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PubMed1996

Distinct sub-populations of carcinoma-associated MUC1 mucins as detected by the monoclonal antibody 9H8 and antibodies against the sialyl-Lewis a and sialyl-Lewis x epitopes in the circulation of breast-cancer patients.

The cancer-associated epitope defined by the monoclonal antibody (MAb) 9H8 was shown to be closely related to the T antigen (Thomsen-Friedenreich antigen) by its sensitivity to 0-glycanase treatment of a mucin glycopeptide known to express this epitope. The reactivity with this glycopeptide increased upon neuraminiclase treatment, and among several MAbs tested for ability to block binding of the 9H8 antibody, the one specific for the T antigen was the most efficient. Out of 41 serum samples from breast-cancer patients, 11 showed elevated levels of the 9H8 epitope, and several sera also showed elevated levels of the cancer-associated carbohydrate epitopes sialyl-Lewis a and sialyl-Lewis x. By the use of antibodies specific for the MUC1 apoprotein (Ma552 and HMFG-2) it could be shown that these epitopes were attached to the MUC1 apoprotein in at least 4 of the cases. By combining antibodies specific to 9H8, sialyl-Lewis a and sialyl-Lewis x in catcher and tracer positions in several types of immunofluorometric assays, it was shown that the 9H8 epitope was rarely co-expressed with sialyl-Lewis a or sialyl-Lewis x epitopes an the same molecule, though all were expressed on MUC1 mucins. In fact, they can be considered as mutually exclusive epitopes, suggesting that these sera contained different populations of MUC1 mucins distinguishable by different sets of oligosaccharides. The existence of mutually exclusive carbohydrate epitopes on different MUC1 mucins in one and the same patient should be taken into account when designing immunoassays exploiting MUC1-reactive antibodies.

Antibodies↗
PubMed1995

The genotypic distribution of shared-epitope DRB1 alleles suggests a recessive mode of inheritance of the rheumatoid arthritis disease-susceptibility gene.

OBJECTIVE: To test whether the genotypic distribution of rheumatoid arthritis (RA)-associated DRB1 alleles suggests that the DRB1-associated disease-susceptibility gene has a recessive or additive (dominant) mode of inheritance. METHODS: Caucasian patients with RA and control subjects were recruited from a faculty outpatient practice. DRB1 typing was done by several DNA-based techniques: polymerase chain reaction (PCR), followed by dot-blot hybridization with sequence-specific oligonucleotides, conventional and PCR-based restriction fragment length polymorphisms (RFLPs), and a multiplex amplification-refractory mutation RFLP system. The genotypic distribution of shared-epitope DRB1 alleles was analyzed by antigen genotype frequency among patients. The analytical method postulates a linkage-disequilibrium model with a disease locus close to a marker locus and a marker allele in linkage disequilibrium with the disease-susceptibility allele. In this instance, the marker allele was defined alternatively by any DR4-group allele, by any DR4-group or DR1-group allele, by any DR4-group shared-epitope allele, by any DR4-group shared-epitope allele plus DRB1*0101, or by any shared-epitope DRB1 allele. Observed numbers were compared with those predicted for recessive mode or additive (dominant) mode of inheritance of the DRB1-associated RA disease-susceptibility gene. RESULTS: The genotypic distribution of shared-epitope DRB1 alleles (DRB1*0401, *0404, *0405, *0408, *0101, *0102, or *1001) fit that predicted for a recessive mode of inheritance and was significantly different from that predicted for an additive (dominant) mode. When the analysis was restricted to shared-epitope DR4 alleles alone (DRB1*0401, *0404, *0405, or *0408), the observed genotype numbers fit the recessive mode best. When DR1-group alleles were added to DR4-group alleles, or alternatively, when the major shared-epitope DR1 allele (*0101) was added to DR4-group shared-epitope alleles, there was a less significant deviation from the additive mode of inheritance. The reason for this was derived by comparison of observed genotype frequencies to those expected under Hardy-Weinberg equilibrium; there was a deficit of persons with DRB1*0401, *0101 and an excess of *0101,X. CONCLUSION: The genotypic distribution of shared-epitope DRB1 marker alleles suggests that the mode of inheritance of the DRB1-associated disease susceptibility gene must be recessive and not additive (dominant).

