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PubMed2002

Identification of citrullinated rheumatoid arthritis-specific epitopes in natural filaggrin relevant for antifilaggrin autoantibody detection by line immunoassay.

OBJECTIVE: To identify immunodominant epitopes in natural filaggrin that are reactive with antifilaggrin autoantibodies (AFA) in the sera of patients with rheumatoid arthritis (RA) and to explore their use in a diagnostic assay format. METHODS: Based on the results of epitope mapping of human natural filaggrin as well as molecular modeling and computational chemistry, synthetic peptides together with recombinant citrullinated filaggrin were evaluated by a line immunoassay (LIA) for AFA detection. Diagnostic performance was assessed using 336 RA and 253 disease control sera and was compared with that of reference methods. RESULTS: Several immunoreactive epitopes were identified in natural filaggrin, all of which contained at least 1 citrulline residue. Three antigenic substrates, including 2 synthetic peptides and recombinant citrullinated filaggrin showing maximal reactivity on LIA, were finally selected. Using the 3-antigen LIA3, overall sensitivity, specificity, and positive predictive value for RA were 65.2%, 98.0%, and 89.1%, respectively, compared with 61.9%, 98.8%, and 92.8% using the 2-antigen LIA2 (without recombinant protein). Thirty-seven percent of the rheumatoid factor (RF)-negative RA samples (30 of 81) were AFA-positive by LIA2, and 52 of 54 RF-positive control samples had no AFA detected on LIA2. Higher specificity and sensitivity were obtained by LIA2 versus anti-RA33 immunoblot, whereas good agreement was observed with antikeratin antibody testing. LIA performed significantly better than AFA immunoblotting using natural filaggrin, at a specificity level of 99% (P = 0.0047). CONCLUSION: Citrullinated residues are present in immunoreactive epitopes of natural human filaggrin. AFA can be readily detected by citrullinated peptides in an LIA-based test, resulting in high specificity and positive predictive value for RA. The LIA could serve as a user-friendly alternative to existing immunofluorescence tests and AFA immunoblot techniques. Given its complementarity to RF, this test can be a valuable tool in the differential diagnosis of arthritis.

Amino Acid Sequence↗
PubMed2003

Induction of pemphigus phenotype by a mouse monoclonal antibody against the amino-terminal adhesive interface of desmoglein 3.

Pemphigus vulgaris (PV) is a life-threatening autoimmune blistering disease that is caused by IgG autoantibodies against the cadherin-type adhesion molecule desmoglein (Dsg)3. Previously, we have generated an active mouse model for PV by adoptive transfer of Dsg3(-/-) splenocytes. In this study, we isolated eight AK series, anti-Dsg3 IgG mAbs from the PV mouse model, and examined their pathogenic activities in induction of blister formation. Intraperitoneal inoculation of the AK23 hybridoma, but not the other AK hybridomas, induced the virtually identical phenotype to that of PV model mice or Dsg3(-/-) mice with typical histology of PV. Epitope mapping with domain-swapped and point-mutated Dsg1/Dsg3 molecules revealed that AK23 recognized a calcium-dependent conformational epitope on Dsg3, which consisted of the V3, K7, P8, and D59 Dsg3-specific residues that formed the adhesive interface between juxtaposed Dsg, as predicted by the crystal structure. The epitopes of the mAbs that failed to show apparent pathogenic activity were mapped in the middle to carboxyl-terminal extracellular region of Dsg3, where no direct intermolecular interaction was predicted. These findings demonstrate the pathogenic heterogeneity among anti-Dsg3 IgG Abs due to their epitopes, and suggest the direct inhibition of adhesive interaction of Dsg as an initial molecular event of blister formation in pemphigus.

Acantholysis↗
PubMed2000

Applicability of three anti-PrP peptide sera including staining of tonsils and brainstem of sheep with scrapie.

