Standardization and validation of assays determining cellular immune responses against influenza.
Influenza vaccine efficacy does not always correlate with humoral immune responses. Recent reports indicate that the cellular immune response also contributes to protection, however robust assays are lacking. We standardized and validated assays for detection of human influenza-specific cellular responses in four international laboratories. The production of granzyme B as marker of T cell-mediated cytotoxicity and release of Th1 and Th2 cytokines were evaluated. The granzyme B and cytokine assays were specific, accurate, precise, and robust. Replicate stimulations with PBMC from the same donors showed an intra-laboratory robustness (coefficient of variation) for quantitation of granzyme B of 33% and for cytokines - including IFN-gamma, TNF-alpha, IL-2, IL-10, IL-4, IL-13, GM-CSF and including the log IFN-gamma/IL-10 ratio - of 52%. The inter-laboratory robustness for detection of granzyme B was 29% and for detection of all cytokines was 49%. The assays can now be used for determining cell-mediated immunity and explored as correlates of protection. Moreover, the precision and robustness of these cellular assays allow the reliable detection of cellular responses even in small study populations.
Gijzen
Karlijn
K
Vaccine Research, Netherlands Vaccine Institute (NVI), Bilthoven, The Netherlands.
Liu
Wai Ming
WM
Visontai
Ildikó
I
Oftung
Fredrik
F
van der Werf
Sylvie
S
Korsvold
Gro Ellen
GE
Pronk
Inge
I
Aaberge
Ingeborg S
IS
Tütto
Anna
A
Jankovics
Istvan
I
Jankovics
Mate
M
Gentleman
Beth
B
McElhaney
Janet E
JE
Soethout
Ernst C
EC
D016207:Cytokines / Q000378:metabolism; D053804:Granzymes / Q000096:biosynthesis; D006801:Humans; D007118:Immunoassay / Q000592:standards; D007252:Influenza Vaccines / Q000276:immunology*; D007251:Influenza, Human / Q000517:prevention & control*; D012680:Sensitivity and Specificity; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology
D016207:Cytokines; D007252:Influenza Vaccines; D053804:Granzymes