Article Activation-Induce Markers Detect -Specific CD4+ Responses Not Measured by Assays Conventionally Used in Clinical Trials

Georgina Bowyer * ID , Tommy Rampling ID , Jonathan Powlson ID , Richard Morter ID , Daniel Wright, Adrian V.S. Hill and Katie J. Ewer ID

The Jenner Institute, University of Oxford, Oxford OX3 7DQ, UK; [email protected] (T.R.); [email protected] (J.P.); [email protected] (R.M.); [email protected] (D.W.); [email protected] (A.V.S.H.); [email protected] (K.J.E.) * Correspondence: [email protected]; Tel.: +44-1865-617-646

 Received: 25 June 2018; Accepted: 30 July 2018; Published: 31 July 2018 

Abstract: Immunogenicity of T cell-inducing vaccines, such as viral vectors or DNA vaccines and Bacillus Calmette-Guérin (BCG), are frequently assessed by -based approaches. While these are sensitive methods that have shown correlates of protection in various vaccine studies, they only identify a small proportion of the vaccine-specific T cell response. Responses to vaccination are likely to be heterogeneous, particularly when comparing prime and boost or assessing vaccine performance across diverse populations. Activation-induced markers (AIM) can provide a broader view of the total -specific T cell response to enable a more comprehensive evaluation of vaccine immunogenicity. We tested an AIM assay for the detection of vaccine-specific CD4+ and CD8+ T cell responses in healthy UK adults vaccinated with viral vectored Ebola vaccine candidates, ChAd3-EBO-Z and MVA-EBO-Z. We used the markers, CD25, CD134 (OX40), CD274 (PDL1), and CD107a, to sensitively identify vaccine-responsive T cells. We compared the use of OX40+CD25+ and OX40+PDL1+ in CD4+ T cells and OX40+CD25+ and CD25+CD107a+ in CD8+ T cells for their sensitivity, specificity, and associations with other measures of vaccine immunogenicity. We show that activation-induced markers can be used as an additional method of demonstrating vaccine immunogenicity, providing a broader picture of the global T cell response to vaccination.

Keywords: vaccines; activation-induced markers; antigen-specific T cells; viral vector; adenovirus; modified vaccinia virus Ankara; Ebola

1. Introduction The majority of currently licensed vaccines mediate protection by inducing responses [1]. Immunogenicity of these vaccines is most often measured by enzyme-linked immunosorbent assays (ELISAs) that measure total antigen-specific antibody titers or assays that measure antibody function. These well-established vaccination programs often have gold-standard assays with a threshold value known to provide clinical protection [2]. Many vaccines currently being developed target pathogens for which neutralizing are difficult to produce (e.g., HIV) [3,4], that have multiple serotypes (staphylococci and streptococci) [5], or are intracellular (tuberculosis (TB), liver-stage malaria) [6,7]. Additionally, many new vaccines are being developed to target non-communicable diseases, such as autoimmunity and cancer [8,9]. It may be necessary for vaccines against many of these targets to induce potent T cell responses instead of, or in addition to, humoral responses to elicit protection [10]. This new generation of vaccines requires a set of assays that effectively capture antigen-specific T cell responses.

Vaccines 2018, 6, 50; doi:10.3390/vaccines6030050 www.mdpi.com/journal/vaccines Vaccines 2018, 6, 50 2 of 19

A number of assays are conventionally used to measure the quantity and quality of antigen-specific T cells in humans [11]. The assays most frequently used for this purpose in clinical vaccine trials are the enzyme-linked immunospot (ELISpot) and intracellular cytokine staining (ICS) assays [12–14]. The ELISpot assay involves stimulating peripheral blood mononucleocyte (PBMC) with antigen in 96-well plates with membranes coated in the anti-cytokine capture antibody. produced by antigen-specific T cells binds to the capture antibody and are detected using an enzyme-conjugated secondary antibody and a chromogenic development substrate. This method is highly sensitive (cells producing fewer than 100 cytokine molecules can be detected [15]) and the lower limit of detection can be under ten cytokine-producing cells per million [16]. ELISpots are currently one of the most frequently used and highly validated assays for the detection of antigen-specific T cell responses in clinical trials [16–18]. The major disadvantages of using this assay to investigate vaccine responses are a limit in the number of parameters than can be investigated, lack of phenotypic information, and preferential detection of effector cells. This likely results in an underestimation of the total antigen-specific T cell response. An alternative type of assay that is frequently used to provide further information on the quantity and quality of vaccine-induced T cells is ICS. Antigen-stimulated PBMC are stained with fluorescently labelled anti-cytokine antibodies and analyzed by flow cytometry. This allows detailed phenotypic and functional analysis of antigen-specific T cell populations. However, ICS assays are also limited in the number of parameters that can be assessed and these must be pre-determined, therefore, these assays can be biased towards the detection of a particular type of T cell. Standard panels in clinical trials often use IFNγ, IL2, and TNFα and, therefore, detect Th1-biased responses [19–21]. T cell responses to vaccination and infection can be highly heterogeneous and, therefore, detection based on the expression of one or more cytokines may significantly underestimate the frequency of antigen-specific cells [22,23]. Multiple groups have overcome these limitations by developing T cell assays that define antigen-specificity based on the upregulation of TCR-stimulated surface markers—termed activation-induced markers (AIM). These include an assay based on the detection of OX40 and CD25 co-expression on CD4+ T cells in whole blood [24] or PBMC [25,26], and assays detecting CD40L [27,28], or co-expression of CD40L and CD69 [29]. Similarly, activation-induced markers, such as CD107a and CD137 (4-1BB), have been used to identify antigen-specific CD8+ T cells [30,31]. Additional markers of activation, such as OX40 and CD25 are expressed on activated CD8+ T cells and could be used in a similar way [32]. The advantages of these assays over conventional methods are that they do not rely on prior knowledge of the epitope or HLA type and they are not limited by pre-determination of the cytokine/s to be analysed. AIM assays can provide a broader picture of the overall antigen-specific T cell response. Increasing knowledge about the total quantity and quality of these responses could significantly aid clinical development, regulatory approval and licensure applications for vaccine candidates [10]. AIM assays have been used to detect vaccine-specific T cell responses in humans [25,33], and we routinely use CD107a as a marker of antigen-specific degranulation in our clinical trials [34–36]. However, AIM assays have not been fully evaluated and compared with cytokine-based methods for their use within the clinical trial setting. In this study, we investigated the use of AIM assays to detect antigen-specific CD4+ and CD8+ T cell responses to vaccination. We then compared the sensitivity and specificity to assays we currently use to investigate vaccine immunogenicity—an IFNγ ELISpot and ICS assay measuring the frequencies of CD4+ and CD8+ T cells producing IFNγ, IL2, and TNFα, or expressing CD107a. AIM assays sensitively and specifically detected antigen-responsive CD4+ and CD8+ T cells after vaccination. AIM assays had a comparable (CD4+) or lower (CD8+) background and detected higher levels of antigen-specific T cells than cytokine-based methods. Additionally, the frequency of AIM+ CD4+ T cells did not correlate with the frequency of cytokine-production measured by ICS or ELISpot, suggesting that the AIM assay can provide novel information about the antigen-specific CD4+ T cell response that is not detected by conventional clinical trial methods. Vaccines 2018, 6, 50 3 of 19

2. Materials and Methods

2.1. Clinical Vaccine Trial The samples used in this study were collected as part of a Phase Ia clinical vaccine trial assessing the safety and immunogenicity of viral vectored Ebola vaccine candidates (EBL04, ClinicalTrials.gov NCT02451891, EudraCT number: 2015-000593-35). The recombinant chimpanzee adenovirus type 3 vectored Ebola Zaire vaccine (ChAd3-EBO-Z) is a replication-deficient chimpanzee adenovirus serotype 3 (ChAd3) vector expressing wild-type (WT) Zaire strain Ebola glycoprotein (GP). The modified vaccinia Ankara virus vectored Ebola Zaire vaccine (MVA-EBO-Z) is a replication-deficient, attenuated vaccinia Ankara virus vector expressing the wild-type Ebola glycoprotein (GP) of the Zaire Mayinga strain. This study uses samples from all 16 volunteers from Group 2 of the EBL04 study. These individuals received 3.6 × 1010 viral particles (vp) ChAd3-EBO-Z and were boosted with 1.0 × 108 plaque forming units (PFU) MVA-EBO-Z seven days later. All vaccinations were administered intramuscularly into the deltoid region of the arm. All volunteers were healthy adults between the ages of 18 and 50 years recruited at the Centre for Clinical Vaccinology and Tropical Medicine at the University of Oxford and the Wellcome Trust Clinical Research Facility at the Imperial College, London, both in the United Kingdom.

2.2. Ethics and Regulatory Approval All participants provided written informed consent prior to enrolment. The study was conducted according to the principles of the Declaration of Helsinki (2008) and the International Conference on Harmonization Good Clinical Practice guidelines. The study protocol and associated documents for the Phase Ia trial were reviewed and approved by the UK National Research Ethics Service (Committee South Central-Oxford A, reference 15/SC/0108, IRAS project ID 174444, approval date: 25/02/2015) and the Medicines and Healthcare Products Regulatory Agency (Ref.: 21584/0341/001-0001, approval date: 24/04/2015). Further details of the study are provided in the clinical trial protocol available with the clinical trial report (Venkatraman et al. manuscript in preparation).