Three rabbit antibodies (R521, R505, R524) were produced, and raised to synthetic peptides corresponding to residues 94-105, 100-111, and 223-234, respectively, of the sheep prion protein (PrP). Epitope mapping analysis revealed the monospecific character of antisera R505 and R524. In addition to the amino acid sequence against which it was raised, R521 also recognized other small epitopes. ELISA and radio-immunoprecipitation were used to assess the relative immunoreactivities of the antisera to the normal sheep prion protein (PrP(c)). Highest reactivity was found for R521, followed by R505 and R524. According to Western blot analysis, all three sera specifically reacted with the prion proteins PrP(Sc) and PrP27-30, extracted from the brain stem of a scrapie-affected sheep. Yet, with R505 not all of the lower molecular weight deglycosylated forms could be detected. Contrary to the immunoreactivities found with the PrP(Sc) and PrP27-30 isoforms, only R521 recognised PrP(c) from a healthy sheep. The usefulness of all three anti-peptide sera in the immunohistochemical detection of PrP(Sc) in brain stem and tonsils of scrapie-affected sheep was demonstrated and compared with an established rabbit anti-PrP serum.

Amino Acid Sequence↗
PubMed2003

An abnormal but functionally active complement component C9 protein found in an Irish family with subtotal C9 deficiency.

Two independently segregating C9 genetic defects have previously been reported in two siblings in an Irish family with subtotal C9 deficiency. One defect would lead to an abnormal C9 protein, with replacement of a cysteine by a glycine (C98G). The second defect is a premature stop codon at amino acid 406 which would lead to a truncated C9. However, at least one of two abnormal proteins was present in the circulation of the proband at 0.2% of normal C9 concentration. In this study, the abnormal protein was shown to have a molecular weight approximately equal to that of normal C9, and to carry the binding site for monoclonal antibody (mAb) Mc42 which is known to react with an epitope at amino acid positions 412-426, distal to 406. Therefore, the subtotal C9 protein carries the C98G defect. The protein was incorporated into the terminal complement complex, and was active in haemolytic, bactericidal and lipopolysaccharide release assays. A quantitative haemolytic assay indicated even slightly greater haemolytic efficiency than normal C9. Epitope mapping with six antihuman C9 mAbs showed the abnormal protein to react to these antibodies in the same way as normal C9. However, none of these mAbs have epitopes within the lipoprotein receptor A module, where the C98G defect is located. The role of this region in C9 functionality is still unclear. In conclusion, we have shown that the lack of a cysteine led to the production of a protein present in the circulation at very much reduced levels, but which was fully functionally active.

Aged↗
PubMed1996

Identification of surface-exposed linear B-cell epitopes of the nonfimbrial adhesin CS31A of Escherichia coli by using overlapping peptides and antipeptide antibodies.

As a first step toward the design of an epitope vaccine, by using the nonfimbrial adhesin CS31A of Escherichia coli as a carrier, a low-resolution topological and epitope map of the CS31A subunit was developed by using solid-phase peptide synthesis and polyclonal rabbit antibodies raised against both native and denatured proteins. Peptides constituting antigenic epitopes on the major subunit (ClpG) of the multimeric CS31A antigen were identified by examining the binding of the antibodies to 249 overlapping nonapeptides covering the amino acid sequence of ClpG. With antibodies raised against denatured ClpG subunit, seven major epitope regions, corresponding to residues 10 to 18, 45 to 58, 88 to 107, 148 to 172, 187 to 196, 212 to 219, and 235 to 241, were located. Most of the epitopes were hydrophilic and were located in variable regions, residing largely in loop regions at the boundaries of secondary structural elements of ClpG. In contrast, antibodies raised against native CS31A antigen reacted only with the peptide AVNPNA (positions 179 to 184), demonstrating that this peptide was the only linear B-cell epitope of the native protein. The different immunogenic profiles of native CS31A antigen and denatured ClpG indicated that the denaturation process resulted in marked conformational changes in the protein, which could expose epitopes hidden or absent in native CS31A. To identify the surface-exposed epitopes, nine peptides covering the dominant antigenic regions of ClpG were synthesized and used to prepare site-specific antibodies. Antipeptide antibodies were tested, in a competitive enzyme-linked immunosorbent assay (ELISA), for cross-reactivity with native CS31A and denatured ClpG subunit. Four of these antipeptide antibodies bound to the native protein in an accessibility ELISA, indicating that residues 44 to 56, 174 to 190, 185 to 199, and 235 to 249 were surface exposed on CS31A. These data indicate that an immunodominant surface-exposed linear epitope was present in the region from positions 179 to 184 of ClpG in the native CS31A antigen on intact bacterial cells and suggest that the four surface-exposed epitopes constitute potential sites for insertions or substitutions with heterologous peptides.

