An immunodominant, cross-reactive B-cell epitope region is located at the C-terminal part of the hamster polyomavirus major capsid protein VP1

The VP1 represents the major capsid protein of the hamster polyomavirus (HaPV). Here we describe the mapping of epitopes along the VP1 using Escherichia coli-expressed VP1-dihydrofolate reductase (DHFR) fusion proteins and PepScan analysis. By use of DHFR fusion proteins an immunodominant region was localized in the C-terminal part of VP1 between amino acids 320-384. Further epitopes are located in the regions amino acids 1-133 and amino acids 133-320, respectively. There were no obvious differences in the reactivity between sera of tumor-bearing and papilloma-free naturally HaPV-infected hamsters. In contrast, PepScan analysis revealed linear epitopes in the regions amino acids 79-97 and amino acids 353-367 for tumor-bearing animals and amino acids 101-113 and amino acids 165-179 for papilloma-free animals. The region between amino acids 320-384 of HaPV-VP1 was found to be involved in cross-reactivity of VP1 from HaPV and other polyomaviruses. Previously we have demonstrated that heterologous expression of HaPV-VP1 allowed the formation of virus-like particles (VLPs). From epitope mapping data and structural predictions it has been suggested that HaPV-VP1-VLPs may tolerate foreign peptides in the region amino acids 81-88 and the C-terminal part of VP1.     

Characterization of the B cell response of patients with anti-liver cytosol autoantibodies in type 2 autoimmune hepatitis

Anti-liver cytosol type 1 (LC1) autoantibody is detected in 30% of sera from patients with type 2 autoimmune hepatitis (AIH), and is the only circulating autoantibody in 10% of cases. Human formiminotransferase cyclodeaminase (FTCD) has been shown to be the specific liver antigen recognized by anti-LC1 autoantibodies. The aim of this study was to identify the dominant epitope on human FTCD and to analyze antigenic-site sequences for clues on the development of AIH. Recombinant proteins and peptides covering the entire cDNA of human FTCD were tested against anti-LC1 autoantibodies. Conformational epitopes were found throughout the protein but linear epitopes were found exclusively in the C-terminal 146 amino acids. Two groups of sera with different reactivities were found: 69%of the sera recognized two specific linear epitopes at positions 428-434 (NTPEEKD) and 440-447 (LQEGLRRA) of human FTCD; others reacted only with a discontinuous epitope between the amino acids at position 395 and 528. FTCD autoantibody production is thus a polyclonal-antigen-driven B cell response. Autoantibodies against conformational or discontinuous epitopes were found in all patients and two-thirds also recognized linear epitopes on human FTCD.     

Relationship between autoepitope and DNA-binding site on a histone H1 molecule.

Autoepitope and DNA-binding domain on a histone H1 molecule were compared using truncated histone H1 peptides as antigens. At least two epitopes (epitope A, N-terminal side; epitope B, C-terminal side) were found both of which were composed of approximately 20 amino acids. IgM from all 17 anti-histone H1-positive SLE sera reacted with epitope A. IgG from 12 sera reacted with epitope A and IgG from 4 sera reacted with epitope B. In one case, no IgG anti-histone H1 reactivities were found while IgM from the same patient reacted with epitope A. Epitope A had the ability to bind DNA. The reactivities against histone H1 of affinity-purified antiepitope A autoantibodies were inhibited by DNA. These data suggest that some anti-histone H1 antibodies are directed against a histone H1 DNA-binding site, raising the possibility that an idiotype/anti-idiotype network, at least in part, is involved in the generation of anti-histone H1 autoantibodies.     

