Gerhard Hunsmann

Association between fulminant hepatic failure and a strain of GBV virus C

BACKGROUND The GB virus C (GBV-C) and the hepatitis G virus (HGV) have been detected in patients with acute indeterminant hepatitis and post-transfusion hepatitis. However, the role of the new hepatitis viruses in the aetiology of fulminant hepatitis is little understood. We investigated the presence of GBV-C/HGV in patients with fulminant hepatic failure. METHODS Serum samples from 22 German patients with fulminant hepatic failure and 106 symptom-free blood donors (controls) were studied for presence of GBV-C RNA by seminested reverse transcriptase PCR. Primer sequences were derived from the published gene sequences of the conserved NS3 region of the GBV-C prototype and the published isolates. Nucleotide and amino acid sequences of GBV-C-positive isolates, the control RNA, and the published HGV and GBV-C prototype sequences were compared by multiple sequence alignment. We also compared the GBV-C sequences of virus-positive patients who had fulminant hepatic failure with those of 19 patients with chronic hepatitis from our centre. In addition, we searched databases and published papers for further GBV-C helicase sequences in patients with non-fulminant hepatitis. FINDINGS GBV-C RNA was detected in 11 (50%) of the 22 patients with fulminant hepatic failure and in five (4.7%) of 106 control-group blood donors. Among the patients with fulminant hepatic failure, six of seven with fulminant hepatitis B and five of ten with fulminant non-A-E hepatitis were positive for GBV-C RNA. Analysis of nucleic acid sequences showed six mutations at defined positions in all 11 patients with fulminant hepatic failure who were positive for GBV-C. None of these mutations were found in the five GBV-C-positive control-group blood donors. Of the six nucleotide changes, four caused no amino acid changes, whereas two mutations at position 100 (G to T) and 102 (T to C) led to an alanine to serine change in the predicted translation product. However, comparison with GBV-C sequences of patients with non-fulminant hepatitis showed that this amino acid mutation was not specific for fulminant hepatic failure. The sequence-motif containing the six nucleotide mutations detected in all patients with fulminant hepatic failure was found in only two of 19 German patients with chronic hepatitis from our centre, and in only one of 88 GBV-C sequences from non-fulminant patients reported by others. INTERPRETATION The frequency of GBV-C RNA is higher in fulminant hepatic failure than in any other group of patients with hepatitis, particularly in patients with fulminant hepatitis B or fulminant non-A-E hepatitis. A specific strain of GBV-C may occur in serum of German patients with fulminant hepatic failure.

Generation of Monoclonal Antibodies against Human Prion Proteins in PrP0/0 Mice

BackgroundPrion diseases belong to a group of neurodegenerative disorders affecting humans and animals. The human diseases indude kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI). The pathogenic mechanisms of the prion diseases are not yet understood. Monoclonal antibodies provide valuable tools in the diagnosis, as well as in the basic research, of several diseases; however, monospecific antisera or monoclonal antibodies (mAbs) against human prion proteins were, until now, not available.Materials and MethodsWe have developed an immunization protocol based on nucleic acid injection into nontolerant PrP0/0 mice. DNA or RNA coding for different human prion proteins including the mutated sequences associated with CJD, GSS, and FFI were injected into muscle tissue. Mice were primarily inoculated with DNA plasmids encoding the prion protein (PRNP) gene and boosted either with DNA, RNA, or recombinant Semliki Forest Virus particles expressing PRNP. Hybridomas were then prepared.ResultsDifferent mAbs against human prion proteins were obtained, and their binding behavior was analyzed by peptide enzyme-linked immunosorbent assay. Western blot, immunofluorescence, and immunoprecipita-tion. Their cross-reactivity with prion protein from other species was also determined. Our mAbs are directed against four different linear epitopes and may also recognize discontinuous regions of the native prion protein.ConclusionsThese antibodies should allow us to address questions concerning the nature of the prion protein as well as the initiation and progression of prion diseases. Moreover, these mAbs can now be used for the diagnosis of prion diseases of humans and animals.

