Enumeration of latently infected CD4 + T cells from HIV-1-infected patients using an HIV-1 antigen ELISPOT assay

Abstract Background: Latently infected resting CD4 + T cells carrying replication-competent HIV-1 are present in naive, chronically infected individuals as well as in those who are receiving highly active antiretroviral therapy (HAART). These cells serve as a potential source of reactivation of viral replication and remain a major obstacle for the eradication of HIV-1. Objectives: The enzyme-linked immunospot (ELISPOT) assay was adapted to the detection and the enumeration of HIV-1 antigen-secreting cells at the single cell level. We applied this test to count latently HIV-1-infected CD4 + T cells. Study design: Latently infected CD4 + T cells were assessed in an in vitro model of HIV-1-infected resting CD4 + T cells as well as in eighteen HAART-treated and in four HIV-1-infected untreated patients. Enriched CD4 + T cells were cultured with or without antibodies against CD3 and CD28 T cell receptors and with irradiated peripheral blood mononuclear cells from HIV-1 seronegative individuals. At the term of the cell culture, CD4 + T lymphocytes were tested using the HIV-1 antigen ELISPOT assay. Results: In the experimental HIV-1 infection model, 5579±4190 CD4 + T cells secreting HIV-1 antigen were enumerated after polyclonal activation. In contrast, only 15±6 HIV-1 immunospots were obtained from unstimulated T cells. In all patients tested, induced HIV-1 antigen-secreting cells were measured at a frequency of 55±108/10 6 CD4 + T cells. Conclusion: As each immunospot represents one HIV-1 antigen-secreting cell, the HIV-1 ELISPOT assay is a powerful to enumerate circulating CD4 + T lymphocytes latently infected with HIV-1.