Genotypic tropism testing by massively parallel sequencing: qualitative and quantitative analysis

Martin Dumer 0 Rolf Kaiser 2 Rolf Klein 0 Thomas Lengauer 1 Bernhard Thiele 0 Alexander Thielen 0 1 0 Institute of Immunology and Genetics , Pfaffplatz 10, 67655 Kaiserslautern , Germany 1 Max Planck Institute for Informatics , Stuhlsatzenhausweg E1.4, 66123, Saarbrucken , Germany 2 Institute of Virology, University of Cologne , Furst-Puckler-Str. 56, 50935 Cologne , Germany Background: Inferring viral tropism from genotype is a fast and inexpensive alternative to phenotypic testing. While being highly predictive when performed on clonal samples, sensitivity of predicting CXCR4-using (X4) variants drops substantially in clinical isolates. This is mainly attributed to minor variants not detected by standard bulk-sequencing. Massively parallel sequencing (MPS) detects single clones thereby being much more sensitive. Using this technology we wanted to improve genotypic prediction of coreceptor usage. Methods: Plasma samples from 55 antiretroviral-treated patients tested for coreceptor usage with the Monogram Trofile Assay were sequenced with standard population-based approaches. Fourteen of these samples were selected for further analysis with MPS. Tropism was predicted from each sequence with geno2pheno[coreceptor]. Results: Prediction based on bulk-sequencing yielded 59.1% sensitivity and 90.9% specificity compared to the trofile assay. With MPS, 7600 reads were generated on average per isolate. Minorities of sequences with high confidence in CXCR4-usage were found in all samples, irrespective of phenotype. When using the default falsepositive-rate of geno2pheno[coreceptor] (10%), and defining a minority cutoff of 5%, the results were concordant in all but one isolate. Conclusions: The combination of MPS and coreceptor usage prediction results in a fast and accurate alternative to phenotypic assays. The detection of X4-viruses in all isolates suggests that coreceptor usage as well as fitness of minorities is important for therapy outcome. The high sensitivity of this technology in combination with a quantitative description of the viral population may allow implementing meaningful cutoffs for predicting response to CCR5-antagonists in the presence of X4-minorities. - Background Treatment of HIV infection has progressed significantly in the last decade. Amongst other factors this improvement is also based on the development of new drugs which are becoming more and more potent and which nowadays are given in combination [1]. This so-called highly active antiretroviral therapy (HAART) aims at inhibiting the viral replication as strongly as possible by using antiretroviral drugs from usually two different drug classes. While mortality and morbidity decreased substantially, long-term side effects and suboptimal drug potency are still major obstacles [2]. Moreover, the emergence of drug-resistant variants from minor populations may lead to early therapy-failures despite apparently effective treatment regimes [3]. With the approval of maraviroc (MVC, Celsentry/ Selzentry, Pfizer) [4] in 2007, a further new class of antiretrovirals was introduced into anti-HIV treatment. In contrast to previously approved drugs, drugs from this class do not bind to viral proteins but to a specific coreceptor which is expressed by the host cell [5,6]. Two coreceptors CCR5 and CXCR4 have been shown to be relevant in vivo. The mode of action of coreceptor antagonists led to the assumption that administration of such drugs would impede the adaptation of the virus and consequently lower the risk of developing resistance. However, the major problem of coreceptor antagonists is that there are two types of viruses: 1) so-called R5-viruses which use the CCR5 coreceptor for cell entry and which are usually dominant in the beginning of infection, and 2) dual-tropic and X4viruses, which can also use another coreceptor (CXCR4) to gain entry into cells. Due to the fact, that maraviroc binds to CCR5, viruses which are capable of using CXCR4 are not harmed by the drug. Therefore, coreceptor usage, also known as viral tropism, has to be determined before administration of a regimen containing maraviroc. Currently such tests are performed with a phenotypic assay, usually the trofile assay (nowadays the enhanced sensitivity version) from Monogram Biosciences (San Francisco, CA, USA) [7]. In this assay a recombinant replication-defective virus carrying the tropism-determining gp120 V3 region of a clinical isolate, is analyzed for entry on either CCR5or CXCR4 expressing cells. The assay has been used in all maraviroc and vicriviroc (another coreceptor antagonist which is not in development anymore, Merck) clinical trials and has therefore become the de facto gold standard for measuring coreceptor-tropism. The main disadvantages of phenotypic assays are that they are very time-consuming and cost-intensive [8]. Additionally, samples with viral loads below 1,000 copies/ml or certain non-B subtypes exhibited an extraordinarily high failure rate in the original Trofile assay. Here the genotypic approach poses an alternative. It is characterized by the experimental determination and computational interpretation of the viral genome. Genotypic prediction of HIV-1 tropism is an inexpensive and fast alternative to phenotypic assays [8,9]. However, standard sequencing approaches afford only a low sensitivity of X4-detection, especially in clinical isolates [10-12]. They generally work well when applied to clonal data [10,12] which led to the conclusion that false predictions are mainly attributed to the lack of detection of minor populations of CXCR-using variants. To overcome this disadvantage, a new approach to genotypic tropism testing, the so-called massively parallel sequencing [13] was introduced. This technology enables generating an unprecedented number of sequences on the basis of single molecule sequencing, thus increasing the probability of identifying minority variants in heterogeneous gene families or virus populations. Therefore, coreceptor usage prediction from genotype is generally regarded as a promising application of ultra-deep sequencing, which detects single clones, thereby being much more sensitive than classical population-based Sanger sequencing. In this study we address the question whether massively parallel sequencing can be successfully combined with bioinformatic approaches in order to afford improved qualitative and a quantitative prediction of coreceptor usage from the V3-loop. was shipped on dry ice for phenotypic tropism testing (original trofile tropism assay, Monogram Biosciences, South San Francisco). Plasma viral load was determined using the M2000 system (Abbott Molecular). Results were documented as either CCR5-tropic (R5), CXCR4tropic (X4) or dual-/mixed-tropic (D/M). X4- and D/M isolates are pooled together and called X4 from here on. The investigation was performed within the German RESINA-study for which the patients signed an informed consent (BMG 310/4476/02/3). RNA extr (...truncated)