Rational design of next-generation filovirus vaccines combining glycoprotein stabilization and nanoparticle display with glycan modification

Domain: neuroscience
Published: 2025-12-12
Editorial outcome: major_revision

Abstract
Filoviruses pose a significant threat to human health due to frequent outbreaks and high mortality. Although two vector-based vaccines are available for Ebola virus, a broadly protective filovirus vaccine remains elusive. Here, we evaluate a general strategy for stabilizing glycoproteins (GP) from Ebola, Sudan, and Bundibugyo orthoebolaviruses, as well as Ravn ortho-marburgvirus. A 3.2 Å crystal structure provides atomic-level details of the redesigned Ebola virus GP, while cryo-electron microscopy reveals how a pan-orthoebolavirus neutralizing antibody targets a conserved site on the stabilized Sudan virus GP (3.13 Å resolution), along with a low-resolution model of antibody-bound Ravn virus GP. A self-assembling protein nanoparticle (SApNP), I3-01v9, is redesigned at the N terminus to enable optimal surface display of filovirus GP trimers. Following detailed in vitro characterization, we examine the lymph node dynamics of Sudan virus GP and GP-presenting SApNPs in mice. Compared with the soluble trimer, SApNPs exhibit ~112-fold longer retention in lymph node follicles, up to 28-fold greater presentation on follicular dendritic cell dendrites, and up to 3-fold stronger germinal center reactions. Functional antibody responses induced by filovirus GP trimers and SApNPs bearing wild-type or modified glycans are assessed in mice. This study provides a foundation for next-generation filovirus vaccine development.

Introduction
The first documented case of human filovirus infection occurred in 1967 in Marburg and Frankfurt, Germany, when laboratory workers were infected with Marburg virus (MARV) by non-human primates (NHPs) imported from Africa1. The largest filovirus outbreak on record was caused by Ebola virus (EBOV), first identified in 1976 in Zaire, Central Africa2,3. The 2013–2016 EBOV epidemic resulted in approximately 28,600 cases and 11,325 deaths4. Both MARV and EBOV cause viral hemorrhagic fever (VHF) in humans, with case fatality rates of up to 90%5,6. Filoviruses are classified into nine genera, of which Orthoebolavirus and Orthomarburgvirus are the most clinically relevant7–9.

Orthoebolavirus includes pathogenic species such as EBOV, Bundibugyovirus (BDBV), and Sudan virus (SUDV), as well as other species that are either non-pathogenic or rarely infect humans. Orthomarburgvirus comprises a single species containing two members: MARV and Ravn virus (RAVV). The single-stranded, negative-sense RNA genome of filoviruses encodes structural proteins, including nucleoprotein, virion proteins, glycoprotein (GP), and RNA polymerase. As a class I viral fusion protein10–13, GP functions as a trimer of heterodimers, mediating host cell attachment, membrane fusion, and viral entry14. GP is recognized by the host immune system during natural infection15–17 and represents the primary target for the development of prophylactic vaccines and antibody therapeutics18–21.

Substantial progress has been made in filovirus research22. First, our understanding of filovirus pathogenesis and infection mechanisms has advanced considerably23–25. Niemann-Pick C1 (NPC1) was identified as the endosomal receptor for filovirus entry, indicating that NPC1 inhibitors may have antiviral potential26–28. Second, neutralizing antibodies (NAbs) have been established as effective therapeutics for treating filovirus infections29–32. Compared to the monoclonal antibody (mAb) cocktail ZMapp33, which initially showed promise34, REGN-EB3 (a three-mAb cocktail35) and mAb11436 demonstrated superior efficacy against EBOV in a randomized controlled trial37. The 2013–2016 EBOV outbreak led to sustained efforts to identify anti-filovirus antibodies38. Panels of mAbs were isolated from human survivors and immunized animals, revealing multiple sites of vulnerability on GP16,17,36,39–54. At the peak of this effort, the Viral Hemorrhagic Fever Immunotherapeutic Consortium analyzed 171 mAbs targeting diverse epitopes on GP, including the base, glycan cap, fusion loop, GP1/core, GP1/head, GP1/2 interface, heptad repeat 2 (HR2), and mucin-like domain (MLD)55. Third, X-ray crystallography and cryo-electron microscopy (cryo-EM) have determined atomic structures of filovirus GP bound to NPC128, small-molecule ligands56–58, and mAbs16,39,40,42,43,47,49–52,59,60. Notably, negative-stain EM (nsEM) remains an effective tool for validating GP conformation and mapping mAb epitopes17,41,46–48,53–55. These structural insights have advanced our understanding of filovirus neutralization and supported the development of antibody therapeutics and vaccines18,61,62. Fourth, two viral vector-based EBOV vaccines have been approved for human use. ERVEBO63 is a single-dose vaccine based on recombinant vesicular stomatitis virus (rVSV) expressing EBOV GP, whereas Zabdeno/Mvabea64 delivers two doses–an Adenovirus 26 (Ad26) vector encoding EBOV GP followed by a modified Vaccinia Ankara (MVA) vector expressing multiple filovirus GPs. Additional EBOV vaccines using DNA65 and viral vectors66–71 have been tested in humans. Recent efforts have expanded to mRNA, protein subunit, and nanoparticle (NP) platforms72–75.

Despite this progress, critical innovations are still needed to advance next-generation filovirus vaccines76. Although correlates of protection remain undefined77, in-depth B cell analysis revealed diverse yet convergent polyclonal NAb responses in a Phase 1 trial of the rVSV-ZEBOV vaccine78. A follow-up study elucidated how NAbs from multiple individuals used the same germline genes to target the receptor-binding site (RBS) on GP79, reinforcing the central role of GP as the primary vaccine antigen. Over the past decade, antibody-guided, structure-based antigen design has transformed vaccine development for important targets such as HIV-180–83 and respiratory syncytial virus (RSV)84. Rational design strategies have been applied to stabilize the HIV-1 envelope (Env)85–87, the RSV fusion (F) protein88–91, and, more recently, the SARS-CoV-2 spike92,93 in their prefusion conformations. Many of these constructs have progressed from preclinical testing to human trials94,95, with multiple COVID-19 vaccines96,97 and three RSV vaccines now approved for human use98. By contrast, only two studies have reported rationally stabilized filovirus GP designs, both with limited in vivo evaluation99,100. Most recently, Xu et al. proposed an immunofocusing approach by engineering additional N-linked glycans onto GP74. Notably, nearly all current filovirus vaccine candidates101 rely on wild-type GP, which contains a highly variable MLD that shields conserved NAb epitopes and facilitates immune evasion102,103. A mucin-deleted GP (GPΔmuc) could, in principle, elicit NAbs more effectively than wild-type GP, but it has yet to be evaluated in humans. In addition to antigen format, the choice of delivery platform may also influence vaccine-induced immunity. Although viral vectors remain prominent, as reflected in the licensure of two EBOV vaccines, mRNA platforms represent a compelling alternative given their success during the COVID-19 pandemic104,105. In the realm of protein-based vaccines, NPs have emerged as a promising platform due to their virus-like size, multivalent surface display, and ability to elicit strong and durable NAb responses106–111. Although two EBOV GP-presenting NP vaccines have recently been reported74,100, neither has been evaluated in animal challenge models. Finally, glycan modification may be a key strategy for optimizing filovirus vaccine antigens. Glycan trimming has improved NAb responses in vaccine candidates targeting influenza hemagglutinin (HA), SARS-CoV-2 spike, and HIV-1 Env112–115. Whether this approach applies to filovirus GP, which is covered with up to 17 surface glycans per protomer, remains to be determined.

