Incorporating temporal dynamics of mutations to enhance the prediction capability of antiretroviral therapy's outcome for HIV-1.

MOTIVATION: In predicting HIV therapy outcomes, a critical clinical question is whether using historical information can enhance predictive capabilities compared with current or latest available data analysis. This study analyses whether historical knowledge, which includes viral mutations detected in all genotypic tests before therapy, their temporal occurrence, and concomitant viral load measurements, can bring improvements. We introduce a method to weigh mutations, considering the previously enumerated factors and the reference mutation-drug Stanford resistance tables. We compare a model encompassing history (H) with one not using this information (NH). RESULTS: The H-model demonstrates superior discriminative ability, with a higher ROC-AUC score (76.34%) than the NH-model (74.98%). Wilcoxon test results confirm significant improvement of predictive accuracy for treatment outcomes through incorporating historical information. The increased performance of the H-model might be attributed to its consideration of latent HIV reservoirs, probably obtained when leveraging historical information. The findings emphasize the importance of temporal dynamics in acquiring mutations. However, our result also shows that prediction accuracy remains relatively high even when no historical information is available. AVAILABILITY AND IMPLEMENTATION: This analysis was conducted using the Euresist Integrated DataBase (EIDB). For further validation, we encourage reproducing this study with the latest release of the EIDB, which can be accessed upon request through the Euresist Network.

Prevalence and Phenotypic Susceptibility to Doravirine of the HIV-1 Reverse Transcriptase V106I Polymorphism in B and Non-B Subtypes.

BACKGROUND: Limited data are available regarding the susceptibility of the reverse transcriptase V106 polymorphism to doravirine. METHODS: Doravirine susceptibility was measured in site-directed mutants (SDMs) containing V106I, V106A, V106M, and Y188L mutations in subtype B (NL4-3, HXB2) and CRF02_AG background and in recombinant viruses with RT harboring V106I alone derived from 50 people with HIV. RESULTS: HIV-1 B subtype was detected in 1523 of 2705 cases. Prevalence of V106I was 3.2% in B and 2.5% in non-B subtypes, and was higher in subtype F (8.1%) and D (14.3%). Fold-changes (FC) in susceptibility for SDMs were below doravirine biological cutoff (3.0) for V106I, but not for V106A, V106M, and Y188L. Clinically derived viruses tested included 22 B (median FC, 1.2; interquartile range [IQR], 0.9-1.6) and 28 non-B subtypes (median FC, 1.8; IQR, 0.9-3.0). Nine (18%) viruses showed FC values equal or higher than the doravirine biological FC cutoff. CONCLUSIONS: The prevalence of the HIV-1 RT V106I polymorphism in MeditRes HIV consortium remains low, but significantly more prevalent in subtypes D and F. V106I minimally decreased the susceptibility to doravirine in SDMs and most clinical isolates. Reduced susceptibility seems to occur at increased frequency in subtype F1; however, the clinical impact remains to be investigated. CLINICAL TRIALS REGISTRATION: NCT04894357.

Polypharmacy, anticholinergic burden and drug-drug interaction assessment in people with four-class-resistant HIV: data from the PRESTIGIO registry.