Adult↗
PubMed1991

T cell reactivity to an epitope of the mycobacterial 65-kDa heat-shock protein (hsp 65) corresponds with arthritis susceptibility in rats and is regulated by hsp 65-specific cellular responses.

Adjuvant arthritis (AA) can be induced in genetically susceptible rats by immunization with heat-killed mycobacteria suspended in mineral oil. From our analysis of arthritogenic T cell clone A2b, obtained from an arthritic Lewis rat and specific for the 180-188 epitope of mycobacterial 65-kDa heat-shock protein (hsp 65), the possible origin of AA was explained by the existence of a molecular mimicry of the 180-188 epitope with a cartilage-associated self antigen. We now have shown that Lewis rats respond to the 180-188 epitope after Mycobacterium tuberculosis immunization and that arthritis-resistant Fisher and (Lewis x Fisher)F1 rats, although major histocompatibility complex class II identical with Lewis, do not respond to this epitope. However, in rare cases of arthritis in Fisher rats, responses to the epitope were seen. We obtained no evidence for a defect at the level of antigen processing and presentation or for suppression in Fisher rats. Thus, non-responsiveness in Fisher rats was likely due to a difference at the level of the T cell repertoire. Previously, we have reported that pretreatment with hsp 65 in experimental arthritis, and not only in AA, caused resistance to arthritis induction. We now present evidence that immunization with hsp 65 or in vitro stimulation with hsp 65 may lead to inhibition of responses specific for epitope 180-188. Thus the hsp 65-induced resistance to arthritis is probably caused by the induction of regulatory control specifically targeted at the 180-188 epitope. Especially in rats that tend to focus their responses on the critical 180-188 sequence, such as Lewis, regulation seems to develop following immunization with hsp 65. Since recent evidence suggests that hsp 65 and also the 180-188 epitope have a role in human arthritic conditions, the present findings are expected to contribute to further experimentation directed at exploiting hsp 65 or its epitopes for the development of new therapeutical approaches in humans.

Animals↗
PubMed1997

MUC1 peptide epitopes associated with five different H-2 class I molecules.

We have previously described the induction of murine CD8+ major histocompatibility complex (MHC) class I-restricted cytotoxic T cells (CTL) recognizing the 20-amino acid repeat region of the human mucin 1 (MUC1) variable number of tandem repeats region (VNTR), a mucin greatly increased in expression in breast cancer and proposed as a target for immunotherapy. In that study, CTL could detect MUC1 peptides associated with the MHC of all nine strains examined, and we now report the different epitopes presented by five different MHC class I molecules. The epitopes were defined in CTL assays using peptide-pulsed phytohemagglutinin blasts or MHC class I-transfected L cells as targets; in addition, peptide binding assays and T cell proliferation studies were performed. Within the 20-amino acid VNTR, nine potential epitopes could be defined. The epitopes for the four MHC class I molecules [Kb (three epitopes), Dd, Ld and Kk] were closely related, all containing the amino acids PDTRPAP. For Db, three epitopes were identified, all containing APGSTAP. Most of the epitopes did not contain a consensus motif for the particular MHC class I allele, and bound with low 'affinity', compared with known high-affinity peptides. CD8+ T cell proliferation also occurred to the same MHC class I-presented epitopes. Finally, when conventional anchor residues were introduced into the peptides, peptide binding increased, whereas CTL recognition was either retained (Kb) or lost (Db) depending on the epitope.

Amino Acid Sequence↗
PubMed2003

Characterization of H+,K+-ATPase T cell epitopes in human autoimmune gastritis.