In our previous study, we stabilized EBOV GP with mutations in the HR1 bend and HR2 stalk and displayed one of two lead designs, termed GPΔmuc-WL2P2, on self-assembling protein nanoparticles (SApNPs) as EBOV vaccine candidates100. Here, we first extended the GPΔmuc-WL2P2 and WL2P4 construct designs from EBOV to SUDV and BDBV and determined a crystal structure of EBOV GPΔmuc-WL2P4 at 3.2 Å resolution. We also obtained a 3.13 Å cryo-EM reconstruction of SUDV GPΔmuc-WL2P4 bound to the CA45 Fab47. The nearly identical backbones of SUDV and EBOV GPΔmuc-WL2P4 demonstrate a broadly applicable strategy for stabilizing orthoebolavirus GPs. We next displayed EBOV, SUDV, and BDBV GPΔmuc-WL2P4 trimers on SApNPs derived from E2p and I3-01v9, the latter of which was computationally redesigned at the N terminus. A similar design approach was applied to RAVV GP, resulting in a promising trimer construct termed GPΔmuc-P2CT. We then examined the lymph node delivery and immunological responses of SUDV GPΔmuc-WL2P4 trimer and SApNPs at the tissue, cellular, and subcellular levels in a mouse model. Compared to soluble trimers, 60-meric SApNPs exhibited prolonged retention and sustained presentation on follicular dendritic cell (FDC) dendrites, along with more robust and durable germinal center (GC) reactions in lymph node follicles. Finally, we immunized mice with a panel of filovirus GP vaccines to assess antibody responses. While the EBOV and SUDV GPΔmuc-WL2P4 trimer and SApNP vaccines elicited serum neutralization limited to orthoebolaviruses, the RAVV GPΔmuc-P2CT trimer induced detectable cross-genus NAb responses. Both multivalent display and glycan modification influenced the immunogenicity of GP-based filovirus vaccines. Although no universal glycan modification strategy may apply to all viral GPs, enriching the GP glycan shield with oligomannose-type glycans improved filovirus vaccine-induced NAb responses in mice. Together, these findings provide a foundation for developing broadly effective vaccines to combat future filovirus outbreaks.

Results
A universal design strategy for orthoebolavirus GP stabilization
GPs within the Orthoebolavirus genus share a high degree of sequence and structural similarity59,116. These viral GPs consist of conserved functional domains, including GP1, MLD, internal fusion loop (IFL), and GP2, which contain heptad repeat 1 (HR1), heptad repeat 2 (HR2), and the transmembrane domain (TMD). Previously, we stabilized EBOV GPΔmuc by introducing a T577P (P2) or L579P (P4) mutation in the HR1 bend, a W615L mutation in the neck region of the HR2 stalk, and a C-terminal extension in HR2 (D632–D637)100 (Fig. 1a). A foldon trimerization motif was appended to the C terminus of HR2. These modifications enhanced the structural and thermal stability of EBOV GPΔmuc trimers compared with wild-type GP.

Here, we applied the same design principles to SUDV and BDBV GPs and characterized two constructs, GPΔmuc-WL2P2 and GPΔmuc-WL2P4, across all three orthoebolaviruses (Fig. S1a). Following our previous protocol100, all six GPΔmuc constructs were transiently expressed in HEK293F cells and purified using two methods. In addition to immobilized nickel affinity chromatography, pan-orthoebolavirus NAbs ADI-15878117 and CA45118, expressed in immunoglobulin G (IgG) form in ExpiCHO cells, were used to prepare immunoaffinity chromatography (IAC) columns. The IAC method was established in our prior study, where mAb100 and mAb11436 columns were used to purify EBOV GP trimers and SApNPs100. The IAC- or nickel-purified orthoebolavirus GP samples were then characterized by size-exclusion chromatography (SEC) on a Superdex 200 column following ADI-15878 (Fig. 1b) and CA45 (Fig. S1b) purification. All SEC profiles displayed an aggregation peak around 8.0 ml and a consistent trimer peak at ~11.0 ml, with minimal evidence of dimer or monomer species.

Compared to IAC, nickel purification resulted in higher aggregation for EBOV and SUDV GPs (Fig. S1b). Of the two broadly neutralizing antibodies (bNAbs) tested, ADI-15878 captured orthoebolavirus GPs more efficiently than CA45, as indicated by stronger ultraviolet absorbance at 280 nm (UV280) in the elution fractions (Fig. 1b and S1b). These results support the use of ADI-15878 as a universal affinity ligand for tag-free purification of orthoebolavirus GP immunogens. Blue native polyacrylamide gel electrophoresis (BN-PAGE) confirmed that SEC fractions eluting at ~11 ml corresponded to GPΔmuc trimers, with bands consistent with theoretical molecular weights (Fig. 1c). Thermostability was assessed by differential scanning calorimetry (DSC). For EBOV, the GPΔmuc-WL2P2 trimer showed two melting temperatures at 60.9 °C (Tm1) and 67.7 °C (Tm2), whereas GPΔmuc-WL2P4 had a single, broadened melting peak at 64.4 °C (Fig. 1d), consistent with our previous findings100. In contrast, all four SUDV and BDBV GPΔmuc trimers generated similar DSC curves with a single denaturing state and tightly clustered Tm values (62.9–63.6 °C). Among the three orthoebolaviruses, SUDV GPΔmuc exhibited the highest onset temperatures (Tonset, 54.8 °C and 56.0 °C), followed by EBOV and BDBV GP constructs (50.1–54.2 °C). Based on these biochemical and biophysical profiles, GPΔmuc-WL2P4 was selected as the lead design candidate for pan-orthoebolavirus GP stabilization.

The HIV-1 Env spike employs a glycan shield as a primary defense mechanism to evade host NAb responses113. Similarly, orthoebolavirus GPs contain a glycan cap that shields the receptor binding site (RBS)100, along with a heavily glycosylated MLD, which is removed in GPΔmuc constructs. These glycans play a critical role in both immune evasion and modulating antibody interactions with GP. To generate site-specific glycosylation profiles, the purified EBOV, SUDV, and BDBV GPΔmuc-WL2P4 proteins were digested with trypsin, chymotrypsin, and α-lytic protease to produce glycopeptides, each containing a single N-linked glycan site. Site-specific composition and occupancy were analyzed by liquid chromatography-mass spectrometry (LC-MS), as previously described113. All glycosylation sites on the three orthoebolavirus GPΔmuc-WL2P4 proteins predominantly carried complex-type glycans, except at positions N563 and N257, where mixed glycan forms were detected. At N563, ~30% of glycans were oligomannose-type for EBOV, compared to ~60% for both SUDV and BDBV. At N257, ~15% oligomannose-type glycans were observed for EBOV (Fig. 1e and Fig. S1c). Due to technical limitations, glycan compositions could not be assessed accurately at all sites.

