Long Dissociation of Bictegravir from HIV-1 Integrase-DNA Complexes.

The HIV integrase (IN) strand transfer inhibitor (INSTI) bictegravir (BIC) has a long dissociation half-life (t1/2) from wild-type IN-DNA complexes: BIC 163 hr > dolutegravir (DTG) 96 hr > raltegravir (RAL) 10 hr > elvitegravir (EVG) 3.3 hr. In cells, BIC had more durable antiviral activity against wild-type HIV after drug washout than RAL or EVG. BIC also had a longer t1/2 and maintained longer antiviral activity after drug washout than DTG with the clinically relevant resistance IN mutant G140S+Q148H. Structural analyses indicate that BIC makes more contacts with the IN-DNA complex than DTG mainly via its bicyclic ring system which may contribute to more prolonged residence time and resilience against many resistance mutations.

Forgiveness of INSTI-Containing Regimens at Drug Concentrations Simulating Variable Adherence In Vitro.

The integrase strand transfer inhibitor (INSTI)-based regimens bictegravir/emtricitabine/tenofovir alafenamide (BIC/FTC/TAF), dolutegravir (DTG)+FTC/TAF, DTG/lamivudine (3TC), and DTG/rilpivirine (RPV) are all approved for treatment of HIV-infected patients, with various limitations. Here, time to in vitro viral breakthrough (VB) and resistance barrier using simulated human drug exposures at either full or suboptimal treatment adherence to each regimen were compared. At drug concentrations corresponding to full adherence and 1 missed dose (Cmin and Cmin-1), no VB occurred with any regimen. At Cmin-2, VB occurred only with DTG+3TC, with emergent resistance to both drugs. At Cmin-3, VB occurred with all regimens: 100% of DTG+3TC cultures had VB by day 12, and <15% of BIC+FTC+TAF, DTG+FTC+TAF, and DTG+RPV cultures had VB. Emergent reverse transcriptase (RT) or integrase (IN) resistance was seen with DTG+RPV and DTG+3TC but not with BIC+FTC+TAF or DTG+FTC+TAF. At Cmin-4, 100% VB occurred with DTG+3TC and DTG+FTC+TAF by day 12, while 94% VB occurred with DTG+RPV by day 25 and only 50% VB occurred with BIC+FTC+TAF by day 35. Emergent Cmin-4 drug resistance was seen with all regimens but at differing frequencies; DTG+RPV had the most cultures with resistance. Emergent resistance was consistent with clinical observations. Overall, under high adherence conditions, no in vitro VB or resistance development occurred with these INSTI-based regimens. However, when multiple missed doses were simulated in vitro, BIC+FTC+TAF had the highest forgiveness and barrier to resistance of all tested regimens. Compared to DTG+3TC and DTG+FTC+TAF, DTG+RPV had higher forgiveness but lower resistance barrier after several simulated missed doses.

Three-year study of pre-existing drug resistance substitutions and efficacy of bictegravir/emtricitabine/tenofovir alafenamide in HIV-1 treatment-naive participants.