Human autoimmune gastritis (AIG) is an organ-specific inflammatory disorder leading to gastric atrophy and pernicious anemia. Gastric H+,K(+)-ATPase was identified as the autoantigen in both human disease and experimental murine AIG (EAIG). Studies of EAIG significantly contributed to current knowledge of human AIG, but to what extent EAIG mimics AIG is still debated, and the autoantigenic epitopes in AIG are yet unknown. This study aimed to identify the H+,K(+)-ATPase epitopes recognized by gastric T cell clones from AIG patients, to define their TCR Vbeta usage and epitope-induced cytokine response. Sixteen H+,K(+)-ATPase-reactive CD4+ gastric T cell clones of four AIG patients were tested for proliferation to overlapping 15-mer peptides spanning the a and beta chains of H+,K(+)-ATPase. We identified 6 epitopes in the a chain and 5 in the beta chain; TCR Vbeta usage was not restricted. Four (36%) of the 11 H+,K(+)-ATPase epitopes recognized in AIG were found to overlap with epitopes that are relevant in EAIG, including a previously described gastritogenic epitope. Gastric T cell recognition of the peptide epitopes resulted in secretion of Th1 cytokines. Our data suggest a striking similarity between human AIG and EAIG, at the epitope level, with regard to cytokine secretion and likely also with regard to pathogenic mechanisms.

Adult↗
PubMed2001

Diagnosis of human parvovirus B19 infections by detection of epitope-type-specific VP2 IgG.

In the B19 VP2 molecule an immunodominant heptapeptide epitope has been detected, recently for which IgG antibodies are synthesized exclusively in the acute phase of B19 infection. Using this acute-phase-specific epitope (KYVTGIN) a 2(nd)-generation epitope-type EIA was developed, which compares serum IgG activity for native VP2 capsids exhibiting conformational VP2 epitopes with IgG activity for the KYVTGIN epitope. In this study the diagnostic performance (clinical sensitivity and specificity) of the 1st and 2nd-generation epitope-type EIAs and of a peptide-based EIA utilising as antigen the KYVTGIN epitope alone was assessed in comparison with various high-quality IgM- and IgG- based B19 assays. Serum samples from 489 patients with B19-related symptoms and asymptomatic controls from three countries were studied. Among 323 patients with B19-IgG, 20% were diagnosed as acute infection, 73% had past immunity and 7% were not classified due to contradictory results among the different assays. The unclassified samples were explored for viral strain diversity by PCR and DNA sequencing but all sequences obtained were B19-like with variance of only a few per cent. The 2nd-generation epitope-type EIA had a diagnostic sensitivity of 98% and a diagnostic specificity of 94%. In combination with conventional approaches, the epitope-type assays increase greatly the accuracy of B19 serodiagnosis.

Acute Disease↗
PubMed1990

Mapping of the Alz 50 epitope in microtubule-associated proteins tau.

Alz 50 and seven other monoclonal antibodies have been shown to react with both tau and Alzheimer brain proteins of molecular mass 60-70 kDa. The location of some of the epitopes of these antibodies (Alz 50, Tau-2, NP14, Ab 636.7) on the tau molecule is unknown, whereas those of others (Tau 60, Tau 14, Tau-1, Tau 46) have recently been demonstrated in fetal human tau at amino acid residues 60-72, 83-120, 131-140, and 315-352. To determine the location of the unknown epitopes, human tau was digested with chymotrypsin and trypsin, and the bovine microtubule fraction was incubated with chymotrypsin. Comparison of the immunoblots of chymotryptic digested tau with those of untreated preparations showed that the Alz 50 epitope was more sensitive than other tau epitopes to proteolysis. Cleavage of a 3-4 kDa polypeptide from the periphery of tau was sufficient to remove the Alz 50 epitope, but not the epitopes of Tau 46 (C-end) or Tau 60 (N-end). The distribution of the Alz 50 epitope in endogenously degraded, chymotrypsin or trypsin digested tau fragments was different from that of the Tau 46 epitope known to be located within 38 residues from the C-terminus of the tau molecule. Based on these observations Alz 50 epitope was considered to be located within 3-4 kDa of the N-terminus of tau. A comparison of immunoblots of different tau-reactive antibodies showed similarities between Tau 60 and Tau-2, and between Tau 14, Tau-1, NP14, and Ab 636.7.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Myelin basic protein specific T cell lines and clones derived from the CNS of rats with EAE only recognize encephalitogenic epitopes.

