PubMed Health⌕ Search

Biomedical subjects

W Lüke

Publications and source records attributed to W Lüke.

At least 55 records · Page 3Linked to original sources

Immunization with tween-ether-treated SIV adsorbed onto aluminum hydroxide protects monkeys against experimental SIV infection.

In order to examine the efficiency of an AIDS vaccine potentially acceptable for human use we have investigated a split vaccine. Since such vaccines are safe and efficient, they have been in use for many years to protect man against enveloped RNA viruses, e.g., influenza and measles. Seven rhesus monkeys were immunized at Week 0, 4, 8, and 16 by im injection of 2 ml of vaccine containing 140 micrograms of Tween-ether-disrupted SIVmac251/32H adsorbed onto aluminum hydroxide. The immunized animals and three nonvaccinated control monkeys were challenged 2 weeks after the last immunization by iv injection of 10 to 50 minimal monkey infectious doses of SIVmac251/32H. Four of seven immunized animals did not show any signs of virus replication and therefore appeared to be protected. Nonvaccinated control animals and the vaccine failures showed a rise in their urinary neopterin concentrations 1 to 2 weeks after infection. At the end of the second week and thereafter, cocultures and polymerase chain reaction of their peripheral blood lymphocytes were positive. After the challenge, control animals and infected vaccinees showed a primary or secondary antibody response while antibody titers declined in virus-negative animals. Specific cytotoxic T-lymphocytes were not present prior to challenge, but were present in some animals thereafter. Therefore, these seem to reflect a response to viral replication rather than to immunization. Prior to challenge the CD4-positive lymphocytes of the peripheral blood of the four virus-negative animals only proliferated after exposure to the immunizing antigen. Thus, this reaction appears to predict protection.

Adjuvants, Immunologic↗

Isolation and characterization of a retrovirus from the fish genus Xiphophorus.

A cell line (BsT) established from neoplastic embryonal tissues of the platyfish (Xiphophorus maculatus) released spontaneously retrovirus-like particles. The particles have a buoyant density of 1.16 g/ml, a mean diameter of 100 nm and the morphology of immature retroviruses. The particle-associated proteins p70, p65, and p28 react with an antiserum directed against the major internal feline leukemia virus structural protein p27. The particles are associated with a reverse transcriptase. The purified enzyme has a molecular weight of about 70 kDa and prefers the template primers poly(rA):oligo(dT), poly(dC):oligo(dG), and poly(rC):oligo(dG) in the presence of Mn2+. The enzyme activity is inhibited by antibodies directed against the reverse transcriptase of feline leukemia virus and simian sarcoma virus. The particles contain a ribonucleic acid of about 70 S. In an endogenous reverse transcriptase reaction nucleic acids in the range of 0.2 to 0.4 kb were synthesized. In Northern blots with these nucleic acids as probe, three transcripts of about 8.5, 4.2, and 1.5 kb were detected in BsT cells. Southern blot analysis with the same probe demonstrates related sequences in the DNA of BsT cells and the platyfish and swordtail (Xiphophorus helleri). Hybridization experiments with the LTR-gag region of the feline leukemia virus show homologous sequences in the Xiphophorus genome.

Animals↗

Characterization of the endogenous reverse transcriptase reaction products of SIVagmTYO-7, a simian immunodeficiency virus isolated from an African green monkey.

The products of the endogenous reverse transcriptase reaction of SIVagmTYO-7 were characterized after the reaction conditions had been optimized. The major reaction product in the presence of actinomycin D and oligo(dT) was a DNA with a size of 300 bases. Without actinomycin D two additional reaction products with 600 or 700 bases appeared. The 300 base product was identified as the (-)strong-stop DNA, whereas the 700 base product is the (+)strong-stop DNA. The 600 base product appeared only after oligo(dT) priming. The (-)strong-stop DNA hybridized specifically with a 9 kb RNA found in virus particles and three RNA species of 1.8, 4.8 and 9 kb isolated from SIVagmTYO-7 infected cells.

