Rational design of flavivirus E protein vaccine optimizes immunogenicity and mitigates antibody dependent enhancement risk

Domain: biology
Published: 2025-12-22
Editorial outcome: minor_revision

Abstract
Flaviviruses are a family of related viruses that cause substantial global morbidity and mortality. Vaccination against one flavivirus can sometimes exacerbate disease caused by related viruses through antibody-dependent enhancement (ADE) or interfere with the efficacy of subsequent vaccines. To address this challenge, we develop a vaccine strategy by introducing G5C/G102C mutations into the flavivirus envelope (E) glycoprotein. These mutations promote E dimerization through the formation of an inter-chain disulfide bond that conceals the immunodominant and ADE-prone fusion loop epitope (FLE). We validate this design on E proteins from multiple flaviviruses through biochemical, antigenic, and structural analyses. The resulting vaccine candidate, CC_FLEsE, derived from the Zika virus (ZIKV) and formulated with an advanced supramolecular adjuvant, provides significant protection in female mice challenged with ZIKV and prevents ADE caused by a related flavivirus, Dengue virus. In genetically modified mice expressing diverse human immunoglobulin loci, ZIKV CC_FLEsE induces robust neutralizing antibody responses targeting key ZIKV E protein epitopes, including the E-dimer–dependent epitope (EDE), indicating that ZIKV CC_FLEsE can elicit protective antibody responses within the human naïve B cell repertoire. Therefore, CC_FLEsE represents a promising strategy for developing flavivirus vaccines that minimize ADE risk while maintaining high protective efficacy.

Introduction
Human pathogenic flaviviruses are a family of related viruses responsible for significant morbidity and mortality worldwide. They are transmitted primarily by mosquitoes, including yellow fever virus (YFV), dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and Zika virus (ZIKV), as well as ticks such as the tick-borne encephalitis virus (TBEV)1. In 2016, ZIKV spread to the Americas and emerged as an infectious agent which caused a global health crisis2,3. ZIKV infection can cause Guillain–Barré syndrome in adults, and severe fetal neuro-malformations and fetal death during pregnancy3. ZIKV infection primarily occurs through mosquito bites, but sexual and vertical (mother-to-fetus) transmissions have also contributed to the recent epidemic2. Despite significant efforts, there is currently no licensed ZIKV vaccine for disease prevention. However, FDA-approved live-attenuated and/or inactivated vaccines are available for YFV, JEV, TBEV, and DENV4.

Flaviviruses possess an ~11 kb positive-sense RNA genome, which is translated into a single polyprotein. This polyprotein is cleaved into three structural proteins: capsid (C), pre-membrane/membrane (prM/M), and envelope (E), along with several non-structural proteins (NS)5. The E protein, which is the major target of neutralizing antibody (nAb) responses and a key focus of vaccine development, consists of three ectodomains (DI, DII, and DIII). The ZIKV E protein adopts a homodimeric conformation on mature virion, similar to other flaviviruses. Notable nAb epitopes on the ZIKV E protein include the quaternary E-dimer epitope (EDE)6,7, the domain III lateral ridge (DIIILR) epitope8,9, and other epitopes that have been identified at the dimer–dimer interface represented by mAb ZIKV-11710,11 and elsewhere on the E protein12–15.

There is significant sequence homology and structural similarity between the ZIKV E protein and those of other flaviviruses, such as DENV. An immunodominant but suboptimal epitope, the fusion loop epitope (FLE), spans 12 amino acid residues within DII and is highly conserved in flavivirus E proteins, eliciting abundant antibodies that demonstrate broad flavivirus cross-reactivity, but limited neutralization capacity9,16,17. Such cross-reactive antibody responses to flaviviruses, acquired through infection or immunization, can significantly affect the immune response and clinical outcomes of subsequent infections or immunizations. These effects may occur through mechanisms such as antibody-dependent enhancement of infection (ADE)18 or immune imprinting19,20.

ADE was first observed in DENV infections, where cross-reactive antibodies facilitate infection by different DENV serotypes via Fcγ receptor (FcγR)-mediated virus entry into myeloid cells21–23. Previous clinical studies on DENV vaccines have shown that DENV-naïve children, after vaccination, became more susceptible to severe dengue disease upon natural infection, likely due to ADE by vaccine-induced antibodies that bind but do not neutralize other DENV serotypes24,25. Moreover, animal studies26–28 and clinical research29 suggest that cross-reactive anti-ZIKV antibodies, primarily stimulated by ZIKV infection, may enhance the severity of subsequent DENV infection through ADE, mediated by cross-reactive antibodies.

Cross-reactive ADE-prone antibodies target conserved epitopes across flaviviruses, including those on the prM protein and the FLE of the E protein30,31. The prM and E proteins, expressed together as prM-E, are common antigens in current ZIKV vaccine candidates2. However, these vaccine candidates may elicit diverse antibody responses, including protective antibodies targeting nAb epitopes and weak or non-neutralizing antibodies, such as FLE-specific antibodies, that can cross-react with DENV E proteins and potentially exacerbate DENV infections15,26, given that a majority of regions experiencing ZIKV epidemics are mostly endemic for DENV. Additionally, flavivirus cross-reactive antibody responses can reduce the effectiveness of vaccines for other flaviviruses due to immune imprinting19,20. For example, preexisting TBEV immunity has been shown to affect the quality of the immune response to subsequent YFV vaccination32 and vice versa33. Furthermore, previous immunization with JEV or YFV vaccines can significantly inhibit the immune response to a subsequent inactivated ZIKV vaccine34,35.

To ensure the safety and efficacy of future flavivirus vaccines, it is essential to address the potential risks associated with ADE and the inhibition of vaccine responses to heterologous flaviviruses and implement effective countermeasures. Several strategies have been proposed to mitigate the induction of ADE-prone antibodies, particularly those targeting the FLE on the viral E protein. One approach involves using ZIKV prM-E mutants with specific point mutations in the FLE to reduce its immunogenicity36. Another strategy includes engineering dimeric soluble E protein (sE) with FLE “stapled” to an adjacent DIII to minimize FLE exposure37. However, these modified immunogens have so far demonstrated suboptimal protection efficacy36,37, as evidenced by significant viral loads observed in immune animals following ZIKV challenges.

In this study, we employ a structure-based approach to design flavivirus vaccine candidates with improved immunogenicity and reduced ADE potential. Our lead vaccine candidate, CC_FLEsE, which incorporates mutations to facilitate a disulfide bond linkage between the FLE and the N-terminus of the flavivirus E glycoprotein is characterized biochemically and antigenically for several flaviviruses including JEV, WNV, and ZIKV. These mutations result in an enhanced E-dimeric context and minimized FLE exposure. We have demonstrated the protective efficacy of the ZIKV CC_FLEsE design in mice, showing no ADE effect for DENV infection compared to the WT sE. Furthermore, we isolated potent monoclonal ZIKV nAbs from ZIKV CC_FLEsE-immunized human Ig loci transgenic mice that target major ZIKV E protein nAb determinants, including the quaternary EDE. To guide future vaccine development, we have also determined the structure of OZ-D4, a representative EDE nAb, in complex with the ZIKV sE dimer at atomic resolution and characterized its clonal affinity maturation.

Our CC_FLE design strategy could be instrumental in developing a range of flavivirus vaccines by reducing undesirable FLE-directed antibody responses while retaining protection efficacy.

Results
Structure-based vaccine design of ZIKV E proteins
Numerous studies suggest that the flavivirus FLE is the major ADE-prone epitope. On the mature virion, the ZIKV E protein is arranged as antiparallel homodimers and recognized by a class of quaternary epitope EDE-directed nAbs. Recombinant soluble forms of the ZIKV E protein ectodomain, known as sE, which lack the stem and anchor portions, are expressed predominantly as monomers37. These monomers bind poorly to EDE nAbs but show high affinity for FLE-directed, ADE-prone non-neutralizing antibodies37. At high in vitro concentrations, dimeric ZIKV sE can appreciably form16,38. The structure of the dimeric sE16,38 suggests that in a closed dimeric context, the ZIKV FLE is heavily occluded by the DI, including the N-terminus, and DIII elements of the adjacent E protein protomer39,40 (Fig. 1A, upper left panel). In contrast, the FLE is readily exposed in the monomeric context or in dynamically opened E dimers (Fig. 1A, lower left panel), a phenomenon known as “viral breathing”39. The accessible surface area (ASA) of the entire FLE in the dimer context is ~459 Å2 (38 Å2/residue) (Fig. 1A, right). In contrast, in the monomer context, the FLE shows a substantially increased exposure, with an ASA of 836 Å2 (70 Å2/residue), representing an >80% increase (Fig. 1A, right). Among the 12 amino acid residues of FLE, three residues - D98, W101, and F108- exhibit ASA values increased by >50% (Fig. 1A, right) due to the dimer-monomer transition, consistent with the observation that residues W101 and F108 are major contact points for FLE-specific ADE-prone antibodies16,36.

Conceptually, the monomeric form of sE is not an ideal immunogen for stimulating protective immune responses due to: (i) the poor presentation of quaternary EDE nAb epitopes, which are more readily formed in a dimeric context (Fig. 1A), and (ii) the extensive exposure of weak/non-neutralizing FLE epitope, which could robustly elicit ADE-prone antibodies (Fig. 1A). Therefore, we first sought to engineer stable ZIKV sE dimer vaccine candidates to mimic the native E protein conformation by introducing one or two inter-protomer disulfide bonds in sE, based on structural modeling of sE (PDB: 5JHM) (Fig. 1B). The corresponding recombinant E protein constructs were designated as CC_FLE sE carrying mutations G5C/G102C, and CC_Core sE bearing mutation A264C (Fig. 1B, C) that has been described by previous studies to promote sE dimerization37,41–43, respectively. Of note, the mutations in CC_FLE located within the FLE could further reinforce the occlusion of major FLE residues such as W101 and F108, thus dampening the antigenicity of FLE by tethering the FLE on one protomer to the N-terminus of the other protomer (Fig. 1B) and reducing the ADE effect.

Additionally, we adopted an alternative strategy to prevent potential ADE antibody elicitation by deleting DII (residues 51–133, and 195–284) or DI & DII (residues 1–302), which contain more conserved amino acid residues to form ADE-prone epitopes. This approach resulted in the constructs DI-DIII and DIII, respectively (Fig. 1C). To enhance the immunogenicity of DIII, we fused its C-terminus with the N-terminus of a gene fragment encoding a self-assembling virus-like nanoparticle (NP), ferritin44,45, creating the vaccine candidate construct DIII-NP (Fig. 1C).

Antigenicity, thermostability, and structure of rationally designed ZIKV E-based immunogens
We cloned the genes encoding ZIKV E-based immunogens listed in Fig. 1C into an insect expression vector and transformed Drosophila S2 cells for protein expression. The dimer-prone mutants E proteins were purified by His-tag column followed by size exclusion chromatography (SEC) (Fig. S1A) to remove aggregates and monomeric proteins that failed to form inter-protomer disulfide bond. These two purification steps resulted in dimeric CC_FLE and CC_Core sE proteins with high homogeneity (Figs. 2A, and S1B), as expected. Differential scanning calorimetry profiles demonstrate substantially improved thermostability of CC_FLE sE compared to WT sE, with the thermal transition temperature (Tm) increased by 15 °C (from 43.5 °C to 58.7 °C) (Fig. 2B). In contrast, CC_Core sE showed a moderate (3 °C) increase in Tm compared to WT sE (Fig. 2B). To confirm the dimeric configuration of CC_FLE sE, we determined the cryogenic-electron microscopy (cryo-EM) structure of the complex of CC_FLE sE and a ZIKV neutralizing antibody SMZAb246 at resolution of 4.09 Å, enabling detailed structural characterization. The resulting structure (Figs. 2C and S2A–G, and Table S1) revealed a symmetric CC_FLE dimer, with overall structure identical to previously published ZIKV sE dimer (Table S2, and Fig. S2L), engaged by two SMZAb2 Fab molecules. The 150-loop (residues 147–161)47 of the E dimer lacks sufficient electron density, indicating that this region is disordered, similar to the wild-type ZIKV E dimer (PDB: 5LBS), and it does not appear to engage in direct contact with SMZAb2 in the structure. The Fabs bind across the dimer interface, contacting both sE protomers and targeting a quaternary epitope that spans domains DII and DIII of the E protein. Epitope analysis reveals that the SMZAb2 epitope significantly overlaps with that of EDE1-C8, a well-characterized broadly neutralizing antibody (Fig. S2I–K). Of the 32 residues engaged by SMZAb2, 27 are shared with the EDE1-C8 epitope (Fig. S2I), underscoring SMZAb2’s classification within the EDE antibody family. The presence of the engineered disulfide bond (C5–C102) linking the protomers is clearly resolved, confirming successful covalent stabilization of the dimer interface (Fig. 2D). As expected, exposure of the FLE loop, including residues W101 and F108, is markedly reduced due to occlusion by the adjacent E protomer in the dimer context (Fig. S2J, M).

To characterize the antigenicity of our immunogens, we assessed their binding affinity for a panel of reference mAbs, in comparison with WT sE. Consistent with the design rationale, WT sE, primarily in monomeric form, binds EDE quaternary epitope mAbs poorly. CC_FLE binds both EDE mAbs, EDE1-C8 and SMZAb2 well, while CC_Core only binds the former mAb well but the latter poorly (Fig. 2E). The DIIILR epitopes are well retained in both CC_FLE and CC_Core, as they bind DIIILR epitope mAbs, ZV67 and Z004, similarly to WT (Fig. 2E). Lastly, as expected, CC_FLE has nearly abolished binding to FLE mAbs 2A10G6 and E60 (Fig. 2E), while CC_Core shows significantly diminished binding compared to WT. We assessed the reactivity of more flavivirus FLE cross-reactive but weak or non-neutralizing mAbs and consistently noted that our dimeric CC_FLE sE, which carries an engineered inter-protomer disulfide bond locking the potential ADE epitope FLE to the N-terminus of the adjacent sE protomer (i) remarkably show enhanced EDE nAb epitope presentation compared to WT sE; (ii) well retain nAb epitopes on domain III; and (iii) all substantially dampen the display of FLE epitopes (Fig. 2F). Of note, the G5C and G102C mutations alone are not sufficient to abolish FLE–mAb binding, as a minor monomeric fraction of ZIKV CC_FLE sE still binds the FLE–mAb E60 (Fig. S1C), similar to WT sE. This suggests that the reduced FLE–mAb binding of CC_FLE sE results from occlusion of the FLE caused by the inter-chain disulfide linkage formed by the G102C/G5C mutations. As expected, DI-DIII and DIII immunogens bind a panel of DIII-specific nAbs, including ZV67 and Z004 (Fig. S1D–H) in a manner similar to WT sE protein, indicating well-retained antigenicity, with abolished binding to FLE mAb, 4G2 (Fig. S1D–H).

CC_FLE linkage can potentially be applied to other flavivirus sEs to prevent ADE
Sequence homology analysis of the N-terminus and FLE regions of the sEs from various mosquito-borne flaviviruses revealed that residues G5 and G102, which were mutated to cysteine (C) in the ZIKV CC_FLE design to form the N-term/FLE disulfide linkage, are conserved in the corresponding sEs of these viruses (Fig. 3A). The potential applicability of this disulfide bond linkage in the sEs of other flaviviruses, such as JEV and WNV, was further supported by structural analysis of their envelope proteins (PDB: 3P54 & 3IYW). This analysis predicted a high likelihood for the formation of this disulfide bond. We then expressed and characterized the CC_FLE version of JEV and WNV sE, respectively, which showed a dimeric configuration after purification (Fig. 3B), in contrast to the predominantly monomeric JEV or WNV WT sE (Fig. 3B). Cryo-EM analysis of JEV and WNV CC_FLE sE proteins confirmed their dimeric configurations (Fig. 3C, Tables S1 and S2, and Fig. S3). We were able to further visualize the disulfide bond linkage facilitated by mutations G5C/G102C in JEV CC_FLE sE (Fig. 3D). As expected, in the JEV CC_FLE sE dimer, the ADE-prone epitope FLE is highly occluded by DI and DII from the adjacent sE protomer (Fig. 3E), similar to its occlusion in the sE dimers displayed on JEV virions48. Consistently, JEV and WNV CC_FLE sE displayed 7- to 300-fold decreased binding to FLE-specific mAbs, in comparison with the WT sE (Fig. 3F, G). Therefore, the CC_FLE design strategy could potentially be applied to prevent ADE of sE-based vaccines of other flaviviruses.

CC_FLE elicit potent nAb responses in immune mice
We set to test the immunogenicity of the engineered ZIKV vaccine candidates described above including the CC-FLE sE (Fig. 1C), formulated with Adjuplex, a lecithin-based adjuvant49 in C57BL/6 mice (Fig. 4A). After two immunizations on days 0 and 28, respectively, sera from mice immunized with CC_FLE showed geometric mean of ID50 titers (~104) against ZIKV reporter virus particle (RVP) of H/PF/2013 strain, similar to WT sE (Fig. 4B). Sera from mice immunized with DIII or DIII-NP/DIII (priming with DIII-NP/boost with DIII) showed tenfold lower ID50 titers than WT sE (Fig. 4B), while immune sera resulted from DI-DIII immunizations showed no neutralization activity (Fig. 4B), suggesting that the deletion of DI and/or DII of sE leads to attenuated immunogenicity. This is possibly due to protein misfolding or reduced stability resulting from the deletion. Surprisingly, the CC_Core dimer has elicited low nAb titers nearly equivalent to PBS inoculated mice group (Fig. 4B) after two immunizations, indicating mutation A264C impedes immunogenicity. Nevertheless, the observed robust nAb response from the immune mice inoculated with ZIKV CC_FLE dimer indicated premise for further development.