MHC Class I Alleles Influence Set-Point Viral Load and Survival Time in Simian Immunodeficiency Virus-Infected Rhesus Monkeys

In HIV-infected humans and SIV-infected rhesus macaques, host genes influence viral containment and hence the duration of the disease-free latency period. Our knowledge of the rhesus monkey immunogenetics, however, is limited. In this study, we describe partial cDNA sequences of five newly discovered rhesus macaque (Mamu) class I alleles and PCR-based typing techniques for the novel and previously published Mhc class I alleles. Using 15 primer pairs for PCR-based typing and DNA sequence analysis, we identified at least 21 Mhc class I alleles in a cohort of 91 SIV-infected macaques. The results confirm the presence of multiple class I genes in rhesus macaques. Of these alleles, Mamu-A*01 was significantly associated with lower set-point viral load and prolonged survival time. Mamu-A*1303 was associated with longer survival and a “novel” Mhc class I allele with lower set-point viral load. The alleles are frequent in rhesus macaques of Indian origin (12–22%). In addition, survival probability of individual SIV-infected rhesus monkeys increased with their number of alleles considered to be associated with longer survival. The results contribute to improve the interpretation and quality of preclinical studies in rhesus monkeys.

Analysis of the Systemic and Intrathecal Humoral Immune Response in Progressive Multifocal Leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML) is a subacute viral infection of oligodendrocytes by JC virus occurring almost exclusively in immunocompromised patients. By use of partially purified recombinant VP1 as antigen, the IgG response was analyzed by a quantitative ELISA of paired cerebrospinal fluid (CSF) and serum samples. An intrathecal immune response to VP1, defined as an antibody-specificity index of CSF to serum antibody titers > or =1.5, was found in 76% of PML patients (47/62) but in only 3.2% of controls (5/155) (P < .001). Intra-blood-brain barrier synthesis of VP1-specific IgG antibodies is 76% sensitive and 96.8% specific for the diagnosis of PML. Furthermore, the excellent correlation (r = .985) between the plasma cell count in brain tissue and the humoral intrathecal immune response to VP1 in PML patients suggests a role for B cells in this disorder.

Shedding and interspecies type sero-reactivity of the envelope glycopolypeptide gp120 of the human immunodeficiency virus

Two glycopolypeptides with molecular weights 160,000 and 120,000 (gp120) are regularly recognized by human immunodeficiency virus (HIV)-specific antisera in lysates of cells persistently infected with HIV. In the present study, gp120 was characterized as the major envelope glycopolypeptide of HIV. Gp120 was identified as the external viral glycoprotein by radiosequencing and by its presence in purified virus. However gp120 was predominantly shed as a soluble protein into the culture fluid. Furthermore gp120 was precipitated by sera from horses infected with equine infectious anaemia virus (EIAV), but not by sera from uninfected animals. This may indicate conserved epitopes common to the envelopes of HIV and EIAV.

Properties of mouse leukemia viruses. IX. Active and passive immunization of mice against Friend leukemia with isolated viral GP71 glycoprotein and its corresponding antiserum.

Abstract Mice could be protected against Friend leukemia virus-induced leukemia by inoculation with the major viral glycoprotein GP71 or its antiserum.

Sera from adult T-cell leukemia patients react with envelope and core polypeptides of adult T-cell leukemia virus.

Sera from five Japanese patients with adult T-cell leukemia (ATL) showed in the immunofluorescence test for ATL-associated antigen (ATLA) titers ranging from 320 to 1280. Control sera from three healthy adults were negative. These eight sera were used to immunoprecipitate radiolabeled polypeptides from three cell lines infected with adult T-cell leukemia virus (ATLV) and two noninfected human cell lines. Cells were labeled either metabolically with [35S]cysteine, [35S]methionine, or [3H]glucosamine, or chemically with 125-iodine. Immunoprecipitates from cells, virus, and concanavalin A-enriched supernatants were analyzed by polyacrylamide gel electrophoresis. Cells producing ATLV contain gp68, the putative precursor to ATLV envelope polypeptides, gp46, and possibly p15, in addition to several nonglycosylated polypeptides between 40 to 70 kDa. Gp46 is shed into the culture medium and appears to be loosely attached to the viral and cellular surface. After purification on density gradients viral particles contain immunoreactive p24, p19, p15, and small amounts of gp46. Kinetics of synthesis, distribution, size, biochemical characteristics, and immunoreactivity of these polypeptides strongly suggest that most of them are structural components of ATLV or their precursors. Apparently, the intracellular ATLA complex predominantly represents precursors of viral structural polypeptides and gp46 is the viral envelope glycopolypeptide.