OBJECTIVES: To evaluate polypharmacy, anticholinergic burden (ACB) and drug-drug interactions (DDIs) in people with four-class-resistant HIV (4DR-PWH). METHODS: We performed a cross-sectional study, including 4DR-PWH from the PRESTIGIO Registry taking at least one non-antiretroviral drug. Polypharmacy was defined as taking five or more non-antiretroviral drugs. ACB was calculated using the ACB scale: 0 = no AC effect, 1-2 = low/moderate risk, ≥3 = high AC risk. Participants' characteristics by ACB score were compared using the Kruskal-Wallis test, and Spearman's correlation coefficient was used to assess linear relationships. DDIs were evaluated using the Liverpool database. RESULTS: Overall, 172 4DR-PLWH were evaluated: 75.6% males, median age 49.9 years (IQR = 45.6-56), 62 (27.1%) on polypharmacy, 124 (72.1%) using a boosting agent and 72 (41.8%) with four or more antiretrovirals. Based on ACB, 128 (74.45%), 33 (19.2%) and 11 (6.4%) had a no, low/moderate and high AC risk, respectively. The most common AC drugs were ß-blockers (12.2%), diuretics (8.7%) and antidepressants (8.7%). The high ACB was significantly related to the number of drugs/person (r = 0.33, P < 0.0001) and the number of clinical events (r = 0.222, P = 0.004). Overall, 258 DDIs were found between antiretrovirals and co-medications in 115 (66.8%) PWH, and 14 (8.1%) PWH received contraindicated drug combinations. CONCLUSIONS: In 4DR-PWH, polypharmacy, DDIs and the proportion of people with moderate/high AC burden were high. In 4DR-PWH undetectability achievement and maintenance is the priority and use of boosted PIs is common. A strict collaboration (infectious diseases specialists, virologists, pharmacologists) is needed to limit the risk of ACB and DDIs and to explore the advantages of new antiretrovirals.

Use of next-generation sequencing on HIV-1 DNA to assess archived resistance in highly treatment-experienced people with multidrug-resistant HIV under virological control: data from the PRESTIGIO Registry.

BACKGROUND: To clarify whether next-generation sequencing (NGS) can be useful for resistance assessment in virologically suppressed highly treatment-experienced (HTE) individuals with MDR HIV. METHODS: Ninety-one participants from the PRESTIGIO Registry were included. NGS was performed on HIV-DNA at 1%, 5% and 20% cut-offs; major drug resistance mutations (DRMs) were evaluated and compared with those detected in historical plasma genotypic resistance testing (h-GRT). APOBEC editing was also characterized. RESULTS: Participants had a complex and long treatment history [median 23 (IQR 21-25) years of ART exposure) and had been virologically suppressed since a median of 3 (IQR 2-5) years. Among all major DRMs detected by HIV-DNA NGS and/or h-GRT, 30% were exclusively found through NGS. The highest detection rate of historical major DRMs was reached with NGS set at 1%, but unusual substitutions and extensive APOBEC hypermutations suggest technical issues and poor clinical relevance in the 1%-5% interval. At NGS set at 5%, 67.2% of historical major DRMs were detected. The number of major DRMs detected exclusively by DNA-NGS as minority variants (frequency 5%-20%) was significantly higher in individuals who later experienced virological rebound compared with those who maintained virological control [median 2 (IQR 1-3) versus 1 (0-2), P = 0.030] and positively correlated with viraemia levels at rebound (rho = 0.474, P = 0.030). CONCLUSIONS: In non-viraemic people with an MDR virus, HIV-1 DNA NGS set at 5% is an acceptable technical cut-off that might help to reveal mutations with a potential clinical relevance. Moreover, the number of minority resistance mutations additionally detected by NGS might be associated with loss of virological control.

Switching the three-component Biginelli-like reaction conditions for the regioselective synthesis of new 2-amino[1,2,4]triazolo[1,5-a]pyrimidines.

Among the eight different triazolopyrimidine isomers existing in nature, 1,2,4-triazolo[1,5-a]pyrimidine (TZP) is one of the most studied and used isomers in medicinal chemistry. For some years, our group has been involved in developing regioselective one-pot procedures for the synthesis of 2-amino-7-aryl-5-methyl- and 2-amino-5-aryl-7-methyl-TZPs of interest in the preparation of antiviral agents. In this work, taking advantage of a Biginelli-like multicomponent reaction (MCR), we report the identification of finely tunable conditions to regioselectively synthesize C-6 ester-substituted amino-TZP analogues, both in dihydro and oxidized forms. Indeed, the use of mild acidic conditions is strongly directed toward the regioselective synthesis of 5-aryl-7-methyl C-6-substituted TZP analogues, while the use of neutral ionic liquids shifted the regioselectivity towards 7-aryl-5-methyl derivatives. In addition, the novel synthesized scaffolds were functionalized at the C-2 position and evaluated for their antiviral activity against RNA viruses (influenza virus, flaviviruses, and SARS-CoV-2). Compounds 25 and 26 emerged as promising anti-flavivirus agents, showing activity in the low micromolar range.

