Nationwide Study of Drug Resistance Mutations in HIV-1 Infected Individuals under Antiretroviral Therapy in Brazil.

The success of antiretroviral treatment (ART) is threatened by the emergence of drug resistance mutations (DRM). Since Brazil presents the largest number of people living with HIV (PLWH) in South America we aimed at understanding the dynamics of DRM in this country. We analyzed a total of 20,226 HIV-1 sequences collected from PLWH undergoing ART between 2008-2017. Results show a mild decline of DRM over the years but an increase of the K65R reverse transcriptase mutation from 2.23% to 12.11%. This increase gradually occurred following alterations in the ART regimens replacing zidovudine (AZT) with tenofovir (TDF). PLWH harboring the K65R had significantly higher viral loads than those without this mutation (p < 0.001). Among the two most prevalent HIV-1 subtypes (B and C) there was a significant (p < 0.001) association of K65R with subtype C (11.26%) when compared with subtype B (9.27%). Nonetheless, evidence for K65R transmission in Brazil was found both for C and B subtypes. Additionally, artificial neural network-based immunoinformatic predictions suggest that K65R could enhance viral recognition by HLA-B27 that has relatively low prevalence in the Brazilian population. Overall, the results suggest that tenofovir-based regimens need to be carefully monitored particularly in settings with subtype C and specific HLA profiles.

Pharmacology of HIV Cure: Site of Action.

Despite significant advances in HIV treatment over the past 30 years, critical barriers to an HIV cure persist. The HIV reservoir, defined at both the cellular and anatomical level, constitutes the main barrier to cure. While the mechanisms underlying the reservoir are not yet well understood, one theory to explain persistence at the anatomical level is that subtherapeutic exposure to antiretroviral therapy (ART) within certain tissue compartments permits ongoing replication. Characterizing ART pharmacology throughout the body is important in the context of these potential pharmacologic sanctuaries and for maximizing the probability of success with forthcoming cure strategies that rely on latency reversal and require ART to prevent reseeding the reservoir. In this review, we provide a comprehensive overview of ART and latency reversal agent distribution at the site of action for HIV cure (i.e., anatomical sites commonly associated with HIV persistence, such as lymphoid organs and the central nervous system). We also discuss methodologic approaches that provide insight into HIV cure pharmacology, including experimental design and advances within the computational, pharmaceutical, and analytical chemistry fields. The information discussed in this review will assist in streamlining the development of investigational cure strategies by providing a roadmap to ensure therapeutic exposure within the site of action for HIV cure.

Antibody-based strategies in HIV therapy.

Antibody-based strategies have been introduced for a number of disease states, but represent a novel approach in the management of human immunodeficiency virus (HIV). Ibalizumab and leronlimab are monoclonal antibodies with unique mechanisms as a CD4-directed post-attachment inhibitor and a C-C chemokine receptor type 5-directed inhibitor, respectively. These antibody-based strategies are generally well tolerated, have a favourable pharmacokinetic profile allowing for less-frequent dosing, and have a high barrier to resistance. Ibalizumab is currently approved by the US Food and Drug Administration (US FDA) for management of multi-drug-resistant (MDR) HIV infection in patients who are failing their current regimens. Clinical data demonstrated impressive antiretroviral activity with ibalizumab among a complex HIV population in combination with an optimized background regimen, where limited therapeutic options exist. To date, leronlimab has not been granted approval by the US FDA, but has been designated fast-track status. Leronlimab is being studied as a maintenance monotherapy agent in virologically suppressed patients, as well as for treatment of MDR HIV infection in patients who are failing their current regimens. Currently available data in both of these potential areas appear promising for leronlimab. The mechanism of action, pharmacokinetic profile, efficacy and safety of these novel antibody-based strategies represent an advance in the management of HIV. Future studies and post-marketing experience will further determine longer-term clinical efficacy, safety and resistance data for ibalizumab and leronlimab.

The Basis of Peracetic Acid Inactivation Mechanisms for Rotavirus and Tulane Virus under Conditions Relevant for Vegetable Sanitation.