Supramolecular polyphosphazene-based adjuvant formulation potentiates vaccine efficacy in mice
Polyphosphazene (PPZ) immunoadjuvants (Fig. S4) are well-defined water-soluble macromolecules with phosphorus-nitrogen backbone and organic side groups, which have been proven to dramatically potentiate immune responses to diverse viral and bacterial vaccine antigens50,51. The unique feature of these synthetic macromolecules is that, upon mixing in aqueous solutions, they are capable of self-assembling with vaccine antigens into nano-scale virus-mimicking complexes. The first-generation PPZ adjuvant, poly[di(carboxylatophenoxy)phosphazene] (PCPP), has shown a dose-dependent immunopotentiation effect and excellent safety profile in five clinical trials51. Its structural homologue, poly[di(carboxylatoethylphenoxy)phosphazene] (PCEP), demonstrated further ability to both enhance and modulate quality of the immune response52,53. A complementary immunoadjuvant, small molecule R848 (resiquimod), which is a clinical-grade TLR7/8 agonist, activates immune responses in TLR7/8 dependent mechanism and induces superior cytokine secretion, macrophage activation, and enhancement of cellular immunity54–56. Both PPZ and R848 technologies have been recently synergized by co-assembling antigen-PCPP complexes with R848, which resulted in non-covalently assembled supramolecular constructs that mimic virus-like features desirable for stimulating potent immune response: (i) 60–80 nm diameter, (ii) repetitive/multiple antigen display, and (iii) decoration with danger signals recognized by pattern recognition receptors57.

Here, we attempted to test if PPZ in conjunction with R848 could better potentiate the immunogenicity of sE protein than Adjuplex, the adjuvant we used earlier. We demonstrated the formation of nano-scale ternary CC_FLE-PCEP-R848 complex in a unimodal size distribution with a z-average hydrodynamic diameter of 60 nm (Fig. S4) and the ability of complexed antigen to bind antibodies (Fig. S5) using Asymmetric Flow Field-Flow Fractionation (AF4) and dynamic light scattering (DLS) methods. We then focused on investigating the immunogenicity of our lead immunogen CC_FLE in various PPZ formulations including (i) PCEP, (ii) PCEP-R848, (iii) PCPP, (iv) PCPP-R848, (v) R848, and (vi) Adjuplex as control. We also added WT sE formulated with Adjuplex as a reference group, in the immunization study depicted in Fig. 5A. 1 week after the second immunization, sera from mice immunized with WT sE formulated with Adjuplex showed neutralization ID50 titers against ZIKV H/PF/2013 RVP around 104, similar to mice immunized with CC_FLE formulated with Adjuplex, PCEP, or PCEP-R848 (Fig. 5B), while sera from immune mice inoculated with CC_FLE formulated with PCPP or PCPP-R848 displayed 20–30 fold lower ID50 titers (Fig. 5B). No neutralization activity was detectable in sera from mice immunized with CC_FLE formulated with R848, or CC_FLE alone (no adjuvant).

In addition to ZIKV RVP neutralization assay, we determined the neutralization capacity of selected sera from the immune mice group against an authentic virus, ZIKV FSS13025, by PRNT assay. Consistent with the RVP neutralization data, sera from mice immunized with CC_FLE formulated in either PCEP-R848 or Adjuplex all displayed potent neutralization capacity against ZIKV FSS13025, similar to sera from mice immunized with WT sE formulated in Adjuplex (Fig. 5C). We then sought to determine protective efficacy of these selected potent immune sera in a passive serum transfer study (Fig. 5D). AG129 mice (IFNα/β/γR−/−), immunocompromised and highly susceptible to ZIKV infection, were infused with donor C57BL/6 mouse immune sera of day 42 (Fig. 5D), challenged with ZIKV FSS13025 1-h post serum infusion, and were monitored for 20 days post infection (DPI). AG129 mice receiving sera from immune mice inoculated with CC_FLE formulated with PCEP+R848 adjuvant displayed significantly longer survival time (median, 15 days) upon challenge than from animals inoculated with PBS (median, 11 days) (*p<0.05, Mantel–Cox log-rank test) or WT sE formulated with Adjuplex (median, 11 days) (Fig. 5D). AG129 mice receiving immune sera from donor mice immunized with CC_FLE formulated with Adjuplex also showed median survival time of 13 days (Fig. 5D), longer than PBS- or WT sE+Adjuplex inoculated group, although this is not statistically significant. AG129 mice receiving immune sera from CC_FLE and PCEP-R848 adjuvant-immunized animals also showed less body weight loss (Fig. S6A, statistically not significant) and lower health score (multiple t test, ***p<0.001, days 11 and 12 post infection, Fig. S6B) than from PBS inoculated animals. Although the nAb titers of immune sera from animals inoculated with WT sE+Adjuplex, CC_FLE sE+Adjuplex, or CC_FLE sE+PCEP-R848 were similar, the better protection efficacy conferred by the sera from the mice immunized with CC_FLE sE+PCEP-R848 indicated that other protection mechanisms, such as antibody effector functions58 including antibody dependent cytotoxicity (ADCC) may play a role in this AG129 mouse model.

In summary, formulation of our immunogen, CC_FLE into nano-scale ternary complexes with PCEP-R848 is beneficial for potentiating immunogen protection efficacy, indicated by (i) potent nAb titers of immune sera (Fig. 5B, C) and (ii) in vivo protection for ZIKV-challenged AG129 mice conferred by passive immune sera transfer (Fig. 5D).

CC_FLE linkage can potentially be applied to other flavivirus sEs to prevent ADE
To further evaluate the potential of CC_FLE inducing cross-reactive ADE-prone Abs, we incubated DENV serotype 1 (DENV-1) or serotype 2 (DENV-2) reporter virus particle with sera from ZIKV CC_FLE or WT sE immune mice (Fig. 5A), followed by co-incubation with K562 cells (FcγRIIA+) to mimic Fc receptor-mediated ADE for DENV infection. As expected, most sera from mice immunized with ZIKV WT sE formulated with Adjuplex showed peak enhancement titer (PET) for DENV-1 or DENV-2 around 200 (Fig. 5E, F), and enhanced magnitude of infectivity for DENV-1 or DENV-2 above 200-fold (Fig. 5E, F) at PET. In contrast, most sera from mice immunized with ZIKV CC-FLE formulated with Adjuplex or PCEP-based adjuvant showed no significant enhancement for DENV-1 or DENV-2 infection (Fig. 5E, F) consistent with the design rationale of CC_FLE which aims at abolishing the elicitation of ADE-prone antibody responses. The in vitro ADE data were confirmed by an in vivo study in which the enhancing effects of ZIKV immune serum on DENV-2 infection in AG129 mice were examined (Fig. 5G). Recipient AG129 mice were passively transferred with pooled donor serum from C57BL/6 mice vaccinated with ZIKV WT or CC_FLE sE formulated in Adjuplex (Fig. 5E, F), followed by challenge with a partial-lethal dose of DENV-2 (strain DS210). We found that AG129 mice receiving WT immune sera showed more severe mortality (**p<0.01, Fig. 5G, left) and higher DENV-2 virus load (*p<0.05, Fig. 5G, right) than PBS-treated mice, indicating a WT immune sera-mediated ADE effect. In contrast, the mice receiving CC_FLE immune sera showed a survival curve (Fig. 5G, left) and terminal tissue (lymph node) DENV-2 virus load (Fig. 5G, right) similar to those receiving PBS buffer, suggesting no ADE effect from the CC_FLE immune sera.

ZIKV dimeric CC_FLE sE elicits nAb responses with distinctive quality compared to monomeric WT sE
To map the specificity of the nAb response elicited by ZIKV CC_FLE sE, we performed immune serum antibody depletion followed by ZIKV RVP neutralization assay. In this assay, pooled day 42 sera (Fig. 5A) from mice immunized with WT or CC_FLE sE formulated with Adjuplex were pre-mixed with sE protein variant antigens to deplete cognate serum neutralizing antibodies prior to ZIKV RVP incubation (Fig. S7). mAbs ZV67 (DIII-specific) and EDE1-C8 (quaternary epitope-specific) were used as controls (Fig. S7).

We found that the neutralization activity of immune sera from mice immunized with WT sE was reduced to below 50% RVP inhibition after depletion with all sE antigens, including WT, CC_FLE and DIII (Fig. S7), which all contain epitopes on DIII, similar to the DIII-specific mAb, ZV67 (Fig. S7). This suggests that the nAb responses elicited by WT sE are DIII-directed.

In contrast, immune sera from mice immunized with CC_FLE showed a distinct specificity profile: only dimeric CC-FLE could efficiently deplete the serum nAb activity (Fig. S7), similar to the quaternary epitope-specific mAb, EDE1-C8 (Fig. S7). Depletion with WT or DIII sE antigen only partially reduced serum neutralization capacity (Fig. S7), indicating additional DIII-directed nAb specificity.

Therefore, dimeric CC-FLE sE elicits multi-layers of nAb specificities, including those directed at quaternary epitopes and DIII, which are more sophisticated than the mainly DIII-focused nAb responses stimulated by WT sE. This suggests that the superior quality of nAb responses elicited by dimeric CC-FLE over WT sE immunogen may translate to enhanced protection efficacy.

ZIKV CC_FLE sE immunization confers nearly complete protection in immunocompetent mice
We aimed to evaluate the protective efficacy of our lead immunogen, ZIKV CC_FLE sE, using an immunocompetent mouse model59,60 that has been validated and widely used in numerous preclinical studies evaluating the efficacy of ZIKV vaccine candidates61–64. We immunized immunocompetent BALB/c mice with CC_FLE formulated in the optimal adjuvant system PCEP-R848, using WT sE as a control, followed by ZIKV challenge (Fig. 6A). To evaluate the effect of individual adjuvants, we also immunized mice with CC_FLE formulated in either PCEP or R848. All mice were immunized and challenged as depicted in Fig. 6A.

We chose BALB/c mice as the model animal for vaccine efficacy here based on our in-house observation that after ZIKV challenge, BALB/c mice in general develop higher titers of viremia than C57BL/6 mice (used in our earlier immunogenicity studies), suggesting BALB/c mouse as a suitable model animal for evaluating vaccine efficacy.

We first assessed the nAb titers of the day 35 immune mouse sera. Sera from mice inoculated with WT sE formulated in PCEP-R848 displayed moderate ID50 titers (geometric mean = 178) (Fig. 6B). In contrast, sera from CC_FLE sE formulated in PCEP-R848 group showed significantly higher ID50 titers (geomean ID50 ~5,012), 28-fold higher than the WT sE group (**p<0.01, Mann–Whitney test) (Fig. 6B). Furthermore, sera from CC_FLE formulated with PCEP-R848 group showed significantly more potent neutralizing activity than sera from animals immunized with CC_FLE formulated with either PCEP (geomean ID50 ~403, **p<0.01, Mann–Whitney test) or R848 (geomean ID50 <100, ***p<0.001, Mann–Whitney test) adjuvant alone (Fig. 6B), indicating the beneficial effect of the supramolecular adjuvant system.

While earlier studies (Figs. 4 and 5) showed similar nAb titers elicited by WT and CC_FLE in C57BL/6 mice, we observed a discrepancy here in BALB/c mice. The variation may stem from the use of different mouse strains. Nonetheless, the nAb titers in BALB/c mice inoculated with monomer WT sE in our study are consistent with historic data from the literature37. This suggests that CC_FLE formulated in PCEP-R848 elicits nAb responses superior to WT in the same adjuvant in BALB/c mice, consistent with observations in C57BL/6 mice. Therefore, CC_FLE sE formulated in PCEP-R848 represents an immunization regimen capable of consistently eliciting nAb responses across various mouse strains, outperforming the monomeric WT sE.

To determine if the observed potent nAb titers induced by CC_FLE formulated in PCEP-R848 could confer protection, we challenged the immune mice on day 56 (1 month after the 2nd immunization) (Fig. 6A) with ZIKV Puerto Rico 2015 and monitored serum virus load (VL) thereafter. As a negative control for immunization, sera from mice inoculated with PBS on day 1 post infection (DPI) showed an average of 104 copies of ZIKV RNA/ml. In contrast, 83% (5/6) of mice immunized with CC_FLE sE formulated in PCEP-R848 showed no detectable serum VL (Fig. 6C). WT sE formulated in PCEP-R848 and CC_FLE sE formulated in PCEP alone provided partial protection, with nearly all mouse sera displaying detectable ZIKV RNA (geomean ~103 ZIKV RNA copies/ml), which was tenfold lower than PBS-inoculated mice (Fig. 6C). Consistent with the serum VL results at 1 DPI, ZIKV RNA was detectable in lymph nodes (Fig. 6D), in all animal groups (average of 104 ZIKV RNA copies/ml) except the group immunized with CC_FLE sE formulated in PCEP-R848 (Fig. 6D), where only one mouse exhibited detectable ZIKV RNA. Furthermore, there is an inverse correlation between pre-infection serum nAb titers and 1 DPI serum virus load (Fig. 6E), indicating that the nAb response is a key factor contributing to protection efficacy.

ZIKV CC_FLE sE protection efficacy in pregnancy
We evaluated the efficacy of CC_FLE sE against ZIKV during pregnancy (Fig. S8A). Female BALB/c mice received two immunizations with either WT or CC_FLE sE formulated in PCEP+R848, with PBS serving as the control. Consistent with previous observations, the geometric mean of ID50 neutralizing antibody titer in sera from CC_FLE sE–immunized mice was 3240, which was sevenfold higher than that from WT sE–immunized mice (ID50 geometric mean = 446) (Fig. S8B). Following immunization, the mice were mated to establish pregnancy and subsequently challenged with ZIKV Puerto Rico 2015 via the retro-orbital route. ZIKV viral loads in serum were measured 1 day post infection (1 DPI). Mice immunized with WT or CC_FLE sE showed 10-fold and 20-fold reductions in viral load, respectively, compared to PBS controls (Fig. S8C, E), indicating that sE immunization reduced ZIKV replication in vivo. On day 8 post challenge, viral loads in maternal uteri were assessed. PBS-inoculated mice showed a geometric mean ZIKV RNA load of 4.7 × 103 copies/gram tissue (Fig. S8D, E). One mouse from the WT sE–immunized group had detectable uterine ZIKV RNA, whereas none of the mice immunized with CC_FLE sE showed any detectable viral RNA, confirming the protective efficacy of the vaccine in maternal tissues. Furthermore, ZIKV RNA was undetectable in both placentas and fetuses of mice immunized with WT or CC_FLE sE (Fig. S8E). These results demonstrate that CC_FLE sE provided effective protection against ZIKV in both pregnant mice and their fetuses. Takentogether, our lead immunogen, ZIKV CC_FLE sE formulated in PCEP-R848 supramolecular adjuvant complex, demonstrates the capacity to elicit potent nAb responses, thereby conferring robust protection in ZIKV-challenged mice and fetus (Figs. 6 and S8), while minimizing the stimulation of ADE-prone Ab responses (Fig. 5). It is likely that CC_FLE sE may elicit a more focused nAb response targeting major nAb epitopes such as the quaternary EDE and DIII LR (Fig. S7), while avoiding the stimulation of non-neutralizing ADE-prone antibody responses. This outcome aligns with the initial rationale behind the structure-based design (Fig. 1).

High-resolution analysis of ZIKV CC_FLE sE-elicited antibody/B cell response
To delineate the nAb response induced by CC_FLE sE at a high resolution, we utilized a transgenic mouse model known as OmniMouse® (OmniAb, Inc.). These mice lack endogenous J and C genes, preventing the production of endogenous immunoglobulins. Instead, they carry transgenes containing un-rearranged human antibody Ig light chain kappa and lambda loci, along with recombinant transgenes comprising human VH, DH and JH genes with rat heavy chain constant regions. With 44 functional VH, 20 functional VK and 15 functional VL genes, OmniMouse animals closely mimic the naïve human B repertoire, facilitating the characterization of human B cell repertoire in response to immunization in preclinical setting.

We immunized two OmniMouse mice with ZIKV CC_FLE sE two and three times, respectively (Fig. 7A). After the second and third immunizations, the mouse sera showed potent neutralization ID50 titers against ZIKV H/PF/2013 reporter virus particles, ~600 and 2000, respectively (Fig. S9A, B). The neutralization specificity mapping of OmniMouse immune sera closely resembled that of the dimer-context-specific mAb EDE1-C8 (Fig. 7B and S9C), as well as the sera from CC_FLE sE-immunized C57BL/6 mice (Fig. S7). We subsequently sacrificed the immune mice and employed a FACS-based antigen-specific single B cell sorting and cloning methodology65–67 developed in-house, to isolate CC_FLE binding mAbs from mouse splenocytes.

In an initial attempt, we utilized ZIKV CC_FLE-PE conjugate as the antigen probe to sort CC_FLE memory B cells binding to CC_FLE at single-cell density (Figs. 7C and S10). We then amplified the Ig heavy (HC) and light chain (LC) encoding genes of each cell. From the obtained 29 cells with both HC and LC amplified, we randomly picked four individual cells with matched HC/LC pairs to express full-length mAbs in human IgG1 form68 for functional analysis.

As anticipated, all four mAbs, named OZ-D4, OZ-B11, OZ-A1, and OZ-B9 (Table S3), showed strong binding to the ZIKV dimeric sE antigen CC_FLE (Fig. 7D). However, OZ-D4 and OZ-B11 exhibited poor binding to monomeric WT sE (Fig. 7D), resembling the quaternary EDE mAb, EDE1-C8, indicating dimer-context specificity. Additionally, while OZ-A1 showed strong binding to WT sE, it displayed no binding to DIII. Conversely, OZ-B9 bound to both WT and DIII sE, akin to the binding profile of DIIILR mAb Z004 (Fig. 7D). Of note, none of these mAbs displayed binding to E proteins of DENV-1 or -2 (not shown), indicating no cross-reactivity.

