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Objective: Persistence of the viral reservoir is the main barrier to curing HIV. Initiation of ART during acute HIV infection can limit the size and diversity of the reservoir. In depth characterization of the reservoir in individuals who initiate ART during acute infection will be critical for clinical trial design and cure strategies. Methods: Four cohortswith participantswhoinitiatedARTduring acute infection or during chronic infectionwere enrolled in a cross-sectional, noninterventional study.Viral reservoir was evaluated by the Intact Proviral DNA Assay (IPDA), the Total HIVDNA Assay (THDA) and the Quantitative Viral Outgrowth Assay (QVOA). Viral diversity and susceptibility to V3-glycan bNAbs were determined by genotyping of the viral envelope gene. Results: Participants who initiated ART during the acute Fiebig I-IV stages had lower level of total HIV DNA than participants who initiated ART during chronic infection whereas no difference was observed in intact HIV DNA or outgrowth virus. Participants who initiated ART during Fiebig I-IV also had lower viral diversity and appeared to have higher susceptibility to bNAbs than participants initiating ART during chronic infection. Conclusion: Individuals initiating ART during Fiebig I-IV had small viral reservoirs, low viral diversity, and high susceptibility to bNAbs, and would be an optimal target population for proof-of-concept HIV cure trials.

Background Lenacapavir (LEN), a potent first-in-class inhibitor of HIV-1 capsid function, is in development as a long-acting agent for treatment and prevention of HIV-1. Methods CAPELLA is an ongoing, phase 2/3 study in heavily treatment-experienced (HTE) people with HIV-1 (PWH) with multidrug-resistance. 36 participants were randomized (2:1) to add oral LEN or placebo to their failing regimen. At D15, those on oral LEN received subcutaneous (SC) LEN 927 mg (Q6M); those on placebo started the oral LEN lead-in, followed by SC Q6M. All randomized participants initiated an investigator-selected, optimized background regimen (OBR) at D15. An additional 36 participants started OBR concurrent with LEN (oral lead-in → SC) in a non-randomized cohort. We report the Week (W) 52 efficacy and safety results from both cohorts. Results Of 72 participants enrolled, 25% were female, 38% Black, median age 52 years, 64% had CD4 < 200 cells/µL, 46% had HIV-1 resistant to all 4 major classes (NRTI, NNRTI, PI, INSTI), and 53% had OBR with 1 or no fully active agents. At W52, 78% (56/72) achieved VL< 50 c/mL and 82% (59/72) achieved VL< 200 c/mL via FDA Snapshot algorithm. CD4 count increased by a median 84 cells/µL (Q1 to Q3: 21 to 153) and the proportion of participants with CD4 count ≥200 cells/ul increased from 36% at baseline to 68% at W52. Ten participants had emergent LEN resistance (8 previously reported); 4 of 10 subsequently suppressed. The median (range) duration of follow up on LEN was 71 (13–111) weeks. One participant discontinued due to injection site nodule (Grade 1). The most common injection site reaction (ISR) was swelling (28% [20/72] and 17% [12/70] after the 1st and 2nd SC doses, respectively). Most ISRs were mild or moderate. The most common AEs (excluding injection site reactions) were nausea and diarrhea (14% each). Conclusion In HTE PWH, subcutaneous LEN was well tolerated and in combination with OBR led to high and sustained rate of virologic suppression at W52. These results support the potential role for LEN for treatment of multi-drug resistant HIV-1 infection. Disclosures Sorana Segal-Maurer, MD, Gilead Sciences: Advisor/Consultant|Gilead Sciences: Grant/Research Support|Gilead Sciences: Honoraria|JANSSEN THERAPEUTICS: Honoraria|ViiV: Honoraria Benoit Trottier, MD, Gilead Sciences: Advisor/Consultant|Gilead Sciences: Honoraria|Merck: Advisor/Consultant|Merck: Honoraria|ViiV Healthcare: Advisor/Consultant|ViiV Healthcare: Honoraria Jason Brunetta, M.D., Gilead Canada: Advisor/Consultant|Gilead Canada: Honoraria|Gilead Canada: Conference Attendance Sponsorship|Viiv Canada: Advisor/Consultant Hui Wang, PhD, Gilead Sciences: Employment|Gilead Sciences: Stocks/Bonds Nicolas A. Margot, MA, Gilead Sciences: Employment|Gilead Sciences: Stocks/Bonds Hadas Dvory-Sobol, PhD, Gilead Sciences: Employment|Gilead Sciences: Stocks/Bonds Martin S Rhee, MD, Gilead Sciences: Stocks/Bonds Jared Baeten, MD, PhD, Gilead Sciences: Employee|Gilead Sciences: Stocks/Bonds Jean-Michel Molina, MD; PhD, GIlead: Board Member|GIlead: Grant/Research Support|Merck: Board Member|Merck: Expert Testimony|ViiV: Board Member.

Patients with multidrug-resistant human immunodeficiency virus type 1 (HIV-1) infection have limited treatment options. Lenacapavir is a first-in-class capsid inhibitor that showed substantial antiviral activity in a phase 1b study.In this phase 3 trial, we enrolled patients with multidrug-resistant HIV-1 infection in two cohorts, according to the change in the plasma HIV-1 RNA level between the screening and cohort-selection visits. In cohort 1, patients were first randomly assigned in a 2:1 ratio to receive oral lenacapavir or placebo in addition to their failing therapy for 14 days; during the maintenance period, starting on day 15, patients in the lenacapavir group received subcutaneous lenacapavir once every 6 months, and those in the placebo group received oral lenacapavir, followed by subcutaneous lenacapavir; both groups also received optimized background therapy. In cohort 2, all the patients received open-label oral lenacapavir with optimized background therapy on days 1 through 14; subcutaneous lenacapavir was then administered once every 6 months starting on day 15. The primary end point was the percentage of patients in cohort 1 who had a decrease of at least 0.5 logA total of 72 patients were enrolled, with 36 in each cohort. In cohort 1, a decrease of at least 0.5 logIn patients with multidrug-resistant HIV-1 infection, those who received lenacapavir had a greater reduction from baseline in viral load than those who received placebo. (Funded by Gilead Sciences; CAPELLA ClinicalTrials.gov number, NCT04150068.).

Background Vesatolimod (VES; GS-9620) is a Toll-like receptor 7 (TLR7) agonist that directly activates human plasmacytoid dendritic cells (pDCs) and B lymphocytes resulting in direct and indirect production of cytokines and immune activation. VES is being evaluated in HIV-1-infected people as part of an HIV remission strategy. Here we investigated the potential of VES to trigger indirect activation of HIV-specific CD8+ T-cells using immune cell cultures derived from HIV+ donors. Methods Peripheral blood mononuclear cell (PBMC) cultures derived from HIV+ donors virologically suppressed on stable antiretroviral therapy ( n=31) were isolated and treated with VES or vehicle for 24 h. Cells were stained with surface and intracellular fluorescent conjugated antibodies and HIV-specific pentamers, and analysed by flow cytometry. Results Treatment of PBMCs with VES resulted in all 31 donors demonstrating a concentration dependent increase in CD8+ T-cell activation (CD69+) of up to 88%. Of these donors, 20 of 31 donors displayed a concentration-dependent increase in HIV-specific CD8+ T-cell activation due to VES with a maximum of 20.8%. Intracellular staining was performed in a subset of donors ( n=14), 5 of which displayed VES-induced activation of functional HIV-specific CD8+ T-cells as assessed by CD107a and/or tumour necrosis factor (TNF)-α upregulation. Conclusions This study demonstrates that VES treatment can induce the activation of functional HIV-specific CD8+ T-cells in donor derived PBMCs. These data support the potential use of VES to activate functional HIV-specific CD8+ T-cells as part of an HIV remission strategy.

