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PubMed2001

Selection and fading of resistance mutations in women and infants receiving nevirapine to prevent HIV-1 vertical transmission (HIVNET 012).

OBJECTIVE: To examine the emergence and fading of NVP resistance (NVP(R)) mutations in HIV-1-infected Ugandan women and infants who received single dose NVP to prevent HIV-1 vertical transmission. DESIGN: We examined NVP(R) in women and infants who received NVP in the HIVNET 012 clinical trial, including 41 out of 48 women with infected infants, 70 randomly-selected women with uninfected infants, and 33 out of 49 infected infants. METHODS: Plasma HIV-1 was analyzed using the Applied Biosystems ViroSeq HIV-1 Genotyping System. RESULTS: NVP(R) mutations were detected in 21 out of 111 (19%) women tested 6-8 weeks after delivery. The rate of NVP(R) was similar among women whose infants were or were not HIV-1 infected. K103N was the most common mutation detected. NVP(R) mutations faded from detection within 12-24 months in all 11 evaluable women. High baseline viral load and low baseline CD4 cell count were associated with development of NVP(R). NVP(R) mutations were detected in 11 out of 24 (46%) evaluable infants who were infected by 6-8 weeks of age. The most common NVP(R) mutation detected in infants was Y181C. Those mutations faded from detection by 12 months of age in all seven evaluable infants. Of nine evaluable infants with late HIV-1 infection, only one had evidence of NVP(R). CONCLUSIONS: NVP(R) was detected more frequently in infants than women following NVP prophylaxis, and different patterns of NVP(R) mutations were detected in women versus infants. NVP(R) was detected infrequently in infants with late HIV-1 infection. NVP-resistant HIV-1 faded from detection in women and infants over time.

Anti-HIV Agents↗
PubMed2000

Antiretroviral therapy during primary immunodeficiency virus infection can induce persistent suppression of virus load and protection from heterologous challenge in rhesus macaques.

A limited period of chemotherapy during primary immunodeficiency virus infection might provide a long-term clinical benefit even if treatment is initiated at a time point when virus is already detectable in plasma. To evaluate this strategy, we infected rhesus macaques with the pathogenic simian/human immunodeficiency virus RT-SHIV and treated them with the antiretroviral drug (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA) for 8 weeks starting 7 or 14 days postinfection. PMPA treatment suppressed viral replication efficiently in all of the monkeys. After chemotherapy ended, virus replication rebounded and viral RNA in plasma reached levels comparable to that of the controls in four of the six monkeys. However, in the other two animals, virus loads peaked only moderately after withdrawal of the drug and then declined to low or even undetectable levels. These low levels of viremia remained stable for at least 31 weeks after cessation of therapy. At this time point, these two monkeys were challenged with SIV(8980) to evaluate whether the host responses which were able to keep RT-SHIV replication under control were also sufficient to protect against infection with a highly pathogenic heterologous virus. Both monkeys proved to be protected against the heterologous virus. In one of the two animals, low levels of SIV(8980) replication were detected. Thus, by chemotherapy during the acute phase of pathogenic virus replication, we could achieve not only persistent virus load suppression in two out of six monkeys but also protection from subsequent heterologous challenge. By this chemotherapeutic attenuation, the replication kinetics of attenuated viruses could be mimicked and a vaccination effect similar to that induced by live attenuated simian immunodeficiency virus vaccines was achieved.

Adenine↗
PubMed2003

Impact of genotypic resistance testing on selection of salvage regimen in clinical practice.