2.3. Sample Handling Blood was collected in heparin-treated vacutainers for peripheral blood mononucleocyte (PBMC) separation and processed within six hours of blood draw to ensure sample quality. All processing was conducted under sterile conditions in a class II microbiological safety cabinet. PBMCs were separated by density gradient isolation using Lymphoprep® (STEMCELL Technologies, Vancouver, BC, Canada) in Leucosep® (Sigma-Aldrich, Saint Louis, MO, USA) tubes. Washes were conducted using R0 medium (RPMI-1640 medium with 5% L-Glutamine and 5% Penicillin/Streptomycin). Cell culture and assays were performed using R10 medium (RPMI-1640 medium, 5% L-Glutamine, 5% Penicillin/Streptomycin and 10% Fetal Calf Serum (FCS)). Ex vivo IFNγ ELISpot assays and ICS were performed using freshly separated PBMC. AIM assays were conducted using thawed cryopreserved PBMC. PBMC remaining after ELISpot and ICS set up were frozen at 6–10 × 106 cells/mL in FCS with 10% dimethyl sulfoxide (DMSO) with controlled cooling using CoolCell® Cell freezing containers (BioCision, San Rafael, CA, USA) and stored in liquid nitrogen until use. For AIM assays, cryopreserved cells were thawed in a 37 ◦C water bath. Cells were washed in pre-warmed R10, then resuspended in 2 mL R10 with 5 µL benzonase nuclease ◦ (250 units/µL, ≥90%, Novagen, Darmstadt, Germany). Cells were rested at 37 C and 5% CO2 for 2 h before washing and resuspension in R10 to the concentration required for the assay.

2.4. IFNγ ELISpot Ex vivo (18 h stimulation) ELISpot assays were performed using Multiscreen IP ELISpot plates (Millipore), human IFNγ SA-ALP antibody kits (Mabtech, Stockholm, Sweden), and BCIP NBT-plus Vaccines 2018, 6, 50 4 of 19 chromogenic substrate (Moss Inc., Pasadena, MD, USA). Cells were cultured in R10 and were tested in triplicate. To measure GP-specific responses, we used 187 peptides covering the length of the Ebola Zaire glycoprotein (Mayinga strain). GP peptides were mostly 15 amino acids in length, overlapping by 11 amino acids. The sequences were provided by the National Institutes of Health (NIH), USA and are the same as those used in a previous study [37]. There were seven pools consisting of peptides from the glycoprotein chain 1 (GP1, amino acids 33–501) and two pools containing peptides from the glycoprotein chain 2 (GP2, amino acids 504–676). Each peptide pool was added to the plate at a final concentration of 2.5 µg/mL in 150 µL with 200,000 PBMC. A mixture of Staphylococcal enterotoxin B (SEB, 0.02 µg/mL) and phytohaemmagglutinin-L (PHA, 10 µg/mL) was used as a positive control in all ELISpot assays. Negative control wells with no peptide were included in all assays to measure the background. Peptides used in the ELISpot, ICS, and AIM assays were resuspended in DMSO. Negative controls did not include DMSO. However, we have shown in previous experiments that DMSO only causes increases in the background when the concentration is >0.1% (unpublished data). In all of the experiments run here, the peptide pools had final DMSO concentrations of 0.0012% or lower. Plates were developed the following day according to the manufacturer’s instructions. Plates were counted using an automated ELISpot counter (AID Diagnostika (Strausberg, Germany) with identical settings for all plates. Counts were only adjusted to remove artefacts. The average spot forming cells per million PBMC (SFC/106 PBMC) was calculated for each pool on the plate. Responses in unstimulated (negative control) wells were subtracted and then responses in individual pools were summed to give a total response against Ebola GP. ELISpot QC criteria were >800 SFC/106 PBMC in the positive control wells and <80 SFC/106 in the negative control wells. Responses greater than the median + 2 standard deviations of the negative control wells were considered positive. The lower limit of detection for this assay is defined as the lowest number of spots that is precisely distinguishable from the unstimulated control well and is calculated as the median plus 2 standard deviations of all unstimulated control wells run for that study. The detection limit for this study was 50 SFC/106 PBMC.

2.5. Intracellular Cytokine Staining (ICS) Stimulations were set up for ICS in parallel with the ELISpot, using freshly isolated PBMC. Cells were stimulated with a single megapool of the same GP peptides used in the ELISpot, at a final concentration of 2.5 µg/mL. An unstimulated condition and a positive control stimulated with 1 µg/mL SEB were run for each sample. Each stimulation was set up in a 5 mL polystyrene FACS tube containing 2 × 106 PBMC in 1 mL of R10. Anti-CD28 and anti-CD49d (eBioscience, San Diego, CA, USA) at final concentrations of 1 µg/mL and 2 µL of αCD107a-PE-Cy5 (eBioH4A3, eBioscience) ◦ were added to each tube. Samples were stimulated for 18 h at 37 C and 5% CO2. Brefeldin A and Monensin (eBioscience), both at 1 µg/mL, were added for the last 16 h. Responses were assessed by a nine-colour staining panel. Cells were incubated for 20 min at room temperature with a dead cell discrimination dye (AQUA 1:2000, Invitrogen) diluted in FACS buffer (PBS with 1% BSA and 0.1% NaN3). PBMC were permeabilized using fixation/permeablization solution (BD Biosciences), then stained intracellularly at room temperature (RT) for 30 min with the following antibodies: αCD3-AF700 (UCHT1, 1/1000, eBioscience), αCD4-APC (RPA-T4, 1/500, eBioscience), αCD8-APC-AF780 (RPA-T8, 1/200, eBioscience), αCD14-eF450 (61D3, 1/1000, eBioscience), αCD19-eF450 (HIB19, 1/1000, eBioscience), αIFNγ-FITC (4S.B3, 1/2000, eBioscience), αIL2-PE (MQ1-17H12, 1/1000, eBioscience), and αTNFα-PE-Cy7 (Mab11, 1/10,000, eBioscience). Cells were then washed and fixed in 1% paraformaldehyde. Compensation was performed using single-stained One-Comp beads (eBioscience) for monoclonal antibodies and ARC beads for AQUA (Life Technologies, Carlsbad, CA, USA). Acquisition was performed on the day of staining on a BD LSRII and FACSDiva v6.2 (BD Biosciences, San Jose, CA, USA). Data was prepared and analysis performed using FlowJo v10.1 (Treestar Inc., Ashland, OR, USA). Cells were gated on , singlets, live CD3+, CD8-CD4+, or CD4-CD8+ and then IFNγ, IL2, TNFα and CD107a. Dead cells (AQUA+), monocytes (CD14+), and B cells (CD19+) were excluded from the analysis. The ICS gating strategy (Supplementary Figure S1) is the same as Vaccines 2018, 6, 50 5 of 19 previously published [38]. Responses to peptide were determined after subtraction of the response in the unstimulated control for each sample. Responses were considered positive if the count was >20 and frequency greater than two times the autologous unstimulated control and greater than the lower limit of detection (LLOD). The LLOD was calculated as the reciprocal of the minimum number of CD4+ (21,971) and CD8+ (21,528) T cells collected. This resulted in LLOD values of 0.005% for both CD4+ and CD8+ populations for this dataset. Samples failed quality control and were excluded from data analysis if the background-subtracted response of cytokine positive CD4+ or CD8+ T cells to the positive control was <1%. In this study 3/16 of the samples either did not have ICS results or did not pass the QC criteria. Therefore the final ICS dataset for this assay included responses from 13 individuals.

2.6. Activation-Induced Markers (AIM) Assay The activation-induced markers assay was conducted in a similar way to previously published work [25,26,39]. PBMC were stimulated overnight (20 h at 37 ◦C) with 1–2 × 106 cells per well in a 96-well U-bottom plate. A stimulation time of 20 h was chosen because this was used in previous studies and longer incubations were shown to be optimal for OX40 expression [39,40]. Additionally, the clinical trial ICS SOP we use is a 16–20 h stimulation and having incubations of the same length would allow the assays to be easily run in parallel in clinical trials if required. Despite this longer incubation, we did not see evidence of bystander activation. It has been demonstrated that bystander activation only had a minimal effect on the AIM assay despite a 20 h incubation [39]. Cells were stimulated with 2 µg/mL of the same megapool of Ebola GP peptides used for the ICS assay. An unstimulated well and a positive control well stimulated with 1 µg/mL SEB were included for each sample. Cells were stained with αCD107a-PE-Cy5 (eBioH4A3, 1/2000, eBioscience) during the stimulation. After overnight stimulation, cells were washed twice in FACS buffer (PBS with 1% BSA and 0.1% NaN3), then stained for 20 min at RT with 100 µL per well of a mixture containing the following fluorescently conjugated antibodies: αCD45RA-eV605 (HI100, 1/200, eBioscience), αCD14-eF506 (61D3, 1/200, eBioscience), αCD19-eF506 (HIB19, 1/200, eBioscience), αCD4-APC-eF780 (RPA-T4, 1/100, eBioscience), αCD3-AF700 (UCHT1, 1/133, eBioscience), αOX40-PE/Cy7 (Ber-ACT35, 1/100, Biolegend, San Diego, CA, USA), αCD25-FITC (M-A251, 1/100, Biolegend), αPDL1-APC (-H1, 1/133, Biolegend), αCD8-BV711 (RPA-T8, 1/400, Biolegend), and AQUA live/dead stain (1/2000, Invitrogen, Carlsbad, CA, USA). Cells were washed twice as before, fixed with 4% paraformaldehyde for 10 min at 4 ◦C, washed twice again, resuspended in 100 µL FACS buffer, and acquired immediately on a BD LSRII. Data analysis was conducted in FlowJo version 10.1 (Treestar Inc.). The LLODs for the assay were determined in the same way as the LLODs for the ICS assay (minimum number of events collected were CD3: 87213, CD4: 32730 and CD8: 19172). The LLODs were 0.001% for total T cells, 0.003% for CD4+ T cells, and 0.005% for CD8+ T cells. Compensation was performed as for the ICS assay.

