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Background Data on persistence of NS5A resistance-associated substitutions (RASs) may have implications for resistance testing approaches and selection of initial and retreatment strategies. Methods Long-term persistence of NS5A RASs in HCV genotype (GT) 1 infected subjects ( n=76) who did not achieve sustained virological response after receiving ledipasvir (LDV) without sofosbuvir (SOF) and were subsequently enrolled in an ongoing 3-year follow-up registry study was investigated by population or deep sequencing. Results Of the 76 subjects enrolled, 67 and 9 subjects had GT1a and GT1b infection, respectively. At pretreatment, NS5A RASs were detected in 14% of subjects (11/76) by population sequencing, with three subjects having >1 RAS. All RASs that were detected at pretreatment persisted and were observed at the 96 week visit in the follow-up study (FU96). For the remaining subjects with no detectable RASs at pretreatment, RASs were detected in 98% (63/64) of subjects at virological failure in the parent study and persisted at detectable levels through FU96 in 86% of subjects by deep sequencing (1% cutoff). However, a decline in the quasispecies frequency of most RASs and the number of RASs per subject was observed over time. Phenotypic analysis demonstrated that the majority of NS5A RASs confer similar levels of resistance to LDV and daclatasvir. Conclusions The majority of NS5A RASs can persist at detectable levels for >96 weeks post-treatment in subjects who failed treatment with regimens containing an NS5A inhibitor without SOF, suggesting relatively high fitness of NS5A RASs even in the absence of drug pressure.

Background Voxilaprevir (VOX; GS-9857) is a pangenotypic HCV NS3/4A protease inhibitor (PI) with potent antiviral activity against HCV genotypes (GTs) 1–6 and improved coverage of GT1 NS3 resistance-associated substitutions (RAS) associated with other HCV PIs. In a 3-day Phase Ib monotherapy study in patients infected with HCV GT1a, 1b, 2, 3 and 4, VOX was well-tolerated and resulted in maximal mean viral load reduction >3 log10 IU/ml at the 100 mg dose across all genotypes evaluated. This report characterizes the HCV NS3 RAS in the study. Methods The NS3 gene was amplified and successfully deep sequenced using MiSeq for 66 patients at baseline and 61 patients post-baseline using 15% and 1% assay cutoffs. Results With a 15% assay cutoff, pretreatment HCV NS3 RAS were present in the HCV of 38% (9/24) of patients with GT1a and 5% (1/19) with GT3a; there were no pre-treatment NS3 RAS present in patients with GT1b ( n=6), GT2 ( n=7) or GT4 ( n=4). In patients with and without pretreatment NS3 RAS, ≥3.4 log10 mean maximal viral load reductions over 3 days of VOX administration were observed. The majority of patients did not have detectable treatment-emergent NS3 RAS and only 12% (7/53) and 26% (14/53) had emergent NS3 RAS using 15% and 1% cutoffs, respectively. No NS3 RAS were detected in patients with GT2 or GT4. A156T or A156V were the most prevalent emergent NS3 RAS in patients with GT1a or GT1b infection, but were not observed in patients with GT3 infection. Conclusions The lack of selection of NS3 RAS in the majority of patients demonstrates a high resistance barrier for VOX. ClinicalTrails.gov identifier NCT02185794.

Background Sofosbuvir is a nucleoside analogue inhibitor of the HCV NS5B polymerase approved for treatment of HCV-infected patients in combination with ribavirin or with other antivirals. It has activity against all genotypes of HCV. Resistance to sofosbuvir in genotype-1 and -2 HCV is conferred by the S282T substitution in NS5B. Methods To begin to define the correlates of resistance to sofosbuvir in other genotypes, we performed selection experiments in cell culture using cell lines containing subgenomic replicons derived from genotypes-1b, -2a, -3a and -4a, or chimeric replicons in a genotype-1b background but encoding genotype-2b, -5a and -6a NS5B polymerase. Results In every case, S282T was selected following passage in the presence of increasing concentrations of sofosbuvir for 10 to 15 weeks. When introduced as a site-directed mutant, S282T conferred reductions in sofosbuvir susceptibility of between 2.4 and 19.4-fold. Other substitutions observed during the selections had relatively less impact on susceptibility, such as N237S in genotype-6a (2.5-fold). Replication capacity was affected by the introduction of S282T in all genotypes to variable extents (3.2% to 22% of wild type). Conclusions These results confirm that S282T is the primary sofosbuvir resistance-associated substitution and that replication capacity is reduced when it is present in all genotypes of HCV.

