title: Standardization and validation of assays determining cellular immune responses against influenza.
abstract: 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.
authors.affiliation: Vaccine Research, Netherlands Vaccine Institute (NVI), Bilthoven, The Netherlands.
authors.forename: Karlijn
authors.initials: K
authors.lastname: Gijzen
authors.forename: Wai Ming
authors.initials: WM
authors.lastname: Liu
authors.forename: Ildikó
authors.initials: I
authors.lastname: Visontai
authors.forename: Fredrik
authors.initials: F
authors.lastname: Oftung
authors.forename: Sylvie
authors.initials: S
authors.lastname: van der Werf
authors.forename: Gro Ellen
authors.initials: GE
authors.lastname: Korsvold
authors.forename: Inge
authors.initials: I
authors.lastname: Pronk
authors.forename: Ingeborg S
authors.initials: IS
authors.lastname: Aaberge
authors.forename: Anna
authors.initials: A
authors.lastname: Tütto
authors.forename: Istvan
authors.initials: I
authors.lastname: Jankovics
authors.forename: Mate
authors.initials: M
authors.lastname: Jankovics
authors.forename: Beth
authors.initials: B
authors.lastname: Gentleman
authors.forename: Janet E
authors.initials: JE
authors.lastname: McElhaney
authors.forename: Ernst C
authors.initials: EC
authors.lastname: Soethout
mesh_terms: 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
chemical_list: D016207:Cytokines; D007252:Influenza Vaccines; D053804:Granzymes