Biomedical subjects
D D Richman
Publications and source records attributed to D D Richman.
Recombination leads to the rapid emergence of HIV-1 dually resistant mutants under selective drug pressure.
The potential contribution of recombination to the development of HIV-1 resistance to multiple drugs was investigated. Two distinct viruses, one highly resistant to a protease inhibitor (SC-52151) and the other highly resistant to zidovudine, were used to coinfect T lymphoblastoid cells in culture. The viral genotypes could be distinguished by four mutations conferring drug resistance to each drug and by other sequence differences specific for each parental virus. Progeny virions recovered from mixed infection were passaged in the presence and absence of both zidovudine and SC-52151. Dually resistant mutants emerged rapidly under selective conditions, and these viruses were genetic recombinants. These results emphasize that genetic recombination could contribute to high-level multiple-drug resistance and that this process must be considered in chemotherapeutic strategies for HIV infection.
Viral dynamics of HIV: implications for drug development and therapeutic strategies.
The ability to quantitate human immunodeficiency virus (HIV) in blood and tissues from patients at all stages of disease has provided new insights into the pathogenesis of HIV disease. There is a dynamic equilibrium between HIV production and clearance even during the period of clinical latency, which may permit resistant virus to emerge with the imposition of drug pressure. Disruption of the equilibrium with effective drugs reduces circulating levels of HIV within 1 week, thus allowing the rapid assessment of new candidate drugs. To maximize the magnitude and durability of HIV RNA suppression, therapeutic strategies must be implemented that are effective against high levels of rapidly replicating virus that consist of many genetic variants.
HIV-induced apoptosis requires the CD4 receptor cytoplasmic tail and is accelerated by interaction of CD4 with p56lck.
The roles of the CD4 receptor and the src kinase p56lck were examined in the process of HIV-induced apoptosis of CD4+ T lymphocytes. The presence of the CD4 cytoplasmic tail was found to be essential in delivering an apoptotic signal, and interaction of CD4 with p56lck potentiated HIV-induced apoptosis. Apoptosis, but not HIV replication, was abrogated by deleting the NH2-terminal intracytoplasmic tail of CD4, or by mutating the two critical cysteines in this tail that are responsible for CD4-p56lck interaction. Introduction of p56lck in C8166-45 or MT-2 cells, CD4+ T cell lines deficient for this protein, greatly increased HIV-induced apoptosis and syncytium formation. The ability of p56lck to deliver an apoptotic signal did not depend on its kinase function, since a kinase-deficient mutant was as effective as its normal counterpart in inducing apoptosis, suggesting that p56lck may act as an adapter to anchor other proteins to transduce the death signal.
Antiretroviral drug resistance: mechanisms, pathogenesis, clinical significance.
The contribution of HIV drug resistance to treatment failure, the relationship of drug resistance to pathogenesis and the impact of resistance in the role of the promising new class of protease inhibitors remain areas of active investigation. A more precise understanding of these aspects of antiretroviral drug resistance will permit the more effective use of available drugs and the design of new drugs and drug regimens.
Consensus symposium on combined antiviral therapy. International Society for Antiviral Research and the National Institutes of Allergy and Infectious Diseases.
Explore the source record for details and available documents.
The implications of drug resistance for strategies of combination antiviral chemotherapy.
Explore the source record for details and available documents.
Lipid prodrugs of phosphonoacids: greatly enhanced antiviral activity of 1-O-octadecyl-sn-glycero-3-phosphonoformate in HIV-1, HSV-1 and HCMV-infected cells, in vitro.
