HIV-1 Clinical Isolates Research Articles

Preclinical toxicity analyses of lentiviral vectors expressing the HIV-1 LTR-specific designer-recombinase Brec1.

Drug-based antiretroviral therapies (ART) efficiently suppress HIV replication in humans, but the virus persists as integrated proviral reservoirs in small numbers of cells. Importantly, ART cannot eliminate HIV from an infected individual, since it does not target the integrated provirus. Therefore, genome editing-based strategies that can inactivate or excise HIV genomes would provide the technology for novel curative therapies. In fact, the HIV-1 LTR-specific designer-recombinase Brec1 has been shown to remove integrated proviruses from infected cells and is highly efficacious on clinical HIV-1 isolates in vitro and in vivo, suggesting that Brec1 has the potential for clinical development of advanced HIV-1 eradication strategies in people living with HIV. In line with the preparation of a first-in-human advanced therapy medicinal product gene therapy trial, we here present an extensive preclinical evaluation of Brec1 and lentiviral vectors expressing the Brec1 transgene. This included detailed functional analysis of potential genomic off-target sites, assessing vector safety by investigating vector copy number (VCN) and the risk for potential vector-related insertional mutagenesis, as well as analyzing the potential of Brec1 to trigger an undesired strong T cell immune response. In conclusion, the antiviral designer-recombinase Brec1 is shown to lack any detectable cytopathic, genotoxic or T cell-related immunogenic effects, thereby meeting an important precondition for clinical application of the therapeutic lentiviral vector LV-Brec1 in novel HIV-1 curative strategies.

Rational Design of Sulfonyl-γ-AApeptides as Highly Potent HIV-1 Fusion Inhibitors with Broad-Spectrum Activity.

The HIV-1 epidemic has significant social and economic implications for public health. Developing new antivirus drugs to eradicate drug resistance is still urgently needed. Herein, we demonstrated that sulfonyl-γ-AApeptides could be designed to mimic MTSC22EK, one potent HIV fusion inhibitor derived from CHR. The best two sequences revealed comparable activity to MTSC22EK in an authentic HIV-1 infection assay and exhibited broad-spectrum anti-HIV-1 activity to many HIV-1 clinical isolates. Furthermore, sulfonyl-γ-AApeptides show remarkable resistance to proteolysis and favorable permeability in PAMPA-GIT and PAMPA-BBB assays, suggesting that both sequences could control HIV-1 within the central nervous system and possess promising oral bioavailability. Mechanistic investigations suggest that these sulfonyl-γ-AApeptides function by mimicking the CHR of gp41 and tightly bind with NHR, thereby inhibiting the formation of the 6-HB structure necessary for HIV-1 fusion. Overall, our results suggest that sulfonyl-γ-AApeptides represent a new generation of anti-HIV-1 fusion inhibitors. Moreover, this design strategy could be adopted to modulate many of the PPIs.

Antiviral activity of tenofovir alafenamide (TAF) against HIV-1 clinical isolates harboring K65R.

Tenofovir alafenamide (TAF) is a prodrug of the nucleoside reverse transcriptase (RT) inhibitor tenofovir (TFV). Compared to the earlier TFV prodrug, TFV disoproxil fumarate (TDF), TAF achieves more than fourfold-higher intracellular levels of its active metabolite TFV diphosphate (TFV-DP) in clinical studies, while achieving a significant reduction of TFV systemic exposure. Resistance to TFV has been well established, with the K65R mutation inRTas the signature mutation.Here we evaluated the in vitro activity of TAF and TDF in patient-derived HIV-1 isolates harboring the K65R mutation.Clinical isolates containing K65R were cloned into the pXXLAI construct (n = 42). In vitro phenotypic susceptibility of the constructs to TAF and TDF was evaluated in an MT-2 cell HIV assay and in viral breakthrough assays modeling physiological concentrations of TAF and TDF.TAF and TDF susceptibility were highly correlated in K65R-containing mutants, ranging from 2.7- to 3.0-fold (K65R alone) and 1.2- to 27.6-fold (K65R+ other RT mutations) relative to wild-type. In viral breakthrough assays mimicking differences in physiological concentrations, TAF inhibited breakthrough of 40 of 42 clinical isolates, while the TDF equivalent only inhibited 32 of 42 isolates tested.TAF displayed a higher barrier to resistance than TDF in this panel of K65R-containing clinical isolates.

