Japanese encephalitis virus-primed CD8+ T cells prevent antibody-dependent enhancement of Zika virus pathogenesis

Abstract

Cross-reactive anti-flaviviral immunity can influence the outcome of infections with heterologous flaviviruses. However, it is unclear how the interplay between cross-reactive antibodies and T cells tilts the balance toward pathogenesis versus protection during secondary Zika virus (ZIKV) and Japanese encephalitis virus (JEV) infections. We show that sera and IgG from JEV-vaccinated humans and JEV-inoculated mice cross-reacted with ZIKV, exacerbated lethal ZIKV infection upon transfer to mice, and promoted viral replication and mortality upon ZIKV infection of the neonates born to immune mothers. In contrast, transfer of CD8+ T cells from JEV-exposed mice was protective, reducing the viral burden and mortality of ZIKV-infected mice and abrogating the lethal effects of antibody-mediated enhancement of ZIKV infection in mice. Conversely, cross-reactive anti-ZIKV antibodies or CD8+ T cells displayed the same pathogenic or protective effects upon JEV infection, with the exception that maternally acquired anti-ZIKV antibodies had no effect on JEV infection of the neonates. These results provide clues for developing safe anti-JEV/ZIKV vaccines.

Graphical abstract

Figure 1.

Sera from JEV-exposed humans and mice contain JEV-EDIII- and ZIKV-EDIII-reactive IgG. (A–C) Human serum samples were prepared from JEV-naive 6-mo-old infants (n = 8) or JEV-naive, JEV-vaccinated children or young adults aged 7 (n = 6), 8 (n = 6), 9 (n = 6), 10 (n = 6), 11 (n = 6), and 20 (n = 6) yr. (D and E) Mouse serum samples were prepared from JEV-naive (mock-immune, n = 8) and JEV-immune (n = 8) mice. Mouse anti-His mAb was used as positive control (n = 6). JEV-EDIII– and ZIKV-EDIII–reactive IgG levels were measured by indirect ELISA. Data are presented as the mean ± SEM. *, P < 0.05; **, P < 0.01 by two-tailed Mann–Whitney U test.

Figure 2.

Transfer of ZIKV-immune mouse sera increases the survival of ZIKV-infected mice but decreases that of JEV-infected mice. (A and B) Mouse serum samples were prepared from ZIKV-naive (mock-immune, n = 6) and ZIKV-immune (n = 6) mice. ZIKV-EDIII– and JEV-EDIII–reactive IgG levels were measured by indirect ELISA. Mouse anti-His mAb was used as positive control. Data are presented as the mean ± SEM. (C–F) 1-d-old naive C57BL/6 mice were injected s.c. with 0.05 µl (C and D) or 10 µl (E and F) of mock-immune (n = 19, n = 7, respectively) or ZIKV-immune (n = 16, n = 7, respectively) mouse sera, and 6 h later, the mice were injected s.c. with JEV (12 PFU). Mouse weights and survival were recorded daily for 14 d. Data are presented as the mean ± SD and are pooled from two experiments, each with 3–10 mice per group. (G–J) 1-d-old naive C57BL/6 mice were injected s.c. with 0.05 µl (G and H) or 10 µl (I and J) of serum from mock-immune (n = 12, n = 13, respectively) or ZIKV-immune (n = 13, n = 12, respectively) mice, and injected s.c. with ZIKV (10² FFU) 6 h later. Weights and survival were recorded daily for 28 d. Data are presented as the mean ± SD and are pooled from two experiments, each with six or seven mice/group. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by two-tailed Mann–Whitney U test (B, C, E, G, and I) or log-rank test (D, F, H, and J).

Figure S1.

Modest neutralization but efficient enhancement of ZIKV infection in vitro by JEV-neutralizing, JEV-immune sera from humans and mice. (A–D) Human serum samples isolated from JEV-naive infants (6 mo of age, n = 6) or JEV-immune subjects (n = 6 for all age groups), and mouse serum samples (n = 6 for all groups) were tested for neutralization of ZIKV (A and B) or JEV (C and D) infection of Vero cells. Mouse ZIKV-immune serum was used as the positive control. (E and F) Enhancement of ZIKV infection by JEV-immune sera from humans and mice was evaluated in U937 cells. Data are expressed as the mean ± SEM.

Figure 3.

