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Nat Protoc. 2010 Jun;5(6):1033-41. doi: 10.1038/nprot.2010.73. Epub 2010 May 13.

Ex vivo T cell-based HIV suppression assay to evaluate HIV-specific CD8+ T-cell responses.

Author information

  • 1Unité de Régulation des Infections Rétrovirales, Institut Pasteur, Paris, France. asier.saez-cirion@pasteur.fr

Abstract

To advance T cell-based HIV vaccine development, it is necessary to evaluate the immune correlates of a protective CD8(+) T-cell response. We have developed an assay that assesses the capacity ex vivo of HIV-specific CD8(+) T cells to suppress HIV-1 infection of autologous CD4(+) T cells. This assay directly reflects the ultimate effector function of CD8(+) T cells, the elimination of infected cells, and accurately differentiates the effective CD8(+) T-cell response in spontaneous HIV controllers from ineffective responses in other patients. In this article, we describe all the steps from cell purification to assessment of viral replication by HIV-p24 ELISA and analysis, along with conditions for cell culturing, and how to choose the viral infectious dose that gives the most reliable results. We also depict the conditions of a rapid assay on the basis of flow cytometry analysis of intracellular HIV-Gag products. These procedures take 14-17 d when the p24 ELISA assay is used, or 6 d with the intracellular Gag assay.

PMID:
20539279
[PubMed - indexed for MEDLINE]
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