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PubMed2002

Immunodominant epitopes in Babesia bovis rhoptry-associated protein 1 that elicit memory CD4(+)-T-lymphocyte responses in B. bovis-immune individuals are located in the amino-terminal domain.

Babesia bovis rhoptry-associated protein 1 (RAP-1), which confers partial protection against B. bovis challenge, is recognized by antibodies and T lymphocytes from cattle that have recovered from infection and are immune to subsequent challenge. RAP-1 is a 60-kDa protein with an N-terminal (NT) region that contains four cysteine residues conserved among all Babesia RAP-1 family members and a C-terminal (CT) region that contains multiple, degenerate, tandem 23-amino-acid (aa) repeats. To define the location of CD4(+)-T-cell epitopes for vaccine development using a recombinant protein or minigene construct, a series of truncated recombinant RAP-1 proteins and peptides were tested for stimulation of T-cell lines derived from B. bovis-immune cattle. CD4(+)-T-cell lines from three B. bovis-immune cattle with different DRB3 haplotypes responded to the NT region of RAP-1, whereas T cells from only one animal responded weakly to the CT region. T-cell lines from the three individuals recognized two to six NT-region peptides spanning aa 134 to 316 and representing at least four dominant epitopes. Using RAP-1-specific CD4(+)-T-cell clones, two NT-region epitopes, EYLVNKVLYMATMNYKT (aa 187 to 203) and EAPWYKRWIKKFR (aa 295 to 307), and one CT-region repeat epitope, FREAPQATKHFL, which is present twice at aa positions 391 to 402 and 414 to 425, were identified. Several peptides representing degenerate repeats of the agonist CT-region peptide FREAPQATKHFL neither stimulated responses of T-cell clones specific for this peptide nor inhibited responses to the agonist peptide. Upon stimulation with specific antigen, T-cell clones specific for NT or CT epitopes produced gamma interferon. The presence of T-helper-cell epitopes in the NT domain of RAP-1, which is highly conserved among otherwise antigenically different strains of B. bovis, supports the inclusion of this region in vaccine constructs to be tested in cattle.

Amino Acid Sequence↗
PubMed1996

Increasing incidence of CD44v7/8 epitope expression during uterine cervical carcinogenesis.

Splice variants of the cell surface glycoprotein CD44 (CD44v) have been implicated in the progression of various human tumors. In the present study, we have examined the expression pattern of a CD44v epitope encoded by the adjacent variant exons v7 and v8 during human cervical carcinogenesis. While only l/ll normal cervical squamous epithelia was positive for this epitope by immunohistochemistry, 4/21 samples of low-grade squamous intra-epithelial lesions (LSIL), 17/35 samples of high-grade squamous intra-epithelial lesions (HSIL), 11/12 samples of the HSIL subgroup of carcinomas in situ and 17/17 cases of invasive cervical carcinoma showed CD44v7/8 epitope expression. In addition to CD44 variant expression, we have analyzed 67 lesions for the presence of HPV16/18-DNA using PCR. Most of the samples expressing the v7/8 epitope were also HPV16-positive (29/32), whereas only 17/35 of the v7/8-negative samples were HPV16-positive. HPV18 DNA was found in only one invasive carcinoma. Our data suggests that high-risk HPV infection may precede CD44v7/8 expression and that the number of samples expressing the CD44v7/8 epitope increases during carcinogenesis and reaches nearly 100% at the carcinoma in situ stage. This CD44 epitope could, therefore, serve as a diagnostic marker of cervical squamous cell carcinomas and as a possible target for CD44v7/8 epitope-directed therapies.

Alternative Splicing↗
PubMed1997

The HNK-1 Carbohydrate Epitope and the Human Eye in Health and Disease.

