Resistant Subtypes Research Articles

ERCC1, XPF and XPA-locoregional differences and prognostic value of DNA repair protein expression in patients with head and neck squamous cell carcinoma.

Nucleotide excision repair protein expression has been claimed to be responsible for platinum-based chemotherapy resistance. ERCC1, XPF and XPA, core proteins in DNA repair, were evaluated regarding their prognostic value in patients with head and neck squamous cell carcinoma by looking at overall survival and time to recurrence. Tissue microarrays were constructed from 453 cases of HNSCC, including 222 oral (49%), 126 oropharyngeal (27.8%) and 105 laryngeal (23.2%) tumours. There were 284 XPF, 293 XPA and 294 ERCC1 specimens evaluable for protein expression analysis after immunohistochemical workup. Expression levels were dichotomised into high- and low-expressing groups. Outcomes for overall survival (OS) and time to recurrence (TTR) were analysed using the Kaplan-Meier method. No correlation between ERCC1, XPA and XPF expression and OS was found by looking at the overall patient cohort. However, subsite analysis revealed that high ERCC1 expression was associated with a significantly inferior OS in patients with SCC of the oral cavity (p = 0.028) and showed an independent predictive value in multivariate analysis (p = 0.0123). High XPA expression showed a significantly increased OS in patients with oropharyngeal SCC (p = 0.0386). Regarding XPF, no impact on OS in any subsite could be shown. While high ERCC1 expression functions as a predictive marker with decreased OS in patients with squamous cell carcinoma of the oral cavity, high XPA expression shows an inverse effect in the subsite of the oropharynx, which has not been described previously. ERCC1 and XPA might be candidates to overcome chemotherapy resistance in subtypes of HNSCC.

Abstract 4938: Molecular mechanisms of intrinsic resistance to taxanes in gastric cancer

Abstract Gastric cancer (GC) is histologically divided into intestinal (INT) and diffuse (DIF) clinical subtypes. Even though these two GC groups are structurally and biologically different, this classification is not used to inform choice of treatment. Taxanes (paclitaxel, docetaxel (DTX) and cabazitaxel) are widely used for cancer treatment and for GC specifically the TAX-325 study revealed therapeutic benefit when DTX was added to the standard chemotherapy regimen. Despite these improvements, however, patients often exhibit intrinsic or acquired resistance to DTX adversely affecting patient survival. Yet, the molecular basis of clinical drug resistance remains poorly elucidated posing a major barrier for the effective treatment of GC patients. We performed a post-hoc analysis of the TAX-325 study to examine the potential influence of GC subtypes in clinical response to DTX. We classified randomized patients as diffuse or non-diffuse histology and correlated histology with clinical outcomes using a Cox proportional hazards model. Non-diffuse GC showed a significant improvement in overall survival with the addition of DTX (12.1 v 8.8 mo, p = 0.002), whereas diffuse histology was not associated with an improvement in survival (8.3 v 8.5 mo, p = 0.66). To investigate the molecular mechanism of GC DIF subtype resistance to taxanes, we used a panel of 12 GC cell lines representative of both subtypes (4 intestinal subtype, 8 diffuse subtype). DTX cytotoxicity assays revealed that similarly to what we observed clinically, 63% (5/8) of the DIF GC cell lines were resistant (IC50 > 600 nM) to DTX compared to 25% (1/4) of the INT GC cell lines. Further functional studies revealed that there was minimal DTX drug-target engagement in the DIF GC cells, assessed by confocal microscopy of the microtubule (MT) network and tubulin polymerization assays. These results suggested that DTX interaction with its target, MT, was impaired in the DIF GC cell lines. To rule out multi-drug resistance (MDR) as potential cause of intrinsic DTX resistance in these cells we performed flow cytometric evaluation of P-glycoprotein and found that all of the DIF GC cell lines were negative. Additionally, drug accumulation studies with C-14 radiolabeled DTX revealed that the drug accumulated intracellularly in all of GC cell lines in our panel. Next generation sequencing of our panel of untreated or DTX-treated GC cell lines revealed 84 significantly differentially expressed genes in drug-sensitive cell lines but no changes in resistant cells. Our analysis showed a significant enrichment and a transcriptional co-regulation after treatment of genes encoding for kinesins, motor proteins associated with MT, in DTX-sensitive cells but not in DTX-resistant cells. These studies will provide novel insights into mechanism of drug resistance and sensitivity and will ultimately allow us to design more effective targeted therapies to overcome chemo-resistance and eventually prolong patient survival. Citation Format: Giuseppe Galletti, Chao Zhang, Kyle Cleveland, Doron Betel, Manish Shah, Paraskevi Giannakakou. Molecular mechanisms of intrinsic resistance to taxanes in gastric cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4938.

