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.

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.

HIV-1 transmitted drug resistance in newly diagnosed individuals in Italy over the period 2015-21.

BACKGROUND: Transmitted drug resistance (TDR) is still a critical aspect for the management of individuals living with HIV-1. Thus, its evaluation is crucial to optimize HIV care. METHODS: Overall, 2386 HIV-1 protease/reverse transcriptase and 1831 integrase sequences from drug-naïve individuals diagnosed in north and central Italy between 2015 and 2021 were analysed. TDR was evaluated over time. Phylogeny was generated by maximum likelihood. Factors associated with TDR were evaluated by logistic regression. RESULTS: Individuals were mainly male (79.1%) and Italian (56.2%), with a median (IQR) age of 38 (30-48). Non-B infected individuals accounted for 44.6% (N = 1065) of the overall population and increased over time (2015-2021, from 42.1% to 51.0%, P = 0.002). TDR prevalence to any class was 8.0% (B subtype 9.5% versus non-B subtypes 6.1%, P = 0.002) and remained almost constant over time. Overall, 300 transmission clusters (TCs) involving 1155 (48.4%) individuals were identified, with a similar proportion in B and non-infected individuals (49.7% versus 46.8%, P = 0.148). A similar prevalence of TDR among individuals in TCs and those out of TCs was found (8.2% versus 7.8%, P = 0.707).By multivariable analysis, subtypes A, F, and CFR02_AG were negatively associated with TDR. No other factors, including being part of TCs, were significantly associated with TDR. CONCLUSIONS: Between 2015 and 2021, TDR prevalence in Italy was 8% and remained almost stable over time. Resistant strains were found circulating regardless of being in TCs, but less likely in non-B subtypes. These results highlight the importance of a continuous surveillance of newly diagnosed individuals for evidence of TDR to inform clinical practice.

Characterization of HIV-1 Reservoirs in Children and Adolescents: A Systematic Review and Meta-Analysis Toward Pediatric HIV Cure.

OBJECTIVE: To conduct a comprehensive, systematic review of the profile of HIV-1 reservoirs in children and adolescents with perinatally acquired HIV infection. STUDY DESIGN: Randomized and nonrandomized trials, cohort studies, and cross-sectional studies on HIV reservoirs in pediatric populations, published between 2002 and 2022, were included. Archived-drug resistance mutations (ADRMs) and the size of reservoirs were evaluated. Subgroup analyses were performed to characterize further the data, and the meta-analysis was done through random effect models. RESULTS: Overall, 49 studies from 17 countries worldwide were included, encompassing 2356 perinatally infected participants (48.83% females). There are limited data on the quantitative characterization of viral reservoirs in sub-Saharan Africa, with sensitive methodologies such as droplet digital polymerase chain reaction rarely employed. The overall prevalence of ADRMs was 37.80% (95% CI 13.89-65.17), with 48.79% (95% CI 0-100) in Africa, 42.08% (95% CI 6.68-82.71) in America, 23.88% (95% CI 14.34-34.90) in Asia, and 20.00% (95% CI 10.72-31.17) in Europe, without any difference between infants and adolescents (P = .656). Starting antiretroviral therapy (ART) before 2 months of age limited the levels of HIV-1 DNA (P = .054). Participants with long-suppressed viremia (>5 years) had lower levels of HIV-1 DNA (P = .027). Pre- and post-ART CD4 ≤29% and pre-ART viremia ≥5Log were all found associated with greater levels of HIV-1 DNA (P = .038, P = .047, and P = .041, respectively). CONCLUSIONS: The pooled prevalence of ADRMs is high in perinatally infected pediatric population, with larger proviral reservoir size driven by delayed ART initiation, a shorter period of viral suppression, and immunovirological failures. Thus, strategies for pediatric HIV functional cure should target children and adolescents with very early ART initiation, immunocompetence, and long-term viral suppression.

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.

Genotypic Resistance Testing of HIV-1 DNA in Peripheral Blood Mononuclear Cells.

