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PubMed1995

CD8+ T cells suppress human immunodeficiency virus replication by inhibiting viral transcription.

CD8+ cells from human immunodeficiency virus (HIV)-infected individuals suppress HIV replication in cultured CD4+ cells by a noncytolytic mechanism that involves a secreted CD8(+)-cell antiviral factor (CAF). The results of this study suggest that CD8+ cells, as well as CAF, arrest HIV replication at the level of viral transcription. Culturing naturally infected CD4+ cells actively producing HIV with autologous CD8+ cells or a 50% dilution of culture fluids from these cells results in a > 80% reduction in the number of cells expressing HIV antigens and RNA. This effect was observed within 2 days after exposure to CD8+ cells but required 6 days in the presence of CAF-containing culture fluids to reach the same extent of HIV suppression. Northern blot analysis of CD4+ cell extracts revealed that all viral RNA species (unspliced and single and double spliced) were reduced in quantity to a similar extent. CAF-containing culture fluids also had a direct inhibitory effect on HIV long terminal repeat (LTR)-driven transcription in HIV-infected 1G5 cells carrying an LTR-luciferase construct. Suppression of basal levels of LTR-driven transcription was not detected. Thus, the results suggest that the noncytolytic CD8+ cell antiviral activity observed in HIV infection exerts its effects, at least in part, by specifically interrupting HIV transcription. These findings could help in developing therapies for HIV infection.

Antiviral Agents↗
PubMed1996

Lamivudine or stavudine in two- and three-drug combinations against human immunodeficiency virus type 1 replication in vitro.

Two- and three-drug combinations of lamivudine or stavudine with other antiretroviral drugs were evaluated for activity against human immunodeficiency virus type 1 (HIV-1) activity in peripheral blood mononuclear cells. Other agents included zidovudine, didanosine, nevirapine, and saquinavir. Paired zidovudine-sensitive and -resistant clinical HIV-1 isolates were used. Additive or synergistic interactions were observed against the zidovudine-sensitive isolate with the following combinations: lamivudine-zidovudine, lamivudine-stavudine, lamivudine-saquinavir, lamivudine-nevirapine, stavudine-zidovudine, stavudine-didanosine, stavudine-saquinavir, stavudine-nevirapine, lamivudine-zidovudine-saquinavir, lamivudine-zidovudine-stavudine, stavudine-zidovudine-nevirapine, lamivudine-zidovudine-nevirapine, and stavudine-zidovudine-saquinavir. Against the zidovudine-resistant isolate, additive or synergistic interactions were seen with most two- and three-drug combinations, but the combination of stavudine-zidovudine was antagonistic. The clinical implications of these in vitro observations should be explored.

Antiviral Agents↗
PubMed2001

Inhibition of human immunodeficiency virus type 1 reverse transcriptase, protease and integrase by bovine milk proteins.

Different proteins have been isolated from bovine milk including lactoferrin, lactoperoxidase, glycolactin, angiogenin-1, lactogenin, alpha-lactalbumin, lactoglobulin and casein. These proteins have been assayed for inhibitory activity against human immunodeficiency virus type 1 (HIV-1) reverse transcriptase, protease and integrase, enzymes crucial to the HIV-1 life cycle. It was found that different milk proteins inhibited the three aforementioned HIV enzymes to different extents. Lactoferrin strongly inhibited HIV-1 reverse transcriptase but only slightly inhibited HIV-1 protease and integrase. On the other hand, alpha-lactalbumin, beta-lactoglobulin and casein inhibited HIV-1 protease and integrase to an appreciable extent but did not inhibit HIV-1 reverse transcriptase. Glycolactin and angiogenin-1 suppressed the activity of HIV-1 reverse transcriptase by a moderate extent but more powerfully inhibited HIV-1 protease and integrase. In comparison with the other milk proteins glycolactin was a strong inhibitor of HIV-1 protease and integrase and a moderate inhibitor of HIV-1 reverse transcriptase. Lactogenin was a strong inhibitor of HIV-1 integrase, a moderate inhibitor of HIV-1 reverse transcriptase and a weak inhibitor of HIV-1 protease.

Animals↗
PubMed1995

HIV-1 reverse transcriptase-associated RNase H cleaves RNA/RNA in arrested complexes: implications for the mechanism by which RNase H discriminates between RNA/RNA and RNA/DNA.