OBJECTIVE: To determine whether genotypic resistance testing leads to selection of more potent drug regimens when compared to regimens based on treatment history only. DESIGN: Prospective, tertiary care centre-based study. PATIENTS: One-hundred-and-forty-five HIV-infected adults on stable antiretroviral therapy (ART) for >6 months experiencing virological failure. METHODS: The physicians' decision-making process when choosing a salvage regimen was prospectively documented: at time of virological failure, on 'failing ART', genotyping was performed and a hypothetical 'clinical expert ART' based upon patient's drug history was documented. Subsequently, data on resistance mutations, rating by a decision support software and drug history were used to define 'genotyping ART'. After discussion with the patient, final treatment, 'new personalized ART' was chosen and prescribed. To compare the relative potency of the four ART regimens in a standardized manner, a resistance score ranging from 1 (best) to 8 (worst) based on drug ranking by decision support software was attributed to each ART regimen. Virological and immunological outcomes were analysed based on the magnitude of the resistance score. RESULTS: Median follow-up was 1.5 years. In all 145 patients, median resistance scores for the stepwise selected ART regimens were: 'failing ART': 4.5, 'clinical expert ART': 1.8, 'genotyping ART': 1.5 and 'new personalized ART': 2. The latter was 1.5 in patients who effectively switched to 'new personalized ART' (n=89). Lower resistance scores translated into significantly improved virological response after initiation of 'new personalized ART'. In multivariable analysis, lower resistance scores, lower baseline HIV RNA levels and use of novel antiretroviral drugs were associated with the probability of reducing plasma viraemia to <50 copies/ml. CONCLUSIONS: This study suggests that treatment choices including genotype and decision support software were virologically superior to those based on drug history only.

Adult↗
PubMed1998

Chimeric HIV-1 and feline immunodeficiency virus reverse transcriptases: critical role of the p51 subunit in the structural integrity of heterodimeric lentiviral DNA polymerases.

The reverse transcriptase (RT) of HIV-1 and feline immunodeficiency virus (FIV) consist of two subunits of 51 kDa (p51) and 66 kDa (p66). In order to elucidate the role of p51 in the heterodimer, chimeric HIV-1/FIV RT heterodimers were constructed and characterized. The FIV RT p51/HIV-1 RT p66 chimera showed a 2.5-fold higher RNase H activity than the natural HIV-1 RT, a 50% lower strand displacement DNA synthesis activity and resistance to the two RT inhibitors 3'-azido-3'-deoxythymidine triphosphate (AZTTP) and Nevirapine. The HIV-1 RT p51/FIV RT p66 chimera on the other hand had very similar properties to the natural FIV RT. The differences observed upon exchange of the p51 subunits suggest that the three-dimensional structure of the p51 subunit in the RT heterodimers is not completely conserved between the human and the feline lentiviruses. Finally, our data suggest an important role for the p51 subunit in maintaining the optimal structural integrity of the RT heterodimer. The different effects of the small subunits on the sensitivity to known RT inhibitors might be of importance in the development of novel drugs against HIV-1 RT.

Antiviral Agents↗
PubMed2001

Restoration of wild-type infectivity to human immunodeficiency virus type 1 strains lacking nef by intravirion reverse transcription.

Human immunodeficiency virus type 1 (HIV-1) Nef protein exerts several effects, both on infected cells and as a virion protein, which work together to enhance viral replication. One of these activities is the ability to enhance infectivity and the formation of proviral DNA. The mechanism of this enhancement remains incompletely understood. We show that virions with nef deleted can be restored to wild-type infectivity by stimulating intravirion reverse transcription. Particle composition and measures of reverse transcriptase activity remain the same for Nef(+) and Nef(-) virions both before and after natural endogenous reverse transcription (NERT) treatment. The effect of NERT treatment on virions pseudotyped with murine leukemia virus envelope protein was similar to that on particles pseudotyped with HIV-1 envelope protein. However, virions pseudotyped with vesicular stomatitis virus G envelope protein showed no influence of Nef on NERT enhancement of infectivity. These observations suggest that Nef may function at a level prior to reverse transcription. Since NERT treatment results in partial disassembly of the viral core, we speculate that Nef may function at the level of core particle disassembly.

DNA, Viral↗
PubMed1995

New virologic tools for the design and analysis of clinical trials.

