View All Abstracts for Session 18
75 Measurement of Vaccination-Induced Cell-Mediated Immunity by in Vitro CMI
J. B. Woodcock*1, N. Hooper1, R. Kowalski1, A.-C. Leandersson2, B. Wahren2, and J. Britz1
1Cylex Inc., Columbia, MD, USA and 2Karolinska Inst., Stockholm, Sweden
Background: Lymphoproliferation in response to specific antigens or mitogens has traditionally been used to measure T-cell function. A rapid alternative method is described. The CD4 in vitro CMI assay detects T-cell activation by quantifying increases in ATP following stimulation of whole blood or PBMCs with mitogens and viral or bacterial antigens. The assay uses a bioluminescent readout, eliminating the use of radioactivity.
Methods: Immune responses of HIV-infected and noninfected subjects vaccinated against infective agents such as HIV or Hepatitis B were assessed by the CD4 in vitro CMI assay. Responses to other related antigens in these subjects were also determined. Diluted whole blood samples of PBMCs were placed in 96-well microtiter plates and incubated with the antigens for 4 or 15(18 hours. After the incubation CD4 cells were immunoselected using antibody-coated magnetic particles, washed to remove unbound cells, and lysed to release intracellular ATP. Released ATP was detected using firefly luciferin/luciferase and quantified using an ATP calibration curve.
Results: Increases in ATP in response to antigenic stimuli were detected in individuals after vaccination. In gp160-vaccinated individuals, ATP response to stimulation with gp160 was 60(70 ng/mL, whereas their nonstimulated values were less than 20 ng/mL ATP. Magnitude of ATP increase after vaccination varied among individuals. Cellular response to HBsAg stimulation was monitored in a Hepatitis B vaccinated subject. ATP levels increased from 10 ng/mL pre-vaccination to 45 ng/mL post-vaccination.
Conclusions: The data demonstrate the utility of the CD4 in vitro CMI assay for monitoring the T-cell-specific activation in vaccinated subjects. Unlike traditional methods, this technology allows rapid in vitro assessment of cell-mediated immunity to antigenic challenges.
Contact Author about this Abstract
