Minor Resistance Mutations Research Articles

Drug resistance mutations in protease gene of HIV-1 subtype C infected patient population.

Failure of antiretroviral therapy (ART) in HIV-1 infection is a critical issue for the physicians treating HIV patients. The major cause of drug failure is the development of resistance mutations in reverse transcriptase (RT) and/or protease (PR) genes. Mutations associated with drug resistance decrease drug effectiveness. This study was conducted to assess drug resistance profile of the entire PR gene in 90 HIV-1 patients consisting of 23 ART non-responsive, 32 ART responsive and 35 drug naive patients. It was observed that the majority of the sequences (94.4%) belonged to subtype C and (5.5%) to subtype A1. The ART non-responsive and responsive patients were treated with either first line of ART regimen (two NRTI and one NNRTI) or second line of ART regimen that included additional one protease inhibitor (PI). All the patients in each group except one responsive patient had various minor resistance mutations. Thus, drug failures in ART non-responsive patients may not always be due to drug resistance mutations instead other factors may also be responsible for drug failures such as non-compliance, suboptimal dose or drug interaction. The presence of minor drug resistance mutations in drug naive patients is suggestive of transmitted resistance mutations.

Predictors of Virological Failure and Time to Viral Suppression of First-Line Integrase Inhibitor-Based Antiretroviral Treatment.

BackgroundIntegrase strand transfer inhibitors (InSTIs) are recommended for first-line treatment of persons with human immunodeficiency virus (HIV). We identified risk factors, including baseline minor InSTI resistance mutations, for treatment failure of InSTI-based regimens.MethodsWe studied time-to-treatment failure and time to viral suppression among 1419 drug-naive patients in the Swiss HIV Cohort Study. We performed Cox regression models adjusted for demographic factors, baseline HIV RNA/CD4 cell counts, AIDS-defining events, and the type of InSTI. In 646 patients with a baseline genotypic resistance test of the integrase, we studied the impact of minor integrase resistance mutations.ResultsWe observed 121 virological failures during 18 447 person-years of follow-up. A baseline viral load ≥100 000 copies/mL (multivariable hazard ratio [mHR], 2.2; 95% confidence interval [CI], 1.3–3.6) and an AIDS-defining event (mHR, 1.8; 95% CI. 1.1–3.0) were associated with treatment failure. CD4 counts between 200 and 500 cells/µL (mHR, 0.5; 95% CI, .3–.8) and >500 cells/µL (mHR, 0.4; 95% CI, .2–.7) were protective. Time to suppression was shorter in lower viral load strata (mHR, 0.7; 95% CI, .6–.8) and in dolutegravir-based therapy (mHR, 1.2; 95% CI, 1.0–1.4). Minor resistance mutations were found at baseline in 104 of 646 (16%) patients with no effect on treatment outcome.ConclusionsFactors associated with treatment failure on InSTI-based first-line regimen remained similar to those of older treatments, in particular high viral load and low CD4 counts.

Polymorphisms and drug resistance analysis of HIV-1 isolates from patients on first line antiretroviral therapy (ART) in South-eastern Nigeria.

Acquisition of resistance mutations by HIV-1 isolates causes treatment failure among infected patients receiving antiretroviral therapy (ART). This study determined patterns of drug-resistance mutations (DRMs) among HIV-1 isolates from patients receiving first-line ART in South-eastern Nigeria. Blood samples were collected from HIV-1 infected patients accessing antiretroviral treatment centers at General Hospital Awo-Omamma, Imo state, State Hospital Asaba, Delta state and St Joseph's Catholic Hospital Adazi, Anambra state and used for HIV-1 DNA sequencing and phylogenetic analysis. DRMs were scored using combination of Stanford algorithm and the 2015 International Antiviral Society-USA list while drug susceptibility was predicted using Stanford algorithm. Twenty eight of the HIV-1 isolates were sequenced and identified as subtypes G (35.7%), CRF02_AG (57.1%) and unclassifiable, UG (7.1%). Major PI resistance-associated mutations were identified at two sites including M46L (16.7% of subtype G/UG) and V82L (6.3% of CRF02_AG). Minor PI resistance-associated mutations identified among subtype G/UG are L10V/I (8.3%) and K20I (100%) while L10V/I (50%), K20I (100%), L33F (6.3%) and N88D (6.3%) were identified among CRF02_AG. Other polymorphisms found include; I13V/A, E35Q, M36I/L, N37D/S/E/H, R57K/G, L63T/P/S/Q, C67E/S, H69K/R, K70R, V82I and L89M in the range of 28.6% to 100% among the different subtypes. Interpretation based on Stanford algorithm showed that Darunavir/ritonavir is the only regimen whose potency was not compromised by the circulating mutations. Identification of major and minor PI resistance mutations in this study underscores the need for drug resistance testing prior to initiation of second line antiretroviral therapy in Nigeria.