Background Tenofovir alafenamide (TAF) is a novel prodrug of the nucleotide reverse transcriptase inhibitor tenofovir (TFV) that loads lymphocytes with TFV-diphosphate more efficiently than tenofovir disoproxil fumarate (TDF). The single-tablet regimen (STR) comprising elvitegravir, cobicistat, emtricitabine and TAF (E/C/F/TAF) has demonstrated non-inferiority to the STR of E/C/F/TDF in clinical studies, with high proportions of subjects achieving HIV-1 RNA Methods Genotypic and phenotypic susceptibility to antiretrovirals (ARVs) was evaluated for subjects with HIV-1 RNA ≥400 copies/ml at time of virological failure (VF) or early discontinuation. Results Through week 96, VF resistance analyses were conducted for 24 subjects in each arm (2.8%, 24/866 and 2.8%, 24/867; for E/C/F/TAF and E/C/F/TDF arms, respectively). Primary resistance development to ARVs of the regimen occurred in 10 of 24 subjects in the E/C/F/TAF arm, and 8 of 24 subjects in the E/C/F/TDF arm (E/C/F/TAF: M184V/I, n=9; integrase strand-transfer inhibitor resistance-associated mutations [INSTI-RAMs], n=8; K65R/N, n=2; E/C/F/TDF: M184V/I, n=6; INSTI-RAMs, n=5; K65R/N, n=3). The emergent resistance mutations were similar between the treatment arms. Conclusions E/C/F/TAF achieved a high level of virological suppression in HIV-1 treatment-naive subjects through 96 weeks of treatment, with infrequent resistance development and comparable genotypic changes across both the E/C/F/TAF and E/C/F/TDF treatment groups.

Background The clinical impact of transient episodes of HIV viraemia (viral blips) on virological failure and resistance development is not fully understood. Here we investigated the blip frequency and virological outcomes of HIV-1-infected subjects experiencing viral blips among treatment-naive subjects initiating therapy on rilpivirine (RPV)/emtricitabine (FTC)/tenofovir disoproxil fumarate (TDF) or efavirenz (EFV)/FTC/TDF through 96 weeks of treatment. Methods Subjects treated with at least one dose of study drug and with at least one post-baseline HIV-1 RNA value were included in this analysis. All on-drug HIV-1 RNA data points and FDA snapshot outcome data through week 96 were utilized. A viral blip was defined as after achieving confirmed suppression (two consecutive HIV-1 RNA values Results Of the 717 subjects with confirmed suppression, 67 (9.3%) experienced ≥1 blip through week 96 with similar blip frequencies occurring in both treatment arms (10.7% RPV/FTC/TDF versus 8.0% EFV/FTC/TDF; P=0.25). A significantly higher proportion of subjects with baseline HIV-1 RNA >100,000 copies/ml experienced blips compared to subjects with baseline HIV-1 RNA ≤100,000 copies/ml and this was observed in both arms. Of 72 total blip events, 61 (85%) were low-level (50-199 copies/ml). Overall, among subjects with blips, 79% were virological successes at week 96, similar to those subjects without blips (83%; P=0.50). More subjects with blips ≥200 copies/ml experienced virological failure compared to subjects with blips Conclusions Viral blips were infrequent and similar among subjects treated with RPV/FTC/TDF or EFV/FTC/TDF. Most blips were low-level and most subjects with blips remained virologically suppressed through week 96 without experiencing virological failure.

