Measuring Enzymatic HIV-1 Susceptibility to Two Reverse Transcriptase Inhibitors as a Rapid and Simple Approach to HIV-1 Drug-Resistance Testing

Simple and cost-effective approaches for HIV drug-resistance testing are highly desirable for managing increasingly expanding HIV-1 infected populations who initiate antiretroviral therapy (ART), particularly in resource-limited settings. Non-nucleoside reverse trancriptase inhibitor (NNRTI)-based regimens with an NRTI backbone containing lamivudine (3TC) or emtricitabine (FTC) are preferred first ART regimens. Failure with these drug combinations typically involves the selection of NNRTI- and/or 3TC/FTC- resistant viruses. Therefore, the availability of simple assays to measure both types of drug resistance is critical. We have developed a high throughput screening test for assessing enzymatic resistance of the HIV-1 RT in plasma to 3TC/FTC and NNRTIs. The test uses the sensitive “Amp-RT” assay with a newly-developed real-time PCR format to screen biochemically for drug resistance in single reactions containing either 3TC-triphosphate (3TC-TP) or nevirapine (NVP). Assay cut-offs were defined based on testing a large panel of subtype B and non-subtype B clinical samples with known genotypic profiles. Enzymatic 3TC resistance correlated well with the presence of M184I/V, and reduced NVP susceptibility was strongly associated with the presence of K103N, Y181C/I, Y188L, and G190A/Q. The sensitivity and specificity for detecting resistance were 97.0% and 96.0% in samples with M184V, and 97.4% and 96.2% for samples with NNRTI mutations, respectively. We further demonstrate the utility of an HIV capture method in plasma by using magnetic beads coated with CD44 antibody that eliminates the need for ultracentifugation. Thus our results support the use of this simple approach for distinguishing WT from NNRTI- or 3TC/FTC-resistant viruses in clinical samples. This enzymatic testing is subtype-independent and can assist in the clinical management of diverse populations particularly in resource-limited settings

Prevention of Rectal SHIV Transmission in Macaques by Daily or Intermittent Prophylaxis with Emtricitabine and Tenofovir

Using a repeat-exposure macaque model, Walid Heneine and colleagues find that pre-exposure prophylaxis with combination antiretroviral drugs provides protection against rectal challenge with a SHIV virus

Genital inflammation undermines the effectiveness of tenofovir gel in preventing HIV acquisition in women.

CAPRISA, 2018.Abstract available in pdf

Centrality evolution of the charged-particle pseudorapidity density over a broad pseudorapidity range in Pb-Pb collisions at root s(NN)=2.76TeV

Peer reviewe

The Fitness Cost of Mutations Associated with Human Immunodeficiency Virus Type 1 Drug Resistance Is Modulated by Mutational Interactions

It is generally accepted that the fitness cost of resistance mutations plays a role in the persistence of transmitted drug-resistant human immunodeficiency virus type 1 and that mutations that confer a high fitness cost are less able to persist in the absence of drug pressure. Here, we show that the fitness cost of reverse transcriptase (RT) mutations can vary within a 72-fold range. We also demonstrate that the fitness cost of M184V and K70R can be decreased or enhanced by other resistance mutations such as D67N and K219Q. We conclude that the persistence of transmitted RT mutants might range widely on the basis of fitness and that the modulation of fitness cost by mutational interactions will be a critical determinant of persistence

Transmitted Human Immunodeficiency Virus Type 1 Carrying the D67N or K219Q/E Mutation Evolves Rapidly to Zidovudine Resistance In Vitro and Shows a High Replicative Fitness in the Presence of Zidovudine