The HNK-1 carbohydrate epitope is part of many cell membrane and extracellular matrix molecules, several of which have been implicated in cell adhesion. It is a versatile tool in eye research. In the human eye this epitope is present in the retina, the optic and ciliary nerves, the ciliary and iris epithelia, the zonular lamella, and the sclera. It is phylogenetically conserved, but the positive cell types vary from species to species. In addition to revealing interspecies differences in the vertebrate retina, the HNK-1 epitope has been used to identify a novel cell type in the eye: the subepithelial matrix cells that reside in the inner connective tissue layer (ICTL) of the ciliary body. Although these cells resemble fibroblasts in ultrastructure, they form a distinct cell population that differs in antigenic profile from fibroblasts in other tissues. The HNK-1 epitope is also associated with the elastic fiber system of the ICTL, which may be produced by the subepithelial matrix cells. It may help to structurally stabilize the ciliary body and the retina. The HNK-1 epitope is also involved in many important eye diseases. The subepithelial matrix cells seem to be susceptible to irrreversible atrophy as a result of glaucoma, thermal injury, and tissue compression. On the other hand, the HNK-1 epitope is found in the extracellular matrix of secondary cataracts and may contribute to its pathogenesis. Finally, this epitope has proved to be useful in identifying deposits of exfoliation material, and in tracing neuroepithelial derivatives in developmental anomalies and tumors of the eye.

Journal Article↗
PubMed1992

The adenoma-carcinoma sequence in the colorectum--early appearance of a hierarchy of small intestinal mucin antigen (SIMA) epitopes and correlation with malignant potential.

The colorectal adenoma-carcinoma sequence was examined in relation to the ectopic expression of the oncofoetal Small Intestinal Mucin Antigen (SIMA), to the development of morphologic changes in the adenoma and perineoplastic mucosa and to indices of malignant potential. Four anti-SIMA MAbs, which define a novel hierarchy of SIMA epitopes in the normal small intestine and adjacent to colorectal cancers, were used in a retrospective immunohistochemical study of Familial Adenomatous Polyposis (FAP, n = 183) and non-familial (n = 44) adenomas. Inappropriate expression of SIMA epitopes was first detected in mucosa adjacent to minute microadenomas larger than three glands, and with increase in size, in increasing amounts within adenomas themselves, but not with microadenomas smaller than three glands or regions of flat mucosa free of adenomas. SIMA epitope expressed in mucosa adjacent to adenomas preceded changes in perineoplastic morphology, which progressed with adenoma growth to resemble transitional mucosa (TM) adjacent to cancers. Thus, the onset of both SIMA expression and morphological changes in TM were consistent with reactive rather than pre-existing field change phenomena. The previously reported hierarchy of four SIMA epitopes (5C5, 3D4, 4D3, 6C5) was also consistently observed in the adenoma-carcinoma sequence, and applied to (i) the order of epitope detection, (ii) the number of positive adenomas and (iii) extent of staining; (iv) the height in the crypt and (v) distance from the adenoma to which epitopes were expressed in perineoplastic mucosa. These observations are consistent with a progression of changes in mucin composition with adenoma development. The percentage of positive adenomas and reactivity scores for each anti-SIMA MAb correlated with increasing adenoma size, degree of dysplasia and growth pattern. SIMA expression appears to predate the earliest reported oncogene and tumour suppressor gene changes, was persistent and increased throughout adenoma development. SIMA epitopes are thus markers of very early neoplastic change, whose expression correlates with malignant potential and may contribute to the accumulation of changes necessary for tumourigenesis.

Adenoma↗
PubMed2000

In Alzheimer's disease, heme oxygenase is coincident with Alz50, an epitope of tau induced by 4-hydroxy-2-nonenal modification.

In this study, we compared the neuronal induction of the antioxidant heme oxygenase-1 (HO-1) in Alzheimer's disease with abnormalities in tau marked by antibodies recognizing either phosphorylation (AT8) or conformational change (Alz50). The epitope recognized by Alz50 shows a complete overlap with HO-1-containing neurons, but AT8 recognized these neurons as well as neurons not displaying HO-1. These findings suggest that tau phosphorylation precedes the HO-1 response and that HO-1 is coincident with the Alz50 epitope. This led us to consider whether oxidative damage plays a role in forming the Alz50 epitope. We found that 4-hydroxy-2-nonenal (HNE), a highly reactive product of lipid peroxidation, reacts with normal tau and induces the Alz50 epitope in tau. It is important that the ability of HNE to create the Alz50 epitope not only is dependent on lysine residues of tau but also requires tau phosphorylation because neither methylated, recombinant, nor dephosphorylated tau reacts with HNE to create the Alz50 epitope. Supporting the in vivo relevance of this observation, endogenous paired helical filament-tau isolated from subjects with Alzheimer's disease was immunoreactive with an antibody to a stable HNE-lysine adduct, as were all vulnerable neurons in subjects with Alzheimer's disease but not in control individuals. Together, these findings support the involvement of oxidative damage early in neurofibrillary tangle formation in Alzheimer's disease and also suggest that HNE modification contributes to the generation of the tau conformation defining the Alz50 epitope. These findings provide evidence that an interplay between phosphorylation of tau and neuronal oxidative stress-induced pathology is important in the formation of neurofibrillary tangles.