HIV-1 DNA exists in nonintegrated linear and circular episomal forms and as integrated proviruses. In patients with plasma viremia, most peripheral blood mononuclear cell (PBMC) HIV-1 DNA consists of recently produced nonintegrated virus DNA while in patients with prolonged virological suppression (VS) on antiretroviral therapy (ART), most PBMC HIV-1 DNA consists of proviral DNA produced months to years earlier. Drug-resistance mutations (DRMs) in PBMCs are more likely to coexist with ancestral wild-type virus populations than they are in plasma, explaining why next-generation sequencing is particularly useful for the detection of PBMC-associated DRMs. In patients with ongoing high levels of active virus replication, the DRMs detected in PBMCs and in plasma are usually highly concordant. However, in patients with lower levels of virus replication, it may take several months for plasma virus DRMs to reach detectable levels in PBMCs. This time lag explains why, in patients with VS, PBMC genotypic resistance testing (GRT) is less sensitive than historical plasma virus GRT, if previous episodes of virological failure and emergent DRMs were either not prolonged or not associated with high levels of plasma viremia. Despite the increasing use of PBMC GRT in patients with VS, few studies have examined the predictive value of DRMs on the response to a simplified ART regimen. In this review, we summarize what is known about PBMC HIV-1 DNA dynamics, particularly in patients with suppressed plasma viremia, the methods used for PBMC HIV-1 GRT, and the scenarios in which PBMC GRT has been used clinically.

Transmitted Drug Resistance to Integrase-Based First-Line Human Immunodeficiency Virus Antiretroviral Regimens in Mediterranean Europe.

BACKGROUND: We evaluated the prevalence of transmitted drug resistance (TDR) to integrase strand-transfer inhibitors (INSTIs) and nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) and of clinically relevant resistance (CRR) in newly diagnosed people with human immunodeficiency virus (HIV; PWH) naive to antiretroviral therapy (ART) in Europe. METHODS: MeditRes is a consortium that includes ART-naive PWH newly diagnosed in France, Greece, Italy, Portugal, and Spain during 2018-2021. Reverse transcriptase and INSTI sequences were provided by participating centers. To evaluate the prevalence of surveillance drug resistance mutations (SDRM), we used the calibrated population resistance tools from the Stanford HIV website. To evaluate CRR, defined as any resistance level ≥3, we used the Stanford HIV Drug Resistance Database v.9.1 algorithm. RESULTS: We included 2705 PWH, 72% men, median age of 37 years (interquartile range, 30-48); 43.7% were infected by non-B subtypes. The prevalence of INSTI-SDRMs was 0.30% (T66I, T66A, E92Q, E138T, E138K, Y143R, S147G, R263K; all n=1) and the prevalence of NRTI-SDRMs was 5.77% (M184V: 0.85%; M184I: 0.18%; K65R/N: 0.11%; K70E: 0.07%; L74V/I: 0.18%; any thymidine analog mutations: 4.36%). INSTI-CRR was 2.33% (0.15% dolutegravir/bictegravir, 2.29% raltegravir/elvitegravir) and 1.74% to first-line NRTIs (0.89% tenofovir/tenofovir alafenamide, 1.74% abacavir, 1.07% lamivudine/emtricitabine). CONCLUSIONS: We present the most recent data on TDR to integrase-based first-line regimens in Europe. Given the low prevalence of CRR to second-generation integrase inhibitors and to first-line NRTIs during 2018-2021, it is unlikely that newly diagnosed PWH in MeditRes countries would present with baseline resistance to a first-line regimen based on second-generation integrase inhibitors.

Evaluation of integrase resistance in individuals who failed a regimen containing dolutegravir in French and Italian clinical settings.