Allergenic and antigenic determinants of latex allergen Hev b 1: peptide mapping of epitopes recognized by human, murine and rabbit antibodies

Background The rubber elongation factor in Hevea rubber (Hev b 1) is one of the most important latex allergen and is leading cause oflatex type 1 hypersensitivity in children with spina bifida. Objective The aim of this study was to define the allergenic and antigenic epitopes of Hev b 1. Methods The immunoglobulin- (Ig)E and IgG antibody binding sites on Hev b 1 allergen were delineated by enzyme linked immunosorbent assay (ELISA) using synthetic overlapping peptides covering the whole Hev b 1 sequence. In order to improve the binding capacity and specificity all peptides were biotinylated at the N-terminal end via a 6-aminohexanoic acid as spacer and then adsorbed to streptavidin pre-coated microtitre plates. Fine mapping to define the essential amino acid residues for the antibody binding was achieved by using overlapping peptides with one amino acid offset. Results It was demonstrated that the IgE epitopes were located in different regions of Hev b 1 including the C-terminal segment (121–137) and the segments with amino acid residues of 30–49 and 46–64. Two monoclonal antibodies (MoAbs) II2F3 and II4G9 raised against purified Hev b 1 recognized the C-terminal segment only. The results of epitope mapping with three rabbit antisera revealed that five positive peptides, including the epitope peptides 31–49, 46–64 and 121–137, were involved in the antibody-binding sites. Eine mapping on the segments 46–64 and 121–137 showed that the two MoAbs reacted with the peptide 125–134 in the C-terminal region, whereas the peptide with amino acids 124–134 was essential for recognition by human IgE antibodies. Epitopes to rabbit polyclonal IgG and human IgE were also found to be involved in the amino acid residues of 47–59. Conclusion Our results indicate that the most allergenic/antigenic portions of Hev b 1 allergen are the C-terminal region and the region with amino acid residues of 31–64. In both regions, the minimal IgE-binding epitope is almost identical with the IgG-binding epitope.     

Autoantibodies to a Novel Early Endosome Antigen 1

Autoantibodies to EEA1, an antigen on early endosomes, were first reported in the serum of a patient with subacute cutaneous lupus erythematosus (SCLE). Here we have examined 38 sera selected for investigation of autoantibodies to EEA1 on the basis of cytoplasmic vesicle-like reactivity by immunofluorescence. Ten of the sera were reactive to a HeLa cell protein of approximately the sameM_ras human EEA1. Eight of these sera belonged to the IgG₁subclass. Five of the sera reacted with fusion proteins incorporating either the amino (from amino acids 1 to 209) or the carboxyl (incorporating the most C-terminal 300 amino acids) terminus of the human EEA1 protein. Antigens reactive with these 5 sera colocalized with internalized transferrin receptors, indicating their association with early endosomes. The other 5 sera which did not react to both fusion proteins did not colocalize with internalized transferrin receptors. We conclude that 5 of the 38 patients (13%) have autoantibodies to EEA1. None of these patients have SCLE, but have generalized joint pain, polyarthritis, rheumatoid arthritis, or circulating rheumatoid factors.     

Analysis of the autoimmune epitopes on human testicular NASP using recombinant and synthetic peptides

The human nuclear autoantigenic sperm protein, NASP, is a testicular histone-binding protein of 787 amino acids to which most vasectomized men develop autoantibodies. In this study to define the boundaries of antigenic regions and epitope recognition pattern, recombinant deletion mutants spanning the entire protein coding sequence and a human NASP cDNA sublibrary were screened with vasectomy patients' sera. Employing panel sera from 21 vasectomy patients with anti-sperm antibodies, a heterogeneous pattern of autoantibody binding to the recombinant polypeptides was detected in ELISA and immunoblotting. The majority of sera (20/21) had antibodies to one or more of the NASP fusion proteins. Antigenic sites preferentially recognized by the individual patients' sera were located within aa 32-352 and aa 572-787. Using a patient's serum selected for its reactivity to the whole recombinant protein in Western blots, cDNA clones positive for the C-terminal domain of the molecule were identified. The number and location of linear epitopes in this region were determined by synthetic peptide mapping and inhibition studies. The epitope-containing segment was delimited to the sequence aa 619-692 and analysis of a series of 74 concurrent overlapping 9mer synthetic peptides encompassing this region revealed four linear epitopes: amino acid residues IREKIEDAK (aa 648-656), KESQRSGNV (aa 656-664), AELALKATL (aa 665-673) and GFTPGGGGS (aa 680-688). All individual patients' sera reacted with epitopes within the sequence IREellipsis.GGS (aa 648-688). The strongest reactivity was displayed by peptides corresponding to the sequence AELALKATL (aa 665-673). Thus, multiple continuous autoimmune epitopes in NASP involving sequences in the conserved C-terminal domain as well as in the less conserved testis-specific N-terminal region comprising the histone-binding sites, as predicted for an antigen-driven immune response, may be a target of autoantibodies in vasectomized men and may provide a relevant laboratory variable to describe more accurately the spectrum of autoantibody specificities associated with the clinical manifestation of vasectomy.     