Phenotype, Genotype, and Drug Resistance in Subtype C HIV-1 Infection.

Virologic failure in subtype C is characterized by high resistance to first-line antiretroviral (ARV) drugs, including efavirenz, nevirapine, and lamivudine, with nucleoside resistance including type 2 thymidine analog mutations, K65R, a T69del, and M184V. However, genotypic algorithms predicting resistance are mainly based on subtype B viruses and may under- or overestimate drug resistance in non-B subtypes. To explore potential treatment strategies after first-line failure, we compared genotypic and phenotypic susceptibility of subtype C human immunodeficiency virus 1 (HIV-1) following first-line ARV failure. AIDS Clinical Trials Group 5230 evaluated patients failing an initial nonnucleoside reverse-transcriptase inhibitor (NNRTI) regimen in Africa and Asia, comparing the genotypic drug resistance and phenotypic profile from the PhenoSense (Monogram). Site-directed mutagenesis studies of K65R and T69del assessed the phenotypic impact of these mutations. Genotypic algorithms overestimated resistance to etravirine and rilpivirine, misclassifying 28% and 32%, respectively. Despite K65R with the T69del in 9 samples, tenofovir retained activity in >60%. Reversion of the K65R increased susceptibility to tenofovir and other nucleosides, while reversion of the T69del showed increased resistance to zidovudine, with little impact on other NRTI. Although genotype and phenotype were largely concordant for first-line drugs, estimates of genotypic resistance to etravirine and rilpivirine may misclassify subtype C isolates compared to phenotype.

Genetic Changes in HIV-1 Gag-Protease Associated with Protease Inhibitor-Based Therapy Failure in Pediatric Patients.

Studies have shown a low frequency of HIV-1 protease drug resistance mutations in patients failing protease inhibitor (PI)-based therapy. Recent studies have identified mutations in Gag as an alternate pathway for PI drug resistance in subtype B viruses. We therefore genotyped the Gag and protease genes from 20 HIV-1 subtype C-infected pediatric patients failing a PI-based regimen. Major protease resistance mutations (M46I, I54V, and V82A) were identified in eight (40%) patients, as well as Gag cleavage site (CS) mutations (at codons 373, 374, 378, 428, 431, 449, 451, and 453) in nine (45%) patients. Four of these Gag CS mutations occurred in the absence of major protease mutations at PI failure. In addition, amino acid changes were noted at Gag non-CS with some predicted to be under HLA/KIR immune-mediated pressure and/or drug selection pressure. Changes in Gag during PI failure therefore warrant further investigation of the Gag gene and its role in PI failure in HIV-1 subtype C infection.

Time trends in HIV-1 transmitted drug resistance mutation frequency in Poland.