We determined the disinfection efficacy and inactivation mechanisms of peracetic acid (PAA)-based sanitizer using pH values relevant for vegetable sanitation against rotavirus (RV) and Tulane virus (TV; a human norovirus surrogate). TV was significantly more resistant to PAA disinfection than RV: for a 2-log10 reduction of virus titer, RV required 1 mg/liter PAA for 3.5 min of exposure, while TV required 10 mg/liter PAA for 30 min. The higher resistance of TV can be explained, in part, by significantly more aggregation of TV in PAA solutions. The PAA mechanisms of virus inactivation were explored by quantifying (i) viral genome integrity and replication using reverse transcription-quantitative PCR (RT-qPCR) and (ii) virus-host receptor interactions using a cell-free binding assay with porcine gastric mucin conjugated with magnetic beads (PGM-MBs). We observed that PAA induced damage to both RV and TV genomes and also decreased virus-receptor interactions, with the latter suggesting that PAA damages viral proteins important for binding its host cell receptors. Importantly, the levels of genome-versus-protein damage induced by PAA were different for each virus. PAA inactivation correlated with higher levels of RV genome damage than of RV-receptor interactions. For PAA-treated TV, the opposite trends were observed. Thus, PAA inactivates each of these viruses via different molecular mechanisms. The findings presented here potentially contribute to the design of a robust sanitation strategy for RV and TV using PAA to prevent foodborne disease.IMPORTANCE In this study, we examined the inactivation mechanisms of peracetic acid (PAA), a sanitizer commonly used for postharvest vegetable washing, for two enteric viruses: Tulane virus (TV) as a human norovirus surrogate and rotavirus (RV). PAA disinfection mechanisms for RV were mainly due to genome damage. In contrast, PAA disinfection in TV was due to damage of the proteins important for binding to its host receptor. We also observed that PAA triggered aggregation of TV to a much greater extent than RV. These studies demonstrate that different viruses are inactivated via different PAA mechanisms. This information is important for designing an optimal sanitation practice for postharvest vegetable washing to minimize foodborne viral diseases.

Lopinavir/ritonavir use in Covid-19 infection: is it completely non-beneficial?

Covid-19 infection caused by the novel coronavirus SARS-COV-2 continues to be a major global health challenge. Till date, no drug has been approved for the treatment of this infection. A number of medications have been proposed and there are ongoing clinical trials around the world to find a suitable treatment. A recent randomised control trial compared lopinavir/ritonavir with standard care among 199 patients with severe Covid-19 infection and concluded that there was no significant reduction in mortality rate with lopinavir/ritonavir. However, there are a few important lessons which may be learnt from the study apart from the statistical reduction in mortality rate. There was a numerical reduction in mortality rate, less intensive care unit stay and less complications in the lopinavir-ritonavir group. This article points out some of those important lessons with some suggestions for future clinical trials.

Discovery of a Silicon-Containing Pan-Genotype Hepatitis C Virus NS5A Inhibitor.

We describe a study leading to the discovery of compound 11, a pan-genotypic hepatitis C virus (HCV) nonstructural protein 5A (NS5A) inhibitor with excellent potency, metabolic stability, and pharmacokinetics (PK). Compound 11 incorporating a 4-silapiperidine group was discovered by further optimizing our previous lead with a triethylsilyl moiety. It displayed great potency against genotype 1 subtype a (GT1a), -1b, -2a, -3a, -4a, -5a, and -6a with an EC50 range of 0.33-17 pM and improved potency against the resistance-associated variant GT1a_M28T. Pharmacokinetics (PK) study indicated that compound 11 has reasonable PK exposures with a high liver distribution in preclinical animal species (mouse, rat, and dog). The results of a 14 day repeat-dose toxicity study identified the safety of compound 11.

Systematic review with meta-analysis: impact of baseline resistance-associated substitutions on the efficacy of glecaprevir/pibrentasvir among chronic hepatitis C patients.

