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. 2018 Oct 1:9:2225.
doi: 10.3389/fimmu.2018.02225. eCollection 2018.

Cross-Reactivity and Anti-viral Function of Dengue Capsid and NS3-Specific Memory T Cells Toward Zika Virus

Affiliations

Cross-Reactivity and Anti-viral Function of Dengue Capsid and NS3-Specific Memory T Cells Toward Zika Virus

Mei Qiu Lim et al. Front Immunol. .

Abstract

Zika virus (ZIKV), a flavivirus with homology to dengue virus (DENV), is spreading to areas of DENV hyper-endemicity. Heterologous T cell immunity, whereby virus-specific memory T cells are activated by variant peptides derived from a different virus, can lead to enhanced viral clearance or diminished protective immunity and altered immunopathology. In mice, CD8+ T cells specific for DENV provide in vivo protective efficacy against subsequent ZIKV infection. In humans, contrasting studies report complete absence or varying degrees of DENV/ZIKV T cell cross-reactivity. Moreover, the impact of cross-reactive T cell recognition on the anti-viral capacity of T cells remains unclear. Here, we show that DENV-specific memory T cells display robust cross-reactive recognition of ZIKV NS3 ex vivo and after in vitro expansion in respectively n = 7/10 and n = 9/9 dengue-immune individuals tested. In contrast, cross-reactivity toward ZIKV capsid is low or absent. Cross-reactive recognition of DENV or ZIKV NS3 peptides elicits similar production of the anti-viral effector mediators IFN-γ, TNF-α, and CD107a. We identify 9 DENV/ZIKV cross-reactive epitopes, 7 of which are CD4+ and 2 are CD8+ T cell epitopes. We also show that cross-reactive CD4+ and CD8+ T cells targeting novel NS3 epitopes display anti-viral effector potential toward ZIKV-infected cells, with CD8+ T cells mediating direct lyses of these cells. Our results demonstrate that DENV NS3-specific memory T cells display anti-viral effector capacity toward ZIKV, suggesting a potential beneficial effect in humans of pre-existing T cell immunity to DENV upon ZIKV infection.

Keywords: T cells; Zika virus (ZIKV); anti-viral response; cross-reactive immune response; dengue virus (DENV); heterologous immunity.