PRESTIGIO RING: "An 80-year-old man living with HIV resistant to all four antiretroviral classes and desiring treatment simplification".

People aging with 4 antiretroviral class resistant HIV are a very challenging population. It is difficult to build up a fully suppressive regimen, and the high prevalence of comorbidities and polypharmacy may cause drug-drug interactions and put adherence at risk. We herein present the case of an 80-year-old man, participating in the PRESTIGIO registry, asking for a reduction in his antiretroviral burden while on polypharmacy for his comorbidities.

PRESTIGIO RING "a 59-year-old man with multidrug resistant HIV-1 infection failing a regimen including dolutegravir, rilpivirine, atazanavir/cobicistat: successful treatment tailoring based on genotypic and phenotypic resistance tests".

Management of virological failure in heavily treatment-experienced people with multidrug-resistant (MDR) HIV infection is a serious clinical challenge. New drugs with novel mechanisms of action have recently been approved, and their use has improved the outcome of subjects with limited treatment options (LTO). In this setting, the choice of antiretroviral therapy (ART) should be tailored based on the pattern of resistance, treatment history and patients' individual characteristics. While genotypic resistance testing is the reference method for analysing residual drug susceptibility, phenotypic resistance testing can provide additional support when facing LTO. Herein, we present the case of a patient with MDR HIV-1 infection on virological failure enrolled in the PRESTIGIO Registry. The salvage ART regimen, which included drugs with novel mechanisms of action (MoA), was tailored to the patient's clinical characteristics and on the resistance pattern explored with genotypic and phenotypic investigation, allowing the achievement of viro-immunological success. The use of recently approved drugs with novel MoA, combined with an optimized background regimen, may also achieve virological suppression in people with LTO.

Serendipitous Identification of Azine Anticancer Agents Using a Privileged Scaffold Morphing Strategy.

The use of privileged scaffolds as a starting point for the construction of libraries of bioactive compounds is a widely used strategy in drug discovery and development. Scaffold decoration, morphing and hopping are additional techniques that enable the modification of the chosen privileged framework and better explore the chemical space around it. In this study, two series of highly functionalized pyrimidine and pyridine derivatives were synthesized using a scaffold morphing approach consisting of triazine compounds obtained previously as antiviral agents. Newly synthesized azines were evaluated against lymphoma, hepatocarcinoma, and colon epithelial carcinoma cells, showing in five cases acceptable to good anticancer activity associated with low cytotoxicity on healthy fibroblasts. Finally, ADME in vitro studies were conducted on the best derivatives of the two series showing good passive permeability and resistance to metabolic degradation.

Virologic Failure Following Low-level Viremia and Viral Blips During Antiretroviral Therapy: Results From a European Multicenter Cohort.

BACKGROUND: It is unclear whether low-level viremia (LLV), defined as repeatedly detectable viral load (VL) of <200 copies/mL, and/or transient viremic episodes (blips) during antiretroviral therapy (ART), predict future virologic failure. We investigated the association between LLV, blips, and virologic failure (VF) in a multicenter European cohort. METHODS: People with HIV-1 who started ART in 2005 or later were identified from the EuResist Integrated Database. We analyzed the incidence of VF (≥200 copies/mL) depending on viremia exposure, starting 12 months after ART initiation (grouped as suppression [≤50 copies/mL], blips [isolated VL of 51-999 copies/mL], and LLV [repeated VLs of 51-199 copies/mL]) using Cox proportional hazard models adjusted for age, sex, injecting drug use, pre-ART VL, CD4 count, HIV-1 subtype, type of ART, and treatment experience. We queried the database for drug-resistance mutations (DRM) related to episodes of LLV and VF and compared those with baseline resistance data. RESULTS: During 81 837 person-years of follow-up, we observed 1424 events of VF in 22 523 participants. Both blips (adjusted subhazard ratio [aHR], 1.7; 95% confidence interval [CI], 1.3-2.2) and LLV (aHR, 2.2; 95% CI, 1.6-3.0) were associated with VF, compared with virologic suppression. These associations remained statistically significant in subanalyses restricted to people with VL <200 copies/mL and those starting ART 2014 or later. Among people with LLV and genotype data available within 90 days following LLV, 49/140 (35%) had at least 1 DRM. CONCLUSIONS: Both blips and LLV during ART are associated with increased risk of subsequent VF.