Animals↗

CD4-binding of gp130 micelles isolated from SIVagmTYO-7.

The binding and binding inhibition of the SIVagmTYO-7 external glycoprotein gp130 in micellar form to the CD4 molecule on human Molt-4 clone 8 cells was investigated. The best binding of gp130 to Molt-4 clone 8 cells occurred at pH 5.5 to 6.5 at 37 degrees C after 4 h or at room temperature after 10 h. The dissociation constant of this reaction was 0.2-0.4 nM, with both soluble CD4 or CD4 on Molt-4 clone 8 cells. This value is close to 0.15 nM determined for the antihuman CD4 monoclonal antibody 30F16H5. After partial deglycosylation of gp130, a 90 kD product arose which still bound to CD4. Fully deglycosylated gp130 of 60 kD was still immunoprecipitable, but had lost the CD4 binding activity. Lens culinaris agglutinin was able to inhibit the gp130-CD4 interaction very efficiently, while the agglutinin of Phaseolus vulgaris was half as efficient and Canavalia ensiformis was inefficient. CD4 binding of gp130 micelles was also inhibited with several anti CD4 monoclonal antibodies directed against the OKT4a epitope as well as with soluble recombinant CD4.

Antibodies, Monoclonal↗

Immunization of rhesus monkeys with high- and low-dose Tween-ether-disrupted SIVMAC.

Two vaccine trials were conducted with low- and high-dose purified Twen-ether-treated SIVmac adsorbed onto aluminum hydroxide using rhesus macaques. In the first experiment 7 macaques were immunized with a total amount of 560 micrograms protein and 3 animals served as controls. After the immunization period the vaccinees exhibited ELISA titers up to 1:1280 and 5 immunized animals showed an antigen-specific proliferative response. After the virus challenge the 3 control animals and 3 vaccinees became infected. Four of the infected animals developed a cytotoxic T-cell response beginning 8 weeks postchallenge. The 4 protected animals were rechallenged 16 weeks later and all became infected. For the high-dose experiment 5 immunized animals receiving 2 mg of antigen and 2 control animals were used. The ELISA titers of the vaccinees reached 1:20480 and 4 animals exhibited an antigen-specific proliferative response. In response to virus challenge the 2 control and 1 immunized animal became infected. From these data it can be concluded that the high-dose immunization scheme elicited higher antibody titers and increased the fraction of protected animals.

Adjuvants, Immunologic↗

Virus-specific cytotoxic T-lymphocytes in HIV-2-infected cynomolgus macaques.

OBJECTIVE: Specific cytotoxic T-lymphocytes (CTL) are induced in humans or monkeys after infection with HIV-1 or SIVmac, respectively. Since, like HIV-1, HIV-2 causes AIDS, our objective was to determine the characteristics of the HIV-2-specific CTL response. DESIGN: Since it is rarely possible to study cellular immunity in individuals, because of the small number of HIV-2-infected patients available in Europe and the necessity for co-operation in the performance of sequential CTL assays, cynomolgus macaques were infected with HIV-2. Autologous transformed B-lymphoblastoid cell lines infected with recombinant vaccinia viruses were used as target cells for cytotoxicity assays. METHODS: Recombinant vaccinia viruses expressing HIV-2 genes were constructed to infect B-lymphoblastoid cell lines from macaques. These cells were used as target cells for cytotoxicity assays with peripheral blood mononuclear cells from HIV-2BEN-infected cynomolgus macaques. To characterize the effector cells, CD8+ cells were separated with immunomagnetic beads. Major histocompatibility complex (MHC) restriction of the cytotoxic cells was determined by incubation with matched or mismatched target cells. RESULTS: HIV-2BEN-infected cynomolgus macaques raised CTL against proteins of the three major viral structural genes, gag, pol and env. The cytotoxic cells were CD8+ and their activity was MHC class I-restricted. In contrast to SIVmac-infected macaques, env-specific lysis was mediated exclusively by CD8+ cells. CTL from individual animals recognized different viral proteins and the recognition pattern varied over time. CONCLUSIONS: Like HIV-1 and SIVmac, HIV-2 induces virus-specific CTL. The variation of antigen recognition between individual animals and over time indicates that sequential experiments are necessary to determine the complete spectrum of the CTL response of infected animals. HIV-2-infected macaques represent a suitable model for investigations into the cellular immune response against HIV.

