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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.

This review summarizes available data for lenacapavir, an investigational first-in-class agent that disrupts functioning of HIV capsid protein across multiple steps in the viral life cycle.Lenacapavir demonstrated picomolar potency in vitro with no cross resistance to existing antiretroviral classes and potent antiviral activity in persons with HIV-1. In persons with HIV-1, there was no preexisting resistance to lenacapavir regardless of treatment history. Lenacapavir can be administered orally either daily or weekly and subcutaneously up to every 6 months. In heavily treatment-experienced persons with multidrug-resistant HIV-1 and in treatment-naive persons with HIV-1, lenacapavir in combination with other antiretroviral agents led to high rates of virologic suppression and was well tolerated.Ongoing studies are evaluating long-acting dosing of lenacapavir for treating HIV-1 in combination with other antiretrovirals and preventing HIV-1 as a single agent.

Because no currently available vaccine can prevent HIV infection, pre-exposure prophylaxis (PrEP) with antiretrovirals (ARVs) is an important tool for combating the HIV pandemic1,2. Long-acting ARVs promise to build on the success of current PrEP strategies, which must be taken daily, by reducing the frequency of administration3. GS-CA1 is a small-molecule HIV capsid inhibitor with picomolar antiviral potency against a broad array of HIV strains, including variants resistant to existing ARVs, and has shown long-acting therapeutic potential in a mouse model of HIV infection4. Here we show that a single subcutaneous administration of GS-CA1 provides long-term protection against repeated rectal simian–human immunodeficiency virus (SHIV) challenges in rhesus macaques. Whereas all control animals became infected after 15 weekly challenges, a single 300 mg kg−1 dose of GS-CA1 provided per-exposure infection risk reduction of 97% for 24 weeks. Pharmacokinetic analysis showed a correlation between GS-CA1 plasma concentration and protection from SHIV challenges. GS-CA1 levels greater than twice the rhesus plasma protein-adjusted 95% effective concentration conferred 100% protection in this model. These proof-of-concept data support the development of capsid inhibitors as a novel long-acting PrEP strategy in humans., A single dose of a small-molecule HIV capsid inhibitor provides long-term protection from repeated simian–human immunodeficiency virus challenges in macaques and might serve as a novel strategy for HIV prevention in humans.

Background HIV-1 recent infection testing algorithms (RITAs) use recency assays to estimate population level HIV incidence rates (IRs), and are currently being employed in PrEP trials to estimate background HIV incidence rates (bHIV-IR). The SIENA study was conducted to determine the HIV incidence rate among young women in and around central and mid-western Uganda, and to assess the suitability of different recency assay platforms for use in determining bHIV-IR in future PrEP trials. Methods Diagnosis of HIV was confirmed by positive results on both the Alere Determine HIV-1/2 and Oraquick HIV-1/2 rapid tests. Positive samples were analyzed for recent infection using the Sedia HIV-1 Limiting Antigen Avidity Enzyme ImmunoAssay (LAg-EIA; Sedia Biosciences, Beaverton, OR) and the Sedia Asante HIV-1 Rapid Recency Assay (Asante; Sedia Biosciences). The Asante assay was performed by electronic reader (Asante-Reader) or visual read (Asante-Visual). VL was determined by COBAS TaqMan HIV-1 Test (LabCorp, Indianapolis, IN). HIV incidence was calculated based on previously determined MDRI and FRRs that were specific for the study population. Results Of 743 women screened, 191 were diagnosed with HIV, of whom 44 (23%) had a viral load of < 75 copies/mL. The 3 recency assays identified between 43 and 57 samples as recent and between 35 and 37 samples when the VL cutoff of < 75 copies/mL was used (Table). When no VL cutoff was used the calculated bHIV-IR was 17.9/100 person-years (PY) for the LAg-EIA, 12.8/100PY for the Asante-Reader and 20.3/100PY for the Asante-Visual. Using the VL cutoff, the calculated bHIV-IR was 11.4/100 PY with the LAg-EIA assay and 10.9/100 PY with the Asante-Reader. No MDRI or FRR for the Asante-Visual with a VL cutoff was available for calculation of the Asante-Visual HIV incidence. Conclusion When the RITA included a VL cutoff of 75 cp/mL, the LAg-EIA and Asante assays classified the number of recent and long-term infections similarly, resulting in comparable bHIV-IR results. Overall, these analyses support the use of these recency assays in the RITA to estimate the bHIV-IR in future PrEP trials. Our results demonstrate extremely high prevalence and incidence of HIV in young women in central and mid-western Uganda, highlighting the need for expanded HIV prevention options in these areas. Disclosures Stephanie Cox, BS, Gilead Sciences: Paid employee|Gilead Sciences: Stocks/Bonds Shelley N. Facente, PhD, MPH, CDC-funded TRACE program: Grant/Research Support|Gilead Sciences: Advisor/Consultant|Sedia Biosciences Corporation: Grant/Research Support Eduard Grebe, PhD, CDC-funded TRACE program: Grant/Research Support|Gilead Sciences: Advisor/Consultant|Sedia Biosciences Corp: Grant/Research Support Ramin Ebrahimi, MS, Gilead Sciences: Paid employee|Gilead Sciences: Stocks/Bonds Christoph C. Carter, MD, PhD, Gilead Sciences: Employee|Gilead Sciences: Employee|Gilead Sciences: Stocks/Bonds|Gilead Sciences: Stocks/Bonds Christian Callebaut, PhD, Gilead Sciences: Paid employee|Gilead Sciences: Stocks/Bonds Jared Baeten, MD, PhD, Gilead Sciences: Employee|Gilead Sciences: Stocks/Bonds Moupali Das, MD, Gilead Sciences: Employee|Gilead Sciences: Employee|Gilead Sciences: Stocks/Bonds|Gilead Sciences: Stocks/Bonds.

