Kurt Van Baelen

HIV-1 resistance patterns to integrase inhibitors in antiretroviral-experienced patients with virological failure on raltegravir-containing regimens

BACKGROUND Our aim was to study the in vivo viral genetic pathways for resistance to raltegravir, in antiretroviral-experienced patients with virological failure (VF) on raltegravir-containing regimens. METHODS We set up a prospective study including antiretroviral-experienced patients receiving raltegravir-based regimens. Integrase (IN) genotypic resistance analysis was performed at baseline. IN was also sequenced at follow-up points in the case of VF, i.e. plasma HIV-1 RNA>400 copies/mL at month 3 and/or >50 copies/mL at month 6. For phenotyping, the IN region was recombined with an IN-deleted HXB2-based HIV-1 backbone. A titrated amount of IN recombinant viruses was used for antiviral testing against raltegravir and elvitegravir. RESULTS Among 51 patients, 11 (21.6%) had VF. Four different patterns of IN mutations were observed: (i) emergence of Q148H/R with secondary mutations (n=5 patients); (ii) emergence of N155H, then replaced by a pattern including Y143C/H/R (n=3); (iii) selection of S230N (n=1); and (iv) no evidence of selection of IN mutations (n=2). The median raltegravir and elvitegravir fold changes (FCs) were 244 (154-647) and 793 (339-892), respectively, for the Q148H/R pattern, while the median raltegravir and elvitegravir FCs were 21 (6-52) and 3 (2-3), respectively, with Y143C/H/R. The median plasma raltegravir Cmin was lower in patients with selection of the N155H mutation followed by Y143C/H/R compared with patients with Q148H/R and with patients without emerging mutations or without VF. CONCLUSIONS Diverse genetic profiles can be associated with VF on raltegravir-containing regimens, including the dynamics of replacement of mutational profiles. Pharmacokinetic parameters could be involved in this genetic evolution.

Susceptibility of Human Immunodeficiency Virus Type 1 to the Maturation Inhibitor Bevirimat Is Modulated by Baseline Polymorphisms in Gag Spacer Peptide 1

ABSTRACT In this study, we evaluated baseline susceptibility to bevirimat (BVM), the first in a new class of antiretroviral agents, maturation inhibitors. We evaluated susceptibility to BVM by complete gag genotypic and phenotypic testing of 20 patient-derived human immunodeficiency virus type 1 isolates and 20 site-directed mutants. We found that reduced BVM susceptibility was associated with naturally occurring polymorphisms at positions 6, 7, and 8 in Gag spacer peptide 1.

Performance of a population-based HIV-1 tropism phenotypic assay and correlation with V3 genotypic prediction tools in recent HIV-1 seroconverters.

Background:Pure X4 and X4R5 dual-tropic viruses may be recognized in ∼15% of drug-naive HIV-1-positive patients. CCR5 antagonists are active against R5 viruses; therefore, HIV tropism should be known before their prescription. Patients and Methods:A population-based phenotypic assay was performed in 61 recent HIV-1 seroconverters. The results were compared with those obtained using 8 different predictor software programs (C4.5, C4.5 with 8 and 12, PART, SVM, Charge Rule, PSSMsinsi, PSSMx4r5, and geno2pheno), which are freely available at 3 different Web sites and use V3 sequences derived from patients viruses. Results:Phenotypic testing reported X4R5 dual-tropic viruses in 10 (16.4%) patients. CD4 cell counts and viral loads were significantly lower in X4R5 dual-tropic (450 cells/μL and 3.9 log HIV RNA copies/mL) than in R5 viruses (629 cells/μL, 4.5 log HIV RNA copies/mL) (P < 0.05). The overall concordance of genotype and phenotype was relatively good (>80%). Although specificity was >90% using all but 1 genotypic predictor (geno2pheno), however, the sensitivity for the detection of X4 variants was low (<30%), except for SVM and geno2pheno (70%). Conclusions:The prevalence of X4 and X4/R5 dual-tropic viruses in recent HIV seroconverters is 16%. Current genotypic algorithms need to be improved for the estimation of HIV-1 coreceptor use before moving to the clinic. This information is crucial for the selection of candidates to receive CCR5 antagonists in places where phenotypic tropism assays may not be feasible.

Clade-specific HIV-1 integrase polymorphisms do not reduce raltegravir and elvitegravir phenotypic susceptibility.

The contribution of clade-specific polymorphisms in the HIV-1 integrase gene towards integrase inhibitor phenotypic susceptibility was tested on 137 clinical isolates, of which 60 were non-clade B strains. Control Q148R mutant virus showed fold change values of 17.85 ± 2.77 and 88.94 ± 9.02 for raltegravir and elvitegravir, respectively, whereas the average fold change for the clinical samples was 0.91 ± 0.40, and 0.84 ± 0.37. Phenotypic testing proved that clade-specific integrase polymorphisms do not contribute to reduced susceptibility towards integrase inhibitors.