2.7. Statistical Analysis The data were non-parametric, therefore, Mann-Whitney analyses were used for comparison between two groups and Kruskal-Wallis analyses with Dunn’s post-tests were used for comparison across multiple groups. Linear regression analyses were conducted using Spearman’s . Where responses were compared at two different time points within a group, Wilcoxon matched pairs analyses were used. An alpha-level of 0.05 was considered significant and all p-values are 2-tailed. Analyses were performed in GraphPad Prism version 7. (GraphPad Software Inc., La Jolla, CA, USA). Medians and inter-quartile ranges (IQR) are presented. * p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Vaccines 2018, 6, 50 6 of 19

3. Results 3. Results 3.1. Detection of Vaccine-Specific T cells Using Activation-Induced Markers 3.1. Detection of Vaccine-Specific T cells Using Activation-Induced Markers + + + The expression of combinations of activation-induced markers on CD4 +(OX40 CD25+ and+ TheOX40 expression+PDL1+) and ofCD8 combinations+ (OX40+CD25+ of and activation-induced CD25+CD107a+) T cells markers were assessed on CD4 by (OX40flow cytometryCD25 and + + + + + + + OX40usingPDL1 the) gating and CD8 strategy(OX40 definedCD25 in Figureand CD251. CD107a ) T cells were assessed by flow cytometry using the gating strategy defined in Figure1.

Figure 1. Activation-induced markers (AIM) gating strategy. Cells were gated on single lymphocytes Figure 1. Activation-induced markers (AIM) gating strategy. Cells were gated on single lymphocytes + + basedbased on size, on size, then then dead dead cells, cells, CD14 CD14,+ and, and CD19 CD19+ cells were were excluded. excluded. T cell T cell subsets subsets were were gated gated as as + - + - CD4 CD4CD8+CD8or CD8- or CD8CD4+CD4and- and then then the the expressionexpression of of activation activation-induced-induced markers markers was was measured measured within within each subset.each subset. Gates Gates displayed displayed are are representative representative ofof thethe top quartile quartile of of Ebola Ebola glycoprotein glycoprotein (GP) (GP)-specific-specific responsesrespon toses clearly to clearly demonstrate demonstrate where where these these populations populations sit. sit.

Very little CD107a expression was detected in CD4+ T cells and PDL1 expression on CD8+ T cells Very little CD107a expression was detected in CD4+ T cells and PDL1 expression on CD8+ T cells was also low, therefore these markers were not included in the analysis of antigen-specific CD4+ and + was alsoCD8 low,+ T cell therefore responses, these respectively. markers Vaccine were not-specific included T cell in responses the analysis could of clearly antigen-specific be detected CD4in the and + CD8 CD4T cell+ T responses,cell subset as respectively. OX40+CD25+ Vaccine-specific or OX40+PDL1+ and T cell in the responses CD8+ T cell could subset clearly as OX40 be detected+CD25+ or in the + + + + + + + + CD4 CD25T cell+CD107a subset+. as For OX40 each CD25sample, oran OX40 unstimulatedPDL1 controland in was the CD8run toT determine cell subset background as OX40 CD25AIM or CD25expression+CD107a+ and. For an eachSEB-stimulated sample, an positive unstimulated control was control included. was Representative run to determine FACS background plots of AIM+ AIM expressionpopulations and an in SEB-stimulatedeach condition are positive shown in control Figure was2A. included. Representative FACS plots of AIM+ populationsFrequencies in each condition of AIM expression are shown in in GP Figure-stimulated2A. PBMC were significantly higher than the Frequenciescorresponding of background AIM expression for all four in of GP-stimulated the AIM populations PBMC measured were significantly (Figure 2B,C, p higher < 0.0001 than for the all populations). Within the CD4+ T cell subset, background levels of AIM expression in unstimulated corresponding background for all four of the AIM populations measured (Figure2B,C, p < 0.0001 for all cells were generally low and were comparable between the OX40+CD25+ and OX40+PDL1+ populations). Within the CD4+ T cell subset, background levels of AIM expression in unstimulated cells populations (Figure 2B, median + inter-quartile range (IQR) OX40+CD25+: 0.110% (0.069–0.172) and + + + + wereOX40 generally+PDL1 low+: 0.102% and were(0.044 comparable–0.131), p = 0.468). between The background the OX40 CD25was alsoand low OX40 in the CD8PDL1+ subsetpopulations and (Figure2B, median + inter-quartile range (IQR) OX40 +CD25+: 0.110% (0.069–0.172) and OX40+PDL1+: Vaccines 2018, 6, x; doi: FOR PEER REVIEW www.mdpi.com/journal/vaccines 0.102% (0.044–0.131), p = 0.468). The background was also low in the CD8+ subset and comparable between the two AIM populations (Figure2C, OX40 +CD25+: 0.021% (0.010–0.033) and CD25+CD107a+: 0.020% (0.012–0.036), p = 0.934). Frequencies of GP-specific CD4+ T cells measured using OX40+CD25+ or OX40+PDL1+ were comparable (Figure2B, OX40 +CD25+: 0.870% (0.493–1.088) and OX40+PDL1+: Vaccines 2018, 6, x FOR PEER REVIEW 2 of 14 comparable between the two AIM populations (Figure 2C, OX40+CD25+: 0.021% (0.010–0.033) and VaccinesCD25+CD107a2018, 6, 50+: 0.020% (0.012–0.036), p = 0.934). Frequencies of GP-specific CD4+ T cells measured7 of 19 using OX40+CD25+ or OX40+PDL1+ were comparable (Figure 2B, OX40+CD25+: 0.870% (0.493–1.088) and OX40+PDL1+: 0.736% (0.389–1.088), p = 0.773). Similar frequencies of GP-specific CD8+ T cells were 0.736% (0.389–1.088), p = 0.773). Similar frequencies of GP-specific CD8+ T cells were detected and were detected and were also comparable for the two different AIM populations in this subset (Figure 2C, also comparable for the two different AIM populations in this subset (Figure2C, OX40 +CD25+: 0.633% OX40+CD25+: 0.633% (0.319–0.837) and CD25+CD107a+: 0.882% (0.406–1.258), p = 0.224). Due to the (0.319–0.837) and CD25+CD107a+: 0.882% (0.406–1.258), p = 0.224). Due to the lower background in the lower background in the CD8+ subset, the fold-change in the frequency of AIM+ cells (GP- CD8+ subset, the fold-change in the frequency of AIM+ cells (GP-stimulated/unstimulated) was higher stimulated/unstimulated) was higher for the CD8+ subset than the CD4+ subset (Figure 2D, for the CD8+ subset than the CD4+ subset (Figure2D, OX40 +CD25+ CD4+: 9 (4–14), OX40+PDL1+ OX40+CD25+ CD4+: 9 (4–14), OX40+PDL1+ CD4+: 9 (4–26), OX40+CD25+ CD8+: 31 (12–73), CD25+CD107a+ CD4+: 9 (4–26), OX40+CD25+ CD8+: 31 (12–73), CD25+CD107a+ CD8+: 47 (17–68)). However, there CD8+: 47 (17–68)). However, there was no difference between the marker combinations in either of was no difference between the marker combinations in either of the subsets (CD4+: p = 0.662, CD8+: the subsets (CD4+: p = 0.662, CD8+: p = 0.616). p = 0.616).

Figure 2. Detection of vaccine antigen-specific T cells: (A) Representative flow cytometry plots detailing Figure 2. Detection of vaccine antigen-specific T cells: (A) Representative plots AIM+ populations in unstimulated, GP-stimulated and Staphylococcal enterotoxin B (SEB)-stimulated detailing AIM+ populations in unstimulated, GP-stimulated and Staphylococcal enterotoxin B (SEB)- CD4+ and CD8+ T cells; (B) AIM+ responses in CD4+ T cells; and (C) AIM+ responses in CD8+ T stimulated CD4+ and CD8+ T cells; (B) AIM+ responses in CD4+ T cells; and (C) AIM+ responses in CD8+ cells. Mann-Whitney analyses between stimulation conditions within each population and between T cells. Mann-Whitney analyses between stimulation conditions within each population and between the same stimulation conditions in different populations. Medians and inter-quartile range (IQR) the same stimulation conditions in different populations. Medians and inter-quartile range (IQR) shown. **** p < 0.0001, ns: Not significant (p > 0.05); (D) fold change in frequency of AIM+ cells shown. **** p < 0.0001, ns: Not significant (p > 0.05); (D) fold change in frequency of AIM+ cells (GP- (GP-stimulated/unstimulated conditions). Individuals below the dashed line did not have responses greater than the background. Vaccines 2018, 6, 50 8 of 19

