Extensive reviews on how to perform T-cell assays to assess vaccine immunogenicity have been published previously [
39,
40] and, therefore, we will not discuss these methods in detail. Instead we will describe briefly the advantages and disadvantages of well-established methods used in vaccine studies and explore more thoroughly newer technologies which are currently being used in, and which could be applied to, vaccine studies.
4.1. Advantages and Disadvantages of Established Methods to Assess T-Cell Immunogenicity
The ELISpot assay is a gold standard method for quantifying antigen-specific cellular responses after vaccination in clinical trials [
41]. The most widely used ELISpot in vaccine studies is the IFN-γ assay. Advantages of this assay include: It is high throughput, robust and economical, it can be easily standardised and validated, it can be adapted to use in pre-clinical i.e. mouse or non-human primate (NHP) studies and in human trials, both cells and supernatant can be recovered for further analysis, data is obtained from single cells, fine epitope mapping can be performed, and data analysis is straightforward [
39,
42]. Disadvantages include the lack of information about cell phenotype and the fact that it is normally a single parameter (IFN-γ) readout. Two adaptations to the traditional IFN-γ ELISpot can be used to circumvent these disadvantages; the cultured ELISpot and fluorospot respectively [
43]. The cultured ELISpot involves 10 days culture of lymphocytes in the presence of peptide and Il-2, compared with the standard ex vivo 18 h culture and, therefore, the cultured assay is thought to assess mainly T
CM [
44]. Dual and triple colour fluorospot assays allow analysis of multiple cytokines, typically IFN-γ and IL-2, and commercial kits are available. However, they are more costly than standard ELISpot and require an automated reader with the relevant filters [
45]. One application of the ELISpot into a diagnostic test is the T-SPOT, which is used in TB diagnosis and vaccine development to screen for TB [
46].
Intracellular cytokine staining (ICS) and analysis by flow cytometry provides an advantage over ELISpot as they allow both multiparameter cytokine analysis and phenotyping from potentially millions of single cells [
47]. Typically CD4
+ and CD8
+ T-cell populations are assessed along with cytokine expression allowing quantification of subsets, such as % of cytokine positive CD4
+ cells, which is not achievable by ELISpot. Disadvantages of ICS and flow cytometry compared with ELISpot are that it is much more labour intensive, costs more per sample, requires fixation of cells such that they cannot be used for further assays, requires expensive specialist equipment to run samples, complex panel optimisation, more detailed data analysis using specific software, and is more difficult to validate [
48]. Over 10 years ago, Seder and colleagues reviewed Th1 memory and implications for vaccine development, recommending the analysis of IFN-γ, TNF-α, and IL-2 by multiparameter flow cytometry to assess both effector and memory T-cells [
49]. Additional cell surface markers can be utilised to assess memory phenotypes, for example CD45RO or CD45RA to phenotype naïve and memory T-cells in human PBMCs, and CCR7 or CD62L to differentiate between T
CM and T
EM [
50]. CD69, an activation marker, can be used alongsideCD103 to identify T
RM in both mice and man [
51].
Another flow cytometry-based assay, which has recently been described, is the activation-induced marker (AIM) assay. This assay can be performed in a similar way to ICS (see
Figure 1). However, rather than staining for intracellular cytokines, staining is performed for T-cell receptor-induced cell-surface markers including PDL1, CD25 and OX40. Different combinations of these markers were assessed for their ability to identify AIM
+ T-cell populations [
52]. Therefore, detection of antigen-specific T-cells is not dependent upon cytokine production, which may be very low in some T-cell subsets. The assay was initially developed to detect T-follicular helper cells, which are difficult to detect by ICS [
53], but can also be used for CD4
+ T-cell assessment after exposure to antigen by vaccination or infection [
52,
53]. The use of an AIM assay in a vaccine clinical trial setting showed comparable sensitivity and specificity to ELISpot and ICS [
54]. This study showed that AIM assays can be used to detect both CD4
+ and CD8
+ T-cells. Whilst CD8
+ AIM responses correlated with CD8
+ ELISpot and ICS responses, CD4
+ AIM responses did not. This is likely due to the fact that AIM assays detect more CD4
+ subpopulations, including Tregs and NKTs, compared with ICS which primarily detects CD4
+ T
EM. This study indicates that AIM assays can be used in vaccine studies to detect novel memory T-cell responses which are hard to enumerate by ICS alone.