IntroductionIn Poland, the HIV epidemic has shifted recently from being predominantly related to injection drug use (IDU) to being driven by transmissions among men-who-have-sex-with-men (MSM). The number of new HIV cases has increased in the recent years, while no current data on the transmitted drug resistance associated mutations (tDRM) frequency trend over time are available from 2010. In this study, we analyze the temporal trends in the spread of tDRM from 2008 to 2013.Materials and MethodsPartial pol sequences from 833 antiretroviral treatment-naive individuals of European descent (Polish origin) linked to care in 9 of 17 Polish HIV treatment centres were analyzed. Drug resistance interpretation was performed according to WHO surveillance recommendations, subtyping with REGA genotyping 2.0 tool. Time trends were examined for the frequency of t-DRM across subtypes and transmission groups using logistic regression (R statistical platform, v. 3.1.0).ResultsFrequency of tDRM proved stable over time, with mutation frequency change from 11.3% in 2008 to 8.3% in 2013 [OR: 0.91 (95% CI 0.80–1,05), p=0.202] (Figure 1a). Also, no significant differences over time were noted for the subtype B (decrease from 8.4% 2008 to 6.2% in 2013 [OR: 0.94 (95% CI 0.79–1.11), p=0.45] and across non-B variants [change from 22.6% 2008 to 23.1% in 2013, OR: 0.94 (95% CI 0.75–1.19), p=0.62]. When patient groups were stratified according to transmission route, in MSM there was a trend for a NNRTI t-DRM decrease (from 6.8% 2008 to 1% in 2013, OR: 0.61 (95% CI 0.34–1.02), p=0.0655, slope −0.74%/year) (Figure 1b), related to the subtype B infected MSM (decrease from 7% 2008 to 1% in 2013, OR: 0.61 (95% CI 0.34–1.03), p=0.0662, slope −0.75%/year). Overall tDRM frequency decrease was also noted for the heterosexually infected patients [from 17.6% 2008 to 10.3% in 2013, OR: 0.83 (95% CI 0.67–1.02, p=0.077, slope −2.041%/year)] but did not associate with drug class (Figure 1c). In IDUs, the trends in t-DRM frequency were not significant over time (change from 1.9% in 2008 to 0 in 2013 [OR:1.24 (95% CI 0.73–2.26), p=0.4)].ConclusionsThe frequency of t-DRM in Poland is generally stable over time. Decrease in the overall tDRM frequency in heterosexual infected cases and NNRTI resistance in subtype B infected MSM may be related to the higher treatment efficacy of current cART.

Abstract 897: Elucidating the molecular basis of intrinsic taxane resistance in gastric cancer

Abstract Gastric cancer (GC) is histologically divided into intestinal (INT) and diffuse (DIF) clinical subtypes. Even though these two GC groups are structurally and biologically different, this classification is not used to inform choice of treatment. The taxanes (i.e., docetaxel and cabazitaxel) are widely used for the treatment of cancer and for GC specifically the TAX-325 study revealed a significantly improved survival when docetaxel (DTX) was added to cisplatin and fluorouracil. Despite this advancement, patients still exhibit intrinsic or acquired resistance to DTX. Yet, the molecular basis of drug resistance remains unknown. We performed a post-hoc analysis of the TAX-325 study to examine the potential influence of GC subtypes in clinical response to DTX. We classified randomized patients as diffuse or non-diffuse histology and correlated histology with clinical outcomes using a Cox proportional hazards model. Non-diffuse GC showed a significant improvement in overall survival with the addition of DTX (12.1 v 8.8 mo, p=0.002), whereas diffuse histology was not associated with an improvement in survival (8.3 v 8.5 mo, p=0.66). To investigate the molecular mechanism of GC DIF subtype resistance to taxanes, we used a panel of 12 GC cell lines representative of both subtypes (4 intestinal subtype, 8 diffuse subtype). DTX cytotoxicity assays revealed that similarly to what we observed clinically, 5/8 (63%) DIF GC cell lines were resistant (IC50 > 600 nM) to DTX compared to 1/4 (25%) INT GC cell lines. Further functional studies revealed that there was minimal DTX drug-target engagement in the DIF GC cells, as, assessed by confocal microscopy of the microtubule network and tubulin polymerization assays. These results suggested that DTX interaction with its target, microtubule, was impaired in the DIF GC cell lines. To rule out multi-drug resistance (MDR) as potential cause of intrinsic DTX resistance in these cells we performed flow cytometric evaluation of P-glycoprotein and found that all of the DIF GC cell lines were negative. Additionally, drug accumulation studies with C-14 radiolabeled DTX revealed that the drug accumulated intracellularly in all of GC cell lines in our panel. Next generation sequencing of our panel of untreated or DTX-treated GC cell lines revealed a number of genes/pathways that are differentially expressed in the drug-sensitive versus the resistant cell lines. Gene set enrichment analysis is currently ongoing in order to identify a subset of genes/pathways implicated in intrinsic DTX resistance in GC. These studies will provide novel insights into mechanism of intrinsic drug resistance and will ultimately allow us to design more effective targeted therapies to overcome chemo-resistance and eventually prolong patient survival. Citation Format: Giuseppe Galletti, Kyle Cleveland, Chao Zhang, Ada Gjyrezi, Alexandre Matov, Doron Betel, Manish A. Shah, Paraskevi Giannakakou. Elucidating the molecular basis of intrinsic taxane resistance in gastric cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 897. doi:10.1158/1538-7445.AM2014-897