Human autoantibodies directed against the RNA recognition motif of La (SS-B) bind to a conformational epitope present on the intact La (SS-B)/Ro (SS-A) ribonucleoprotein particle.

In systemic autoimmunity, the human B cell response to the La (SS-B) autoantigen is polyclonal and directed to both conserved and human-specific epitopes. This study has further characterized the B cell epitope(s) present within the conserved central region of the La protein, LaC (amino acids 111-242) containing the RNA recognition motif (RRM, aa 111-187). Ten overlapping and non-overlapping protein fragments spanning LaC were expressed in bacteria as NH2-terminal fusions with glutathione-S-transferase. The fusion proteins were tested by ELISA for reactivity with a panel of human anti-La sera in order to define the nature of the epitopes. Ninety-two percent of patient sera containing anti-La antibodies reacted with the region of La containing the RRM. Fine mapping of this reactivity using deletion mutants indicated that the deletion of 19 amino acids from either the NH2-terminal or COOH-terminal region of the RRM was associated with loss of antibody reactivity, suggesting that the immunodominant epitope expressed in this region is discontinuous. Autoantibodies affinity-purified from the La RRM fragment to remove other specificities immunoprecipitated newly synthesized native La (SS-B)/Ro (SS-A) complexes, providing additional evidence that autoantibodies were recognizing a conformational epitope. The findings indicate that the human autoantibody response to La involves recognition of a conformational determinant involving the conserved RRM region without necessarily interfering with the RNA-dependent association of the La/Ro ribonucleoprotein.     

Fine-specificity of the anti-CENP-A B-cell autoimmune response

The major targets recognized by anti-centromere autoantibodies are the three centromere-associated proteins (CENPs) A, B, and C, with apparent molecular masses of 19, 80, and 140 kDa, respectively. Previously a major epitope region on the 19-kDa CENP-A antigen was identified by synthesis of a soluble synthetic 15-mer peptide (amino acids 3-17) to be used in enzyme-linked immunosorbent assay and western blot competition assays. However, no systematic experimental scanning for epitope regions on the CENP-A autoantigen has yet been performed. In this study we scanned the complete CENP-A amino acid sequence for epitopes using 19 previously characterized autoimmune-sera. Overlapping peptides 15 amino acids in length and offset by three amino acids were synthesized on activated membranes, covering the whole CENP-A autoantigen. Probing of the membranes with various anti-centromere sera showed that all epitopes are clustered in the N-terminal 45 amino acids. For fine-mapping of this autoreactive region the N-terminus of CENP-A (amino acids 1-45) was scanned again by probing overlapping 15-mer, 12-mer, 10-mer, 8-mer, 7-mer, 6-mer, and 5-mer peptides, all offset by one amino acid, with anti-centromere sera. In this way we localized two epitope core regions within the N-terminal 45 amino acids, one covering amino acids 2-17, recognized by 17 sera, and the other covering amino acids 22-38, recognized by 18 sera. One serum did not react with CENP-A at all. Several sera seem to recognize overlapping individual epitopes within these two epitope core regions. All sera, however, recognize a sequence motif G/A-P-R/S-R-R.     

Characterization of B cell epitopes on the 16K antigen of Mycobacterium tuberculosis.

