Nonnucleoside Reverse Transcriptase Inhibitor Susceptibility Research Articles

HIV Drug Resistance Mutations in Non-B Subtypes After Prolonged Virological Failure on NNRTI-Based First-Line Regimens in Sub-Saharan Africa.

To determine drug resistance mutation (DRM) patterns in a large cohort of patients failing nonnucleoside reverse transcriptase inhibitor (NNRTI)-based first-line antiretroviral therapy regimens in programs without routine viral load (VL) monitoring and to examine intersubtype differences in DRMs. Sequences from 787 adults/adolescents who failed an NNRTI-based first-line regimen in 13 clinics in Uganda, Kenya, Zimbabwe, and Malawi were analyzed. Multivariable logistic regression was used to determine the association between specific DRMs and Stanford intermediate-/high-level resistance and factors including REGA subtype, first-line antiretroviral therapy drugs, CD4, and VL at failure. The median first-line treatment duration was 4 years (interquartile range 30-43 months); 42% of participants had VL ≥100,000 copies/mL and 63% participants had CD4 <100 cells/mm. Viral subtype distribution was A1 (40%; Uganda and Kenya), C (31%; Zimbabwe and Malawi), and D (25%; Uganda and Kenya), and recombinant/unclassified (5%). In general, DRMs were more common in subtype-C than in subtype-A and/or subtype-D (nucleoside reverse transcriptase inhibitor mutations K65R and Q151M; NNRTI mutations E138A, V106M, Y181C, K101E, and H221Y). The presence of tenofovir resistance was similar between subtypes [P (adjusted) = 0.32], but resistance to zidovudine, abacavir, etravirine, or rilpivirine was more common in subtype-C than in subtype-D/subtype-A [P (adjusted) < 0.02]. Non-B subtypes differ in DRMs at first-line failure, which impacts on residual nucleoside reverse transcriptase inhibitor and NNRTI susceptibility. In particular, higher rates of etravirine and rilpivirine resistance in subtype-C may limit their potential utility in salvage regimens.

A376S in the Connection Subdomain of HIV-1 Reverse Transcriptase Confers Increased Risk of Virological Failure to Nevirapine Therapy

The clinical relevance of mutations in the connection subdomain and the ribonuclease (RNase) H domain of HIV-1 reverse transcriptase (RT) is uncertain. The risk of virological failure to nonnucleoside RT inhibitor (NNRTI)-based antiretroviral therapy (ART) was evaluated in NNRTI-naive patients who started NNRTIs in the EuroSIDA study after July 1997 according to preexisting substitutions in the connection subdomain and the RNase H domain of HIV-1 RT. An observed association between A376S and virological failure was further investigated by testing in vitro NNRTI susceptibility of single site-directed mutants and patient-derived recombinant viruses. Enzymatic assays also determined the effects of A376S on nevirapine and template-primer binding to HIV-1 RT. Virological failure occurred in 142 of 287 (49%) individuals: 77 receiving nevirapine (67%) and 65 receiving efavirenz (38%) (P < .001). Preexisting A376S was associated with an increased risk of virological failure to nevirapine (relative hazard [RH] = 10.4; 95% confidence interval [CI], 2.0-54.7), but it did not affect efavirenz outcome the same way (RH = 0.5; 95% CI, 0.1-2.2) (P value for interaction = .013). A376S conferred selective low-level nevirapine resistance in vitro, and led to greater affinity for double-stranded DNA. The A376S substitution in the connection subdomain of HIV-1 RT causes selective nevirapine resistance and confers an increased risk of virological failure to nevirapine-based ART.