BACKGROUND: The effect of baseline resistance-associated substitutions (RAS) on the sustained virologic response at 12 weeks (SVR12) among chronic hepatitis C (CHC) patients receiving the second generation, pan-genotypic glecaprevir/pibrentasvir (G/P) regimen is unclear. AIM: To assess the effect of RAS on the SVR12 in CHC patients treated with G/P regimen. METHODS: The EMBASE, MEDLINE and Cochrane central register of controlled trials databases were searched for relevant studies published before 1 March 2019. The principal outcome was to compare the SVR12 in CHC patients with and without baseline RAS, particularly in genotype-1, genotype-3 and direct-acting anti-virals (DAAs) failure patients. The outcomes were pooled using a random-effects model and odds ratio (OR) was calculated. The risk of bias was assessed using the Cochrane risk of bias tools for randomised and nonrandomised interventional studies. RESULTS: After initially identifying 410 studies, 3302 patients from 17 studies were included. Among 50 cases of virologic failures, 48% had genotype-3 infection, 44% genotype-1 infection and 36% DAA-failure patients. Baseline RAS were present in 44(88%) patients. The most common NS5a and NS3 mutations were Y93H and A166S respectively. The odds of SVR12 were significantly reduced in patients with any baseline RAS (NS3 and/or NS5a) (OR 0.32, 95%C I[0.15, 0.65], I2  = 0%) and NS5a substitutions (OR 0.36, 95%CI [0.18,0.73]). The impact of RAS on SVR12 was significant among genotype-3 patients, but not among genotype-1 or DAA-failure cases. The presence of Y93H and A30K mutations significantly impacted SVR12 rates in genotype-3 patients. CONCLUSION: Baseline NS3 or NS5a RAS, especially the NS5a substitutions-A30K, Y93H, decrease the odds of achieving SVR12 in genotype-3 CHC patients.

Impact of Suboptimal APOBEC3G Neutralization on the Emergence of HIV Drug Resistance in Humanized Mice.

HIV diversification facilitates immune escape and complicates antiretroviral therapy. In this study, we take advantage of a humanized-mouse model to probe the contribution of APOBEC3 mutagenesis to viral evolution. Humanized mice were infected with isogenic HIV molecular clones (HIV-WT, HIV-45G, and HIV-ΔSLQ) that differ in their abilities to counteract APOBEC3G (A3G). Infected mice remained naive or were treated with the reverse transcriptase (RT) inhibitor lamivudine (3TC). Viremia, emergence of drug-resistant variants, and quasispecies diversification in the plasma compartment were determined throughout infection. While both HIV-WT and HIV-45G achieved robust infection, over time, HIV-45G replication was significantly reduced compared to that of HIV-WT in the absence of 3TC treatment. In contrast, treatment responses differed significantly between HIV-45G- and HIV-WT-infected mice. Antiretroviral treatment failed in 91% of HIV-45G-infected mice, while only 36% of HIV-WT-infected mice displayed a similar negative outcome. Emergence of 3TC-resistant variants and nucleotide diversity were determined by analyzing 155,462 single HIV reverse transcriptase gene (RT) and 6,985 vif sequences from 33 mice. Prior to treatment, variants with genotypic 3TC resistance (RT-M184I/V) were detected at low levels in over a third of all the animals. Upon treatment, the composition of the plasma quasispecies rapidly changed, leading to a majority of circulating viral variants encoding RT-184I. Interestingly, increased viral diversity prior to treatment initiation correlated with higher plasma viremia in HIV-45G-infected animals, but not in HIV-WT-infected animals. Taken together, HIV variants with suboptimal anti-A3G activity were attenuated in the absence of selection but displayed a fitness advantage in the presence of antiretroviral treatment.IMPORTANCE Both viral (e.g., RT) and host (e.g., A3G) factors can contribute to HIV sequence diversity. This study shows that suboptimal anti-A3G activity shapes viral fitness and drives viral evolution in the plasma compartment in humanized mice.

Virological Outcome of Patients With HIV Drug Resistance Attending an Urban Outpatient Clinic in Uganda: A Need for Structured Adherence Counseling and Third-Line Treatment Options.