Aged↗
PubMed1994

Epitope mapping and topology of baculovirus-expressed HIV-1 gp160 determined with a panel of murine monoclonal antibodies.

To define protein folding patterns of HIV-1 Env subunit vaccines, we have isolated a set of 30 monoclonal antibodies (MAbs) from BALB/c mice immunized with a recombinant gp160 vaccine (rgp160) expressed in a baculovirus system. This article describes epitope mapping for the MAb panel and topology of the epitopes for rgp160 and a recombinant gp120 (rgp120) also expressed in a baculovirus system. The following results are reported: (1) rgp160 harbors a minimum of 4 antigenic domains, 3 mapping to the C1, C2, and C3/V4 regions of gp120 and 1 mapping to the cytoplasmic tail of gp41; (2) there are at least 3 adjacent or overlapping epitopes in each antigenic domain; (3) a minimum of 14 independent epitopes were mapped, all of which are continuous sites; (4) each of the epitopes is exposed on rgp160 without prior manipulation of the protein; and (5) by contrast, 6 of the 8 epitopes mapping to the C1, C2, and C3/V4 regions are not exposed on rgp120, but become exposed when the protein is denatured. Taken together, these results show that rgp160 and rgp120 are folded differently, illustrating the use of this MAb panel to compare epitope topographies of recombination HIV-1 Env proteins. This MAb panel may aid in the refinement of HIV-1 Env subunit vaccines.

AIDS Vaccines↗
PubMed1998

A sensitive assay for measuring alpha-Gal epitope expression on cells by a monoclonal anti-Gal antibody.

BACKGROUND: The assessment of a-gal epitope (Galalpha1-3Galbeta1-4GlcNAc-R) expression on various cells and tissues is important for the prediction of anti-Gal-mediated immune rejection of xenografts. This study describes an enzyme-linked immunosorbent assay (ELISA inhibition assay) developed for this purpose, which uses the monoclonal anti-Gal antibody M86. METHODS: Cells at various concentrations were incubated overnight with M86 at 1/100 dilution. The cells and bound antibody were removed, and the residual antibody in the supernatant was measured in an ELISA assay with a-gal-bovine serum albumin as a solid phase antigen. The extent of a-gal epitope expression on cells correlates with the subsequent inhibition of M86 binding in ELISA. The inhibition binding curves at various cell concentrations were compared with those of a standard cell line with a known number of epitopes per cell. RESULTS AND CONCLUSIONS: The mouse IgM M86 monoclonal antibody was highly specific for a-gal epitopes. Using this antibody in an ELISA inhibition assay with cells at a wide range of concentrations enables the detection of at least 5 x 10(4) and up to more than 5 x 10(7) a-gal epitopes per cell. This assay can be used also for the detection of a-gal epitopes on membranes from tissue homogenates, and thus it enables the determination of the extent of decrease in a-gal epitope expression in animals that are genetically manipulated to alter their carbohydrate make-up.

Animals↗
PubMed1997

Influence of the HLA-DR beta shared epitope on susceptibility to and clinical expression of rheumatoid arthritis in Chilean patients.

