WP5 - Work performed since the beginning of the project

 Overview work package


To reiterate the status at the end of the first reporting period, the initial plan to standardise assays between the laboratories (to allow samples from clinical trials to be analysed at different sites) was changed when it became clear that standardisation  of multi-parametric cell-mediated immune responses (i.e. T-cell responses) by flow cytometry in multiple centres would be practically impossible. As the crucial aspect of WP5 is a direct side-by-side comparison of the immune responses induced by the different vaccines, it was decided to instead designate centralised laboratories to carry out the most important of the assays on clinical samples or a large subset thereof.

As the putative correlates of protection for vaccines designed to induce predominantly T-cell responses remain unknown, it was considered important to analyse samples using an approach that would maximise the information generated. To this end, a strategy for the multi-parametric FACS analysis of immune responses in PBMC was developed during the first year in which cells stimulated with different immunogens are evaluated with regard to the induction of multiple intracellular cytokine responses in CD4+ and CD8+ T-cells. This approach had already been established and validated for use in large scale AIDS vaccine trials in humans and was adapted for use in the UNISEC clinical trials as a group effort between the WP5 partners. The final protocol was included in the portfolio of standardised protocols disseminated to the group.

The decision to designate centralised laboratories demanded that the processes for sampling, processing, freezing and transport of PBMC and plasma samples at the different clinical sites be precisely standardised to ensure conformity in the quality of the material. Based on consultations concerning the equipment and expertise available at the different sites, a standardised protocol was developed and distributed.

Although able to deliver an extensive amount of information in one assay, the 'core' FACS-based T-cell assay will need to be supplemented with other assays that measure critical aspects of the cellular and humoral immune responses if the putative correlates of protection are to be identified. To this end, secondary assays for T-cell immunity (e.g. ELISpot, cytokine RNA PCR) and antibody-based assays such as ELISA, hemagglutination-inhibition and microneutralisation were established and standardized in WP5 partner laboratories that will act as centralised facilities for testing at least a subset of the samples generated.

Although most of the efforts in establishing standardised/centralised assays concentrated on the critical requirements of the clinical trials, it was also important that the immune responses induced by vaccination of animals in preclinical studies be measured using comparable assays. To this end, the protocol for the multi-parametric FACS analysis of cytokine responses in human PBMC following in vitro stimulation was adapted and tested for measuring T-cell responses in spleen cells from immunised mice. These protocols were included in the disseminated plan for the analysis of samples generated in animal studies.

Refinement and validation of the multi-parametric FACS protocol for evaluating T-cell responses

The original strategy developed involved antigenic stimulation of thawed and rested PBMC for six hours in the presence of co-stimulatory antibodies and protein transport inhibitors. Cells were then stained for surface markers, exposed to a fixable viability dye, fixed and permeabilised and then stained for cytoplasmic cytokines before FACS analysis.

The initial protocol had been distributed to WP5 members who suggested a number of alterations to improve the handling and accuracy of the analyses and to bring the protocol in line with GCLP guidelines. The strategy was therefore very much a group effort involving numerous telephone conferences and email exchanges. 

However, the results of the inter-laboratory assay comparison suggested that additional refinements were needed. Furthermore, partners requested that additional markers of activation/suppression should be included in the FACS based ICS assay. A series of experiments were therefore carried out in which the following parameters were compared and refined: (a) time of stimulation (6hr vs 24hr); (b) period of stimulation before addition of protein-transport inhibitor; (c) choice of stimulant for positive control (SEB vs CSC); (d) presence or absence of FcR-blocking reagents; (e) measurement of IL4 vs IL17 responses (or both); (f) inclusion of anti-CD107a (cytotoxicity marker) in the panel of cytokine specific antibodies; (g) impact of adding the anti-CD107a antibody during the stimulation and then staining for intracellular and extracellular markers at the same time.

The outcome was that a final protocol was created that allows for stimulation with six different antigens to be carried out simultaneously, plus positive (SEB) and negative controls, in the presence of anti-CD107a antibody. After stimulation, cells are stained with fixable viability dye, fixed and permeablilised, and stained for CD3, CD4, CD8, intracellular IFNg, IL-2, TNF-a, IL-4 and IL-17. After acquisition of samples by flow cytometry, the cells are gated (lymphocytes/viable cells/CD3+/CD4+ or CD8+). Using the unstimulated cells to set gates for positive cells, the number of cells in each population staining positive for each of the stimulation markers is determined.

Although the potential problem of inter-laboratory variability had been solved by concentrating assays in centralised facilities, it was important to validate the ICS assay for intra- and inter-assay consistency. For this, multiple aliquots of PBMC from different donors were frozen in liquid nitrogen and thawed on three separate days to be analysed using the ICS protocol.