Vaccines 2018, 6, x FOR PEER REVIEW 8 of 19 3.2. Comparison of Different Activation-Induced Markers for Detection of Vaccine-Specific T Cells The frequency of GP-specific T cell responses was compared between the different AIM+ subsets The frequency of GP-specific T cell responses was compared between the different AIM+ after subtracting the corresponding background for each sample (AIM+ frequency in the subsets after subtracting the corresponding background for each sample (AIM+ frequency in the unstimulated condition, Figure 3A,B). Frequencies of OX40+CD25+ and OX40+PDL1+ in CD4+ T cells unstimulated condition, Figure3A,B). Frequencies of OX40 +CD25+ and OX40+PDL1+ in CD4+ T cells were comparable (0.753% (0.445–0.924) and 0.700% (0.259–0.961), respectively, p = 0.876). All, but one were comparable (0.753% (0.445–0.924) and 0.700% (0.259–0.961), respectively, p = 0.876). All, but one individual (15/16), had responses above the LLOD (0.003%) in both AIM populations. The frequencies individual (15/16), had responses above the LLOD (0.003%) in both AIM populations. The frequencies of AIM+ cells detected by either of the marker combinations in the CD8+ subset were also comparable of AIM+ cells detected by either of the marker combinations in the CD8+ subset were also comparable (OX40+CD25+: 0.601% (0.304–0.826) and CD25+CD107a+: 0.861% (0.359–1.219), p = 0.196). Almost all (OX40+CD25+: 0.601% (0.304–0.826) and CD25+CD107a+: 0.861% (0.359–1.219), p = 0.196). Almost all vaccines had detectable levels of AIM+ CD8+ T cells measured by both marker combinations. One vaccines had detectable levels of AIM+ CD8+ T cells measured by both marker combinations. One individual did not have a CD25+CD107a+ response above the LLOD (0.005%), although they did have individual did not have a CD25+CD107a+ response above the LLOD (0.005%), although they did have a detectable OX40+CD25+CD8+ population. Frequencies of GP-specific T cells measured by each of the a detectable OX40+CD25+CD8+ population. Frequencies of GP-specific T cells measured by each of combinations of AIM markers was highly correlated in both CD4+ (Figure 3C, Spearman r = 0.83, p = the combinations of AIM markers was highly correlated in both CD4+ (Figure3C, Spearman r = 0.83, 0.0001) and CD8+ T cells (Figure 3D, Spearman r = 0.91, p < 0.0001). p = 0.0001) and CD8+ T cells (Figure3D, Spearman r = 0.91, p < 0.0001).

AB P=0.876 P=0.196 10 10

1 1

0.1 0.1 (GP-unstim) (GP-unstim) + +

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6 4 T cells T + T cells T + 3 4 in CD8 + in CD4 in 2 +

2 (GP-unstim) (GP-unstim) PDL1 1 + CD107a +

0 0

%OX40 012345 0.00.51.01.52.0 %CD25 %OX40+CD25+ in CD4+ T cells %OX40+CD25+ in CD8+ T cells (GP-unstim) (GP-unstim) Spearman r: 0.83 Spearman r: 0.91 P= 0.0001 (***) P< 0.0001 (****)

Figure 3. Comparison of activation-induced markers for the detection of Ebola GP-specific T cells:Figure (A )3. Proportion Comparison of of AIM activation-induced+ cells in GP-stimulated markers for CD4 the+ detectionand; (B) of CD8 Ebola+ subsets GP-specific after T removal cells: (A) + + + ofProportion background of (frequencyAIM cells in in GP-stimulated unstimulated CD4 condition). and; (B) Dashed CD8 subsets linesindicate after removal the lower of background limit of detection(frequency (LLOD). in unstimulated Mann-Whitney condition). analyses Dashed used for lines comparisons indicate the between lower markers;limit of detection (C) relationship (LLOD). betweenMann-Whitney the frequency analyses of GP-specific used for CD4comparisons+ T cells measured between bymarkers; OX40+CD25 (C) +relationshipand OX40+ PDL1between+; and the (Dfrequency) relationship of betweenGP-specific the CD4 frequency+ T cells of GP-specific measured CD8by +OX40T cells+CD25 measured+ and byOX40 OX40+PDL1+CD25+; and+ and (D) CD25relationship+CD107a +between. the frequency of GP-specific CD8+ T cells measured by OX40+CD25+ and CD25+CD107a+. 3.3. Relationship Between AIM Assays and Other Measures of Vaccine Immunogenicity 3.3. Relationship Between AIM Assays and Other Measures of Vaccine Immunogenicity The relationships between the frequency of GP-specific T cells detected by the AIM assay and other methodsThe relationships used in the between clinical the vaccine frequency trial wereof GP-specific compared. T Atcells the detected peak time by pointthe AIM (seven assay days and other methods used in the clinical vaccine trial were compared. At the peak time point (seven days after MVA boost, M+7), GP-specific T cell responses were measured using IFNγ ELISpot. Responses were further dissected using ICS to analyse the frequency of CD4+ and CD8+ T cells producing IFNγ, IL2, and TNFα, or expressing CD107a. Frequencies of AIM+ CD8+ T cells measured using either

Vaccines 2018, 6, 50 9 of 19 after MVA boost, M+7), GP-specific T cell responses were measured using IFNγ ELISpot. Responses Vaccines 2018, 6, x FOR PEER REVIEW 9 of 19 were further dissected using ICS to analyse the frequency of CD4+ and CD8+ T cells producing IFNγ, IL2, and TNFα, or expressing CD107a. Frequencies of AIM+ CD8+ T cells measured using either combination of markers were correlated with all other methods used to detect GP-specific CD8+ T cell combination of markers were correlated with all other methods used to detect GP-specific CD8+ T cell responses at this time point (Figure 4). In contrast, the frequency of GP-specific OX40+CD25+ CD4+ T + + + responsescells did not at this correlate time point with (Figureany of 4th).e In other contrast, measures the frequency of GP-specific of GP-specific CD4+ responses OX40 CD25and thereCD4 wereT + cellsonly did weak not associations correlate with between any of GP-specific the other measures OX40+PDL1 of GP-specific+ and IFNγ+ CD4 (Spearmanresponses r = 0.57, and p there = 0.047) were or + + γ+ r p onlyTNF weakα+ (Spearman associations r = 0.59, between p = 0.039) GP-specific CD4+ T OX40 cells measuredPDL1 and by IFN ICS (Figure(Spearman 5). = 0.57, = 0.047) or TNFα+ (Spearman r = 0.59, p = 0.039) CD4+ T cells measured by ICS (Figure5).

A B

10 10

1 1 (GP-unstim) (GP-unstim) + + 0.1 0.1 of CD8 of

0.01 CD8 of 0.01 + +

%AIM 0.001 0.001 %AIM 10 100 1000 10000 0.001 0.01 0.1 1 10 M+7 IFNγ ELISpot (SFC/106 PBMC) %IFNγ+ CD8+

+ + + CD25+CD107a+ CD8+ r: 0.51 P=0.046 (*) CD25 CD107a CD8 r: 0.65 P=0.019 (*) + + + OX40+CD25+ CD8+ r: 0.58 P=0.020 (*) OX40 CD25 CD8 r: 0.76 P=0.003 (**) C D

10 10

1 1 (GP-unstim) (GP-unstim) + 0.1 + 0.1 of CD8 of CD8 of 0.01 0.01 + +

0.001 0.001 %AIM %AIM 0.001 0.01 0.1 1 0.001 0.01 0.1 1 %IL2+ in CD8+ %TNFα+ CD8+ + + + CD25+CD107a+ CD8+ r: 0.63 P=0.025 (*) CD25 CD107a CD8 r:0.72 P=0.007 (**) + + + E OX40+CD25+ CD8+ r: 0.75 P=0.004 (**) OX40 CD25 CD8 r:0.82 P=0.001 (***)

10

1 (GP-unstim) + 0.1

of CD8 of 0.01 +

0.001 %AIM 0.001 0.01 0.1 1 %CD107a+ in CD8+

CD25+CD107a+ CD8+ r: 0.83 P=0.0007 (***) OX40+CD25+ CD8+ r: 0.71 P=0.0083 (**)

Figure 4. Relationships between activation-induced markers on CD8+ T cells and other measures of Figure 4. Relationships between activation-induced markers on CD8+ T cells and other measures of vaccine immunogenicity: (A) IFNγ enzyme-linked immunospot (ELISpot); (B) frequency of IFNγ+ vaccine immunogenicity: (A) IFNγ enzyme-linked immunospot (ELISpot); (B) frequency of IFNγ+ CD8+ T cells measured by intracellular cytokine staining (ICS); (C) frequency of IL2+ CD8+ T cells CD8+ T cells measured by intracellular cytokine staining (ICS); (C) frequency of IL2+ CD8+ T cells measured by ICS; (D) frequency of TNFα+ CD8+ T cells measured by ICS; and (E) frequency of + + CD107ameasured+ CD8 by +ICS;T cells (D) measured frequency by of ICS. TNF SFC/10α CD86 PBMC: T cells Spot-forming measured by cells ICS; per and million (E) frequency peripheral of + + 6 bloodCD107a mononucleocytes. CD8 T cells measured Spearman’s by rankICS. analyses.SFC/10 PBMC: Spot-forming cells per million peripheral blood mononucleocytes. Spearman’s rank analyses.

