PCR sequencing with the aid of detergents.
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Detergents↗
Biomedical subjects
W Lüke
Publications and source records attributed to W Lüke.
Clinical implications of nucleic acid amplification methods for the diagnosis of viral infections of the nervous system.
Amplification of viral nucleic acids from the cerebrospinal fluid (CSF) has considerably improved the diagnosis of several acute, subacute and chronic viral infections of the nervous system. In herpes simplex virus (HSV) encephalitis (HSE) the polymerase chain reaction (PCR) has become the method of choice for the rapid, non invasive diagnosis. Other herpes virus associated diseases which can now be reliably diagnosed are encephalitis, ventriculoencephalitis, polymyeloradiculitis, myelitis and an inflammatory polyradiculoneuropathy caused by cytomegalovirus (CMV), HSV, varicella-zoster virus (VZV) or Epstein-Barr virus (EBV), EBV associated primary B-cell-lymphoma of the brain, acute aseptic meningitis in young adults allied with VZV, and meningoencephalitis with recurrent seizures due to human herpes virus type 6 (HHV-6). In AIDS patients, PCR has helped to differentiate lesions either due to the human immunodeficiency virus (HIV) itself or to opportunistic infections such as progressive multifocal leukoencephalopathy (PML) caused by JC virus (JCV) or CMV related complications. HIV can be detected early in the course of infection in the CSF and the amount of proviral DNA in CSF cells seems to be correlated with the severity and/or progression of neurological signs and symptoms. Acute epidemic aseptic meningitis caused by enterovirus infections can now be reliably diagnosed and typed by reverse transcriptase PCR (RT-PCR). Meningitis cases caused by vaccination with the Jeryl Lynn and Urabe vaccine strain of mumps virus have been identified using RT-PCR and sequencing of the amplified products (amplicon).
Central Nervous System↗
Expression and characterization of the reverse transcriptase enzyme from type 1 human immunodeficiency virus using different baculoviral vector systems.
To produce the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) in amounts required to study its structure and function, the p66 enzyme subunit was expressed using two different baculovirus vectors in Sf158 insect host cells. Both vectors permitted an efficient HIV-1 RT expression. The resulting products were purified up to 90% homogeneity, characterized, and investigated for their susceptibility to digestion with various proteases. The recombinant baculoviral RT obtained with the pAc373 expression vector was purified as a p66/p60 heterodimer. The recombinant His-RT was expressed with the pBlueBacHis vector. Thereby, the protein was tagged with an N-terminal hexahistidine peptide and it was purified as a p70/p70 homodimer. The two enzymes differed in their specific activity, kinetic properties, and in vitro activation by viral and non-viral proteases. The recombinant His-RT exhibited a lower specific activity than the recombinant RT. The latter yielded enzyme activities as high as an Escherichia coli-expressed RT. Removal of the hexahistidine tag from the recombinant His-RT by digestion with enterokinase resulted in a complete loss of enzyme activity. Thus, the hexahistidine tag might be an intrinsic part of the active recombinant His-RT.
Animals↗
Follicular dendritic cells productively infected with immunodeficiency viruses transmit infection to T cells.
Lymphoid organs have been proposed to function as the major reservoir for the human immunodeficiency virus type 1 (HIV-1). Within lymphatic tissues germinal centers represent foci of rapidly proliferating B cells governed by the interaction between B and T cells and follicular dendritic cells (FDC). Accumulating evidence suggests an important role of FDC in the pathophysiology of the acquired immunodeficiency syndrome. Direct proof for the infectibility of FDC with HIV-1 has been lacking until recently when we were able to demonstrate a CD4-independent infection of FDC in vitro. Here we report that in vitro HIV-1-infected human FDC do not only contain proviral DNA, but also produce the virus, and transmit the infection to T cells. Furthermore, electron microscopical studies on ex vivo isolated FDC from simian immunodeficiency virus (SIV)-infected rhesus monkeys revealed typical virus budding. In addition, FDC from SIV-infected rhesus monkeys transmitted the infection to T cells in vitro. Due to this central role within the immune response FDC may serve as preferential targets for HIV both by trapping of virions on their surfaces and by productive infection. During disease FDC become productively infected and may, thus, be regarded as crucial elements in viral dissemination.
Animals↗
Repeated exposure of rhesus macaques to low doses of simian immunodeficiency virus (SIV) did not protect them against the consequences of a high-dose SIV challenge.