Oral antiretroviral agents provide life-saving treatments for millions of people living with HIV, and can prevent new infections via pre-exposure prophylaxis1–5. However, some people living with HIV who are heavily treatment-experienced have limited or no treatment options, owing to multidrug resistance6. In addition, suboptimal adherence to oral daily regimens can negatively affect the outcome of treatment—which contributes to virologic failure, resistance generation and viral transmission—as well as of pre-exposure prophylaxis, leading to new infections1,2,4,7–9. Long-acting agents from new antiretroviral classes can provide much-needed treatment options for people living with HIV who are heavily treatment-experienced, and additionally can improve adherence10. Here we describe GS-6207, a small molecule that disrupts the functions of HIV capsid protein and is amenable to long-acting therapy owing to its high potency, low in vivo systemic clearance and slow release kinetics from the subcutaneous injection site. Drawing on X-ray crystallographic information, we designed GS-6207 to bind tightly at a conserved interface between capsid protein monomers, where it interferes with capsid-protein-mediated interactions between proteins that are essential for multiple phases of the viral replication cycle. GS-6207 exhibits antiviral activity at picomolar concentrations against all subtypes of HIV-1 that we tested, and shows high synergy and no cross-resistance with approved antiretroviral drugs. In phase-1 clinical studies, monotherapy with a single subcutaneous dose of GS-6207 (450 mg) resulted in a mean log10-transformed reduction of plasma viral load of 2.2 after 9 days, and showed sustained plasma exposure at antivirally active concentrations for more than 6 months. These results provide clinical validation for therapies that target the functions of HIV capsid protein, and demonstrate the potential of GS-6207 as a long-acting agent to treat or prevent infection with HIV. The small molecule GS-6207, which disrupts the function of the HIV capsid protein, shows potential as a long-acting therapeutic agent for the treatment and prevention of HIV infection.

Summary Background Tenofovir alafenamide shows high antiviral efficacy and improved renal and bone safety compared with tenofovir disoproxil fumarate when used for HIV treatment. Here, we report primary results from a blinded phase 3 study evaluating the efficacy and safety of pre-exposure prophylaxis (PrEP) with emtricitabine and tenofovir alafenamide versus emtricitabine and tenofovir disoproxil fumarate for HIV prevention. Methods This study is an ongoing, randomised, double-blind, multicentre, active-controlled, phase 3, non-inferiority trial done at 94 community, public health, and hospital-associated clinics located in regions of Europe and North America, where there is a high incidence of HIV or prevalence of people living with HIV, or both. We enrolled adult cisgender men who have sex with men and transgender women who have sex with men, both with a high risk of acquiring HIV on the basis of their self-reported sexual behaviour in the past 12 weeks or their recent history (within 24 weeks of enrolment) of bacterial sexually transmitted infections. Participants with current or previous use of PrEP with emtricitabine and tenofovir disoproxil fumarate were not excluded. We used a computer-generated random allocation sequence to randomly assign (1:1) participants to receive either emtricitabine (200 mg) and tenofovir alafenamide (25 mg) tablets daily, with matched placebo tablets (emtricitabine and tenofovir alafenamide group), or emtricitabine (200 mg) and tenofovir disoproxil fumarate (300 mg) tablets daily, with matched placebo tablets (emtricitabine and tenofovir disoproxil fumarate group). As such, all participants were given two tablets. The trial sponsor, investigators, participants, and the study staff who provided the study drugs, assessed the outcomes, and collected the data were masked to group assignment. The primary efficacy outcome was incident HIV infection, which was assessed when all participants had completed 48 weeks of follow-up and half of all participants had completed 96 weeks of follow-up. This full analysis set included all randomly assigned participants who had received at least one dose of the assigned study drug and had at least one post-baseline HIV test. Non-inferiority of emtricitabine and tenofovir alafenamide to emtricitabine and tenofovir disoproxil fumarate was established if the upper bound of the 95·003% CI of the HIV incidence rate ratio (IRR) was less than the prespecified non-inferiority margin of 1·62. We prespecified six secondary bone mineral density and renal biomarker safety endpoints to evaluate using the safety analysis set. This analysis set included all randomly assigned participants who had received at least one dose of the assigned study drug. This trial is registered with ClinicalTrials.gov , NCT02842086 , and is no longer recruiting. Findings Between Sept 13, 2016, and June 30, 2017, 5387 (92%) of 5857 participants were randomly assigned and received emtricitabine and tenofovir alafenamide (n=2694) or emtricitabine and tenofovir disoproxil fumarate (n=2693). At the time of the primary efficacy analysis (ie, when all participants had completed 48 weeks and 50% had completed 96 weeks) emtricitabine and tenofovir alafenamide was non-inferior to emtricitabine and tenofovir disoproxil fumarate for HIV prevention, as the upper limit of the 95% CI of the IRR, was less than the prespecified non-inferiority margin of 1·62 (IRR 0·47 [95% CI 0·19–1·15]). After 8756 person-years of follow-up, 22 participants were diagnosed with HIV, seven participants in the emtricitabine and tenofovir alafenamide group (0·16 infections per 100 person-years [95% CI 0·06–0·33]), and 15 participants in the emtricitabine and tenofovir disoproxil fumarate group (0·34 infections per 100 person-years [0·19–0·56]). Both regimens were well tolerated, with a low number of participants reporting adverse events that led to discontinuation of the study drug (36 [1%] of 2694 participants in the emtricitabine and tenofovir alafenamide group vs 49 [2%] of 2693 participants in the emtricitabine and tenofovir disoproxil fumarate group). Emtricitabine and tenofovir alafenamide was superior to emtricitabine and tenofovir disoproxil fumarate in all six prespecified bone mineral density and renal biomarker safety endpoints. Interpretation Daily emtricitabine and tenofovir alafenamide shows non-inferior efficacy to daily emtricitabine and tenofovir disoproxil fumarate for HIV prevention, and the number of adverse events for both regimens was low. Emtricitabine and tenofovir alafenamide had more favourable effects on bone mineral density and biomarkers of renal safety than emtricitabine and tenofovir disoproxil fumarate. Funding Gilead Sciences.

