Human antibody response to Zika targets type-specific quaternary structure epitopes

Abstract

The recent Zika virus (ZIKV) epidemic in the Americas has revealed rare but serious manifestations of infection. ZIKV has emerged in regions endemic for dengue virus (DENV), a closely related mosquito-borne flavivirus. Cross-reactive antibodies confound studies of ZIKV epidemiology and pathogenesis. The immune responses to ZIKV may be different in people, depending on their DENV immune status. Here, we focus on the human B cell and antibody response to ZIKV as a primary flavivirus infection to define the properties of neutralizing and protective antibodies generated in the absence of preexisting immunity to DENV. The plasma antibody and memory B cell response is highly ZIKV type-specific, and ZIKV-neutralizing antibodies mainly target quaternary structure epitopes on the viral envelope. To map viral epitopes targeted by protective antibodies, we isolated 2 type-specific monoclonal antibodies (mAbs) from a ZIKV case. Both mAbs were strongly neutralizing in vitro and protective in vivo. The mAbs recognize distinct epitopes centered on domains I and II of the envelope protein. We also demonstrate that the epitopes of these mAbs define antigenic regions commonly targeted by plasma antibodies in individuals from endemic and nonendemic regions who have recovered from ZIKV infections.

Keywords: Adaptive immunity; B cells; Immunoglobulins; Immunology; Virology.

Figure 1. Primary serologic response to ZIKV.

(A) Plasma from 4 primary ZIKV cases (DT168, DT172, DT206, and DT244) were tested for IgG binding to ZIKV (top) and DENV (bottom) over the dilution series indicated on the x axis. (B) Primary ZIKV plasma and primary (DT001) and secondary (DT000) DENV plasma were tested for IgG binding to ZIKV recombinant E (ZIKV E80), DENV recombinant E (DENV E80), ZVEDI, and ZVEDIII. (C) Neutralization assays were also performed for each primary ZIKV plasma as well as a secondary (2°) DENV control. NHS, naive human plasma (negative binding control for ELISA).

Figure 2. Antibodies against quaternary epitopes are the predominant mediators of ZIKV neutralization.

(A) Depletion of ZIKV E80–binding IgG in primary ZIKV plasma was confirmed by direct antigen-coating ELISA comparing ZIKV E80–binding IgG in depleted (red bars) to MBP-control-depleted (white bars) or undepleted (black bars) plasma. Mean optical density values from flavivirus-uninfected plasma (background) are subtracted from each group. (B) IgG binding to ZIKV in depleted plasma was tested by antigen-capture ELISA. Mean optical density values from flavivirus-uninfected plasma (background) are subtracted from each group. (C) FRNT assays were performed for ZIKV E80–depleted plasma and controls using ZIKV H/PF/2013. (D) FRNT assays were performed for ZIKV VLP–depleted plasma and controls using ZIKVH/PF/2013.

Figure 3. Frequency of ZIKV-specific and cross-reactive MBCs.

MBCs were transduced using the 6XL method and culture supernatants assessed for ZIKV- and DENV-binding IgG. Pie charts show the proportion of ZIKV-specific and cross-reactive wells for 2 donors with prior primary ZIKV infection. The table below delineates the raw numbers used to calculate the proportions shown in pie charts and the total frequency of ZIKV-reactive MBCs for each donor. ZIKV-TS, wells were designated ZIKV-type specific when IgG ELISA result for that well was positive for ZIKV and negative for DENV antigen. ZIKV-CR, ZIKV cross-reactive, wells IgG positive for both ZIKV and DENV antigen.

Figure 4. mAbs from primary ZIKV case exhibit potent ZIKV-specific neutralization and confer in vivo protection against lethal ZIKV challenge.

(A) Antigen-capture ELISA for IgG binding was performed for 2 candidate ZIKV mAbs and 2 control mAbs (C10, ZIKV and DENV neutralizing; 2D22, DENV2 neutralizing) against DENV4 (left) and ZIKV (right). (B) Binding to ZIKV E monomers and EDI and EDIII was assessed for each mAb. (C) Competition assays (BOB) with a panel of mAbs with known binding specificities was performed to localize the epitopes of A9E and G9E. (D and E) FRNT assays against a panel of ZIKV strains and related flaviviruses were performed for A9E (D) and G9E (E). (F and G) Four- to 6-week-old Ifnar^–/– mice were treated with 200 μg of indicated A9E, G9E, or polyclonal human IgG as a negative control on day –1 and challenged with 1,000 FFU of ZIKV (H/PF/2013). Weight loss (F) and mortality (G) were monitored for 14 days after infection. Results represent 6 to 7 mice per group combined from 2 independent experiments. Weights are shown as mean ± SEM and were censored upon the first death in the group. NA, not applicable.

Figure 5. Epitope mapping of ZIKV-neutralizing mAb.

(A–C) Escape mutants for A9E were generated from PRVABC59. (A) Binding of indicated mAb (left) and plasma (right) against A9E escape mutants from 2 independent experiments is shown. (B) Neutralization of 2 A9E escape mutants from 2 independent experiments by indicated mAb (top) and plasma (bottom) is shown. (C) ZIKV E homodimer with escape mutations indicated. (D) Amino acid residues critical for A9E mAb and G9E Fab binding were determined by alanine scanning shotgun mutagenesis. Plots show the binding of A9E and G9E versus control mAbs. The data point in red corresponds to the alanine mutant that significantly reduces probe mAb binding compared with loading control mAbs. (E) Critical residues (green spheres) discovered in alanine mutagenesis mapping are represented on a 3-dimensional model from a ZIKV cryo-EM structure (PDB ID: 5IRE). The fusion loop of E domain II is in cyan, domain I is in red, domain II is in yellow, and domain III is in blue.

Figure 6. A9E and G9E epitope-binding IgGs are widely represented in polyclonal plasma following natural ZIKV infection.

(A) Blockade of binding (BOB) against A9E and G9E was tested among plasma at a 1:20 dilution from ZIKV and DENV cases from the UNC Traveler’s study, Nicaragua, and Sri Lanka, as was done for mAbs in Figure 4C. (B) The ZIKV cases were subdivided into primary (1°) and secondary (2°) ZIKV (ZIKV infection in a DENV-immune host). (C) Paired plasma specimens at 1:10 dilution from symptomatic ZIKV cases in Nicaragua were analyzed by BOB at early (day 21 after symptom onset) versus late (6 months after symptom onset) convalescence. An unpaired Student’s t test was performed to determine differences in means between groups as indicated by bars. **P < 0.01; ***P < 0.001; ****P < 0.0001. ns, not significant.