184V Mutation Research Articles

Short Communication: Lamivudine Plus a Boosted-Protease Inhibitor as Simplification Strategy in HIV-Infected Patients: Proof of Concept

There are scarce data about the use of a dual therapy with lamivudine plus a ritonavir-boosted protease inhibitor (PI/r) as simplification strategy. We performed a retrospective cohort study of 44 HIV-infected patients on suppressive triple therapy PI/r-based HAART, HBV negative, who discontinued one nucleoside analogue (NA) due to toxicity, and continued dual therapy with lamivudine plus the PI/r. The median time of HIV infection was 18.6 years, and CD4(+) count nadir was 150 cells/μl (6-470). They had received a mean of four regimens before (2-20), and 55% had a previous AIDS diagnosis. In eight cases, a previous resistance test showed two to seven secondary mutations in the protease gene, without resistance to the PI/r. The 184V mutation was not observed, but previous failure with a lamivudine-including regimen had been observed in four patients (9%). At baseline, patients had viral suppression (<50 copies/ml) for a median time of 794 days (235-2344), while receiving atazanavir/r in five cases, lopinavir/r in 14, and darunavir/r in 25. They discontinued mainly tenofovir (19, 43%) and didanosine (15, 34%). During 101.5 patient-years of follow-up, only two patients failed (5%), because of incomplete adherence. CD4(+) count increase at 48 weeks was 55 cells/μl. No new adverse events were observed, although total cholesterol and triglycerides increased significantly during the first 6 months after simplification. In conclusion, dual therapy with lamivudine plus a boosted PI was safe and effective as simplification strategy in patients with toxicity to NA. This combination could be an alternative to mono or triple therapy in hard-to-treat patients.

Tenofovir DF en pautas de rescate

As with other nucleoside analogues, tenofovir (TDF) can be affected by several mutations in the reverse transcriptase gene. Most nucleoside analogue mutations (NAMs) are not induced specifically by TDF, although they can affect the activity of this drug. The impact of thymidine analogue mutations (TAMs) on tenofovir varies and, as with the remaining nucleoside analogue reverse transcriptase inhibitors, largely depends on the type and number present. Thus, the greater the number of TAMs, and the greater the number of type 1 TAMs, the more TDF activity will be affected. The 41L and 210W mutations have the greatest effect. The incidence of the 65R mutation was slight before the clinical introduction of TDF. This mutation was selected by treatments with zalcitabine monotherapy. However, after TDF came on to the market, the 65R mutation began to be more frequently reported and is currently the signature mutation of this drug. TDF has been shown to be safe and effective in patients with prior virological failure and resistance mutations in the reverse transcriptase gene. In these patients, the presence of the 41L and 210W mutations is associated with a worse response to rescue therapy containing TDF. In contrast, the presence of type 2 TAMs (67N, 70R and 219Q/E/N) has little effect on TDF activity in these patients. Importantly, in TDF therapy, the presence of the 184V mutation is associated with a more favorable virologic response than the absence of this mutation, with any of the distinct combinations of mutations present.

HIV‐1 replication capacity and genotype changes in patients undergoing treatment interruption or lamivudine monotherapy

The objective of this study was to investigate the mechanisms underlying the virological and immunological changes occurring in failing HIV-1 infected patients undergoing treatment interruption or lamivudine monotherapy (the E-184V Study). Associations were sought between the de-selection of individual reverse transcriptase and protease resistance mutations and replication capacity recovery, HIV-RNA changes, and immunological changes. The replication capacity recovery was defined as the ratio between the replication capacity at weeks 24 or 48, and that measured at baseline. The replication capacity recovery, which was evaluable in 21 patients at week 24 and in 18 at week 48, was significantly higher in the treatment interruption than in the lamivudine group at week 24 (P = 0.002). Forty-eight week replication capacity recovery was greater when the 184V (P = 0.023), the 41L (P = 0.02), or the 215Y mutation (P = 0.037) were deselected at week 12. A greater reduction in the CD4+/CD8+ ratio at week 48 (P = 0.038) was observed as the 184V mutation was deselected and the de-selection of the 184V mutation at week 12 was the only independent predictor of the change of the CD4+/CD8+ ratio at week 48 from baseline at multivariable analysis (F-value = 6.72, P = 0.021). In conclusion, among patients undergoing treatment interruption or lamivudine monotherapy, the recovery of HIV-1 replication capacity was associated with the de-selection of reverse transcriptase mutations. The de-selection of the 184V mutation predicts independently a reduction in the CD4+/CD8+ ratio.