Background The selection of antiretroviral (ARV) drugs for treatment of HIV-1 infection is based on several factors including potency, toxicity, resistance and ease of administration. Emtricitabine (FTC) or lamivudine (3TC), components of recommended initial ARV regimens, are structurally related and share the same resistance mutation (M184V/I). However they differ with respect to potency and incidence of M184V/I. Methods Resistance-associated mutation (RAM) prevalence data were obtained from genotype test results performed in a large reference laboratory from 2003–2010; subsets of data were defined by mutation pattern to resemble those following failure of non-nucleoside reverse transcriptase inhibitor (NNRTI)-based combination therapy. Mutational trend data were compared to contemporaneous ARV prescription information. Results In the unfiltered data set ( n=107,231), the prevalence in 2010 decreased compared to 2003 for all nucleoside/nucleotide reverse transcriptase inhibitor (NRTI) RAMs, such as M184V/I (44.0% to 17.9%), T215Y (22.7% to 4.1%), and K65R (4.3% to 2.1%). Among samples resembling those typical of first-line NNRTI-based failures, prevalence of K103N increased slightly, but prevalence of M184V/I decreased (49.8% to 36.8%), as did other NRTI RAMs. These decreases were coincident with a shift in ARV prescriptions away from zidovudine and 3TC towards tenofovir and FTC, and an increase in use of fixed-dose combinations. Conclusions RAM prevalence decreased substantially since 2003 among samples submitted for resistance testing in the US. The causes of this decrease are multifactorial, but our results suggest a possible role of increased use of potent ARVs that are available as fixed-dose combinations or as single-tablet regimens.

Background Antiretroviral therapy that targets HIV type-1 (HIV-1) reverse transcriptase (RT) can be linked to mutations in the thumb–connection (amino acids [AA] 241–426) and RNase H (AA 427–560) domains, which could affect drug resistance. Methods Genotypical and statistical analyses were performed on HIV-1 RT from 100 antiretroviral treatment-naive and 248 antiretroviral treatment-experienced patients, the majority of whom were infected with HIV-1 subtype B. The RT region was analysed in three parts: the polymerase (AA 1–240), thumb–connection (AA 241–426) and RNase H (AA 427–560) domains. Results The polymerase domain had statistically significant changes between the two groups at 24 AA positions that are known resistance sites. Within the thumb–connection domain, R284 and N348 had statistically significant changes between the groups ( P=0.007 and P≤0.001, respectively). In treatment-experienced patients, 17.3% had R284K, whereas 24.5% had N348I substitutions. Both R284 and N348 were 100% conserved in treatment-naive patients. Within the RNase H domain, only K451 showed a statistically significant change ( P≤0.001), with K451R present in 11% of treatment-experienced patients but remaining 100% conserved among treatment-naive patients. Conclusions RT mutations at three positions outside of the polymerase region were associated with antiretroviral therapy: R284K, N348I and K451R. Both R284K and K451R interact with the phosphate backbone of the template or primer in HIV-1 RT crystal structures and could potentially influence the positioning of the primer strand, thus affecting polymerization, the efficiency of nucleoside reverse transcriptase inhibitor excision and/or RNase H activity.

BackgroundNucleoside reverse transcriptase inhibitor (NRTI)-associated mutations (NAMs) can affect response to treatment with NRTIs and might also result in HIV-1 with reduced replication capacity.MethodsA large commercial HIV-1 database ( n=60,487) was analysed for the prevalence of NAMs, antiviral drug susceptibilities and viral replication capacity.ResultsThymidine analogue mutations (TAMs) and M184V were the most commonly observed NAMs (>25%). L74V/I was detected in 11% of isolates. K65R was detected in 3.3% of isolates and its frequency remained stable from 2003 to 2006, similar to trends observed for other NAMs. TAMs were rarely observed in combination with K65R, but frequently associated with L74V/I. HIV-1 with K65R or L74V/I alone were fully susceptible to zidovudine and stavudine. K65R was associated with reduced susceptibility to tenofovir, didanosine, abacavir and lamivudine; L74V/I was associated with reduced susceptibility to abacavir and didanosine. The addition of M184V to either K65R or L74V/I improved susceptibility to tenofovir, zidovudine and stavudine, but reduced susceptibility to abacavir, didanosine and lamivudine. Other NAMs commonly associated with K65R were A62V, S68G and Y115F; their NRTI susceptibilities were similar to those of viruses containing K65R alone. The replication capacity for HIV-1 with M184V/I or K65R was significantly reduced compared with wild-type (median 68% and 72%, respectively; PConclusionsThese results demonstrate a relative stability in the prevalence of HIV-1 clinical isolates with NAMs from 2003 to 2006. Differences between the genotypic patterns, phenotype and replication capacity associated with common NAMs are described.

