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150 Natural Killer Cells Are Targets for HIV-1 Infection in Vivo: Use of Real-Time PCR for the Quantification of Cell-Associated Viral Load
A. Valentin*1, M. Rosati1, D. Patenaude1, R. Yarchoan2, L. Kostrikis3, A. Hatzakis4, and G. N. Pavlakis1
1NCI, Frederick, MD USA; 2NCI, Bethesda, MD, USA; 3The Aaron Diamond AIDS Res. Ctr., New York, NY, USA; and 4Univ. of Athens, Greece
Background: The new combination therapies against HIV do not eradicate the virus, suggesting the existence of long-lived HIV-infected cells that act as viral reservoirs and are responsible for the maintenance of chronic HIV infection even under HAART. The identification of these reservoirs has become critical for understanding AIDS pathogenesis and the development of new strategies to eradicate HIV infection.
Methods: A 4-color flow cytometry assay was used to monitor CD3-CD56+ NK cells for the expression of CCR5 and CXCR4, the main HIV coreceptors, as well as CD4.
Viral DNA was measured by a method that combines positive selection of specific cell populations from HIV-1-infected individuals together with a highly sensitive quantitative real-time PCR protocol. We use HIV-1 primers that recognize a wide range of clinical isolates together with fluorescent probes for monitoring the amplification profile in each PCR cycle. Results are quantified within the linear range of internal PCR standards.
Results: We have identified a subpopulation of CD4+ NK cells. Most of the CD3-TCR-CD4+CD56+ NK cells express CCR5 and/or CXCR4. Real-time PCR analysis demonstrated the presence of HIV-1 proviral DNA in highly purified NK cells from 31 HIV-1-infected individuals (cross-sectional study). Analysis of 10 additional samples from patients prior to HAART and sequential samples (up to 18(24 months) after initiation of therapy, showed that the viral load in the NK compartment is very stable even in patients with undetectable plasma viral load.
Conclusions: These results strongly suggest that the defects in natural immunity present in HIV-infected patients may be the result of direct HIV infection and identify NK cells as an important viral reservoir for the maintenance of chronic HIV-1 infection even under HAART.
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