Reverse Transcriptase Inhibitor-resistant Viruses Research Articles

Second-generation mother-to-child HIV transmission in South Africa is characterized by poor outcomes.

The worldwide incidence of pregnancy for women living with perinatal HIV infection is increasing. Subsequently, there is growing risk of second-generation mother-to-child HIV transmission. The infant clinical outcomes for such a phenomenon have yet to be described. As part of a wider observational study in KwaZulu-Natal, South Africa, six infants with in-utero HIV infection were identified as being born to mothers with perinatal HIV infection. Blood results and clinical data were collected in the first 3 years of life. In two cases, sample availability allowed confirmation by phylogenetic analysis of grandmother-to-mother-to-child HIV transmission. Outcomes were poor in all six cases. All six mothers had difficulty administering twice daily combination antiretroviral therapy to their infants due to difficulties with acceptance, disclosure, poor health and being themselves long-term nonprogressors. Nonnucleoside reverse transcriptase inhibitor-resistant virus was detected in all mothers tested. None of the infants maintained suppression of viraemia on combination antiretroviral therapy. One infant died, and another was lost to follow-up. As the numbers of second-generation mother-to-child transmissions increase, it is important to highlight that this mother-infant dyad represents an extremely vulnerable group. In order for them to survive and thrive, these infants' mothers require their specific needs to be addressed and given intensive support.

Antiretroviral potency of 4'-ethnyl-2'-fluoro-2'-deoxyadenosine, tenofovir alafenamide and second-generation NNRTIs across diverse HIV-1 subtypes.

4'-Ethnyl-2'-fluoro-2'-deoxyadenosine (EFdA) is a novel translocation-defective reverse transcriptase inhibitor. We investigated the virological and biochemical inhibitory potentials of EFdA against a broad spectrum of subtype-specific chimeric viruses and compared it with tenofovir alafenamide, nevirapine, efavirenz, rilpivirine and etravirine. pNL4.3 chimeric viruses encoding gag-pol from treatment-naive patients (n = 24) and therapy-failure patients (n = 3) and a panel of reverse transcriptase inhibitor-resistant strains (n = 7) were used to compare the potency of reverse transcriptase inhibitor drugs. The phenotypic drug susceptibility assay was performed using TZM-bl cells. In vitro inhibition assays were done using patient-derived reverse transcriptase. IC50 values of NNRTIs were calculated using a PicoGreen-based spectrophotometric assay. Steady-state kinetics were used to determine the apparent binding affinity (Km.dNTP) of triphosphate form of EFdA (EFdA-TP) and dATP. Among the chimeric treatment-naive viruses, EFdA had an ex vivo antiretroviral activity [median (IQR) EC50 = 1.4 nM (0.6-2.1 nM)] comparable to that of tenofovir alafenamide [1.6 nM (0.5-3.6 nM)]. Subtype-specific differences were found for etravirine (P = 0.004) and rilpivirine (P = 0.017), where HIV-1C had the highest EC50 values. EFdA had a greater comparative efficiency [calculated by dividing the efficiency of monophosphate form of EFdA (EFdA-MP) incorporation (kcat.EFdA-TP/Km.EFdA-TP) over the efficiency of dATP incorporation (kcat.dATP/Km.dATP)] compared with the natural substrate dATP, with a fold change of between 1.6 and 3.2. Ex vivo analysis on reverse transcriptase inhibitor-resistant strains showed EFdA to have a higher potency. Despite the presence of rilpivirine DRMs, some non-B strains showed hypersusceptibility to rilpivirine. Our combined virological and biochemical data suggest that EFdA inhibits both WT and reverse transcriptase inhibitor-resistant viruses efficiently in a subtype-independent manner. In contrast, HIV-1C is least susceptible to etravirine and rilpivirine.

IFN-λ Inhibits Drug-Resistant HIV Infection of Macrophages.

Type III interferons (IFN-λs) have been demonstrated to inhibit a number of viruses, including HIV. Here, we further examined the anti-HIV effect of IFN-λs in macrophages. We found that IFN-λs synergistically enhanced anti-HIV activity of antiretrovirals [azidothymidine (AZT), efavirenz, indinavir, and enfuvirtide] in infected macrophages. Importantly, IFN-λs could suppress HIV infection of macrophages with the drug-resistant strains, including AZT-resistant virus (A012) and reverse transcriptase inhibitor-resistant virus (TC49). Mechanistically, IFN-λs were able to induce the expression of several important anti-HIV cellular factors, including myxovirus resistance 2 (Mx2), a newly identified HIV post-entry inhibitor and tetherin, a restriction factor that blocks HIV release from infected cells. These observations provide additional evidence to support the potential use of IFN-λs as therapeutics agents for the treatment of HIV infection.