The epitope specificities of myelin basic protein (BP) specific T cell lines derived from the spinal cords (SC) and lymph nodes (LN) of rats with experimental autoimmune encephalomyelitis (EAE) were compared. To induce EAE, Lewis rats were immunized with guinea pig (GP)-BP and complete Freund's adjuvant. Mononuclear cells from the SC and LN of these animals proliferated in response to BP and the purified protein derivative (PPD) of mycobacterium. After initially being cultured in growth medium, SC mononuclear cells had an enhanced response to BP and lost their response to PPD. LN cells cultured in identical conditions lost their response to both BP and PPD. LN-derived BP specific cell lines recognized only two epitopes of GP-BP: an encephalitogenic epitope in residues 72-89 and a non-encephalitogenic epitope in residues 43-68. SC-derived BP specific cell lines and clones recognized the 72-89 epitope and a second encephalitogenic epitope contained in residues 87-99 but not the non-encephalitogenic 43-68 epitope. Unlike those from LN, BP-specific T cell lines and clones derived from the CNS appear to recognize only encephalitogenic epitopes, including epitopes not recognized by LN lines.

Animals↗
PubMed1999

High-resolution autoreactive epitope mapping and structural modeling of the 65 kDa form of human glutamic acid decarboxylase.

The smaller isoform of the GABA-synthesizing enzyme, glutamic acid decarboxylase 65 (GAD65), is unusually susceptible to becoming a target of autoimmunity affecting its major sites of expression, GABA-ergic neurons and pancreatic beta-cells. In contrast, a highly homologous isoform, GAD67, is not an autoantigen. We used homolog-scanning mutagenesis to identify GAD65-specific amino acid residues which form autoreactive B-cell epitopes in this molecule. Detailed mapping of 13 conformational epitopes, recognized by human monoclonal antibodies derived from patients, together with two and three-dimensional structure prediction led to a model of the GAD65 dimer. GAD65 has structural similarities to ornithine decarboxylase in the pyridoxal-5'-phosphate-binding middle domain (residues 201-460) and to dialkylglycine decarboxylase in the C-terminal domain (residues 461-585). Six distinct conformational and one linear epitopes cluster on the hydrophilic face of three amphipathic alpha-helices in exons 14-16 in the C-terminal domain. Two of those epitopes also require amino acids in exon 4 in the N-terminal domain. Two distinct epitopes reside entirely in the N-terminal domain. In the middle domain, four distinct conformational epitopes cluster on a charged patch formed by amino acids from three alpha-helices away from the active site, and a fifth epitope resides at the back of the pyridoxal 5'-phosphate binding site and involves amino acid residues in exons 6 and 11-12. The epitopes localize to multiple hydrophilic patches, several of which also harbor DR*0401-restricted T-cell epitopes, and cover most of the surface of the protein. The results reveal a remarkable spectrum of human autoreactivity to GAD65, targeting almost the entire surface, and suggest that native folded GAD65 is the immunogen for autoreactive B-cells.

Amino Acid Sequence↗
PubMed1994

Immunohistochemical localization of chondroitin sulfate proteoglycan and tenascin in the human eye compared with the HNK-1 epitope.