Characterization of the dynamics of human cytomegalovirus resistance to antiviral drugs by ultra-deep sequencing

Prophylactic or preemptive treatment strategies are required to prevent human cytomegalovirus (CMV) infections in transplant recipients. However, treatment failure occurs when CMV resistant-associated variants (RAVs) are selected. Although the diversity of CMV is lower than that of RNA viruses, CMV appears to show some genetic instability, with possible minor emerging resistance that may be undetectable by Sanger sequencing. We aimed to examine CMV-resistance mutations over time by ultra-deep sequencing (UDS) and Sanger sequencing in a kidney transplant recipient experiencing CMV infection. This patient showed a transient response to three different antiviral drugs (valganciclovir, foscarnet, and maribavir) and four episodes of CMV resistance over two years. The full-length UL97 (2.3kpb) and partial UL54 (2.4kpb) CMV genes were studied by UDS and Sanger sequencing and linkage mutations calculated to determine RAVs. We detected four major and five minor resistance mutations. Minor resistant variants (2–20%) were detected by UDS, whereas major resistance substitutions (>20%) were identified by both UDS and Sanger method. We detected cross-resistance to three drugs, despite high CMV loads, suggesting that the fitness of the viral mutants was not impaired. In conclusion, CMV showed complex dynamic of resistance under antiviral drug pressure, as described for highly variable viruses. The emergence of successive RAVs constitutes a clinically challenging complication and contributes to the difficulty of therapeutic management of patients.

Molecular evolution of HIV-1 integrase during the 20\xa0years prior to the first approval of integrase inhibitors

BackgroundDetailed knowledge of the evolutionary potential of polymorphic sites in a viral protein is important for understanding the development of drug resistance in the presence of an inhibitor. We therefore set out to analyse the molecular evolution of the HIV-1 subtype B integrase at the inter-patient level in Germany during a 20-year period prior to the first introduction of integrase strand inhibitors (INSTIs).MethodsWe determined 337 HIV-1 integrase subtype B sequences (amino acids 1–278) from stored plasma samples of antiretroviral treatment-naïve individuals newly diagnosed with HIV-1 between 1986 and 2006. Shannon entropy was calculated to determine the variability at each amino acid position. Time trends in the frequency of amino acid variants were identified by linear regression. Direct coupling analysis was applied to detect covarying sites.ResultsTwenty-two time trends in the frequency of amino acid variants demonstrated either single amino acid exchanges or variation in the degree of polymorphy. Covariation was observed for 17 amino acid variants with a temporal trend. Some minor INSTI resistance mutations (T124A, V151I, K156 N, T206S, S230 N) and some INSTI-selected mutations (M50I, L101I, T122I, T124 N, T125A, M154I, G193E, V201I) were identified at overall frequencies >5%. Among these, the frequencies of L101I, T122I, and V201I increased over time, whereas the frequency of M154I decreased. Moreover, L101I, T122I, T124A, T125A, M154I, and V201I covaried with non-resistance-associated variants.ConclusionsTime-trending, covarying polymorphisms indicate that long-term evolutionary changes of the HIV-1 integrase involve defined clusters of possibly structurally or functionally associated sites independent of selective pressure through INSTIs at the inter-patient level. Linkage between polymorphic resistance- and non-resistance-associated sites can impact the selection of INSTI resistance mutations in complex ways. Identification of these sites can help in improving genotypic resistance assays, resistance prediction algorithms, and the development of new integrase inhibitors.