Dynamics of 103K/N and 184M/V HIV-1 drug resistant populations: Relative comparison in plasma virus RNA versus CD45RO+T cell proviral DNA

Background Viral populations defined by 103K/N and 184M/V as linked or single mutations in the HIV-1 reverse transcriptase gene were investigated in plasma samples and compared with previous findings in the CD45RO +T cell compartment. Objective To develop an ARMS assay for plasma virions and to investigate the expression of resistance mutations (103N and 184V) and dynamic interactions between proviral DNA and plasma virions. Study design A clinical cross-sectional study, including 11 patients on lamivudine efavirenz and/or nevirapine therapy. The viral populations were determined by an assay based on real-time PCR and amplification refractory mutation system (ARMS). Results The 103N and 184V mutations were not detected in patients with stable low viremia. Patients previously exposed to mono or dual therapy often carried minor viral populations of either one or both mutations in plasma. The viral population with linked mutations (103N and 184V) was detected in two patients after more than 2 years of non-NNRTI HAART. Conclusion The ARMS assay is useful for detecting viral quasi-species containing efavirenz and lamivudine resistant mutations in plasma virions and in proviral DNA. Data suggest an unequal distribution of linked-mutation populations in plasma and CD45RO +T cells. Furthermore, the linked 103N-184V mutation may be more fit than the single 184V mutation and this linked population emerges rapidly under inadequate drug pressure.

HIV-1 Reverse Transcriptase Gene 103K/N and 184M/V Combinations in Tandem

The proviral HIV-1 reverse transcriptase gene for the 103K/N and 184M/V combinations were studied in tandem. The CD45RO T (memory) cell compartment was investigated. A new double-ARMS (amplification refractory mutation system) real-time polymerase chain reaction assay was developed to detect and quantify 4 populations (103K-184M, 103K-184V, 103N-184M, and 103N-184V) in the CD45RO T-cell compartment. Twenty-one patients, 18 lamivudine and efavirenz/nevirapine experienced, were enrolled in a cross-sectional study. None of the mutation combinations were detected in patients on highly active antiretroviral therapy (HAART) (naive at start) with viremia suppression below detection limits. Conversely, all patients exposed to mono- or dual therapy (prior to HAART) carried at least 1 mutation combination regardless of viral load. In 9 patients, 17 mutations were detected in a mosaic of combinations. This study provides definite evidence of the existence of 103N and 184V mutation quasi-populations in tandem, and separately in combination with the wild-type codons, 184M and 103K, in the CD45RO T-cell compartment. The initiation and continuation of potent antiretroviral therapy effectively hinders the appearance of 103N and 184V mutations alone or in tandem in memory cells. When switching therapies because of failure, caution should be exercised with drugs associated with single-mutation threshold; they can appear in tandem with contemporary resistant virus populations, leading to multidrug resistance.

The L74V Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Counteracts Enhanced Excision of Zidovudine Monophosphate Associated with Thymidine Analog Resistance Mutations

Thymidine analog mutations (TAMs) in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) confer resistance to zidovudine (AZT) by increasing the rate of ATP-dependent phosphorolysis of the terminal nucleotide monophosphate (primer unblocking). By contrast, the L74V mutation, which confers resistance to didanosine, sensitizes HIV-1 to AZT and partially restores AZT susceptibility when present together with one or more TAMs. To compare rates of primer unblocking in RTs carrying different clusters of TAMs and to explore the biochemical mechanism by which L74V affects AZT susceptibility, ATP-mediated rescue of AZT-blocked DNA synthesis was assayed using a series of purified recombinant RTs. Rates of primer unblocking were higher in the 67N/70R/219Q RT than in the 41L/210W/215Y enzyme and were similar to rates observed with an RT carrying six TAMs (41L/67N/70R/210W/215Y/219Q). The presence of 74V in an otherwise wild-type RT reduced the rate of primer unblocking to a degree similar to that observed with the M184V mutation for lamivudine resistance, which also sensitizes HIV-1 to AZT. Introduction of 74V into RTs carrying TAMs partially counteracted the effect of TAMs on the rate of primer unblocking. The effect of 74V was less marked than that of the 184V mutation in the 67N/70R/219Q and 41L/210W/215Y RTs but similar in the RT carrying six TAMs. These results demonstrate that L74V enhances AZT susceptibility by reducing the extent of its removal by ATP-dependent phosphorolysis and provides further evidence for a common mechanism by which mutations conferring resistance to didanosine and lamivudine sensitize HIV-1 to AZT.