As part of an in vivo titration study of the macaque simian immunodeficiency virus (SIVmac) strain 251/spl, macaques were inoculated intravenously with various dilutions of this infectious SIVmac. Seven animals received dilutions from 10(-3) to 10(-6) of SIVmac251/spl. Two monkeys infected with the 10(-3) dilution of SIVmac exhibited a productive infection as indicated by seroconversion, detection of genomic RNA and proviral DNA and positive virus isolation. These animals showed a cytotoxic T cell (CTL) response against different SIVmac proteins without any measurable T cell proliferation. The five macaques receiving higher virus dilutions did not seroconvert and were negative for both viral RNA and for infectious virus, although proviral DNA was detected in their peripheral blood mononuclear cells. In contrast to the animals receiving the 10(-3) virus dilution, these five silently infected monkeys developed an SIV-specific proliferative T cell response but SIV-specific CTL could not be observed. The SIV-specific T cell proliferation of the silently infected animals could be boosted by a second low-dose exposure with a 10(-4) or 10(-5) dilution of SIVmac251/spl. The virological status of the animals was not changed following this second virus inoculation. Four months later these macaques were challenged intravenously with 2 ml of a 10(-4) dilution of SIVmac251/32H containing 10 monkey ID50. After this challenge all SIV-pre-exposed animals and three naive controls became productively infected. In addition, all infected animals developed typical signs of an immunodeficiency within 6 months after infection. These observations indicate that macaques infected silently by a low-dose exposure to infectious virus generated a virus-specific cellular immune response. However, SIV-specific T cell proliferation alone could not protect the monkeys against an intravenous challenge with SIVmac and the subsequent development of AIDS-like symptoms.
Animals↗
Virus-specific helper T cell response of macaques immunized with inactivated whole SIV is not correlated with reactivity against cellular components of vaccine preparation.
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Animals↗
Specific diagnosis of progressive multifocal leukoencephalopathy by polymerase chain reaction.
Using polymerase chain reaction (PCR), 34 cerebrospinal fluid (CSF) samples from 28 patients with progressive multifocal leukoencephalopathy (PML) were analyzed. As controls, 116 samples were evaluated from 82 human immunodeficiency virus type 1 (HIV-1)-infected patients and 1 HIV-1-negative patient. Of the HIV-1-positive patients, 23 had cerebral toxoplasmosis, 10 had HIV leukoencephalopathy, and 49 had other neurologic complications. Detection of JC virus (JCV) DNA in CSF was increased 10-fold by the addition of carrier DNA before phenol-chloroform-isoamyl alcohol extraction. The primer pair JC 26/29, from the VP1/large T region, had a limit of detection of 10(5) JCV DNA molecules/100 microL. The primer pair JC 36/39, located in the large T gene region, had a 100-fold lower limit of detection. With JC 26/29, the sensitivity was 43% (12/28) and specificity was 100%. Using JC 36/39, sensitivity increased to 82% (23/28), and false-positive results were not observed. Diagnosis of PML is greatly aided by PCR analysis of CSF.
Base Sequence↗
Progressive multifocal leukoencephalopathy diagnosed by amplification of JC virus-specific DNA from cerebrospinal fluid.
OBJECTIVE: To study the diagnostic sensitivity and specificity of polymerase chain reaction (PCR) for the non-invasive diagnosis of progressive multifocal leukoencephalopathy (PML) in HIV-1-infected individuals. DESIGN: Retrospective analysis of stored cerebrospinal fluid (CSF) samples by PCR of HIV-1-infected patients. METHODS: Results of the PCR analysis of the CSF of three AIDS patients with autopsy-proven PML were compared with the results in 15 neurologically asymptomatic HIV-1-infected patients and with 15 AIDS patients with other opportunistic infections of the central nervous system (CNS). A polyclonal antiserum to simian virus 40 (SV40) cross-reacting with JC virus (JCV) late antigens was used for immunocytochemical confirmation of the diagnosis. Two different primer pairs, one taken from the VP1/large T gene and the other from the large T gene, were used to amplify JCV-specific DNA sequences from CSF. RESULTS: Five CSF samples were analysed and JCV-specific DNA found in three patients with autopsy-proven PML. No JCV-specific DNA was detected in 47 CSF samples, including serial samples from 14 of the 30 non-PML patients. The diagnosis of PML was confirmed in all three cases by immunocytochemistry. CONCLUSION: PML can be diagnosed by PCR analysis of CSF. The sensitivity and specificity of the method depends on the sensitivity of the primers used for amplification. Using a primer pair from the large T gene, JCV-specific DNA was amplified in three cases with PML as early as the day of presentation with the first neurological symptom of PML.
