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Biomedical subjects

R Kurth

Publications and source records attributed to R Kurth.

At least 19 recordsLinked to original sources
PubMed1992

Development of a quasispecies of human immunodeficiency virus type 1 in vivo.

During treatment with one specific batch of blood clotting factor IX, a number of hemophilia B patients in Germany recently became infected with human immunodeficiency virus type 1 (HIV-1). The nucleotide sequences of cloned HIV-1 envelope gene regions including the variable V3 loop and the V4 region derived from short-term virus cultures and directly from peripheral blood cells of these patients were shown to be highly homologous. Based on the assumption that the corresponding consensus sequence (termed HIV-1MBK) was identical to the genotype of the initially infecting virus, we were able to construct phylogenetic trees of the developing quasispecies in two patients studied in detail. True intermediates between input and multiply mutated genotypes were found in individual blood samples. Except for the initially infecting variant HIV-1MBK, variants found at 11 months postinfection had replaced those seen at 5 months postinfection. Variability early after infection was shown to cluster in two small regions located 3' of the V3 loop (i.e., outside the loop) and within the V4 region. This communication therefore describes the evolution of an HIV-1 quasispecies in humans starting from a single genotype.

Amino Acid Sequence↗
PubMed1992

AIDS vaccine: present problems and future perspectives.

Vaccination has proved to be an effective means for the prevention of infectious diseases. Advances in our understanding of the human immunodeficiency virus (HIV) and the immune system of the host may lay the foundation for the development of an AIDS vaccine. Current attempts to develop vaccines focus on the development of substances that will produce a different type of immune response from that which occurs naturally. Progress has been made in understanding the mechanisms by which the AIDS virus stimulates an neutralizing antibody response and triggers specific cytotoxic T lymphocytes in the host. The pressing need for a vaccine has prompted the testing of several candidate vaccines based on the simian immunodeficiency virus (closely related to HIV) in the macaque animal model for AIDS. The lessons learned from these trials will be valuable for developing future vaccines.

AIDS Vaccines↗
PubMed1992

Vaccination against HIV.

The spread of AIDS progresses unrelentingly despite all efforts of public education and the only real hope of epidemiological control lies in the development of an effective vaccine. The very nature of the AIDS virus (HIV) and the manner with which it interacts with the host makes development of a practical vaccine very difficult. Recent successes using whole inactivated virus as immunogen in the SIVmac animal model system now show that it is possible to protect against infection. However, due to supposed limited efficacy in the field of such a vaccine and the fact that large scale production and administration of a multi-shot whole HIV-based product would be technically impossible, it is now important to identify which of the proteins are responsible and which immune response is protective. With this knowledge a recombinant or synthetic vaccine could be produced on a large scale. However, despite recent protection of a few chimpanzees against HIV infection itself there is currently no proven effective and practical vaccine even in the laboratory and it will be many years before such a product is available to the general public.

Animals↗
PubMed1992

Quantitation of a lentivirus in its natural host: simian immunodeficiency virus in African green monkeys.

We have examined the viral load in the peripheral blood of simian immunodeficiency virus (SIV)-infected African green monkeys with a view to the unexplained apathogenicity of African green monkey SIV (SIVagm) in its natural host. By using polymerase chain reaction, viral DNA was detected in fresh peripheral blood mononuclear cells (PBMC) of each of nine seropositive animals. The virus DNA load was variable among the monkeys tested, ranging from 5 to 50 (mean = 15) copies per 10(5) PBMC, which is comparable to that of human immunodeficiency virus type 1 (HIV-1) in humans. The level of infectious SIVagm in PBMC was measured by endpoint dilution cultures. SIVagm was recovered from PBMC from 14 of 17 antibody-positive monkeys (82%), and the mean SIVagm titer in PBMC of seropositive African green monkeys was 10 tissue culture infectious doses per 10(6) cells, similar to the titer shown for HIV in asymptomatic carriers. Free infectious virus was isolated from the plasma of 4 of 17 monkeys (24%), and SIVagm expression in peripheral blood in vivo, as demonstrated by in situ hybridization, was detectable only in those animals which were viremic. SIVagm replication is therefore not totally suppressed in vivo, and SIVagm has a viral load equivalent to that seen for HIV-1 in asymptomatic humans.

Animals↗
PubMed1992

B-cell epitope mapping of the entire SIVagm envelope glycoprotein including fine mapping of immunogenic regions.