N348I in HIV-1 Reverse Transcriptase Can Counteract the Nevirapine-mediated Bias toward RNase H Cleavage during Plus-strand Initiation

Drug resistance-associated mutations in HIV-1 reverse transcriptase (RT) can affect the balance between polymerase and ribonuclease H (RNase H) activities of the enzyme. We have recently demonstrated that the N348I mutation in the connection domain causes selective dissociation from RNase H-competent complexes, whereas the functional integrity of the polymerase-competent complex remains largely unaffected. N348I has been associated with resistance to the non-nucleoside RT inhibitor (NNRTI), nevirapine; however, a possible mechanism that links changes in RNase H activity to changes in NNRTI susceptibility remains to be established. To address this problem, we consider recent findings suggesting that NNRTIs may affect the orientation of RT on its nucleic acid substrate and increase RNase H activity. Here we demonstrate that RNase H-mediated primer removal is indeed more efficient in the presence of NNRTIs; however, the N348I mutant enzyme is able to counteract this effect. Efavirenz, a tight binding inhibitor, restricts the influence of the mutation. These findings provide strong evidence to suggest that N348I can thwart the inhibitory effects of nevirapine during initiation of (+)-strand DNA synthesis, which provides a novel mechanism for resistance. The data are in agreement with clinical data, which demonstrate a stronger effect of N348I on susceptibility to nevirapine as compared with efavirenz.

The "Connection" Between HIV Drug Resistance and RNase H.

Currently, nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs) are two classes of antiretroviral agents that are approved for treatment of HIV-1 infection. Since both NRTIs and NNRTIs target the polymerase (pol) domain of reverse transcriptase (RT), most genotypic analysis for drug resistance is limited to the first ∼300 amino acids of RT. However, recent studies have demonstrated that mutations in the C-terminal domain of RT, specifically the connection subdomain and RNase H domain, can also increase resistance to both NRTIs and NNRTIs. In this review we will present the potential mechanisms by which mutations in the C-terminal domain of RT influence NRTI and NNRTI susceptibility, summarize the prevalence of the mutations in these regions of RT identified to date, and discuss their importance to clinical drug resistance.

Rare mutations at codon 103 of HIV-1 reverse transcriptase can confer resistance to non-nucleoside reverse transcriptase inhibitors

The K103N mutation in HIV-1 reverse transcriptase (RT) confers high-level resistance to current non-nucleoside reverse transcriptase inhibitors (NNRTI). The prevalence and resistance profile of HIV-1 with other substitutions at RT codon 103 is less well documented. K103 substitutions among over 70,000 clinical samples submitted for routine antiretroviral resistance testing at two independent centres were examined. Phenotypic resistance profiles of isolates harboring rare K103 variants in the absence of known NNRTI-associated resistance mutations were retrieved from Virco's correlative genotype/phenotype database. Genotyped samples with known treatment histories were retrieved from the British Columbia Centre for Excellence in HIV/AIDS database. Site-directed mutants containing K103 variants were constructed and phenotyped. K103N, R and S were observed in 29, 1.8, and 0.9% of Virco isolates and in 16, 1.5 and 0.4% of British Columbia isolates. K103T/Q/H substitutions were observed only rarely (<0.2%). The prevalence of unusual codon 103 substitutions remained stable over 5 years, except K103S, which increased over fourfold in both datasets. K103R/Q-containing clinical isolates remained phenotypically susceptible to NNRTI, whereas K103S/T/H-containing isolates showed over 10-fold decreased NNRTI susceptibility. Among patients with a known treatment history, K103S/T/H were observed primarily in individuals failing NNRTI-containing regimens. Site-directed mutants confirmed decreased susceptibility to NNRTI in K103S/T/H-containing recombinants. Variants at HIV RT codon 103 other than K103N are observed relatively rarely in clinical isolates, but K103 S, T and H confer decreased susceptibility to NNRTI. These data are relevant for interpretive genotype algorithms and in the design of assays specific to RT codon 103 mutations.