AIDS-Related Opportunistic Infections↗
Early helper T-cell dysfunction in simian immunodeficiency virus but not in human immunodeficiency virus type-2-infected macaques.
Both naive and vaccinated macaques acquired a virus-specific proliferative helper T-cell reactivity in response to infection with the nonpathogenic human immunodeficiency virus type 2 (HIV-2). In contrast, macaques infected with the pathogenic simian immunodeficiency virus of the macaque strain (SIVmac) did not develop a helper T-cell response. Furthermore, a vaccine-induced preexisting T-cell reactivity was abrogated after SIVmac infection in vaccine failures. These differences may reflect the different pathogenicity of the two closely related viruses.
AIDS Vaccines↗
Increasing melanoma incidence: putatively explainable by retrotransposons. Experimental contributions of the xiphophorine Gordon-Kosswig melanoma system.
The worldwide accelerating increase of neoplasia in humans is difficult to explain. We use the Xiphophorus tumor model to approach this problem by melanoma provocation with X-rays. Melanoma develops following inappropriate expression of x-erb B-conducted developmental genes and their controllers. These oncodeterminants are inherited according to Mendelian rules. We detected a new type of oncodeterminants that, following a single treatment of embryos with X-rays, generates a self-generating non-Mendelian melanoma transmission and accelerating increase of its incidence in succeeding generations (e.g., 0-->18-->33-->52%). To localize these oncodeterminants, we crossed nonirradiated fish having half of their chromosomes irradiated with nonirradiated fish having none of, half of, or all of their chromosomes irradiated. Because tumor rate and expression in the following generations correspond to the rates of treated chromosomes, we conclude that the new oncodeterminants are distributed over the chromosomes of the fish, where they may increase in the changing generations. By means of xiphophorine-specific retroviral DNA, we isolated two retrotransposons that behave hereditarily like the new transgenerational oncodeterminants. Sequence analysis revealed three ORFs flanked by LTRs containing motives of regulatory sequences typical for known retroviral and retrotransposal LTRs. Pol- and env-resembling sequences are lacking. Southern and in situ hybridization showed their multiple and repetitive nature distributed throughout the chromosomes and indications for their capability to increase in number without further treatment. Their transcripts are expressed in concert with those of most of the other known xiphophorine tumor determinants. Their expression is extremely high in cell cultures from tumorous embryos derived from ancestors treated as embryos with X-rays.
Animals↗
Variability of the env gene in cynomolgus macaques persistently infected with human immunodeficiency virus type 2 strain ben.
The sequence variability of distinct regions of the proviral env gene of human immunodeficiency virus type 2 strain ben (HIV-2ben) isolated sequentially over 3 to 4 years from six experimentally infected macaques was studied. The regions investigated were homologous to the V1, V2, V3, V4, V5, and V7 hypervariable regions identified in the env genes of HIV-1 and simian immunodeficiency virus SIVmac, respectively. In contrast to findings with HIV-1 and SIVmac, the V1- and V2-homologous regions were found to be highly conserved during the course of the HIV-2ben infection in macaques. The V3-homologous region showed a degree of variation comparable to that of HIV-1 but not of SIV. In the V4-, V5-, and V7-homologous regions, mutation hot spots were detected in most reisolates of the infected monkeys. Most of these mutations occurred during the first 10 weeks after infection. After 50 weeks, new mutations were rarely detected. At most mutation sites, a dynamic equilibrium between the mutated viral isotype and the infecting predominant wild type was present. This equilibrium might prevent an accumulation of mutations in isolates later in the course of infection.
Amino Acid Sequence↗
Immunization with virion-derived glycoprotein 130 from HIV-2 or SIV protects macaques against challenge virus grown in human or simian cells or prepared ex vivo.
