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B Wahren

Publications and source records attributed to B Wahren.

At least 127 records · Page 7Linked to original sources
PubMed1996

[Composition of HIV-1 B-epitopes and heterologous T-cell epitopes. II. Combined analysis of immunogenicity of composite peptides in rabbits].

Rabbits were immunized with a mixture of linear cojoined peptides (composites) combining an immunodominant B-cell epitope from gp41 of HIV-1 (amino acids 593-604) and a heterogeneous T-cell epitope from tetanus toxoid or hepatitis B core antigen. HIV-1 B-epitope in the context of the composites induced HIV-1 specific antibody response, while no antibodies were produced in response to the peptide representing single HIV-1 B-epitope. Competition ELISA and ELISA of HIV-1 peptide omission analogous demonstrated that immunization induced antibodies of the correct specificity. T-Cell epitopic components also elicited potent antibody production. T-Cell epitopes appeared to be immunodominant. However, HIV-1 specific antibody response was not suppressed, on the contrary, antibody response against HIV-1 B- and heterologous T-cell epitopes developed in parallel. Cellular responses against composite peptides and/or their components were induced in all animals. The ability to develop cellular response against T-cell epitopes from the composites determined the efficacy of humoral response against the composites including HIV-1 specific one. Animals with strong cellular responses rapidly developed maximum antipeptide antibody titers. The immunization results imply that colinear fusion of B- and T-cell epitopes yielded bivalent multiepitopic products in which the immunological properties of the individual components were maintained and desired immunogenic properties were obtained. Immunogenicity of the composite peptides corresponded to that expected from the study of the composite antigenic properties.

Animals↗
PubMed1995

Increased mRNA expression of IL-6, IL-10, TNF-alpha, and perforin in blood mononuclear cells in human HIV infection.

Evidence has been presented for the involvement of various cytokines, including interleukin (IL)-6, IL-10, and tumor necrosis factor (TNF)-alpha, in the pathogenesis of human immunodeficiency virus (HIV) infection. Since measured plasma levels may poorly reflect in vivo production of cytokines, we adopted in situ hybridization with cDNA oligonucleotide probes to enumerate blood mononuclear cells (MNCs) expressing mRNA for IL-6, IL-10, TNF-alpha, and perforin. The HIV-infected patients had elevated levels of MNCs expressing mRNA for all four cytokines compared to healthy controls. Numbers of IL-6 mRNA-expressing cells were higher in patients with clinical AIDS than in asymptomatic seropositive patients, and correlated inversely with CD4+ cell counts in blood, reflecting the involvement of IL-6 in later stages of HIV infection. The described approach could be an alternative way to study cytokines in HIV infection.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Cytotoxicity of 3'-azido-3'-deoxythymidine correlates with 3'-azidothymidine-5'-monophosphate (AZTMP) levels, whereas anti-human immunodeficiency virus (HIV) activity correlates with 3'-azidothymidine-5'-triphosphate (AZTTP) levels in cultured CEM T-lymphoblastoid cells.

Activation of the anti-human immunodeficiency virus (HIV) compound 3'-azido-3'-deoxythymidine (AZT) is dependent on its 5'-phosphorylation by cellular nucleoside and nucleotide kinases. Azidothymidine 5'-triphosphate (AZTTP) is considered to be the metabolite responsible for both the anti-HIV effect of AZT, via inhibition of reverse transcriptase, and cytoxicity by interference with cellular DNA polymerases. During the characterization of AZT metabolism in cultured human T-lymphoblastoid CEM cells, a spontaneously occurring variant cell line, CEM/Ag-1, was found that showed approximately 10-fold resistance to AZT growth inhibition as compared to wild type (wt) cells (EC50 = 2 mM as compared to 350 microM for wt cells). CEM/Ag-1 cells had a 3-fold reduced capacity to accumulate azidothymidine monophosphate (AZTMP) compared to wt cells whereas similar levels of AZTTP were found in both cell lines. The intracellular half-life of AZTMP was approximately 70 min in both wt and CEM/Ag-1 cells. A 3-fold lower specific activity of cytoplasmic thymidine kinase was observed in CEM/Ag-1 extracts as compared to wt. The reduced thymidine kinase activity was not correlated to a decreased level of thymidine kinase mRNA. Syncytium formation of CEM/Ag-1 cells infected with HIV-2 as well as HIV-1 antigen production was inhibited at the same concentrations of AZT (approx. 0.01 microM) as were HIV-1 and HIV-2 infected wt cells. Thus, minor decreases in cellular thymidine kinase levels may markedly affect the cytoxicity of AZT but have no major effect on the antiviral activity of AZT. Our results strongly suggest that AZTMP is responsible for a major part of the growth inhibitor effects, while AZTTP mainly mediates the antiviral activity of AZT.

