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PubMed1990

Immune response and epitope mapping of a candidate HIV-1 p17 vaccine HGP30.

A thirty amino acid synthetic peptide (HGP30) representing the conserved region of HIV-1 p17 induced high titer antibodies to the native p17 in rabbits. This immune sera neutralized HIV-1 replication in cell culture and one of the high titer antisera also inhibited CD4-dependent cell fusion. Pepscan analysis with overlapping nonapeptides derived from the sequence of HIV-1 p17 identified the sequence (KE) ALDKIEE (EQ) as the major antibody binding site. Sera of 9% of AIDS patients (7/76) and 18% of HIV-1 seropositive healthy homosexuals (40/223) were positive for HGP30 antibodies. Decline in HIV-1 p17 antibodies has been shown to be related to disease progression in both children and adults, suggesting that HIV-1 p17 antibodies may be protective. Hence, a synthetic HIV-1 p17 peptide, representing the immunodominant epitope, could be useful as a candidate vaccine for immunization of HIV-1 seronegative or seropositive healthy homosexuals.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Epitope mapping of monoclonal antibodies to the paired helical filaments of Alzheimer's disease: identification of phosphorylation sites in tau protein.

Tau is a neuronal phosphoprotein the expression of which is developmentally regulated. A single tau isoform is expressed in fetal human brain but six isoforms are expressed in adult human brain, with the fetal isoform corresponding to the shortest adult isoform. Phosphorylation is also developmentally regulated, as fetal tau is phosphorylated at more sites than adult tau. In Alzheimer's disease, the six adult tau isoforms become hyperphosphorylated and form the paired helical filament (PHF), the major fibrous component of the neurofibrillary lesions. One way to identify phosphorylated sites in tau is to use antibodies that recognize phosphorylated residues within a specific amino acid sequence. We here characterize the two novel phosphorylation-dependent anti-tau antibodies AT270 and AT180 and identify their epitopes as containing phosphorylated Thr-181 and Thr-231 respectively. With these antibodies we show that these two threonine residues are partially phosphorylated in fetal and adult tau and almost fully phosphorylated in PHF tau. This result contrasts with previous studies of Ser-202 and Ser-396 which are partially phosphorylated in fetal tau, unphosphorylated in adult tau but almost fully phosphorylated in PHF tau.

Alzheimer Disease↗
PubMed1994

Epitope mapping for epidermolysis bullosa acquisita autoantibody by molecularly cloned cDNA for type VII collagen.

Epidermolysis bullosa acquisita is a subepidermal blistering disease in which patients have autoantibodies against the non-collagenous domain of type VII collagen. Starting with previously isolated 1-kilobase pair (Kb) cDNA for this autoantigen, we isolated overlapping cDNAs with a combined open reading frame of approximately 3.2 Kb, encoding most (approximately 115 kilodaltons [KDa]) of the N-terminal non-collagenous domain of type VII collagen. To localize immunogenic domains, we produced maltose-binding fusion proteins with cDNA encoding different portions of this autoantigen. These cDNA fragments scan from 5' to 3' of this non-collagenous domain and overlap each other. An immunoblot analysis of these fusion proteins with eight epidermolysis bullosa acquisita patient sera demonstrated that each patient serum binds to different regions of this molecule and that epitopes for these patient sera locate throughout this autoantigen. These data suggest that multiple epitopes on the N-terminal non-collagenous domain of type VII collagen are recognized by circulating autoantibodies in patients with epidermolysis bullosa acquisita.

Autoantibodies↗
PubMed1993

The globular head domain of titin extends into the center of the sarcomeric M band. cDNA cloning, epitope mapping and immunoelectron microscopy of two titin-associated proteins.

