Kurt Hertogs

Genotypic Correlates of Phenotypic Resistance to Efavirenz in Virus Isolates from Patients Failing Nonnucleoside Reverse Transcriptase Inhibitor Therapy

ABSTRACT Efavirenz (also known as DMP 266 or SUSTIVA) is a potent nonnucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity and of HIV-1 replication in vitro and in vivo. Most patients on efavirenz-containing regimens have sustained antiviral responses; however, rebounds in plasma viral load have been observed in some patients in association with the emergence of mutant strains of HIV-1. Virus isolates from the peripheral blood mononuclear cells (PBMCs) of patients with such treatment failures, as well as recombinant viruses incorporating viral sequences derived from patient plasma, show reduced in vitro susceptibility to efavirenz in association with mutations in the RT gene encoding K103N, Y188L, or G190S/E substitutions. Patterns of RT gene mutations and in vitro susceptibility were similar in plasma virus and in viruses isolated from PBMCs. Variant strains of HIV-1 constructed by site-directed mutagenesis confirmed the role of K103N, G190S, and Y188L substitutions in reduced susceptibility to efavirenz. Further, certain secondary mutations (V106I, V108I, Y181C, Y188H, P225H, and F227L) conferred little resistance to efavirenz as single mutations but enhanced the level of resistance of viruses carrying these mutations in combination with K103N or Y188L. Viruses with K103N or Y188L mutations, regardless of the initial selecting nonnucleoside RT inhibitor (NNRTI), exhibited cross-resistance to all of the presently available NNRTIs (efavirenz, nevirapine, and delavirdine). Some virus isolates from nevirapine or delavirdine treatment failures that lacked K103N or Y188L mutations remained susceptible to efavirenz in vitro, although the clinical significance of this finding is presently unclear.

A randomized trial assessing the impact of phenotypic resistance testing on antiretroviral therapy

Objective To compare the effect of treatment decisions guided by phenotypic resistance testing (PRT) or standard of care (SOC) on short-term virological response. Design A prospective, randomized, controlled clinical trial conducted in 25 university and private practice centers in the United States. Participants A total of 272 subjects who failed to achieve or maintain virological suppression (HIV-1-RNA plasma level > 2000 copies/ml) with previous exposure to two or more nucleoside reverse transcriptase inhibitors and one protease inhibitor. Interventions Randomization was to antiretroviral therapy guided by PRT or SOC. Main outcome measures The percentage of subjects with HIV-1-RNA plasma levels less than 400 copies/ml at week 16 (primary); change from baseline in HIV-1-RNA plasma levels and number of ‘active’ (less than fourfold resistance) antiretroviral agents used (secondary). Results At week 16, using intent-to-treat (ITT) analysis, a greater proportion of subjects had HIV-1-RNA levels less than 400 copies/ml in the PRT than in the SOC arm (P = 0.036, ITT observed;P = 0.079, ITT missing equals failure). An ITT observed analysis showed that subjects in the PRT arm had a significantly greater median reduction in HIV-1-RNA levels from baseline than the SOC arm (P = 0.005 for 400 copies/ml;P = 0.049 for 50 copies/ml assay detection limit). Significantly more subjects in the PRT arm were treated with two or more ‘active’ antiretroviral agents than in the SOC arm (P = 0.003). Conclusion Antiretroviral treatment guided prospectively by PRT led to the increased use of ‘active’ antiretroviral agents and was associated with a significantly better virological response.

Resistance Mutations in Human Immunodeficiency Virus Type 1 Integrase Selected with Elvitegravir Confer Reduced Susceptibility to a Wide Range of Integrase Inhibitors

ABSTRACT Integration of viral DNA into the host chromosome is an essential step in the life cycle of retroviruses and is facilitated by the viral integrase enzyme. The first generation of integrase inhibitors recently approved or currently in late-stage clinical trials shows great promise for the treatment of human immunodeficiency virus (HIV) infection, but virus is expected to develop resistance to these drugs. Therefore, we used a novel resistance selection protocol to follow the emergence of resistant HIV in the presence of the integrase inhibitor elvitegravir (GS-9137). We find the primary resistance-conferring mutations to be Q148R, E92Q, and T66I and demonstrate that they confer a reduction in susceptibility not only to elvitegravir but also to raltegravir (MK-0518) and other integrase inhibitors. The locations of the mutations are highlighted in the catalytic sites of integrase, and we correlate the mutations with expected drug-protein contacts. In addition, mutations that do not confer reduced susceptibility when present alone (H114Y, L74M, R20K, A128T, E138K, and S230R) are also discussed in relation to their position in the catalytic core domain and their proximity to known structural features of integrase. These data broaden the understanding of antiviral resistance against integrase inhibitors and may give insight facilitating the discovery of second-generation compounds.