Negative-stain EM (nsEM) was used to characterize orthoebolavirus GPΔmuc-WL2P4 structures, either alone or bound to bNAb ADI-15878 (Fig. S1d). For EBOV and BDBV, crystal structures of wild-type GPΔmuc in complex with ADI-15878 (Protein Data Bank [PDB] IDs: 7JPH100, 6EA7, and 6EA5116) were fitted into three-dimensional (3D) EM reconstructions. For SUDV, the GPΔmuc and ADI-15878 structures from two separate complexes (PDB IDs: 3S8859 and 6DZM117) were fitted individually (Fig. 1f and Fig. S1e–g). Overall, the low-resolution nsEM models of three orthoebolavirus GPΔmuc trimers displayed a three-lobed chalice shape consistent with the native-like structure, although the HR2 stalk was unresolved. The ADI-15878-bound nsEM models were highly similar across all three species, revealing an epitope that spans two GP protomers and overlaps the conserved regions of the IFL and HR1 in GP248,117.

The antigenicity of six stabilized orthoebolavirus GPΔmuc trimers was assessed using four pan-orthoebolavirus bNAbs–CA4547, ADI-15878, ADI-15946119, and BDBV-28952–alongside MR78, a MARV-specific NAb40. In enzyme-linked immunosorbent assays (ELISA), the proline mutation in HR1c (P2 or P4) had no effect on antibody binding (Fig. 1g and Fig. S1h). Among the four bNAbs, ADI-15946 and BDBV-289 showed the lowest binding affinity for SUDV GPΔmuc trimers, 2.5- to 6-fold weaker than those for EBOV and BDBV GPΔmuc trimers, as indicated by the half-maximal effective concentration (EC50) values. The NHP-derived bNAb CA45 exhibited 1.5- to 2.5-fold stronger binding to EBOV GPΔmuc than to its SUDV and BDBV counterparts. As expected, the negative control MR78 showed no detectable binding to any of the GPΔmuc trimers from the Orthoebolavirus genus. These findings were confirmed by biolayer interferometry (BLI) (Fig. 1h and Fig. S1i). Among the six orthoebolavirus antigens tested, CA45 bound most strongly to EBOV GPΔmuc, whereas ADI-15946 and BDBV-289 interacted least favorably with SUDV GPΔmuc. Consistent with ELISA, BLI revealed negligible or no binding between orthoebolavirus GPΔmuc trimers and MARV-specific NAb MR78.

Structural characterization of rationally designed orthoebolavirus GPΔmuc trimers
Previously, we determined the crystal structure of EBOV GPΔmuc-WL2P2100. Here, we sought to obtain atomic details for the more stable EBOV GPΔmuc-WL2P4. To this end, EBOV GPΔmuc-WL2P4 was transiently expressed in HEK293S cells and purified by IAC (mAb100) and SEC, as previously described100 (Fig. S2a). Crystallization under conditions similar to those used for GPΔmuc-WL2P2 resulted in comparable packing in the P321 space group (Table S1). The final structure was resolved at 3.2 Å. Each asymmetric unit contained one GP protomer, with the glycan cap at top GP1 and the belt-forming IFL of GP2 clearly visible in the electron density (Fig. 2a). Three GP1-GP2 heterodimers assembled into a chalice-shaped trimer in the crystal lattice, with the GP2 subunits forming a base that cradles the outward-facing GP1 lobes (Fig. S2b). Four N-linked glycans (N228, N257, N563, and N618) were identified in the structure. The most complete carbohydrate chain was observed at N563 (with Man4GlcNAc2 density), part of the ADI-15878 epitope in HR1a116, whereas only a single N-acetylglucosamine (GlcNAc) was visible at the other three sites. The backbone of GPΔmuc-WL2P4 closely resembled that of GPΔmuc-WL2P2, with a global Cα root-mean-square deviation (RMSD) of 0.48 Å across 243 aligned Cα atoms and a local Cα-RMSD of 0.68 Å for the HR1c bend following protomer superposition (Fig. 2a and Fig. S2c). In GPΔmuc-WL2P4, the amine group of the K95 side chain formed hydrogen bonds with the backbone carbonyls of L573 and T576 (~3.0 Å) – interactions absent in GPΔmuc-WL2P2. These interprotomer hydrogen bonds at the trimer center likely contributed to the enhanced thermostability of GPΔmuc-WL2P4. Similar interactions were observed in the EBOV GPΔmuc-WL2 structure (PDB ID: 7JPI), where K95 formed hydrogen bonds with L573 and T576 at 2.7 and 2.9 Å, respectively. This structural analysis is consistent with the DSC data, in which EBOV GPΔmuc-WL2P4 exhibited a single denaturation transition with a Tm of 64.4 °C100.

Cryo-EM plays an important role in understanding how viral GPs interact with antibodies120–122. Several cryo-EM models have been reported for EBOV and BDBV GPs43,52,117, but only two crystal structures59,123 and no cryo-EM models are currently available for SUDV GP. Here, we determined a cryo-EM structure of SUDV GPΔmuc-WL2P4 in complex with the CA45 Fab. SUDV GP was transiently expressed in HEK293F cells and purified using an ADI-15878 IAC column, prior to complexation with CA45 Fab and SEC on a Superose 6 16/600 GL column (Fig. S2d). The complex fraction at ~72 ml was imaged on a Krios G4 transmission electron microscope (TEM), with data processed in CryoSPARC124 (Fig. S2e). The final 3D reconstruction reached 3.13 Å resolution, with an EMRinger score of 2.60125 and less optimal but within-range map-model correlation coefficients (Fig. S2f). In this map, the SUDV GPΔmuc-WL2P4 trimer flanked by three CA45 Fabs was well resolved (Fig. 2b). Three regions in GP – GP1 (A187–L218), GP2 (E522–N525), and the C-terminal stalk (P612–N637) – were excluded from the final model due to limited local resolution (Fig. S2g). The glycan cap and GP2 stalk became partially visible when the density threshold was lowered beyond 4.0 Å (Fig. S2e). The constant domains of the CA45 Fab heavy (S113–T211) and light (T109–S215) chains were also excluded because their density was poorly resolved due to intrinsic flexibility of these domains, but this did not affect the structural quality of the GP-CA45 interface or its biological interpretation. Crystal structures of EBOV GPΔmuc-WL2P2 (PDB ID: 7JPH)100 and CA45 variable domains (PDB ID: 6EAY)118 were used as starting models for EM fitting, followed by manual refinement. The final SUDV GPΔmuc-WL2P4 model, lacking the glycan cap and GP2 stalk, was superimposed onto the EBOV GPΔmuc-WL2P4 crystal structure, yielding a Cα-RMSD of 0.66 Å for 154 matching Cα atoms (Fig. S2h, left). The three HR1 bends of SUDV GPΔmuc-WL2P4 were also superimposed onto those of EBOV GPΔmuc-WL2P4 from this study and BDBV GPΔmuc (PDB ID: 7KFE)52, with Cα-RMSDs of 0.34 and 0.53 Å for the 24 aligned Cα atoms, respectively (Fig. 2c, right). Compared with the crystal structure of the EBOV GPΔmuc/CA45 complex (PDB ID: 6EAY)118, cryo-EM revealed a highly similar antibody epitope in the SUDV GPΔmuc-WL2P4/CA45 complex, with a Cα-RMSD of 0.69 Å for 147 aligned Cα atoms after superposition of a single GP/CA45 component from each complex (Fig. S2h, right). Specifically, the CA45-GP2 interface was stabilized by a backbone hydrogen bond between CA45 HC-Y32 and GP2-E545 (~3.0 Å), a side chain hydrogen bond between the CA45 HC-Y33 carboxyl group and the GP2-H516 imidazole ring (~3.4 Å), and additional hydrogen bonds involving the CA45 LC-Y49 and LC-N53 side chains with the GP2-N550 backbone (~3.3 Å and ~3.8 Å, respectively), as well as the CA45 LC-S56 backbone with the GP2-N514 side chain (~2.9 Å) (Fig. 2c). Although interpretation of these interactions may be limited by the cryo-EM resolution, similar contacts were observed in the 3.7 Å-resolution crystal structure of the EBOV GPΔmuc/CA45 complex (PDB ID: 6EAY)(Fig. S2i)118, including those between CA45 HC-Y32 and GP2-E545 (~3.2 Å), CA45 HC-D101 and GP2-H549 (~3.9 Å), and the same CA45 LC-GP2 residue pairs with nearly identical distances. Snapshots of the cryo-EM density at the GP-CA45 interface were inspected fragment by fragment for anisotropy, demonstrating that the modeled side chain contacts are supported by well-defined density (Fig. S2j and S2k). Furthermore, we compared our SUDV GPΔmuc-WL2P4/CA45 complex with previously reported cryo-EM models of EBOV GPΔmuc bound to ADI-15878 (PDB ID: 6DZL and EMD-8935117)(Fig. S2l), rEBOV-515 and rEBOV-442 (PDB ID: 7M8L and EMD-23719126)(Fig. S2m), and rEBOV-520 and rEBOV-548 (PDB ID: 6PCI and EMD-2030150)(Fig. S2n), demonstrating comparable model quality.