Phosphonoformate (PFA) effectively inhibits viral polymerases but is relatively ineffective in virus-infected cells in tissue culture. A lipid prodrug of phosphonoformate was synthesized by coupling the phosphonate residue of phosphonoformate to the sn-3 hydroxyl of 1-O-octadecyl-sn-glycerol. This prodrug, 1-O-octadecyl-sn-glycero-3-phosphonoformate (ODG-PFA), was 93-fold more active than phosphonoformate in cells infected with the AD169 strain of cytomegalovirus (CMV), and 111-147-fold more active in cells infected with three human clinical isolates of CMV. The compound was also 44-fold more active in human immunodeficiency virus-1 (HIV-1) infected cells and 43-fold more active in cells infected with herpes simplex virus (HSV). Studies of the mechanisms of increased antiviral activity indicate that 1-O-octadecyl-sn-glycero-3-[14C]phosphonoformate is taken up more extensively than the free drug by the host MRC-5 human lung fibroblasts. Intracellular enzymes convert 1-O-octadecyl-sn-glycero-3-phosphonoformate to phosphonoformate. This conversion does not occur in the tissue culture medium containing fetal bovine serum (FBS) or in MRC-5-conditioned medium. In view of its greatly increased in vitro potency and selectivity, 1-O-octadecyl-sn-glycero-3-phosphonoformate may be useful in treating viral diseases.
HIV viral load markers in clinical practice.
Plasma HIV RNA determinations are an important prognostic marker of disease progression and, when used appropriately, provide a valuable tool for the management of individual patients. But what constitutes appropriate use?
Extensive polymorphisms observed in HIV-1 clade B protease gene using high-density oligonucleotide arrays.
Naturally occurring mutations in HIV-1-infected patients have important implications for therapy and the outcome of clinical studies. However, little is known about the prevalence of mutations that confer resistance to HIV-1 protease inhibitors in isolates derived from patients naive for such inhibitors. In the first clinical application of high-density oligonucleotide array sequencing, the sequences of 167 viral isolates from 102 patients have been determined. The DNA sequence of USA HIV-1 clade B proteases was found to be extremely variable and 47.5% of the 99 amino acid positions varied. This level of amino acid diversity is greater than that previously known for all worldwide HIV-1 clades combined (40%). Many of the amino acid changes that are known to contribute to drug resistance occurred as natural polymorphisms in isolates from patients who had never received protease inhibitors.
Prognostic value of plasma human immunodeficiency virus type 1 (HIV-1) RNA levels in patients with advanced HIV-1 disease and with little or no prior zidovudine therapy. AIDS Clinical Trials Group Protocol 116A/116B/117 Team.
The association of plasma human immunodeficiency virus type 1 (HIV-1) RNA level at study entry and over time with clinical progression was evaluated in 187 patients from AIDS Clinical Trials Group protocol 116A who had little or no prior zidovudine treatment. Three-fold-higher HIV-1 RNA levels at study entry and 3-fold increases by week 8 were associated with progression (relative hazard [RH], 1.67; 95% confidence limits [CL], 1.20, 2.32; and RH, 1.45; CL, 1.02, 2.05, respectively). Having 3-fold-higher CD4 cell count at entry was independently associated with a 52% reduction in risk for progression (adjusted RH, 0.48; CL, 0.33, 0.70). When stratified by length of prior zidovudine therapy, RNA level was predictive in drug-naive patients (adjusted RH, 1.87; CL, 1.23, 2.85) but not predictive in patients with up to 16 weeks of prior therapy (adjusted RH, 1.11; CL, 0.70, 1.76). Analysis suggests that the acquisition of mutations at HIV-1 reverse transcriptase codons 215 and 74 is associated with subsequent increases in HIV-1 RNA level (relative risk, 7.00; CL, 0.86, 56.90).
Association of plasma human immunodeficiency virus type 1 RNA level with risk of clinical progression in patients with advanced infection. AIDS Clinical Trials Group (ACTG) 116B/117 Study Team. ACTG Virology Committee Resistance and HIV-1 RNA Working Groups.
Human immunodeficiency virus (HIV)-1 RNA level in plasma was evaluated as a surrogate marker for disease progression in a clinical trial of advanced HIV-1 infection. Baseline HIV-1 RNA level was an independent predictor of disease progression (relative hazard [RH] for each doubling of HIV-1 RNA level, 1.26; 95% confidence interval [CI], 1.03-1.54; P = .02), after adjusting for the week 4 change in HIV-1 RNA level, baseline CD4 cell count, syncytium-inducing phenotype, clinical status at study entry, and therapy randomization. A 50% reduction in HIV-1 RNA level was associated with a 27% decrease in the adjusted risk of disease progression during the study (RH, 0.73; 95% CI, 0.52-1.02; P = .07). The partial validation of HIV-1 RNA as a predictor for clinical end points has implications for the use of HIV-1 RNA in clinical trials and practice.