Reverse transcription of human immunodeficiency virus type 1 (HIV-1) is primed by tRNA(Lys3), which forms an 18 base pair RNA homoduplex with its 3' terminus and the primer binding site (PBS) of the viral genome. Using an in vitro system mimicking initiation of minus strand DNA synthesis, we analyzed the mechanism by which HIV-1 reverse transcriptase (RT)-associated ribonuclease H (RNase H) distinguishes between RNA/DNA and RNA/RNA (dsRNA). tRNA(Lys3) was hybridized to a PBS-containing RNA template and extended by addition of deoxynucleoside triphosphates (dNTPs). In the presence of all four dNTPs, initial cleavage of the RNA template occurred immediately downstream of the tRNA-DNA junction, reflecting RNase H specificity for RNA in a RNA/DNA hybrid. However, in the absence of DNA synthesis, or limiting this by chain termination, the PBS was cleaved at a constant distance of 18 nucleotides upstream of the nascent primer 3' terminus. The position of cleavage remained in register with the position of DNA synthesis arrest, indicating that hydrolysis of homoduplex RNA is spatialy co-ordinated with DNA synthesis. Kinetic studies comparing cleavage rates of an analogous DNA primer/PBS heteroduplex and the tRNA(Lys3)/PBS homoduplex showed that while the former is cleaved as rapidly as RT polymerizes, the latter proceeds 30-fold slower. Although the RNase H domain hydrolyzes dsRNA when RT is artificially arrested, specificity for RNA/DNA hybrids is maintained when DNA is actively synthesized, since residency of the RNase H domain at a single base position is not long enough to allow significant cleavage on dsRNA.

Base Sequence↗
PubMed2005

2-(Aryl)-3-furan-2-ylmethyl-thiazolidin-4-ones as selective HIV-RT inhibitors.

A series of 4-thiazolidinones were evaluated as selective inhibitors of the HIV-RT enzyme. Our attempt in correlating the derived physicochemical properties with the HIV-RT inhibitory activity resulted in some statistically significant QSAR models with good predictive ability. The QSAR studies indicated the role of lipophilicity, dipole moment and out-of-plane potential energy of the compounds in rationalizing the activity. One of the compounds, 1, inhibited the enzyme at 0.204 microM concentration with minimal toxicity to MT-4 cells.

Anti-HIV Agents↗
PubMed1997

Anti-HIV activity of extracts from Calendula officinalis flowers.

Extracts of dried flowers from Calendula officinalis were examined for their ability to inhibit the human immunodeficiency virus type 1 (HIV-1) replication. Both organic and aqueous extracts were relatively nontoxic to human lymphocytic Molt-4 cells, but only the organic one exhibited potent anti-HIV activity in an in vitro MTT/tetrazolium-based assay. In addition, in the presence of the organic extract (500 micrograms/mL), the uninfected Molt-4 cells were completely protected for up to 24 h from fusion and subsequent death, caused by cocultivation with persistently infected U-937/HIV-1 cells. It was also found that the organic extract from Calendula officinalis flowers caused a significant dose- and time-dependent reduction of HIV-1 reverse transcription (RT) activity. An 85% RT inhibition was achieved after a 30 min treatment of partially purified enzyme in a cell-free system. These results suggested that organic extract of flowers from Calendula officinalis possesses anti-HIV properties of therapeutic interest.

Anti-HIV Agents↗
PubMed2005

Convenient route to both enantiomers of a highly functionalized trans-disubstituted cyclopentene. Synthesis of the carbocyclic core of the nucleoside BCA.

[structures: see text] Synthesis of both enantiomers of a highly functionalized cyclopentenol derivative, versatile building block for a vast array of biologically active compounds, is described. The key steps involve stereocontrolled synthesis of a diene with two syn-disposed substituents from a (R)-(+)-glyceraldehyde derivative, ring-closing metathesis of this diene, and functional group manipulation of the resulting trans-disubstituted cyclopentene. One of the enantiomers of the cyclopentenol thus obtained has been converted to an amino cyclopentene, the carbocyclic core of the nucleoside (-)-BCA, a potent inhibitor of HIV reverse transcriptase.

Adenine↗
PubMed1996

Positive selection system to screen for inhibitors of human immunodeficiency virus-1 transcription.

