Detection of vaccinia virus-specific IFNγ and IL-10 secretion from human PBMCs and CD8 ⁺ T cells by ELISPOT

High-throughput in vitro assays, which rapidly and succinctly assess the immune status of large cohorts of individuals, are essential tools for conducting population-based studies, including vaccine research. The enzyme-linked immunospot (ELISPOT) assay has emerged as a sensitive, reliable high-throughput tool to measure functional recall immunity by assessing the frequency of antigen-specific cytokine-secreting lymphocytes present in peripheral blood mononuclear cells (PBMCs). For the past 10 years, ELISPOT method has been the dominant platform and a standard for the cell-mediated immune (CMI) assays. ELISPOT assays are used extensively as a measure of CMI response to vaccines, including smallpox (vaccinia), following primary or secondary vaccination. Here, we present detailed methodology for using ELISPOT assays to detect the frequency of cytokine secreting vaccinia-specific lymphocytes including optimized protocols for growing, titrating, and inactivating vaccinia virus; isolating, cryopreserving, and thawing human PBMCs; and finally, detecting vaccinia-specific IL-10 and IFNγ secreting lymphocytes, as well as CD8 ⁺ IFNγ T cells following in vitro stimulation of PBMCs with vaccinia virus. The methods presented below, although optimized for vaccinia virus, emphasize principles that can be generally applied to create ELISPOT assays capable of assessing the immune status as well as antiviral CD8 ⁺ T cell response of individuals following primary or secondary vaccination with other licensed or novel vaccines.