Cryopreservation-related loss of antigen-specific IFNγ producing CD4+ T-cells can skew immunogenicity data in vaccine trials: Lessons from a malaria vaccine trial substudy

Abstract

Ex vivo functional immunoassays such as ELISpot and intracellular cytokine staining (ICS) by flow cytometry are crucial tools in vaccine development both in the identification of novel immunogenic targets and in the immunological assessment of samples from clinical trials. Cryopreservation and subsequent thawing of PBMCs via validated processes has become a mainstay of clinical trials due to processing restrictions inherent in the disparate location and capacity of trial centres, and also in the need to standardize biological assays at central testing facilities. Logistical and financial requirement to batch process samples from multiple study timepoints are also key. We used ELISpot and ICS assays to assess antigen-specific immunogenicity in blood samples taken from subjects enrolled in a phase II malaria heterologous prime-boost vaccine trial and showed that the freeze thaw process can result in a 3–5-fold reduction of malaria antigen-specific IFNγ-producing CD3+CD4+ effector populations from PBMC samples taken post vaccination. We have also demonstrated that peptide responsive CD8+ T cells are relatively unaffected, as well as CD4+ T cell populations that do not produce IFNγ. These findings contribute to a growing body of data that could be consolidated and synthesised as guidelines for clinical trials with the aim of increasing the efficiency of vaccine development pipelines.

Susceptibility of A. fumigatus‐specific T‐cell assays to pre‐analytic blood storage and PBMC cryopreservation greatly depends on readout platform and analytes

“…Despite of minor protocol changes compared with our previously published method including the use of lithium heparin anticoagulation and Leucosep ™ tubes to standardise cell isolation, the results of the present study confirmed previous evidence that cryopreservation of PBMCs in RPMI supplemented with autologous serum facilitates excellent recovery of CD154 + A. fumigatus‐ specific T cells . By contrast, flow cytometric detection of IFN‐γ + cells showed lower concordance of fresh and frozen cells and a tendency towards reduced detection rates in frozen cells, matching previous observations on Plasmodium falciparum TRAP peptide reactive T‐helper cells . Cryopreservation of PBMCs using AIM‐V or FCS‐supplemented RPMI resulted in poor performance of CD154‐ and IFN‐γ‐based‐specific T‐cell quantification, with high unspecific background and weak correlation with immediately processed samples.…”

“…Differences in susceptibility of T‐cell subsets to cryopreservation may explain the broad bandwidth of assay performance in thawed samples . In particular, CD4 + cells were reported to exhibit greater susceptibility to cryopreservation than CD8 + cells .…”

“…Differences in susceptibility of T‐cell subsets to cryopreservation may explain the broad bandwidth of assay performance in thawed samples . In particular, CD4 + cells were reported to exhibit greater susceptibility to cryopreservation than CD8 + cells . Freezing also differently affects T‐helper cell subsets, with a significant decline of naive and central memory cells, suggesting that antigens eliciting a prominent response of these cell types enhance susceptibility of assay performance to cryopreservation.…”

Activation-induced Markers Detect Vaccine-Specific CD4+ T Cell Responses Not Measured by Assays Conventionally Used in Clinical Trials

“…Additionally, a recent study showed that the frequency of antigen-specific CD4 + T cells detected by cytokine secretion was significantly reduced (between three and five-fold) after cryopreservation; ICS or ELISpot using thawed cells showed lower and more CD8-biased responses than those using PBMC ex vivo [ 50 ]. The ability to perform T cell assays using cryopreserved cells is crucial in many clinical trials—particularly those involving multiple centres.…”

Methods for Measuring T-Cell Memory to Vaccination: From Mouse to Man

“…This is because there is evidence from a malaria vaccine study that antigen-specific IFNγ producing CD4 + T-cells were reduced three to five-fold after freeze-thaw compared with fresh. However, CD8 + populations remained mostly unaffected [ 94 ]. This effect may not be seen for viral antigens, as fresh and frozen PBMCs were used in ELIspot assays in an HIV vaccination study [ 95 ].…”