Evaluation of canarypox-induced CD8 + responses following immunization by measuring the effector population IFNγ production

Abstract

CD8 + cytolytic activity is traditionally measured by detecting the release of 51Cr after incubation of effector cells with HLA-matched, infected, radiolabeled targets. An alternative method to detect CD8+ activity is to measure the production of intracellular interferon gamma (IFNγ) after antigen-specific stimulation, either by ELISPOT or by flow cytometry. Studies were performed in 19 volunteers enrolled in a phase 1 trial of candidate canarypox HIV-1 vaccines that encoded multiple HIV-1 genes. The vaccines including vCP205 (Env, Gag, and protease), vCP1433 (Env, Gag, protease, and CTL epitope-rich regions of pol and nef) and vCP1452 (equivalent to vCP1433 with additional immunomodulatory genes of vaccinia). PBMCs were stimulated in vitro with vaccinia constructs encoding env and gag or a lacZ control, and the effectors 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 IFNγ cells in the CD3 +CD8 +CD45RO + gate after subtracting both the isotype control and the lacZ control stimulation. CD4 memory IFNγ production was simultaneously determined in the CD3 +CD8 −CD45RO + gate. Using these techniques in blinded studies, we found that CD8 + IFNγ activity could be measured in the majority of volunteers given four immunizations. Specifically, the responses 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 immunization. Flow cytometric techniques hold promise as a surrogate measure of CTL and for ease of phenotyping of the effector population.