Lenacapavir (LEN; GS-6207) is a potent first-in-class inhibitor of HIV-1 capsid with long-acting properties and the potential for subcutaneous dosing every 3 months or longer. In the clinic, a single subcutaneous LEN injection (20 mg to 750 mg) in people with HIV (PWH) induced a strong antiviral response, with a >2.3 mean log10 decrease in HIV-1 RNA at day 10. HIV-1 Gag mutations near protease (PR) cleavage sites have emerged with the use of protease inhibitors (PIs). Here, we have characterized the activity of LEN in mutants with Gag cleavage site mutations (GCSMs) and mutants resistant to other drug classes. HIV mutations were inserted into the pXXLAI clone, and the resulting mutants (n = 70) were evaluated using a 5-day antiviral assay. LEN EC50 fold change versus the wild type ranged from 0.4 to 1.9 in these mutants, similar to that for the control drug. In contrast, reduced susceptibility to PIs and maturation inhibitors (MIs) was observed. Testing of isolates with resistance against the 4 main classes of drugs (n = 40) indicated wild-type susceptibility to LEN (fold change ranging from 0.3 to 1.1), while reduced susceptibility was observed for control drugs. HIV GCSMs did not impact the activity of LEN, while some conferred resistance to MIs and PIs. Similarly, LEN activity was not affected by naturally occurring variations in HIV Gag, in contrast to the reduced susceptibility observed for MIs. Finally, the activity of LEN was not affected by the presence of resistance mutations to the 4 main antiretroviral (ARV) drug classes. These data support the evaluation of LEN in PWH with multiclass resistance.

Background: The ability of elvitegravir/cobicistat/emtricitabine/tenofovir alafenamide (E/C/F/TAF) to maintain virologic suppression in participants with M184V and/or M184I resistance mutations from historical genotypic reports when switching from a tenofovir disoproxil fumarate-based or abacavir (ABC)-based regimen was investigated. Setting: Phase IIIb, 48-week, open-label, single-arm, multicenter, clinical trial (NCT02616029). Methods: Virologically suppressed adults with HIV and documented M184V/I on historical genotypic records switched to E/C/F/TAF from a tenofovir disoproxil fumarate-based or ABC-based regimen. The primary end point was HIV-1 RNA of = 50 copies per milliliter (week 12); confirmatory HIV-1 RNA was

Background GS-6207 is a first-in-class HIV capsid inhibitor, targeting several functions of the HIV capsid in the viral cycle, including viral particle assembly, capsid formation and nuclear entry. GS-6207 has demonstrated picomolar potency in vitro, activity confirmed by high potency in a Phase 1 clinical study, with a long-acting antiretroviral profile with potential dosing every 6 months. In vitro resistance selections previously conducted with increasing doses of GS-6207 have identified capsid variants with reduced susceptibility to GS-6207. Objectives To study the prevalence of capsid mutations associated with in vitro resistance to GS-6207 in people living with HIV (PLWH). Methods Plasma samples from ART-naive or -experienced PLWH, including PI-experienced people, were sequenced and analysed for the presence of capsid variants identified during in vitro resistance selection: L56I, M66I, Q67H, K70N, N74D, N74S and T107N. Results Among the samples from the 1500 patients studied, none of the seven GS-6207 resistance mutations identified during in vitro selection experiments was detected, regardless of HIV subtype or PLWH treatment history. Conclusions Out of the seven HIV capsid substitutions previously selected in vitro and shown to confer phenotypic resistance to GS-6207, none of these seven mutations was observed in this large dataset, suggesting that neither PLWH with previous PI failure nor PLWH with emergence of PI resistance mutations are anticipated to impact GS-6207 activity in these diverse HIV-infected populations.

BACKGROUND The M184V/I reverse transcriptase mutation, which confers major resistance to lamivudine and emtricitabine, is still quite frequent in people living with HIV. The underlying presence of the M184V/I mutation may undermine virological outcomes of ART, particularly in the context of proposed treatment with two-drug combinations that include drugs affected by M184V, such as lamivudine. In suppressed patients for whom historical data are seldom available, resistance assays evaluating integrated viral DNA can help select a fully active switch regimen. OBJECTIVES To compare detectability of M184V/I in historical HIV-1 RNA analyses versus HIV-1 DNA sequencing. METHODS We analysed the detection of the M184V/I mutation in a prospective study and compared HIV historical genotypes (plasma) versus integrated HIV DNA (PBMCs) obtained via a validated commercial proviral HIV DNA assay. Eligible participants had HIV-1 RNA

Resistance selection experiments using HIV-1 isolates harboring pre-existing tenofovir (TFV)-resistance (K65R, 3TAMs, and Q151M complex) were carried out with the novel tenofovir prodrug tenofovir alafenamide (TAF) as well as with tenofovir (TFV), to investigate the potential for additional resistance development in the presence of TAF or TFV. Extended resistance selection of these TFV resistance associated mutations (RAMs)-containing viruses with TAF or TFV did not lead to the accumulation of additional known RAMs, or significant additional phenotypic resistance, after 6 months in culture. Two new mutations were found during the selections (L429I, T69I) that were further characterized, and found to have very limited or no role in resistance to TAF or TFV. Notably, viral survival in the presence of drug increases could not be sustained and led to viral cure in cell culture, suggesting a lack of alternative resistance pathways for the mutant viruses.

Plasma HIV-1 RNA (viral load, VL) is measured routinely in HIV-infected persons with FDA-approved commercially available assays such as the Cobas-TaqMan HIV-1 Assay v2.0. This assay provides quantification of viremia ≥20 copies/mL. More sensitive methods, able to quantify low-level persistent viremia below the detection limit of commercially available assays, are needed to assess the impact of current HIV cure strategies on viremia.The novel integrase HIV-1 RNA single-copy assay (iSCA) was evaluated for measurement of low-level persistent viremia in clinical trial samples (n = 151) from subjects participating in Gilead HIV clinical research.Paired plasma samples from HIV-1-infected patients treated with combination ART were assessed using both HIV-1 Cobas-TaqMan and iSCA; results from the two assays were compared.Paired Cobas-TaqMan/iSCA data were obtained for 151 HIV-infected adults. Most samples (117/151, 77%) had non-quantifiable Cobas-TaqMan result, either20 copies/mL ("20") or "Target Not Detected" (TND). All 117 non-quantified samples were quantified with iSCA and showed higher HIV-1 RNA levels in samples with20 than TND Cobas-TaqMan results (p 0.0001).In this large sample collection from virologically suppressed HIV-infected adults, use of iSCA led to quantification of low-level viremia below the limit of detection of the Cobas-TaqMan assay in all 117 previously non-quantifiable plasma samples. These data confirm the value of the iSCA as a helpful addition to the classical HIV VL assays and its potential for use in HIV cure studies to assess whether experimental interventions alter viremia.

The development of resistance to human immunodeficiency virus 1 (HIV-1) integrase strand-transfer inhibitors (INSTI) has been documented; however, knowledge of the impact of pre-existing integrase (IN) mutations on INSTI resistance (INSTI-R) is still evolving. The frequency of HIV-1 IN mutations in 2177 treatment-naive subjects was investigated, along with the INSTI susceptibility of site-directed mutant viruses containing major and minor INSTI-R mutations. Total 6 of 39 minor INSTI-R mutations (M50I, S119P/G/T/R, and E157Q) were found in >1% of IN-treatment-naive subjects with no impact on INSTI susceptibility. When each combined with major INSTI-R mutation, M50I, S119P, and E157Q led to decreased susceptibility to elvitegravir but remained sensitive to dolutegravir and bictegravir.