Rational design and characterization of orthoebolavirus GPΔmuc-presenting nanoparticles
In our previous EBOV study, a 10GS linker was used to connect GPΔmuc to the N-termini of I3-01v9, which formed a triangle of 55.0 Å100. This SApNP exhibited variability in yield, purity, and stability, suggesting that further optimization might be necessary100. In a more recent study, we redesigned the I3-01v9 SApNP to display monomeric antigens127. The resulting I3-01v9a scaffold featured a smaller triangle of 43.9 Å between the N-termini (Fig. 3a, left). Here, using I3-01v9a as a template, we aimed to improve the design of an I3-01v9 scaffold optimized for surface display of filovirus GP trimers, which contain an arrow stalk. After removing the first 4 amino acids (aa) of I3-01v9a, we fused a 9-aa helix to the truncated I3-01v9a N-terminal helix via a 4-aa turn. The resulting model was computationally optimized – first in structure and then in sequence – to generate two I3-01v9 variants, termed I3-01v9b and I3-01v9c (Fig. S3a), in which the N-termini form a triangle of 12.9 Å (Fig. 3a, right). The trimeric forms of I3-01v9b/c, termed I3-01v9b/c-T, were generated by interrupting the particle-forming interface based on the structure of bacterial aldolase (PDB ID: 1VLW), from which I3-01 was derived. Two “scaffolded” EBOV GP constructs, termed GPΔmuc-WL2P4-I3-01v9b-T and GPΔmuc-WL2P4-I3-01v9c-T, were designed to test whether the I3-01v9b/c N-termini could accommodate the GP2 stalk (Fig. S3b). Both constructs were transiently expressed in HEK293S cells, purified using a mAb100 column, and analyzed by SEC (Fig. S3c). The trimer fractions were validated by BN-PAGE (Fig. S3d) prior to nsEM analysis, which yielded two-dimensional (2D) classes consistent with native-like GPΔmuc-WL2P4 trimers anchored to I3-01v9b/c-T (Fig. S3e and f). The GP2 stalk, although not visible by EM, was likely well formed, as the GPΔmuc trimer displayed the classic chalice shape (Fig. 3b and Fig. S3g). Computational modeling suggested that EBOV GPΔmuc-WL2P4-I3-01v9b would form a particle with a diameter of 49.1 nm, measured at GP1-T269 (Fig. 3c).

Six SApNP constructs – displaying EBOV, SUDV, and BDBV GPΔmuc-WL2P4 trimers on the E2p and I3-01v9b scaffolds (Fig. S3h) – were transiently expressed in ExpiCHO cells and purified by IAC using an ADI-15878 column, followed by SEC on a Superose 6 Increase 10/300 GL column. All constructs exhibited a peak at ~8.7 ml, corresponding to assembled protein particles (Fig. 3d). For EBOV and BDBV, higher yields were observed for the E2p SApNPs, whereas for SUDV, I3-01v9b produced a more prominent particle peak in the SEC profile. The structural integrity of these SApNPs was validated by nsEM (Fig. 3e). Most micrographs showed well-formed protein particles displaying arrays of GP trimers on the outer surface. Particle size distributions were analyzed in solution by dynamic light scattering (DLS) (Fig. 3f). For EBOV SApNPs, E2p and I3-01v9b exhibited sizes of 55.7 nm and 84.0 nm, respectively (Fig. 3f). The unusually large DLS size for I3-01v9b may reflect SApNP aggregation, although this form was rarely observed by EM. For SUDV and BDBV SApNPs, particle sizes ranged from 61.1 to 71.9 nm and from 51.3 to 53.8 nm, respectively. This variation may be attributed to the intrinsic structural flexibility of the glycan cap, which likely affects the hydrodynamic radius measured by DLS. Site-specific glycosylation profiles were generated for SUDV GPΔmuc-presenting SApNPs (Fig. 3g and Fig. S3i). An increase in oligomannose-type glycans was observed on SApNPs compared to soluble trimers. At site N563, both the soluble GPΔmuc trimer and SApNPs presented predominantly oligomannose-type glycans (Fig. 3g).

The antigenicity of orthoebolavirus GPΔmuc-WL2P4-presenting SApNPs was assessed against the same antibody panel. In ELISA, SApNPs exhibited broadly similar antibody-binding patterns to the corresponding soluble trimers (Fig. 3h and Fig. S3j). For example, SUDV SApNPs bound less favorably to ADI-15946 and BDBV-289 than EBOV and BDBV SApNPs, with 5.0- to 16.8-fold higher EC50 values. Between the two NP scaffolds, E2p outperformed I3-01v9b, showing 1.4- to 1.9-fold lower EC50 values for both bNAbs. A similar trend was observed in SUDV SApNP binding to CA45, with a 2.5-fold difference in EC50 between the two NP platforms. Interestingly, the opposite pattern was seen for EBOV and BDBV SApNPs, where I3-01v9b bound more tightly than E2p. BLI revealed a somewhat different antigenic profile (Fig. 3i and Fig. S3k). Most notably, SUDV and BDBV SApNPs exhibited stronger binding signals to ADI-15878 and ADI-15946 than EBOV SApNPs, whereas SUDV SApNPs showed the weakest binding to BDBV-289 among the three orthoebolaviruses. For each GP antigen, binding signals to individual bNAbs were generally similar between E2p and I3-01v9b, except in the cases of SUDV SApNPs with CA45 and BDBV SApNPs with CA45 and BDBV-289, where scaffold-dependent differences were apparent.

In this study, we displayed the stabilized GPΔmuc-WL2P4 trimer on the multilayered E2p and I3-01v9b SApNP scaffolds for three representative orthoebolaviruses. Particle properties were broadly comparable to those of our HIV-1 Env SApNPs113, while glycan and antigenic analyses revealed distinct features, including an increased proportion of oligomannose-type glycans. As in our previous SApNP studies100,113,127,128, BLI assays were performed with IgG to mimic native antibody binding under physiological conditions. The resulting binding profiles therefore represent apparent rather than intrinsic affinities. Supporting this approach, Fab analyses showed minimal binding to SApNPs (Fig. S3l), reflecting restricted access of shortened antibodies to densely arrayed GPΔmuc trimers on NP surfaces. Together, these findings support the GPΔmuc-WL2P4 trimer and SApNPs as a general vaccine framework for orthoebolaviruses.

