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


OBJECTIVE
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.


METHODS
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.


CONCLUSIONS
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.
Fondere
Jean-Michel
JM
Departement de Virologie Clinique, Hôpital Lapeyronie, 371 Avenue du Doyen Giraud, 34295 Montpellier edex 5, France.
Planas
Jean-François
JF
Huguet
Marie-France
MF
Baillat
Vincent
V
Bolos
Frederic
F
Reynes
Jacques
J
Vendrell
Jean-Pierre
JP
D018791:CD4 Lymphocyte Count; D015496:CD4-Positive T-Lymphocytes / Q000821:virology*; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D015488:HIV Antigens / Q000276:immunology; D015658:HIV Infections / Q000188:drug therapy / Q000276:immunology* / Q000821:virology*; D015497:HIV-1 / Q000302:isolation & purification / Q000502:physiology*; D006801:Humans; D008213:Lymphocyte Activation; D008297:Male; D017735:Virus Latency
D015488:HIV Antigens