Blotting, Northern↗
PubMed1995

Mimicking the humoral immune response in vitro results in antigen-specific isotype switching supported by specific autologous T helper cells: generation of human HIV-1-neutralizing IgG monoclonal antibodies from naive donors.

Molecular and cellular requirements for antigen-specific isotype switch of human B cells have been investigated by mimicking signaling occurring in germinal centers. Peripheral blood mononuclear cells from healthy seronegative blood donors were first primary immunized in vitro, using a synthetic immunogen containing both a T and B cell epitope, which generated specific IgM-secreting B cells. We used the apex of the V3 loop of gp120 as B cell epitope linked to a promiscuous T helper epitope from tetanus toxin. In parallel, CD4+ T helper cell clones specific for the T epitope of the immunogen were established. In a secondary in vitro stimulation period, we co-cultured the antigen-specific T and B cells on CD32-transfected fibroblasts, together with an anti-CD40 monoclonal antibody. This resulted in isotype switching and human antigen-specific, IgG-secreting B cells were detected. This response was strictly dependent upon the presence of autologous T helper cells and the immunogen. Antigen-specific human B cells derived from this primary and secondary in vitro immunization were subsequently subjected to electrofield-induced somatic cell hybridization and hybridomas secreting human anti-V3 IgG monoclonal antibodies were isolated. One human antibody was further characterized and shown to be specific for the immunizing antigen with an affinity constant of 24 nM. This antibody also effectively neutralized different isolates of HIV-1, achieving a 50% neutralization at 0.46 microgram/ml.

Amino Acid Sequence↗
PubMed1995

Molecular characterization of a human anti-HIV 1 monoclonal antibody revealed a CD26-related motif in CDR2.

Genes encoding the immunoglobulin variable regions of a human anti-HIV-1 IgG1 kappa monoclonal antibody were rescued from a hybridoma, derived from a sero-negative donor, using PCR cloning and expression in Escherichia coli. The ELISA binding results obtained from the expressed Fab fragment confirmed the anti-V3 loop specificity for HIV-1 (LAI) of the original antibody. In addition, an amino acid sequence derived from the second complementarity determining region (CDRH2) of the heavy chain was found to be very similar to the catalytic motif of CD26, a T-cell activation antigen. Furthermore, synthetic peptides containing both the catalytic domain of CD26 and CDRH2 of the antibody showed specific binding to an HIV peptide representing the V3 region in a dose-dependent manner. This suggests an involvement of CD26 as a possible coreceptor for HIV-1.

Amino Acid Sequence↗
PubMed1995

Continuous detection of CMV DNA in plasma of patients with advanced HIV infection implies the poorest prognosis.

BACKGROUND: The contribution of cytomegalovirus (CMV) to progressive HIV infection is still controversial. OBJECTIVE: The occurrence of CMV DNA in the plasma of patients with advanced HIV infection was studied in relation to the development of clinical disease. STUDY DESIGN: Plasma samples were collected every 2 weeks for 6 months. The patients have thereafter been followed clinically at least every 3 months. CMV DNA was extracted and analysed by a nested PCR. RESULTS: CMV DNA was repeatedly detected in the plasma of five patients for more than 45 days (group 1). These patients also had very low CD4+ cell counts 51+/-41 x 10(6) /l). Three patients in group 1 developed CMV complications and CMV was the cause of death in two cases. Two patients with anti-CMV IgM responses did not develop CMV complications. All five patients died at a mean of 17 months after CMV DNA became continuously detectable by PCR. In another six patients, CMV DNA was not or only sporadically detected (group 2). In these six patients, four are still alive after more than 3.5 years and only one patient developed CMV retinitis 3 years later. CONCLUSIONS: The presence of CMV DNA in plasma was frequent in 11 patients with advanced HIV infection. The continued presence of CMV DNA in plasma of the patients with low CD4+ cell counts was related to the development of clinical CMV disease and carried the poorest prognosis.

Journal Article↗
PubMed1995

Monoclonal antibodies to p24-core protein of HIV-1 mediate ADCC and inhibit virus spread in vitro.

BACKGROUND: Certain antigens of the HIV-1, e.g., gp120-envelop proteins, can be expressed on the membrane of HIV-infected cells. Little is known about the membrane expression of other HIV-antigens and their interaction with specific antibodies. OBJECTIVE: To develop murine monoclonal antibodies (mAbs) to the p24-core protein of HIV-1 and to characterise their binding sites and biological activities on HIV-infected T cells. METHODS: Monoclonal antibodies were developed from mice hyperimmunised with a recombinant p24-core protein from HIV-1. Two mAbs were epitope-mapped on overlapping peptides and characterised for their reactivity with non-fixed HIV-infected T cells by immunofluorescence staining and flow cytometric analysis. Their biological activities were studied for antibody-dependent cellular cytotoxicity (ADCC) and suppression of viral spread in vitro. RESULTS: The epitopes of two selected mAbs were located on the amino terminal region of p24 in the regions 147-152 aa and 178-187 aa, respectively. The antibodies were able to react with living HIV-1 infected cells. The expression of the antigens was time-dependent after the infection of certain cell lines by HIV-1. The mAbs mediated a strong HIV-1-specific ADCC and were able to delay the spread of HIV-1 for about 6 days in cell cultures. CONCLUSIONS: Certain epitopes of the p24-core protein of HIV-1 can be expressed on living, HIV-infected T cells and are recognised by specific antibodies. Such antibodies can destroy infected cells by ADCC or delay the virus spread, and therefore, should be considered in immunisation strategies against HIV.

