Protease Resistance Mutations Research Articles

HIV protease resistance mutations in patients receiving second-line antiretroviral therapy in Libreville, Gabon

IntroductionIn 2022, the WHO reported that 29.8 million people around the world were living with HIV (PLHIV) and receiving antiretroviral treatment (ART), including 25‌ 375 people in Gabon (54% of all those living with HIV in the country). The literature reports a frequency of therapeutic failure with first-line antiretrovirals (ARVs) of between 20% and 82%. Unfortunately, data relating to the failure of second-line ARVs are scarce in Gabon. This study aims to determine the profiles of HIV drug resistance mutations related to protease inhibitors in Gabon.MethodologyPlasma from 84 PLHIV receiving ARVs was collected from 2019 to 2021, followed by RNA extraction, amplification, and sequencing of the protease gene. ARV resistance profiles were generated using the Stanford interpretation algorithm version 8.9-1 (https://hivdb.stanford.edu) and statistical analyses were performed using EpiInfo software version 7.2.1.0 (CDC, USA).ResultsOf 84 HIV plasma samples collected from 45 men and 39 women, 342 mutations were detected. Of these, 43.3% (148/342) were associated with nucleoside reverse transcriptase inhibitors (NRTIs), 30.4% (104/342) with non-nucleoside reverse transcriptase inhibitors (NNRTIs), and 26.3% (90/342) with protease inhibitors (PIs). Most NRTI mutations were associated with thymidine analogues (TAMs) (50.7%; 75/148), including T215F/V (14.9%; 22/148), D67DN/E/G/N/T (10.1%; 15/148), M41L (9.5%; 14/148), and K70E/KN/S/R (9.5%; 14/148). Resistance mutations related to non-TAM NRTIs (33.1%; 49/148) were M184V (29.1%; 43/148), and L74I/V (8.1%; 12/148). NNRTI mutations were predominantly K103N/S (32.7%; 34/104), V108I (10.6%; 11/104), A98G (10.6%; 11/104), and P225H (9.6%; 10/104). Minor mutations associated with PIs (60.0%; 54/90) were predominantly K20I (15.6%; 14/90) and L10F/I/V (14.5%; 13/90). The major mutations associated with PIs (40.0%; 36/90) were M41L (12.2%; 11/90), I84V (6.7%; 06/90), and V82A (6.7%; 06/90). The four most prescribed therapeutic regimens were TDF + 3TC + LPV/r (20.3%; 17/84), ABC + DDI + LPV/r (17.9%; 15/84), TDF + FTC + LPV/r (11.9%; 10/84), and ABC + 3TC + LPV/r (11.9%; 10/84).ConclusionThis study revealed that HIV drug resistance mutations are common in Gabon. The major mutations associated with PIs were M41L, I84V, and V82A. There is a need for access to new NRTIs, NNRTIs, and PIs for a better therapeutic management of PLHIV in Gabon.

No transmitted drug resistance to HIV integrase strand-transfer inhibitors after their scale-up in Estonia in 2017

In Eastern Europe HIV-1 transmitted drug resistance (TDR) data, especially in integrase (IN) region is limited. In Estonia INSTI (Integrase Strand Transfer Inhibitors) TDR has been studied only prior to the INSTI scale-up in late 2010s. Current study aimed to determine the levels of protease (PR), reverse transcriptase (RT) and IN surveillance drug resistance mutations (SDRMs) among newly diagnosed patients in Estonia in 2017. Study included 216 newly diagnosed HIV-1 individuals from January 1 until December 31, 2017 in Estonia. Demographic and clinical data were obtained from the Estonian Health Board, Estonian HIV Cohort Study (E-HIV) and clinical laboratories' databases. The PR-RT and IN regions were sequenced and analyzed for SDRMs and subtype determination. Seventy-one percent (151/213) of available HIV-positive samples were successfully sequenced. The overall level of TDR was 7.9% (12/151; 95% CI 4.4-13.8%); no dual or triple class resistance was detected. No major INSTI mutations were found. The distribution of SDRMs for NNRTI, NRTI and PI was 5.9% (9/151), 1.3% (2/151) and 0.7% (1/151), respectively. The predominant NNRTI mutation was K103N. CRF06_cpx was predominant variant (59%) in Estonian HIV-1 population followed by subtype A (9%) and subtype B (8%). Although no major INSTI mutations were found, close monitoring of INSTI SDRMs is needed considering the extensive use of the first and second-generation INSTIs. PR-RT TDR is slowly rising in Estonia indicating the need for continuous surveillance in the future. Low genetic barrier NNRTIs should be avoided in the treatment regimens.