BACKGROUND: This work aims to evaluate integrase resistance and its predictors in HIV-1 infected combined antiretroviral therapy (cART) experienced individuals failing a dolutegravir-based regimen. METHODS: Major resistance mutations (MRM) and genotypic susceptibility score (GSS) of dolutegravir companion drugs were evaluated on plasma genotypic resistance test (GRT) performed at dolutegravir failure. Logistic regression was used to evaluate factors associated to the risk of integrase strand-transfer inhibitors (INSTI)-resistance at dolutegravir failure. RESULTS: We retrospectively analysed 467 individuals. At failure GRT, individuals had been under dolutegravir for a median (IQR) time of 11 (5-20) months; around half of them had never been exposed to INSTI (52%) and 10.7% were at first-line regimen. Fifty-eight (12.4%) individuals showed ≥1 INSTI MRM. Among them, people INSTI-exposed showed significantly higher prevalence of INSTI resistance compared to those who were INSTI naïve [46 (21.2%) versus 9 (3.9%), P < 0.001].N155H was the most prevalent MRM (5.4%), followed by G140S (4.5%) and Q148H (4.3%). These MRM were more probably present in INSTI-experienced individuals compared to those INSTI naïve. Despite failure, 89.5% of individuals harboured viral strains fully susceptible to dolutegravir and bictegravir and 85.0% to all INSTI. No INSTI exposure before receiving dolutegravir [OR: 0.35 (0.16-0.78), P < 0.010] and a GSS for companion drugs ≥2 (OR: 0.09 [0.04-0.23], P < 0.001) were negatively associated with INSTI resistance at failure. CONCLUSIONS: In a large set of individuals failing dolutegravir in real-life, INSTI resistance was low and mainly related to previous first-generation INSTI exposure. Surveillance of integrase resistance remains crucial to preserve efficacy of INSTI class in the future.

Evaluation of archived drug resistance mutations in HIV-1 DNA among vertically infected adolescents under antiretroviral treatment in Cameroon: Findings during the COVID-19 pandemic.

BACKGROUND: With the success of antiretroviral therapy (ART), children born with HIV are more likely to reach adolescence. However, frequent non-adherence to ART in adolescents living with HIV (ALHIV) leads to viral replication. Notably, a viraemic infection might lead to archived drug resistance mutations (ADRMs). Hence, within the context of the COVID-19 pandemic, we aimed to compare the patterns of ADRMs in viraemic and non-viraemic vertically infected ALHIV and to assess their immunity to and diagnosis of SARS-CoV-2. METHODS: A comparative study was conducted among COVID-19-unvaccinated ALHIV receiving ART in Yaoundé-Cameroon over the period October 2021 to March 2022. Plasma HIV-RNA was measured using Abbott® m2000rt; HIV-1 genotyping was performed on buffy-coat (HIV-1 DNA) and ADRMs were interpreted using HIVdb.v9.0.1. Patterns of HIV-1 ADRMs were compared between viraemic (≥ 1.60 log10 HIV-1 RNA copies/ml) and non-viraemic (< 1.60 log10 copies/ml) individuals. SARS-CoV-2 antibodies were assessed on whole blood using Abbott Panbio COVID-19 immunoglobulin G/M (IgG/IgM) rapid test and COVID-19 polymerase chain reaction test was performed using nasopharyngeal swab samples. RESULTS: Of the 60 ALHIV [aged 17 (16-19) years, 51.6% female], median ART duration was 14 (12-16) years; 31/55 (56.3%) were exposed to nonnucleoside reverse transcriptase inhibitor (NNRTI)-based first-line ART (of whom 19/31 transitioned to dolutegravir-based ART in 2020) and 24/55 (43.6%) were on second-line ART. Forty-two out of 60 (70.0%) ALHIV were non-viraemic; 43/60 (71.6%) were successfully sequenced. Overall the ADRM rate was 62.7% (27/43), with 69.2% (9/13) viraemic and 60.0% (18/30) non-viraemic (p = 0.56). NNRTI-ADRMs were significantly higher among viraemic ALHIV (69.2% vs. 46.7%, p = 0.030). Regarding immunity, those with CD4 nadir < 350 cells/µl had significantly higher rates of ADRMs [adjusted odds ratio (aOR) = 3.20 (1.36-95.53), p = 0.03]. In relation to COVID-19 immunity, overall SARS-CoV-2 IgG seropositivity was 28.3% (17/60), whereas 0% (0/60) were seropositive to IgM; in particular, those with CD4 count nadir ≥ 350 cells/µl had higher odds of SARS-CoV-2 IgG seropositivity [OR =7.85 (2.03-30.28), p < 0.01]. No significant association was found between SARS-CoV-2 IgG seropositivity and HIV-RNA (non-viraemic, 33.3%; viraemic, 16.7%; p = 0.18). SARS-CoV-2 RNA prevalence was 4.5% (2/44). The two positive participants were with low-levels of viral load (Ct > 30) and seropositive to IgG. CONCLUSION: In the context of virological success, the majority of ALHIV harbour ADRMs, essentially driven by NNRTI mutations and low CD4 nadir. During the current pandemic, about one-third of ALHIV were previously exposed to SARS-CoV-2. However, some children might have been exposed and uninfected and others might have been infected but showed no serological response at sampling. These findings support the use of NNRTI-sparing regimens and the implementation of COVID-19 barrier measures targeting ALHIV during such a pandemic.