OBJECTIVES: Two Phase 3, randomized, double-blind, active-controlled studies of initial HIV-1 treatment demonstrated that bictegravir/emtricitabine/tenofovir alafenamide (B/F/TAF) was non-inferior to dolutegravir/abacavir/lamivudine (DTG/ABC/3TC; Study 1489) or to DTG+F/TAF (Study 1490) through 144 weeks. In both studies, there was no emergent resistance to study drugs. Here, the 3 year resistance analysis and impact of baseline resistance substitutions on treatment response are described. METHODS: Population sequencing of HIV-1 protease and reverse transcriptase (RT) was performed at screening. Retrospective baseline next generation sequencing of protease, RT and integrase (IN) was analysed at a ≥ 15% cutoff. Resistance analyses were performed on participants with confirmed viral rebound of HIV-1 RNA ≥200 copies/mL through Week 144 or last visit who did not resuppress to <50 copies/mL while on study drug. RESULTS: Transmitted primary drug resistance substitutions were present in the following proportions of participants: integrase strand transfer inhibitor (INSTI) resistance (-R) in 1.3% (17/1270) of participants; NRTI-R in 2.7% (35/1274); NNRTI-R in 14.1% (179/1274); and PI-R in 3.5% (44/1274). These pre-existing resistance substitutions not associated with study drug did not affect treatment outcomes. One participant in the B/F/TAF group had pre-existing bictegravir and dolutegravir resistance substitutions (Q148H+G140S in integrase) at baseline and suppressed and maintained HIV-1 RNA <50 copies/mL through Week 144. In total, 21 participants qualified for resistance testing [1.3% (8/634) B/F/TAF; 1.9% (6/315) DTG/ABC/3TC; 2.2% (7/325) DTG+F/TAF]; none had emergent resistance to study drugs. CONCLUSIONS: Treatment with B/F/TAF, DTG/ABC/3TC, or DTG+F/TAF achieved high, durable rates of virological suppression in HIV-1 treatment-naive participants. The presence of pre-existing resistance substitutions did not affect treatment outcomes, and there was no treatment-emergent resistance.

Resistance Analysis of Bictegravir-Emtricitabine-Tenofovir Alafenamide in HIV-1 Treatment-Naive Patients through 48 Weeks.

In clinical studies GS-US-380-1489 (study 1489) and GS-US-380-1490 (study 1490), bictegravir-emtricitabine-tenofovir alafenamide (B-F-TAF), dolutegravir-abacavir-lamivudine (DTG-ABC-3TC), and dolutegravir plus emtricitabine-tenofovir alafenamide (DTG+F-TAF) treatment achieved high rates of virologic suppression in HIV-1 treatment-naive participants through week 48. Preexisting primary drug resistance was present at levels of 1.3% integrase strand transfer inhibitor resistance (INSTI-R), 2.7% nucleoside reverse transcriptase inhibitor resistance (NRTI-R), 14.1% nonnucleoside reverse transcriptase inhibitor resistance (NNRTI-R), and 3.5% protease inhibitor resistance (PI-R) in the 1,274 participants from these studies. These mutations did not affect treatment outcomes. Resistance analyses in 13 virologic failures found no emergent resistance to study drugs.

Switching to bictegravir/emtricitabine/tenofovir alafenamide maintained HIV-1 RNA suppression in participants with archived antiretroviral resistance including M184V/I.

OBJECTIVES: Studies 1878 and 1844 demonstrated non-inferior efficacy of switching suppressed HIV-1-infected adults to bictegravir/emtricitabine/tenofovir alafenamide (BIC/FTC/TAF) versus continuing boosted PI-based triple regimens or dolutegravir/abacavir/lamivudine (DTG/ABC/3TC). Here, detailed analyses of pre-existing resistance in the two BIC/FTC/TAF switch studies and efficacy at week 48 are described. METHODS: Pre-existing resistance was assessed from historical genotypes (documented resistance to study drugs was excluded) and by retrospective baseline proviral archive DNA genotyping from whole blood. Outcomes were based on HIV-1 RNA at week 48 with missing values imputed using the last on-treatment observation carried forward method. RESULTS: Cumulative pre-existing resistance data from historical and proviral genotypes were obtained for 95% (543/570) of participants who switched to BIC/FTC/TAF. Altogether, 40% (217/543) had one or more pre-existing primary resistance substitutions in protease, reverse transcriptase and/or integrase. Pre-switch NRTI resistance was detected in 16% (89/543) of BIC/FTC/TAF-treated participants, with M184V or M184I detected by proviral genotyping in 10% (54/543). At week 48, 98% (561/570) of all BIC/FTC/TAF-treated participants versus 98% (213/217) with pre-existing resistance and 96% (52/54) with archived M184V/I had HIV-1 RNA <50 copies/mL. No BIC/FTC/TAF-treated participants developed treatment-emergent resistance to study drugs. CONCLUSIONS: Pre-existing resistance substitutions, notably M184V/I, were unexpectedly common among suppressed participants who switched to BIC/FTC/TAF. High rates of virological suppression were maintained in the overall study population and in those with pre-existing resistance, including M184V/I, for up to 48 weeks of BIC/FTC/TAF treatment with no resistance development. These results indicate that BIC/FTC/TAF is an effective treatment option for suppressed patients, including those with evidence of archived NRTI resistance.

