, and greater than two-thirds of targeted regions were directed at: TGTEELRSLYNTVATLY (p17, 35%); GPKEPFRDYVDRFFKTLRAEQATQDV (p24, 19%); and RGGKLDKWEKIRLRPGGKKHYMLKHL (p17, 15%). After alignment of these epitopic regions with consensus M and a consensus subtype C sequence from the cohort, it was evident that the regions targeted were highly conserved. Fine epitope mapping revealed that five of nine identified optimal Gag epitopes were novel: HLVWASREL, LVWASRELERF, LYNTVATLY, PFRDYVDRFF, and TLRAEQATQD, and were restricted by unique HLA-Cw*08, HLA-A*30/B*57, HLA-A*29/B*44, and HLA-Cw*03 alleles, respectively. Notably, three of the mapped epitopes were restricted by more than one HLA allele. Although these epitopes were novel and restricted by unique HLA, they overlapped or were embedded within previously described CTL epitopes from subtype B HIV-1 infection. These data emphasize the promiscuous nature of epitope binding and support our hypothesis that HLA diversity between populations can shape fine epitope identity, but may not represent a constraint for universal recognition of Gag in highly conserved domains.

Africa, Southern↗
PubMed1999

Relative cytotoxic activity of immunotoxins reactive with different epitopes on the extracellular domain of the c-erbB-2 (HER-2/neu) gene product p185.

Different epitopes on the extracellular domain of the HER-2 receptor can serve as distinct targets for immunotoxins. To determine the optimal epitope target for immunotoxin therapy, 7 anti-HER-2 ricin A chain murine monoclonal immunotoxins, each reactive with different epitopes of HER-2 receptor, were tested for cytotoxic activity. The immunotoxins produced 1.2-4.6 logs of cytotoxicity in limiting dilution clonogenic assays with 2 breast cancer cell lines that overexpressed HER-2. Cytotoxicity did not correlate with immunoglobulin isotype, binding affinity, relative position of epitopes or internalization of the anti-HER-2 immunotoxins. Interestingly, the most and least effective immunotoxins bound to epitopes in very close proximity. Competitive binding assays with unconjugated antibodies have previously indicated that our antibodies recognized epitopes that are arranged in a linear array. To orient this relative epitope map, deletions were prepared in the HER-2/neu gene and these mutant constructs were expressed in NIH3T3 cells. Epitope expression was determined by antibody binding and radioimmunoassay. Epitopes targeted by the PB3, 454C11 and NB3 antibodies are localized N-terminal to the epitopes recognized by ID5, BD5, 741F8 and 520C9 antibodies. The 2 non-conformational epitopes PB3 and NB3 were localized to regions corresponding to amino acides 78-242 of the p185(HER-2) protein.

3T3 Cells↗
PubMed2005

High epitope density in a single protein molecule significantly enhances antigenicity as well as immunogenicity: a novel strategy for modern vaccine development and a preliminary investigation about B cell discrimination of monomeric proteins.

Although early studies have shown a close correlation between epitope density and epitope-specific humoral immune responses, few attempts have been made to quantitatively compare the antigenic and immunogenic differences between protein molecules bearing low or high degrees of epitope density, nor have studies quantitatively investigated the mechanism of B cell discrimination of monomeric antigens. In this study, we prepared glutathione S-transferase (GST) fusion proteins bearing various copies of the M2e epitope from the influenza virus M2 protein [GST-(M2e)8, GST-(M2e)4 and GST-(M2e)1], which were used to detect and compare the real-time kinetic binding with M2e-specific mAb by surface plasma resonance. Our data show clearly that fusion proteins bearing higher M2e epitope density resulted in higher average avidity for M2e-specific mAb. Furthermore, it was observed that fusion proteins bearing high M2e epitope density could induce polyclonal antibodies (pAb) with enhanced an average affinity constant (KA) for M2e epitope peptide compared to fusion proteins bearing low epitope density. The average KA of pAb induced by GST-(M2e)8 (3.08 x 10(8) M(-1) or 9.96 x 10(8) M(-1)) was up to two orders of magnitude greater than the average KA of pAb induced by GST-(M2e)1 (2.00 x 10(6) M(-1) or 3.43 x 10(6) M(-1)). Thus, the data presented here demonstrate that high epitope density in a single protein molecule significantly enhances antigenicity and immunogenicity. These findings enrich our knowledge of how epitope density might relate to the recognition, activation and antibody production processes of epitope-specific immature B cells.