Analysis of mutational history of multidrug-resistant genotypes with a mutagenetic tree model.

Human immunodeficiency virus (HIV) can develop resistance to all antiretroviral drugs. Multidrug resistance, however, is a rare event in modern HIV treatment, but can be life-threatening, particular in patients with very long therapy histories and in areas with limited access to novel drugs. To understand the evolution of multidrug resistance, we analyzed the EuResist database to uncover the accumulation of mutations over time. We hypothesize that the accumulation of resistance mutations is not acquired simultaneously and randomly across viral genotypes but rather tends to follow a predetermined order. The knowledge of this order might help to elucidate potential mechanisms of multidrug resistance. Our evolutionary model shows an almost monotonic increase of resistance with each acquired mutation, including less well-known nucleoside reverse transcriptase (RT) inhibitor-related mutations like K223Q, L228H, and Q242H. Mutations within the integrase (IN) (T97A, E138A/K G140S, Q148H, N155H) indicate high probability of multidrug resistance. Hence, these IN mutations also tend to be observed together with mutations in the protease (PR) and RT. We followed up with an analysis of the mutation-specific error rates of our model given the data. We identified several mutations with unusual rates (PR: M41L, L33F, IN: G140S). This could imply the existence of previously unknown virus variants in the viral quasispecies. In conclusion, our bioinformatics model supports the analysis and understanding of multidrug resistance.

Pillars of long-term antiretroviral therapy success.

BACKGROUND: Meeting the challenge of antiretroviral therapy (ART) whose efficacy can last a lifetime requires continuous updating of the virological, pharmacological, and quality of life outcomes to be pursued and a continuous review of literature data on the efficacy and tolerability of new drugs and therapeutic strategies. METHODS: With the aim of identifying open questions and answers about the current controversies in modern ART, we adapted the Design Thinking methodology to the needs of the design phase of a scientific article, involving a team of experts in HIV care. RESULTS: Five main pillars of treatment success were discussed: sustained virologic suppression over time; immunological recovery; pharmacological attributes; long-term tolerability and safety of ART; and people's satisfaction and quality of life. The definition of the outcomes to be achieved in each thematic area and the tools to achieve them were reviewed and discussed. CONCLUSIONS: Long-term treatment success should be intended as a combination of HIV-RNA suppression, immune recovery, and high quality of life. To achieve this, the regimen should be well-tolerated, with high potency, genetic barrier, and forgiveness, and should be tailored by a person-centered perspective, based on individual needs, preferences, and therapeutic history.

Generating synthetic clinical data that capture class imbalanced distributions with generative adversarial networks: Example using antiretroviral therapy for HIV.