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.

The effects of orally administered lactoferrin in the prevention and management of viral infections: A systematic review.

It has been demonstrated that lactoferrin (LF) plays a role in host defence, but evidence on its potential antiviral property from clinical studies is fragmented. Our systematic review aimed at identifying the effects of orally administered LF against virus infections. The systematic search was conducted on PubMed, Scopus, Web of Science, BioRxiv.org and ClinicalTrials.gov from database inception to 7th January 2021. Eligible articles investigated any virus family and provided data on the effects of orally administered LF of any origin in the prevention and/or management of confirmed viral infections in people of any age. A narrative synthesis of the results was performed. Quality was assessed with the Cochrane Risk-Of-Bias and ROBINS-1 tools. A total of 27 records were included, nine of which were registered protocols. We found data on Flaviviridae (n = 10), Retroviridae (n = 3), Coronaviridae (n = 2), Reoviridae (n = 2) and Caliciviridae (n = 1). Most published trials were at high risk of bias. The findings were heterogeneous across and within viral families regarding virological, immunological and biological response, with no clear conclusion. Some weak but positive results were reported about decrease of symptom severity and duration, or reduction in viral loads. Despite high tolerability, the effects of LF as oral supplement are still inconsistent, both in preventing and managing viral infections. Small sample sizes, variety in recruitment and treatment protocols, and low study quality may have contributed to such heterogeneity. Better-designed studies are needed to further investigate its potential benefits against viral infections, including SARS-CoV-2.

EuCARE-POSTCOVID Study: a multicentre cohort study on long-term post-COVID-19 manifestations.

BACKGROUND: Post-COVID-19 condition refers to persistent or new onset symptoms occurring three months after acute COVID-19, which are unrelated to alternative diagnoses. Symptoms include fatigue, breathlessness, palpitations, pain, concentration difficulties ("brain fog"), sleep disorders, and anxiety/depression. The prevalence of post-COVID-19 condition ranges widely across studies, affecting 10-20% of patients and reaching 50-60% in certain cohorts, while the associated risk factors remain poorly understood. METHODS: This multicentre cohort study, both retrospective and prospective, aims to assess the incidence and risk factors of post-COVID-19 condition in a cohort of recovered patients. Secondary objectives include evaluating the association between circulating SARS-CoV-2 variants and the risk of post-COVID-19 condition, as well as assessing long-term residual organ damage (lung, heart, central nervous system, peripheral nervous system) in relation to patient characteristics and virology (variant and viral load during the acute phase). Participants will include hospitalised and outpatient COVID-19 patients diagnosed between 01/03/2020 and 01/02/2025 from 8 participating centres. A control group will consist of hospitalised patients with respiratory infections other than COVID-19 during the same period. Patients will be followed up at the post-COVID-19 clinic of each centre at 2-3, 6-9, and 12-15 months after clinical recovery. Routine blood exams will be conducted, and patients will complete questionnaires to assess persisting symptoms, fatigue, dyspnoea, quality of life, disability, anxiety and depression, and post-traumatic stress disorders. DISCUSSION: This study aims to understand post-COVID-19 syndrome's incidence and predictors by comparing pandemic waves, utilising retrospective and prospective data. Gender association, especially the potential higher prevalence in females, will be investigated. Symptom tracking via questionnaires and scales will monitor duration and evolution. Questionnaires will also collect data on vaccination, reinfections, and new health issues. Biological samples will enable future studies on post-COVID-19 sequelae mechanisms, including inflammation, immune dysregulation, and viral reservoirs. TRIAL REGISTRATION: This study has been registered with ClinicalTrials.gov under the identifier NCT05531773.

