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1.  Comparative impact of antiretroviral drugs on markers of inflammation and immune activation during the first two years of effective therapy for HIV-1 infection: an observational study 
BMC Infectious Diseases  2014;14:122.
Background
Few studies have compared the impact of different antiretroviral regimens on residual immune activation and inflammation with discordant results. Aim of the study was to investigate the impact of various antiretroviral regimens on markers of immune activation and inflammation during the first two years of effective therapy.
Methods
We studied HIV-infected antiretroviral-naïve patients who began cART with either abacavir/lamivudine or tenofovir/emtricitabine, combined with ritonavir-boosted lopinavir (LPV/r), atazanavir (ATV/r) or efavirenz (EFV). All the patients had a virological response within 6 months, which was maintained for 2 years with no change in their ART regimen. C-reactive protein (hs-CRP), interleukin-6 (IL-6), soluble CD14 (sCD14), monokine induced by interferon-γ (MIG) and interferon-γ-inducible protein-10 (IP-10) were measured in stored plasma obtained at cART initiation and 24 months later. Mean changes from baseline were analyzed on loge-transformed values and multivariable linear regression models were used to study the effect of the treatment components, after adjusting for factors that might have influenced the choice of ART regimen or biomarker levels. Differences were expressed as the mean fold change percentage difference (Δ).
Results
Seventy-eight patients (91% males) with a median age of 43 years met the inclusion criteria. Their median baseline CD4 cell count was 315/mm3 and HIV-1 RNA level 4.6 log10 copies/ml. During the 2-years study period, IL-6, IP-10 and MIG levels fell significantly, while hs-CRP and sCD14 levels remained stable. IP-10 and MIG levels declined significantly less strongly with ATV/r than with EFV (IP-10Δ -57%, p = 0.011; MIGΔ -136%, p = 0.007), while no difference was noted between LPV/r and EFV. The decline in IL-6 did not differ significantly across the different treatment components.
Conclusions
After the first 2 years of successful cART, IL-6, IP-10 and MIG fell markedly while hs-CRP and sCD14 levels remained stable. The only impact of ART regimen was a smaller fall in markers of immune activation with ATV/r than with EFV. Our results suggest that these markers could be worthwhile when evaluating new antiretroviral drugs.
doi:10.1186/1471-2334-14-122
PMCID: PMC3945800  PMID: 24589015
HIV; cART; Immune activation; Inflammation; Markers
2.  Characterization of HIV-1 antiretroviral drug resistance after second-line treatment failure in Mali, a limited-resources setting 
Journal of Antimicrobial Chemotherapy  2012;67(12):2943-2948.
Objectives
We describe the outcomes of second-line drug resistance profiles and predict the efficacy of drugs for third-line therapy in patients monitored without the benefit of plasma HIV-1 RNA viral load (VL) or resistance testing.
Methods
We recruited 106 HIV-1-infected patients after second-line treatment failure in Mali. VL was determined by the Abbott RealTime system and the resistance by the ViroSeq HIV-1 genotyping system. The resistance testing was interpreted using the latest version of the Stanford algorithm.
Results
Among the 106 patients, 93 had isolates successfully sequenced. The median age, VL and CD4 cells were respectively 35 years, 72 000 copies/mL and 146 cells/mm3. Patients were exposed to a median of 4 years of treatment and to six antiretrovirals. We found 20% of wild-type viruses. Resistance to etravirine was noted in 38%, to lopinavir in 25% and to darunavir in 12%. The duration of prior nucleos(t)ide reverse transcriptase inhibitor exposure was associated with resistance to abacavir (P < 0.0001) and tenofovir (P = 0.0001), and duration of prior protease inhibitor treatment with resistance to lopinavir (P < 0.0001) and darunavir (P = 0.06).
Conclusion
Long duration of therapy prior to failure was associated with high levels of resistance and is directly related to limited access to VL monitoring and delayed switches to second-line treatment, precluding efficacy of drugs for third-line therapy. This study underlines the need for governments and public health organizations to recommend the use of VL monitoring and also the availability of darunavir and raltegravir for third-line therapies in the context of limited-resource settings.
doi:10.1093/jac/dks310
PMCID: PMC3584968  PMID: 22888273
resistance; third-line; Africa
3.  A Polymorphism at Position 400 in the Connection Subdomain of HIV-1 Reverse Transcriptase Affects Sensitivity to NNRTIs and RNaseH Activity 
PLoS ONE  2013;8(10):e74078.