Background Lenacapavir in vitro resistance selections identified seven mutations in HIV-1 capsid protein (CA) associated with reduced susceptibility. Objectives To analyse lenacapavir activity against lenacapavir-associated resistance mutations in multiple assays. We also report Day 10 resistance analyses conducted in a Phase 1b study of lenacapavir (Study 4072) in people with HIV (PWH). Methods Mutations were inserted in a proviral DNA clone by site-directed mutagenesis, and viruses (n = 12) were generated by transfection. Sequences were used to generate single-cycle (SC) test vectors that were evaluated in a Gag-Pro assay, and replicative viruses were tested in a multicycle (MC) MT-2 assay to determine lenacapavir susceptibility. Study 4072 was a Phase 1b, double-blinded, placebo-controlled, dose-ranging, randomized study of lenacapavir in untreated PWH. Participants received a single dose of lenacapavir (up to 750 mg) or placebo (10 day monotherapy). CA resistance was characterized using genotypic and/or phenotypic assays. Results Lenacapavir susceptibility in the SC assay showed an inverse relationship between replication capacity and resistance. In Study 4072, all 29 participants receiving lenacapavir showed a robust virological response with no rebound. At baseline, no participant had resistance mutations to lenacapavir, and all had WT susceptibility to lenacapavir. Post-monotherapy analyses revealed the emergence of CA mutation Q67H at Day 10 in two participants. Conclusions In vitro assays confirmed that increased resistance to lenacapavir was associated with decreased replication capacity of mutant viruses. In the clinical study no pre-existing lenacapavir resistance was detected. Emergence of Q67H occurred at exposures below the dose used in current Phase 2/3 studies. These results support development of lenacapavir as an antiretroviral agent.

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.

BACKGROUND HIV envelope (env) diversity represents a significant challenge for the use of broadly neutralizing antibodies (bNAbs) in HIV treatment and cure studies. Screening for viral sensitivity to bNAbs to select eligible trial participants will be important to improve clinical efficacy; however, no universal approach has been established. METHODS Pre-antiretroviral therapy plasma virus from participants in the Zurich Primary HIV Infection (ZPHI) study was genotyped and phenotyped for sensitivity to the bNAbs elipovimab (EVM, formerly GS-9722) and 3BNC117. The genotyping and phenotyping assessments were performed following the Clinical Laboratory Improvement Amendments of 1988 guidelines as required for entry into clinical trials. The genotypic-based prediction of bNAb sensitivity was based on HIV env amino acid signatures identified from a genotypic-phenotypic correlation algorithm using a subtype B database. RESULTS Genotyping the plasma virus and applying env sensitivity signatures, ZPHI study participants with viral sensitivity to EVM and 3BNC117 were identified. ZPHI study participants with virus sensitive to EVM and 3BNC117 were also identified by phenotyping the plasma virus. Comparison of the genotypic and phenotypic sensitivity assessments showed strong agreement between the 2 methodologies. CONCLUSIONS The genotypic assessment was found to be as predictive as the direct measurement of bNAb sensitivity by phenotyping and may, therefore, be preferred because of more rapid turnaround time and assay simplicity. A significant number of the participants were predicted to have virus sensitive to EVM and 3BNC117 and could, thus, be potential participants for clinical trials involving these bNAbs.