Immunoelectron microscopical results have shown that the Z and M bands of the sarcomere are interconnected by the long titin molecules. Here we have characterized by monoclonal antibodies, cDNA cloning and immunoelectron microscopy the two titin-associated proteins (190 and 165 kDa proteins), which seem responsible for the formation of a head structure on one end of the 0.9 micron long titin string. The human 165 kDa (1465 residues) and 190 kDa (1451 residues) proteins have unique N-terminal domains some 110 residues in length. Both proteins show 12 repeat domains with strong homology to either fibronectin type III (motif I) or immunoglobulin C2 (motif II) domains, which are arranged in the order II-II-I-I-I-I-I-II-II-II-II-II. Over these repeat domains the two proteins share 50% sequence identity (70% similarity). Epitopes situated in the C-terminal 138 or in the preceding 206 residues of the 165 kDa protein locate in immunoelectron microscopy to stripes situated 18 or 15 nm from the center of the M band. An epitope situated 277 to 129 residues prior to the C-terminus of the 190 kDa protein (i.e. repeats 10 and 11) locates to the center of the M band. Thus the head structure of the titin molecule extends into the center of the M band. Microsequence data on peptides from the titin-associated bovine 165 kDa protein and from conventionally purified bovine M-protein argue together with the reactivity of the antibodies that 165 kDa protein and M-protein are identical. The integrating structure of the sarcomere, which is based on titin and its side-on (C-protein and 86 kDa protein) or end-on (190 kDa protein and 165 kDa protein) associated proteins arises from muscle-specific members of the superfamily of immunoglobulin-like proteins.

Amino Acid Sequence↗
PubMed2001

Prevention of hepatitis C virus infection in a chimpanzee by vaccination and epitope mapping of antiserum directed against hypervariable region 1.

We previously reported on a chimpanzee immunized with both putative envelope glycoproteins (E1 and E2) of hepatitis C virus (HCV), strain HCV-N2, and synthetic peptides of hypervariable region 1 (HVR1) of a different isolate, HCV-#6. The chimpanzee showed complete protection against HCV-#6 infection only when the titer of anti-HVR1 increased, suggesting that an immune response to the HVR1 is more essential in protecting a chimpanzee from HCV infection than an immune response to E1 and E2. In this study, we immunized this chimpanzee with only synthetic HVR1 peptides after anti-E1 and anti-E2 antibody levels dropped and then rechallenged with 10 infectious chimpanzee doses of HCV. The immunized animal was protected, and neutralization of HCV with the antiserum from the protected animal was achieved by inoculating another chimpanzee with HCV preneutralized by this antiserum mixture. Epitope analysis of HVR1 by Pin-ELISA using this antiserum seemed to demonstrate that the antibody response was directed mainly against the C terminus of HVR1. Moreover, our results showed that, if a part of the sequences was conserved, a broad cross-reactivity of the antiserum could be observed, even if amino-acid sequences in this epitope were substituted for those of other HCV strains.

Journal Article↗
PubMed1997

Plasmodium vivax: epitope mapping of monoclonal antibodies against the N-terminal region of the merozoite surface protein 1.

Plasmodium vivax is the most widely distributed human malaria with an estimate of 35 million cases per year. The deduced amino acid sequence comparisons of the Merozoite Surface Protein 1 (MSP1) from several plasmodial species, including that of P. vivax (PvMSP1), revealed the existence of highly conserved blocks and polymorphic blocks. We had previously shown that sequences within conserved blocks from the N-terminal region of the PvMSP1 were poorly immunogenic in natural human infections. These results suggest that these regions code for important and unknown structural and/or functional features and thus they could potentially be tested as a sub-unit PvMSP1 vaccine. In the present study, a battery of monoclonal antibodies (Mabs) was produced against the N-terminal region of the PvMSP1 in an attempt to determine whether these N-terminal ICBs contained all the epitopes exposed on the native molecule. The results suggest that the most terminal ICB2 and ICB3 blocks are not exposed on the surface of the PvMSP1 native molecule and clearly eliminate the possibility of considering the N-terminal domains as unique components of a sub-unit PvMSP1 vaccine candidate.

Amino Acid Sequence↗
PubMed1993

Epitope mapping and characterization of antigenic determinants of heat-stable enterotoxin (STh) of enterotoxigenic Escherichia coli by using monoclonal antibodies.

