Elispot Assay

This assay depends on T cells responding specifically to antigen by the synthesis and secretion of cytokines, particularly IFN-y. The principle of the ELISPOT assay is that secreted IFN-y from stimulated antigen-specific T cells is captured by an anti-IFN-y monoclonal antibody immobilized on a nitrocellulose membrane. The captured IFN-y is then detected by a second anti-IFN-y antibody conjugated to an enzyme; "cytokine spots" on the membrane which represent individual IFN-y-secreting T cells are visualized and enumerated following enzyme-substrate reactions (Fig. 6.1).

The IFN-y ELISPOT is relatively straightforward to perform, is reasonably sensitive, and has found a place for measuring T-cell response in vaccine trials [56, 57]. However, it is now perceived to have several shortcomings. First, the enumeration of antigen-specific T cells is merely a reflection of T-cell reactivity to recombinant antigens (not as expressed on antigen-presenting cells, APC, in vivo during a natural HIV-1 infection), and does not necessarily correspond to CTL cytotoxicity against HIV-infected cells. Second, IFN-y has little anti-HIV-1 activity per se and, therefore, may not be the best cytokine to be tested [58]. Third, although the IFN-y ELISPOT works well for strong immune responses to acute viral infections - including Epstein-Barr virus (EBV), cytomegalovirus (CMV), influenza virus, and HIV-1 - responses to vaccines are often far weaker, and this

Enzyme Linked Immunospot Assay

Fig. 6.1 Schematic diagram illustrating the enzyme-linked immunospot (ELISPOT) assay. Antigenically stimulated T lymphocytes are triggered to produce cytokines, for example, IFN-y. The secreted cytokine is captured by an immobilized anti-cytokine antibody on the surface of a nitrocellulose membrane; the bound cytokine is detected by a second anti-cytokine antibody and cytokine spots are visualized by enzyme/ substrate.

Fig. 6.1 Schematic diagram illustrating the enzyme-linked immunospot (ELISPOT) assay. Antigenically stimulated T lymphocytes are triggered to produce cytokines, for example, IFN-y. The secreted cytokine is captured by an immobilized anti-cytokine antibody on the surface of a nitrocellulose membrane; the bound cytokine is detected by a second anti-cytokine antibody and cytokine spots are visualized by enzyme/ substrate.

results in decreased sensitivity. Finally, variations in cell sampling times following immunization, variations in the reagents, the length of time required to develop the assay plates, and variations in environmental conditions can each significantly affect both the size and number of spots and the level of "background" response [59]. The lack of IFN-y ELISPOT standardization/harmonization reagents and conditions may, to some degree, account for differences in the results reported for HIV-antigen-stimulated T-cell responses. For example, the magnitude of IFN-y CD8+ T-cell responses to the gag and p24 HIV-1 proteins in chronically infected patients has been reported to correlate inversely with viral loads and directly to absolute CD4+ T-cell counts [60]. However, in other studies it has been shown that the frequencies of HIV-1-specific IFN-y-secreting T cells correlated positively with viral loads [32, 61, 62]; also, measures of disease progression did not correlate with the magnitude of HIV-1-specific IFN-y responses [63-65]. Therefore, exvivo measurements of IFN-y secretion may not necessarily correspond to CTL cytotoxicity [80, 84] or to containment of the HIV infective process during the chronic phase [43]. In the cases of preventive vaccines against HIV-1, it is possible to demonstrate for most that, using the IFN-y ELISPOT assay, they do stimulate the development of HIV-1 antigen-specific CD8+ T cells [56, 57] (see Table 6.1). Such measurements would serve as a measure of vaccine activity or potency in vivo, but since the "jury is out" on whether IFN-y-secreting CD8+ T cells play a significant protective role against infection by HIV-1, they do not in themselves predict the protective efficacy of a vaccine [118].

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