Kinetic analysis of intravirion reverse transcription in the blood plasma of human immunodeficiency virus type 1-infected individuals: direct assessment of resistance to reverse transcriptase inhibitors in vivo.
Intravirion reverse transcripts have been identified in the blood plasma of human immunodeficiency virus type 1 (HIV-1)-infected individuals. In the present studies, the kinetic processes of intravirion HIV-1 reverse transcription, in the blood plasma of HIV-1-infected persons treated with nevirapine, were investigated. Nevirapine is a nonnucleoside inhibitor of reverse transcriptase (RT) which decreases the level of HIV-1 viral particles in the blood plasma of infected individuals. By analyzing HIV-1 virions at different time points prior to and after initiation of nevirapine therapy in vivo, the levels of intravirion reverse transcripts have been demonstrated to be dramatically susceptible to this anti-RT agent, out of proportion to effects on plasma virion load. The intravirion reverse transcripts were also documented to rebound to the pretreatment levels, concomitant with the development of resistant viral mutants. In addition, the infectivity of HIV-1 virions dramatically decreased after nevirapine treatment, further indicating that the effects of this anti-RT agent begin within the cell-free virions. Since the levels of intravirion reverse transcripts were altered according to the susceptibility or resistance of the HIV-1 RT enzyme to this inhibitor, these data demonstrate that the formation of intravirion reverse transcripts is a dynamic process in vivo. Moreover, because the alteration in ratios between intravirion HIV-1 reverse transcripts and viral genomic RNA directly reflects the efficiency of reverse transcription, we propose that the determination of these ratios in the blood plasma of HIV-1-positive patients may be a useful and, most importantly, a direct assay to monitor the efficacy of anti-RT agents in vivo.
Nevirapine-resistant human immunodeficiency virus: kinetics of replication and estimated prevalence in untreated patients.
The nonnucleoside reverse transcriptase inhibitor nevirapine rapidly selects for mutant human immunodeficiency virus (HIV) in vivo. The most common mutation occurs at amino acid residue 181 in patients receiving monotherapy. After the initiation of nevirapine therapy, plasma and peripheral blood mononuclear cell samples were collected at frequent intervals and assayed for HIV RNA levels and the proportion of virus containing a mutation at residue 181. HIV RNA levels remained stable for the first 24 h after initiation of therapy and rapidly declined between 1 and 7 days. There was a consistent maximum decrease of 2 log10 HIV RNA copies per ml of plasma (range, 1.96 to 2.43) from baseline after 2 weeks in all monotherapy subjects. The estimated median half-life of HIV RNA was 1.11 days (range, 0.63 to 1.61). After 14 days of therapy, HIV RNA levels began to increase and 181 mutant virus was detected. The estimated doubling time of the emerging virus population ranged from 1.80 to 5.73 days. Viral DNA in peripheral blood mononuclear cells turned over from wild type to the mutant with a mutation at residue 181 significantly more slowly than did HIV RNA in plasma. In two subjects, the calculated prevalence of the 181 mutant virus prior to treatment was 7 and 133 per 10,000 copies of plasma HIV RNA.
Producer-cell modification of human immunodeficiency virus type 1: Nef is a virion protein.