Characterization of a Novel CD4 Mimetic Compound YIR-821 against HIV-1 Clinical Isolates.

Small CD4-mimetic compound (CD4mc), which inhibits the interaction between gp120 with CD4, acts as an entry inhibitor and induces structural changes in the HIV-1 envelope glycoprotein trimer (Env) through its insertion within the Phe43 cavity of gp120. We recently developed YIR-821, a novel CD4mc, that has potent antiviral activity and lower toxicity than the prototype NBD-556. To assess the possibility of clinical application of YIR-821, we tested its antiviral activity using a panel of HIV-1 pseudoviruses from different subtypes. YIR-821 displayed entry inhibitor activity against 53.5% (21/40) of the pseudoviruses tested and enhanced neutralization mediated by coreceptor binding site (CoRBS) antibodies in 50% (16/32) of these. Furthermore, when we assessed the antiviral effects using a panel of pseudoviruses and autologous plasma IgG, enhancement of antibody-mediated neutralization activity was observed for 48% (15/31) of subtype B strains and 51% (28/55) of non-B strains. The direct antiviral activity of YIR-821 as an entry inhibitor was observed in 53% of both subtype B (27/51) and non-B subtype (40/75) pseudoviruses. Enhancement of antibody-dependent cellular cytotoxicity was also observed with YIR-821 for all six selected clinical isolates, as well as for the transmitted/founder (T/F) CH58 virus-infected cells. The sequence diversity in the CD4 binding site as well as other regions, such as the gp120 inner domain layers or gp41, may be involved in the multiple mechanisms related to the sensitive/resistant phenotype of the virus to YIR-821. Our findings may facilitate the clinical application of YIR-821. IMPORTANCE Small CD4-mimetic compound (CD4mc) interacts with the Phe43 cavity and triggers conformational changes, enhancing antibody-mediated neutralization and antibody-dependent cellular cytotoxicity (ADCC). Here, we evaluated the effect of YIR-821, a novel CD4mc, against clinical isolates, including both subtype B and non-B subtype viruses. Our results confirm the desirable properties of YIR-821, which include entry inhibition, enhancement of IgG-neutralization, binding, and ADCC, in addition to low toxicity and long half-life in a rhesus macaque model, that might facilitate the clinical application of this novel CD4mc. Our observation of primary viruses that are resistant to YIR-821 suggests that further development of CD4mcs with different structural properties is required.

P-selectin glycoprotein ligand-1 (PSGL-1/CD162) is incorporated into clinical HIV-1 isolates and can mediate virus capture and subsequent transfer to permissive cells

BackgroundP-selectin glycoprotein ligand-1 (PSGL-1/CD162) has been studied extensively for its role in mediating leukocyte rolling through interactions with its cognate receptor, P-selectin. Recently, PSGL-1 was identified as a novel HIV-1 host restriction factor, particularly when expressed at high levels in the HIV envelope. Importantly, while the potent antiviral activity of PSGL-1 has been clearly demonstrated in various complementary model systems, the breadth of PSGL-1 incorporation across genetically diverse viral isolates and clinical isolates has yet to be described. Additionally, the biological activity of virion-incorporated PSGL-1 has also yet to be shown.ResultsHerein we assessed the levels of PSGL-1 on viruses produced through transfection with various amounts of PSGL-1 plasmid DNA (0–250 ng), compared to levels of PSGL-1 on viruses produced through infection of T cell lines and primary PBMC. We found that very low levels of PSGL-1 plasmid DNA (< 2.5 ng/well) were necessary to generate virus models that could closely mirror the phenotype of viruses produced via infection of T cells and PBMC. Unique to this study, we show that PSGL-1 is incorporated in a broad range of HIV-1 and SIV isolates and that virions with incorporated PSGL-1 are detectable in plasma from viremic HIV-1-infected individuals, corroborating the relevance of PSGL-1 in natural infection. Additionally, we show that PSGL-1 on viruses can bind its cognate selectin receptors, P-, E-, and L-selectins. Finally, we show viruses with endogenous levels of PSGL-1 can be captured by P-selectin and transferred to HIV-permissive bystander cells, highlighting a novel role for PSGL-1 in HIV-1 infection. Notably, viruses which contained high levels of PSGL-1 were noninfectious in our hands, in line with previous findings reporting the potent antiviral activity of PSGL-1.ConclusionsOur results indicate that levels of PSGL-1 incorporation into virions can vary widely among model systems tested, and that careful tailoring of plasmid levels is required to recapitulate physiological systems when using pseudovirus models. Taken together, our data suggest that PSGL-1 may play diverse roles in the physiology of HIV-1 infection, particularly due to the functionally active state of PSGL-1 on virion surfaces and the breadth of PSGL-1 incorporation among a wide range of viral isolates.

