Identification of the seven critical residues that control ZIKV-DENV cross-reactivity to engineer a non-cross-reactive ZIKV vaccine

Abstract

The development of a Zika virus (ZIKV) vaccine is complicated by the high homology between ZIKV and dengue virus (DENV) envelope (E) proteins, resulting in immunological cross-reactivity that can exacerbate disease through antibody-dependent enhancement (ADE). Here, we screen 121 anti-DENV monoclonal antibodies (mAbs) for cross-reactivity with ZIKV E proteins. We identify 70 cross-reactive mAbs, 66 of which have epitopes that included at least one of seven E protein residues conserved among DENV1-DENV4 and ZIKV (R73, E79, W101, L107, F108, K110, and W212), establishing these residues as the key determinants of DENV-ZIKV cross-reactivity. Using these data, we engineer a ZIKV E protein variant with 10 mutations ("ZIKVm10") that reduces cross-reactivity with DENV mAbs in vitro and minimizes the induction of anti-DENV antibodies in immunized mice. Passive serum transfer from ZIKVm10-immunized mice confers near-complete protection against lethal ZIKV challenge and reduced ADE for DENV infection, providing a pathway for improved ZIKV vaccine design.

Keywords: CP: Immunology; CP: Microbiology; antibody enhancement; antigen engineering; epitope mapping; immunogenic cross-reactivity; virus envelope.

Figure 1.. Epitope mapping of 171 mAbs on DENV E glycoprotein

The DENV E glycoprotein consists of five major antigenic regions: the prM protein; DI, DII, and DIII; and the fusion loop of the E protein. (A) Insets show epitope residues (red) identified by shotgun mutagenesis, mapped onto the DENV structure highlighted in each region. The epitopes shown are a summary of mAbs described and mapped previously.^– 82 residues of DENV prM/E protein are recognized by these mAbs. (B and C) 73 distinct residues on E protein form the epitopes of the 133 mapped DENV anti-E mAbs, shown on a DENV2 E monomer (B) and homodimer (C) (PDB: 1OAN). (B–E) Epitope residues are shown as spheres. DI is colored red, DII yellow, and DIII blue. Also shown are the 9 distinct residues shown in dark blue on prM (PDB: 3C5X) that form the epitopes of the 38 mapped DENV anti-prM mAbs. (D) Epitope residues for anti-DENV mAbs that do not cross-react with ZIKV are shown as blue spheres mapped onto a DENV2 monomer (PDB: 1OAN). (E) Epitope residues for anti-DENV mAbs that are cross-reactive with ZIKV are shown as green spheres.

Figure 2.. Identification of anti-DENV mAbs that cross-react with ZIKV prM/E

mAb cross-reactivity against HEK-293T cells expressing ZIKV prM/E was determined for anti-DENV mAbs targeting the fusion loop, DI, DII, DIII, fusion loop/DII, bc loop, and prM. Reactivity is reported as fluorescence signal over background. Data represent the mean and range of two replicate measurements. The x axis indicates individual mAbs ordered by ZIKV reactivity values.

Figure 3.. Summary of cross-reactive epitopes

Cross-reactive epitopes of DENV mAbs were characterized based on (A) neutralization status; mAb critical residues are shown as ZIKV cross-neutralizing mAbs (XNAbs), broadly neutralizing mAbs (bNAbs) for DENV1–DENV4, and DENV-neutralizing mAbs (NAbs). (B–D) Conformational binding, determined from epitope mapping data or prior denaturing western blot experiments (B), conserved (or non-conserved) residue identity across all DENV serotypes and ZIKV (C), and the number of mAbs that bind to each epitope residue (D).

Figure 4.. Engineering of ZIKV E protein reduces DENV mAb cross-reactivity

(A) Key residues on ZIKV E protein that determine cross-reactivity with anti-DENV mAbs were mutated. Fusion loop residues W101, L107, and F108; fusion loop/DII residue K110; bc loop residues R73 and E79; and DII residue W217 (W212 on DENV) were each mutated to 16 different amino acids (shown on ZIKV E: PDB: 5IRE). Mutation to Cys and Pro was avoided due to probable structural perturbations, and mutation to Ala was unnecessary here due to prior Ala-scan mutational studies. Residue numbering aligns with ZIKV. (B) SVP production for each mutant was measured by ELISA. Data points are the average of 3 replicates and are shown as a percentage of non-mutated WT ZIKV SVP production. (C) Cross-reactivity with anti-DENV mAbs that target the specific mutated residue on ZIKV E was evaluated by flow cytometry. Results are shown as a percentage of binding to the WT and are an average of 2 replicates. (D) Cells expressing the engineered ZIKV prM/E construct (ZIKVm10) showed high reactivity with conformational anti-ZIKV mAbs (ZIKV-195, ZIKV-394, and ZIKV-161) binding to different epitopes of E protein and low reactivity with anti-DENV mAbs targeting DII fusion loop (3F21) and the DII bc loop (1C19) but high activity with mAb 1N18 targeting DII at non-engineered ZIKV residue W217. Binding was evaluated by flow cytometry; results are shown as a percentage of binding to the WT and show the average and standard deviation of 4 replicates. (E) SVPs were produced, incorporating WT or ZIKVm10 prM/E, with expression confirmed by ELISA, using titration with a conformational anti-DII mAb ZIKV-117 (after capture by conformational anti-DIII mAb ZV-56). Each data point is the average and range of 2 replicates.

Figure 5.. Immunization with ZIKVm10 SVPs reduces DENV cross-reactivity while maintaining protection from live ZIKV

(A–E) Average reactivity of serum (shown as signal:background) from 15 naive CD-1 mice or mice immunized with WT or ZIKVm10 SVPs, tested by flow cytometry against cells expressing prM/E from (A) ZIKV and (B) DENV2 (*p < 0.05 vs. naive, **p < 0.01, ***p < 0.001; data are represented as mean ± SEM). AG129 mice then received passive immunizations of serum from naive mice or mice immunized with WT SVPs or ZIKVm10 SVPs and were then challenged with live ZIKV. The AG129 mice were monitored for (C) survival, (D) body weight (data represented as mean ± SD), and (E) health score (data are represented as mean ± SD). (F) Viral load in serum from the AG129 mice was determined 4 days post infection (line at median). Tukey’s multiple comparisons test was used to compare differences between experimental groups for antibody binding. One-way ANOVA and Dunn’s multiple comparison test were used to compare viremia load. Kaplan-Meier survival curves were analyzed using the Mantel-Cox log rank test. Results were considered statistically significant if p < 0.05.

Figure 6.. Sera from mice immunized with ZIKVm10 SVPs show reduced ADE of DENV infectivity

(A) The serum dilution at which maximum ADE was observed (maximum of the fitted curve) is plotted for all samples that demonstrate ADE (WT ZIKV, n = 6; ZIKVm10, n = 8). WT ZIKV sera facilitated ADE at more dilute concentrations (higher log reciprocal values), while ZIKVm10 sera required significantly higher concentrations for maximum ADE (*p < 0.05 by Student’s t test, line at mean ± SD). (B and C) ADE-mediated infectivity of DENV2 and ZIKV reporter virus particles (RVPs) in K562 cells (which express the Fc-gamma receptor) after incubation with serum from individual mice immunized with WT ZIKV SVPs (B) or ZIKVm10 SVPs (C). All infectivity values for ZIKV RVPs are shown on the left y axis, while DENV RVPs are shown on the right y axis, with axis ranges set based on the maximum values for each dataset. Data were fit to a Gaussian distribution and are plotted as the mean ± range from duplicate assays.