A panel of monoclonal antibodies (MAbs) specific for the heat-stable enterotoxin (STh) of enterotoxigenic Escherichia coli was produced. All four MAbs (8G7, 53-4, 11C, and SH1) bound to native STh in an enzyme-linked immunosorbent assay to various degrees, with clone SH1 showing the best affinity. The MAbs were screened for neutralizing and guanylate cyclase-inhibiting activities by the suckling mouse assay and the cyclic GMP assay using T84 cells, respectively. The contact amino acid residues governing the reactivity of the four MAbs were precisely determined by using several chemically synthesized analogs of the various heat-stable enterotoxins (STa's). Three distinct antigenic sites of STh sufficiently removed from each other, one near the N terminus, another in the core functional region of the toxin, and the third in the C-terminal region, were recognized by the different MAbs. MAb SH1, which recognized Asn at position 4 and Tyr at position 5 from the N terminus was 100 times more potent in neutralizing the bioactivity of STh in the suckling mouse assay than was MAb 11C, which recognized Thr at position 16 and Tyr at position 19 from the N terminus of the STh molecule. The MAbs which recognized Leu at position 9 from the N terminus (MAb 53-4) and Tyr at position 19 from the N terminus (MAb 8G7) showed intermediate activities in the neutralization assay. The guanylate cyclase-inhibiting activities of SH1 and 11C essentially paralleled the results for the neutralization of bioactivity, while MAbs 53-4 and 8G7 exhibited reverse activity. These results indicate that MAbs that recognize the N-terminal residues which have been shown not to be essential for toxic activity have a potent protective capacity. None of the MAbs reacted with reduced and carboxy-methylated native STh. This suggests that all of the MAbs mediate their effect by reacting with conformation-dependent antigenic determinants.

Amino Acid Sequence↗
PubMed1990

Fine epitope mapping of the human SS-B/La protein. Identification of a distinct autoepitope homologous to a viral gag polyprotein.

To analyze the autoepitopes on the SS-B/La protein, a cDNA covering the entire region coding the protein was isolated from a human cDNA library. The cDNA was subcloned into an expression plasmid vector, pEX, to express its protein product as a fusion protein with cro-beta-galactosidase in Escherichia coli. A recombinant pEX plasmid expressing three-fourths of the protein (amino acid 112-408) was also constructed. The antigenicities of these recombinant proteins were confirmed with a patient's serum. Their various deletion mutants were produced with exonuclease III treatment from the 3' ends of the cDNAs without changing the proper translational frame. Immunoblot analysis and enzyme-linked immunosorbent assay were used to evaluate the reactivities of the recombinant proteins with patients' sera to determine the autoepitopes. A narrow segment (amino acid 88-101) and the region where several epitopes were located (amino acid 283-338) on the SS-B/La protein were universally recognized by all the sera with anti-SS-B/La antibodies examined. An additional epitope region (amino acid 179-220) was recognized by some patients' sera. Computer analysis revealed that the most distinct autoepitope, amino acid 88-101, had a striking homology to a retroviral gag polyprotein. These findings indicate that exogenous or endogenous retroviruses may play a role in initiation of the anti-SS-B/La autoimmunity.

Amino Acid Sequence↗
PubMed1991

Murine and human B cell epitope mapping of the Mycobacterium tuberculosis 10-kD heat shock protein using overlapping peptides.

The human immune response to the 10-kD M. tuberculosis protein was studied by a competition ELISA using monoclonal antibody (MoAb) SA-12. Twenty-five per cent of the sera from 20 patients with tuberculosis and none from 21 control subjects inhibited binding of SA-12 to the 10-kD antigen. To characterize the antigenic parts of the 10-kD antigen, overlapping decapeptides according to the amino acid sequence of the 10-kD protein were synthesized. In total, 91 sequential decapeptides, with an overlap of nine amino acids, were tested in ELISA with MoAb SA-12, human and murine sera (PEP scan). SA-12 recognized the amino acid sequence WDEDGEK (amino acid 50-56). All human sera, from patients with tuberculosis as well as from control subjects, gave almost identical undulating patterns of reactivity with the decapeptides. No relationship was found between the ability of the patients' sera to inhibit binding of MoAb SA-12 and the binding of these sera to the decapeptides comprising the epitope recognized by SA-12 in the PEP scan. Apparently, antibodies in patients' sera against the 10-kD protein are predominantly directed against discontinuous epitopes and, consequently, the continuous epitopes as presented in the PEP scan are not suitable to discriminate between patients with tuberculosis and control subjects. In the PEP scan, sera from BALB/c mice, both non-immunized and immunized with either live M. tuberculosis or the 10-kD protein gave similar patterns of reactivity, albeit different from the patterns obtained with the human sera. However, after immunization of the mice, clearly increased levels of antibodies to primary structures of the 10-kD protein were observed.

