Article Figures & Data
Figures
- FIG 1
CD8+ T cell responses from LF survivors from Nigeria (N) and Sierra Leone (SL). (A) PBMCs were incubated with rscVSVs encoding EGFP (negative control), GP1, GP2, or ∼60-amino acid polypeptides derived from GPC sequences overnight in the presence of brefelin A. Cells were surface stained for CD3e and CD8 before fixation, permeabilization, and staining with antibodies against TNF-α and IFN-γ. Graphs indicate the percentage of CD3+ CD8+ cells positive for both TNF-α and IFN-γ by flow cytometry. Positive gates were set at a mean fluorescence intensity of >1.2 log10 over the median negative control, and dotted horizontal lines indicate levels of highest negative-control sample (no stim or enhanced green fluorescent protein [EGFP]). (B) Same as panel A except PBMCs were incubated with rscVSVs encoding NP and associated polypeptides. CD8+ T cell responses to NP using PBMCs from N-07 and 3568610 were considered negative, and responses to NP fragments were not assessed.
- FIG 2
CD8+ T cell responses to rscVSV encoding EGFP and LASV antigens GP1, GP2, and NP, as well as unstimulated controls, were assessed in two U.S. donors with no history of travel to West Africa and three Sierra Leoneans seronegative for LASV.
- FIG 3
CD8+ T cell responses to NP antigens from a Nigerian (N-13) and Sierra Leonean (2848950) LF survivor. PBMCs from N-13 (A) and 2848950 (B) were incubated with rscVSVs encoding LASV NP and NP fragments (designated by an “f”) and evaluated by intracellular staining of IFN-γ and TNF-α flow cytometry. The ability of T cells to produce IFN-γ and TNF-α after engagement of the TCR was evaluated by incubation with antibodies against CD3 and CD28. Candidate peptide epitopes were identified by in silico MHC-1 prediction algorithms using reactivity to NP fragments and HLAs expressed by each LF survivor. The top predicted peptide epitopes were incubated with PBMCs from N-13 (C) and 2848950 (D) for 5 h in the presence of brefeldin A, and CD3+ CD8+ cells were evaluated by intracellular staining of IFN-γ and TNF-α flow cytometry.
- FIG 4
CD8+ T cell responses to GPC antigens from a Nigerian (N-07) and Sierra Leonean (3568610) LF survivor. PBMCs from N-07 (A) and 3568610 (B) were incubated with rscVSVs encoding LASV GPC and GPC fragments (designated by an “f”) and evaluated by intracellular staining of IFN-γ and TNF-α flow cytometry. Candidate peptide epitopes were identified as explained in Fig. 2. The top predicted peptide epitopes were incubated with PBMCs from N-07 (C) and 3568610 (D) for 5 h in the presence of brefeldin A, and CD3+ CD8+ cells were evaluated by intracellular staining of IFN-γ and TNF-α flow cytometry.
- FIG 5
CD8+ T cell responses to GPC antigens from a Nigerian (N-13) and two Sierra Leonean (2889600, 2848950) LF survivors. PBMCs from N-13 (A), 2889600 (B), and 2848950 (C) were incubated with rscVSVs encoding LASV GPC and GPC fragments (designated by an “f”) and evaluated by intracellular staining of IFN-γ and TNF-α flow cytometry. Candidate peptide epitopes were identified as explained in Fig. 2. The top predicted peptide epitopes were incubated with PBMCs from N-13 (D), 2889600 (E), and 2848950 (F) for 5 h in the presence of brefeldin A, and CD3+ CD8+ cells were evaluated by intracellular staining of IFN-γ and TNF-α flow cytometry.
- FIG 6
An additional seven 10-aa peptide epitopes discovered from LF survivors. (A) Candidate peptide epitopes were identified through in silico prediction of MHC-1 binding using reactivity to NP or GPC fragments and HLAs expressed by each LF survivor. Unstimulated and positive epitopes are shown. Epitopes were considered positive if we observed ≥3 events double positive for IFN-γ and TNF-α over the unstimulated controls. (B) Graph depicting percentages of CD3+ CD8+ cells double positive for IFN-γ and TNF-α in response to all candidate epitopes tested for each individual. Red empty circles indicate negative epitopes. Data from each patient are from a single experiment. GPC epitopes are denoted by a circle with a black outline, while NP epitopes are not outlined. Common epitopes are denoted by common colors, while epitopes only found in a single individual are gray. (C) CD8+ T cell responses to epitopes GPC233-242, GPC235-244, and GPC60-68 from independent experiments. (D) CD8+ T cell responses in duplicate from LF survivor 2889600 to a positive epitope, NP554-563, and a negative epitope, GPC442-451.
Tables
- TABLE 1
Amino acid positions encoded by rscVSVs expressing LASV antigens