In Vitro Characterization of a Simian Immunodeficiency Virus-Human Immunodeficiency Virus (HIV) Chimera Expressing HIV Type 1 Reverse Transcriptase To Study Antiviral Resistance in Pigtail Macaques

Antiviral resistance is a significant obstacle in the treatment of human immunodeficiency virus type 1 (HIV-1)-infected individuals. Because nonnucleoside reverse transcriptase inhibitors (NNRTIs) specifically target HIV-1 reverse transcriptase (RT) and do not effectively inhibit simian immunodeficiency virus (SIV) RT, the development of animal models to study the evolution of antiviral resistance has been problematic. To facilitate in vivo studies of NNRTI resistance, we examined whether a SIV that causes immunopathogenesis in pigtail macaques could be made sensitive to NNRTIs. Two simian-human immunodeficiency viruses (SHIVs) were derived from the genetic background of SIV(mne): SIV-RT-YY contains RT substitutions intended to confer NNRTI susceptibility (V181Y and L188Y), and RT-SHIV(mne) contains the entire HIV-1 RT coding region. Both mutant viruses grew to high titers in vitro but had reduced fitness relative to wild-type SIV(mne). Although the HIV-1 RT was properly processed into p66 and p51 subunits in RT-SHIV(mne) particles, the RT-SHIV(mne) virions had lower levels of RT per viral genomic RNA than HIV-1. Correspondingly, there was decreased RT activity in RT-SHIV(mne) and SIV-RT-YY particles. HIV-1 and RT-SHIV(mne) were similarly susceptible to the NNRTIs efavirenz, nevirapine, and UC781. However, SIV-RT-YY was less sensitive to NNRTIs than HIV-1 or RT-SHIV(mne). Classical NNRTI resistance mutations were selected in RT-SHIV(mne) after in vitro drug treatment and were monitored in a sensitive allele-specific real-time RT-PCR assay. Collectively, these results indicate that RT-SHIV(mne) may be a useful model in macaques for the preclinical evaluation of NNRTIs and for studies of the development of drug resistance in vivo.

Effects of the G190A substitution of HIV reverse transcriptase on phenotypic susceptibility of patient isolates to delavirdine.

Cross resistance is common among the non-nucleoside reverse transcriptase inhibitors (NNRTIs). G190A appears in 5-15% of the patients treated with nevirapine or efavirenz who develop clinical resistance. In this study we investigated the effect of G190A and other NNRTI substitutions on the phenotypic susceptibility to this class of drugs. We identified 15 individuals, who after treatment with NNRTIs (nevirapine or efavirenz; median exposure of 20 months), developed isolated G190A, G190A in combination with K103N, or K103N alone. Phenotypic and genotypic analyses of stored plasma specimens were performed before and after the mutations occurred to assess NNRTI susceptibility. All isolates that developed only G190A substitution became less susceptible to nevirapine (median: 125-fold) and efavirenz (median: 10-fold) but were 2.5-fold more sensitive to delavirdine (Wilcoxon P = 0.06). In the group with only K103N substitution, acquisition of resistance to all NNRTIs was observed. In the group with the double substitutions, G190A and K103N, delavirdine susceptibility decreased 13-fold, while resistance to nevirapine and efavirenz decreased by 239- and 154-folds, respectively (Kruskal-Wallis H P = 0.009). The data suggest that the presence of a G190A substitution attenuates the phenotypic resistance associated with a K103N substitution, although resistance is still present. The in vivo significance of the increased phenotypic susceptibility to delavirdine is not known but could be evaluated in a clinical trial.

Susceptibility of feline immunodeficiency virus/human immunodeficiency virus type 1 reverse transcriptase chimeras to non-nucleoside RT inhibitors.