OBJECTIVE: To analyse the influence of shared epitope positive HLA-DRB1 alleles (QKRAA or QRRAA)) on rheumatoid arthritis (RA) susceptibility and severity in Chileans, a population that exhibits a weak association with HLA-DR4. METHODS: Prevalence of alleles DRB1*01 and DRB1*04 alleles was determined by polymerase chain reaction amplification and sequence specific oligonucleotide hybridisation in 129 RA patients with defined clinical features and in 97 healthy controls. RESULTS: The shared epitope was found in 70 (54%) of the RA patients and in 29 (30%) of controls (odds ratio (OR) = 3; 95% confidence intervals (CI) = 1.5, 5.1; p = 0.0004), and was present in a double dose in 20% of patients versus 4% of controls (OR = 6; 95% CI = 2, 21; p = 0.0009). HLA-DRB1*0403 was the most prevalent DR4 subtype in controls (19%). HLA-DRB1*0403 or *0408 were the alleles most prominently associated with RA, 19% versus 6% in controls (OR = 3; 95% CI = 1.3, 10; p = 0.01). The risk of RA in those carrying a double dose of the shared epitope was 7.5 times that seen in patients lacking the epitope. Disease severity was moderate: 33% had extra-articular manifestations. The double dose was associated with an increased risk of vasculitis or extra-articular manifestations. However, 59 patients (46%) did not carry the shared epitope and 18 of them (31%) had extra-articular manifestations. CONCLUSIONS: The weak association of RA with DR4 in Chileans seems to relate to a relatively high frequency of the DRB1*0403 allele among DR4 subtypes. As in other populations, the shared epitope in double dose is associated with RA development, especially in its more severe forms. However, both development and expression of severe forms of the disease were independent of the shared epitope in a high proportion of patients, thus emphasising the genetic heterogeneity of the disease and the possible involvement of other genetic elements.

Adult↗
PubMed2006

Improved method for predicting linear B-cell epitopes.

BACKGROUND: B-cell epitopes are the sites of molecules that are recognized by antibodies of the immune system. Knowledge of B-cell epitopes may be used in the design of vaccines and diagnostics tests. It is therefore of interest to develop improved methods for predicting B-cell epitopes. In this paper, we describe an improved method for predicting linear B-cell epitopes. RESULTS: In order to do this, three data sets of linear B-cell epitope annotated proteins were constructed. A data set was collected from the literature, another data set was extracted from the AntiJen database and a data sets of epitopes in the proteins of HIV was collected from the Los Alamos HIV database. An unbiased validation of the methods was made by testing on data sets on which they were neither trained nor optimized on. We have measured the performance in a non-parametric way by constructing ROC-curves. CONCLUSION: The best single method for predicting linear B-cell epitopes is the hidden Markov model. Combining the hidden Markov model with one of the best propensity scale methods, we obtained the BepiPred method. When tested on the validation data set this method performs significantly better than any of the other methods tested. The server and data sets are publicly available at http://www.cbs.dtu.dk/services/BepiPred.

Journal Article↗
PubMed1992

[The human chorionic gonadotropin (hCG) receptor: a new class within the family of GTP protein coupled receptors. Epitope mapping of receptor-bound agonistic and antagonistic forms of hCG].

Human chorionic gonadotropin (hCG) is a member of the glycoprotein hormone family. It is composed of an alpha and a beta subunit, the latter being closely homologous to that of human luteinizing hormone (hLH). HCG and hLH bind to the same receptor. The molecular mass of hCG is 38 kD, up to 30% of which is contributed by the carbohydrate moieties linked to each of the two subunits. Structure-function relationship studies have indicated that both subunits interact with the receptor and that altogether four different peptide domains (two on each subunit) are responsible for high-affinity receptor binding. The carbohydrate units are responsible for expression of agonist activity: deglycosylated hCG (degly-hCG) is unable to induce a biological response (cAMP increase), despite high-affinity binding. We have previously mapped the antigenic surface of hCG and described 14 different epitopes that can be recognized by specific monoclonal antibodies (MCA), i.e. five epitopes on the alpha subunit, five on the beta subunit and four epitopes which are formed through association of the two subunits (conformational or alpha beta epitopes). The number and topography of epitopes was found to be the same on degly-hCG. This framework of immunological coordinates was then applied to probe the orientation of hCG, as well as that of the competitive antagonist degly-hCG in their receptor-bound states. With a receptor-hormone-125I-MCA sandwich approach we found that while hCG still presented two of the 14 epitopes, i.e. beta 3 and beta 5, no epitope was accessible on receptor-bound degly-hCG. This differential pattern thus correlates with the signal transduction-competence of the respective ligand. Overall, these data indicate that most of the surface of hCG is masked by moieties of the receptor. Accordingly, that moiety must itself be folded in a way that it provides a large contact surface. No portion other than the 341-residue long extracellular domain would seem capable of doing that. Computer-assisted secondary structure predictions support this view. This domain is the novel feature that distinguishes glycoprotein-hormone receptors from the other members of the G protein-coupled receptor superfamily.