Early selection of resistance-associated mutations in HIV-1 RT C-terminal domains across different subtypes: role of the genetic barrier to resistance.

Interpretation of drug resistance mutation (DRM) has been based solely on HIV-1 subtype B. Reverse transcriptase (RT) C-terminal domains have been disregarded in resistance interpretation, as their clinical relevance is still controversial. We determined the emergence of DRM in RT C-terminal domains of different HIV-1 subtypes, the genetic barrier for the acquisition of these DRM and their temporal appearance with 'classical' RT inhibitor (RTI) mutations. HIV-1 RT sequences were obtained from information from 6087 treatment-naive and 3795 RTI-treated patients deposited in the Stanford HIV Resistance Database, including all major subtypes. DRM emergence was evaluated for subtype B, and was correlated with the number of DRM in the polymerase domain. Genetic barrier was calculated for each DRM studied and in each subtype. N348I, T369I and A360V were found at low prevalence in treatment-naive isolates of all subtypes. A371V was common to treatment-naive isolates. N348I was observed in all subtypes, while T369I was only selected in subtype C. A360V and T369V were selected by RTI treatment in several subtypes. A371V was selected in subtypes B and C, but is a signature in subtype A. RT C-terminal mutations were correlated with early drug resistance in subtype B. All subtypes have a low calculated genetic barrier towards C-terminal DRM acquisition, despite a few disparities having been observed. C-terminal mutations were selected in all HIV-1 subtypes, while some represent subtype-specific signatures. The selection of C-terminal DRMs occurs early in RTI resistance failure in subtype B.

Resistance of Subtype C HIV-1 Strains to Anti-V3 Loop Antibodies

HIV-1's subtype C V3 loop consensus sequence exhibits increased resistance to anti-V3 antibody-mediated neutralization as compared to the subtype B consensus sequence. The dynamic 3D structure of the consensus C V3 loop crown, visualized by ab initio folding, suggested that the resistance derives from structural rigidity and non-β-strand secondary protein structure in the N-terminal strand of the β-hairpin of the V3 loop crown, which is where most known anti-V3 loop antibodies bind. The observation of either rigidity or non-β-strand structure in this region correlated with observed resistance to antibody-mediated neutralization in a series of chimeric pseudovirus (psV) mutants. The results suggest the presence of an epitope-independent, neutralization-relevant structural difference in the antibody-targeted region of the V3 loop crown between subtype C and subtype B, a difference that we hypothesize may contribute to the divergent pattern of global spread between these subtypes. As antibodies to a variable loop were recently identified as an inverse correlate of risk for HIV infection, the structure-function relationships discussed in this study may have relevance to HIV vaccine research.

