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PubMed1996

Epitope maps of the Escherichia coli heat-labile toxin B subunit for development of a synthetic oral vaccine.

Linear B- and T-cell epitopes spanning all 103 amino acids of the Escherichia coli heat-labile toxin B subunit (LT-B) were assessed in mice orally immunized with native LT or with recombinant Salmonella enteritidis expressing LT-B. Oral administration of native LT induced mucosal immunoglobulin A (IgA) antibodies reactive with an epitope at residues 85 to 91, while IgA induced by recombinant Salmonella LT-B reacted with an epitope at residues 36 to 44. Serum IgG anti-LT-B antibodies from mice orally immunized with either LT or with recombinant Salmonella LT-B were directed to both epitopes. A single T-cell epitope spanning residues 34 to 42 was identified by T-cell proliferative and cytokine responses. When a 20-mer peptide (residues 26 to 45) with B- and T-cell epitopes was given orally to BALB/c (H-2(d)) and B10 congenic (I-A(d), I-A(b), and I-A(k)) mice, significant fecal IgA and serum IgG anti-LT-B antibodies were induced. The peptide also induced LT-B-specific T-cell proliferative responses in these mice. Orally administered LT-B peptide (residues 26 to 45) induced a cytokine profile indicative of both T helper 1- and 2-type cells. The remarkable immunogenicity of this 20-mer peptide makes it a candidate for a vaccine to protect against enterotoxigenic E. coli.

Administration, Oral↗
PubMed1995

Epitope mapping of monoclonal antibodies to tumor necrosis factor-alpha by synthetic peptide approach.

A monoclonal antibody (mAb) against human tumor necrosis factor-alpha (TNF-alpha), designated 3B10, neutralizes biological activity of TNF-alpha, while another anti-TNF-alpha mAb 10F10 does not. In Western blot analysis, both mAbs bound to SDS-denatured TNF-alpha, indicating that the epitopes recognized by the mAbs are sequential but not conformational. To map precisely the epitopes of the mAbs, 76 overlapping octapeptides corresponding to an entire sequence of TNF-alpha were synthesized and their abilities to react with the mAbs were examined by enzyme-linked immunosorbent assay (ELISA). 3B10 bound to only one peptide at position 81-88 of TNF-alpha, SRIAVSYQ, whereas 10F10 was reactive with three overlapping peptides, ANALLANG (33-40), ALLANGVE (35-42), and LANGVELR (37-44). These results demonstrate that the 81-88 and 37-40 regions are important for the recognition of TNF-alpha by 3B10 and by 10F10, respectively. In solid-phase ELISA, 3B10 inhibited the binding of TNF-alpha to soluble TNF receptors, sTNF-RI and sTNF-RII. In contrast, 10F10 exerted little effect on the binding. TNF-alpha was detected by sandwich-type ELISA where 3B10 alone was used for both capture and detection, suggesting that 3B10 did not interfere with the trimer formation of TNF-alpha. The results obtained in this study suggest that the 81-88 region of TNF-alpha may participate in the receptor binding and that 3B10 neutralizes the activities of TNF-alpha by blocking the region.

Amino Acid Sequence↗
PubMed2003

Epitope mapping of pneumococcal surface protein A of strain Rx1 using monoclonal antibodies and molecular structure modelling.

Pneumococcal surface protein A (PspA) is an antigenic variable vaccine candidate of Streptococcus pneumoniae. Epitope similarities between PspA from the American vaccine candidate strain Rx1 and Norwegian clinical isolates were studied using PspA specific monoclonal antibodies (mAbs) made against clinical Norwegian strains. Using recombinant PspA/Rx1 fragments and immunoblotting the epitopes for mAbs were mapped to two regions of amino acids, 1-67 and 67-236. The discovered epitopes were visualized by modelling of the PspA:Fab part of mAb in three dimensions. Flow cytometric analysis showed that the epitopes for majority of mAbs were accessible for antibody binding on live pneumococci. Also, the epitopes for majority of the mAbs are widely expressed among clinical Norwegian isolates.

Antibodies, Monoclonal↗
PubMed1995

Characterization and epitope mapping of four HLA class II reactive mouse monoclonal antibodies using transfected L cells and human cells transfected with mutants of DQB1*0302.

