VZV Foundation - A Decade Of Leadership In The Fight Against VZV Infections

	Oral Presentation Abstracts: 19 [19] DEVELOPMENT OF AN INTERFERON-y ELISPOT ASSAY FOR QUANTITATION OF CELLULAR IMMUNE RESPONSES TO VZV *J.G. Smith (1), X. Liu (1), R.M. Kaufhold (1), J. Clair (2), P. Keller (1) & M.J. Caulfield (1) (1) Virus & Cell Biology, (2) Vaccine Biometrics, Merck and Co., Inc. West Point, PA, USA Cell mediated immunity (CMI) appears to be critical for the prevention and control of Varicella-zoster virus (VZV) infection and complications arising from zoster. Current laboratory assays of VZV-specific CMI are performed by limiting dilution responder cell frequency (RCF) analysis or cytolytic T lymphocyte (CTL) assays. These methods are cumbersome and lack sensitivity. Newer flow cytometry-based assays for detection of intracellular cytokine (e.g. interferon-y) production are an improvement but may not have the sensitivity needed for detection of weak responses. We have developed an interferon-y ELISPOT assay that provides a direct measure of the number of T cells secreting cytokine following stimulation with killed VZV as a source of antigen. This assay is extremely sensitive and specific with the ability to detect interferon-y spot-forming cells (SFC) in the range of 10 - 1000 SFC per million PBMCs. Cell depletion studies indicate that the response is mediated almost entirely by CD4 T cells. The following attributes make the ELISPOT assay particularly useful for monitoring clinical trials: (1) responses from fresh-frozen PBMCs are equivalant to those from freshly isolated cells. (2) Frozen PBMCs can be shipped on dry ice for up to 48 hours without loss of activity. (3) Frozen PBMC samples can be stored in liquid nitrogen over long periods (>22 months) without any significant change in response. (4) The number of ELISPOTs can be counted using a computer-based imaging system. Frozen PBMC samples can be cycled through multiple changes in storage between liquid nitrogen and dry ice without any change in response 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 during Herpes Zoster (HZ) disease outbreak or following vaccination. Corresponding Author: J.G. Smith, Department of Virus & Cell Biology, Merck & Co., Inc., WP26B-1144, P.O. Box 4, West Point, PA 19486, USA

[19]

DEVELOPMENT OF AN INTERFERON-y ELISPOT ASSAY FOR QUANTITATION OF CELLULAR IMMUNE RESPONSES TO VZV

*J.G. Smith (1), X. Liu (1), R.M. Kaufhold (1), J. Clair (2), P. Keller (1) & M.J. Caulfield (1) (1) Virus & Cell Biology, (2) Vaccine Biometrics, Merck and Co., Inc. West Point, PA, USA

Cell mediated immunity (CMI) appears to be critical for the prevention and control of Varicella-zoster virus (VZV) infection and complications arising from zoster. Current laboratory assays of VZV-specific CMI are performed by limiting dilution responder cell frequency (RCF) analysis or cytolytic T lymphocyte (CTL) assays. These methods are cumbersome and lack sensitivity. Newer flow cytometry-based assays for detection of intracellular cytokine (e.g. interferon-y) production are an improvement but may not have the sensitivity needed for detection of weak responses. We have developed an interferon-y ELISPOT assay that provides a direct measure of the number of T cells secreting cytokine following stimulation with killed VZV as a source of antigen. This assay is extremely sensitive and specific with the ability to detect interferon-y spot-forming cells (SFC) in the range of 10 - 1000 SFC per million PBMCs. Cell depletion studies indicate that the response is mediated almost entirely by CD4 T cells. The following attributes make the ELISPOT assay particularly useful for monitoring clinical trials: (1) responses from fresh-frozen PBMCs are equivalant to those from freshly isolated cells. (2) Frozen PBMCs can be shipped on dry ice for up to 48 hours without loss of activity. (3) Frozen PBMC samples can be stored in liquid nitrogen over long periods (>22 months) without any significant change in response. (4) The number of ELISPOTs can be counted using a computer-based imaging system. Frozen PBMC samples can be cycled through multiple changes in storage between liquid nitrogen and dry ice without any change in response 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 during Herpes Zoster (HZ) disease outbreak or following vaccination.

Corresponding Author: J.G. Smith, Department of Virus & Cell Biology, Merck & Co., Inc., WP26B-1144, P.O. Box 4, West Point, PA 19486, USA