Reverse transcriptase (RT) plays an essential role in HIV-1 replication, and inhibition of this enzyme is a key component of HIV-treatment. However, the use of RT inhibitors can lead to the emergence of drug-resistant variants. Until recently, most clinically relevant resistance mutations were found in the polymerase domain of RT. Lately, an increasing number of resistance mutations has been identified in the connection and RNaseH domain. To further explore the role of these domains we analyzed the complete RT sequence of HIV-1 subtype B patients failing therapy. Position A/T400 in the connection subdomain is polymorphic, but the proportion of T400 increases from 41% in naïve patients to 72% in patients failing therapy. Previous studies suggested a role for threonine in conferring resistance to nucleoside RT inhibitors. Here we report that T400 also mediates resistance to non-nucleoside RT inhibitors. The susceptibility to NVP and EFV was reduced 5-fold and 2-fold, respectively, in the wild-type subtype B NL4.3 background. We show that substitution A400T reduces the RNaseH activity. The changes in enzyme activity are remarkable given the distance to both the polymerase and RNaseH active sites. Molecular dynamics simulations were performed, which provide a novel atomistic mechanism for the reduction in RNaseH activity induced by T400. Substitution A400T was found to change the conformation of the RNaseH primer grip region. Formation of an additional hydrogen bond between residue T400 and E396 may play a role in this structural change. The slower degradation of the viral RNA genome may provide more time for dissociation of the bound NNRTI from the stalled RT-template/primer complex, after which reverse transcription can resume.
doi:10.1371/journal.pone.0074078
PMCID: PMC3788777  PMID: 24098331
4.  Scoring Methods for Building Genotypic Scores: An Application to Didanosine Resistance in a Large Derivation Set 
PLoS ONE  2013;8(3):e59014.
Background
Several attempts have been made to determine HIV-1 resistance from genotype resistance testing. We compare scoring methods for building weighted genotyping scores and commonly used systems to determine whether the virus of a HIV-infected patient is resistant.
Methods and Principal Findings
Three statistical methods (linear discriminant analysis, support vector machine and logistic regression) are used to determine the weight of mutations involved in HIV resistance. We compared these weighted scores with known interpretation systems (ANRS, REGA and Stanford HIV-db) to classify patients as resistant or not. Our methodology is illustrated on the Forum for Collaborative HIV Research didanosine database (N = 1453). The database was divided into four samples according to the country of enrolment (France, USA/Canada, Italy and Spain/UK/Switzerland). The total sample and the four country-based samples allow external validation (one sample is used to estimate a score and the other samples are used to validate it). We used the observed precision to compare the performance of newly derived scores with other interpretation systems. Our results show that newly derived scores performed better than or similar to existing interpretation systems, even with external validation sets. No difference was found between the three methods investigated. Our analysis identified four new mutations associated with didanosine resistance: D123S, Q207K, H208Y and K223Q.
Conclusions
We explored the potential of three statistical methods to construct weighted scores for didanosine resistance. Our proposed scores performed at least as well as already existing interpretation systems and previously unrecognized didanosine-resistance associated mutations were identified. This approach could be used for building scores of genotypic resistance to other antiretroviral drugs.
doi:10.1371/journal.pone.0059014
PMCID: PMC3605419  PMID: 23555613
5.  Description of the L76V Resistance Protease Mutation in HIV-1 B and “Non-B” Subtypes 
PLoS ONE  2013;8(1):e54381.
Objective
To describe the prevalence of the L76V protease inhibitors resistance-associated mutation (PI-RAM) in relation with patients’ characteristics and protease genotypic background in HIV-1 B- and “non-B”-infected patients.
Methods
Frequency of the L76V mutation between 1998 and 2010 was surveyed in the laboratory database of 3 clinical centers. Major PI-RAMs were identified according to the IAS-USA list. Fisher’s and Wilcoxon tests were used to compare variables.
Results
Among the overall 29,643 sequences analyzed, the prevalence of L76V was 1.50%, while was 5.42% in PI-resistant viruses. Since 2008 the prevalence of L76V was higher in “non-B”-infected than in B-infected patients each year. Median time since diagnosis of HIV-1 infection and median time under antiretroviral-based regimen were both shorter in “non-B”- than in B-infected patients (8 vs 11 years, P<0.0001; and 7 vs 8 years, P = 0.004). In addition, “non-B”-infected patients had been pre-exposed to a lower number of PI (2 vs 3, P = 0.016). The L76V was also associated with a lower number of major PI-RAMs in “non-B” vs B samples (3 vs 4, P = 0.0001), and thus it was more frequent found as single major PI-RAM in “non-B” vs B subtype (10% vs 2%, P = 0.014).
Conclusions
We showed an impact of viral subtype on the selection of the L76V major PI-RAM with a higher prevalence in “non-B” subtypes observed since 2008. In addition, in “non-B”-infected patients this mutation appeared more rapidly and was associated with less PI-RAM.
doi:10.1371/journal.pone.0054381
PMCID: PMC3548776  PMID: 23349869
6.  In Silico and In Vitro Comparison of HIV-1 Subtypes B and CRF02_AG Integrases Susceptibility to Integrase Strand Transfer Inhibitors 
Advances in Virology  2012;2012:548657.