Journal Article↗
PubMed1995

Enzyme-linked immunosorbent assay-based inhibition test for neutralizing antibodies to polioviruses as an alternative to the neutralization test in tissue culture.

A poliovirus-binding inhibition test (PoBI test) was established for the quantitative determination of antibodies to polioviruses and was evaluated in comparison with the conventional neutralization test (NT). The first step of the PoBI test is an incubation of serial dilutions of test samples with inactivated poliovirus followed by the detection of free viral epitopes by a double antibody sandwich enzyme-linked immunosorbent assay with type-specific capture polyclonal antisera and type-specific neutralizing monoclonal indicator antibodies. A comparison of the PoBI test with the conventional NT for antibodies to all three types in 100 human serum samples showed excellent correlations (r > 0.95) over a wide range of antibody concentrations. The PoBI test, not necessitating live virus and tissue culture facilities, could be a simple alternative to the NT, and the principle of the assay is potentially applicable to other microbial systems.

Animals↗
PubMed1994

Enzyme immunoassay (ELISA) for the evaluation of antibodies directed to the CD4 receptor-binding site of the HIV gp120 molecule.

The interaction between the HIV envelope glycoprotein gp120 and the CD4 molecule is probably the most important primary event determining HIV infection. Reactivity with the native viral envelope has been difficult to measure due to the lack of gp120 ligand purified directly from primary virus cultures. We have developed an ELISA, utilizing Galanthus nivalis agglutinin (GNA) which selectively binds native HIV envelope gp120 in culture medium. The GNA-based ELISA eliminates the need for isotope-labelled reagents, live cells and recombinant non-natively glycosylated envelope proteins and offers an easy way of using gp120 directly from crude HIV culture medium. The reactivities of sera from several categories of HIV infected individuals were assayed for inhibition of the HIV-1 gp120-CD4 binding. 19/32 (59.3%) sera from asymptomatic individuals and 7/10 (70%) sera from ARC/AIDS patients blocked the CD4-gp120 binding. 20 serum samples from uninfected individuals showed a gp120-CD4 interaction blocking capacity of 0-15%. Two monoclonal antibodies, T4.2 directed to CD4 and 1171 directed to the CD4 binding site of gp120 were used as positive controls. Both Mabs inhibited CD4-gp120 binding by 66-90%.

Adolescent↗
PubMed1994

Identification of hepatitis B virus core protein regions exposed or internalized at the surface of HBcAg particles by scanning with monoclonal antibodies.

Hepatitis B virus (HBV) core antigen (HBcAg) particles purified from Escherichia coli were probed in a competition enzyme immunoassay (EIA) with a panel of 16 murine monoclonal antibodies (MAbs) directed to different forms of core protein. The linear binding sites of the MAbs were mapped by combination of solid-phase and competition EIA using synthetic peptides covering the complete sequence of HBV core protein. Relative accessibilities of the linear binding sites at the HBcAg surface were investigated by comparing reactivities in solution of the MAbs to (i) two genetic variants of particulate HBcAg, (ii) denatured core protein, and (iii) synthetic peptides mimicking the appropriate linear binding sites. Further, accessibilities of HBV preS1 and preS2 epitopes (introduced into core protein at positions 77 or 144) at the surface of chimeric HBcAg particles were investigated. The previously described surface localization of core protein region 78-83 at the core particle surface was confirmed. In addition, another region, encompassing residues 127-133, was found to occupy a surface position at particulate HBcAg, whereas regions 9-20 and 133-145 were exposed after denaturation of the core protein and at synthetic peptides but not at particulate HBcAg.

Amino Acid Sequence↗
PubMed1994

Immune recognition of linear antigenic regions within the hepatitis B pre-C and C-gene translation products using synthetic peptides.