We have compared in the macaque model the efficacy of the virion-derived glycoprotein of HIV-2ben (HIV-2 gp130) with that of SIVmac251/32H (SIV gp130). The latter vaccination trial was in part combined with vaccinia virus (VV) priming. Both antigen preparations induced a strong humoral, but a weak cellular, immune response. The first challenge was performed with autologous virus grown on a human T cell line. More than 50% of the monkeys immunized with HIV-2 gp130 (five of nine) and 63% of the monkeys immunized with SIV gp130 (five of eight) were protected. All such protected animals received one or two booster immunizations before they were rechallenged either with heterologous HIV-2SBL6669 grown on monkey peripheral blood mononuclear cells or with an ex vivo stock of SIVmac251/32H prepared from the spleen of an SIV-infected macaque and not passaged in vitro. Immunization with HIV-2 gp130 did not protect against the second challenge, but one animal showed limited infection as indicated by positive PCR only. Challenge of the SIV gp130-immunized monkeys with the spleen-derived virus led to infection of three animals; remarkably, one of these was only PCR positive. Two animals were completely protected. Thereby we can exclude the influence of cellular proteins on protective immunity. Priming with VV was not superior to immunization with gp130 alone. Neither at the first nor at the second challenge were the virus-specific humoral and cellular immune responses of the vaccinees clearly correlated with protection. However, neutralizing antibodies may have been important in the SIV gp130-immunized animals at first challenge.
AIDS Vaccines↗
Biochemical and immunological characterization of micellar complexes of the envelope glycoprotein of a simian immunodeficiency virus isolated from an African green monkey.
The external envelope glycoprotein gp130 of a simian immunodeficiency virus isolated from an African green monkey (SIVagmTYO-7) was purified as micellar complexes. The molecular weight of the gp130 micelles was about 700 K. On electron microscopy, the micelles appeared as spherical particles with a diameter of 15 to 20 nm. Such aggregates consisted of about 4 to 5 gp130 monomers. Hyperimmune sera raised in rabbits and rhesus monkeys against these gp130 micelles exhibited titers between 10(5) and 10(6). Such sera inhibit the CD4 binding of gp130 and neutralize SIVagmTYO-7 and SIVmac251 but not HIV-2ben.
Animals↗
Chemical modifications of aminonaphthalenesulfonic acid derivatives increase effectivity and specificity of reverse transcriptase inhibition and change mode of action of reverse transcriptase and DNA polymerase alpha inhibition.
The reverse transcriptase (RT) inhibition and the specificity of 15 aminonaphthalenesulfonic acid derivatives were examined with RT of a simian immunodeficiency virus derived from an African green monkey (SIVagmTYO-7). The two compounds with the strongest RT inhibition (NF415) or the highest specificity (NF345), together with suramin, were evaluated against polymerase alpha-primase complex from calf thymus. We have also compared the kinetics of inhibition of the viral and the cellular polymerase by these three compounds. While RT inhibition followed a mixed competitive and non-competitive mechanism, inhibition of the DNA polymerase alpha was competitive for suramin and non-competitive for NF415 and NF345. Certain structural characteristics appeared to be common for specific RT inhibitors.
Animals↗
Protection of cynomolgus macaques (Macaca fascicularis) against infection with the human immunodeficiency virus type 2 strain ben (HIV-2ben) by immunization with the virion-derived envelope glycoprotein gp130.
We have investigated the efficiency of a subunit vaccine consisting of native gp130 micelles of HIV-2ben mixed with keyhole limpet hemocyanin (KLH). Over a period of 52 weeks, nine cynomolgus monkeys (Macaca fascicularis) were immunized with seven intramuscular injections of gp130-KLH, equivalent to a total of about 1.1 mg of purified gp130 per animal. The first three applications were formulated in Freund's incomplete adjuvant. Because of the effects of Freund's incomplete adjuvant, aluminum hydroxide was used for the four subsequent immunizations. Each of the nine vaccinated animals along with six controls were challenged with 10 monkey infectious doses (MID50) of live HIV-2ben. At the time of challenge, the vaccinees had developed anti-gp130 titers ranging from 1 to 1.5 x 10(5). Four animals exhibited neutralizing antibodies. After iv challenge with 10 MID50 of HIV-2ben the nine vaccinees showed neither a secondary immune response nor a transient viremia. However, in four of the nine immunized animals proviral sequences were sporadically detected by polymerase chain reaction (PCR) and one of these four animals developed cytotoxic T lymphocytes. All six control animals developed a primary antibody response to HIV-2ben and became PCR positive. Four animals showed cytotoxic T cell activity and two developed a transient viremia. The five vaccinees with no sign of virus infection were reimmunized once and challenged with 10 MID50 of the heterologous virus HIV-2SBL-6999. Four weeks later all animals were PCR positive. A naive control animal and four of the vaccinees showed primary or secondary antibody responses and transient viremia. One of the revaccinated animals did not become viremic, and viral antibodies did not increase.
