Native Epitopes Research Articles

Immunoreactivity of human and rabbit antisera to hepatitis A virus.

Rabbit antibodies produced by immunization with complete hepatitis A virions (HAV) recognized all the viral structural proteins and neutralized HAV infectivity in cell culture. Rabbit antibodies to chromatographically purified individual viral proteins and to synthetic peptides representing epitopes on the structural viral protein VP1 neither recognized whole virus nor neutralized infectivity, indicating that native epitopes on the virus surface are necessary for virus recognition and neutralization. Human anti-HAV-positive sera of the acute and convalescent phase of disease recognized and neutralized viral particles. Analysis of the immunoreactivity of these human sera in immunoblot showed that the IgM antibody preferentially recognizes the structural viral proteins VP0 and VP3 of HAV, whereas IgA and IgG antibodies reacted more strongly with VP1.

A simple ELISA for the classification of monoclonal antibodies according to their recognition of native epitopes

A sandwich ELISA for testing whether pairs of monoclonal antibodies recognize the same native antigenic site was developed. The assay was performed with murine anti-lysozyme monoclonal antibodies. A monoclonal antibody, adsorbed onto a microtiter plate, was used as a capture antibody for native lysozyme. After the reaction with the antigen a second monoclonal antibody,the test antibody, was added. The amount of bound antibody was quantitatively measured using rabbit anti-mouse immunoglobulin serum conjugated to alkaline phosphatase. The simultaneous binding of pairs of monoclonal antibodies to lysozyme was further substantiated by structural considerations.

Structure and Function of the Erythrocyte Receptor CD2 on Human T Lymphocytes: A Review

The human CD2 molecule is a 50kd surface glycoprotein expressed on greater than 95% of thymocytes and all peripheral T lymphocytes which mediates both adhesion between T cells and their targets, and subsequent T cell activation events. Molecular cloning of human CD2 cDNAs predicts a mature CD2 protein of 327 amino acids, with an extracellular segment of 185 amino acids, a transmembrane domain of 24 amino acids and an intracytoplasmic region of 117 amino acids. Genomic cloning shows that the extracellular segment is encoded by two exons, the transmembrane segment by a single exon and the intracytoplasmic region by a single exon. Expression and biochemical analysis of a soluble extracellular domain CD2 molecule reveal that it expresses native CD2 epitopes and contains a stable 15kd NH2-terminal fragment corresponding to a single exon. Binding analyses of the soluble CD2 molecule indicate that it binds specifically to a known cell-surface ligand for CD2 at a relatively low affinity, thus suggesting that T cell-target adhesion mediated by CD2 and its ligand depends on multimeric attachment between an array of CD2 molecules and their cognate ligands.

Heterogeneity of human immunodeficiency virus cell-associated antigens and demonstration of virus type specificities of human antibody responses

We examined the antigens of human immunodeficiency viruses (HIV) expressed on infected H9 cells using live-cell membrane immunofluorescence and immunofluorescence absorption. Application of this nondenaturing serological method permitted analysis of HIV antigenic determinants maintained in their native configurations on the cell surface. Sera from infected individuals were found to react variably with H9 cells productively infected with nine different HIV isolates, and certain sera were completely unreactive with some isolates. Absorption of the sera prior to use in immunofluorescence revealed extensive heterogeneity of HIV cell-surface antigens and multiple type-specific antibodies in patients' sera. Immunoprecipitation and SDS-PAGE analysis of radiolabeled cell-surface proteins indicated that the predominant serological reactions were to env-encoded proteins. The observed antigenic and antibody heterogeneity likely reflects env sequence heterogeneity which has been previously reported for different HIV isolates. The demonstration of antigenic diversity among HIVs and the importance of defining the native antigenic epitopes, particularly those most widely shared, are important issues that must be considered in vaccine development.

Immune response to human immunodeficiency virus. In vivo administration of anti-idiotype induces an anti-gp160 response specific for a synthetic peptide.