Impact of nucleotide polymorphisms at drug resistance sites on genetic barrier in human immunodeficiency virus type 1 subtype C resistance evolution

Genetic diversity is the hallmark of human immunodeficiency virus (HIV), the causative agent of acquired immune deficiency syndrome (AIDS). This diversity has resulted in a spectrum of different subtypes within the group M viruses that is responsible for the AIDS pandemic. Nucleotide substitutions and polymorphisms at codons known to confer drug resistance in subtype B viruses were compared with similar substitutions in subtype C viruses. Genetic barrier was determined on viruses isolated from drug naive patients infected with subtype C viruses. We found a reduced genetic barrier in subtype C viruses at codon V106M (GTA to ATG) and an increased barrier at codon L210W (TTA/CTG/CTA to TGG) when compared to subtype B consensus. The highest genetic barrier in subtype C viruses is found at codon Q151M where two transversions or each one of transition and transversion are needed for the resistance evolution.

Impact of nucleotide polymorphisms at drug resistance sites on genetic barrier in human immunodeficiency virus type 1 subtype C resistance evolution

Genetic diversity is the hallmark of human immunodeficiency virus (HIV), the causative agent of acquired immune deficiency syndrome (AIDS). This diversity has resulted in a spectrum of different subtypes within the group M viruses that is responsible for the AIDS pandemic. Nucleotide substitutions and polymorphisms at codons known to confer drug resistance in subtype B viruses were compared with similar substitutions in subtype C viruses. Genetic barrier was determined on viruses isolated from drug naive patients infected with subtype C viruses. We found a reduced genetic barrier in subtype C viruses at codon V106M (GTA to ATG) and an increased barrier at codon L210W (TTA/CTG/CTA to TGG) when compared to subtype B consensus. The highest genetic barrier in subtype C viruses is found at codon Q151M where two transversions or each one of transition and transversion are needed for the resistance evolution.

Replicative Fitness Costs of Nonnucleoside Reverse Transcriptase Inhibitor Drug Resistance Mutations on HIV Subtype C

Single-dose nevirapine (NVP) is quite effective in preventing transmission of the human immunodeficiency virus (HIV) from mother to child; however, many women develop resistance to NVP in this setting. Comparing outcomes of clinical studies reveals an increased amount of resistance in subtype C relative to that in other subtypes. This study investigates how nonnucleoside reverse transcriptase inhibitor (NNRTI) drug resistance mutations of subtype C affect replication capacity. The 103N, 106A, 106M, 181C, 188C, 188L, and 190A drug resistance mutations were placed in a reverse transcriptase (RT) that matches the consensus subtype C sequence as well as the HXB2 RT, as a subtype B reference. The replicative fitness of each mutant was compared with that of the wild type in a head-to-head competition assay. The 106A mutant of subtype C would not grow in the competition assay, making it the weakest virus tested. The effect of the 106M mutation was weaker than those of the 181C and 188C mutations in the consensus C RT, but in subtype B, this difference was not seen. To see if the 106A mutation in a different subtype C background would have a different replicative profile, the same NNRTI resistance mutations were added to the MJ4 RT, a reference subtype C molecular clone. In the context of MJ4 RT, the 106A mutant was not the only mutant that showed poor replicative fitness; the 106M, 188C, and 190A mutants also failed to replicate. These results suggest that NNRTIs may be a cost-effective alternative for salvage therapy if deleterious mutations are present in a subtype C setting.

Factors Affecting Template Usage in the Development of K65R Resistance in Subtype C Variants of HIV Type-1