Detection and quantification of proviral HIV-1 184M/V in circulating CD4 + T cells of patients on HAART with a viremia less than 1000 copies/ml

Background Highly active anti-retroviral therapy (HAART) effectively reduces HIV replication but does not completely hinder it. Sub-optimal therapy leads to HIV resistance to the drugs administered. However, the role of low-level viremia (viral-load less than 1000 copies/ml) on mutation genesis and incorporation of resistant forms in the long-lived CD4 + T cellular DNA compartment is not clear. Objective To investigate the relationship between lamivudine associated mutant-type 184V and the wild-type 184M proviral forms in the circulating CD4 + T cells of patients and low-level viremia. Study design Cross-sectional study of 50 patients on long-term HAART, with a viremia of less than 1000 copies/ml. Patients were stratified into three groups; on lamivudine, group I (viral load <20 copies/ml), group II (viral load 20–1000 copies/ml) and as lamivudine experienced, group III (viral load <1000 copies/ml). 184M and 184V proviral HIV-1 was detected and quantified by a specific and sensitive assay combining a TaqMan real-time PCR analysis with the amplification-refractory mutation system (ARMS) principle. Results Fifty-six percent of patients with low-level viremia had 184V in the CD4 + T cellular DNA compartment as compared to only 8% in those with undetectable viremia. The presence of 184V was significantly associated with a higher viral load ( P = 0.001). Patients with low-level viremia without 184V in the CD4 + T cellular DNA compartment, had a median plasma viral load of 135 copies/ml, while patients harbouring 184V had a median viral load of 498 copies/ml ( P = 0.006). No significant differences between the groups were observed in proviral HIV-1 DNA load. Conclusions The frequency of the 184V mutation was significantly lower, in the CD4 + T cellular compartment of patients with a viral load of less than 20 copies/ml as compared to patients with a viremia of 20–1000 copies/ml. Viremia, sustained below 20 copies/ml may prevent the appearance of 184V mutation in this reservoir and therefore should be the objective of treatment.

Impact of a treatment including tenofovir plus didanosine on the selection of the 65R mutation in highly drug-experienced HIV-infected patients

Data from 20 highly drug-experienced HIV-infected patients receiving tenofovir plus didanosine as part of a salvage regimen were analysed. At baseline, all but one patient harboured a virus bearing at least one nucleoside excision mutation (NEM); in 13 cases (65%) three or more NEM were detectable. After a median of 26 weeks of treatment, two patients (10%) selected the 65R mutation. These results support the hypothesis that NEM hinder the selection of this mutation.

Factores asociados a la resistencia a los inhibidores de la proteasa del virus de la inmunodeficiencia humana

To assess the risk factors associated with genotypic resistance to protease inhibitors (PI) in HIV-infected subjects with virologic failure despite highly active antiretroviral treatment (HAART). Retrospective chart review including 47 consecutive patients with virologic failure despite PI-based HAART who had undergone a genotypic HIV-1 testing. The prevalence of genotypic resistance to PI was determined and several demographic, clinical and laboratory variables were compared between patients with and without genotypic resistance to those drugs. The entire nucleotide sequence of the protease gene was obtained in 43 of the 47 patients; 18 of them had genotypic resistance to PI. Genotypic resistance to PI was associated with a previous therapy with suboptimal antiretroviral regimens (OR = 10.2; 95% CI, 1.05-245.1; P = 0.02), duration of antiretroviral therapy longer than 18 months (OR = 13.3; 95% CI, 1.23-340.85; P = 0.01), greater number of antiretroviral regimens and drugs before the virologic failure (p < 0.01) and presence of the 184V mutation in the reverse transcriptase gene (OR = 5.6; 95% CI, 1.2-29.2; P = 0.02). There was no relationship between PI resistance and the risk group, viral load or CD4 cell count. In the multivariate analysis, previous therapy with suboptimal antiretroviral regimens was the better predictor of PI resistance (OR = 11.1; 95% CI, 1.04-117.47; P = 0.046). Patients treated with suboptimal antiretroviral activity regimens before starting HAART can be at greater risk of developing genotypic resistance to protease inhibitors.