Transfer of JEV-immune human sera increases the survival of JEV-infected mice but decreases that of ZIKV-infected mice. (A–J) 1-d-old naive C57BL/6 mice were injected s.c. with the indicated volumes of JEV-naive or JEV-immune human sera. 6 h later, the mice were injected s.c. with (A–F) ZIKV (10² FFU) or (G–J) JEV (12 PFU). Mouse weights and survival were recorded daily for up to 28 d. Data are presented as the mean ± SD and are pooled from two experiments, each with 5–10 mice per group (A–F) or 3–6 mice/group (G–J). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by two-tailed Mann–Whitney U test (A, C, E, G, and I) or log-rank test (B, D, F, H, and J).

Figure 4.

Transfer of JEV-immune mouse sera increases the survival of JEV-infected mice but decreases that of ZIKV-infected mice. (A–J) 1-d-old naive C57BL/6 mice were injected s.c. with the indicated volumes of mock- or JEV-immune mouse sera, and 6 h later, the mice were injected s.c. with (A–F) ZIKV (10² FFU) or (G–J) JEV (12 PFU). Mouse weights and survival were recorded daily for up to 28 d. Data are presented as the mean ± SD and are pooled from two experiments, each with five to eight mice per group (A–F) or two or three mice per group (G–J). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by two-tailed Mann–Whitney U test (A, C, E, G, and I) or log-rank test (B, D, F, H, and J).

Figure S2.

Transfer of JEV-immune IgG exacerbates lethal ZIKV infection in mice. (A–D) 1-d-old naive C57BL/6 mice were injected s.c. with (A and B) 75 µg of IgG purified from JEV-naive (n = 12) or JEV-immune (n = 8) human sera or (C and D) 12 µg of IgG purified from mock-immune (n = 15) or JEV-immune (n = 13) mice and injected s.c. with ZIKV (10² FFU) 6 h later. Weights and survival were recorded daily for up to 28 d. Data are presented as the mean ± SD and are pooled from two experiments, each with four to eight mice/group. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by two-tailed Mann–Whitney U test (A and C) or log-rank test (B and D).

Figure 5.

Pups born to JEV-immune mothers have decreased survival and increased viral burden upon ZIKV challenge compared with pups born to naive mothers. 6-wk-old female C57BL/6 mice were i.p. injected with 1 mg anti-Ifnar1 mAb (MAR1-5A3) 1 d before r.o. injection with JEV (10² PFU). 4 wk later, naive or JEV-immune females were mated with 10-wk-old naive male mice. (A and B) 1-d-old C57BL/6 pups were sacrificed, and sera were prepared. JEV-EDIII and ZIKV-EDIII–reactive IgG were measured by indirect ELISA. Mouse anti-His mAb was used as positive control. Data are presented as the mean ± SEM. (C–H) 1-d-old pups were injected s.c. with (C–F) ZIKV (10² FFU) or (G and H) JEV (12 PFU). (C, D, G, and H) Mouse weights and survival were recorded daily for up to 28 d. Data are presented as the mean ± SD and are pooled from three experiments totaling 20 infected mice born to 3 naive mothers and 17 infected mice born to 3 JEV-immune mothers (C and D), or two experiments totaling 16 infected mice born to 2 naive mothers and 12 infected mice born to 2 JEV-immune mothers (G and H). (E and F) Mice were sacrificed 3 d after ZIKV infection, and blood and brains were collected for measurement of viral burden using the FFA. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by two-tailed Mann–Whitney U test (B, C, E, and G) or log-rank test (D and H).

Figure S3.

JEV- and ZIKV-reactive IgG in the serum of mice 6 h after transfer of mouse JEV-immune serum. (A–F) 1-d-old naive C57BL/6 mice were injected s.c. with 3, 15, or 75 µl of mock- or JEV-immune mouse sera (n = 5 for all groups). 6 h later, the mice were sacrificed, and sera were collected. JEV-EDIII– and ZIKV-EDIII–reactive IgG levels were measured by indirect ELISA. Data are presented as the mean ± SEM. **, P < 0.01 by two-tailed Mann–Whitney U test.

Figure S4.

Maternally acquired anti-ZIKV Abs have no effect on the survival of JEV-infected pups born to ZIKV-immune mothers. 6-wk-old female C57BL/6 mice were i.p. injected with 1 mg anti-Ifnar1 mAb (MAR1-5A3) 1 d before r.o. injection with ZIKV (10² FFU). 4 wk later, naive or ZIKV-immune females were mated with 10-wk-old naive male mice. (A and B) 1-d-old C57BL/6 pups were sacrificed, and sera were prepared. ZIKV-EDIII and JEV-EDIII–reactive IgG were measured by indirect ELISA. Mouse anti-His mAb was used as positive control. Data are presented as the mean ± SEM. (C and D) 1-d-old C57BL/6 pups were injected s.c. with JEV (12 PFU). Mouse weights and survival were recorded daily for 14 d. Data are presented as the mean ± SD and are pooled from four experiments totaling 23 infected mice born to 4 naive mothers and 25 infected mice born to 4 ZIKV-immune mothers. *, P < 0.05; ****, P < 0.0001 by two-tailed Mann–Whitney U test.