We describe a transcription-based assay system to screen for antiviral drugs in vivo. The system consists of two transcription units, a cytomegalovirus promoter driving a reporter gene physically linked to an HIV-1 promoter oriented in the opposite direction. Based on the arrangement of the transcription units, enhanced HIV-1 promoter activity in the presence of the viral transactivating Tat protein downregulates reporter gene expression initiated from the CMV promoter. Inhibitors of HIV-1 transcription relieve the suppression and are identified by an increase in reporter gene expression. This positive selection system allows discrimination between drugs that nonspecifically block cellular functions by cytotoxicity and molecules that specifically repress HIV-1 promoter activity.

Alkaline Phosphatase↗
PubMed2001

MIKADO: a multicentre, open-label pilot study to evaluate the antiretroviral activity and safety of saquinavir with stavudine and zalcitabine.

BACKGROUND: Since eradication of HIV is unlikely, long-term management of the disease necessitates careful evaluation of the combinations of currently available drugs to determine the most potent and useful rational sequencing of regimens. OBJECTIVE: To determine the antiretroviral efficacy and tolerability of saquinavir soft gelatin capsule (SQV-SGC) plus zalcitabine (ddC) and stavudine (d4T), as first-line treatment in HIV-infected patients. DESIGN: Multicentre, open-label, non-comparative study. PATIENTS AND METHODS: Thirty-five asymptomatic, HIV-infected adults with no prior antiretroviral treatment, a CD4 count > or =250 cells/microL and baseline > or = 5000 HIV RNA copies/mL were included in the study. Patients received SQV-SGC 1200 mg three times a day (tid), ddC 0.75 mg tid and d4T 30 or 40 mg twice a day (bid) for 24 weeks. Plasma HIV RNA, CD4 and CD8 cell counts, HIV reverse transcriptase and protease resistance genotypes, SQV plasma concentration and tolerability were evaluated. RESULTS: At baseline, median HIV RNA (interquartile range) was 4.99 (4.81-5.48) log10 copies/mL, and median CD4 count was 370 (318-504) cells/microL (n = 35). At week 24, the median decrease in HIV RNA was 3.05 (2.19-3.68) log10 copies/mL. A viral load below the level of quantification (200 copies/mL and 20 copies/mL) was achieved in 63% and 34% of patients, respectively (intent-to-treat analysis). The only mutations detected were L90M substitutions in two patients. At week 24, the median CD4 count increased (P < 0.0001), and CD8 cell counts decreased (P < 0.0001), relative to baseline. In total, there were five cases of peripheral neuropathy (14%). Mean triglyceride and cholesterol levels remained within normal ranges. CONCLUSIONS: Triple therapy with SQV-SGC plus ddC and d4T is a reasonably well tolerated regimen that markedly and rapidly reduces viral load with immunological improvement. This combination is an effective additional therapeutic option, with an efficacy that compares favourably to other triple regimens used in HIV treatment.

Adult↗
PubMed1998

Comparative performance of high-density oligonucleotide sequencing and dideoxynucleotide sequencing of HIV type 1 pol from clinical samples.

The performance of the high-density oligonucleotide array methodology (GeneChip) in detecting drug resistance mutations in HIV-1 pol was compared with that of automated dideoxynucleotide sequencing (ABI) of clinical samples, viral stocks, and plasmid-derived NL4-3 clones. Sequences from 29 clinical samples (plasma RNA, n = 17; lymph node RNA, n = 5; lymph node DNA, n = 7) from 12 patients, from 6 viral stock RNA samples, and from 13 NL4-3 clones were generated by both methods. Editing was done independently by a different investigator for each method before comparing the sequences. In addition, NL4-3 wild type (WT) and mutants were mixed in varying concentrations and sequenced by both methods. Overall, a concordance of 99.1% was found for a total of 30,865 bases compared. The comparison of clinical samples (plasma RNA and lymph node RNA and DNA) showed a slightly lower match of base calls, 98.8% for 19,831 nucleotides compared (protease region, 99.5%, n = 8272; RT region, 98.3%, n = 11,316), than for viral stocks and NL4-3 clones (protease region, 99.8%; RT region, 99.5%). Artificial mixing experiments showed a bias toward calling wild-type bases by GeneChip. Discordant base calls are most likely due to differential detection of mixtures. The concordance between GeneChip and ABI was high and appeared dependent on the nature of the templates (directly amplified versus cloned) and the complexity of mixes.