Animals↗
PubMed2002

Viral vector-mediated delivery of competing glycosyltransferases modifies epitope expression cell specifically.

The glycoconjugate epitopes 3-fucosyl-N-acetyllactosamine (CD15) and sulfoglucuronylcarbohydrate (SGC) mediate cell adhesion events in several systems, and are regulated both spatially and temporally during cerebellar development. In cotransfection studies using COS-1 cells, competition between glycosyltransferases that utilize a common precursor involved in the final synthetic steps of these epitopes, can modulate epitope expression. For example, cotransfection of rat alpha1,3-fucosyltransferase IV (Fuc-TIV) and either rat glucuronic acid transferase P (GlcAT) or pig alpha1,3-galactosyltransferase (GalT) resulted in the dominance of either SGC or GalalphaGal epitope expression, respectively, with blockage of CD15 epitope expression. Viral vectors expressing these glycosyltransferases were used to determine whether competition plays a role in establishing epitope dominance in cerebellar cells, and whether overexpression of competing glycosyltransferases could be used to block epitope expression. Infection of cerebellar astrocytes with viral vectors expressing either Fuc-TIV, or Fuc-TIX, caused dramatic increases in CD15 expression in the presence of continued endogenous SGC epitope expression. Likewise, viral transduction with GalT resulted in GalalphaGal expression without affecting endogenous CD15 or SGC expression. Thus, competition between these enzymes does not appear to play a role in establishing epitope expression in astrocytes, and transduction of these enzymes does not provide a method of blocking the expression of endogenous epitopes. In contrast to what was observed for astrocytes, infection with viral vectors expressing either Fuc-T, GlcAT, or GalT did not result in significant expression of the relevant epitopes (CD15, SGC or GalalphaGal, respectively) on granule neurons. These results suggest a different complement of precursors are present in granule neurons and astrocytes, presumably due to the presence of different complements of glycosyltransferases in these cells.

Animals↗
PubMed1996

Induction and persistence of a cytotoxic T lymphocyte (CTL) response against a herpes simplex virus-specific CTL epitope expressed in a cellular protein.

CD8+ cytotoxic T-lymphocytes recognize small epitope peptides in association with MHC class I molecules expressed on the cell surface. In this study, we have determined whether an 8 amino acid viral CTL epitope, when expressed in a cellular protein, can be appropriately processed, presented, and recognized by the corresponding epitope-specific CTL and whether it is capable of inducing a CTL response in vivo. An H-2Kb-restricted CTL epitope from herpes simplex virus type 1 (HSV-1) glycoprotein B (gB epitope, residues 498-505) was cloned into the mouse dihydrofolate reductase protein (DHFR) at amino acid position 87. The recombinant DHFRs were expressed in vaccinia virus recombinants. To distinguish the recombinant DHFR proteins from the endogenous DHFR, an antibody epitope, recognized by monoclonal antibody PAb 901 and derived from simian virus 40 (SV40) T antigen was tagged to the C-termini of recombinant DHFR proteins. In vivo expression of recombinant DHFR was demonstrated by immunoprecipitation with the monoclonal antibody PAb 901. The H-2b cells infected with recombinant vaccinia virus expressing the recombinant DHFR were specifically lysed by gB epitope-specific CTL. Furthermore, the recombinant DHFR was functional in inducing a long lasting HSV gB epitope-specific CTL response upon immunization of C57BL/6 (B6) mice. These results indicate that a viral epitope expressed in a cellular protein can be efficiently processed, presented, and recognized by epitope-specific CTL and show that cellular proteins expressing CTL epitopes can be used for induction of CD8+ T lymphocyte responses.

Animals↗
PubMed1999

Natural variation of equine infectious anemia virus Gag protein cytotoxic T lymphocyte epitopes.

