Development and validation of a gamma interferon ELISPOT assay for quantitation of cellular immune responses to varicella-zoster virus

Cell-mediated immunity appears to be critical for the prevention and contro l of varicella-zoster virus (VZV) infection and complications arising from zoster. Current assays of VZV-specific cell-mediated immunity are cumbersom e or lack sensitivity. We have developed a gamma interferon ELISPOT assay t hat provides a direct measure of the number of T cells secreting a cytokine following stimulation with antigen. This assay is extremely sensitive and specific, with the ability to detect gamma interferon spot-forming cells (S FC) in the range of 10 to 1,000 SFC per million peripheral blood mononuclea r cells (PBMCs). This assay has been validated by demonstrating the followi ng: (i) the response detected is mediated almost entirely by CD4(+) T cells , (ii) ELISPOT responses from fresh-frozen PBMCs are equivalent to those fr om freshly isolated cells, (iii) frozen PBMCs can be shipped on dry ice for up to 48 h without loss of activity, (iv) frozen PBMC samples can be store d in liquid nitrogen over long periods (>22 months) without any significant change in response, and (v) the numbers of ELISPOTs counted using a comput er-based imaging system are equivalent to those counted by humans but have lower variability. The ability to use frozen cells is facilitated by the us e of a recombinant nuclease (Benzonase) that can prevent cell clumping when samples are thawed. Frozen PBMC samples can be cycled through multiple cha nges in storage between liquid nitrogen and dry ice without any change in r esponse being detected. This facilitates collection of samples at one site and testing performed at a remote location. This VZV ELISPOT assay provides a new versatile tool for monitoring cellular immune responses either durin g a herpes zoster disease outbreak or following vaccination.