Background Adefovir dipivoxil is a nucleotide prodrug approved for the treatment of chronic hepatitis B. During clinical trials, ADV-associated mutations were observed in 0, 3, 11, 18 and 29% of patients after 48, 96, 144, 192 and 240 weeks of therapy, respectively. Hepatitis B virus (HBV) polymerase mutations associated with virological breakthrough to ADV include rtA181V and rtN236T, which occur alone or in combination. The rtA181T mutation has also been observed at low frequency, alone or in combination with rtN236T. Methods To investigate the in vitro activity of adefovir and other anti-HBV agents against these mutants, we generated five stable cell lines that each expressed one of the following HBV mutants: rtN236T, rtA181V, rtA181V+rtN236T, rtA181T+rtN236T and rtA181T. Using these cell lines, we quantified in vitro changes in drug susceptibility for eight nucleotide/nucleoside analogues. Results The rtN236T mutant had 7-fold resistance to adefovir but remained sensitive to entecavir, telbivudine and torcitabine (≤3.2-fold reduced susceptibility). The A181V mutant had 4.3-fold resistance to adefovir and had reduced susceptibility to multiple other agents ranging from 3.2-fold (tenofovir) to >191-fold (clevudine). The A181V+rtN236T double mutant was the most highly resistant showing 18-fold resistance to adefovir and higher levels of resistance to other tested drugs with the exception of tenofovir (10-fold reduced susceptibility). Our results and preliminary clinical data suggest that patients with rtN236T or rtA181V remain susceptible to tenofovir, entecavir and lamivudine. Further clinical data are necessary to precisely define in vitro cutoffs indicative of clinically-relevant resistance, particularly for drugs in development such as emtricitabine, telbivudine, torcitabine and clevudine.

The HIV-1 nucleoside reverse transcriptase inhibitors (NRTIs) tenofovir (TFV), abacavir, didanosine and stavudine can select for K65R, whereas zidovudine (AZT) and stavudine can select for thymidine analogue mutations (TAMs) in HIV-1 reverse transcriptase (RT). HIV-1 with TAMs shows reduced susceptibility to all NRTIs, most notably AZT, whereas HIV-1 with K65R shows reduced susceptibility to all NRTIs except AZT. K65R and TAMs rarely occur together in patients. However, when present together, K65R can restore susceptibility to AZT. This study characterizes the underlying mechanisms of resistance of these RT mutants to TFV and AZT. K65R mediated decreased binding/incorporation of TFV and AZT (increased Ki/Kmof 7.1- and 4.3-fold, respectively), but also decreased excision of TFV and AZT (0.7- and 0.3-fold, respectively) when compared with wild-type RT. By contrast, TAMs mediated increased TFV and AZT excision (11- and 5.4-fold, respectively), and showed no changes in binding/incorporation. When these mutations were combined, K65R reversed TAM-mediated AZT resistance by strongly reducing AZT excision. Molecular modelling studies suggest that K65R creates additional hydrogen bonds that reduce the conformational mobility of RT, resulting in reduced polymerization and excision. Thus, consistent with clinical HIV-1 genotyping data, there appears to be no net NRTI resistance benefit for TAMs and K65R to develop together in patients taking AZT and TFV disoproxil fumarate, where the TAM pathway alone provides the greatest resistance for both drugs.

Several next-generation nucleoside and nucleotide analogues are currently in clinical development for the treatment of chronic hepatitis B. However, the efficacy of newer agents against lamivudine-resistant hepatitis B virus (HBV) has not been fully explored. To investigate this in vitro, we generated novel stable cell lines expressing HBV encoding the four major patterns of lamivudine resistance mutations (rtL180M+rtM204V, rtV173L+rtL180M+rtM204V, rtM204I and rtL180M+ rtM204I). Using these cell lines, we assessed the susceptibility of all four strains of lamivudine-resistant HBV to eleven nucleoside analogues in various stages of clinical development. Our studies indicate that lamivudine-resistant HBV remain sensitive to acyclic phosphonate nucleotides (adefovir, tenofovir, and alamifovir), have reduced susceptibility to entecavir, and have high-level cross-resistance to all L-nucleosides tested including emtricitabine, telbivudine, clevudine, and torcitabine.