Effectiveness of Ritonavir-Boosted Protease Inhibitor Monotherapy in Clinical Practice Even with Previous Virological Failures to Protease Inhibitor-Based Regimens.

Background and ObjectiveSignificant controversy still exists about ritonavir-boosted protease inhibitor monotherapy (mtPI/rtv) as a simplification strategy that is used up to now to treat patients that have not experienced previous virological failure (VF) while on protease inhibitor (PI) -based regimens. We have evaluated the effectiveness of two mtPI/rtv regimens in an actual clinical practice setting, including patients that had experienced previous VF with PI-based regimens.MethodsThis retrospective study analyzed 1060 HIV-infected patients with undetectable viremia that were switched to lopinavir/ritonavir or darunavir/ritonavir monotherapy. In cases in which the patient had previously experienced VF while on a PI-based regimen, the lack of major HIV protease resistance mutations to lopinavir or darunavir, respectively, was mandatory. The primary endpoint of this study was the percentage of participants with virological suppression after 96 weeks according to intention-to-treat analysis (non-complete/missing = failure).ResultsA total of 1060 patients were analyzed, including 205 with previous VF while on PI-based regimens, 90 of whom were on complex therapies due to extensive resistance. The rates of treatment effectiveness (intention-to-treat analysis) and virological efficacy (on-treatment analysis) at week 96 were 79.3% (CI95, 76.8−81.8) and 91.5% (CI95, 89.6–93.4), respectively. No relationships were found between VF and earlier VF while on PI-based regimens, the presence of major or minor protease resistance mutations, the previous time on viral suppression, CD4+ T-cell nadir, and HCV-coinfection. Genotypic resistance tests were available in 49 out of the 74 patients with VFs and only four patients presented new major protease resistance mutations.ConclusionSwitching to mtPI/rtv achieves sustained virological control in most patients, even in those with previous VF on PI-based regimens as long as no major resistance mutations are present for the administered drug.

Method Comparison between the Siemens VERSANT® HIV-1 RNA 1.5 Assay (kPCR)* and the Abbott REALTIME HIV-1 Assay

Integrase inhibitors are recently prescribed to multi-class drug resistant HIV-1 patients in Hong Kong. Unlike pol gene, there are no FDA-approved genotypic resistance tests available for int. Limited studies compared the performance between an in-house and commercial integrase genotyping system. Information on baseline polymorphisms was also insufficient in our region.To compare integrase genotyping data obtained from an in-house and ViroSeq™ Integra48 assay, and to illustrate integrase polymorphisms on HIV-1 B and non-B subtypes in Hong Kong.A total of 283 HIV-1 patients were recruited during 2006–2012 in Hong Kong. All samples were genotyped by an in-house assay for pol and int separately, and 46 of them were further genotyped by ViroSeq™ Integra48. Polymorphisms and resistance mutations were analyzed by Stanford HIV Drug Resistance Database.The included patients were mainly infected by HIV-1 subtype B (38.9%) and CRF01_AE (43.1%), followed by CRF07_BC (5.3%), C (3.9%), CRF02_AG (2.8%), D (1.4%), CRF08_BC (1.1%) or others (3.5%). Of 46 samples genotyped by ViroSeq™ and the in-house assays, all major and minor resistance mutations were concordant. Integrase major resistance mutations were identified in two CRF01_AE raltegravir-treated patients. Integrase minor resistance mutations were observed in subtypes B and CRF01_AE.With 25% of the commercial cost, the in-house integrase genotyping assay managed to regenerate over 96% concordant results as good as the RUO ViroSeq™ assay. Further investigations are required to understand the effect on integrase minor mutations, which are present in many subtype B and CRF01_AE samples.

Characterization of natural polymorphic sites of the HIV-1 integrase before the introduction of HIV-1 integrase inhibitors in Germany.