BACKGROUND: HIV drug resistance and suboptimal adherence are the main reasons for treatment failure among HIV-infected individuals. As genotypic resistance testing is not routinely available in resource-limited settings such as Uganda, data on transmitted and acquired resistance are sparse. METHODS: This observational follow-up study assessed the virological outcomes of patients diagnosed with virological failure or transmitted HIV drug resistance in 2015 at the adults' outpatient clinic of the Infectious Diseases Institute in Kampala, Uganda. Initially, 2430 patients on antiretroviral therapy (ART) underwent virological monitoring, of which 190 had virological failure and were subsequently eligible for this follow-up study. Nine patients diagnosed with transmitted drug resistance were eligible. In patients with a viral load > 1000 copies/mL, genotypic resistance testing was performed. RESULTS: Of 190 eligible patients, 30 (15.8%) had either died or were lost to follow-up. A total of 148 (77.9%) were included, of which 98 had had a change of ART regimen, and 50 had received adherence counseling only. The majority was now on second-line ART (N = 130, 87.8%). The median age was 39 years (interquartile range: 32-46), and 109 (73.6%) were women. Virological failure was diagnosed in 29 (19.6%) patients, of which 24 (82.8%) were on second-line ART. Relevant drug resistance was found in 25 (86.2%) cases, of which 12 (41.3%) carried dual and 7 (24.1%) triple drug resistance. CONCLUSION: Two years after initial virological failure, most patients followed up by this study had a successful virological outcome. However, a significant proportion either continued to fail or died or was lost to follow-up.

Human Immunodeficiency Virus Drug Resistance: 2018 Recommendations of the International Antiviral Society-USA Panel.

Background: Contemporary antiretroviral therapies (ART) and management strategies have diminished both human immunodeficiency virus (HIV) treatment failure and the acquired resistance to drugs in resource-rich regions, but transmission of drug-resistant viruses has not similarly decreased. In low- and middle-income regions, ART roll-out has improved outcomes, but has resulted in increasing acquired and transmitted resistances. Our objective was to review resistance to ART drugs and methods to detect it, and to provide updated recommendations for testing and monitoring for drug resistance in HIV-infected individuals. Methods: A volunteer panel of experts appointed by the International Antiviral (formerly AIDS) Society-USA reviewed relevant peer-reviewed data that were published or presented at scientific conferences. Recommendations were rated according to the strength of the recommendation and quality of the evidence, and reached by full panel consensus. Results: Resistance testing remains a cornerstone of ART. It is recommended in newly-diagnosed individuals and in patients in whom ART has failed. Testing for transmitted integrase strand-transfer inhibitor resistance is currently not recommended, but this may change as more resistance emerges with widespread use. Sanger-based and next-generation sequencing approaches are each suited for genotypic testing. Testing for minority variants harboring drug resistance may only be considered if treatments depend on a first-generation nonnucleoside analogue reverse transcriptase inhibitor. Different HIV-1 subtypes do not need special considerations regarding resistance testing. Conclusions: Testing for HIV drug resistance in drug-naive individuals and in patients in whom antiretroviral drugs are failing, and the appreciation of the role of testing, are crucial to the prevention and management of failure of ART.

Modern Human Immunodeficiency Virus Therapy: Progress and Prospects.

Safe and effective lifelong treatment to control human immunodeficiency virus (HIV) infection is one of the greatest scientific and public health achievements of the past century. The majority of infected individuals able to maintain a daily oral regimen now have a normal or near-normal life expectancy. More than 30 approved drugs and dozens of formulations have produced thousands of possible drug combinations used clinically in the past, but today most patients receive only a handful of high priority and rigorously tested regimens. Unique features of antiretroviral therapy include the need for lifelong treatment to control virus replication and the possibility of rapid emergence of permanent drug resistance if these agents are not properly used. Although three-drug combination oral regimens have radically altered the course of this epidemic, the future will include long-acting injectable and implantable drugs and devices to treat and prevent infection.

Evolutionary pathways to NS5A inhibitor resistance in genotype 1 hepatitis C virus.

