Universität zu Köln

Sierra, Saleta, Kaiser, Rolf, Luebke, Nadine, Thielen, Alexander, Schuelter, Eugen, Heger, Eva, Daeumer, Martin, Reuter, Stefan, Esser, Stefan, Faetkenheuer, Gerd, Pfister, Herbert, Oette, Mark and Lengauer, Thomas (2011). Prediction of HIV-1 Coreceptor Usage (Tropism) by Sequence Analysis using a Genotypic Approach. J. Vis. Exp. (58). CAMBRIDGE: JOURNAL OF VISUALIZED EXPERIMENTS. ISSN 1940-087X

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Abstract

Maraviroc (MVC) is the first licensed antiretroviral drug from the class of coreceptor antagonists. It binds to the host coreceptor CCR5, which is used by the majority of HIV strains in order to infect the human immune cells (Fig. 1). Other HIV isolates use a different coreceptor, the CXCR4. Which receptor is used, is determined in the virus by the Env protein (Fig. 2). Depending on the coreceptor used, the viruses are classified as R5 or X4, respectively. MVC binds to the CCR5 receptor inhibiting the entry of R5 viruses into the target cell. During the course of disease, X4 viruses may emerge and outgrow the R5 viruses. Determination of coreceptor usage (also called tropism) is therefore mandatory prior to administration of MVC, as demanded by EMA and FDA. The studies for MVC efficiency MOTIVATE, MERIT and 1029 have been performed with the Trofile assay from Monogram, San Francisco, U.S.A. This is a high quality assay based on sophisticated recombinant tests. The acceptance for this test for daily routine is rather low outside of the U.S.A., since the European physicians rather tend to work with decentralized expert laboratories, which also provide concomitant resistance testing. These laboratories have undergone several quality assurance evaluations, the last one being presented in 2011(1). For several years now, we have performed tropism determinations based on sequence analysis from the HIV env-V3 gene region (V-3)(2). This region carries enough information to perform a reliable prediction. The genotypic determination of coreceptor usage presents advantages such as: shorter turnover time (equivalent to resistance testing), lower costs, possibility to adapt the results to the patients' needs and possibility of analysing clinical samples with very low or even undetectable viral load (VL), particularly since the number of samples analysed with VL<1000 copies/mu l roughly increased in the last years (Fig. 3). The main steps for tropism testing (Fig. 4) demonstrated in this video: 1. Collection of a blood sample 2. Isolation of the HIV RNA from the plasma and/or HIV proviral DNA from blood mononuclear cells 3. Amplification of the env region 4. Amplification of the V3 region 5. Sequence reaction of the V3 amplicon 6. Purification of the sequencing samples 7. Sequencing the purified samples 8. Sequence editing 9. Sequencing data interpretation and tropism prediction

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Sierra, Saleta UNSPECIFIED UNSPECIFIED UNSPECIFIED Kaiser, Rolf UNSPECIFIED UNSPECIFIED UNSPECIFIED Luebke, Nadine UNSPECIFIED UNSPECIFIED UNSPECIFIED Thielen, Alexander UNSPECIFIED UNSPECIFIED UNSPECIFIED Schuelter, Eugen UNSPECIFIED UNSPECIFIED UNSPECIFIED Heger, Eva UNSPECIFIED UNSPECIFIED UNSPECIFIED Daeumer, Martin UNSPECIFIED UNSPECIFIED UNSPECIFIED Reuter, Stefan UNSPECIFIED UNSPECIFIED UNSPECIFIED Esser, Stefan UNSPECIFIED UNSPECIFIED UNSPECIFIED Faetkenheuer, Gerd UNSPECIFIED UNSPECIFIED UNSPECIFIED Pfister, Herbert UNSPECIFIED UNSPECIFIED UNSPECIFIED Oette, Mark UNSPECIFIED UNSPECIFIED UNSPECIFIED Lengauer, Thomas UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-484904
DOI:	10.3791/3264
Journal or Publication Title:	J. Vis. Exp.
Number:	58
Date:	2011
Publisher:	JOURNAL OF VISUALIZED EXPERIMENTS
Place of Publication:	CAMBRIDGE
ISSN:	1940-087X
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language MARAVIROC Multiple languages Multidisciplinary Sciences Multiple languages
URI:	http://kups.ub.uni-koeln.de/id/eprint/48490