Human Ebola virus infection results in substantial immune activation

Abstract

Four Ebola patients received care at Emory University Hospital, presenting a unique opportunity to examine the cellular immune responses during acute Ebola virus infection. We found striking activation of both B and T cells in all four patients. Plasmablast frequencies were 10-50% of B cells, compared with less than 1% in healthy individuals. Many of these proliferating plasmablasts were IgG-positive, and this finding coincided with the presence of Ebola virus-specific IgG in the serum. Activated CD4 T cells ranged from 5 to 30%, compared with 1-2% in healthy controls. The most pronounced responses were seen in CD8 T cells, with over 50% of the CD8 T cells expressing markers of activation and proliferation. Taken together, these results suggest that all four patients developed robust immune responses during the acute phase of Ebola virus infection, a finding that would not have been predicted based on our current assumptions about the highly immunosuppressive nature of Ebola virus. Also, quite surprisingly, we found sustained immune activation after the virus was cleared from the plasma, observed most strikingly in the persistence of activated CD8 T cells, even 1 mo after the patients' discharge from the hospital. These results suggest continued antigen stimulation after resolution of the disease. From these convalescent time points, we identified CD4 and CD8 T-cell responses to several Ebola virus proteins, most notably the viral nucleoprotein. Knowledge of the viral proteins targeted by T cells during natural infection should be useful in designing vaccines against Ebola virus.

Keywords: Ebola infection; T cells; human immune response; immune activation; plasmablasts.

Fig. 1.

Representative flow cytometry plots of plasmablasts and activated CD4 and CD8 T-cell responses in Ebola patients. Samples from a healthy donor and from each Ebola virus-infected patient are depicted. EVD patient samples were obtained during the acute phase of disease: samples from EVD2 were obtained 16 d after symptom onset; from EVD5 at day 17; from EVD9 at day 17; and from EVD15 at day 3.

Fig. 2.

Kinetics of plasmablasts, activated CD4 and CD8 T-cell responses, and viral load in EVD patients. Percentages of plasmablasts (CD27⁺CD38⁺CD19⁺), activated CD4 T cells (HLA-DR⁺CD38⁺CD3⁺CD4⁺), activated CD8 T cells (HLA-DR⁺CD38⁺CD3⁺CD8⁺), and viral load are graphed as a function of time. Responses from a healthy control are shown in red. The dotted lines represent the limit of detection for the viral load assay. TCID₅₀, 50% tissue culture infective dose.

Fig. 3.

Phenotyping activated CD8 T cells in EVD patients. The expression of various effector cell markers on CD3⁺CD8⁺ T cells from EVD9 on day 31 after onset of symptoms (A) and in EVD15 3–9 d after onset of symptoms (B). The Ki-67 histogram is derived from the population depicted in red in the first plot.

Fig. 4.

CD4 and CD8 T-cell responses in convalescent EVD patients. Representative flow plots from EVD2 at two time points after discharge (first follow-up was 1 mo post discharge and the second follow-up was 3 mo post discharge) from the hospital demonstrating activated CD4 and CD8 T-cell populations. Days noted in all panels are days post symptom onset.

Fig. 5.

B-cell responses during acute and convalescent EVD. (A) Nucleoprotein-specific antibody titers using ELISA and viral load are graphed as a function of time. Dotted lines represent the limit of detection for each assay. Open boxes represent IgM titers, filled black circles represent IgG titers, and filled red triangles indicate viral load. (B) A representative image of an ELISpot assay to detect Ebola virus (EBOV)-specific plasmablasts from a sample obtained from EVD2 at the first follow-up visit (59 d post onset of symptoms). (C) Graphical representation of antigen-specific ASCs, obtained from EVD2 (at day 59) and EVD5 (at day 60) at their first follow-up visit, as a percentage of total IgG-secreting cells. Whole-cell lysate of Ebola virus-infected Vero-E6 cells was used as antigen.

Fig. 6.

Antigen specificity of CD4 and CD8 T cells in EVD patients. Flow cytometry plots showing IFN-γ and TNF-α expression in response to stimulation with the indicated peptide pools from Ebola viral proteins. Assays were performed on PBMCs obtained from EVD2, EVD5, and EVD9 at 28, 144, and 71 d post onset of symptoms, respectively.