OBJECTIVE: Clinical data's confidential nature often limits the development of machine learning models in healthcare. Generative adversarial networks (GANs) can synthesise realistic datasets, but suffer from mode collapse, resulting in low diversity and bias towards majority demographics and common clinical practices. This work proposes an extension to the classic GAN framework that includes a variational autoencoder (VAE) and an external memory mechanism to overcome these limitations and generate synthetic data accurately describing imbalanced class distributions commonly found in clinical variables. METHODS: The proposed method generated a synthetic dataset related to antiretroviral therapy for human immunodeficiency virus (ART for HIV). We evaluated it based on five metrics: (1) accurately representing imbalanced class distribution; (2) the realism of the individual variables; (3) the realism among variables; (4) patient disclosure risk; and (5) the utility of the generated dataset for developing downstream machine learning models. RESULTS: The proposed method overcomes the issue of mode collapse and generates a synthetic dataset that accurately describes imbalanced class distributions commonly found in clinical variables. The generated data has a patient disclosure risk of 0.095%, lower than the 9% threshold stated by Health Canada and the European Medicines Agency, making it suitable for distribution to the research community with high security. The generated data also has high utility, indicating the potential of the proposed method to enable the development of downstream machine learning algorithms for healthcare applications using synthetic data. CONCLUSION: Our proposed extension to the classic GAN framework, which includes a VAE and an external memory mechanism, represents a promising approach towards generating synthetic data that accurately describe imbalanced class distributions commonly found in clinical variables. This method overcomes the limitations of GANs and creates more realistic datasets with higher patient cohort diversity, facilitating the development of downstream machine learning algorithms for healthcare applications.

Potential role of doravirine for the treatment of HIV-1-infected persons with transmitted drug resistance.

BACKGROUND: Doravirine has a unique resistance profile but how this profile might increase its usefulness beyond first-line therapy in persons with susceptible viruses has not been well studied. We sought to determine scenarios in which doravirine would retain activity against isolates from ART-naïve persons with transmitted drug resistance (TDR) and to identify gaps in available doravirine susceptibility data. METHODS: We analyzed published in vitro doravirine susceptibility data and applied the results to 42,535 RT sequences from ART-naïve persons published between 2017 and 2021. NNRTI drug resistance mutations (DRMs) were defined as those with a Stanford HIV Drug Resistance Database doravirine penalty score either alone or in combination with other mutations. RESULTS: V106A, Y188L, F227C/L, M230L, and Y318F were associated with the greatest reductions in doravirine susceptibility. However, several NNRTI DRMs and DRM combinations lacking these canonical resistance mutations had > tenfold reduced susceptibility including G190E, one isolate with G190S, three isolates with L100I + K103N, one isolate with K103N + P225H, and isolates with L100I + K103N + V108I and K101E + Y181C + G190A. Of the 42,535 ART-naïve sequences, 3,374 (7.9%) contained a NNRTI DRM of which 2,788 (82.6%) contained 1 DRM (n = 33 distinct mutations), 426 (12.6%) contained 2 DRMs (79 distinct pairs of mutations), and 143 (4.2%) contained ≥ 3 DRMs (86 distinct mutation patterns). Among the 2,788 sequences with one DRM, 112 (4.0%) were associated with ≥ 3.0-fold reduced doravirine susceptibility while 2,625 (94.2%) were associated with < 3.0-fold reduced susceptibility. Data were not available for individual NNRTI DRMs in 51 sequences (1.8%). Among the 426 sequences with two NNRTI DRMs, 180 (42.3%) were associated with ≥ 3.0 fold reduced doravirine susceptibility while just 32 (7.5%) had < 3.0 fold reduced susceptibility. Data were not available for 214 (50.2%) sequences containing two NNRTI DRMs. CONCLUSIONS: First-line therapy containing doravirine plus two NRTIs is expected to be effective in treating most persons with TDR as more than 80% of TDR sequences had a single NNRTI DRM and as more than 90% with a single DRM were expected to be susceptible to doravirine. However, caution is required for the use of doravirine in persons with more than one NNRTI DRM even if none of the DRMs are canonical doravirine-resistance mutations.

PRESTIGIO RING: "A 59-year-old HIV-1 positive, highly treatment-experienced woman failing darunavir/ ritonavir plus raltegravir".

