Japanese Encephalitis Virus Vaccination Elicits Cross-Reactive HLA-Class I-Restricted CD8 T Cell Response Against Zika Virus Infection

Abstract

Japanese encephalitis virus (JEV) exposure or vaccination could elicit cross-reactive CD8 T cell immunity against heterologous flaviviruses in humans. In addition, cross-reactive CD8 T cells induced by dengue virus (DENV) have been shown to play a protective role against Zika virus (ZIKV). However, how JEV exposure or vaccination affects ZIKV infection in humans remains unclear. In this report, epitope prediction algorithms were used to predict the cross-reactive CD8 T cell epitope restricted to human HLA between JEV and ZIKV. We found that these predicted CD8 T cell epitopes are immunogenic and cross-reactive in humanized HLA transgenic mice. Moreover, JEV vaccine immunization provided cross-protection against ZIKV infection. Furthermore, CD8 T cells were involved in the protection against ZKIV infection in vivo. Our results have an important clinical implication that vaccination with JEV SA14-14-2 may provide protection against ZIKV infection in humans.

Keywords: CD8 T cells; HLA-A2 transgenic mice; JEV; Zika; cross-reactive; epitope; heterologous immunity.

FIGURE 1

Prediction of JEV and ZIKV-derived cross-reactive HLA-B*58:01, HLA-A*02:01, HLA-A*11:01 and HLA-A*24:02-restricted epitope candidates. (A) Sequence of predicted HLA-A*02:01-restricted JEV SA14, ZIKV MR766, ZIKVSPH2015 and ZIKVSZWIV01 cross-reactive epitopes. (B) Number of predicted JEV-derived HLA-B*58:01, HLA-A*02:01, HLA-A*11:01 and HLA-A*24:02-restricted epitopes and their comparison with ZIKV MR766-derived epitopes. (C) Location of predicted cross-reactive epitopes on the JEV polyprotein sequence and IEDB percentage ranks. *Starting position in MR766 (-/) and SPH2015/SZWIV01 (/-); **Starting position in MR766 (-/), SPH2015/SZWIV01 (/-/) and SA14 (/-).

FIGURE 2

Identification of JEV and ZIKV epitopes recognized by JEV- and ZIKV-specific CD8⁺ T cells in HHD mice. (A) IFNγ ELISpot assay of splenocytes from JEV SA14-14-2-immunized HHD mice (n = 5) stimulated with the indicated peptides. (B) IFNγ ELISpot assay of splenocytes from ZIKV SZWIV01-infected HDD mice stimulated with the indicated peptides. (C) Intracellular staining of splenocytes from ZIKV SZWIV01-infected HDD mice. Splenocytes were stimulated with J/Z11, J12, Z12 and Z12’ peptides. ZIKV SZWIV01-infected WT mice served as negative controls. Cells were gated on both CD8 and TCRβ positive. (D) HLA A2-J12 and Z12 tetramer staining of splenocytes from ZIKV SZWIV01-infected HDD mice.

FIGURE 3

JEV-specific CD8 T cells are recalled upon ZIKV infection in HHD mice. (A,B) IFNγ ELISpot assay of indicated epitopes with splenocytes from HHD mice (n = 5 each) that were immunized without (A) or with (B) the JEV SA14-14-2 vaccine once and then infected with ZIKV. (C,D) Intracellular staining of splenocytes from HHD mice (n = 5 each) that were immunized with or without the JEV SA14-14-2 vaccine once and then infected with ZIKV. The splenocytes were cultured with indicated peptides before intracellular staining. The averages of IFNγ⁺ (C) and IFNγ⁺TNFα⁺ (D) gated on CD8 T cells. (E,F) Representative plot of IFNγ and TNFα expression in CD8 T cells.

FIGURE 4

JEV immunization provides protection against ZIKV infection in HHD Ifnar1^–/– mice (A,B). Body weight (A) and survival rate (B) of HHD Ifnar1^–/– mice (n = 5 each) that were injected with JEV SA14-14-2 vaccine (n = 7) or mock (n = 5) two times on days 0 and 28 and then infected with ZIKV on day 14 after the boost.

FIGURE 5

JEV-specific CD8 T cells contribute to the cross-protection against ZIKV in HHD Ifnar1^–/– mice (A,B). Body weight (A) and survival (B) of HHD Ifnar1^–/– mice (n = 5 each) that were injected with JEV SA14-14-2 vaccine (n = 7) or mock (n = 5) two times on days 0 and 28. CD8 T cells were purified and adoptively transferred to naïve mice before ZIKV infection, body weight (A) and survival rate (B) were monitored. (C,D) CD8 T cells in JEV SA14-14-2-immunized mice were depleted with antibody before ZIKV challenge, body weight (C) and survival rate (D) of HHD Ifnar1^–/– mice (n = 6 each) were monitored.