To delineate the epitope specificity of the generated mAbs, we performed a BLI-based Ab competition assay, evaluating the competition between the four identified mAbs and a panel of ZIKV reference mAbs (Fig. 7E). The results revealed that these mAbs can be categorized into two competition clusters: (i) OZ-D4 and OZ-B11, which compete mutually and with EDE mAbs, and (ii) OZ-A1 and OZ-B9, which exhibit mutual competition and compete with the DIII CC-loop mAb, ZV64. Notably, OZ-B9 also competes with DIIILR mAb Z004. Based on these results, we speculate that OZ-D4 and OZ-B11 likely target the quaternary epitope EDE, while the epitope of OZ-B9 overlaps with both the DIIILR and DIII CC-loop. The epitope of OZ-A1 may partially overlap with DIII and potentially involve moieties from other domains such as DI.

Importantly, all ZIKV CC_FLE sE-elicited mAbs demonstrated neutralization activity against the ZIKV reporter virus particle (Fig. 7F). OZ-D4 showed an IC50 titer of 0.048 μg/ml, comparable to that of EDE1-C8, while OZ-B9 displayed a potency (IC50 = 0.031 μg/ml) similar to that of the DIIILR mAb, ZV67 (Fig. 7F). Consistently, OZ-D4, OZ-A1, and OZ-B9 exhibited neutralizing activity against authentic ZIKV strain FSS13025 in a plaque reduction neutralization test (PRNT) (Fig. 7G), albeit with reduced potency compared to the ZIKV RVP assay, while OZ-B11 showed no detectable neutralizing activity. These results underscore the ability of CC_FLE sE to elicit neutralizing antibody responses targeting the key nAb epitopes, including the quaternary EDE and DIII LR epitopes in OmniMouse mice that express naïve human Ig loci (Table S3). OZ-D4, identified as an immunization-elicited EDE potent nAb, was selected for further studies.

Structure determination of ZIKV nAb isolated from OmniMouse animal
To gain detailed information on the cognate epitopes of selected nAbs (Fig. 7) isolated from OmniMouse, we attempted to solve the structures of the nAbs complexed with ZIKV sE dimer antigens using cryo-EM single particle reconstruction. Initially, our efforts using ZIKV CC_FLE sE as the antigen yielded data of suboptimal resolution, likely due to cryo-EM technical issues. We then employed ZIKV CC_Core sE, a dimeric sE variant bearing the A264C mutation, which presents nAb epitopes effectively (Fig. 2) and exhibits similar binding affinity to OmniMouse mAb as CC_FLE sE (Fig. 7D), as the antigen instead. A structure with a resolution of 3.45 Å was obtained for the OZ-D4:ZIKV CC_Core sE complex (Figs. 8A, B and S11, and Table S4), revealing a ZIKV E dimer comprised of two protomers (designated as protomer A and B, respectively) in a single conformation bound to two OZ-D4 Fab fragments. Each OZ-D4 Fab binds an E dimer quaternary epitope site encompassing the DII and DIII (Fig. 8B), partially overlapping with the footprint of the reference mAb EDE1-C8. Overall, OZ-D4 binds to DIII of protomer A and DII of protomer B, including part of FLE (Fig. 8C). The OZ-D4 epitope residues on the E dimer include: (i) light chain contact residue clusters 313-318 (F314, T315, & I317) and 396-398 (I396, H398) of DIII on protomer A (Fig. 8D, left), (ii) heavy chain contact residues including the end of β-strand b and adjacent loop residues 73–78, the fusion loop residues G106 and L107 (with 80 Å2 buried surface area) on DII of protomer B, and I317 on DIII of protomer A (Fig. 8D, right). Note that hydrophobic residue I317 is engaged in both OZ-D4 heavy/light chain interactions, accounting for nearly 18% of the E dimer/OZ-D4 interface (Fig. 8D), while the other prominent hydrophobic contact residue, F314, contributes to 20% of this interface by engaging the OZ-D4 light chain (Fig. 8D).

OZ-D4 CDR-L1, CDR-L3, and CDR-H3 make the largest contribution to the contact surface area with the ZIKV E dimer. Light chain residues Y30 and Y91 interact with F314 and I317, respectively (Fig. 8D, left), while OZ-D4 CDR-H3 residue W99 is involved in hydrophobic interaction with E dimer residues I317 and L107 at the E protomer interface (Fig. 8D, right). In summary, these data indicate that OZ-D4 has the major features of dimer context dependent EDE-class nAbs (Fig. 8E, and Table S6).

However, the difference in epitope location between OZ-D4 and classical EDE mAbs, including EDE1-C8 and SMZAb2, is well appreciated (Fig. 8E, F, and Table S6). Specifically, OZ-D4 contacting residues 396-398 of ZIKV E are divergent from the corresponding residues on the DENV E protein and are absent in the epitopes of EDE1-C8 and SMZAb2 (Fig. 8E), consistent with the observation that OZ-D4 is not cross-reactive with DENV-1 or DENV-2 E protein. Additionally, the E dimer approach angle utilized by OZ-D4 is distinct from that of classical EDE mAbs. Superposition of mAb–E dimer complexes (Fig. 8G) shows that the angles between the classical EDE mAb planes and the E dimer plane are 68° for EDE1-C8 and 73° for SMZAb2—both close to vertical. In contrast, the angle for OZ-D4 is 56°, which is more lateral. This more lateral approach, to some extent, causes steric hindrance from the viral membrane, consistent with the observation that OZ-D4 exhibits reduced neutralization potency against authentic ZIKV in the PRNT assay (Fig. 7G). Future work using electron microscopy–based polyclonal epitope mapping (EMPEM)69,70 and isolating additional mAbs from immunized mice will be informative for further characterizing the epitopes and approach angles of EDE-targeting antibodies elicited by CC_FLE sE.

Note that classical EDE mAbs were isolated from DENV-infected individual71. Therefore, to our knowledge, OZ-D4 represents immunization-elicited potent nAbs encoded by human Ig gene segments that targets the quaternary neutralizing epitope of ZIKV E protein investigated at atomic resolution.

OZ-D4 clonal affinity maturation and elicitation by immunization
The elicitation of a specific antibody involves critical steps, including the activation of naïve B cell germline precursor by an immunogen, followed by clonal affinity maturation. This process is characterized by the accumulation of somatic hypermutations (SHMs) in the antibody variable domain genes, driven by affinity selection. These SHM mutations cause divergence from the naïve germline precursor V(D)J sequences, leading to increased affinity for the immunogen72,73. Comparison of the heavy chain (HC) and light chain (LC) sequences of OZ-D4 and the corresponding inferred human germline gene segments, IGHV4-59, and IGLV2-23 (Table S3, and Fig. 9A), respectively, reveals low to moderate level of SHM in the OZ-D4 variable domain genes. The amino acid sequence of the OZ-D4 HC is identical to the germline counterpart, while the OZ-D4 LC shows 8.1% SHM (Table S3, and Fig. 9A). Moreover, most of the residues within the OZ-D4 paratope contacting the sE dimer are identical to the germline version (Fig. 9A), suggesting that extensive accumulation of SHM (e.g., above 10%) is not required for OZ-D4 functionality.

We generated germline-reverted OZ-D4 (OZ-D4_GL) based on the inferred germline sequences to examine the affinity for selected sE immunogens and the contribution of SHM to OZ-D4 function. As expected, the monomeric WT sE shows no binding to either OZ-D4 or OZ-D4_GL, whereas both ZIKV dimeric CC_Core and CC_FLE sEs display high apparent affinity, KD, for OZ-D4 or OZ-D4_GL at pico-molar and nano-molar scale (Fig. 9B), respectively, consistent with the immunogen design rationale that dimeric sE immunogens are superior for engaging and activating EDE epitope-directed naïve B cell precursors over the monomeric sE WT. Additionally, for OZ-D4_GL, CC_FLE sE shows slightly higher affinity, with ~1.5-fold and 3-fold decreases in the dissociation constant (KD) and off-rate (koff), respectively, compared with CC_Core sE (Fig. 9B). This enhanced affinity may be attributed to the engineered disulfide bond in CC_FLE sE, which tethers the FLE to the sE N-terminus, improving the presentation of the antibody epitope. Furthermore, OZ-D4_GL can neutralize ZIKV H/PF/2013 RVP, albeit with lower potency than the mature OZ-D4 (Fig. 9C). This observation confirms that the recognition surface between the naïve precursor OZ-D4_GL and the ZIKV E protein is largely preformed prior to immunization. Collectively, our data suggest that dimeric immunogens, such as CC_FLE sE can readily activate the EDE epitope-directed naïve precursor B cells represented by OZ-D4 and drive sufficient affinity maturation to confer protection through immunization.

Methods
Structural analysis of flavivirus E proteins and design of inter-protomer disulfide bond
The structure visualization of the ZIKV E protein (PDB: 5JHM), JEV E protein (PDB: 3P54), and WNV E protein (PDB: 3IYW) was performed using Chimera, ChimeraX78 or Pymol (PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC). Buried surface areas (BSAs), accessible surface areas (ASAs), and residue contacts were calculated using the PDBePISA server79, Chimera80, or MAPIYA81. Inter-protomer disulfide bond design was conducted using Disulfide by Design 2.082.

DNA constructs and protein expression
The DNA encoding ZIKV WT sE, contains amino acid residues 1-404 of the ZIKV E protein from the Brazilian Zika SPH2015 strain (GenBank: KU321639), a prototypical circulating strain during the 2015–2016 Americas outbreak. The JEV sE construct contains residues 1–406 of the JEV E from the SA-14-14-2 strain83, while the WNV sE construct contains residues 1–400 of WNV E protein84. All DNA constructs were synthesized by GenScript (Piscataway NJ) or Twist Bioscience (South San Francisco, CA) and cloned into pMT/BiP Vector Version B (Thermo Fisher), with a myc tag at the N-terminus and a 6 × His Tag at the C-terminus. Mutagenesis to construct ZIKV sE dimer variants were performed using standard Gibson assembly cloning with primers containing the mutant sequence for CC_Core (A264C) and CC_FLE (G5C/G102C). For the construction of DI-DIII sE, domain II (residues 51–133 and 195-287) was replaced by two linkers, GGGGS or GGGGSGGGGS, respectively. The DIII sE construct contains residues 303–404. To generate the DIII-NP sE construct, DIII was connected with a ferritin nanoparticle (NP) moiety via a linker. The protein constructs were expressed in Drosophila Schneider S2 cells (Thermo Fisher, CAT# R69007) according to the manufacturer’s instruction. Protein purification was performed as previously described85, with additional purification and analysis by size-exclusion chromatography using a Superose 6 column or Blue-Native gel analysis.

Plasmids encoding the heavy and light chains of antibodies (Table S5) EDE1-C8, SMZAb246, 2A10G6, E16, E53, Z004, Z006, and ZV67, in a human IgG1 expression vector66,86, and 4G2 and 9E10 in a mouse IgG2 expression vector, were transiently transfected into FreeStyle™ 293F cells (Thermo Fisher, CAT# R79007) and purified as described previously66,87. E60 was kindly provided by Dr. Michael Diamond.

Differential scanning calorimetry (DSC)
The thermal transition temperature (Tm) of the ZIKV WT sE and variants were determined by DSC using a MicroCal VP-Capillary DSC instrument (Malvern Panalytics). The protein samples were dialyzed in PBS, pH 7.4, and 400 µl of the sample at concentration of 0.27 mg/mL was loaded into the instrument. PBS buffer was used as the reference solution. The DSC experiments were performed at a scanning rate of 1 K/min under 3.0 atmospheres of pressure. The data were analyzed after buffer correction, normalization, and baseline subtraction using MicroCal VP-Capillary DSC analysis software provided by the manufacturer (Origin 7 SR4 v 7.0522). The assays were repeated at least two times.

BioLayer light interferometry
Biolayer light interferometry (BLI) was performed using an Octet RED96 instrument (ForteBio, Pall Life Sciences) as described previously66,87. The antibody was captured onto anti-human Fc biosensors at a concentration of 10 μg/ml as ligand, with test sample of sE proteins diluted in two fold series ranging from 250 nM to 62.5 nM unless otherwise specified. Biosensors, pre-hydrated in binding buffer (phosphate buffered saline (PBS), 0.01% BSA, and 0.2% Tween-20) for 10 min, were first immersed in binding buffer for 60 s to establish a baseline. They were then submerged in a solution containing the antibody ligand for 60 s to capture it, followed by a 60-s wash in binding buffer. The biosensors were subsequently immersed in solutions with various concentrations of analyte for 120 s to monitor analyte/ligand association, followed by 120 s in binding buffer to assess dissociation. Binding affinity constants, including the dissociation constant (KD), on-rate (kon), off-rate (koff), were determined using Octet Analysis software.

ELISA binding assays
mAbs were coated onto 96-well Maxisorb ELISA plates at 200 ng/well in PBS and incubated overnight at 4 °C. The following day, the plates were washed four times with 300 μL/well of 1× PBST (PBS/0.05% Tween-20). The wells were blocked with a blocking buffer (2% dry milk / 5% fetal bovine serum in PBS) for 1 h at 37 °C. After blocking, the plates were washed again, and antigen proteins were added, starting at 10 μg/ml and diluted in a five fold series, followed by incubation for 1 h at 37 °C. After another wash step, a 1:5000 dilution of Goat anti-His-HRP conjugate (Jackson ImmunoResearch) in PBST was added to each well and incubated for 1 h at room temperature. The antibody-antigen binding signal was developed by adding 100 μl/well of 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate (Life Technologies) and incubating at room temperature for 5 min, before stopping the reaction with 50 μl of 3% H2SO4. The optical density (OD) signal was measured at 450 nm to quantify the binding interaction.

Viruses
The stock of ZIKV FSS13025 (Genbank: JN860885)88 which is identical or highly related to the America circulating strains was used for challenging AG129 mice that received immune serum transfer. It was the third passage of the virus isolate in Vero cells obtained from the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA) at the University of Texas Medical Branch.

The stock of ZIKV Puerto Rico 2015 strain (GenBank: KU501215.1) that is identical or highly related to the circulating strains during the 2015–2016 Americas outbreak was used for the challenge protection studies of immunized mice. It was prepared as described previously89. Briefly, the seed virus obtained from the Division of Vector-borne Diseases, Centers for Disease Control and Prevention, followed by two passages in Vero 76 cells maintained with Leibovitz’s L-15 medium supplemented with 10% fetal bovine serum (v/v) 10% tryptose phosphate broth (v/v), L-glutamine at 2 mM, penicillin at 100 units/ml, and streptomycin at 100 μg/ml. For each passage, virus stocks were harvested at 5 days post infection, when ~70% of cells developed cytopathic effect.

DENV strain D2S10 (GenBank accession number JN796245) was originally generated by ten alternating passages of the Taiwanese clinical isolate PL046 between C6/36 Aedes albopictus cells and serum of 129/Sv mice90. A passage-three virus was kindly provided by Dr. Sujan Shresta and subsequently passed on a confluent monolayer of C6/36 cells in Leibovitz L-15 medium to produce a tissue-culture stock virus. Tissue culture virus at a tissue culture infectious dose 50% (TCID50, log10) titer of ~6.0 was provided for these studies.

Animal experiments
Animal experiments were carried out in compliance with all relevant U.S. National Institutes of Health regulations and were approved by the Institutional Animal Care and Use Committees (IACUC) at NOBLE LIFE SCIENCES, INC. (IACUC approval number: SVL-197), BIOQUAL (IACUC approval number 20-026), and University of Maryland School of Medicine (IACUC approval number: #0219005). Unless specified, all animals were housed and handled using Biosafety Level 1 containment. Animal room illumination is automatically controlled in 12-h day/night cycle, with temperature maintained between 20 and 26 oC, and humidity maintained between 30 and 70%. For the immunogenicity study, 8-to 9-week-old female C57BL/6 female mice (Charles River) were inoculated subcutaneously at two sites. Each animal received a single dose of 20 μg protein immunogen in 100 μl of PBS (pH 7.4), containing either 5 μl of Adjuplex (Advanced Bioadjuvants, LLC), formulated according to the manufacture’s manual, or polyphosphazene (PPZ) adjuvants51. The PPZ adjuvants used were poly[di(carboxylatophenoxy)phosphazene] (PCPP) and poly[di(carboxylatoethylphenoxy)phosphazene] (PCEP), as well as their supramolecular nano-scale complexes with R848 (resiquimod). PPZ-R848 complexes were prepared by mixing either PCEP or PCPP with R848 at 2:1 (w/w) ratio in PBS (pH 7.4), as described previously57. Each PPZ adjuvanted formulation contained either 50 μg of PPZ or 70 μg of PPZ-R848 complex (comprising 50 μg PPZ and 20 μg R848). The formation of the ternary complex (CC_FLE-PPZ-R848) was monitored using Asymmetric Flow Field Flow Fractionation (AF4) and dynamic light scattering (DLS). AF4 was performed with a Postnova AF2000 MT series system (Postnova Analytics GmbH, Landsberg, Germany), equipped with two PN1130 isocratic pumps, a PN7520 solvent degasser, a PN5120 injection bracket, and a UV-Visible detector (SPD-20A/20AV, Shimadzu Scientific Instruments, Columbia, MD). A regenerated cellulose membrane with a 10 kDa molecular weight cutoff and a 350 μm spacer was used in a separation micro-channel with both laminar and cross flows of PBS (pH 7.4) as the eluent. Data were processed using AF2000 software (Postnova Analytics GmbH). DLS was conducted using a Malvern Zetasizer Nano series, ZEN3600, and the data were analyzed using Malvern Zetasizer 7.10 software (Malvern Instruments Ltd, Worcestershire, UK). Samples were prepared in PBS (pH 7.4) and filtered through Millex 0.22 μm filters before analysis.

For the serum passive transfer protection efficacy study, 6-to 8-week-old female AG129 mice were infused intraperitoneally (IP) with pooled immune sera 1 h prior to subcutaneous (SC) viral challenge with 1×104 PFU/mouse of ZIKV strain FSS13025. The mice were housed and handled using Biosafety Level 2 containment, and monitored daily for weight loss, health score, and survival for 20 days post challenge.