We have shown that the K65R resistance mutation in HIV type-1 (HIV-1) reverse transcriptase (RT) is selected more rapidly in subtype C than subtype B HIV-1 in biochemical, cell culture and clinical studies. Template-usage experiments demonstrated that subtype C nucleotide coding sequences caused RT to preferentially pause, leading to K65R acquisition. This new study now further establishes the basis for differential occurrence of both K65R and thymidine analogue mutations (TAMs) between subtypes. Gel-based nucleotide extension assays were used to study the homopolymeric sequence surrounding K65. When positive double-stranded DNA synthesis was evaluated from a negative single-stranded DNA template, pausing at the 67 region, which is linked to occurrence of TAMs, was alleviated with both subtype B and C templates at high dCTP concentrations, but this alleviation was more pronounced with the subtype C template. By contrast, pausing at the 65 region on the subtype C but not subtype B template always occurred and was not alleviated at high levels of nucleotide triphosphates or by other means. Furthermore, templates containing repeats of the homopolymeric sequence spanning codons 64-66 of pol showed corresponding pausing repeats at the 65 region with the subtype C template only. Inverted RNA and DNA templates both displayed pausing at position K65 for the subtype C template and a ladder of pausing events culminating at codon 67 for the subtype B templates. These results further establish a mechanistic basis for the exclusion of both K65R and TAMs on single templates as well as the preferential acquisition of K65R in subtype C viruses.

Effect of human immunodeficiency virus type 1 protease inhibitor therapy and subtype on development of resistance in subtypes B and G

Europe is currently observing a significant rise in non-B subtypes. Consequently, the effect of genetic variability on therapy response or genotypic resistance interpretation algorithms is an emerging concern. The purpose of this study is to investigate the amino acid substitutions selected under drug pressure in the protease of human immunodeficiency virus type 1 (HIV-1) subtypes B and G, and determine if there are any significant differences. We investigated therapy-related and subtype-related substitutions in the protease, considering subtype, overall protease inhibitor treatment and individual drug exposure. Many mutations were significantly related to protease inhibitor (PI) therapy, with mutations exclusive to subtype B or subtype G. Some mutations are at positions related to resistance in both subtypes, but the amino acid substitution is different. Other mutations were significantly associated with subtype and PI selective pressure ( p < 0.05), pointing towards a differential selective pressure in both subtypes. We confirmed previous reports on the subtype-dependent selection of D30N and 89I, and identified a new mutation with such differential selective pressure: 37D was preferentially selected by lopinavir in subtype B. Other novel mutations found under therapy pressure were 13A, 35N, K55R, I66F, I72L/T, T74S, 82M and 89I/V. Our study indicates that even though in general, drug selective pressure and resistance pathways are relatively similar between subtypes B and G, some differences do occur, leading to subtype-dependent substitutions.

Effects of the K65R and K65R/M184V reverse transcriptase mutations in subtype C HIV on enzyme function and drug resistance

BackgroundWe investigated the effects of mutations K65R and K65R plus M184V on enzymatic function and mechanisms of drug resistance in subtype C reverse transcriptase (RT).MethodsRecombinant subtype C HIV-1 RTs containing K65R or K65R+M184V were purified from Escherichia coli. Enzyme activities and tenofovir (TFV) incorporation efficiency by wild-type (WT) and mutant RTs of both subtypes were determined in cell-free assays. Efficiency of (-) ssDNA synthesis and initiation by subtype C RTs was measured using gel-based assays with HIV-1 PBS RNA template and tRNA3Lys as primer. Single-cycle processivity was assayed under variable dNTP concentrations. Steady-state analysis was performed to measure the relative inhibitory capacity (ki/km) of TFV-disphosphate (TFV-DP). ATP-dependent excision and rescue of TFV-or ZDV-terminated DNA synthesis was monitored in time-course experiments.ResultsThe efficiency of tRNA-primed (-)ssDNA synthesis by subtype C RTs was: WT > K65R > K65R+M184V RT. At low dNTP concentration, K65R RT exhibited lower activity in single-cycle processivity assays while the K65R+M184V mutant showed diminished processivity independent of dNTP concentration. ATP-mediated excision of TFV-or ZDV-terminated primer was decreased for K65R and for K65R+M184V RT compared to WT RT. K65R and K65R+M184V displayed 9.8-and 5-fold increases in IC50 for TFV-DP compared to WT RT. The Ki/Km of TFV was increased by 4.1-and 7.2-fold, respectively, for K65R and K65R+M184V compared to WT RT.ConclusionThe diminished initiation efficiency of K65R-containing RTs at low dNTP concentrations have been confirmed for subtype C as well as subtype B. Despite decreased excision, this decreased binding/incorporation results in diminished susceptibility of K65R and K65R+M184 RT to TFV-DP.