An human immunodeficiency virus (HIV)-1 integrase (IN) genotyping assay was developed to evaluate clinical samples from patients infected with HIV-1 subtype AE, a subtype highly prevalent in Asia. The HIV-1 IN gene was amplified from plasma-derived HIV-1 viral RNA via reverse transcription polymerase chain reaction followed by population sequencing. Assay sensitivity was also evaluated using serially diluted plasma samples. The genotyping assay successfully amplified the IN gene from patient plasma samples with HIV-1 RNA as low as 500 copies/mL. This assay is suitable for IN genotyping to identify IN drug resistance mutations in HIV-1 patients harboring subtype AE virus.

Differences in emergence of HIV resistance between subtypes B and C in vitro and potential implications on tenofovir alafenamide efficacy in vivo were evaluated. Dose escalation resistance selections showed K65R emerging earlier for subtype C viruses in vitro, as previously reported. Viral breakthrough experiments at therapeutic drug concentrations, however, showed no difference in time to breakthrough between these subtypes. Finally, clinical trial data found no evidence of greater K65R emergence in patients harboring subtype C HIV.

We previously demonstrated superior efficacy and safety advantages in HIV-infected, virologically suppressed adults switched to a regimen containing tenofovir alafenamide (TAF) as compared with those remaining on a tenofovir disoproxil fumarate (TDF) regimen through week 48. We now report long-term data through week 96. In this randomized, active-controlled, multicenter, open-label, noninferiority trial (ClinicalTrials.gov No. NCT01815736), we randomized virologically suppressed (HIV-1 RNA50 copies/ml) adults (2:1) to receive a once-daily, single-tablet regimen containing elvitegravir (EVG), cobicistat (COBI), emtricitabine (FTC), and TAF group or to continue one of four TDF-containing regimens (TDF group) for 96 weeks. We evaluated efficacy (HIV-1 RNA50 copies/ml using the FDA snapshot algorithm) and prespecified bone and renal endpoints at week 96. We randomized and treated 1,436 participants in this study (TAF n = 959, TDF n = 477). At week 96, TAF was superior to TDF in virologic efficacy, with 93% on TAF and 89% on TDF having HIV-1 RNA50 copies/ml (difference 3.7%, 95% confidence interval: 0.4%-7.0%). Improvements in hip and spine bone mineral density for those assigned to TAF versus TDF continued through week 96 (p .001). Significant improvements in urine protein or albumin to creatinine ratios were also seen among those in the TAF group versus TDF through week 96 (p .001). There were no cases of investigator-reported proximal renal tubulopathy in the TAF group as compared with one case in the TDF group. Switching to EVG/COBI/FTC/TAF (E/C/F/TAF) was associated with statistically significant efficacy and safety advantages over remaining on a standard-of-care TDF-based regimen.

T97A is an HIV-1 integrase polymorphism associated with integrase strand transfer inhibitor (INSTI) resistance. Using pooled data from 16 clinical studies, we investigated the prevalence of T97A (pre-existing and emergent) and its impact on INSTI susceptibility and treatment response in INSTI-naive patients who enrolled on elvitegravir (EVG)- or raltegravir (RAL)-based regimens. Prior to INSTI-based therapy, primary INSTI resistance-associated mutations (RAMs) were absent and T97A pre-existed infrequently (1.4%; 47 of 3367 integrase sequences); most often among non-B (5.3%) than B (0.9%) HIV-1 subtypes. During INSTI-based therapy, few patients experienced virologic failure with emergent INSTI RAMs (3%; 122 of 3881 patients), among whom T97A emerged infrequently in the presence (n = 6) or absence (n = 8) of primary INSTI RAMs. A comparison between pre-existing and emergent T97A patient populations (i.e., in the absence of primary INSTI RAMs) showed no significant differences in EVG or RAL susceptibility in vitro. Furthermore, among all T97A-containing viruses tested, only 38-44% exhibited reduced susceptibility to EVG and/or RAL (all of low magnitude

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 presence of transmitted drug resistance mutations (TDRMs) in antiretroviral treatment (ART)-naive patients can adversely affect the outcome of ART. Methods Resistance testing was conducted in 6704 ART-naive subjects predominantly from the United States and Europe in 9 clinical studies conducted by Gilead Sciences from 2000 to 2013. Results The presence of TDRMs increased during this period (from 5.2% to 11.4%), primarily driven by an increase in nonnucleoside reverse-transcriptase (RT) inhibitor (NNRTI) resistance mutations (from 0.3% to 7.1%), particularly K103N/S (increase from 0.3% to 5.3%). Nucleoside/nucleotide RT inhibitor mutations were found in 3.1% of patients. Only 1 patient had K65R (0.01%) and 7 had M184V/I (0.1%), despite high use of tenofovir disoproxil fumarate (TDF), emtricitabine, and lamivudine and potential transmission of resistance to these drugs. At least 1 thymidine-analogue mutations was present in 2.7% of patients with 0.07% harboring T215Y/F and 2.7% harboring T215 revertant mutations (T215rev). Patients with the combination of M41L + L210W + T215rev showed full human immunodeficiency virus RNA suppression while receiving a TDF- or tenofovir alafenamide-containing regimen. Conclusions There was an overall increase of TDRMs among patients enrolling in clinical trials from 2000 through 2013, driven primarily by an increase in NNRTI resistance. However, the presence of common TDRMs, including thymidine-analogue mutations/T215rev, showed no impact on response to TDF- or tenofovir alafenamide-containing regimens.

Tenofovir alafenamide (TAF) is a new oral prodrug of tenofovir (TFV) recently approved for the treatment of HIV-1 as part of the single-tablet regimen containing elvitegravir, cobicistat, emtricitabine, and TAF. Clinical dosing with TAF vs. tenofovir disoproxil fumarate (TDF) has shown improved bone and kidney safety, and has been associated with an increased concentration of the anti-HIV active moiety tenofovir diphosphate (TFV-DP) in the PBMCs of treated patients and a reduction of TFV systemic exposure. We have studied the potential benefit of this increased concentration of TFV-DP observed clinically in an in vitro model system. Using a newly developed virus breakthrough assay with TAF exposure set at physiological concentrations, we show that HIV-1 clinical isolates harboring TFV resistance mutations such as K65R, 3 or 4 thymidine-analog mutations (TAMs), Q151M/K65R, or T69 insertion complex could be inhibited by TAF, but not by TFV when used at clinically relevant concentrations for TDF. These data suggest that the inhibitory quotient (IQ) of TAF is projected to be higher than the IQ of TDF, and that TAF has the potential to inhibit viruses containing TDF resistance in the clinic.