In the in vivo ADE study, 6-to 8-week-old female AG129 mice were passively transferred with 200 μl of PBS containing 10 μl of pooled donor sera, as described previously36 via the IP route. One day later, the animals were challenged with 1×105 TCID50 of DENV-2 strain DS210, and housed using Biosafety Level 2 containment, monitored daily for weight loss, health score, and survival for 14 days post challenge.

In the vaccine protection efficacy study, 6-to 8-week-old female BALB/c mice (Jackson Laboratory) were inoculated SC over the intrascapular area with immunogen/PPZ adjuvant on study days 0 and 28. On study day 56, 4 weeks post second immunization, the mice were challenged with ZIKV strain Puerto Rico 2015 (2×106 TCID50) via subcutaneous route, a prototype of the circulating strains in Americas, and housed using Biosafety Level 2 containment. Post-challenge, the animals were monitored daily for 10 days for weight loss and clinical manifestation of disease. Blood was collected for serum viral load detection, while spleen, lymph nodes, and brain were harvested on the terminal day for tissue viral load analysis.

In the pregnant mice protection study, immunization of female BALB/c mice was performed in the same manner as described in the vaccine protection efficacy study, with PCEP+R848 as adjuvant. Following immunization, the mice were mated and observed daily for the detection of a copulatory plug (E0.5) indicating pregnancy. At embryonic day 9.5 (E9.5), female mice were challenged with ZIKV Puerto Rico 2015 (2×106 TCID50) via retro-orbital route and euthanized 8 days after infection (E17.5). Maternal sera on 1 and 8 DPI, and tissues including maternal uterus, placentas, and fetuses were collected for ZIKV virus RNA detection by quantitative RT-PCR assay.

In the immunogenicity study with OmniMouse mice, which express naïve human Ig loci, two female OmniMouse mice (OmniAb, CA), designated OM5.1 and OM5.2, aged 20–21 weeks, were immunized with 20 μg of CC_FLE sE formulated in TiterMax Gold (Sigma) adjuvant via the SC route on weeks 0 and 4. Mouse OM5.1 received an additional immunization with 20 μg of CC_FLE sE formulated in Sigma Adjuvant System (Sigma) via the IP route on week 36 and was terminated on week 38. Mouse OM5.2 received another immunization with 20 μg of CC_FLE sE without any adjuvant via the IP route 4 days before termination on week 6.

ZIKV reporter virus particle (RVP) construction, production, and neutralization assay
To quantify ZIKV mAb neutralization capacity and neutralizing antibody titers in serum samples, we utilized a previously described high-throughput plate-based ZIKV reporter virus particle (RVP) assay89. RVPs were generated by complementing a WNV sub-genomic replicon that expresses a luciferase reporter gene (Renilla luciferase) with a plasmid encoding the ZIKV CprME structural proteins (pZIKV/HPF/CprME, ZIKV French Polynesian strain H/PF/2013). Briefly, plasmids pWNVII-Rep-REN-IB and pZIKV/HPF/CprME were co-transfected at a 1:3 ratio into 293T cells (ATCC, CAT# CRL-3216) using the Fugen6 transfection reagent (Promega). After 48–96 h of incubation at 30 °C, supernatants containing RVPs were collected, filtered through a 0.45-micron filter, and stored at −80 °C. RVPs were titrated on Vero cells (ATCC, CAT# CRL-81) in duplicate to determine the appropriate dilution for the neutralization assay, aiming for ~1–5× 104 relative luminescence units (RLU) in the absence of serum or antibody, which was 1000 times higher than background (cells only).

To measure neutralization, heat-inactivated serum, starting from a 100-fold dilution with five fold serial dilutions, was incubated with diluted RVP in Eagle’s Minimal Essential Medium (EMEM) containing 10% fetal bovine serum (FBS) for 1 h at 37 °C, before infecting Vero cells at 37 °C and 5% CO2 for 1 h. The next day, cells were lysed with lysis buffer (Promega) for 40 min at room temperature with shaking, followed by the addition of Renilla Luciferase Assay System substrate (Promega) according to the manufacturer’s instructions. Luminesce was measured immediately using a Molecular Devices reader. Percent neutralization was calculated based on measurements of wells infected by RVPs in the absence of serum and or uninfected cells. Data were fitted to a 4PL curve in GraphPad Prism 8, with the top and bottom percent neutralization constrained at 100 and 0, respectively. ID50 values represented the reciprocal of the serum dilution that resulted in 50% virus entry inhibition compared to RVP alone.

For the serum neutralizing depletion assay, a dilution of pooled sera was pre-mixed with various E variant proteins at a concentration of 0.4 μg/ml for 0.5 h at 37 °C prior to RVP incubation. A dilution of antibodies (ZV67 and EDE1-C8), starting from 0.1 μg/ml, was used as control antibodies. The mixture of RVP with serum or antibody depleted by soluble protein was subsequently added to Vero cells to quantify RVP entry signal (luciferase activity) as described above.

Plaque reduction neutralization test (PRNT)
The neutralization activity of selected serum samples was measured using a standard plaque reduction neutralization test (PRNT) on VERO cells. Twelve two fold serial dilutions of pooled sera starting from 25-fold dilution, were added to 500 plaque forming units (PFUs) of ZIKV FSS13025 strain in duplicate and incubated for 1 h at 37 °C. The virus/serum mixtures were then added to Vero cells in 24-well plates for an additional hour of incubation, followed by the addition of an overlay of 0.8% methylcellulose. After incubation for 3 days at 37 °C with 5% CO2, the cells were fixed and stained to enumerate plaques using crystal violet staining. PRNT50 values were determined as the serum dilution that resulted in 50% of the number of plaques obtained with no serum treatment as control.

ADE assays
In vitro ADE assays were performed following a published method91. Briefly, serial dilutions of heat-inactivated sera, starting at a 60-fold dilution, were mixed with DENV-1 RVPs (Western Pacific strain with GenBank accession number U88535.1) or DENV-2 RVPs (16681 strain with GenBank accession number M84727.1), produced as described above by replacing pZIKV/HPF/CprME with pDENV/CprME. The mixture was incubated for 1 h at 37 °C in duplicate prior to addition to K562 cells (ATCC, CAT# CCL-243) for 2 days. Cells were lysed, and RVP infection was measured for Renilla Luciferase signal. To normalize the magnitude of enhancement across independent experiments, results are displayed relative to the maximum infectivity observed with a control cross-reactive WNV mAb E6092 run in parallel.

Quantitative RT-PCR assay for ZIKV and DENV RNA
The quantity of ZIKV or DENV RNA copies per milliliter of plasma or per gram of tissue was determined using the TAQMAN assay93. Briefly, viral RNA was extracted from serum using the QIAamp MinElute Virus Spin kit (Qiagen, Frederick, MD) or from tissue samples using the RNA STAT-60 kit (Tel-Test Inc., Friendswood, TX). The RT-PCR assays were performed using the SensiFAST Probe Lo-ROX One-Step Kit (Bioline BIO-78005, Taunton, MA). Primers and probe were designed to amplify a conserved region of the ZIKV capsid gene from the BeH815744 strain, including the primers ZIKV PR15 FWD (GGAAAAAAGAGGCTATGGAAATAATAAAG) and ZIKV PR15 REV (CTCCTTCCTAGCATTGATTATTCTCA), and the probe ZIKV PR15 PRB (AGTTCAAGAAAGATCTGGCTG), as described previously93. The sequences for the DENV primer/probe set, which target a conserved region of the polyprotein gene, are as follows: Denv-2 16681 FWD (GCGGGAAAGACGAAGAGATAC), Denv-2 16681 REV (CTCTAAGGGCTTCCTCCATTTC), and Denv-2 16681 Probe (5’–/56-FAM/CTTCCGGCC/ZEN/ATAGTCAGAGAAGCT/3IABkFQ/–3’).

To generate a control for the amplification reaction, RNA was isolated from the applicable virus stock using the same procedure. The standard curve was used to calculate the ZIKV or DENV RNA copy number of each test sample, conducted in triplicate.

Flow cytometry & Ag-specific B cell sorting
Cryopreserved OmniMouse animal splenocytes were quickly thawed in a 37 °C water bath, followed by treatment with DNase I (10,000 μ/ml, Roche, 1000-fold dilution) in RPMI 1640 with 10% FBS at room temperature for 5 min, and washed with chilled PBS. After staining with Aqua Dead Cell Staining dye (Life Technologies, Cat# L349660, LOT# 1941440, 400-fold dilution) in PBS for 15 min, followed by two steps of staining procedure, each lasting 1 h, cells were washed with PBS between these two staining procedures. Since OmniMouse mice carry a rat Ig constant region encoding sequences adjacent to the human heavy chain variable gene segments, most of the class-switched B cells would bear rat IgG constant region. The first staining medium contains polyclonal goat anti-rat IgG PE/Dazzle 594 conjugate (Biolegend, CAT# 405432, LOT# B319517, 100-fold dilution) to identify class-switched B cells.

The second staining medium contained a cocktail of antibodies and antigen for identifying antigen-specific B cells, as described previously with minor modifications67. Briefly, a cocktail of antibodies diluted in RPMI 1640/10% FBS, which contains CD3-PerCp/Cy5.5 (clone 17A2, Biolegend, CAT# 100218, LOT# B233420, 500-fold dilution), F4/80-PerCp/Cy5.5 (clone BM8, Biolegend, CAT# 123128, LOT# B222447, 1000-fold dilution), Gr1-PerCp/Cy5.5 (clone RB6-8C5, Biolegend, CAT# 108428, LOT# B245570, 2000-fold dilution), CD19-APC-Cy7 (clone 1D3, BD Pharmingen, CAT# 557655, LOT# 6070644, 500-fold dilution), B220- Alexa Fluor® 700 (clone RA3-6B2, BioLegend, CAT# 103232, LOT# B242183, 2000-fold dilution), mouse IgG2a-FITC (clone RMG2a-62, Biolegend, CAT# 407106, LOT# B199948, 500-fold dilution), mouse IgG2b-FITC (clone R12-3, BD Pharmingen, CAT# 553395, LOT# 7096543, 500-fold dilution), mouse IgG1-FITC (clone A85-1, BD Pharmingen, CAT# 553443, LOT# 7020839, 500-fold dilution), mouse IgD-Pacific Blue (clone RA3-6B2, BioLegend, CAT# 405712, LOT# B209676, 1000-fold dilution), and CC_FLE–streptavidin-PE conjugate (at 2.5 μg/ml) was used to stain the cells. CC_FLE-Avi carrying an Avi-tag at the c-terminus were biotinylated with BirA 500 biotin ligase (Avidity, AviTag™ Technology, Aurora, CO), followed by mixing with streptavidin-PE conjugate (Invitrogen, CAT# S21388, LOT# 1784902) at 1:1 molar ratio to form CC_FLE–streptavidin-PE conjugate as described previously66. As shown in Fig. S10, the stained cells were applied to a BD FACS Aria III cell sorter (BD Biosciences) to sort for antigen-specific class-switched B cells with BD FACSDiva software V8.02 using the phenotype of CD19+/B220+/CD3-Gr1-F4/80-/mouse IgD-IgG-/rat IgG+/CC_FLE+ into 96-well plates at single-cell density, followed by single-cell reverse transcription and PCR reactions to amplify human Ig V(D)J gene segments as previously described86,94,95. The sequences of the PCR primers are provided as Supplementary Data 1 file in the Supplementary Information File folder.

The resultant human Ig gene segments were analyzed by IMGT/V-Quest using the human Ig germline reference database, with the variable domains of selected heavy/light (VH/VL) chain matched clones being cloned into corresponding expression vectors, as described previously86, followed by co-transfection of VH/VL expression plasmids into 293F cells66 to reconstitute human antibodies in IgG1 format. The expressed human antibodies were purified by Protein A Sepharose columns (GE Healthcare). To produce an antibody Fab fragment, the heavy chain variable domain was cloned into the Fab expression vector described previously96, followed by co-transfection with light chain expression vector in 293F cells and purification using cOmplete His-Tag Purification Resin (Roche).

OmniMouse Zika E-specific mAb competition assay
The following experiment was performed using Bio-Layer Interferometry (BLI) with an Octet RED96 platform (ForteBio; Pall Life Sciences) to determine if antibodies compete for binding to ZIKV E-dimer. ZIKV E-dimer CC-FLE, purified by size exclusion chromatography at 20 nM, was immobilized on the pre-wet Octet® Ni-NTA (NTA) Biosensors (Sartorius) for 3 min, followed by a wash in 1× kinetics buffer (1× PBS, 0.2% Tween-20). The biosensors were then submerged in wells containing the ligand mAb at 10 μg/ml in kinetics buffer for 10 min, followed by a 60-s wash step. Subsequently, the biosensors were immersed in wells containing analysate mAb at 10 μg/ml in kinetics buffer for 10 min, followed by a dissociation step in which the biosensors were held in 1× kinetics buffer for another 10 min. In each step, BLI signals were collected at 24 °C with orbital shaking at 1000 rpm in 96-well black flat bottom plates (Greiner, VWR). The maximal analysate mAb signals were obtained in the absence of any ligand mAb (kinetics buffer only). The relative analysate mAb binding signals were calculated as 100*(Analysate mAb signal in the presence of ligand Ab / Maximal analysate mAb signal in the absence of ligand Ab).

Cryo-EM sample preparation, data collection, model refinement and analysis
SMZAb2 Fab was incubated with the ZIKV CC_FLE sE protein at a 2:1 molar ratio per E protomer overnight at 4 °C. The concentrations of the CC_FLE sE:SMZAb2 complex, or unliganded JEV and WNV CC_FLE sEs were adjusted to 1 mg/ml in PBS, and 3 μl of the sE was added to a Quantifoil grid (R2/2 Cu 200 mesh or R1.2/1.3 Cu 300 mesh; Electron Microscopy Services) that had been freshly glow-discharged using a PELCO easiGLOW (Ted Pella). Samples were vitrified in 100% liquid ethane using a Mark IV Vitrobot (Thermo Fisher) at 100% humidity and 12 oC. The micrographs were collected on a Thermo Fisher-FEI Glacios transmission electron microscope operating at 200 kV, using custom scripts in SerialEM software version 4.197. Movies were obtained on a Gatan K3 direct electron detector at a magnification of 56,200×, using a 0.4–2.6 μm or 0.5–2.7 μm defocus range. The micrographs were processed with cryoSPARC version 3.3.098. For the CC_FLE sE:SMZAb2 complex, a total of 141,948 particles were selected for final local 3D refinement with C2 symmetry applied, resulting in a reconstruction at 4.09 Å resolution. For the JEV CC_FLE sE, the final 3D reconstruction and refinement of 172,829 particles with imposed C2 symmetry resulted in a 4.18 Å resolution map. The structure of the JEV E dimer (PDB: 3P54) was used as the initial template. The individual chains of the initial model were docked into Cryo-EM density maps with UCSF Chimera (version 1.18)80, followed by the iterative cycles of building and refinement with Coot (version 0.9.8.1)99, Rosetta (version 3.13)100, and Phenix (version 1.20)101. For the WNV CC_FLE sE, the final 3D reconstruction and refinement of 14,474 particles with imposed C2 symmetry resulted in a 7.96 Å resolution map. No attempts to build a structural model of WNV CC_FLE sE were made.

The OZ-D4 Fab was incubated with ZIKV CC_Core sE in a 1.5:1 molar ratio (per E protomer) overnight at 4 oC. The concentration of the complex was adjusted to 1 mg/ml in PBS, and followed by grid preparation, vitrification, and data collection as described above. The final local 3D refinement of 710,140 particles with imposed C1 symmetry resulted in a 3.45 Å global resolution map (Fig. S11). The structure of ZIKV E dimer (PDB: 5LBV) was used as the initial template for ZIV CC_Core. The OZ-D4 Fab variable region was generated with SAbPred102. The individual chains of the initial models were docked into Cryo-EM density maps with UCSF Chimera80, followed by the iterative cycles of building and refinement with Coot99, Rosetta100, and Phenix101. The structure building, refinement, and visualizations of the E:mAb complex were performed using similar tools and approaches described earlier.