Two defined cytotoxic T lymphocyte (CTL) epitopes from equine infectious anemia virus (EIAV)-infected horses, equine leukocyte alloantigen (ELA)-A5.1-restricted epitope 18a, and ELA-A9-restricted epitope 28b-1 were evaluated for conservation among three wild-type EIAV strains. Epitope 18a variation occurred in all three wild-type EIAV strains, while epitope 28b-1 varied in one strain. Further, 12% amino acid changes occurred in the Gag proteins of a recently isolated wild-type strain, documenting a much greater Gag protein variation than previously reported. Evaluation of epitope 18a among two virus isolates from sequential disease episodes in a single horse, H513 (ELA-A5.1/A8), demonstrated that no variation that affected CTL recognition occurred. H513 PBMC had CTLm to epitope 18a before the occurrence of disease episodes caused by viruses expressing epitope 18a; however, the frequencies were low (5-15/10(6) PBMC). Later in infection there was an absence of disease episodes associated with an increase in CTLm frequency to EIAV(WSU5)-infected targets, but not epitope 18a-pulsed targets. Therefore, if CTLm to EIAV epitopes were involved in maintaining the carrier state in H513, they recognized epitopes other than 18a.

Amino Acid Sequence↗
PubMed1996

In vivo induction of cytotoxic T lymphocytes specific for a single epitope introduced into an unrelated molecule.

Cytotoxic T lymphocytes (CTLs) recognise antigenic peptides in the context of major histocompatibility complex (MHC) class I molecules on virus-infected cells. The formation and transportation of antigenic peptides to class I MHC in the cells are multi-step reactions known as antigen processing. In order to design a good DNA vaccine, it is important to dissect the specificity of antigen processing. Here we describe the construction of an epitope-based plasmid vector as a device to investigate antigen processing in transfected cells. The epitope-based plasmid vector was constructed by insertion of an epitope-encoding minigene into the lacZ gene. We used a CTL epitope on influenza A virus nucleoprotein (NP366-374 epitope) as a model. Upon transfection, the epitope-based plasmid vector induced the expression of NP epitope antigenically as well as immunogenically. Immunization of mice with plasmid-transfected cells was able to induce NP epitope-specific CTLs in vivo. Moreover, the plasmid vector functioned as a gene vaccine; NP epitope-specific CTLs were primed in vivo upon transfection of the vector into dermis by electroporation. The results suggest that this epitope-based DNA delivery system may provide a new strategy for in vivo induction of epitope-specific CTLs to investigate antigen processing and presentation.

Amino Acid Sequence↗
PubMed1995

Induction of a cellular immune response to a defined T-cell epitope as an insert in the flagellin of a live vaccine strain of Salmonella.

Attenuated strains of Salmonella have been used as vaccines to deliver heterologous antigens mainly to generate a humoral immune response. However, little is known about their ability to induce a cell-mediated immune response to the T-cell epitopes of another infectious agent or how optimally to deliver these epitopes to the host immune system. In order to study this question, a well defined MHC class II-restricted epitope (residues 88-103) from moth cytochrome C (MCC) was inserted into the central hypervariable domain of the flagellin of an attenuated strain of Salmonella dublin. The resulting flagellin was exported to the bacterial surface and polymerized into flagellar filaments that contained multiple copies of the MCC epitope. When flanked by Lys-Lys cathepsin B cleavage sites to facilitate its proteolytic release within the endosomal compartment of antigen-presenting cells, the MCC-chimeric flagellin epitope was efficiently processed in vitro by mouse peritoneal macrophages and presented to 2B4 T-hybridoma cells (specific for the MCC epitope 88-103). Stable expression of the epitope and a higher immune response was obtained in H-2k mice by integrating the chimeric flagellin gene into the chromosome of the vaccine strain. Bacteria with MCC-chimeric flagellins that were expressed from a stable chromosomal locus and flanked by cathepsin B cleavage sites were cleared more rapidly from the livers and spleens of transgenic mice with T-cell receptor (TCR) alpha and beta chains specific for the MCC epitope than were bacteria lacking the epitope. Antigen processing and presentation of class II-restricted epitopes expressed as chimeric proteins by attenuated bacterial vaccine vectors may be facilitated by the presence of endosomal protease cleavage sites on each side of the epitope and by chromosomal integration of the coding sequence.

Amino Acid Sequence↗
PubMed2006

Involvement of Ssp-4-related carbohydrate epitopes in mammalian cell invasion by Trypanosoma cruzi amastigotes.