Amino Acid Sequence↗
PubMed1998

A protective cytotoxic T cell response to a subdominant epitope is influenced by the stability of the MHC class I/peptide complex and the overall spectrum of viral peptides generated within infected cells.

This study identifies instability of MHC class I/peptide complexes and intermolecular competition for MHC class I presentation as factors responsible for the subdominance of cytotoxic T lymphocyte (CTL) epitopes. This evidence is based on the characterization of a new CTL epitope derived from the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV). This epitope, peptide GP117-125 (GP117) is presented to T cells by the mouse MHC class I molecule, H-2Db. In short-term experiments induction of GP117-specific CTL by vaccination rendered C57BL/6 mice only partially resistant to infection with wild-type LCMV (LCMV-WE) but completely resistant to challenge with a previously described LCMV variant. The variant virus, LCMV-8.7B23, bears point mutations within both known LCMV-GP, H-2 Db-restricted epitopes GP33-41 (GP33) and GP276-286 (GP276) resulting in a valine to leucine change at position 35 in peptide GP33 (V35L) and an asparagine to serine change at position 280 in peptide GP276 (N280S). Although variant peptide GP33/V35L stimulates a weak CTL response, GP276/N280S does not. Elution of peptide GP117 from both LCMV-WE- and LCMV-8.7B23-infected cells revealed that the difference in the capacity of GP117-specific CTL to protect against LCMV-WE and the virus variant LCMV-8.7B23 was due to differences in the level of GP117 presentation on the surface of both types of cells. Thus, it appears that the protective capacity of CTL specific for the subdominant epitope GP117 is influenced by the extent of presentation of other immunodominant peptide epitopes present within infected cells.

Animals↗
PubMed1997

Identification of epitopes on a mutant form of Pseudomonas exotoxin using serum from humans treated with Pseudomonas exotoxin containing immunotoxins.

PE38 is a 38-kDa derivative of the 66-kDa Pseudomonas exotoxin (PE) in which the cell binding domain of PE (domain Ia, amino acids 1-252) and a portion of domain Ib (amino acids 365-380) are deleted. The immunotoxins LMB-1 and LMB-7 contain PE38 and kill cancer cells by exploiting the cytotoxic action of PE38. The major human B cell epitopes of PE38 were mapped by measuring the reactivity of 45 serum samples from patients treated with the PE38-containing immunotoxins LMB-1 or LMB-7 to two panels of overlapping synthetic peptides representing the sequence of PE38. One panel of peptides is ten amino acids long and overlap by seven amino acids, and the second panel of peptides is twenty amino acids long and overlap by ten. Five major epitopes were identified: amino acids 274-283, 470-492, 531-540, 555-564, and the C-terminal amino acids 596-609. Two minor epitopes were identified as well: amino acids 501-510 and 582-589. These epitopes are predominantly located on the surface of the protein. The amino acids believed to be critical for binding are highly solvent-accessible residues. The results of the human antibody response to peptides are compared to the pattern of reactivity previously identified with serum samples obtained from monkeys administered LMB-1 and LMB-7. The epitopes between monkey and human are almost identical, demonstrating similarity in the response of antibody repertoires between the two species and providing further support that these are the immunodominant epitopes. This information is critical for genetically engineering less immunogenic immunotoxins and provides a foundation for the development of a vaccine against pseudomonal infections which plague immunocompromised individuals and individuals with cystic fibrosis.

ADP Ribose Transferases↗
PubMed1994

Different antibody response to a neutralizing epitope of human cytomegalovirus glycoprotein B among seropositive individuals.