Adhesins, Bacterial↗
PubMed1997

Immunization with viral antigens: infectious pancreatic necrosis.

Clinical IPN has traditionally been observed in brook trout (Salvelinus fontinalis) and rainbow trout (Oncorhynchus mykiss). However, during the past 10 years outbreaks of IPN have been reported frequently in farmed Atlantic salmon (Salmo salar L.) in Norway. Acute IPN with high mortality is observed in fry at start feeding and in smolt in the first six months after transfer to seawater. Today IPN is the most important infectious disease in Norway in farmed fish, giving economic losses of about 60 million USD yearly. A prophylactic strategy against this disease is strongly needed. Different strategies for developing IPN vaccines have been tested since the virus was first isolated in 1960. Vaccination with live virus has not been successful and is probably not an acceptable strategy for environmental risk reasons. Vaccination with inactivated virus has been tested in rainbow trout given by the oral route, by immersion and injection. Protection against challenge was obtained only by injection. IPN vaccines based on inactivated virus may be effective but are expensive. A subunit vaccine produced by fermentation is a more realistic strategy for fish vaccine production if the actual protective epitopes can be identified. Protective IPNV epitopes may include both B- and T-cell epitopes, but only B-cell epitopes have been examined so far. Epitope mapping with neutralising monoclonal antibodies indicates that the internal variable region of VP2 (aa 200-350) folds into an immunodominant structure including both serotype specific and conserved neutralisation epitopes that can renaturate spontaneously from E. coli-expressed recombinant VP2 (rVP2). Analysis with an IPNV group-A specific neutralising monoclonal antibody indicates that immunisation with recombinant protein containing the segment (aa 86-210) might induce protection against all IPNV serotypes. Subunit vaccines based on E. coli-expressed IPNV proteins have been tested in rainbow trout and Atlantic salmon. Vaccination by immersion in rainbow trout fry with bacterial lysate from E. coli expressing the IPNV Sp strain gene segment A induced protection against challenge with the IPNV Buhl strain. By injection of Atlantic salmon parr with partly purified E. coli-expressed rVP2 (N1 strain), increased resistance against IPN infection was demonstrated by challenge. In field trials it is shown that vaccination of pre-smolt with rVP2 included in a commercial oil/glucan adjuvanted multivalent bacterial vaccine gives protection against IPN in natural outbreaks, compared to fish vaccinated with the same vaccine without the IPNV component.

Animals↗
PubMed2005

Solution structure of the immunodominant domain of protective antigen GNA1870 of Neisseria meningitidis.

GNA1870, a 28-kDa surface-exposed lipoprotein of Neisseria meningitidis recently discovered by reverse vaccinology, is one of the most potent antigens of Meningococcus and a promising candidate for a universal vaccine against a devastating disease. Previous studies of epitope mapping and genetic characterization identified residues critical for bactericidal response within the C-terminal domain of the molecule. To elucidate the conformation of protective epitopes, we used NMR spectroscopy to obtain the solution structure of the immunodominant 18-kDa C-terminal portion of GNA1870. The structure consists of an eight-stranded antiparallel beta-barrel overlaid by a short alpha-helix with an unstructured N-terminal end. Residues previously shown to be important for antibody recognition were mapped on loops facing the same ridge of the molecule. The sequence similarity of GNA1870 with members of the bacterial transferrin receptor family allows one to predict the folding of this class of well known bacterial antigens, providing the basis for the rational engineering of high affinity B cell epitopes.

Amino Acid Sequence↗
PubMed2005

Molecular and biological characterization of human monoclonal antibodies binding to the spike and nucleocapsid proteins of severe acute respiratory syndrome coronavirus.

