Unmasking of an unusual myelin basic protein epitope during the process of myelin degeneration in humans: a potential mechanism for the generation of autoantigens.

A rabbit antiserum (anti-EP), induced against a synthetic peptide corresponding to residues 68 to 86 of guinea pig myelin basic protein, powerfully immunostained abnormal-appearing oligodendrocytic processes and cell bodies in demyelinating areas associated with multiple sclerosis plaques. However, it failed to recognize any structures in normal human, rat, or guinea pig brain. The antiserum recognized the synthetic peptide QDENPVV, which corresponds to human myelin basic protein residues 82 to 88. Immunoabsorption with this peptide eliminated immunohistochemical staining. By contrast, several commercial antibodies recognizing nearby sequences of human myelin basic protein intensely stained all myelinated structures in both normal and multiple sclerosis tissue. The unusual epitope recognized by anti-EP appears to be accessible only in areas of myelin degeneration. If insults occur that repeatedly expose a region of MBP normally sheltered from immunosurveillance, a self-sustaining immune reaction might result.     

Isolation characterization and idiotype of Lewis rat antibodies against peptide 68-88 of guinea pig myelin basic protein

Antibodies against the 19 amino acid encephalitogenic peptide )residues 68-88) of guinea pig myelin basic protein (GPBP) were raised in Lewis (Le) rats. Anti-peptide antibodies were isolated from immune ascitic fluids by affinity chromatography using peptide 43-88-Sepharose 4B. The purified antibodies were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and isoelectric focusing. Immunoglobulin class was determined by radioimmunoassay. Anti-idiotypic (anti-ID) antibodies were raised in a rabbit using purified anti-peptide antibodies from a single rat. The results of these experiments showed antibody heterogeneity both within an individual anti-peptide antiserum and between antisera from different rats. Antibody activity was found in IgG1, IgG2, and IgE immunoglobulin classes. Isoelectric focusing revealed multiple bands within a population of purified antibodies with significant pattern variation from one antiserum to another. Idiotypic characterization showed various levels of cross-reactive idiotypes present in some sera while these were absent in others.     

T cell response to myelin basic protein epitopes in multiple sclerosis patients and healthy subjects.

T cell lines and clones specific for human myelin basic protein (BP) were selected from three multiple sclerosis (MS) patients and two healthy subjects and tested for their proliferative responses to a battery of synthetic peptides, 9 to 21 amino acid residues long. The combined amino acid sequence of the peptides spanned the complete sequence of the human BP. The results suggest the development of T cells sensitized to at least four independent regions of the human BP, indicating some diversity of the human T cell repertoire to BP. However, an immunodominant T cell epitope was located in the C-terminal region, defined by residues 149-162. This epitope was recognized by T cells from three subjects out of five (one MS patient and both healthy controls) in the context of different DR specificities. Another epitope (located in the 57-75 region) which triggered one MS patient's T cell response was also recognized by a mycobacteria-specific T cell clone cross-reacting with BP.     

Naturally occurring anti-Escherichia coli protein antibodies in the sera of healthy humans cause analytical interference in a recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay for serodiagnosis of severe acute respiratory syndrome

We reported the analytical interference of anti-Escherichia coli protein (EP) antibodies in human sera and residual EP in a recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay as a possible source of false positives in severe acute respiratory syndrome serodiagnosis. The rate of false positives was significantly reduced by adding mouse anti-EP antiserum in the blocking step.     

Usefulness of the phage display technology for the identification of a hepatitis C virus NS4A epitope recognized early in the course of the disease

A dodecapeptide phage-displayed library was screened with the mouse monoclonal antibody (mAb) 2E3C2 which competed with human antibodies for the binding to the HCV c100 recombinant protein. Four mimotopes shared a consensus motif with the HCV 1701-1707 sequence corresponding to the carboxyl-terminal domain of the non-structural protein NS4A. However, these mimotopes reacted with 2E3C2 only, whereas the corresponding NS4 epitope defined at the sequence 1698-1709 and displayed on phage was recognized by both 2E3C2 and sera from HCV infected patients. Using the Spot method of multiple peptide synthesis and alanine replacement analysis, the respective reactivities of mAb 2E3C2 and anti-NS4A human antibodies against NS4 were shown to be directed against two slightly different overlapping minimal linear sequences and to involve different critical residues. The phage clone displaying the NS4 epitope was used to study the specific recognition of this epitope by different individual HCV positive sera as well as by two seroconversion panels of sera from HCV infected patients. Compared with the detection by RIBA of the different HCV antigens and c100 particularly, these results indicated that the antibodies directed against the NS4 (1698-1709) epitope were produced early during the course of the disease and decreased later.     