Inhibition of human immunodeficiency virus type I reverse transcriptase by suramin-related compounds

Ninety analogues of suramin have been examined for their ability to inhibit the exogenous reverse transcriptase (RT) of human immunodeficiency virus type I (HIV-I). Of these compounds, 57 inhibited the poly(rC).oligo(dG)-dependent RT activity. Three classes of dose-response curves could be discriminated. Allocation of a compound to one class did not correspond with obvious structural features. Twenty-four substances were superior to suramin in our RT inhibition assay. The RT-inhibitory activity of these compounds did not correlate with their effect against filariae or trypanosomes. Preliminary antiviral evaluation in susceptible human T cells inoculated with HIV-I demonstrated in vitro therapeutic efficacy for some compounds with lower drug-related cellular toxicity than suramin. Certain structural features relevant for the RT-inhibitory effect of these compounds were recognized. Predictions are made for the design of more effective RT inhibitors. Such compounds will help to understand the molecular mechanism of reverse transcription and might be useful in the therapy of retroviral infections.

Induction of antibodies against human prion proteins (PrP) by DNA-mediated immunization of PrP00 mice

Prion diseases are neurodegenerative disorders, affecting humans and animals. The human diseases include kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI). To generate monospecific antisera against human prion proteins we have immunized mice with DNA coding for different human prion proteins. We constructed immunization vectors expressing individual genotypes of either the cellular prion gene (PRNP) or mutant forms under appropriate promoters. This approach avoids the preparation of infectious material for immunization. To circumvent immunological tolerance prion protein-deficient PrP0/0 mice were used for the DNA-mediated immunization. Thereby monospecific sera were raised capable of specifically precipitating in vitro synthesized human prion proteins. With prion protein-specific peptide ELISAs, we found that antibodies are predominantly directed against the octapeptide repeat region and to a lesser extent to regions comprising the signal peptide, the neurotoxic domain or the GPI anchor. In contrast, prion gene-positive (PrP+/+) BALB/c mice immunized under the same experimental conditions as the PrP0/0 mice did not respond with antibody formation against the human prion protein. This is the first report clearly showing that immune competent prion protein-deficient mice react with a vigorous polyclonal immune response after DNA-mediated immunization with human prion gene sequences.

Rapid development of vaccine protection in macaques by live-attenuated simian immunodeficiency virus.

Convincing data on experimental vaccines against AIDS have been obtained in the simian immunodeficiency virus (SIV) macaque model by preinfection with a virus attenuated by a nef deletion. To investigate the efficacy of a nef deletion mutant of SIVmac32H called pC8 as a live-attenuated vaccine after shorter preinfection periods and to learn more about the nature of the immune protection induced, eight rhesus monkeys were infected intravenously with the pC8 virus. All monkeys became persistently infected, exhibiting low cell-associated viral loads, but strong cellular and, in terms of binding antibodies, strong humoral antiviral responses. Two of eight pC8-infected monkeys developed an immunodeficiency and were not challenged. Sequence analysis of their nef revealed complete replenishment of the deletion. The other six monkeys, two preinfected for 42 weeks and four for 22 weeks, were challenged with pathogenic spleen-derived SIV. Complete protection was achieved in four vaccinees. Virus was consistently detected in two vaccinees from the 22-week-group challenge, however, they remained clinically healthy over a prolonged period. Protection from challenge virus infection or a delayed disease development seemed to be associated with a sustained SIV-specific T helper cell response after challenge. Thus, a sterilizing immunity against superinfection with pathogenic SIV can be induced even after a relatively short waiting period of 22 weeks. Nevertheless, such a vaccine raises severe safety concerns because of its potential to revert to virulence.