Evaluation of canarypox-induced CD8(+) responses following immunization bymeasuring the effector population IFN gamma production

CD8(+) cytolytic activity is traditionally measured by detecting the releas e of Cr-51 after incubation of effector cells with HLA-matched, infected, r adiolabeled targets. An alternative method to detect CD8+ activity is to me asure the production of intracellular interferon gamma (IFN gamma) after an tigen-specific stimulation, either by ELISPOT or by flow cytometry. Studies were performed in 19 volunteers enrolled in a phase 1 trial of candidate c anarypox HIV-1 vaccines that encoded multiple HIV-1 genes. The vaccines inc luding vCP205 (Env, Gag, and protease), vCP1433 (Env, Gag, protease, and CT L epitope-rich regions of pol and nef) and vCP1452 (equivalent to vCP1433 w ith additional immunomodulatory genes of vaccinial. PBMCs were stimulated i n vitro with vaccinia constructs encoding enu and gag or a lacZ control, an d the effecters were cultured for 12-14 days. EBV-transformed B cell lines were infected overnight with the vaccinia vectors, and then incubated with the effector cells for 4 h in the presence of monensin. CD8+ gene-specific activity was determined as a percentage of IFN gamma cells in the CD3(+)CD8 (+)CD45RO(+) gate after subtracting both the isotype control and the lacZ c ontrol stimulation. CD4 memory IFN gamma production was simultaneously dete rmined in the CD3(+)CD8(-)CD45RO(+) gate. Using these techniques in blinded studies, we found that CD8(+) IFN gamma activity could be measured in the majority of volunteers given four immunizations. Specifically, the response s to the gag gene were control - 0/2; vCP205 - 2/4; vCP1433 - 5/6; vCP1452 - 4/7. Most of the positive responses were detected after the fourth immuni zation. Flow cytometric techniques hold promise as a surrogate measure of C TL and for ease of phenotyping of the effector population. (C) 2001 Elsevie r Science B.V. All rights reserved.