Human inhalable antibody fragments neutralizing SARS-CoV-2 variants for COVID-19 therapy.

As of December 2021, coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a global emergency, and novel therapeutics are urgently needed. Here we describe human single-chain variable fragment (scFv) antibodies (76clAbs) that block an epitope of the SARS-CoV-2 spike protein essential for ACE2-mediated entry into cells. 76clAbs neutralize the Delta variant and other variants being monitored (VBMs) and inhibit spike-mediated pulmonary cell-cell fusion, a critical feature of COVID-19 pathology. In two independent animal models, intranasal administration counteracted the infection. Because of their high efficiency, remarkable stability, resilience to nebulization, and low cost of production, 76clAbs may become a relevant tool for rapid, self-administrable early intervention in SARS-CoV-2-infected subjects independently of their immune status.

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.

Evaluation of HIV-1 capsid genetic variability and lenacapavir (GS-6207) drug resistance-associated mutations according to viral clades among drug-naive individuals.

OBJECTIVES: We evaluated the HIV-1 capsid genetic variability and lenacapavir drug resistance-associated mutations (DRMs) among drug-naive individuals across HIV-1 clades. METHODS: A total of 2031 HIV-1 sequences from drug-naive patients were analysed for capsid amino acid modification and the prevalence of lenacapavir DRMs. Amino acid positions with <5% variability were considered as conserved and variability was analysed by HIV-1 clades. RESULTS: Overall, 63% (148/232) of amino acid positions were conserved in the capsid protein. Of note, conservation was consistent in specific binding residues of cellular factors involved in viral replication [CypA (G89, P90), CPSF6 (Q4, N57, N74, A77, K182) and TRIM-NUP153 (R143)], while N183 (12.31%) was the only non-conserved lenacapavir binding residue. The overall prevalence (95% CI) of lenacapavir DRMs was 0.14% (0.05-0.44) (3/2031), with M66I (0.05%) and Q67H (0.05%) observed in subtype C, and T107N (0.05%) observed in CRF01_AE. Moreover, polymorphic mutations M66C (n = 85; 4.18%), Q67K (n = 78; 3.84%), K70R (n = 7; 0.34%), N74R (n = 57; 2.81%) and T107L (n = 82; 4.03%) were observed at lenacapavir resistance-associated positions. CONCLUSIONS: The low level of lenacapavir DRMs (<1%) supports its predicted effectiveness for treatment and prevention, regardless of HIV-1 clades. The established conserved regions hence serve as a hallmark for the surveillance of novel mutations potentially relevant for lenacapavir resistance.

Archiving of mutations in HIV-1 cellular reservoirs among vertically infected adolescents is contingent with clinical stages and plasma viral load: Evidence from the EDCTP-READY study.