Anti-idiotypic antibodies (anti-Id) to chimpanzee antibodies directed against a synthetic peptide corresponding to a native epitope associated with gp41 of human immunodeficiency virus (HIV) envelope glycoprotein were produced in rabbits. The peptide was analogous to amino acid sequences 735 to 752 from the human T cell leukemia virus-IIIB isolate of HIV. Characteristics of the anti-Id preparation included: 1) detection of a shared determinant present on a second chimpanzee and one of three rabbit antibody preparations directed against the synthetic peptide, 2) failure to recognize an idiotype (Id) in BALB/c mouse antisera to the peptide, and 3) partial inhibition of the homologous chimpanzee Id preparation from binding either peptide or a recombinant HIV gp160 preparation. Immunization of BALB/c mice with the anti-Id induced an antipeptide response which bound a recombinant gp160 preparation without subsequent peptide or gp160 exposure. The anti-gp160 containing sera from mice immunized with anti-Id were able to inhibit the Id-anti-Id reaction indicating that an Id-positive antibody response was induced. This Id is not normally expressed in the murine anti-gp 160 immune response to the synthetic peptide and suggests that this anti-Id may activate normally silent clones. This study indicates that Id networks may be operational during the immune response to HIV epitopes. Alternatively, anti-Id may be useful in altering the serologic characteristics of an antibody response to HIV and may offer potential for modulating the immune response in this viral infection.

Conformational equilibria in the gamma chain COOH terminus of human fibrinogen.

The conformations of the gamma chain COOH terminus of intact fibrinogen and various fragments containing this region have been compared by an immunochemical analysis. Location of a major epitope in the sequence gamma 391-405 was successfully predicted from a hydrophobicity profile. An antibody population specific for the native epitope within the gamma 391-405 segment was isolated by immunoadsorption. Between 19.2 and 22.8% of antibodies were obtained from three different antisera, indicating that this region represents one of the major epitopes of native fibrinogen. Anti-gamma 391-405(N) antibodies were used to determine the value of Kconf, the equilibrium constant for the interconversion of the non-native and native conformations of this epitope. The measurements were done using native fibrinogen, fragments D1 and DD, gamma chain, and gamma 391-405. In addition, the effect of 5 M guanidine HCl on the conformation of fragments D1 and DD, which is known to abolish their antipolymerizing activity, was studied. Radioiodinated fibrinogen was used in the determination of Kconf, CI50%, and CIs (quantitative analytical parameters calculated from competitive inhibition radioimmunoassays) by measuring the competition between 125I-fibrinogen and the fibrinogen derivatives under study for binding to the immunochemically purified antibody. The measurements indicated that the epitope is unperturbed by iodination of fibrinogen and that 38.5% of fragment D1, 8.9% of fragment DD, 3.6% of the gamma chain, and less than 0.008% of the gamma 391-405 molecules adopt in aqueous solution the native conformation within the epitope. Denaturation of fragment D1 with 5 M guanidine HCl affected only slightly the conformation of this gamma chain determinant. More significant changes in the conformation were observed when fragment DD was denatured. The results suggest that long-range interactions are necessary for the stabilization of the native structure in the region of fibrinogen that interacts with the antibody and which is in close vicinity to the polymerization site, cross-linking site, and platelet recognition site.

Turnover and tissue sites of degradation of glucosylated low density lipoprotein in normal and immunized rabbits