Human monoclonal antibodies (MAbs) were selected from semisynthetic antibody phage display libraries by using whole irradiated severe acute respiratory syndrome (SARS) coronavirus (CoV) virions as target. We identified eight human MAbs binding to virus and infected cells, six of which could be mapped to two SARS-CoV structural proteins: the nucleocapsid (N) and spike (S) proteins. Two MAbs reacted with N protein. One of the N protein MAbs recognized a linear epitope conserved between all published human and animal SARS-CoV isolates, and the other bound to a nonlinear N epitope. These two N MAbs did not compete for binding to SARS-CoV. Four MAbs reacted with the S glycoprotein, and three of these MAbs neutralized SARS-CoV in vitro. All three neutralizing anti-S MAbs bound a recombinant S1 fragment comprising residues 318 to 510, a region previously identified as the SARS-CoV S receptor binding domain; the nonneutralizing MAb did not. Two strongly neutralizing anti-S1 MAbs blocked the binding of a recombinant S fragment (residues 1 to 565) to SARS-CoV-susceptible Vero cells completely, whereas a poorly neutralizing S1 MAb blocked binding only partially. The MAb ability to block S1-receptor binding and the level of neutralization of the two strongly neutralizing S1 MAbs correlated with the binding affinity to the S1 domain. Finally, epitope mapping, using recombinant S fragments (residues 318 to 510) containing naturally occurring mutations, revealed the importance of residue N479 for the binding of the most potent neutralizing MAb, CR3014. The complete set of SARS-CoV MAbs described here may be useful for diagnosis, chemoprophylaxis, and therapy of SARS-CoV infection and disease.

Amino Acid Sequence↗
PubMed1997

Structure-function analysis of the integrin beta 7 subunit: identification of domains involved in adhesion to MAdCAM-1.

Beta 7 integrins serve special roles in mucosal immunity. Alpha 4 beta 7-mediated adhesion to mucosal addressin cell adhesion molecule-1 (MAdCAM-1) directs lymphocyte homing to the gut, and alpha E beta 7 mediates binding of lymphocytes to E-cadherin on epithelial cells. Since alpha 4 beta 7 mediates adhesion to MAdCAM-1 but alpha 4 beta 1 does not, we used beta 7/beta 1 chimeras to directly assess the importance of specific regions of beta 7 in MAdCAM-1 binding. We found a region of beta 7 (residues 46-386) that accounts for specificity of alpha 4 beta 7 binding to MAdCAM-1. We also used human/mouse and human/rat chimeric beta 7 subunits to map epitopes recognized by fifteen anti-beta 7 mAbs. Six of seven Abs that block adhesion to MAdCAM-1 and E-cadherin (Fib 21, 22, 27, 30, 504; Act-1) mapped to amino acid residues 176-250. Residues 176-250 lie within the region of beta 7 that specifies MAdCAM-1 binding and also within a region that has a predicted structure homologous to the metal ion-dependent adhesion site (MIDAS) domains of the integrin subunits alpha L and alpha M. Three new Abs that recognize beta 7 in the presence of Mn2+, but not Ca2+, and promote adhesion to MAdCAM-1, mapped to amino acids 46-149. One blocking and five other Abs mapped to other regions (amino acids 387-725). We conclude that a MIDAS-like domain serves a critical role in beta 7 integrin-mediated adhesion.

Animals↗
PubMed1995

Characterization of murine monoclonal antibodies against 60-kD Ro/SS-A and La/SS-B autoantigens.

Small cytoplasmic ribonucleoproteins (scRNPs) are important autoantigens in patients with systemic lupus erythematosus and Sjögren's syndrome. MoAbs against these proteins were made by immunization of BALB/c mice with purified human recombinant 60-kD Ro/SS-A or 50-kD La/SS-B proteins. Five stable hybridoma cell lines were obtained, of which four secreted anti-Ro/SS-A antibodies (clones 1D8, 1D11, 2G10 and 6G8) and one produced anti-La/SS-B antibodies (clone 7F6). The MoAbs were further characterized using four different immunoassays: immunofluorescence, immunoblotting, RNA precipitation combined with Northern blotting, and recombinant protein precipitation. All four MoAbs against Ro/SS-A recognized the native protein and one of them (2G10) recognized also intact scRNP particles. Interestingly, hY3-RNA was reproducibly not efficiently precipitated by MoAb 2G10. Epitope mapping using deletion mutants of the 60-kD Ro/SS-A antigen showed that MoAb 1D8 recognized the C-terminal part of this protein, while 1D11 and 2G10 recognized distinct epitopes in the region between the RNP motif and the putative zinc finger domain. The epitopes recognized by these MoAbs are highly conserved among species, and the epitope recognized by MoAb 2G10 may be identical to an autoepitope recognized by sera of patients. This is the first report describing the isolation and characterization of MoAbs of the IgG class against the 60-kD Ro/SS-A and La/SS-B autoantigens obtained by immunization with purified human recombinant proteins. These MoAbs can be of great use in studying the cellular processes in which scRNPs are involved, and may help to determine why these scRNPs become autoantigenic in autoimmune diseases.