Amino Acid Sequence↗
PubMed1991

Epitope mapping of two monoclonal antibodies to the central portion of human osteonectin.

In this study preliminary characterization of two monoclonal antibodies against osteonectin was undertaken. One monoclonal originally raised against bovine bone osteonectin cross reacts with human bone and platelet osteonectin. The other monoclonal antibody has been reported to react with osteonectin derived from human bone and bovine bone but not to the same extent with that from platelets. Initial mapping of the antigenic determinants for both monoclonals was done by testing their ability to bind to the expressed forms of osteonectin in two overlapping SaOS-2 lambda gt11 osteonectin cDNA clones. One clone contains a 0.54 kb insert and is comprised of 50 nucleotides of 5' noncoding and a coding segment for a 17 amino acid signal peptide and 146 amino acids of the N-terminal region of the mature protein. The other clone has a 1.9 kb insert, and includes amino acid no. 18 to the C-terminus of the molecule (amino acid no. 286), a single termination codon, and 1115 nucleotides of 3' noncoding sequence. Both monoclonals recognized expressed osteonectin from the two lambda gt11 SaOS-2 cDNA clones. These results localize the epitope to a region between amino acids 18-146 of osteonectin.

Amino Acid Sequence↗
PubMed1992

Linear epitope mapping of an Sm B/B' polypeptide.

Autoantibodies binding the Sm B/B' peptides are commonly associated with SLE. IgG antibodies binding overlapping octapeptides of Sm B/B' have been evaluated in 10 patients with anti-Sm and anti-nRNP precipitins, 5 patients with other autoimmune serology, and 4 normal human sera. Neither normal controls nor patients without an anti-Sm precipitin significantly bind any of the Sm B/B' octapeptides. All sera tested containing an anti-Sm precipitin strongly bind octapeptides from eight regions of the Sm B/B' sequence. Three of these eight regions share the same octapeptide sequences (PPPGMRPP) that are consistently the most immunoreactive octapeptides from Sm B/B'. Binding of the similar PPPGIRGP, as well as binding to deletion and substitution peptides, suggest that the motif PPPG(I,M) (R,K) appears to best define this binding. Interestingly, PAPGMRPP in the nRNP C peptide is as antigenic as PPPGMRPP and may provide a partial explanation for the cross-reactivity shown between Sm and nRNP autoantibodies. However, the sequence, PPPGMIPP, from nRNP A is not antigenic. These data define the linear sequence autoantigenicity of the Sm B/B' protein. They also demonstrate that the predominant autoimmune epitope is a proline-rich sequence from which limited variance is permitted before antigenicity is destroyed.

Amino Acid Sequence↗
PubMed1994

Comprehensive T-cell epitope mapping of HIV-1 env antigens reveals many areas recognized by HIV-1-seropositive and by low-risk HIV-1-seronegative individuals.

Peripheral blood mononuclear cells from 12 asymptomatic human immunodeficiency virus (HIV)-1-seropositive and nine HIV-1-seronegative donors were screened for proliferative T-lymphocyte responses to peptides derived from a consensus sequence of the HIV-1 env gene products from 25 HIV-1 isolates. Two hundred seventy-eight overlapping 17mer peptides, incremented by three residues each, were pooled into groups, each containing eight sequential peptides, for use in proliferation tests. Thirty-eight additional peptides containing variant amino acid residues also were tested. Proliferation data were analyzed using an algorithm that reduced subjective bias and estimated the responding cell frequencies. Peripheral blood mononuclear cells from a majority of donors, regardless of HIV-1 status, recognized peptides within two pools derived from the gp120 sequence and peptides from one pool in gp41. Pool 25 peptides from gp41 (centered around residue 600 of the gp160 consensus sequence) were recognized most frequently. The observed inability to differentiate between responses of HIV-1-seropositive and HIV-1-seronegative individuals implies either a lack of HIV-1 disease-related immunodominant env epitopes or functional abrogation of HIV-1 env-specific T-helper lymphocyte responses soon after infection. The observed proliferation of T lymphocytes from noninfected, low-risk individuals questions the origin of the responses to HIV-1 env-derived peptides and suggest that preexisting, cross-reactive immunity could influence responses to HIV-1.

