Assay optimisation and technology transfer for multi-site immuno-monitoring in vaccine trials.

Steven G Smith,K Barry Walker,Hazel M Dockrell,Helen Mcshane,Iman Satti,Donna Bryan,Mei Mei Ho,Stephanie A Harris

doi:10.1371/journal.pone.0184391

Abstract

Cellular immunological assays are important tools for the monitoring of responses to T-cell-inducing vaccine candidates. As these bioassays are often technically complex and require considerable experience, careful technology transfer between laboratories is critical if high quality, reproducible data that allows comparison between sites, is to be generated. The aim of this study, funded by the European Union Framework Program 7-funded TRANSVAC project, was to optimise Standard Operating Procedures and the technology transfer process to maximise the reproducibility of three bioassays for interferon-gamma responses: enzyme-linked immunosorbent assay (ELISA), ex-vivo enzyme-linked immunospot and intracellular cytokine staining. We found that the initial variability in results generated across three different laboratories reduced following a combination of Standard Operating Procedure harmonisation and the undertaking of side-by-side training sessions in which assay operators performed each assay in the presence of an assay ‘lead’ operator. Mean inter-site coefficients of variance reduced following this training session when compared with the pre-training values, most notably for the ELISA assay. There was a trend for increased inter-site variability at lower response magnitudes for the ELISA and intracellular cytokine staining assays. In conclusion, we recommend that on-site operator training is an essential component of the assay technology transfer process and combined with harmonised Standard Operating Procedures will improve the quality, reproducibility and comparability of data produced across different laboratories. These data may be helpful in ongoing discussions of the potential risk/benefit of centralised immunological assay strategies for large clinical trials versus decentralised units.

Highlights

Immune bioassays are essential tools with which to measure the immunogenicity of vaccines
Following an initial review of different versions of assay standard operating procedure (SOP) used in each participating laboratory, we found that SOPs for the same assay varied considerably across the groups
enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot (ELISpot), CD4+ T-cell intracellular cytokine staining (ICS) and CD8+ T-cell ICS assay results are shown in Fig 1A– 1D

Summary

Introduction

Immune bioassays are essential tools with which to measure the immunogenicity of vaccines. Some of these are well established and can indicate the presence or absence of protection in vaccinated individuals where the assay detects a reliable correlate of protection such as neutralising antibody [1]. Bioassays used to determine the immunogenicity of T-cell-inducing vaccines are often more complex and require a greater degree of operator expertise and experience. Some T-cell assays can simultaneously measure more than one parameter of interest such as responder cell phenotype together with cytokine secretion profile using flow cytometric assays or multiple secreted effector molecules using multiplex bead array or dual/triple colour enzyme-linked immunospot (ELISpot). Others only measure one effector function (e.g. single cytokine ELISpot or enzyme-linked immunosorbent assay (ELISA) for interferon-gamma (IFNγ))

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Conclusion