Brian Wynhoven

Predictors of HIV Drug-Resistance Mutations in a Large Antiretroviral-Naive Cohort Initiating Triple Antiretroviral Therapy

OBJECTIVE The objective of this study was to systematically characterize the incidence and determinants of antiretroviral resistance in the HOMER (Highly Active Antiretroviral Therapy [HAART] Observational Medical Evaluation and Research) cohort of 1191 human immunodeficiency virus-infected, antiretroviral-naive adults initiating HAART in British Columbia, Canada. METHODS All plasma samples with plasma virus loads (pVLs) >1000 copies/mL collected during the first 30 months of follow-up were genotyped for drug resistance. The primary outcome measure was time to the first detection of major drug-resistance mutation(s). Cox proportional hazard regression was used to identify factors significantly associated with the detection of drug-resistance mutations. RESULTS Drug-resistance mutations were detected in 298 subjects (25%). Factors significantly associated with detection of drug-resistance mutations included high baseline pVL (multivariate hazard ratio [HR], 1.59; P<.001) and adherence (estimated using prescription-refill data and/or untimed plasma drug-concentration measurements). When compared with subjects with low (0%-<20%) prescription-refill percentages, subjects at an elevated risk of harboring drug-resistance mutations were those with relatively high but imperfect prescription-refill percentages (80%-<90%; multivariate HR, 4.15; P<.001) and those with essentially perfect (>/=95%) refill percentages but with 2 plasma drug concentrations below the steady-state trough concentration minus 1 standard deviation (multivariate HR, 4.57; P<.001). Initial use of nonnucleoside reverse-transcriptase inhibitor-based HAART was significantly associated with multiclass drug resistance (multivariate HR, 1.84; P=.001). CONCLUSION High baseline pVLs and substantial but imperfect levels of adherence were major predictors of antiretroviral resistance.

N348I in the connection domain of HIV-1 reverse transcriptase confers zidovudine and nevirapine resistance

Background The catalytically active 66-kDa subunit of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) consists of DNA polymerase, connection, and ribonuclease H (RNase H) domains. Almost all known RT inhibitor resistance mutations identified to date map to the polymerase domain of the enzyme. However, the connection and RNase H domains are not routinely analysed in clinical samples and none of the genotyping assays available for patient management sequence the entire RT coding region. The British Columbia Centre for Excellence in HIV/AIDS (the Centre) genotypes clinical isolates up to codon 400 in RT, and our retrospective statistical analyses of the Centres database have identified an N348I mutation in the RT connection domain in treatment-experienced individuals. The objective of this multidisciplinary study was to establish the in vivo relevance of this mutation and its role in drug resistance. Methods and Findings The prevalence of N348I in clinical isolates, the time taken for it to emerge under selective drug pressure, and its association with changes in viral load, specific drug treatment, and known drug resistance mutations was analysed from genotypes, viral loads, and treatment histories from the Centres database. N348I increased in prevalence from below 1% in 368 treatment-naïve individuals to 12.1% in 1,009 treatment-experienced patients (p = 7.7 × 10−12). N348I appeared early in therapy and was highly associated with thymidine analogue mutations (TAMs) M41L and T215Y/F (p < 0.001), the lamivudine resistance mutations M184V/I (p < 0.001), and non-nucleoside RTI (NNRTI) resistance mutations K103N and Y181C/I (p < 0.001). The association with TAMs and NNRTI resistance mutations was consistent with the selection of N348I in patients treated with regimens that included both zidovudine and nevirapine (odds ratio 2.62, 95% confidence interval 1.43–4.81). The appearance of N348I was associated with a significant increase in viral load (p < 0.001), which was as large as the viral load increases observed for any of the TAMs. However, this analysis did not account for the simultaneous selection of other RT or protease inhibitor resistance mutations on viral load. To delineate the role of this mutation in RT inhibitor resistance, N348I was introduced into HIV-1 molecular clones containing different genetic backbones. N348I decreased zidovudine susceptibility 2- to 4-fold in the context of wild-type HIV-1 or when combined with TAMs. N348I also decreased susceptibility to nevirapine (7.4-fold) and efavirenz (2.5-fold) and significantly potentiated resistance to these drugs when combined with K103N. Biochemical analyses of recombinant RT containing N348I provide supporting evidence for the role of this mutation in zidovudine and NNRTI resistance and give some insight into the molecular mechanism of resistance. Conclusions This study provides the first in vivo evidence that treatment with RT inhibitors can select a mutation (i.e., N348I) outside the polymerase domain of the HIV-1 RT that confers dual-class resistance. Its emergence, which can happen early during therapy, may significantly impact on a patients response to antiretroviral therapies containing zidovudine and nevirapine. This study also provides compelling evidence for investigating the role of other mutations in the connection and RNase H domains in virological failure.