Secondary Integrase Resistance Mutations Found in HIV-1 Minority Quasispecies in Integrase Therapy-Naive Patients Have Little or No Effect on Susceptibility to Integrase Inhibitors

ABSTRACT The goal of this study was to explore the presence of integrase strand transfer inhibitor (InSTI) resistance mutations in HIV-1 quasispecies present in InSTI-naïve patients and to evaluate their in vitro effects on phenotypic susceptibility to InSTIs and their replication capacities. The RT-RNase H-IN region was PCR amplified from plasma viral RNA obtained from 49 HIV-1 subtype B-infected patients (21 drug naïve and 28 failing highly active antiretroviral therapy [HAART] not containing InSTIs) and recombined with an HXB2-based backbone with RT and IN deleted. Recombinant viruses were tested against raltegravir and elvitegravir and for replication capacity. Three-hundred forty-four recombinant viruses from 49 patients were successfully analyzed both phenotypically and genotypically. The majority of clones were not phenotypically resistant to InSTIs: 0/344 clones showed raltegravir resistance, and only 3 (0.87%) showed low-level elvitegravir resistance. No primary resistance mutations for raltegravir and elvitegravir were found as major or minor species. The majority of secondary mutations were also absent or rarely present. Secondary mutations, such as T97A and G140S, found rarely and only as minority quasispecies, were present in the elvitegravir-resistant clones. A novel mutation, E92G, although rarely found in minority quasispecies, showed elvitegravir resistance. Preexisting genotypic and phenotypic raltegravir resistance was extremely rare in InSTI-naïve patients and confined to only a restricted minority of secondary variants. Overall, these results, together with others based on population and ultradeep sequencing, suggest that at this point IN genotyping in all patients before raltegravir treatment may not be cost-effective and should not be recommended until evidence of transmitted drug resistance to InSTIs or the clinical relevance of IN minor variants/polymorphisms is determined.

Comparison of phenotypic and genotypic tropism determination in triple-class-experienced HIV patients eligible for maraviroc treatment

Background Determination of HIV-1 tropism is a pre-requisite to the use of CCR5 antagonists. This study evaluated the potential of population genotypic tropism tests (GTTs) in clinical practice, and the correlation with phenotypic tropism tests (PTTs) in patients accessing routine HIV care. Methods Forty-nine consecutive plasma samples for which an original TrofileTM assay was performed were obtained from triple-class-experienced patients in need of a therapy change. Viral tropism was defined as the consensus of three or more tropism calls obtained from the combination of two independent population PTT assays (Trofile Biosciences, San Francisco, CA, USA, and Virco, Beerse, Belgium), population GTTs and GTTs based on ultra-deep sequencing. If no consensus was reached, a clonal PTT was performed in order to finalize the tropism call. This two-step approach allowed the definition of a reference tropism call. Results According to the reference tropism result, 35/49 samples were CCR5 tropic (R5) (patients eligible for maraviroc treatment) and 14/49 were assigned as non-R5 tropic. The non-R5 samples [patients not eligible for maraviroc treatment according to the FDA/European Medicines Agency (EMEA) label] group included both the CXCR4 (X4) samples and the dual and mixed CCR5/CXCR4 (R5/X4) samples. Compared with TrofileTM population PTTs, population GTTs showed a higher sensitivity (97%) and a higher negative predictive value (91%), but almost equal specificity and an equal positive predictive value. Conclusions In line with recent reports from clinical trial data, our data support the use of population genotypic tropism testing as a tool for tropism determination before the start of maraviroc.

A combined genotypic and phenotypic human immunodeficiency virus type 1 recombinant virus assay for the reverse transcriptase and integrase genes

With the approval of the first HIV-1 integrase inhibitor raltegravir and a second one in phase III clinical development (elvitegravir), genotypic and phenotypic resistance assays are required to guide antiretroviral therapy and to investigate treatment failure. In this study, a genotypic and phenotypic recombinant virus assay was validated for determining resistance against integrase inhibitors. The assays are based on the amplification of a region encompassing not only HIV-1 integrase, but also reverse transcriptase and RNAseH. The overall amplification success was 85% (433/513) and increased to 93% (120/129) for samples with a viral load above 3 log(10) copies/ml. Both B and non-B HIV-1 subtypes could be genotyped successfully (93%; 52/56 and 100%; 49/49, respectively) and reproducibly. The phenotypic assay showed a high success rate (96.5%; 139/144) for subtype B (100%; 19/19) and non-B subtypes (92%; 45/49), and was found to be accurate and reproducible as assessed using well-characterized integrase mutants. Using both assays, baseline resistance to raltegravir and elvitegravir in subtype B and non-B HIV-1 strains selected at random was not observed, although integrase polymorphisms were present at varying prevalence. Biological cutoff values were found to be 2.1 and 2.0 for raltegravir and elvitegravir, respectively. In summary, a genotypic and phenotypic integrase resistance assay was validated successfully for accuracy, reproducibility, analytical and clinical sensitivity, and dynamic range.