BACKGROUND: A previous study revealed the HNK-1 epitope in the human ciliary body beneath the ciliary epithelium. The molecules bearing this 3-sulphoglucuronic acid-containing oligosaccharide epitope in the eye remain unknown. As chondroitin sulphate proteoglycan (CSPG) and tenascin are potential candidates as bearers of the HNK-1 epitope, their distribution in the human eye was compared with that of the HNK-1 epitope. METHODS: Fifty-five formalin-fixed, paraffin-embedded human eyes, including 20 normal eyes and 35 eyes with exfoliation syndrome or glaucoma, were studied immunohistochemically with monoclonal antibody (MAb) CS-56 to CSPG, MAb TN2 to tenascin, and MAbs HNK-1 and VC1.1 to the HNK-1 epitope. Additionally, four frozen lens capsules with exfoliation material were studied by indirect immunofluorescence. RESULTS: A population of dendritic cells in the inner connective tissue layer of the ciliary body and exfoliation material were immunoreactive with antibodies to the HNK-1 epitope, but no labelling for CSPG and tenascin was seen in them, including frozen sections. The inner surface of the nonpigmented ciliary epithelium was reactive for the HNK-1 epitope, and at the ora serrata also for CSPG. In some eyes with glaucoma, immunoreaction for CSPG and tenascin was seen beneath the epithelium and endothelium of the cornea. The nerve fibre layer of the retina was labelled for tenascin. In the sclera, all antibodies labelled the ground substance, and in some large blood vessels immunoreaction for CSPG and tenascin was seen subendothelially. CONCLUSION: Apart from the sclera, the distribution of CSPG and tenascin was different form that of the HNK-1 epitope, suggesting that this carbohydrate epitope may not be borne by these molecules in the human ciliary body.

Adult↗
PubMed1992

Immunocytochemistry of neurofibrillary tangles with antibodies to subregions of tau protein: identification of hidden and cleaved tau epitopes and a new phosphorylation site.

Antibodies to multiple epitopes spanning the length of the tau molecule were used to study Alzheimer neurofibrillary tangles (NFT) using immunocytochemical methods and several different methods of fixation and tissue processing, including staining of vibratome sections, hydrated autoclaving of paraffin sections and immunofluorescence of NFT isolated from fresh brain tissue. Smears and sections were pretreated with trypsin and/or phosphatase to further characterize antibody binding. In tissue fixed briefly in periodate-lysine-paraformaldehyde, tau immunoreactivity was detected in astrocytes, but only a few tau epitopes were detected in NFT with this fixation method. In contrast, all tau epitopes were detected in NFT in tissue fixed in formaldehyde for prolonged periods of time. In the hippocampus, the number of NFT detected in the dentate fascia was in proportion to the duration of dementia, as we previously noted. Dentate fascia NFT were intracellular (i-NFT) and were reactive with antibodies recognizing epitopes in both the carboxy- and amino-terminal regions of tau, but not the microtubule-binding domain of tau, suggesting that microtubule-binding domain epitopes are hidden in i-NFT. In contrast, NFT in the subiculum and layer II of the parahippocampal cortex were mostly extracellular (e-NFT), especially in severe cases of long duration. e-NFT were immunoreactive with antibodies to the microtubule-binding domain, but only weakly reactive with antibodies to carboxy- or amino-terminal epitopes, suggesting that e-NFT may contain fragments of tau. In both isolated NFT and NFT in sections, amino-terminal epitopes, including the Alz-50 epitope, were sensitive to trypsin proteolysis, which suggests that the lack of staining of e-NFT by antibodies to the amino-terminal regions of tau is due to proteolysis. Antibodies reactive with amino-terminal epitopes also stained fewer NFT following hydrated autoclaving, while those reacting with the carboxy half of tau stained more NFT after hydrated autoclaving. Thus, although carboxy-terminal regions are not detected in e-NFT, they are probably masked, rather than proteolytically cleaved, since they can be revealed by hydrated autoclaving. Finally, phosphatase treatment of isolated NFT revealed enhanced immunostaining not only with Tau-1, as in previous studies demonstrating abnormal phosphorylation of tau proteins in NFT, but also with an antibody to exon 2, which reveals yet another phosphorylation site in tau of NFT.

Aged↗
PubMed2006

The use of reverse immunology to identify HLA-A2 binding epitopes in Tie-2.

