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Biomedical subjects

G Hunsmann

Publications and source records attributed to G Hunsmann.

At least 73 records · Page 4Linked to original sources
PubMed1997

No reactivation of attenuated immunodeficiency viruses in rhesus macaques after vaccinia virus-induced immune activation.

Live-attenuated simian immunodeficiency virus (SIV) protects macaques against challenge with pathogenic SIV. To evaluate the safety of such vaccines, an investigation of whether or not nef-deleted SIV could be reactivated in vivo by immune activation of the host was conducted. In addition, monkeys infected with apathogenic SIV/HIV-1 chimeric viruses, and two control monkeys that had suppressed replication of pathogenic SIV were examined. During the infection virus became undetectable or persisted at a low level of replication in all monkeys. At this time-point 11 monkeys were immune-activated by a vaccinia virus (VV) superinfection. After VV infection up to 80% of their lymphocytes showed expression of the activation markers CD25 and CD69 over 2 weeks. However, only the two non-progressing monkeys infected with pathogenic SIV showed a noticeable but transient enhancement of SIV replication and increased SIV antibody titres. By contrast, in monkeys infected with apathogenic immunodeficiency viruses no change in virus load was observed. Therefore, attenuated immunodeficiency viruses cannot be reactivated in vivo by a VV-induced immune activation.

Animals↗
PubMed1997

Intestinal manifestations of experimental SIV-infection in rhesus monkeys (Macaca mulatta): a histological and ultrastructural study.

Intestinal lesions were studied in 32 rhesus monkeys experimentally infected with different strains of simian immunodeficiency virus SIVmac (251/32H, 251/32H-SPL and 251/MPBL) by light microscopy, transmission and scanning electron microscopy. A spectrum of primary and secondary manifestations of SIV-infection were detected. Primary changes included 'SIV-enteropathy' in 12 monkeys and virus-induced syncytial giant cell formation (GCF) of the intestine in two animals. A primary virus-induced enteropathy occurred both as only histologically visible 'SIV-enteropathy' and as 'AIDS-enteropathy' accompanied by clinical signs of enteritis. Secondary opportunistic infections (Balantidium coli, Cryptosporidium, Trichuris, Trichomonas, Spironucleus, Mycobacteria and Cytomegalovirus) were identified in 27 animals and three monkeys developed malignant lymphomas involving the intestinal tract. Compared to intestinal lesions in HIV-infected patients, differences were found concerning the incidence of GCF and the range of opportunistic infections, with cryptosporidium, cytomegalovirus and mycobacteria occurring in both SIV-infected macaques and AIDS patients. The present observations revealed that SIV-infected rhesus monkeys provide an excellent model both for studies on the pathogenesis of HIV-enteropathy and opportunistic infections and for the development of therapies against cryptosporidial, cytomegalovirus and mycobacteria infection. Comparison of three SIV-strains revealed differences in primary and secondary lesions observed: SIVmac251/MPBL was correlated with severe primary SIV-induced pathologic changes and SIVmac251-SPL-infected animals showed a higher incidence of malignant lymphomas.

Animals↗
PubMed1997

Identification of the V1 region as a linear neutralizing epitope of the simian immunodeficiency virus SIVmac envelope glycoprotein.