Animals↗
PubMed1999

Cross-subtype neutralizing antibodies induced in baboons by a subtype E gp120 immunogen based on an R5 primary human immunodeficiency virus type 1 envelope.

Global human immunodeficiency virus type 1 (HIV-1) diversity may require engineering vaccines to express antigens representing strains prevalent in the target population of vaccine testing. The majority (90%) of incident infections in Thailand are genetic subtype E, with a small percentage of subtype B infections in the intravenous drug user populations. We have evaluated and compared the binding and HIV-1 neutralizing properties of serum antibodies induced in baboons by CHO cell-expressed monomeric gp120 derived from a CCR5-using (R5) subtype E primary HIV-1CM235 or a CXCR4-using (X4) subtype B T-cell line-adapted (TCLA) HIV-1SF2 isolate. In contrast to the subtype-specific HIV-1 neutralizing antibodies induced with recombinant HIV-1SF2 gp120 (rgp120SF2), rgp120CM235 immunization induced antibodies capable of neutralizing both subtype E and subtype B TCLA HIV-1 isolates. However, neither immunogen induced antibodies capable of neutralizing primary HIV-1 isolates. Antibody induced by rgp120CM235 preferentially bound natively folded gp120 and retained strong cross-reactivity against multiple gp120 strains within subtype E as well as subtype B. In contrast, antibody responses to rgp120SF2 were directed predominantly to linear epitopes poorly exposed on native gp120 and had more limited cross-recognition of divergent gp120. Fine epitope mapping revealed differences in antibody specificities. While both rgp120CM235 and rgp120SF2 induced antibodies to regions within C1, V1/V2, V3, and C5, unique responses were induced by rgp120CM235 to multiple epitopes within C2 and by rgp120SF2 to multiple epitopes within C3, V4, and C4. These data demonstrate that strain and/or phenotypic differences of HIV-1 subunit gp120 immunogens can substantially alter antibody binding specificities and subsequent HIV-1 neutralizing capacity.

AIDS Vaccines↗
PubMed1996

Mapping of monoclonal antibody- and receptor-binding domains on human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) using a surface plasmon resonance-based biosensor.

An automated surface plasmon resonance (SPR)-based biosensor system has been used for mapping antibody and receptor-binding regions on the recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) molecule. A rabbit antimouse IgG1-Fc antibody (RAM.Fc) was coupled to an extended carboxymethylated-hydrogel matrix attached to a gold surface in order to capture an anti-rhGM-CSF monoclonal antibody (MAb) injected over the sensing layer. rhGM-CSF was subsequently injected and allowed to bind to this antibody. Multisite binding assays were then performed, by flowing sequentially other antibodies and peptides over the surface, and the capacity of the latter to interact with the entrapped rhGM-CSF in a multimolecular complex was monitored in real time with SPR. Eleven MAb (all IgG1K), were analyzed: respectively, four antipeptide MAb raised against three distinct epitopes of the cytokine (two clones against residues 14-24, that includes part of the first alpha-helix toward the N-terminal region; one clone against peptide 30-41, an intrahelical loop; and one clone against residues 79-91, including part of the third alpha-helix) and seven antiprotein MAbs raised against the entire rhGM-CSF, whose target native epitopes are still undetermined. In addition, the binding capacity to rhGM-CSF of a synthetic peptide, corresponding to residues 238-254 of the extracellular human GM-CSF receptor alpha-chain, endowed with rhGM-CSF binding activity, was tested. The results from experiments performed with the biosensor were compared with those obtained by a sandwich enzyme-linked immunosorbent assay (ELISA), using the same reagents. The features of the biosensor technology (fully automated, measure in real time, sharpened yes/no response, less background disturbances, no need for washing step or labeling of the reagent) offered several advantages in these studies of MAb immunoreactivity and epitope mapping, giving a much better resolution and enabling more distinct epitopes to be identified over ELISA.

