T cell immunity rather than antibody mediates cross-protection against Zika virus infection conferred by a live attenuated Japanese encephalitis SA14-14-2 vaccine

Abstract

Zika virus (ZIKV) and Japanese encephalitis virus (JEV) are closely related to mosquito-borne flaviviruses. Japanese encephalitis (JE) vaccine SA14-14-2 has been in the Chinese national Expanded Program on Immunization since 2007. The recent recognition of severe disease syndromes associated with ZIKV, and the identification of ZIKV from mosquitoes in China, prompts an urgent need to investigate the potential interaction between the two. In this study, we showed that SA14-14-2 is protective against ZIKV infection in mice. JE vaccine SA14-14-2 triggered both Th1 and Th2 cross-reactive immune responses to ZIKV; however, it was cellular immunity that predominantly mediated cross-protection against ZIKV infection. Passive transfer of immune sera did not result in significant cross-protection but did mediate antibody-dependent enhancement in vitro, though this did not have an adverse impact on survival. This study suggests that the SA14-14-2 vaccine can protect against ZIKV through a cross-reactive T cell response. This is vital information in terms of ZIKV prevention or precaution in those ZIKV-affected regions where JEV circulates or SA14-14-2 is in widespread use, and opens a promising avenue to develop a novel bivalent vaccine against both ZIKV and JEV. KEY POINTS: • JEV SA14-14-2 vaccine conferred cross-protection against ZIKV challenge in mice. • T cell immunity rather than antibody mediated the cross-protection. • It provides important information in terms of ZIKV prevention or precaution.

Keywords: Cross-protection; Cross-reactivity; Japanese encephalitis virus; SA14-14-2 vaccine; T cell; Zika virus.

Fig. 1

Cross-reactive protection against ZIKV in SA14-14-2-immunized Ifnar1^-/- mice. a Schedule. Female Ifnar1^-/- mice were immunized three times at 3-week intervals. Three weeks after the final vaccination, the Ifnar1^-/- mice were challenged i.c. with a lethal dose of JEV or ZIKV. Body weight and mortality were monitored daily for 14 consecutive days. b Percentage changes in body weight. Data were expressed as mean ± SD. c Survival rate was shown as the percentage of survivors (n = 6). Mice exhibiting more than 25% loss in weight were humanely euthanized for ethical reasons. Each experiment was independently repeated three times. Asterisk indicates P < 0.05; double asterisk indicates P < 0.01; triple asterisk indicates P < 0.001

Fig. 2

Cross-reactive nAb responses in mouse sera. a Schedule. Female adult C57BL/6 mice were immunized as previously described. Sera were collected and mixed with either JEV (Beijing-1 strain) or ZIKV (SMGC-1 strain). Then, serum/virus mixture was gently injected i.c. into neonatal C57BL/6 mice. b Serum cross-reactive nAb titers assayed by PRNT₅₀ (n = 7). NAb titers are expressed as GMT + SD. c, d In vitro neutralizing and passive cross-protective effects of immune sera (n = 8, 8, 10, 12, respectively). The mice were monitored daily for body weight and survival rate for 31 consecutive days. c Body weight was expressed as mean ± SD. d Survival rate was shown as the percentage of survivors. Triple asterisk indicates P < 0.001; NS, non-significant

Fig. 3

Cross-reactive IgG and its subclass responses and ADE in mouse sera. a Schedule of mouse immunization and serum collection. Female adult C57BL/6 mice were immunized three times at 3-week intervals. Sera were collected 3 weeks after the final immunization. b Cross-reactive IgG responses detected by ELISA (n = 7). Ab titers are recorded as GMT + SD. c Cross-reactive IgG subclass responses determined by ELISA (n = 7). d ADE in sera measured by plaque forming (n = 7 per dilution). Asterisk indicates P < 0.05; double asterisk indicates P < 0.01; triple asterisk indicates P < 0.001

Fig. 4

Cross-reactive cytokine responses in mouse splenocytes. a Schedule. Three weeks after the final immunization, splenocytes from adult C57BL/6 mice were collected. b The cytokines IL-2, IL-4, and IFN-γ secreted by splenocytes were determined using ELISPOT (n = 7). The numbers of cytokine-positive cells are reported as the mean SFU/3 × 10⁵ splenocytes + SD. Asterisk indicates P < 0.05; double asterisk indicates P < 0.01; triple asterisk indicates P < 0.001

Fig. 5

Adoptive transfer of splenocytes from SA14-14-2-vaccinated C57BL/6 mice and cross-protection of splenocytes in Ifnar1^-/- mice. a Schedule. Splenic lymphocytes (3 × 10⁶ cells per mouse) collected 3 weeks post final immunization were adoptively transferred r.o. to naïve adult mice, and 1 day later mice were challenged with a lethal dose of either JEV or ZIKV. As a control, splenic lymphocytes from PBS-treated mice were transferred to naïve mice prior to challenge. Results were evaluated for b body weight change and c survival rate of mice 14 consecutive days post challenge (n = 6). Mice exhibiting more than 25% loss in weight were humanely euthanized for ethical reasons. Each experiment was independently repeated three times. Results are expressed as mean ± SD. Asterisk indicates P < 0.05; double asterisk P < 0.001