The sequence variability of viral structure polypeptides has been associated with immune escape mechanisms. The V1 region of simian immunodeficiency virus (SIV) is a highly variable region of the SIVmac env gene. Here, we describe the V1 region as a linear neutralizing epitope. V1 region-specific neutralizing antibodies (NAb) were first demonstrated in a rabbit infected with a recombinant vaccinia virus carrying the env gene of human immunodeficiency virus type 2 strain ben (HIV-2ben). Since we detected in this animal V1 region-specific NAb that were able to neutralize not only human immunodeficiency virus type 2 but also SIVmac32H, we investigated whether a similar immune response is evoked in macaques (Macaca mulatta) either infected with SIVmac or immunized with the external glycoprotein (gp130) of the same virus. Distinctly lower NAb titers were found in the SIVmac-infected animals than in the gp130-immunized macaques. Since the NAb titers in both groups were high enough for competition experiments, we used five overlapping peptides encompassing the whole V1 region for a detailed identification of the epitope. In each of the 12 macaques investigated, we detected a high level of NAb reacting with at least one peptide located in the central part of the V1 region. The relatively high degree of divergence, especially within the central part of the V1 region, which characterized the evolution of the retroviral sequences from the original inoculum in the infected macaques suggests the development of escape mutants. Furthermore, 3 of 12 animals developed NAb directed against the amino-terminal end of the V1 region epitope. Sequence analysis, however, revealed relatively low levels of genetic drift and genetic variability within this part of the V1 region. The induction of V1 env-specific NAb not only in gp130-immunized macaques but also in SIVmac-infected animals in combination with the increased genetic variability of this region in vivo indicates a marked biological significance of this epitope for the virus.

Amino Acid Sequence↗
PubMed1997

Construction, replication, and immunogenic properties of a simian immunodeficiency virus expressing interleukin-2.

To study the effect of interleukin-2 (IL-2) on simian immunodeficiency virus (SIV) replication, pathogenesis, and immunogenicity, we replaced the nef gene of SIVmac239 by the IL-2 coding region. The virus, designated SIV-IL2, stably expressed high levels of IL-2 in cell culture. In comparison to SIVmac239, SIV-IL2 replicated more efficiently in peripheral blood mononuclear cells in the absence of exogenously added IL-2. To determine whether this growth advantage would be of relevance in vivo, four juvenile rhesus monkeys were infected with SIV-IL2 and four monkeys were infected with a nef deletion mutant of SIV (SIVdeltaNU). After a peak in the cell-associated viral load 2 weeks postinfection, the viruses could barely be isolated 3 to 7 months postinfection. Mean capsid antigen levels were higher in the SIV-IL2 group than in the nef deletion group 2 weeks postinfection. Viruses reisolated from the SIV-IL2-infected animals expressed high levels of IL-2 during the acute phase of infection. Deletions in the IL-2 coding region of SIV-IL2 were observed in two of the SIV-IL2-infected macaques 3 months postinfection. Urinary neopterin levels, a marker for unspecific immune stimulation, were higher in the SIV-IL2-infected macaques than in SIVdeltaNU-infected animals during the acute phase of infection. The SIV-specific T-cell-proliferative response and antibody titers were similar in both groups. Cytotoxic T cells directed against viral antigens were detected in all SIV-IL2-infected macaques and in two of the SIVdeltaNU-infected animals. Expression of IL-2 did not seem to alter the attenuated phenotype of nef deletion mutants fundamentally, although there might have been a slight increase in virus replication and immune stimulation during the acute phase of infection. Deletion of the viral IL-2 gene 3 months postinfection could be a consequence of a selective disadvantage due to local coexpression of viral antigen and IL-2 in the presence of an antiviral immune response.

Animals↗
PubMed1996

Induction of antibodies against human prion proteins (PrP) by DNA-mediated immunization of PrP0/0 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.

Amino Acid Sequence↗
PubMed1996

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.

Adult↗
PubMed1996

The effect of simian immunodeficiency virus infection in vitro and in vivo on the cytokine production of isolated microglia and peripheral macrophages from rhesus monkey.

