Research ArticleIMMUNOLOGY

Maternal immunity and antibodies to dengue virus promote infection and Zika virus–induced microcephaly in fetuses

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Science Advances  27 Feb 2019:
Vol. 5, no. 2, eaav3208
DOI: 10.1126/sciadv.aav3208
  • Fig. 1 Sustained ZIKV infection in female immunocompetent mice.

    ZIKV infection was quantified in peripheral tissues in (A) the cells isolated from the peritoneal cavity, (B) the spleen, (C) the mesenteric lymph node (LN), and (D) iliac lymph nodes, each day for 1 week following infection of female mice with 1 × 106 plaque-forming units (PFU) of ZIKV, strain H/PF/2013. (E) Serum from uninfected or ZIKV-infected dams was harvested 72 hours after infection and absorbed onto BHK-21 cells for focus-forming assays to show replicating virus. Many cells appear infected, staining above baseline levels of uninfected serum, with ZIKV-infected serum diluted 1:2. Individual foci of infection (red circles) surrounded by a few single infected cells can be observed at higher 1:5 dilution of ZIKV serum. Additional images from multiple time points are provided in fig. S1. (F) ZIKV genome copies in the spleen are plotted with the splenic mass. (G) ZIKV was not detected (ND) in the brain of any animals by PCR. n = 5 female mice per group. Error bars represent the SEM.

  • Fig. 2 Maternal DENV immunity increases ZIKV microcephaly in fetuses.

    (A) Schematic of the experimental time course showing that female naïve mice or mice that were infected with DENV2 3 weeks before or mice that were passively transferred the monoclonal antibody 4G2 (50 μg, intraperitoneally) were each crossed to male mice. Dams were infected on E7 after conception, and the fetal development was assessed on E18. (B) ELISAs were performed against equivalent concentrations of DENV or ZIKV antigen using serum isolated 21 days after DENV infection. Serum from DENV-immune animals binds significantly more to DENV than to ZIKV. Graph represents the geometric mean titer twofold over naïve controls, and the error bars represent the SEM. Significance was determined by comparing endpoint dilutions by Student’s paired t test (P = 0.001). IgG, immunoglobulin G. (C) Representative images of fetal mice on a 1-mm2 grid are provided to show that the DENV2-immune and 4G2-injected groups were visually smaller than controls. (D) Cross-sections of embryos stained with toluidine blue to reveal the reduced size of fetuses of naïve, DENV2-immune, and 4G2-injected ZIKV-infected dams. Additional representative images are presented in fig. S3. (E) Mass and (F) head circumference of fetal mice on E18 are presented. (G) Incidence of mice having microcephaly, defined as a head size in the third percentile or less, calculated from the SD of fetuses from naïve dams without ZIKV infection. For (E) and (F), **P < 0.01, ***P < 0.001, and ****P < 0.0001, by one-way analysis of variance (ANOVA) with Holm-Sidak’s multiple comparison test. Error bars represent the SEM. Variances do not differ significantly. For (E), n = 22 (naïve-uninfected), n = 28 (naïve-ZIKV), n = 15 (DENV2-ZIKV), n = 18 (4G2-ZIKV). For (F), n = 17 (naïve-uninfected), n = 18 (naïve-ZIKV), n = 15 (DENV2-ZIKV), n = 11 to 18 (4G2-ZIKV). Groups contain the combined data from all fetuses of three independent dams. (Photo credit: Wilfried A. A. Saron, Duke-NUS Medical School)

  • Fig. 3 Impaired cortical development in ZIKV fetuses is enhanced by maternal DENV immunity.

    (A) Images of embryonic brain sections showing reduced cortical thickness in ZIKV-infected embryos. Tissue sections were cut to 15-μm thickness and Nissl-stained using cresyl violet before imaging by light microscopy. Labels indicate the lateral ventricle (LV) and cortical regions: ventricular zone (VZ), subventricular zone (SVZ), intermediate zone (IZ), cortical plate (CP), and marginal zone (MZ). Additional representative images are included in fig. S6. (B) Quantification of cortical thickness from images. Error bars represent the SEM of average cortical thickness for individual fetuses obtained from three independent pregnancies; n = 5 (naïve-uninfected), n = 6 (naïve-ZIKV), n = 7 (DENV2-ZIKV), and n = 8 (4G2-ZIKV). (C) Normalized ratio of cortical thickness to body mass for fetal mice. Additional controls are provided in fig. S7. (D) Relative expression of transcriptional factors critical for neuronal growth in the fetal mouse brains (normalized to Actin) as determined by real-time reverse transcription PCR (RT-PCR). Error bars represent the SEM and n = 5 fetuses per group. For (B) to (D), *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, as determined by one-way ANOVA with Holm-Sidak’s multiple comparison test. Variances do not differ among groups.