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.

ObjectivesCurrent prophylaxis options for people at risk for HIV infection include two US FDA-approved daily pre-exposure prophylaxis (PrEP) regimens and guidelines for a 2-1-1 event-driven course specifically for men who have sex with men. Despite this, PrEP use rates remain suboptimal, and additional PrEP options may help to improve uptake among diverse populations. Here, we evaluated protective efficacy of two-dose PrEP and two-dose postexposure prophylaxis (PEP) schedules with emtricitabine (FTC)/tenofovir alafenamide (TAF) with or without bictegravir (BIC) in an SHIV macaque model.MethodsMacaques received one oral dose of 200 mg emtricitabine, 25 mg tenofovir alafenamide and 25-100 mg of bictegravir to establish pharmacokinetic profiles of each drug either in the plasma or the peripheral blood mononuclear cells. Protective efficacy of multiple two-dose PrEP and PEP schedules with FTC/TAF with or without bictegravir was then assessed in two repeat low-dose rectal SHIV challenge studies.ResultsThe data revealed over 95% per-exposure risk reduction with FTC/TAF PrEP initiated 2 h before the exposure, but a loss of significant protection with treatment initiation postexposure. In contrast, FTC/TAF plus BIC offered complete protection as PrEP and greater than 80% per-exposure risk reduction with treatment initiation up to 24 h postexposure.ConclusionsTogether, these results demonstrate that two-dose schedules can protect macaques against SHIV acquisition and highlight the protective advantage of adding the integrase inhibitor bictegravir to the reverse transcriptase inhibitors emtricitabine and tenofovir alafenamide as part of event-driven prophylaxis.

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 CD8Peripheral 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.Treatment of PBMCs with VES resulted in all 31 donors demonstrating a concentration dependent increase in CD8This study demonstrates that VES treatment can induce the activation of functional HIV-specific CD8

Tenofovir alafenamide (TAF) and tenofovir disoproxil fumarate (TDF) are prodrugs of the HIV-1 nucleotide reverse transcriptase inhibitor tenofovir (TFV). In vivo, TAF achieves >4-fold-higher intracellular levels of TFV diphosphate (TFV-DP) compared to TDF. Since thymidine analog-associated mutations (TAMs) in HIV-1 confer reduced TFV susceptibility, patients with TAM-containing HIV-1 may benefit from higher TFV-DP levels delivered by TAF. Moreover, the presence of the M184V mutation increases TFV susceptibility during TDF- or TAF-based therapy. The susceptibilities to antiviral drugs of site-directed mutants (SDMs) and patient-derived mutants containing combinations of TAMs (M41L, D67N, K70R, L210W, T215Y, and K219Q) with or without the M184V mutation (TAMs±M184V) were evaluated using either 5-day multicycle (MC; n = 110) or 2-day single-cycle (SC; n = 96) HIV assays. The presence of M184V in TAM-containing HIV-1 SDMs (n = 48) significantly increased TAF sensitivity compared to SDMs without M184V (n = 48). The comparison of TAF and TDF resistance profiles was further assessed in viral breakthrough (VB) experiments mimicking clinically relevant drug concentrations. A total of 68 mutants were assayed at physiological concentration in VB experiments, with 15/68 mutants breaking through with TDF (TFV, the in vitro equivalent of TDF, was used in these experiments), and only 3 of 68 mutants breaking through under TAF treatment. Overall, in the VB assay mimicking the 4-fold-higher intracellular levels of TFV-DP observed clinically with TAF versus TDF, TAF inhibited viral breakthrough of most TAM-containing HIV-1, whereas TDF did not. These results indicate that TAF has a higher resistance threshold than TDF and suggest that higher resistance cutoffs should be applied for TAF compared to TDF in genotypic and phenotypic resistance algorithms.

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.To compare detectability of M184V/I in historical HIV-1 RNA analyses versus HIV-1 DNA sequencing.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 RNA50 copies/mL for ≥6 consecutive months prior to screening. A plasma historical genotypic report (HGR) showing the presence of M184V/I was required for all participants and proviral HIV DNA analysis was conducted prior to enrolment.All 84 participants had evidence of M184V or M184I in their HGR (100%), whereas the mutation was detected in only 40/84 participants by proviral HIV DNA sequencing analysis (48%). Differential detection of M184V/I was not associated with timing differences between the HGR and proviral HIV DNA sampling, the overall duration of ART, or CD4 cell counts and HIV-1 viral load at baseline.Our results suggest that undetected M184V/I should be considered when switching virologically suppressed patients to new regimens, particularly two-drug lamivudine- or emtricitabine-containing combinations.

