Phenotypic resistance to lenacapavir and monotherapy efficacy in a proof-of-concept clinical study.

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.

Absence of Lenacapavir (GS-6207) Phenotypic Resistance in HIV Gag Cleavage Site Mutants and in Isolates with Resistance to Existing Drug Classes.

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.

Antiviral Activity of Tenofovir Alafenamide against HIV-1 with Thymidine Analog-Associated Mutations and M184V.

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.

Differential detection of M184V/I between plasma historical HIV genotypes and HIV proviral DNA from PBMCs.

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 <50 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. RESULTS: 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. CONCLUSIONS: 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.

Antiviral activity of HIV-1 integrase strand-transfer inhibitors against mutants with integrase resistance-associated mutations and their frequency in treatment-naïve individuals.

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-naïve 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-naïve 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.

Activation of HIV-specific CD8+ T-cells from HIV+ donors by vesatolimod.

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.

Randomized study evaluating the efficacy and safety of switching from an an abacavir/lamivudine-based regimen to an elvitegravir/cobicistat/emtricitabine/tenofovir alafenamide single-tablet regimen.

OBJECTIVE: To evaluate the efficacy and safety of switching from an abacavir/lamivudine (ABC/3TC)-based regimen to an elvitegravir/cobicistat/emtricitabine/tenofovir alafenamide (E/C/F/TAF) single-tablet regimen in virologically suppressed, HIV-1-infected adults. DESIGN: Randomized, open-label, noninferiority study. METHODS: Participants with HIV-1 RNA levels less than 50 copies/ml receiving ABC/3TC plus a third agent for at least 6 months were randomized 2 : 1 to switch immediately to E/C/F/TAF (immediate-switch group) for 48 weeks or to continue receiving ABC/3TC plus a third agent for 24 weeks followed by E/C/F/TAF for 24 weeks (delayed-switch group). The primary endpoint was HIV-1 RNA less than 50 copies/ml at Week 24 by Food and Drug Administration Snapshot algorithm (-12% noninferiority margin). RESULTS: Baseline characteristics of 274 participants (183 in immediate-switch group and 91 in delayed-switch group) were similar. Virologic response was maintained at Week 24 by 93.4 and 97.8% of participants in the immediate-switch and delayed-switch groups, respectively, with a treatment difference of -4.4% (95% confidence interval: -9.4 to 1.9%), confirming noninferiority. Adverse events of any grade were similar between groups through Week 24 (66% E/C/F/TAF, 64% ABC/3TC); adverse event-related drug discontinuations occurred in 4% of participants switching to E/C/F/TAF (no discontinuations because of renal events) and no participants continuing ABC/3TC. Renal biomarkers of urine albumin:creatinine and beta-2-microglobulin:creatinine ratios significantly improved on E/C/F/TAF. Self-reported treatment satisfaction was significantly higher with E/C/F/TAF. CONCLUSION: Switching to E/C/F/TAF was noninferior to continuing ABC/3TC plus a third agent for maintenance of HIV RNA suppression at Week 24. This study supports E/C/F/TAF as an efficacious and well tolerated option for participants switching from ABC/3TC-based regimens.

A highly potent long-acting small-molecule HIV-1 capsid inhibitor with efficacy in a humanized mouse model.

People living with HIV (PLWH) have expressed concern about the life-long burden and stigma associated with taking pills daily and can experience medication fatigue that might lead to suboptimal treatment adherence and the emergence of drug-resistant viral variants, thereby limiting future treatment options1-3. As such, there is strong interest in long-acting antiretroviral (ARV) agents that can be administered less frequently4. Herein, we report GS-CA1, a new archetypal small-molecule HIV capsid inhibitor with exceptional potency against HIV-2 and all major HIV-1 types, including viral variants resistant to the ARVs currently in clinical use. Mechanism-of-action studies indicate that GS-CA1 binds directly to the HIV-1 capsid and interferes with capsid-mediated nuclear import of viral DNA, HIV particle production and ordered capsid assembly. GS-CA1 selects in vitro for unfit GS-CA1-resistant capsid variants that remain fully susceptible to other classes of ARVs. Its high metabolic stability and low solubility enabled sustained drug release in mice following a single subcutaneous dosing. GS-CA1 showed high antiviral efficacy as a long-acting injectable monotherapy in a humanized mouse model of HIV-1 infection, outperforming long-acting rilpivirine. Collectively, these results demonstrate the potential of ultrapotent capsid inhibitors as new long-acting agents for the treatment of HIV-1 infection.

Evolution of tenofovir-resistant HIV-1 isolates exposed to tenofovir alafenamide dose escalation.

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.

Lack of impact of pre-existing T97A HIV-1 integrase mutation on integrase strand transfer inhibitor resistance and treatment outcome.

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; <11-fold), while all maintained susceptibility to dolutegravir. Of the patients with pre-existing T97A, 17 had available clinical follow-up: 16 achieved virologic suppression and 1 maintained T97A and INSTI sensitivity without further resistance development. Overall, T97A is an infrequent integrase polymorphism that is enriched among non-B HIV-1 subtypes and can confer low-level reduced susceptibility to EVG and/or RAL. However, detection of T97A does not affect response to INSTI-based therapy with EVG or RAL. These results suggest a very low risk of initiating INSTI-based therapy in patients with pre-existing T97A.