Most antiretroviral medical treatments were developed and tested principally on HIV-1 B nonrecombinant strain, which represents less than 10% of the worldwide HIV-1-infected population. HIV-1 circulating recombinant form CRF02_AG is prevalent in West Africa and is becoming more frequent in other countries. Previous studies suggested that the HIV-1 polymorphisms might be associated to variable susceptibility to antiretrovirals. This study is pointed to compare the susceptibility to integrase (IN) inhibitors of HIV-1 subtype CRF02_AG IN respectively to HIV-1 B. Structural models of B and CRF02_AG HIV-1 INs as unbound enzymes and in complex with the DNA substrate were built by homology modeling. IN inhibitors—raltegravir (RAL), elvitegravir (ELV) and L731,988—were docked onto the models, and their binding affinity for both HIV-1 B and CRF02_AG INs was compared. CRF02_AG INs were cloned and expressed from plasma of integrase strand transfer inhibitor (INSTI)-naïve infected patients. Our in silico and in vitro studies showed that the sequence variations between the INs of CRF02_AG and B strains did not lead to any notable difference in the structural features of the enzyme and did not impact the susceptibility to the IN inhibitors. The binding modes and affinities of INSTI inhibitors to B and CRF02_AG INs were found to be similar. Although previous studies suggested that several naturally occurring variations of CRF02_AG IN might alter either IN/vDNA interactions or INSTIs binding, our study demonstrate that these variations do affect neither IN activity nor its susceptibility to INSTIs.
doi:10.1155/2012/548657
PMCID: PMC3398581  PMID: 22829822
7.  Investigation of Super Learner Methodology on HIV-1 Small Sample: Application on Jaguar Trial Data 
AIDS Research and Treatment  2012;2012:478467.
Background. Many statistical models have been tested to predict phenotypic or virological response from genotypic data. A statistical framework called Super Learner has been introduced either to compare different methods/learners (discrete Super Learner) or to combine them in a Super Learner prediction method. Methods. The Jaguar trial is used to apply the Super Learner framework. The Jaguar study is an “add-on” trial comparing the efficacy of adding didanosine to an on-going failing regimen. Our aim was also to investigate the impact on the use of different cross-validation strategies and different loss functions. Four different repartitions between training set and validations set were tested through two loss functions. Six statistical methods were compared. We assess performance by evaluating R2 values and accuracy by calculating the rates of patients being correctly classified. Results. Our results indicated that the more recent Super Learner methodology of building a new predictor based on a weighted combination of different methods/learners provided good performance. A simple linear model provided similar results to those of this new predictor. Slight discrepancy arises between the two loss functions investigated, and slight difference arises also between results based on cross-validated risks and results from full dataset. The Super Learner methodology and linear model provided around 80% of patients correctly classified. The difference between the lower and higher rates is around 10 percent. The number of mutations retained in different learners also varys from one to 41. Conclusions. The more recent Super Learner methodology combining the prediction of many learners provided good performance on our small dataset.
doi:10.1155/2012/478467
PMCID: PMC3324131  PMID: 22550568
8.  Raltegravir Concentrations in the Genital Tract of HIV-1-Infected Women Treated with a Raltegravir-Containing Regimen (DIVA 01 Study)▿ 
We studied the penetration of raltegravir and HIV shedding in the genital tract among 14 HIV-1-infected women receiving a raltegravir-containing regimen who had <40 copies/ml blood plasma (BP) HIV RNA. None of the cervicovaginal fluid (CVF) samples showed detectable HIV RNA. Median raltegravir concentrations were 235 ng/ml in BP and 93 ng/ml in CVF, with a CVF/BP ratio of approximately 2.3. This good penetration of raltegravir may contribute to the control of viral replication in the female genital tract.
doi:10.1128/AAC.01460-10
PMCID: PMC3101453  PMID: 21444705
9.  Positive Impact of HIV-1 gag Cleavage Site Mutations on the Virological Response to Darunavir Boosted with Ritonavir▿  
We assessed the roles of baseline gag and gag-pol cleavage site mutations (CSM) on the virological outcome of a darunavir-based regimen in highly antiretroviral-experienced patients. We showed the association, in multivariate analysis, between the A431V gag CSM and the virological response, defined as a reduction in plasma HIV-1 RNA to <50 copies/ml at month 3 (P = 0.028). Our results suggest that a specific gag CSM might have a role on protease inhibitor susceptibility in an inhibitor-specific manner.
doi:10.1128/AAC.01049-10
PMCID: PMC3067137  PMID: 21282435
10.  Low Frequency of Intermittent HIV-1 Semen Excretion in Patients Treated with Darunavir-Ritonavir at 600/100 Milligrams Twice a Day plus Two Nucleoside Reverse Transcriptase Inhibitors or Monotherapy▿  
Antimicrobial Agents and Chemotherapy  2010;54(11):4910-4913.