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

We address a data gap identified by Adams et al., of virological efficacy outcomes and emergent drug resistance in HIV-1-infected, antiretroviral treatment (ART)-naive participants with baseline (BL) viral loads (VL) >1,000,000 copies/ml. Initial ART regimens including an integrase strand transfer inhibitor (INSTI) cause a rapid decline in VL. In two cases described, the VL response on the single tablet regimen (STR) of co-formulated elvitegravir, cobicistat, emtricitabine and tenofovir disoproxil fumarate (EVG/COBI/FTC/TDF) was

Background Lenacapavir (LEN) is a first-in-class HIV-1 capsid (CA) inhibitor in clinical development for treatment and prevention of HIV-1 infection. CALIBRATE is an ongoing, phase 2 clinical study evaluating subcutaneous (SC) or oral LEN, in combination with other antiretrovirals, in treatment-naïve people with HIV-1. High rates of virologic success (HIV-1 RNA < 50 copies/mL) were achieved with LEN-based regimens by FDA Snapshot analysis at Week 28. Here, we present interim resistance analyses through Week 28. Methods Participants were randomized (2:2:2:1) to treatment groups (TG) (Figure): SC LEN + oral daily emtricitabine/tenofovir alafenamide (F/TAF); at Week 28, participants switch F/TAF to oral TAF (TG-A) or bictegravir (B, BIC) (TG-B); oral daily LEN + F/TAF (TG-C), or oral daily B/F/TAF (TG-D). Genotypic analyses (population sequencing) of HIV-1 reverse transcriptase and integrase, and genotypic (deep sequencing)/phenotypic analyses for CA were performed at screening; genotypic and phenotypic analyses were conducted at confirmed virologic failure. Figure CALIBRATE Study Design Results 182 participants were randomized and dosed in TG-A to D (n=52, 53, 52, 25). Most participants had subtype B HIV-1 (92.9%). Sequence analysis of baseline samples found 65% of amino acid residues were conserved with < 1% variation across CA overall, and 55% of residues were fully conserved. No mutations were detected at 6 positions in CA associated with reduced susceptibility to LEN in vitro; residues were fully conserved at 5 positions (L56, M66, Q67, K70, N74), and < 2% variation was observed at 1 position (T107). Three participants met the criteria for resistance analysis: 2 participants resuppressed to < 50 copies/mL while continuing treatment. One participant on SC LEN + F/TAF developed emergent resistance to LEN (Q67H+K70R) and emtricitabine (M184M/I), followed by resuppression after starting dolutegravir, zidovudine + lamivudine, tenofovir disoproxil fumarate. Conclusion Emergent resistance to LEN was uncommon in treatment-naïve participants receiving SC or oral LEN (0.6%, 1/157). These interim resistance findings support the ongoing evaluation of LEN for treatment and prevention of HIV. Disclosures Laurie VanderVeen, PhD, Gilead Sciences (Employee, Shareholder) Nicolas Margot, MA, Gilead Sciences (Employee, Shareholder) Vidula Naik, MSc, Gilead Sciences (Employee, Shareholder) Silvia Chang, Masters, Gilead Sciences, Inc (Employee, Shareholder) Ross Martin, PhD, Gilead Sciences, Inc (Employee, Shareholder) Hadas Dvory-Sobol, PhD, Gilead Sciences (Employee, Shareholder) Martin Rhee, MD, Gilead Sciences (Employee, Shareholder) Christian Callebaut, PhD, Gilead Sciences (Employee, Shareholder)

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.

HIV-infected patients treated with certain nucleoside reverse transcriptase inhibitors (NRTIs) have experienced adverse effects due to drug-related mitochondrial toxicity. Tenofovir alafenamide (TAF) is a novel prodrug of the NRTI tenofovir (TFV) with an improved safety profile compared to tenofovir disoproxil fumarate (TDF). Prior in vitro studies have demonstrated that the parent nucleotide TFV has no significant effects on mtDNA synthesis. This study investigated whether clinically relevant TAF and TDF exposures affect mtDNA content in human lymphocytes. First, activated or resting peripheral blood mononuclear cells (PBMCs), as well as MT-2 and Jurkat T-cell lines, were continuously treated with ddC for 10 days to establish their susceptibility to mtDNA depletion. PBMCs had low sensitivity to NRTI-mediated mtDNA depletion in vitro. In contrast, ddC treatment of rapidly dividing MT-2 and Jurkat cells resulted in a dose-dependent decrease in mtDNA. Therefore, these two T-cell lines were selected for evaluating TAF and TDF treatment effects. MT-2 and Jurkat cells were pulse-treated with TAF or TDF every 24 h for 10 days to mimic pharmacologically relevant drug exposures. Pulse treatment of cells with 3.3 μM TAF or 1.1 μM TDF for 10 days resulted in 2- to 7-fold greater steady-state intracellular TFV-diphosphate (TFV-DP) levels than those observed clinically in TAF- or TDF-treated patients. At these concentrations, no significant TAF- (106.7% and 84.1% of control; p = 0.77 and 0.12 for MT-2 and Jurkat, respectively) or TDF- (100.6% and 91.0% of control; p = 0.91 and 0.37, respectively) associated reduction in mtDNA content was observed compared with untreated control cells. This study demonstrates that, despite delivering higher intracellular levels of TFV-DP than TDF, TAF does not inhibit mtDNA synthesis in vitro at concentrations exceeding the clinically relevant intracellular drug exposures. Thus, TAF has a low potential for mitochondrial toxicity in T-cells of HIV-infected patients.