Vaccines 2018, 6, 50 10 of 19 Vaccines 2018, 6, x FOR PEER REVIEW 10 of 19

A B 10 10

1 1 (GP-unstim) (GP-unstim) + + 0.1 0.1 of CD4 of

of CD4 of 0.01 0.01 + +

0.001 %AIM 0.001 %AIM 10 100 1000 10000 0.001 0.01 0.1 1 10 M+7 IFNγ ELISpot (SFC/106 PBMC) %IFNγ+ CD4+

+ + + OX40+PDL1+ CD4+ r: 0.20 P=0.463 OX40 PDL1 CD4 r:0.57 P=0.047 (*) + + + OX40+CD25+ CD4+ r: 0.44 P=0.091 OX40 CD25 CD4 r:0.48 P=0.101 C D 10 10

1 1 (GP-unstim) (GP-unstim) + 0.1 + 0.1 of CD4 of 0.01 CD4 of 0.01 + +

%AIM 0.001 0.001 %AIM 0.001 0.01 0.1 1 10 0.001 0.01 0.1 1 10 %IL2+ in CD4+ %TNFα+ CD4+ + + + OX40+PDL1+ CD4+ r: 0.48 P=0.100 OX40 PDL1 CD4 r:0.59 P=0.039 (*) + + + OX40+CD25+ CD4+ r: 0.32 P=0.282 OX40 CD25 CD4 r:0.50 P=0.083

Figure 5. Relationships between activation-induced markers on CD4+ T cells and other measures Figure 5. Relationships between activation-induced markers on CD4+ T cells and other measures of of vaccine immunogenicity: (A) IFNγ ELISpot; (B) frequency of IFNγ+ CD4+ T cells measured by vaccine immunogenicity: (A) IFNγ ELISpot; (B) frequency of IFNγ+ CD4+ T cells measured by ICS; (C) ICS; (C) frequency of IL2+ CD4+ T cells measured by ICS; and (D) frequency of TNFα+ CD4+ T cells frequency of IL2+ CD4+ T cells measured by ICS; and (D) frequency of TNFα+ CD4+ T cells measured 6 measuredby ICS. by ICS.SFC/10 SFC/106 PBMC: PBMC:Spot-forming Spot-forming cells per million cells per peripheral million blood peripheral mononucleocytes. blood mononucleocytes. Spearman’s Spearman’srank analyses. rank analyses.

The specificityThe specificity of the of AIM the assayAIM assay for detecting for detecting GP-specific GP-specific T cells T cells was was compared compared with with the the ICS ICS assay + (Figureassay6A,B). (Figure The frequency 6A,B). The of frequency AIM expression of AIM inexpression unstimulated in unstimulated CD4 + T cells CD4 was T notcells significantly was not significantly different to the frequency of cytokine production by unstimulated cells in the ICS assay different to the frequency+ of+ cytokine production by unstimulated+ + cells in the ICS assay (Figure6A, +(Figure+ 6A, OX40 CD25 : 0.110 (0.069–0.172),+ OX40+ PDL1 : 0.102 (0.044–0.131), ICS “any of three”: OX40 0.064CD25 (0.045–0.096),: 0.110 (0.069–0.172), Kruskal-Wallis OX40 p = 0.092).PDL1 The: frequency 0.102 (0.044–0.131), of AIM expression ICS in “any unstimulated of three”: CD8 0.064+ + (0.045–0.096),T cells was Kruskal-Wallis lower than thep frequency= 0.092). of Thecytokine frequency production of AIM in unstimulated expression CD8 in unstimulated+ T cells in the ICS CD8 + T cellsassay was lower (Figure than 6B, theOX40 frequency+CD25+: 0.021 of cytokine (0.010–0.033), production CD25+CD107a in unstimulated+: 0.020 (0.012–0.036), CD8 T cellsICS “any in the of ICS assay (Figurethree”: 60.033B, OX40 (0.020–0.048),+CD25+ :ICS 0.021 “any (0.010–0.033), of four”: 0.067 CD25 (0.053–0.150),+CD107a Kruskal-Wallis+: 0.020 (0.012–0.036), p = 0.0001). ICS “any of three”: 0.033The (0.020–0.048), sensitivity of ICSthe AIM “any assay of four”: for detecting 0.067 (0.053–0.150), GP-specific T Kruskal-Wallis cells was comparedp = 0.0001). with the ICS Theassay sensitivity (Figure 6C,D). of the Within AIM assayCD4+ T for cells, detecting this was GP-specific defined as th Te cells frequency was comparedproducing withany of the the ICS assay (Figurethree cytokines6C,D). Withintested: IFN CD4γ, IL2,+ T or cells, TNF thisα, “any was of defined three”. Within as the CD8 frequency+ T cells, producing this was defined anyof as the three cytokineseither “any tested: of three” IFN orγ as, IL2, the frequency or TNFα ,producin “any ofg three”. any of the Within three CD8cytokines+ T cells, or expressing this was CD107a, defined as “any of four”. For each of these assays, GP-specific T cell frequency was calculated by subtracting the either “any of three” or as the frequency producing any of the three cytokines or expressing CD107a, frequency in the unstimulated condition from that in the GP-stimulated condition. Within the CD4+ “any ofsubset, four”. the For LLOD each for of thesethe AIM assays, assay GP-specificwas lower (0.003%) T cell frequency than for the was ICS calculated assay (0.005%) by subtracting and the the frequencyfrequency in of the GP-specific unstimulated CD4+ conditionT cells detected from thatwas inhigher the GP-stimulated (OX40+CD25+: 0.753% condition. (0.445–0.924), Within the + CD4 subset,OX40+PDL1 the+ LLOD: 0.700% for (0.259–0.961)) the AIM assay than that was detected lower (0.003%) by ICS “any than of for three” the (0.265% ICS assay (0.069–0.527)). (0.005%) and the frequencyAdditionally of GP-specific there were three CD4+ individualsT cells detected whose wasresponses higher were (OX40 below+CD25 the LLOD+: 0.753% in the (0.445–0.924), ICS assay, OX40+whilstPDL1 +there: 0.700% was only (0.259–0.961)) one individual than whose that detectedresponse was by ICS below “any the ofLLOD three” in the (0.265% AIM assay. (0.069–0.527)). In the AdditionallyCD8+ subset, there the were LLOD three was individuals the same for whose both assays responses (0.005%). were Again, below the frequency the LLOD of in GP-specific the ICS assay, T + + whilstcells there detected was only by the one AIM individual assay was whose higher response than by the was ICS below assay (OX40 the LLODCD25 in: 0.601% the AIM (0.304–0.826), assay. In the CD25+CD107a+: 0.861% (0.359–1.219), ICS “any of three”: 0.298% (0.210–0.456), ICS “any of four”: CD8+ subset, the LLOD was the same for both assays (0.005%). Again, the frequency of GP-specific T cells detected by the AIM assay was higher than by the ICS assay (OX40+CD25+: 0.601% (0.304–0.826), CD25+CD107a+: 0.861% (0.359–1.219), ICS “any of three”: 0.298% (0.210–0.456), ICS “any of four”: 0.492% (0.341–0.676)). The AIM assay using CD25 and CD107a markers detected a significantly higher VaccinesVaccines2018, 6 2018, 50 , 6, x FOR PEER REVIEW 11 of 1119 of 19

0.492% (0.341–0.676)). The AIM assay using CD25 and CD107a markers detected a significantly frequencyhigher of frequency GP-specific of GP-specific CD8+ T cells CD8 than+ T cells the ICSthan “any the ICS ofthree”, “any of but three”, there but were there no were other no significant other differencessignificant across differences the four across measures the four (Kruskal-Wallis measures (Kruskal-Wallisp = 0.027). p = 0.027).

AB

*** 10 10 ***

1 1 T cells T T cells T 0.1 0.1 + +

+ CD8 CD4 0.01 ICS LLOD 0.01 CD8 AIM and ICS LLOD CD4+ AIM LLOD Frequency in unstimulated in Frequency Frequency in unstimulated in Frequency 0.001 0.001 + + ) + + ) ) 3 8 8 3 4 D4 D4 f D D f f + C + C o + C + C o o y y y 1 5 a n n L n 2 7 + (a 0 + (a + (a D25 D D 1 + C + P + C D 8 8 D4 0 + D D 40 40 + C 4 C + C + C 5 X e X 2 e e O OX in O D in in k C k k to to yto y y C C C CD * 10 10

1 1

0.1 0.1 (GP-unstim) (GP-unstim) 0.01 0.01 CD8+ AIM LLOD ICS LLOD and ICS LLOD CD4+ AIM LLOD FrequencyT cell of subset Frequency of T cell subset Frequency of 0.001 0.001 + + + + ) 4 4 3) 3 4) D D f D8 D8 + + C + C o C + C of of a 7 25 0 D D + (any D25 1 + (any + (any + C + P 4 + C D 8 8 D 40 + C 40 40 + + CD + CD X X C 5 O O e OX D2 e in ine k C kin to to tok y y y C C C