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.

Drug Susceptibility and Viral Fitness of HIV-1 with Integrase Strand Transfer Inhibitor Resistance Substitution Q148R or N155H in Combination with Nucleoside/Nucleotide Reverse Transcriptase Inhibitor Resistance Substitutions.

In clinical trials of coformulated elvitegravir (EVG), cobicistat (COBI), emtricitabine (FTC), and tenofovir disoproxil fumarate (TDF), emergent drug resistance predominantly involved the FTC resistance substitution M184V/I in reverse transcriptase (RT), with or without the tenofovir (TFV) resistance substitution K65R, accompanied by a primary EVG resistance substitution (E92Q, N155H, or Q148R) in integrase (IN). We previously reported that the RT-K65R, RT-M184V, and IN-E92Q substitutions lacked cross-class phenotypic resistance and replicative fitness compensation. As a follow-up, the in vitro characteristics of mutant HIV-1 containing RT-K65R and/or RT-M184V with IN-Q148R or IN-N155H were also evaluated, alone and in combination, for potential interactions. Single mutants displayed reduced susceptibility to their corresponding inhibitor classes, with no cross-class resistance. Viruses with IN-Q148R or IN-N155H exhibited reduced susceptibility to EVG (137- and 40-fold, respectively) that was not affected by the addition of RT-M184V or RT-K65R/M184V. All viruses containing RT-M184V were resistant to FTC (>1,000-fold). Mutants with RT-K65R had reduced susceptibility to TFV (3.3- to 3.6-fold). Without drugs present, the viral fitness of RT and/or IN mutants was diminished relative to that of the wild type in the following genotypic order: wild type > RT-M184V ≥ IN-N155H ≈ IN-Q148R ≥ RT-M184V + IN-N155H ≥ RT-M184V + IN-Q148R ≥ RT-K65R/M184V + IN-Q148R ≈ RT-K65R/M184V + IN-N155H. In the presence of drug concentrations approaching physiologic levels, drug resistance counteracted replication defects, allowing single mutants to outcompete the wild type with one drug present and double mutants to outcompete single mutants with two drugs present. These results suggest that during antiretroviral treatment with multiple drugs, the development of viruses with combinations of resistance substitutions may be favored despite diminished viral fitness.

Antiviral Activity of Bictegravir (GS-9883), a Novel Potent HIV-1 Integrase Strand Transfer Inhibitor with an Improved Resistance Profile.