Supplemental Digital Content is Available in the Text., Background: HIV-infected, treatment-experienced adults with a history of prior resistance and regimen failure can be virologically suppressed but may require multitablet regimens associated with lower adherence and potential resistance development. Methods: We enrolled HIV-infected, virologically suppressed adults with 2-class to 3-class drug resistance and at least 2 prior regimen failures into this phase 3, open-label, randomized study. The primary endpoint was the percentage of participants with HIV-1 RNA

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.

BackgroundTenofovir alafenamide (TAF)-based regimens are being evaluated for pre-exposure prophylaxis (PrEP). We used a macaque model of repeated exposures to simian human immunodeficiency virus (SHIV) to investigate whether TAF alone or the combination of TAF and emtricitabine (FTC) can prevent vaginal infection.MethodsPigtail macaques were exposed vaginally to SHIV162p3 once a week for up to 15 weeks. Animals received clinical doses of FTC/TAF (n = 6) or TAF (n = 9) orally 24 hours before and 2 hours after each weekly virus exposure. Infection was compared with 21 untreated controls.ResultsFive of the 6 animals in the FTC/TAF and 4 of the 9 animals in the TAF alone group were protected against infection (P = .001 and P = .049, respectively). The calculated efficacy of FTC/TAF and TAF was 91% (95% confidence interval [CI], 34.9%–98.8%) and 57.8% (95% CI, −8.7% to 83.6%), respectively. Infection in FTC/TAF but not TAF-treated macaques was delayed relative to controls (P = .005 and P = .114). Median tenofovir diphosphate (TFV-DP) levels in peripheral blood mononuclear cells (PBMCs) were similar among infected and uninfected macaques receiving TAF PrEP (351 and 143 fmols/106 cells, respectively; P = .921).ConclusionsEmtricitabine/TAF provided a level of protection against vaginal challenge similar to FTC/TFV disoproxil fumarate combination in the macaque model. Our results support the clinical evaluation of FTC/TAF for PrEP in women.

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.

Tenofovir alafenamide (TAF) is an investigational oral prodrug of the HIV-1 nucleotide reverse transcriptase inhibitor tenofovir (TFV). Tenofovir disoproxil fumarate (TDF) is another TFV prodrug, widely used for the treatment of HIV-1 infection. TAF is converted mostly intracellularly to TFV and, in comparison to TDF, achieves higher tenofovir diphosphate (TFV-DP) levels in peripheral blood mononuclear cells. As a result, TAF has demonstrated potent anti-HIV-1 activity at lower doses than TDF in monotherapy studies. Here, the in vitro virology profile of TAF was evaluated and compared to that of TDF. TAF displayed potent antiviral activity against all HIV-1 groups/subtypes, as well as HIV-2. TAF exhibited minimal changes in the drug concentration needed to inhibit 50% of viral spread (EC 50 ) upon removal of the prodrug, similar to TDF, demonstrating intracellular antiviral persistence. While TAF and TDF exhibited comparable potencies in the absence of serum pretreatment, TAF maintained activity in the presence of human serum, whereas TDF activity was significantly reduced. This result demonstrates TAF's improved plasma stability over TDF, which is driven by the different metabolic pathways of the two prodrugs and is key to TAF's improved in vivo antiviral activity. The activity of TAF is specific for HIV, as TAF lacked activity against a large panel of human viruses, with the exception of herpes simplex virus 2, where weak TAF antiviral activity was observed, as previously observed with TFV. Finally, in vitro combination studies with antiretroviral drugs from different classes showed additive to synergistic interactions with TAF, consistent with ongoing clinical studies with TAF in fixed-dose combinations with multiple other antiretroviral drugs for the treatment of HIV.

OBJECTIVES To evaluate the safety and efficacy of the novel tenofovir prodrug, tenofovir alafenamide (TAF), as part of the first protease inhibitor-based single-tablet regimen (STR) for initial treatment of HIV-1 infection. METHODS Antiretroviral therapy (ART)-naive adults with estimated glomerular filtration rate ≥ 70 mL/min were randomized 2:1 to receive the darunavir/cobicistat/emtricitabine/tenofovir alafenamide (D/C/F/TAF) STR (TAF: N = 103) or darunavir + cobicistat + emtricitabine/tenofovir disoproxil fumarate (TDF: N = 50) once daily with matched placebos for 48 weeks. RESULTS At week 24, viral suppression (HIV-1 RNA

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.