Ibalizumab shows in-vitro activity against group A and group B HIV-2 clinical isolates.

Treatment of multidrug-resistant HIV-2 is an emerging issue, because of the rapid selection of mutations at time of virological failure and the low number of antiretrovirals active on HIV-2. The aim of this study was to determine the susceptibility of HIV-2 primary isolates to ibalizumab, a long-acting monoclonal antibody that binds to CD4 that is approved for the treatment of MDR HIV-1. In-vitro phenotypic susceptibility of 16 HIV-2 primary isolates was measured using a modified version of the ANRS peripheral blood mononuclear cells (PBMC) assay. Susceptibility to ibalizumab was assessed through 50% inhibitory concentrations and maximum percentage inhibitions (MPI), and gp105 was sequenced to look for determinants of reduced susceptibility. Ibalizumab inhibited viral replication of all 16 isolates, with a median IC 50 value of 0.027 μg/ml (range = 0.001-0.506 μg/ml), and a median MPI of 93%. Although two isolates presented higher IC 50 (above 0.1 μg/ml), they did not exhibit a loss of potential N-linked glycosylation sites in V5 loop, as reported in HIV-1 strains with reduced susceptibility. However, both presented shorter V1 and V2 loops than the HIV-2 reference strain. Ibalizumab inhibits HIV-2 replication, with IC 50 and MPI in the range of those reported for HIV-1. These in vitro data support the use of ibalizumab in patients with MDR HIV-2, in combination with an optimized background regimen.

The impact of lentiviral vector genome size and producer cell genomic to gag-pol mRNA ratios on packaging efficiency and titre

Lentiviral vectors are showing success in the clinic, but producing enough vector to meet the growing demand is a major challenge. Furthermore, next-generation gene therapy vectors encode multiple genes resulting in larger genome sizes, which is reported to reduce titers. A packaging limit has not been defined. The aim of this work was to assess the impact of genome size on the production of lentiviral vectors with an emphasis on producer cell mRNA levels, packaging efficiency, and infectivity measures. Consistent with work by others, vector titers reduced as genome size increased. While genomic infectivity accounted for much of this effect, genome sizes exceeding that of clinical HIV-1 isolates result in low titers due to a combination of both low genomic infectivity and decreased packaging efficiency. Manipulating the relative level of genomic RNA to gag-pol mRNA in the producer cells revealed a direct relationship between producer cell mRNA levels and packaging efficiency yet could not rescue packaging of oversized genomes, implying a de facto packaging defect. However, independent of genome size, an equimolar ratio between wild-type gag-pol mRNA and vector genomic RNA in producer cells was optimal for titer.

Computer-aided drug design: from discovery of novel pharmaceutical agents to systems pharmacology

New drug discovery is based on the analysis of public information about the mechanisms of the disease, molecular targets, and ligands, which interaction with the target could lead to the normalization of the pathological process. The available data on diseases, drugs, pharmacological effects, molecular targets, and drug-like substances, taking into account the combinatorics of the associative relations between them, correspond to the Big Data. To analyze such data, the application of computer-aided drug design methods is necessary. An overview of the studies in this area performed by the Laboratory for Structure-Function Based Drug Design of IBMC is presented. We have developed the approaches to identifying promising pharmacological targets, predicting several thousand types of biological activity based on the structural formula of the compound, analyzing protein-ligand interactions based on assessing local similarity of amino acid sequences, identifying likely molecular mechanisms of side effects of drugs, calculating the integral toxicity of drugs taking into account their metabolism, have been developed in the human body, predicting sustainable and sensitive options strains and evaluating the effectiveness of combinations of antiretroviral drugs in patients, taking into account the molecular genetic characteristics of the clinical isolates of HIV-1. Our computer programs are implemented as the web-services freely available on the Internet, which are used by thousands of researchers from many countries of the world to select the most promising substances for the synthesis and determine the priority areas for experimental testing of their biological activity.

