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.
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.
Boaz
Mark J
MJ
International AIDS Vaccine Initiative, New York, New York, USA.
Hayes
Peter
P
Tarragona
Tony
T
Seamons
Laura
L
Cooper
Andrew
A
Birungi
Josephine
J
Kitandwe
Paul
P
Semaganda
Aloysius
A
Kaleebu
Pontiano
P
Stevens
Gwynneth
G
Anzala
Omu
O
Farah
Bashir
B
Ogola
Simon
S
Indangasi
Jackton
J
Mhlanga
Patrick
P
Van Eeden
Melanie
M
Thakar
Madhuri
M
Pujari
Ashwini
A
Mishra
Shadri
S
Goonetilleke
Nilu
N
Moore
Stephen
S
Mahmoud
Abdul
A
Sathyamoorthy
Pattabiraman
P
Mahalingam
Jayashri
J
Narayanan
Paranji R
PR
Ramanathan
Vadakkuppattu D
VD
Cox
Josephine H
JH
Dally
Len
L
Gill
Dilbinder K
DK
Gilmour
Jill
J
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
D016915:AIDS Vaccines; D007371:Interferon-gamma