HIV-1 RNA level and darunavir concentration in the genital tract were measured in 45 men receiving darunavir-ritonavir mono- or tritherapy. At week 48, a low frequency (3/45) of HIV-1 RNA shedding was observed in patients (1 on monotherapy and 2 on triple therapy), although they had undetectable HIV-1 RNA in plasma. The median darunavir seminal plasma concentration was close to the blood plasma free fraction, demonstrating a good penetration of darunavir into the male genital tract.
doi:10.1128/AAC.00725-10
PMCID: PMC2976146  PMID: 20713677
11.  Evaluation of the Genotypic Prediction of HIV-1 Coreceptor Use versus a Phenotypic Assay and Correlation with the Virological Response to Maraviroc: the ANRS GenoTropism Study▿  
Genotypic algorithms for prediction of HIV-1 coreceptor usage need to be evaluated in a clinical setting. We aimed at studying (i) the correlation of genotypic prediction of coreceptor use in comparison with a phenotypic assay and (ii) the relationship between genotypic prediction of coreceptor use at baseline and the virological response (VR) to a therapy including maraviroc (MVC). Antiretroviral-experienced patients were included in the MVC Expanded Access Program if they had an R5 screening result with Trofile (Monogram Biosciences). V3 loop sequences were determined at screening, and coreceptor use was predicted using 13 genotypic algorithms or combinations of algorithms. Genotypic predictions were compared to Trofile; dual or mixed (D/M) variants were considered as X4 variants. Both genotypic and phenotypic results were obtained for 189 patients at screening, with 54 isolates scored as X4 or D/M and 135 scored as R5 with Trofile. The highest sensitivity (59.3%) for detection of X4 was obtained with the Geno2pheno algorithm, with a false-positive rate set up at 10% (Geno2pheno10). In the 112 patients receiving MVC, a plasma viral RNA load of <50 copies/ml was obtained in 68% of cases at month 6. In multivariate analysis, the prediction of the X4 genotype at baseline with the Geno2pheno10 algorithm including baseline viral load and CD4 nadir was independently associated with a worse VR at months 1 and 3. The baseline weighted genotypic sensitivity score was associated with VR at month 6. There were strong arguments in favor of using genotypic coreceptor use assays for determining which patients would respond to CCR5 antagonist.
doi:10.1128/AAC.00148-10
PMCID: PMC2916345  PMID: 20530226
12.  International Cohort Analysis of the Antiviral Activities of Zidovudine and Tenofovir in the Presence of the K65R Mutation in Reverse Transcriptase▿  
A K65R mutation in HIV-1 reverse transcriptase can occur with the failure of tenofovir-, didanosine-, abacavir-, and, in some cases, stavudine-containing regimens and leads to reduced phenotypic susceptibility to these drugs and hypersusceptibility to zidovudine, but its clinical impact is poorly described. We identified isolates with the K65R mutation within the Stanford Resistance Database and a French cohort for which subsequent treatment and virological response data were available. The partial genotypic susceptibility score (pGSS) was defined as the genotypic susceptibility score (GSS) excluding the salvage regimen's nucleoside reverse transcriptase inhibitor (NRTI) component. A three-part virologic response variable was defined (e.g., complete virologic response, partial virologic response, and no virologic response). Univariate, multivariate, and bootstrap analyses evaluated factors associated with the virologic response, focusing on the contributions of zidovudine and tenofovir. Seventy-one of 130 patients (55%) achieved a complete virologic response (defined as an HIV RNA level of <200 copies/ml). In univariate analyses, pGSS and zidovudine use in the salvage regimen were predictors of the virologic response. In a multivariate analysis, pGSS and zidovudine and tenofovir use were associated with the virologic response. Bootstrap analyses showed similar reductions in HIV RNA levels with zidovudine or tenofovir use (0.5 to 0.9 log10). In the presence of K65R, zidovudine and tenofovir are associated with similar reductions in HIV RNA levels. Given its tolerability, tenofovir may be the preferred agent over zidovudine even in the presence of the K65R mutation.
doi:10.1128/AAC.01380-09
PMCID: PMC2849386  PMID: 20124005
13.  Impact of Y143 HIV-1 Integrase Mutations on Resistance to Raltegravir In Vitro and In Vivo▿  
Integrase (IN), the HIV-1 enzyme responsible for the integration of the viral genome into the chromosomes of infected cells, is the target of the recently approved antiviral raltegravir (RAL). Despite this drug's activity against viruses resistant to other antiretrovirals, failures of raltegravir therapy were observed, in association with the emergence of resistance due to mutations in the integrase coding region. Two pathways involving primary mutations on residues N155 and Q148 have been characterized. It was suggested that mutations at residue Y143 might constitute a third primary pathway for resistance. The aims of this study were to investigate the susceptibility of HIV-1 Y143R/C mutants to raltegravir and to determine the effects of these mutations on the IN-mediated reactions. Our observations demonstrate that Y143R/C mutants are strongly impaired for both of these activities in vitro. However, Y143R/C activity can be kinetically restored, thereby reproducing the effect of the secondary G140S mutation that rescues the defect associated with the Q148R/H mutants. A molecular modeling study confirmed that Y143R/C mutations play a role similar to that determined for Q148R/H mutations. In the viral replicative context, this defect leads to a partial block of integration responsible for a weak replicative capacity. Nevertheless, the Y143 mutant presented a high level of resistance to raltegravir. Furthermore, the 50% effective concentration (EC50) determined for Y143R/C mutants was significantly higher than that obtained with G140S/Q148R mutants. Altogether our results not only show that the mutation at position Y143 is one of the mechanisms conferring resistance to RAL but also explain the delayed emergence of this mutation.
