Rapid Screening of Phenotypic Resistance to Nevirapine by Direct Analysis of HIV Type 1 Reverse Transcriptase Activity in Plasma

Lerma, J. Gerardo García; Yamamoto, Shinji; Switzer, William M.; Havlir, Diane V.; Folks, Thomas M.; Richman, Douglas D.; Heneine, Walid

doi:10.1089/088922299310287

Cited by 16 publications

(16 citation statements)

References 32 publications

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“…One Amp-RT assay with 50 M nevirapine was used in this screening. This concentration of nevirapine completely inhibits WT HIV-1 RT activity (IC 50 , 4 M) (49). In contrast to what was observed with WT HIV-1, no inhibition of PERV RT was observed in this test, indicating lack of activity of nevirapine on PERV RT (data not shown).…”

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confidence: 57%

“…The susceptibility of PERV RT to the RTIs was evaluated enzymatically in the Amp-RT assay to determine the 50 and 90% inhibitory concentrations (IC 50 s and IC 90 s, respectively) (20,52). Results obtained in triplicate were compared with those determined from reference wild-type (WT) and drug-resistant HIV-1 isolates using methodologies previously described (15,49). The RTIs tested were nevirapine; the triphosphorylated nucleoside analogs of zidovudine (AZT-TP), lamivudine (2Ј,3Ј-deoxy-3Ј-thiacytidine; 3TC-TP), zalcitabine (dideoxycytosine Two sources of PERV were used, the first from culture supernatant of a porcine embryonic kidney cell line (PK15) and the second from PERV-infected human embryonic kidney cell line 293 (PERV-293) (33).…”

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confidence: 99%

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Susceptibility of the Porcine Endogenous Retrovirus to Reverse Transcriptase and Protease Inhibitors

Qari

Magre

Garcı́a-Lerma

et al. 2001

J Virol

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Porcine xenografts may offer a solution to the shortage of human donor allografts. However, all pigs contain the porcine endogenous retrovirus (PERV), raising concerns regarding the transmission of PERV and the possible development of disease in xenotransplant recipients. We evaluated 11 antiretroviral drugs licensed for human immunodeficiency virus type 1 (HIV-1) therapy for their activities against PERV to assess their potential for clinical use. Fifty and 90% inhibitory concentrations (IC 50 s and IC 90 s, respectively) of five nucleoside reverse transcriptase inhibitors (RTIs) were determined enzymatically for PERV and for wild-type (WT) and RTI-resistant HIV-1 reference isolates. In a comparison of IC 50 s, the susceptibilities of PERV RT to lamivudine, stavudine, didanosine, zalcitabine, and zidovudine were reduced >20-fold, 26-fold, 6-fold, 4-fold, and 3-fold, respectively, compared to those of WT HIV-1. PERV was also resistant to nevirapine. Tissue culturebased, single-round infection assays using replication-competent virus confirmed the relative sensitivity of PERV to zidovudine and its resistance to all other RTIs. A Gag polyprotein-processing inhibition assay was developed and used to assess the activities of protease inhibitors against PERV. No inhibition of PERV protease was seen with saquinavir, ritonavir, indinavir, nelfinavir, or amprenavir at concentrations >200-fold the IC 50 s for WT HIV-1. Thus, following screening of many antiretroviral agents, our findings support only the potential clinical use of zidovudine.

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confidence: 57%

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confidence: 99%

Susceptibility of the Porcine Endogenous Retrovirus to Reverse Transcriptase and Protease Inhibitors

Qari

Magre

Garcı́a-Lerma

et al. 2001

J Virol

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“…Similarly, no development of R5 viral resistance was observed following treatment with single IV doses of PRO 140, which produced significant antiviral effects for two weeks (16). In contrast, phenotypic and/or genotypic resistance has been reported within two weeks of monotherapy with some non-nucleoside reverse transcriptase inhibitors (32–35). The PRO 140 results are especially notable in that monotherapy was followed by slow washout of drug.…”

Section: Discussionmentioning

confidence: 99%

Anti–HIV‐1 Activity of Weekly or Biweekly Treatment with Subcutaneous PRO 140, a CCR5 Monoclonal Antibody

Jacobson¹,

Thompson²,

Lalezari³

et al. 2010

J INFECT DIS

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Background PRO 140 is a humanized CCR5 monoclonal antibody that has demonstrated potent antiviral activity when administered intravenously to adults infected with CCR5-tropic (R5) human immunodeficiency virus type 1 (HIV-1). This study is the first to evaluate subcutaneous (SC) administration. Methods A randomized, double-blind, placebo-controlled study was conducted in 44 subjects with HIV-1 RNA > 5,000 copies/mL, CD4+ cells > 300/μL, no antiretroviral therapy for ≥12 weeks, and only R5 HIV-1 detectable. Subjects received placebo, 162mg PRO 140, or 324mg PRO 140 weekly for three weeks or 324mg PRO 140 every other week for two doses by SC infusion. Subjects were monitored for 58 days for safety, antiviral effects and PRO 140 serum concentrations. Results SC PRO 140 demonstrated potent and prolonged antiretroviral activity. Mean log10 reductions in HIV-1 RNA were 0.23, 0.99 (p=0.0093), 1.37 (p=0.0001), and 1.65 (p<0.0001) for the placebo, 162mg weekly, 324mg biweekly and 324mg weekly dose groups, respectively. Viral loads remained suppressed between successive doses. Treatment was generally well tolerated. Conclusions This trial is the first to demonstrate proof of concept for a mAb administered subcutaneously in HIV-1 infected subjects. SC PRO 140 offers the potential for significant dose-dependent HIV-1 RNA suppression and infrequent patient self-administration. Trial registration Clinicaltrials.gov register NCT00642707

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“…Like other RT assays that are broadly reactive on all retroviruses, Amp-RT can detect generically RT activity from diverse retrovirus groups including HIV-1 non-subtype B and HIV-2 [15], [16]. We have previously demonstrated that this technology can also be used for HIV drug-resistance testing and have shown that biochemical detection of NNRTI and 3TC resistance by this method correlates well with genotypic and replication-based phenotypic assays [9], [17]. Here, we describe the development of improved Amp-RT assays that use real-time PCR detection and simplified virus capture from plasma.…”

Section: Introductionmentioning

confidence: 95%

“…EFV and NVP have overlapping resistance profiles conferred by a number of mutations. K103N and Y188L confer high-level resistance to NVP and EFV, while Y181C/I/V and G190A mainly reduce susceptibility to NVP [7]–[9]. Virologic failure with NNRTI-containing regimens generally associates with the emergence of NNRTI- and/or 3TC/FTC-resistant viruses [10], [11].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2011

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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.

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Rapid Screening of Phenotypic Resistance to Nevirapine by Direct Analysis of HIV Type 1 Reverse Transcriptase Activity in Plasma

Cited by 16 publications

References 32 publications

Susceptibility of the Porcine Endogenous Retrovirus to Reverse Transcriptase and Protease Inhibitors

Susceptibility of the Porcine Endogenous Retrovirus to Reverse Transcriptase and Protease Inhibitors

Anti–HIV‐1 Activity of Weekly or Biweekly Treatment with Subcutaneous PRO 140, a CCR5 Monoclonal Antibody

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

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