Immunological mechanisms have been implicated in the atherogenic process since immunoglobulins are frequently found in the atherosclerotic aorta. We have previously shown that modifications of homologous low density lipoproteins (LDL) make it immunogenic. In particular we have demonstrated that immunization with homologous nonenzymatically glucosylated LDL (glcLDL) results in the generation of antibodies specific to the derivatized lysine residue, and that such antibodies do not react with native LDL epitopes. In the present study we immunized rabbits with reductively glucosylated rabbit LDL and then determined the effects of the circulating antibodies on the rates of plasma clearance and on the sites of degradation of LDL in which varying degrees of glucosylation had been achieved. In normal chow-fed animals, the plasma clearance of glcLDL was retarded in proportion to the extent of lysine derivatization. In contrast, in immunized animals the clearance of glcLDL was greatly accelerated. When 10% or more of lysine residues were derivatized, clearance of glcLDL was accelerated 50- to 100-fold. Even when only 5% of lysines were derivatized, plasma clearance was accelerated 2- to 3-fold. Cholesterol feeding inhibited LDL clearance from plasma and decreased LDL uptake of LDL receptor-rich tissues. In a similar manner, glucosylation of LDL inhibited its ability to bind to the LDL receptor and redirected sites of LDL degradation away from LDL receptor-rich tissues. Thus degradation of glcLDL by liver and adrenal was markedly diminished. The presence of antibodies to glcLDL also redirected sites of degradation of the modified LDL, primarily to the reticuloendothelial cells of the liver. There was no evidence for specific targeting of glcLDL-immunoglobulin complexes to the aorta; instead they were targeted to the liver. These data suggest that the presence of humoral antibodies to modified LDL acts to rapidly remove such LDL from plasma and specifically targets such complexes to reticuloendothelial cells, primarily in the liver. In this manner such antibodies may serve a useful purpose.

Immune response to intermediate filament-associated, Epstein-Barr virus-induced early antigen.

A murine monoclonal antibody (MAb) (15TD3) was found to recognize a unique filamentous structure in Epstein-Barr virus (EBV)-producing lymphoblastoid cell lines. By immunofluorescent morphology, in comparison with a control MAb to vimentin, the 15TD3 filamentous structure was judged to be associated with intermediate filaments of the cytoskeleton. Expression of the 15TD3 antigen and vimentin was induced simultaneously in some EBV genome-positive cell lines either by EBV superinfection or by 12-0-tetradecanoyl-1-phorbol-13-acetate (TPA) and n-butyrate treatment. The 15TD3 antigen was considered to be a restricted component of the EBV-induced early antigen (EA) complex. The 15TD3 antigen was expressed only in EBV genome-activated cells after either spontaneous EBV genome activation, EBV superinfection, or TPA and n-butyrate treatment. The expression of 15TD3 antigen paralleled the induction of EA in several models of induction of EBV antigens, and was detected only in EA+ cells which were stained with anti-EA+ human sera. The reactivity of 15TD3 MAb was blocked with anti-EA+ human serum, but not with anti-EA- serum. The synthesis of 15TD3 antigen was not inhibited with phosphonoacetic acid, was resistant to acetone fixation, and was sensitive to ethanol (or methanol) fixation. Human lymphoblastoid cells from patients with acute infectious mononucleosis were cloned for the production of antibodies which detected EBV-specific or -nonspecific epitopes on filamentous structures. Two human MAb were defined by two-color immunofluorescence to react to the 15TD3 determinants on intermediate filaments of EBV+ cells. This study supports the following views: that EBV genome activation induces a structure associated with intermediate filaments, and that antibodies against both the EBV-specific, intermediate filament-associated epitope and native intermediate filament epitopes are produced by some EBV-transformed lymphoblastoid cell lines from patients with infectious mononucleosis.

Partly native epitopes are already present on early intermediates in the folding of tryptophan synthase.