Characterization of brain-isolated rat encephalitogenic T cell lines

In the present study, we have isolated and characterized five myelin basic protein (MBP)-reactive T cell lines directly from the brains of Lewis rats during the early paralytic phase of experimental autoimmune encephalomyelitis (EAE). Each T cell line responded to the dominant encephalitogenic epitope spanning residues 68–88, and did not react against the conserved encephalitogenic epitope [MBP(87–99)] or the nonencephalitogenic MBP epitope [MBP(50–69)]. We determined the T cell receptor (TcR) β chain usage by polymerase chain reaction, DNA sequencing analysis and by generation of MBP-reactive hybridomas from one of the T cell lines (BT74). The results revealed that brain-infiltrating, MBP-reactive T cells freshly isolated early in the course of the disease exhibit TcR diversity.     

Immunocytochemical method to identify basic protein in myelin-forming oligodendrocytes of newborn rat C.N.S.

Summary An immunocytochemical method for detecting myelin basic protein in oligodendrocytes and myelin of newborn rat C.N.S. is described. C.N.S. tissue is perfused and fixed in HgCl₂-formaldehyde and 20 m Vibratome sections are treated with antibodies to myelin basic protein using the peroxidase-antiperoxidase method. Oligodendrocytes in the newborn rat are intensely stained by antiserum to basic protein and multiple stained processes extend from the perikaryon to myelin sheaths. With this procedure it is possible to demonstrate the geometric relationships between a single oligodendrocyte and multiple myelin sheaths. Stained oligodendrocytes and myelin are present in newborn cervical spinal cord, medulla oblongata, pons and midbrain. By 25 days of age, staining in oligodendrocytes is less intense than in newborn rats and differences in amount of staining can be detected in areas that are myelinating at different rates. With anticerebroside serum, cerebroside, of newborn and developing rat C.N.S. tissue is localized only in myelin. In the developing P.N.S., myelin basic protein is localized in Schwann cell cytoplasm and myelin sheaths of the trigeminal ganglion. Cerebroside is found only in myelin.     

Epitope mapping and use of epitope-specific antisera to characterize the VP5* binding site in rotavirus SA11 NSP4

Rotavirus (RV) is the leading cause of infantile gastroenteritis worldwide. RV nonstructural protein 4 (NSP4), the first characterized viral enterotoxin, is a 28-kDa glycoprotein that has pleiotropic functions in RV infection and pathogenesis. NSP4 has multiple forms enabling it to perform its different functions. Dissecting such functions could be facilitated by use of epitope-specific antibodies. This work mapped the epitopes for the monoclonal antibody B4-2/55 and three polyclonal antisera generated against synthetic SA11 NSP4 peptides corresponding to residues 114-135, 120-147, and 150-175. The epitope for B4-2/55 mapped to residues 100-118, wherein residues E105, R108 and E111 are critical for antibody binding. Antiserum generated to two peptides (aa114-135 and aa120-147) with enterotoxin activity each recognize a single but distinct epitope. The epitope for the peptide antiserum to aa114-135 was mapped to residues 114-125 with highly conserved residues T117/T118, E120, and E122 being critical for antibody binding. The peptide antiserum to aa120-147 binds to NSP4 at residues 130-140 and residues Q137-T138 are critical for this epitope. Finally, the epitope for the antiserum to peptide aa150-175 mapped to residues 155-170, wherein residues E160 and E170 are critical for antibody binding. Knowledge of the binding sites of domain-specific antibodies can aid in further characterizing different functions of NSP4. To demonstrate this, we characterized the interaction between NSP4 and VP5() [K(D)=0.47 microM] and show that binding of NSP4 to VP5* is blocked by antibody to NSP4 aa114-135 and aa120-147, but not aa150-175. The use of single epitope-specific antibodies to differentially block functions of NSP4 is a feasible approach to determine the functional domain structure of this important RV virulence factor.     

Detection of B-lymphocyte (B-cell)-associated antigens on human leukemic lymphocytes. Masking of membrane antigens.

