The World Health Organization declared the clusters of microcephaly and neurological disorders and their association with ZIKV infection to be a global public health emergency on February 1, 2016. ZIKV is believed to cause neuropathology in developing fetuses by crossing the placenta and targeting cortical neural progenitor cells9,10,11,12,13,14, leading to impaired neurogenesis and resulting in microcephaly and other congenital malformations. ZIKV has also been associated with neurologic conditions in adults, such as Guillain-Barré syndrome15.

Vaccines have been developed for other flaviviruses, including yellow fever virus, Japanese encephalitis virus, tick-borne encephalitis virus, and dengue viruses, but no vaccine currently exists for ZIKV. To develop preclinical challenge models for candidate ZIKV vaccines, we obtained low-passage ZIKV isolates from northeast Brazil (Brazil/ZKV2015; University of São Paulo)11 and Puerto Rico (PRVABC59; US Centers for Disease Control and Prevention) (Extended Data Fig. 1). We expanded these viruses in Vero cells to generate preclinical challenge stocks, which we termed ZIKV-BR and ZIKV-PR, respectively. These ZIKV strains are part of the Asian ZIKV lineage16 and differ from each other by five amino acids in the polyprotein (Extended Data Fig. 2). The Brazil/ZKV2015 strain has also recently been reported to recapitulate key clinical manifestations, including fetal microcephaly and intrauterine growth restriction, in wild-type SJL mice11. Similarly, the related French Polynesian H/PF/2013 strain has been shown to induce placental damage and fetal demise in Ifnar−/− C57BL/6 mice as well as in wild-type C57BL/6 mice following IFN-α receptor blockade10.

We designed ZIKV prM-Env immunogens based on the Brazil BeH815744 strain (Extended Data Fig. 2) and optimized them for increased antigen expression. We also designed deletion mutants lacking prM and/or lacking the transmembrane region (ΔTM) or the full stem (Δstem) of Env (Fig. 1a). Plasmid DNA vaccines encoding these antigens were produced, and transgene expression was verified by western blot (Fig. 1b). To assess the immunogenicity of these vaccines, groups of Balb/c mice (n = 5–10 per group) received a single immunization of 50 μg of each DNA vaccine by the intramuscular (i.m.) route at week 0. Env-specific antibody responses were evaluated at week 3 by ELISA. The prM-Env DNA vaccine elicited higher Env-specific antibody titers than did the Env DNA vaccine and all of the ΔTM and Δstem deletion mutants (Fig. 1c), indicating the importance of including prM as well as the full-length Env sequence. No prM-specific antibody responses were detected (Extended Data Fig. 3). The prM-Env DNA vaccine also induced ZIKV-specific neutralizing antibodies after a single immunization (Table 1), as measured by a virus-specific microneutralization assay17. In addition, the prM-Env DNA vaccine induced Env-specific CD8+ and CD4+ T-lymphocyte responses, as assessed by IFNγ ELISPOT and multiparameter intracellular cytokine staining assays (Fig. 1d, e).

Figure 1: Construction and immunogenicity of DNA vaccines. a, Schema of ZIKV prM-Env immunogens and deletion mutants. b, Western blot of transgene expression from (1) prM-Env, (2) prM-Env(ΔTM), (3) prM-Env(Δstem), (4) Env, (5) Env(ΔTM), (6) Env(Δstem), and (7) sham DNA vaccines transfected in 293T cells. Balb/c mice (n = 5 per group) received a single immunization with 50 μg of these DNA vaccines by the i.m. route. c, Humoral immune responses were assessed at week 3 following vaccination by Env-specific ELISA. Red bars reflect medians. d, e, Cellular immune responses were assessed by IFNγ ELISPOT assays (d) and multi-parameter intracellular cytokine staining assays (e). Error bars reflect s.e.m. PowerPoint slide Full size image

Table 1 ZIKV-specific neutralizing antibody titers Full size table

To assess the protective efficacy of these DNA vaccines against ZIKV challenge, we infected vaccinated or sham control Balb/c mice at week 4 by the intravenous (i.v.) route with 105 viral particles (102 plaque-forming units (PFU)) of ZIKV-BR or ZIKV-PR. Viral loads following ZIKV challenge were quantitated by RT–PCR18. Sham-vaccinated mice inoculated with ZIKV-BR developed approximately 6 days of detectable viraemia with a mean peak viral load of 5.42 log copies per ml (range 4.55–6.57 log copies per ml; n = 10) on day 3 after challenge (Fig. 2a). In contrast, a single immunization with the prM-Env DNA vaccine provided complete protection against ZIKV-BR challenge with no detectable viraemia (<100 copies per ml) at any time point (n = 10). Complete protection was also observed when vaccinated mice were challenged at week 8 (data not shown). The prM-Env DNA vaccine also afforded complete protection against ZIKV-PR challenge (n = 5). ZIKV-PR replicated to slightly lower levels (mean peak viral load 4.96 log copies per ml; range 4.80–5.33 log copies per ml; n = 5) than did ZIKV-BR in sham controls. In contrast with the prM-Env DNA vaccine, the DNA vaccines lacking prM as well as the ΔTM and Δstem deletion mutants did not provide complete protection against ZIKV-BR challenge, although viral loads were still reduced in these animals as compared with sham controls (Fig. 2b).