Bictegravir (BIC; GS-9883), a novel, potent, once-daily, unboosted inhibitor of HIV-1 integrase (IN), specifically targets IN strand transfer activity (50% inhibitory concentration [IC50] of 7.5 ± 0.3 nM) and HIV-1 integration in cells. BIC exhibits potent and selective in vitro antiretroviral activity in both T-cell lines and primary human T lymphocytes, with 50% effective concentrations ranging from 1.5 to 2.4 nM and selectivity indices up to 8,700 relative to cytotoxicity. BIC exhibits synergistic in vitro antiviral effects in pairwise combinations with tenofovir alafenamide, emtricitabine, or darunavir and maintains potent antiviral activity against HIV-1 variants resistant to other classes of antiretrovirals. BIC displayed an in vitro resistance profile that was markedly improved compared to the integrase strand transfer inhibitors (INSTIs) raltegravir (RAL) and elvitegravir (EVG), and comparable to that of dolutegravir (DTG), against nine INSTI-resistant site-directed HIV-1 mutants. BIC displayed statistically improved antiviral activity relative to EVG, RAL, and DTG against a panel of 47 patient-derived HIV-1 isolates with high-level INSTI resistance; 13 of 47 tested isolates exhibited >2-fold lower resistance to BIC than DTG. In dose-escalation experiments conducted in vitro, BIC and DTG exhibited higher barriers to resistance than EVG, selecting for HIV-1 variants with reduced phenotypic susceptibility at days 71, 87, and 20, respectively. A recombinant virus with the BIC-selected M50I/R263K dual mutations in IN exhibited only 2.8-fold reduced susceptibility to BIC compared to wild-type virus. All BIC-selected variants exhibited low to intermediate levels of cross-resistance to RAL, DTG, and EVG (<8-fold) but remained susceptible to other classes of antiretrovirals. A high barrier to in vitro resistance emergence for both BIC and DTG was also observed in viral breakthrough studies in the presence of constant clinically relevant drug concentrations. The overall virologic profile of BIC supports its ongoing clinical investigation in combination with other antiretroviral agents for both treatment-naive and -experienced HIV-infected patients.

Clinical Outcomes of Virologically-Suppressed Patients with Pre-existing HIV-1 Drug Resistance Mutations Switching to Rilpivirine/Emtricitabine/Tenofovir Disoproxil Fumarate in the SPIRIT Study.

OBJECTIVES: Antiretroviral regimen switching may be considered for HIV-1-infected, virologically-suppressed patients to enable treatment simplification or improve tolerability, but should be guided by knowledge of pre-existing drug resistance. The current study examined the impact of pre-existing drug resistance mutations on virologic outcomes among virologically-suppressed patients switching to Rilpivirine (RPV)/emtricitabine (FTC)/tenofovir disoproxil fumarate (TDF). METHODS: SPIRIT was a phase 3b study evaluating the safety and efficacy of switching to RPV/FTC/TDF in virologically-suppressed HIV-1-infected patients. Pre-existing drug resistance at baseline was determined by proviral DNA genotyping for 51 RPV/FTC/TDF-treated patients with known mutations by historical RNA genotype and matched controls and compared with clinical outcome at Week 48. RESULTS: Drug resistance mutations in protease or reverse transcriptase were detected in 62.7% of patients by historical RNA genotype and in 68.6% by proviral DNA genotyping at baseline. Proviral DNA sequencing detected 89% of occurrences of NRTI and NNRTI resistance-associated mutations reported by historical genotype. Mutations potentially affecting RPV activity, including E138A/G/K/Q, Y181C, and H221Y, were detected in isolates from 11 patients by one or both assays. None of the patients with single mutants had virologic failure through Week 48. One patient with pre-existing Y181Y/C and M184I by proviral DNA genotyping experienced virologic failure. Nineteen patients with K103N present by historical genotype were confirmed by proviral DNA sequencing and 18/19 remained virologically-suppressed. DISCUSSION: Virologic success rates were high among virologically-suppressed patients with pre-existing NRTI and NNRTI resistance-associated mutations who switched to RPV/FTC/TDF in the SPIRIT study. While plasma RNA genotyping remains preferred, proviral DNA genotyping may provide additional value in virologically-suppressed patients for whom historical resistance data are unavailable.

A cell-based strategy to assess intrinsic inhibition efficiencies of HIV-1 reverse transcriptase inhibitors.

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 of p by 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.

Reduced viral fitness and lack of cross-class resistance with integrase strand transfer inhibitor and nucleoside reverse transcriptase inhibitor resistance mutations.

