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

Jean-Michel Fondere^a,  Vincent Baillat^b,  Gaël Petitjean^a, Jacques Reynes^b, Jean-Pierre Vendrell^a*; ^aDepartement de Virologie Clinique, Hôpital Lapeyronie, 371 Avenue du Doyen Giraud , 34295 Cedex 5, Montpellier, France, ^bService des Maladies Infectieuses et Tropicales, Hôpital Gui de Chauliac, 34295 Montpellier Cedex 5 (France).

Background: The presence of latently infected, resting CD4⁺ T cells carrying replication-competent HIV-1 is induced 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. The enzyme-linked immunospot (ELISPOT) is adapted to detecting and enumerating HIV-1-antigen secreting cells at the single level. We applied the HIV-1 ELISPOT assay to the enumeration of latently HIV-1-infected CD4⁺ T cells. Material and Methods: Latent 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 patients and in four untreated patients. Enriched CD4⁺ T cells were cultured with or without antibodies directed to CD3 and to CD28 T-cell receptors plus irradiated peripheral blood mononuclear cells from HIV-1–seronegative individuals. At term of culture, CD4⁺ T cells were tested using HIV-1 ELISPOT assay. Results: CD4⁺ T cells secreting HIV-1 antigens after polyclonal activation were found in the experimental model to be 5,579 ± 4,190 per million CD4⁺ T cells and as low as 15 ± 6  spontaneously. In 22 patients, induced HIV-1 antigen-secreting cells were measured at 55 ± 108 per million CD4⁺ T cells. In contrast, spontaneous HIV-1 antigen-secreting cells (1.6 ± 3 per million) were only detected in the four untreated patients, in two patients with an incomplete response to therapy and in two responders to HAART. Conclusion: As each immunospot represents one HIV-1-secreting cell, the HIV-1 ELISPOT assay is an eligible assay to enumerate HIV-1 latently infected CD4⁺ T lymphocytes from peripheral blood. This sensitive assay could become a useful tool for precisely evaluating latently infected CD4⁺ T cell frequencies.