doi:10.1128/AAC.01075-09
PMCID: PMC2798554  PMID: 19901095
14.  Tetherin Restricts Productive HIV-1 Cell-to-Cell Transmission 
PLoS Pathogens  2010;6(6):e1000955.
The IFN-inducible antiviral protein tetherin (or BST-2/CD317/HM1.24) impairs release of mature HIV-1 particles from infected cells. HIV-1 Vpu antagonizes the effect of tetherin. The fate of virions trapped at the cell surface remains poorly understood. Here, we asked whether tetherin impairs HIV cell-to-cell transmission, a major means of viral spread. Tetherin-positive or -negative cells, infected with wild-type or ΔVpu HIV, were used as donor cells and cocultivated with target lymphocytes. We show that tetherin inhibits productive cell-to-cell transmission of ΔVpu to targets and impairs that of WT HIV. Tetherin accumulates with Gag at the contact zone between infected and target cells, but does not prevent the formation of virological synapses. In the presence of tetherin, viruses are then mostly transferred to targets as abnormally large patches. These viral aggregates do not efficiently promote infection after transfer, because they accumulate at the surface of target cells and are impaired in their fusion capacities. Tetherin, by imprinting virions in donor cells, is the first example of a surface restriction factor limiting viral cell-to-cell spread.
Author Summary
Tetherin is a cell surface “restriction factor” that acts as an innate antiviral defense. Tetherin prevents newly produced particles of HIV-1 and other enveloped viruses from escaping the surface of infected cells. HIV-1 encodes the protein Vpu to counteract this host defense. We have studied here if HIV-1 particles trapped at the cell surface may be transmitted to neighboring uninfected cells. Direct transmission through cell-to-cell contacts is indeed an efficient means for viral spread. Virological synapses may be formed between infected donor cells and target cells, allowing rapid and massive transmission of viruses. We show that tetherin inhibits productive cell-to-cell transmission of Vpu-deleted HIV to target cells, and impairs that of wild-type virus. Tetherin accumulates with Gag at the contact zone between infected and target cells, but does not prevent the formation of virological synapses. With tetherin, viruses are then mostly transferred to targets as abnormally large patches that are impaired in their fusion capacities. These results represent the first example of a surface restriction factor limiting viral cell-to-cell spread, acting in donor cells, but inhibiting infection after transfer of viral material to novel recipient cells.
doi:10.1371/journal.ppat.1000955
PMCID: PMC2887479  PMID: 20585562
15.  Genotypic Resistance Analysis of the Virological Response to Fosamprenavir-Ritonavir in Protease Inhibitor-Experienced Patients in CONTEXT and TRIAD Clinical Trials▿  
Antimicrobial Agents and Chemotherapy  2008;52(12):4251-4257.
The aim of this study was to identify human immunodeficiency virus (HIV) protease mutations associated with virological response (VR) to fosamprenavir-ritonavir (FPV/r) in 113 protease inhibitor (PI)-experienced patients randomized in both CONTEXT and TRIAD clinical trials and receiving the same dose (700/100 mg twice daily) of FPV/r. The impact of each protease mutation on the VR to FPV/r, defined as the decrease in HIV RNA at week 12, was investigated with nonparametric analyses. A step-by-step procedure was done using a Jonckheere-Terpstra (JT) test that retains the group of mutations most strongly associated with the VR. Mutations at the following 14 codons were associated with a reduced VR to FPV/r: 10, 15, 33, 46, 54, 60, 62, 63, 72, 73, 82, 84, 89, and 90. The JT procedure led to selecting the CONTEXT/TRIAD genotypic set of mutations, I15V, M46I/L, I54L/M/V, D60E, L63P/T, and I84V, as providing the strongest association with the VR (P = 1.45 × 10−11). In the nine patients with zero mutations within this set, the median decrease in HIV RNA was −2.63 log copies/ml, and was −2.22 (n = 45), −1.50 (n = 26), −0.58 (n = 23), −0.47 (n = 6), −0.13 (n = 3), and 0.04 (n = 1) log copies/ml in those with one, two, three, four, five, and six mutations, respectively. This study identified six mutations associated with VR to FPV/r. Some of these mutations are shared with the current FPV/r Agence Nationale de Recherches sur le SIDA (ANRS) resistance score, which has been cross-validated in the CONTEXT/TRIAD data set, suggesting that the current ANRS FPV/r score is a useful tool for the prediction of VR to FPV/r in PI-experienced patients.