Statistical analysis
All statistical analyses are performed using GraphPad Prism V10.1.0, unless otherwise specified. Significance thresholds are set at p<0.05. Two-tailed Mann–Whitney U tests (95% confidence level) are used for comparisons of serum neutralizing titers (Figs. 5B and 6B), viral loads (Figs. 5G, 6C–D and S8B–D), and ID50 titers (Fig. S8B–D). Serum ADE for DENV-1 and DENV-2 (Fig. 5E–F) is analyzed using a one-way ANOVA (Kruskal–Wallis) test. Survival curves (Fig. 5D, G) are compared using the Mantel–Cox log-rank test. Health scores (Fig. S6) are analyzed using multiple t tests, and correlations between serum neutralizing titers and log10-transformed viral loads (Fig. 6E) are assessed using the Spearman nonparametric test.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Wang and colleagues present in this paper the characterization of an engineered dimer of the Zika virus (ZIKV) envelope protein (E). The Flavivirus E dimer has been shown to be target of potent cross-neutralizing dengue and Zika antibodies (E-dimer epitope antibodies – EDE). The dimer also hide the fusion loop epitope (FLE) which is targeted by antibodies that cause antibody-dependent enhancement (ADE) of infection. FLE antibodies are cross-reactive but poorly neutralizing and ADE is a risk factor for severe pathology during Flavivirus secondary infection. EDE antibodies instead are cross-reactive and highly neutralizing, and several strategies have been developed to engineer antigens able to expose EDE epitopes and hide FLE epitopes. Here, the authors have introduced a G5C/G102C mutations (CC_FLE construct) that generate a disulphide bond between the N-term of domain I (DI) of one protomer and the fusion loop (FL) of the opposite protomer resulting in a locked ZIKV E dimer. They compare this construct to an E-monomer and a E-dimer with a single mutation A264C (CC_Core construct). The latter construct has already been reported in the literature (doi: 10.1074/jbc.RA119.007443; doi: 10.1038/s41598-017-01097-5). The three constructs have been tested for antigenicity and protection in an AG129 mouse and in immunocompetent mice model. In addition, the authors used the CC_FLE antigen to immunize Omni-mouse animals (carrying human Ig-loci) from which 4 antibodies have been cloned and one EDE-like humanized antibody (OZ-D4) has been characterized. The engineered dimer alone or in complex with OZ-D4 has been structurally characterized. The authors gathered an impressive amount of data that however need to be thoroughly discussed in comparison to the already published constructs to highlight the added value of the proposed strategy. Immunological cross-reactivity between Flaviviruses circulating in the same endemic population makes vaccine design challenging because the antigen to be used in a vaccine formulation, in addition to expose protective epitopes, needs to mask enhancing epitopes and avoid uncovering cryptic epitopes. The strategy to engineer stabilized E-dimer for dengue or Zika has been first proposed by Rouvinski et al. in 2017 (doi: 10.1038/ncomms15411) and used for vaccine design and protection studies by several authors (doi.org/10.1016/j.jbc.2022.102079; doi.org/10.1038/s41590-019-0477-z; doi.org/10.1038/s41590-021-00966-6; doi 10.1074/jbc.RA119.007443).\nFrom the structural point of view there are few points that are not clear:\n1) How the introduced mutations affect the overall structure of the E-dimer? In the E dimer, the N-term of DI, leans towards DIII and the DI-150loop is juxtaposed to the FL. In the TBE dimer, for example, amino acids H5 and G102 form an electrostatic bond only at low pH when the 150-loop is in an “open lid” position away from the FL, but at neutral pH the 150-loop is in a “closed lid” position interacting with G102 while the DI N-term shifts slightly away from FL (doi.org/10.1038/s41467-022-31111-y). The newly created C-C bond between G5 and G102 would force DI N-term to stay close to the FL and affect the 150-loop position and probably also DIII overall conformation. From figure S2A and S2C The CC_FLE dimer looks open in the middle with an altered intradimer interface and a bulgy DIII. The PDB model shows a more closed dimer but the fitting of the model in the density map could be slightly adjusted to assure a more precise reconstruction (within the limits of the relative low resolution).\nHow this structure overlaps with the unlocked dimer (for example from the cryoEM structure of the Zika or JEV virus)? In figure S2D the map density is barely visible, a more intense colour would help to appreciate the amino acids fit in the density. X-ray crystallography for both locked dimers would help in defining the impact of the mutations on the overall structure of the dimer. Structure of the dimer in complex with known human antibodies would allow to verify if the protective epitopes are correctly exposed.\n2) What is the rationale for the strategy reported in lines 169-175? Has the structure of DI-DIII construct been analyzed? DI may not be correctly folded, and this antigen would focus the response only to DIII and probably also to DIII epitopes that usually are not exposed in the dimer.\n3) Fig.8: The footprint of OZ-D4 antibody does not overlap with the ones of the EDE antibodies. OZ-D4 binds DII and DIII but not DI (differently from the EDEs) and approaches the dimer from the side. This binding would clash with the herringbone arrangement of the dimer on the virus. A docking of this antibody on the cryoEM of the Zika particle would allow to check the accessibility of the E dimer to OZ-D4. Neutralization has been tested with the RVP system but not with authentic virus. It would be interesting to verify the affinity of OZ-D4 for the dimer in the context of the herringbone organization.\nFor the antigenicity studies:\n1) Could the authors clarify the epitope targeted by antibody SMZAb2? Is this the antibody isolated by Magnani et al. (doi: 10.1126/scitranslmed.aan8184)? SMZAb2 in this publication has been classified as an FL antibody, is binding in ELISA to a ZIKV E protein and is competing with FL antibody 4G2. Here the authors classify SMZAb2 as an EDE that does not recognize the WT monomer construct (Fig. 2C). In addition to EDE-C8 have the authors tested EDE-C10? This antibody has shown to be more sensible to the costraints induced in the E-dimer by engineered C-C bonds and could give information about the flexibility of the C5/C102 E-dimer.\n2) Some of the FLE antibodies, like 2AG106, bind G102. Have the authors considered the possibility that the lack of binding of the CC_FLE antigen could be due to the mutation of this amino acid more than a general non-exposure of the FL?\n3) Why the binding of FLE antibodies (Fig.2C) is different between WT and CC_Core? The FL should be exposed equally in both constructs because the dimer is “breathing”.\n4) The neutralization data reported in Fig.4B are not clear. How C264 mutation would affect antigenicity? The impact of this mutation on the structure does not explain the different response obtained compared to WT construct. Also, DI-DIII and DIII construct bind similarly to DIII antibodies, but the neutralization profile is different. Have these sera been tested against authentic virus? May be the organization of the E protein on the RVP plays a role.\n5) Fig.5G: How was the amount of serum to be passively transferred chosen? How would a positive control (Virus + FLE antibody at enhancing concentration) behave in this system?\n6) Fig.S6A: For the depletion experiments graphs, I would add in the legend a column indicating the Abs specificities left (FLE, DIII, EDE). This would facilitate the reading of the graphs.\n7) I would move Fig.S6B to main Fig.6 because it shows the landscape of antibody specificities elicited by the different antigens. I would give more information on how to read the graph. My understanding is that a high Y axis value corresponds to a drop of neutralization activity (i.e. the antigen depletion has removed antibodies important for neutralization). It is interesting that the sera obtained from WT immunization show the highest reduction in neutralization when depleted with the CC_FLE and not when depleted with WT. It would suggest that the immunization with WT has induced also some EDE antibodies (may be because the soluble E protein in solution is in monomer-dimer equilibrium and can also expose dimer epitopes?). These antibodies specificities should be analyzed more in detail and discussed.\n8) The OmniMouse model is interesting. Why have the authors not performed a comparison between WT and CC_FLE antigens? I see the interest in cloning specific antibodies from these mice, but it would be also informative to look at the B cell repertoire induced by the two types of antigens to appreciate the superiority of the CC_FLE antigen.\n9) Why was the OmniMouse 5.2 terminated at 6 weeks p.i., much earlier than the OmniMouse 5.1 (38 weeks)? From the shape of the neutralization curves, the sera do not show a strong neutralizing activity. Have the sera been tested for presence of FLE antibodies?\n10) It is interesting that the complex of OZ-D4 with CC_FLE did not result in high resolution data while the complex with CC_Core did. The antibody binds the two antigens with comparable affinity (Fig.7D).\nMinor points:\nLine 173: What is (32-34) referring to? The reference number does not match with “DIII immunogenicity” described in the text.\nLines 179-181: It would be informative to show the SEC of profile of the proteins after affinity purification (may be as a supplementary figure) to appreciate the heterogeneity of the forms produced by the constructs.\nLines 191-192: Please list all the antibodies that have been tested with their epitope specificity and recognition of the antigens tested. A figure and a summary table in supplementary data would be informative.\nLines 196-199: This sentence is redundant. These results have been presented in the previous paragraphs.\nFig.5: B and C show neutralization assays results (RVP and authentic virus). I would plot both in the same way (either ID50 or % of neutralization).\nLine 364: the ID50 titer increase is not 2 logs (5012 vs 178 = 28 folds).\nLines 399-400: I do not understand the sentence “surpassing the nascent WT sE”.\nFig.8: 8A – show the HC and LC chains with more contrasting colors. 8F – show a top view of the dimer to highlight the antibody footprint. The actual side view is not informative.", "reviewer_2": "The present manuscript by Wang et al presents a novel vaccine design strategy targeting the E protein of the Zika virus (ZIKV), aimed at bolstering immune responses and mitigating the risk of Antibody-dependent enhancement (ADE). The authors have engineered two mutations, G5C/G102C, into the flavivirus E protein, resulting in the formation of inter-chain disulfide bonds and the creation of E-dimers. The E-dimer formation not only augments the immunogenicity of the vaccine candidate but also masks the ADE-associated site FLE, thereby diminishing the likelihood of ADE. The vaccine candidate, CC-FLE sE, is primarily constructed based on the ZIKV E protein sequence. Upon immunization in mice, it elicits potent neutralizing antibodies that confer protection against ZIKV infection and attenuate the risk of ADE following infections with various flaviviruses. Furthermore, the article delineates the structural and functional analysis of the E-dimer-dependent epitope (EDE) monoclonal antibody OZ-D4, highlighting the high affinity of CC-FLE sE for the natural precursor of OZ-D4. This suggests that the robust viral epitope recognition and neutralization by CC-FLE sE are achievable without extensive somatic mutations, indicating its potential to elicit an immune response within the human B cell repertoire. Consequently, CC-FLE sE emerges as a promising vaccine design strategy for flaviviruses, addressing the dual challenges of immune protection and ADE reduction that are central to flavivirus vaccine development. However, to bolster the rigor and comprehensiveness of the study's conclusions, the authors are advised to address the following key points prior to further consideration:\n1) Immunogenicity data: The presented data regarding immunogenicity exhibit inconsistencies and lack persuasiveness. To enhance credibility, it is imperative to conduct PRNT (Plaque Reduction Neutralization Test) titers for Figures 4-6. While Figure 4C illustrates the outcomes of the PRNT, it is crucial to express these findings as either PRNT50 or PRNT90 values for meaningful comparison. Moreover, a statistical correlation analysis between the RVP system results and the PRNT titers must be performed to strengthen the validity of the conclusions drawn. Of particular concern is the discrepancy observed in Figure 5D, where negative outcomes in mortality from passive transfer experiments contradict the reported high levels of neutralizing antibodies. This apparent paradox necessitates a thorough explanation and nuanced discussion within the text.\n2) Vaccine efficacy assay: At lines 357-360, the authors' decision to employ Balb/c mice for assessing vaccine efficacy is unconventional and warrants additional justification. Given industry standards, incorporating challenge data from A129 or AG129 mouse models would significantly bolster the study's comprehensiveness. Furthermore, evaluating the immunogenicity and protective efficacy of the vaccine candidate in pregnant mice is highly recommended to address potential concerns related to maternal immunization strategies.\n3)Figure 6 depicts solely the effects of adjuvants, which could be more appropriately relocated to the supplementary materials section.", "reviewer_3": "Wang and colleagues employed a structure-based approach to design the envelope (E) dimer of various flaviviruses using intra- and inter-chain disulfide bonds to improve stability and minimize exposure of the antibody-dependent enhancement (ADE)-prone fusion loop epitope (FLE). The designed constructs were produced as recombinant proteins and their immunogenicity was evaluated in mice, in conjunction with several adjuvants used either alone or in combination. One candidate, CC_FLE sE, derived from the Zika virus (ZIKV) and formulated with an advanced supramolecular adjuvant, emerged as the most potent in inducing a desirable antibody response and providing protection against live ZIKV challenge. The same immunogen also induced monoclonal antibodies targeting the E-dimer-dependent epitope (EDE) in a transgenic mouse model expressing human antibody genes. The authors propose that their designed immunogen, CC_FLE sE, represents a promising strategy for developing ZIKV vaccines that minimize ADE risks while maintaining high protective efficacy. Furthermore, the design strategies may be applicable to vaccines targeting other flaviviruses such as Japanese encephalitis virus (JEV) and tick-borne encephalitis virus (TBEV).\nThis study is significant. The design of CC_FLE sE is scientifically robust, most of the experiments are well-executed, and the conclusions are largely supported by the data. However, there are several major gaps in understanding the structural, immunological, and protective features of CC_FLE sE, which render some parts of the study less convincing and compelling. Specific questions and suggestions are detailed below:\n1. Structural Confirmation: While the antigenicity of CC_FLE sE and CC_Core was evaluated using mAbs specific to EDE, DIII, and FLE, obtaining the crystal structure of CC_FLE sE would confirm that the introduced disulfide bonds are correctly positioned and do not disrupt the overall E-dimer structure.\n2. Biophysical Stability: Thermal stability comparisons of CC_FLE sE and CC_Core to the wild-type (WT) protein would provide valuable insights into the enhanced stability conferred by the design.\n3. Neutralizing Response (Figure 4): The unexpectedly low neutralizing response induced by CC_Core warrants an explanation. Are there structural or immunological factors that might account for this outcome?\n4. Adjuvant Selection (Figure 5B): The neutralizing antibody response induced by CC_FLE with Adjuplex is similar to that with RCEP+R848. Why did the authors prioritize the use of RCEP+R848, which is a complex and mechanistically unclear adjuvant? Additionally, it is surprising that CC_FLE formulated with R848, or CC_FLE alone, failed to induce any detectable neutralizing antibody response.\n5. Protective Efficacy (Figure 5D): The absence of protective differences between WT and PBS is puzzling, given the high levels of neutralizing antibodies in WT compared to the absence of detectable antibodies in PBS (Figures 5B and 5D). This discrepancy requires clarification.\n6. ADE-Mediated Enhancement (Figures 5E-G): While the results suggest potential ADE-mediated enhancement of DENV-1 and DENV-2 infection in vitro and DENV-2 in vivo, these findings cannot definitively be attributed to FLE-specific antibodies. Competition assays to quantify the proportion of FLE-specific antibodies in the sera and depletion experiments to remove FLE-specific antibodies before conducting in vitro and in vivo studies would strengthen the conclusions.\n7. Mouse Model Discrepancies (Figure 6): Substantial differences in neutralizing antibody responses were observed between BALB/c and C57BL/6 mice. Additionally, the authors shifted from using Adjuplex to focusing solely on RCEP+R848. What is the rationale for this change, and how can the discrepancies observed in C57BL/6 mice be reconciled?\n8. Monoclonal Antibody Competition (Figure 7): The isolation and characterization of four mAbs (OZ-D4, B11, A1, and B9) with distinct epitope specificities is interesting. Competing immune sera with these mAbs to estimate the relative proportion of similar antibodies in the sera would be a critical experiment to demonstrate that CC_FLE sE indeed induces higher levels of EDE antibodies compared to WT.\n9. ADE Potential of Isolated mAbs (Figure 8): The structure of OZ-D4 indicates it has four-residue contacts within FLE. Investigating whether OZ-D4, along with OZ-B11, A1, and B9, contributes to ADE effects on DENV-1 and DENV-2 in vitro would add valuable insights.\n10. Germline Reversion (Figure 9): While the germline reversion experiment is intriguing, the results may be influenced by the specific nature of the transgenic mouse model used. Overstatements regarding these findings should be avoided."}, "author_response": "Point-by-Point Response to Reviewer’s Comments\nWe appreciate the overall positive and constructive comments from the reviewers. We have\nperformed additional experiments and added more data into the manuscript including (i) cryoEM\nstructure of ZIKV CC_FLE sE complexed with antibody SMZAb2 (EDE1 class), (ii)\nthermostability characterization of ZIKV CC_FLE sE, and (iii) ZIKV CC_FLE sE efficacy in\npregnancy.\nAfter carefully revising our manuscript, with the edited text highlighted in a word file containing\ntracked changes, we are providing point-by-point response to their comments as below.