To map the determinants of the lack of susceptibility of feline immunodeficiency virus (FIV) reverse transcriptase (RT) to anti human immunodeficiency virus type 1 (HIV-1) non-nucleoside RT inhibitors (NNRTIs), a variety of chimeric HIV-1/FIV RTs were constructed. The majority of chimeric RTs had an affinity (Km) for their natural substrates comparable with that of the wild-type HIV-1 and FIV RTs, but their catalytic efficacy was decreased. Whereas HIV-1 RT could be made entirely insensitive to NNRTIs by exchanging the amino acid sequence 97 through 205 of FIV RT, none of the reverse FIV/HIV-1 RT chimeras gained susceptibility to NNRTIs. The amino acids that are thought to be involved in NNRTI susceptibility and that are different from those in HIV-1 RT have also been introduced in FIV RT. These mutant RTs gained virtually no susceptibility to efavirenz or capravirine. Vice versa, when these HIV-1-specific amino acids were replaced by their FIV RT counterparts in HIV-1 RT, susceptibility to the NNRTIs was lost. Thus, replacing segments or substituting relevant amino acids in FIV RT by their HIV-1 RT counterparts did not suffice to make FIV RT sensitive toward NNRTIs and was often accompanied by a decrease or even total loss of polymerase activity. It is postulated that, in contrast to the results found for HIV-1/HIV-2 RT chimeras and supported by the crystal structure of HIV-2 RT, there exist significant differences in the structure and/or flexibility of FIV RTs that may prevent NNRTIs from interacting with the FIV RT.

Hypersusceptibility to non-nucleoside reverse transcriptase inhibitors in HIV-1: clinical, phenotypic and genotypic correlates.

The routine use of phenotypic drug resistance testing in patient management has revealed that many HIV-1 strains possess significantly increased drug sensitivity, or 'hypersusceptibility' compared with wild-type viruses. This study describes hypersusceptibility to non-nucleoside reverse transcriptase inhibitors (NNRTI) and was designed to determine the prevalence of and viral characteristics associated with NNRTI hypersusceptibility in patient-derived viruses. Retrospective analyses were performed on a large clinical laboratory dataset containing phenotypic drug susceptibility and genotypic sequence results from HIV-1 patient isolates. Genetically engineered viruses were used to confirm the role of certain nucleoside reverse transcriptase inhibitor (NRTI)-resistance mutations in NNRTI hypersusceptibility. Hypersusceptibility to delavirdine, efavirenz and nevirapine was detected in 10.7, 10.8 and 8.0% of more than 17,000 consecutive plasma samples submitted for phenotypic susceptibility testing. In analyses limited to a subset of viruses derived from patients with known treatment histories, NNRTI hypersusceptibility was observed significantly more frequently among viruses from NRTI experienced/NNRTI-naive patients compared with viruses from NRTI/NNRTI-naive patients. Significant inverse correlations between NRTI and NNRTI susceptibility exist among the viruses from NRTI-experienced patients. Analyses of viruses classified according to their NNRTI susceptibility identified 18 positions in reverse transcriptase where substitutions were significantly associated with NNRTI hypersusceptibility. NNRTI hypersusceptibility is common among patient HIV-1 isolates, especially in NRTI-resistant viruses. Genotypic correlates of hypersusceptibility are complex and not easily defined by a simple analysis of NRTI-associated resistance mutations. NNRTI hypersusceptibility may provide an explanation for the superior virologic response to NNRTI-containing salvage regimens observed in NRTI-experienced patients in several clinical trials.

Modest Decreases in Nnrti Susceptibility Do Not Influence Virological Outcome in Patients Receiving Initial Nnrti-Containing Triple Therapy