Improved Virological Outcomes in British Columbia Concomitant with Decreasing Incidence of HIV Type 1 Drug Resistance Detection

BACKGROUND There have been limited studies evaluating temporal changes in the incidence of detection of drug resistance among human immunodeficiency virus type 1 (HIV-1) isolates and concomitant changes in plasma HIV load for treated individuals in a population-wide setting. METHODS Longitudinal plasma viral load and genotypic resistance data were obtained from patients receiving antiretroviral therapy from the British Columbia Drug Treatment Program from July 1996 through December 2008. A total of 24,652 resistance tests were available from 5422 individuals. The incidence of successful plasma viral load suppression and of resistance to each of 3 antiretroviral categories (nucleoside/nucleotide reverse-transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors, and protease inhibitors) was calculated for the population receiving therapy. RESULTS There has been a drastic decrease in the incidence of new cases of HIV-1 drug resistance in individuals followed during 1996-2008. In 1997, the incidence rate of any newly detected resistance was 1.73 cases per 100 person-months of therapy, and by 2008, the incidence rate had decreased >12-fold, to 0.13 cases per 100 person-months of therapy. This decrease in the incidence of resistance has occurred at an exponential rate, with half-times on the order of 2-3 years. Concomitantly, the proportion of individuals with plasma viral load suppression has increased linearly over time (from 64.7% with HIV RNA levels <50 copies/mL in 2000 to 87.0% in 2008; R2=0.97; P<.001). CONCLUSIONS Our results suggest an increasing effectiveness of highly active antiretroviral therapy at the populational level. The vast majority of treated patients in British Columbia now have either suppressed plasma viral load or drug-susceptible HIV-1, according to their most recent test results.

Clinical and immunological impact of HIV envelope V3 sequence variation after starting initial triple antiretroviral therapy

Background: The HIV-1 envelope third variable loop (V3 loop) is an important determinant of viral phenotype and co-receptor usage. We wished to determine the impact of specific V3 genotypes associated with viral phenotype and co-receptor usage on response to initial triple antiretroviral therapy. Methods: Pre-therapy plasma samples from the HOMER cohort of 1191 antiretroviral-naive, HIV-infected adults who initiated triple therapy in British Columbia, Canada between August 1996 and September 1999 were genotyped for V3 loop sequence. V3 sequences were dichotomized by the presence or absence of positively charged residues at codons 11 and/or 25 (an ‘11/25’ genotype). Neural network (NN) and Position Specific Scoring Matrix (PSSM) approaches were used as alternative V3 sequence interpretation methods. The association of V3 genotypes with clinical endpoints was assessed over a median of 43 months of follow up. Results: One-hundred and eighteen (10.9%) of the 1085 isolates successfully genotyped for V3 displayed the 11/25 genotype. In multivariate analyses, this genotype was associated with a more rapid CD4 decline [risk ratio, (RR), 1.38; P = 0.012] and earlier mortality (RR, 1.70; P = 0.027), despite comparable viral load suppression below 500 HIV RNA copies/ml. We observed no influence of the 11/25 genotype on time to viral rebound or the development of drug resistance. PSSM-based sequence categories were similarly predictive of outcomes. NN sequence categories were not associated with any endpoints. Conclusion: The 11/25 genotype of the HIV V3 loop is an independent predictor of poor immunological response and more rapid mortality even after starting triple antiretroviral therapy. These results may prove to be useful for the clinical management of HIV-infected individuals.