Antibodies, Monoclonal↗
PubMed1997

[A recombinant autoantigen U 1 RNP 70,000: expression in E. coli. and its serodiagnostic application].

OBJECTIVE: To express the recombinant autoantigen U 1 RNP 70,000 and to evaluate its serodiagnostic value. METHOD: We used molecular cloning techniques and immunoblotting technique. RESULTS: The recombinant proteins had authentic U 1 RNP 70,000 antigenicity, with the molecular weight corresponding to their theoretical value. 112 of 115(97.4%) anti-70,000 positive sera(IBT) could be detected by r 70,000. CONCLUSIONS: r 70,000 has good specificity and can be used in further detailed epitopes mapping. r 70,000 can be put into use for serodiagnostic detection.

Autoantigens↗
PubMed1995

Mapping of T cell epitopes of the 30-kDa alpha antigen of Mycobacterium bovis strain bacillus Calmette-Guérin in purified protein derivative (PPD)-positive individuals.

The fibronectin-binding 30-kDa alpha Ag is a major secretory protein of growing mycobacteria that stimulates in vitro lymphocyte blastogenesis in most healthy purified protein derivative-positive individuals, but only a minority of patients with active tuberculosis. T cell epitopes of the alpha Ag were assessed using blastogenic responses of PBMC from 12 healthy purified protein derivative-positive subjects to a set of synthetic peptides based on the 325-amino acid sequence of the alpha Ag of Mycobacterium bovis BCG. Because epitope-specific precursor cells are infrequent and randomly distributed, we used Poisson analysis to determine positive responses to 10 micrograms/ml of each peptide in 12 replicate culture wells. Seven immunodominant regions of the alpha Ag were identified. Each subjects responded to at least one of the two most dominant epitopes, which correspond to amino acids 131-155 and 233-257 (from N terminus). Peptides of these two epitopes induced production of IFN-gamma by sorted CD4+ T cells. The immunodominant peptides may have use a components of a vaccine and as tools to study the evolution of the immune response to M. tuberculosis. The two most dominant epitopes both occur in regions of the alpha Ag that differ from those of the atypical pathogens M. avium and M. kansasii. In addition, the M. bovis epitope of amino acids 133-155 differs from that of M. tuberculosis by a single amino acid. It may be possible to exploit the sequence differences for development of diagnostic tests with increased specificity.

Adult↗
PubMed1997

A novel antibody-dependent cellular cytotoxicity epitope in gp120 is identified by two monoclonal antibodies isolated from a long-term survivor of human immunodeficiency virus type 1 infection.

Two monoclonal antibodies (MAbs), 42F and 43F, were isolated some 14 months apart from a single long-term survivor of human immunodeficiency virus type 1 (HIV-1) infection. These MAbs were found to be indistinguishable in terms of their isotypes, specificities, affinities, and biological activities. Both 42F and 43F directed substantial antibody-dependent cellular cytotoxicity (ADCC) against cells infected with four divergent lab-adapted strains of HIV-1, but no neutralizing activity against these strains was detectable. The ability of MAbs 42F and 43F, as well as that of MAbs against two other gp120 epitopes, to direct ADCC against uninfected CD4+ cells to which recombinant gp120SF2 had been adsorbed (i.e., "innocent bystanders") was demonstrated to be less efficient by at least an order of magnitude than their ability to direct ADCC against HIV-1-infected cells. Flow cytometry analyses showed that 42F and 43F also bind to native primary isolate Envs from clades B and E expressed on cell surfaces. By direct binding and competition assays, it was demonstrated that the 42F/43F epitope lies in a domain of gp120 outside the previously described CD4-binding site and V3 loop ADCC epitope clusters. Immunoblot analysis revealed that the 42F/43F epitope is not dependent on disulfide bonds or N-linked glycans in gp120. Epitope mapping of 42F and 43F by binding to linear peptides demonstrated specificity of these MAbs for a sequence of 10 amino acids in the C5 domain comprising residues 491 to 500 (Los Alamos National Laboratory numbering for the HXB2 strain). Thus, 42F and 43F define a new ADCC epitope in gp120. Because of the relative conservation of this epitope and the fact that it appears to have been significantly immunogenic in the individual from which these MAbs were derived, it may prove to be a useful component of HIV vaccines. Furthermore, these MAbs may be used as tools to probe the potential importance of ADCC as an antiviral activity in HIV-1 infection.