Anti-HIV Agents↗
PubMed2004

HIV type 1 BF recombinant strains exhibit different pol gene mosaic patterns: descriptive analysis from 284 patients under treatment failure.

Different complex structures of the pol gene have been identified in 284 HIV-1 B/F recombinant sequences obtained from a group of 587 patients under treatment failure from Argentina. To analyze the mosaic structures of these viral sequences and to determine their phylogenetic relationship, the 284 partial pol gene sequences of BF recombinant viruses were amplified by RT-PCR and sequenced. Intersubtype breakpoints were analyzed by bootscanning. Phylogenetic relationships were determined by means of neighbor-joining trees. The analysis of the sequences showed multiple phylogenetic topologies clustering within intersubtype BF reference sequences. At least three different mosaic patterns were found compared to previously described BF-type viruses with unequal distribution in the studied population. The analysis also showed that HIV-1 BF recombinant viruses with diverse mosaic structures are phylogenetically related in their F segments and in selected B fragments with the F1 subtype and with BF recombinant viruses from Brazil, respectively, suggesting a common recombinant ancestor. No association was observed between the prevalence of each mosaic pattern and the frequency of major drug-resistance mutations in PR and RT.

Anti-HIV Agents↗
PubMed2006

Lamivudine monotherapy in HIV-1-infected patients harbouring a lamivudine-resistant virus: a randomized pilot study (E-184V study).

OBJECTIVE: We compared the immunological and clinical outcomes of lamivudine monotherapy and complete therapy interruption in the treatment of HIV-1-infected patients harbouring lamivudine-resistant virus. METHODS: This 48-week, open-label pilot study randomly assigned HIV-infected patients receiving lamivudine-containing HAART and harbouring the M184V mutation to monotherapy with lamivudine 300 mg once daily (lamivudine group) or the discontinuation of all antiretroviral drugs (TI group). The primary endpoint was the occurrence of immunological or clinical failure; immunological failure was defined as the first report of a CD4 T-cell count less than 350 cells/microl, and clinical failure as the occurrence of a Centers for Disease Control and Prevention grade B or C event. The data were analysed on the basis of the intention-to-treat principle. RESULTS: By week 48, 20 of 29 patients in the TI group (69%; 95% CI 51-83%) and 12 of 29 in the lamivudine group (41%; 95% CI 26-59%) had discontinued the study because of immunological or clinical failure, which was significantly delayed in the lamivudine group (P = 0.018). Only patients in the TI group (6/29, 20.7%) experienced grade 3-4 clinical adverse events at least possibly related to HIV-1 (P = 0.02). The mean decline in CD4 cell percentage, viral rebound and recovery of HIV-1 replication capacity were significantly lower in the lamivudine group. The 24-week virological and immunological response after therapy resumption in patients who prematurely discontinued the study was similar in the two groups. CONCLUSION: In HIV-1-infected patients harbouring a lamivudine-resistant virus, lamivudine monotherapy may lead to a better immunological and clinical outcome than complete therapy interruption.

Adult↗
PubMed2002

Discordance between genotypic and phenotypic drug resistance profiles in human immunodeficiency virus type 1 strains isolated from peripheral blood mononuclear cells.

The aim of the study was to analyze the relationship between genotypic and phenotypic drug resistance profiles of human immunodeficiency virus type 1 (HIV-1) strains isolated from patients during double-analogue nucleoside therapy. A drug-resistant HIV strain was isolated from 20 out of 25 patients, with 16 (64%) subjects carrying a virus with multiple drug resistance mutations. The most frequent resistance mutations were M184V (18 isolates) and M41L (7 isolates). Discordance between the genotypic and phenotypic profile for at least one drug was detected in 16 out of 25 strains. Particularly, eight isolates had a discordant genotypic-phenotypic resistance pattern for two drugs and one isolate had such a pattern for three drugs. A genotypic resistance pattern with a phenotypic sensitivity profile was detected in six isolates (four resistant to zidovudine and two resistant to lamivudine). On the other hand for several strains a genotypic pattern of sensitivity pattern to abacavir (10 strains), didanosine (7 strains), stavudine (3 strains), zidovudine (2 strains), and lamivudine (1 strain) with a phenotypic resistance profile was detected. After a follow-up period of 8 months, an impairment of virological and immunological parameters was detected only in subjects with an HIV-1 isolate with a phenotypic resistance profile in despite of the genotypic results. Predicting resistance phenotype from genotypic data has important limitations. Despite the low number of patients and the short follow-up period, this study suggests that during failing therapy with analogue nucleosides, a phenotypic analysis could be performed in spite of an HIV genotypic sensitivity pattern.