To characterize the antigenic parts of the 16K protein of Mycobacterium tuberculosis, overlapping peptides according to the amino acid sequence of the 16K protein were synthesized. In total, 14 peptides of 20 amino acids in length with an overlap of 10 amino acids and two additional decapeptides (amino acids 31-40 and 61-70) were tested with eight anti-16K MoAbs and human sera. The common recognition site of MoAbs F67-8 and F67-16 was LRPTFDTRLM (amino acids 31-40) and of MoAbs F159-1 and F159-11 DPDKDVDIMV (amino acids 61-70). However, for binding of the MoAbs to these peptides additional amino acids were required at either the N- or C-terminus, suggesting that some kind of conformation is required. The recognition sites of the MoAbs F23-41, F23-49, F24-2 and TB68 could not be identified using the peptides, indicating that the MoAbs only bound to conformational epitopes and not to peptides which may contain parts of these epitopes. The MoAbs bound to beta-galactosidase fusion proteins comprising parts of the 16K protein, indicating that some kind of native conformation is present on the recombinant proteins. Sera from 14 of 19 patients with tuberculosis and none from 19 controls reacted with the purified 16K protein. Sera from four of these 14 patients reacted with two overlapping peptides (amino acids 71-100). Apparently, antibodies in patients' sera against the 16K protein are predominantly directed against conformational epitopes.     

Mapping of epitopes of VP2 protein of chicken anemia virus using monoclonal antibodies

To map the epitopes of VP2 protein of chicken anemia virus (CAV), VP2 was expressed as a fusion protein in Escherichia coli BL21 (DE3). The Western blot demonstrated that recombinant VP2 protein could be recognized by sera of chickens infected with CAV. Female BALB/c mice were immunized with purified recombinant VP2 produced in E. coli BL21 (DE3) and seven VP2-specific monoclonal antibodies (MAbs) were developed. The results of Western blot showed that all the seven MAbs recognized the recombinant VP2 protein expressed in the baculovirus and reacted with MDCC-MSB1 cells infected with CAV by indirect immunofluorescence assay. The VP2 protein was dissected into 21 overlapping fragments, expressed as fusion peptides in E. coli and used for epitope mapping by pepscan analysis. ELISA and Western blot assays indicated that most of MAbs reacted with the 12th and 13th fragments (amino acids 111-136) and one of them reacted with the 3rd fragment (amino acids 21-36). The linear immunodominant epitope of VP2 was located mainly in amino acid residues 111-126 and 121-136.     

Elucidation of linear epitopes of pertussis toxin using overlapping synthetic decapeptides: identification of a human B-cell determinant in the S1 subunit indicative of acute infections

To identify relevant linear epitopes within the immunodominant ADP-ribosyl transferase (S1 subunit) of pertussis toxin (PT), its complete amino acid sequence was synthesized as consecutive, overlapping decapeptides on solid phase and probed for seroreactivity with pertussis specific human antisera in 'peptide scans'. Comparison of the resulting antigenic profiles revealed two distinct types of human antisera, though amino acids 140-200 could not be assessed as the corresponding peptides reacted non-specifically with the detection system. Human anti-pertussis sera predominantly recognized linear immunodominant epitopes located in three separated segments spanning amino acids 3-16, 21-30, and 211-222. Antisera originating from infants with acute B. pertussis infections (type I) identified determinants in all three segments, while type-II antisera from convalescent patients only recognized epitopes in the N-terminal regions. The binding of pertussis specific antisera—both type I and type II—to the holotoxin was inhibited by preincubation of antibodies with synthetic peptides corresponding to two linear determinants located at the N-terminus of S1: R 3-16 and R 21-30. However, competitive binding of antibodies to PT and to synthetic peptides equivalent to the third epitope (R 211-222) was only observed with type I antisera. Thus, the linear immunogenic determinant identified at the C-terminus of the A-protomer represents a human epitope which is apparently specific for antisera from pertussis patients with acute infections. The possible application of this determinant in serologic diagnosis will be a valuable tool to detect and distinguish acute Bordetella pertussis infections.     