Figure 6.

Identification of cross-reactive responses to JEV or ZIKV epitopes by CD8⁺ T cells from primed mice. 6-wk-old female C57BL/6 mice were i.p. injected with 1 mg anti-Ifnar1 mAb (MAR1-5A3) 1 d before r.o. injection with (A and C) JEV (10³ PFU) or (B and D) ZIKV (10³ FFU). 7 or 28 d later, splenocyte-derived CD8⁺ T cells were isolated and stimulated in vitro with the indicated CD8⁺ T cell epitopes from ZIKV or the corresponding JEV variants. The frequencies of IFN-γ–producing CD8⁺ T cells (SFCs) were detected using IFN-γ ELISpot assays. Data are presented as the mean ± SEM and are pooled from two independent experiments, each with three or four mice per group. Dotted lines represent the cutoff value. Asterisk indicates a positive response, as defined in the Materials and methods.

Figure 7.

Transfer of JEV-elicited CD8⁺ T cells increases the survival of ZIKV-infected mice in the absence or presence of JEV-immune serum. (A and B) 1-d-old naive C57BL/6 mice were r.o. injected with 2 × 10⁶ CD8⁺ T cells from EV71-VP1–immunized mice (n = 18) or JEV-exposed mice injected r.o. with JEV (10² PFU) 28 d earlier (n = 18). 2 h later, the neonates were injected s.c. with ZIKV (10² FFU). Weights and survival were recorded daily for 28 d. (C and D) Mice were treated as described for A and B except that immediately following infusion of neonatal mice with CD8⁺ T cells isolated from EV71-VP1–immunized mice (n = 18) or JEV-exposed mice (n = 17), recipient mice were s.c. injected with 3 µl of JEV-immune mouse sera and then s.c. inoculated with ZIKV (10² FFU) 6 h after the infusion of T cells and sera. Data are presented as the mean ± SD and are pooled from two experiments, each with nine mice/group (A and B) or from two experiments, each with 8 or 9 mice per group (C and D). *, P < 0.05; **, P < 0.01; ***, P < 0.001 by two-tailed Mann–Whitney U test (A and C) or log-rank test (B and D).

Figure 8.

Transfer of ZIKV-elicited CD8⁺ T cells increases the survival of JEV-infected mice. (A and B) 1-d-old naive C57BL/6 mice were r.o. injected with 2 × 10⁶ CD8⁺ T cells from EV71-VP1–immune mice that had been s.c. immunized with EV71-VP1 (50 µg/mouse/each; twice, 2 wk apart) 28 d earlier (n = 9) or ZIKV-exposed mice injected r.o. with ZIKV (10² PFU) 28 d earlier (n = 9). 2 h later, the neonates were injected s.c. with JEV (12 PFU). Mouse weights (A) and survival (B) were recorded daily for 14 d. Data are presented as the mean ± SD and are pooled from two experiments, each with four or five mice/group. *, P < 0.05; ***, P < 0.001 by two-tailed Mann–Whitney U test (A) or log-rank test (B).

Figure 9.

Transfer of JEV-elicited CD8⁺ T cells modulates anti-JEV Ab-mediated ADE of ZIKV infection. (A and D) 1-d-old C57BL/6 naive mice were s.c. injected with 3 µl of mock-immune or JEV-immune mouse serum. 6 h later, mice were injected s.c. with ZIKV (10² FFU). (B and E) 1-d-old C57BL/6 naive mice were r.o. injected with 2 × 10⁶ CD8⁺ T cells from EV71-VP1–immune or JEV-exposed mice and injected s.c. with ZIKV (10² FFU) 2 h later. (C and F) Mice were treated as described for B and E except that the CD8⁺ T cells were from EV71-VP1–immune or JEV-exposed mice injected s.c. with 3 µl of JEV-immune mouse sera before s.c. injection of ZIKV (10² FFU). ZIKV viral loads in serum and brain were measured using the FFA at 3 or 8 d after infection. Data are presented as the mean ± SEM and are pooled from two experiments, each with three to six mice/group. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by two-tailed Mann–Whitney U test.