We examined whether the expression of Ssp-4-related carbohydrate epitopes defined by monoclonal antibodies 1D9 and 2B7 was related to cell invasion by Trypanosoma cruzi amastigotes from different isolates and whether the highest expression of the epitope defined by MAb 1D9 would confer greater infectivity. Confocal microscopy showed that both epitopes localize to the membrane of amastigotes from 569, 588, 573, 587 and SC2005 isolates, similar to the G isolate, whereas the CL isolate showed a punctate and diffuse staining. Flow cytometry revealed inter- and intra-isolate variable expression of these epitopes. Apart from the lower expression of MAb 2B7 epitope by intracellular amastigotes of the SC2005 isolate, amastigotes from chagasic patient isolates expressed both epitopes similar to the G isolate, in contrast to CL isolate, that showed lower expression of both epitopes. MAb 1D9 did not react with CL isolate on immunoblots and reacted poorly with 588 and 587 parasites. MAb 2B7 preferentially reacted with an epitope on an 84 kDa component in G and 573 isolates. Invasion assays revealed that despite the fact that amastigotes from chagasic patient isolates displayed high levels of the epitope defined by MAb 1D9, only isolate 588 invaded host cells in levels comparable to that of isolate G. Both MAbs specifically inhibited cell invasion by G and 588, but not CL. These results suggested that the highest expression of MAb 1D9 epitope was not sufficient to confer higher infectivity on the isolate, and besides the two epitopes, other factors may modulate the invasiveness of extracellular amastigotes from the different isolates.

Adult↗
PubMed1998

A phosphatase activity in Xenopus oocyte extracts preferentially dephosphorylates the MPM-2 epitope.

MPM-2 antigens are a large family of mitotic phosphoproteins that contain similar phosphoepitopes recognized by the anti-phosphoepitope antibody MPM-2 (MPM-2 epitopes). These proteins are phosphorylated during M phase induction and dephosphorylated from the onset of anaphase through interphase. Since biochemical characterization of the MPM-2 epitope phosphatase requires a specific assay for its activity, we tested different methods for measurement of the MPM-2 epitope phosphatase activity in crude cell lysates. First, an ELISA-based assay was designed that measured the phosphatase-induced reduction of the MPM-2 reactivity in crude M phase cell lysates. Using this assay to follow the phosphatase activity during sequential chromatography of Xenopus oocyte extracts, one predominant peak of phosphatase activity was detected which was separated from the majority of PP1 and PP2A activities. This phosphatase activity dephosphorylated the MPM-2 epitope on multiple MPM-2 antigens. The second method measured dephosphorylation of cdc25, a known MPM-2 antigen. Two major peaks of cdc25 dephosphorylating activities were detected during the sequential chromatography, one that copurified with the major peak of MPM-2 epitope phosphatase activity, and the other with the major peak of PP2A activity. Finally, we examined whether GST-MPM2, a fusion protein between glutathione S-transferase and a 19-residue peptide that contained two representative MPM-2 epitope sequences, could be dephosphorylated efficiently and specifically by the major MPM-2 epitope phosphatase activity in Xenopus oocyte extracts. Neither the crude extract nor the partially purified MPM-2 epitope phosphatase activity efficiently dephosphorylated the MPM-2 epitope on GST-MPM2. These results demonstrate that the ELISA-based assay preferentially detects the MPM-2 epitope phosphatase activity in crude cell lysates which may represent a physiological MPM-2 epitope phosphatase.

Animals↗
PubMed2003

Specificity of autoantibodies to epitopes of myelin proteins in multiple sclerosis.

An autoimmune response to one or more myelin-protein components is thought to be part of the pathogenesis of multiple sclerosis (MS). The immunodominant-autoantibody epitope may be localized on a linear peptide segment, on a conformation-sensitive epitope, or on an epitope resulting from post-translational modifications. Primary, secondary, and tertiary structures of myelin proteins may determine the specific site for binding of autoantibodies. A myelin protein-specific autoantibody can bind to either a linear or conformational epitope, whereas all of the T cell epitopes are linear. At present, the conformational epitopes of myelin proteins have not been identified; most of the methods used to identify the myelin-protein epitopes corresponding to the pathogenesis of multiple sclerosis are involved in the linear epitope mapping. Polymorphism or mutations may cause inappropriate expression of the myelin proteins with alterations to their linear and/or conformational epitopes, and make them susceptible to autoantibody binding, especially if these changes occur at the surface of the protein. This review focuses on the specificity of autoantibodies to the epitopes of myelin proteins and correlates this to the structures of proteins. Factors that influence the expression of myelin-protein epitopes such as the alpha-helical or beta-sheet structure of the protein, the tri-proline site, and the post-translational modifications as well as physicochemical properties of amino acid changed are included.