Two monoclonal antibodies directed against the native beta 2 subunit of Escherichia coli tryptophan synthase [L-serine hydro-lyase (adding indoleglycerol-phosphate), EC 4.2.1.20], one recognizing the C-terminal F1 domain and the other the N-terminal F2 domain, were used to detect immunoreactive intermediates in the folding of the protein. For that purpose, the association of the monoclonal antibodies with either the beta 2 subunit or its isolated domains was studied by using fluorescence energy transfer between tryptophan residues of the antibodies and a dansyl group covalently linked to the antigen. It is shown that the association of both monoclonal antibodies with the antigen occurs within a few seconds after initiation of the renaturation, whereas complete refolding of the beta 2 subunit requires several minutes under the same experimental conditions. Thus, immunoreactive intermediates appear to be formed at an early stage of the folding process. While the isolated F1 domain alone is able to rapidly refold into a conformational intermediate already well recognized by the anti-native-beta 2 antibody, it cannot, in the absence of the F2 domain, reach its native conformation. However, its association with the anti-native-beta 2 antibody induces a structural change of F1 that brings it closer to the conformation it normally adopts when interacting with F2 inside the native beta 2 subunit.

Envelope proteins of human T cell leukemia virus type I: characterization by antisera to synthetic peptides and identification of a natural epitope.

Three peptides corresponding to selected regions of the env gene products of human T cell leukemia virus type I were synthesized by solid-phase Merrifield techniques. The sequence of peptide designated SP-65 was identical to the predicted C-terminal 12 residues of the transmembrane protein p21env, and peptide SP-74 was inferred from a region shown to be highly conserved among mammalian retroviruses. The third peptide, SP-70, was derived from a C-terminal region of the surface glycoprotein gp46. Antibodies to each peptide were raised in rabbits and were used to identify and further characterize the proteins coded by the env gene. Despite being present at very low levels in purified viral preparations, these proteins were chromatographed by reverse-phase high pressure liquid chromatography and were located by Western blot analysis of the column fractions. Anti-SP-70 recognized the surface glycoprotein (gp46) and also its C-terminal cleavage fragment (gp16). Anti-SP-65 and anti-SP-74 both reacted with the hydrophobic transmembrane protein (p21) and provided evidence that this protein does not undergo apparent C-terminal processing during viral maturation, unlike the trans-membrane protein of murine leukemia virus. As expected, anti-SP-74 also reacted with homologous proteins from other Type C and Type D viruses, confirming that peptide SP-74 corresponds to a broadly conserved region of retroviral transmembrane proteins. SP-70, which is predicted to be quite near the C terminus of the major surface glycoprotein, was also reactive with sera of HTLV-I-positive patients, indicating that this peptide corresponds to, or is part of, a native epitope recognized by the natural host.

Antiserum to a synthetic peptide recognizes the HTLV-III envelope glycoprotein.

In a study performed to determine which regions of the human T-cell lymphotrophic virus type III (HTLV-III) may represent vaccine candidates to prevent the acquired immune deficiency syndrome (AIDS), a synthetic peptide corresponding to amino acid sequence 735 to 752 of the precursor envelope glycoprotein of HTLV-III was used to immunize rabbits. The resulting rabbit antiserum to the synthetic peptide specifically recognized the precursor envelope glycoprotein (gp160) of HTLV-III. Human sera positive for antibody to HTLV-III reacted with this peptide. These findings indicate that synthetic peptides can be used to induce an immune response directed against a native envelope glycoprotein epitope of HTLV-III. The data are discussed in terms of using synthetic peptides to identify antigenic determinants involved in the induction of protective immunity and possibly as vaccine candidates against the etiologic agent of AIDS.

An asparagines–rich protein from blood stages ofPlasmodium falciparumshares determinants with sporozoites

We describe a cDNA clone derived from mRNA of asexual blood-stages of the malaria parasite Plasmodium falciparum. This clone, designated Ag319, expresses a P.falciparum antigen fused to beta-galactosidase in Escherichia coli. Human antibodies from Papua New Guinea were affinity-purified by adsorption to extracts of Ag319 immobilized on CNBr-Sepharose. The antibodies reacted predominantly with P. falciparum polypeptides of Mr 220,000 and 160,000, and a number of ill-defined lower molecular weight species. Antibodies reacted in indirect immunofluorescence with all asexual blood-stages although the antigen appeared to be most abundance in the schizont. Surprizingly the antibodies also reacted with sporozoites. The amino acid sequence predicted from the complete nucleotide sequence of this clone is remarkable because 40% of the residues are Asn, and so the antigen has been termed the Asparagine-Rich Protein (ARP). Like other P. falciparum antigens, ARP contains tandemly repetitive sequences, based on the tetrapeptide Asn-Asn-Asn-Met and we have confirmed that these represent natural epitopes by reaction of the corresponding synthetic peptides with human antibodies. Surprisingly, ARP is also rich in Asn outside the tandem repeats.