The M184I/V and K65R nucleoside resistance mutations in HIV-1 prevent the emergence of resistance mutations against dolutegravir.

Recommended treatments for newly diagnosed HIV-positive individuals now focus on the integrase strand transfer inhibitors, raltegravir (RAL), elvitegravir (EVG) and dolutegravir (DTG). In treatment-naive individuals, cases of RAL-based and EVG-based virological failure, although rare, are associated with the occurrence of resistance mutations in integrase and/or reverse transcriptase coding sequences. In such cases, common resistance substitutions in reverse transcriptase that were associated with nucleos(t)ide reverse transcriptase inhibitors included M184I/V and K65R and these occurred together with various mutations in integrase. In some instances, these mutations in reverse transcriptase preceded the emergence of mutations in integrase. In contrast, no resistance substitutions in either integrase or reverse transcriptase have been observed to date in viruses isolated from treatment-naive individuals who experienced treatment failure with DTG-based regimens. The objective of this study was to determine the effects of the M184I/V and K65R substitutions in reverse transcriptase on the ability of HIV-1 to become resistant against RAL, EVG or DTG. We performed tissue culture selection experiments using reverse transcriptase inhibitor-resistant viruses containing resistance substitutions at positions K65R, M184I or M184V in the presence of increasing concentrations of RAL, EVG or DTG and monitored changes in integrase sequences by genotyping. Selections using EVG and RAL led to the emergence of resistance mutations in integrase. In contrast, only the wild-type virus was able to acquire resistance mutations for DTG. Resistance mutations against nucleos(t)ide reverse transcriptase inhibitors antagonized the development of HIV-1 resistance against DTG but not RAL or EVG.

Predicted levels of HIV drug resistance

There is concern that the expansion of antiretroviral roll-out may impact future drug resistance levels and hence compromise the benefits of antiretroviral therapy (ART) at an individual and population level. We aimed to predict future drug resistance in South Africa and its long-term effects. The previously validated HIV Synthesis model was calibrated to South Africa. Resistance was modeled at the level of single mutations, transmission potential, persistence, and effect on drug activity. We estimate 652 000 people (90% uncertainty range: 543 000-744 000) are living with nonnucleoside reverse transcriptase inhibitor (NNRTIs)-resistant virus in South Africa, 275 000 in majority virus [Non-nucleoside reverse transcriptase inhibitor resistant virus present in majority virus (NRMV)] with an unsuppressed viral load. If current diagnosis and retention in care and eligibility criteria are maintained, in 20 years' time HIV incidence is projected to have declined by 22% (95% confidence interval, CI -23 to -21%), and the number of people carrying NNRTI resistance to be 2.9-fold higher. If enhancements in diagnosis and retention in care occur, and ART is initiated at CD4 cell count less than 500 cells/μl, HIV incidence is projected to decline by 36% (95% CI: -37 to -36%) and the number of people with NNRTI resistance to be 4.1-fold higher than currently. Prevalence of people with viral load more than 500 copies/ml carrying NRMV is not projected to differ markedly according to future ART initiation policy, given the current level of diagnosis and retention are maintained. Prevalence of resistance is projected to increase substantially. However, introduction of policies to increase ART coverage is not expected to lead to appreciably higher prevalence of HIV-positive people with resistance and viral load more than 500 copies/ml. Concern over resistance should not stop expansion of treatment availability.

Extracts of the medicinal herb Sanguisorba officinalis inhibit the entry of human immunodeficiency virus-1

Highly active antiretroviral therapy (HAART) has been successful in reducing human immunodeficiency virus (HIV)-1-associated morbidity and mortality since its introduction in 1996. However, it fails to eradicate HIV-1 infection. The high cost of life-long highly active antiretroviral therapy and the emergence of drug resistance among HIV-1-infected individuals have brought renewed pressure for the discovery of novel antivirals and alternative medicines. Traditional Chinese medicine (TCM) is a complementary and alternative medicine, and serves as a rich resource for new drug development. Despite the almost 100 plant-derived compounds that are in clinical trials, few target HIV-1 infection. In this study, we discovered that Sanguisorba officinalis extract (SOE) has anti-HIV-1 properties. Using a cell-based assay and single-cycle luciferase reporter viruses pseudotyped with envelopes from HIV-1 or control viruses, we found that SOE exhibited significant inhibitory ability against both CCR5 and CXCR4 tropic HIV-1 (ADA and HXB2), with respective IC50 values of 1.91 ± 0.16 μg/mL and 3.70 ± 0.53 μg/mL. SOE also inhibited simian immunodeficiency virus infection but failed to block vesicular stomatitis virus, severe acute respiratory syndrome coronavirus, and influenza H5N1 pseudoviruses. Furthermore, we showed that SOE had no effect on postentry events of HIV-1 replication. Because SOE pretreatment with the virus but not with cell lines expressing viral receptors showed the maximal inhibitory activity, we can state that SOE probably blocks entry by acting on the viral envelope directly. In addition, SOE was able to inhibit reverse transcriptase inhibitor resistant viruses (K103N, Y188L, and K103N/Y188L/G190A) and a protease inhibitor resistant strain (PI-2840). Our findings demonstrate SOE as a novel and specific entry inhibitor, which sheds light on the discovery of anti-HIV-1 drugs from traditional herbal medicines.