Figure 2: Protective efficacy of DNA vaccines. a, Balb/c mice (n = 5 or 10 per group) received a single immunization by the i.m. route with 50 μg prM-Env DNA vaccine or a sham vaccine and were challenged at week 4 by the i.v. route with 105 viral particles (102 PFU) ZIKV-BR or ZIKV-PR. Serum viral loads are shown. b, Mice (n = 5 per group) received a single immunization with 50 μg of various DNA vaccines and were challenged with ZIKV-BR. c, d, Correlates of protective efficacy (c) and day 3 viral loads (d) are shown. Red bars reflect medians. P values and R2 values reflect t-tests and Spearman rank-correlation tests. PowerPoint slide Full size image

The varying degrees of protection obtained with this set of DNA vaccines allowed for an analysis of immune correlates of protection. Protective efficacy correlated with Env-specific binding antibody titers (P = 0.0005 comparing protected versus infected animals; Fig. 2c) as well as ZIKV-specific neutralizing antibody titers >10 (Table 1). In addition, peak viral loads on day 3 were inversely correlated with antibody titers (P < 0.0001, R2 = 0.72; Fig. 2d). These data suggest that Env-specific antibodies were critical for the protective efficacy of DNA vaccines against ZIKV-BR challenge. Mice that received two immunizations with the prM-Env DNA vaccine at week 0 and week 4 developed high neutralizing antibody titers of 1,022 at week 8 (Table 1) and were also protected against ZIKV-BR challenge (data not shown).

The prM-Env DNA vaccine also provided complete protection against ZIKV-BR challenge in SJL mice (Extended Data Fig. 4) and against both ZIKV-BR and ZIKV-PR challenge in C57BL/6 mice (Extended Data Figs 5 and 6). ZIKV-BR replicated efficiently in SJL mice, consistent with a previous study11, although at slightly lower levels (mean peak viral load 4.70 log copies per ml; range 3.50–5.92 log copies per ml; n = 5) than in Balb/c mice (Fig. 2a). In contrast, both ZIKV-BR and ZIKV-PR replicated poorly in C57BL/6 mice (Extended Data Fig. 5), also consistent with previous reports, potentially as a result of robust IFN-α-mediated innate immune restriction in this strain of mice10,11,19,20.

To investigate the immunological mechanism of protection against ZIKV-BR challenge, we purified IgG from serum from Balb/c mice vaccinated with prM-Env DNA. Passive infusion of varying quantities of purified IgG by the i.v. route resulted in median Env-specific log serum antibody titers of 2.82 (high), 2.35 (mid) and 1.87 (low) in recipient mice following adoptive transfer (Fig. 3a). All recipient mice with log serum antibody titers of 2.35 or higher were protected against ZIKV-BR challenge (Fig. 3b, c), demonstrating that protection can be mediated by vaccine-elicited IgG alone and confirming that the magnitude of Env-specific antibody titers correlates with protective efficacy (P < 0.0001, Fig. 3b). In contrast, only 1 out of 5 recipient mice that received low levels of Env-specific IgG were protected, although they still exhibited reduced viral loads compared with sham controls (Extended Data Fig. 7). These data define the minimum threshold of Env-specific antibody titers required for protection in this model.

Figure 3: Mechanistic studies. a, Env-specific serum antibody titers in recipient Balb/c mice (n = 5 per group) following adoptive transfer of varying amounts (high, mid, low) of IgG purified from serum from mice vaccinated with prM-Env DNA or naive mice (sham). b, Correlates of protective efficacy. c, Serum viral loads in mice that received adoptive transfer of purified IgG from vaccinated mice and were challenged with ZIKV-BR. d, Serum viral loads in prM-Env-DNA-vaccinated mice that were depleted of CD4+ and/or CD8+ T lymphocytes before challenge with ZIKV-BR. Red bars reflect medians. P values reflect t-tests. PowerPoint slide Full size image

We next depleted CD4+ and/or CD8+ T lymphocytes in prM-Env-vaccinated mice on day −2 and day −1 before challenge (>99.9% efficiency; Extended Data Fig. 8). Depletion of these T-lymphocyte subsets did not detectably abrogate the protective efficacy of the prM-Env DNA vaccine against ZIKV-BR challenge (Fig. 3d). These data indicate that Env-specific T-lymphocyte responses were not required for protection in this model, although these findings do not exclude the possibility that ZIKV-specific cellular immune responses may be beneficial in other settings.