Adult↗
PubMed1988

Epitope mapping of human luteinizing hormone using monoclonal antibodies.

To establish structure-function relationships for human (h) LH, 14 murine monoclonal antibodies (MABs) to hLH were characterized in terms of their affinity of binding (Ka), their specificity for intact glycoproteins and their subunits, their paratopic relationships, and their ability to interfere with the biological activity of hLH. The Ka values obtained ranged between 2.4 x 10(6) and 1.0 x 10(10) for intact hLH and between 2.3 x 10(6) and 7.5 x 10(8) liters/M for the free alpha- and beta-subunits, indicating that, in general, the antibodies showed higher avidity for the intact hormone. Six MAB recognized both the intact and free alpha-subunit of hLH, cross-reacted with intact hCG, hFSH, and hTSH, and thus appeared to be alpha-directed. Four MAB were beta-directed, recognizing both intact hLH and its free beta-subunit. One of these beta-directed MABs also cross-reacted with intact hCG, hFSH, and hTSH, while two others recognized both intact and free beta-subunits of hLH and hCG. The fourth beta-directed MAB was quite specific for intact hLH and its beta-subunit. The remaining four MABs recognized epitopes only on the intact hormone; three recognized intact hLH and hCG, and the fourth was specific for intact hLH. Their paratopic relationships tested in competitive binding studies resulted in either mutual competition or complementarity, sometimes with cooperativity. Biointerference, defined as the ability to inhibit hLH-induced testosterone biosynthesis in dispersed rat Leydig cells, indicated that three of the alpha- and one of the beta-directed antibodies neutralized the biological response of hLH in this bioassay in a dose-responsive manner. Their ability to inhibit hLH bioactivity largely paralleled their affinity constants. Our data have allowed us to establish a tentative topographic relationship of epitopes to the biological region of the molecule of hLH, foreshadowing (in additive binding studies) some of the possible combinations of antibodies that might allow us to design two- or multiple-site immunometric assays in which measurement of immunoactive LH reflects biological activity. In addition, these studies suggest that both the alpha- and beta-subunits participate in LH receptor binding and/or biological activity.

Animals↗
PubMed1988

Characteristics and epitope mapping of a cloned human autoantigen La.

The La (SS-B) polypeptide is a ribonucleoprotein against which high titer antinuclear antibodies (ANA) react in the human autoimmune disease primary Sjögren's syndrome. To identify the autoepitopes with which the ANA anti-La (anti-SS-B) reacts, we isolated a 1.4-kb cDNA clone for La from a lambda gt10 library made from a human Burkitt's cell line. This clone contained an open reading frame of 1065 bp, encoding a 40.1-kDa polypeptide that corresponded to the carboxyl-terminal end of the La protein. The predicted polypeptide sequence of the recombinant protein was highly charged and unrelated to any previously published sequence. We also compared this clone to a previously published cDNA sequence for La and demonstrated significant differences, particularly that the open reading frame in our cDNA continued for 926 additional bases 3' to a putative termination codon in the previously reported sequence. The recombinant La protein was expressed in Escherichia coli and tested for reactivity with 200 sera containing ANA of various specificities. Only the sera containing anti-La antibodies reacted with the cloned La. By expressing subclones of the La cDNA as fusion proteins with beta-galactosidase, we have localized at least one epitope for the binding of anti-La antibodies to the carboxyl-terminal 103 amino acids of the La protein. No anti-La binding could be demonstrated to the region of the La protein that had previously been predicted to contain an autoepitope for the binding of anti-La (SS-B) antibodies. Studies of cloned autoepitopes could provide important clues to the role ANA play in disease and lead to targeted intervention in the treatment of primary Sjögren's syndrome.

Amino Acid Sequence↗
PubMed1991

Genetic control of antibody response to bovine rhodopsin in mice: epitope mapping of rhodopsin structure.