Mechanism of action and in vitro activity of 1',3'-dioxolanylpurine nucleoside analogues against sensitive and drug-resistant human immunodeficiency virus type 1 variants.

(-)-Beta-D-1',3'-Dioxolane guanosine (DXG) and 2,6-diaminopurine (DAPD) dioxolanyl nucleoside analogues have been reported to be potent inhibitors of human immunodeficiency virus type 1 (HIV-1). We have recently conducted experiments to more fully characterize their in vitro anti-HIV-1 profiles. Antiviral assays performed in cell culture systems determined that DXG had 50% effective concentrations of 0.046 and 0.085 microM when evaluated against HIV-1(IIIB) in cord blood mononuclear cells and MT-2 cells, respectively. These values indicate that DXG is approximately equipotent to 2', 3'-dideoxy-3'-thiacytidine (3TC) but 5- to 10-fold less potent than 3'-azido-2',3'-dideoxythymidine (AZT) in the two cell systems tested. At the same time, DAPD was approximately 5- to 20-fold less active than DXG in the anti-HIV-1 assays. When recombinant or clinical variants of HIV-1 were used to assess the efficacy of the purine nucleoside analogues against drug-resistant HIV-1, it was observed that AZT-resistant virus remained sensitive to DXG and DAPD. Virus harboring a mutation(s) which conferred decreased sensitivity to 3TC, 2',3'-dideoxyinosine, and 2',3'-dideoxycytidine, such as a 65R, 74V, or 184V mutation in the viral reverse transcriptase (RT), exhibited a two- to fivefold-decreased susceptibility to DXG or DAPD. When nonnucleoside RT inhibitor-resistant and protease inhibitor-resistant viruses were tested, no change in virus sensitivity to DXG or DAPD was observed. In vitro drug combination assays indicated that DXG had synergistic antiviral effects when used in combination with AZT, 3TC, or nevirapine. In cellular toxicity analyses, DXG and DAPD had 50% cytotoxic concentrations of greater than 500 microM when tested in peripheral blood mononuclear cells and a variety of human tumor and normal cell lines. The triphosphate form of DXG competed with the natural nucleotide substrates and acted as a chain terminator of the nascent DNA. These data suggest that DXG triphosphate may be the active intracellular metabolite, consistent with the mechanism by which other nucleoside analogues inhibit HIV-1 replication. Our results suggest that the use of DXG and DAPD as therapeutic agents for HIV-1 infection should be explored.

Combination of mutations in human immunodeficiency virus type 1 reverse transcriptase required for resistance to the carbocyclic nucleoside 1592U89.

The carbocyclic nucleoside 1592U89 is a selective inhibitor of the human immunodeficiency virus (HIV), targeting the reverse transcriptase (RT). In vitro selection studies were undertaken to generate resistant variants with both HIV type 1 (HIV-1) wild-type strain HIV-1(HXB2) and 3'-azido-3'-deoxythymidine (AZT)-resistant strain HIV-1(RTMC). At least two or three mutations in RT were required to produce a 10-fold reduction in susceptibility. The first RT mutation selected was at codon 184, methionine (M) to valine (V), for HIV-1(HXB2) and HIV-1(RTMC), conferring two- and fivefold resistance, respectively. Two additional mutations were selected with HIV-1(HXB2), either leucine (L) 74 to V and lysine (K) 65 to arginine (R) (first-passage series) or L74 to V and tyrosine (Y) 115 to phenylalanine (F) (second-passage series). Cloned variants, obtained from the 1592U89 selection, were either double RT mutants 65R/184V and 74V/184V or triple RT mutant 74V/115Y/184V. Molecular clones were constructed with single, double, and triple combinations of these mutations for resistance analysis with different RT inhibitors. Each individual mutation conferred only low-level resistance (two- to fourfold) to 1592U89 in the HXB2 background. Double mutants containing the 184V mutation and triple mutants showed slightly greater levels of resistance to 1592U89 (7- to 11-fold). Some of the 1592U89-resistant variants were cross-resistant with 2',3'-dideoxycytidine, 2',3'-dideoxyinosine, and (-)-2'-deoxy-3'-thiacytidine, but none were resistant to 2',3'-didehydro-3'-deoxythymidine or AZT.