title: Evaluation of canarypox-induced CD8(+) responses following immunization by measuring the effector population IFNgamma 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 (IFNgamma) 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 IFNgamma cells in the CD3(+)CD8(+)CD45RO(+) gate after subtracting both the isotype control and the lacZ control stimulation. CD4 memory IFNgamma production was simultaneously determined in the CD3(+)CD8(-)CD45RO(+) gate. Using these techniques in blinded studies, we found that CD8(+) IFNgamma 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.
authors.affiliation: Infectious Diseases Unit, Suite 500, Department of Medicine, University of Rochester School of Medicine and Dentistry, PSSB 4150 V St., Sacramento, CA 95817, USA. tgevans@ucdavis.edu
authors.forename: T G
authors.initials: TG
authors.lastname: Evans
authors.forename: E G
authors.initials: EG
authors.lastname: Kallas
authors.forename: M
authors.initials: M
authors.lastname: Campbell
authors.forename: J
authors.initials: J
authors.lastname: Andrews
authors.forename: D
authors.initials: D
authors.lastname: Schwartz
authors.forename: M
authors.initials: M
authors.lastname: Keefer
authors.forename: P
authors.initials: P
authors.lastname: Caudrelier
mesh_terms: D016915:AIDS Vaccines / Q000276:immunology*; D000328:Adult; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D004311:Double-Blind Method; D015658:HIV Infections / Q000517:prevention & control*; D015497:HIV-1 / Q000276:immunology*; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis*; D011446:Prospective Studies; D050378:T-Lymphocytes, Regulatory / Q000276:immunology*; D014611:Vaccination; D014614:Vaccines, Synthetic / Q000276:immunology*
chemical_list: D016915:AIDS Vaccines; D014614:Vaccines, Synthetic; D007371:Interferon-gamma