The most common pattern of emergent resistance in the phase III clinical trials of coformulated elvitegravir (EVG)-cobicistat (COBI)-emtricitabine (FTC)-tenofovir disoproxil fumarate (TDF) was the EVG resistance substitution E92Q in integrase (IN) with the FTC resistance substitution M184V in reverse transcriptase (RT), with or without the tenofovir (TFV) resistance substitution K65R. In this study, the effect of these IN and RT substitutions alone and in combination in the same genome on susceptibility to antiretroviral inhibitors and viral replication fitness was characterized. Single resistance substitutions (E92Q in IN [IN-E92Q], M184V in RT [RT-M184V], and K65R in RT [RT-K65R]) specifically affected susceptibility to the corresponding inhibitor classes, with no cross-class resistance observed. The IN-E92Q mutant displayed reduced susceptibility to EVG (50-fold), which was not impacted by the addition of RT-M184V or RT-K65R/M184V. Viruses containing RT-M184V had high-level resistance to FTC (>1,000-fold) that was not affected by the addition of IN-E92Q or RT-K65R. During pairwise growth competitions, each substitution contributed to decreased viral fitness, with the RT-K65R/M184V + IN-E92Q triple mutant being the least fit in the absence of drug. In the presence of drug concentrations approaching physiologic levels, however, drug resistance offset the replication defects, resulting in single mutants outcompeting the wild type with one drug present, and double and triple mutants outcompeting single mutants with two drugs present. Taken together, these results suggest that the reduced replication fitness and phenotypic resistance associated with RT and IN resistance substitutions are independent and additive. In the presence of multiple drugs, viral growth is favored for viruses with multiple substitutions, despite the presence of fitness defects.

96-week resistance analyses of the STaR study: rilpivirine/emtricitabine/tenofovir DF versus efavirenz/emtricitabine/tenofovir DF in antiretroviral-naive, HIV-1-infected subjects.

BACKGROUND: STaR (GS-US-264-0110) was a 96-week phase 3b study evaluating the safety and efficacy of two single-tablet regimens, rilpivirine/emtricitabine/tenofovir DF (RPV/FTC/TDF) and efavirenz/emtricitabine/tenofovir DF (EFV/FTC/TDF) in treatment-naive, HIV-1-infected subjects. METHODS: Genotypic analyses (population sequencing) of HIV-1 protease (PR) and reverse transcriptase (RT) were performed at screening; subjects with pre-existing resistance to study drugs were excluded. The protocol-defined resistance analysis population had genotypic/phenotypic analyses at failure and baseline for PR and RT. RESULTS: Through week 96, the resistance analysis population included 24/394 subjects (6.1%) receiving RPV/FTC/TDF and 9/392 subjects (2.3%) receiving EFV/FTC/TDF. In the RPV/FTC/TDF arm, HIV-1 isolates from 21/394 subjects (5.3%) developed non-nucleoside reverse transcriptase inhibitor (NNRTI) and/or nucleoside reverse transcriptase inhibitor (NRTI) resistance mutations and 20/21 isolates had both NNRTI and NRTI genotypic and/or phenotypic resistance. In the EFV/FTC/TDF arm, isolates from 4/392 subjects (1.0%) developed NNRTI and/or NRTI resistance mutations. Resistance development after week 48 was infrequent (1.0% RPV/FTC/TDF; 0.3% EFV/FTC/TDF). When stratified by baseline HIV-1 RNA ≤ or >100 000 copies/ml, 9/260 (3.5%) versus 12/134 (9.0%) RPV/FTC/TDF-treated subjects and 3/250 (1.2%) versus 1/142 (0.7%) EFV/FTC/TDF-treated subjects developed resistant isolates, respectively. Pre-existing NRTI- and NNRTI-associated resistance mutations (not related to study drugs) did not impact treatment response to either regimen. CONCLUSIONS: Resistance development to RPV/FTC/TDF consisted of NNRTI and NRTI mutations and was more frequent than resistance development to EFV/FTC/TDF through week 96. Emergent resistance after week 48 was infrequent in both arms. Within the RPV/FTC/TDF arm, resistance development was more frequent in subjects with baseline HIV-1 RNA >100 000 copies/ml compared to baseline HIV-1 RNA ≤ 100 000 copies/ml.