4.2. Multiplex Cytokine Analysis
Whilst both ELIspot and ICS have a clear role in quantifying T-cell responses, there are a range of newer tools which can be applied to assess T-cell responses to vaccination, both pre-clinically and in clinical trials. A number of these tools assess multiple parameters, rather than one, two or three cytokines. The datasets generated by such analyses require more detailed analysis compared to ELISpot or triple cytokine analysis by ICS, however, the data generated by such analyses can provide much greater information about vaccine responses and helps to generate a bigger picture.
Here we discuss three platforms which measure soluble analytes, essentially providing a multiplex ELISA. Luminex, LegendPlex and Meso-Scale Discovery (MSD) systems can all be used to quantify cytokine levels in cell culture supernatant, tissue homogenate, plasma or serum. A summary of the technical aspects of each of these platforms is provided in
Table 2. In terms of T-cell responses after immunisation, multiple cytokine analysis of supernatant from PBMCs stimulated with vaccine antigen peptides, such as that from ELISpot assay, would provide a valuable dataset. We also discuss two platforms based on flow cytometry, which analyse markers in cellular samples, CHIP Cytometry and CyTOF. A summary of the technical aspects of both of these platforms is provided in
Table 3.
4.2.1. Luminex
Luminex assays allow multiple cytokine analysis using different fluorescent (red and infrared) beads. Each bead subset is conjugated to capture an antibody specific for a cytokine, chemokine or disease biomarker. Samples such as serum or cell supernatant, are added, and the relevant analyte binds to the relevant antibody coated bead. Biotinylated detection antibody, also specific for the relevant analyte, is applied. The reporter, streptavidin conjugated PE, is then added. Samples are run on a dedicated machine which allows detection of the analyte through the bead fluorescence and quantity of the analyte through PE detection [
55]. A number of companies offer kits and machines to allow assessment of cytokines via Luminex, including MerckMillipore, Bio-Rad and ThermoFisher Scientific. Depending on the instrument and kit used, a maximum of 50–80 cytokines can be investigated. Luminex technology has been used in pre-clinical and clinical studies to aid exploration of cytokine profiles induced by vaccination. For example, King and colleagues describe different cytokine patterns induced by co-administration through different immunisation routes of an HIV vaccine in mice. These results indicate that different routes of administration could be used to elicit the desired cellular response from the vaccine [
56]. During the development of a vaccine for grass-pollen allergy, cytokine profiles in PBMCs from allergy sufferers were compared after stimulation with the vaccine proteins compared to grass pollen extract. The vaccine induced reduced release of pro-inflammatory cytokines compared to pollen, showing promise for this vaccine in allergy sufferers [
57]. Cytokine networks were investigated after vaccination with increasing doses of VSV-ZEBOV in a Phase I trial, and increasing cytokine activity was seen with increasing dose, which will help inform dose selection for future studies [
58].
4.2.2. LegendPlexTM
The principle behind the LegendPlex
TM assay, made by BioLegend, is similar to that of Luminex in that it uses beads with differing levels of APC fluorescence conjugated to an analyte-specific antibody. It also uses biotinylated capture antibody and strepdavidin-PE reagent for detection of a sample. LegendPlex
TM also uses two different sized beads, so different analytes are distinguished based on colour and size [
59]. Unlike Luminex, the assay does not require a dedicated machine; it can be run on a flow cytometer, although it does require the purchase of specialist analysis software. However, fewer analytes can be multiplexed compared to Luminex platform. This platform provides an attractive alternative to Luminex for a laboratory which already has a dedicated flow cytometer and does not have funds to invest in a new piece of hardware. For example, Copland et al. used LegendPlex
TM to assess T-cell cytokine levels in stimulated splenocytes from mice vaccinated with a fusion protein TB candidate. Cytokine levels were higher in mice vaccinated with the new candidate compared with BCG, showing increased immunogenicity of the new vaccine [
60].
