Development of a chimeric Zika vaccine using a licensed live-attenuated flavivirus vaccine as backbone

Abstract

The global spread of Zika virus (ZIKV) and its unexpected association with congenital defects necessitates the rapid development of a safe and effective vaccine. Here we report the development and characterization of a recombinant chimeric ZIKV vaccine candidate (termed ChinZIKV) that expresses the prM-E proteins of ZIKV using the licensed Japanese encephalitis live-attenuated vaccine SA14-14-2 as the genetic backbone. ChinZIKV retains its replication activity and genetic stability in vitro, while exhibiting an attenuation phenotype in multiple animal models. Remarkably, immunization of mice and rhesus macaques with a single dose of ChinZIKV elicits robust and long-lasting immune responses, and confers complete protection against ZIKV challenge. Significantly, female mice immunized with ChinZIKV are protected against placental and fetal damage upon ZIKV challenge during pregnancy. Overall, our study provides an alternative vaccine platform in response to the ZIKV emergency, and the safety, immunogenicity, and protection profiles of ChinZIKV warrant further clinical development.

Fig. 1

Construction and characterization of ChinZIKV. a Strategy for construction of the chimeric ZIKV vaccine ChinZIKV and schematic representation of its genome. b Immunostaining of BHK-21 cells infected with ChinZIKV-, ZIKV-, or mock-infected at an MOI of 0.01 at 48 h post-infection with ZIKV patient anti-serum and JEV NS1 antibodies. Scale bar: 100 μm. c Plaque morphology of ZIKV, the recovered ChinZIKV, and ChinZIKV at passages 3 and 6 on BHK-21 cells. BHK-21 cells were infected with the indicated viruses, and plaques were developed after 96 h. d Growth curves of the recovered ChinZIKV and ChinZIKV at passage 6 in Vero cells. Vero cells were infected with the indicated viruses at an MOI of 2 and the cell supernatants were collected at the indicated times for determination of virus titers by plaque assay using BHK-21 cells. The data are representative of at least three independent experiments and error bars indicate the SD

Fig. 2

ChinZIKV is highly attenuated in mice and non-human primates. a, b Viral load in serum in mice inoculated with ChinZIKV or ZIKV. Four-week-old male mice were infected with 10³ (A129) or 10⁵ PFU (BALB/c) of the indicated viruses by the s.c. or i.p. routes, respectively. Viral RNA loads were determined by reverse transcription-quantitative PCR (RT-qPCR). Dotted lines indicate the detection limit. The data are representative of at least three independent experiments, and error bars indicate the SD. Significance was calculated using a two-way ANOVA with multiple comparison tests a, b (**P-value < 0.01; ****P-value < 0.0001). Viral load in serum c, urine d, and saliva e of monkeys infected with ChinZIKV or ZIKV. Monkeys were infected with 10⁵ PFU of ChinZIKV or ZIKV by the s.c. route. Blood and tested fluids were collected at the indicated times. Viral RNA amounts in the tested samples were determined by RT-qPCR

Fig. 3

ChinZIKV elicits ZIKV-specific humoral and cellular immune responses in non-human primates. a, b Monkeys were immunized s.c. with a single dose of ChinZIKV (n = 5) or PBS as a control (n = 3). ZIKV-specific IgG and neutralizing antibody titers were determined by ELISA and PRNT₅₀, respectively. Dotted lines indicate the limits of detection. c PBMCs from immunized monkeys were collected at the indicated times. The production of IFN-γ by PMBCs stimulated with the ZIKV E protein was measured by ELISPOT assay and expressed as spot-forming units (SFU) per 10⁶ PBMCs. Experiments were performed in duplicate (error bars represent SD). Significance was calculated using a one-way ANOVA with multiple comparison tests (*, P-value < 0.05; **, P-value < 0.01). d Heat map of the cytokine profile in sera from the monkeys inoculated with ChinZIKV or ZIKV at day 7 post inoculation. Each sample was analyzed using a Monkey Cytokine Magnetic 29-Plex Panel kit. Each cytokine level is summarized as the log₁₀ of the ratio relative to baseline (Day 0 post infection)

Fig. 4

A single dose of ChinZIKV confers complete protection against ZIKV challenge in mice and monkeys. a Viral load in the sera of immunized mice after ZIKV challenge. The ChinZIKV immunized mice were challenged i.p. with 10³ PFU of ZIKV. Viral RNA loads were determined by RT-qPCR. Dotted lines indicate the detection limit. The data are representative of at least three independent experiments, and error bars indicate the SD. Significance was calculated using a two-way ANOVA with multiple comparison tests (****P-value < 0.0001). b Four-week-old female BALB/c mice were immunized with 10⁴ PFU of ChinZIKV (n = 3) or PBS (n = 2) as a control. On day 60 post immunization, the immunized mice were mated to 10-week-old male BALB/c mice. At embryonic day 13.5 (E13.5), the pregnant mice were infected with 10⁵ PFU of ZIKV by the i.p. route. Viral RNA loads at days 1–3 post-infection were determined by RT-qPCR. Dotted lines indicate the detection limit. The data are representative of at least three independent experiments, and error bars indicate the SD. Significance was calculated using a two-way ANOVA with multiple comparison tests (****P-value < 0.0001). One-day-old suckling mice born to the ChinZIKV-immunized (n = 15) or PBS-immunized (n = 9) dams were challenged i.c. with 100 PFU of ZIKV. The mice were then monitored for clinical symptoms and mortality for 21 days. Asterisks indicate values that are statistically significant (***P-value < 0.001). c The PBS- or ChinZIKV-immunized monkeys were challenged s.c. with 10³ PFU of ZIKV. Serum and body fluids were collected at the indicated times. Viral RNA amounts were determined by RT-qPCR. Dotted lines indicate the detection limit. d Cytokine amounts in the sera of the PBS- or ChinZIKV-immunized monkeys (n = 3, each group) at day 7 post challenge determined by a Monkey Cytokine Magnetic 29-Plex Panel kit. Experiments were performed in duplicate (error bars represent SD). Significance was calculated using the Student’s t test (**P-value < 0.01; ****P-value < 0.0001)

Fig. 5

ChinZIKV immunization prevents vertical ZIKV transmission and fetus injury during pregnancy. a Scheme of immunization of BALB/c female mice with 10⁴ PFU of ChinZIKV or PBS. b–e At day 210 post immunization, vaccinated female mice were mated with BALB/c males. Pregnant mice (n = 3) were administered 2.0 mg of anti-Ifnar1-blocking antibody on E5 and 1 day later (E6) challenged with 10⁵ PFU of ZIKV. Viral RNA loads in serum at days 1–3 post challenge were determined by RT-qPCR b. On E13, animals were killed; maternal spleens and brains c, placenta d, and fetal heads e were collected and viral RNA levels were quantified by RT-qPCR. The data are representative of at least three independent experiments and error bars indicate the SD. f, g Outcome of fetuses from PBS or ChinZIKV vaccinated dams. Scale bar: 1 cm. f Representative images of hemorrhagic uterus (left) and moribund fetus (right) recovered from PBS-immunized moribund dams at E18. g Representative images of pups delivered at term to ChinZIKV vaccinated dams. Dotted lines indicate the detection limit. Significance was calculated using a two-way ANOVA with multiple comparison tests b (**P-value < 0.01; ***P-value < 0.001) or a Student’s t-test c–e (**P-value < 0.01; ****P-value < 0.0001)