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

	Oral Presentation Abstracts: 61 [61] MEASUREMENT OF CELL-MEDIATED IMMUNE-RESPONSE TO VARICELLA-ZOSTER VIRUS USING FLOW CYTOMETRIC ANALYSIS OF ACTIVATED WHOLE-BLOOD *A. Svahn, H. Gaines, R. Thorstensson, A. Linde Swedish Institute for Infectious Disease Control, Solna, Sweden The knowledge of the role of cell-mediated immunity (CMI) for the control of Varicella-zoster virus (VZV) in man is limited. The interest in CMI against VZV has increased since the introduction of a VZV vaccine. However, there has been no simple assay available for the measurement of CMI. The conventional method, using detection of incorporated ³H thymidine as a marker for DNA synthesis and proliferation of cultured peripheral blood mononuclear cells is labour-intensive, technically complex, and subject to high intra- and inter-test variation. We have therefore developed a new, simple method for measurement of CMI, using FACS analysis for examination of whole blood cultures. Seventy-seven children, age 1-14 years, with or without a history of chickenpox were included in the study; sixty-one were VZV antibody positive and 16 were VZV antibody negative, as determined by ELISA and immunofluorescence assay testing. Whole blood was diluted 1:10 in culture medium, incubated with VZV nucleocapsid antigen for 6-7 days, immunostained with monoclonal antibodies (anti-CD3, -CD4, -CD45RO, -HLA-Dr), lysed, and analysed for six parameters using a FACSCalibur flow cytometer. Cultures in the presence of superantigens staphylococcus enterotoxin A and B, or with medium only, were included as positive and negative controls. Results were expressed as % stimulation [=100 (test - negative) / (positive - negative)] and a cut off for reactivity was set at 0.1%, by which 95% of VZV-antibody positive children and 94% of VZV-antibody negative children were found to be VZV-CMI positive and negative, respectively. Culture supernates were tested for the presence of IFN-gamma, IL-5, and IL-10, and the majority of VZV-CMI positive cultures were found to contain IFN-gamma only. A highly sensitive and specific method for measuring CMI to VZV, has been established. The new method is less labour-intensive than the conventional thymidine uptake assay, can be performed with only 0.5mL of blood, and can easily be run in large scale. Corresponding Author: A. Svahn, MD, Department of Virology, Swedish Institute for Infectious Disease Control, SE-171 82 Solna, Sweden

[61]

MEASUREMENT OF CELL-MEDIATED IMMUNE-RESPONSE TO VARICELLA-ZOSTER VIRUS USING FLOW CYTOMETRIC ANALYSIS OF ACTIVATED WHOLE-BLOOD

*A. Svahn, H. Gaines, R. Thorstensson, A. Linde
Swedish Institute for Infectious Disease Control, Solna, Sweden

The knowledge of the role of cell-mediated immunity (CMI) for the control of Varicella-zoster virus (VZV) in man is limited. The interest in CMI against VZV has increased since the introduction of a VZV vaccine. However, there has been no simple assay available for the measurement of CMI. The conventional method, using detection of incorporated ³H thymidine as a marker for DNA synthesis and proliferation of cultured peripheral blood mononuclear cells is labour-intensive, technically complex, and subject to high intra- and inter-test variation. We have therefore developed a new, simple method for measurement of CMI, using FACS analysis for examination of whole blood cultures.
Seventy-seven children, age 1-14 years, with or without a history of chickenpox were included in the study; sixty-one were VZV antibody positive and 16 were VZV antibody negative, as determined by ELISA and immunofluorescence assay testing. Whole blood was diluted 1:10 in culture medium, incubated with VZV nucleocapsid antigen for 6-7 days, immunostained with monoclonal antibodies (anti-CD3, -CD4, -CD45RO,
-HLA-Dr), lysed, and analysed for six parameters using a FACSCalibur flow cytometer. Cultures in the presence of superantigens staphylococcus enterotoxin A and B, or with medium only, were included as positive and negative controls.
Results were expressed as % stimulation [=100 (test - negative) / (positive - negative)] and a cut off for reactivity was set at 0.1%, by which 95% of VZV-antibody positive children and 94% of VZV-antibody negative children were found to be VZV-CMI positive and negative, respectively. Culture supernates were tested for the presence of IFN-gamma, IL-5, and IL-10, and the majority of VZV-CMI positive cultures were found to contain IFN-gamma only. A highly sensitive and specific method for measuring CMI to VZV, has been established. The new method is less labour-intensive than the conventional thymidine uptake assay, can be performed with only 0.5mL of blood, and can easily be run in large scale.

Corresponding Author: A. Svahn, MD, Department of Virology, Swedish Institute for Infectious Disease Control, SE-171 82 Solna, Sweden