The evolution of the HIV-1 protease folding stability.

The evolution of structural proteins is generally constrained by the folding stability. However, little is known about the particular capacity of viral proteins to accommodate mutations that can potentially affect the protein stability and, in general, the evolution of the protein stability over time. As an illustrative model case, here, we investigated the evolution of the stability of the human immunodeficiency virus (HIV-1) protease (PR), which is a common HIV-1 drug target, under diverse evolutionary scenarios that include (1) intra-host virus evolution in a cohort of seventy-five patients sampled over time, (2) intra-host virus evolution sampled before and after specific PR-based treatments, and (3) inter-host evolution considering extant and ancestral (reconstructed) PR sequences from diverse HIV-1 subtypes. We also investigated the specific influence of currently known HIV-1 PR resistance mutations on the PR folding stability. We found that the HIV-1 PR stability fluctuated over time within a constant and wide range in any studied evolutionary scenario, accommodating multiple mutations that partially affected the stability while maintaining activity. We did not identify relationships between change of PR stability and diverse clinical parameters such as viral load, CD4+ T-cell counts, and a surrogate of time from infection. Counterintuitively, we predicted that nearly half of the studied HIV-1 PR resistance mutations do not significantly decrease stability, which, together with compensatory mutations, would allow the protein to adapt without requiring dramatic stability changes. We conclude that the HIV-1 PR presents a wide structural plasticity to acquire molecular adaptations without affecting the overall evolution of stability.

Protease and gag diversity and drug resistance mutations among treatment-naive Mexican people living with HIV

IntroductionIn Mexico, HIV genotyping is performed in people living with HIV (PLWH) failing their first-line antiretroviral (ARV) regimen; it is not routinely done for all treatment-naive PLWH before ARV initiation. The first nationally representative survey published in 2016 reported that the prevalence of pretreatment drug mutations in treatment-naive Mexican PLWH was 15.5% to any antiretroviral drug and 10.6% to non-nucleoside reverse transcriptase inhibitors (NNRTIs) using conventional Sanger sequencing. Most reports in Mexico focus on HIV pol gene and nucleoside and non-nucleoside reverse transcriptase inhibitor (NRTI and NNRTI) drug resistance mutations (DRMs) prevalence, using Sanger sequencing, next-generation sequencing (NGS) or both. To our knowledge, NGS has not be used to detect pretreatment drug resistance mutations (DRMs) in the HIV protease (PR) gene and its substrate the Gag polyprotein.MethodsTreatment-naive adult Mexican PLWH were recruited between 2016 and 2019. HIV Gag and protease sequences were obtained by NGS and DRMs were identified using the WHO surveillance drug resistance mutation (SDRM) list.ResultsOne hundred PLWH attending a public national reference hospital were included. The median age was 28 years-old, and most were male. The median HIV viral load was 4.99 [4.39–5.40] log copies/mL and median CD4 cell count was 150 [68.0–355.78] cells/mm3. As expected, most sequences clustered with HIV-1 subtype B (97.9%). Major PI resistance mutations were detected: 8 (8.3%) of 96 patients at a detection threshold of 1% and 3 (3.1%) at a detection threshold of 20%. A total of 1184 mutations in Gag were detected, of which 51 have been associated with resistance to PI, most of them were detected at a threshold of 20%. Follow-up clinical data was available for 79 PLWH at 6 months post-ART initiation, seven PLWH failed their first ART regimen; however no major PI mutations were identified in these individuals at baseline.ConclusionsThe frequency of DRM in the HIV protease was 7.3% at a detection threshold of 1% and 3.1% at a detection threshold of 20%. NGS-based HIV drug resistance genotyping provide improved detection of DRMs. Viral load was used to monitor ARV response and treatment failure was 8.9%.