The combined anti-HIV-1 activities of emtricitabine and tenofovir plus the integrase inhibitor elvitegravir or raltegravir show high levels of synergy in vitro.

Highly active antiretroviral therapy (HAART) involves combination treatment with three or more antiretroviral agents. The antiviral effects of combinations of emtricitabine (FTC) plus tenofovir (TFV) plus antiretroviral agents of all the major drug classes were investigated. Combinations of FTC and TFV with a nonnucleoside reverse transcriptase inhibitor (NNRTI) (efavirenz or rilpivirine) or with a protease inhibitor (PI) (atazanavir, lopinavir, or darunavir) showed additive to synergistic anti-HIV-1 activity. FTC-TFV with an HIV-1 integrase strand transfer inhibitor (INSTI) (elvitegravir or raltegravir) showed the strongest synergy. Anti-HIV-1 synergy suggests enhancement of individual anti-HIV-1 activities within cells that may contribute to potent treatment efficacy and open new areas of research into interactions between reverse transcriptase (RT) and integrase inhibitors.

Week 144 resistance analysis of elvitegravir/cobicistat/emtricitabine/tenofovir DF versus atazanavir+ritonavir+emtricitabine/tenofovir DF in antiretroviral-naïve patients.

OBJECTIVE: To describe baseline and emergent HIV-1 resistance to elvitegravir/ cobicistat/emtricitabine/tenofovir DF (EVG/COBI/FTC/TDF) and ritonavir-boosted atazanavir/emtricitabine/tenofovir DF (ATV+RTV+FTC/TDF) in HIV-1-infected, treatment-naïve subjects through 144 weeks. METHOD: This was a randomized, double-blind, phase 3 study. HIV-1 protease (PR) and reverse transcriptase (RT) were sequenced at screening. Genotypic and phenotypic analyses were performed at virologic failure confirmation and retrospectively at baseline for PR, RT, and integrase (IN) for patients with virologic failure through week 144. RESULTS: In the EVG/ COBI/FTC/TDF group through week 144, HIV-1 from 8 patients (2.3%; 8/353 treated patients) developed primary IN strand transfer inhibitor (INSTI) (n = 6) and/or nucleoside RT inhibitor (NRTI) resistance substitutions (n = 7). The emergence of resistance decreased after the first year, with 5 patients developing HIV-1 resistance through week 48, 1 from weeks 48-96, and 2 from weeks 96-144. Emergent substitutions were E92Q, N155H, or Q148R (n = 2 each) and T66I or T97A (n = 1 each) in IN and M184V/I (n = 7) and K65R (n = 1) in RT. All 8 isolates had reduced susceptibility to EVG, FTC, or TDF. Virus with EVG phenotypic resistance showed cross-resistance to raltegravir. In the ATV+RTV+FTC/TDF group, HIV-1 from 2 patients (0.6%; 2/355 treated patients; both at week 144) developed the resistance substitution M184V/I in RT. CONCLUSIONS: Resistance development to EVG/COBI/FTC/TDF was infrequent (2.3%) through 144 weeks of therapy and decreased over time, consistent with durable efficacy.

Impact of primary elvitegravir resistance-associated mutations in HIV-1 integrase on drug susceptibility and viral replication fitness.