Tenofovir alafenamide (TAF), a novel prodrug of the NtRTI tenofovir (TFV), delivers TFV-diphosphate (TFV-DP) to target cells more efficiently than the current prodrug, tenofovir disoproxil fumarate (TDF), with a 90% reduction in TFV plasma exposure. TAF, within the fixed dose combination of elvitegravir /cobicistat / emtricitabine (FTC)/TAF (E/C/F/TAF), has been evaluated in one Phase 2 and two Phase 3 randomized, double-blinded studies in HIV-infected treatment-naive patients, comparing E/C/F/TAF to E/C/F/TDF. In these studies, the TAF-containing group demonstrated non-inferior efficacy to the TDF-containing comparator group with 91.9% of E/C/F/TAF patients having50 copies/mL of HIV-1 RNA at week 48. An integrated resistance analysis across these three studies was conducted, including HIV-1 genotypic analysis at screening, and genotypic/phenotypic analysis for patients with HIV-1 RNA400 copies/mL at virologic failure. Pre-existing primary resistance-associated mutations (RAMs) were observed at screening among the 1903 randomized and treated patients: 7.5% had NRTI-RAMs, 18.2% had NNRTI-RAMs, and 3.4% had primary PI-RAMs. Pre-treatment RAMs did not influence treatment response at Week 48. In the E/C/F/TAF group, resistance development was rare; seven patients (0.7%, 7/978) developed NRTI-RAMs, five of whom (0.5%, 5/978) also developed primary INSTI-RAMs. In the E/C/F/TDF group, resistance development was also rare; seven patients (0.8%, 7/925) developed NRTI-RAMs, four of whom (0.4%, 4/925) also developed primary INSTI-RAMs. An additional analysis by deep sequencing in virologic failures revealed minimal differences compared to population sequencing. Overall, resistance development was rare in E/C/F/TAF-treated patients, and the pattern of emergent mutations was similar to E/C/F/TDF.

BACKGROUND Study GS-01-934 was a randomized open-label phase III study comparing efavirenz and tenofovir/emtricitabine to efavirenz and zidovudine/lamivudine in treatment-naive HIV-1-infected individuals. Through 144 weeks, 50 of 487 participants without baseline nonnucleoside reverse transcriptase inhibitor resistance by population sequencing (efavirenz/tenofovir/emtricitabine, n = 19; efavirenz/zidovudine/lamivudine, n = 31) experienced virologic failure (>400 copies/ml). Here, we analyzed whether the presence of low levels of K103N at baseline correlated with virologic failure. METHODS Available baseline plasma samples (n = 485) were amplified and tested for K103N using an allele-specific PCR (AS-PCR) assay with a lower detection cut-off of 0.5%. RESULTS Sixteen of 476 (3.4%) evaluable participants had low-level K103N at baseline by AS-PCR (0.8-15%). The abundance of the K103N subpopulation at baseline distinguished individuals with virologic failure from those who responded durably to efavirenz-containing therapy. Among six participants with at least 2000 K103N copies/ml before treatment, five experienced virologic failure, compared with only one virologic failure among 10 who had less than 2000 K103N copies/ml (P = 0.008). Multivariate logistic regression analysis showed that K103N viral load at least 2000 copies/ml increased the risk of virologic failure with an odds ratio of 47.4 (95% confidence interval 5.2-429.2, P = 0.0006). CONCLUSION The presence of K103N mutant virus in plasma above 2000 copies/ml prior to therapy in treatment-naive individuals correlated with increased risk of virologic failure of these efavirenz-containing triple-drug regimens.

Background: Prior abacavir (ABC) or didanosine (ddI) therapy can result in the L74V/I or K65R mutation in HIV-1 reverse transcriptase. Preexisting K65R may have an impact on the treatment response to tenofovir disoproxil fumarate (TDF). Methods: An allele-specific polymerase chain reaction (AS-PCR) assay was developed to detect K65R with a lower limit of quantitation of 0.5%. Results: Among baseline plasma samples from 63 treatment-naive patients, no K65R was detected by AS-PCR. Among baseline samples from 154 treatment-experienced patients, 8 had K65R and 44 had L74V/I by population sequencing. Low-level K65R was detected in an additional 11 patients by AS-PCR, 3 of whom subsequently developed full K65R. Baseline K65R correlated with absence of thymidine analog mutations (TAMs; P = 0.003) and use of ABC or ddI (P = 0.004). Patients with full or low-level K65R at baseline or with L74V/I showed a diminished TDF response. Multivariate analyses confirmed that multiple TAMs, K65R, and L74V/I were independent predictors of diminished TDF response. Conclusions: Prior therapy with ABC or ddI can result in a population genotype that shows K65R or L74V/I but does not reveal low-level K65R present in some patients. Subsequent treatment intensification with TDF resulted in a poor virologic response and may result in expansion of the preexisting K65R mutant.

The ATP-dependent phosphorolytic excision of nucleoside analogue reverse transcriptase inhibitors can diminish their inhibitory effects on human immunodeficiency virus replication. Previous studies have shown that excision can occur only when the reverse transcriptase complex exists in its pretranslocational state. Binding of the next complementary nucleotide causes the formation of a stable dead-end complex in the posttranslocational state, which blocks the excision reaction. To provide mechanistic insight into the excision of the acyclic phosphonate nucleotide analog tenofovir, we compared the efficiencies of the reaction in response to changes in the translocation status of the enzyme. We found that rates of excision of tenofovir with wild-type reverse transcriptase can be as high as those seen with 3′-azido-3′-deoxythymidine monophosphate (AZT-MP). Thymidine-associated mutations, which confer >100-fold and 3-fold decreased susceptibility to AZT and tenofovir, respectively, caused substantial increases in the efficiency of excision of both inhibitors. However, in contrast to the case for AZT-MP, the removal of tenofovir was highly sensitive to dead-end complex formation. Site-specific footprinting experiments revealed that complexes with AZT-terminated primers exist predominantly pretranslocation. In contrast, complexes with tenofovir-terminated primers are seen in both configurations. Low concentrations of the next nucleotide are sufficient to trap the complex posttranslocation despite the flexible, acyclic character of the compound. Thus, the relatively high rate of excision of tenofovir is partially neutralized by the facile switch to the posttranslocational state and by dead-end complex formation, which provides a degree of protection from excision in the cellular environment.

Tenofovir alafenamide (TAF) is an investigational prodrug of the HIV-1 nucleotide reverse transcriptase (RT) inhibitor (NtRTI) tenofovir (TFV), with improved potency and drug delivery properties over the current prodrug, tenofovir disoproxil fumarate (TDF). TAF is currently in phase 3 clinical studies for the treatment of HIV-1 infection, in combination with other antiretroviral agents. Phase 1 and 2 studies have shown that TAF was associated with increased peripheral blood mononuclear cell (PBMC) drug loading and increased suppression of HIV-1 replication compared to treatment with TDF. In this study, selection ofin vitroresistance to both TAF and the parent compound, TFV, led to the emergence of HIV-1 with the K65R amino acid substitution in RT with 6.5-fold-reduced susceptibility to TAF. Although TAF is more potent than TFVin vitro, the antiviral susceptibilities to TAF and TFV of a large panel of nucleoside/nucleotide RT inhibitor (NRTI)-resistant mutants were highly correlated (R2= 0.97), indicating that the two compounds have virtually the same resistance profile when assessed as fold change from the wild type. TAF showed full antiviral activity in PBMCs against primary HIV-1 isolates with protease inhibitor, nonnucleoside RT inhibitor (NNRTI), or integrase strand transfer inhibitor resistance but reduced activity against isolates with extensive NRTI resistance amino acid substitutions. However, the increased cell loading of TFV with TAF versus TDF observedin vivosuggests that TAF may retain activity against TDF-resistant mutant viruses.