Fine specificity of the B-cell epitopes recognized in HIV-1 NEF by human sera.

We have previously used partially overlapping synthetic nonapeptides to characterize the human natural antibody response against HIV-1 negative regulatory factor (NEF), and identified nine 5 to 13 amino acid long regions that were recognized by sera of HIV-1-infected individuals. In this report we define the minimal size of these epitopes with the use of shorter, from 3 to 8 amino acid long partially overlapping peptides covering the complete sequence of the previously identified reacting regions and the N- and C-terminal flanking sequences. We also introduce a new method for the analysis of the reactivities obtained with peptides of different lengths. In six of the antigenic regions the epitopes were found to be noncontiguous and to consist of multiple, down to three amino acid long separate reactive stretches (epitope 1: WSK, VGW, TVRERMRR; epitope 3A: PLRPM, SHFLK; epitope 3B: SQRRQD, DLW; epitope 3C: IYHT, QGYFPDWQN; epitope 4: SLL, VSL; epitope 5: EVLEWRFDSR, VAR). Three epitopes were clearly linear (epitope 2: CAWLE; epitope 3D: LTFGWC; epitope 6: PEYF). Interestingly, five of the minimized B-cell epitopes (1, 3A, 3C, 3D, 5) recognized by human sera overlap totally or partly with the previously identified T-cell epitopes in HIV-1 NEF. Also, only three of the epitopes (3C, 3D, 5) were in a computer-based homology search shown to contain strictly NEF-specific sequences.     

Mapping of antigenic sites on the nucleocapsid protein of the severe acute respiratory syndrome coronavirus

Antigenic sites on the nucleocapsid (N) protein of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) were mapped by Pepscan analysis with overlapping peptides that span the N protein sequence. Two major immunodominant epitopes located in the C-terminal region (amino acids [aa] 362 to 412) and middle region (aa 153 to 178) reacted with more than 75% of sera from SARS patients. Several minor immunodominant epitopes were reactive with about 50% of the SARS sera. Antisera from mice immunized with inactivated SARS-CoV recognized the two major immunodominant epitopes and one antigenic site located adjacent to the N-terminal region (aa 76 to 101), which did not react with the sera from SARS patients. Several monoclonal antibodies against SARS-CoV bound to the N- or C-terminal antigenic sites. These results suggest that the above antigenic sites on the N protein are important in eliciting humoral immune response against SARS-CoV in humans and animals and can be used as antigens for developing diagnostic tests.     

Mapping of novel autoreactive epitopes of the diabetes-associated autoantigen IA-2

IA-2, a member of the tyrosine phosphatase family, has been identified as a dominant autoantigen in type 1 diabetes. To define humoral IA-2 epitopes, we generated a panel of IA-2 deletion mutants and chimeric proteins using the highly homologous tyrosine phosphatase-like protein IA-2beta. Analysis of autoantibody reactivity in 111 IA-2 antibody positive sera from patients with type 1 diabetes revealed that humoral epitopes cluster to several domains of the intracytoplasmic part of IA-2 [IA-2ic, amino acid (aa) 604-979]. Immunodominant epitopes were found in the first N-terminal 73 amino acids (56% positive), in the middle domain residing between residues 699-874 (45% positive) and the C-terminus depending on the presence of aa 931-979 (at least 37% positive). Competition experiments with overlapping peptides revealed that autoantibody binding towards the N-terminus was dependent on residues 621-628. In the C-terminal domain, two novel conformation-dependent epitopes were identified. The first epitope requires the presence of the C-terminal part of IA-2 (aa 933-979) and an IA-2-specific region between residues 771-932. Reactivity against the second epitope was dependent on intact C-terminal domains as well as residues in the middle (aa 887-932) and N-terminal regions (aa 604-771) which are conserved in IA-2 and IA-2beta. We here defined novel autoantigenic determinants in the N-terminus of IA-2 and characterized conformational epitopes residing in the C-terminal region or spanning from C-terminal residues to the N-terminal domain of IA-2ic. The identification of dominant target regions of diabetes-specific autoantibodies may help to elucidate the molecular mechanisms involved in the autoimmunity towards IA-2.     

