Neutralizing human antibodies prevent Zika virus replication and fetal disease in mice

Abstract

Zika virus (ZIKV) is an emerging mosquito-transmitted flavivirus that can cause severe disease, including congenital birth defects during pregnancy. To develop candidate therapeutic agents against ZIKV, we isolated a panel of human monoclonal antibodies from subjects that were previously infected with ZIKV. We show that a subset of antibodies recognize diverse epitopes on the envelope (E) protein and exhibit potent neutralizing activity. One of the most inhibitory antibodies, ZIKV-117, broadly neutralized infection of ZIKV strains corresponding to African and Asian-American lineages. Epitope mapping studies revealed that ZIKV-117 recognized a unique quaternary epitope on the E protein dimer-dimer interface. We evaluated the therapeutic efficacy of ZIKV-117 in pregnant and non-pregnant mice. Monoclonal antibody treatment markedly reduced tissue pathology, placental and fetal infection, and mortality in mice. Thus, neutralizing human antibodies can protect against maternal-fetal transmission, infection and disease, and reveal important determinants for structure-based rational vaccine design efforts.

Extended Data Figure 1. Binding of human mAbs to Zika E protein, E DIII or E-FLM

mAbs are organized by competition binding groups A to D. Purified mAbs were tested for binding to different antigens as indicated in ELISA as described in Methods. Non-linear regression analysis of the data was performed, and the data plotted are the mean and s.d.

Extended Data Figure 2. High resolution epitope mapping of ZIKV mAbs

a, An alanine scanning mutation library for ZIKV envelope protein was constructed, in which each amino acid of prM/E was mutated individually to alanine (and alanine to serine) and expression constructs arrayed into 384-well plates, one mutation per well. Each clone in the ZIKV prM/E mutation library, expressed in HEK-293T cells, was tested for immunoreactivity with five mAbs from competition groups A–D, measured using an Intellicyt high-throughput flow cytometer. Shown here for each of the five mAbs is the reactivity with the ZIKV E protein mutants that identified the epitope residues for these mAbs. mAb reactivity for each alanine mutant are expressed as percent of the reactivity of mAb with wild-type ZIKV prM/E. Clones with reactivity < 30% relative to wild-type ZIKV prM/E were identified as critical for mAb binding. Bars represent the mean and range of at least two replicate data points. Binding of group B mAbs, ZIKV-116 to wild-type ZIKV E DIII (b) or DIII LR mutant (c) was compared with mouse mAbs ZV-2 and ZV-54. Binding of ZIKV-116 was decreased by mutations in DIII-LR. Data plotted are mean ± s.d.

Extended Data Figure 3. Binding of human mAbs to permeabilized DENV-infected C6/36 cells

C6/36 cells were infected with DENV-1, DENV-2, DENV-3, DENV-4 or mock infected. Cells were stained with the indicated anti-ZIKV mAbs, an isotype negative control (hCHK-152), or a positive control (a cross-reactive antibody to DENV; chimeric human E60 (chE60)) and processed by flow cytometry. The data are representative of two independent experiments. The numbers in the box indicate the fraction of cells that stained positively.

Extended Data Figure 4. Detection of human IgG in placenta or fetal head tissues in ZIKV-117- or PBS-treated pregnant mice

As described in Fig. 3, wild-type female mice were mated with wild-type sires and monitored for pregnancy. At E5.5, dams were treated with anti-Ifnar1 mAb and PBS or 250 μg of ZIKV-117. One day later (E6.5), dams were inoculated with 10³ FFU of ZIKV-Dakar. Fetuses and placentas (n = 4 each) were collected on E13.5, homogenized, and tested for human IgG by ELISA. Human antibody in tissues was captured on ELISA plates coated with ZIKV E protein and detected using goat anti-human IgG (Fc-specific) antibody. The quantity of antibody was determined by comparison with a standard curve constructed using purified ZIKV-117 in a dilution series. Four replicate measurements were performed for each mouse tissue and the results were averaged. The graphs represent the mean + s.e.m. from 3 mice per group.