doi:10.1128/AAC.00514-08
PMCID: PMC2592865  PMID: 18852278
16.  Antiretroviral Therapy with a Twice-Daily Regimen Containing 400 Milligrams of Indinavir and 100 Milligrams of Ritonavir in Human Immunodeficiency Virus Type 1-Infected Women during Pregnancy▿  
We evaluated the safety and efficacy of a twice daily regimen containing 400 mg of indinavir and 100 mg of ritonavir in 32 human immunodeficiency virus (HIV)-infected women during pregnancy. The median indinavir trough concentration was 208 ng/ml during the third trimester. At delivery, 26 of 28 women on indinavir-ritonavir had HIV RNA levels of <200 copies/ml. No infant was HIV infected. These data are encouraging for the use of this combination for prevention of mother-to-child transmission of HIV.
doi:10.1128/AAC.01301-07
PMCID: PMC2292558  PMID: 18250187
17.  Mutations Associated with Failure of Raltegravir Treatment Affect Integrase Sensitivity to the Inhibitor In Vitro▿  
Raltegravir (MK-0518) is a potent inhibitor of human immunodeficiency virus (HIV) integrase and is clinically effective against viruses resistant to other classes of antiretroviral agents. However, it can select mutations in the HIV integrase gene. Nine heavily pretreated patients who received salvage therapy including raltegravir and who subsequently developed virological failure under raltegravir therapy were studied. For each patient, the sequences of the integrase-coding region were determined and compared to that at the beginning of the treatment. Four different mutation profiles were identified in these nine patients: E92Q, G140S Q148H, N155H, and E157Q mutations. For four patients, each harboring a different profile, the wild-type and mutated integrases were produced, purified, and assayed in vitro. All the mutations identified altered the activities of integrase protein: both 3′ processing and strand transfer activities were moderately affected in the E92Q mutant; strand transfer was markedly impaired in the N155H mutant; both activities were strongly impaired in the G140S Q148H mutant; and the E157Q mutant was almost completely inactive. The sensitivities of wild-type and mutant integrases to raltegravir were compared. The E92Q and G140S Q148H profiles were each associated with a 7- to 8-fold decrease in sensitivity, and the N155H mutant was more than 14-fold less sensitive to raltegravir. At least four genetic profiles (E92Q, G140S Q148H, N155H, and E157Q) can be associated with in vivo treatment failure and resistance to raltegravir. These mutations led to strong impairment of enzymes in vitro in the absence of raltegravir: strand transfer activity was affected, and in some cases 3′ processing was also impaired.
doi:10.1128/AAC.01228-07
PMCID: PMC2292515  PMID: 18227187
18.  Factors Associated with the Selection of Mutations Conferring Resistance to Protease Inhibitors (PIs) in PI-Experienced Patients Displaying Treatment Failure on Darunavir▿  
The objective of this study was to characterize the mutations selected by darunavir (DRV) use in protease inhibitor (PI)-experienced patients and the associated factors. We analyzed treatment failure in 54 PI-experienced human immunodeficiency virus (HIV)-infected patients on a DRV- and ritonavir-containing regimen. Viral genotyping was carried out at the baseline, at between 1 and 3 months of treatment, and at between 3 and 6 months of treatment to search for the selection of mutations conferring resistance to PIs. The median baseline HIV RNA level was 4.9 log10 copies/ml, and the median CD4 count was 87 cells/mm3. At the baseline, the median numbers of resistance mutations were as follows: 3 DRV resistance mutations, 4 major PI resistance mutations, and 10 minor PI resistance mutations. The most common mutations that emerged at rebound included V32I (44%), I54M/L (24%), L33F (25%), I84V (21%), and L89V (12%). Multivariate analysis showed that higher baseline HIV RNA levels and smaller numbers of nucleoside reverse transcriptase inhibitor simultaneously used with DRV were associated with a higher risk of DRV resistance mutation selection. By contrast, L76V, a known DRV resistance mutation, was found to decrease the risk of selection of another DRV resistance mutation. The occurrence of virological failure while a patient was on DRV was associated with the selection of mutations that increased the level of DRV resistance without affecting susceptibility to tipranavir (TPV). In these PI-treated patients who displayed treatment failure while they were on a DRV-containing regimen, we confirmed the set of emerging mutations associated with DRV failure and identified the factors associated with the selection of these mutations. TPV susceptibility does not seem to be affected by the selection of a DRV resistance mutation.
doi:10.1128/AAC.00909-07
PMCID: PMC2224714  PMID: 18039922
20.  Superior control of HIV-1 replication by CD8+ T cells is reflected by their avidity, polyfunctionality, and clonal turnover 
The Journal of Experimental Medicine  2007;204(10):2473-2485.