Anti-HIV Agents↗
PubMed2005

Synthesis and QSAR studies on thiazolidinones as anti-HIV agents.

Selected 4-thiazolidinone have been synthesized and tested as anti-HIV activity. The results of the in vitro tests showed that one of the compounds, 5, inhibited the enzyme at 0.204 microM concentration with minimal toxicity to MT-4 cell. Furthermore, the QSAR studies indicated the role of PMIZ, Ovality and Total energy content of the compounds in rationalizing the activity.

Animals↗
PubMed2003

Salvage therapy with abacavir in HIV-1-infected patients with previously documented M184V mutation: a possibility of NRTI recycling.

We evaluated in an open-label, randomized, controlled, pilot trial if the re-emergence of previously selected resistant strains, harbouring M184V mutation, could be modulated by the use of different drug associations as components of the new antiretroviral regimens. In addition, we assessed the clinical relevance of this mutation on the management of heavily pretreated HIV-infected patients. The primary end-point of the study was the reselection of M184V mutation. Secondary end-points were the variation over time of HIV RNA plasma levels and CD4 cell counts and the progression of HIV disease. The primary population for efficacy analysis was the intention-to-treat exposed population. After a run-in phase consisting in a new treatment regimen excluding either lamivudine (3TC) or abacavir (ABC) so as to clear the previously documented M184V mutation, 18 patients with an HIV RNA plasma level greater than 10000 copies/ml were randomized to receive an antiretroviral drug regimen (at least three drugs) including either ABC or the association of ABC+3TC. All patients were naive to ABC. The M184V mutation reappeared in 1/9 patients in the ABC group and in 8/9 patients in the ABC+3TC group (P<0.003, 95% CI: 0.5-1). In the ABC group we observed a rapid decrement of viral load that was maintained throughout all the study period (P<0.05). On the contrary, in the ABC+3TC group, after a transient decrement at 2 months, a progressive increment towards baseline values was observed. The proportion of patients with a viral load reduction of at least 0.5 logs at 12 months was significantly higher in the ABC group: 8/9 patients vs 3/9 (P=0.05, 95% CI: 0.2-0.92). Similarly, from an immunological point-of-view, the increase at all time points (since randomization) in CD4 cell count was statistically significant in the ABC group (P<0.01), while no difference was observed in the ABC+3TC group. The possibility of a successful use of ABC in salvage regimens opens alternative therapeutic options for heavily pretreated patients with previously documented M184V mutation. Further studies should clarify whether this is true for other drugs of the nucleoside analogues class.

Adult↗
PubMed2003

Drug resistance mutations during structured treatment interruptions.

BACKGROUND: We assessed whether treatment interruptions induce selection of mutations associated with drug resistance in the Swiss-Spanish Intermittent Treatment Trial (SSITT). Patients had been on HAART without previous failure and had undetectable viraemia for at least 6 months. Their HAART was interrupted for 2 weeks and restarted for 8 weeks. After four of these cycles, treatment was definitively interrupted at week 40. METHODS: Genotypic resistance testing was performed in 87/97 Swiss patients: in those failing treatment before week 40, at the time of first viral rebound > 500 copies/ml off treatment and preceding failure to reach RNA < 50 copies/ml after 8 weeks of re-treatment; for patients without virological failure, on the first sample with HIV-1 RNA > 1000 copies/ml after week 40. RESULTS: Mutations associated with drug resistance were detected in 9/25 (36%) patients with virological failure during the first 40 weeks and in 6/59 (10%) patients after week 40. Overall, drug resistance mutations were detected in 17% of patients, all but two with the 184V/I mutation. Among the 74 patients receiving lamivudine, the M184V/I mutation was detected in 13/74 (17.6%) patients. A wild-type codon at position 184 was detected in previous samples in all but two. The relative risk for virological failure was 2.55-fold higher in patients with the M184V/I mutation than in patients without detectable mutation (P=0.007). CONCLUSIONS: The M184V/I mutation is frequently selected during repeated treatment interruptions.

Anti-HIV Agents↗