Tipranavir resistance associated mutations in protease inhibitor-naïve patients with HIV-1 subtype A/E infection

Background Tipranavir-resistance associated mutations (TPV-RAMs) are often observed among patients with HIV-1 subtype A/E infection. Data regarding TPV resistance in subtype A/E is still limited. Objectives To determine the prevalence of TPV-RAMs among protease inhibitor-naïve, HIV-1 subtype A/E infected patients. Study design Genotypic resistance testing was conducted among HIV-1-infected patients who were PI-naïve. Results We studied 112 patients (mean age, 40.7 years; 58% male). Median CD4 cell count and HIV-1 RNA were 192 cells/mm 3 and 4.2 log copies/mL, respectively. Ninety-three patients (83%) infected with subtype A/E; the others had subtype B. The most common TPV-RAMs were M36I (88%), H69K (61%), and I13V (48%). Median number of TPV-RAMs was 3 mutations. Patients with subtype A/E had higher prevalence of I13V (54% vs. 21%, P = 0.011), M36I (96% vs. 53%, P < 0.001), H69K (68% vs. 26%, P = 0.001), and >2 TPV-RAMs (62% vs. 21%, P = 0.002). In multivariate analysis, only subtype A/E was associated with the occurrence of >2 TPV-RAMs (OR 9.83; 95%CI, 1.95–39.57; P = 0.006). Conclusions TPV-RAMs previously described by IAS-USA are commonly observed in PI-naïve patients with HIV-1 subtype A/E infection. Further studies to define virologic response of subtype A/E to TPV and clinical validation of TPV-RAMs in HIV-1 subtype A/E are essentially needed.

GENOTYPIC RESISTANCE PROFILE OF HIV-1 PROTEASE GENE: A PRELIMINARY REPORT FROM VELLORE, SOUTH INDIA

HIV-1 subtypes other than B are responsible for most new HIV infections worldwide; virus sequence data for drug resistance is described only from a limited number of non-B subtype HIV-1. This study is on mutations and polymorphisms of HIV-1 protease gene that can predict drug resistance in subtype C. The genotypic resistance assay was carried out on 38 HIV-1 strains with their plasma RNA and in nine, the proviral protease gene was sequenced. The treatment naïve strains showed minor resistance mutations, there were no major resistance mutations in the protease gene. We suggest the use of resistance testing to monitor individuals on therapy and also before initiation of therapy, gathering more sequence information for a data bank of Indian strains.

Impact of HIV-1 protease mutations A71V/T and T74S on M89I/V-mediated protease inhibitor resistance in subtype G isolates

Non-B human immunodeficiency virus (HIV)-1 subtypes possess several amino acid signatures in the viral protease that distinguish them from subtype B, some of which are reported as secondary drug-related mutations. We have previously shown a strong statistical interdependency of residues 71, 89 and 90 in subtype G, but the impact of substitutions on protease inhibitor (PI) resistance is unknown. We selected subtype G viruses from patients with diverse amino acid combinations at codons 71 (A/T), 74 (T/S), 89 (I/L/M/V) and 90 (L/M). Viral protease genes were inserted into an HIV molecular clone (HXB2). PI drug susceptibilities of chimeric viruses were determined. In isolates displaying 89I/V in combination with A71 or T74, a reversal to subtype G wild-type 89M was observed after growth in the absence of PI. The presence of 71T in one isolate and 74S in another allowed the persistence of 89I. Mutation 90M conferred intermediate but significant degrees of drug resistance to ritonavir and nelfinavir in subtype G viruses. The combination of 71T or 74S, 89I and 90M resulted in higher levels of resistance to those PIs. Our results point to the hypothesis that 71T or 74S stabilizes 89I in the protease of subtype G, whose association was previously seen by Bayesian network analyses. The association of 89I with 90M may further increase the PI resistance of subtype G viruses when compared with 90M alone, highlighting novel mutational profiles for drug resistance in this non-B subtype.

