Isolation and characterization of human monoclonal antibodies against hepatitis C virus envelope glycoproteins.

The isolation and characterization of human monoclonal antibodies (humAbs) against the hepatitis C Virus (HCV) glycoproteins E1 and E2 are described. B-cells from blood donors with anti-HCV were transformed with Epstein-Barr virus. The supernatants of the resulting lymphoblastoid clones were screened by ELISA with an extract of cells infected with a recombinant vaccinia virus RMPA95 expressing the envelope proteins E1 and E2 of an HCV genotype 1a virus (H strain). Positive clones were fused to the heteromyeloma cell line K6H6/B5. Fifteen heterohybridoma cell lines have been established. The specificity of the isolated humAbs was determined both by ELISA and Western blot assays. Several recombinant extracts expressing either the E1 or E2 protein or truncated forms were used in an attempt to map the epitopes on the viral glycoproteins. Some of the humAbs were used successfully for immunofluorescence investigation of transfected cells. Seven specific anti-E2 humAbs, which react with the envelope protein 2 of genotype 1a and 1b isolates, were characterized.

Human monoclonal antibody with T-cell-like specificity recognizes MHC class I self-peptide presented by HLA-DR1 on activated cells.

Alloreactive T cells recognize peptides presented in the binding groove of major histocompatibility complex molecules (MHCs), whereas B cells mainly recognize the MHCs independent of bound peptides. Here, we demonstrate that the human B-cell repertoire comprises B cells which can be stimulated during pregnancy to produce antibodies reacting with MHCs in a way similar to T cells. The human monoclonal antibody UL-5A1 recognizes DR1(DRA/DRB1*0101) molecules on lymphoblastoid cell lines only if they co-express HLA-A2 or if they have been loaded with HLA-A2-derived peptides. The effect of the HLA-A2 peptide 105-117 on UL-5A1 reactivity was specific, time and dose-dependent. Reactivity increased when naturally processed peptides were removed from DR1 molecules before the HLA-A2 peptide 105-117 was loaded. UL-5A1 reacted specifically with cells that had been activated. The results imply a role of activation of cells in peptide processing and/or loading.

Characterization of two human IgM monoclonal antibodies reactive with HLA-B27.

We describe here the generation and characterization of two human monoclonal IgM antibodies (UL-4F11 and UL-F6) reactive with HLA-B27. The monoclonal antibody (mAb) UL-4F11 is cytotoxic for peripheral mononuclear cells and, therefore, useful as typing reagent for HLA-B27 and HLA-B38. Protein chemistry showed that the mAb UL-4F11 precipitates HLA-B27 molecules. Epitope mapping analysis suggests that the amino acids 45, 67, 82 and 83 (alpha-1 domain) of the HLA-B27 sequence are necessary for mAb UL-4F11 reactivity. The mAb UL-F6 is suitable for complement dependent lysis of lymphoblastoid cell lines and stimulated peripheral blood mononuclear cells with HLA-B27 (B*2701, B*2702, B*2703, B*2705, B*2707), B13, B40 (60,61), B47 and B48 specificities. Its reactivity indicates that the amino acid valine in position 152 and glutamic acid in position 163 of the alpha-2 domain are crucial for the binding epitope.

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.

The serology of hepatitis C virus (HCV) infection: antibody crossreaction in the hypervariable region 1.

We determined the NS1/E2 N-terminal sequence including the hypervariable region 1 (HVR1) from five individuals chronically infected with HCV: two from the Czech Republic and three from Germany. From each sequence, six 12-mer overlapping peptides were synthesized and used in a peptide scan to evaluate seroreactivity of each of those patients, as well as three anti-HCV positive blood donors to the different isolates. We could show the general presence of antibodies to multiple HVR1 specific sequences reflecting the existence of multiple variants in infected persons. Finally, we observed the persistance of HCV infections in all individuals despite an active humoral response directed against the virus.

Isotype-specific immune response to a single hepatitis C virus core epitope defined by a human monoclonal antibody: diagnostic value and correlation to PCR.