The amino-terminal portion of human cytomegalovirus glycoprotein B (HCMV-gB) was expressed as a fusion protein to analyze the neutralizing epitope recognized by human monoclonal antibody C23 and the humoral immune response to this epitope. The linear neutralizing epitope was further localized to the peptide within 17 amino acids (position 68-84) which were conserved between two HCMV laboratory strains. Ten out of 17 HCMV-seropositive human sera contained the antibody against this epitope. Although seven sera were negative for reacting with the fusion protein, the viruses isolated from the same patients retained the epitope. The immunogenicity of the epitope and the possible application of C23 human monoclonal antibody for passive immunization against HCMV infections are discussed.

Amino Acid Sequence↗
PubMed1998

Epitope mapping of mouse monoclonal antibody EP-5C7 which neutralizes both human E- and P-selectin.

The epitope of mouse monoclonal antibody (mAb) EP-5C7, which binds to and blocks both human E- and P-selectin, was mapped onto the protein structure of E-selectin. Analyses with E- and L-selectin chimeric proteins and randomly mutagenized E-selectins demonstrated that the EP-5C7 epitope consists of the amino acid residues at positions 21, 22, 23, 119 and 120 of E-selectin. The binding of three neutralizing anti-E-selectin mAb's (E-1E4, H18/7 and CL2), whose epitopes were found to overlap with the E-selectin binding site for carbohydrate ligands, was not affected by the amino acid substitutions at these five positions. Inspection of the three-dimensional structure of E-selectin indicated that the EP-5C7 epitope is located near the junction between the lectin and EGF-like domains. The ligand binding site was distant from the EP-5C7 epitope, suggesting that the amino acid residues in the EP-5C7 epitope play an important role other than ligand binding in selectin-mediated cell adhesion.

Animals↗
PubMed2001

Conformational and sequential epitopes on the human granulocyte-colony stimulating factor molecule (hG-CSF) and their role in binding to human placenta receptors.

Monoclonal antibodies (mAbs) named 8C2 and 6E3, directed against the recombinant human granulocyte colony-stimulating factor (hG-CSF), were used as probes to study the cytokine orientation on its binding to receptors from human placenta. Competition enzyme linked immunoabsorbent assays (ELISA) revealed that mAb 8C2 would be directed to a linear epitope, whereas mAb 6E3 would delimit a more assembled epitope. Gel-filtration high performance liquid chromatography (HPLC) of the immune complexes formed by incubating [(125)I]hG-CSF with each mAb showed that epitope 8C2, but not 6E3, was altered after cytokine iodination. In addition, mAb 6E3 completely inhibited [(125)I]hG-CSF binding to human placental microsomes. Although [(125)I]mAb 6E3 was unable to bind to preformed hG-CSF-receptor complexes, [(125)I]mAb 8C2 did recognize hG-CSF previously bound to receptors, suggesting that epitope 8C2 would remain accessible in the hG-CSF-receptor complex. To identify the cytokine region defined by mAbs, hG-CSF was digested with different proteolytic enzymes: Arg-C, Glu-C, trypsin and alpha chymotrypsin. Immunoreactivity of the resulting peptides was examined by Western blot and their sequences were established by Edman degradation. Results showed that mAb 6E3 would be directed to a conformation-dependent epitope located close to the hG-CSF binding domain and included into the sequence 1-122/123, whereas mAb 8C2 recognized the region 41-58, which represents a linear epitope left exposed after cytokine binding to receptors from human placenta.

Amino Acid Sequence↗
PubMed1995

Comprehensive epitope analysis of monoclonal anti-proenkephalin antibodies using phage display libraries and synthetic peptides: revelation of antibody fine specificities caused by somatic mutations in the variable region genes.