\nReviewer #1 (Remarks to the Author):\nComment\nWang and colleagues present in this paper the characterization of an engineered dimer of the\nZika virus (ZIKV) envelope protein (E). The Flavivirus E dimer has been shown to be target of\npotent cross-neutralizing dengue and Zika antibodies (E-dimer epitope antibodies – EDE). The\ndimer also hide the fusion loop epitope (FLE) which is targeted by antibodies that cause\nantibody-dependent enhancement (ADE) of infection. FLE antibodies are cross-reactive but\npoorly neutralizing and ADE is a risk factor for severe pathology during Flavivirus secondary\ninfection. EDE antibodies instead are cross-reactive and highly neutralizing, and several\nstrategies have been developed to engineer antigens able to expose EDE epitopes and hide FLE\nepitopes. Here, the authors have introduced a G5C/G102C mutations (CC_FLE construct) that\ngenerate a disulphide bond between the N-term of domain I (DI) of one protomer and the fusion\nloop (FL) of the opposite protomer resulting in a locked ZIKV E dimer. They compare this\nconstruct to an E-monomer and a E-dimer with a single mutation A264C (CC_Core construct).\nThe latter construct has already been reported in the literature (doi: 10.1074/jbc.RA119.007443;\ndoi: 10.1038/s41598-017-01097-5). The three constructs have been tested for antigenicity and\nprotection in an AG129 mouse and in immunocompetent mice model. In addition, the authors\nused the CC_FLE antigen to immunize Omni-mouse animals (carrying human Ig-loci) from\nwhich 4 antibodies have been cloned and one EDE-like humanized antibody (OZ-D4) has been\ncharacterized. The engineered dimer alone or in complex with OZ-D4 has been structurally\ncharacterized. The authors gathered an impressive amount of data that however need to be\nthoroughly discussed in comparison to the already published constructs to highlight the added\nvalue of the proposed strategy.\nImmunological cross-reactivity between Flaviviruses circulating in the same endemic population\nmakes vaccine design challenging because the antigen to be used in a vaccine formulation, in\naddition to expose protective epitopes, needs to mask enhancing epitopes and avoid uncovering\ncryptic epitopes. The strategy to engineer stabilized E-dimer for dengue or Zika has been first\nproposed by Rouvinski et al. in 2017 (doi: 10.1038/ncomms15411) and used for vaccine design\nand protection studies by several authors (doi.org/10.1016/j.jbc.2022.102079;\ndoi.org/10.1038/s41590-019-0477-z; doi.org/10.1038/s41590-021-00966-6; doi\n10.1074/jbc.RA119.007443).\nResponse:\nWe have cited these references in the text.\nComment\n1) How the introduced mutations affect the overall structure of the E-dimer? In the E dimer, the\nN-term of DI, leans towards DIII and the DI-150loop is juxtaposed to the FL. In the TBE\ndimer, for example, amino acids H5 and G102 form an electrostatic bond only at low pH\nwhen the 150-loop is in an “open lid” position away from the FL, but at neutral pH the 150-\nloop is in a “closed lid” position interacting with G102 while the DI N-term shifts slightly\naway from FL (doi.org/10.1038/s41467-022-31111-y). The newly created C-C bond between\nG5 and G102 would force DI N-term to stay close to the FL and affect the 150-loop position\nand probably also DIII overall conformation. From figure S2A and S2C The CC_FLE dimer\nlooks open in the middle with an altered intradimer interface and a bulgy DIII. The PDB\nmodel shows a more closed dimer but the fitting of the model in the density map could be\nslightly adjusted to assure a more precise reconstruction (within the limits of the relative low\nresolution).\nResponse:\nCryoEM structures of the ZIKV CC_FLE (new structure, Fig. 2C) and JEV CC_FLE (Fig. 3C,\n3D) constructs confirm the integrity of the engineered dimers. The engineered G5C/G102C\ndisulfide bonds stabilizing the dimers are visualized in both structures (Fig2D., Fig3D CC bond\nwith map). In ZIKV CC_FLE structure the 150-loop is disordered. In JEV CC-FLE, the 150-loop\nadopts very similar configuration (yellow, with glycan 154) as in unmodified JEV dimer (pdb\n3p54, blue) and very different from TBEV E dimer structure (pdb 7qre, red) (see Figure below)\nComment\nHow this structure overlaps with the unlocked dimer (for example from the cryoEM structure of\nthe Zika or JEV virus)? In figure S2D the map density is barely visible, a more intense colour\nwould help to appreciate the amino acids fit in the density. X-ray crystallography for both locked\ndimers would help in defining the impact of the mutations on the overall structure of the dimer.\nStructure of the dimer in complex with known human antibodies would allow to verify if the\nprotective epitopes are correctly exposed.\nResponse:\nWe compared (Table S2) several structural parameters of Zika CC_FLE dimer with several\nunmodified wild-type (WT) dimers (e.g., PDB: 5LBS) and virus-derived dimers (e.g., PDB:\n6CO8). Zika CC_FLE maintained a similar interprotomer distance (24.5 Å) and crossing angle\n(8.8°) compared to the virus-derived dimer but exhibited a higher buried surface area (BSA:\n1689.8 Å², close to average BSA: 1618 Å² for virus-derived dimer) than the WT dimer (average\nwhile providing a stronger dimer presentation, better mimicking the virus-derived dimer (PDB:\n6CO8).\nFor JEV, structural comparisons showed that the wild-type dimer (e.g., PDB: 3p54) had the\nlargest interprotomer distance (26.5 Å) with a smaller BSA (842.7 Å²) (Table S2). The virus-\nderived dimer (PDB: 5wsn) had a more compact structure with interprotomer distance 25.4 Å,\nand a higher BSA (1129.1 Å²). The modified JEV_FLE dimer closely resembled the virus-derived\ndimer, sharing the same interprotomer distance (25.4 Å) but with an intermediate crossing angle\n(7.4°) and increased BSA (1191.8 Å²). This suggests that JEV_FLE version of the dimer shifts\ntoward a virus-like conformation, enhancing its stability and displaying more natural virus-like\nantigenic presentation.\nWe have enhanced the map density of Fig. 2D and Fig. 3D, which is more visible. We have\nobtained Cryo-EM structure of the ZIKV CC_FLE-sE protein complex with known human\nSMZAb2 Fab (Fig. 2C, 2D, Fig. S2), which verified that the EDE1 epitope is correctly presented\nin our modified dimer.\nComment\n2) What is the rationale for the strategy reported in lines 169-175? Has the structure of DI-\nDIII construct been analyzed? DI may not be correctly folded, and this antigen would focus the\nresponse only to DIII and probably also to DIII epitopes that usually are not exposed in the\ndimer.\nResponse:\nThe rationale is to delete DII (residues 51–133 and 195–284) or DI and DII (residues 1–302),\nwhich contain more conserved amino acid residues including the FLE that form ADE-prone\nepitopes, in order to prevent the potential elicitation of ADE-inducing antibodies. Since the\nimmunogenicity outcome of DI-DIII is suboptimal, we therefore did not further investigate the\nstructure.\nComment\n3) Fig.8: The footprint of OZ-D4 antibody does not overlap with the ones of the EDE antibodies.\nOZ-D4 binds DII and DIII but not DI (differently from the EDEs) and approaches the dimer\nfrom the side. This binding would clash with the herringbone arrangement of the dimer on the\nvirus. A docking of this antibody on the cryoEM of the Zika particle would allow to check the\naccessibility of the E dimer to OZ-D4. Neutralization has been tested with the RVP system but\nnot with authentic virus. It would be interesting to verify the affinity of OZ-D4 for the dimer in\nthe context of the herringbone organization.\nResponse:\nsE dimer in the herringbone arrangement at a more lateral\nangle (figure on right) (OZ-D4 in red), whereas SMZAb2\n(in green) at a more vertical angle. OZ-D4 demonstrated\nneutralization activity against authentic ZIKV virus (new\ndata, Fig. 7G), as well as the RVP, despite with reduced\npotency compared to the RVP, suggesting that it can\noverall overcome the steric hindrance on the virus surface.\nFor the antigenicity studies:\nComment:\n1) Could the authors clarify the epitope targeted by antibody SMZAb2? Is this the antibody\nisolated by Magnani et al. (doi: 10.1126/scitranslmed.aan8184)? SMZAb2 in this publication has\nbeen classified as an FL antibody, is binding in ELISA to a ZIKV E protein and is competing\nwith FL antibody 4G2. Here the authors classify SMZAb2 as an EDE that does not recognize the\nWT monomer construct (Fig. 2C). In addition to EDE-C8 have the authors tested EDE-C10?\nThis antibody has shown to be more sensible to the costraints induced in the E-dimer by\nengineered C-C bonds and could give information about the flexibility of the C5/C102 E-dimer.\nResponse\nWe have obtained cryoEM structure of ZIKV CC_FLE sE complexed with SMZAb2 (Fig. 2C,\nFig. S2, Table S1), with epitope analysis reveals that the SMZAb2 epitope significantly overlaps\nwith EDE1-C8, a well-characterized broadly neutralizing antibody (Fig. S2F-G, Fig. 8E). Of\nthe 32 residues engaged by SMZAb2, 27 are shared with the EDE1-C8 epitope (Fig. S2G),\nunderscoring SMZAb2’s classification within the EDE1 antibody family.\nWe have tested the binding of EDE-C10 to our dimeric immunogens by BLI, with an overall\naffinity of ~100 nM, indicating that all soluble form of ZIKV sE dimers impose flexibility\nconstraints for the C10 epitope presentation.\nComment:\n2) Some of the FLE antibodies, like 2A10G6, bind G102. Have the authors considered the\npossibility that the lack of binding of the CC_FLE antigen could be due to the mutation of this\namino acid more than a general non-exposure of the FL?\nResponse:\nMutation G102C by itself is not sufficient to abolish FLE-mAb binding, as minor monomeric\nfraction of ZIKV CC_FLE sE could still bind FEL-mAbs, like WT sE. Therefore, the reduced\nFLE-mAb binding of CC_FLE is caused by the non-exposure of the FL resulting from the\nG102C/G5C mutations.\nComment:\n3) Why the binding of FLE antibodies (Fig.2C) is different between WT and CC_Core? The FL\nshould be exposed equally in both constructs because the dimer is “breathing”.\nResponse:\nBriefly, The FL in monomeric WT sE is fully exposed, while the CC_Core dimer is “breathing”\n(Tm increased by only 3 oC compared to WT, Fig. 2B) with FL partially exposed, the CC_FLE\ndimer is much “locked” with FL heavily occluded.\n4) The neutralization data reported in Fig.4B are not clear. How C264 mutation would affect\nantigenicity? The impact of this mutation on the structure does not explain the different response\nobtained compared to WT construct. Also, DI-DIII and DIII construct bind similarly to DIII\nantibodies, but the neutralization profile is different. Have these sera been tested against\nauthentic virus? May be the organization of the E protein on the RVP plays a role.\nResponse:\nAntigenicity (antibody binding profile) of a given immunogen (tested in vitro) does not always\ntranslate to in vivo immunogenicity (e.g. neutralizing antibody titers), this is often observed in\nimmunogen design studies. Some other factors, such as protein stability, could also play a role in\nimpacting the presentation of neutralizing epitopes. In the case of the different immunogenicity\noutcome of CC_FLE and CC_Core immunogen, the substantially higher thermostability of\nCC_FLE may enable this E dimer presenting neutralizing antibody epitopes in the germinal\ncenter for a longer duration than the CC_Core. Additionally, the “beathing” dimer nature of\nCC-Core may also partially affect the presentation of DIII LR epitope that is well exposed in the\nmonomeric sE. This may help to explain the poor immunogenicity of CC_core immunogen after\ntwo immunizations. In a different study, after the third immunization, CC_Core immunized\nanimals started to show higher neutralizing antibody titers. Therefore, CC_Core does not\nimprove immunogenicity compared to the WT sE.\nThe DI-DIII configuration may lead to a decreased protein stability due to the deletion of DII,\nthus having a different immunogenicity outcome from DIII.\nComment:\n5) Fig.5G: How was the amount of serum to be passively transferred chosen? How would a\npositive control (Virus + FLE antibody at enhancing concentration) behave in this system?\nResponse:\nThe amount of serum to be passively transferred followed the previous publications (PMID:\n28340344). We have not used any FLE antibody as control. The WT sE polyclonal sera in our\nstudy showed behave very consistently with previously published studies (PMID: 28340344).\nComment:\n6) Fig.S6A: For the depletion experiments graphs, I would add in the legend a column indicating\nthe Abs specificities left (FLE, DIII, EDE). This would facilitate the reading of the graphs.\nResponse:\nWe have added the Ab specificity legend column in this figure (now Fig. S7)\nComment:\n7) I would move Fig.S6B to main Fig.6 because it shows the landscape of antibody specificities\nelicited by the different antigens. I would give more information on how to read the graph. My\nunderstanding is that a high Y axis value corresponds to a drop of neutralization activity (i.e. the\nantigen depletion has removed antibodies important for neutralization). It is interesting that the\nsera obtained from WT immunization show the highest reduction in neutralization when depleted\nwith the CC_FLE and not when depleted with WT. It would suggest that the immunization with\nWT has induced also some EDE antibodies (may be because the soluble E protein in solution is\nin monomer-dimer equilibrium and can also expose dimer epitopes?). These antibodies\nspecificities should be analyzed more in detail and discussed.\nWe appreciate the reviewer’s suggestion. However, the polyclonal antibody response specificity\nanalysis data is linked to Fig.5, which is already quite full. Therefore, we would leave it in the\nnew Fig. S7. We have similar analysis data from the OmniMouse animal shown in Fig. 7B,\nwhich well represents the Ab response specificity of CC_FLE sE immunization.\nIn the figure legend, we have listed how the depletion effect was calculated.\nThe Y-axis indicates the degree of response to antigen depletion, expressed as the percentage\ndecrease compared to the mock condition (no antigen depletion). For WT sE immune sera, all\nthree antigens showed strong depletion effects, similar to the DIII-specific antibody ZV67,\nsuggesting predominant DIII specificity. Since CC_FLE, WT sE, and DIII all present the DIII\nlateral ridge (LR) epitope well, ZV67 (DIII-specific) showed strong depletion by CC_FLE,\nsimilar to the WT sE sera. Notably, a strong response to CC_FLE depletion alone does not imply\nhigh EDE specificity. In contrast, EDE1-C8 was only depleted by CC_FLE, but not by DIII or\nWT sE, confirming its EDE specificity. The CC_FLE immune sera were primarily depleted by\nCC_FLE, but showed minimal depletion by WT sE and DIII, resembling the behavior of EDE1-\nC8. We plan to further analyze sera specificity in future studies, for example, using sE mutants\nwith selectively altered epitopes.\nComment:\n8) The OmniMouse model is interesting. Why have the authors not performed a comparison\nbetween WT and CC_FLE antigens? I see the interest in cloning specific antibodies from these\nmice, but it would be also informative to look at the B cell repertoire induced by the two types of\nantigens to appreciate the superiority of the CC_FLE antigen.\nResponse:\nThanks to the reviewer’s suggestion to compare the WT vs. CC_FLE sE antibody response in the\nOmniMouse animal model. Since such investigation has been performed in wildtype mouse\nmodels, we then focus on isolating the dimer-specific neutralizing antibodies elicited by the CC-\nFLE sE reported in the current manuscript. We will perform such investigation in Omnimouse\nanimals in follow-up studies in the future.\nComment:\n9) Why was the OmniMouse 5.2 terminated at 6 weeks p.i., much earlier than the OmniMouse\n5.1 (38 weeks)? From the shape of the neutralization curves, the sera do not show a strong\nneutralizing activity. Have the sera been tested for presence of FLE antibodies?\nResponse:\nOmniMouse 5.2 was terminated at 6 weeks post-immunization (p.i.), as its serum neutralizing\ntiter at 5 weeks p.i. was 594—above the preset cutoff of 500 for termination and antibody\nisolation. OmniMouse 5.1 continued to receive additional immunizations, as its serum\nneutralizing titer at 5 weeks p.i. was below 500. We have mapped the neutralizing antibody\nspecificity to epitopes including EDE and DIII. Due to limited serum volume, we will assess the\npresence of FLE-directed antibodies in a follow-up study using a larger number of animals and\nWT sE as a control immunogen to support this investigation.\nComment:\n10) It is interesting that the complex of OZ-D4 with CC_FLE did not result in high resolution\n6\ndata while the complex with CC_Core did. The antibody binds the two antigens with comparable\naffinity (Fig.7D).\nResponse:\nWe now have obtained cryoEM structure of SMZAb2 in complex with CC_FLE sE (Fig. 2C).\nMinor points:\nLine 173: What is (32-34) referring to? The reference number does not match with “DIII\nimmunogenicity” described in the text.\nResponse: We have removed the corresponding text.\nLines 179-181: It would be informative to show the SEC of profile of the proteins after affinity\npurification (may be as a supplementary figure) to appreciate the heterogeneity of the forms\nproduced by the constructs.\nResponse: We have added the SEC profile after affinity purification in Fig. S1A\nLines 191-192: Please list all the antibodies that have been tested with their epitope specificity\nand recognition of the antigens tested. A figure and a summary table in supplementary data\nwould be informative.\nResponse: We have generated a Table S5 for this purpose.\nLines 196-199: This sentence is redundant. These results have been presented in the previous\nparagraphs.\nResponse: We have removed this sentence.\nFig.5: B and C show neutralization assays results (RVP and authentic virus). I would plot both in\nthe same way (either ID50 or % of neutralization).\nResponse: We have listed ID50 values for each pooled serum sample in Fig. 5C.\nLine 364: the ID50 titer increase is not 2 logs (5012 vs 178 = 28 folds).\nResponse: We have made the correction as suggested.\nLines 399-400: I do not understand the sentence “surpassing the nascent WT sE”.\nResponse: It means “CC_FLE sE is better than the unmodified WT sE”.\nFig.8: 8A – show the HC and LC chains with more contrasting colors. 8F – show a top view of\nthe dimer to highlight the antibody footprint. The actual side view is not informative.\nResponse: We have modified Fig. 8A to show the surface rendering of the electron density\ncolored based on the Fab chains and antigen regions. Fig. 8F is now shown as a top view of the\ndimer to illustrate the antibody footprint.\nReviewer #2 (Remarks to the Author):\nThe present manuscript by Wang et al presents a novel vaccine design strategy targeting the E\nprotein of the Zika virus (ZIKV), aimed at bolstering immune responses and mitigating the risk\nof Antibody-dependent enhancement (ADE). The authors have engineered two mutations,\nG5C/G102C, into the flavivirus E protein, resulting in the formation of inter-chain disulfide\n7\nbonds and the creation of E-dimers. The E-dimer formation not only augments the\nimmunogenicity of the vaccine candidate but also masks the ADE-associated site FLE, thereby\ndiminishing the likelihood of ADE. The vaccine candidate, CC-FLE sE, is primarily constructed\nbased on the ZIKV E protein sequence. Upon immunization in mice, it elicits potent neutralizing\nantibodies that confer protection against ZIKV infection and attenuate the risk of ADE following\ninfections with various flaviviruses. Furthermore, the article delineates the structural and\nfunctional analysis of the E-dimer-dependent epitope (EDE) monoclonal antibody OZ-D4,\nhighlighting the high affinity of CC-FLE sE for the natural precursor of OZ-D4. This suggests\nthat the robust viral epitope recognition and neutralization by CC-FLE sE are achievable without\nextensive somatic mutations, indicating its potential to elicit an immune response within the\nhuman B cell repertoire. Consequently, CC-FLE sE emerges as a promising vaccine design\nstrategy for flaviviruses, addressing the dual challenges of immune protection and ADE\nreduction that are central to flavivirus vaccine development. However, to bolster the rigor and\ncomprehensiveness of the study's conclusions, the authors are advised to address the following\nkey points prior to further consideration:\n1) Immunogenicity data: The presented data regarding immunogenicity exhibit inconsistencies\nand lack persuasiveness. To enhance credibility, it is imperative to conduct PRNT (Plaque\nReduction Neutralization Test) titers for Figures 4-6. Moreover, a statistical correlation analysis\nbetween the RVP system results and the PRNT titers must be performed to strengthen the\nvalidity of the conclusions drawn.