The key attributes of CD8+ T cell protective immunity in human immunodeficiency virus (HIV) infection remain unclear. We report that CD8+ T cell responses specific for Gag and, in particular, the immunodominant p24 epitope KK10 correlate with control of HIV-1 replication in human histocompatibility leukocyte antigen (HLA)–B27 patients. To understand further the nature of CD8+ T cell–mediated antiviral efficacy, we performed a comprehensive study of CD8+ T cells specific for the HLA-B27–restricted epitope KK10 in chronic HIV-1 infection based on the use of multiparametric flow cytometry together with molecular clonotypic analysis and viral sequencing. We show that B27-KK10–specific CD8+ T cells are characterized by polyfunctional capabilities, increased clonal turnover, and superior functional avidity. Such attributes are interlinked and constitute the basis for effective control of HIV-1 replication. These data on the features of effective CD8+ T cells in HIV infection may aid in the development of successful T cell vaccines.
doi:10.1084/jem.20070784
PMCID: PMC2118466  PMID: 17893201
21.  Predictive Genotypic Algorithm for Virologic Response to Lopinavir-Ritonavir in Protease Inhibitor-Experienced Patients▿  
Several genotypic resistance algorithms have been proposed for quantitation of the degree of phenotypic resistance to the human immunodeficiency virus (HIV) protease inhibitor (PI) lopinavir (LPV), including the original LPV mutation score. In this study, we retrospectively evaluated 21 codons in HIV protease known to be associated with PI resistance in a large antiretroviral agent-experienced observational patient cohort, “Autorisation Temporaire d'Utilization” (ATU), to assess whether a more optimal algorithm could be derived by using virologic response data from patients treated with LPV in combination with ritonavir (LPV/r). Five of the 11 mutations constituting the LPV mutation score were not associated with a virologic response, while 4 additional mutations not included in this score demonstrated an association. Therefore, the LPV ATU score, which includes mutations at codons 10, 20, 24, 33, 36, 47, 48, 54, 82, and 84, was constructed and shown in two different types of multivariable analyses of the ATU cohort to be a better predictor of the virologic response than the LPV mutation score. The LPV ATU score was also more strongly associated with a virologic response when it was applied to independent clinical trial populations of PI-experienced patients receiving LPV/r. This study provides the basis for a new genotypic resistance algorithm that is useful for predicting the antiviral activities of LPV/r-based regimens in PI-experienced patients. The refined algorithm may be useful in making clinical treatment decisions and in refining genetic and pharmacologic methods for assessing the activity of LPV/r.
doi:10.1128/AAC.00388-07
PMCID: PMC2043245  PMID: 17576846
22.  Virological and Pharmacological Parameters Predicting the Response to Lopinavir-Ritonavir in Heavily Protease Inhibitor-Experienced Patients 
The genotypic inhibitory quotient (GIQ) has been proposed as a way to integrate drug exposure and genotypic resistance to protease inhibitors and can be useful to enhance the predictivity of virologic response for boosted protease inhibitors. The aim of this study was to evaluate the predictivity of the GIQ in 116 protease inhibitor-experienced patients treated with lopinavir-ritonavir. The overall decrease in human immunodeficiency virus type 1 (HIV-1) RNA from baseline to month 6 was a median of −1.50 log10 copies/ml and 40% of patients had plasma HIV-1 RNA below 400 copies/ml at month 6. The overall median lopinavir study-state Cmin concentration was 5,856 ng/ml. Using univariate linear regression analyses, both lopinavir GIQ and the number of baseline lopinavir mutations were highly associated with virologic response through 6 months. In the multivariate analysis, only lopinavir GIQ, baseline HIV RNA, and the number of prior protease inhibitors were significantly associated with response. When the analysis was limited to patients with more highly mutant viruses (three or more lopinavir mutations), only lopinavir GIQ remained significantly associated with virologic response. This study suggests that GIQ could be a better predictor of the virologic response than virological (genotype) or pharmacological (minimal plasma concentration) approaches used separately, especially among patients with at least three protease inhibitor resistance mutations. Therapeutic drug monitoring for patients treated by lopinavir-ritonavir would likely be most useful in patients with substantially resistant viruses.