AIDS-Related Complex↗

Potential significance of the cellular immune response against the macaque strain of simian immunodeficiency virus (SIVMAC) in immunized and infected rhesus macaques.

The cellular immune response of seven rhesus macaques immunized with Tween-ether-treated macaque strain of simian immunodeficiency virus (SIVMAC) and three non-vaccinated control animals was investigated. Immunization elicited antigen-specific proliferating CD4+ cells in five of seven monkeys. Proliferating T cells were found in all animals protected from a first virus challenge. Cytotoxic T lymphocytes (CTLs) were not induced by the immunization. After the second challenge, the four formerly protected animals became infected, despite a strong proliferative CD4+ cell activity in three of them. All animals lost their proliferative activity 2 weeks after infection. After the first challenge four of the six infected animals exhibited a CTL response and after the second challenge, one of four newly infected macaques acquired a CTL response. The five animals with a CTL activity against SIVMAC proteins were protected from severe thrombocytopenia, which appeared in the five CTL-negative animals after infection. Our data show the induction of proliferative T cells by immunization with soluble SIVMAC antigen. This T cell reactivity was found in all animals protected from the first virus challenge, but did not confer protection from the second challenge. Interestingly, the proliferative T cell reactivity disappeared 2 weeks after virus infection. Furthermore a CTL response against viral proteins seems to protect infected animals from severe thrombocytopenia which is an early sign of AIDS in monkeys.

Animals↗

An anti-CD4 antibody for treatment of chronic inflammatory arthritis.

Monoclonal antibodies (mAb) to the CD4 surface molecule inhibit the function of CD4+ T cells in vitro and have been used for treatment of autoimmune diseases in several animal models. Recently, an anti-CD4 mAb has been described that improved the clinical situation of rheumatoid arthritis (RA) patients although no change in laboratory parameters could be observed. Here, we report on a different high-affinity anti-CD4 mAb (MAX.16H5) and its use for treatment of RA. Reduction of the Ritchie index, morning stiffness and the number of swollen joints demonstrated the clinical benefits of the therapy. In addition, laboratory parameters like ESR, CRP, and rheumatoid factor were reduced in 6/12 treatments. A rapid depletion of CD4+ T cells was observed in all patients which reached a minimum 1 hour after administration. However, efficacy of treatment did not correlate with T cell depletion. The antibody accumulates at the site of inflamed joints as detected by 99m-Tc-labelling. Affected digital joints were detected earlier by virtue of helper T cell imaging than by conventional bone scans.

Antibodies↗

Structural comparison of the external glycoproteins of human and simian immunodeficiency virus.

The structural variability of the external glycoproteins of primate immunodeficiency viruses, has, so far, been investigated exclusively by sequence comparison of the respective proviral genomes. We have examined the structural relationship amount the external glycoproteins from three specific human immunodeficiency viruses (HIF-1, HIV-2), three specific simian immunodeficiency viruses from macaques (SIVmac) and three specific SIV from African green monkeys (SIVagm) by peptide mapping. Differences among glycoproteins were most pronounced between HIV-1 and SIVmac, as well as HIV-2. Two specific glycoproteins from independent SIVagm isolates were closely related to HIV-1, whereas the glycoprotein from a third SIVagm isolate was more similar to those of SIVmac and HIV-2. Our analysis reflects the classification of primate immunodeficiency viruses into three groups, the HIV-2 and SIVmac viruses, the green monkey isolates and HIV-1.

Amino Acid Sequence↗

Detection of RNA-dependent DNA polymerase activity in the Xiphophorus melanoma system.