Filamentous phage libraries, displaying 6, 12 or 20 amino acid residue peptides at the N terminus of coat protein pIII were used to define and localize the epitopes of 15 monoclonal antibodies raised against human proenkephalin, a neuropeptide precursor. Eight monoclonal antibodies (PE14 to PE19, PE23 and PE25), which inhibit each other's binding to proenkephalin, recognized phage clones selected by PE14, PE15, PE19, PE23 and PE25. With the peptide sequences DLL(X)(X)LL (12mer library) and DLL(X)(X)L (6mer library) shared by most of the phage clones it was possible to define the putative antibody epitope 155DLLKELL161 on human proenkephalin. For five antibodies (PE13, PE20 to PE22 and PE24) belonging to another inhibition group, a common consensus motif G(X)D(X)E(X)(X)V(X)(X)R could be defined with help of a 20mer library. The corresponding minimum epitope sequence has been found to be 175GSDNEEEVSKR185. Antibody PE1, raised in a separate fusion, was able to select phage clones from a 12mer and 20mer library, revealing that the sequence 187GGFMRG192 is probably the antibody epitope. The assumed localization of the epitopes was confirmed by screening a set of overlapping synthetic peptides, covering the region of human proenkephalin thought to contain all antibody binding sites. It was found that antibodies, although recognizing the same epitope, gave different binding patterns with the selected phage clones. By analysing the VH chain sequences of these antibodies it could be shown that a varying number of somatic mutations is likely to be the reason for the observed differences in antibody fine specificity.

Amino Acid Sequence↗
PubMed2000

Identification of an epitope in the C terminus of normal prion protein whose expression is modulated by binding events in the N terminus.

We have characterized the epitopes of a panel of 12 monoclonal antibodies (Mabs) directed to normal human cellular prion protein (PrP(C)) using ELISA and Western blotting of recombinant PrP or synthetic peptide fragments of PrP. The first group of antibodies, which is represented by Mabs 5B2 and 8B4, reacts with PrP(23-145), indicating that the epitopes for these Mabs are located in the 23 to 145 N-terminal region of human PrP. The second group includes Mabs 1A1, 6H3, 7A9, 8C6, 8H4, 9H7 and 2G8. These antibodies bind to epitopes localized within N-terminally truncated recombinant PrP(90-231). Finally, Mabs 5C3, 2C9 and 7A12 recognize both PrP(23-145) and PrP(90-231), suggesting that the epitopes for this group are located in the region encompassing residues 90 to 145. By Western blotting with PepSpot(TM), only three of Mabs studied (5B2, 8B4 and 2G8) bind to linear epitopes that are present in 13-residue long synthetic peptides corresponding to human PrP fragments. The remaining nine Mabs appear to recognize conformational epitopes. Two N terminus-specific Mabs were found to prevent the binding of the C terminus-specific Mab 6H3. This observation suggests that the unstructured N-terminal region may influence the local conformation within the folded C-terminal domain of prion protein.

Amino Acid Sequence↗
PubMed1997

The signal sequence of lymphocytic choriomeningitis virus contains an immunodominant cytotoxic T cell epitope that is restricted by both H-2D(b) and H-2K(b) molecules.

Infection of H-2b mice with lymphocytic choriomeningitis virus (LCMV) generates three well-characterized H-2D(b)-restricted immunodominant epitopes delineated in the NP, GP1, and GP2 proteins. Here we report that the H-2D(b)-restricted GP1 epitope GP33-41/43 (KAVYNFATC/GI) located in the signal sequence of LCMV is also the immunodominant epitope recognized by CTL at the surface of the same infected cells in the context of H-2K(b) restriction. The GP1 epitope bound to H-2D(b) and H-2K(b) molecules with comparable affinities. The respective binding processes involved different sets of peptide anchoring residues and required dramatically different conformations of the peptide backbone as well as rearrangement of residue side chains. The 10-mer peptide GP34-43 (AVYNFATCGI) was the optimal H-2K(b)-binding sequence and the 8-mer peptide GP34-41 (AVYNFATC) the minimal sequence for optimal H-2K(b)-restricted CTL recognition. Comparison of lytic activities of primary splenic anti-LCMV CTL from C57BL/6 (D(b+)/K(b+)), B10A.[5R] (D(b-)/K(b+)), and B10A.[2R] (D(b+)/K(b-)) mice against LCMV-infected or peptide-coated target cells expressing either one or the two MHC alleles revealed that the H-2K(b)-restricted component of the anti-GP1 CTL response was mounted independently of but as efficiently as its H-2D(b) counterpart. Analysis of the immune response against a GP1 variant that escapes CTL recognition showed that the GP1 epitope: (i) was likely the only immunodominant LCMV epitope in the context of H-2K(b), and (ii) could efficiently evade H-2D(b) and H-2K(b)-restricted CTL mediated lysis.

Alleles↗