Multicentre comparison of assays measuring T-cell responses

There are numerous approaches to measuring the T-cell response to a stimulant, all of which have their advantages and disadvantages with regards to handling, expense, sensitivity and the amount of information delivered. For example, the multiparametric intracellular cytokine staining assay selected for analysing the UNISEC trial samples is relatively expensive and labour-intensive but delivers a large amount of information concerning the nature of the response. The IFNg-ELISpot is simpler, cheaper and potentially more sensitive, but is relatively limited in the breadth of information gained. The measurement of cytokine RNA by PCR has the potential advantage of sensitivity and can be used to measure responses in species for which the necessary anti-cytokine antibodies are not available, an important factor for animal models such as ferrets and cotton rats.

In order to compare these three assays, multiple aliquots of PBMC from three healthy donors were frozen using the UNISEC protocol and shipped to the partner laboratories in which the assays had been established (RKI:ICS-FACS NIPH:ELISpot, NCE: RNA-PCR). A standardized panel of stimulants (antigens and mitogens) were also distributed to the partner laboratories. As far as possible, the PBMC were stimulated in the different laboratories under the same conditions and the T-cell responses measured using the relevant assay. The results of this multicentre comparison (which constitute D5.3) demonstrated that, indeed, the ELISpot and the RNA-PCR were somewhat more sensitive in detecting low-level T-cell responses than the multiparametric FACS analysis, a finding which prompted the further refinement of the ICS-FACS (see above).

Development and validation of additional assays

In addition to the 'core' assay for T-cell response described above, a suite of additional assays that can yield valuable information concerning the nature of the immune response to the different vaccines has been developed and validated. WP5 partners were polled for their capacity to act as centralised facilities for such additional assays. In addition to the FACS-based T-cell assays performed at the RKI, partners at the NIPH and NCE will, where appropriate, carry out these crucial additional assays for the UNISEC consortium. These partners have provided detailed protocols for the assays established in their laboratories and these have been disseminated to other UNISEC participants via the web server.

Partners at NIPH have established and standardized a number of antibody- and T cell-based assays for evaluating immunogenicity of universal influenza vaccine candidates tested in clinical trials (WP6). One example is amultiplex assay for IgG subclasses. This is needed because IgG subclasses have different abilities to facilitate FcR mediated phagocytosis and ADCC, making subclass analysis of crucial importance for evaluating putative effector functions of vaccine induced antibody responses. IgG3 antibodies are considered the most potent for protective effector functions against virus infections, including influenza.

The Elispot assay has a number of advantages (cost, ease of use, sensitivity) and accumulating evidence supports Elispot-based enumeration of antigen specific IFN-γ secreting T cells for the clinical evaluation of CMI-inducing influenza vaccines. The NIPH has therefore introduced a methodological platform that allows validated dual Elispot with different combination of cytokines and other cellular markers. IFN-γ and granzyme B were selected because of their relative importance as markers for Th1 responses and T cell mediated cytotoxic responses, respectively. Validated assay procedures and computer assisted image analysis, involving software for spot size and intensity auto-gating, ensures high reproducibility of the results (CV< 6.7 %). Spot detection has been improved by using an ISO-certified and GLCP approved Elispot reader for clinical trials (CTL-ImmunoSpot S6 reader system). This setup also provides the possibility for comparison with well characterized reference cell material which can serve as internal standards. Standard operating procedures (SOPs) covering dual Elispot assays and data handling for analyzing PBMC samples from clinical trials (WP6) has been revised accordingly.

Furthermore, the applicability of IFN-γ Elispot assays to evaluate and dissect cellular responses with regard to T cell subsets and antigen targets was demonstrated at NIPH by analysis of clinical samples from healthy normal individuals (baseline) and patients recruited during the 2009 pandemic. To facilitate this, optimized panels of conserved CD4 and CD8 T cell epitopes were developed to be used as efficient tools to evaluate universal cellular immune responses against influenza in different settings. Elispot responses obtained with such peptide panels correlated to responses obtained with inactivated whole virus as seen for both IFN-γ and granzyme B. This approach has also been used by NIPH to characterize T cell responses in A(H1N1)pdm09 infected pregnant women with and without recorded clinical symptoms. The results showed that asymptomatic individuals had significantly higher frequency of IFN-γ producing CD8+ T cells recognizing epitopes from internal antigens as compared to symptomatic individuals. Further characterisation of this cell population by multi-parameter flow-cytometry analysis suggested that a subset of late effector memory CD8+ T cells (Temra: CCR7-, CD45RA+ cells) may serve as a putative post-infection correlate of protection against symptomatic influenza disease

Partners at the NCE have established and provided protocols for the measurement of cytokine responses at the mRNA level following antigenic stimulation of responding cells and used this approach in the inter-laboratory comparison of T-cell assays. This approach will be particularly instrumental for the measurement of responses in vaccinated laboratory animals for which the full repertoire of immunological reagents is not available.

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