Comparable In Vitro Activities of Second-Generation HIV-1 Integrase Strand Transfer Inhibitors (INSTIs) on HIV-1 Clinical Isolates with INSTI Resistance Mutations.

Second-generation HIV-1 integrase strand transfer inhibitors (INSTIs) dolutegravir (DTG), bictegravir (BIC), and cabotegravir (CAB) showed a high genetic barrier to resistance and limited cross-resistance with first-generation INSTIs raltegravir (RAL) and elvitegravir (EVG). In this study, DTG, BIC, and CAB demonstrated a comparable activity on a panel of INSTI-resistant strains isolated from patients exposed to RAL, EVG, and/or DTG, with a significantly reduced susceptibility only with the pathway Q148H/K/R plus one to two additional INSTI mutations.

Long-Acting HIV-1 Fusion Inhibitory Peptides and their Mechanisms of Action.

The clinical application of HIV fusion inhibitor, enfuvirtide (T20), was limited mainly because of its short half-life. Here we designed and synthesized two PEGylated C34 peptides, PEG2kC34 and PEG5kC34, with the PEG chain length of 2 and 5 kDa, respectively, and evaluated their anti-HIV-1 activity and mechanisms of action. We found that these two PEGylated peptides could bind to the HIV-1 peptide N36 to form high affinity complexes with high α-helicity. The peptides PEG2kC34 and PEG5kC34 effectively inhibited HIV-1 Env-mediated cell–cell fusion with an effective concentration for 50% inhibition (EC50) of about 36 nM. They also inhibited infection of the laboratory-adapted HIV-1 strain NL4-3 with EC50 of about 4–5 nM, and against 47 HIV-1 clinical isolates circulating in China with mean EC50 of PEG2kC34 and PEG5kC34 of about 26 nM and 32 nM, respectively. The plasma half-life (t1/2) of PEG2kC34 and PEG5kC34 was 2.6 h and 5.1 h, respectively, and the t1/2 of PEGylated C34 was about 2.4-fold and 4.6-fold longer than C34 (~1.1 h), respectively. These findings suggest that PEGylated C34 with broad-spectrum anti-HIV-1 activity and prolonged half-life can be further developed as a peptide fusion inhibitor-based long-acting anti-HIV drug for clinical use to treat HIV-infected patients who have failed to respond to current anti-retrovirus drugs.

539. GS-CA2: A Novel, Potent, and Selective First-In-class Inhibitor of HIV-1 Capsid Function Displays Nonclinical Pharmacokinetics Supporting Long-Acting Potential in Humans