In this study we tested the seroreactivity of 223 selected anti-HCV-reactive blood donors to the human B-cell epitope N-VYLLPR-C (C34-39) of the hepatitis C virus core antigen. The epitope was recently identified and characterized by the human monoclonal IgG antibody Ul/F10 and is located within the amino acid residues 34-39 of the aminoterminal core region. The blood donor sera were selected from anti-HCV ELISA (Ortho, 2nd generation)-reactive samples. Sixty-seven of these sera were further reactive in RIBA (Ortho, 2nd generation). According to their RIBA pattern, these samples were divided into four groups. Samples in the first group (n = 18) reacted to all four recombinant HCV antigens. The samples of the second (n = 9) and third group (n = 8) reacted to c22-3/c33c and c22-3/c100-3, respectively. Sera from group 4 (n = 32) showed a RIBA indeterminate pattern with reactivity only to c22-3. All 223 samples were analyzed for anti-C34-39 antibodies by ELISA, and the 67 RIBA-reactive samples were additionally tested for the presence of HCV RNA by RT/PCR. In groups 1 and 2, over 80% of the samples showed anti-C34-39 reactivity which was restricted to the IgG1 isotype. In contrast, in groups 3 and 4, antibodies to the epitope C34-39 were detected in less than 10% of the samples. Interestingly, the anti-C34-39 response correlates with the presence of HCV RNA; 95.5% of the samples had coincident results in all subgroups. None of the RIBA-negative sera showed a specific seroreaction to the C34-39 peptide.

Generation and characterization of a human monoclonal IgG antibody specific for HLA-B12.

We describe here the generation and characterization of a human monoclonal IgG antibody (UL/F14) specific for HLA-B12. The antibody is suitable for complement-dependent lysis on lymphoblastoid cell lines and stimulated peripheral blood mononuclear cells. UL/F14 competes for antigen binding with an HLA-B12 human monoclonal IgM antibody and with a specific alloantiserum. Protein chemistry shows that the monoclonal antibody UL/F14 can precipitate solubilized HLA-B12 molecules.

Isolation and epitope characterization of human monoclonal antibodies to hepatitis C virus core antigen.

In this study we describe the establishment of two hybridoma cell lines secreting human monoclonal antibodies to the 22-kD nucleocapsid protein (core, p22) of the hepatitis C virus (HCV). For this purpose we isolated B lymphocytes from an anti-HCV positive blood donor and infected them with Epstein-Barr (EBV). We obtained several lymphoblastoid cell clones secreting antibodies to the recombinant HCV core protein. The B-cell cultures were oligoclonally expanded and two of them were fused with the (mouse:human) heteromyeloma cell line K6H6/B5. The resulting stable hybridomas produce antibodies of the IgG1/kappa (U1/F10) and the IgM/kappa (Ul/F11) isotype reacting specifically with the recombinant core protein p22. To identify the epitopes recognized by these antibodies we synthesized overlapping peptides (13-mer and 6-mer) from the amino terminus of the core amino acid sequence. Antibody reactivity to these peptides was analyzed in an immunoblot assay. Finally, we were able to define a linear epitope recognized by the Ul/F10 antibody on the nucleocapsid protein. The antibody shows specificity to the sequence N-VYLLPR-C, which corresponds to the amino acids 34-39 of the core sequence.

[Recombinant antibodies in serologic diagnosis]. / Rekombinante Antikörper in der serologischen Diagnostik.

In this paper we discuss the value of a new methodology for the production of monoclonal antibodies by recombinant techniques. This approach is especially useful for human monoclonal antibodies due to instability of human antibody-producing cell lines. As an example, we present a recombinant human antibody to the hepatitis C virus (HCV) core protein.

[Pulmonary manifestations in anaphylactoid purpura (Henoch-Schönlein Syndrome) (author's transl)]. / Lungenbeteiligung bei anaphylaktoider Purpura (Schönlein-Henoch-Syndrom).

Pulmonary manifestations in anaphylactoid purpura (Henoch-Schönlein-syndrome). Radiological observations are reported on 2 female adults with Henoch-Schönlein-syndrome accompanied by reversible pulmonary disease. This form of anaphylactoid purpura has rare been reported until now. The pulmonary opacities are regarded as intraalveolar bleeding probably with edem and interstitial perivascular infiltrations. They developed at the same time as the skin lesions and are probably part of the disease. These pulmonary changes are characterized by the following radiological criteria..: 1. parahilar, butterfly-shaped opacities, 2. air-bronchograms, 3. reticulo-nodular pattern, 4. rapid change.