Tipranavir inhibits broadly protease inhibitor-resistant HIV-1 clinical samples.

ObjectiveAlthough the use of HIV-1 protease inhibitors (PI) has substantially benefited HIV-1-infected individuals, new PI are urgently needed, as broad PI resistance and therapy failure is common. MethodsThe antiviral activity of tipranavir (TPV), a non-peptidic PI, was assessed in in vitro culture for 134 clinical isolates with a wide range of resistance to currently available peptidomimetic PI. The susceptibility of all 134 variants was then re-tested with the four PI simultaneously with TPV, using the AntivirogramTM assay. ResultsOf 105 viruses with more than tenfold resistance to three or four PI and an average of 6.1 PI mutations per sample, 95 (90%) were susceptible to TPV; eight (8%) had four- to tenfold resistance to TPV and only two (2%) had more than tenfold resistance. ConclusionsThe substantial lack of PI cross-resistance to TPV shown by highly PI-resistant clinical isolates makes TPV an attractive new-generation HIV inhibitor.

Prevalence of genotypic and phenotypic resistance to anti-retroviral drugs in a cohort of therapy-naïve HIV-1 infected US military personnel.

ObjectiveWhile transmission of drug-resistant HIV-1 has been reported, estimates of prevalence of resistance in drug-naïve populations are incomplete. We investigated the prevalence of genotypic mutations and phenotypic antiretroviral resistance in a cohort of HIV-1 infected U.S. military personnel prior to the institution of antiretroviral therapy. DesignCross-sectional cohort study. MethodsPlasma was obtained from 114 recently HIV-1 infected subjects enrolled in an epidemiological study. Genotypic resistance was determined by consensus sequencing of a PCR product from the HIV-1 pol gene. Sequences were interpreted by a phenotypic–genotypic correlative database. Resistance phenotypes were determined by a recombinant virus cell culture assay. ResultsGenotypic mutations and phenotypic resistance were found at a higher than expected frequency. Resistance to non-nucleoside reverse transcriptase inhibitors was most common, with a prevalence of 15% of 95 subjects by genotype and 26% of 91 subjects by phenotype. Genotypic and phenotypic resistance respectively were found in 4% and 8% of subjects for nucleoside reverse transcriptase inhibitors and in 10% and 1% for protease inhibitors. One subject harbored virus with resistance to all three drug classes. ConclusionsA substantial frequency of resistance to antiretroviral drugs was identified in a therapy-naïve U.S. cohort. In most cases, the genotypic and phenotypic assays yielded similar results, although the genotypic assay could detect some protease inhibitor resistance-associated mutations in the absence of phenotypic resistance. These data suggest the need for optimization of treatment guidelines based on current estimates of the prevalence of drug resistance in HIV-1 seroconverters.

Binding Kinetics of Darunavir to Human Immunodeficiency Virus Type 1 Protease Explain the Potent Antiviral Activity and High Genetic Barrier

ABSTRACT The high incidence of cross-resistance between human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) limits their sequential use. This necessitates the development of PIs with a high genetic barrier and a broad spectrum of activity against PI-resistant HIV, such as tipranavir and darunavir (TMC114). We performed a surface plasmon resonance-based kinetic study to investigate the impact of PI resistance-associated mutations on the protease binding of five PIs used clinically: amprenavir, atazanavir, darunavir, lopinavir, and tipranavir. With wild-type protease, the binding affinity of darunavir was more than 100-fold higher than with the other PIs, due to a very slow dissociation rate. Consequently, the dissociative half-life of darunavir was much higher (>240 h) than that of the other PIs, including darunavirs structural analogue amprenavir. The influence of protease mutations on the binding kinetics was tested with five multidrug-resistant (MDR) proteases derived from clinical isolates harboring 10 to 14 PI resistance-associated mutations with a decreased susceptibility to various PIs. In general, all PIs bound to the MDR proteases with lower binding affinities, caused mainly by a faster dissociation rate. For amprenavir, atazanavir, lopinavir, and tipranavir, the decrease in affinity with MDR proteases resulted in reduced antiviral activity. For darunavir, however, a nearly 1,000-fold decrease in binding affinity did not translate into a weaker antiviral activity; a further decrease in affinity was required for the reduced antiviral effect. These observations provide a mechanistic explanation for darunavirs potent antiviral activity and high genetic barrier to the development of resistance.

Baseline HIV drug resistance profile predicts response to ritonavir-saquinavir protease inhibitor therapy in a community setting.