BackgroundGS-CA2, an analog of GS-CA1, is a novel and selective inhibitor of HIV-1 capsid function. Safety and pharmacokinetics (PK) of GS-CA2 is currently being evaluated in healthy human subjects. Herein, we present the anti-HIV activity and nonclinical PK of GS-CA2, demonstrating its potential as a first-in-class long-acting antiretroviral agent.MethodsGS-CA2 antiviral activity was evaluated in MT-4 cells and in human peripheral blood mononuclear cells (PBMCs) acutely infected with HIV-1 (IIIb) and clinical HIV-1 isolates, respectively. Standard in vitro methods were used to characterize compound lipophilicity (LogD), solubility and relative binding to cell culture and plasma proteins. Metabolic stability was assessed in cryopreserved hepatocytes. GS-CA2 PK parameters following intravenous and subcutaneous (SC) administration were assessed in rat and dog. GS-CA2 plasma concentrations were determined by HPLC-MS/MS.ResultsGS-CA2 showed potent and selective anti-HIV activity in MT-4 cells (EC50 = 0.1 nM; CC50 = 26.6 µM). In PBMCs, GS-CA2 displayed a mean EC50 of 0.05 nM (0.02–0.16 nM) against 23 HIV-1 clinical isolates representing all major subtypes. GS-CA2 is highly lipophilic (LogD of 3.7) with low aqueous solubility (<0.01 mg/mL) and low predicted clearance (CL) in human hepatocytes (0.01 L/h/kg). In rat and dog, GS-CA2 demonstrated low CL (<4% of liver blood flow). GS-CA2 PK in rat and dog exhibited sustained and slow drug release following a single SC administration. Factors including species, formulation, concentration, dose, volume, and number of injections were examined for the effect on systemic exposure over time. GS-CA2 plasma concentrations in dogs (Figure 1) were maintained above the human plasma protein binding-adjusted EC95 (4 nM) for the entire study duration (16 weeks).ConclusionGS-CA2 is a selective and first-in-class HIV capsid inhibitor with picomolar potency and potential to be clinically effective against a broad range of HIV-1 strains. In animals following a single SC injection, GS-CA2 maintained therapeutically relevant concentrations for >3 months. These nonclinical data support clinical development of GS-CA2 as a novel long-acting antiretroviral agent suitable for the treatment of HIV-1 infection. Disclosures J. Zheng, Gilead Sciences, Inc.: Employee, Salary. S. R. Yant, Gilead Sciences, Inc.: Employee, Salary. S. Ahmadyar, Gilead Sciences, Inc.: Employee, Salary. T. Y. Chan, Gilead Sciences, Inc.: Employee, Salary. A. Chiu, Gilead Sciences, Inc.: Employee, Salary. T. Cihlar, Gilead Sciences, Inc.: Employee, Salary. J. O. Link, Gilead Sciences, Inc.: Employee, Salary. B. Lu, Gilead Sciences, Inc.: Employee, Salary. J. Mwangi, Gilead Sciences, Inc.: Employee, Salary. W. Rowe, Gilead Sciences, Inc.: Employee, Salary. S. D. Schroeder, Gilead Sciences, Inc.: Employee, Salary. G. J. Stepan, Gilead Sciences, Inc.: Employee, Salary. K. W. Wang, Gilead Sciences, Inc.: Employee, Salary. R. Subramanian, Gilead Sciences, Inc.: Employee, Salary. W. C. Tse, Gilead Sciences, Inc.: Employee, Salary.

A New Mechanism of Resistance of Human Immunodeficiency Virus Type 2 to Integrase Inhibitors: A 5-Amino-Acid Insertion in the Integrase C-Terminal Domain.

Integrase strand transfer inhibitors (INSTIs) are crucial for the treatment of human immunodeficiency virus (HIV) type 2 infection, due to limited available therapeutic options. Recently, bictegravir has been approved for HIV-1, but no data are currently available for HIV-2. We assessed the phenotypic susceptibility of 12 HIV-2 clinical isolates, obtained from 2 antiretroviral-naive and 10 antiretroviral-experienced patients, to 5 INSTIs (bictegravir, cabotegravir, dolutegravir, elvitegravir, and raltegravir) at the virological failure of an INSTI-based regimen. The 50% inhibitory concentrations (IC50s) were determined. Phenotypic inhibitory quotients were determined using trough INSTI plasma concentrations. Wild-type viruses were susceptible to the 5 INSTIs, with IC50s in the nanomolar range. Bictegravir had a lower IC50 than the other INSTIs on those HIV-2 isolates bearing major, resistance-associated mutations (codons 143, 148, and 155). We identified a new resistance profile-a 5-amino-acid insertion at codon 231 of the HIV-2 integrase (231INS)-in 6 patients at the virological failure of a raltegravir-based regimen. Those patients had adequate raltegravir concentrations, but harbored multiresistant viruses with low genotypic susceptibility scores (median = 1.5). This insertion rendered isolates highly resistant to raltegravir and elvitegravir, and moderately resistant to dolutegravir and cabotegravir. Regarding bictegravir, 2 isolates remained susceptible and 2 had a slight increase in IC50 (3- to 5-fold change). Our results confirm the potency of INSTI on HIV-2 clinical isolates with wild-type integrase. In addition, we identified a new resistance pathway, 231INS, selected in antiretroviral-experienced patients with multiresistant HIV-2 viruses. This highlights the need of close follow-up of those patients initiating an INSTI-based regimen.