Amino Acid Sequence↗
PubMed1998

An epitope structure for the C-terminal domain of dystrophin and utrophin.

The muscular dystrophy protein, dystrophin, and the closely related protein, utrophin, are large cytoskeletal proteins which link actin microfilaments to the plasma membrane. A panel of 38 monoclonal antibodies (mAbs) has been produced against the C-terminal domains of dystrophin and utrophin. This domain interacts with both dystrobrevins, via their "leucine zipper" coiled-coil helices, and syntrophins, adaptor proteins which also interact with nitric oxide synthetase and transmembrane sodium channels. The amino acid sequences recognized by the mAbs have now been identified using a variety of epitope mapping techniques, including fragmentation by transposon mutagenesis, synthetic peptides, phage-displayed peptide libraries, and mutant dystrophins expressed in transgenic mice. In addition to defining antibody recognition sites, mapping was sufficiently precise to provide structural information, since individual amino acids accessible on the surface of the native protein were identified in many cases. In two regions of the domain, short linear epitopes were found in proline-rich sequences which may form surface loops, turns, or linkers, but these were separated by a third region which contained mainly conformational epitopes. The results are consistent with a loose and flexible structure for much of the C-terminal domain, especially around the highly conserved second leucine zipper or coiled-coil helix (CC-H2), but there is evidence for denaturation-resistant tertiary structure in the syntrophin-binding region and the first coiled-coil helix (CC-H1).

Amino Acid Sequence↗
PubMed2004

Characterization of immunodominant linear B-cell epitopes on the carboxy terminus of the rinderpest virus nucleocapsid protein.

The nucleocapsid (N) protein of rinderpest virus (RPV) is one of the most abundant and immunogenic viral proteins expressed during natural or experimental infection. To identify immunogenic epitopes on the N protein, different forms of RPV N protein, including the full-length protein (N(1-525)), an amino-terminal construct (N(1-179)), and a carboxy-terminal construct (N(414-496)), were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins. The antigenicity of each recombinant protein was evaluated by Western immunoblotting. All recombinants were recognized by hyperimmune RPV bovine antisera, indicating that immunoreactive epitopes may be present at both ends of the N protein. However, GST-N(414-496) was much more antigenic than GST-N(1-179) when tested with sera from vaccinated cattle, suggesting that an immunodominant or highly immunogenic epitope(s) may be located at the carboxy terminus of the N protein. Epitope mapping with overlapping peptides representing different regions of the carboxy terminus (amino acids 415 to 524) revealed three nonoverlapping antigenic sites in regions containing the residues (440)VPQVRKETRASSR(452) (site 1), (479)PEADTDPL(486) (site 2), and (520)DKDLL(524) (site 3). Among these, antigenic site 2 showed the strongest reactivity with hyperimmune anti-RPV bovine sera in a peptide enzyme-linked immunosorbent assay but did not react with hyperimmune caprine sera raised against peste-des-petits-ruminants virus, which is antigenically closely related to RPV. Identification of an immunodominant linear antigenic site at the carboxy terminus of the N protein may provide an antigen basis for designing diagnostics specific for RPV.

Animals↗
PubMed2001

Analysis of the proliferative responses to peptides in individuals with vigorous Gag protein-specific proliferation.

Proliferative responses to recombinant HIV proteins in infected individuals may represent a correlate of protection from disease progression. In this study, the proliferative responses to HIV p24, p55 and gp120 were evaluated in infected subjects. Whereas, vigorous proliferative responses directed at the Gag proteins were detected in several individuals, Env-specific proliferation was observed in only one subject. Epitope mapping using overlapping peptides demonstrated proliferative responses of PBMC to Gag peptides. Responses were broadly directed at multiple peptides in some subjects. Although several of the peptides that induced proliferative responses also contain CTL epitopes potentially relevant in the particular individuals, many additional Gag T cell epitopes were present in each subject. This finding may be relevant for the design and testing of HIV candidate vaccines.

AIDS Vaccines↗