DNA polymerases have been isolated from muscle and melanoma tissues of Xiphophorus, which are similar to retroviral RNA-dependent DNA polymerases as they prefer RNA to DNA templates. They appear to associate with submicroscopic structures which exhibit a density of about 1.13 g/ml after sucrose-density-gradient centrifugation. The RNA-dependent-DNA-polymerase-like enzymes could be separated from the DNA-dependent DNA polymerases by DEAE-cellulose chromatography. Further purification on phosphocellulose revealed that the muscle enzyme eluted at the void volume and at about 0.6 M KCl, whereas most of the melanoma enzyme eluted at 0.1 M KCl. Comparison of the template primer specificities of the muscle and melanoma enzymes with those of known DNA polymerases showed obvious similarities to the RNA-dependent DNA polymerase isolated from Rous sarcoma virus.

Animals↗

Partial purification and characterization of the reverse transcriptase of the simian immunodeficiency virus TYO-7 isolated from an African green monkey.

The reverse transcriptase (RT) was partially purified by a newly developed procedure from the simian immunodeficiency virus TYO-7 isolated from an African green monkey (SIVagmTYO-7). The method comprised lysis of the virus with nonionic detergent followed by two centrifugations in isopycnic sucrose density gradients and one velocity sedimentation in a glycerol gradient. The enzyme exhibited a purity of 70-80% and showed an exceptional high specific activity of 135 nmol incorporation of dTMP per milligram of protein in 1 h with poly(rA).oligo(dT) as template-primer (TP). The molecular weight of the native enzyme was estimated by velocity sedimentation analysis as 120K-130K. Investigation of the RT by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the active enzyme is a heterodimer composed of a 64- and a 50-kDa subunit. The two subunits were identified to be RT specific by Western blot analysis. In activity gels, both subunits exhibited enzymatic activity, whereby the 64-kDa subunit showed the predominant activity. The RT preferred the TP poly(rA).oligo(dT) over poly(rC).oligo(dG). With poly(rCm).oligo(dG), only marginal activity was detected, and no activity was measured with poly(dA).oligo(dT). The TP specificity was influenced by the reaction temperature. The highest activity was measured around the melting temperature of the TP used. Furthermore, the enzyme activity was more thermolabile when measured with poly(rA).oligo(dT) than with poly(rC).oligo(dG). To compare the specificity of RT inhibitors, their inhibition efficiency (IE) was defined as the ratio of the 50% inhibiting concentration (ID50) obtained with the RT in viral lysates to the ID50 of purified RT.

Animals↗

A novel proviral clone of HIV-2: biological and phylogenetic relationship to other primate immunodeficiency viruses.

Infectious molecular clones of the human immunodeficiency virus type 2 (HIV-2) will be valuable tools for the study of regulatory gene functions and the development of an animal model for the human acquired immunodeficiency syndrome (AIDS). To this end, we have cloned and sequenced a novel HIV-2 isolate, HIV-2BEN. One clone, designated MK6, is infectious for various human T-cell lines and for human and macaque peripheral blood lymphocytes (PBL), allowing molecular studies of HIV-2 infection and replication. Since MK6 is highly cytopathic in MT-2 and Molt-4 clone 8 cells, antiviral agents and neutralizing sera may be tested. Cluster analysis of HIV-1, HIV-2, and simian immunodeficiency virus (SIV) env and gag genes revealed that HIV-2BEN yielded the earliest node of phylogenetic divergence for all reported HIV-2 sequences. Noise analysis showed that, with the current data, no specification of any branching order can be made among the four groups of primate lentiviruses, HIV-1, HIV-2/SIVSMM/MAC, SIVAGM, and SIVMND.

Animals↗

Isolation and characterization of HIV-2ben obtained from a patient with predominantly neurological defects.