Drug-naive patients infected with drug-resistant human immunodeficiency virus type 1 (HIV-1) who initiate antiretroviral therapy show a shorter time to virologic failure than patients infected with wild-type (WT) viruses. Resistance-related HIV genotypes not commonly seen in treated patients, which likely result from reversion or loss of primary resistance mutations, have also been recognized in drug-naive persons. Little work has been done to characterize the patterns of mutations in these viruses and the frequency of occurrence, their association with phenotypic resistance, and their effect on fitness and evolution of resistance. Through the analysis of resistance mutations in 1082 newly diagnosed antiretroviral-naive persons from the United States, we found that 35 of 48 (72.9%) persons infected with HIV-1 containing thymidine analog mutations (TAMs) had viruses that lacked a primary mutation (T215Y/F, K70R, or Q151M). Of these viruses, 9 (25.7%) had only secondary TAMs (D67N, K219Q, M41L, or F77L), and all were found to be sensitive to zidovudine (AZT) and other drugs. To assess the impact of secondary TAMs on the evolution of AZT resistance, we generated recombinant viruses from cloned plasma-derived reverse transcriptase sequences. Two viruses had D67N, three had D67N and K219Q/E, and three were WT. Four site-directed mutants with D67N, K219Q, K219E, and D67N/K219Q were also made in HIV-1(HXB2). In vitro selection of AZT resistance showed that viruses with D67N and/or K219Q/E acquired AZT resistance mutations more rapidly than WT viruses (36 days compared to 54 days; P = 0.003). To investigate the factors associated with the rapid selection of AZT mutations in these viruses, we evaluated fitness differences among HXB2(WT) and HXB2(D67N) or HXB2(D67N/K219Q) in the presence of AZT. Both HXB2(D67N/K219Q) and HXB2(D67N) were more fit than HXB2(WT) in the presence of either low or high AZT concentrations, likely reflecting low-level resistance to AZT that is not detectable by phenotypic testing. In the absence of AZT, the fitness cost conferred by D67N or K219Q was modest. Our results demonstrate that viruses with unique patterns of TAMs, including D67N and/or K219Q/E, are commonly found among newly diagnosed persons and illustrate the expanding diversity of revertant viruses in this population. The modest fitness cost conferred by D67N and K219Q supports persistence of these mutants in the untreated population and highlights the potential for secondary transmission. The faster evolution of these mutants toward AZT resistance is consistent with the higher viral fitness in the presence of AZT and shows that these viruses are phenotypically different from WT HIV-1. Our study emphasizes the need for clinical studies to better define the impact of these mutants on treatment responses and evolution of resistance

Evidence of a Role for the Q151L Mutation and the Viral Background in Development of Multiple Dideoxynucleoside-Resistant Human Immunodeficiency Virus Type 1

The majority of human immunodeficiency virus type 1 (HIV-1)-infected patients treated with zidovudine (AZT) plus zalcitabine (ddC) and didanosine (ddI) develop AZT resistance mediated by mutations such as T215Y and M41L. Only a small proportion of patients develop multiple dideoxynucleoside resistance (MDNR) mediated by the Q151M mutation. To gain insight into the factors responsible for the low frequency of selection of Q151M, we evaluated the replication capabilities of recombinant viruses carrying two possible intermediates (151L or 151K) of the Q151M mutation generated in different reverse transcriptase (RT) genetic backgrounds. The 151L and 151K mutations were introduced by site-directed mutagenesis in RTs from two patient-derived HIV-1 isolates that had either wild type (WT) Q or the Q151M (posttreatment isolate) mutation. For comparison, both mutations were also introduced in a laboratory-adapted HIV-1 strain (HIV-1(HXB2)). Analysis of replication capabilities showed that both 151L and 151K were lethal in RT genetic backgrounds of the WT isolate and in HIV-1(HXB2). In contrast, 151L but not 151K allowed virus replication in RT backgrounds of the posttreatment isolate. Three mutations (V35I, S68G, and I178M) were present in the RT background of the posttreatment isolate but not in the WT isolate. Introduction of S68G in the RT of both the WT isolate and HIV-1(HXB2) partially restored replication capacity of recombinants carrying the 151L mutation. The S68G mutation alone did not confer a significant replicative disadvantage in WT viruses. Like HIV-1(151M), HIV-1(151L) RT was found to have six- to eightfold resistance to AZT-triphosphate (TP), ddA-TP, and ddC-TP, indicating an MDNR phenotype. However, HIV-1(151L) was found to be less fit than HIV-1(151M), which may explain the preferential selection of HIV-1(151M) observed in vivo. The demonstrated ability of HIV-1(151L/68G) to replicate and the associated MDNR suggest that 151L is a potential intermediate of Q151M. The dependence of HIV-1(151L) on other mutations, such as S68G, for replication may explain the low frequency of the Q151M-mediated pathway of resistance