OBJECTIVES To evaluate the safety and efficacy of the novel tenofovir prodrug, tenofovir alafenamide (TAF), as part of a single-tablet regimen (STR) for the initial treatment of HIV-1 infection. DESIGN Phase 2, randomized, double-blind, double-dummy, multicenter, active-controlled study. METHODS Antiretroviral naive adults with HIV-1 RNA ≥5000 copies per milliliter and a CD4 count ≥50 cells per microliter were randomized 2:1 to receive an STR of elvitegravir/cobicistat/emtricitabine/tenofovir alafenamide (E/C/F/TAF) or elvitegravir/cobicistat/emtricitabine/tenofovir disoproxil fumarate (E/C/F/TDF), plus placebo for 48 weeks. RESULTS Patients on both E/C/F/TAF (n = 112) and E/C/F/TDF (n = 58) had high rates of virologic suppression (

GS-8374 is a potent HIV protease inhibitor (PI) with a unique diethyl-phosphonate moiety. Due to a balanced contribution of enthalpic and entropic components to its interaction with the protease (PR) active site, the compound retains activity against HIV mutants with high-level multi-PI resistance. We report here the in vitro selection and characterization of HIV variants resistant to GS-8374. While highly resistant viruses with multiple mutations in PR were isolated in the presence of control PIs, an HIV variant displaying moderate (14-fold) resistance to GS-8374 was generated only after prolonged passaging for >300 days. The isolate showed low-level cross-resistance to darunavir, atazanavir, lopinavir, and saquinavir, but not other PIs, and contained a single R41K mutation in PR combined with multiple genotypic changes in the Gag matrix, capsid, nucleocapsid, and SP2 domains. Mutations also occurred in the transframe peptide and p6* domain of the Gag-Pol polyprotein. Analysis of recombinant HIV variants indicated that mutations in Gag, but not the R41K in PR, conferred reduced susceptibility to GS-8374. The Gag mutations acted in concert, since they did not affect susceptibility when introduced individually. Analysis of viral particles revealed that the mutations rendered Gag more susceptible to PR-mediated cleavage in the presence of GS-8374. In summary, the emergence of resistance to GS-8374 involved a combination of substrate mutations without typical resistance mutations in PR. These substrate changes were distributed throughout Gag and acted in an additive manner. Thus, they are classified as primary resistance mutations indicating a unique mechanism and pathway of resistance development for GS-8374.

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.

Adverse effects induced by HIV protease inhibitors (PIs) are a significant factor in limiting their clinical success. PIs directly contribute to peripheral insulin resistance and alterations in lipid metabolism. GS-8374 is a novel PI with potent antiretroviral activity and a favorable resistance profile. Here we report on the potential of GS-8374 to adversely affect glucose and lipid homeostasis. Acute effects of GS-8374 and control PIs on glucose uptake and lipid accumulation were assessed in vitro in mouse OP9 and primary human adipocytes, respectively. GS-8374 and atazanavir showed no effect on insulin-stimulated deoxyglucose uptake, whereas ritonavir and lopinavir caused significant reductions. Similarly, in vitro lipid accumulation was not significantly affected in adipocytes treated with either GS-8374 or atazanavir. In euglycemic-hyperinsulinemic clamp experiments performed in rats during acute infusion of therapeutic levels of PIs, sustained serum GS-8374 levels of 8 μM had no effect on peripheral glucose disposal (similar to the findings for atazanavir). Comparable serum levels of lopinavir and ritonavir produced acute 19% and 53% reductions in in vivo glucose disposal, respectively. In conclusion, similar to atazanavir, but unlike ritonavir and lopinavir, GS-8374 neither affects insulin-stimulated glucose uptake in adipocytes in culture nor acutely alters peripheral glucose disposal in a rodent model system. These results dissociate the antiretroviral activity of GS-8374 from adverse effects on insulin sensitivity observed with some of the first-generation PIs and provide further support for the use of these experimental systems in the preclinical evaluation of novel PIs.

GS-8374 is a novel bis-tetrahydrofuran HIV-1 protease (PR) inhibitor (PI) with a unique diethylphosphonate moiety. It was selected from a series of analogs containing various di(alkyl)phosphonate substitutions connected via a linker to the para position of a P-1 phenyl ring. GS-8374 inhibits HIV-1 PR with high potency ( K i = 8.1 pM) and with no known effect on host proteases. Kinetic and thermodynamic analysis of GS-8374 binding to PR demonstrated an extremely slow off rate for the inhibitor and favorable contributions of both the enthalpic and entropic components to the total free binding energy. GS-8374 showed potent antiretroviral activity in T-cell lines, primary CD4 + T cells (50% effective concentration [EC 50 ] = 3.4 to 11.5 nM), and macrophages (EC 50 = 25.5 nM) and exhibited low cytotoxicity in multiple human cell types. The antiviral potency of GS-8374 was only moderately affected by human serum protein binding, and its combination with multiple approved antiretrovirals showed synergistic effects. When it was tested in a PhenoSense assay against a panel of 24 patient-derived viruses with high-level PI resistance, GS-8374 showed lower mean EC 50 s and lower fold resistance than any of the clinically approved PIs. Similar to other PIs, in vitro hepatic microsomal metabolism of GS-8374 was efficiently blocked by ritonavir, suggesting a potential for effective pharmacokinetic boosting in vivo . In summary, results from this broad in vitro pharmacological profiling indicate that GS-8374 is a promising candidate to be further assessed as a new antiretroviral agent with potential for clinical efficacy in both treatment-naïve and -experienced patients.

