Dengue virus-reactive CD8+ T cells mediate cross-protection against subsequent Zika virus challenge

Abstract

Zika virus (ZIKV) and dengue virus (DENV) are antigenically related flaviviruses that share cross-reactivity in antibody and T cell responses, and co-circulate in increasing numbers of countries. Whether pre-existing DENV immunity can cross-protect or enhance ZIKV infection during sequential infection of the same host is unknown. Here, we show that DENV-immune Ifnar1 ^-/- or wild-type C57BL/6 mice infected with ZIKV have cross-reactive immunity to subsequent ZIKV infection and pathogenesis. Adoptive transfer and cell depletion studies demonstrate that DENV-immune CD8⁺ T cells predominantly mediate cross-protective responses to ZIKV. In contrast, passive transfer studies suggest that DENV-immune serum does not protect against ZIKV infection. Thus, CD8⁺ T cell immunity generated during primary DENV infection can confer protection against secondary ZIKV infection in mice. Further optimization of current DENV vaccines for T cell responses might confer cross-protection and prevent antibody-mediated enhancement of ZIKV infection.

Fig. 1

ZIKV tissue burden in DENV-immune Ifnar1 ^−/− mice in the presence or absence of CD8⁺ T cells. Ifnar1 ^−/− mice were immunized intraperitoneally with DENV2 (2 × 10² FFU) for 28 days. Naive and DENV2-immune mice were injected intraperitoneally with isotype control Ab and anti-mouse CD8 Ab at 3 days and 1 day before ZIKV challenge. Three days post ZIKV challenge, peptide-specific CD3⁺CD8⁺IFNγ⁺ T cells (a–d; a, c naive mice; b, d DENV2-immune mice) and infectious ZIKV levels in sera (e) and indicated organs (f–i) were detected using ICS assay and FFA, respectively. Data were pooled from two independent experiments with n = 3–5 mice per group per experiment and expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. A Kruskal–Wallis one-way ANOVA was used for a–d while two-tailed Mann–Whitney test was used for e–i. Supplementary Tables 1 and 2 provide exact values of n and p

Fig. 2

The effect of DENV2-immune sera on ZIKV infection in vitro and in Ifnar1 ^−/− mice. Ifnar1 ^−/− mice were immunized intraperitoneally with DENV2 (2 × 10² FFU) for 28 days. Naive Ifnar1 ^−/− mouse sera (n = 10) and DENV2-immune Ifnar1 ^−/− mouse sera (n = 10) were examined for their ability to neutralize DENV2 or ZIKV using U937 DC-SIGN cells and a flow cytometry-based neutralization assay (a). Data were pooled from two independent experiments with n = 5 mouse sera per group and expressed as mean ± SEM. Different volumes of naive and DENV2-immune Ifnar1 ^−/− mouse sera were passively transferred (retro-orbital route) to naive recipient Ifnar1 ^−/− mice (b, c). Three days post retro-orbital challenge with 1 × 10⁴ FFU of ZIKV FSS13025, viral burdens in sera and indicated organs were measured using FFA. In b, data are pooled from two independent experiments (n = 3–4 mice per group per experiment), while results in c represent one experiment. Data are expressed as mean ± SEM. **p < 0.01. b A Kruskal–Wallis one-way ANOVA. c Two-tailed Mann–Whitney test. Supplementary Tables 1 and 2 provide exact values of n and p

Fig. 3

Reduced ZIKV burden in Ifnar1 ^−/− mice adoptively transferred with DENV2-exposed CD8⁺ T cells. Naive (1 × 10⁷) and DENV2-exposed CD8⁺ T cells (1 × 10⁷) from Ifnar1 ^−/− mice were adoptively transferred to naive recipient Ifnar1 ^−/− mice. Three days post ZIKV challenge (1 × 10⁴ FFU of ZIKV FSS13025, retro-orbital route), cross-reactive peptide-specific CD8⁺ T cell responses (a) and ZIKV levels in sera and indicated organs (b) were detected by ICS assay and FFA, respectively. Data are pooled from two independent experiments (n = 3–4 mice per group per experiment) and expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001,****p < 0.0001. a A Kruskal–Wallis one-way ANOVA. b Two-tailed Mann–Whitney test. Supplementary Tables 1 and 2 provide exact values of n and p

Fig. 4

Reduced ZIKV burden in WT mice adoptively transferred with DENV2-exposed CD8⁺ T cells. Naive or DENV2-exposed CD8⁺ T cells (1 × 10⁷) from WT mice were adoptively transferred to naive recipient WT mice that were intraperitoneally injected with 2 mg of the IFNAR1-blocking mAb MAR1-5A3 1 day before ZIKV challenge. Three days post ZIKV challenge (1 × 10⁶ FFU of FSS13025, retro-orbital route), cross-reactive peptide-specific CD8⁺ T cell responses (a) and ZIKV levels in serum and liver (b) were detected by ICS assay and FFA, respectively. Data are pooled from two independent experiments (n = 4–6 mice per group per experiment) and expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. a A Kruskal–Wallis one-way ANOVA. b Two-tailed Mann–Whitney test. Supplementary Tables 1 and 2 give exact values of n and p

Fig. 5

Decreased ZIKV-induced morbidity and mortality in Ifnar1 ^−/− mice adoptively transferred with DENV2-exposed CD8⁺ T cells. Naive or DENV2-exposed CD8⁺ T cells (1 × 10⁷) from Ifnar1 ^−/− mice were adoptively transferred via the retro-orbital route to recipient Ifnar1 ^−/− mice. One day later, mice were retro-orbitally challenged with 1 × 10³ FFU of ZIKV FSS13025. Clinical score (a, b), weight loss (c), and survival rates (d) were recorded daily. Mice having >20% weight loss and/or exhibiting paralysis of two hind limbs were euthanized according to Animal Protocols. This experiment was performed twice with n = 5 mice per group per experiment. Data represent pooled data from 10 mice per group and expressed as mean ± SEM. *p < 0.05, ***p < 0.001, ****p < 0.0001. c Two-way ANOVA. d Log rank test. Supplementary Tables 1 and 2 give exact values of n and p