Detection of Gag C-terminal mutations among HIV-1 non-B subtypes in a subset of Cameroonian patients

Response to ritonavir-boosted-protease inhibitors (PI/r)-based regimen is associated with some Gag mutations among HIV-1 B-clade. There is limited data on Gag mutations and their covariation with mutations in protease among HIV-1 non-B-clades at PI/r-based treatment failure. Thus, we characterized Gag mutations present in isolates from HIV-1 infected individuals treated with a PI/r-regimen (n = 143) and compared them with those obtained from individuals not treated with PI/r (ART-naïve [n = 101] or reverse transcriptase inhibitors (RTI) treated [n = 118]). The most frequent HIV-1 subtypes were CRF02_AG (54.69%), A (13.53%), D (6.35%) and G (4.69%). Eighteen Gag mutations showed a significantly higher prevalence in PI/r-treated isolates compared to ART-naïve (p < 0.05): Group 1 (prevalence < 1% in drug-naïve): L449F, D480N, L483Q, Y484P, T487V; group 2 (prevalence 1–5% in drug-naïve): S462L, I479G, I479K, D480E; group 3 (prevalence ≥ 5% in drug-naïve): P453L, E460A, R464G, S465F, V467E, Q474P, I479R, E482G, T487A. Five Gag mutations (L449F, P453L, D480E, S465F, Y484P) positively correlated (Phi ≥ 0.2, p < 0.05) with protease-resistance mutations. At PI/r-failure, no significant difference was observed between patients with and without these associated Gag mutations in term of viremia or CD4 count. This analysis suggests that some Gag mutations show an increased frequency in patients failing PIs among HIV-1 non-B clades.

Drug resistance mutations in protease gene of HIV-1 subtype C infected patient population.

Failure of antiretroviral therapy (ART) in HIV-1 infection is a critical issue for the physicians treating HIV patients. The major cause of drug failure is the development of resistance mutations in reverse transcriptase (RT) and/or protease (PR) genes. Mutations associated with drug resistance decrease drug effectiveness. This study was conducted to assess drug resistance profile of the entire PR gene in 90 HIV-1 patients consisting of 23 ART non-responsive, 32 ART responsive and 35 drug naive patients. It was observed that the majority of the sequences (94.4%) belonged to subtype C and (5.5%) to subtype A1. The ART non-responsive and responsive patients were treated with either first line of ART regimen (two NRTI and one NNRTI) or second line of ART regimen that included additional one protease inhibitor (PI). All the patients in each group except one responsive patient had various minor resistance mutations. Thus, drug failures in ART non-responsive patients may not always be due to drug resistance mutations instead other factors may also be responsible for drug failures such as non-compliance, suboptimal dose or drug interaction. The presence of minor drug resistance mutations in drug naive patients is suggestive of transmitted resistance mutations.

Understanding the co-evolutionary molecular mechanisms of resistance in the HIV-1 Gag and protease

Due to high human immunodeficiency virus type 1 (HIV-1) subtype C infections coupled with increasing antiretroviral treatment failure, the elucidation of complex drug resistance mutational patterns arising through protein co-evolution is required. Despite the inclusion of potent protease inhibitors Lopinavir (LPV) and Darunavir (DRV) in second- and third-line therapies, many patients still fail treatment due to the accumulation of mutations in protease (PR) and recently, Gag. To understand the co-evolutionary molecular mechanisms of resistance in the HIV-1 PR and Gag, we performed 100 ns molecular dynamic simulations on multidrug resistant PR’s when bound to LPV, DRV or a mutated A431V NC|p1 Gag cleavage site (CS). Here we showed that distinct changes in PR’s active site, flap and elbow regions due to several PR resistance mutations (L10F, M46I, I54V, L76V, V82A) were found to alter LPV and DRV drug binding. However, binding was significantly exacerbated when the mutant PRs were bound to the NC|p1 Gag CS. Although A431V was shown to coordinate several residues in PR, the L76V PR mutation was found to have a significant role in substrate recognition. Consequently, a greater binding affinity was observed when the mutated substrate was bound to an L76V-inclusive PR mutant (Gbind: −62.46 ± 5.75 kcal/mol) than without (Gbind: −50.34 ± 6.28 kcal/mol). These data showed that the co-selection of resistance mutations in the enzyme and substrate can simultaneously constrict regions in PR’s active site whilst flexing the flaps to allow flexible movement of the substrate and multiple, complex mechanisms of resistance to occur. Communicated by Ramaswamy H. Sarma

In Vivo Emergence of a Novel Protease Inhibitor Resistance Signature in HIV-1 Matrix.