Human immunodeficiency virus type 1 (HIV-1) resistance development was evaluated in vitro by using combinations of the drugs tenofovir and emtricitabine or abacavir and lamivudine, as well as by using the compounds individually. Emtricitabine- and lamivudine-resistant HIV-1 isolates with the M184I or M184V mutation in reverse transcriptase were readily selected in the cultures with emtricitabine alone, lamivudine alone, and the two drug combinations and conferred high-level resistance to emtricitabine and lamivudine. Tenofovir-resistant HIV-1 isolates with the K65R mutation occurred in both the culture with tenofovir alone and the culture with the combination of emtricitabine and tenofovir. The S68N and S68K mutations were also observed in the tenofovir cultures, with no detectable impact on resistance, suggesting a possible compensatory role in viral fitness. At low concentrations of emtricitabine and tenofovir, the M184I mutation appeared first, followed by the K65R mutation, in a subset of viruses. At intermediate concentrations of emtricitabine and tenofovir, viruses harboring the K65R mutation or a novel K65N and K70R double mutation grew before they gave rise to mutants with K65R and M184V/I double mutations at higher emtricitabine concentrations. Abacavir resistance was characterized by the accumulation of the M184V, Y115F, and K65R mutations in the abacavir culture, while the M184V and L74V mutations were selected in combination with lamivudine. In the presence of the abacavir resistance mutations, viral growth was strong even in the presence of high concentrations of abacavir. In contrast, viral growth was markedly impaired in the cultures with high tenofovir concentrations, even in the presence of K65R. In conclusion, these studies show that HIV-1 mutants with a K65R and M184V genotype are generated under maximum selection pressure from the combination of tenofovir and emtricitabine.

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.

Objective: To determine the mechanisms of resistance of K65R mutant reverse transcriptase (RT) to the currently approved nucleoside and nucleotide RT inhibitors (NRTI). Methods: Susceptibilities of K65R mutant HIV-1 to NRTI were determined in cell culture. The K i /K m values were measured to determine the relative binding or incorporation of the NRTI, and ATP-mediated excision of incorporated NRTI was measured to determine NRTI stability as chain terminators. Results: K65R HIV-1 had decreased susceptibility to most NRTI, but increased susceptibility to zidovudine (ZDV). K i /K m values were increased 2- to 13-fold for K65R compared to wild-type RT for all NRTI, indicating decreased binding or incorporation. However, K65R also showed decreased excision of all NRTI compared to wild-type, indicating greater stability once incorporated. At physiological nucleotide concentrations, excision of ZDV, carbovir (the active metabolite of abacavir; ABC), stavudine (d4T), and tenofovir was further decreased, while excision of didanosine (ddl), zalcitabine (ddC), lamivudine (3TC), and emtricitabine (FTC) was unchanged. The decreased binding or incorporation of ZDV by K65R appeared counteracted by decreased excision resulting in overall increased susceptibility to ZDV in cell culture. For ABC, tenofovir, and d4T, despite having decreased excision, decreased binding or incorporation resulted in reduced susceptibilities to K65R. For ddl, ddC, 3TC, and FTC, decreased binding or incorporation by K65R appeared responsible for the decreased susceptibilities in cell culture. Conclusions: NRTI resistance in cells can consist of both altered binding or incorporation and altered excision of the NRTI. For K65R, the combination of these opposing mechanisms results in decreased susceptibility to most NRTI but increased susceptibility to ZDV.

In human immunodeficiency virus (HIV)-infected patients successfully treated with highly active antiretroviral therapy (HAART), a low level of HIV RNA persists in plasma at steady state for years and varies among patients. To understand predictors of residual viremia, we measured HIV RNA levels

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.

To study the potential development of genotypic and phenotypic resistance to tenofovir disoproxil fumarate (tenofovir DF) when used as a part of a 96-week HIV-1 treatment regimen for antiretroviral treatment-experienced HIV-infected patients.Clinical trial GS-98-902 was a placebo-controlled, 48-week phase 2 study of three doses of tenofovir DF when added to stable antiretroviral therapy for 189 treatment-experienced HIV-infected patients (mean of 4.6 years of prior antiretroviral treatment; 94% had nucleoside reverse transcriptase [RT] inhibitor [NRTI]-associated mutations). There was a statistically significant reduction in the mean HIV-1 RNA level at week 24 (average change in HIV-1 RNA level of -0.58 log(10) through week 24) with 300 mg of tenofovir DF once daily that was durable through week 48 (average change in HIV-1 RNA level of -0.62 log(10) through week 48). At week 48, 135 patients enrolled in an open-label, 48-week extension phase with 300 mg of tenofovir DF once daily added to their antiretroviral therapy. Genotypic analysis of plasma HIV-1 was performed for all patients after 96 weeks of study or upon early termination. Phenotypic analyses were performed at week 96 for patients with increases in HIV-1 RNA levels ofor =0.5 log(10) from week 48 to week 96.Genotypic results were obtained for 96 of 135 patients. NRTI-associated mutations developed in 41 (30%) of 135 patients from week 48 to week 96. Those mutations were primarily thymidine analog-associated mutations (33/41 patients) and developed while patients were receiving either stavudine or zidovudine. Two patients (1.5%) developed the K65R RT mutation (selected by tenofovir in vitro) but maintained HIV-1 suppression (-0.39 log(10)). These 96-week results were analogous to the 48-week results, in which 33% (n = 63) and 2.1% (n = 4) of patients developed thymidine analog-associated mutations or the K65R mutation, respectively. Although most patients maintained HIV-1 RNA suppression, an analysis of patients with increases in HIV-1 RNA levels ofor =0.5 log(10) (n = 21) from week 48 to week 96 was performed. For eight of 21 patients, development of primary protease inhibitor-associated or non-NRTI-associated resistance mutations was likely responsible for the HIV-1 RNA rebound. The remaining patients developed either no mutation (n = 3) or a new NRTI-associated mutation (n = 10) and were analyzed phenotypically. No phenotypic changes for tenofovir were observed in these analyses. In addition, no new mutations potentially associated with tenofovir DF therapy were identified. Overall, patients had a similar reduction in HIV-1 RNA levels at week 96 and at week 48 compared with baseline (-0.55 and -0.60 log(10), respectively).Adding tenofovir DF (300 mg) to existing antiretroviral therapy for highly treatment-experienced patients with preexisting resistance mutations showed significant and durable reductions in HIV-1 RNA levels through week 96. Through 96 weeks of tenofovir DF therapy, 48 weeks of which included suboptimal doses of tenofovir DF, there was infrequent development of RT mutations associated with tenofovir DF therapy (K65R mutation, 3%), consistent with the durability of the observed HIV-1 RNA responses.

Removal of nucleoside chain terminator inhibitors mediated by human immunodeficiency virus (HIV) reverse transcriptase (RT) using ATP as an acceptor molecule has been proposed as a novel mechanism of HIV resistance. Recombinant wild-type and mutant HIV type 1 (HIV-1) RT enzymes with thymidine analog resistance mutations D67N, K70R, and T215Y were analyzed for their ability to remove eight nucleoside reverse transcriptase inhibitors in the presence of physiological concentrations of ATP. The order for the rate of removal of the eight inhibitors by the mutant RT enzyme was zidovudine (AZT) > stavudine (d4T) ≫ zalcitabine (ddC) > abacavir > amdoxovir (DAPD) > lamivudine (3TC) > didanosine (ddI) > tenofovir. Thymidine analogs AZT and d4T were the most significantly removed by the mutant enzyme, suggesting that removal of these inhibitors by the ATP-dependent removal mechanism contributes to the AZT and d4T resistance observed in patients with HIV expressing thymidine analog resistance mutations. ATP-dependent removal of tenofovir was 22- to 35-fold less efficient than removal of d4T and AZT, respectively. The addition of ATP and the next complementary deoxynucleoside triphosphate caused a reduction of ATP-mediated removal of d4T, ddC, and DAPD, while AZT and abacavir removal was unaffected. The reduction of d4T, ddC, and DAPD removal in the presence of the deoxynucleoside triphosphate could explain the minor changes in susceptibility to these drugs observed in conventional in vitro phenotypic assays using cells that have higher deoxynucleoside triphosphate pools. The minimal removal of abacavir, ddC, DAPD, 3TC, ddI, and tenofovir is consistent with the minor changes in susceptibility to these drugs observed for HIV mutants with thymidine analog resistance mutations.