IntroductionThe aim of our study was to analyze the occurrence and evolution of HIV-1 integrase polymorphisms during the HIV-1 epidemic in Germany prior to the introduction of the first integrase inhibitor raltegravir in 2007.Materials and MethodsPlasma samples from drug-naïve HIV-1 infected individuals newly diagnosed between 1986 and 2006 were used to determine PCR-based population sequences of the HIV-1 integrase (amino acids 1–278). The HIV-1 subtype was determined using the REGA HIV-1 subtyping tool. We calculated the frequency of amino acids at each position of the HIV-1 integrase in 337 subtype B strains for the time periods 1986–1989, 1991–1994, 1995–1998, 1999–2002, and 2003–2006. Positions were defined as polymorphic if amino acid variation was >1% in any period. Logistic regression was used to identify trends in amino acid variation over time. Resistance-associated mutations were identified according to the IAS 2013 list and the HIVdb, ANRS and GRADE algorithms.ResultsOverall, 56.8% (158/278) amino acid positions were polymorphic and 15.8% (25/158) of these positions exhibited a significant trend in amino acid variation over time. Proportionately, most polymorphic positions (63.3%, 31/49) were detected in the N-terminal zinc finger domain of the HIV-1 integrase. Motifs and residues essential for HIV-1 integrase activity were little polymorphic, but within the minimal non-specific DNA binding region I220-D270 up to 18.1% amino acid variation was noticed, including four positions with significant amino acid variation over time (S230, D232, D256, A265). No major resistance mutations were identified, and minor resistance mutations were rarely observed without trend over time. E157Q considered by HIVdb, ANRS, and GRADE algorithms was the most frequent resistance-associated polymorphism with an overall prevalence of 2.4%.ConclusionsDetailed knowledge of the evolutionary variation of HIV-1 integrase polymorphisms is important to understand the development of resistance in the presence of the drug. Our results will contribute to define the relevance of integrase polymorphisms in HIV-strains resistant to integrase inhibitors and to improve resistance interpretation algorithms.

Hepatitis C virus NS3 mutations in haemophiliacs

Haemophiliacs have high hepatitis C virus (HCV) exposure risk from blood products that did not undergo heat inactivation or disease-specific screening prior to 1987. Repeated exposure to infected factor concentrates predisposes haemophiliacs to higher likelihood of HCV from multiple sources. HIV coinfection could result in impaired clearance of less fit variants resulting in enrichment of quasispecies carrying resistance mutations. We postulated that haemophiliacs demonstrate increased prevalence of baseline signature mutations in the HCV NS3/4 serine protease coding domain. We examined the prevalence of putative HCV protease inhibitor mutations, mutations, subclassified into dominant mutations if changes conferred resistance, and minor variants not associated with drug resistance, in patients with haemophilia A or B, infected with HCV or HCV/HIV, prior to HCV PI exposure. A total of 151 subjects were evaluated, including 22 haemophiliacs and 129 non-haemophilic controls. Of the 58 mutations detected, 55 (95%) were resistance mutations and three (5%) were minor variants. Dominant mutations were detected in 10 (45.5%) haemophiliacs and in 43 (33.3%) controls (OR 1.67, 95% CI 0.67-4.16). There was no statistical difference in proportion of dominant mutations (P=0.27) or minor variants (P=0.47) between groups, despite adjustment for HIV status (P=0.44). No significant differences in dominant or minor resistance mutations between haemophiliacs and non-haemophiliacs were observed. HIV presence or prior HAART exposure did not affect baseline distribution. We conclude that haemophiliacs are not at higher risk for pre-existing HCV PI mutations, and prospective studies of response to PI-based regimens with HCV activity are indicated.