Protease inhibitors (PIs) are the second- and last-line therapy for the majority of HIV-infected patients worldwide. Only around 20% of individuals who fail PI regimens develop major resistance mutations in protease. We sought to explore the role of mutations in gag-pro genotypic and phenotypic changes in viruses from six Nigerian patients who failed PI-based regimens without known drug resistance-associated protease mutations in order to identify novel determinants of PI resistance. Target enrichment and next-generation sequencing (NGS) with the Illumina MiSeq system were followed by haplotype reconstruction. Full-length Gag-protease gene regions were amplified from baseline (pre-PI) and virologic failure (VF) samples, sequenced, and used to construct gag-pro-pseudotyped viruses. Phylogenetic analysis was performed using maximum-likelihood methods. Susceptibility to lopinavir (LPV) and darunavir (DRV) was measured using a single-cycle replication assay. Western blotting was used to analyze Gag cleavage. In one of six participants (subtype CRF02_AG), we found 4-fold-lower LPV susceptibility in viral clones during failure of second-line treatment. A combination of four mutations (S126del, H127del, T122A, and G123E) in the p17 matrix of baseline virus generated a similar 4-fold decrease in susceptibility to LPV but not darunavir. These four amino acid changes were also able to confer LPV resistance to a subtype B Gag-protease backbone. Western blotting demonstrated significant Gag cleavage differences between sensitive and resistant isolates in the presence of drug. Resistant viruses had around 2-fold-lower infectivity than sensitive clones in the absence of drug. NGS combined with haplotype reconstruction revealed that resistant, less fit clones emerged from a minority population at baseline and thereafter persisted alongside sensitive fitter viruses. We used a multipronged genotypic and phenotypic approach to document emergence and temporal dynamics of a novel protease inhibitor resistance signature in HIV-1 matrix, revealing the interplay between Gag-associated resistance and fitness.

Reviewing HIV-1 Gag Mutations in Protease Inhibitors Resistance: Insights for Possible Novel Gag Inhibitor Designs.

HIV protease inhibitors against the viral protease are often hampered by drug resistance mutations in protease and in the viral substrate Gag. To overcome this drug resistance and inhibit viral maturation, targeting Gag alongside protease rather than targeting protease alone may be more efficient. In order to successfully inhibit Gag, understanding of its drug resistance mutations and the elicited structural changes on protease binding needs to be investigated. While mutations on Gag have already been mapped to protease inhibitor resistance, there remain many mutations, particularly the non-cleavage mutations, that are not characterized. Through structural studies to unravel how Gag mutations contributes to protease drug resistance synergistically, it is thus possible to glean insights to design novel Gag inhibitors. In this review, we discuss the structural role of both novel and previously reported Gag mutations in PI resistance, and how new Gag inhibitors can be designed.

Characterisation of protease resistance mutations in a South African paediatric cohort with virological failure, 2011 - 2017.

Advances in HIV management have improved treatment outcomes in the HIV-infected population. However, these advances have not been without multifaceted challenges. In sub-Saharan Africa, their impact is reflected in the increased emergence of HIV drug resistance mutations. With the rise in exposure of children to protease inhibitors (PIs), the possibility of increasing PI resistance remains a concern. To describe a group of antiretroviral-experienced children with PI drug resistance mutations after failure on first- or second-line regimens in a public sector setting in South Africa. This was a retrospective cohort study of 22 children perinatally infected with HIV who had HIV genotyping conducted between January 2011 and December 2017. Of the 236 children who had HIV genotyping conducted, 22 (9.3%) had evidence of HIV PI resistance mutations. Twenty-one of the 22 children (95.5%) had major mutations in the protease region of the HIV genome. Of these children, 66.7% (14/21) had loss of response to both boosted lopinavir and atazanavir, with boosted darunavir remaining susceptible in only 12 (57.1%). The most frequent major PI mutations were V82A (76.2%), M46I/M46L (76.2%), I54V (62.0%) and L76V (33.3%). We observed a high rate of PI resistance mutations, with a resulting loss of PIs that could be used in construction of third-line regimens. To build on improvements from the introduction of antiretroviral therapy, increased efforts are needed by both health professionals and caregivers to improve adherence measures in children perinatally infected with HIV.

Gag-protease coevolution shapes the outcome of lopinavir-inclusive treatment regimens in chronically infected HIV-1 subtype C patients.