An indirect immunofluorescent technic has been used for rabbit antisera (anti-EP) that demonstrated complement-dependent cytotoxicity against human leukemic lymphocytes but not against normal blood lymphocytes. With the immunofluorescent technic, the antisera were found to react with 2--23% of normal blood lymphocytes. Simultaneous staining of normal cells with anti-EP and for surface immunoglobulins (SIg) on bone-marrow-derived B-cells showed that the proportions of stained cells were similar to percentages of cells stained by anti-EP alone or for SIg alone. The percentage of anti-EP reactive cells also approximated the percentages of cells reactive to a known antiserum to human B-cell associated, or Ia-like, antigens. The anti-EP reacted with lymphoblastoid cells from two B-cell lines lacking the Epstein-Barr viral genome. The antigens detected by anti-EP probably are B-cell-associated. The anti-EP intensely stained neoplastic cells of acute or chronic lymphocytic leukemia and lymphosarcoma-cell leukemia. Cells from two patients with chronic lymphocytic leukemia and from two patients with acute lymphocytic leukemia showed increased intensity of anti-EP staining and/or increased proportions of stained cells following overnight incubation in culture medium, compared with the preincubation samples. This observation suggests the presence of a "blocking component(s)" on cell surfaces, which interfered with anti-EP reactivity. After overnight incubation, the component might have been removed from the antigenic sites on cell surfaces. Further studies of leukemia lymphocytes using anti-EP for the cell-bound "blocking component" may reveal important pathogenetic mechanisms.     

Cells producing antibodies specific for myelin basic protein region 70-89 are predominant in cerebrospinal fluid from patients with multiple sclerosis.

Cells secreting antibodies against guinea pig myelin and synthetic myelin basic protein (MBP) peptides were evaluated in cerebrospinal fluid (CSF) and blood from patients with multiple sclerosis (MS) and a variety of other neurological diseases (OND). The peptides used, reproducing amino acid sequences 1-20, 70-89, 108-126, or 157-166 of MBP, were selected on the basis of their hydrophilic and encephalitogenic properties. Low numbers of cells secreting IgG antibodies against myelin or each of the MBP peptides (about 1 per 50,000) were detected in peripheral blood, with no difference between MS and OND. In CSF, cells secreting IgG antibodies to MBP 70-89 were more frequently (p = 0.007) detected in patients with MS (1/380 IgG-secreting cells on average) than in patients with OND (1/2083 IgG-secreting cells on average). The frequencies of cells secreting antibodies against myelin or the three other MBP peptides were similar in MS and OND. Thus, evaluation of B cell immunity at the cellular level indicates that MBP 70-89 is an immunodominant B cell epitope in MS. It is not clear whether this intrathecal anti-MBP 70-89 IgG antibody response has any pathogenetic relevance in MS or is the result of myelin breakdown.     

Natural antibodies against alliinase in human serum and polyclonal antibodies elicited in rabbit share the same immunogenic determinants

Human serum contains natural antibodies against alliinase, a protein abundantly found in garlic (Allium sativum) cloves. In order to study the epitope(s) of this protein recognized by anti-alliinase antibodies, we used a random hexapeptide library displayed on filamentous M13 phage. Analysis of the phagotopes selected on rabbit anti-alliinase antibodies revealed that the motif-GKXVXX- was common for all peptides. The most frequent phage displaying -GKHVAV- sequence has a 50% identity with the original alliinase sequence (amino acid residues 156-161). The position of this epitope is only nine amino acids apart from the oligosaccharide chain attached to the N146. The rabbit anti-alliinase immunoglobulin G (IgG), which bound the phages displaying this phagotope, also bound the corresponding peptide derived from the alliinase sequence. Affinity-purified natural antibodies against alliinase, present in normal human serum (which can specifically recognize the native and denaturated protein) also bound the selected phagotope. Thus, our results indicate that specific natural anti-dietary protein antibodies presented in human serum can have the same. or overlapping. epitopes with the IgG evoked during the active (experimental) immunization in animals.     

Human monoclonal antibodies for the immunological characterization of a highly conserved protein domain of the hepatitis C virus glycoprotein E1.