No Clinically Significant Drug-Resistance Mutations in HIV-1 Subtype C–Infected Women After Discontinuation of NRTI-Based or PI-Based HAART for PMTCT in Botswana

Risk of developing drug resistance after stopping antiretroviral regimens to prevent mother-to-child HIV-1 transmission is unknown. The Mma Bana Study randomized treatment-naive pregnant women with CD4 ≥200 cells per cubic millimeter to receive either abacavir/zidovudine/lamivudine [triple nucleoside reverse transcriptase inhibitor (NRTI) arm] or lopinavir/ritonavir/zidovudine/lamivudine [protease inhibitor (PI) arm]. Drugs were discontinued after 6 months of breastfeeding. One month after discontinuation, 29 NRTI arm samples and 25 PI arm samples were successfully genotyped. No clinically significant antiretroviral resistance mutations were detected. Eight minor resistance mutations were found among 11 (20%) women (3 from NRTI arm and 8 from PI arm), occurring at similar frequencies to those reported in HIV-1 subtype C treatment-naive cohorts.

Performance comparison of an in-house integrase genotyping assay versus the ViroSeq™ Integra48, and study of HIV-1 integrase polymorphisms in Hong Kong

BackgroundIntegrase inhibitors are recently prescribed to multi-class drug resistant HIV-1 patients in Hong Kong. Unlike pol gene, there are no FDA-approved genotypic resistance tests available for int. Limited studies compared the performance between an in-house and commercial integrase genotyping system. Information on baseline polymorphisms was also insufficient in our region. ObjectivesTo compare integrase genotyping data obtained from an in-house and ViroSeq™ Integra48 assay, and to illustrate integrase polymorphisms on HIV-1 B and non-B subtypes in Hong Kong. Study designA total of 283 HIV-1 patients were recruited during 2006–2012 in Hong Kong. All samples were genotyped by an in-house assay for pol and int separately, and 46 of them were further genotyped by ViroSeq™ Integra48. Polymorphisms and resistance mutations were analyzed by Stanford HIV Drug Resistance Database. ResultsThe included patients were mainly infected by HIV-1 subtype B (38.9%) and CRF01_AE (43.1%), followed by CRF07_BC (5.3%), C (3.9%), CRF02_AG (2.8%), D (1.4%), CRF08_BC (1.1%) or others (3.5%). Of 46 samples genotyped by ViroSeq™ and the in-house assays, all major and minor resistance mutations were concordant. Integrase major resistance mutations were identified in two CRF01_AE raltegravir-treated patients. Integrase minor resistance mutations were observed in subtypes B and CRF01_AE. ConclusionsWith 25% of the commercial cost, the in-house integrase genotyping assay managed to regenerate over 96% concordant results as good as the RUO ViroSeq™ assay. Further investigations are required to understand the effect on integrase minor mutations, which are present in many subtype B and CRF01_AE samples.

Deep Sequencing Reveals Minor Protease Resistance Mutations in Patients Failing a Protease Inhibitor Regimen

Standard genotypic antiretroviral resistance testing, performed by bulk sequencing, does not readily detect variants that comprise <20% of the circulating HIV-1 RNA population. Nevertheless, it is valuable in selecting an antiretroviral regimen after antiretroviral failure. In patients with poor adherence, resistant variants may not reach this threshold. Therefore, deep sequencing would be potentially valuable for detecting minority resistant variants. We compared bulk sequencing and deep sequencing to detect HIV-1 drug resistance at the time of a second-line protease inhibitor (PI)-based antiretroviral regimen failure. Eligibility criteria were virologic failure (HIV-1 RNA load of >500 copies/ml) of a first-line nonnucleoside reverse transcriptase inhibitor-based regimen, with at least the M184V mutation (lamivudine resistance), and second-line failure of a lopinavir/ritonavir (LPV/r)-based regimen. An amplicon-sequencing approach on the Roche 454 system was used. Six patients with viral loads of >90,000 copies/ml and one patient with a viral load of 520 copies/ml were included. Mutations not detectable by bulk sequencing during first- and second-line failure were detected by deep sequencing during second-line failure. Low-frequency variants (>0.5% of the sequence population) harboring major protease inhibitor resistance mutations were found in 5 of 7 patients despite poor adherence to the LPV/r-based regimen. In patients with intermittent adherence to a boosted PI regimen, deep sequencing may detect minority PI-resistant variants, which likely represent early events in resistance selection. In patients with poor or intermittent adherence, there may be low evolutionary impetus for such variants to reach fixation, explaining the low prevalence of PI resistance.