In vitro effect of glycan modification on orthoebolavirus GPΔmuc trimers and SApNPs
Previously, we investigated the effect of glycan modification on rationally designed HIV-1 Env trimer and SApNP vaccines113. Supplementing ExpiCHO cultures with kifunensine (Kif) – a class I α-mannosidase inhibitor that blocks ER glycan processing and leads to accumulation of high mannose – did not affect NAb responses. In contrast, glycan trimming with endoglycosidase H (Endo H) significantly enhanced NAb elicitation, as reflected by a higher vaccine responder rate113. The benefit of glycan trimming has also been reported for influenza HA and SARS-CoV-2 spike vaccines112,114. These findings highlight the importance of examining how glycan modification may affect filovirus vaccines in vivo. However, the glycan-modified filovirus GP immunogens first needed to be generated and characterized in vitro.

Here, we adopted a similar strategy113 to produce orthoebolavirus GPΔmuc trimers carrying either oligomannose-only glycans or a single layer of mono-N-acetylglucosamine (mono-GlcNAc) glycans. EBOV and SUDV GPΔmuc-WL2P4 were transiently expressed in HEK293F cells in the presence of Kif, which yielded oligomannose-only glycans, and were purified using an ADI-15878 column followed by SEC. The SEC profiles showed a trimer peak at ~10.7 ml, similar to wild-type trimers (Fig. 4a). SEC-purified, Kif-treated GPΔmuc (e.g., 1 mg) was then treated with Endo H (termed Kif/EndoH) to reduce the glycan shield to mono-GlcNAc stumps, with the enzymes subsequently removed by SEC. The resulting profiles showed a trimer peak at ~11.1 ml and an EndoH peak at ~13.9 ml (Fig. 4a). Sodium dodecyl sulfate (SDS)-PAGE and BN-PAGE confirmed that Kif/EndoH-treated GPΔmuc had a lower molecular weight than both wild-type and Kif-treated GPΔmuc (Fig. S4a). DSC revealed a differential effect of glycan modification on GPΔmuc thermostability (Fig. 4b). The Kif-treated EBOV GPΔmuc-WL2P4 trimer exhibited two denaturation states, with Tm1 at 58.2 °C and Tm2 at 64.3 °C. EndoH treatment further reduced Tm1 and Tm2 by 5.2 °C and 2.6 °C, respectively. In contrast, the Kif-treated SUDV GPΔmuc-WL2P4 trimer remained thermostable, displaying the same Tm (63.6 °C) as the wild-type trimer, with Tonset measured at 54.7 °C. EndoH treatment slightly reduced the Tm to 62.6 °C and lowered the Tonset to 48.9 °C. Antigenicity was assessed using the same antibody panel. In the ELISA (Fig. 4c and Fig. S4b), EndoH treatment enhanced trimer binding to CA45, ADI-15878, and ADI-15946, which recognize a quaternary epitope near the trimer base with few glycan contacts, but reduced binding to BDBV-289, which targets the glycan cap and relies on glycans at N238 and N268. However, the EC50 differences were small (1.5- to 1.9-fold) and not specific to either orthoebolavirus. In the BLI analysis, glycan-modified trimers generated slightly higher binding signals than wild-type trimers (Fig. 4d and Fig. S4c). Overall, glycan modification had only a modest impact on the biochemical, biophysical, and antigenic properties of stabilized orthoebolavirus GPΔmuc trimers, except for thermostability in the EBOV constructs.

We next characterized glycan-modified GPΔmuc-WL2P4-presenting SApNPs. For SUDV, both E2p and I3-01v9 SApNPs were included in this analysis, whereas for EBOV, only the E2p scaffold was tested due to the low yield of I3-01v9b SApNPs. Notably, an N655D mutation was introduced to eliminate an N-glycan site at the base of the SUDV GP2 stalk that may interfere with SApNP assembly. This design variant, termed SUDV GPΔmuc-WL2P4-PD, will be used hereafter (Fig. S4d). EBOV and SUDV SApNPs were transiently expressed in ExpiCHO cells with Kif and purified by IAC (ADI-15878) followed by SEC. SEC-purified, Kif-treated SApNPs were trimmed with EndoH and further purified by SEC. The resulting SEC profiles showed a main peak at ~8.5-9.0 ml corresponding to SApNPs, and following EndoH treatment, an additional peak at ~16.8 ml corresponding to the enzyme (Fig. 4e). The structural integrity of glycan-modified EBOV and SUDV GPΔmuc-presenting SApNPs was validated by nsEM (Fig. 4e). SDS-PAGE confirmed that Kif/EndoH-treated SApNPs had a lower molecular weight than Kif-treated SApNPs (Fig. S4e). Because of the low yield of EBOV SApNPs, only SUDV SApNPs were subjected to glycan profiling and antigenic characterization. Site-specific glycan profiles confirmed that all complex glycans in SUDV GPΔmuc were replaced by oligomannose-type glycans in the Kif-treated material (Fig. 4f and Fig. S4f). As expected, the N655 glycan was absent in the N655D mutant construct. In the ELISA (Fig. 4g and Fig. S4g), the antigenic profiles of SApNPs appeared to be largely consistent with those of the trimers, though a greater difference in EC50 values between Kif and Kif/EndoH-treated samples (1.0- to 7.9-fold) was observed for CA45, ADI-15878, and ADI-15946. In the BLI analysis, all pan-orthoebolavirus bNAbs showed increased binding signals to Kif/EndoH-treated SApNPs, which displayed only a single layer of mono-GlcNAc stumps on the GPΔmuc trimer (Fig. 4h and Fig. S4h). In summary, these results suggest that glycan trimming enhances antibody access to conserved GP epitopes on the SApNP surface.

Rational design of a prefusion-stabilized RAVV GPΔmuc trimer
RAVV is one of the two known members of the Orthomarburgvirus genus in the filovirus family40,49. Similar to orthoebolavirus GPs, RAVV GP contains GP1 with a heavily glycosylated MLD and GP2 (Fig. 5a). Here, we applied a similar strategy to stabilize the prefusion RAVV GP trimer, using a mucin-deleted construct (ΔN257-T425), termed GPΔmuc. Additional mutations – T578P (P2) or E580P (P4) in the HR1c bend, and E631F/Q632V at the C terminus (termed CT) – were also evaluated. The five RAVV GP constructs tested were: (1) GPΔTM (truncation at residue 637), (2) GPΔmuc, (3) GPΔmuc-P2, (4) GPΔmuc-P4, and (5) GPΔmuc-P2CT, each containing a C-terminal foldon motif and His tag (Fig. S5a). All constructs were expressed in ExpiCHO cells and purified using a nickel column followed by SEC. Both GPΔTM and GPΔmuc yielded low expression levels with multiple GP species, including aggregates (~9-10 ml), trimers (~10.5 ml), dimers (~13.6 ml), and monomers (~14.7 ml) (Fig. 5b). The P2 mutation substantially improved the SEC profiles of GPΔmuc constructs, producing a predominant trimer peak at ~10.5 ml, along with visible dimer and monomer peaks. In contrast, the P4 mutation resulted in significant aggregation, indicated by a peak at ~8.6 ml (Fig. S5b). The GPΔmuc-P2CT construct demonstrated an optimal SEC profile, comparable in yield to GPΔmuc-P2 but with reduced dimer and monomer content. When MARV NAbs MR78 and MR19149 were used in IAC purification, they failed to capture GPΔmuc-P2 and GPΔmuc-P2CT (Fig. S5c). Nevertheless, distinct trimer bands were observed by SDS-PAGE after nickel/SEC purification (Fig. 5c and Fig. S5d). In DSC, GPΔmuc-P2 exhibited two melting transitions at 51.0 °C and 60.0 °C, while GPΔmuc-P2CT displayed three transitions at 54.5 °C, 58.3 °C, and 66.6 °C (Fig. 5d). Although CT appeared to enhance trimer thermostability, this double mutation introduced an additional unfolding intermediate.