  • Fig. 4 Enhanced ZIKV infection of fetuses in DENV-immune dams.

    (A to C) ZIKV genome copies were quantified by PCR after injection of ultraviolet (UV) inactivation of the virus compared to injection of live virus in tissues including the (A) dams’ spleens (**P = 0.0047), (B) placentas, or (C) fetuses on E10, 3 days after infection. Input UV-ZIKV could be detected only in the dam’s spleen but at significantly lower genome copy numbers than live virus. PCR specific for the ZIKV negative strand was performed using tissue (D) from the mother’s spleen days 1 and 3 after infection and (E) from embryos or placentas on E10, 3 days after infection. For (D) and (E), uninfected and ZIKV-infected vero cell lysates were used as negative and positive controls, and uninfected tissues were used as negative controls. Expected band size is 188 base pairs (bp). The gels confirm active ZIKV replication in mother and fetal mice. (F) Real-time RT-PCR was used to quantify the ZIKV genome copies in the mouse fetuses of DENV-naïve uninfected dams (n = 10), DENV-naïve ZIKV-infected dams (n = 12), DENV2-immune ZIKV-infected dams (n = 17), and 4G2-injected ZIKV-infected dams (n = 10) derived from two to three independent experiments. Fetuses were harvested on E10 (3 days after maternal infection) or on E18 (11 days after maternal infection) for RNA isolation. Fetuses from DENV2-immune and 4G2-injected dams showed significant increases in ZIKV compared to those of naïve dams by one-way ANOVA with Holm-Sidak’s multiple comparison test (*P < 0.01, **P < 0.001). (G) Quantification of ZIKV infection in the spleen of dams 3 days after infection when the fetuses were harvested on E10 (n = 3 per group). *P < 0.05. Error bars represent the SEM. (H) Quantification of ZIKV infection in the placentas at E10. ns, not significant. (I) Representative flow cytometry plots showing intracellular staining for ZIKV E protein in vimentin+ fetal endothelial cells from the placenta at E10, following maternal ZIKV infection at E7. FSC, forward scatter. (J) ZIKV E protein was detectable in a significantly larger proportion of vimentin+ cells in the placentas from DENV-immune compared to naïve dams infected with ZIKV (n = 5 per group). (K) Representative flow cytometry plots showing intracellular staining for ZIKV E protein in cytokeratin+ syncytiotrophoblast cells from the placenta at E10, following maternal ZIKV infection at E7. (L) E protein was detectable in a significantly larger proportion of cytokeratin+ cells in the placentas from DENV-immune compared to naïve dams infected with ZIKV (n = 5 per group). (J) and (L): ****P < 0.0001.

  • Fig. 5 Maternal DENV immunity leads to FcRN-dependent enhancement of fetal ZIKV infection.