The antibody recognition of linear regions within the amino acid (aa) sequence of hepatitis B (HB) core antigen (HBcAg), e antigen (HBeAg), and pre-C region was investigated in 46 patients infected with hepatitis B virus (HBV), and one immunized rabbit. Peptide analogues were synthesized to cover the complete product of the C-gene, including the pre-C region using various synthetic methods. Two carriers of hepatitis B surface antigen (HBsAg) with anti-HBe, recognized pin-bound decapeptides covering amino acid (aa) 76-83 of HBc/eAg, and the most essential residues were found to be Asp78, Pro79, Arg82, and Asp83. Pre-C peptides were recognized by IgG1 or IgG3 in sera from two out of ten cases with acute HB, in four out of twelve sera from HBeAg-positive carriers of HBsAg, and in two out of twelve sera from anti-HBe-positive carriers of HBsAg. Two sera from the cases of acute HB showed strong reactivity of the IgG3 isotype with HBc/eAg peptides 61-85. Five of the sera from HBeAg-positive carriers of HBsAg were weakly reactive with peptides 41-60, 61-85, 121-140, and/or 141-160. Eight of the sera from anti-HBe-positive carriers of HBsAg recognized aa 121-140 of HBc/e with IgG1, IgG3, and/or IgG4 isotypes. IgG from one immunized rabbit recognized peptides 1-20, 61-85, and 71-90, and the T-cells recognized peptides 1-20 and 71-90. Thus, human and rabbit antibodies recognize linear antigenic regions within the pre-C, and within regions 1-20, 41-60, 61-85, 121-140, and 141-160 of HBcAg.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Locations of antibody binding sites within conserved regions of the hepatitis C virus core protein.

The binding sites for human antibodies recognising antigenic domains within the hepatitis C virus (HCV) core protein were analyzed using synthetic peptides. Omission peptide analogues where a pair of residues was sequentially omitted were produced corresponding to the regions 1-18, 11-28, 21-38, 51-68, and 101-118. The N-terminal part of HCV core was found to contain three distinct antibody binding sites, which includes the previously reported one at residues 9-16. The other two were located at residues 19-26 and residues 29-34. Within the region 51-68 two overlapping sites were found, the first at residues 51-60 and the second at residues 59-68. Within the region 101-118, residues 107-114 were identified as the binding site, which contains two residues differing between genotypes I/II and III/VI. Thus the HCV core protein contains at least six distinct linear antibody binding sites, located at regions highly conserved between the genotypes of HCV. The human recognition of these regions show a variation with respect to the amino- and carboxy-terminal extension of each individual binding site. These findings will have implications for the prediction of the structure of the HCV core protein, since these antibody binding sequences are likely to be more or less accessible from the exterior of either, or both, of the native HCV core and its precursor polyprotein.

Amino Acid Sequence↗
PubMed1994

Immunogenic combinations of HIV-1 B- and heterologous T-cell epitopes.

Peptides were synthesized which combined HIV-1 B-epitopes from gp41, p34pol and heterologous T-cell epitopes from hepatitis B virus (HBV) core or tetanus toxoid. Mixtures of these composite peptides and peptides representing single HIV-1 B-epitopes were used to immunize rabbits in an adjuvant-free immunization regimen. Fusion to T-cell epitopes made the HIV-1 B-epitopes immunogenic and high titers of anti-HIV-1 antibodies were reached. Efficient antibody response against an immunorecessive HIV-1 B-epitope from p34 pol introduced as a B+T-composite also developed in rabbits pre-immunized by composites of the same T-cell epitopes but with a B-epitope from gp41. Fusion changed the fine antigenicity of the epitopes, but at least part of the antibodies against gp41-containing B+T composites recognized whole viral gp160. Composite peptides stimulated T-cells in the majority of the immunized animals.

Amino Acid Sequence↗
PubMed1994

Autoreactivity in HIV-infected individuals does not increase during vaccination with envelope rgp160.

The HIV-1 envelope protein contains several regions with amino acid homology to HLA class I and class II molecules. We evaluated possible changes in antibody responses to those regions during vaccination with rgp 160 produced in a baculovirus system. Forty asymptomatic HIV-infected patients with CD4 cell counts above 400 were vaccinated with rgp 160. Twenty-one patients were tissue-typed as HLA A2. Sixty-two percent of these patients exhibited cytotoxic lymphocyte antibodies directed to CD8+, HLA A2 cells. This cytotoxicity decreased during HIV gp160 vaccination. In order to further characterize the specificity of these responses, analogues of HLA class I and HLA-DR peptides were chemically synthesized together with their correct HIV-1 gp160 sequences. Enzyme-linked immunosorbent assays (ELISA) with sera from before, during and after immunization were performed with HIV proteins, peptides and their homologues. All patients showed an increase in their previously poor specific T-cell activation to gp160. Fourteen patients developed increased avidities or titres to HIV proteins and/or peptides. Contrarily, serum IgG titers to the HLA homologous peptides were initially low and decreased further during the course of vaccination. This decrease occurred in the majority of patients, 35-40 of the 40 individuals, depending on the antigen. Independent measures of autoantibodies to Ro/SS-A and La/SS-B remained undetectable.

AIDS Vaccines↗