Figure 6. Specificity and sensitivity of the AIM assay compared with trials ICS protocol: (A) Frequency of AIM+ cells or cells producing at least one of the three cytokines measured (IL2, TNFα, or IFNγ) Figure 6. Specificity and sensitivity of the AIM assay compared with trials ICS protocol: (A) Frequency + + by ICSof inAIM unstimulated+ cells or cells producing CD4 T cells. at least Kruskal-Wallis one of the three pcytokines=0.092; measured (B) frequency (IL2, TNF ofα AIM, or IFNcells,γ) by cells producingICS in at unstimulated least one of CD4 the+three T cells. cytokines Kruskal-Wallis measured p =0.092; (IL2, (B TNF) frequencyα, or IFN of AIMγ), or+ cells, producing/expressing cells producing + any ofat the least four one (IL2, of the TNF threeα ,cytokines IFNγ or measured CD107a) (IL2, in unstimulated TNFα, or IFN CD8γ), or producing/expressingT cells. Kruskal-Wallis anyp of= the 0.0001; (C) frequencyfour (IL2, ofTNF GP-specificα, IFNγ or AIMCD107a)+ cells in inunstimulated CD4+ T cell CD8 subset+ T cells. producing Kruskal-Wallis at least p one= 0.0001; of the (C three) cytokinesfrequency measured of GP (IL2,-specific TNF AIMα or+ cells IFN inγ ).CD4 Kruskal-Wallis+ T cell subset producingp = 0.058; andat least (D )one frequency of the three of AIMcytokines+ cells in CD8+measuredT cell subset, (IL2, TNF producingα or IFN atγ). least Kruskal-Wallis one of the p three = 0.058; cytokines and (D) measuredfrequency of (IL2, AIM TNF+ cellsα ,in or CD8 IFN+ Tγ ), or producing/expressingcell subset, producing any ofat theleast four one (IL2, of TNFthe threeα, IFN cytokinesγ, or CD107a). measured Kruskal-Wallis (IL2, TNFα, por= 0.027.IFNγ), Dashedor lines showproducing/expressing LLOD for ICS assaysany of the (0.005 four for(IL2, both TNF CD4α, IFN+ andγ, or CD8CD107a).+ Tcells), Kruskal-Wallis dotted lines p = 0.027. show Dashed LLOD for lines show LLOD for ICS assays (0.005 for both CD4+ and CD8+ T cells), dotted lines show LLOD for AIM assays (0.003 for CD4+ and 0.005 for CD8+). * p < 0.05, *** p < 0.001. AIM assays (0.003 for CD4+ and 0.005 for CD8+). * p < 0.05, *** p < 0.001.

AIM+AIMpopulations+ populations within within CD4 CD4++ and CD8 CD8+ T+ cellsT cells were were combined combined (frequency (frequency of AIM+ of CD4 AIM+ + AIM+ CD4+ + + AIM+CD8CD8+ +inin CD3 CD3+) to+) compare to compare the thespecificity specificity and sensitivity and sensitivity of AIM of and AIM ELISpot and ELISpot assays for assays detecting for detecting the the frequencyfrequency of of antigen-specific antigen-specific totaltotal T cells (Figure 77).). The frequency frequency of of AIM AIM+ cells+ cells within within CD3 CD3+ cells+ cells was calculatedwas calculated using using each each of theof the combinations combinations of of CD4CD4 and CD8 CD8 AIM AIM markers markers and and an anaverage average taken taken across the combinations of markers. The frequency of AIM+CD3+ within PBMC was also calculated across the combinations of markers. The frequency of AIM+CD3+ within PBMC was also calculated for for a more direct comparison with the ELISpot assay, for which the input is PBMC and therefore the a more direct comparison with the ELISpot assay, for which the input is PBMC and therefore the result is result is the frequency of IFNγ+ PBMC. The LLOD for AIM+ total T cells was 0.001%. There were no γ+ + the frequencysignificant of differences IFN PBMC. in the The level LLOD of forbackground AIM total AIM T cells+ T cells was 0.001%.between Therethe different were no marker significant + differences in the level of background AIM T cells between the different marker combinations (Figure7A, OX40 +CD25+CD4+ and OX40+CD25+CD8+: 0.073 (0.047–0.116), OX40+CD25+CD4+ and CD25+CD107a+CD8+: 0.072 (0.046–0.122), OX40+PDL1+CD4+ and OX40+CD25+CD8+: 0.061 (0.030–0.091), OX40+PDL1+CD4+ and CD25+CD107a+CD8+: 0.063 (0.034–0.095), Kruskal-Wallis Vaccines 2018, 6, x FOR PEER REVIEW 12 of 19 combinations (Figure 7A, OX40+CD25+CD4+ and OX40+CD25+CD8+: 0.073 (0.047–0.116), VaccinesOX40+2018CD25, 6,+ 50CD4+ and CD25+CD107a+CD8+: 0.072 (0.046–0.122), OX40+PDL1+CD4+ 12 ofand 19 OX40+CD25+CD8+: 0.061 (0.030–0.091), OX40+PDL1+CD4+ and CD25+CD107a+CD8+: 0.063 (0.034– + p0.095),= 0.677 Kruskal-Wallis). The average p AIM = 0.677).+ frequency The average from theseAIM populationsfrequency from was these compared populations with the was background compared 6 inwith the the ELISpot background (Figure7 inB). the The ELISpot ELISpot (Figure results were7B). The converted ELISpot from results SFC/10 were6 PBMCconverted to percentages from SFC/10 for + thesePBMC analyses. to percentages The background for these level analyses. of AIM The+ T cellsbackground was significantly level of AIM higher Tthan cells the was background significantly in thehigher ELISpot than the assay, background in which noin the individuals ELISpot assay, had responses in which aboveno individuals the LLOD had of responses 50 SFC/10 above6 PBMC the 6 + + orLLOD 0.005% of 50 (%AIM SFC/10+ inPBMC T cells: or 0.005% 0.067 (%AIM (0.040–0.104), in T cells: %AIM 0.067+ T(0.040–0.104), cells in PBMC: %AIM 0.026 T cells (0.016–0.039) in PBMC: ELISpot:0.026 (0.016–0.039) 0.005, Kruskal-Wallis ELISpot: 0.005,p < 0.0001). Kruskal-Wallis The frequency p < 0.0001). of antigen-specific The frequency total of antigen-specific T cells (frequency total in GP-stimulatedT cells (frequency after in backgroundGP-stimulated subtraction) after background was compared subtraction) for each was thecompared marker for combinations each the marker and + + + therecombinations were no significantand there differences were no (Figuresignificant7C OX40 differences+CD25+ CD4(Figure+ and 7C OX40 OX40+CD25CD25+CD8CD4+: 0.593 and + + + + + + + + + (0.399–0.804),OX40 CD25 CD8 OX40: +0.593CD25 (0.399–0.804),+CD4+ and CD25 OX40+CD107aCD25+CD4CD8+ :and 0.641 CD25 (0.477–0.866),CD107a CD8 OX40: +0.641PDL1 +(0.477–CD4+ + + + + + + + + + and0.866), OX40 OX40+CD25PDL1+CD8CD4+: 0.590 and (0.343–0.803), OX40 CD25 OX40CD8 :+ PDL10.590+ CD4(0.343–0.803),+ and CD25 OX40+CD107aPDL1+CD8CD4+: 0.661and + + + (0.434–0.850),CD25 CD107a Kruskal-WallisCD8 : 0.661 (0.434–0.850),p = 0.902). TheKruskal-Wallis frequency ofp = antigen-specific 0.902). The frequency cells within of antigen-specific total T cells detectedcells within by thetotal AIM T cells assay detected was significantly by the AIM greater assay was than significantly the antigen-specific greater responsethan the antigen-specific detected by the ELISpot.response However detected theby the frequency ELISpot. of However antigen-specific the freq PBMCuency detected of antigen-specific by each assay PBMC was detected not significantly by each + + differentassay was (Figure not significantly7D, %AIM + differentin T cells: (Figure 0.633 (0.443–0.820), 7D, %AIM in %AIM T cells:+ T 0.633 cells in(0.443–0.820), PBMC: 0.227 %AIM (0.111–0.364), T cells + %IFNin PBMC:γ+ PBMC 0.227 measured(0.111–0.364), by ELISpot: %IFNγ 0.159PBMC (0.120–0.275), measured byp ELISpot:= 0.0009). 0.159 Comparisons (0.120–0.275), of the p AIM= 0.0009). and cytokine-basedComparisons of assays the AIM are and summarized cytokine-based in Table assays1. are summarized in Table 1.

AB ns **** 10 10 * ***

1 1 (unstim)

+ 0.1 0.1 or PBMC (unstim) + 0.01 0.01 ELISpot LLOD % of % of CD3

0.001 0.001 AIM CD3+ LLOD % of CD3 % of t 1 2 3 4 C o IM IM IM IM M A A A A B in CD3 P LISp + E in IM 3 A + CD

AIM

CD** ns 10 10 * ns

1 1

(GP-unstim) 0.1 0.1 + or PBMC (GP-unstim)

0.01 + 0.01 ELISpot LLOD % of CD3 0.001 0.001 AIM CD3+ LLOD 1 2 3 4 3 t

% of % of CD3 C IM IM IM IM D M po A A A A B in C P LIS + E IM 3 in A D

+ C IM A AIM1 = OX40+CD25+CD4+ + OX40+CD25+CD8 + AIM2 = OX40+CD25+CD4+ + CD25+CD107a+CD8+ AIM3 = OX40+PDL1+CD4+ + OX40+CD25+CD8 + AIM4 = OX40+PDL1+CD4+ + CD25+CD107a+CD8+

FigureFigure 7.7. SpecificitySpecificity and and sensitivity sensitivity of of AIM AIM assay assay compared compared with with ELISpot ELISpot using using cells cells at M+7: at M+7: (A) + + (FrequencyA) Frequency of AIM of AIM+ cellscells within within total total T cells T cells using using each each combination combination of AIM of AIM markers markers in CD4 in+ CD4 and CD8and+ + CD8T cellsT (Kruskal-Wallis cells (Kruskal-Wallis p = 0.677).p = 0.677).(B) Average (B) Average of the four of the AIM four combinations AIM combinations as a frequency as a frequency of CD3+ + ofT cells CD3 andT cells as a andfrequency as a frequency of PBMC of compared PBMC compared with the withbackground the background in the ELISpot in the assay ELISpot (% assayIFNγ+ + + (%PBMC) IFNγ (Mann-WhitneyPBMC) (Mann-Whitney p < 0.0001).p (C<) 0.0001).Frequency (C )of Frequency antigen-specific of antigen-specific T cells (AIM+ Tin cellsGP-stimulated (AIM in GP-stimulatedcells after background cells after subtraction) background detected subtraction) using detectedeach of the using marker each combinations of the marker (Kruskal-Wallis combinations + (Kruskal-Wallisp = 0.902). (D) Averagep = 0.902). of (Dthese) Average combinations of these as combinations a frequency as of a CD3 frequency+ T cells of and CD3 asT a cellsfrequency and as of a + frequencyPBMC compared of PBMC with compared the ELISpot with (% the IFN ELISpotγ+ PBMC) (% IFN resultsγ PBMC) (Kruskal-Wallis results (Kruskal-Wallis p = 0.0018). Dashedp = 0.0018). lines Dashedshow LLOD lines showfor ELISpot LLOD for(0.005), ELISpot dotted (0.005), lines dotted showlines LLOD show for LLODAIM in for total AIM T in cells total (0.001). T cells (0.001).ns: No ns:significance, No significance, * p <0.05, * p **<0.05, p <0.01, ** p ***<0.01, p <0.001, *** p <0.001,**** p <0.0001. **** p <0.0001.