A HIV-2 strain named HIV-2ben was isolated from peripheral blood lymphocytes of a patient who, since 1984, had developed neurological symptoms such as Raynaud's syndrome, followed by paresthesia of extremities and ataxia, and finally paraparesis of the legs and incontinence. This new isolate could be distinguished from HIV-2rod by antibody-binding epitopes, peptide maps of core p24 and p18 polypeptides and restriction endonuclease cleavage pattern.

Animals↗

Experimental infection of macaques with HIV-2ben, a novel HIV-2 isolate.

Ten rhesus (Macaca mulatta) and six fascicularis (Macaca fascicularis) macaques were inoculated with HIV-2ben using three different virus preparations and two routes of inoculation. Thirteen of the 16 inoculated macaques seroconverted 2-6 weeks after infection. Three M. mulatta remained seronegative. The seroconverted animals developed antibody titres from 80 to 40,000. Their antibodies reacted with gp160 and gp130 and, in varying degrees, with gp32 and core proteins. Virus could be re-isolated from 11 of the 16 macaques. M. mulatta were transiently viraemic 6-14 weeks after infection whereas all M. fascicularis were persistently viraemic 2-7 weeks after infection onwards. In the 6-18 months after infection one M. mulatta lost 20% of its body weight and two M. fascicularis showed transient lymphadenopathy and splenomegaly; the other animals remained clinically normal. A re-isolated virus from a M. mulatta was indistinguishable from the inoculated HIV-2ben by genomic restriction enzyme analysis. M. mulatta and M. fascicularis are infectable by a single intravenous injection of cell-free HIV-2ben. Persistent viraemia in M. fascicularis represents a valuable and reliable parameter for studies on antivirals and vaccines.

Animals↗

Urinary neopterin concentrations in rhesus monkeys after infection with simian immunodeficiency virus (SIVmac 251).

Two rhesus monkeys were infected with SIVmac 251. Elevated urinary neopterin concentrations were observed as the first sign of infection. Virus-specific antibodies were detected 14 days after infection, when neopterin concentrations were already decreasing. The neopterin levels of one animal remained elevated and the virus was repeatedly isolated. Urinary or serum neopterin concentrations appear to be early markers for SIV infection and viremia in rhesus monkeys.

Animals↗

Structural variability of the external glycoprotein of simian immunodeficiency virus propagated in cell cultures.

We examined the structural variability of the external glycoprotein (gp130) of a cloned simian immunodeficiency virus (SIV) in culture. Cloned SIVmac142 was either permanently propagated in HUT-78 cells or sequentially passaged in MT-2 cells. After 12, 24 and 60 passages of permanent or lytic cell-culture systems, virus was harvested, gp130 was isolated and peptide mapped. Comparison of gp130 peptide maps of SIVmac142 by computer graphic analysis revealed a variation in relative spot intensity of up to 20%. No major variability of gp130 was observed in SIVmac142 propagated in HUT-78 cells (i.e. without cytopathic effect induction) in up to 60 passages. However, in MT-2 cells, which are lysed by this virus, gp130 exhibited significant variability and displayed six additional fragments in peptide maps after 24 passages. Peptide map of SIVmac142 gp130 obtained after 60 passages in MT-2 cells was comparable to that after 24 passages. Alterations in the intensity of certain spots indicated changes in the composition of the replicating virus population.

Genetic Variation↗

Experimental infection of rhesus monkeys with SIV isolated from African green monkeys.

To establish an animal model for AIDS, 6 juvenile rhesus monkeys were infected intravenously with cell-free SIVagmTYO-1, 5, or 7. One animal was infected with SIV of known pathogenicity isolated from a sooty mangabey (SIVsmm). The 2 animals infected with TYO-1, 1 of 2 infected with TYO-7 and the 1 infected with SIVsmm seroconverted within 4-8 weeks after infection and infectious virus could be recovered 8-10 or 44 weeks after infection. Three of the 4 seroconverted monkeys developed a persistent lymphadenopathy 12 weeks after infection. Rhesus monkeys infected with SIVagm could serve as a model to study HIV infection and for vaccine trials.

Acquired Immunodeficiency Syndrome↗