Discordances between Interpretation Algorithms for Genotypic Resistance to Protease and Reverse Transcriptase Inhibitors of Human Immunodeficiency Virus Are Subtype Dependent

ABSTRACT The major limitation of drug resistance genotyping for human immunodeficiency virus remains the interpretation of the results. We evaluated the concordance in predicting therapy response between four different interpretation algorithms (Rega 6.3, HIVDB-08/04, ANRS [07/04], and VGI 8.0). Sequences were gathered through a worldwide effort to establish a database of non-B subtype sequences, and demographic and clinical information about the patients was gathered. The most concordant results were found for nonnucleoside reverse transcriptase (RT) inhibitors (93%), followed by protease inhibitors (84%) and nucleoside RT inhibitor (NRTIs) (76%). For therapy-naive patients, for nelfinavir, especially for subtypes C and G, the discordances were driven mainly by the protease (PRO) mutational pattern 82I/V + 63P + 36I/V for subtype C and 82I + 63P + 36I + 20I for subtype G. Subtype F displayed more discordances for ritonavir in untreated patients due to the combined presence of PRO 20R and 10I/V. In therapy-experienced patients, subtype G displayed a lot of discordances for saquinavir and indinavir due to mutational patterns involving PRO 90 M and 82I. Subtype F had more discordance for nelfinavir attributable to the presence of PRO 88S and 82A + 54V. For the NRTIs lamivudine and emtricitabine, CRF01_AE had more discordances than subtype B due to the presence of RT mutational patterns 65R + 115 M and 118I + 215Y, respectively. Overall, the different algorithms agreed well on the level of resistance scored, but some of the discordances could be attributed to specific (subtype-dependent) combinations of mutations. It is not yet known whether therapy response is subtype dependent, but the advice given to clinicians based on a genotypic interpretation algorithm differs according to the subtype.

Connection Domain Mutations N348I and A360V in HIV-1 Reverse Transcriptase Enhance Resistance to 3′-Azido-3′-deoxythymidine through Both RNase H-dependent and -independent Mechanisms

Thymidine analogue-associated mutations (TAMs) in reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) cause resistance to 3′-azido-3′-deoxythymidine (AZT) through excision of the incorporated monophosphate. Mutations in the connection domain of HIV-1 RT can augment AZT resistance. It has been suggested that these mutations compromise RNase H cleavage, providing more time for AZT excision to occur. However, the underlying mechanism remains elusive. Here, we focused on connection mutations N348I and A360V that are frequently observed in clinical samples of treatment-experienced patients. We show that both N348I and A360V, in combination with TAMs, decrease the efficiency of RNase H cleavage and increase excision of AZT in the presence of the pyrophosphate donor ATP. The TAMs/N348I/A360V mutant accumulates transiently formed, shorter hybrids that can rebind to RT before the template is irreversibly degraded. These hybrids dissociate selectively from the RNase H-competent complex, whereas binding in the polymerase-competent mode is either not affected with N348I or modestly improved with A360V. Both connection domain mutations can compensate for TAM-mediated deficits in processive DNA synthesis, and experiments with RNase H negative mutant enzymes confirm an RNase H-independent contribution to increased levels of resistance to AZT. Moreover, the combination of diminished RNase H cleavage and increased processivity renders the use of both PPi and ATP advantageous, whereas classic TAMs solely enhance the ATP-dependent reaction. Taken together, our findings demonstrate that distinct, complementary mechanisms can contribute to higher levels of excision of AZT, which in turn can amplify resistance to this drug.

Improved detection of CXCR4-using HIV by V3 genotyping: application of population-based and "deep" sequencing to plasma RNA and proviral DNA.

Background:Tropism testing should rule out CXCR4-using HIV before treatment with CCR5 antagonists. Currently, the recombinant phenotypic Trofile assay (Monogram) is most widely utilized; however, genotypic tests may represent alternative methods. Methods:Independent triplicate amplifications of the HIV gp120 V3 region were made from either plasma HIV RNA or proviral DNA. These underwent standard, population-based sequencing with an ABI3730 (RNA n = 63; DNA n = 40), or “deep” sequencing with a Roche/454 Genome Sequencer-FLX (RNA n = 12; DNA n = 12). Position-specific scoring matrices (PSSMX4/R5) (−6.96 cutoff) and geno2pheno[coreceptor] (5% false-positive rate) inferred tropism from V3 sequence. These methods were then independently validated with a separate, blinded dataset (n = 278) of screening samples from the maraviroc MOTIVATE trials. Results:Standard sequencing of HIV RNA with PSSM yielded 69% sensitivity and 91% specificity, relative to Trofile. The validation dataset gave 75% sensitivity and 83% specificity. Proviral DNA plus PSSM gave 77% sensitivity and 71% specificity. “Deep” sequencing of HIV RNA detected >2% inferred-CXCR4-using virus in 8/8 samples called non-R5 by Trofile, and <2% in 4/4 samples called R5. Conclusions:Triplicate analyses of V3 standard sequence data detect greater proportions of CXCR4-using samples than previously achieved. Sequencing proviral DNA and “deep” V3 sequencing may also be useful tools for assessing tropism.