A potential target for a cancer vaccine would be receptors, such as Tie-2 which are over expressed on tumour endothelium. Using computer aided motif predictions for possible HLA class I epitopes, we have identified peptides from Tie-2 that should bind with a range of affinities to HLA-A*0201. No direct correlation between predicted values and actual binding affinities was observed. Although, the programs did produce a number of false positives, two epitopes were predicted that bound with relatively high affinity when compared with an influenza peptide. We have previously identified a Tie-2 epitope and shown that it was only immunogenic when we substituted preferred amino acids at key anchor residues to increase binding affinity. In this study we used a similar approach to generate modified epitopes. When HLA-A2 transgenic mice were immunised with peptides, CTL killing of the target cells was only achieved when the wild type epitope was presented at moderate levels. Moreover, the efficiency of immunisation was increased when we linked CD4 epitopes to CD8 epitopes. Caution should therefore be employed in the use of both reverse immunology and anchor modification of CTL epitopes in the identification of CTL epitopes for cancer vaccines.

Algorithms↗
PubMed2004

Expression of multiple foreign epitopes presented as synthetic antigens on the surface of Potato virus X particles.

We describe the construction of recombinant Potato virus X (PVX) vectors expressing two different epitopes, ep4 and ep6, from Beet necrotic yellow vein virus (BNYVV). The seven-amino-acid epitopes were expressed as N-terminal coat protein fusions and were displayed on the surface of PVX particles. Particle assembly into full virions was successful even though no wild type coat protein subunits were present, and the epitopes could be detected in crude extracts and purified virus preparations with appropriate antibodies. A construct containing both epitope sequences in tandem was also prepared. The resulting PVX particles could be detected by antibodies against ep4 and ep6, either individually or simultaneously, showing that both epitopes were accessible. In addition mixed infections with PVX vectors containing the individual ep4 and ep6 sequences were carried out. This resulted in the formation of PVX particles displaying ep4 alone, ep6 alone, or both epitopes. These experiments demonstrate for the first time that PVX can be utilized to present multiple epitopes, either tandemly on every coat protein subunit or as heteromultimeric assemblies, both of which could be useful vaccination strategies. The production of epitope-presenting viruses in which every coat protein subunit contains a foreign epitope allows the high-level expression of defined numbers of foreign antigen sites, making such viruses useful standards for immune detection.

Antigens, Viral↗
PubMed2004

Recognition of HLA-A2-restricted mammaglobin-A-derived epitopes by CD8+ cytotoxic T lymphocytes from breast cancer patients.

A breast cancer-associated antigen, mammaglobin-A, is specifically expressed in 80% of primary breast tumors. The definition of immune responses against this highly expressed breast cancer-specific antigen should be of great value in the development of new therapeutic strategies for breast cancer. Thus, the purpose of this study was to identify HLA-A2-restricted mammaglobin-A-derived epitopes recognized by CD8+ cytotoxic T lymphocytes (CTL). We identified seven mammaglobin-A-derived candidate epitopes that bind the HLA-A2 molecule (Mam-A2.1-7) by means of a HLA class I-peptide binding computer algorithm from the Bioinformatics & Molecular Analysis Section of the National Institutes of Health. Subsequently, we determined that CD8+ CTLs from breast cancer patients reacted to the Mam-A2.1 (83-92, LIYDSSLCDL), Mam-A2.2 (2-10, KLLMVLMLA), Mam-A2.3 (4-12, LMVLMLAAL), Mam-A2.4 (66-74, FLNQTDETL), and Mam-A2.7 (32-40, TINPQVSKT) epitopes using an IFN-gamma ELISPOT assay. Interestingly, healthy individuals also showed high reactivity to the Mam-A2.2 epitope. Two CD8+ CTL lines generated in vitro against TAP-deficient T2 cells loaded with the candidate epitopes showed significant cytotoxic activity against the Mam-A2.1-4 epitopes. These CD8+CTL lines recognized a HLA-A2+breast cancer cell line expressing the Mam-A2.1 epitope. In addition, DNA vaccination of HLA-A2+/human CD8+ double-transgenic mice with a DNA construct encoding the Mam-A2.1 epitope and the HLA-A2 molecule induced a significant expansion of epitope-specific CD8+ CTLs that recognize the same HLA- A2+/Mam-A2.1+ breast cancer cell line. In conclusion, these results demonstrate the immunotherapeutic potential of mammaglobin-A for the treatment and prevention of breast cancer.