To allow the precise definition of the anti-lentiviral immune response in the natural host and to facilitate the development of an alternative animal model for vaccine development, we are identifying the immunogenic domains of SIVagm proteins. First, a total of 173 synthetic 15-mer peptides with an overlap of 10 amino acids were produced spanning the entire envelope glycoprotein of the molecular clone SIVagm3. These peptides were used as antigen in enzyme-linked immunosorbent assays for identifying regions recognized by antibodies from naturally infected African green monkeys and monkeys infected with a molecular clone. Regions corresponding to the HIV-1 V3, the transmembrane protein (TMP) "Gnann peptide", and the C-terminal area of the outer envelope protein were shown to be immunodominant. These regions were re-synthesized as 15-mer peptides with an overlap of 14 amino acids and used to precisely map the epitopes recognized. Sera from 93 captive and 61 wild animals were tested by SIVagm-specific Western blot (WB) and for ELISA reactivity against the immunodominant TMP peptide. One hundred percent (76/76) of the WB-positive captive animals and 98% (41/42) wild WB-positive animals also reacted against the peptide. In contrast, only 62% of the WB-positive sera reacted with the "V3" epitope and 46% with the gp130 C-terminal epitope.

Amino Acid Sequence↗
PubMed1992

Vaccine protection against SIVmac infection by high- but not low-dose whole inactivated virus immunogen.

Eight rhesus macaques were immunized intramuscularly four times (0, 1, 2, and 4 months) over a period of 4 months with a formalin-inactivated whole Simian immunodeficiency virus (SIV) vaccine in the presence of muramyl dipeptide (MDP) as adjuvant. Four animals received 0.5 mg and the other four received 0.1 mg immunogen per injection. Three weeks after the final immunization, the vaccinated monkeys along with two control monkeys were challenged intravenously with 50 MID50 of SIVmac251-32H. At the time of challenge, one monkey from the high dose group had a high titer (1:761) of antibody able to neutralize in vitro the homologous 32H strain of SIV mac. All other animals had lower but measurable titers (1:57-1:453), although there was no correlation between the levels of neutralizing antibody and subsequent protection. Upon challenge, three of four animals from the low dose group (plus the nonvaccinated control animals) became infected as demonstrated by reisolation of virus from peripheral blood mononuclear cells, by SIVmac-specific polymerase chain reaction, and by the development of a strong anamnestic response. In the sera from these animals the titer of neutralizing antibody rose to over 1:5,100. All other animals (one from low dose group and all four of the high dose group) remained negative by all parameters at 4 months post challenge. These data indicate that when used in conjunction with MDP, the amount of immunogen required per immunization for protection against challenge is between 0.1 and 0.5 mg.

Animals↗
PubMed1991

Development of vivo of genetic variability of simian immunodeficiency virus.

Rapid development of genetic variability may contribute to the pathogenicity of lentiviruses as it may allow escape from immune surveillance and/or from suppression of virus replication. Although apathogenic in African green monkeys, simian immunodeficiency virus isolated from African green monkeys is shown to display extensive genetic variability and defectiveness in the V1- and V2-like variable domains of the external envelope protein comparable to that known for human immunodeficiency virus. However, in contrast to the situation in human immunodeficiency virus-infected individuals, a predominant major virus variant was detected neither in a monkey naturally infected for more than 10 years nor in two monkeys infected with a molecular virus clone for 15-20 months. Extensive variability evolves from a single genotype with a maximal rate of 7.7 mutations per 1000 nucleotides per year. A remarkable selection for nonsynonymous mutations that accounts for 92% of all changes indicates continuous selection of variants.

Amino Acid Sequence↗
PubMed1991

Fidelity of reverse transcriptase of the simian immunodeficiency virus from African green monkey.

The in vitro fidelity of highly purified recombinant reverse transcriptase from simian immunodeficiency virus of African green monkeys (SIVagm) was determined. By using the phi X174am16 reversion assay an overall error rate of 1/19,000 was determined. This is 2.4-fold higher than the overall accuracy of purified recombinant HIV-1 reverse transcriptase, measured in parallel. The evaluation of error frequencies from nucleotide pool bias studies suggest an even higher accuracy for the SIVagm-derived reverse transcriptase. T:dGMP mismatches were formed most frequently with an error rate of 1/155,000, followed by G:dGMP (1/230,000), A:dGMP (1/315,000), G:dAMP (1/340,000), T:dCMP (1/540,000), T:dTMP (1/790,000), and A:dCMP (1/1,050,000) mispairs. Thus, according to pool bias effects and depending on the mismatch under consideration SIVagm reverse transcriptase appears to be 2 to 20-fold more accurate than the homologous enzyme from the human immunodeficiency virus type 1. This higher accuracy is not due to a co-purifying exonuclaease activity. Like the enzyme from HIV-1, the simian monkey-derived enzyme was found to be devoid of a proofreading 3' to 5' exonuclease.