Microglia are the major target for human immunodeficiency virus (HIV) infection within the central nervous system. Because only a few cells are productively infected, it has been suggested that an aberrant cytokine production by this cell population may be an indirect mechanism leading to the development of neurological disorders in HIV-infected patients. Therefore we decided to study the secretion pattern of several interleukins (IL) by microglial cells and peripheral blood macrophages isolated from uninfected and simian immunodeficiency virus (SIV)-infected Rhesus monkeys. We found that uninfected, unstimulated primate microglia produce more IL-6 and less TNF alpha than peripheral blood macrophages, but generate comparable levels of IL-1 beta and IL-8. After infection with SIV in vitro, synthesis of all cytokines tested is increased compared to uninfected cultures and to peripheral blood macrophages. Microglia isolated from infected animals produce more IL-8 and TNF alpha than the uninfected cultures and display a strongly increased capacity to secrete TNF alpha upon stimulation with lipopolysaccharide. In addition, production of IL-6 by in vivo-infected microglia increases with time in culture to very high levels despite the fact that only a few cells contained replicating virus. These findings clearly show that the cytokine production of microglia is impaired after SIV infection both in vitro and in vivo and that a low level of viral replication is sufficient for these alterations to occur. In conclusion, the results of this study further support a possible role of cytokines in the pathogenesis of neuro-AIDS.

Animals↗
PubMed1996

Simian immunodeficiency virus (SIV) gp130 oligomers protect rhesus macaques (Macaca mulatta) against the infection with SIVmac32H grown on T-cells or derived ex vivo.

The efficacy of three SIVmac32H gp130 vaccines was compared in rhesus monkeys. Three rhesus monkeys were each immunized over a period of 20 weeks with a total of 600 microgram virion-derived gp130 oligomers (O-gp130) mixed with keyhole limpet hemocyanin and emulsified with incomplete Freund's adjuvant. Three other monkeys were infected with 5 x 10(8) PFU of vaccinia virus wild type (VV-wt) while three additional animals received an equivalent dose of VV expressing the gp130 of SIVmac (VV-gp130). At Week 8, the two VV-wt animals received an additional immunization with 100 microgram O-gp130 each. All VV-infected animals then received booster immunizations at Weeks 12, 16, and 20 with a total of 300 microgram O-gp130 per animal. All animals along with two controls were challenged iv with 50 MID50 of T-cell-grown SIVmac32H at Week 22. Four weeks after the challenge and thereafter, both controls and one animal from either VV group were infected as demonstrated by polymerase chain reaction (PCR), virus isolation, and antibody response. In contrast, all O-gp130 animals and one animal each from the VV-wt and the VV-gp130 group were completely protected as shown by negative PCR and virus reisolation. One animal of the VV-gp130 group was partially protected, since it remained virus isolation negative but became PCR positive. All protected animals did not develop a secondary antibody response. Six months after the first challenge, the five completely protected animals were reimmunized twice 4 weeks apart with a total of 200 microgram O-gp130 per animal. Two weeks later, all animals were challenged with 5 MID50 of the SIVmac32H/spI prepared from the spleen of an immunized, but unprotected SIV-infected rhesus monkey. After the second challenge, all three control animals and one of the vaccinees become productively infected. In contrast, two animals were completely protected, one from the former O-gp130 and one from the former VV-gp130 group. One animal from the former VV-wt group was only DNA-PCR positive and thus partially protected. Therefore, immunization with virion-derived gp130 oligomers of SIVmac32H can confer protection against the infection with T-cell-grown SIVmac32H as well as the ex vivo isolate SIVmac32H/spI.

Animals↗
PubMed1996

Cell-mediated immune response of macaques immunized with low doses of simian immunodeficiency virus (SIV).