The lead design, RAVV GPΔmuc-P2CT, was complexed with MR78 for nsEM analysis (Fig. 5e and Fig. S5e). We hypothesized that if the nsEM model, despite its low resolution, matched the crystal structure (PDB ID: 5UQY)40, it would validate our design and confirm presentation of a conserved NAb epitope. Indeed, 2D class averages revealed well-formed RAVV GPΔmuc/MR78 complexes, with the crystal structure fitting well into the nsEM density map (Fig. 5e, left). Notably, our model also aligned closely with a previously reported nsEM reconstruction of the same complex42 (Fig. 5e, right). Site-specific glycan analysis revealed distinct patterns relative to orthoebolavirus GPs (Fig. 5f and Fig. S5f). Specifically, N-glycan sites N94, N171, N219, and N564 carried primarily oligomannose-type glycans. In contrast, sites N190, N202, and N487 presented either oligomannose- or complex-type glycans, while N475 and N619 were mostly occupied by complex-type glycans. In ELISA (Fig. 5g and Fig. S5g), RAVV GPΔmuc-P2 and GPΔmuc-P2CT trimers bound to MARV NAbs MR78 and MR191 with similar EC50 values, consistent with the BLI data (Fig. 5h and Fig. S5h). RAVV GPΔmuc-P2CT was subsequently displayed on E2p and I3-01v9b SApNPs and analyzed by nsEM following ExpiCHO expression and IAC purification (Fig. S5i). However, well-formed protein particles were not observed in the micrographs. Taken together, these findings support the use of RAVV GPΔmuc-P2CT as a native-like, prefusion-stabilized GP construct for orthomarburgvirus vaccine development, although further optimization is needed for multivalent display on SApNPs.

Distribution and retention of SUDV GPΔmuc trimers and SApNPs in lymph nodes
In our previous studies, we observed a positive correlation between the thermostability of a viral antigen (i.e., Tm) displayed on E2p or I3-01v9 SApNPs and the retention time of the resulting SApNP vaccine antigens in lymph node follicles113,127,129. Given the comparable Tm values (~63-66 °C) of SUDV GPΔmuc and HIV-1 Env trimers, we anticipated that SUDV GPΔmuc-presenting SApNPs would exhibit similar in vivo behavior. Following our previously established protocol113,127,129, we immunized mice with SUDV GPΔmuc-WL2P4 trimer and SApNPs (including both E2p and I3-01v9b) to investigate their interaction with cellular components in lymph nodes and analyze vaccine-induced immunological responses.

To induce a robust humoral response, vaccine antigens must be transported through the lymphatic system, accumulate in lymph node follicles, and effectively engage B cell receptors (BCRs) to stimulate B cell activation130–133. We first examined the distribution of E2p and I3-01v9b SApNPs in lymph nodes. SApNPs were administered intradermally to mice via footpads (4 footpads, 10 μg/footpad). The brachial and popliteal sentinel lymph nodes were isolated from both sides of each mouse 12 h after a single-dose injection for immunohistological studies. Four NAbs – ADI-15878116, ADI-15946119, CA45118, and mAb100134 – were used to stain lymph node sections for antigen detection (Fig. S6a). Overall, the immunostaining images from CA45 exhibited the best signal-to-noise ratio. Based on these data, CA45 was selected to study the distribution and trafficking of SUDV GPΔmuc vaccines in lymph nodes. Consistent with our previous studies of SARS-CoV-2 spike129, HIV-1 Env113, and influenza M2ex3127 SApNPs, SUDV GPΔmuc-presenting E2p and I3-01v9b SApNPs accumulated in the centers of lymph node follicles (Fig. 6a and b, images on the left; schematics on the right).

We next assessed the trafficking and retention patterns of SUDV GPΔmuc-WL2P4 trimer and SApNPs in lymph node follicles over a period of 8 weeks following a single-dose injection (4 footpads, 10 μg/footpad) (Fig. 6c). Histological images showed that all SUDV immunogens were transported into lymph nodes and accumulated in the subcapsular sinus within 2 h (Fig. 6c). While the soluble trimer was transported into lymph node follicles within 2 h and completely cleared by 48 h, both E2p and I3-01v9b SApNPs appeared in follicles at 2 h, reached peak accumulation at 48 h, and were retained in follicles over the entire 8-week period (Fig. 6c). Next, the CA45-stained area was quantified in a time-dependent manner, revealing a ~112-fold longer retention for E2p and I3-01v9b SApNPs compared with the soluble trimer (Fig. 6c and d). The area under the curve (AUC) indicated that the exposure of SUDV GPΔmuc presented on the SApNP surface was 14-28 times higher than the same antigen in soluble trimer form (Fig. 6e). At 48 h, the two large SApNPs displaying 20 GPΔmuc trimers also showed 19-33 times greater accumulation compared with the soluble GPΔmuc trimer (Fig. 6f). These findings are consistent with our previous studies113,127,129, in which individual antigens were cleared from follicles within 48 h, whereas large antigen-presenting SApNPs exhibited prolonged follicular retention lasting 2–8 weeks. Notably, the retention time of SUDV GPΔmuc SApNPs fell within the same range as HIV-1 Env and influenza M2ex3 SApNPs, all of which had Tm values of 62-66 °C. Together, these data support the conclusion that stabilized antigens with high thermostability (Tm > 62 °C) result in prolonged SApNP retention in lymph node follicles for at least 8 weeks.

FDCs are resident stromal cells that form a network structure in lymph node follicles and play an essential role in antigen retention and presentation to stimulate B cell responses130–132,135. FDC networks retain soluble antigens, immune complexes, viruses, and bacteria on their surfaces and dendrites to initiate and maintain GC responses136–138. Our previous studies demonstrated that FDC networks are the primary follicular components responsible for retaining SARS-CoV-2 spike, HIV-1 Env, and influenza M2ex3 SApNPs113,127,129. To test whether this was also true for SUDV GPΔmuc SApNPs, we isolated sentinel lymph nodes at the peak of antigen accumulation (48 h), as well as a series of timepoints (2 h to 8 weeks) after intradermal injection (Fig. 6g and Fig. S6b–g). Lymph node sections were stained with the NAb CA45 (white)118 for SUDV GPΔmuc, anti-CD21 antibodies (green) for FDCs, and anti-CD169 antibodies (red) for subcapsular sinus macrophages. To confirm the identity of FDC networks, co-staining was performed using the more specific FDC marker, anti-FDC-M1. Immunofluorescence imaging showed that SApNPs colocalized with FDC networks labeled by both CD21 and FDC-M1 at 48 h post-injection (Fig. 6g and Fig. S6h), supporting the conclusion that FDC networks mediate the accumulation and retention of SUDV GPΔmuc-presenting SApNP vaccines.