Although both envelope glycoproteins of the hepatitis C virus, E1 and E2/NS1, show a high degree of sequence variation, the E1 protein includes a well conserved domain, which may be functionally important. We have analysed the human B cell response to a peptide fragment from amino acid residues 314-330 (EP3) covering the central conserved sequence of this domain. Anti-hepatitis C virus-positive blood donors were screened for anti-EP3 antibodies with an ELISA based on immobilized peptide. Thirty out of 92 (32%) RIBA-confirmed donors displayed a significant antibody response to EP3. From three of these blood donors we established four anti-EP3-producing heterohybridoma cell lines: Ul/F30 and Ul/F31 produced IgM-kappa, whereas Ul/F32 and Ul/F33 secreted the isotypes IgG1-lambda and IgG1-kappa, respectively. Epitope analysis with overlapping nonapeptides suggests the existence of different antigenic determinants within the EP3 fragment. Although both IgG antibodies Ul/F32 and Ul/F33 have dissociation constants to the peptide of approximately 10(-9) M, binding to recombinant E1 protein expressed in COS-7 cells was different. Only Ul/F33 detected envelope protein of approximately 24-35 kD in Western blot. This human MoAb will be useful for further investigations on the hepatitis C virus glycoprotein E1.     

Monoclonal antibodies to cyanogen bromide fragments of glycophorin A

Eleven mouse monoclonal antibodies directed against epitopes on CNBr peptides of the major sialoglycoconjugate of the human red blood cell, glycophorin A, have been produced by hybridomas derived from P3-X63-Ag8.653 myeloma cells and spleen cells from BALB/c mice immunized with purified glycophorin. The monoclonal antibodies could be divided into four groups according to their reactivities with CNBr peptides in a direct ELISA assay: one antibody (6B5) that binds solely to the aminoterminal octapeptide (CNBr3); two antibodies (8F10 and 9C3) that bind to CNBrl (residues 9-81); two antibodies (3D2 and 4C6) that are reactive with CNBr2, The C-terminal portion of the molecule (residues 82-131); six antibodies (1B4, 4C3, 4E7, 7B10, 7C11 and 9D6) which are cross-reactive with an epitope on both CNBr1 and CNBR3 glycopeptides. This cross-reactive epitope(s) appears to involve both carbohydrate and protein residues.     

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.     

HCV core immunodominant region analysis using mouse monoclonal antibodies and human sera: Characterization of major epitopes useful for antigen detection

Monoclonal antibodies (MAbs) were generated by immunizing mice with a truncated recombinant protein corresponding to the immunodominant region (residues 1–120) of hepatitis C virus (HCV) nucleocapsid protein. The specific recognition by either human sera or mouse monoclonal antibodies of overlapping peptides spanning the core region 1–120 as well as the comparison with epitopes described earlier allowed the fine mapping of HCV core. Within the region 1–120, the major antigenic domain could be restricted to the first 45 amino acids. Indeed, the peptide S42G (residues 2–45) allowed the detection of an anti-HCV core response by all anticore-positive human sera examined. According to their epitope localization, three groups of mouse MABs could be evidenced that were directed against different regions of core. Group II MAbs recognized a strictly linear epitope (QDVKF, residues 20–24), whereas group I MABs were directed against a conformational epitope mainly located at the amino acid residues (QIVGG, 29–33). The epitope of group III MABs was also conformational (PRGRRQPI, residues 58–65). These three epitopes appeared close but different from the three major human epitopes RKTKRNTN, VYLLPR, and GRTWAQPGYPWPLY (residues 7–17, 34–39, and 73–86, respectively). Group II MAB 7G12A8 and group I MAB 19D9D6 were used in a sandwich ELISA for the capture and the detection, respectively, of viral core antigen in sera of patients with chronic HCV infection. After treatment of sera with triton × 100 in acidic conditions, amounts of viral antigen as low as 20 pg/ml of sera could be detected. J. Med. Virol. 56:300–309, 1998 . © 1998 Wiley-Liss, Inc.     

Three separate epitopes on human IFN-alpha variants defined by monoclonal antibodies and their role in the binding to receptors.

Two monoclonal antibodies (mAbs) designed 9-1-1 and 2-2-1, produced by murine hybridoma clones, raised to recombinant IFN-alpha 2c and one mAb, designed 3A3-2, raised to recombinant IFN-alpha 88, have been characterized with respect to neutralization of IFN antiviral and antiproliferative activities. The regions of IFN molecule which these antibodies are directed against have been defined by analyzing cross- reactivity with four IFN-alpha subtypes (IFN-alpha 88, IFN-alpha 2a, IFN-alpha 2b, IFN-alpha 2c) and two fragments of IFN-alpha 88. An analysis of cross-reactivity patterns with IFN-alpha 88 and IFN-alpha 2c indicated that 3A3-2 mAb was directed to an epitope on IFN-alpha 88 not overlapping with epitopes of other mAbs, however its localization was not exactly defined. The epitope recognized by 9-1-1 mAb was present on IFN-alpha 88 and IFN-alpha 2c, and also was not overlapping the epitopes of other mAbs. Using another variant IFN-alpha 2a, containing Lys instead of Arg at position 23, for competitive binding study it was shown that Arg 23 was implicated in the epitope recognized by 9-1-1 mAb. The competition study with IFN-alpha 88 fragments allowed to locate the epitope recognized by 2-2-1 mAb between amino acid residues 51 and 166. The epitope blocking test indicated that 2-2-1 mAb was directed to an epitope overlapping that of previously reported for mAb NK-2, located around Glu at position 113.(ABSTRACT TRUNCATED AT 250 WORDS)     

