Cargando…

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

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)....

Descripción completa

Detalles Bibliográficos
Autores principales: Waerlop, Gwenn, Leroux-Roels, Geert, Lambe, Teresa, Bellamy, Duncan, Medaglini, Donata, Pettini, Elena, Cox, Rebecca Jane, Trieu, Mai-Chi, Davies, Richard, Bredholt, Geir, Montomoli, Emanuele, Gianchecchi, Elena, Clement, Frédéric
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9493492/
https://www.ncbi.nlm.nih.gov/pubmed/36159843
http://dx.doi.org/10.3389/fimmu.2022.984642
_version_ 1784793733018943488
author Waerlop, Gwenn
Leroux-Roels, Geert
Lambe, Teresa
Bellamy, Duncan
Medaglini, Donata
Pettini, Elena
Cox, Rebecca Jane
Trieu, Mai-Chi
Davies, Richard
Bredholt, Geir
Montomoli, Emanuele
Gianchecchi, Elena
Clement, Frédéric
author_facet Waerlop, Gwenn
Leroux-Roels, Geert
Lambe, Teresa
Bellamy, Duncan
Medaglini, Donata
Pettini, Elena
Cox, Rebecca Jane
Trieu, Mai-Chi
Davies, Richard
Bredholt, Geir
Montomoli, Emanuele
Gianchecchi, Elena
Clement, Frédéric
author_sort Waerlop, Gwenn
collection PubMed
description 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.
format Online
Article
Text
id pubmed-9493492
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher Frontiers Media S.A.
record_format MEDLINE/PubMed
spelling pubmed-94934922022-09-23 Harmonization and qualification of an IFN-γ Enzyme-Linked ImmunoSpot assay (ELISPOT) to measure influenza-specific cell-mediated immunity within the FLUCOP consortium Waerlop, Gwenn Leroux-Roels, Geert Lambe, Teresa Bellamy, Duncan Medaglini, Donata Pettini, Elena Cox, Rebecca Jane Trieu, Mai-Chi Davies, Richard Bredholt, Geir Montomoli, Emanuele Gianchecchi, Elena Clement, Frédéric Front Immunol Immunology 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. Frontiers Media S.A. 2022-09-08 /pmc/articles/PMC9493492/ /pubmed/36159843 http://dx.doi.org/10.3389/fimmu.2022.984642 Text en Copyright © 2022 Waerlop, Leroux-Roels, Lambe, Bellamy, Medaglini, Pettini, Cox, Trieu, Davies, Bredholt, Montomoli, Gianchecchi and Clement https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Immunology
Waerlop, Gwenn
Leroux-Roels, Geert
Lambe, Teresa
Bellamy, Duncan
Medaglini, Donata
Pettini, Elena
Cox, Rebecca Jane
Trieu, Mai-Chi
Davies, Richard
Bredholt, Geir
Montomoli, Emanuele
Gianchecchi, Elena
Clement, Frédéric
Harmonization and qualification of an IFN-γ Enzyme-Linked ImmunoSpot assay (ELISPOT) to measure influenza-specific cell-mediated immunity within the FLUCOP consortium
title Harmonization and qualification of an IFN-γ Enzyme-Linked ImmunoSpot assay (ELISPOT) to measure influenza-specific cell-mediated immunity within the FLUCOP consortium
title_full Harmonization and qualification of an IFN-γ Enzyme-Linked ImmunoSpot assay (ELISPOT) to measure influenza-specific cell-mediated immunity within the FLUCOP consortium
title_fullStr Harmonization and qualification of an IFN-γ Enzyme-Linked ImmunoSpot assay (ELISPOT) to measure influenza-specific cell-mediated immunity within the FLUCOP consortium
title_full_unstemmed Harmonization and qualification of an IFN-γ Enzyme-Linked ImmunoSpot assay (ELISPOT) to measure influenza-specific cell-mediated immunity within the FLUCOP consortium
title_short Harmonization and qualification of an IFN-γ Enzyme-Linked ImmunoSpot assay (ELISPOT) to measure influenza-specific cell-mediated immunity within the FLUCOP consortium
title_sort harmonization and qualification of an ifn-γ enzyme-linked immunospot assay (elispot) to measure influenza-specific cell-mediated immunity within the flucop consortium
topic Immunology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9493492/
https://www.ncbi.nlm.nih.gov/pubmed/36159843
http://dx.doi.org/10.3389/fimmu.2022.984642
work_keys_str_mv AT waerlopgwenn harmonizationandqualificationofanifngenzymelinkedimmunospotassayelispottomeasureinfluenzaspecificcellmediatedimmunitywithintheflucopconsortium
AT lerouxroelsgeert harmonizationandqualificationofanifngenzymelinkedimmunospotassayelispottomeasureinfluenzaspecificcellmediatedimmunitywithintheflucopconsortium
AT lambeteresa harmonizationandqualificationofanifngenzymelinkedimmunospotassayelispottomeasureinfluenzaspecificcellmediatedimmunitywithintheflucopconsortium
AT bellamyduncan harmonizationandqualificationofanifngenzymelinkedimmunospotassayelispottomeasureinfluenzaspecificcellmediatedimmunitywithintheflucopconsortium
AT medaglinidonata harmonizationandqualificationofanifngenzymelinkedimmunospotassayelispottomeasureinfluenzaspecificcellmediatedimmunitywithintheflucopconsortium
AT pettinielena harmonizationandqualificationofanifngenzymelinkedimmunospotassayelispottomeasureinfluenzaspecificcellmediatedimmunitywithintheflucopconsortium
AT coxrebeccajane harmonizationandqualificationofanifngenzymelinkedimmunospotassayelispottomeasureinfluenzaspecificcellmediatedimmunitywithintheflucopconsortium
AT trieumaichi harmonizationandqualificationofanifngenzymelinkedimmunospotassayelispottomeasureinfluenzaspecificcellmediatedimmunitywithintheflucopconsortium
AT daviesrichard harmonizationandqualificationofanifngenzymelinkedimmunospotassayelispottomeasureinfluenzaspecificcellmediatedimmunitywithintheflucopconsortium
AT bredholtgeir harmonizationandqualificationofanifngenzymelinkedimmunospotassayelispottomeasureinfluenzaspecificcellmediatedimmunitywithintheflucopconsortium
AT montomoliemanuele harmonizationandqualificationofanifngenzymelinkedimmunospotassayelispottomeasureinfluenzaspecificcellmediatedimmunitywithintheflucopconsortium
AT gianchecchielena harmonizationandqualificationofanifngenzymelinkedimmunospotassayelispottomeasureinfluenzaspecificcellmediatedimmunitywithintheflucopconsortium
AT clementfrederic harmonizationandqualificationofanifngenzymelinkedimmunospotassayelispottomeasureinfluenzaspecificcellmediatedimmunitywithintheflucopconsortium