Introduction : COBI, a PK enhancer with no ARV activity is a more selective cytochrome P450 (CYP)3A inhibitor than ritonavir (RTV), does not induce CYP isozymes, and thus has less potential for drug-drug interactions. COBI boosts DRV PK as effectively as RTV in healthy volunteers. Materials and Methods : This 48-week, phase IIIb, open-label, single-arm, US multicentre study (NCT01440569) included HIV-infected treatment-nave and experienced adults with no DRV RAMs, viral load (VL) ≥1000 c/mL, eGFR ≥80 mL/min and genotypic sensitivity to investigator-selected N[t]RTIs. Patients received DRV/COBI 800/150 mg qd (as single agents) plus two fully active N[t]RTIs. The primary endpoint was any treatment-emergent grade 3 or 4 AEs through Week 24. We report 48-week safety, efficacy and PK/PD results in treatment-nave patients. Results : Of 313 ITT patients, 295 were treatment-nave (94%). In the treatment-nave cohort, 90% were male, 60% white and 294 (99.7%) received a TDF-containing regimen. Median baseline (BL) VL was 4.8 log 10 c/mL and CD4 + 370 cells/mm 3 . Treatment-emergent grade 3 or 4 AEs regardless of causality were reported in 21 (7%) patients. AEs regardless of causality (any grade; ≥10% of patients) were: diarrhoea (27%), nausea (23%), URTI (15%) and headache (12%). Sixteen (5%) patients had AEs leading to study drug discontinuation, most frequently rash (three patients), hypersensitivity and nausea (two patients each). Consistent with the known inhibition of tubular creatinine secretion by COBI, there was a mean increase from BL in serum creatinine by week 2 (0.09 mg/dL), remaining stable through week 48 (mean 0.10 mg/dL increase from BL). At week 48, 83% of patients achieved VL

OBJECTIVE: To determine whether genotypic changes in HIV-1 (HIV) reverse transcriptase (RT) occur during adefovir dipivoxil (ADV) therapy that may alter the susceptibility of HIV to adefovir or the related nucleotide inhibitor, tenofovir. DESIGN AND METHODS: GS-96-408 was a 1:1 randomized, double-blind, phase III clinical trial assessing the safety and efficacy of 120-mg daily ADV compared with placebo for the treatment of HIV when added to stable background antiretroviral therapy (ART). Of 442 patients enrolled, 142 were prospectively selected for a virology substudy. Baseline and posttreatment (weeks 24-48) plasma samples were genotypically analyzed in HIV RT. HIV from ADV-treated patients who developed RT mutations at week 24 were also phenotypically analyzed. RESULTS: Nucleoside-associated RT mutations arose with similar frequency among the ADV-and placebo-treated patients, 32% (n = 23) and 28% (n = 20), respectively, during the 24-week blinded treatment phase. RT mutations previously selected by adefovir in vitro (K70E or K65R) did not develop in any patient. Most mutations were typical zidovudine (ZDV)-resistance mutations (e.g., M41L, D67N, K70R, T215Y) in patients taking ZDV or stavudine (d4T) concomitantly, demonstrating directly in the placebo arm that d4T is able to select for these mutations. There appeared to be more patients developing D67N and K70R mutations in the ADV arm versus more T215Y mutations in the placebo arm. Between weeks 24 and 48, 19 of 50 patients (38%) in the ADV arm developed similar RT mutations. The mean HIV RNA responses at weeks 24 and 48 among the ADV-treated patients developing RT mutations were -0.68 log(10) copies/ml (n = 23) and -0.52 log(10) copies/ml (n = 19), respectively, similar to the overall week-24 and week-48 responses (-0.53 and 0.48 log(10) copies/ml, respectively). Patient-derived HIV expressing the observed RT mutations showed insignificant decreases in adefovir susceptibility compared with wild-type in 12 of 16 cases (< threefold). HIV from 1 patient showed significantly reduced susceptibility to tenofovir, which was in association with a double insertion mutation after codon 69 that was also present at baseline. CONCLUSIONS: HIV RT changes that arose during ADV therapy appear attributable to the patient's background ART. ADV therapy may have influenced the pattern of ZDV-associated resistance mutations that developed, but this did not result in an observed loss of viral load suppression. There was a trend toward decreased phenotypic susceptibility to adefovir in ADV-treated patients, with 4 of 16 analyzed patients showing mild, but significantly decreased susceptibility associated with the additional ZDV-associated mutations. Decreased susceptibility to the related nucleotide analog, tenofovir, was not observed to develop in ADV-treated patients.

Pyrophosphorolysis, the removal of nucleoside chain-terminators by a pyrophosphate (PPi) acceptor molecule, and a similar mechanism (nucleotide-dependent chain-terminator removal) which uses ATP as an acceptor molecule have been proposed as mechanisms of zidovudine (AZT) resistance. Recombinant HIV-1 wild-type reverse transcriptase (RT) and a mutant RT enzyme containing the AZT/thymidine analog resistance mutations D67N/K70R/T215Y were analyzed for pyrophosphorolysis and nucleotide-dependent chain-terminator removal activities. Our results confirm that pyrophosphorolysis and nucleotide-dependent chain-terminator removal are potential mechanisms of AZT and d4T resistance. However, tenofovir is less efficiently removed by pyrophosphorolysis and by nucleotide-dependent mechanisms. These results are consistent with the minor changes in susceptibility to tenofovir of the AZT/thymidine analog-resistant HIV RT mutants and the corresponding resistance of these mutants to AZT. The inability to remove tenofovir efficiently by these mechanisms may contribute to the durability of the HIV RNA response observed in patients treated with the oral prodrug, tenofovir disoproxil fumarate.

Simian immunodeficiency virus (SIV) infection of newborn macaques is a useful animal model of human pediatric AIDS to study pathogenesis and to develop intervention strategies aimed at preventing infection or delaying disease progression. In previous studies, we demonstrated that 9-[2-( R )-(phosphonomethoxy)propyl]adenine (PMPA; tenofovir) was highly effective in protecting newborn macaques against infection with virulent wild-type (i.e., drug-susceptible) SIVmac251. In the present study, we determined how reduced drug susceptibility of the virus inoculum affects the chemoprophylactic success. SIVmac055 is a virulent isolate that has a fivefold-reduced in vitro susceptibility to PMPA, associated with a K65R mutation and additional amino acid changes (N69T, R82K, A158S, S211N) in reverse transcriptase (RT). Eight newborn macaques were inoculated orally with SIVmac055. The three untreated control animals became SIVmac055 infected; these animals had persistently high viremia and developed fatal immunodeficiency within 3 months. Five animals were treated once daily with PMPA (at 30 mg/kg of body weight) for 4 weeks, starting 24 h prior to oral SIVmac055 inoculation. Two of the five PMPA-treated animals had no evidence of infection. The other three PMPA-treated infant macaques became infected but had a delayed viremia, enhanced antiviral antibody responses, and a slower disease course (AIDS in 5 to 15 months). No reversion to wild-type susceptibility or loss of the K65R mutation was detected in virus isolates from any of the PMPA-treated or untreated SIVmac055-infected animals. Several additional amino acid changes developed in RT, but they were not exclusively associated with PMPA therapy. The results of this study suggest that prophylactic administration of PMPA to human newborns and to adults following exposure to human immunodeficiency virus will still be beneficial even in the presence of viral variants with reduced susceptibility to PMPA.