Objective An anti-HIV regimen composed of the nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) tenofovir (TFV) disoproxil fumarate (TDF), abacavir (ABC) and lamivudine (3TC) has performed poorly in patients. This study evaluated the combination of TFV, ABC and 3TC for metabolic or antiviral antagonism in vitro. Design: Procedures were developed to evaluate the in vitro metabolism and antiviral activity of drug combinations of TFV, ABC and 3TC in cell types relevant for HIV infection. Methods Anabolism of combinations of TFV and ABC were studied over a 24 h period in the human T leukaemic CEM lymphoblast cell line and human primary peripheral blood mononuclear cells (PBMCs) stimulated with human interleukin-2 and phytohaemagglutinin. The anti-HIV activity of combinations of TFV and ABC in the presence or absence of 3TC was studied in stimulated PBMCs infected with the HXB2 strain of HIV-1. Results Levels of the active metabolites produced from TFV and ABC after incubation with CEM or PBMCs showed no significant change upon introduction of the other NRTI. Moreover, the pool sizes for the natural substrates of 2′-deoxyadenosine triphosphate and 2′-deoxyguanosine triphosphate were also unchanged. In anti-HIV assays in PBMCs, the combination of TFV and ABC was found to be additive with respect to inhibition of HIV replication. Addition of 3TC to the combination did not result in synergistic or antagonistic effects. Conclusions The poor efficacy of the triple NRTI regimen of TDF, ABC and 3TC is probably not due to a metabolic drug interaction resulting in antagonism of antiviral activity.

BackgroundTenofovir disoproxil fumarate (TDF) and emtricitabine (FTC) are nucleoside reverse transcriptase inhibitors (NRTIs) with once-daily dosing approved for use in HIV-1 infection. We evaluated this combination for anti-HIV activity and intracellular metabolic interactions in vitro.MethodsIntracellular anabolism of combinations of FTC and tenofovir (TFV) were studied in the human T leukaemic cell line CEM and human peripheral blood mononuclear cells (PBMCs). The anti-HIV activity of the combination was studied in the human T leukaemic MT-2 cell line against wild-type or mutant virus (K65R and M184V) and in PBMCs against wild-type virus. Antiviral synergy was quantitated by isobologram and MacSynergy methods.ResultsBoth TFV and FTC were efficiently converted to their active metabolites in PBMCs and CEM cells. In CEM cells, there was a statistically significant increase in the levels of TFV diphosphate ( P=0.047) and FTC triphosphate ( P=0.0069) following a 24 h incubation with the combination compared with the levels seen with the individual drugs. In PBMCs, similar levels of active metabolites were observed for each drug when they were incubated individually or in combination. The combination of TFV and FTC displayed additive to synergistic activity against HIV replication in PBMCs and resulted in strongly synergistic anti-HIV activity in MT-2 cells against both wild-type and mutant virus.ConclusionsThe combination of TFV and FTC showed additive to synergistic anti-HIV activity in vitro, which correlated with the levels of intracellular phosphorylation observed. These results support the use of these drugs as a dual NRTI backbone in combination therapy for the treatment of HIV-1.

In patients coinfected and treated for both HIV-1 and hepatitis C virus (HCV), administration of ribavirin (RBV) may result in altered intracellular drug levels of nucleoside reverse transcriptase inhibitors through inhibition of inosine 5′-monophosphate dehydrogenase. Drug interactions between tenofovir and RBV were studied in vitro in order to provide insights into the safety of co-administration of tenofovir disoproxil fumarate (DF) and RBV in HCV/HIV-1-coinfected patients. In accordance with previous in vitro studies, strongly increased anti-HIV activity was observed when RBV was combined with didanosine (ddI). In contrast, low-level anti-HIV antagonism was observed when RBV was combined with either tenofovir or abacavir. Significantly stronger anti-HIV antagonism was observed when RBV was combined with either zidovudine, stavudine, emtricitabine or lamivudine. Thus, although tenofovir and ddI are both adenosine analogues, their in vitro interactions with RBV are markedly different. These results suggest a low potential for increased toxicity upon co-administration of teno-fovir DF with RBV in patients.