Prevalence of Antiretroviral Drug Resistance Mutations and HIV-1 Subtypes among Newly-diagnosed Drugnaïve Persons Visiting A Voluntary Testing and Counselling Centre in Northeastern South Africa

Data on antiretroviral drug resistance among drug-naive persons are important in developing sentinel and surveillance policies. This study was conducted to determine the prevalence of antiretroviral drug resistance mutations among drug-naïve HIV-infected individuals attending a voluntary testing and counselling centre at the Mankweng Hospital in northeastern South Africa. In total, 79 drug-naïve HIV-positive individuals were sequentially recruited during February 2008-December 2008. Drug resistance mutations were determined using the calibrated population resistance tool available on the Stanford HIV drug resistance database. Viral DNA was obtained from 57 (72%) of the 79 individuals. Reliable nucleotide sequences were obtained for 54 reverse transcriptase (RT) and 54 protease (PR) gene regions from 54 individuals. Overall, five sequences (9.3%) harboured drug resistance mutations (95% confidence interval -1.53 to 16.99). Four (7.4%) of these were nucleoside RT inhibitor mutations (D67G, D67E, T69D, and T215Y), and one (1.9%) was a PR inhibitor mutation (M46I). No major non-nucleoside RT resistance mutation was detected. Several minor resistance mutations and polymorphisms common in subtype C viruses were observed in the PR and RT genes. Phlyogenetic analysis of the partial pol sequences showed that 52 (96%) of the 54 isolates were HIV-1 subtype C. One isolate (08MB08ZA) was HIV-1 subtype B while another (08MB26ZA) was related to HIV-1 subtype J. HIV-1 subtype recombination analysis with REGA assigned the pol sequence to HIV subtype J (11_cpx) with a bootstrap value of 75%. The prevalence of drug resistance mutations observed in the population studied was relatively higher than previously reported from other parts of South Africa. In addition, this is apparently the first report of an HIV-1 subtype J-like virus from northeastern South Africa.

Single Genome Sequencing of HIV-1 Gag and Protease Resistance Mutations at Virologic Failure during the Ok04 Trial of Simplified versus Standard Maintenance Therapy

Ritonavir-boosted lopinavir (LPV/RTV) alone has been evaluated as simplified maintenance therapy for HIV-1 infection, but there are concerns about greater potential for emergence of protease inhibitor (PI) resistance. The OK04 trial evaluated maintenance therapy with LPV/RTV alone versus standard therapy (ST) with two NRTIs plus LPV/RTV in 205 patients, of whom 15 had virological rebound by week 48 (11 versus 4 patients, respectively). We developed a single genome sequencing (SGS) assay of HIV-1 gag and protease to assess the emergence of low frequency drug-resistant variants during virological rebound. Plasma samples from 15 subjects at virological rebound were analysed by SGS of HIV-1 gag and protease genes. A total of 45 SGS sequences were planned per sample, providing 90% power to detect variants comprising >5% of the virus population. Overall, 521 single sequences obtained from 13 patients (range 4-48 sequences/patient) revealed similar frequencies of major protease resistance mutations in samples from the LPV/RTV alone (3/11) and ST (3/4) arms (P=0.10), with a median number of minor protease resistance mutations of 3.0 versus 3.5, respectively (P=0.23). Median number of gag PI resistance mutations were similar between the LPV/RTV alone and ST arms at cleavage sites (3.0 versus 2.5; P=0.83), non-cleavage sites (21 versus 16.5; P=0.71) and the transframe protein-p6 pol region cleavage sites (4.0 versus 3.0; P=0.6). Although more subjects with simplified maintenance therapy with LPV/RTV alone had virological rebound compared to the ST arm, this was not associated with more frequent emergence of variants encoding PI resistance mutations in gag or protease detected by SGS.