AIDS Vaccines↗
Protection of monkeys by a split vaccine against SIVmac depends upon biological properties of the challenge virus.
OBJECTIVE: To investigate the role of the anti-cellular immune response in the protection of rhesus macaques against infection with the simian immunodeficiency virus SIVmac. To determine the biological differences between SIV challenge stocks grown either on human T-cell lines or on monkey peripheral blood mononuclear cells (MPBMC). DESIGN: A protective SIVmac split vaccine was administered to rhesus macaques and their anti-, B- and T-cell response monitored. Vaccinees and controls were challenged with SIVmac grown either on human or on monkey cells. The in vivo replication rate of, and the immune response to, the two viruses was compared. METHODS: Five rhesus macaques were immunized with a total of 2 mg each of purified SIVmac251/32H grown on the human C8166 T-cell line. The antibody and proliferative T-cell responses were evaluated by enzyme-linked immunosorbent assay and T-cell proliferation assay, respectively. Four protected animals and four controls were reboosted and challenged with MPBMC-grown SIVmac251 (SIVmac251/MPBMC). Cell-free virus load was determined by titration of plasma for SIV infectivity on C8166 cells and antigen with a core antigen capture assay. RESULTS: Protection from virus challenge with C8166-grown SIVmac251/32H or SIVmac251/MPBMC did not correlate with anti-cellular antibodies or proliferative T-cell reactivities. Control animals infected with SIVmac251/MPBMC showed high persistent antigenaemia and high plasma virus titres. Both were absent in controls infected with complement C8166-grown SIVmac251/32H. Whereas the latter always seroconverted against the full panel of viral polypeptides, SIVmac251/MPBMC-infected animals showed a drastically decreased antibody response. CONCLUSIONS: Neither the antibody nor the proliferative T-cell response to SIVmac correlates with protection from virus challenge. In contrast to SIVmac251/32H grown on C8166 cells, the MPBMC-grown challenge virus SIVmac251 appears to belong to the 'rapid-high' phenotype, possibly explaining the lack of protection against this SIV.
Animals↗
Differences in the B and T cell immune response to the envelope glycoprotein 130 (gp130) of the macaque strain of simian immunodeficiency virus (SIVmac), induced by immunization of rhesus macaques with virus-derived or vaccinia virus-expressed gp130.
Rhesus macaques were immunized with purified virus-derived simian immunodeficiency virus of macaques (SIVmac) 251/32H glycoprotein 130 (gp130) or primed with recombinant vaccinia virus (VV) expressing the env gene of the SIVmac BK28 clone and boosted subsequently with virus-derived gp130. High antibody titres of at least 10(4) against recombinant gp140 were induced with both vaccines. Analysis of the antibody specificity with a peptide ELISA revealed that different linear epitopes were recognized after administration of virus-derived gp130 compared with those after priming with VV. Antibodies to some epitopes (peptides 10 and 49), which were also found in SIV-infected animals, were induced with both vaccines, whereas antibodies to other regions were induced by only one vaccine preparation. The analysis of the helper T cell response revealed a poor immunogenicity of the virus-derived gp130, whereas priming with VV induced a considerable helper T cell activity in all three vaccinees after the second VV infection. Using synthetic peptides, several epitopes were identified. Our observations show that immunization with a virus-derived gp130 or live recombinant VV induces a considerably different antibody and helper T cell response. These differences in immunogenicity might have important implications for further vaccine development.
Amino Acid Sequence↗
Characterization of monoclonal antibodies to the envelope proteins of an immunodeficiency virus of African green monkeys, SIVagmTYO-7.
Five monoclonal antibodies (mabs) specific for the envelope proteins of a simian immunodeficiency virus of African green monkeys (SIVagm) have been raised. Two mabs were directed against distinct epitopes on the transmembrane protein gp41. A conformational epitope on the gp130 was recognized by three mabs. This is the first report on mabs specific for SIVagm-gp130. Studies of the cross-reactivities revealed that the epitopes recognized by the env-directed mabs are conserved species-specifically in SIVagm isolates. Therefore, these mabs can be used to distinguish SIVagm strains from other virus groups.
Animals↗