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.

Patients with on-going HIV-1 replication and a K65R mutation in HIV-1 RT were assessed for further development of RT mutations while taking tenofovir disoproxil fumarate and other antiretroviral drugs. K65R was observed in 10 out of 536 treatment-experienced patients entering the study. K65R became undetectable in two patients, and the development of additional resistance mutations was minimal. Over 18 months, no patient developed multinucleoside resistance (Q151M or T69 insertions) and plasma viral loads were stable (median +0.04 log10 copies/ml).

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

This 96-week, double-blind, active-controlled, phase 3 study, randomized subjects to elvitegravir once daily or raltegravir twice daily with a fully active, ritonavir-boosted protease inhibitor plus a third agent. The proportion of subjects randomized to elvitegravir that achieved and maintained HIV-1 RNA < 50 copies/mL through week 96 was 47.6% (167/351) compared with 45.0% (158/351) for raltegravir with a treatment difference of 2.6% (95% confidence interval: 4.6% to 9.9%). Both regimens were well tolerated, with comparable rates of adverse events and laboratory abnormalities through week 96. Once-daily elvitegravir was noninferior to twice-daily raltegravir, showed durable long-term efficacy, and was well tolerated in HIV+ treatment-experienced patients.

The global scale-up of antiretroviral (ARV) therapy has dramatically reduced human immunodeficiency virus (HIV) morbidity and mortality. Stavudine (d4T) has been among the most commonly used ARVs in resource-limited settings because of its efficacy, short-term tolerability, low cost, and availability in coformulated form. However, many countries are transitioning away from d4T, mainly because of mitochondrial toxicities associated with long-term d4T use. In 2010, the World Health Organization (WHO) recommended phasing out d4T even in patients without documented virological failure [1]. There are two distinct mutational pathways of d4T resistance with different implications for zidovudine (AZT) and tenofovir (TDF) cross-resistance, the primary candidates for replacing d4T [2–4]. However, genotypic resistance testing is usually not available in the resource-limited settings where d4T-containing regimens are most commonly used. It is therefore important to determine the extent of nucleoside reverse-transcriptase inhibitor (NRTI) cross-resistance in viruses from individuals with virological failure on d4T-containing first-line therapy undergoing genotypic resistance testing. In this study, we combined genetic sequence data from 35 different studies to characterize the patterns of NRTI resistance mutations in viruses from individuals with virological failure on the 2 widely used d4T-containing ARV regimens: d4T/lamivudine(3TC)/nevirapine(NVP) and d4T/3TC/efavirenz(EFV). We also examine the effects of HIV-1 subtype, duration of therapy, and concomitant nonnucleoside reverse-transcriptase inhibitor (NNRTI) on the selection of specific mutations. We focused our analysis on NRTI resistance mutations with differential effects on the residual activity of AZT compared with TDF. The implications of these data for optimal replacement of d4T and subsequent NRTI options are discussed.

Elvitegravir is a strand transfer inhibitor of HIV-1 integrase that is currently undergoing phase 3 clinical testing. The two predominant metabolites of elvitegravir, M1 and M4 (elvitegravir hydroxide and elvitegravir glucuronide), have been shown to inhibit HIV-1 integrase in vitro. While they are markedly less potent than elvitegravir and present only at low levels in plasma clinically, we investigated their potential to select for elvitegravir resistance in vitro. Resistance selection experiments using metabolites M1 and M4 led to the development of the previously reported elvitegravir integrase resistance mutations H51Y, T66A, E92G, and S147G, as well as a novel S153F substitution. Additional resistance selection experiments using elvitegravir led to the development of previously reported integrase inhibitor resistance mutations (T66I, F121Y, and S153Y) as well as a novel R263K integrase mutation. Phenotypic analyses of site-directed mutants with these mutations demonstrated broad cross-resistance between elvitegravir and its M1 and M4 metabolites with more limited cross-resistance to the integrase inhibitor raltegravir. Overall, our in vitro studies demonstrate that the resistance profile of the M1 and M4 metabolites of elvitegravir overlaps with that of the parent molecule elvitegravir; as such, their presence at low levels is not considered clinically relevant.

Bevirimat (BVM) is the first of a new class of anti-HIV drugs with a novel mode of action known as maturation inhibitors. BVM inhibits the last cleavage of the Gag polyprotein by HIV-1 protease, leading to the accumulation of the p25 capsid-small peptide 1 (SP1) intermediate and resulting in noninfectious HIV-1 virions. Early clinical studies of BVM showed that over 50% of the patients treated with BVM did not respond to treatment. We investigated the impact of prior antiretroviral (ARV) treatment and/or natural genetic diversity on BVM susceptibility by conductingin vitrophenotypic analyses of viruses made from patient samples. We generated 31 recombinant viruses containing the entiregagand protease genes from 31 plasma samples from HIV-1-infected patients with (n= 21) or without (n= 10) prior ARV experience. We found that 58% of the patient isolates tested had a >10-fold reduced susceptibility to BVM, regardless of the patient's ARV experience or the level of isolate resistance to protease inhibitors. Analysis of mutants with site-directed mutations confirmed the role of the V370A SP1 polymorphism (SP1-V7A) in resistance to BVM. Furthermore, we demonstrated for the first time that a capsid polymorphism, V362I (CA protein-V230I), is also a major mutation conferring resistance to BVM. In contrast, none of the previously defined resistance-conferring mutations in Gag selectedin vitro(H358Y, L363M, L363F, A364V, A366V, or A366T) were found to occur among the viruses that we analyzed. Our results should be helpful in the design of diagnostics for prediction of the potential benefit of BVM treatment in HIV-1-infected patients.

Study 934 was an open-label, randomized Phase III study of emtricitabine + tenofovir DF + efavirenz (FTC + TDF + EFV) compared with lamivudine + zidovudine + efavirenz (3TC + ZDV + EFV) in antiretroviral therapy-naïve HIV-1 infected subjects. Baseline genotyping revealed the presence of primary nonnucleoside reverse transcriptase inhibitor resistance (NNRTI-R) in 22 of 509 enrolled patients (4.3%, 11 subjects in each group). The 487 subjects without baseline NNRTI-R formed the primary efficacy population (modified intent-to-treat population). Through 144 weeks, 50 of 487 modified intent-to-treat subjects (FTC + TDF + EFV, n = 19; 3TC + ZDV + EFV, n = 31) were analyzed for resistance development after virologic failure. NNRTI-R, primarily the K103N mutation, was the most common form of resistance that developed in both groups. No subject on FTC + TDF + EFV developed the K65R mutation. Significantly fewer subjects on FTC + TDF + EFV compared with 3TC + ZDV + EFV developed the M184V/I mutation (two versus 10, respectively, P = 0.021). Thymidine analog mutations developed in two subjects on 3TC + ZDV + EFV. Subjects with baseline NRTI genotypic resistance (TAMs, n = 13) or non-B HIV-1 subtypes (n = 28) showed no evidence of reduced treatment responses in either group. Nine of 22 patients with baseline NNRTI-R experienced virologic failure (FTC + TDF + EFV, n = 4; 3TC + ZDV + EFV, n = 5); seven of nine developed M184V/I and/or additional NNRTI-R, but none developed K65R. Baseline NNRTI-R was significantly associated with virologic failure in both groups (P0.001).