Development and Performance of Conventional HIV-1 Phenotyping (Antivirogram®) and Genotype-Based Calculated Phenotyping Assay (virco®TYPE HIV-1) on Protease and Reverse Transcriptase Genes to Evaluate Drug Resistance

Objectives: A wide array of monitoring tests is commercially available to gauge HIV-1 disease progression and the overall health status of an HIV-1-infected patient. Viral load tests provide a picture of viral activity, while CD4 cell counts shed light on the immune status and can help physicians to prevent the development of opportunistic infections in patients. On the other hand, genotypic and phenotypic resistance testing and therapeutic drug monitoring help to optimize HIV-1 antiretroviral therapy. Resistance testing is currently recommended within the standard of care guidelines to aid the choice of new drug regimens following treatment failure(s). Methods: Genotypic testing described here is based on the amplification and sequencing of an HIV-1 protease (PR) and reverse transcriptase (RT) region from a patient sample to identify resistance mutations associated with PR and RT inhibitor resistance. A genotypic test takes a week to perform and the results are reported as a list of detected mutations. The virco®TYPE HIV-1 report uses genotypic data to predict phenotypic susceptibility by linear regression modeling that uses a large correlative database of genotype-phenotype pairs. Phenotypic testing measures the ability of the virus to replicate in the presence of a drug and provides a direct measurement of drug susceptibility in vitro. Since phenotypic analysis is laborious and time consuming (28 days), genotypic resistance testing is currently the standard reference method used for HIV-1 resistance testing. However, a phenotypic test is important when a patient harbors virus with complex genetic patterns, or when the mutational resistance profile for a particular drug is not well-characterized. Results and Conclusions: Some of the currently used resistance tests are partially automated enabling laboratories to increase overall efficiency. However, maximum automation and standardization of the process, instruments and software that we have described here can overcome many of the problems encountered with current tests and aims at having a compliant, high-throughput, diagnostic laboratory, which can guarantee sample integrity from sample reception to result reporting. We also describe in detail the development and performance of virco®TYPE HIV-1 (genotype) and Antivirogram® (phenotype) assay on PR and RT genes to evaluate antiretroviral resistance.

Minor Variant Detection at Different Template Concentrations in HIV-1 Phenotypic and Genotypic Tropism Testing

The clinical trials of maraviroc showed that treatment failure was mostly associated with lack of X4 virus detection at baseline. The detection limit for X4 in tropism assays is ill defined around 10%. In the current study, quantification of X4-tropic minority populations was assessed on artificial mixed samples and 38 clinical isolates. These mixtures were subjected to tropism “clonal genotyping” or “population phenotyping”. The detection of minority variants was dependant on the input of amplifiable copies. At VL > 4 log IU/ml, X4 quantification was deemed reliable. PCR founder effect and clonal resampling might result in misrepresentation of the minority species concentration at VL < 4 log. Fourteen of the clinical isolates contained dual/mixed X4-tropic virus, 5 of which were below 10% of the virus population. Currently, there is no indication what level of X4 would lead to treatment failure. Assays aiming for the detection of minority species should express results in function of VL.

Mutation Q95K enhances N155H-mediated integrase inhibitor resistance and improves viral replication capacity

OBJECTIVES The genetic barrier to development of raltegravir resistance is considered to be low, requiring at least one primary integrase mutation: Y143C, Q148H/K/R or N155H to confer raltegravir therapy failure. However, during continued raltegravir treatment failure, additional mutations may be selected. In a patient failing raltegravir therapy, we investigated the impact of multiple integrase mutations on resistance and viral replication. Furthermore, in vivo fitness was investigated during failure of raltegravir-containing highly active antiretroviral therapy and after raltegravir was discontinued from the regimen. METHODS Patient-derived viral integrase genes were cloned into a reference strain. These recombinant viruses were used to determine the contribution of individual integrase mutations to raltegravir resistance and replication capacity in vitro. To determine in vivo fitness, the relative proportion of specific integrase mutations was monitored over time by in-depth clonal analysis of the viral integrase at baseline, during and after raltegravir treatment. RESULTS Raltegravir therapy failure was associated with the initial selection of primary resistance mutation N155H. This mutation conferred a 3.8-fold reduction in raltegravir susceptibility and a severe reduction in viral replication. Acquisition of integrase mutation Q95K increased resistance (6.2-fold) and partly restored viral replication. Selection of a third mutation, V151I, further increased raltegravir resistance (20-fold), but decreased viral replication. After prolonged raltegravir interruption, raltegravir resistance mutations were lost, demonstrating the reduced replication capacity of the resistant virus. CONCLUSIONS We describe selection of Q95K as a secondary resistance mutation during raltegravir therapy failure. In the background of N155H, Q95K enhances raltegravir and elvitegravir resistance and improves the impaired replication of the virus.