Extended Data Figure 5. Comparison of wild-type and LALA-mutated antibodies

a, Binding to recombinant human FcγR1. The functional abrogation of the binding of the LALA variant IgG was confirmed in an ELISA binding assay with recombinant human FcγRI. ZIKV-117 wild-type bound to FcγRI, whereas the ZIKV-117 LALA antibody did not. Wild-type and LALA versions of another human mAb, CKV063, were used as controls. Binding to human FcγRI is one representative experiment of two, and error bars indicate s.e.m. of triplicate technical replicates. b, Neutralization assays. Wild-type ZIKV-117 and LALA antibodies exhibited equivalent neutralizing activity in vitro to each other and to the hybridoma-derived antibody. Neutralization assays are representative of two independent experiments completed in triplicate.

Extended Data Figure 6. In situ hybridization of Ifnar1^+/− placenta after inoculation with ZIKV-Brazil and treatment with ZIKV-117

As described in Fig. 3a, Ifnar1^−/− female mice were mated with wild-type sires and monitored for pregnancy. At E5.5, dams were treated with 250 μg of either hCHK-152 isotype control or ZIKV-117. At E6.5, dams were inoculated with 10³ FFU of ZIKV-Brazil. Collected placentas were fixed in 10% neutral buffered formalin at ambient temperature and embedded in paraffin. At least three placentas from different litters with the indicated treatments were sectioned for in situ hybridization staining using negative or ZIKV-specific RNA probes. Low (scale bar, 500 μm) and high (scale bar, 50 μm) power images are presented in sequence.

Figure 1. Human antibody and B-cell response to ZIKV infection

a, b, Serum samples from humans with a previous ZIKV infection were tested for binding to ZIKV E protein in ELISA (a) (with two technical replicates) and neutralization of ZIKV (b) (at least two independent repeats in triplicate). Subject 1001 had the highest endpoint titre in the binding assay and displayed potent neutralizing activity. Subject 657 was a control without history of exposure to ZIKV. c, Supernatants of Epstein–Barr virus (EBV)-transformed B-cell cultures from subject 1001 were tested for binding to ZIKV E or DIII of ZIKV E or related flavivirus E proteins; the WNV-reactive clone and all but one DENV-reactive B-cell line also reacted with ZIKV E protein. The frequency of antigen-specific cells against each viral protein was determined with a threshold absorbance value at 405 nm (A_{405 nm}) of 1.5 as indicated. d, In four additional separate B-cell transformation experiments, the frequency of B cells reactive with intact ZIKV E or E-FLM was determined.

Figure 2. Characterization of anti-ZIKV mAbs

a, We tested 29 mAbs in binding, neutralization, and competition binding assays. The EC₅₀ against ZIKV E and the IC₅₀ (by focus reduction neutralization test) against H/PF/2013 strain for neutralizing antibodies (highlighted in blue) are shown. The mAbs are displayed in four groups (A, B, C or D) based on a competition binding assay. The values are the percentage of binding that occurred during competition compared to non-competed binding, which was normalized to 100% and the range of competition is indicated by the box colours. Black filled boxes indicate strongly competing pairs (residual binding < 30%), grey filled boxes indicate intermediate competition (residual binding 30–69%), and white filled boxes indicate non-competing pairs (residual binding ≥ 70%). The IC₅₀ against H/PF/2013 strain for neutralizing antibodies is shown with neutralizing clones highlighted in blue. b, A ribbon diagram of three protomers of ZIKV E (DI in red, DII in yellow and DIII in blue) is shown with critical residues highlighted as spheres from epitope mapping experiments for representative antibodies in each of the competition binding groups. The colours of the critical residues correspond to the competition group designation as in a. The mutations in the E-FLM and DIII-LR mutants are indicated by black and silver spheres, respectively. c, Representative mAbs from each competition binding group are listed with the domains and residues critical for binding. FL, fusion loop. d, Two mAbs were tested for neutralization of five strains of ZIKV. The concentrations (ng ml⁻¹) at which 50% or 90% neutralization occurred are listed in e. The neutralization data are pooled from at least three independent experiments performed in triplicate.