Direct-acting antivirals (DAAs) targeting NS5A are broadly effective against hepatitis C virus (HCV) infections, but sustained virological response rates are generally lower in patients infected with genotype (gt)-1a than gt-1b viruses. The explanation for this remains uncertain. Here, we adopted a highly accurate, ultra-deep primer ID sequencing approach to intensively study serial changes in the NS5A-coding region of HCV in gt-1a- and gt-1b-infected subjects receiving a short course of monotherapy with the NS5A inhibitor, elbasvir. Low or undetectable levels of viremia precluded on-treatment analysis in gt-1b-infected subjects, but variants with the resistance-associated substitution (RAS) Y93H in NS5A dominated rebounding virus populations following cessation of treatment. These variants persisted until the end of the study, two months later. In contrast, while Y93H emerged in multiple lineages and became dominant in subjects with gt-1a virus, these haplotypes rapidly decreased in frequency off therapy. Substitutions at Q30 and L31 emerged in distinctly independent lineages at later time points, ultimately coming to dominate the virus population off therapy. Consistent with this, cell culture studies with gt-1a and gt-1b reporter viruses and replicons demonstrated that Y93H confers a much greater loss of replicative fitness in gt-1a than gt-1b virus, and that L31M/V both compensates for the loss of fitness associated with Q30R (but not Y93H) and also boosts drug resistance. These observations show how differences in the impact of RASs on drug resistance and replicative fitness influence the evolution of gt-1a and gt-1b viruses during monotherapy with an antiviral targeting NS5A.

Prediction of HIV Drug Resistance by Combining Sequence and Structural Properties.

Drug resistance is a major obstacle faced by therapist in treating HIV infected patients. The reason behind these phenomena is either protein mutation or the changes in gene expression level that induces resistance to drug treatments. These mutations affect the drug binding activity, hence resulting in failure of treatment. Therefore, it is necessary to conduct resistance testing in order to carry out HIV effective therapy. This study combines both sequence and structural features for predicting HIV resistance by applying SVM and Random Forests classifiers. The model was tested on the mutants of HIV-1 protease and reverse transcriptase. Taken together the features we have used in our method, total contact energies among multiple mutations have a strong impact in predicting resistance as they are crucial in understanding the interactions of HIV mutants. The combination of sequence-structure features offers high accuracy with support vector machines as compared to Random Forests classifier. Both single and acquisition of multiple mutations are important in predicting HIV resistance to certain drug treatments. We have discovered the practicality of these features; hence, these can be used in the future to predict resistance for other complex diseases.

Estimation of discrete survival function for error-prone diagnostic tests.

The product limit or Kaplan-Meier (KM) estimator is commonly used to estimate the survival function in the presence of incomplete time to event. Application of this method assumes inherently that the occurrence of an event is known with certainty. However, the clinical diagnosis of an event is often subject to misclassification due to assay error or adjudication error, by which the event is assessed with some uncertainty. In the presence of such errors, the true distribution of the time to first event would not be estimated accurately using the KM method. We develop a method to estimate the true survival distribution by incorporating negative predictive values and positive predictive values, into a KM-like method of estimation. This allows us to quantify the bias in the KM survival estimates due to the presence of misclassified events in the observed data. We present an unbiased estimator of the true survival function and its variance. Asymptotic properties of the proposed estimators are provided, and these properties are examined through simulations. We demonstrate our methods using data from the Viral Resistance to Antiviral Therapy of Hepatitis C study.

Elbasvir-grazoprevir: A new direct-acting antiviral combination for hepatitis C.

PURPOSE: The chemistry, pharmacology, pharmacodynamics, pharmacokinetics, efficacy, safety, dosage, administration, and role of elbasvir-grazoprevir in the treatment of hepatitis C virus (HCV) infection are reviewed. SUMMARY: Elbasvir-grazoprevir was recently approved by the Food and Drug Administration for the treatment of chronic HCV genotype 1 or 4 infections with or without ribavirin in patients with or without compensated cirrhosis. Elbasvir exhibits antiviral activity against HCV genotypes 1a, 1b, 2a, 3a, and 4a. Elbasvir-grazoprevir undergoes fecal excretion, does not require dosage adjustment in patients with renal impairment, and is contraindicated in moderate and severe hepatic impairment. In Phase II and III clinical trials, elbasvir-grazoprevir administered orally for 12 weeks was shown to achieve a high sustained virological response 12 weeks after the end of treatment. Elbasvir-grazoprevir is a once-daily, fixed-dose combination tablet that can be taken without regard to food. The adverse drug reactions most commonly reported include fatigue, headache, and nausea. Elbasvir-grazoprevir is indicated with ribavirin for treatment-naive and treatment-experienced patients with genotype 1a with baseline NS5A polymorphisms, for treatment-experienced patients with genotype 1b, and for treatment-experienced patients with genotype 4. CONCLUSION: Elbasvir-grazoprevir achieves a high cure rate in the treatment of patients with chronic HCV with a once-daily oral regimen and without serious adverse effects; however, it requires close monitoring of liver function values. It is an effective option for patients with HCV genotype 1a, 1b, or 4 with or without compensated cirrhosis and is a particularly attractive option in patients with chronic kidney disease receiving hemodialysis and in patients with HIV coinfection.