An antigenic determinant is shared by psoriasis-associated p27 antigen and the Fc part of human IgG.

Rabbit antisera against the major internal protein, p27, of retrovirus-like particles from psoriatic urine, and against the serologically cross-reacting antigen, pso p27, from psoriatic scale, reacted with the Fc part of human IgG. Evidence indicating that the p27 antigen and the pso p27 antigen are identical has been presented in previous reports. A commercial antiserum against human IgG recognized a component in the pso p27-containing solution used as the source of antigen for immunization of the rabbits. By means of monoclonal antibodies against the pso p27 antigen, it was demonstrated that the Fc-reacting antibodies, and the antiserum against human IgG, recognized an epitope on the pso p27 antigen. The data indicated that an antigenic determinant is shared by the p27 antigen(s) and human IgG, suggesting that p27 antigen(s) may act as antigen(s) eliciting the production of antibodies with rheumatoid factor activity in psoriatic patients.     

Characterization of three novel monoclonal antibodies against hepatitis C virus core protein

Three novel monoclonal antibodies (MAbs) were established against a recombinant hepatitis C virus (HCV) core protein derived from cloned genotype 1b HCV cDNA. MAbs C7-50 and C8-59 recognize a conserved linear epitope represented by amino acid residues 21 to 40 of the nucleocapsid protein. MAb C8-48 is directed against a strain-specific conformational epitope located within the first 82 amino acids. A sensitive two-site MAb-based immunoradiometric assay was established using antibodies directed against distinct epitopes on the nucleocapsid protein. Processed 21 kDa core protein was detected by immunoblotting in human hepatocellular carcinoma cell lines and primary adult rat hepatocytes transfected with a cytomegalovirus promoter-driven expression construct. Immunofluorescence microscopy studies revealed a granular and vesicular cytoplasmic staining pattern. MAb C7-50 was used successfully to detect HCV core antigen in chronically infected chimpanzee liver tissue. These MAbs represent important reagents for the study of HCV biology and for the development of immunodiagnostic assays. © 1996 Wiley-Liss, Inc.     

Lack of cross-reaction between myelin basic proteins and putative demyelinating virus envelope proteins

No cross-reaction could be detected between purified myelin basic proteins (MBP) from mouse, rat or human origins and envelope proteins of viruses suspected of inducing demyelinating processes. In the experimental model using Theiler's murine encephalomyelitis virus, competition radioimmunoassay failed to detect any cross-reaction between MBP and VP1, VP2 and VP3 envelope antigens. In the human situation, antibodies against SV5 and measles viruses, both etiologically linked with multiple sclerosis, also failed to recognize MBP. These results rule out molecular mimicry as a cause of demyelination.     

Antibodies against a Class II HLA–Peptide Complex Raised by Active Immunization of Mice with Antigen Mimicking Peptides

Multiple sclerosis (MS) is an autoimmune disease linked to the human leucocyte antigen (HLA) class II genes DRB1*1501, DRB5*0101 and DQB1*0602. T cells reactive towards the DRB1*1501 in complex with various peptides derived from myelin basic protein (MBP), which is the major component of myelin, have been found in the peripheral blood of MS patients. These autoreactive T cells are believed to play a role in the pathogenesis of MS. In this article, antibodies against the HLA complex DR2b (DRA1*0101/DRB1*1501) in complex with the MBP-derived peptide MBP_85-99 have been generated by immunization of NMRI mice with three different antigen mimicking peptides displayed on M13 bacteriophages. The peptides mimick the epitope of a monoclonal antibody specific for the DR2b–MBP_85-99 complex. The mice developed IgG antibodies not only against the peptides injected, but they also developed antibodies against the DR2b complex and specific antibodies against the DR2b–MBP_85-99 complex. These data open up the possibility of designing antigen mimicking peptides for vaccination against MS.