Background: The K65R mutation in human immunodeficiency virus type 1 reverse transcriptase can be selected by abacavir, didanosine, tenofovir, and stavudine in vivo resulting in reduced susceptibility to these drugs and decreased viral replication capacity. In clinical isolates, K65R is frequently accompanied by the A62V and S68G reverse transcriptase mutations. Methods: The role of A62V and S68G in combination with K65R was investigated using phenotypic, viral growth competition, pre-steady-state kinetic, and excision analyses. Results: Addition of A62V and S68G to K65R caused no significant change in human immunodeficiency virus type 1 resistance to abacavir, didanosine, tenofovir, or stavudine but partially restored the replication defect of virus containing K65R. The triple mutant K65R+A62V+S68G still showed some replication defect compared with wild-type virus. Pre-steady-state kinetic analysis demonstrated that K65R resulted in a decreased rate of incorporation (k pol ) for all natural dNTPs, which were partially restored to wild-type levels by addition of A62V and S68G. When added to K65R and S68G, the A62V mutation seemed to restore adenosine triphosphate-mediated excision of tenofovir to wild-type levels. Conclusions: A62V and S68G serve as partial compensatory mutations for the K65R mutation in reverse transcriptase by improving the viral replication capacity, which is likely due to increased incorporation efficiency of the natural substrates.

The HIV-1 reverse transcriptase (RT) resistance mutations K65R and M184V occur individually and in combination, and can contribute to decreased treatment responses in patients. In order to understand how these mutations interact with one another to confer drug resistance, the susceptibilities and underlying resistance mechanisms of these mutants to nucleoside RT inhibitors (NRTIs) were determined. Virus carrying K65R have reduced susceptibility to most NRTIs, but retain full susceptibility to zidovudine (AZT). M184V mutants have reduced susceptibility to lamivudine (3TC), emtricitabine (FTC) and didanosine (ddI), and contribute to reduced susceptibility to abacavir; however, they remain fully susceptible to tenofovir (TFV), AZT and stavudine (d4T). In cell culture, the K65R+M184V virus showed slightly increased susceptibility to TFV, AZT and d4T compared with K65R alone, but showed further decreases in susceptibility to 3TC, FTC, ddI and abacavir. There are two major biochemical mechanisms of resistance: altered NRTI binding/incorporation and altered NRTI excision after incorporation. For most NRTIs, the primary mechanism of resistance by K65R, M184V and K65R+M184V mutant RTs is to disrupt the NRTI- binding/incorporation steps. In the case of AZT, however, decreased binding/incorporation by K65R and K65R+M184V was counteracted by decreased AZT excision resulting in wild-type susceptibility. For TFV, decreased excision by K65R and K65R+M184V may partially counteract the K65R-driven decrease in incorporation relative to wild-type resulting in only low levels of TFV resistance. The K65R-mediated effect on decreasing NRTI excision was stronger than for M184V. These studies show that both mechanisms of resistance (binding/incorporation and excision) must be considered when defining resistance mechanisms.

Objective Study 903 was a 144-week, randomized, double-blind, active-controlled study of tenofovir disoproxil fumarate (TDF) therapy in treatment-naive HIV-1-infected patients. Patients received either TDF (n=299) or stavudine (d4T) (n=301) with lamivudine (3TC) and efavirenz (EFV). Resistance analyses were performed at baseline and at virological failure to determine the effects of baseline resistance and the patterns of resistance at virological failure. Methods Plasma HIV-1 from patients at baseline and at virological failure (>400 HIV-1 RNA copies/mL at week 144 or early discontinuation) was analysed phenotypically and by population sequencing. Results Sixteen per cent of patients were classified as having virological failure (47 on TDF and 49 on d4T; P=0.91). Patients with non-B HIV-1 subtypes or baseline nucleoside reverse transcriptase inhibitor (NRTI)-associated mutations responded similarly to the overall population. Resistance to EFV (K103N and others) or 3TC (M184V) developed most frequently (8.3% and 5.8%, respectively) and similarly in the two arms. In the d4T arm, a variety of NRTI mutations developed: K65R (n=2), L74V (n=2), V75M (n=1), and T69A+Y115H (n=1). K65R developed in eight TDF patients (2.7%); in seven of these eight patients, within 48 weeks. All eight patients began new regimens with a protease inhibitor (PI) and NRTIs, including two patients who remained on TDF; five of the eight patients achieved HIV RNA

Emtricitabine (FTC) and lamivudine (3TC) are deoxycytidine analogues with potent and selective inhibition of human immunodeficiency virus (HIV) and hepatitis B virus (HBV) replication. The K65R mutation in the HIV reverse transcriptase (RT) confers reduced susceptibility to 3TC, ddC, ddI, abacavir, and tenofovir in vitro. The Q151M mutation confers reduced susceptibility to many of the approved anti-HIV nucleoside analogues with the exception of 3TC and tenofovir. The double mutation K65R/Q151M has been shown to be more resistant to many NRTIs than either of the single mutations alone. In this study, we measured the antiviral activity of FTC and 3TC against HIV-1 containing K65R, Q151M, and K65R/Q151M mutations. We also studied the steady-state kinetic properties for the inhibition of dCTP incorporation by FTC 5'-triphosphate (TP) and 3TC-TP In addition, we measured the incorporation of dCTP, FTC-TP, and 3TC-TP into a random sequence DNA/DNA primer/template by the HIV-1 RTs using pre-steady-state kinetic analysis. Finally, we studied the incorporation of these deoxycytidine analogues into a HIV-1 genomic DNA/DNA primer/template by K65R HIV-1 RT to address certain concerns associated with DNA sequence specificity. Overall, this study demonstrated that K65R and K65R/Q151M related drug resistance to FTC and 3TC was mainly due to a significant decrease in the rate of incorporation. There was little to no effect on the binding affinities of the mutant HIV-1 RTs for the deoxycytidine analogues. The Q151M mutation remained sensitive to both FTC and 3TC in both cell culture and enzymatic assays. At a molecular level, FTC-TP was incorporated at least as efficiently as 3TC-TP for all of the HIV-1 RT and primer/templates tested.

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.

Study GS-99-907 was a 48-week, phase 3, double-blind, placebo-controlled intensification trial of tenofovir disoproxil fumarate (tenofovir DF). Antiretroviral-experienced patients added tenofovir DF 300 mg once daily to their existing regimen. The patterns of HIV-1 resistance development and the corresponding virologic responses were evaluated in a virology substudy at week 48. Although 94% of these treatment-experienced patients had nucleoside-associated resistance mutations (NAMs) at baseline, addition of tenofovir DF resulted in a mean reduction in viral load of -0.59 log10 copies/mL after 24 weeks that was durable through 48 weeks. Relative to the placebo-controlled arm, patients in the tenofovir DF arm had a reduced frequency of development of resistance mutations to all classes of HIV-1 inhibitors, with reduction in new protease inhibitor (PI)-associated mutations achieving statistical significance. The K65R mutation, which occurred in 8 patients (3%), was the only emergent mutation directly associated with tenofovir DF therapy. New thymidine analogue-associated mutations (TAMs) emerged in 19% of patients by week 48. Other than K65R, the patterns of mutations that developed were not significantly different between the tenofovir DF and placebo control arms, suggesting that the background therapies caused their development. The K65R mutation emerged only in patients with no detectable TAMs at baseline, whereas new TAMs developed similarly between patients with or without TAMs at baseline. Development of K65R was associated with mostly low-level changes in phenotypic susceptibility to tenofovir DF and other nucleoside reverse transcriptase inhibitors and was not associated with viral load rebound. No novel patterns of genotypic or phenotypic resistance to tenofovir were identified. Therefore, intensification with once-daily tenofovir DF therapy resulted in a sustained reduction in HIV-1 viral load and a low risk for development of the K65R mutation in this treatment-experienced patient population.