A defucosylated bispecific multivalent molecule exhibits broad HIV-1-neutralizing activity and enhanced antibody-dependent cellular cytotoxicity against reactivated HIV-1 latently infected cells.

Current treatments cannot completely eradicate HIV-1 owing to the presence of latently infected cells, which harbor transcriptionally silent HIV-1. However, defucosylated antibodies can readily kill latently infected cells after their activation to express envelope glycoprotein (Env) through antibody-dependent cellular cytotoxicity (ADCC). We herein aimed to test a defucosylated bispecific multivalent molecule consisting of domain-antibody and single-domain CD4, LSEVh-LS-F, for its HIV-1 neutralizing activity and ADCC against the reactivated latently infected cells, compared with the nondefucosylated molecule LSEVh-LS. LSEVh-LS-F's neutralizing activity against a panel of newly characterized Chinese HIV-1 clinical isolates was assessed by using TZM-bl-based and PBMC-based assays. LSEVh-LS-F-mediated ADCC in the presence of natural killer cells against cell lines that stably express Env proteins, HIV-1-infected cells and LRA-reactivated HIV-1 latent cells, was measured using a lactate dehydrogenase (LDH) cytotoxicity assay or flow cytometry. LSEVh-LS-F and LSEVh-LS were equally effective in neutralized infection of all HIV-1 isolates tested with IC50 and IC90 values 3∼4-fold lower than those of VRC01. LSEVh-LS-F was more effective in natural killer-mediated killing of HIV-1 Env-expressing cell lines, HIV-1-infected cells, latency reactivation agents-reactivated ACH2 cells and reactivated latently infected resting CD4+ T cell line as well as resting CD4+ T lymphocytes isolated from patients receiving HAART. LSEVh-LS-F exhibits broad HIV-1 neutralizing activity and enhanced ADCC against HIV-1-infected cells, reactivated latently infected cell lines and primary CD4+ T cells, thus being a promising candidate therapeutic for eradicating the HIV-1 reservoir.

CXCR7/ACKR3-targeting ligands interfere with X7 HIV-1 and HIV-2 entry and replication in human host cells

Chemokine receptors CCR5 and CXCR4 are considered the main coreceptors for initial HIV infection, replication and transmission, and subsequent AIDS progression. Over the years, other chemokine receptors, belonging to the family of G protein-coupled receptors, have also been identified as candidate coreceptors for HIV entry into human host cells. Amongst them, CXCR7, also known as atypical chemokine receptor 3 (ACKR3), was suggested as a coreceptor candidate capable of facilitating both HIV-1 and HIV-2 entry in vitro. In this study, a cellular infection model was established to further decipher the role of CXCR7 as an HIV coreceptor. Using this model, CXCR7-mediated viral entry was demonstrated for several clinical HIV isolates as well as laboratory strains. Of interest, the X4-tropic HIV-1 HE strain showed rapid adaptation towards CXCR7-mediated infection after continuous passaging on CD4- and CXCR7-expressing cells. Furthermore, we uncovered anti-CXCR7 monoclonal antibodies, small molecule CXCR7 inhibitors and the natural CXCR7 chemokine ligands as potent inhibitors of CXCR7 receptor-mediated HIV entry and replication. Even though the clinical relevance of CXCR7-mediated HIV infection remains poorly understood, our data suggest that divergent HIV-1 and HIV-2 strains can quickly adapt their coreceptor usage depending on the cellular environment, which warrants further investigation.

Mutation Frequencies in RNAi Targets in HIV-1 Genomes Obtained from Blood Plasma of Patients Receiving Anti-Retroviral Therapy