Animals↗
PubMed1991

Neutralizing antibodies and antigens in AIDS.

Until recently, much of the effort put into development of an AIDS vaccine has focussed on the elicitation of a neutralizing antibody response. The viral target of neutralization, HIV envelope glycoprotein, has been produced in bulk through recombinant techniques, but has had little success as a vaccine. The specific epitopes to which neutralizing antibodies bind have been mapped, and although the major epitope is hypervariable, others are conserved. This allows the design of second generation vaccines. Meanwhile, vaccine studies in the SIV animal model simply using inactivated virus as immunogen have demonstrated that an effective vaccine is at least possible. A variety of HIV vaccine preparations are now under investigation and the outlook for the future is promising.

Acquired Immunodeficiency Syndrome↗
PubMed1991

The quest for an AIDS vaccine: the state of the art and current challenges.

Despite intense efforts worldwide, using state-of-the-art methods and techniques and despite ever-increasing knowledge about the molecular and structural make-up of HIV, a practical vaccine against acquired immunodeficiency syndrome (AIDS) has yet to be developed. The increasing use of recombinant DNA techniques and synthetic peptide technology has allowed many groups to identify at the epitope level the regions of HIV proteins which act as targets for (and stimulate) the immune response. Epitopes which stimulate and bind neutralizing antibodies have been examined in detail and an ever-increasing number of antibody-dependent cellular cytotoxicity (ADCC) and cytotoxic T lymphocytes (CTL) epitopes are being defined, as are potentially harmful (immunosuppressive or enhancing) domains. It still is not clear which of the different immune responses (or combinations thereof) it will be necessary to stimulate in order to protect from infection. Infected humans develop neutralizing antibodies, ADCC-inducing antibodies and CTL responses against a variety of viral proteins but it is not known which of these can control or prevent infection in vivo. The extensive knowledge of HIV and the immune response it elicits is being used to design and produce a wide variety of putative vaccines, ranging from whole inactivated virus, through recombinant organisms/proteins, to synthetic peptides although each has its inherent advantages and disadvantages. The very nature of HIV makes vaccine development difficult at best. However, recent successes using whole inactivated virus or virus-infected cells in the macaque simian immunodeficiency virus (SIVmac) model system at least show that protection against lethal lentivirus infection can be achieved.(ABSTRACT TRUNCATED AT 250 WORDS)

Acquired Immunodeficiency Syndrome↗
PubMed1991

Inhibition of HIV-1 replication and NF-kappa B activity by cysteine and cysteine derivatives.

HIV-1 proviral DNA contains two binding sites for the transcription factor NF-kappa B. HIV-1-infected individuals have, on average, abnormally high levels of tumour necrosis factor alpha (TNF alpha) and abnormally low plasma cysteine levels. We therefore investigated the effects of cysteine and related thiols on HIV-1 replication and NF-kappa B expression. The experiments in this report show that cysteine or N-acetylcysteine (NAC) raise the intracellular glutathione (GSH) level and inhibit HIV-1 replication in persistently infected Molt-4 and U937 cells. However, inhibition of HIV-1 replication appears not to be directly correlated with GSH levels. Cysteine and NAC also inhibit NF-kappa B activity as determined by electrophoretic mobility shift assays and chloramphenicol acetyl-transferase (CAT) gene expression under control of NF-kappa B binding sites in uninfected cells. This suggests that the cysteine deficiency in HIV-1-infected individuals may cause an over-expression of NF-kappa B-dependent genes and enhance HIV-1 replication. NAC may be considered for the treatment of HIV-1-infected individuals.

Acetylcysteine↗
PubMed1991

Lack of HIV-1 V3 region sequence diversity in two haemophiliac patients infected with a putative biologic clone of HIV-1.

Peripheral blood mononuclear cells (PBMC) from two haemophilia B patients, who presumably became infected with a putative HIV-1 biological clone following treatment with the same suspected batch of commercial factor, were used to clone and sequence the hypervariable V3 region of the HIV-1 envelope protein. In 10 of 12 clones the V3 region was identical and two (one from each patient) had a single non-synonymous point mutation. Viable reisolates (shown to be authentic by sequencing of V3) currently appear to be monocyte tropic. These results strongly indicate that the patients were infected from a common source with a very low number of infectious particles and indicate that variability under these conditions is limited.