Commonly, protease inhibitor failure is characterized by the development of multiple protease resistance mutations (PRMs). While the impact of PRMs on therapy failure are understood, the introduction of Gag mutations with protease remains largely unclear. Here, we utilized phylogenetic analyses and Bayesian network learning as tools to understand Gag-protease coevolution and elucidate the pathways leading to Lopinavir failure in HIV-1 subtype C infected patients. Our analyses indicate that while PRMs coevolve in response to drug selection pressure within protease, the Gag mutations added to the existing network while specifically interacting with known Lopinavir failure PRMs. Additionally, the selection of mutations at specific positions in Gag-protease suggests that these coevolving mutational changes occurs to maintain structural integrity during Gag cleavage. Supplementary data are available at Bioinformatics online.

Robust and sensitive subtype-generic HIV-1 pol genotyping for use with dried serum spots in epidemiological studies

HIV-1 genotyping of larger pol-fragments from dried serum/plasma spots (DSS/DPS) is often hindered by RNA-degradation during transportation at ambient temperature. We evaluated an in-house HIV-1 DSS/DPS-genotyping assay designed in two shorter overlapping fragments covering all resistance mutations in protease and reverse transcriptase. Validation criteria such as specificity, detection limit, accuracy, reproducibility and storage conditions were assessed using reference plasma samples prepared as DPS and clinical DSS from the German molecular HIV-1 surveillance processed under real-life transportation conditions. The specificity was 100% for both samples types, and the experimental DPS detection limit of 1000 copies/ml yielded a 98.7% (3,329/3373) success rate for DSS (including all subtypes) above this detection limit. Accuracy for DPS compared to the gold standard was 99.1% and the reproducibility was 100% for DPS replicates and 99.9% for DSS pairs. Storage of DPS at room temperature was possible for 90 or 30 days and at −20 °C for at least 180 or 90 days at viral loads of 10,000 or 1000 copies/ml, respectively. The HIV-1 pol-genotyping assay presented here is a sensitive, robust and subtype generic tool for a large-scale population-based HIV-1 drug resistance surveillance for the use of DSS/DPS.

Prevalence of Drug Resistance Mutations in Protease, Reverse Transcriptase, and Integrase Genes of North Central Mexico HIV Isolates.

This study set out to determine the frequency of antiretroviral drug resistance mutations in treatment-naive subjects of the north central Mexican state of San Luis Potosí. Mexican studies of antiretroviral drug resistance mutations have focused mainly on large metropolitan areas and border towns subjected to intense international migrations. This study set forth to describe the frequency of these mutations in a Mexican region less subjected to such migratory influences and more representative of smaller Mexican cities. Thirty-eight full-length pol sequences spanning the protease, reverse-transcriptase, and integrase-encoding regions were obtained from 42 treatment-naive human immunodeficiency virus (HIV)-infected subjects. Most exhibited subtype B homology, but CRF02_AG was also detected. Evidence of APOBEC3 hypermutation was seen in two samples. Calibrated population analysis revealed a surveillance drug resistance mutation prevalence of 4.9% for protease inhibitors, of 2.7% for nucleoside reverse transcriptase inhibitors, of 8.1% for non-nucleoside reverse transcriptase inhibitors, and an overall prevalence of 9.5%. This corresponds to an intermediate level of transmitted drug resistance according to the World Health Organization. The identification of integrase mutations suggests that transmitted drug mutations are being imported, as inhibitors targeting integrase have not been widely used in Mexico. Our results provide a greater understanding of HIV diversity in Mexico and highlight the way internal migrations allow HIV mutations and genetic features to permeate regions less subjected to international migrations. The implications of these findings will become more evident as Mexico hosts increased repatriations of migrants in the coming years.

Structural Analysis of Darunavir Analogs Against Primary Resistance Mutations in HIV Protease

Antiretroviral therapies (ARTs) involve combinations of small molecule inhibitors that suppress the viral load of Human Immunodeficiency Virus (HIV), the causative agent of Acquired Immune Deficiency Symptom (AIDS). ARTs, currently the only defense against HIV, target critical proteins necessary for viral replication, such as HIV‐1 protease. The most potent FDA approved protease inhibitor, darunavir (DRV), is still susceptible to resistance if the protease acquires mutations in and around the active site. One method to avoid resistance is to optimize the inhibitor to fit the consensus volume that the protease's natural substrates occupy. Using this strategy, DRV analogs with larger moieties at the P1’ position were designed and synthesized to leverage unexploited space and create additional protease‐inhibitor interactions, resulting in inhibitors that are more potent and less susceptible to resistance. To elucidate the molecular mechanisms of active site resistance mutations against these inhibitors, we used a multidisciplinary approach, including biochemical assays and x‐ray crystallography. Results suggest that modifications to the DRV scaffold result in inhibitors with potency comparable to DRV, but structural analysis highlights differences in intermolecular and intramolecular interactions, which will inform future drug design.Support or Funding InformationThis work was supported in part by a grant from the National Institute of General Medical Sciences, National Institutes of Health (P01‐GM109767).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Next generation sequencing of HIV-1 protease in the PIVOT trial of protease inhibitor monotherapy