Automating HIV drug resistance genotyping with RECall, a freely accessible sequence analysis tool

ABSTRACT Genotypic HIV drug resistance testing is routinely used to guide clinical decisions. While genotyping methods can be standardized, a slow, labor-intensive, and subjective manual sequence interpretation step is required. We therefore performed external validation of our custom software RECall, a fully automated sequence analysis pipeline. HIV-1 drug resistance genotyping was performed on 981 clinical samples at the Stanford Diagnostic Virology Laboratory. Sequencing trace files were first interpreted manually by a laboratory technician and subsequently reanalyzed by RECall, without intervention. The relative performances of the two methods were assessed by determination of the concordance of nucleotide base calls, identification of key resistance-associated substitutions, and HIV drug resistance susceptibility scoring by the Stanford Sierra algorithm. RECall is freely available at http://pssm.cfenet.ubc.ca. In total, 875 of 981 sequences were analyzed by both human and RECall interpretation. RECall analysis required minimal hands-on time and resulted in a 25-fold improvement in processing speed (∼150 technician-hours versus ∼6 computation-hours). Excellent concordance was obtained between human and automated RECall interpretation (99.7% agreement for >1,000,000 bases compared). Nearly all discordances (99.4%) were due to nucleotide mixtures being called by one method but not the other. Similarly, 98.6% of key antiretroviral resistance-associated mutations observed were identified by both methods, resulting in 98.5% concordance of resistance susceptibility interpretations. This automated sequence analysis tool provides both standardization of analysis and a significant improvement in data workflow. The time-consuming, error-prone, and dreadfully boring manual sequence analysis step is replaced with a fully automated system without compromising the accuracy of reported HIV drug resistance data.

Rates of antiretroviral resistance among HIV-infected patients with and without a history of injection drug use.

Background:There exist concerns regarding the potential for elevated rates of antiretroviral resistance among HIV-infected injection drug users (IDUs) prescribed highly active antiretroviral therapy (HAART), however, no population-based study has examined if IDUs have elevated rates of antiretroviral resistance in comparison to non-IDUs. Objective:To evaluate the time to the development of antiretroviral resistance among antiretroviral-naive patients with and without a history of injection drug use. Methods:In British Columbia there is a province-wide HIV/AIDS treatment program that provides antiretrovirals free of charge. We examined all antiretroviral-naive patients initiating HAART between 1 August 1996 and 30 September 2000 and who were followed to 31 March 2002. The main outcome measure was the time to class-specific antiretroviral resistance. Cumulative antiretroviral resistance rates among IDUs and non-IDUs were evaluated using Kaplan–Meier methods and relative hazards were estimated using Cox regression. Results:Overall, 1191 antiretroviral-naive patients initiated HAART during the study period. Resistance mutations were observed in 298 (25%) subjects during the first 30 months of HAART. In comparison with non-IDUs, the risk of protease inhibitor resistance [relative hazard (RH), 0.9; 95% confidence interval (CI), 0.5–1.6] and non-nucleoside reverse transcriptase inhibitor resistance (RH, 1.5; 95% CI, 1.0–2.2) were similar among IDUs, and there were no differences in the rates of resistance to the sub-classes of nucleoside reverse transcriptase inhibitors. Conclusions:Resistance to all major classes of antiretrovirals were similar among IDUs and non-IDUs after 30 months of follow-up. These findings should help to allay fears that prescribing HAART to IDUs may result in elevated rates of resistance.

A Mutation in the 3′ Region of the Human Immunodeficiency Virus Type 1 Reverse Transcriptase (Y318F) Associated with Nonnucleoside Reverse Transcriptase Inhibitor Resistance

ABSTRACT 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.