INTRODUCTION: Globally, HIV-related adolescent deaths have increased about 50%, especially for those who are vertically infected. This could be driven by archived drug resistance mutations (DRMs) as children grow up, which might jeopardize antiretroviral therapy (ART). Our objective was to compare HIV-1 genotypic variation between plasma RNA and proviral DNA of vertically infected adolescents (aged 10-19 years) failing ART. METHODS: A comparative study was conducted in 2019 among 296 adolescents with perinatal HIV infection (ALPHI) failing ART in health facilities of the Centre Region of Cameroon. The WHO clinical stage, CD4 count and plasma viral load (PVL) were measured. For those failing ART (PVL ≥ 1000 copies/mL), RNA (plasma) and proviral DNA (buffy coat) were sequenced in the pol gene at Chantal BIYA International Reference Centre (CIRCB), Yaoundé, Cameroon. HIV-1 subtypes and DRMs were interpreted using Stanford HIVdb v.8.8 and MEGA-X. RESULTS: Of the 30% (89/296) failing ART, 81 had both RNA and DNA sequences generated and three were excluded for APOBEC mutations: the mean age was 16 ± 3 years; female-to-male ratio was 3:5; median PVL was 46 856 copies/mL [interquartile range (IQR): 19 898-271 410]; median CD4 count was 264 cells/µL (IQR: 131-574); and 42% were at WHO clinical stage 3/4. Subtype concordance between RNA and DNA viral strains was 100%, with CRF02_AG being predominant (65%) and two potential new recombinants found (A1/G/K; F1/G). Adolescents with DRMs were significantly higher in plasma than in proviral DNA (92% vs. 86%, p < 0.0001). Prevalent DRMs by drug class (RNA vs. DNA respectively) were at position M184 (74% vs. 67%) for nucleoside reverse transcriptase inhibitors (NRTIs), K103 (63% vs. 59%) for non-NRTIs, and V82, L76 and M46 (2% vs. 2%) for protease inhibitors. A total of 35% (27/78) of adolescents had concordant DRM profiles in RNA and DNA, while 27% (21/78) had DRMs only in proviral DNA. The presence of archived DRMs was associated with advanced clinical stage 3/4 (OR = 0.14, p = 0.0003) and PVL < 5 Log (Copies/mL) (OR: 4.88, p = 0.006). CONCLUSIONS: Although plasma RNA remains more sensitive for detecting HIV-1 DRMs, about a quarter of ALPHI experiencing ART failure in an African setting might have archived DRMs in viral reservoirs, indicating clinically occult resistance. Thus, to ensure effective ART success, proviral DNA profiling (alongside RNA genotyping) would provide additional DRMs for adolescents with advanced clinical stages and/or moderate PVL.

Targeting SARS-CoV-2 nsp13 Helicase and Assessment of Druggability Pockets: Identification of Two Potent Inhibitors by a Multi-Site In Silico Drug Repurposing Approach.

The SARS-CoV-2 non-structural protein 13 (nsp13) helicase is an essential enzyme for viral replication and has been identified as an attractive target for the development of new antiviral drugs. In detail, the helicase catalyzes the unwinding of double-stranded DNA or RNA in a 5' to 3' direction and acts in concert with the replication-transcription complex (nsp7/nsp8/nsp12). In this work, bioinformatics and computational tools allowed us to perform a detailed conservation analysis of the SARS-CoV-2 helicase genome and to further predict the druggable enzyme's binding pockets. Thus, a structure-based virtual screening was used to identify valuable compounds that are capable of recognizing multiple nsp13 pockets. Starting from a database of around 4000 drugs already approved by the Food and Drug Administration (FDA), we chose 14 shared compounds capable of recognizing three out of four sites. Finally, by means of visual inspection analysis and based on their commercial availability, five promising compounds were submitted to in vitro assays. Among them, PF-03715455 was able to block both the unwinding and NTPase activities of nsp13 in a micromolar range.

Genotypic HIV-1 tropism determination might help to identify people with exhausted treatment options and advanced disease.