To assess the prevalence of modest (< 10-fold) decreases in baseline non-nucleoside reverse transcriptase inhibitor (NNRTI) susceptibility and their impact on virological response to NNRTI-containing triple therapy in drug-naive individuals. Baseline HIV resistance phenotype, genotype and response to therapy were examined retrospectively for all antiretroviral-naive individuals initiating therapy with two nucleoside analogues and an NNRTI in British Columbia, Canada, between 05/1997 and 08/1999 (n = 279), followed until July 31 2001. Time to viral suppression (first of at least two consecutive plasma viral loads < 400 copies HIV RNA copies/ml) and viral rebound (to > or = 400 copies/ml after first pVL < 400 copies HIV RNA copies/ml), were estimated by Kaplan-Meier methods. Multivariate analyses were performed using Cox proportional hazards regression. Nevirapine was the most commonly prescribed NNRTI (96%). Four- to 10-fold decreased susceptibility to NNRTIs was observed in > 30% of untreated individuals at baseline, an observation strongly driven by decreased susceptibility to delavirdine (22.4%). A > 10-fold decrease in susceptibility to any NNRTI was observed only rarely (< 2%). There was no association between four- and 10-fold decreased baseline susceptibility to NNRTIs and virological outcome (P > 0.05). In multivariate analyses, the strongest predictors of poor virological response to NNRTI-based therapy were baseline plasma viral load and the proportion of time on therapy in the first year of follow-up. There was no relationship between the presence of previously reported mutations associated with decreased NNRTI susceptibility (at codons 135 and 283 in HIV reverse transcriptase) and virological response. These data suggest that the clinically significant level of resistance to NNRTIs, particularly nevirapine, in drug-naive individuals is likely greater than four- to 10-fold.

A mutation in the 3' region of the human immunodeficiency virus type 1 reverse transcriptase (Y318F) associated with nonnucleoside reverse transcriptase inhibitor resistance.

The Y318F substitution in the 3' region of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) has been linked to nonnucleoside RT inhibitor (NNRTI) resistance in vitro. A systematic search of a large phenotypic-genotypic database (Virco) linked the Y318F substitution with a >10-fold decrease in NNRTI susceptibility in >85% of clinically derived isolates. There was a significant association between Y318F and use of delavirdine (P = 10(-11)) and nevirapine (P = 10(-6)) but not efavirenz (P = 0.3). Site-directed HIV-1 Y318F mutants in an HXB2 background displayed 42-fold-decreased susceptibility to delavirdine but <3-fold-decreased susceptibility to nevirapine or efavirenz. Combinations of Y318F with K103N, Y181C, or both resulted in decreased efavirenz susceptibility of 43-, 3.3-, and 84-fold, respectively, as well as >100- and >60-fold decreases in delavirdine and nevirapine susceptibility, respectively. These results indicate the importance of the Y318F substitution in HIV-1 drug resistance.

Prevalence of nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance-associated mutations and polymorphisms in NNRTI-naïve HIV-infected patients

BACKGROUND: The efficacy of treatment containing nonnucleoside reverse transcriptase inhibitors (NNRTIs) could be compromised in NNRTI-naÏve patients already harboring a virus resistant to NNRTIs. On the contrary, hypersusceptibility to NNRTIs in patients having failed nucleoside reverse transcriptase inhibitor (NRTI)-containing regimens has been described and has been associated with improved outcome. METHOD: We assessed the prevalence of NNRTI resistance-associated mutations or polymorphisms in 146 antiretroviral-naÏve patients and in 181 HIV-infected patients who were given an NNRTI-based regimen. We phenotypically evaluated the NNRTI susceptibility of 41 strains presenting with amino acid substitutions at positions involved in NNRTI resistance. RESULTS: In the 268 genotypically analyzable samples, the overall prevalence of NNRTI resistance-associated mutations was 2% (6/268 patients). The prevalence of strains with amino acid substitutions at reverse transcriptase (RT) gene positions (A98, K101, K103, V106, V108, V179) involved in NNRTI resistance was 15%. Hypersusceptibility to NNRTI was rare (2%, 1/41) in those samples. RT substitutions at positions involved in NNRTI resistance were not associated with a significantly worse virologic outcome in NNRTI-treated patients. Our understanding of small shifts in IC50 values (higher or lower) toward NNRTI is very limited. The significance of many RT mutations on NNRTI susceptibility is not clear. CONCLUSION: In contrast to resistance mutations, RT substitutions at positions involved in NNRTI resistance are frequent. They are not associated with a worse virologic outcome or with decreased phenotypic susceptibility to NNRTIs. It may be prudent not to rule out the use of NNRTIs in patients with small shifts in IC50 values or poorly understood mutations.