Gene therapy for AIDS based on RNA interference (RNAi) is currently looked upon as a promising alternative to conventional antiretroviral chemotherapy. The high variability of HIV-1 is the main challenge in developing new approaches to AIDS therapy. To date, about 18 million HIV-1 infected individuals receive antiretroviral therapy worldwide. As of 2017, about 44% of individuals with AIDS received antiretroviral therapy in Russia. Since the RNAs used for efficient RNAi and the corresponding targets in the viral transcript should be perfectly complementary to each other, it is necessary to continuously monitor the nucleotide sequences of clinical HIV-1 isolates obtained from blood and cells of naïve patients and patients receiving antiretroviral therapy. Comprehensive analysis of the mutation frequencies in the viral genome is only possible with deep sequencing approaches. The present paper reports on an analysis of the mutation frequencies in six 100 bp genome regions in clinical HIV-1 isolates obtained from blood plasma of four Russian AIDS patients who have been receiving antiretroviral therapy for several years. These regions contain efficient RNAi targets. The average frequencies of all possible transversions and transitions within the RNAi targets and in their proximity have been estimated. It has been demonstrated that reverse transcriptase inhibition decreases the frequency of a number of reverse mutations. It has been found that mutations in RNAi targets are rarer (5-75 times lower than the mutation frequency for different nucleotide substitutions) than in the adjacent sequences. Our findings speak in favor of these conservative targets for developing new approaches to gene therapy of AIDS.

Innate Immune Activation by cGMP-AMP Nanoparticles Leads to Potent and Long-Acting Antiretroviral Response against HIV-1

HIV-1 evades immune detection by the cGAS-STING cytosolic DNA-sensing pathway during acute infection. STING is a critical mediator of type I IFN production, and STING agonists such as cGMP-AMP (cGAMP) and other cyclic dinucleotides elicit potent immune and antitumor response. In this article, we show that administration of cGAMP, delivered by an ultra-pH-sensitive nanoparticle (NP; PC7A), in human PBMCs induces potent and long-acting antiretroviral response against several laboratory-adapted and clinical HIV-1 isolates. cGAMP-PC7A NP requires endocytosis for intracellular delivery and immune signaling activation. cGAMP-PC7A NP-induced protection is mediated through type I IFN signaling and requires monocytes in PBMCs. cGAMP-PC7A NPs also inhibit HIV-1 replication in HIV+ patient PBMCs after ex vivo reactivation. Because pattern recognition receptor agonists continue to show more clinical benefits than the traditional IFN therapy, our data present important evidence for potentially developing cGAMP or other STING agonists as a new class of immune-stimulating long-acting antiretroviral agents.

The HIV-1 integrase E157Q polymorphism per se does not alter susceptibility to raltegravir and dolutegravir in vitro.

: The HIV-1 integrase E157Q natural polymorphism has been reported to cause one case of raltegravir (RAL) and dolutegravir (DTG) failure. Six recombinant viruses were constructed containing integrase from clinical HIV-1 isolates found to harbour E157Q as the only integrase strand inhibitor (INSTI) resistance-related mutation. Phenotypic analysis showed that E157Q results in minimal changes in RAL and DTG susceptibility. Together with data retrieved from the Stanford HIV database, our results indicate that E157Q is not a relevant INSTI resistance mutation per se. The previously reported case of E157Q-based resistance must have resulted from combined as yet unidentified integrase polymorphisms.

GRL-09510, a Unique P2-Crown-Tetrahydrofuranylurethane -Containing HIV-1 Protease Inhibitor, Maintains Its Favorable Antiviral Activity against Highly-Drug-Resistant HIV-1 Variants in vitro

We report that GRL-09510, a novel HIV-1 protease inhibitor (PI) containing a newly-generated P2-crown-tetrahydrofuranylurethane (Crwn-THF), a P2′-methoxybenzene, and a sulfonamide isostere, is highly active against laboratory and primary clinical HIV-1 isolates (EC50: 0.0014–0.0028 μM) with minimal cytotoxicity (CC50: 39.0 μM). Similarly, GRL-09510 efficiently blocked the replication of HIV-1NL4-3 variants, which were capable of propagating at high-concentrations of atazanavir, lopinavir, and amprenavir (APV). GRL-09510 was also potent against multi-drug-resistant clinical HIV-1 variants and HIV-2ROD. Under the selection condition, where HIV-1NL4-3 rapidly acquired significant resistance to APV, an integrase inhibitor raltegravir, and a GRL-09510 congener (GRL-09610), no variants highly resistant against GRL-09510 emerged over long-term in vitro passage of the virus. Crystallographic analysis demonstrated that the Crwn-THF moiety of GRL-09510 forms strong hydrogen-bond-interactions with HIV-1 protease (PR) active-site amino acids and is bulkier with a larger contact surface, making greater van der Waals contacts with PR than the bis-THF moiety of darunavir. The present data demonstrate that GRL-09510 has favorable features for treating patients infected with wild-type and/or multi-drug-resistant HIV-1 variants, that the newly generated P2-Crwn-THF moiety confers highly desirable anti-HIV-1 potency. The use of the novel Crwn-THF moiety sheds lights in the design of novel PIs.