To study epitopes of HLA class II molecules, four mouse monoclonal antibodies (mAbs) 13B6, 17F8, 19A1 and 12G6 were made using HLA-DQ8, DP2 and DP4 expressing mouse transfectants for immunization. Three of the mAbs, 13B6, 17F8 and 19A1, bound to all DQ1, 4, 8 or 9 positive B-lymphoblastoid cell lines (B-LCLs) and transfectants tested, i.e. cells carrying the DQB1 genes 0302-3, 0401-2, 0501-3, 0601-4 and 0609 irrespective of the accompanying DQA1 gene. These DQB1 genes code for the shared amino acids (aa) GVY in position 45-47 of the DQ beta chain. DQ1+4+8+9 specific (IIB3) and DQ3 specific (IVD12) reference mAbs inhibited binding of all three mAbs. Testing 13B6, 17F8 and 19A1 with cells made using aa substitutions in various positions of DQ beta 1*0302 indicated involvement of aa 45 in the epitopes of all three mAbs. The last mAb (12G6) bound to all B-LCLs and all DP transfected cells. However, only some DR transfectants and a single DQ transfectant (carrying DQA1*0201 and DQB1*0202) bound mAb 12G6. This reactivity pattern correlates with a shared sequence of aa (RFDSDVGE) in position 39-46 of DR- and DQ- and 37-44 of DP beta chains.

Amino Acid Sequence↗
PubMed1993

A novel LFA-1 activation epitope maps to the I domain.

A panel of 21 alpha-subunit (CD11a) and 10 beta-subunit (CD18) anti-LFA-1 mAbs was screened for ability to activate LFA-1. A single anti-CD11a mAb, MEM-83, was identified which was able to directly induce the binding of T cells to purified ICAM-1 immobilized on plastic. This ICAM-1 binding could be achieved by monovalent Fab fragments of mAb MEM-83 at concentrations equivalent to whole antibody, was associated with appearance of the "activation reporter" epitope detected by mAb 24, and was completely inhibited by anti-ICAM-1 and LFA-1 blocking mAbs. The epitope recognized by mAb MEM-83 was distinct from that recognized by mAb NKI-L16, an anti-CD11a mAb previously reported to induce LFA-1 activation, in that it was constitutively present on freshly isolated peripheral blood mononuclear cells and was not divalent cation dependent for expression. The ICAM-1 binding activity induced by mAb MEM-83 was, however, dependent on the presence of Mg2+ divalent cations. Using an in vitro-translated CD11a cDNA deletion series, we have mapped the MEM-83 activation epitope to the "I" domain of the LFA-1 alpha subunit. These studies have therefore identified a novel LFA-1 activation epitope mapping to the I domain of LFA-1, thereby implicating this domain in the regulation of LFA-1 binding to ICAM-1.

Antibodies, Monoclonal↗
PubMed1998

Epitope mapping of human centromere autoantigen centromere protein C (CENP-C); heterogeneity of anti-CENP-C response in rheumatic diseases.

OBJECTIVE: To analyze the autoantigenic epitopes of centromere protein C (CENP-C) recognized by anti-centromere antibodies (ACA). METHODS: A series of truncated peptides of human CENP-C were expressed in Escherichia coli and immunoblotting analysis was performed with 45 ACA positive sera obtained from patients with different types of autoimmune diseases. RESULTS: Although 9 epitopes were scattered over the entire molecule, the N terminus was immunodominant for IgG and IgM classes and the C terminus was dominant for IgG class. Both epitopes were separately located within the instability and centromere targeting domains in vivo, or the oligomerization-accessible and homodimerization domains in vitro, respectively. In contrast, minor epitopes were clustered at the internal DNA binding domain. A number of patterns of immunoreactivities against 3 representative antigenic sites by IgG or IgM class antibodies were found. CONCLUSION: The results indicated the existence of different anti-CENP-C responses in rheumatic diseases and a possible correlation between antigenic regions and functional sites in the CENP-C antigen.

Autoantigens↗
PubMed1992

Epitope mapping of monoclonal antibodies to bovine prolactin.