Many uninfected people at high risk of HIV infection developed an HIV-specific cellular immune response despite their lack of seroconversion. Therefore, they must have been exposed to HIV without subsequent infection. It has been concluded from these data, that cell-mediated immunity (CMI) rather than humoral immunity might confer protection to HIV infection. Therefore, we tried to induce such a strong CMI in macaques by different immunization strategies. Five or seven animals were immunized with high or low doses of a whole SIV split vaccine. The lower dose of the vaccine provoked a stronger T-helper cell (TH) proliferation than the higher dose, which led to a pronounced humoral immune response. To induce a strong CMI without any specific antibody response, five macaques were inoculated with low doses of infectious SIV. None of these animals seroconverted but each animal developed a SIV-specific TH response. Interestingly, we could neither detect an SIV-specific CTL activity in the animals nor did we find typical TH1- or TH2-like cytokine profiles investigating stimulated bulk-cultures from SIV-exposed animals by RT-PCR. 24 weeks after the first low dose SIV exposure the animals were boosted by a second low dose of SIV followed by a subsequent intravenous challenge with a high dose of SIV 12 weeks later. Unexpectedly, none of the animals was found to be protected against infection and the development of AIDS-like symptoms.

Animals↗
PubMed1996

Attenuated SIV imparts immunity to challenge with pathogenic spleen-derived SIV but cannot prevent repair of the nef deletion.

To date, some success has been achieved with several experimental vaccines against AIDS in the available animal models. In the simian immunodeficiency virus (SIV) macaque model protection against superinfection was obtained by preinfection with a virus attenuated by a deletion in nef. To investigate the efficacy of SIVmac32H(pC8), a nef deletion mutant of SIVmac251, as a live-attenuated vaccine, rhesus monkeys were infected intravenously (i.v.) with this virus. All monkeys became productively infected by the pC8 virus. The animals had low cell-associated viral loads but developed a strong cellular and humoral antiviral immune response. Two out of eight preinfected monkeys developed signs of immunodeficiency and were excluded from the challenge. Sequence analysis of reisolates from one of them revealed a complete repair of the nef deletion. The remaining six monkeys, two preinfected for 42 weeks and four for 22 weeks, were challenged i.v. with a pathogenic SIV derived ex vivo from the spleen of a SIV infected macaque. Four of the monkeys challenged resisted the second infection whereas in two monkeys preinfected for 22 weeks full length nef was detectable. All monkeys maintained a virus-specific CD4-cell proliferative response after challenge. Thus, even after short preinfection periods with an attenuated SIV sterilising immunity against a challenge with a pathogenic SIV can be obtained. However, such a vaccine is unsafe since the attenuated virus frequently reverts to a more virulent form.

AIDS Vaccines↗
PubMed1996

GBV-C/HGV is not the major cause of autoimmune hepatitis.

Recently, GBV-C and HGV-two isolates of the same new flavivirus-were identified in serum samples of patients with indeterminate hepatitis and posttransfusion hepatitis, respectively. The pathogenic relevance of these viruses is still uncertain. As viral infections are presumed to trigger autoimmune processes, we investigated GBV-C in autoimmune hepatitis as well as in cryptogenic hepatitis, and compared the prevalences to patients with chronic viral hepatitis and those of blood donors. We found only a slightly higher prevalence of the virus in cryptogenic (12%) and autoimmune hepatitis type I-III (6.7%, 10%, and 12.5%) compared to blood donors (4.7%). In contrast, patients with viral hepatitis B, C, and D were more frequently infected with GBV-C (16%, 20%, 36%). These results suggest that GBV-C is not a major cause for inducing autoimmunity and leading to autoimmune hepatitis. We analyzed the nucleic acid sequences of a representative number of GBV-C positive patients (24/42) and found a broad range of nucleotide similarity in the NS3 helicase region (74-100%) among the isolates and the prototype sequences. However, we could not identify a specific sequence, which would point to a certain strain or subtype of the virus associated with autoimmune or cryptogenic liver disease.

Autoimmune Diseases↗
PubMed1996

Chimeric viruses between SIVmac and various HIV-1 isolates have biological properties that are similar to those of the parental HIV-1.

