Vaccine Mediated Protection Against Zika Virus-Induced Congenital Disease

Abstract

The emergence of Zika virus (ZIKV) and its association with congenital malformations has prompted the rapid development of vaccines. Although efficacy with multiple viral vaccine platforms has been established in animals, no study has addressed protection during pregnancy. We tested in mice two vaccine platforms, a lipid nanoparticle-encapsulated modified mRNA vaccine encoding ZIKV prM and E genes and a live-attenuated ZIKV strain encoding an NS1 protein without glycosylation, for their ability to protect against transmission to the fetus. Vaccinated dams challenged with a heterologous ZIKV strain at embryo day 6 (E6) and evaluated at E13 showed markedly diminished levels of viral RNA in maternal, placental, and fetal tissues, which resulted in protection against placental damage and fetal demise. As modified mRNA and live-attenuated vaccine platforms can restrict in utero transmission of ZIKV in mice, their further development in humans to prevent congenital ZIKV syndrome is warranted.

Keywords: Vaccine; antibody; fetus; flavivirus; immunity; microcephaly; pregnancy; transmission.

Figure 1. ZIKV prM-E mRNA LNP vaccine protects pregnant C57BL/6 mice and their developing fetuses

A. Scheme of immunization and boosting of WT C57BL/6 female mice with 10 µg of prM-E or placebo mRNA LNP vaccines. B. Serum was collected at day 49 and analyzed for neutralizing activity (Dowd et al., 2016a). Representative neutralization curves are shown. Error bars denote the range of duplicate technical replicates. C-D. EC50 (C) and EC90 (D) values were calculated for individual animals in each group (n = 19 to 20). The dashed lines indicate the limit of detection of the assay. Asterisks indicate statistically significant differences (Mann-Whitney test: ****, P < 0.001). E-H. At day 56, vaccinated female mice were mated with WT C57BL/6 males. A subset of the mice developed vaginal plugs, and pregnant mice (n = 7 or 8 depending on group pooled from two independent experiments) were administered 2 mg of anti-Ifnar1 blocking antibody on E5, and one day later (E6) challenged with 10⁵ FFU of mouse-adapted ZIKV Dakar 41519. At E13, animals were euthanized and maternal spleen (E), maternal brain (F), placenta (G), and fetal heads (H) were harvested and analyzed for levels of ZIKV RNA. The dashed line indicates the limit of detection of the assay and asterisks indicate significant differences (Mann-Whitney test: **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

Figure 2. Development and characterization of a live-attenuated ZIKV vaccine with mutations in the NS1 gene

A. Scheme of ZIKV genome with mutations in the NS1 gene. Mutated amino acids and their coding nucleotides are indicated in red. B. Western blotting of lysates from Vero cells infected with parental WT, N130Q, N207Q, or N130Q+S132A+N207Q+T209V (DKO) ZIKV with an anti-NS1 antibody. Where indicated, PNGase F treatment was performed on lysates to remove N-linked glycans. Results are representative of several experiments. C-E. Attenuated growth of ZIKV-NS1-LAV (DKO). Plaque assays (C), replication kinetics (D), and transient replicon (E) assays were performed in Vero cells. D. Multi-step growth curves of parental WT and ZIKV-NS1-LAV in Vero cells. Results are the average of two independent experiments, and the error bars indicate standard deviations (SD). E. Replication of parental WT or ZIKV-NS1-LAV subgenomic replicons encoding a luciferase reporter gene after transfection of in vitro derived RNA into Vero cells. Results are the average of two independent experiments, and the error bars indicate SD. F. Scheme of vaccination and challenge of three week-old Ifnar1^−/− A129 male mice with parental and ZIKV-NS1-LAV. G-H. Weight measurements (G) and mortality (H) over the first two weeks after immunization with mock vaccine (G only, n = 4), parental WT (G, n = 5: H, n = 10) or ZIKV-NS1-LAV (n = 5). Arrows (G) and asterisks (H) indicate statistically significant differences: ((G) Two-way ANOVA with Bonferroni multiple comparison test: day 7 and 8, ***, P < 0.001; days 9–12, ****, P < 0.0001; day 13, **, P < 0.01; (H) Log-rank test: *, P < 0.05). I. Viremia measurements at days 1 through 4 after inoculation with parental (n = 5) and ZIKV-NS1-LAV (n = 3) as determined by plaque assay. Dotted line indicates limit of detection of assay. Asterisks indicate statistical significance (Mann-Whitney test: *, P < 0.05; **, P < 0.01; ****, P < 0.0001). J. Blood was collected at day 28 and analyzed for serum neutralizing activity. K. A129 mice that were initially inoculated with placebo (mock-vaccinated) (n = 4), parental WT (n = 4) or ZIKV-NS1-LAV (n = 5) were challenged at day 30 with 10⁶ PFU of ZIKV strain PRVABC59. At day 2 after challenge, viremia was measured.