Impact of HIV-1 Group O Genetic Diversity on Genotypic Resistance Interpretation by Algorithms Designed for HIV-1 Group M

HIV-1 group O (HIV-O) is characterized by a high genetic divergence from HIV-1 group M viruses. Little is known about the therapeutic impact of this diversity. The aim of this study was to assess in a large series of samples (1) the genotypic impact of natural polymorphism of the HIV-O reverse transcriptase and protease genes; and (2) the predictive value of resistance interpretation algorithms developed for HIV-1 group M when used for highly mutated HIV-O viruses. Sixty-eight antiretroviral-naive and 9 highly antiretroviral-experienced HIV-O-infected patients were included. The viruses were sequenced and resistance-associated mutations were identified using 3 different algorithms (Agence Nationale de Recherches sur le SIDA et les hépatites virales, Rega, Stanford). All HIV-O samples naturally exhibited the A98G and V179E resistance mutations in the reverse transcriptase region; 54% of samples presented the Y181C mutation, conferring resistance to nonnucleoside reverse transcriptase inhibitors. Twelve minor resistance mutations, present in more than 75% of the protease sequences, led to the different algorithms giving discrepant results for nelfinavir and saquinavir susceptibility. A marked virological response was observed in 8 of the 9 antiretroviral-experienced patients, despite the prediction of limited activity of the combination for 5 to 8 patients according to the algorithm used. The high level of natural polymorphism in HIV-O genes, and the important discrepancies between genotypic resistance interpretation and the virological response, emphasize the need for resistance algorithm rules better adapted to HIV-O.

Short Communication: Antiretroviral Therapy Resistance Mutations Present in the HIV Type 1 Subtype CpolandenvRegions from Therapy-Naive Patients in Zambia

The prevalence of antiretroviral therapy (ART) resistance mutations present in HIV-1 subtype C pol and env regions of the proviral DNA was analyzed and compared from therapy-naive individuals before (Cohort A) and after (Cohort B) the availability of free ART in Zambia. Mutations present in sequences published in a previous study from Zambian ART-naive individuals infected with subtype C were analyzed using current parameters for the classification of ART drug resistance and compared with Cohorts A and B. No statistically significant differences were observed when comparing mutations present in the pol and env of these cohorts. However, an increase in the number of minor, borderline, or partial resistance mutations as well as the presence of major resistance mutations were observed in Cohort B. These results suggest there is an increasing trend of drug resistance-associated mutations that could be a result of the availability of free ART in Zambia. Moreover, the high prevalence of resistance mutations observed for maraviroc and vicriviroc in both cohorts may suggest a limited efficacy of entry inhibitors on HIV-1 subtype C.

Tissue culture drug resistance analysis of a novel HIV-1 protease inhibitor termed PL-100 in non-B HIV-1 subtypes

PL-100 is a novel HIV-1 protease inhibitor (PI) that maintains activity against viruses that are resistant to other PIs. To further characterize this compound, we used it to select for drug resistance in tissue culture, using two non-B HIV-1 subtypes, viz. subtype C and a CRF01_AE recombinant virus. PL-100 selected for both minor and major PI resistance mutations along either of two distinct pathways. One of these involved the V82A and L90M resistance mutations while the other involved a mutation at position T80I, with other mutations being observed at positions M46I/L, I54M, K55R, L76F, P81S and I85V. The resistance patterns in both subtype C and CRF01_AE were similar and an accumulation of at least three mutations in the flap and active sites were required in each case for high-level resistance to occur, demonstrating that PL-100 has a high genetic barrier against the development of drug resistance.