A Triazinone Derivative Inhibits HIV‐1 Replication by Interfering with Reverse Transcriptase Activity

A novel HIV-1 inhibitor, 6-(tert-butyl)-4-phenyl-4-(trifluoromethyl)-1H,3H-1,3,5-triazin-2-one (compound 1), was identified from a compound library screened for the ability to inhibit HIV-1 replication. EC50 values of compound 1 were found to range from 107.9 to 145.4 nm against primary HIV-1 clinical isolates. In in vitro assays, HIV-1 reverse transcriptase (RT) activity was inhibited by compound 1 with an EC50 of 4.3 μm. An assay for resistance to compound 1 selected a variant of HIV-1 with a RT mutation (RTL100I ); this frequently identified mutation confers mild resistance to non-nucleoside RT inhibitors (NNRTIs). A recombinant HIV-1 bearing RTL100I exhibited a 41-fold greater resistance to compound 1 than the wild-type virus. Compound 1 was also effective against HIV-1 with RTK103N , one of the major mutations that confers substantial resistance to NNRTIs. Computer-assisted docking simulations indicated that compound 1 binds to the RT NNRTI binding pocket in a manner similar to that of efavirenz; however, the putative compound 1 binding site is located further from RTK103 than that of efavirenz. Compound 1 is a novel NNRTI with a unique drug-resistance profile.

Preclinical evaluation of a mercaptobenzamide and its prodrug for NCp7-targeted inhibition of human immunodeficiency virus

Although the effective use of highly active antiretroviral therapy results in the suppression of virus production in infected individuals, it does not eliminate the infection and low level virus production in cells harboring virus in sanctuary sites. Thus, the continued search for new antiretroviral agents with unique and different mechanisms of HIV inhibition remains critical, and compounds that can reduce the level of virus production from cells already infected with HIV, as opposed to preventing de novo infection, would be of great benefit. A mercaptobenzamide (MDH-1-38) and its prodrug (NS1040) are being developed as potential therapeutic compounds targeting the zinc finger of HIV nucleocapsid. In the presence of esterase enzymes, NS1040 is designed to be converted to MDH-1-38 which has antiviral activity. While we presume that NS1040 is rapidly converted to MDH-1-38 in all experiments, the two compounds were tested side-by-side to determine whether the presence of a prodrug affects the antiviral activity or mechanism of action. The two compounds were evaluated against a panel of HIV-1 clinical isolates in human PBMCs and monocyte-macrophages and yielded EC50 values ranging from 0.7 to 13 μM with no toxicity up to 100 μM. MDH-1-38 and NS1040 remained equally active in human PBMCs in the presence of added serum proteins as well as against HIV-1 isolates resistant to reverse transcriptase, integrase or protease inhibitors. Cell-based and biochemical mechanism of antiviral action assays demonstrated MDH-1-38 and NS1040 were virucidal at concentrations of 15 and 50 μM, respectively. Cell to cell transmission of HIV in multiple passages was significantly reduced in CEM-SS and human PBMCs by reducing progeny virus infectivity at compound concentrations greater than 2 μM. The combination of either MDH-1-38 or NS1040 with other FDA-approved HIV drugs yielded additive to synergistic antiviral interactions with no evidence of antiviral antagonism or synergistic toxicity. Serial dose escalation was used in attempts to select for HIV strains resistant to MDH-1-38 and NS1040. Virus at several passages failed to replicate in cells treated at increased compound concentrations, which is consistent with the proposed mechanism of action of the virus inactivating compounds. Through 14 passages, resistance to the compounds has not been achieved. Most HIV inhibitors with mechanism of antiviral action targeting a viral protein would have selected for a drug resistant virus within 14 passages. These studies indicate that these NCp7-targeted compounds represent new potent anti-HIV drug candidates which could be effectively used in combination with all approved anti-HIV drugs.