Genotypic resistance profile of HIV-1 protease gene: A preliminary report from Vellore, south India

HIV-1 subtypes other than B are responsible for most new HIV infections worldwide; virus sequence data for drug resistance is described only from a limited number of non-B subtype HIV-1. This study is on mutations and polymorphisms of HIV-1 protease gene that can predict drug resistance in subtype C. The genotypic resistance assay was carried out on 38 HIV-1 strains with their plasma RNA and in nine, the proviral protease gene was sequenced. The treatment naïve strains showed minor resistance mutations, there were no major resistance mutations in the protease gene. We suggest the use of resistance testing to monitor individuals on therapy and also before initiation of therapy, gathering more sequence information for a data bank of Indian strains.

Increasing HIV Type 1 Polymorphic Diversity But No Resistance to Antiretroviral Drugs in Untreated Patients from Central African Republic: A 2005 Study

As the HIV-1 pandemic becomes increasingly complex and as new countries acceed to antiretroviral drugs, the molecular characterization of HIV-1 strains circulating has important implications for vaccine research and for the efficacy of treatments. To follow the evolution of HIV-1 diversity in African countries, we have carried out a molecular analysis of HIV-1 strains collected from 150 HIV-1-positive pregnant women recruited in Bangui, Central African Republic (CAR). We have sequenced reverse transcriptase (RT) and protease (PROT) genes to (1) characterize the subtypes and CRFs, (2) describe the polymorphism of RT and PROT, particularly at the positions of drug resistance mutations in subtype B, and (3) observe potential drug resistance mutations and evaluate the prevalence of isolates bearing such mutations in this untreated population. The results showed that there is a very high and increasing diversity of HIV-1 strains circulating in CAR; out of 117 samples sequenced, we have observed 45 CRF11_cpx, 22 subtypes A1, 13 subtypes G, 7 subtypes CRF01_AE, 3 subtypes B, 3 subtypes CRF02_AG, 2 of each subtype F2 and CRF09_cpx, and one of each subtype D, J, C, H, CRF06_cpx, CRF13_cpx, and CRF19_cpx; the remaining 13 strains showed discordant genomic results suggesting multiple recombinations leading to mosaic viruses. The polymorphism of RT and PROT was high compared to subtype B, particularly at some positions that have been involved in antiretroviral resistance in subtype B, but we could not observe any major resistance mutation in this sample of untreated patients. The prevalence of drug resistance mutations in this population was therefore clearly under the WHO 5% threshold.

Nucleotide and amino acid polymorphisms at drug resistance sites in non-B-subtype variants of human immunodeficiency virus type 1.

We have compared nucleotide substitutions and polymorphisms at codons known to confer drug resistance in subtype B strains of human immunodeficiency virus type 1 (HIV-1) with similar substitutions in viruses of other subtypes. Genotypic analysis was performed on viruses from untreated individuals. Nucleotide and amino acid diversity at resistance sites was compared with a consensus subtype B reference virus. Among patients with non-subtype B infections, polymorphisms relative to subtype B were observed at codon 10 in protease (PR). These included silent substitutions (CTC-->CTT, CTA, TTA) and an amino acid mutation, L10I. Subtype A viruses possessed a V179I substitution in reverse transcriptase (RT). Subtype G viruses were identified by silent substitutions at codon 181 in RT (TAT-->TAC). Similarly, subtype A/G viruses were identified by a substitution at position 67 in RT (GAC-->GAT). Subtype C was distinguished by silent substitutions at codons 106 (GTA-->GTG) and 219 (AAA-->AAG) in RT and codon 48 (GGG-->GGA) in PR. Variations relative to subtype B were seen at RT position 215 (ACC-->ACT) for subtypes A and A/E. These substitutions and polymorphisms reflect different patterns of codon usage among viruses of different subtypes. However, the existence of different subtypes may only rarely affect patterns of drug resistance-associated mutations.