Analysis of HIV Type 1 gp41 and Enfuvirtide Susceptibility among Men in the United States Who Were HIV Infected Prior to Availability of HIV Entry Inhibitors

We analyzed HIV gp41 from 195 men in the United States who were HIV-1 infected between 1999 and 2002, before enfuvirtide (ENF) was approved for clinical use in the United States. gp41 genotyping results were obtained for 175 samples. None of the samples had major ENF resistance mutations. Six (3.4%) samples had minor ENF resistance mutations in the HR1 region (V38G, N43K, L44M, L45M). Twenty-eight (16%) samples had the N42S polymorphism, which is associated with ENF hypersusceptibility. Accessory mutations in the HR2 region were identified in some samples (E137K, S138A). Five of the six samples with HR1 resistance mutations were analyzed with a phenotypic assay; one sample had reduced ENF susceptibility (a sample with N42S +L44M + E137K). Prior to the availability of ENF, some men in the United States were infected with HIV that contained mutations associated with ENF resistance or hypersusceptibility. However, most of the mutations were not associated with phenotypic ENF resistance.

Positive Selection Pressure Introduces Secondary Mutations at Gag Cleavage Sites in Human Immunodeficiency Virus Type 1 Harboring Major Protease Resistance Mutations

Human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) specifically target the HIV-1 protease enzyme. Mutations in the enzyme can result in PI resistance (termed PI mutations); however, mutations in the HIV-1 gag region, the substrate for the protease enzyme, might also lead to PI resistance. We analyzed gag and pol sequence data from the following 313 HIV-1-infected patients: 160 treatment-naïve patients, 93 patients failing antiretroviral treatment that included a PI (with no major PI mutations), and 60 patients failing antiretroviral treatment that included a PI (with major PI mutations). Additional sequences from 13 patients were included for longitudinal analysis. We assessed positive selection pressure on the gag/protease region using a test for the overall influence of positive selection and a total of five tests to identify positively selected single codons. We found that positive selection pressure was the driving evolutionary force for the gag region in all three patient groups. An increase in positive selection was observed in gag cleavage site regions p7/p1/p6 only after the acquisition of major PI mutations, suggesting that amino acids in gag cleavage sites under positive selection pressure could function as compensatory mutations for major PI mutations in the protease region. Isolated gag mutations did not appear to confer PI resistance, but mutations in the gag cleavage sites could substitute for minor PI resistance mutations in the protease region.

Differences in resistance mutations among HIV-1 non-subtype B infections: a systematic review of evidence (1996-2008).

Ninety percent of HIV-1-infected people worldwide harbour non-subtype B variants of HIV-1. Yet knowledge of resistance mutations in non-B HIV-1 and their clinical relevance is limited. Although a few reviews, editorials and perspectives have been published alluding to this lack of data among non-B subtypes, no systematic review has been performed to date.With this in mind, we conducted a systematic review (1996–2008) of all published studies performed on the basis of non-subtype B HIV-1 infections treated with antiretroviral drugs that reported genotype resistance tests. Using an established search string, 50 studies were deemed relevant for this review.These studies reported genotyping data from non-B HIV-1 infections that had been treated with either reverse transcriptase inhibitors or protease inhibitors. While most major resistance mutations in subtype B were also found in non-B subtypes, a few novel mutations in non-B subtypes were recognized. The main differences are reflected in the discoveries that: (i) the non-nucleoside reverse transcriptase inhibitor resistance mutation, V106M, has been seen in subtype C and CRF01_AE, but not in subtype B, (ii) the protease inhibitor mutations L89I/V have been reported in C, F and G subtypes, but not in B, (iii) a nelfinavir selected non-D30N containing pathway predominated in CRF01_AE and CRF02_AG, while the emergence of D30N is favoured in subtypes B and D, (iv) studies on thymidine analog-treated subtype C infections from South Africa, Botswana and Malawi have reported a higher frequency of the K65R resistance mutation than that typically seen with subtype B.Additionally, some substitutions that seem to impact non-B viruses differentially are: reverse transcriptase mutations G196E, A98G/S, and V75M; and protease mutations M89I/V and I93L.Polymorphisms that were common in non-B subtypes and that may contribute to resistance tended to persist or become more frequent after drug exposure. Some, but not all, are recognized as minor resistance mutations in B subtypes. These observed differences in resistance pathways may impact cross-resistance and the selection of second-line regimens with protease inhibitors. Attention to newer drug combinations, as well as baseline genotyping of non-B isolates, in well-designed longitudinal studies with long duration of follow up are needed.