While a large number of candidate drugs and drug combinations are being evaluated for treating human immunodeficiency virus (HIV) infection, clinicians are confronted by the problem of how to optimally use those antiretroviral drugs already approved for clinical use. While new techniques are being developed for studying HIV pathogenesis, clinicians are asking whether these new techniques can be used to develop improved strategies for treating the HIV-infected individual. In the current issue of the Journal, Kojima et al. from the National Cancer Institute (NCI) used two research techniques to analyze the results of a phase I/II study comparing alternating versus simultaneous therapy with zidovudine and didanosine. The authors used a quantitative polymerase chain reaction (PCR) assay for measuring plasma virus RNA and a selective PCR assay for detecting specific HIV reverse transcriptase (RT) drug-resistance mutations. Their study demonstrates the potential for effective combination therapy if drugs are optimally combined and suggests that assays for measuring HIV burden and HIV drug resistance can be used to better understand the results of preliminary clinical trials. In addition, their study raises questions about the potential role for measurements of virus burden and the detection of HIV drug resistance in larger clinical trials and clinical practice.

Antiviral Agents↗
PubMed1996

Determination of HIV-1 susceptibility to reverse transcriptase (RT) inhibitors by a quantitative cell-free RT assay.

BACKGROUND: Mutations in the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) gene confer resistance to antiviral drugs acting on RT. Current methods employed to detect such resistance require time-consuming culture techniques during which selective pressures may affect the outcome of the test. OBJECTIVES: We sought to determine whether drug-susceptible and drug-resistant HIV-1 derived from clinical specimens could be distinguished by the effects of the active form of the drug on the enzyme activity in a quantitative, cell-free RT assay. STUDY DESIGN: Polyethylene glycol (PEG)-precipitated virus was obtained from 7-day culture supernatants. RT activity in the lysed viral extracts was measured in the presence of increasing concentrations of the active form of the drug being tested. IC50 (50% inhibitory concentration) values were determined by application of the median effect equation. RESULTS: Assays from nine post-nevirapine therapy isolates gave IC50 values at least 2 logs greater than pre-nevirapine isolates. The method also correctly distinguished between isolates sensitive and resistant to 2',3'-dideoxyinosine (ddI), but not between the ZDV-sensitive and ZDV-resistant isolates tested. The results agreed with data obtained by sequencing and by culture-based susceptibility assays.

Cell-Free System↗
PubMed1991

Comparative study of the anti-HIV activities of ascorbate and thiol-containing reducing agents in chronically HIV-infected cells.

To elucidate the action of vitamin C on pathogenic human retroviruses, we investigated and compared the effects of noncytoxic concentrations of ascorbic acid (AA), its calcium salt (Ca-ascorbate), and two thiol-based reducing agents [glutathione (GSH) and N-acetyl-L-cysteine (NAC)] against human immunodeficiency virus (HIV)-1 replication in chronically infected T lymphocytes. Ca-ascorbate reduced extracellular HIV reverse transcriptase (RT) activity by about the same magnitude as the equivalent dose of AA. Long-term experiments showed that continuous presence of ascorbate was necessary for HIV suppression. NAC (10 mmol/L) caused less than twofold inhibition of HIV RT and conferred a synergistic effect (approximately eightfold inhibition) when tested simultaneously with AA (0.426 mmol/L). In contrast, nonesterified GSH (less than or equal to 1.838 mmol/L) had no effect on RT concentrations and did not potentiate the anti-HIV effect of AA. These results further support the potent antiviral activity of ascorbate and suggest its therapeutic value in controlling HIV infection in combination with thiols.

Acetylcysteine↗
PubMed2000

Phosphorylation of AZT-resistant human immunodeficiency virus type 1 reverse transcriptase by casein kinase II in vitro: effects on inhibitor sensitivity.

Casein kinase II (CKII) phosphorylates wild-type (WT) recombinant reverse transcriptase (RT) mainly in the p66 subunit in vitro. Phosphorylation of T215F RT and D67N/K70R/T215F/K219Q RT (AZT-resistant RT) in vitro increases discrimination against AZTTP 2. 5- and 3.6-fold, respectively. This in vitro resistance can be reversed by treatment of phosphorylated AZT-resistant RT with phosphatase. Phosphorylation has no effect on WT RT. Terminal transferase activity of RT is selectively suppressed on phosphorylated AZT-resistant RT. Resistance to phosphonoformic acid (PFA, foscarnet) increases 3-fold upon phosphorylation of AZT-resistant RT. Although T215, the most important residue for AZT-resistance, is part of a CKII consensus target site, serines are primarily phosphorylated relative to threonines. Mutational analysis shows that phosphorylation can be reduced to 10% that of WT when amino-acid changes are introduced both in the "fingers" subdomain and motif D. These results suggest that phosphorylation of RT might be one factor involved in drug resistance in vivo.