The epitopes recognized by three monoclonal antibodies generated to sheep prolactin were determined by evaluating their cross-reactivities by immunodot analysis with 14 mutants of bovine prolactin, in which individual amino acids had been deleted or substituted. Mutations were made throughout the molecule and included disruption of the amino-terminal, carboxyl-terminal, and central disulfide loops. Lack of immunoreactivity was taken as an indication that the site of mutation was part of the epitope. Antibody 6F11 reacted with all bovine prolactin mutants tested, except those in which the carboxyl-terminal cysteine (position 199) was substituted by a serine. Antibodies 5G2 and 4C10 reacted with all of the bovine prolactin mutants, except those in which the amino-terminal cysteine (position 4) was substituted by a serine. Western blot analysis of sheep, squirrel monkey, and rat prolactins with the monoclonal antibodies revealed that 5G2 and 4C10 were specific for sheep prolactin, whereas antibody 6F11 cross-reacted with prolactins from all three species. The mitogenic activity of sheep or rat prolactin in the Nb2 bioassay was determined in the presence of the antibodies to determine whether the epitopes were part of the functional domains of these prolactins. The bioactivity of sheep prolactin (0.4 ng/ml) was unaffected by the monoclonal antibodies [0.01-1 microgram immunoglobulin G (IgG)/ml], whereas the bioactivity of rat prolactin (1.25 ng/ml) was inhibited by 6F11 with an apparent 50% inhibitory concentration of 0.25 microgram IgG/ml. These results indicate that monoclonal antibodies 5G2 and 4C10 cross-react with a species-specific region of the amino-terminal disulfide loop of bovine prolactin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2003

Epitope mapping on E1alpha subunit of pyruvate dehydrogenase complex with autoantibodies of patients with primary biliary cirrhosis.

BACKGROUND: A major mitochondrial autoantigen recognized by sera of patients with primary biliary cirrhosis (PBC) is dihydrolipoamide acetyltransferase (E2) of the pyruvate dehydrogenase complex (PDH). The alpha subunit of pyruvate decarboxylase (E1alpha) of PDH is also recognized in some E2-reactive PBC sera, suggesting that the occurrence of autoimmunity against Elalpha is subsequent to that against E2. METHODS. To investigate the mechanism inducing autoimmunity against E1alpha, we surveyed immunoreactive sequences of E1alpha by ELISA with synthesized oligopeptides, and determined minimum amino acid residues for each determinant. RESULTS: The major determinants of E1alpha appeared to reside in its N-terminal region, apparently forming 'nested epitopes', and all E1alpha-reactive PBC sera tested recognized these regions. Minor epitopes were also found scattered throughout the entire sequence. The reactivities of these minor epitopes to individual PBC sera were proportional to those of the major epitopes. All the epitopes were located in hydrophilic regions of E1alpha, and many of them were out of the known functional domains (TPP-binding domain, subunit interaction site, and phosphorylation sites) whose structures are phylogenically well conserved. Furthermore, the sequences of many epitopes appeared to be specific to humans. CONCLUSION: These observations suggest that determinant spreading might underlie the autoimmunity against Elalpha.

Amino Acid Sequence↗
PubMed1997

Peptide epitope mapping in vaccine development: introduction.

Protection from infectious disease by the host immune response requires specific molecular recognition of unique antigenic determinants of a given pathogen. An epitope is an antigenic determinant which: 1) specifically stimulates the immune response (either B or T cell mediated); and 2) is acted upon by the products of these protective mechanisms. In B cell immunity, antibodies produced from stimulation by specific epitopes recognize and bind to these same antigenic structures. Identification of protective epitopes is extremely valuable to successful vaccine development. In order to be protective these antibodies must, in addition to recognition and binding, interfere with some vital step in pathogenesis such as adherence or toxin action. Protein B cell epitopes are frequently composed of the side chains (R-groups) of the amino acids found at solvent-exposed surfaces. These epitopes are classified as continuous (also linear or sequential) if composed of a single antibody-recognizing element located at a single locus of the primary structure. They are discontinuous (or assembled) if more than one physically separated entity is involved. T cell epitopes are peptides on the surface of antigen-presenting cells (macrophages, dendritic cells, and B cells) that are bound to major histocompatibility proteins; the T cell recognizes this peptide-MHC complex.

Animals↗
PubMed1988

Canine host range and a specific epitope map along with variant sequences in the capsid protein gene of canine parvovirus and related feline, mink, and raccoon parvoviruses.

Canine parvovirus (CPV) is a recently recognized pathogen of dogs that is similar to the long-recognized feline, mink, and raccoon parvoviruses. Relationships between the viruses determined from DNA sequences of the capsid protein genes of 10 virus isolates showed the CPV isolates to be closely related to the other viruses, although comprising a distinct group. No immediate ancestor of CPV was observed amongst the mink, cat, or raccoon viruses examined. Three different directly repeated sequences were present within the noncoding region downstream from the capsid protein genes. Analysis of recombinants between CPV and feline panleukopenia virus at restriction sites within the capsid protein genes mapped a CPV-specific neutralization epitope on the virus capsid, differences in the pH dependence of hemagglutination, and part of the determinant of canine host range between 59 and 64 genome map units (m.u.). Those differences were therefore the result of up to three nucleotide or predicted amino acid sequence differences in that region. A second region between 64 and 73 m.u., which may affect the viability of certain recombinant viruses, contained four nucleotide differences, one of which was a coding change.