Vaccines 2018, 6, 50 13 of 19

Table 1. Comparison of sensitivity and specificity of activation-induced markers (AIM) and cytokine-based assays.

Antigen-Specific Antigen-Specific Lower Limit Frequency in Signal at M+7 Signal at M+84 Assay/Markers of Detection Unstimulated (% (GP-Unstim, % of (GP-Unstim, % of (% of Subset) of Subset) Subset) Subset) ELISpot 0.005 0.005 0.159 (0.120–0.275) 0.015 (0.010–0.032) ICS: CD4+ “any of three” 0.005 0.064 (0.045–0.096) 0.265 (0.069–0.527) NA ICS: CD8+ “any of three” 0.005 0.033 (0.020–0.048) 0.298 (0.210–0.456) NA ICS: CD8+ “any of four” 0.005 0.067 (0.053–0.150) 0.492 (0.341–0.676) NA AIM: CD4+ OX40+CD25+ 0.003 0.110 (0.069–0.172) 0.753 (0.445–0.924) 0.153 (0.064–0.231) AIM: CD4+ OX40+PDL1+ 0.003 0.102 (0.044–0.131) 0.700 (0.259–0.961) 0.230 (0.020–0.443) AIM: CD8+ OX40+CD25+ 0.005 0.021 (0.010–0.033) 0.601 (0.304–0.826) 0.056 (0.026–0.101) AIM: CD8+ CD25+CD107a+ 0.005 0.020 (0.012–0.036) 0.861 (0.359–1.219) 0.048 (0.024–0.112) AIM: CD3+ (average of marker 0.001 0.067 (0.040–0.104) 0.633 (0.443–0.820) 0.150 (0.039–0.236) combinations) AIM+CD3+ (average of marker 0.001 0.026 (0.016–0.039) 0.227 (0.111–0.364) 0.058 (0.012–0.095) combinations) in PBMC Medians (inter-quartile ranges), NA—not applicable—assay not conducted at this time point.

3.4. Activation-Induced Markers as a Measure of Durable Vaccine Responses The durability of vaccine responses in this clinical trial were measured by IFNγ ELISpot three months after MVA-EBO-Z vaccination (M+84). The frequency of the GP-specific T cell response detected at this time point by the ELISpot and AIM assays was compared. GP-specific T cell responses measured by all methods were significantly reduced from peak to three months post-boost (Figure8A,B). However, almost all individuals still had detectable frequencies of AIM + cells within the CD4 and CD8 subsets and total T cells. GP-specific CD8+ responses were detectable for 14/16 individuals, whilst GP-specific CD4+ responses were detectable for 13/16 individuals by OX40 and PDL1 markers or for 14/16 individuals by OX40 and CD25 markers. At M+84, there was a higher frequency of GP-specific CD4+ T cells than CD8+ T cells measured by the AIM assay (Figure8A, OX40 +CD25+CD4+: 0.153% (0.064–0.231), OX40+PDL1+CD4+: 0.230% (0.020–0.443), OX40+CD25+CD8+: 0.056% (0.026–0.101), CD25+CD107a+CD8+: 0.048% (0.024–0.112)). IFNγ ELISpot results at this time point, which include both CD4+ and CD8+ responses, were ten-fold lower than at the peak (Figure8B, 0.0155% (0.010–0.032) compared with peak responses of 0.159% (0.120–0.275)). Additionally, 2/16 volunteers did not have detectable ELISpot responses at this time point. In comparison, AIM+ cells as a frequency of the total T cells were only four-fold lower than at the peak (M+84: 0.150% (0.039–0.236), M+7: 0.633% (0.443–0.820)) and AIM+ T cells as a frequency of PBMC were also four-fold lower than at the peak (M+84: 0.058% (0.012–0.095), M+7: 0.227% (0.111–0.364)). There was no correlation between the AIM assay and ELISpot at this late time point (Figure8C–E, frequency of AIM + cells in CD4+, CD8+ or CD3+, or frequency of AIM+CD3+ cells in PBMC). VaccinesVaccines2018 2018, 6,, 50 6, x FOR PEER REVIEW 14 14of of19 19

ABAIM+ %IFNγ+ AIM+ T cells in PBMC in T cells PBMC (ELISpot) *** ** **** **** *** ** **** 10 10

1

or PBMC 1 + 0.1 0.1

(GP-unstim) 0.01 CD8+ LLOD + 0.01

frequency T cellof subset CD4 LLOD ELISpot LLOD + 0.001 +

AIM 4 4 0.001 AIM CD3 LLOD +7 84 +7 8 +7 8 +7 Frequency ofCD3 + + + +84 M M M M 7 4 7 4 7 4 M M M M + 8 + 8 + 8 M + M + M + M M M OX40+CD25+ OX40+PDL1+ OX40+CD25+ CD25+CD107a+

CD4+ T cells CD8+ T cells

CDE

10 1 1

1 0.1 0.1 (GP-unstim) (GP-unstim) + 0.1 + at M+84 at at M+84 at 0.01 0.01 of CD4 of (GP-unstim) at M+84 at (GP-unstim) 0.01 CD8 of + + + %AIM

%AIM 0.001

0.001 %AIM 0.001 0.001 0.01 0.1 1 0.001 0.01 0.1 1 0.001 0.01 0.1 1 % IFNγ+ PBMC at M+84 (ELISpot) % IFNγ+ PBMC at M+84 (ELISpot) % IFNγ+ PBMC at M+84 (ELISpot)

OX40+PDL1+ CD4+ r: 0.25 P=0.349 CD25+CD107a+ CD8+ r: 0.46 P=0.071 M+84 AIM+ in CD3+ r:0.26 P=0.330 + + + OX40 CD25 CD4 r: 0.16 P=0.556 OX40+CD25+ CD8+ r: 0.36 P=0.168 M+84 AIM+ CD3+ in PBMC r:0.36 P=0.175

Figure 8. Detection of AIM+ T cells at late time points: (A) Frequency of AIM+ cells within the CD4+ Figure 8. Detection of AIM+ T cells at late time points: (A) Frequency of AIM+ cells within the CD4+ and CD8+ T cell subsets three months after MVA-EBO-Z vaccination (M+84) compared with peak and CD8+ T cell subsets three months after MVA-EBO-Z vaccination (M+84) compared with peak responses seven days after vaccination (M+7); (B) GP-specific T cells measured by ELISpot (%IFNγ+ responses seven days after vaccination (M+7); (B) GP-specific T cells measured by ELISpot (%IFNγ+ PBMC) or AIM+ cells in the total T cells or PBMC at M+7 and M+84. Wilcoxon matched-pairs analyses PBMC) or AIM+ cells in the total T cells or PBMC at M+7 and M+84. Wilcoxon matched-pairs analyses for comparisons across time points. Relationship between the proportion of GP-specific T cells at M+84 for comparisons across time points. Relationship between the proportion of GP-specific T cells at γ C + + D + measuredM+84 measured by IFN byELISpot IFNγ ELISpot and; ( )and; frequency (C) frequency of AIM ofCD4 AIM+ TCD4 cells+ T atcells M+84; at M+84; ( ) frequency (D) frequency of AIM of + + + CD8AIMT+ cellsCD8+ at T M+84;cells at andM+84; (E and) frequency (E) frequency of AIM of AIMcells+ cells within within T cells T cells or AIMor AIMT+ T cells cells within within PBMC PBMC at M+84.at M+84. ** p < ** 0.01, p < 0.01, *** p ***< 0.001,p < 0.001, **** ****p < p 0.0001. < 0.0001.