4.2.3. Meso Scale Discovery
Meso Scale Discovery (MSD) assays work using electrochemiluminescence. Capture antibody is bound to carbon electrodes, sample is applied, and detection antibody conjugated to electrochemiluminescent labels. Samples are run on a dedicated plate reader; electricity is applied to the plate results in light emission from the conjugate, which is measured for quantification. Multiplexing is achieved by having up to 10 spots within the wells of the 96-well plate, each of which has a different detection antibody conjugated to it [
61]. A greater number of analytes can be assessed by using multiple plates and plates are customisable. In our experience, MSD assays are much quicker and simpler to run compared with Luminex, especially in terms of the time spent reading plates on the machine; an MSD plate takes only a couple of minutes rather than up to an hour for Luminex. Sample volumes required are similar and cost for a similar number of cytokines is similar. One disadvantage of MSD is that 10 cytokines can be done per plate, so multiplexing of more cytokines requires more plates. Data analysis for MSD is slightly more time consuming compared with Luminex, for which much of the data analysis is done in real time on the machine. One advantage of MSD is that the software is free; so plates can be read and then analysed off site at a later date. Both MSD and Luminex technologies have been validated and compared for human cytokine profiling [
62]. MSD technology has been used to assess cytokine responses to influenza vaccination with different adjuvants in a mouse study [
63].
4.2.4. CHIP Cytometry
Chipcytometry allows storage of cellular samples in a small chip which can be analysed by a microscopic flow cytometric method at a later date. Cycles of staining, imaging and bleaching allow multiple rounds of analysis, leading to multiplexing [
64]. Advantages of this method over flow cytometry include the fact that samples can be stored in their chips for up to 2 years either before analysis or after initial analysis for further analysis at a later date without the loss of biomarkers [
65]. This means that samples from a clinical trial could all be biobanked in chips, and all analysed for multiple cytokine and cell immunophenotyping markers, rather than processing each sample on a day-by-day basis, as is the case for many laboratories, with flow cytometry. This technology could have potential in assessing T-cell phenotypes in clinical trials to assess vaccine immunogenicity. It has been used recently in a comparison against flow cytometry methods in assessing cerebrospinal fluid samples from a cohort of patients [
66].
4.2.5. CyTOF
Time of flight (TOF) mass cytometry allows the evaluation of more than 40 markers per cell. Instead of labelling with fluorescently conjugated antibodies, cells are labelled with different metal isotope-tagged antibodies. When running samples on the dedicated mass cytometer, each cell is vaporized and metal ions are resolved to produce a mass spectrum. Data from each metal ion relates to the cytokine or cellular parameter in the same way that a detected signal within an emission spectrum does in flow cytometry [
67]. One disadvantage of this technology is that it does not have the capacity for forward or side scatter and so cell size and density cannot be assessed. However, there is little compensation required and autofluorescence does not occur. Single cell CyTOF has been used to analyse T-cell functionality in a subset of volunteers in a trial assessing T-cell memory to Hepatitis C virus vaccination. These authors showed a progressive increase in CD8
+ polyfunctionality post-vaccination. They also validated this CyTOF approach, showing that it correlated to flow cytometric methods [
68].
4.3. Omics Approaches
Omics approaches have the potential to aid the discovery of novel correlates of protection in combination with traditional analyses such as cytokine levels [
69]. Omics technologies add to the “systems vaccinology” approach to enable further understanding of the immune responses to vaccination [
70].
DNA and protein microarrays have been used previously for the evaluation of vaccine-specific induced responses [
71]. However, these assays require prior knowledge of the protein or DNA targets of interest and technical expertise for assay set up, when compared to RNA-seq or proteomics. For a bacterial pathogen such as TB which expresses thousands of proteins, a protein microarray is a valuable tool [
72]. It can be used as a correlate of protection to investigate how responses differ after BCG vaccination [
73].
In terms of immunogenicity, RNA-seq analysis post vaccination will add valuable information regarding up and down regulation of parameters other than cytokines and cell surface markers, such as transcription factors, as there is no selection bias—all transcripts within a cell are analysed [
74]. However, one challenge with RNA-seq approaches is the skill set and huge time involved in analysing these datasets, compared with cytokine analyses described above. The cost and application of RNA-seq analysis to clinical trial settings is a huge commitment; however, these analyses will become standard in the coming years. Using a single cell RNA-seq approach, Afik and colleagues describe subpopulations of yellow fever virus specific CD8
+ T-cells exhibiting ‘naïve-like’ and T
EM profiles in a volunteer vaccinated against yellow fever [
75]. This kind of study allows in-depth analysis of T-cell populations that are not possible though cytokine analyses alone, as other cellular pathways can also be explored. Transcriptional signatures and epigenetics of T-cell memory populations have been described [
76]; there are some examples of transcriptomic signatures in vaccine studies which predict vaccine efficacy [
77]. Immunopeptidomics, which involves analysing the peptides presented by MHC molecules using mass spectrometry, could help identify T-cell antigens for inclusion in new vaccines and T-cell subsets generated by particular vaccines [
78].