Tenofovir diphosphate concentrations and prophylactic effect in a macaque model of rectal simian HIV transmission

ObjectivesThis study evaluated the relationship between intracellular tenofovir diphosphate concentrations in peripheral blood mononuclear cells and prophylactic efficacy in a macaque model for HIV pre-exposure prophylaxis (PrEP).MethodsMacaques were challenged with simian HIV (SHIV) via rectal inoculation once weekly for up to 14 weeks. A control group (n=34) received no drug, a second group (n=6) received oral tenofovir disoproxil fumarate/emtricitabine 3 days before each virus challenge and a third group (n=6) received the same dosing plus another dose 2 h after virus challenge. PBMCs were collected just before each weekly virus challenge. The relationship between tenofovir diphosphate in PBMCs and prophylactic efficacy was assessed with a Cox proportional hazards model.ResultsThe percentages of animals infected in the control, one-dose and two-dose groups were 97, 83 and 17, respectively. The mean (SD) steady-state tenofovir diphosphate concentration (fmol/10(6) cells) was 15.8 (7.6) in the one-dose group and 30.7 (10.1) in the two-dose group. Each 5 fmol tenofovir diphosphate/10(6) cells was associated with a 40% (95% CI 17%-56%) reduction in risk of SHIV acquisition, P=0.002. The tenofovir diphosphate concentration associated with a 90% reduction in risk (EC90) was 22.6 fmol/10(6) cells (95% CI 13.8-60.8).ConclusionsThe prophylactic EC90 for tenofovir diphosphate identified in macaques exposed rectally compares well with the EC90 previously identified in men who have sex with men (MSM; 16 fmol/10(6) cells, 95% CI 3-28). These results highlight the relevance of this model to inform human PrEP studies of oral tenofovir disoproxil fumarate/emtricitabine for MSM

Antiretroviral Drug Activity in Macaques Infected during Pre-Exposure Prophylaxis Has a Transient Effect on Cell-Associated SHIV DNA Reservoirs

<div><p>Background</p><p>Pre-exposure prophylaxis (PrEP) with emtricitabine and tenofovir disoproxil fumarate (FTC/TDF) is a novel HIV prevention strategy. Suboptimal PrEP adherence and HIV infection creates an opportunity for continued antiretroviral drug activity during undiagnosed infection. We previously showed that macaques infected with SHIV during PrEP with FTC/TDF display reduced acute plasma viremias and limited virus diversity. We investigated the effect of PrEP on acute SHIV DNA dynamics and on the size of the persistent virus reservoir in lymphoid tissues.</p><p>Design</p><p>Cell-associated SHIV DNA levels in PBMCs were measured in 8 macaques infected during PrEP with FTC/TDF or single-agent TAF and was compared to those seen in untreated infections (n = 10). PrEP breakthrough infections continued treatment with 1–2 weekly drug doses to model suboptimal drug exposure during undiagnosed HIV infection in humans. SHIV DNA was also measured in lymphoid tissues collected from FTC/TDF PrEP breakthroughs after 1 year of infection.</p><p>Results</p><p>Compared to untreated controls, PrEP infections had reduced plasma RNA viremias both at peak and throughout weeks 1–12 (p<0.005). SHIV DNA levels were also reduced at peak and during the first 12 weeks of infection (p<0.043) but not throughout weeks 12–20. At 1 year, SHIV DNA reservoirs in lymphoid tissues were similar in size among macaques that received PrEP or placebo.</p><p>Conclusions</p><p>Antiviral drug activity due to PrEP limits acute SHIV replication but has only a transient effect on cell-associated SHIV DNA levels. Our model suggests that suboptimal drug exposure in persons that are taking PrEP and become infected with HIV may not be sufficient to reduce the pool of HIV-infected cells, and that treatment intensification may be needed to sustain potential virological benefits from the PrEP regimen.</p></div