Management of heavily treatment experienced (HTE) people with HIV remains a challenge. Tailored antiretroviral therapy (ART) is needed in this fragile population who almost invariably harbor viral quasispecies with resistance-associated mutations (RAMs). The reference method for HIV genotypic resistance testing (GRT) has long been Sanger sequencing (SS), but next-generation sequencing (NGS), following recent progress in workflow and cost-effectiveness, is replacing SS because of higher sensitivity. From the PRESTIGIO Registry, we present a case of a 59-year-old HTE woman who failed darunavir/ritonavir plus raltegravir at low-viremia levels due mainly to high pill burden and poor adherence. NGS-GRT was performed on HIV-RNA at failure and the results were compared to all past SS-GRT data available (historical genotype). In this case, NGS-GRT did not detect any minority drug-resistant variants. After discussing several therapeutic options, the treatment was changed to dolutegravir 50 mg twice daily plus doravirine 100 mg once a day, based on clinical history, adherence issues, and pill burden, as well as the historical SS-GRT and the latest NGS-GRT results. At six months follow-up visit, the patient had HIV-RNA below 30 copies/ml and CD4+ T cell count increased from 673 cells/ mm3 to 688 cells/ mm3. Close follow-up of this patient is ongoing.

Effectiveness of integrase strand transfer inhibitors in HIV-infected treatment-experienced individuals across Europe.

OBJECTIVES: To explore the effectiveness and durability of integrase strand transfer inhibitor (INSTI)-based regimens in pre-treated subjects. METHODS: Treatment-experienced individuals starting an INSTI-based regimen during 2012-2019 were selected from the INTEGRATE collaborative study. The time to virological failure [VF: one measurement of viral load (VL) ≥ 1000 copies/mL or two ≥ 50 copies/ml or one VL measurement ≥ 50 copies/mL followed by treatment change] and to INSTI discontinuation were evaluated. RESULTS: Of 13 560 treatments analysed, 4284 were from INSTI-naïve, non-viraemic (IN-NV) individuals, 1465 were from INSTI-naïve, viraemic (IN-V) individuals, 6016 were from INSTI-experienced, non-viraemic (IE-NV) individuals and 1795 were from INSTI-experienced, viraemic (IE-V) individuals. Major INSTI drug resistance mutations (DRMs) were previously detected in 4/519 (0.8%) IN-NV, 3/394 (0.8%) IN-V, 7/1510 (0.5%) IE-NV and 25/935 (2.7%) IE-V individuals. The 1-year estimated probabilities of VF were 3.1% [95% confidence interval (CI): 2.5-3.8] in IN-NV, 18.4% (95% CI: 15.8-21.2) in IN-V, 4.2% (95% CI: 3.6-4.9) in IE-NV and 23.9% (95% CI: 20.9-26.9) in IE-V subjects. The 1-year estimated probabilities of INSTI discontinuation were 12.1% (95% CI: 11.1-13.0) in IN-NV, 19.6% (95% CI: 17.5-21.6) in IN-V, 10.8% (95% CI: 10.0-11.6) in IE-NV and 21.7% (95% CI: 19.7-23.5) in IE-V subjects. CONCLUSIONS: Both VF and INSTI discontinuation occur at substantial rates in viraemic subjects. Detection of DRMs in a proportion of INSTI-experienced individuals makes INSTI resistance testing mandatory after failure.

Early versus delayed antiretroviral therapy based on genotypic resistance test: Results from a large retrospective cohort study.