Antiretroviral Drug Resistance Surveillance among Treatment-Naive Human Immunodeficiency Virus Type 1-Infected Individuals in Angola: Evidence for Low Level of Transmitted Drug Resistance

The prevalence of transmitted human immunodeficiency virus type 1 drug resistance in Angola in 2001 in 196 untreated patients was investigated. All subtypes were detected, along with unclassifiable and complex recombinant strains. Numerous new polymorphisms were identified in the reverse transcriptase and protease. Two (1.6%) unrelated patients harbored nucleoside reverse transcriptase inhibitor- and nonnucleoside reverse transcriptase inhibitor-resistant viruses (mutations: M41L, D67N, M184V, L210W, T215Y or T215F, and K103N). Continued surveillance of drug resistance is required for maximization of ART efficacy in Angola.

Stable frequency of HIV-1 transmitted drug resistance in patients at the time of primary infection over 1996–2006 in France

Transmission of drug-resistant variants is influenced by several factors, including the HIV-1 RNA levels in HIV-1-infected patients. Our study describes the transmitted drug-resistant virus among 1446 French patients diagnosed at the time of primary infection and included from 1996 to 2006 along with the proportion of chronically infected treated patients in the French Hospital Database on HIV (FHDH). Genotypic resistance tests were performed at the time of primary infection. The proportion of patients with viral load <500 copies/ml among treated patients, enrolled in the FHDH, was calculated. Over 1996-2006, the proportion of transmitted resistant viruses to at least one antiretroviral was estimated as 10.9%. When considering class resistance, there was an increase in transmission of nonnucleoside reverse transcriptase inhibitor-resistant virus from 0.6% in 1996-1998 to 4.4% in 1999 (P = 0.034), whereas no change was evidenced for either nucleoside/nucleotide reverse transcriptase inhibitor or protease inhibitor resistance. In the FHDH, the proportion of patients receiving combination antiretroviral therapy (cart) increased from 27.7% in 1996 to 81.4% in 2006 and the proportion of viral load <500 copies/ml in treated patients increased from 17.0% in 1996 to 85.3% in 2006. Phylogenetic analysis revealed that 25.5% of patients harboured HIV-1-non-B virus at the time of primary infection in 2005-2006 compared to 10% in 1996-1998. In this large study of patients at the time of primary infection, the frequency of acquired resistant virus was stable over time, over 5% for nucleoside/nucleotide reverse transcriptase inhibitor and nonnucleoside reverse transcriptase inhibitor. One explanation for this stability may be the increasing number of treated patients in virological success.

Analysis of Near Full-Length Genomic Sequences of Drug-Resistant HIV-1 Spreading among Therapy-Naïve Individuals in Nagoya, Japan: Amino Acid Mutations Associated with Viral Replication Activity

We analyzed a total of 12 near full-length genomes of drug-resistant HIV-1 spreading among therapy-naïve individuals in Nagoya, Japan. Genomes comprised seven protease inhibitor (PI)-resistant viruses possessing an M46I (n = 6) or L90M mutation (n = 1) and five non-nucleoside reverse transcriptase inhibitor-resistant viruses possessing a K103N mutation. All 12 viruses conserved both an H87Q mutation in the cyclophilin A-binding site of Gag p24 (capsid) and a T23N mutation in the cysteine-rich domain of Tat protein. PI-resistant viruses commonly possessed two cleavage site mutations in the p6(Pol)/protease of Pol polyprotein (F48L in p6(Pol)) and the anchor/core domains of Nef protein (L57V). These amino acid mutations represent candidates for enhancing replication activity of drug-resistant viruses and supporting expansion of such viruses in therapy-naïve individuals.