4. Discussion4. Discussion Antigen-specificAntigen-specific T T cell cell responses responses inin clinicalclinical trialstrials have classically been been determined determined using using cytokine-basedcytokine-based approaches, approaches, such such as as ELISpot ELISpot and and ICSICS assaysassays [[12–14].12–14]. These These assays assays allow allow for for sensitive sensitive andand specific specific detection detection of of antigen-responsive antigen-responsive TT cellscells withoutwithout the need need for for labor-intensive labor-intensive HLA HLA typing. typing. However,However, these these methods methods require require pre-determination pre-determination of of thethe cytokinescytokines to be be analyzed, analyzed, which which may may cause cause a biasa bias in thein the type type of of T cellsT cells detected, detected, and and likely likely underestimateunderestimate the frequency of of the the antigen-specific antigen-specific response.response. Memory Memory cells cells that that are are produced produced inin responseresponse to both infection and and vaccination vaccination consist consist of of multiplemultiple populations populations with with different different gene expression profiles profiles and functional and functi capacityonal capacity [41,42]. Crucially,[41,42]. theseCrucially, cells differ these in cells their differ ability in their to secrete ability cytokines to secrete [cytokines43]. In particular, [43]. In particular, in several in studies,several studies, it has been it has been observed that the majority of antigen-specific central memory CD4+ T cells were not observed that the majority of antigen-specific central memory CD4+ T cells were not secreting TNFα secreting TNFα or IFNγ and, therefore, were not detected by assays based on cytokine secretion or IFNγ and, therefore, were not detected by assays based on cytokine secretion [44,45]. Especially at [44,45]. Especially at late time points after infection and vaccination, when the ratio of central to late time points after infection and vaccination, when the ratio of central to effector memory T cells is effector memory T cells is likely to be higher, the majority of the antigen-specific T cells may be likely to be higher, the majority of the antigen-specific T cells may be undetectable by cytokine-based undetectable by cytokine-based assays [46]. Therefore, we investigated the use of activation-induced assaysmarkers [46]. for Therefore, the analysis we of investigated antigen-specific the useT cell of activation-inducedresponses to vaccination markers in a forclinical the analysistrial and of antigen-specificcompared the Tsensitivity cell responses and specificity to vaccination of this method in a clinical with IFN trialγ andELISpot compared and ICS the assays. sensitivity and specificityOne of possible this method issue with IFNthe AIMγ ELISpot assay andis the ICS background assays. caused by bystander activation. This hasOne been possible investigated issue within a study the AIM that assay found is the the level background of bystander caused activation by bystander to be relatively activation. low This [39]. has been investigated in a study that found the level of bystander activation to be relatively low [39]. We found that the AIM assay provided a high level of specificity, with background levels comparable or Vaccines 2018, 6, 50 15 of 19 lower than those in the ICS assay, and typically with frequencies lower than 0.15% of the CD4+ or CD8+ T cell subset. This is lower than, or comparable to, the level of background observed for these and other combinations of AIMs previously investigated [25,30,39]. An issue with the use of OX40 and CD25 markers is the inclusion of antigen-responsive Tregs. This was addressed in a previous study by the addition of PDL1 to discriminate between antigen-responsive Tregs and non-Tregs [39]. In this study by Reiss and colleagues, several marker combinations were directly compared in T cells stimulated with tetanus peptides. In each of these populations, only a minority of the cells were Tregs (based on FOXP3 expression): 1.6% of OX40+PDL1+ CD4+ T cells, 6.8% of CD69+CD40L+ CD4+ T cells, and 16% of the OX40+CD25+ CD4+ T cells. These data suggest that OX40/CD25 are useful markers for identifying the maximal antigen-specific population, whilst OX40/PDL1 or CD69/CD40L can be used to discriminate antigen-specific non-Tregs. In our study, AIM frequencies in GP-stimulated CD4+ T cells detected using either of the marker combinations tested were comparable and the two populations were highly correlated. However, this may not be the case for all vaccine antigens at all time points or in all populations. Therefore, an assay including OX40, CD25, and PDL1 could be of use in clinical trials to measure both the maximal antigen-specific response and to discriminate non-Tregs. This may be particularly useful in some circumstances where antigen-specific Tregs are thought to play an important role, such as in malaria-exposed populations [47–49]. All of the AIM marker combinations used in our study sensitively detected antigen-specific T cells. The lower limit of detection for the AIM assay in this study was either comparable to (CD8+ AIM 0.005%) or lower than (CD4+ AIM 0.003%) the ICS assay (0.005% for CD4+ and CD8+ T cells). Antigen-specific T cells detected using the AIM assay were 2.6–2.8-fold higher than ICS for CD4+ T cells and 1.2–1.8-fold higher than ICS for CD8+ T cells at the peak time point. Although ELISpot assays are highly specific, the AIM assay tested here was much more sensitive and had a higher signal-to-noise ratio. However it should be noted that the LLOD for the AIM and ICS assays are calculated by the reciprocal of the lowest number of events collected in the appropriate gate (CD4+, CD8+ or CD3+) and will therefore vary depending on the number of cells used in each assay and their viability. In this study 2 × 106 PBMC were used for the ICS, which was conducted as a primary immunological assay in the clinical trial according to an established standard operating procedure. For the AIM assay 1–2 × 106 PBMC were used depending on the number of cryopreserved cells remaining. These assays capture a comparable level of vaccine-specific T cells in the CD8+ subset at the peak time point. However, there was far less agreement between the assays within the CD4+ subset, suggesting that we may be missing a substantial portion of the CD4+ T cell response to vaccination. This was particularly apparent at late time points. Detection of CD4+ T cells and central memory cells not producing cytokine may be an important part of the durability of vaccine responses that may be underestimated. The ELISpot assay is sensitive enough to detect antigen-specific IFNγ-secreting cells in almost all volunteers at least three months after the peak response. ICS is often not performed at these time points in clinical trials as the ability to detect IFNγ-secreting cells by ICS is lower and very few responses are detected [22]. However, the AIM assay results at this late time point indicate that antigen-specific CD4+ T cells may be particularly well maintained, an observation that could not be made from the ELISpot assay. 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. Equally, conducting assays using cryopreserved cells allows samples from multiple time points to be batched, increasing efficiency and reducing experimental variation. Although we have not directly assessed AIM expression on fresh and frozen PBMC, we show here that AIM assays conducted using cryopreserved cells give comparable or higher antigen-specific signals than ICS and ELISpot using PBMC ex vivo. Vaccines 2018, 6, 50 16 of 19

This study is the first in-depth assessment of AIM assays in CD4+ and CD8+ T cells in a clinical trial setting. The ICS and ELISpot assays routinely used in clinical trials are highly effective at identifying antigen-specific effector cells at the peak time point. Here we have directly compared the use of AIM and cytokine-based assays for the identification of vaccine-specific T cell responses in clinical trials. There has been substantial work towards standardizing T cell assays for use in human trials and further studies are required to develop and validate AIM assays to a similar level [51–53]. These would include studies to optimize the concentration of stimulating antigen, choose which markers to use, and compare responses ex vivo with those from thawed PBMC. Studies are also needed to asses assay variability, particularly between different operators. Additionally it will be important to characterise the functionality of these cells and determine their clinical relevance. For many of the vaccines currently in development, there are no clear immunological correlates of protection in humans. Therefore, determination of protective correlates is an important part of the vaccine development process [2,54–56]. The AIM assay is a tool, which in combination with functional measures, could aid the search for immunological correlates of protection. In fact, antigen-specific T cells defined by CD40L expression that were also producing TNFα or IL2 induced by RTS,S/AS vaccination were higher in individuals protected from malaria [57]. However, the markers used in our study have not yet been used to detect antigen-specific T cells in a Phase II vaccine study. It would be useful to include this assay in Phase II clinical studies to determine the clinical relevance of these subsets. We show that AIM assays can be used to detect antigen-specific T cells with a sensitivity and specificity comparable to or higher than the cytokine-based assays conventionally used in clinical vaccine trials. Additionally, AIM assays may provide novel information about non-cytokine-secreting cells and the durability of the response, particularly in the CD4+ subset. AIM assays may provide a more comprehensive account of the total antigen-specific response and, in combination with additional phenotypic and functional markers, could be a valuable tool for more detailed assessments of vaccine-specific responses.

5. Conclusions A combination of AIM assays with traditional cytokine analysis could provide a more complete assessment of the antigen-specific response to vaccination, particularly within CD4+ T cells and when assessing durability. Further validation of these assays would be required for use as a primary immunogenicity measure within clinical trials. However, this could significantly enhance knowledge about vaccine-specific T cell responses and aid vaccine development.

Supplementary Materials: The following are available online at http://www.mdpi.com/2076-393X/6/3/50/s1, Figure S1: Intracellular cytokine staining gating strategy. Cells were gated on single lymphocytes based on size. Dead cells, CD14+ and CD19+ cells were excluded and T cells were identified by CD3 expression. T cell subsets were gated as CD4+ and CD8+ populations. Cytokine expression was quantified by plotting pairs of cytokines against each other and gating positive populations. Author Contributions: A.V.S.H., T.R. and K.E. conceived and designed the clinical trial; T.R. provided clinical support for the trial; G.B. conceived and designed the experiments; G.B., J.P., R.M., and D.W. performed the experiments; G.B., J.P., R.M. and D.W. analyzed the data; A.V.S.H. and K.E. provided supervision of the project; G.B. wrote the paper. All authors reviewed a final version of the manuscript. Funding: The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results. Acknowledgments: The clinical trial was supported by funding from an Enhancement Award to a Wellcome Trust Strategic Award (to AVSH as PI) co-funded by the UK Medical Research Council, the UK Department for International Development and the European and Developing Countries Clinical Trials Partnership, with additional funding from the NIHR Oxford Biomedical Research Centre. The MVA-EBO-Z vaccine was biomanufactured for these trials by Emergent Biosolutions under a contract from Oxford University with funding from the same Enhancement Award. We would like to acknowledge Navin Venkatraman’s contributions towards this manuscript. He helped to design the clinical trial and provided clinical support for the trial but sadly passed away before this manuscript was prepared and was therefore unable to review the final manuscript and fulfil the ICMJE recommendations and COPE standards for authorship. Vaccines 2018, 6, 50 17 of 19

Conflicts of Interest: A.V.S.H. is a named inventor on patents relating to viral vectored vaccines. All other authors declare no conflicts of interest.

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