Amino Acid Motifs↗
PubMed1995

Viral dynamics in human immunodeficiency virus type 1 infection.

The dynamics of HIV-1 replication in vivo are largely unknown yet they are critical to our understanding of disease pathogenesis. Experimental drugs that are potent inhibitors of viral replication can be used to show that the composite lifespan of plasma virus and virus-producing cells is remarkably short (half-life approximately 2 days). Almost complete replacement of wild-type virus in plasma by drug-resistant variants occurs after fourteen days, indicating that HIV-1 viraemia is sustained primarily by a dynamic process involving continuous rounds of de novo virus infection and replication and rapid cell turnover.

Antiviral Agents↗
PubMed2001

A multiple-site-specific heteroduplex tracking assay as a tool for the study of viral population dynamics.

Rapidly evolving entities, such as viruses, can undergo complex genetic changes in the face of strong selective pressure. We have developed a modified heteroduplex tracking assay (HTA) capable of detecting the presence of single, specific mutations or sets of linked mutations. The initial application of this approach, termed multiple-site-specific (MSS) HTA, was directed toward the detection of mutations in the HIV-1 pro gene at positions 46, 48, 54, 82, 84, and 90, which are associated with resistance to multiple protease inhibitors. We demonstrate that MSS HTA is sensitive and largely specific to all targeted mutations. The assay allows the accurate and reproducible quantitation of viral subpopulations comprising 3% or more of the total population. Furthermore, we used MSS HTA in longitudinal studies of pro gene evolution in vitro and in vivo. In the examples shown here, populations turned over rapidly and more than one population was present frequently. To demonstrate the versatility of MSS HTA, we also constructed a probe sensitive to changes at positions 181 and 184 of the RT coding domain. Changes at these positions are involved in resistance to nevirapine and 2',3'-dideoxy-3'-thiacytidine (3TC), respectively. This assay easily detected the evolution of resistance to 3TC. MSS HTA provides a rapid and sensitive approach for detecting the presence of and quantifying complex mixtures of distinct genotypes, including genetically linked mutations, and, as one example, represents a useful tool for following the evolution of drug resistance during failure of HIV-1 antiviral therapy.

Antiviral Agents↗
PubMed2005

[Study of resistance using the TRUGENE HIV-1 genotyping system and analysis of agreement between rule-based algorithms and virtual phenotyping].

INTRODUCTION: This study attempts to describe the results obtained in the HIV resistance study performed with clinical samples obtained from patients receiving "HAART" therapy and to compare the results using different interpretation algorithms. METHODS: 397 samples have been analysed (TRUGENE HIV-1 GENOTYPING kit). The results were interpreted with the algorithms Visible Genetics and Retrogram. The concordance interalgorithm was done for 105 of these samples, including the virtual phenotype interpretation. RESULTS: The samples corresponded to multi regimen failure (39%), first and second failure (30.7% and 27.1% respectively). A 27.6% of the samples were wild type. The more frequent mutations to the ITIAN were T215Y/F (37.2%) and M184V (32.9%) following by other NAMS. The 69 insertion and Q151M complex had low representativity. For the ITINN K103N (25.8%), Y181C (11.2%) and G190A (10.9%). For the IP: key mutations L90M (26.1%), M46I (18.1%) and V82AFTS (12.9%); and accessory mutations L63P (50.5%), A71V (27.2%), L10I (25.2%) and M36I (19.2%). Low correlation was observed between interpretation systems, mainly for ITIAN and IP, being the virtual phenotype more flexible in the assignation of resistance. CONCLUSIONS: The requests for HIV resistance testing were similar for the three groups of patients. Many of the failures were the consequence of a poor adherence to the therapy. The mutation pattern found corresponded with the "TARGA" therapy. The low correlation found between interpretation systems, makes necessary the elaboration of a consensus algorithm.