    Placenta tissue sections from E10 were stained for FcRN, DNA (4′,6-diamidino-2-phenylindole), and either (A) the fetal endothelial cell marker vimentin or (B) the syncytiotrophoblast marker cytokeratin and were imaged by confocal microscopy. Both cytokeratin and vimentin colocalized with FcRN. Scale bars, 50 μm. Images are representative of three independent experiments. (C) FcRN mRNA expression in E8 and E10 mouse placentas. (D) Detection by flow cytometry of FcRN on cells from E8 placentas, gated on vimentin (left) or cytokeratin (right) from uninfected or ZIKV-infected dams, 24 hours after infection at E7. (E) Average mean fluorescence intensity (MFI) of FcRN on cells from E8 placentas, relative to levels on uninfected control endothelial cells (vimentin+); n = 5 per group. (F) Detection by flow cytometry of FcRN on cells from E10 placentas, gated on vimentin (left) or cytokeratin (right) from uninfected or ZIKV-infected dams, 72 hours after infection at E7. (G) Average MFI of FcRN on cells from E8 placentas, relative to levels on uninfected control endothelial cells (vimentin+); n = 5 per group. Flow cytometry gating strategy for (D) to (G) is provided as fig. S11. (H) Schematic showing experimental setup of transwells, where FcRN-expressing HULEC-5a cells were plated on transwell inserts to form a tight monolayer, and trophoblast placenta cells were plated on the bottom chamber. ZIKV or ZIKV + antibody 4G2 were added to the top chamber. (I) Permeability of the monolayers 24 hours after addition of ZIKV or ZIKV with various concentrations of antibodies was measured by quantitating fluorescein isothiocyanate (FITC)–dextran leakage. No differences in leakage were observed compared to uninfected controls. (J) Addition of antibody 4G2 significantly increased the amounts of infection in trophoblast target cells, in a dose-dependent manner, on the opposite side of the transwell insert 24 hours after exposure to ZIKV. (K) Addition of antibody 4G2 significantly increased the detection of ZIKV associated with trophoblast target cells 6 hours after exposure. (L) Addition of DENV-immune human serum (1:500 dilution) with ZIKV promoted enhanced translocation of the virus, determined by plaque assay using supernatants on the opposite side of transwells, 24 hours after exposure. (M) No significant difference was observed in serum endpoint titers fourfold over naïve against DENV2 between WT and FcRN−/− female mice 21 days after infection. P = 0.32 by Student’s unpaired t test; n = 5 per group. (N) Quantification of ZIKV in DENV2-immune (n = 17) or DENV2-naïve (n = 12) WT and DENV2-immune (n = 15) or DENV2-naïve FcRN−/− (n = 21) embryos on E10 derived from three to four independent dams per group. (O) Proportions of PCR-positive embryos for each group in (N). Significance was determined by Fisher’s exact test (*P = 0.01, ns for P = 0.736). (P) Comparison of ZIKV titers for ZIKV+ fetuses alone shows enhanced viral burden in the embryos of DENV2-immune dams for both WT and FcRN−/− groups, determined by one-way ANOVA with Holm-Sidak’s multiple comparison test; *P = 0.0119. n = 6 (DENV-naïve WT), n = 17 (DENV2-immune WT), n = 9 (DENV-naïve FcRN−/−), and n = 8 (DENV2-immune FcRN−/−), corresponding to the PCR-positive embryos from (N). For (N) to (P), all fetal measurements were derived from three to four independent dams. KO, knockout. (Q and R) Blocking antibody against FcRN (4C9) or IC antibodies were injected therapeutically into DENV-immune pregnant mice (n = 3 dams) before ZIKV infection on E7. Head circumference of the fetal mice was measured on E18. (Q) Head circumferences and (R) cortical thicknesses were significantly increased in 4C9-treated fetal mice compared to those given IC (P < 0.0001). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/2/eaav3208/DC1

    Supplementary Materials and Methods

    Fig. S1. Infectious ZIKV can be detected in the serum at multiple time points after infection in mice.

    Fig. S2. DENV-immune serum neutralizes DENV but not ZIKV.

    Fig. S3. Reduced fetal size during ZIKV infection enhanced by DENV immunity.

    Fig. S4. Additional control groups demonstrate the requirement of cross-reactive antibodies and ZIKV for enhanced microcephaly.

    Fig. S5. Immunocompromised dams die during pregnancy because of ZIKV infection.

    Fig. S6. Reduced cortical thickness in ZIKV-infected mice enhanced by maternal DENV immunity.

    Fig. S7. Normalized ratio of cortical thickness to body mass for fetal mice.

    Fig. S8. Confirmation of protein-level reduction of BRN1 and PAX6 in fetal brains during maternal ZIKV infection.

    Fig. S9. ZIKV viral genome copies in pregnant versus nonpregnant female mice.

    Fig. S10. ZIKV antigens detected in the fetal cortex.

    Fig. S11. Flow cytometry gating strategy to identify syncytiotrophoblasts and fetal endothelial cells.

    Fig. S12. IC antibody injection does not affect brain development in uninfected mice.

    References (54, 55)

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Materials and Methods
    • Fig. S1. Infectious ZIKV can be detected in the serum at multiple time points after infection in mice.
    • Fig. S2. DENV-immune serum neutralizes DENV but not ZIKV.
    • Fig. S3. Reduced fetal size during ZIKV infection enhanced by DENV immunity.
    • Fig. S4. Additional control groups demonstrate the requirement of cross-reactive antibodies and ZIKV for enhanced microcephaly.
    • Fig. S5. Immunocompromised dams die during pregnancy because of ZIKV infection.
    • Fig. S6. Reduced cortical thickness in ZIKV-infected mice enhanced by maternal DENV immunity.
    • Fig. S7. Normalized ratio of cortical thickness to body mass for fetal mice.
    • Fig. S8. Confirmation of protein-level reduction of BRN1 and PAX6 in fetal brains during maternal ZIKV infection.
    • Fig. S9. ZIKV viral genome copies in pregnant versus nonpregnant female mice.
    • Fig. S10. ZIKV antigens detected in the fetal cortex.
    • Fig. S11. Flow cytometry gating strategy to identify syncytiotrophoblasts and fetal endothelial cells.
    • Fig. S12. IC antibody injection does not affect brain development in uninfected mice.
    • References (54, 55)

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