Figure 3. ZIKV-NS1-LAV protects pregnant C57BL/6 mice and their fetuses

A. Scheme of immunization of WT C57BL/6 female mice with 10⁵ FFU of ZIKV-NS1-LAV (n = 18) or placebo (n = 11) control. One day prior to immunization, all mice were administered 0.5 mg of anti-Ifnar1. B. Serum was collected at day 28 and analyzed for neutralizing activity. Representative neutralization curves are shown. Error bars denote the range of duplicate technical replicates. C-D. EC50 (C) and EC90 (D) values were calculated for individual animals in each group. The dashed lines indicate the limit of detection of the assay. Asterisks indicate statistically significant differences (Mann-Whitney test: ****, P < 0.0001). E-H. At day 35, vaccinated female mice were mated with WT C57BL/6 males. A subset of mice developed vaginal plugs (n = 6, PBS placebo; n = 6, ZIKV-NS1-LAV). Pregnant mice were challenged with ZIKV as described in Fig 1. At E13, animals were euthanized and maternal spleen (E), maternal brain (F), placenta (G), and fetal heads (H) were harvested and analyzed for levels of ZIKV RNA. The dashed line indicates the limit of detection of the assay, and asterisks indicate significant differences (Mann-Whitney test: (*, P < 0.05; **, P < 0.01; ****, P < 0.0001).

Figure 4. prM-E ZIKV vaccine protects against placental and fetal infection

Pregnant dams vaccinated with placebo ot prM-E mRNA LNPs were treated with anti-Ifnar1 and then inoculated with ZIKV-Dakar at E6 as described in Fig 1. A. Measurements of thickness and indicated areas of placentas from placebo or prM-E mRNA LNPs immunized mice after ZIKV challenge. Each symbol represents data from an individual placenta. Statistical significance was analyzed (Mann-Whitney test: *, P < 0.05; **, P < 0.01). B. In situ hybridization. Low power (scale bar = 100 µm) and high power (scale bar = 20 µm) images are presented in sequence (indicated with a red box) from placebo or prM-E mRNA LNPs (immunized mice after ZIKV challenge. The images in panels are representative of three to four independent placentas from multiple dams. C-E. Outcome of fetuses from placebo or prM-E mRNA LNP vaccinated dams. C. The percentage of offspring that were resorbed (fetuses, prior to delivery) or delivered (pups, at term) (n = 17 for placebo; n = 14 for prM-E mRNA LNP vaccine; chi-square test (****, P < 0.0001). D. Representative images of grossly hemorrhagic uterus (left) and hypomorphic fetus and placenta (right) recovered from placebo-immunized moribund dams at E18. E. Representative images of pups delivered at term to prM-E mRNA LNP vaccinated dams. F. Levels of viral RNA in the heads of placebo-vaccinated and ZIKV challenged (harvested from moribund dams at day E18 or by Caesarean section at term) or prM-E mRNA-vaccinated and ZIKV challenged (harvested at delivery). Each symbol represents data from an individual fetus or pup from at least two independent pregnant dams. Statistical significance was analyzed (Mann-Whitney test: ****, P < 0.0001).

Figure 5. ZIKV-NS1-LAV vaccine protects against placental and fetal infection

Pregnant dams vaccinated with placebo or ZIKV-NS1-LAV were treated with anti-Ifnar1 and then inoculated with ZIKV-Dakar at E6 as described in Fig 1. A. Measurements of thickness and indicated areas of placentas from placebo or ZIKV-NS1-LAV immunized mice after ZIKV challenge. Each symbol represents data from an individual placenta. Statistical significance was analyzed (Mann-Whitney test: *, P < 0.05; **, P < 0.01). B. In situ hybridization. Low power (scale bar = 100 µm) and high power (scale bar = 20 µm) images are presented in sequence (indicated with a red box) from placebo or ZIKV-NS1-LAV immunized mice after ZIKV challenge. The images in panels are representative of three to four independent placentas from multiple dams. C. Fetal resorption rates in placebo or ZIKV-NS1-LAV immunized dams after ZIKV challenge. Data are pooled from multiple dams in independent experiments and reflects the following number of fetuses (n = 32 for placebo and n = 48 for ZIKV-NS1-LAV). Significance for fetal survival was analyzed by the chi-square test (*, P < 0.05).