To extend these observations to a vaccine platform that has historically provided clinical efficacy against other flaviviruses, we explored the immunogenicity and protective efficacy of a ZIKV purified inactivated virus (PIV) vaccine derived from the Puerto Rico PRVABC59 strain. Groups of Balb/c mice (n = 5 per group) received a single immunization of 1 μg of the PIV vaccine with alum or alum alone by the i.m. or subcutaneous (s.c.) routes. Antibody titers were higher in the group that received the PIV vaccine by the i.m. route rather than by the s.c. route, as compared by ELISA (Fig. 4a). The PIV vaccine by both routes also induced ZIKV-specific neutralizing antibodies after a single immunization (Table 1). At week 4, all mice were i.v. challenged with ZIKV-BR as described above. Complete protection was observed in the group that received the PIV vaccine by the i.m. route (Fig. 4b, c). Two mice that received the PIV vaccine by the s.c. route showed brief low levels of viraemia (Fig. 4c), potentially consistent with the lower Env-specific binding antibody titers in this group (Fig. 4b).

Figure 4: Immunogenicity and protective efficacy of the PIV vaccine. Balb/c mice (n = 5 per group) received a single immunization by the i.m. or s.c. route with 1 μg PIV vaccine with alum, or alum alone, and were challenged at week 4 by the i.v. route with 105 viral particles (102 PFU) ZIKV-BR. a, Humoral immune responses were assessed at week 3 following vaccination by Env-specific ELISA. b, Correlates of protective efficacy. c, Serum viral loads are shown following ZIKV-BR challenge. Red bars reflect medians. P values reflect t-tests. PowerPoint slide Full size image

Our data demonstrate that a single immunization with a DNA vaccine or a PIV vaccine provided complete protection against parenteral ZIKV challenge in mice. The prM-Env DNA vaccine afforded protection in three strains of mice and against both ZIKV-BR and ZIKV-PR challenges, suggesting the generalizability of these observations. Protective efficacy was mediated by vaccine-elicited Env-specific antibodies, as evidenced by (1) statistical analyses of immune correlates of protection (Figs 2c, d), (2) adoptive transfer studies with purified IgG from vaccinated mice (Fig. 3a–c), and (3) T-lymphocyte depletion studies in vaccinated mice (Fig. 3d). The adoptive transfer studies also defined the threshold of Env-specific antibody titers required for protection in this model.

It is difficult to extrapolate directly the results from these vaccine studies in mice to potential clinical efficacy in humans. Nevertheless, the robust protection observed in the present studies and the clear immune correlates of protection suggest a path forward for ZIKV vaccine development in humans. Of note, similar antibody-based correlates of protection, including neutralizing antibody titers >10, have been reported for other flavivirus vaccines, including yellow fever virus, tick-borne encephalitis virus, and Japanese encephalitis virus21,22,23. Moreover, the ZIKV-BR challenge isolate used in the present study has been shown in wild-type SJL mice to recapitulate certain key clinical findings of ZIKV infection in humans, including fetal microcephaly and intrauterine growth retardation11. ZIKV-BR did not lead to a fatal outcome in wild-type Balb/c and SJL mice, as has been observed in Ifnar−/− C57BL/6 mice10,19,20, but the magnitude and duration of viraemia in Balb/c and SJL mice appear comparable with that in humans2, suggesting the potential relevance of this model. It is notable that ZIKV-BR replicated efficiently in wild-type Balb/c and SJL mice (Fig. 2a, Extended Data Fig. 4), but replicated poorly in wild-type C57BL/6 mice (Extended Data Fig. 5), which is consistent with previous observations10,11 and indicates important strain-specific differences for ZIKV infectivity. Further investigation into the immunologic mechanisms underlying these differences may lead to insights into innate immune control of ZIKV. Moreover, further characterization of the susceptible Balb/c and SJL murine models may facilitate future studies of ZIKV pathogenesis and the development of antiviral interventions. Future studies will also need to address the potential relevance of cross-reactive antibodies against dengue virus and other flaviviruses on ZIKV vaccine immunogenicity and protective efficacy.

The epidemiology of the current ZIKV outbreak1,2 and the clinical consequences for fetuses in pregnant women who become infected3,4,5,6,7,8 necessitate the urgent development of a ZIKV vaccine. Our data demonstrate that complete protection against ZIKV challenge was reliably and robustly achieved with both DNA vaccines and purified inactivated virus vaccines in susceptible mice. These vaccine platforms have previously been used at comparable doses to develop vaccines for other flaviviruses, including West Nile virus24,25, dengue viruses26,27, tick-borne encephalitis virus28,29, and Japanese encephalitis virus30, and may offer safety advantages over live attenuated and replicating flavivirus vaccines, particularly for pregnant women. Moreover, the magnitude of Env-specific antibody titers that provide complete protection against ZIKV challenge in mice should be readily achievable by DNA vaccines and purified inactivated virus vaccines in humans. Taken together, our findings provide substantial optimism that the development of a safe and effective ZIKV vaccine for humans will probably be feasible.