\nResponse: Thanks to the reviewer for suggesting validation of the RVP method, which has been\nperformed very carefully in a previous study (PMID: 27481466). Shown below is a statistical\ncorrelation analysis from this study between the RVP system results and the PRNT titers.\nComparison of the mean EC50 for all serum samples evaluated with both RVPs and infectious\nvirus revealed remarkable agreement.\n[redacted]\nlisted for comparison, which is consistent with our RVP neutralization data. Moreover, the RVP\nassay system has been wildly used in the field to characterize clinical trial immune response as a\nstandard method currently. To name a few, VRC/NIH used the RVP assay to measure\nneutralization in two clinical trials, and Takeda uses the same assay for its ZIKV and DENV\nvaccine studies. Therefore, our data using a well-established and validated high-through-out\nRVP assay to evaluate immune sera neutralizing titers are solid.\nComment: While Figure 4C illustrates the outcomes of the PRNT, it is crucial to express these\nfindings as either PRNT50 or PRNT90 values for meaningful comparison.\nResponse: We have listed the PRNT50 values of the immune sera for comparison in the revised\nFig. 5C.\nComment:\nOf particular concern is the discrepancy observed in Figure 5D, where negative outcomes in\nmortality from passive transfer experiments contradict the reported high levels of neutralizing\nantibodies. This apparent paradox necessitates a thorough explanation and nuanced discussion\nwithin the text.\nResponse: Thanks to the reviewer who suggests clarification. We have added the following text\nto explain such observation.\n“Although the nAb titers of immune sera from animals inoculated with WT sE + Adjuplex ,\nCC_FLE sE+ Adjuplex, or CC_FLE sE +PCEP-R848 were similar, the better protection\nefficacy conferred by the sera from the mice immunized with CC_FLE sE +PCEP-R848\nindicated that other protection mechanisms, such as antibody effector functions including\nantibody dependent cytotoxicity (ADCC) may play a role in this AG129 mouse model.”\nComment:\n2) Vaccine efficacy assay: At lines 357-360, the authors' decision to employ Balb/c mice for\nassessing vaccine efficacy is unconventional and warrants additional justification. Given industry\nstandards, incorporating challenge data from A129 or AG129 mouse models would significantly\nbolster the study's comprehensiveness.\nResponse: We chose BALB/c mice as the model animal for vaccine efficacy here based on our in-\nhouse observation that after ZIKV challenge, BALB/c mice in general develop higher titers of\nviremia than C57BL/6 mice (used in our earlier immunogenicity studies), suggesting BALB/c\nmouse as a suitable model animal for evaluating vaccine efficacy. In the current study, we have\nused various mouse strains to evaluate immunogenicity and efficacy, including AG129\n(immunocompromised), C57BL/6 (immunocompetent), and BALB/c (immunocompetent) mice, in\norder to avoid bias caused by one strain. Our vaccine candidate, ZIKV CC_FLE sE\ndemonstrated superior performance over the unmodified WT sE in all tested mouse strains. We\nfirmly believe that using an immunocompetent mouse model (e.g., BALB/c mouse model) to\nevaluate protection efficacy would closely mimic the ZIKV natural infection.\nComment:\nFurthermore, evaluating the immunogenicity and protective efficacy of the vaccine candidate in\npregnant mice is highly recommended to address potential concerns related to maternal\nimmunization strategies.\ncandidate in pregnant mice, reported in Fig. S8. These results demonstrate that CC_FLE sE\nprovided effective protection against ZIKV in both pregnant mice and their fetuses, and\noutperformed WT sE in this immunocompetent mouse pregnancy-protection model.\n3) Figure 6 depicts solely the effects of adjuvants, which could be more appropriately relocated\nto the supplementary materials section.\nResponse: We respectively emphasis that Fig. 6 is important to show the critical effect of the\nadjuvant, and the protection corelates as a main figure.\nReviewer #3 (Remarks to the Author):\nComment:\nWang and colleagues employed a structure-based approach to design the envelope (E) dimer of\nvarious flaviviruses using intra- and inter-chain disulfide bonds to improve stability and\nminimize exposure of the antibody-dependent enhancement (ADE)-prone fusion loop epitope\n(FLE). The designed constructs were produced as recombinant proteins and their\nimmunogenicity was evaluated in mice, in conjunction with several adjuvants used either alone\nor in combination. One candidate, CC_FLE sE, derived from the Zika virus (ZIKV) and\nformulated with an advanced supramolecular adjuvant, emerged as the most potent in inducing a\ndesirable antibody response and providing protection against live ZIKV challenge. The same\nimmunogen also induced monoclonal antibodies targeting the E-dimer-dependent epitope (EDE)\nin a transgenic mouse model expressing human antibody genes. The authors propose that their\ndesigned immunogen, CC_FLE sE, represents a promising strategy for developing ZIKV\nvaccines that minimize ADE risks while maintaining high protective efficacy. Furthermore, the\ndesign strategies may be applicable to vaccines targeting other flaviviruses such as Japanese\nencephalitis virus (JEV) and tick-borne encephalitis virus (TBEV).\nThis study is significant. The design of CC_FLE sE is scientifically robust, most of the\nexperiments are well-executed, and the conclusions are largely supported by the data. However,\nthere are several major gaps in understanding the structural, immunological, and protective\nfeatures of CC_FLE sE, which render some parts of the study less convincing and compelling.\nSpecific questions and suggestions are detailed below:\nResponse: We appreciate the reviewer’s overall enthusiasm and have made modifications\naccordingly.\nComment:\n1. Structural Confirmation: While the antigenicity of CC_FLE sE and CC_Core was evaluated\nusing mAbs specific to EDE, DIII, and FLE, obtaining the crystal structure of CC_FLE sE would\nconfirm that the introduced disulfide bonds are correctly positioned and do not disrupt the overall\nE-dimer structure.\nResponse: We have obtained cryoEM structure of the CC_FLE sE complexed with EDE antibody\nSMZAb2 that shows the formation of the engineered interchain disulfide bond and the overall E-\ndimer structure similar to the virus-derived dimers (Fig. 2C, 2D, Fig. S2, Table S1, Table S2).\n2. Biophysical Stability: Thermal stability comparisons of CC_FLE sE and CC_Core to the wild-\ntype (WT) protein would provide valuable insights into the enhanced stability conferred by the\ndesign.\nResponse: We have determined the thermostability of the mutant sEs, in comparison with the WT\nsE (Fig. 2B). We have added the following text to report the data:\n“Differential scanning calorimetry profiles demonstrate substantially improved thermostability\nof CC_FLE sE compared to WT sE, with the thermal transition temperature (Tm) increased by\n15°C (from 43.5°C to 58.7°C) (Fig. 2B). In contrast, CC_Core sE showed a moderate (3°C)\nincrease in Tm compared to WT sE (Fig. 2B).”\nComment:\n3. Neutralizing Response (Figure 4): The unexpectedly low neutralizing response induced by\nCC_Core warrants an explanation. Are there structural or immunological factors that might\naccount for this outcome?\nResponse: Antigenicity (antibody binding profile) of a given immunogen (tested in vitro) does not\nalways translate to in vivo immunogenicity (e.g. neutralizing antibody titers), this is often\nobserved in immunogen design studies. Some other factors, such as protein stability, could also\nplay a role in impacting the presentation of neutralizing epitopes. In the case of the different\nimmunogenicity outcome of CC_FLE and CC_Core immunogen, the substantially higher\nthermostability of CC_FLE may enable this E dimer presenting neutralizing antibody epitopes in\nthe germinal center of B cells for a longer duration than the CC_Core. Additionally, the\n“beathing” dimer nature of CC-Core may also partially affect the presentation of DIII LR\nepitope that is well exposed in the monomeric WT sE. This may help to explain the poor\nimmunogenicity of CC_core immunogen after two immunizations. In a different study, after the\nthird immunization, CC_Core immunized animals started to show increased neutralizing\nantibody titers. Therefore, CC_Core does not improve immunogenicity compared to the WT sE.\nComment:\n4. Adjuvant Selection (Figure 5B): The neutralizing antibody response induced by CC_FLE with\nAdjuplex is similar to that with RCEP+R848. Why did the authors prioritize the use of\nRCEP+R848, which is a complex and mechanistically unclear adjuvant?\nResponse: The selection of an adjuvant was based on its higher potency demonstrated in vivo\n(Fig. 5D, only immune sera from CC_FLE in PCEP+R848 provided protection statistically\nsignificant compared with the immune sera from PBS-injected mice, while protection of sera\nfrom CC_FLE in Adjuplex group was not statistically significant).\nThe following additional reasons justify prioritizing the use of PCEP+R848: (1) PCEP is a\nchemically well-defined molecular adjuvant and vaccine delivery vehicle of nano-dimensions,\nwhich is fully water-soluble and forms clear solutions when complexed with R848 and antigens.\nAs a molecular solution, it can be analyzed by a variety of physico-chemical techniques, such as\nHPLC, DLS, AF4, UV-spectroscopy, etc. In contrast, Adjuplex is a multi-component bi-phasic\nadjuvant, which consists of a carbomer (polyacrylate) and soybean lecithin formulated as\nsubmicron-sized liposomes as main components (doi:10.1128/CVI.00736-14). The formulation\nand characterization of PCEP+R848 are much more detailed and thoroughly conducted.\n11\n(2) In contrast to Adjuplex, which is based on polyacrylate with a hydrolytically and biologically\nstable carbon-carbon backbone, PCEP is fully biodegradable. Furthermore, PCEP is a\nstructural homologue of another polyphosphazene adjuvant – PCPP, which showed efficacy and\nexcellent safety profile in five clinical trials (please see summary on degradability of PCEP, its\nmechanism of action and clinical use of polyphosphazene adjuvants in reference 49 – Andrianov,\nLanger, J Control Release 329, 299-315, doi:10.1016/j.jconrel.2020.12.001 (2021). In contrast,\ndetailed reports on clinical trials of Adjuplex were not available.\n(3). Adjuplex has been commercially unavailable for several years lately.\nComment: Additionally, it is surprising that CC_FLE formulated with R848, or CC_FLE alone,\nfailed to induce any detectable neutralizing antibody response.\nResponse: The lack of adjuvant effect of R848 in the absence of an appropriate delivery vehicle\n(e.g., R848 alone) is well-established. The main reasons for that are the lack of association of\nthis small molecule with the antigen and its rapid clearance from the body (doi:\n10.1586/14760584.6.5.835). These deficiencies are alleviated by the complexation of R848 with\nPCEP in the form of a counterion.\nAdjuvants are a critical component of vaccines, complementing immunogens to enhance and\nshape immune responses. They can influence the affinity, specificity, magnitude, and functional\nprofile of both B and T cell responses. It is commonly observed that antigen alone often fails to\nelicit a detectable neutralizing antibody response—for example, as seen with CC_FLE alone in\nour study. In another case (PMID: 33422991, Fig. 2C), the SARS-CoV-2 spike protein alone\ninduced negligible neutralizing responses in mice, whereas the inclusion of various adjuvants led\nto robust neutralizing antibody titers.\nComment:\n5. Protective Efficacy (Figure 5D): The absence of protective differences between WT and PBS\nis puzzling, given the high levels of neutralizing antibodies in WT compared to the absence of\ndetectable antibodies in PBS (Figures 5B and 5D). This discrepancy requires clarification.\nResponse: We have added the following text to explain the observation: “Although the nAb titers\nof immune sera from animals inoculated with WT sE + Adjuplex , CC_FLE sE+ Adjuplex, or\nCC_FLE sE +PCEP-R848 were similar, the better protection efficacy conferred by the sera from\nthe mice immunized with CC_FLE sE +PCEP-R848 indicated that other protection mechanisms,\nsuch as antibody effector functions including antibody dependent cytotoxicity (ADCC) may play\na role in this AG129 mouse model.”\nComment:\n6. ADE-Mediated Enhancement (Figures 5E-G): While the results suggest potential ADE-\nmediated enhancement of DENV-1 and DENV-2 infection in vitro and DENV-2 in vivo, these\nfindings cannot definitively be attributed to FLE-specific antibodies. Competition assays to\nquantify the proportion of FLE-specific antibodies in the sera and depletion experiments to\nremove FLE-specific antibodies before conducting in vitro and in vivo studies would strengthen\nthe conclusions.\nResponse: We appreciate the reviewer’s suggestion to define that the FLE-specific antibody\ncontributes to the WT sE immune sera ADE effect. We envision that CC-FLE may abolish the\n12\nelicitation of FLE-specific ADE-prone antibodies, but also for antibodies with other ADE-\nepitopes elsewhere including the N-terminus of sE, caused by the “locked” dimeric\nconfiguration. We are going to map the ADE-specificity thoroughly in follow-up studies.\nWe have added the following text in the discussion section to elaborate this point “This\nengineered linkage further stabilizes the sE dimer shown by substantially increased Tm by 15oC\n(Fig. 2B) and occludes the FLE (Fig. 2B-C), as well as the sE N-terminus, which is adjacent to\nanother ADE epitope consisting residues N8-E13 of DENV sE —identical to those in the\ncorresponding regions of ZIKV sE. Future studies aimed at delineating the effects of the\nG5C/G102C mutations on various ADE epitopes would be informative.”\nComment:\n7. Mouse Model Discrepancies (Figure 6): Substantial differences in neutralizing antibody\nresponses were observed between BALB/c and C57BL/6 mice.\nResponse: We chose BALB/c mice as the model animal for vaccine efficacy here based on our in-\nhouse observation that after ZIKV challenge, BALB/c mice in general develop higher titers of\nviremia than C57BL/6 mice (used in our earlier immunogenicity studies), suggesting BALB/c\nmouse as a suitable model animal for evaluating vaccine efficacy. It is very common that a given\nimmunogen can induce different immune responses in different mouse strains. However, in\nC57BL/6 (two studies, Fig. 4, Fig. 5) or BALB/c mice (two studies, Fig. 6, Fig. S8), CC_FLE sE\nwas able to consistently elicit potent neutralizing antibody responses. In contrast, the\nperformance of WT sE varies (two studies in C57BL/6 Fig. 4, Fig. 5 and BALB/c, Fig. 6, Fig.\nS8, respectively). This demonstrates that CC_FLE sE has immunogenicity superior to WT sE, in\nall tested mouse strains.\nComment:\nAdditionally, the authors shifted from using Adjuplex to focusing solely on RCEP+R848.\nWhat is the rationale for this change, and how can the discrepancies observed in C57BL/6 mice\nbe reconciled?\nResponse: The rationale of shifting from Adjuplex to PCEP+R848 is based on (i) Adjuplex\nbecame commercially unavailable recently, and (ii) the supporting data in Fig. 5D (survival\ncurve) and Fig. S6B (health score) that showed sera from CC_FLE sE in PCEP+R848\nbehaved better than CC-FLE sE in Adjuplex. AG129 mice receiving sera from immune mice\ninoculated with CC_FLE formulated with PCEP+R848 adjuvant displayed significantly longer\nsurvival time (median, 15 days) upon challenge than from animals inoculated with PBS (median,\n11 days) (*p <0.05,) or WT sE formulated with Adjuplex (median,11 days) (Fig. 5D). AG129\nmice receiving sera from donor mice immunized with CC_FLE formulated with Adjuplex also\nshowed median survival time of 13 days (Fig. 5D), longer than PBS- or WT sE + Adjuplex\ninoculated group, although this is not statistically significant. AG129 mice receiving immune\nsera from CC_FLE and PCEP-R848 adjuvant-immunized animals also showed lower health\nscore (multiple t test, ***p<0.001, days 11 and 13 post infection, Fig. S6B) than from PBS\ninoculated animals. In contrast, AG129 mice receiving immune sera from CC_FLE in Adjuplex\ndid not show lower health scores statistically significant than the PBS inoculated animals.\nRegarding the discrepancies observed in C57BL/6 mice, as discussed earlier, a given\nimmunogen can elicit different immune responses across mouse strains. Although the mechanism\n13\nis not fully understood, we speculate that the threshold for sE to induce a neutralizing antibody\nresponse is higher in BALB/c mice than in C57BL/6 mice. While CC_FLE sE appears able to\novercome this threshold in both strains, WT sE may struggle to do so in BALB/c mice.\nConsistently, CC_FLE sE elicited potent neutralizing antibody responses in both C57BL/6 and\nBALB/c mice, whereas WT sE showed variable performance for unknown reasons. These\nfindings suggest that CC_FLE sE exhibits more consistent and superior immunogenicity\ncompared to WT sE across the tested mouse strains.\nComment:\n8. Monoclonal Antibody Competition (Figure 7): The isolation and characterization of four\nmAbs (OZ-D4, B11, A1, and B9) with distinct epitope specificities is interesting. Competing\nimmune sera with these mAbs to estimate the relative proportion of similar antibodies in the sera\nwould be a critical experiment to demonstrate that CC_FLE sE indeed induces higher levels of\nEDE antibodies compared to WT.\nResponse: In wild-type mice (Fig. S7), we provided evidence that sera from CC_FLE sE-\nimmunized mice exhibited neutralization specificity resembling both EDE1-C8-like and ZV67-\nlike (DIII LR) epitopes, whereas sera from WT sE-immunized mice showed primarily ZV67-like\n(DIII LR) specificity. In Omnimouse animals (Fig. 7B, Fig. S9C), we demonstrated that the\nneutralizing activity of CC_FLE sE-immunized sera—similar to that of EDE1-C8—could be\nefficiently depleted by CC_FLE sE, but not so by the WT sE antigen. This suggests that CC_FLE\nsE effectively elicits antibodies targeting the EDE epitope. As suggested by the reviewer, we will\npursue follow-up studies in Omnimouse animals using larger cohort sizes, with WT sE-immunized\nmice included as controls to enable a more rigorous comparison.\nComment:\n9. ADE Potential of Isolated mAbs (Figure 8): The structure of OZ-D4 indicates it has four-\nresidue contacts within FLE. Investigating whether OZ-D4, along with OZ-B11, A1, and B9,\ncontributes to ADE effects on DENV-1 and DENV-2 in vitro would add valuable insights.