Immune Monitoring Design within the Developmental Pipeline for an Immunotherapeutic or Preventive Vaccine

Janetzki, Sylvia; Romero, Pedro; Bolton, Diane L.; Jandus, Camilla

doi:10.1002/9781118345313.ch28

Cited by 3 publications

(2 citation statements)

References 102 publications

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“…Immune monitoring is an essential component of immunological research, translational science and even clinical settings. Many different assays are available that allow the assessment of the immune system for a variety of phenotypes and functions [ 1 ]. These assays differ in their sensitivity, specificity, complexity, difficulty of validation and robustness.…”

Section: Introductionmentioning

confidence: 99%

Improvement of IFNg ELISPOT Performance Following Overnight Resting of Frozen PBMC Samples Confirmed Through Rigorous Statistical Analysis

Santos

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Sabri

et al. 2014

Cells

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Immune monitoring of functional responses is a fundamental parameter to establish correlates of protection in clinical trials evaluating vaccines and therapies to boost antigen-specific responses. The IFNγ ELISPOT assay is a well-standardized and validated method for the determination of functional IFNγ-producing T-cells in peripheral blood mononuclear cells (PBMC); however, its performance greatly depends on the quality and integrity of the cryopreserved PBMC. Here, we investigate the effect of overnight (ON) resting of the PBMC on the detection of CD8-restricted peptide-specific responses by IFNγ ELISPOT. The study used PBMC from healthy donors to evaluate the CD8 T-cell response to five pooled or individual HLA-A2 viral peptides. The results were analyzed using a modification of the existing distribution free resampling (DFR) recommended for the analysis of ELISPOT data to ensure the most rigorous possible standard of significance. The results of the study demonstrate that ON resting of PBMC samples prior to IFNγ ELISPOT increases both the magnitude and the statistical significance of the responses. In addition, a comparison of the results with a 13-day preculture of PBMC with the peptides before testing demonstrates that ON resting is sufficient for the efficient evaluation of immune functioning.

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Section: Introductionmentioning

confidence: 99%

Improvement of IFNg ELISPOT Performance Following Overnight Resting of Frozen PBMC Samples Confirmed Through Rigorous Statistical Analysis

Santos

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Sabri

et al. 2014

Cells

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“…These assays differ in their sensitivity to detect low frequency T cells, quantity of information obtainable, ability to determine structural and/or functional features of T cells, and their complexity in qualification and validation demands (17). For years the field of T cell monitoring has been plagued by a seemingly inherent variability in assay results, even between assays evaluating the same sample in the same laboratories or by different laboratories.…”

Section: Immune Assay Harmonization and Variables That Impact On Assamentioning

confidence: 99%

Immunological Monitoring to Rationally Guide AAV Gene Therapy

Britten¹,

Walter²,

Janetzki³

2013

Front. Immunol.

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Recent successes with adeno-associated virus (AAV)-based gene therapies fuel the hope for new treatments for hereditary diseases. Pre-existing as well as therapy-induced immune responses against both AAV and the encoded transgenes have been described and may impact on safety and efficacy of gene therapy approaches. Consequently, monitoring of vector- and transgene-specific immunity is mandated and may rationally guide clinical development. Next to the humoral immune response, the cellular response is central in our understanding of the host reaction in gene therapy. But in contrast to the monitoring of antibodies, which has matured over many decades, sensitive and robust monitoring of T cells is a relatively new development. To make cellular immune assessments fit for purpose, investigators need to know, control and report the critical assay variables that influence the results. In addition, the quality of immune assays needs to be continuously adjusted to allow for exploratory hypothesis generation in early stages and confirmatory hypothesis validation in later stages of clinical development. The concept of immune assay harmonization which includes use of field-wide benchmarks, harmonization guidelines, and external quality control can support the context-specific evolution of immune assays. Multi-center studies pose particular challenges to sample logistics and quality control of sample specimens. Cooperative groups need to define if immune assessments should be performed in one central facility, in peripheral labs or including a combination of both. Finally, engineered reference samples that contain a defined number of antigen-specific T cells may become broadly applicable tools to control assay performance over time or across institutions.

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Enzyme-Linked ImmunoSpot (ELISpot) for Single-Cell Analysis

Janetzki¹,

Rabin²

2015

Methods in Molecular Biology

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The ELISpot, a heterogeneous immunoassay, is widely used for detection of low abundant analytes. It is a reliable and robust assay to monitor responses of the immune system at the single-cell level by capturing secreted molecules of interest with specific, membrane-bound antibodies. Those molecules are then made visible by a cascade of ELISA-related development steps. The final results are distinct spots on the membrane as an imprint of the cell secreting the captured molecules, not only allowing their quantification but also providing insight on the kinetics and strength of secretion. This chapter describes the optimized protocol steps of the ELISpot technique, important improvements and tools available for the community, and the current expansion of the technique into polyfunctional cell analysis.

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Immune Monitoring Design within the Developmental Pipeline for an Immunotherapeutic or Preventive Vaccine

Cited by 3 publications

References 102 publications

Improvement of IFNg ELISPOT Performance Following Overnight Resting of Frozen PBMC Samples Confirmed Through Rigorous Statistical Analysis

Improvement of IFNg ELISPOT Performance Following Overnight Resting of Frozen PBMC Samples Confirmed Through Rigorous Statistical Analysis

Immunological Monitoring to Rationally Guide AAV Gene Therapy

Enzyme-Linked ImmunoSpot (ELISpot) for Single-Cell Analysis

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