Elvitegravir (EVG) is an effective HIV-1 integrase (IN) strand transfer inhibitor (INSTI) in advanced clinical development. Primary INSTI resistance-associated mutations (RAMs) at six IN positions have been identified in HIV-1-infected patients failing EVG-containing regimens in clinical studies: T66I/A/K, E92Q/G, T97A, S147G, Q148R/H/K, and N155H. In this study, the effect of these primary IN mutations, alone and in combination, on susceptibility to the INSTIs EVG, raltegravir (RAL), and dolutegravir (DTG); IN enzyme activities; and viral replication fitness was characterized. Recombinant viruses containing the six most common mutations exhibited a range of reduced EVG susceptibility: 92-fold for Q148R, 30-fold for N155H, 26-fold for E92Q, 10-fold for T66I, 4-fold for S147G, and 2-fold for T97A. Less commonly observed primary IN mutations also showed a range of reduced EVG susceptibilities: 40- to 94-fold for T66K and Q148K and 5- to 10-fold for T66A, E92G, and Q148H. Some primary IN mutations exhibited broad cross-resistance between EVG and RAL (T66K, E92Q, Q148R/H/K, and N155H), while others retained susceptibility to RAL (T66I/A, E92G, T97A, and S147G). Dual combinations of primary IN mutations further reduced INSTI susceptibility, replication capacity, and viral fitness relative to either mutation alone. Susceptibility to DTG was retained by single primary IN mutations but reduced by dual mutation combinations with Q148R. Primary EVG RAMs also diminished IN enzymatic activities, concordant with their structural proximity to the active site. Greater reductions in viral fitness of dual mutation combinations may explain why some primary INSTI RAMs do not readily coexist on the same HIV-1 genome but rather establish independent pathways of resistance to EVG.

Inhibition of 2',5'-oligoadenylate synthetase expression and function by the human cytomegalovirus ORF94 gene product.

The human cytomegalovirus (HCMV) ORF94 gene product has been reported to be expressed during both productive and latent phases of infection, although its function is unknown. We report that expression of pORF94 leads to decreased 2',5'-oligoadenylate synthetase (OAS) expression in transfected cells with and without interferon stimulation. Furthermore, the functional activity of OAS was inhibited by pORF94. Finally, we present evidence of OAS modulation by pORF94 during productive HCMV infection of human fibroblasts. This study provides the first identification of a function for pORF94 and identifies an additional means by which HCMV may limit a critical host cell antiviral response.

Brief Report: Bictegravir/Emtricitabine/Tenofovir Alafenamide Efficacy in Participants With Preexisting Primary Integrase Inhibitor Resistance Through 48 Weeks of Phase 3 Clinical Trials.

BACKGROUND: Preexisting drug resistance limits the utility of HIV antiretroviral therapy. Studies have demonstrated safety and efficacy of bictegravir/emtricitabine/tenofovir alafenamide (B/F/TAF), including in patients with M184V/I substitutions. SETTING: We investigated virologic outcomes through 48 weeks of B/F/TAF treatment in individuals with preexisting primary integrase strand transfer inhibitor resistance (INSTI-R). METHODS: Preexisting INSTI-R was retrospectively evaluated from 7 B/F/TAF studies. INSTI-R was assessed by historical genotypes and/or baseline RNA or DNA sequencing. Viral loads were measured at all visits. RESULTS: Preexisting primary INSTI-R substitutions were detected in 20 of the 1907 participants (1.0%). The 20 participants were predominantly male (75%), were Black (65%), had HIV-1 subtype B (85%), and had baseline median CD4 counts of 594 cells/mm3 and median age of 52 years. Most of the participants (n = 19) were virologically suppressed at baseline and had one primary INSTI-R substitution, E92G, Y143C/H, S147G, Q148H/K/R, N155S, or R263K, +/-secondary substitutions. All suppressed participants maintained virologic suppression throughout 48 weeks without any viral blips. One treatment-naive participant had virus with Q148H+G140S that was fully sensitive to bictegravir but only partially to dolutegravir (phenotype <2.5-fold change and >4-fold change, respectively). With a baseline viral load of 30,000 copies/mL, this participant was virologically suppressed by week 4 and maintained <50 copies/mL through week 48. CONCLUSIONS: This small cohort with primary INSTI-R achieved and/or maintained virologic suppression through 48 weeks of B/F/TAF treatment. Consistent with the potent in vitro activity of bictegravir against most INSTI-R patterns, B/F/TAF may be a potential treatment option for patients with select preexisting INSTI-R, if confirmed by further studies.