Antibody Specificity↗
PubMed2001

A monoclonal antibody specific for a carbohydrate epitope recognizes an IgE-binding determinant shared by taxonomically unrelated allergenic pollens.

Carbohydrate epitopes are capable of binding human IgE from allergic subjects and these epitopes play a role in the cross-reactivity between allergens from unrelated sources. A monoclonal antibody (5E6), specific for a carbohydrate epitope detectable on components of Cupressus arizonica pollen extract, has been produced and characterized. To study the relationship between the epitopes recognized by the monoclonal antibody and by IgE from allergic subjects. To investigate the presence of such carbohydrate IgE determinant in extracts from 21 pollen species belonging to 16 taxonomically related and unrelated families, by means of the monoclonal antibody. IgG-depleted fraction from protein G-purified human allergic serum was obtained. The monoclonal antibody and the IgE from the purified fraction were tested on two glycoproteins, polyamine oxidase and ascorbate oxidase, adsorbed on the ELISA plates. The relationship between the monoclonal- and the IgE-recognized epitopes was investigated by ELISA-competition experiments. Analysis of the distribution of this carbohydrate epitope was performed by direct binding of the monoclonal antibody onto the various extracts. The monoclonal antibody and the IgE were able to bind carbohydrate epitopes on the two plant glycoproteins, ascorbate oxidase and polyamine oxidase. Polyamine oxidase shows only one N-glycosilation site whose carbohydrate moiety seems to be composed of a branched chain of seven ordered sugars, i.e. two N-acetyl-D-glucosamine-, three mannose-, one fucose- and one xylose-residues. This structure bears the epitope recognized by mAb 5E6. Human IgE from the IgG-depleted fraction were found capable of inhibiting the monoclonal antibody binding. The allergenic epitope identified was shared by a large number of extracts with different levels of reactivity (OD490 ranging from 0.110 to 2.060). Our data support the finding that a monoclonal antibody specific for a carbohydrate epitope of Cupressus arizonica pollen extract detects an epitope which is also recognized by IgE from allergic subjects. This characterized reagent could be a useful tool for studying distribution of cross-reactive carbohydrate determinants in allergenic pollen extracts and their components.

Allergens↗
PubMed2002

Linear IgE epitope mapping of the English walnut (Juglans regia) major food allergen, Jug r 1.

BACKGROUND: Peanut and tree nut allergies can be life-threatening, and they appear to be growing in prevalence. Jug r 1, a 2S albumin seed storage protein, was previously characterized as a major English walnut food allergen. OBJECTIVE: We sought to identify the linear IgE-binding epitopes of Jug r 1 and to determine which, if any, amino acids are necessary for this binding to occur. METHODS: Pools of sera from walnut-allergic patients and overlapping peptides synthesized on an activated cellulose membrane were used to screen for IgE-binding epitopes. Mutational analysis of the immunodominant epitope was carried out through single and multisite amino acid substitutions. Inhibition assays were performed through use of affinity-purified IgE, soluble forms of the epitope peptide, and the recombinant 2S albumin, rJug r 1. RESULTS: One immunodominant linear epitope was identified. Amino acid mutations to the epitope demonstrated that the residues RGEE, at positions 36 through 39, were minimally required for IgE binding. Probing of this epitope with sera from each of 20 patients revealed 15 of the sera to be positive. Binding of patients' IgE to the epitope was inhibited with a soluble form of the peptide; however, soluble peptide did not completely inhibit the binding of IgE to the intact rJug r 1. CONCLUSION: One major linear IgE-reactive epitope and its critical core amino acid residues have been identified. Mutation of any of these core amino acids resulted in loss of IgE binding to the epitope, and this points toward the feasibility of reducing allergenicity in genetically modified walnuts. However, strong evidence for the existence of conformational epitopes was also obtained.

Allergens↗