Rapid start of antiretroviral therapy (ART) pending genotypic resistance test (GRT) has been recently proposed, but the effectiveness of this strategy is still debated. The rate of virological success (VS), defined as HIV-RNA < 50 copies/ml, with and without GRT was compared in drug-naïve individuals enrolled in the Italian ARCA cohort who started ART between 2015 and 2018. 521 individuals started ART: 397 without GRT (pre-GRT group) and 124 following GRT (post-GRT group). Overall, 398 (76%) were males and 30 (6%) were diagnosed with AIDS. In the pre-GRT group, baseline CD4+ cell counts were lower (p < 0.001), and viral load was higher (p < 0.001) than in the post-GRT group. The estimated probability of VS in pre-GRT versus post-GRT group was 72.54% (CI95 : 67.78-76.60) versus 66.94% (CI95 : 57.53-74.26) at Week 24 and 92.40% (CI95 : 89.26-94.62) versus 92.92% (CI95 : 86.35-96.33) at Week 48, respectively (p = 0.434). At Week 48, VS was less frequent among individuals with baseline CD4+ cell counts <200 versus >500 (90.33% vs. 97.33%), log viral load <5.00 versus >5.70 log10 cps/ml (97.17% vs 78.16%; p < 0.001), and those treated with protease inhibitors or non-nucleoside reverse transcriptase inhibitors versus those treated with integrase strand transfer inhibitors (p < 0.001). The rate of VS does not seem to be affected by an early ART initiation pending GRT results, but it could be influenced by the composition of the ART regimen, as well as immuno-virological parameters.

Privileged Scaffold Decoration for the Identification of the First Trisubstituted Triazine with Anti-SARS-CoV-2 Activity.

Current therapy against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) are based on the use of Remdesivir 1, Molnupiravir 2, and the recently identified Nirmatrelvir 3. Unfortunately, these three drugs showed some limitations regarding potency and possible drug-drug interactions. A series of derivatives coming from a decoration approach of the privileged scaffold s-triazines were synthesized and evaluated against SAR-CoV-2. One derivative emerged as the hit of the series for its micromolar antiviral activity and low cytotoxicity. Mode of action and pharmacokinetic in vitro preliminary studies further confirm the role as candidates for a future optimization campaign of the most active derivative identified with this work.

Maraviroc as a potential HIV-1 latency-reversing agent in cell line models and ex vivo CD4 T cells.

Recent studies have suggested that the CCR5 antagonist maraviroc (MVC) may exert an HIV-1 latency reversal effect. This study aimed at defining MVC-mediated induction of HIV-1 in three cell line latency models and in ex vivo CD4 T cells from six patients with suppressed viraemia. HIV-1 induction was evaluated in TZM-bl cells by measuring HIV-1 LTR-driven luciferase expression, and in ACH-2 and U1 latently infected cell lines by measuring cell-free (CFR) and cell-associated (CAR) HIV-1 RNA by qPCR. NF-κB p65 was quantified in nuclear extracts by immunodetection. In ex vivo CD4 T cells, CAR, CFR and cell-associated DNA (CAD) were quantified at baseline and 1-7-14 days post-induction (T1, T7, T14). At T7 and T14, the infectivity of the CD4 T cells co-cultured with MOLT-4/CCR5 target cells was evaluated in the TZM-bl assay (TZA). Results were expressed as fold activation (FA) with respect to untreated cells. No LTR activation was observed in TZM-bl cells at any MVC concentration. NF-κB activation was only modestly upregulated (1.6±0.4) in TZM-bl cells with 5 µM MVC. Significant FA of HIV-1 expression was only detected at 80 µM MVC, namely on HIV-1 CFR in U1 (3.1±0.9; P=0.034) and ACH-2 cells (3.9±1.4; P=0.037). CFR was only weakly stimulated at 20 µM in ACH-2 (1.7±1.0 FA) cells and at 5 µM in U1 cells (1.9±0.5 FA). Although no consistent pattern of MVC-mediated activation was observed in ex vivo experiments, substantial FA values were detected sparsely on individual samples with different parameters. Notably, in one sample, MVC stimulated all parameters at T7 (2.3±0.2 CAD, 6.8±3.7 CAR, 18.7±16.7 CFR, 7.3±0.2 TZA). In conclusion, MVC variably induces HIV-1 production in some cell line models not previously used to test its latency reversal potential. In ex vivo CD4 T cells, MVC may exert patient-specific HIV-1 induction; however, clinically relevant patterns, if any, remain to be defined.

In vitro cross-resistance to doravirine in a panel of HIV-1 clones harbouring multiple NNRTI resistance mutations.