OBJECTIVE: To examine the biological properties of HIV-1/SIVmac chimeric viruses from HIV-1 isolates that have different replication rates, cell tropisms and cytopathicities. DESIGN AND METHODS: Four chimeric viruses with gag, pol, vif, vpx, nef and long terminal repeats of SIVmax and vpr, tat, rev, vpu and env of various HIV-1 isolates were constructed and compared in vitro. Cynomolgus monkeys were inoculated with two chimeras that were replicative in monkey peripheral blood mononuclear cells (PBMC). RESULTS: The type-specific neutralization of the chimeras by monoclonal antibodies 0.5 beta and mu 5.5, which recognize V3 of HIV-1IIIB and HIV-1MN respectively, was observed to be similar to those of the parental viruses, HIV-1NL432, HIV-1HAN2 and HIV-1SF13. The chimeras constructed from HIV-1SF2 and HIV-1SF13, which were isolates from the same individual but from different disease stages, reflected their parental properties, that is, the isolate from the later stage was rapid-high replicating, was more cytopathic and had a wider host range. Chimeras constructed from HIV-1HAN2' HIV-1SF13 and HIV-1NL432 were infectious to macaque monkeys, although the monkeys infected with the chimera from HIV-1SF13 showed lower virus loads and shorter viremic periods than those infected with the others. CONCLUSIONS: Chimeras have in vitro properties that are similar to those of their parental HIV-1 isolates, but their growth in macaque PBMC was dependent on which HIV-1 isolate was used. Evaluation of a vaccine by challenging with viruses possessing different antigenicities has become possible in macaque monkeys using newly constructed chimeras.

Animals↗
PubMed1996

T cell apoptosis in human immunodeficiency virus type 2- and simian immunodeficiency virus-infected macaques.

Recent evidence suggests that T cell apoptosis could be involved in the pathogenesis of HIV infection. In addition, lymphocyte apoptosis has been described in SIV-infected macaques that developed simian AIDS. To investigate further the role of apoptosis in AIDS pathogenesis, we studied lymphocytes of HIV-2-infected cynomolgus macaques that did not develop simian AIDS. We compared apoptosis of lymphocytes from animals infected with non-pathogenic HIV-2 to that in macaques infected with pathogenic SIV. Unfractionated peripheral blood mononuclear cells of SIV- and HIV-2-infected macaques showed evidence of apoptosis by electron microscopy, flow cytometry (terminal dUTP nick end labelling) and visualization of DNA fragmentation. Between 30-50% apoptotic cells could be detected in SIV-infected animals, compared to approximately 30% in HIV-2-infected and 5-12% in uninfected monkeys. However, separation of PBMC into T cell subpopulations revealed striking differences in apoptosis between SIV- and HIV-2-infected macaques. In SIV-infected monkeys both CD4 and CD8 cells underwent apoptosis to a large extent. In contrast, in the HIV-2-infected macaques apoptosis was restricted to the CD8 cell compartment. The lack of apoptosis in CD4 cells of healthy HIV-2-infected macaques implies an important role for CD4 cell apoptosis in AIDS pathogenesis.

Animals↗
PubMed1996

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.

Animals↗
PubMed1996

PCR-RFLP-based Mamu-DQB1 typing of rhesus monkeys: characterization of two novel alleles.

Up to now 19 allelic sequences of the rhesus monkey DQB1 locus have been published. Referring to these sequences, we have developed a typing protocol for Mamu-DQB1 alleles which was verified by additional cloning, sequence analysis and segregation studies. The protocol is based on the amplification of the second exon with only one specific primer pair followed by the digestion of the PCR products with up to 10 different restriction endonucleases. The alleles can be identified in homozygous and heterozygous combinations since most amplified second exon sequences give unique hand patterns after digestion with at least one of the selected restriction endonucleases. By the use of this protocol we analyzed DNA-samples from 182 rhesus monkeys. Among these samples two novel Mamu-DQB1 alleles were detected, subsequently cloned and their nucleic sequence determined. Since we typed four complete breeding groups consisting of two generations we were able to identify several DQ haplotypes by segregation analysis using the previously developed typing protocol for DQA1.

Alleles↗