Antibody responses to an immunodominant nonstructural 1 synthetic peptide in patients with dengue fever and dengue hemorrhagic fever

Two flaviviruses, dengue (DEN) virus and Japanese encephalitis (JE) virus, are important because of their global distribution and the frequency of epidemics in tropical and subtropical areas. To study the B-cell epitopes of nonstructural 1 (NS1) glycoprotein and anti-NS1 antibody response in DEN infection, a series of 15-mer synthetic peptides from the predicted B-cell linear epitopes of DEN-2 NS1 protein were prepared. Enzyme-linked immunosorbent assay (ELISA) was performed to analyze antibody responses to these peptides from sera of both DEN and JE patients. One peptide derived from DEN-2 NS1, D2 NS1-P1 (amino acids 1–15), was identified as the immunodominant epitope that reacted with sera from dengue fever (DF) patients but not JE patients. The isotype of D2 NS1-P1-specific antibodies was mainly immunoglobulin M (IgM) in all sera that tested positive. A specificity study demonstrated that sera from all four DEN types reacted with D2 NS1-P1. A dynamics study showed that specific antibodies to this peptide could be detected as early as 2 days after the onset of symptoms. We observed significant anti-D2 NS1-P1 antibody responses in 45% of patients with primary and secondary infections with DF or with dengue hemorrhagic fever. This is the first report demonstrating that significant anti-DEN NS1 antibodies can be induced in the sera of patients with primary DEN infection. J. Med. Virol. 57:1–8, 1999. © 1999 Wiley-Liss, Inc.     

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

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

Evaluation of the allergenicity and antigenicity of bovine-milk alphas1-casein using extensively purified synthetic peptides

Alphas1-Casein (CAS1_BOVIN), the major allergen of cow's milk (CM), is widely used as hydrolysates in infant diet formulae and additive to other processed food items. To date, most of the reported B-cell epitope mapping were performed on polyethylene pins or cellulose-derivative membrane. We sought to locate the motifs critical for human-specific IgE and rabbit polyclonal IgG binding using extensively purified CAS1_BOVIN, synthetic peptides and derivatives. Thirteen overlapping peptides covering the whole CAS1_BOVIN encompassing 17 : 20 amino acid (AA) were synthesized by f-moc AA solid-phase polyamide peptide synthesis. In addition, six cyanogen bromide (CNBr) cleavage fragments were prepared. Limited hydrolysis, oxidized and reduced/alkylated derivatives were also produced. The preparations were purified by ion exchange, gel filtration chromatography, reversed phase and high-performance liquid chromatography. The homogeneity was visualized by sodium dodecyl sulfate (SDS) and poly acryl amide gel electrophoresis (PAGE) followed by IgE and IgG immunoblotting. IgE binding was measured by Biotin Streptavidin (Bio/strep) fluoro enzyme immuno assay (FEIA) or ELISA-inhibition. Eighteen CM allergy (CMA) sera from 45 clinically examined children (Melbourne) and five adults (Bergen) were selected. Individual sera and pools were used for mapping IgE-binding epitopes. Rabbit IgG sera and pools were used for locating the antigenic sites of the molecule. Results indicated that all the individual CMA sera and pools recognized the intact molecule and three of the CNBr fragments as major antibody-binding allergens. The N- and C-terminal peptides (CAS 16-35; CAS 136-155) showed high IgE-binding affinity. CAS 1-18 and CAS 181-199 showed high IgG bindings. Considering the diversity of the antibody specificities, a reasonable agreement between IgE and IgG epitopes were found at the N- and C-terminals of CAS1_BOVIN. Mapping IgE B-cell epitopes by direct Bio/strep FEIA allowed the development of a sensitive modified technique for detecting unlabelled, casein immune dominant peptides in food products.     

