IN-VITRO SEPARATION AND EXPANSION OF CD4 LYMPHOCYTES FROM HIV-INFECTED INDIVIDUALS WITHOUT ACTIVATION OF HIV-INFECTION

In order to offer a gene therapy-based treatment against AIDS, it is l ikely to be necessary to harvest and culture CD4 cells from HIV-positi ve patients without activating the HIV infection. We have used a magne tic cell sorting (MACS) system to enrich CD4 cells. Using positive sel ection, CD4 cells from a total of 14 patients were enriched from a mea n percentage of CD4 cells in PBMC of 18% to 91% CD4 cells in the enric hed cell fraction. Furthermore, we found that this separation did not lead to an increase in viral load. The MACS performed equally well on cells from HIV-positive patients and HIV-negative donors. CD4 cells fr om HIV-positive patients were readily expanded with PHA; 19-fold by da y 10, 50-fold by day 20, and 156-fold by day 25. However, CD4 cells fr om HIV-positive patients grew at a slower rate than CD4 cells from HIV -negative donors. The expanded CD4 cells showed a high degree of CD4 e xpression and no loss of polyclonality. Only in two of six cultures we re we able to detect HIV-antigen production, and using an LTR-PCR and an RT assay, we did not find activation of the HIV infection during th e culture period. Thus, the method described separates and expands CD4 cells from HIV-positive patients without activation of the HIV infect ion.