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Rapid single-cycle assay for human immunodeficiency virus type-1 drug resistance
| Details |
Inventors: Paulous, Sylvie; Charneau, Pierre; Zennou, Veronique; Clavel, Francois;
Assignee: Institut Pasteur (Paris, FR)
Primary Examiner: Stucker; Jeffrey
Assistant Examiner:
Attorney, Agent or Firm: Finnegan, Henderson, Farabow, Garrett & Dunner, L.L.P.
An in vitro, single cycle, recombinant virus assay (RVA) for determining inhibition of HIV replication by a protease inhibitor comprises transfecting a human epithelial cell line with amplified HIV protease sequences of an HIV virus; an HIV, envelope defective, molecular clone having a complete deletion of its protease coding sequence; and a plasmid containing HIV envelope coding sequence under the control of a promoter for phenotypic complementation of the envelope defective molecular clone. The transfected cells are cultured in the presence of a protease inhibitor to produce a testable stock of infectious particles that can be used to infect indicator cells containing an indicator gene without amplification of the infectious particles prior to infecting the indicator cells. Accumulation of indicator gene product is a measure of inhibition of HIV replication by the protease inhibitor. |
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DETAILED DESCRIPTION This invention aids in fulfilling this need in the art by providing an in vitro, single cycle, recombinant virus assay (RVA) for determining inhibition of HIV replication by a protease inhibitor.
The method comprises transfecting a human epithelial cell line with: (1) Amplified HIV protease sequences of an HIV virus to be assayed for resistance to the protease inhibitor; (2) an HIV, envelope defective, molecular clone for recombination, wherein the molecular clone has a complete deletion of its protease coding sequence and an envelope deletion so that no HIV envelope is expressed by the HIV molecular clone; and (3) a plasmid containing VSV-G envelope coding sequence under the control of a promoter for phenotypic complementation of the HIV, envelope defective, molecular clone.
The resulting transfected cells are contacted with the protease inhibitor and cultured to repair the protease coding sequence by homologous recombination between the HIV molecular clone and the amplified HIV protease sequences, and to produce a testable stock of infectious particles by coexpression of the protease-repaired HIV molecular clone and the VSV-G envelope coding sequence, such as a VSV-G envelope coding sequence.
Indicator cells containing an indicator gene are contacted with the testable stock of infectious particles without amplification of the infectious particles prior to infecting the indicator cells.
The indicator gene is expressed in the indicator cells, and accumulation of indicator gene product is determined as a measure of HIV replication.
The susceptibility of the recombinant virus to protease inhibitors is determined by contacting transfected cells with serial dilutions of the inhibitor, allowing to calculate the concentration of inhibitor able to inhibit 50 or 90% of virus infectivity [IC50 and IC90].
This invention provides a single-cycle version of RVA for testing HIV resistance to protease inhibitors.
Similar to the previously described RVA systems, the assay of the invention uses recombination of PCR-amplified HIV sequences from plasma with a deleted HIV molecular clone
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Gene Therapy 