OBJECTIVE To determine whether baseline drug resistance assays could help to predict treatment failure with the protease inhibitor combination ritonavir-saquinavir. METHODS Baseline HIV-1 drug resistance was determined for 76 consecutive patients who started treatment with the dual protease inhibitor combination ritonavir-saquinavir between September 1996 and June 1997 either alone or in combination with other antiviral agents. Resistance to 10 different antiviral agents was assessed by both phenotype (Virco Antivirogram) and genotype (Vircogen). RESULTS Resistance inferred from viral genotype was similar to measured phenotypic resistance for both ritonavir and saquinavir (P<0.01). Baseline drug resistance phenotype was predictive of poor virological response to this dual protease inhibitor combination, despite the confounding effects of other antivirals. Patients were at least four times less likely to achieve a 0.5 log10 decrease in plasma HIV RNA viral load if their viral isolates were resistant to ritonavir or saquinavir. Patients classified as resistant to either drug using either method had median decreases in plasma viral load of 0.05 log10 HIV RNA copies/ml or less, compared to >0.8 log10 for those with sensitive virus. Patients resistant to both drugs never achieved plasma viral loads <100000 copies/ml. As little as fourfold increases in baseline resistance appeared to be sufficient to compromise even dual protease inhibitor therapy. CONCLUSION Baseline resistance to ritonavir or saquinavir or both was associated with a poor antiviral response. Our data suggest that the measurement of drug resistance may assist in optimizing antiretroviral therapy in the clinic.

ANTIVIRAL ACTIVITY OF TENOFOVIR (PMPA) AGAINST NUCLEOSIDE-RESISTANT CLINICAL HIV SAMPLES

The presence of the lamivudine-associated M184V RT mutation increases tenofovir susceptibility in multiple HIV genotypes. Tenofovir is uniquely active against multinucleoside-resistant HIV expressing the Q151M mutation, but shows reduced susceptibility to the T69S insertion mutations. HIV with common forms of zidovudine and lamivudine resistance are susceptible to tenofovir, corroborating phase II clinical results demonstrating the activity of tenofovir DF in treatment-experienced patients.

Indolopyridones Inhibit Human Immunodeficiency Virus Reverse Transcriptase with a Novel Mechanism of Action

ABSTRACT We have discovered a novel class of human immunodeficiency virus (HIV) reverse transcriptase (RT) inhibitors that block the polymerization reaction in a mode distinct from those of the nucleoside or nucleotide RT inhibitors (NRTIs) and nonnucleoside RT inhibitors (NNRTIs). For this class of indolopyridone compounds, steady-state kinetics revealed competitive inhibition with respect to the nucleotide substrate. Despite substantial structural differences with classical chain terminators or natural nucleotides, these data suggest that the nucleotide binding site of HIV RT may accommodate this novel class of RT inhibitors. To test this hypothesis, we have studied the mechanism of action of the prototype compound indolopyridone-1 (INDOPY-1) using a variety of complementary biochemical tools. Time course experiments with heteropolymeric templates showed “hot spots” for inhibition following the incorporation of pyrimidines (T>C). Moreover, binding studies and site-specific footprinting experiments revealed that INDOPY-1 traps the complex in the posttranslocational state, preventing binding and incorporation of the next complementary nucleotide. The novel mode of action translates into a unique resistance profile. While INDOPY-1 susceptibility is unaffected by mutations associated with NNRTI or multidrug NRTI resistance, mutations M184V and Y115F are associated with decreased susceptibility, and mutation K65R confers hypersusceptibility to INDOPY-1. This resistance profile provides additional evidence for active site binding. In conclusion, this class of indolopyridones can occupy the nucleotide binding site of HIV RT by forming a stable ternary complex whose stability is mainly dependent on the nature of the primer 3′ end.

Dual Resistance to Zidovudine and Lamivudine in Patients Treated with Zidovudine-Lamivudine Combination Therapy: Association with Therapy Failure

Human immunodeficiency virus type 1 (HIV-1) strains dually resistant to zidovudine and lamivudine (3TC) may arise during zidovudine-3TC combination therapy. The objective of this cross-sectional study (n = 43 patients) was to test the association between therapy response (clinical and immunologic) to zidovudine-3TC and the level of phenotypic zidovudine resistance and zidovudine resistance-associated genotype of 3TC-resistant isolates. Other variables included were baseline CD4+ cell count, baseline Centers for Disease Control and Prevention (CDC) classification, virus load, and time receiving zidovudine. Phenotypic resistance was assessed using a recombinant virus assay. Genotypic analysis was based on population sequencing of plasma HIV-1. In a univariate analysis using a logistic regression model, it was found that therapy response was significantly associated with phenotypic and genotypic zidovudine resistance, baseline CD4+ cell count, and virus load. After adjustment for all variables, phenotypic resistance to zidovudine remained the only significantly associated factor, independent of baseline CD4+ cell count, baseline CDC classification, and virus load.