Background Here, the baseline and emergent resistance to elvitegravir/cobicistat/emtricitabine/tenofovir disoproxil fumarate (EVG/COBI/FTC/TDF) versus efavirenz (EFV)/FTC/TDF in HIV-1-infected antiretroviral-naive adults through 144 weeks from the randomized, ongoing, Phase III study GS-US-236-0102 is described. Methods HIV-1 protease (PR) and reverse transcriptase (RT) were sequenced at screening; patients with HIV-1 resistant to EFV, FTC or TDF were excluded. Genotypic/phenotypic analyses were performed at virological failure confirmation and baseline for PR, RT and integrase (IN) for patients with virological failure and for patients with HIV-1 RNA≥400 copies/ml at weeks 48, 96, 144 or early study drug discontinuation. Retrospective, baseline, IN genotyping was conducted for EVG/COBI/FTC/ TDF patients. Results In the EVG/COBI/FTC/TDF group through week 144, HIV-1 from 10 patients (2.9%; 10/348 treated patients) developed primary IN strand transfer inhibitor ( n=9) and/or nucleoside RT inhibitor resistance substitutions ( n=10). The emergence of resistance decreased over time with 8, 2 and 0 patients developing HIV-1 resistance through week 48, post-week 48–96 and post-week 96–144, respectively. Emergent substitutions were E92Q ( n=7), N155H ( n=3), Q148R ( n=1) and T66I ( n=1) in IN, and M184V/I ( n=10) and K65R ( n=4) in RT. All 10 isolates had reduced susceptibility to EVG, FTC or TDF. Virus with EVG phenotypic resistance had cross-resistance to raltegravir. In the EFV/ FTC/TDF group, virus from 14 patients (4.0%; 14/352 treated patients; 4 during weeks 96–144) developed a resistance substitution to EFV ( n=14; K103N: n=13), FTC (M184V/I: n=4) or TDF (K65R: n=3). Conclusions Resistance development to EVG/COBI/FTC/ TDF was infrequent through 144 weeks of therapy and decreased over time, consistent with durable efficacy.

The presence of the HIV reverse transcriptase (RT) resistance mutation, M184V, induced by lamivudine and abacavir treatment results in increased tenofovir, adefovir and zidovudine susceptibility for HIV-1 with zidovudine-associated RT mutations in vitro. Treatment with oral prodrugs of tenofovir and adefovir has resulted in substantial HIV-1 RNA reductions in antiretroviral-experienced patient populations who have lamivudine-and zidovudine-resistant HIV-1. An enzymatic analysis was undertaken to elucidate the mechanisms of altered drug susceptibilities of HIV-1 containing zidovudine-associated mutations in the presence or absence of M184V. The inhibition constants (Ki) for the active metabolites of tenofovir, adefovir and zidovudine did not vary significantly between recombinant mutant and wild-type RT enzymes. Although increased removal of chain-terminating inhibitors by pyrophosphorolysis and ATP-dependent unblocking correlated with reduced susceptibility of viruses with zidovudine-associated mutations, a reduction in the removal of chain-terminators was not observed, which would explain the increased drug susceptibility of mutants containing M184V plus zidovudine-associated mutations. However, analyses of single-cycle processivity of the mutant RT enzymes on heteropolymeric RNA templates showed that all M184V-containing mutant RT enzymes were less processive than wild-type RT, most notably for mutants expressing both zidovudine-associated mutations and M184V. Similarly, the in vitro replication capacity of a mutant virus expressing a zidovudine-associated mutation and M184V was significantly reduced compared with wild-type virus. The observed decrease in enzymatic processivity of the M184V-expressing RT enzymes might result in decreased viral replication, which then might contribute to the increased drug susceptibility of HIV-1 expressing these RT mutations.

9-(2-phosphonomethoxypropyl)adenine (PMPA) has demonstrated remarkable anti-simian immunodeficiency virus (SIV) activity in macaque models of SIV infection and transmission prevention. Recently, PMPA and its oral prodrug, bis-POC PMPA, have also shown potent anti-human immunodeficiency virus type 1 (HIV-1) activity in Phase I clinical studies. In vitro experiments were performed to address the resistance properties of PMPA. After eight passages in increasing concentrations of PMPA, HIV-1IIIBwas able to grow in the presence of 2 μM PMPA, fivefold above the IC50of PMPA for wild-type parental virus. Sequence analysis of the reverse transcriptase (RT) genes from four of 15 RT clones demonstrated the presence of a K65R substitution in RT and recombinant HIV expressing the K65R RT mutation showed a threefold to fourfold increase in IC50value for PMPA as compared to wild-type. Additional experiments demonstrated that viruses expressing other nucleoside-associated RT resistance mutations all showed wild-type or