Neurokinin-1 Receptor Antagonist (Aprepitant) Inhibits Drug-Resistant HIV-1 Infection of Macrophages in vitro

Despite the success of antiretroviral therapy in controlling HIV replication, treatment failure may ultimately occur in more than 50% of the individuals on antiretroviral therapy. Cellular targets offer an attractive alternative, as it may be more difficult for HIV to develop resistance to alternative cellular inhibitory pathways. We have previously shown that CP-96,345, a neurokinin-1 receptor (NK-1R) antagonist, inhibits HIV-1 infection of macrophages in vitro by downregulating CCR5 expression (Lai JP, Ho WZ, Zhan GX, Yi Y, Collman RG, Douglas SD 2001). We have now investigated the effects of a Food and Drug Administration (FDA)-approved NK-1R antagonist, aprepitant (Emend), on HIV infection of macrophages in an in vitro system. Aprepitant is in clinical use for the prevention of nausea and vomiting associated with cancer chemotherapy or following surgical procedures. Monocytes isolated from healthy donors were cultured for 7 days and then treated with or without aprepitant (10(-6) M) for 2 h, followed by HIV infection with drug-resistant strains for 2 h. Untreated and HIV-infected macrophages were used as controls. Culture supernatants were harvested for p24 enzyme-linked immunosorbent assay (ELISA) or HIV reverse transcriptase (RT) activity at different time points after infection. R5X4 tropic and AZT-resistant strains (R5X4 tropic: A012 and A018) and RT inhibitor-resistant HIV strains (R5 tropic: TC60 and TC49) were used for infection. Aprepitant suppressed HIV Bal infection of macrophages. Treatment with aprepitant (10(-6) M) inhibited infection of macrophages with the AZT-resistant viruses (A018, A012) by 0.7 log(10). Aprepitant also suppressed infection of macrophages with RT inhibitor-resistant virus (TC 49 and TC 60) by 0.5 log(10). Furthermore, aprepitant significantly enhanced the anti-HIV activity of antiretrovirals (AZT, Efavirenz, and Indinavir) in HIV Bal-infected macrophages, and aprepitant inhibited CCR5 expression on macrophages, ranging from 50.5 to 29.6%. Donor heterogeneity was observed in antiviral activity and CCR5 receptor expression. Aprepitant is active against HIV drug-resistant isolates and enhances the anti-HIV activity of the antiretrovirals. Aprepitant downregulates CCR5 expression on macrophages. NK-1R antagonists merit further investigation as potential HIV therapeutic and immunomodulatory agents.

The role of genotypic heterogeneity in wild type virus populations on the selection of nonnucleoside reverse transcriptase inhibitor-resistant viruses

Virus populations were selected in cell culture using two widely used protocols in order to evaluate the role of selection methodology on the genotype and phenotype of nonnucleoside reverse transcriptase inhibitor resistant viruses. Selection was performed by serial passage of virus in the presence of gradually increasing concentrations of antiviral compound or passage in the presence of a constant high concentration of compound. Using the CEM-SS cell line, the IIIB strain of HIV-1, and identical nonnucleoside reverse transcriptase inhibitors, resistant viruses were obtained and their phenotypic and genotypic properties were defined. Resistant virus populations containing the Y181C amino acid change in the reverse transcriptase were predominantly selected with each of the tested compounds. Several of the compounds selected secondary amino acid changes using both methods. A comparison of the resistant viruses selected in our laboratory using each of the two protocols with viruses reported by a second laboratory employing one of the two methods suggests that genotypic differences in the selected virus isolates may most likely result from variation in the genetic composition of the respective wild type virus pools, rather than the specific selection methodology employed. These results imply that HIV may select a wide variety of amino acid changes to avoid the inhibitory effects of the nonnucleoside reverse transcriptase inhibitors and the selection of compounds for clinical use in combination with agents possessing non-overlapping resistance phenotypes will require evaluation of the agents against virus isolates possessing each of the mutations known to confer drug resistance.

Comparative anti-HIV evaluation of diverse HIV-1-specific reverse transcriptase inhibitor-resistant virus isolates demonstrates the existence of distinct phenotypic subgroups

We have biologically and biochemically evaluated a structurally diverse group of HIV-1-specific reverse transcriptase (RT) inhibitors and determined that the members of this class share many common properties. These include reproducible and selective antiviral activity against a panel of biologically distinct laboratory and clinical strains of HIV-1, activity against HIV-1 in a wide variety of culture and fresh human cells, and potent inhibition of HIV-1 RT when evaluated using a heteropolymeric ribosomal RNA template assay. Each of the HIV-1-specific compounds was capable of inhibiting HIV replication when challenged at high m.o.i., further distinguishing them from the nucleoside analogs 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxycytidine (ddC). When tested in combination with AZT, each of the HIV-1-specific compounds synergistically inhibited the replication of HIV-1. HIV-1 isolates resistant to different HIV-1-specific inhibitors exhibited heterogeneous patterns of cross-resistance to other members of this pharmacologic class. Four distinct phenotypic classes have been defined through the use of drug-resistant virus isolates which derive from distinct mutations in the RT. These results indicate that the various subgroups of HIV-1-specific inhibitors interact differently with HIV-1 RT, suggesting important potential implications for drug combination therapeutic strategies.