GS-9131 is a phosphonoamidate prodrug of the novel ribose-modified phosphonate nucleotide analog GS-9148 that demonstrates potent anti-human immunodeficiency virus type 1 (HIV-1) activity and an excellent resistance profile in vitro. Prodrug moieties were optimized for the efficient delivery of GS-9148 and its active diphosphate (DP) metabolite to lymphoid cells following oral administration. To understand the intracellular pharmacology of GS-9131, incubations were performed with various types of lymphoid cells in vitro. The intracellular accumulation and antiviral activity levels of GS-9148 were limited by its lack of cellular permeation, and GS-9131 increased the delivery of GS-9148-DP by 76- to 290-fold relative to that of GS-9148. GS-9131 activation was saturable at high extracellular concentrations, potentially due to a high-affinity promoiety cleavage step. Once inside the cells, GS-9148 was efficiently phosphorylated, forming similar amounts of anabolites in primary lymphoid cells. The levels of GS-9148-DP formed in peripheral blood mononuclear cells infected with HIV-1 were similar to that in uninfected PBMCs, and approximately equivalent intracellular concentrations of GS-9148-DP and tenofovir (TVF)-DP were required to inhibit viral replication by 90%. Once it was formed, GS-9148-DP was efficiently retained in activated CD4 + cells, with a half-life of 19 h. In addition, GS-9131 showed a low potential for drug interactions with other adenine nucleoside/nucleotide reverse transcriptase inhibitors, based on the lack of competition for anabolism between suprapharmacologic concentrations of GS-9148 and TVF and the lack of activity of GS-9131 metabolites against purine nucleoside phosphorylase, an enzyme involved in the clearance of 2′,3′-dideoxyinosine. Together, these observations elucidate the cellular pharmacology of GS-9131 and illustrate its efficient loading of lymphoid cells, resulting in a prolonged intracellular exposure to the active metabolite GS-9148-DP.

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.

During HIV-1 reverse transcription, there are increasing opportunities for nucleos(t)ide (NRTI) or nonnucleoside (NNRTI) reverse transcriptase (RT) inhibitors to stop elongation of the nascent viral DNA (vDNA). In addition, RT inhibitors appear to influence the kinetics of vDNA synthesis differently. While cell-free kinetic inhibition constants have provided detailed mechanistic insight, these assays are dependent on experimental conditions that may not mimic the cellular milieu. Here we describe a novel cell-based strategy to provide a measure of the intrinsic inhibition efficiencies of clinically relevant RT inhibitors on a per-stop-site basis. To better compare inhibition efficiencies among HIV-1 RT inhibitors that can stop reverse transcription at any number of different stop sites, their basic probability,p, of getting stopped at any potential stop site was determined. A relationship between qPCR-derived 50% effective inhibitory concentrations (EC50s) and this basic probability enabled determination ofpby successive approximation. On a per-stop-site basis, tenofovir (TFV) exhibited 1.4-fold-greater inhibition efficiency than emtricitabine (FTC), and as a class, both NRTIs exhibited an 8- to 11-fold greater efficiency than efavirenz (EFV). However, as more potential stops sites were considered, the probability of reverse transcription failing to reach the end of the template approached equivalence between both classes of RT inhibitors. Overall, this novel strategy provides a quantitative measure of the intrinsic inhibition efficiencies of RT inhibitors in the natural cellular milieu and thus may further understanding of drug efficacy. This approach also has applicability for understanding the impact of viral polymerase-based inhibitors (alone or in combination) in other virus systems.

The maturation of dendritic cells (DCs) is associated with a diminished ability to support human immunodeficiency virus (HIV) replication; however, the precise step in the HIV life cycle impaired by DC maturation remains uncertain. Using an HIV virion-based fusion assay, we now show that HIV fusion to monocyte-derived DCs (MDDCs) both decreases and kinetically slows when DCs are induced to mature with poly(I:C) and tumor necrosis factor alpha. Specifically, laboratory-adapted CCR5-tropic 81A virions fused with markedly lower efficiency to mature MDDCs than immature DCs. In contrast, fusion of NL4-3, the isogenic CXCR4-tropic counterpart of 81A, was low in both immature and mature MDDCs. Fusion mediated by primary HIV envelopes, including seven CCR5- and four CXCR4-tropic envelopes, also decreased with DC maturation. The kinetics of virion fusion were also altered by both the state of DC maturation and the coreceptor utilized. Fusion of 81A and NL4-3 virions was delayed in mature compared to immature MDDCs, and NL4-3 fused more slowly than 81A in both mature and immature MDDCs. Surprisingly, primary envelopes with CXCR4 tropism mediated fusion to immature MDDCs with efficiencies similar to those of primary CCR5-tropic envelopes. This result contrasted with the marked preferential fusion of the laboratory-adapted 81A over NL4-3 in immature MDDCs and in ex vivo Langerhans cells, indicating that these laboratory-adapted HIV strains do not fully recapitulate all of the properties of primary HIV isolates. In conclusion, our results demonstrate that the defect in HIV replication observed in mature MDDCs stems at least in part from a decline in viral fusion.