Inbred strains of mice of independent haplotype were immunized with bovine rhodopsin. All mice tested except SJL developed significant titers of specific antibodies 21 days after a single immunization. Anti-rhodopsin antibody level differed among conventional inbred strains. Comparison of the immune response to rhodopsin of congenic mice on two different genetic backgrounds showed that animals with an A background typically produced higher levels of specific antibody than mice with a B10 background. Titer of specific antibodies in antisera of mice of the same H-2 haplotype but different Igh haplotype differed; e.g. for H-2d haplotype, NZB (Ighn) generated the highest level of antibody with BALB/c (Igha), DBA/2 (Ighc), and B10.D2 (Ighb) strains giving successively lower responses. The location of immunodominant regions of bovine rhodopsin was investigated in primary sera among strains of mice. Sera were tested for their binding of anti-rhodopsin antibodies to synthetic peptides covering the entire primary structure of rhodopsin. From direct binding studies with hydrophilic rhodopsin peptides, the majority of the antigenic binding sites were localized in the sequence of the amino terminus, the II-III loop and the carboxyl terminus. Binding to these antigenic peptides was not strain restricted. Application of the overlapping synthetic peptide strategy of Geysen enabled refinement of these epitopes and determination of an additional major epitope in the hydrophobic sequence 304-310.

Amino Acid Sequence↗
PubMed1993

Mimicking of discontinuous epitopes by phage-displayed peptides, I. Epitope mapping of human H ferritin using a phage library of constrained peptides.

We have constructed a random nonapeptide library in the N-terminal region of the major coat protein VIII of bacteriophage f1, with two cysteines flanking the insert, and preliminary data suggest that many of the clones display at least some of their peptides in cyclized form. This library was used to select oligopeptides binding to the monoclonal antibody (mAb) H107, recognising the assembled native conformation of recombinant human H-subunit ferritin (H Fer), whose three-dimensional structure is known. Comparison of the selected oligopeptides with one another allowed us to derive two consensus sequences characterized by conserved amino acid (aa) residues. Analysis of the distribution of the aa side chains exposed on the surface of H Fer reveals that most of the aa defining both consensus sequences are present either at the end of the big loop or at the end of the A helix. These two regions of the H Fer, though separated in the linear sequence, are very close in the folded molecule. Interestingly, each consensus sequence derived from the selected phage-displayed peptides is characterized by aa present both at the end of the big loop and at the end of the A helix. These two H Fer regions are good candidates for mimicry by the selected peptides and therefore for constituting part of the H107 epitope. To provide support to this hypothesis, we constructed several H Fer mutants carrying point mutations in different positions of these two regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Epitope mapping of annexin I: antibodies that compete with phospholipids and calcium recognize amino acids 42-99.

To understand further the structural basis of phospholipid binding by annexin I, three monoclonal antibodies that compete with Ca2+ and phospholipids for binding of annexin I were used to screen an expression library containing fragments of bovine annexin I cDNA. In all, 15 clones were isolated, and all contain overlapping fragments of the cDNA. The smallest unit common to all of the clones encodes amino acids 42-99 of annexin I, representing a portion of the first repeat domain. This demonstrates that recognition of a single domain of annexin I is sufficient to completely block phospholipid binding, and implies that the first repeat may contribute to phospholipid binding by annexin I.

Amino Acid Sequence↗
PubMed1993

Epitope mapping of the Sta58 major outer membrane protein of Rickettsia tsutsugamushi.

Binding studies of 160 overlapping, synthetic octapeptides from the hydrophilic regions of the Sta58 major outer membrane protein of Rickettsia tsutsugamushi with sera from patients with scrub typhus revealed 15 immunodominant peptides which are recognized by all the sera tested. Further analysis of the specificity of peptide binding with five of these peptides indicated that the peptides showed significantly stronger binding to scrub typhus patients' sera than they did to sera from patients with other febrile illnesses common in the region, i.e., malaria, dengue fever, typhoid fever, and leptospirosis. The main antibody class binding to these peptides appears to be immunoglobulin M, and there appears to be little correlation between reactivity with peptides and antibody titers measured by the indirect immunoperoxidase test.

Amino Acid Sequence↗