Definition of the peptide binding motif within DRB1*1401 restricted epitopes by peptide competition and structural modeling

This study identified the peptide-binding motif of HLA-DRA/DRB1*1401 (DR1401). First, peptides containing DR1401 restricted epitopes were identified using tetramer-guided epitope mapping. Among these, an influenza B peptide was selected for the motif study. After confirming the binding register for this peptide using a set of arginine substitutions, binding affinities were determined for 33 peptides derived from this influenza B sequence with single amino acid substitutions. The DR1401 peptide-binding motif was deduced from the relative binding affinities of these peptides and confirmed by structural modeling. Pocket 1 demonstrated a preference for aliphatic anchor residues and methionine. Pocket 4 accommodated methionine and aliphatic residues, but also allowed some polar and charged amino acids. Pocket 6 preferred basic residues but also allowed some polar and aliphatic amino acids. Pocket 9 preferred aliphatic and aromatic amino acids and tolerated some polar residues but excluded all charged residues. Together these preferences define a distinct set of peptides that can be presented by DR1401. The resulting motif was used to verify T cell epitopes within the novel antigenic peptides identified by tetramer-guided epitope mapping and within peptides from published reports that contain putative DR1401 epitopes.     

IgE-binding epitopes of the American cockroach Per a 1 allergen

Cockroach is one of the major indoor allergens for IgE-mediated allergic respiratory illnesses throughout the world. The American cockroach (Periplaneta americana) Per a 1 allergen is antigenically cross-reactive with the German cockroach (Blattella germanica) Bla g 1 allergen. The aim of this study was to identify linear B cell epitopes of Per a 1 that are recognized by human IgE. Per a 1 deletion mutants were generated from the recombinant Per a 1.0104 allergen (274 amino acid residues), and antigenicities were assessed by immunoblotting, enzyme-linked immunosorbent assay (ELISA), and binding inhibition. Human atopic sera were not able to recognize deletion mutants consisting of amino acids 1-77, 86-205, and 200-266. However, human sera did recognize the N-terminal mutant containing amino acids 1-87 and the C-terminal mutant containing amino acids 200-274, demonstrating positive IgE binding that was heterogeneously distributed among the different sera tested. Amino acid residues 78-85 and 267-274, containing internal repeats, were shown to be required for IgE binding to the Per a 1.0104 protein. Two peptides corresponding to these IgE-binding amino acid sequences were synthesized. Peptide 78-85 showed a positive IgE interaction with 80% of the sera, while peptide 267-274 was capable of IgE binding to all of the sera tested. Moreover, preincubation of atopic sera with IgE-positive recombinants and peptides resulted in marked inhibition of the IgE binding to purified Per a 1.0104 allergen. Amino acid sequences 78LIRALFGL85 and 267IRSWFGLP1274 of the American cockroach Per a 1.0104 allergen were involved in IgE binding. These findings will advance the understanding of the specific reactivity of the epitopes of cockroach allergens, thereby contributing to the development of specific immunotherapies for clinical use.     

Lymphocyte recognition elements on the VP1 protein of Theiler's virus.

Theiler's virus is a murine picornavirus that persists in the central nervous system in susceptible mouse strains, and gives rise to immune mediated demyelinating disease. Antiviral CD4 T cells are necessary to protect from overwhelming virus replication in the acute phase of the disease, and are thought to act by stimulating the antibody response. The present study used overlapping synthetic peptides to map the location of epitopes recognized by CD4 T cells. One T-cell epitope was identified between amino acids 33-47 of VP1, which was recognized by virus-reactive T cells. 'Cryptic' epitopes were also present within VP1 at positions 153-167, 166-180, 225-239 and 233-247. A linear B-cell epitope was identified in the C-terminal region 225-276. Immunization of CBA mice with inactivated virus, but not peptides containing VP1 B- or T-cell epitopes, reduced the virus titre in the CNS in the acute phase of the disease.