Algorithms↗
PubMed2002

Modulation of HIV-1 replication by RNA interference.

RNA interference (RNAi) is the process by which double-stranded RNA (dsRNA) directs sequence-specific degradation of messenger RNA in animal and plant cells. In mammalian cells, RNAi can be triggered by 21-nucleotide duplexes of small interfering RNA (siRNA). Here we describe inhibition of early and late steps of HIV-1 replication in human cell lines and primary lymphocytes by siRNAs targeted to various regions of the HIV-1 genome. We demonstrate that synthetic siRNA duplexes or plasmid-derived siRNAs inhibit HIV-1 infection by specifically degrading genomic HIV-1 RNA, thereby preventing formation of viral complementary-DNA intermediates. These results demonstrate the utility of RNAi for modulating the HIV replication cycle and provide evidence that genomic HIV-1 RNA, as it exists within a nucleoprotein reverse-transcription complex, is amenable to siRNA-mediated degradation.

Cell Line↗
PubMed1999

Total Synthesis of Quinoxapeptin A-C: Establishment of Absolute Stereochemistry.

The relative and absolute stereochemistry of the naturally occurring potent HIV reverse transcriptase (RT) inhibitors 1 and 2, quinoxapeptin A and B, were established by total synthesis. Their synthetic precursor 3 (dubbed quinoxapeptin C) was found to be a more potent HIV-1 RT inhibitor and to lack the potent cytotoxic activity characteristic of 1 and 2.

Journal Article↗
PubMed1997

HIV-1 reverse transcriptase-specific CTL against conserved epitopes do not protect against progression to AIDS.

A small group of HIV-1-infected subjects who either do not progress to AIDS or progress only slowly have sustained HIV-1-specific CTL responses. It has been suggested that the specificities of these responses differ from the CTL responses of rapid progressors due to recognition of epitopes that are under structural or functional constraints. We have, in this respect, studied the CTL response to reverse transcriptase (RT) in long term survivors (LTS) and in HIV-1-infected individuals who progressed to AIDS within 3 to 6 yr. Both LTS and progressors displayed vigorous RT-specific CTL responses of comparable magnitude during the asymptomatic phase. From each individual at least two CTL lines were obtained from blood samples drawn at different time points during follow-up. A total of 19 CTL lines recognized nine different RT-derived epitopes. CTL obtained from progressors recognized epitopes with a similar degree of amino acid conservation as epitopes targeted by CTL from LTS. Furthermore, five of seven epitopes were recognized by both LTS and progressors. Moreover, one of the epitopes recognized by progressors contained the highly conserved YMDD motif that is essential for RT activity. In conclusion, our data imply that neither the magnitude nor the specificity of HIV-1-specific CTL against RT is a major cause of a more protracted course of disease.

Acquired Immunodeficiency Syndrome↗
PubMed2003

[Decrease of HIV-1 replicative capacity after serial passage in MT-4 cells under conditions of the combined antiretroviral therapy].

The replicative activity of HIV-1/IIIB was determined in MT-4 cells under conditions of combined HIV reverse transcriptase and protease inhibitors in the presence of four different drug cocktails: (1) phosphazide, didanosine, nevirapine; (2) stavudine, didanosine, nevirapine; (3) phosphazide, didanosine, nevirapine, indinavir; (4) stavudine, didanosine, nevirapine, indinavir. The concentration of every inhibitor was 10 times higher than the 50% effective concentration. Alpha interferon was used as a natural antiretroviral agent in addition. The virus was subjected to 5 serial passages in the presence of the drug cocktails and then to 5 serial passages without the agents using cocultivation of infected and uninfected cells at ratio 1:5 to increase virus activity. Virus replication in the presence of all the drug combinations resulted in the appearance of HIV-1 variants with low replicative activity that was insignificantly increased during further passages even without the antiretroviral agents. If extrapolated to the clinical practice, these results indicate that all the drug cocktails were effective inhibitors of HIV-1 replication because the virus variants with high replicative activity did not emerge. Moreover, the results showed that the clinical use of the drug cocktails was promising.

Anti-HIV Agents↗