1st International Workshop on HIV Drug Resistance & Treatment Strategies 25-28 June 1997, St. Petersburg, Florida, USA

A 1 WEEK, SINGLE-CYCLE PROTEASE INHIBITOR RESISTANCE ASSAY

Antiviral Therapy 1997;2 (Suppl 5):30 (abstract no. 46)

S Paulous, V Zennou and F Clavel
Institut Pasteur, Paris, France

The recombinant virus assay (RVA) allows phenotypic evaluation of HIV resistance to antiretrovirals following recombination of PCR-amplified HIV sequences with a deleted proviral molecular clone. However, currently used RVA protocols require serial rounds of virus propagation and amplification, a lengthy process that promotes genetic drift of the virus. We present here a single-cycle adaptation of the RVA that can efficiently and reliably evaluate HIV resistance to protease inhibitors in less than 1 week. The product of PCR amplification of HIV protease (PR) sequences from the plasma of infected patients is cotransfected with a PR-deleted, linearized, pNL43-derived plasmid. The cotransfections are performed in a series of small-scale HeLa cell cultures in 24-well plates by the calcium phosphate precipitation method, which is known to be more recombinogenic than most other transfection techniques. The yield of a typical such transfection is 1-10 ng of HIV p24/ml of supernatant after 48h, which corresponds to an efficiency of recombination of about 2%. At 24 h after transfection, the cultures are washed and treated with increasing concentrations of PR inhibitor. After a further 24 h incubation period, the virus produced by each treated culture is immediately titrated on the P4 cell line (HeLa-CD4, LTR-LacZ), which allows rapid titration of HIV infectivity on the basis of a single cycle of virus replication. The titration is conducted in triplicate in microtitre plates and revealed by a simple β-galactosidase colorimetric or luminometric assay. To increase the sensitivity of the assay, the PR-deleted plasmid has been further deleted in the envelope gene, and complemented by separate expression of the vesicular stomatitis virus (VSV) G envelope glycoprotein. HIV pseudotyping by the VSV envelope increases the relative infectivity of HIV particles by 10- to 20-fold and does not affect the resistance phenotype. In addition, the absence of HIV envelope from the system means that replication of HIV in the target cells is limited to a single cycle.

The single-cycle RVA (SC-RVA) allows rapid and reliable measurement of HIV sensitivity to PR inhibitors, yielding excellent correlations between the measured IC₅₀ or IC₉₀ values and the corresponding plasma HIV PR genotypes. Recombination with pretherapy wild-type PR sequences yields an IC₉₀ of 10-20 nM for saquinavir, and of 50-100 nM for ritonavir and indinavir. A typical saquinavir-resistant virus (L63P, A71V, L90M) displays a 10-fold increase in IC₉₀ to saquinavir and a threefold increase in IC₉₀ to indinavir. For a typical ritonavir-resistant virus (K20R, M36I, I54V, V82A), the increase in IC₉₀ to ritonavir is ninefold.

We conclude that the SC-RVA described here is a fast, reliable method for phenotypic assessment of HIV resistance to PR inhibitors. It is currently being extended to RT inhibitor resistance testing and could prove useful for routine monitoring of antiretroviral treatments.

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1997-06-25
46

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