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Figures

Figure 1
Figure 1
Cross-reactive T cell recognition ex vivo of ZIKV peptides in dengue-immune individuals. (A–F) Ex vivo T cell recognition of DENV and ZIKV NS3 and capsid proteins in dengue-immune and dengue-naïve subjects as assessed by intracellular cytokine staining (ICS) for IFN-γ production after a 5-h stimulation of PBMCs with or without overlapping 15-mer peptides spanning DENV and ZIKV NS3 and capsid proteins. (A,B) Dot plots depicting IFN-γ production in CD4+ and CD8+ T cells from one representative dengue-immune (A) and dengue-naïve (B) donor are shown. Plots are gated on live, CD3+, CD4+, or CD8+ T cells (top or bottom panels, respectively). (C–F) CD4+ and CD8+ T cell recognition based on IFN-γ production in the presence of DENV and ZIKV peptides as shown in a/b is summarized for dengue-immune individuals (C: CD4+; D: CD8+, n = 10) and for healthy donors (E: CD4+; F: CD8+; n = 14). Results are expressed as percentage of cytokine positive cells relative to total CD4+ or CD8+ T cells. Statistics are calculated using the non-parametric two-tailed Mann-Whitney test. (G,H) The percentage of dengue-immune individuals that display a CD4+ (G) and CD8+ (H) T cell response to ZIKV capsid and NS3 is summarized.
Figure 2
Figure 2
Strong cross-reactive T cell recognition in vitro of ZIKV NS3 protein in DENV-immune individuals. (A,B) T cells from dengue-immune (A, n = 9) and dengue-naïve subjects (B, n = 14) were expanded in vitro for 10 days with NS3 and capsid peptides from either DENV or ZIKV. T cell recognition of DENV and ZIKV NS3 and capsid peptides was assessed by IFN-γ ELISPOT. Results are expressed as Spot Forming cells (SFC) relative to 105 cells. Values indicate the sum of responses detected for each peptide pool within a single protein. (C) Percentage of peptide pools from DENV and ZIKV proteins that elicit a response in dengue-immune individuals from a, where the total number of peptide pools for DENV NS3, ZIKV NS3, DENV capsid, ZIKV capsid is n = 23, 22, 13, and 10, respectively. (D) Percentage of dengue-immune subjects (n = 9) that respond to at least one DENV and ZIKV NS3 and capsid peptide pool. (E,F) Production of IFN-γ and TNF-α by DENV and ZIKV T cell lines upon stimulation with or without single DENV or ZIKV peptides or with PMA/ionomycin, as assessed by ICS. Results are from two representative patients. Plots are gated on live CD4+ (E,F, top row) or CD8+ T cells (F, bottom row). Statistics are calculated using the non-parametric two-tailed Mann-Whitney test.
Figure 3
Figure 3
In vitro and ex vivo cytokine production by cross-reactive T cells upon stimulation with DENV or ZIKV peptides. (A–D) IFN-γ and TNF-α production after DENV or ZIKV stimulation is assessed by ICS in CD4+ (A) and CD8+ T cell lines (C) in dengue-immune subjects (n = 7) and is shown as percentages of cytokine+ cells in CD4+ or CD8+ T cells, respectively. Each point indicates a single peptide. The ratios of IFN-γ/TNF-α production in CD4+ (B) and CD8+ (D) T cell lines from (A) and (C) are shown for each peptide. (E,F) Expression of CD107a and production of IFN-γ and TNF-α after DENV or ZIKV stimulation is assessed directly ex vivo by ICS in dengue-immune subjects (n = 15). Each point indicates a single subject. All differences are not significant based on the non-parametric two-tailed Mann Whitney test. (G,H) Pearson correlation of CD107a expression after DENV or ZIKV stimulation in CD4+ (G) or CD8+ T cells (H). (I,J) Pearson correlation of CD4+ (I) and CD8+ (J) T cell expression of CD107a and IFN-γ production. Lines inside bars in (A-F) indicate mean.
Figure 4
Figure 4
Anti-viral capacity of cross-reactive CD8+ T cells targeting DENV NS3 108. (A) Production of IFN-γ and TNF-α after restimulation of DENV T cell lines in the presence or absence of DENV NS3 108 and ZIKV NS3 107 peptides or with PMA/ionomycin. Plots are gated on live, CD8+ T cells. (B,C) Characterization of the minimal CD8+ T cell epitope from DENV NS3 108 (B) and ZIKV NS3 107 (C) capable of inducing optimal IFN-γ production by CD8+ T cell lines. The optimal minimal epitope is highlighted in blue or red. (D,E) Production of effector cytokines (D) or cytolytic mediators (E) by the CD8+ T cell line after 5 h co-culture with WGP 48 cells pulsed with serial dilutions of the 9-mers DENV NS3 108 RRG or ZIKV NS3 107 KRG, as assessed by ICS. (F) Production of IFN-γ, TNF-α, granzyme B and CD107a by the CD8+ T cell line upon overnight co-culture with WGP 48 cells that were pulsed with or without ZIKV NS3 107 or were previously infected with ZIKV (strain MR766). (G) Kinetics of cellular lysis of WGP 48 cells after co-culture with CD8+ T cell line, measured by xCELLigence Real-Time Cell Analysis (RTCA). Cell lysis is measured as normalized cell index over a 24 h period of WGP 48 cells that were either left untreated (w/o peptide), pulsed with DENV or ZIKV peptides or previously infected with ZIKV (strain MR766). SD error bars were calculated on duplicates. One representative experiment is shown. (H) Summary from (G) of the percentage of WGP 48 lysis in the presence of the T cell line.
Figure 5
Figure 5
Anti-viral capacity of cross-reactive CD4+ T cells targeting DENV NS3 57. (A) Production of IFN-γ, TNF-α, and CD107a after stimulation of CD4+ T cell lines in the presence or absence of DENV NS3 57 and ZIKV NS3 56 peptides or with PMA/ionomycin. Plots are gated on live, CD4+ T cells. (B,C) Production of effector cytokines (B) or cytolytic mediators (C) of the CD4+ T cell line after co-culture with LKC cells pulsed with serial dilutions of DENV NS3 57 or ZIKV NS3 56 peptides, as assessed by ICS. (D) Production of IL-2, IFN-γ, TNF-α, granzyme B, and CD107a by the CD4+ T cell line upon overnight co-culture with LKC cells that were pulsed with or without ZIKV NS3 56 or were previously infected with ZIKV (strain MR766). (E) Kinetics of cellular lysis of LKC target cells in the presence of the CD4+ T cell line, measured by RTCA. Cell lysis is measured as normalized cell index over a 24 h period in LKC cells that were either left untreated (w/o peptide) or pulsed with DENV or ZIKV peptides. SD error bars are calculated on duplicates. One representative experiment is shown. The peptide sequences of DENV NS3 57 and ZIKA NS3 56 are IIMDEAHFTDPASIA and YIMDEAHFTDPSSIA, respectively.

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