High efficacy of switching to bictegravir/emtricitabine/tenofovir alafenamide in people with suppressed HIV and preexisting M184V/I.

OBJECTIVE: We investigated the prevalence of preexisting M184V/I and associated risk factors among clinical trial participants with suppressed HIV and evaluated the impact of M184V/I on virologic response after switching to bictegravir/emtricitabine/tenofovir alafenamide (B/F/TAF). DESIGN: Participant data were pooled from six clinical trials investigating the safety and efficacy of switching to B/F/TAF in virologically suppressed people with HIV. METHODS: Preexisting drug resistance was assessed by historical genotypes and/or baseline proviral DNA genotyping. Virologic outcomes were determined by last available on-treatment HIV-1 RNA. Stepwise selection identified potential risk factors for M184V/I in a multivariate logistic regression model. RESULTS: Altogether, 2034 participants switched treatment regimens to B/F/TAF and had follow-up HIV-1 RNA data, and 1825 of these participants had baseline genotypic data available. Preexisting M184V/I was identified in 182 (10%), mostly by baseline proviral DNA genotype ( n  = 167). Most substitutions were M184V ( n  = 161) or M184V/I mixtures ( n  = 10). Other resistance substitutions were often detected in addition to M184V/I ( n  = 147). At last on-treatment visit, 98% (179/182) with preexisting M184V/I and 99% (2012/2034) of all B/F/TAF-treated participants had HIV-1 RNA less than 50 copies/ml, with no treatment-emergent resistance to B/F/TAF. Among adult participants, factors associated with preexisting M184V/I included other resistance, black race, Hispanic/Latinx ethnicity, lower baseline CD4 + cell count, advanced HIV disease, longer duration of antiretroviral therapy, and greater number of prior third agents. CONCLUSION: M184V/I was detected in 10% of virologically suppressed clinical trial participants at study baseline. Switching to B/F/TAF demonstrated durable efficacy in maintaining viral suppression, including in those with preexisting M184V/I.

Mechanism of resistance to GS-9148 conferred by the Q151L mutation in HIV-1 reverse transcriptase.

GS-9148 is an investigational phosphonate nucleotide analogue inhibitor of reverse transcriptase (RT) (NtRTI) of human immunodeficiency virus type 1 (HIV-1). This compound is an adenosine derivative with a 2',3'-dihydrofuran ring structure that contains a 2'-fluoro group. The resistance profile of GS-9148 is unique in that the inhibitor can select for the very rare Q151L mutation in HIV-1 RT as a pathway to resistance. Q151L is not stably selected by any of the approved nucleoside or nucleotide analogues; however, it may be a transient intermediate that leads to the related Q151M mutation, which confers resistance to multiple compounds that belong to this class of RT inhibitors. Here, we employed pre-steady-state kinetics to study the impact of Q151L on substrate and inhibitor binding and the catalytic rate of incorporation. Most importantly, we found that the Q151L mutant is unable to incorporate GS-9148 under single-turnover conditions. Interference experiments showed that the presence of GS-9148-diphosphate, i.e., the active form of the inhibitor, does not reduce the efficiency of incorporation for the natural counterpart. We therefore conclude that Q151L severely compromises binding of GS-9148-diphosphate to RT. This effect is highly specific, since we also demonstrate that another NtRTI, tenofovir, is incorporated with selectivity similar to that seen with wild-type RT. Incorporation assays with other related compounds and models based on the RT/DNA/GS-9148-diphosphate crystal structure suggest that the 2'-fluoro group of GS-9148 may cause steric hindrance with the side chain of the Q151L mutant.