doi:10.1128/AAC.49.5.1720-1726.2005
PMCID: PMC1087618  PMID: 15855487
23.  Clinically Relevant Genotype Interpretation of Resistance to Didanosine 
We analyzed the didanosine (ddI) arm of the randomized, placebo-controlled Jaguar trial in order to define a genotypic score for ddI associated with virologic response. In this arm, 111 patients experiencing virologic failure received ddI in addition to their current combination therapy for 4 weeks. The impact of mutations in the reverse transcriptase gene on the virologic response to ddI was studied in univariate analysis. Genotypic score was constructed using step-by-step analyses first including only mutations associated to poorer virologic response (scored as +1), while secondarily, mutations associated to a better response (scored as −1) were also eligible. Eight mutations were associated with a reduced response to ddI, M41L, D67N, T69D, L74V, V118I, L210W, T215Y/F, and K219Q/E, and two mutations were associated with a better response, K70R and M184V/I. The best prediction of the virologic response to ddI was obtained with a composite score comprising mutations added and subtracted (set II, M41L + T69D + L74V+ T215Y/F + K219Q/E − K70R − M184V/I; P = 4.5 × 10−9) and by comparing that to only mutations added (set I, M41L + T69D + L74V + L210W + T215Y/F + K219Q/E; P = 1.2 × 10−7). Patients had a human immunodeficiency virus RNA reduction of 1.24, 0.84, 0.61, 0.40, and 0.07 log10 copies/ml when they were ranked as having a genotypic score II of −2, −1, or 0 or 1 and 2 mutations or more, respectively. In conclusion, we developed and validated a genotypic score, taking into account mutations negatively and positively impacting the virologic response to ddI.
doi:10.1128/AAC.49.5.1739-1744.2005
PMCID: PMC1087657  PMID: 15855490
24.  Clinically Relevant Interpretation of Genotype and Relationship to Plasma Drug Concentrations for Resistance to Saquinavir-Ritonavir in Human Immunodeficiency Virus Type 1 Protease Inhibitor-Experienced Patients 
Antimicrobial Agents and Chemotherapy  2004;48(12):4687-4692.
It has been shown that virological protease inhibitor (PI) resistance mutations present at the initiation of saquinavir (SQV) plus ritonavir (RTV) therapy in PI-experienced patients are the strongest predictors of virological response. But most of the current resistance algorithms are adapted for unboosted SQV regimens. We applied a stepwise methodology for the development and validation of a clinically relevant genotypic resistance score for an SQV (800 mg twice per day [b.i.d.]) plus RTV (100 mg b.i.d.)-containing regimen. PI-experienced patients treated by this regimen achieved a human immunodeficiency virus plasma viral load (VL) of <200 copies/ml at months 3 to 5 for 41.7% of subjects. Adjusted in a multivariate analysis, taking into account all the confounding factors, such as the nucleoside used, five mutations were combined in a resistance score associated with a reduced virological response to an SQV-plus-RTV regimen: L24I, I62V, V82A/F/T/S, I84V, and L90IM. Patients with isolates harboring 0 to 1 mutation among the score achieved −2.20 log10 and −1.23 log10 copies/ml of VL reduction, respectively, while it was −0.27 log10 copies/ml for those with at least two mutations, classifying the isolates as “no evidence of resistance” (0 or 1 mutation) or “resistance ” (≥2 mutations). The minimum concentration in plasma (Cmin) of SQV alone was not associated with the virological response. However, the combination of the SQV Cmin and the genotypic score, expressed as the genotypic inhibitory quotient, was predictive of the virological response, suggesting that the interpretation of SQV concentrations in plasma should be done only in the context of the resistance index provided by viral genotype for PI-experienced patients.
doi:10.1128/AAC.48.12.4687-4692.2004
PMCID: PMC529185  PMID: 15561845
25.  Genotypic Inhibitory Quotient as Predictor of Virological Response to Ritonavir-Amprenavir in Human Immunodeficiency Virus Type 1 Protease Inhibitor-Experienced Patients 
Forty-nine protease inhibitor (PI)-experienced but amprenavir (APV)-naïve patients experiencing virological failure were treated with ritonavir (RTV) (100 mg twice a day [b.i.d.]) plus APV (600 mg b.i.d.). Patients responded to therapy with a median viral load decrease of −1.32 log10 by week 12. The addition of low-dose RTV enhanced the minimal APV concentration in plasma (APV Cmin) up to 10-fold compared with that obtained with APV (1,200 mg b.i.d.) without RTV. Baseline PI resistance mutations (L10F/I/V, K20M/R, E35D, R41K, I54V, L63P, V82A/F/T/S, I84V) identified by univariate analysis and included in a genotypic score and APV Cmin at week 8 were predictive of the virological response at week 12. The response to APV plus RTV was significantly reduced in patients with six or more of the resistance mutations among the ones defined above. The genotypic inhibitory quotient, calculated as the ratio of the APV Cmin to the number of human immunodeficiency virus type 1 protease mutations, was a better predictor than the virological or pharmacological variables used alone. This genotypic inhibitory quotient could be used in therapeutic drug monitoring to define the concentrations in plasma needed to control replication of viruses with different levels of PI resistance, as measured by the number of PI resistance mutations.
doi:10.1128/AAC.47.2.594-600.2003
PMCID: PMC151735  PMID: 12543665

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