Animals↗
PubMed2001

Random-peptide libraries and antigen-fragment libraries for epitope mapping and the development of vaccines and diagnostics.

Random peptide libraries and antigen-fragment libraries (also known as gene-fragment libraries) have been used to identify epitopes on protein antigens. These technologies promise to make significant contributions to diagnostic and vaccine development. Researchers in a number of labs have shown that phage selected from libraries with protective antibodies, raised against whole antigen, can be used as immunogens to stimulate antibody responses that bind native antigen and provide protection in vivo. Others have used the sera of patients with idiopathic diseases to screen libraries, and by this approach have identified candidate antigens involved in immune disease. These may prove useful for diagnosis and, possibly, in determining disease etiology.

Antibodies↗
PubMed1998

Epitope mapping of anti-MUC1 mucin protein core monoclonal antibodies.

A panel of 56 murine monoclonal antibodies against the MUC1 mucin was analysed for reactivity against purified MUC1 mucin and synthetic MUC1 mucin protein core-related peptides. In an ELISA assay, with purified normal urinary mucin as the target antigen, 48/56 (86%) of antibodies showed positive reactivity. A smaller proportion of antibodies, 31/56 (55%), reacted with a bovine serum albumin conjugate containing the synthetic peptide, APDTRPAPG. This peptide represents the hydrophilic region of the tandem repeat sequence of the MUC1 mucin core. These 31 anti-mucin core antibodies were then evaluated for reactivity with a set of overlapping heptamers with sequences based upon that of the 20-amino acid tandem repeat of the MUC1 protein core. For this purpose, the 'Pepscan' procedure was employed to detect antibody binding to peptides tethered at their carboxy termini to the polypropylene pins. Synthetic peptides binding to these antibodies were identified for 29 of the samples provided and epitopes or 'minimum binding units' were deduced for these. Most antibodies bound to epitopes of 4 amino acid residues with the sequence RPAP occurring most frequently. The results confirm that the short hydrophilic region PDTRPAP in the MUC1 mucin core is immunodominant in the induction of antibodies to the MUC1 mucin since half of the antibodies submitted reacted with this peptide.

Amino Acid Sequence↗
PubMed1999

Epitope mapping of Aleutian mink disease parvovirus virion protein VP1 and 2.

Six overlapping fragments of the Aleutian Mink Disease parvoVirus (AMDV) virion protein VP1 and 2 (VP1/2) gene were inserted into the expression vector pMAL-c2. Four of the clones carried large overlapping fragments covering the entire VP1/2 gene. The remaining two clones covered specifically chosen regions within the VP1/2 gene. Using a Western blotting detection system, sera from AMDV-infected mink were tested against the recombinant polypeptides. These studies showed reactions primarily directed against the two AMDV polypeptides ranging from amino acids 297 to 518. Weaker reactions against other regions of the VP1/2 were also observed. The small fusion protein designed to cover the presumed AMDV VP1/2 loop 4 was purified by affinity chromatography and used to develop solid-phase immunoassays. Twelve small synthetic peptides were constructed and used as inhibitors. A peptide covering amino acids S428 to T448 was shown to block the reactivity of a pool of positive mink sera, indicating the presence of one dominant linear epitope.

Aleutian Mink Disease↗
PubMed1992

Monoclonal antibodies to the myogenic regulatory protein MyoD1: epitope mapping and diagnostic utility.

