Rapid infectious viral assay (RIVA) of monocytes in patients with VL< 50 copies/ml of blood
O.A. Ducoudret, N.S. Baichoo, M.R. Ruff, C.B. Pert, L. Agrawal
RAPID Laboratories, Rockville, United States
Background: Detection of infectious HIV particles from patients' Peripheral Blood Mononuclear Cells (PBMC) using classical co-culture methods is time-consuming and focuses on CD4 cells. We propose a 4 day cell-based approach capable of detecting virus in suppressed patients. Rather than lymphocytes, this method monitors the putative clinically-relevant cellular compartment (CRCC):CD14-positive monocytes. In conjunction with this method, real-time PCR (rtPCR) and flow cytometry assays have been developed to monitor CRCC infection in suppressed patients for mDAPTA phase II clinical studies.
Methods: CD8-depleted PBMC were isolated from HIV-negative donors and HIV patients with VL< 50 copies/ml using Dynal beads. Peripheral monocytes (CD14) were used in a rapid infectious assay using TZM-bl coculture technique. We compare this shorter infection assay to classical method of PBMC activation with anti CD3/CD28 antibodies. Major CCR5 blockers were addressed. Presence of infectious virus was also investigated using FACS and rtPCR.
Results: We detected infectious HIV in all studied patients' (VL< 50) purified CD14-monocytes using a Rapid Infectious Viral Assay (RIVA). Virus rescue in RIVA format was comparable to the traditional rescue methods. The virus released was found to be sensitive to CCR5 inhibitors. Our cellular assay confirms that live HIV particles are released by the monocytes, even in absence of stimulating drug. We also found that monocytes constituted a significant source of virus as assessed by PCR and FACS analysis.
Conclusions: We determined that the monocytic compartment is a significant source of the viral burden. Furthermore our 4 day cellular assay confirms that live HIV particles are released by the monocytes. Since we didn't detect any significant increase of infectivity in presence of activators, we propose that this assay provides a direct measurement of the cellular viral burden and not merely the ability to generate de novo infectious particles. This method avoids the confounding factors of evolution/selection in long term cultures.
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