OBJECTIVES: Doravirine is a recently licensed HIV-1 NNRTI with improved efficacy, pharmacokinetics and safety profile compared with efavirenz and limited cross-resistance with rilpivirine and etravirine. In this in vitro study, cross-resistance to doravirine was analysed in a representative panel of NNRTI-resistant clones. METHODS: In vitro phenotypic susceptibility to doravirine was assessed in 10 clinically derived infectious clones with intermediate- to high-level resistance to rilpivirine, etravirine, efavirenz and nevirapine, and in NL4-3 site-directed mutants harbouring K103N, Y181C, M230L or K103N/Y181C NNRTI mutations. RESULTS: Although none of the infectious clones harboured any of the major doravirine resistance-associated mutations (RAMs) included in the IAS-USA reference list, doravirine fold change (FC) values were comparable to or higher than those calculated for other NNRTIs, particularly etravirine and rilpivirine. As expected, single NNRTI mutations K103N and Y181C did not impair doravirine susceptibility (FC 1.4 and 1.8, respectively), while reduced activity was observed with the single M230L or double K103N/Y181C mutations (FC 7.6 and 4.9, respectively). Median FC values increased significantly with increasing numbers of NNRTI RAMs (P = 0.005) and were >10 in 4/4 and 1/4 clones harbouring four and three NNRTI RAMs, respectively. FC values correlated well with predicted susceptibility as inferred by Stanford HIV Drug Resistance Database (HIVdb) and ANRS algorithms (both P < 0.001). CONCLUSIONS: Substantial cross-resistance to doravirine was detected in NNRTI-resistant viruses harbouring complex mutational patterns, even in the absence of major IAS-USA doravirine RAMs. Therefore, based on the simple IAS-USA reference list, doravirine resistance may be underestimated in viruses harbouring multiple NNRTI mutations.

Effectiveness of integrase strand transfer inhibitor-based regimens in HIV-infected treatment-naive individuals: results from a European multi-cohort study.

BACKGROUND: INSTIs have become a pillar of first-line ART. Real-world data are needed to assess their effectiveness in routine care. OBJECTIVES: We analysed ART-naive patients who started INSTI-based regimens in 2012-19 whose data were collected by INTEGRATE, a European collaborative study including seven national cohorts. METHODS: Kaplan-Meier analyses assessed time to virological failure (VF), defined as one viral load (VL) ≥1000 copies/mL, two consecutive VLs ≥50 copies/mL, or one VL ≥50 copies/mL followed by treatment change after ≥24 weeks of follow-up, and time to INSTIs discontinuation (INSTI-DC) for any reason. Factors associated with VF and INSTI-DC were explored by logistic regression analysis. RESULTS: Of 2976 regimens started, 1901 (63.9%) contained dolutegravir, 631 (21.2%) elvitegravir and 444 (14.9%) raltegravir. The 1 year estimated probabilities of VF and INSTI-DC were 5.6% (95% CI 4.5-6.7) and 16.2% (95% CI 14.9-17.6), respectively, and were higher for raltegravir versus both elvitegravir and dolutegravir. A baseline VL ≥100 000 copies/mL [adjusted HR (aHR) 2.17, 95% CI 1.55-3.04, P < 0.001] increased the risk of VF, while a pre-treatment CD4 count ≥200 cells/mm3 reduced the risk (aHR 0.52, 95% CI 0.37-0.74, P < 0.001). Predictors of INSTI-DC included use of raltegravir versus dolutegravir (aHR 3.03, 95% CI 2.34-3.92, P < 0.001), use of >3 drugs versus 3 drugs (aHR 2.73, 95% CI 1.55-4.79, P < 0.001) and starting ART following availability of dolutegravir (aHR 0.64, 95% CI 0.48-0.83, P = 0.001). Major INSTI mutations indicative of transmitted drug resistance occurred in 2/1114 (0.2%) individuals. CONCLUSIONS: This large multi-cohort study indicates high effectiveness of elvitegravir- or dolutegravir-based first-line ART in routine practice across Europe.