Figure 3. Protective activity of ZIKV-117 in adult male and pregnant female mice

a, We treated 4–5-week-old wild-type male mice with 2 mg of anti-Ifnar1 mAb followed by subcutaneous inoculation with 10³ FFU of mouse-adapted ZIKV-Dakar. Mice were treated with a single 100 μg or 250 μg dose of isotype control mAb (hCHK-152) or ZIKV-117 on D+1 or D+5 (n = 10 per group from two independent experiments), respectively. Significance was analysed by the log-rank test (*P < 0.05; **P < 0.01). b, c, Ifnar1^−/− female mice were mated with wild-type sires. At E5.5, dams were treated with 250 μg of either hCHK-152 isotype control mAb or ZIKV-117. Bars indicate the median values and reflect data pooled from four independent experiments. Significance for fetal survival and viral RNA was analysed by chi-square (b; ****P < 0.0001) and Mann–Whitney (c; *P < 0.05) tests, respectively. d–f, Wild-type female mice were mated with wild-type sires. At E5.5, dams were treated with anti-Ifnar1 mAb and one of the following: PBS (d, e), 250 μg (d–f) of hCHK-152 isotype control mAb, 250 μg of ZIKV-117 (d–f) or 250 μg of ZIKV-117 LALA (f). At E6.5, dams were inoculated with 10³ FFU of ZIKV-Dakar. d–f, Fetuses and placentas (d, f) and maternal brain and serum (e) were collected on E13.5 and viral RNA was measured by qRT–PCR. Bars indicate the median values of samples collected from three biological replicates (d, n = 20–36; e, n = 5–9; f, n = 23–28). Significance was analysed by ANOVA with a Dunn’s multiple comparison test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). g, h, Wild-type female mice were mated with wild-type sires. At E5.5, dams were treated with anti-Ifnar1 mAb. At E6.5, dams were inoculated with 10³ FFU of ZIKV-Dakar. At E7.5 (day +1 after infection), dams were treated with PBS, 250 μg of hCHK-152 isotype control mAb, or 250 μg of ZIKV-117. g, h, Fetuses and placentas (g) and maternal brain and serum (h) were collected on E13.5 and viral RNA was measured by qRT–PCR. Bars indicate the median values of samples collected from three biological replicates (g, n = 8–20; h, n = 3–7). Significance was analysed by ANOVA with Dunn’s (g) or Tukey’s (h) multiple comparisons test (*P < 0.05, ***P < 0.001, ****P < 0.0001). Dashed lines indicate the limit of detection of the assay.

Figure 4. Effect of ZIKV-117 treatment on the placenta and the fetus

a, Cartoon depicting murine placental structures and zones. b–e, Pregnant dams were treated with PBS, hCHK-152, or ZIKV-117 as described in Fig. 3d–f before infection with ZIKV-Dakar or mock-infected. b, Haematoxylin and eosin staining of placenta at E13.5. Placental labyrinth zone is marked with a solid line. Low power (scale bar, 1 mm) and high power (scale bar, 50 μm) images are presented in sequence. Black arrows indicate apoptotic trophoblasts in areas corresponding to regions of ZIKV infectivity (see panel d, below). c, Measurements of thickness and indicated areas of placenta and fetus body size. Each symbol represents data from an individual placenta or fetus. Significance was analysed by ANOVA with a Dunn’s multiple comparison test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, P > 0.05, NS, not significant). d, In situ hybridization. Low (scale bar, 500 μm) and high (scale bar, 50 μm) power images are presented in sequence. Black arrows indicate cells positive for ZIKV RNA in the junctional zone of the placenta. The images in panels are representative of several placentas from independent dams. e, Low (scale bar, 50 μm) and high (scale bar, 10 μm) power magnified images of immunofluorescence staining of placentas for vimentin (in green, which marks fetal capillary endothelium) from ZIKV-infected dams treated with PBS or ZIKV-117 or from uninfected pregnant animals. Nuclei are counter-stained blue with DAPI.