Community Based Antiretroviral Treatment in Rural Zimbabwe.

Treatment of HIV has reduced HIV/AIDS-related mortality. Sustaining >90% virologic suppression in sub-Saharan Africa requires decentralized care and prevention services to rural communities. In Zimbabwe, the number of people receiving antiretroviral treatment (ART) has increased rapidly. However, access to treatment monitoring tools such as viral load and drug resistance testing is limited. We assessed virologic treatment outcomes among ART recipients in Nyamutora, a rural community receiving bimonthly ART and prevention services. We enrolled all ART recipients (143) at 6-monthly visits in the Nyamutora community in 2014 and 2015. Whole blood samples were collected in K-EDTA tubes, transported to Harare for CD4 counts and viral load testing, and genotype was obtained in participants with viral loads >1,000 copies/ml. Ages ranged from 2 to 75 years (median 43 years) with a median 42 months on ART at follow-up. Eight of 143 (6%) had viral loads >1,000 copies/ml at one of the 3 visits, 7 on first-line nevirapine (NVP)-based ART and 1 on second-line LPV/r-based ART. Seven participants had sequence data available, and five had drug resistance mutations, K65R, T69N, K101E, K103N, Y181C/I, M184V, and G190A. Virologic failure (p = .001) and drug resistance mutations (p = .01) on first-line NVP-based ART were associated with younger age by univariate exact logistic regression. The participants had high viral suppression (94%) despite less than optimal (NVP based) ART regimens without laboratory monitoring. Virologic failure and drug resistance were higher among children and adolescents. Effective ART delivery to the community achieved high rates of virologic suppression and minimal drug resistance.

Molecular Mechanism Underlying the Action of Influenza A Virus Fusion Inhibitor MBX2546.

Influenza A virus envelop protein hemagglutinin (HA) plays important roles in viral entry. We previously have reported that MBX2546, a novel influenza A virus inhibitor, binds to HA and inhibits HA-mediated membrane fusion. In this report, we show that (i) both binding and stabilization of HA by MBX2546 are required for the inhibition of viral infection and (ii) the binding of HA by MBX2546 represses the low-pH-induced conformational change in the HA, which is a prerequisite for membrane fusion. Mutations in MBX2546-resistant influenza A/PR/8/34 (H1N1) viruses are mapped in the HA stem region near the amino terminus of HA2. Finally, we have modeled the binding site of MBX2546 using molecular dynamics and find that the resulting structure is in good agreement with our results. Together, these studies underscore the importance of the HA stem loop region as a potential target for therapeutic intervention.

Glucocorticoid Insensitivity in Virally Infected Airway Epithelial Cells Is Dependent on Transforming Growth Factor-ß Activity.

Asthma and chronic obstructive pulmonary disease (COPD) exacerbations are commonly associated with respiratory syncytial virus (RSV), rhinovirus (RV) and influenza A virus (IAV) infection. The ensuing airway inflammation is resistant to the anti-inflammatory actions of glucocorticoids (GCs). Viral infection elicits transforming growth factor-ß (TGF-ß) activity, a growth factor we have previously shown to impair GC action in human airway epithelial cells through the activation of activin-like kinase 5 (ALK5), the type 1 receptor of TGF-ß. In the current study, we examine the contribution of TGF-ß activity to the GC-resistance caused by viral infection. We demonstrate that viral infection of human bronchial epithelial cells with RSV, RV or IAV impairs GC anti-inflammatory action. Poly(I:C), a synthetic analog of double-stranded RNA, also impairs GC activity. Both viral infection and poly(I:C) increase TGF-ß expression and activity. Importantly, the GC impairment was attenuated by the selective ALK5 (TGFßRI) inhibitor, SB431542 and prevented by the therapeutic agent, tranilast, which reduced TGF-ß activity associated with viral infection. This study shows for the first time that viral-induced glucocorticoid-insensitivity is partially mediated by activation of endogenous TGF-ß.