OBJECTIVES: To evaluate HIV-1 tropism in 1382 combined antiretroviral therapy (cART)-experienced patients failing therapy to characterize those with exhausted therapeutic options. METHODS: HIV-1 genotypic tropism was inferred through Geno2Pheno by estimating the false-positive-rate (FPR) values. Cumulative resistance and drug activity were evaluated by Stanford algorithm. RESULTS: Overall, median (IQR) CD4 count (cells/mm3) nadir and at last genotypic resistance test (GRT) available were 98 (33-211) and 312 (155-517), respectively. Considering HIV-1 tropism, 30.5% had X4/dual-mixed strains (FPR ≤5%: 22.2%; FPR 5%-10%: 8.3%). By stratifying according to tropism, by decreasing FPR, a significant decrease of CD4 nadir and at last GRT was observed. The proportion of individuals with CD4 count <200 cells/mm3, who were perinatally infected and with a long treatment history significantly increased as FPR levels decreased. Regarding resistance, 933 (67.5%) individuals accumulated at least one class resistance, with 52.7%, 48.2%, 23.5% and 13.2% of individuals showing resistance to NRTIs, NNRTIs, PIs and INIs; while 23.2%, 27.2%, 14.3% and 2.8% harboured resistance to 1, 2, 3 and 4 classes, respectively. Individuals with FPR ≤5% showed a significantly higher level of resistance to PIs, NRTIs and INIs compared with others. The proportion of individuals harbouring strains susceptible to ≤2 active drugs was only about 2%; nonetheless, this proportion doubled (4.6%) in patients infected with FPR ≤5%. CONCLUSIONS: Our findings showed that a small proportion of cART failing individuals have limited therapeutic options. However, tropism determination might help to identify people who have accumulated a high level of resistance and have a greater risk of advanced disease.

Baseline integrase drug resistance mutations and conserved regions across HIV-1 clades in Cameroon: implications for transition to dolutegravir in resource-limited settings.

BACKGROUND: Transition to dolutegravir-based regimens in resource-limited settings (RLS) requires prior understanding of HIV-1 integrase variants and conserved regions. Therefore, we evaluated integrase drug resistance mutations (DRMs) and conserved regions amongst integrase strand transfer inhibitor (INSTI)-naive patients harbouring diverse HIV-1 clades in Cameroon. METHODS: A cross-sectional study was conducted amongst 918 INSTI-naive patients from Cameroon (89 ART-naive and 829 ART-experienced patients). HIV-1 sequences were interpreted regarding INSTI-DRMs using the Stanford HIVdb v8.9-1 and the 2019 IAS-USA list. Amino acid positions with <1% variability were considered as highly conserved. Subtyping was performed by phylogeny. RESULTS: Overall prevalence (95% CI) of INSTI-DRMs was 0.8% (0.4-1.7), with 0.0% (0.0-4.0) amongst ART-naive versus 0.9% (0.5-1.9) amongst ART-experienced patients; P = 0.44. Accessory mutations (95% CI) were found in 33.8% (30.9-37.0), with 38.2% (28.1-49.1) amongst ART-naive versus 33.4% (30.4-36.7) amongst ART-experienced patients; P = 0.21. Of 288 HIV-1 integrase amino acid positions, 58.3% were highly conserved across subtypes in the following major regions: V75-G82, E85-P90, H114-G118, K127-W132, E138-G149, Q168-L172, T174-V180, W235-A239 and L241-D253. Wide genetic diversity was found (37 clades), including groups M (92.3%), N (1.4%), O (6.2%) and P (0.1%). Amongst group M, CRF02_AG was predominant (47.4%), with a significantly higher frequency (95% CI) of accessory mutations compared with non-AG [41.4% (36.8-46.0) versus 27.1% (23.3-31.2) respectively; P < 0.001]. CONCLUSIONS: The low baseline of INSTI-DRMs (<1%) in Cameroon suggests effectiveness of dolutegravir-based regimens. In spite of high conservation across clades, the variability of accessory mutations between major circulating strains underscores the need for monitoring the selection of INSTI-DRMs while scaling up dolutegravir-based regimens in RLS.