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. 2022 Sep 8:13:984642.
doi: 10.3389/fimmu.2022.984642. eCollection 2022.

Harmonization and qualification of an IFN-γ Enzyme-Linked ImmunoSpot assay (ELISPOT) to measure influenza-specific cell-mediated immunity within the FLUCOP consortium

Gwenn Waerlop  1 , Geert Leroux-Roels  1 , Teresa Lambe  2 , Duncan Bellamy  2 , Donata Medaglini  3 , Elena Pettini  3 , Rebecca Jane Cox  4 , Mai-Chi Trieu  4 , Richard Davies  4 , Geir Bredholt  4 , Emanuele Montomoli  5   6 , Elena Gianchecchi  6 , Frédéric Clement  1
Affiliations

Harmonization and qualification of an IFN-γ Enzyme-Linked ImmunoSpot assay (ELISPOT) to measure influenza-specific cell-mediated immunity within the FLUCOP consortium

Gwenn Waerlop et al. Front Immunol. .

Abstract

Influenza continues to be the most important cause of viral respiratory disease, despite the availability of vaccines. Today's evaluation of influenza vaccines mainly focuses on the quantitative and functional analyses of antibodies to the surface proteins haemagglutinin (HA) and neuraminidase (NA). However, there is an increasing interest in measuring cellular immune responses targeting not only mutation-prone surface HA and NA but also conserved internal proteins as these are less explored yet potential correlates of protection. To date, laboratories that monitor cellular immune responses use a variety of in-house procedures. This generates diverging results, complicates interlaboratory comparisons, and hampers influenza vaccine evaluation. The European FLUCOP project aims to develop and standardize assays for the assessment of influenza vaccine correlates of protection. This report describes the harmonization and qualification of the influenza-specific interferon-gamma (IFN-γ) Enzyme-Linked ImmunoSpot (ELISpot) assay. Initially, two pilot studies were conducted to identify sources of variability during sample analysis and spot enumeration in order to develop a harmonized Standard Operating Procedure (SOP). Subsequently, an assay qualification study was performed to investigate the linearity, intermediate precision (reproducibility), repeatability, specificity, Lower and Upper Limits of Quantification (LLOQ-ULOQ), Limit of Detection (LOD) and the stability of signal over time. We were able to demonstrate that the FLUCOP harmonized IFN-γ ELISpot assay procedure can accurately enumerate IFN-γ secreting cells in the analytical range of 34.4 Spot Forming Units (SFU) per million cells up to the technical limit of the used reader and in the linear range from 120 000 to 360 000 cells per well, in plates stored up to 6 weeks after development. This IFN-γ ELISpot procedure will hopefully become a useful and reliable tool to investigate influenza-specific cellular immune responses induced by natural infection or vaccination and can be an additional instrument in the search for novel correlates of protection.

Keywords: IFN-γ ELISpot; assay harmonization; assay qualification; cell-mediated immunity; influenza.

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Conflict of interest statement

Authors EM and EG are employed by VisMederi srl. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Pilot study 1 - unstimulated conditions. Results from each of the 24 samples reported by the 5 labs are given in panel (A) and are expressed as SFU/106 cells. The performance of the 5 labs is shown in panel (B) and is expressed as Z-scores. Panel (C) shows the imprecision, expressed as CV%, for the within-sample mean values, expressed as SFU/106 cells. Here the horizontal dotted line represents the overall mean CV observed (64%) and the vertical dotted line indicates the arbitrary acceptance criterion of 50 SFU/106 cells. Calculations for data shown in panels (B) and (C) were performed with log-transformed data.
Figure 2
Figure 2
Pilot study 1 – Background-subtracted results from the cell cultures stimulated with inactivated split A/California (panels A, B) and B/Phuket (panels C, D). The performance of the 5 labs is shown in the left panels and is expressed as Z-scores. The observed imprecision, expressed as CV%, for the mean within-sample values, expressed as SFU/106 cells, is shown in the right panels. Here the dotted horizontal line indicates the overall mean CV of 63 and 34% for A/California and B/Phuket, respectively. Analyses were performed with log-transformed counts.
Figure 3
Figure 3
Pilot study 2. An IFN-γ ELISpot plate with 84 wells to be scored was distributed to 6 laboratories within one month after preparation. Z-scores per well per lab (A) and the CV per well (B) are shown. The dotted line in the right panel indicates the CV of 40%, commonly applied as a threshold of acceptable variation in CMI assays.
Figure 4
Figure 4
Determination of linearity and proportionality. Mean SFU/well is plotted against the number of cells plated per well for each sample (A–D). The blue zone indicates the range between 120 000 and 360 000 cells/well where recovery values ranged between 50 and 150% and therefore linearity was demonstrated. Correlation curves of the number of plated cells per well and the mean responses (SFU/well) are shown in black with the concerned R2 values. The coefficients of proportionality, calculated as the ratios of the observed number of spots per well versus the number of plated cells, are indicated in green triangles and represented on the right y-axis.
Figure 5
Figure 5
Determination of Lower Limit of Intermediate Precision (LLOIP). (A) Distribution of background-subtracted responses after in vitro stimulation of cells with A/California and tetanus toxin (TT). Each symbol represents a measurement of a certain sample, indicated on the x-axis. A set of 10 samples was tested each in duplicate by 2 operators performing each 2 runs on different days, resulting in 8 measurements per sample. Measurements from operator 1 are indicated with circles and from operator 2 with triangles. Responses obtained after stimulation with A/California are indicated in blue, with TT in green. (B) LLOIP was determined by plotting the mean background-subtracted responses to A/California and TT against the mean inter-assay CV% per sample. The LLOIP was defined as the lowest response with a CV of 40% and was determined at 34.4 SFU/106 cells. (C) The inter-assay, intra-assay and inter-operator variability were assessed by the mean CV% observed for the mean responses of each sample. Each horizontal dotted line represents the cut-off of 40% CV and each vertical dotted line represents the LLOIP determined at 34.4 SFU/106 cells.
Figure 6
Figure 6
Determination of assay specificity. PBMC from 25 paired samples collected before and 7 days after the administration of a seasonal influenza vaccine were stimulated with influenza (split A/California) and control (tetanus toxin; TT) antigens. Background-subtracted values are shown as box plots. Differences in IFN-γ spot forming responses between the pre- and post-vaccination samples following in vitro stimulation with either influenza or TT antigen were examined. Wilcoxon matched-pairs signed-rank test was applied and p < 0.05 was considered statistically significant.
Figure 7
Figure 7
Determination of the ULOQ. PBMC from two samples were plated each on one plate in a serial dilution, ranging from 100 000 to 48 PBMC per well. Data from sample 1 is shown. Each condition was repeated 8 times and all cells were stimulated with SEB. Mean SFU with SD bars is indicated in blue and represented on the left y-axis. The related CV% are shown in black and represented on the right x-axis. The dotted line indicates the acceptance criterion of 40% CV.
Figure 8
Figure 8
Determination of validity of signal stability over time. A set of 9 plates was re-read every other week for 24 weeks. The sum of all antigen-specific IFN-γ ELISpot responses of each plate was evaluated over time. Reading of the plates was performed by 3 different operators, indicated in black (n = 6), blue (n = 8) and red (n = 1).
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