Adenocarcinoma↗
PubMed1988

The binding of a monoclonal antibody reactive with pp60v-src to the rat CNS both in vitro and in vivo: evidence that the epitope is present intracellularly as well as being associated with a number of antigenically related polypeptides located externally in the plasma membrane only in the synaptic region.

A monoclonal antibody, F4, has been produced which reacts with an epitope possessing an unusual subcellular distribution. It binds to the external surface of the neuronal plasma membrane only in the region of the synapse. This is evidenced by binding of F4 to presynaptic terminals in unfixed cultures of rat cerebellum and to preparations of unfixed synaptosomes. In addition, much larger amounts of the epitope are present intracellularly. In fixed nervous tissue, the epitope is found in many neurons, and is associated mainly with presynaptic plasma membranes, synaptic vesicles, postsynaptic densities (cerebral cortex and hippocampus, but not cerebellum), rough endoplasmic reticulum, and the Golgi apparatus. The epitope is especially abundant in large neurons (e.g. pyramidal cells). Similar amounts of epitope are present in the chromaffin cells of the adrenal medulla. It is also expressed in ependymal cells in the brain, and in epithelial cells present in ducts of the medulla, but not cortex, of the kidney. However, the epitope is not found in glial cells in the brain, or in either liver, spleen, skeletal muscle, or testes. F4 is not species specific, as it binds to postmortem adult human cerebral cortex and neonatal cerebellum in a manner as described for the rat. It also binds to homogenates of brains of fish, chicken and mouse. The appearance of the epitope during development of the cerebellum in vivo and in vitro occurs in parallel with the differentiation of neurons and formation of synapses, though small amounts are also present in neuronal precursor cells. The F4 antibody can detect nanogram amounts of pp60v-src on immunodots. The strength of this reaction is high enough that F4 can be used to demonstrate pp60v-src-like immunoreactivity in Rous Sarcoma virus-transformed chick embryo fibroblasts. However, present evidence suggests that it may be premature to assign the immunocytochemical reactivity of F4 in the brain exclusively to pp60c-src. This conclusion is based on the fact that F4 reacts with several polypeptides from synaptic plasma membranes on Western blots of renaturing, two-dimensional gels that are dissimilar in size to pp60c-src, and from the fact that it can cross-react, albeit weakly, with several other serine protein kinases in an immunodot assay. Appreciation of this cross-reactivity, and of the evolutionary conservation of the epitope, as well as its sensitivity to denaturation, has led to our working hypothesis that F4 binds to a conformational epitope present on several polypeptides that may be most perfectly represented by some aspect of the catalytic domain of tyrosine protein kinases.

Animals↗
PubMed1992

Silencing of immunodominant epitopes by contiguous sequences in complex synthetic peptides.

We have previously shown that the T cell response to the synthetic peptide cI12-26:NP365-380 (covalently linked epitopes of lambda repressor (cI) and influenza A nucleoprotein (NP) polypeptides) requires amino acid sequences located in the junctional region between the cI12-26 and NP365-380 epitopes in the H-2d and H-2k haplotypes. In this study, we show that the dominant epitope of cI12-26:NP365-380 in H-2b mice is also located within the junctional region of the peptide, indicating that the same amino acid sequence is immunodominant in three different H-2 haplotypes. Based on results using fixed APC, there was no qualitative difference in epitope recognition due to antigen processing. In addition, antigen presentation by APC expressing mutant I-A molecules constructed by hemiexon shuffling of regions of the molecule containing primarily beta sheet or alpha helix showed that many different substitutions were permissive for at least one of the T hybridomas. More importantly, however, when the junctional sequences are covalently linked in composite synthetic peptides containing additional previously defined T cell epitopes, antigenicity of the immunodominant junctional region was silenced and a new epitope assumed immunodominance. Thus, immunodominance does not correlate with the primary amino acid sequence of the potential epitope. Instead, the immunodominant epitope is determined by complex interactions among the epitopes, which most likely depend on the structural conformation of the composite peptide.