Interaction of SUDV GPΔmuc SApNPs with FDCs and phagocytic cells in lymph nodes
FDC networks form a reservoir in lymph node follicles for antigen sequestration, alignment, and presentation to effectively crosslink BCRs, activate naïve B cells, and stimulate GC reactions130–132,139. Our previous TEM analysis showed that FDC networks retained SARS-CoV-2 spike and HIV-1 Env SApNPs on their surfaces and dendrites through interactions with complement protein 3 (C3) and complement receptor 2 (CR2)113,129. Here, we visualized the interface between FDC dendrites and B cells to understand how FDC networks present SUDV GPΔmuc SApNPs adjuvanted with aluminium phosphate (AP) to engage B cells. To this end, we injected adjuvanted E2p and I3-01v9b SApNPs into the mouse hind footpads (2 footpads, 50 μg/footpad). Fresh popliteal sentinel lymph nodes were isolated 2, 12, and 48 h after a single injection. The lymph node tissues were sectioned and processed for TEM analysis. TEM images revealed the expected, characteristic morphology of long FDC dendrites interacting with B cells in lymph node follicles (Fig. 7a-c). Intact SApNPs (round-shaped granules, yellow arrows) were aligned on FDC dendrites and B cell surfaces at all timepoints, and AP particles did not appear to colocalize with SApNPs (Fig. 7a–c and Fig. S7a–f). These findings demonstrate FDC networks collect and present vaccine antigens (i.e., SUDV GPΔmuc SApNPs) and use their long dendrites to maximize interactions between antigens and BCRs. At the later timepoints (12 and 48 h), SUDV GPΔmuc SApNPs were also found within endolysosomes of B cells (Fig. S7c and e).

Phagocytic cell populations, such as macrophages and dendritic cells, take up and process vaccine antigens to facilitate adaptive immune responses133,140,141. These innate immune cells, located in the subcapsular sinus and medullary sinus of lymph nodes, can capture vaccine antigens, transport them to migrating B cells, and eventually deposit them on FDCs through a complement-dependent transport mechanism131,132,142–146. In this study, we assessed the interaction between phagocytic cells and AP-adjuvanted SApNPs. SUDV GPΔmuc SApNPs, both E2p and I3-01v9b, were found either on the surface or within endolysosomes of phagocytic cells (Fig. 7d, e and Fig. S7g–l), consistent with our previous findings for SARS-CoV-2 spike and HIV-1 Env SApNPs113,129. AP particles tended to aggregate in the extracellular matrix (ECM) 2 h after injection (Fig. 7f and Fig. S7m). Both I3-01v9b SApNPs and AP particles were also observed inside endolysosomes of phagocytic cells at all timepoints (Fig. 7g and Fig. S7n–p). Overall, E2p and I3-01v9b SApNPs exhibited similar patterns of interaction with FDC networks, B cells, and phagocytic cells. FDC networks retained SApNPs on their dendrites and presented native antigens to B cells in lymph node follicles. A substantial amount of SApNPs was also processed inside endolysosomes of phagocytic cells in the presence of AP adjuvant.

Assessment of GC reactions induced by SUDV GPΔmuc trimers and SApNPs in lymph nodes
In lymph node follicles, GCs are the sites where B cell somatic hypermutation, selection, affinity maturation, and class switching occur136,147,148. GC reactions lead to the formation of immune memory and the development of NAb responses following vaccination130,133. As the essential components supporting GC initiation and maintenance, FDC networks and T follicular helper (Tfh) cells present vaccine antigens and stimulate B cells, respectively149–151. Here, we hypothesized that SUDV GPΔmuc SApNPs, due to prolonged retention by FDC networks, would induce more robust and long-lived GC reactions in lymph node follicles compared with the soluble trimers. We first characterized GC reactions induced by I3-01v9b SApNPs after a single-dose injection (4 footpads, 10 μg/footpad). As previously described113,127,129, vaccine-induced GC B cells (GL7+, red) and Tfh cells (CD4+Bcl6+, co-labeled with cyan and red) in lymph nodes were assessed by immunohistological analysis. Robust GCs were formed and attached to FDC networks (CD21+, green), with well-organized dark zone (DZ) and light zone (LZ) compartments in B cell follicles (B220+, blue) (Fig. 8a, left). Tfh cells were observed in the LZ of GCs, sustaining B cell affinity maturation (Fig. 8a, right). We then applied this immunohistological assessment to the SUDV GPΔmuc trimer and SApNPs (E2p and I3-01v9b) at 2, 5, and 8 weeks after a single-dose injection (Fig. 8b and Fig. S8a–c), and at 2 and 5 weeks after the boost (Fig. 8c and Fig. S8d and e). GC reactions were quantified using two metrics: GC/FDC ratio (the frequency of GCs associated with FDC networks) and size of GCs (area occupied in follicles) as previously described113,127,129. All three SUDV GPΔmuc immunogens induced robust GCs, with the I3-01v9b SApNP groups showing the largest GCs at 2 weeks after a single-dose vaccination (Fig. 8b and Fig. S8a). While the GC/FDC ratio decreased significantly over 8 weeks following a single trimer dose, both SApNPs supported long-lived GCs with high GC/FDC ratios (e.g., > 50%) lasting for 8 weeks (Fig. 8b). GC sizes declined over time in both trimer and SApNP groups (Fig. 8b and d). After boosting, all vaccine groups showed restored GC formation, and the GC/FDC ratio in the GPΔmuc trimer group remained comparable (all > 75%) to those in the two SApNP groups (Fig. 8c). Overall, the two SApNPs generated larger GCs than the soluble trimer, 1.3-2.5 times larger after the prime (Fig. 8b and d) and 1.4 to 1.8 times larger after the boost (Fig. 8c and e).

Next, we assessed GC reactions using flow cytometry. We intradermally immunized mice with the three vaccines, and sentinel lymph nodes were isolated at 2, 5, and 8 weeks after a single-dose injection (Fig. 8f and Fig. S9) and 2 and 5 weeks after the boost (Fig. 8g) (four footpads, 10 μg/injection). Fresh lymph node tissues were disaggregated into single-cell suspensions and stained with an antibody cocktail. GC reactions were quantified based on the percentage and number of GC B and Tfh cells (Fig. S9). The I3-01v9b SApNP group yielded the highest percentage and number of GC B cells at 2 weeks post-prime (Fig. 8f), consistent with the immunohistological findings. Among all vaccine groups, GC B cells declined over time, whereas Tfh cell levels remained stable over the 8-week period. A boost enhanced both the frequency and number of GC B and Tfh cells (Fig. 8g). Notably, GC B and Tfh cells induced by I3-01v9b SApNP persisted at relatively high levels over 8 weeks post-boost. Compared with the soluble trimer, the E2p and I3-01v9b SApNPs elicited 1.8/2.4-3.3 times more GC B cells, and 1.5-1.8/2.4-3.2 times more Tfh cells, at 8 weeks after the single-dose/boost injection, respectively (Fig. 8f and g). In summary, the two SUDV GPΔmuc SApNPs generated more robust and long-lived GC reactions than the soluble GPΔmuc trimer, leading to more potent and durable humoral immune responses.