Antiretroviral therapy that targets HIV type-1 (HIV-1) reverse transcriptase (RT) can be linked to mutations in the thumb-connection (amino acids [AA] 241-426) and RNase H (AA 427-560) domains, which could affect drug resistance.Genotypical and statistical analyses were performed on HIV-1 RT from 100 antiretroviral treatment-naive and 248 antiretroviral treatment-experienced patients, the majority of whom were infected with HIV-1 subtype B. The RT region was analysed in three parts: the polymerase (AA 1-240), thumb-connection (AA 241-426) and RNase H (AA 427-560) domains.The polymerase domain had statistically significant changes between the two groups at 24 AA positions that are known resistance sites. Within the thumb-connection domain, R284 and N348 had statistically significant changes between the groups (P=0.007 and Por =0.001, respectively). In treatment-experienced patients, 17.3% had R284K, whereas 24.5% had N348I substitutions. Both R284 and N348 were 100% conserved in treatment-naive patients. Within the RNase H domain, only K451 showed a statistically significant change (P/=0.001), with K451R present in 11% of treatment-experienced patients but remaining 100% conserved among treatment-naive patients.RT mutations at three positions outside of the polymerase region were associated with antiretroviral therapy: R284K, N348I and K451R. Both R284K and K451R interact with the phosphate backbone of the template or primer in HIV-1 RT crystal structures and could potentially influence the positioning of the primer strand, thus affecting polymerization, the efficiency of nucleoside reverse transcriptase inhibitor excision and/or RNase H activity.

Alterations in endogenous nucleotide pools as a result of HIV therapy with nucleoside and nucleotide reverse transcriptase inhibitors (N[t]RTIs) is a proposed mechanism for therapy-related adverse events and drug interactions resulting in treatment failure. In vitro studies were performed in order to understand the effect of N(t)RTIs on endogenous nucleotide pools.The T-cell line CEM-CCRF was treated with control antimetabolites or the N(t)RTIs abacavir, didanosine, lamivudine, tenofovir (TFV) and zidovudine (AZT), either alone or in combination. The levels of natural 2'-deoxynucleoside triphosphates (dNTP) and ribonucleoside triphophosphates were determined by liquid chromatography coupled with triple quadrupole mass spectrometry.Antimetabolites altered nucleotide pools in a manner consistent with their known mechanisms of action. AZT was the only N(t)RTI that significantly altered dNTP pools. incubation of 10 microM AZT, either alone or in combination with other N(t)RTIs, increased 2'-deoxyadenosine triphosphate, 2'-deoxyguanosine triphosphate and thymidine triphosphate levels by up to 1.44-fold the concentrations observed in untreated cells. At higher than pharmacological concentrations of AZT, evidence for inhibition of 2'-deoxycytidylate deaminase and enzymes involved in the salvage of thymidine was also observed. Phosphorylated metabolites of TFV are known to inhibit purine nucleoside phosphorylase (PNP). However, in contrast to a potent PNP inhibitor, TFV was unable to alter intracellular dNTP pools upon addition of exogenous 2'-deoxyguanosine.N(t)RTIs have the potential to alter nucleotide pools; however, at the pharmacologically relevant concentrations, tested N(t)RTI or their combinations did not have an effect on nucleotide pools with the notable exception of AZT.

Nucleoside reverse transcriptase inhibitor (NRTI)-associated mutations (NAMs) can affect response to treatment with NRTIs and might also result in HIV-1 with reduced replication capacity.A large commercial HIV-1 database (n=60,487) was analysed for the prevalence of NAMs, antiviral drug susceptibilities and viral replication capacity.Thymidine analogue mutations (TAMs) and M184V were the most commonly observed NAMs (25%). L74V/I was detected in 11% of isolates. K65R was detected in 3.3% of isolates and its frequency remained stable from 2003 to 2006, similar to trends observed for other NAMs. TAMs were rarely observed in combination with K65R, but frequently associated with L74V/I. HIV-1 with K65R or L74V/I alone were fully susceptible to zidovudine and stavudine. K65R was associated with reduced susceptibility to tenofovir, didanosine, abacavir and lamivudine; L74V/I was associated with reduced susceptibility to abacavir and didanosine. The addition of M184V to either K65R or L74V/I improved susceptibility to tenofovir, zidovudine and stavudine, but reduced susceptibility to abacavir, didanosine and lamivudine. Other NAMs commonly associated with K65R were A62V, S68G and Y115F; their NRTI susceptibilities were similar to those of viruses containing K65R alone. The replication capacity for HIV-1 with M184V/I or K65R was significantly reduced compared with wild-type (median 68%/ and 72%, respectively; P0.0001), whereas replication capacity for HIV-1 with L74V or TAMs was not significantly reduced (88% and 97%, respectively).These results demonstrate a relative stability in the prevalence of HIV-1 clinical isolates with NAMs from 2003 to 2006. Differences between the genotypic patterns, phenotype and replication capacity associated with common NAMs are described.

Tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC) are nucleoside reverse transcriptase inhibitors (NRTIs) with once-daily dosing approved for use in HIV-1 infection. We evaluated this combination for anti-HIV activity and intracellular metabolic interactions in vitro.Intracellular anabolism of combinations of FTC and tenofovir (TFV) were studied in the human T leukaemic cell line CEM and human peripheral blood mononuclear cells (PBMCs). The anti-HIV activity of the combination was studied in the human T leukaemic MT-2 cell line against wild-type or mutant virus (K65R and M184V) and in PBMCs against wild-type virus. Antiviral synergy was quantitated by isobologram and MacSynergy methods.Both TFV and FTC were efficiently converted to their active metabolites in PBMCs and CEM cells. In CEM cells, there was a statistically significant increase in the levels of TFV diphosphate (P=0.047) and FTC triphosphate (P=0.0069) following a 24 h incubation with the combination compared with the levels seen with the individual drugs. In PBMCs, similar levels of active metabolites were observed for each drug when they were incubated iindividually or in combination. The combination of TFV and FTC displayed additive to synergistic activity against HIV replication in PBMCs and resulted in strongly synergistic anti-HIV activity in MT-2 cells against both wild-type and mutant virus.The combination of TFV and FTC showed additive to synergistic anti-HIV activity in vitro, which correlated with the levels of intracellular phosphorylation observed. These results support the use of these drugs as a dual NRTI backbone in combination therapy for the treatment of HIV-1.

An anti-HIV regimen composed of the nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) tenofovir (TFV) disoproxil fumarate (TDF), abacavir (ABC) and lamivudine (3TC) has performed poorly in patients. This study evaluated the combination of TFV, ABC and 3TC for metabolic or antiviral antagonism in vitro.Procedures were developed to evaluate the in vitro metabolism and antiviral activity of drug combinations of TFV, ABC and 3TC in cell types relevant for HIV infection.Anabolism of combinations of TFV and ABC were studied over a 24 h period in the human T leukaemic CEM lymphoblast cell line and human primary peripheral blood mononuclear cells (PBMCs) stimulated with human interleukin-2 and phytohaemagglutinin. The anti-HIV activity of combinations of TFV and ABC in the presence or absence of 3TC was studied in stimulated PBMCs infected with the HXB2 strain of HIV-1.Levels of the active metabolites produced from TFV and ABC after incubation with CEM or PBMCs showed no significant change upon introduction of the other NRTI. Moreover, the pool sizes for the natural substrates of 2'-deoxyadenosine triphosphate and 2'-deoxyguanosine triphosphate were also unchanged. In anti-HIV assays in PBMCs, the combination of TFV and ABC was found to be additive with respect to inhibition of HIV replication. Addition of 3TC to the combination did not result in synergistic or antagonistic effects.The poor efficacy of the triple NRTI regimen of TDF, ABC and 3TC is probably not due to a metabolic drug interaction resulting in antagonism of antiviral activity.

In patients coinfected and treated for both HIV-1 and hepatitis C virus (HCV), administration of ribavirin (RBV) may result in altered intracellular drug levels of nucleoside reverse transcriptase inhibitors through inhibition of inosine 5'-monophosphate dehydrogenase. Drug interactions between tenofovir and RBV were studied in vitro in order to provide insights into the safety of co-administration of tenofovir disoproxil fumarate (DF) and RBV in HCV/HIV-1-coinfected patients. In accordance with previous in vitro studies, strongly increased anti-HIV activity was observed when RBV was combined with didanosine (ddl). In contrast, low-level anti-HIV antagonism was observed when RBV was combined with either tenofovir or abacavir. Significantly stronger anti-HIV antagonism was observed when RBV was combined with either zidovudine, stavudine, emtricitabine or lamivudine. Thus, although tenofovir and ddl are both adenosine analogues, their in vitro interactions with RBV are markedly different. These results suggest a low potential for increased toxicity upon co-administration of tenofovir DF with RBV in patients.