title: Concordant proficiency in measurement of T-cell immunity in human immunodeficiency virus vaccine clinical trials by peripheral blood mononuclear cell and enzyme-linked immunospot assays in laboratories from three continents.
abstract: The gamma interferon (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay is used routinely to evaluate the potency of human immunodeficiency virus (HIV) vaccine candidates and other vaccine candidates. In order to compare candidates and pool data from multiple trial laboratories, validated standardized methods must be applied across laboratories. Proficiency panels are a key part of a comprehensive quality assurance program to monitor inter- and intralaboratory performance, as well as assay performance, over time. Seven International AIDS Vaccine Initiative-sponsored trial sites participated in the proficiency panels described in this study. At each laboratory, two operators independently processed identical sample sets consisting of frozen peripheral blood mononuclear cell (PBMC) samples from different donors by using four blind stimuli. PBMC recovery and viability after overnight resting and the IFN-gamma ELISPOT assay performance were assessed. All sites demonstrated good performance in PBMC thawing and resting, with a median recovery of 78% and median viability of 95%. The laboratories were able to detect similar antigen-specific T-cell responses, ranging from 50 to >3,000 spot-forming cells per million PBMC. An approximate range of a half log in results from operators within or across sites was seen in comparisons of antigen-specific responses. Consistently low background responses were seen in all laboratories. The results of these proficiency panels demonstrate the ability of seven laboratories, located across three continents, to process PBMC samples and to rank volunteers with differential magnitudes of IFN-gamma ELISPOT responses. These findings also illustrate the ability to standardize the IFN-gamma ELISPOT assay across multiple laboratories when common training methods, reagents such as fetal calf serum, and standard operating procedures are adopted. These results are encouraging for laboratories that are using cell-based immunology assays to test HIV vaccines and other vaccines.
authors.affiliation: International AIDS Vaccine Initiative, New York, New York, USA.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Boaz
authors.forename: Peter
authors.initials: P
authors.lastname: Hayes
authors.forename: Tony
authors.initials: T
authors.lastname: Tarragona
authors.forename: Laura
authors.initials: L
authors.lastname: Seamons
authors.forename: Andrew
authors.initials: A
authors.lastname: Cooper
authors.forename: Josephine
authors.initials: J
authors.lastname: Birungi
authors.forename: Paul
authors.initials: P
authors.lastname: Kitandwe
authors.forename: Aloysius
authors.initials: A
authors.lastname: Semaganda
authors.forename: Pontiano
authors.initials: P
authors.lastname: Kaleebu
authors.forename: Gwynneth
authors.initials: G
authors.lastname: Stevens
authors.forename: Omu
authors.initials: O
authors.lastname: Anzala
authors.forename: Bashir
authors.initials: B
authors.lastname: Farah
authors.forename: Simon
authors.initials: S
authors.lastname: Ogola
authors.forename: Jackton
authors.initials: J
authors.lastname: Indangasi
authors.forename: Patrick
authors.initials: P
authors.lastname: Mhlanga
authors.forename: Melanie
authors.initials: M
authors.lastname: Van Eeden
authors.forename: Madhuri
authors.initials: M
authors.lastname: Thakar
authors.forename: Ashwini
authors.initials: A
authors.lastname: Pujari
authors.forename: Shadri
authors.initials: S
authors.lastname: Mishra
authors.forename: Nilu
authors.initials: N
authors.lastname: Goonetilleke
authors.forename: Stephen
authors.initials: S
authors.lastname: Moore
authors.forename: Abdul
authors.initials: A
authors.lastname: Mahmoud
authors.forename: Pattabiraman
authors.initials: P
authors.lastname: Sathyamoorthy
authors.forename: Jayashri
authors.initials: J
authors.lastname: Mahalingam
authors.forename: Paranji R
authors.initials: PR
authors.lastname: Narayanan
authors.forename: Vadakkuppattu D
authors.initials: VD
authors.lastname: Ramanathan
authors.forename: Josephine H
authors.initials: JH
authors.lastname: Cox
authors.forename: Len
authors.initials: L
authors.lastname: Dally
authors.forename: Dilbinder K
authors.initials: DK
authors.lastname: Gill
authors.forename: Jill
authors.initials: J
authors.lastname: Gilmour
mesh_terms: D016915:AIDS Vaccines / Q000276:immunology*; D002470:Cell Survival; D002478:Cells, Cultured; D006678:HIV / Q000276:immunology*; D006801:Humans; D007118:Immunoassay / Q000379:methods* / Q000592:standards; D007371:Interferon-gamma / Q000096:biosynthesis*; D007963:Leukocytes, Mononuclear / Q000276:immunology*; D015588:Observer Variation; D015203:Reproducibility of Results; D013048:Specimen Handling / Q000379:methods
chemical_list: D016915:AIDS Vaccines; D007371:Interferon-gamma