Amino Acid Sequence↗
PubMed1991

Mutational analysis of the simian immunodeficiency virus SIVmac nef gene.

We are using site-directed mutagenesis of single viral genes to identify and analyze the genetic determinants of human and simian immunodeficiency virus pathogenicity. In a first approach, we have constructed a series of simian immunodeficiency virus SIVmac nef mutants by partial deletion and insertions in the nef gene, as this gene is a candidate gene for the establishment and maintenance of latency. nef insertion mutants replicated faster than wild-type SIVmac, suggesting that the nef gene product acts as a negative factor for replication. Surface phenotyping revealed that cultures permanently infected with nef mutants exhibit an enhanced expression of viral proteins on the outer cell surface. We have analyzed the properties of the mutant viruses in cell culture and intend to use rapidly replicating mutants (putatively unable to undergo latency) as model vaccine viruses in the rhesus monkey.

Base Sequence↗
PubMed1991

Epidemiology and pathogenicity of human retroviruses.

The human retroviruses can be divided into oncovirus (HTLV-I and HTLV-II) and lentivirus strains (HIV-1 and -2). The HTLVs are endemic in Central Africa, the Caribbean Islands and in southwest Japan, but now tend to spread through the i.v.-drug user population in the USA and in some countries in Western Europe. HTLV infection is associated with a malignant form of adult T-cell leukemia, tropical spastic paraparesis (TSP) and an associated myelopathy (HAM). The pathogenic mechanisms of HTLV are as yet poorly understood. HIV infection is spreading rapidly almost world-wide and has reached epidemic proportions in Central Africa, parts of South America and in certain populations in industrialized countries that have risk behaviour for contracting venereal or blood-borne infections. The mechanisms of HIV-induced immune suppression are still not entirely clear, as direct T-lymphocyte destruction after viral infection cannot account for the almost complete loss of CD4 T-cells in the final stages of disease. Various indirect mechanisms of HIV-induced immune cell destruction are outlined below.

Biological Products↗
PubMed1990

Demonstration of cross-reactive antibodies able to elicit lysis of both HIV-1- and HIV-2-infected cells.

A total of 100% of sera from a large number of HIV-1-infected patients contained antibodies able to elicit Antibody-dependent cellular cytotoxicity lysis of cells infected with the HIV-1 isolates IIIB or RF. Levels of activity could not be correlated with activities in ELISA or neutralizing antibody assays nor with the clinical status of the patients. Surprisingly, 8 of 156 patients sera could additionally elicit lysis of HIV-2-infected cells, and cold target competition assays demonstrated that the cross-reactivity was apparently mediated via recognition of common epitope(s) expressed on the surface of cells infected with either group of HIV. The ADCC mechanism was shown to be mediated by a CD16+ lymphocyte. This demonstration of an effector mechanism able to attack and eliminate cells infected with a wide range of HIV strains has obvious implications for development of putative vaccines.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1990

Simian immunodeficiency virus reverse transcriptase. Purification and partial characterization.

Native reverse transcriptase from simian immunodeficiency virus was purified from virus with good recovery to near homogeneity. The optimum reaction conditions of the enzyme were determined with respect to divalent cations, pH and ionic strength. The enzyme was shown to possess both RNA-dependent and DNA-dependent DNA synthesis activity. In addition, we could demonstrate an associated RNase H activity. Employing novel assay conditions, activated DNA as a heteropolymeric substrate was used more efficiently than the homopolymeric substrate poly(rA).oligo(dT) which in turn was used twofold more effectively as the template primer than poly(dC).oligo(dG). Other homopolymeric substrates, including poly(rC).oligo(dG), were also tested but were found to be poorly used by the reverse transcriptase. The Miachaelis-Menten constants were determined for each of the four nucleotides needed to elongate a natural template primer. Simultaneously, using dideoxyadenosine triphosphate as nucleotide analogue, we could show that this compound acts as a competitive inhibitor with respect to dATP, whereas it acts as a non-competitive inhibitor with respect to the other nucleotides. Gel electrophoretic analysis showed the enzyme to consist of two polypeptides with apparent molecular masses of 64 and 48 kDa. Using activity gel electrophoresis, we were able to demonstrate that both subunits exhibit DNA synthesis activity.

Animals↗