Type 1 human immunodeficiency viruses encoding mutated nef reading frames are 10- to 30-fold less infectious than are isogenic viruses in which the nef gene is intact. This defect in infectivity causes nef-negative viruses to grow at an attenuated rate in vitro. To investigate the mechanism of Nef-mediated enhancement of viral growth rate and infectivity, a complementation analysis of nef mutant viruses was performed. To provide Nef in trans upon viral infection, a CEM derivative cell line (designated CLN) that expresses Nef under the control of the viral long terminal repeat was constructed. When nef-negative virus was grown in CLN cells, its growth rate was restored to wild-type levels. However, the output of nef-negative virus during the first 72 h after infection of CLN cells was not restored, suggesting that provision of Nef within the newly infected cell does not enhance the productivity of a nef-negative provirus. The genetically nef-negative virions produced by the CLN cells, however, were restored to wild-type levels of infectivity as measured in a syncytium formation assay in which CD4-expressing HeLa cells were targets. These trans-complemented, genetically nef-negative virions yielded wild-type levels of viral output following a single cycle of replication in primary CD4 T cells as well as in parental CEM cells. To define the determinants for producer cell modification of virions by Nef, the role of myristoylation was investigated. Virus that encodes a myristoylation-negative nef was as impaired in infectivity as was virus encoding a deleted nef gene. Because myristoylation is required for both membrane association of Nef and optimal viral infectivity, the possibility that Nef protein is included in the virion was investigated. Wild-type virions were purified by filtration and exclusion chromatography. A Western blot (immunoblot) of the eluate fractions revealed a correlation between peak Nef signal and peak levels of p24 antigen. Although virion-associated Nef was detected in part as the 27-kDa full-length protein, the majority of immunoreactive protein was detected as a 20-kDa isoform. nef-negative virus lacked both 27- and 20-kDa immunoreactive species. Production of wild-type virions in the presence of a specific inhibitor of the human immunodeficiency virus type 1 protease resulted in virions which contained only 27-kDa full-length Nef protein. These data indicate that Nef is a virion protein which is processed by the viral protease into a 20-kDa isoform within the virion particle.
Antiretroviral therapy and clinical trials: what now?
Explore the source record for details and available documents.
Can HIV infection be cured?
Explore the source record for details and available documents.
The impact of zidovudine compared with didanosine on health status and functioning in persons with advanced HIV infection and a varying duration of prior zidovudine therapy. AIDS Clinical Trials Group 116/117 Study Group.
The objective of this study was to compare the effects of zidovudine and didanosine on health-related quality of life in persons with advanced HIV infection and varying duration of prior zidovudine exposure. It was designed as a substudy nested in two similar placebo-controlled active-control-arm randomized trials, using sites of the AIDS Clinical Trials Group participating in the randomized trials of zidovudine versus didanosine (ACTG 116 and 117). The patients comprised 356 participants enrolled in ACTG 116 and 117. All had HIV infection and either a CD4 count of <200 cells/mm3, or a CD4 count of <300 cells/mm3 plus symptoms of HIV disease. Participants were randomized equally within strata defined by duration of prior zidovudine therapy, to receive didanosine sachets at a dose of 500 mg daily (334 mg in subjects weighing <60 kg) or 750 mg daily (500 mg in subjects weighting <60 kg) plus inactive capsules resembling zidovudine, or to receive zidovudine capsules at a dose of 600 mg daily plus inactive sachets resembling didanosine. The main outcome measures were self-reported health-related quality of life, healthcare utilization, disability, work and symptom impact. The results showed no differences in reported symptom impact or healthcare utilization, and most measures of disability were similar. In the group with more than 8 weeks of prior zidovudine therapy, several of the health status scale scores for ongoing participants were significantly better for didanosine recipients, but average differences were small. Use of several different approaches to combining health status and survival showed no differences in the overall quality-time experiences between the treatment groups. Individuals taking zidovudine, low-dose didanosine and high-dose didanosine experienced 33, 34 and 35 weeks, respectively, in at least the typical health state if they had fewer than 8 weeks of previous zidovudine therapy, and had 23, 23 and 26 weeks, respectively, if they had more than 8 weeks previous use of zidovudine. Results did not differ when data were analysed within strata ofpatients who had any versus no prior exposure to zidovudine, or AIDS versus non-AIDS status. In conclusion, functional status and health-related quality of life were substantially similar among persons receiving either zidovudine or didanosine, regardless of the duration of prior zidovudine treatment.