Quantitation of activation of the human terminal complement pathway by ELISA

We have devised an enzyme-linked immunosorbent assay (ELISA) to quantitate fluid phase terminal complement pathway activation. Upon activation to form C5b-9, terminal complement components express neoantigens not present in the unassembled individual components. Expression of one of these neoantigens occurs at the step of C9 activation. C9 neoantigen is present in fluid phase SC5b-9 complexes, membrane-bound MC5b-9 complexes, and in vitro polymerized C9. Under physiologic conditions, the presence of C9 neoantigen indicates that the terminal complement pathway is activated through the terminal component C9. In our assay for C9 neoantigen, we used rabbit antiserum to polymerized C9 rendered specific for C9 neoantigen c determinants by serial absorption with human serum, human C9, and other terminal complement components bound to Sepharose. Using the IgG from this antiserum, we devised a sandwich ELISA to bind SC5b-9 from solution onto polystyrene plates. The ELISA plates were developed with the use of goat antiserum to native C9 epitopes followed by a swine anti-goat IgG-alkaline phosphatase conjugate. Quantitation of SC5b-9 in solution was performed by comparing sample OD to a standard curve generated with human SC5b-9 that was purified from zymosan-activated serum. The assay was sensitive to as little as 100 ng of SC5b-9/ml and should be useful for screening plasma, serum, cerebrospinal fluid, or other biological fluids for the presence of terminal complement pathway activation.

Structural and immunological characterization of Friend murine leukaemia virus glycopolypeptide using synthetic oligopeptides.

Using terminal position, hydrophilicity, predicted reverse turns and type specificity as criteria, five oligopeptides were selected for synthesis from the amino acid sequence of the envelope glycopolypeptide gp70 of Friend murine leukaemia virus. These peptides corresponded to the amino acids 6-12 (pep1), 124-131 (pep2), 256-262 (pep3), 283-290 (pep4) and 434-441 (pep5). After coupling to carriers, bovine serum albumin or keyhole limpet hemocyanin, antisera were prepared in rabbits. All of the five oligopeptides were immunogenic and pep1, pep2, pep4 and pep5 were able to elicit antibodies to the native glycopolypeptide. These sequence-specific antisera distinguished between glycoproteins of different leukaemia viruses. At least three of the selected peptides, the type-specific oligopeptides pep3, pep4 and pep5, were found to be natural epitopes of gp70.

Studies on the mode of action of non-steroid anti-inflammatory drugs.

Background: The recombinant vacuolating cytotoxin (rVacA) of Helicobacter pylori that retains native conformational epitopes was evaluated as a vaccine antigen for anti-H. pylori treatment. Methods: s1m1 vacA gene fraction encoding the mature VacA protein was expressed as a soluble protein in E. coli at low temperature. The efficacy of anti-rVacA antibody against VacA or H. pylori was assessed in vitro using AGS cells and in vivo using a murine model. Results: The rabbit antisera against rVacA completely neutralized the vacuolating activity and partially inhibited the cell death induced by VacA in AGS cells. Oral immunization of C57BL/6 mice with rVacA plus CpG-oligodeoxynucleotide (ODN) as an ajuvant stimulated specific anti-VacA antibody and mucosal immune responses which correlated with decreased systemic immune responses and gastric urease activities (p>0.05). Conclusion: The rVacA antigen possessing conformational epitopes may have potential as a vaccine component and may be useful in serological and histopathological analysis.