A dual color ELISPOT method for the simultaneous detection of IL-2 and IFN-gamma HIV-specific immune responses.

The single color IFN-gamma ELISPOT assay has become a standard for assessing HIV-specific immune responses in HIV-infected subjects. However, recent data suggests that single cytokine detection for immune monitoring of HIV-infected individuals may not be sufficient to fully describe virus-specific immune responses. Here, we have designed and validated a dual color ELISPOT assay capable of detecting both IL-2 and IFN-gamma secreting cells simultaneously in response to HIV antigens. We found that a cell input number of 200,000 cells/well provided a good balance between limited availability of cells due to blood volume restrictions and ability to detect all cytokine secretion patterns. The simultaneous detection of IL-2 and IFN-gamma resulted in a decreased magnitude of IFN-gamma but not IL-2 responses. Measures of intra- and inter-assay variability for the dual color ELISPOT assay were comparable to that seen for single cytokine ELISPOT assay with coefficients of variation below 20% for IL-2, IFN-gamma and dual secretion. Although CD8+ T cells mediated most HIV-specific responses in infected subjects, CD4+ T cells mediated responses to HIV were also detected. Features of this assay such as high throughput, cell number requirement and cytokine choice should make this assay a valuable tool for screening for HIV-specific immune responses in several clinically relevant settings.

Longitudinal changes in HIV-specific IFN-gamma secretion in subjects who received Remune vaccination prior to treatment interruption.

BACKGROUND: Despite the benefits of highly active antiretroviral therapy (HAART) for suppressing viral replication in HIV infection, virus persists and rebounds during treatment interruption (TI). This study explored whether HAART intensification with Remune vaccination before TI can boost HIV-1-specific immunity, leading to improved control of viremia off HAART. METHODS: Ten chronically HIV-infected adults were enrolled in this proof of concept study. After a 6-month HAART intensification phase with didanosine, hydroxyurea, granulocyte-macrophage colony-stimulating factor, (GM-CSF), and a first dose of Remune (HIV-1 Immunogen), HAART was discontinued. Patients continued to receive Remune every 3 months until the end of study. HAART was restarted if viral load did not fall below 50,000 copies/ml of plasma within 3 months or if CD4+ counts decreased to <200 cells/mm3. HIV-specific immunity was monitored with the interferon-gamma (IFN-gamma) ELISPOT assay. RESULTS: All subjects experienced viral rebound during TIs. Although the magnitude and breadth of HIV-specific responses to HLA-restricted optimal peptide panels and Gag p55 peptide pools increased and viral load decreased by 0.44 log10 units from TI#1 to TI#2, no significant correlations between these parameters were observed. The patients spent 50.4% of their 36 months follow up off HAART. CONCLUSION: Stopping HAART in this vaccinated population induced immune responses that persisted after therapy was restarted. Induction of HIV-specific immunity beyond IFN-gamma secretion may be contributing to better control of viremia during subsequent TIs allowing for long periods off HAART.

Human immunodeficiency virus (HIV)-specific gamma interferon secretion directed against all expressed HIV genes: relationship to rate of CD4 decline.

Immune responses to human immunodeficiency virus (HIV) are detected at all stages of infection and are believed to be responsible for controlling viremia. This study seeks to determine whether gamma interferon (IFN-gamma)-secreting HIV-specific T-cell responses influence disease progression as defined by the rate of CD4 decline. The study population consisted of 31 subjects naive to antiretroviral therapy. All were monitored clinically for a median of 24 months after the time they were tested for HIV-specific responses. The rate of CD4+-T-cell loss was calculated for all participants from monthly CD4 counts. Within this population, 17 subjects were classified as typical progressors, 6 subjects were classified as fast progressors, and 8 subjects were classified as slow progressors. Peripheral blood mononuclear cells were screened for HIV-specific IFN-gamma responses to all expressed HIV genes. Among the detected immune responses, 48% of the recognized peptides were encoded by Gag and 19% were encoded by Nef gene products. Neither the breadth nor the magnitude of HIV-specific responses correlated with the viral load or rate of CD4 decline. The breadth and magnitude of HIV-specific responses did not differ significantly among typical, fast, and slow progressors. These results support the conclusion that although diverse HIV-specific IFN-gamma-secreting responses are mounted during the asymptomatic phase, these responses do not seem to modulate disease progression rates.