Monoclonal antibodies (MoAbs) were developed against recombinant wild-type murine MyoD1 protein. Each of 4 MoAbs was immunologically reactive with recombinant MyoD1 protein by enzyme-linked immunosorbent assay, and each specifically stained the nuclei of myogenic cells. Epitopes were mapped using fusion protein constructs with specific deletions of defined regions of the MyoD1 molecule. MoAb 5.2F recognized an epitope in the amino terminal region between amino acid residues (AAR) 3 and 56, whereas epitopes for MoAbs 1.1A, 5.4G, and 5.8A were in the carboxyl terminus (AAR 167-318) of the MyoD1 protein. The epitope for MoAb 5.8A was further delineated to AAR 170-209 by Western analysis and immunoprecipitation of in vivo transcribed and translated MyoD1 protein having specific deletions in the carboxyl terminus. The 5.8A epitope was ultimately localized to the region between AAR 180 and 189 of the protein by enzyme-linked immunosorbent assay using 10-amino acid residue synthetic peptides. This sequence is apparently unique to MyoD1 and has little homology to other myogenic regulatory proteins (myogenin, Myf5, Myf6, and MRF4). Transfection of cDNA for murine MyoD1 into a nonmuscle cell line conferred 5.8A reactivity, confirming the specificity of this reagent. MoAb 5.8A was then used to examine the expression of MyoD1 in normal and malignant human tissues. MyoD1 was not detected in any normal adult tissue but was detected in 25 of 25 histologically confirmed rhabdomyosarcomas. Staining was localized to the nucleus and showed marked heterogeneity between cells as well as differential staining within nuclei. Specific subcellular localization of 5.8A was further determined by immunoelectron microscopy, where antibody was found to localize to electron-dense areas, more frequently associated with the nuclear submembranous region. In addition to rhabdomyosarcomas, MoAb 5.8A stained 2 of 5 Wilms' tumors and one ectomesenchymoma, neoplasms known to contain myogenic elements. The 5.8A reagent was also of value in the accurate histopathological classification of 2 of 4 tumors previously diagnosed as extraosseous Ewing's sarcoma and 2 of 3 tumors diagnosed as undifferentiated sarcomas.

Amino Acid Sequence↗
PubMed1992

T and B cell epitope mapping of SM23, an integral membrane protein of Schistosoma mansoni.

SM23 is an integral membrane protein of the blood-vessel dwelling parasitic worm Schistosoma mansoni. This protein has been detected with antibodies in all stages of the parasite found in the human host, notably the lung stage, and therefore is of interest as a vaccine candidate. In addition SM23 has been shown to be a member of a proposed new superfamily of membrane proteins whose structures do not conform to the previously known classifications. To date there are 13 members including ME491 (CD63, Pltgp40), CD9 (p23), TAPA-1, CD37, CD53, MRC OX-44, CO-029, MRP-1, L6, the gene product of TI-1, the target of mAb AD-1, SM23, and SJ23 (the Schistosoma japonicum homologue). Most of these molecules except for those in the two blood vessel-dwelling parasites are found in membranes of hemopoietic and/or malignant cells and all have unknown function. In this study we used recombinantly expressed full-length and partial molecules as well as synthesized peptides to map T cell and B cell epitopes of SM23. The two predicted external hydrophilic domains were found to be highly immunogenic and contained several B cell epitopes. There were at least four T cell epitopes in the large hydrophilic domain. One segment of 23 amino acids contained both a T cell and B cell epitope as well as the putative glycosylation site. This particular segment was recognized by immune sera and cells of every mouse strain tested. The elucidation of these epitopes demonstrates the immunogenic nature of this molecule and raises questions as to the role of SM23 in the host/parasite relationship.

Amino Acid Sequence↗
PubMed1995

Localization of multiple functional domains on human PECAM-1 (CD31) by monoclonal antibody epitope mapping.

PECAM-1, a cell adhesion molecule of the immunoglobulin gene (Ig) superfamily, has been implicated in white cell transmigration, integrin activation on lymphocytes, and cell-cell adhesion. The purpose of this investigation was to identify specific regions of the PECAM-1 extracellular domain mediating these functions by identifying the location of epitopes of bioactive anti-PECAM-1 monoclonal antibodies. The binding regions of mAbs important in PECAM-1-mediated leukocyte transmigration (Hec 7.2 and 3D2) were mapped to N-terminal Ig-like domains. The epitopes of monoclonal antibodies that activated integrin function on lymphocytes were dispersed over the entire extracellular region, but those that had the strongest activating effect were preferentially localized to the N-terminus of the molecule. The binding regions of mAbs that blocked PECAM-1-mediated heterophilic L-cell aggregation were located either in Ig-like domain 2 (NIH31.4) or Ig-like domain 6 (4G6 and 1.2). Site-directed mutagenesis further pinpointed the epitope of the 4G6 mAb to a hexapeptide, CAVNEG, within Ig-like domain 6. These results demonstrate that PECAM-1 contains multiple functional domains. Regions within N-terminal Ig-like domains appear to be required for transmigration. In contrast, two distinct regions were implicated in L-cell mediated heterophilic aggregation.

Amino Acid Sequence↗