Amino Acid Sequence↗
PubMed1989

Epitope mapping by deletion mutants and chimeras of two vesicular stomatitis virus glycoprotein genes expressed by a vaccinia virus vector.

Deletion mutants and chimeras of the glycoprotein (G) genes of vesicular stomatitis virus serotypes Indiana (VSV-Ind) and New Jersey (VSV-NJ) were cloned in plasmids and vaccinia virus vectors under control of the bacteriophage T7 polymerase promoter for expression in CV-1 cells co-infected with a T7 polymerase-expressing vaccinia virus recombinant. Truncated and chimeric G proteins expressed by these vectors were tested for their capacity to react with VSV-Ind and VSV-NJ epitope-specific monoclonal antibodies (MAbs) by Western blot analysis for those antigenic determinants not affected by disulfide-bond reducing conditions or by immuno dotblot analysis for those that are. These experiments allowed us to create putative epitope maps for glycoproteins of both serotypes based on binding affinity and cross-reactivity of VSV-Ind and VSV-NJ MAbs for truncated or chimeric G proteins of known amino acid sequences. Seven of the 9 VSV-NJ G epitopes, including all 4 epitopes involved in virus neutralization by MAbs, mapped to the center (amino acid sequence 193-289) of the 517 amino acid VSV-NJ G protein. Four of the 11 VSV-Ind G epitopes, including 2 neutralizable epitopes, mapped to the cysteine-rich amino-terminal domain (amino acid sequence 80-183) of the 511 amino acid VSV-Ind G protein; the remaining 7 VSV-Ind G epitopes, including 2 involved in virus neutralization, were clustered in the cysteine-poor carboxy-terminal domain (amino acid sequence 286-428). In site-specific mutants of the VSV-Ind G gene defective in one or both glycosylation sites, only the amino-terminal epitopes of the VSV-Ind G protein were affected by deletion of the carbohydrate chain at residue 179; deletion of the carbohydrate chain at residue 336 did not alter reactivity of the G protein with any of the relevant monoclonal antibodies. These results are discussed in relation to earlier attempts to map the antigenic determinants of VSV-NJ and VSV-Ind G proteins by proteolysis of the G protein and by sequencing the G genes of mutant viruses selected for their resistance to neutralization by epitope-specific monoclonal antibodies.

Animals↗
PubMed1990

Complement activation by individual and combinations of monoclonal antibodies to Actinomyces viscosus T14V fimbriae: a probe for epitope distribution on these polymeric proteins.

The spatial requirements for IgG activation of the classical complement pathway has provided a basis for utilizing complement consumption by individual and pairs of monoclonal antibodies (mAbs) to compare the repeating epitope patterns of the type 1 and type 2 fimbriae of Actinomyces viscosus T14V and to examine the co-operative effects of mAbs against these polymeric proteins. Three of five mAbs specific for the type 1 fimbriae consumed complement when assayed individually. Four patterns of complement consumption were detected with pairs of these mAbs: inhibition, addition, enhancement or synergy. Inhibition occurred when both members of a pair reacted with the same epitope but only one consumed complement. A strictly additive effect was observed if both mAbs consumed complement and, in addition, recognized the same epitope. Complement consumption by mAbs against certain epitopes was enhanced by non-complement consuming mAbs that reacted with different epitopes. Synergy was observed with extremely low concentrations of two mAbs each of which reacted with a different epitope and consumed complement. In contrast to the anti-type 1 mAbs, only one of seven mAbs against the type 2 fimbriae consumed more than 20% of the available complement. Pairs of anti-type 2 mAbs exhibited only inhibition or synergy. The latter effect was particularly striking as pairs containing mAbs that reacted with different epitopes and failed to consume complement or were minimally active when assayed individually were extremely efficient. These data indicated that the spatial arrangements of individual mAbs bound to repeating epitopes in the type 1, but not the type 2, fimbriae were appropriate for activation of complement. Thus, the repeating epitope patterns of the two types of fimbriae apparently differ.

Actinomyces↗