BackgroundThe PIVOT trial examined whether patients with suppressed viral load on combination antiretroviral therapy could be safely switched long-term to ritonavir-boosted protease inhibitor (PI) monotherapy. The main trial publication reported that only one of 296 patients allocated to PI monotherapy experienced a loss of drug options due to protease mutations (identified by local Sanger sequencing resistance tests) likely selected by study drug. ObjectivesTo assess if we had missed low frequency mutations, using a more sensitive methodology. Study designWe performed next generation sequencing (NGS) on all available frozen plasma samples with VL >1000 copies/ml from patients who were randomised to PI monotherapy. Assays were performed at Public Health England laboratories using a previously described method.Median coverage depth was 76,000 and the threshold for detection of minority variants was 2%. Drug susceptibility was predicted using the Stanford HIVdb algorithm. Results17 of 26 potential samples, all from different patients, were identified and successfully tested. The median viral load was 6780 copies/ml and the median time since randomisation was 43 weeks. NGS revealed previously unidentified minority variant protease mutations (G73D, I54T, L89V) in three samples, at frequencies ranging between 2% and 10%. None of these mutations predicted intermediate or high level resistance, the trial primary outcome. DiscussionThis report adds to the body of evidence that ritonavir-boosted PI monotherapy, when used as a switch strategy with prompt detection of viral load rebound and early re-introduction of combination therapy, rarely leads to the development of clinically important protease resistance mutations.

Effectiveness of Protease Inhibitor/Nucleos(t)ide Reverse Transcriptase Inhibitor-Based Second-line Antiretroviral Therapy for the Treatment of Human Immunodeficiency Virus Type 1 Infection in Sub-Saharan Africa: A Systematic Review and Meta-analysis.

In sub-Saharan Africa, 25.5 million people are living with human immunodeficiency virus (HIV), representing 70% of the global total. The need for second-line antiretroviral therapy (ART) is projected to increase in the next decade in keeping with the expansion of treatment provision. Outcome data are required to inform policy. We performed a systematic review and meta-analysis of studies reporting the virological outcomes of protease inhibitor (PI)-based second-line ART in sub-Saharan Africa. The primary outcome was virological suppression (HIV-1 RNA <400 copies/mL) after 48 and 96 weeks of treatment. The secondary outcome was the proportion of patients with PI resistance. Pooled aggregate data were analyzed using a DerSimonian-Laird random effects model. By intention-to-treat analysis, virological suppression occurred in 69.3% (95% confidence interval [CI], 58.2%-79.3%) of patients at week 48 (4558 participants, 14 studies), and in 61.5% (95% CI, 47.2%-74.9%) at week 96 (2145 participants, 8 studies). Preexisting resistance to nucleos(t)ide reverse transcriptase inhibitors (NRTIs) increased the likelihood of virological suppression. Major protease resistance mutations occurred in a median of 17% (interquartile range, 0-25%) of the virological failure population and increased with duration of second-line ART. One-third of patients receiving PI-based second-line ART with continued NRTI use in sub-Saharan Africa did not achieve virological suppression, although among viremic patients, protease resistance was infrequent. Significant challenges remain in implementation of viral load monitoring. Optimizing definitions and strategies for management of second-line ART failure is a research priority. CRD42016048985.

Commonly Transmitted HIV-1 Drug Resistance Mutations in Reverse-Transcriptase and Protease in Antiretroviral Treatment-Naive Patients and Response to Regimens Containing Tenofovir Disoproxil Fumarate or Tenofovir Alafenamide.

The presence of transmitted drug resistance mutations (TDRMs) in antiretroviral treatment (ART)-naive patients can adversely affect the outcome of ART. 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. 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. 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.

