14th International AIDS Conference Barcelona, Spain - July 7-12, 2002

Int Conf AIDS 2002 Jul 7-12; 14:(abstract no.. B10191)

Tanaka R, Saito Y, Hanabusa H, Kato S
Keio University, Tokyo, Japan

BACKGROUND: To develop a clinically applicable phenotypic assay using peripheral blood mononuclear cells (PBMC) as target cells to assess HIV-1 susceptibility to numerous drugs.

METHODS: PBMC were prepared by Percoll centrifugation and depleted of CD8 cells with anti-CD8 antibody-coated magnetic beads (Dynabeads, DYNAL). Virus isolation was carried out by coculture of PBMC from infected and normal individuals after stimulation with anti-CD3 antibody (clone CLB-CD3). PBMC grown for 4 days were infected with the virus inoculum and incubated for 3 days in culture medium containing serially decreasing concentrations of antiretroviral drugs. The p24 concentrations in culture supernatant were measured with mini VIDAS (bioMerieux) and the 50% and 90% inhibition concentrations (IC50 and IC90) were obtained from the dose-response curve. The antiretroviral drugs being assayed were AZT, ddI, 3TC, d4T, ABC, NVP, EFV, IDV, SQV, NFV, APV, and LPV. The HIV-1 LAI isolate was used as reference.

RESULTS: Virus isolation was succeeded from infected individuals with viral loads of 400 copies/ml or more (n = 60). In control experiments using the LAI isolate, the average CV values for all drugs were 29% for IC50 and 18% for IC90. Based on these data, resistance was defined as the 4-fold increase in IC50 and IC90. So far, nine drug-naïve and seven drug-experienced patients were assayed. Drug-resistant virus was not observed in any drug-naïve patient, and found in five drug-experienced patients. The drug-resistant viruses observed showed cross-resistance to 12, 10, 9, 9, and 3 different drugs.

CONCLUSIONS: The phenotypic assay presented are able to assess drug susceptibility in PBMC with a low standard errors. This may be useful for evaluation of the role of drug resistance in lymphocytes in vivo.

020707
B10191

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