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Detection and enumeration of HIV-1-producing cells by ELISPOT (enzyme-linked immunospot) assay
P Corne1, M F Huguet, L Briant
1Laboratoire d'Immunologie des Infections Rétrovirales, Centre National de la Recherche Scientifique, Institut de Biologie, Montpellier, France.
Insights
The enzyme-linked immunospot (ELISPOT) assay can detect HIV-1-producing cells. This method quantified productive HIV-1 infections in cells and patients, offering a new tool for studying HIV-1 replication.
Area of Science:
- Immunology
- Virology
- Cell Biology
Background:
- Detecting and quantifying HIV-1-producing cells is crucial for understanding viral replication and disease progression.
- Existing methods may have limitations in sensitivity or specificity for single-cell analysis.
Purpose of the Study:
- To adapt and validate the enzyme-linked immunospot (ELISPOT) assay for the detection and enumeration of HIV-1-producing cells at the single-cell level.
- To assess the utility of the ELISPOT assay in both in vitro cell cultures and in vivo patient samples.
Main Methods:
- The enzyme-linked immunospot (ELISPOT) assay was employed.
- CEM cells and peripheral blood mononuclear cells (PBMCs) infected in vitro with HIV-1 were analyzed.
- Samples from HIV-1-infected patients in the early disease stage, not on antiretroviral therapy, were examined, including peripheral blood and splenic lymphoid tissue.
Main Results:
- The ELISPOT assay successfully detected HIV-1-producing cells in vitro, identifying productively infected cells within p24 antigen-positive CEM cells (5.4–9.5%) and PBMCs (11.1–23.6%).
- In HIV-1-infected patients, the assay detected up to 4.54 HIV-1-producing cells per 10^6 CD4+ T lymphocytes in peripheral blood.
- Significantly higher levels were found in splenic lymphoid tissue, with up to 277.75 HIV-1-producing cells per 10^6 CD4+ T lymphocytes.
Conclusions:
- The adapted ELISPOT assay is effective for detecting and enumerating HIV-1-producing cells at the single-cell level.
- This assay provides valuable quantitative data on HIV-1 replication in both experimental models and clinical settings.
- The ELISPOT assay represents a promising new tool for in vivo studies of HIV-1 replication.
Abstract:
The enzyme-linked immunospot (ELISPOT) assay was adapted to detect and enumerate HIV-1-producing cells at the single cell level. With CEM cells or peripheral blood mononuclear cells (PBMC) infected in vitro with HIV-1, the ELISPOT assay detected cells that produced HIV-1 antigens and showed that between 5.4% and 9.5% of the p24 antigen-positive CEM cells and 11.1% to 23.6% of the p24 antigen-positive PBMC were productively infected. In HIV-1-infected patients in early stage of the disease and without antiretroviral therapy, up to 4.54 HIV-1-producing cells per 10(6) CD4+ T lymphocytes were detected in peripheral blood and up to 277.75 HIV-1-producing cells per 10(6) CD4+ T lymphocytes were detected in splenic lymphoid tissue. Our results indicate that the ELISPOT assay could represent a new tool to study HIV-1 replication in vivo.