Transient HIV-1 Gag–protease interactions revealed by paramagnetic NMR suggest origins of compensatory drug resistance mutations

Cleavage of the group-specific antigen (Gag) polyprotein by HIV-1 protease represents the critical first step in the conversion of immature noninfectious viral particles to mature infectious virions. Selective pressure exerted by HIV-1 protease inhibitors, a mainstay of current anti-HIV-1 therapies, results in the accumulation of drug resistance mutations in both protease and Gag. Surprisingly, a large number of these mutations (known as secondary or compensatory mutations) occur outside the active site of protease or the cleavage sites of Gag (located within intrinsically disordered linkers connecting the globular domains of Gag to one another), suggesting that transient encounter complexes involving the globular domains of Gag may play a role in guiding and facilitating access of the protease to the Gag cleavage sites. Here, using large fragments of Gag, as well as catalytically inactive and active variants of protease, we probe the nature of such rare encounter complexes using intermolecular paramagnetic relaxation enhancement, a highly sensitive technique for detecting sparsely populated states. We show that Gag-protease encounter complexes are primarily mediated by interactions between protease and the globular domains of Gag and that the sites of transient interactions are correlated with surface exposed regions that exhibit a high propensity to mutate in the presence of HIV-1 protease inhibitors.

Development of a rapid phenotypic test for HCV protease inhibitors with potential use in clinical decisions.

Approximately 185 million people worldwide are chronically infected with hepatitis C virus (HCV). The first-wave of approved NS3 protease inhibitors (PIs) were Telaprevir and Boceprevir, which are currently discontinued. Simeprevir is a second-wave PI incorporated into the Brazilian hepatitis C treatment protocol. Drug resistance plays a key role in patients' treatment regimen. Here, we developed a simple phenotypic assay to evaluate the impact of resistance mutations in HCV NS3 protease to PIs, using a protein expression vector containing wild type NS3 protease domain and NS4A co-factor. We analyzed the impact of five resistance mutations (T54A, V36M, V158I, V170I and T54S+V170I) against Telaprevir, Boceprevir and Simeprevir. Protein purifications were performed with low cost methodology, and enzymatic inhibition assays were measured by FRET. We obtained recombinant proteases with detectable activity, and IC50 and fold change values for the evaluated PIs were determined. The variant T54A showed the highest reduction of susceptibility for the PIs, while the other four variants exhibited lower levels of reduced susceptibility. Interestingly, V170I showed 3.2-fold change for Simeprevir, a new evidence about this variant. These results emphasize the importance of enzymatic assays in phenotypic tests to determine which therapeutic regimen should be implemented.

Effectiveness of Ritonavir-Boosted Protease Inhibitor Monotherapy in Clinical Practice Even with Previous Virological Failures to Protease Inhibitor-Based Regimens.

Background and ObjectiveSignificant controversy still exists about ritonavir-boosted protease inhibitor monotherapy (mtPI/rtv) as a simplification strategy that is used up to now to treat patients that have not experienced previous virological failure (VF) while on protease inhibitor (PI) -based regimens. We have evaluated the effectiveness of two mtPI/rtv regimens in an actual clinical practice setting, including patients that had experienced previous VF with PI-based regimens.MethodsThis retrospective study analyzed 1060 HIV-infected patients with undetectable viremia that were switched to lopinavir/ritonavir or darunavir/ritonavir monotherapy. In cases in which the patient had previously experienced VF while on a PI-based regimen, the lack of major HIV protease resistance mutations to lopinavir or darunavir, respectively, was mandatory. The primary endpoint of this study was the percentage of participants with virological suppression after 96 weeks according to intention-to-treat analysis (non-complete/missing = failure).ResultsA total of 1060 patients were analyzed, including 205 with previous VF while on PI-based regimens, 90 of whom were on complex therapies due to extensive resistance. The rates of treatment effectiveness (intention-to-treat analysis) and virological efficacy (on-treatment analysis) at week 96 were 79.3% (CI95, 76.8−81.8) and 91.5% (CI95, 89.6–93.4), respectively. No relationships were found between VF and earlier VF while on PI-based regimens, the presence of major or minor protease resistance mutations, the previous time on viral suppression, CD4+ T-cell nadir, and HCV-coinfection. Genotypic resistance tests were available in 49 out of the 74 patients with VFs and only four patients presented new major protease resistance mutations.ConclusionSwitching to mtPI/rtv achieves sustained virological control in most patients, even in those with previous VF on PI-based regimens as long as no major resistance mutations are present for the administered drug.