\nResponse: None of these antibodies bind DENV-1 or DENV-2 sE. Therefore, we conclude that\nthey have no ADE effect on DENV.\n10. Germline Reversion (Figure 9): While the germline reversion experiment is intriguing, the\nresults may be influenced by the specific nature of the transgenic mouse model used.\nOverstatements regarding these findings should be avoided.\nResponse: We appreciate the reviewer’s comment. The germline sequence is directly derived\nfrom the human Ig germline database, not from the transgenic mouse. Of course, further clinical\ntrial data would provide more direct evidence. We have edited related text in the discussion\nsection accordingly.\nWe appreciate the positive and constructive comments from reviewers. We have performed\nadditional experiments and added more data into the manuscript and revised certain figures and\nrelated text.\nAfter carefully revising our manuscript, with the edited text highlighted in a word file containing\ntracked changes, we are providing point-by-point response to their comments as below.\nReviewer #1 (previous comment in grey, current comment to the authors' responses are in\nitalic)\nComment\n1) How the introduced mutations affect the overall structure of the E-dimer? In the E dimer, the\nN-term of DI, leans towards DIII and the DI-150loop is juxtaposed to the FL. In the TBE dimer,\nfor example, amino acids H5 and G102 form an electrostatic bond only at low pH when the 150-\nloop is in an “open lid” position away from the FL, but at neutral pH the 150-loop is in a “closed\nlid” position interacting with G102 while the DI N-term shifts slightly away from FL\n(doi.org/10.1038/s41467-022-31111-y). The newly created C-C bond between G5 and G102\nwould force DI N-term to stay close to the FL and affect the 150-loop position and probably also\nDIII overall conformation. From figure S2A and S2C The CC_FLE dimer looks open in the\nmiddle with an altered intradimer interface and a bulgy DIII. The PDB model shows a more\nclosed dimer but the fitting of the model in the density map could be slightly adjusted to assure a\nmore precise reconstruction (within the limits of the relative low resolution).\nResponse:\nCryoEM structures of the ZIKV CC_FLE (new structure, Fig. 2C) and JEV CC_FLE (Fig. 3C,\n3D) constructs confirm the integrity of the engineered dimers. The engineered G5C/G102C\ndisulfide bonds stabilizing the dimers are visualized in both structures (Fig2D., Fig3D CC bond\nwith map). In ZIKV CC_FLE structure the 150-loop is disordered. In JEV CC-FLE, the 150-loop\nadopts very similar configuration (yellow, with glycan 154) as in unmodified JEV dimer (pdb\n3p54, blue) and very different from TBEV E dimer structure (pdb 7qre, red) (see Figure below)\nComment:\nSince the proposed constructs will be used as antigens it is important to verify not only that the\nFLE is not exposed but also that the overall structure of the dimer is not altered. The\ncomparison between the ZIKV CC_FLE dimer structure in complex with an antibody and the wt\nZika dimer or virus-derived dimers is proposed in table S2. This comparison is presented only\nas a table and no structure of the ZIKV CC_FLE alone is presented. A figure showing the\noverlap of the compared structures would be informative.\nResponse: We made an additional supplementary figure, Fig. S2H, showing the comparison\nbetween the ZIKV CC_FLE dimer structure and the WT ZIKV dimer. These two structures are\noverall very similar.\nComment:\nThe 150loop structure (disordered or not) is not mentioned in the text. The comparison of the\n150loop (shown here) between the two JEV structures is odd because 3p54 dimer was\nproduced in bacteria so it is not glycosylated. Why was not compared to the JEV dimer from\n5wsn?\nResponse: We have added in the revised text that the 150-loop is disordered in ZIKV CC_FLE\nstructure. [Lines 194-197]\nWe have added 5wsn structure (purple) for comparison of 150-loop conformations.\nIn ZIKV CC_FLE structure, the 150-loop is disordered. In JEV CC-FLE, the 150-loop adopts\nvery similar configuration (yellow, with glycan 154) as in both unmodified JEV E proteins (pdb\n3p54, blue; and pdb 5wsn, purple) and very different from TBEV E dimer structure (pdb 7qre,\nred) (see Figure below).\nComment:\nThe conformation of the engineered dimer is crucial to determine the type of antibodies that can\nbe generated, and the data presented in this work do not strictly support the superiority of ZIKV\nCC_FLE dimer in comparison to the other described constructs.\nResponse: Structural and functional analyses of our CC_FLE construct suggest that it closely\nmimics the native conformation of the E protein. Our in vivo immunogenicity and virus challenge\nstudies demonstrated that after only two immunizations CC_FLE confers protective efficacy\nagainst ZIKV challenge. Notably, in pregnant mice, those immunized with CC_FLE had virus-\nfree uteri and fetus, in contrast to mice immunized with other constructs published previously,\nwhich often require more than two immunizations and showed high frequency of uterine and\nfetus viral infection.\n2) Comment:\nReduced immunogenicity is probably not the cause of the absence of response for DI-DIII\nconstruct because DIII construct seems to be immunogenic. May be the overall structure is\nmisfolded? I would mention this possibility in the text (line 248-249).\nResponse: we have mentioned this possibility in the revised text. [Line 260]\n3) Comment:\nThe sentence at lines 494-497 need to be completed by the notation that OZ-D4 shows a 23-\nfold reduced capacity of neutralization with the authentic virus compared to the RVP system.\nThis is not the case for C8 (fig.7F-7G) suggesting that OZ-D4 does not behave like an EDE\nantibody and the binding at a more lateral angle and steric hindrance on the virus surface may\nbe the reason for the observed reduced neutralizing activity. It is interesting that OZ-D4 binding\nto CC_FLE resulted in low resolution cryoEM and the structure could be obtained only with\nCC_Core which is more flexible and can probably better adapt to the OZ-D4 binding. These\naspects are not discussed.\nResponse: we have discussed the approaching angle and footprint difference part. [Lines 547-\n554]\nRegarding why OZ-D4 binding to CC_FLE resulted in low-resolution cryoEM data, and why the\nstructure could only be obtained with the more flexible CC_Core, we offer the following\nexplanation. While, in general, more rigid and/or stable protein molecules and complexes are\nexpected to yield higher-resolution structures, this correlation is not always straightforward—\nparticularly in cryoEM. Many other factors influence resolution, with the orientational distribution\nof observed particles being one of the most critical, especially as it relates to the ice properties\nrequired for high-quality data collection. This is a complex issue, and it is generally not possible\nto assert that a more rigid protein complex will necessarily produce higher-resolution data.\nNevertheless, after testing several antibody–antigen combinations, we successfully obtained the\nhigh-resolution structure of neutralizing antibody OZ-D4 complexed with the CC_Core dimer,\nwhich clearly reveals the epitope in detail.\n2) Comment:\nCan the authors show and comment these data? [Mutation G102C by itself is not sufficient to abolish FLE-mAb binding, as minor monomeric fraction of ZIKV CC_FLE sE could still bind FEL-mAbs, like WT sE. Therefore, the reduced FLE-mAb binding of CC_FLE is caused by the non-exposure of the FL resulting from the G102C/G5C mutations.]\nResponse: We have added a new panel as Fig. S1C, showing the monomeric fraction of\nCC_FLE binds FLE-specific mAb E60 by ELISA. [Line 222-226]\n3) Comment:\nIn addition to the increased Tm (due to the presence of the C-C bond), what are the other\nevidences that the “breathing” dimer “partially exposes the FL”? A similar construct described in\nDOI: 10.1038/ncomms15411 showed no difference with WT sE for antibody binding or insertion\ninto liposomes.\nResponse: Thanks to the review for sharing the article link. We have similar observations here.\nIn Fig. 2F, we have shown that the “breathing” dimer CC_Core shows moderate binding to FLE-\nspecific antibodies, which suggests the “breathing” nature of this type of dimer. In contrast, the\nlocked dimer CC_FLE shows abolished FLE-specific antibody binding.\n4) Comment:\nHas stability been tested in other context (cell culture) than Tm increase that is probably due to\nthe presence of C-C bond?\nResponse: We have not assessed stability in the context of cell culture. This is an interesting\nidea that could be explored in future studies. Nevertheless, we believe that the Tm\nmeasurements obtained with purified protein are predictive of stability in other contexts.\n5) Comment:\nPublication PMID:28340344 could be cited in the text to justify the amount of serum used. The\nvirus+FLE antibody control in this context would proof that the reduced enhancing effect of\nCC_FLE immune sera is due to the non-exposure of FLE.\nResponse: We have cited PMID:28340344 as reference for the volume of the transferred sera\n[Lines 806-807].\nOur data confirms that immunization with our CC_FLE mitigates ADE risk in a mouse model in\naddition to the in vitro cell-based ADE assay. The ADE mitigation could be caused by the\nreduced exposure of FLE, but other factors may contribute to this property as well. For\nexample, CC_FLE mutations may reduce the exposure of other ADE-prone epitopes yet to be\nidentified. We will consider adding virus+FLE antibody control in future studies.\n10) Comment:\nMy question was referring to the difference of OZ-D4 epitope exposure in the two constructs.\nThe ELISA data show no difference in binding but the cryoEM data suggest a different (weaker? heterogenous?) binding that affect the resolution of the complex. Could the authors comment on this discrepancy?\nMoreover, it would be useful to show in the figures of the different structures, the contact bonds between amino acids and have a table with a list of the polar and salt bridges contacts to better appreciate the footprint of the different antibodies on the dimer. Please show the entire dimer in fig. 8F for all the antibodies.\nResponse: We don’t observe significant difference for OZ-D4 epitope exposure in the two\nconstructs. As discussed earlier, the different resolution of structure could be resulted from other\nfactors which may influence resolution, with the orientational distribution of observed particles\nbeing one of the most critical, especially as it relates to the ice properties required for high-\nquality data collection. [Lines 516-518]\nWe generated a new supplementary TableS6 to summarize the interactions. [Lines 541-543]\nWe have generated new figure panels (Fig. 8G & 8H) to show dimer approaching angle\ncomparison: OZ-D4: CC_Core complex, EDE1-C8: WT Dimer complex (PDB:5LBS), and\nSMZAb2: CC_FLE complex. [Lines547-554]\nPlease note we have already shown the entire dimer in Fig. 8F.\nAdditional points:\n-Lines 165-166: Has the occlusion of W101 and F108 residues been seen in the cryoEM of\nCC_FLE dimer? The structure of this dimer for Zika is presented only in complex with antibodies\nbut not alone, so it is difficult to compare the impact of these mutations on the overall structure\nof the WT E dimer. The close view shown in fig.1B should be compared to the same position in\nthe context of the WT dimer not the monomer (see legend description).\nResponse: The occlusion of W101 and F108 residues has been demonstrated in the cryoEM of\nCC_FLE dimer [Lines 205-207]. The close view shown in Fig.1B is just a model based on the\nWT dimer. The purpose is to explain the rationale of design, not to compare the structure.\nWe have added Fig. S2I, showing the occlusion of W101 and F108 in the CC_FLE dimer.\n[Lines 205-207]\n-Fig.S1A: SEC profile per se does not proof that the protein is a dimer in solution. Elution\nvolume is correlated to the protein size for globular proteins, but the envelope protein is\nelongated, and it has been shown in the literature to elute at unexpected volumes. A MALS\nanalysis or an SDS-PAGE analysis would be more adequate proof.\nResponse: We have added an SDS-PAGE gel figure (Fig. S1B) showing that, under non-\nreducing conditions, the SEC-purified CC_FLE or CC_Core sE migrated at a molecular weight\nconsistent with a dimer. More decisively, our structural analysis confirmed the dimeric nature of\nour constructs. [Line 185]\n-Fig.S1B: The legend of BLI graph is not shown (different protein concentrations). Ab Z006 is\nnot shown in the graph, and it can be removed from the table (fig.S1D).\nResponse: legend for protein concentration in the BLI graph is now provided (currently Fig.\nS1D). Ab Z006 is omitted from the table (currently Fig. S1F).\n-Fig. 3E (as well S3D and E) need to show an overlap with the unlocked dimer to appreciate the\nocclusion of the fusion loop.\nResponse: Fig. 3E, Fig. S3D & S3E are revised as suggested.\n-Fig. S8: these data do not show superior in vivo protection capacity of the CC_FLE construct in\ncomparison to the WT. There is some inconsistency in the data on adult Balb/c mice at day 1\npost challenge showing higher reduction of challenge viral RNA for CC_FLE vs WT in fig.6C and\nno difference in fig. S8C. Moreover, the challenge viral RNA is equally reduced in the uterus\ntissue for both constructs (1/8 vs 0/8 is not significative) thus the sentence at lines 437-439\nwould need a reformulation because the CC_FLE construct does not “outperform” the WT\nconstruct.\nResponse: The discrepancy between Fig. 6C and Fig. S8C was due to the different virus\nchallenge routes. In Fig. 6C, the virus was administered via the subcutaneous route, which\nmimics the natural transmission of ZIKV by mosquito bites. The negative control mice showed\nserum virus loads below 10^4 viral genome copies/mL. As expected, CC_FLE sE showed better\nefficacy than WT sE. In Fig. S8C, we used the retro-orbital route that directly injected virus into\nthe blood to effectively infect the female mouse uterus, which resulted in much higher viral\nloads, with negative control mice showing levels above 10^5 viral genome copies/mL. The\neffective ZIKV infection in vivo was therefore about 10-fold higher in Fig. S8C than in Fig. 6C,\nwhich explains why we did not observe a statistically significant reduction in viral load between\nWT and CC_FLE sE–immunized mice under such overwhelming viral infection condition.\nNevertheless, none of the uteri from the CC_FLE–immunized mice showed detectable virus,\nwhereas virus was detectable in the uterus of one animal from the WT sE–immunized group.\nWe have revised the related text describing the comparison between the WT and CC_FLE for\nFig. S8C and S8D, by deleting the statement “and outperformed WT-sE in this\nimmunocompetent mouse pregnancy protection model”.\nReviewer #2 (Remarks to the Author):\nComment: The authors have addressed most of the comments raised during the previous round\nof review. The reviewer suggests that the authors cite the references (PMID: 27603093, PMID:\n27855206) that firstly report the use of immunocompetent mice in ZIKV research.\nResponse: We thank the reviewer for the positive feedback and for suggesting references that\nfirst reported the use of immunocompetent mice in ZIKV research; these have been added to\nthe text [Line384].\nReviewer #3 (Remarks to the Author):\nThe authors have satisfactorily addressed all of my concerns.\nResponse: We thank the reviewer for the positive feedback.\nPoint-by-Point Response to Reviewer’s Comments\nReviewer #1 (Remarks to the Author):\nComment: The authors have addressed most of my comments.\nResponse: We thank the reviewer for noting that most comments have been addressed.\nComment: However, I still believe the data do not fully support the immunogenic superiority of\nthe CC_FLE construct for the following reasons: (a) the 150-loop structure appears to be\naffected, as seen in the comparison of the JEV construct with the JEV E dimer from the virus\n(yellow vs. purple);\nResponse: We respectfully disagree with the reviewer’s statement that the 150-loop structure is\naffected in the JEV construct based in the comparison between our JEV CC_FLE sE dimer and the\nsE on the virus (PDB: 5wsn). The 150-loop in both structures are largely identical, with minor\nvariation, which is actually smaller (rmsd 1.19 Å2) than the variation between the JEV sE 150-\nloop in PDB: 5wsn and PDB: 3p54 (rmsd 1.33 Å2).\nFurthermore, the purpose of the CC_FLE sE construct is not to replicate the sE conformation on\nthe virus particle, but rather to achieve strong immunogenicity while mitigating ADE risk. This\nrationale is supported by the results from the ZIKV CC_FLE construct. Future immunogenicity\nstudies with the JEV sE CC_FLE will be informative for evaluating the JEV construct.\nComment: (b) the OZ-D4 antibody generated by this antigen binds the dimer in an \"unnatural\"\nway that impacts neutralization in authentic virus;\nResponse: We acknowledge that the epitope and approach angle of the OZ-D4 antibody are not\nidentical to those of classical EDE-targeting antibodies. However, OZ-D4 represents only one of\nthe monoclonal antibodies elicited by CC_FLE sE. Given the polyclonal serum neutralization\nspecificity presented in this study, which resembles that of EDE1-C8, future work using electron\nmicroscopy–based polyclonal epitope mapping (EMPEM) and isolation of additional mAbs from\nimmunized mice will be informative for further characterizing the epitopes and approach angles\nof EDE-targeting antibodies elicited by CC_FLE sE. We have highlighted this perspective in the\nrevised text. [Lines 552-555]\nComment: (c) regardless of the viral load in the animal model, the difference between 1/8 and\n0/8 is not significant.\nResponse: As stated previously, the retro-orbital route directly delivered the virus into the\nbloodstream, leading to efficient uterine infection and approximately tenfold higher viral loads\nthan in the previous efficacy study. This explains the lack of a statistically significant difference\nbetween WT and CC_FLE sE–immunized groups under such high infection levels. Notably, no\nvirus was detected in the uteri of CC_FLE–immunized mice (0/8), whereas one uterus from the\nWT sE group (1/8) was positive. Although not statistically significant due to the small group size\n(N = 8), this difference may become clearer with larger cohorts. We have therefore removed the\nsentence “surpassing the nascent WT sE.” [Line 452]\nComment: I appreciate the effort put into complementing and revising the text according to my\nsuggestions and have no further concerns.\nResponse: We appreciate the reviewer’s constructive comments, which have indeed led to\nsignificant improvements in this manuscript."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed most of my comments. However, I still believe the data do not fully support the immunogenic superiority of the CC_FLE construct for the following reasons: (a) the 150-loop structure appears to be affected, as seen in the comparison of the JEV construct with the JEV E dimer from the virus (yellow vs. purple); (b) the OZ-D4 antibody generated by this antigen binds the dimer in an \"unnatural\" way that impacts neutralization in authentic virus; and (c) regardless of the viral load in the animal model, the difference between 1/8 and 0/8 is not significant.\nI appreciate the effort put into complementing and revising the text according to my suggestions and have no further concerns."}, "author_response": ""}]

Decision letter