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. 2017 Mar 13:2:17036.
doi: 10.1038/nmicrobiol.2017.36.

Identification of Zika virus epitopes reveals immunodominant and protective roles for dengue virus cross-reactive CD8+ T cells

Affiliations

Identification of Zika virus epitopes reveals immunodominant and protective roles for dengue virus cross-reactive CD8+ T cells

Jinsheng Wen et al. Nat Microbiol. .

Abstract

CD8+ T cells play an important role in controlling Flavivirus infection, including Zika virus (ZIKV). Here, we have identified 25 HLA-B*0702-restricted epitopes and 1 HLA-A*0101-restricted epitope using interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) and intracellular cytokine staining (ICS) in ZIKV-infected IFN-α/β receptor-deficient HLA transgenic mice. The cross-reactivity of ZIKV epitopes to dengue virus (DENV) was tested using IFN-γ-ELISPOT and IFN-γ-ICS on CD8+ T cells from DENV-infected mice, and five cross-reactive HLA-B*0702-binding peptides were identified by both assays. ZIKV/DENV cross-reactive CD8+ T cells in DENV-immune mice expanded post ZIKV challenge and dominated in the subsequent CD8+ T cell response. ZIKV challenge following immunization of mice with ZIKV-specific and ZIKV/DENV cross-reactive epitopes elicited CD8+ T cell responses that reduced infectious ZIKV levels, and CD8+ T cell depletions confirmed that CD8+ T cells mediated this protection. These results identify ZIKV-specific and ZIKV/DENV cross-reactive epitopes and demonstrate both an altered immunodominance pattern in the DENV-immune setting relative to naive, as well as a protective role for epitope-specific CD8+ T cells against ZIKV. These results have important implications for ZIKV vaccine development and provide a mouse model for evaluating anti-ZIKV CD8+ T cell responses of human relevance.

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Conflict of interest statement

Competing interests

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Cytokine secretion pattern of CD8+ T cells directed to HLA-B*0702- and HLA-A*0101-binding ZIKV epitopes identified via IFN-γ ELISPOT
a–d, Splenocytes isolated from Ifnar−/− HLA-B*0702 transgenic mice (a,b) and Ifnar−/− HLA-A*0101 transgenic mice (c,d) 7 days after r.o. infection with 1 ×ばつ 102 FFU of ZIKV strain FSS13025 or MR766 were stimulated with each of the 37 positive HLA-B*0702-binding peptides or 13 positive HLA-A*0101-binding peptides identified via IFN-γ ELISPOT and an ICS assay was then performed. Data represent the average of two independent experiments and are expressed as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001. Two-tailed Mann–Whitney test. P/I denotes phorbol myristate acetate/ionomycin. Supplementary Tables 2 and 3 give exact values of n and P.
Figure 2
Figure 2. Impact of prior DENV2 infection on the ZIKV-specific CD8+ T cell response
a–d, Ifnar−/− HLA-B*0702 transgenic mice were inoculated i.p. with 2 ×ばつ 103 FFU of DENV2 strain S221 for 4 weeks. Naive mice and DENV2 strain S221-immune mice were challenged r.o. with 1 ×ばつ 104 FFU of ZIKV FSS13025 for 3 days and the percentages of peptide-specific IFN-γ+ and/or TNF-α+ CD8+ T cells were detected by ICS assay (a,b). In addition, separate groups of naive mice and DENV2 strain S221-immune mice were challenged r.o. with 1 ×ばつ 104 FFU of ZIKV FSS13025 for 7 days and the percentages of peptide-specific IFN-γ+ and/or TNF-α+ CD8+ T cells were detected by ICS assay (c,d). Data are expressed as mean ± s.e.m. *P < 0.05, **P < 0.01. Two-tailed Mann–Whitney test. Black asterisk indicates ZIKV-specific response; red asterisk represents the ZIKV/DENV cross-reactive response. e, All positive peptides were grouped according to the ZIKV specificity of the immune response. Numbers in parentheses indicate the number of positive peptides in this group. Supplementary Tables 2 and 3 give exact n and P values.
Figure 3
Figure 3. ZIKV-specific and ZIKV/DENV cross-reactive peptide immunization elicited a CD8+ T cell response and mediated protection against ZIKV
a–f, Five-week-old Ifnar−/− HLA-B*0702 transgenic mice were divided into four groups: mock versus ZIKV peptide (a–c) and mock versus ZIKV/DENV cross-reactive peptide (d–f). Peptide groups received corresponding peptide immunizations as described in the Methods. All groups were challenged r.o. with 1 ×ばつ 104 FFU of ZIKV FSS13025 for 3 days. CD3+ CD8+ CD44+ CD62L− T cells were gated and the percentages of IFN-γ+ and/or TNF-α+ cells were determined by ICS. The levels of infectious ZIKV in sera and brains were measured via FFA. Data are expressed as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Two-tailed Mann–Whitney test. Dashed lines represent limit of detection. Supplementary Tables 2 and 3 give exact n and P values.
Figure 4
Figure 4. HLA-B*0702-restricted ZIKV peptide immunization-mediated protection is mediated by CD8+ T cells
a–g, Five-week-old Ifnar−/− HLA-B*0702 transgenic mice were divided into four groups: mock + isotype antibody (Ab) versus mock + anti-CD8 Ab versus peptide-immunized + isotype Ab versus peptide-immunized + anti-CD8 Ab. Peptide-immunized Ifnar−/− HLA-B*0702 transgenic mice were immunized with a cocktail of six peptides, as described in the Methods. Mock-immunized mice and peptide-immunized mice were injected i.p. with isotype control Ab and anti-mouse CD8 Ab 3 days and 1 day before ZIKV challenge. All groups were challenged r.o. with 1 ×ばつ 104 FFU of ZIKV FSS13025 for 3 days. CD3+ CD8+ CD44+ CD62L− T cells were gated and the percentages of IFN-γ+ and/or TNF-α+ cells in mock and peptide groups were determined by ICS. Levels of infectious ZIKV in tissues were measured via FFA. Data are expressed as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Two-tailed Mann–Whitney test. Supplementary Tables 2 and 3 give exact n and P values.
Figure 5
Figure 5. HLA-A*0101-restricted ZIKV peptide immunization-mediated protection is mediated by CD8+ T cells
a–g, Five-week-old Ifnar−/− HLA-A*0101 transgenic mice were divided into four groups: mock + isotype Ab versus mock + anti-CD8 Ab versus peptide-immunized + isotype Ab versus peptide-immunized + anti-CD8 Ab. Peptide-immunized Ifnar−/− HLA-A*0101 transgenic mice were immunized with a cocktail of five peptides as described in the Methods. Mock-immunized mice and peptide-immunized mice were injected i.p. with isotype control Ab and anti-mouse CD8 Ab 3 days and 1 day before ZIKV challenge. All groups were challenged r.o. with 1 ×ばつ 104 FFU of ZIKV FSS13025 for 3 days. CD3+ CD8+ CD44+ CD62L− T cells were gated and the percentages of IFN-γ+ and/or TNF-α+ cells in mock and peptide groups were determined by ICS. The levels of infectious ZIKV in tissues were measured via FFA. Data are expressed as mean ± s.e.m. *P < 0.05, **P < 0.01. Two-tailed Mann–Whitney test. Supplementary Tables 2 and 3 give exact n and P values.

Comment in

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