title: Development and evaluation of a phenotypic assay monitoring resistance formation to protease inhibitors in HIV-1-infected patients.
abstract: A novel phenotypic assay, based on recombinant expression of the HIV-1-protease was developed and evaluated; it monitors the formation of resistance to protease inhibitors. The HIV-1 protease-encoding region from the blood sample of patients was amplified, ligated into the expression vector pBD2, and recombinantly expressed in Escherichia coli TG1 cells. The resulting recombinant enzyme was purified by a newly developed one-step acid extraction protocol. The protease activity was determined in presence of five selected HIV protease inhibitors and the 50% inhibitory concentration (IC(50)) to the respective protease inhibitors determined. The degree of resistance was expressed in terms of x-fold increase in IC(50) compared to the IC(50) value of an HIV-1 wild type protease preparation. The established test system showed a reproducible recombinant expression of each individual patients' HIV-1 protease population. Samples of nine clinically well characterised HIV-1-infected patients with varying degrees of resistance were analysed. There was a good correlation between clinical parameters and the results obtained by this phenotypic assay. For the majority of patients a blind genotypic analysis of the patients' protease domain revealed a fair correlation to the results of the phenotypic assay. In a minority of patients our phenotypic results diverged from the genotypic ones. This novel phenotypic assay can be carried out within 8-10 days, and offers a significant advantage in time to the current employed phenotypic tests.
authors.affiliation: Department of Virology, Max von Pettenkofer-Institute, Ludwig-Maximilians-University (LMU), Pettenkoferstr. 9a, D-80336 Munich, Germany.
authors.forename: Heike
authors.initials: H
authors.lastname: Gehringer
authors.forename: Klaus
authors.initials: K
authors.lastname: Von der Helm
authors.forename: Sigrid
authors.initials: S
authors.lastname: Seelmeir
authors.forename: Benedikt
authors.initials: B
authors.lastname: Weissbrich
authors.forename: Josef
authors.initials: J
authors.lastname: Eberle
authors.forename: Hans
authors.initials: H
authors.lastname: Nitschko
mesh_terms: D000163:Acquired Immunodeficiency Syndrome / Q000188:drug therapy* / Q000276:immunology / Q000821:virology; D023241:Antiretroviral Therapy, Highly Active; D018791:CD4 Lymphocyte Count; D024882:Drug Resistance, Viral; D004926:Escherichia coli / Q000235:genetics; D005838:Genotype; D016333:HIV Protease / Q000187:drug effects* / Q000235:genetics; D015497:HIV-1 / Q000187:drug effects*; D006801:Humans; D010641:Phenotype; D011994:Recombinant Proteins / Q000187:drug effects
chemical_list: D011994:Recombinant Proteins; D016333:HIV Protease