Characterisation of the T-cell response to Ebola virus glycoprotein amongst survivors of the 2013-16 West Africa epidemic

Abstract

Zaire ebolavirus (EBOV) is a highly pathogenic filovirus which can result in Ebola virus disease (EVD); a serious medical condition that presents as flu like symptoms but then often leads to more serious or fatal outcomes. The 2013-16 West Africa epidemic saw an unparalleled number of cases. Here we show characterisation and identification of T cell epitopes in surviving patients from Guinea to the EBOV glycoprotein. We perform interferon gamma (IFNγ) ELISpot using a glycoprotein peptide library to identify T cell epitopes and determine the CD4⁺ or CD8⁺ T cell component response. Additionally, we generate data on the T cell phenotype and measure polyfunctional cytokine secretion by these antigen specific cells. We show candidate peptides able to elicit a T cell response in EBOV survivors and provide inferred human leukocyte antigen (HLA) allele restriction. This data informs on the long-term T cell response to Ebola virus disease and highlights potentially important immunodominant peptides.

Fig. 1. Cellular immune response, using fresh PBMC, to EBOV GP (Mayinga) peptide library as measured by IFNγ ELISpot.

a Schematic representation of the EBOV glycoprotein highlighting notable regions and peptide pools used in ELISpot analysis. SP = Signal peptide. b The summed ELISpot response to all GP peptides in the library amongst 57 EVD survivor and 18 non-exposed, negative, PBMC samples. c ELISpot response amongst 57 EVD survivors to either sGP or exclusive GP portions of the glycoprotein. d The ELISpot response amongst 57 EVD survivors to each peptide pool. For graphs b–d bars represent the median values with the upper 95% confidence interval. Two-tailed Mann–Whitney U test used to look for significance in (b) (p = <0.0001) and two-tailed Wilcoxon test in (c) (p = <0.0001). Dashed black line is the lower limit of detection (LLD) represents the in house cut off value (23 SFU), this is the mean of all negative results in (b) plus 3 standard deviations (SD) and discriminates between a positive and negative responder.

Fig. 2. Cellular immune response to EBOV GP peptides as measured by IFNγ ELISpot.

a Response to 187 individual glycoprotein peptides amongst 15 fresh PBMC samples. Bars are stacked and indicate the sum of all results to each peptide amongst 15 EVD survivors. b Response amongst 6 EVD survivors to individual 15 mer peptides which make up the SP peptide pool. c The response amongst 12 EVD survivors to individual 15 mer peptides that make up the GP1-1 peptide pool. d Response amongst 18 EVD survivors to individual 15 mer peptides which make up the GP1-2 peptide pool. e The response amongst 14 EVD survivors to individual 15 mer peptides that make up the GP1-4 peptide pool. For graphs (b–e) red dots indicate individual data points and bars with error represent the median with the upper 95% confidence interval. Black dashed line represents the lower limit of detection (LLD) for the GP1-2 (19 SFU) or GP1-4 (16 SFU) peptide pool respectively, this was calculated using the mean of the negative samples plus three SD.

Fig. 3. EVD survivor T cell memory response to EBOV glycoprotein GP1-4.

IFNγ ELISpot using EVD survivor PBMC that were depleted of either CD4⁺ (−CD4) or CD8⁺ (−CD8) T cells. a Representative flow cytometry and ELISpot images for survivor C052 showing that PBMC were successfully depleted for the desired T cell population and the corresponding ELISpot response to the various stimulation conditions. b–d Response to GP1-4 peptide sub-pools. (b) shows the response to sub-pool 1 which consists of peptides 69–73, (c) shows the response to sub-pool 2 which contains peptides 74–80 and (d) shows the response to sub-pool 3 which contains peptides 81–88. Data show the mean +SD of 11 EVD survivor samples. One-way ANOVA with repeated measures used for statistical analysis in (c) p = 0.0264 and in (d) p = 0.0255, n = 11 biological independent samples examined over 2 independent experiments.

Fig. 4. Flow cytometry studies to characterise the EVD survivor T cell response.

21 EVD survivor or 7 unexposed, negative, control PBMC were either left untreated or stimulated with EBOV GP peptides overnight. a Phenotype of Antigen Specific T cells following stimulation with EBOV GP peptide pool (187 peptides), representative data from Survivor C070. Cytokine combinations have been overlaid on top of the T cell phenotype. b The cytokine combinations from 21 EVD survivors, produced by either CD8⁺ or CD4⁺ T cells following EBOV glycoprotein peptide pool stimulation. c 6 EVD survivor and 4 unexposed, negative, control PBMC were either left untreated or stimulated with EBOV GP peptide 79 overnight. d 9 EVD survivor and 6 unexposed, negative, control PBMC were either left untreated or stimulated with EBOV GP peptide 82 overnight. Samples were analysed using FlowJo v10 and GraphPad v8. For graphs (b–d) red or blue dots indicate the individual data points and grey bars represent the median value with the upper 95% CI. LLD is specific to the respective cytokine combination and was determined by taking the mean +3 SD of the negative group.