Antibody responses induced by rationally designed filovirus GPΔmuc vaccines in mice
We assessed the immunogenicity of rationally designed filovirus GPΔmuc constructs, including trimers and SApNPs (E2p and I3-01v9b), in mice (Fig. 9a). The filovirus antigen (10 μg) was adjuvanted with aluminium hydroxide (AH) and a Toll-like receptor 9 (TLR9) agonist, CpG ODN1826. Mice were immunized intraperitoneally at weeks 0, 3, 9, and 15 with 6-week intervals between the second and third doses and between the third and fourth doses. Serum was collected two weeks after each dose for serological analysis. EBOV vaccine-induced binding antibody (bAb) responses were measured by ELISA using an EBOV GPΔmuc-WL2P4 (1TD0) antigen (Fig. 9b and Fig. S10a, b). The use of 1TD0, rather than foldon, as the trimerization motif in the coating antigen enabled the detection of GPΔmuc-specific bAb responses. Overall, the EBOV GPΔmuc E2p SApNP elicited the highest bAb titers at all four timepoints, with EC50 values 8.2- and 5.3-fold higher than those of the soluble trimer at weeks 2 and 5, respectively. All vaccine groups reached plateau EC50 titers by week 11, after three immunizations. The EBOV GPΔmuc I3-01v9b SApNP achieved an EC50 titer of 182,253 at week 11, identical to its E2p counterpart and 2.5-fold higher than that elicited by the soluble trimer. Week-17 sera (after four doses) were tested against a SUDV GPΔmuc-WL2P4 (1TD0) antigen (Fig. 9c and Fig. S10c). The EBOV GPΔmuc E2p SApNP again elicited the highest EC50 titer (32,585), 3.5-fold higher than that of the soluble trimer. Cross-reactive bAb responses were also evaluated against a RAVV GPΔmuc-P2CT (1TD0) antigen (Fig. 9d and Fig. S10d). Both E2p and I3-01v9b SApNP groups showed significantly higher serum binding signals, indicated by absorbance at 450 nm (A450), compared with the soluble trimer group. NAb responses were evaluated using a filovirus pseudoparticle (pp) neutralization assay36, with 50% inhibitory dilution (ID50) values calculated for comparison. Week-2 sera served as negative controls for longitudinal analysis. None of the EBOV GPΔmuc vaccines elicited NAb responses at week 2, but ID50 titers were detected at weeks 5, 11, and 17, increasing steadily against EBOV-Makona pseudovirus (Fig. 9e and Fig. S10e and f). Consistent with the bAb results, EBOV GPΔmuc E2p SApNP produced the highest NAb titers at all timepoints, although no statistical significance was observed across groups. Week-17 sera were also evaluated against the SUDV-Gulu pseudovirus (Fig. 9f and Fig. S10g). The EBOV GPΔmuc E2p SApNP yielded the highest ID50 titer (761) among all groups. Notably, the EBOV GPΔmuc trimer group elicited the highest ID50 titer (2,759) at week 17 against a BDBV-Uganda pseudovirus, 4.5- and 4.9-fold higher than those elicited by the E2p and I3-01v9b SApNPs, respectively (Fig. 9g and Fig. S10h). Thus, while soluble trimers can achieve high NAb titers, they may require more doses than SApNPs.

We assessed SUDV vaccine-induced serum bAb responses by ELISA against a SUDV GPΔmuc-WL2P4 (1TD0) antigen (Fig. 9h and Fig. S10i, j). Consistent with the bAb responses elicited by EBOV vaccines, SUDV GPΔmuc E2p SApNP generated the highest EC50 titers of 871, 82,910, and 307,187 at weeks 2, 5 and 11, respectively, 12.4-, 4.5-, and 5.7-fold higher than those induced by the soluble trimer. EC50 titers in both E2p and I3-01v9 SApNP groups plateaued after three doses by week 11, whereas the soluble trimer group reached a peak of 102,959 after four doses at week 17, which remained slightly lower than the titers induced by SApNPs. These results correlated well with the GC reactions generated by SUDV GPΔmuc trimer and SApNPs (Fig. 8). Week-17 sera were then evaluated against an EBOV GPΔmuc-WL2P4 (1TD0) antigen (Fig. 9i and Fig. S10k). The SUDV GPΔmuc E2p and I3-01v9b SApNPs elicited EC50 titers of 27,290 and 25,446, respectively, which were 2.6- and 2.4-fold higher than that of the soluble GPΔmuc trimer. Cross-reactive bAb responses were further tested using week-17 sera against a RAVV GPΔmuc-P2CT (1TD0) antigen (Fig. 9j and Fig. S10l). Both E2p and I3-01v9b SApNP groups exhibited significantly higher serum binding signals (A450) compared with the soluble trimer group. Next, we evaluated SUDV GPΔmuc vaccine-induced autologous NAb responses using a SUDV-Gulu pseudovirus assay. Week-2 sera were used as a negative control for the longitudinal analysis. At this timepoint, none of the vaccines elicited detectable NAb responses, except for one mouse in the E2p group. ID50 titers became detectable at week 5, plateaued at week 11 (after three doses), and remained stable through week 17 (Fig. 9k and Fig. S10m, n). The SUDV GPΔmuc trimer and SApNP vaccines elicited comparable ID50 titers (1,628) at each timepoint after two doses, with no significant differences. Finally, week-17 sera were assessed for cross-neutralizing activity against EBOV-Makona (Fig. 9l and Fig. S10o) and BDBV-Uganda (Fig. 9m and Fig. S10p) pseudoviruses. No statistically significant differences in cross-NAb responses were observed between the SUDV trimer and SApNP groups, as measured by ID50 titers.

Lastly, we assessed RAVV GPΔmuc trimer-induced serum bAb responses in ELISA using a RAVV GPΔmuc-P2CT (1TD0) antigen (Fig. 9n and Fig. S10q). Similar to EBOV and SUDV GPΔmuc trimers, RAVV GPΔmuc trimer generated a strong bAb response after two doses, with the peak EC50 titer of 362,215 at week 5. Cross-reactive bAb responses were evaluated using week-17 sera against two Uganda pseudoviruses. n–p RAVV vaccine-induced bAb responses against (n) RAVV GPΔmuc-P2CT (1TD0), (o) EBOV GPΔmuc-WL2P4 (1TD0), and (p) SUDV GPΔmuc-WL2P4 (1TD0). q RAVV vaccine-induced NAb responses against MARV Angola pseudovirus (using purified IgG) and pseudotyped orthoebolaviruses (using serum). EC50 titers were derived from serum ELISA against coating antigens, and ID50/IC50 titers were determined from pseudovirus neutralization assays. Geometric means are labeled on the plots. ID50/IC50 values were calculated using a % neutralization range of 0.0–100.0%. Color coding indicates ID50 magnitude (green to red: low to high neutralization). Data are presented as the mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison post hoc test at each timepoint. ns (not significant), *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. The schematic in (a) was created in BioRender. Zhang, Y. (2025) https://BioRender.com/l79v499.

Antibody responses induced by glycan-modified filovirus GPΔmuc vaccines in mice
We characterized the immunogenicity of filovirus GPΔmuc vaccines bearing oligomannose-rich (Kif) and trimmed (Kif/EndoH) glycans in mice (Fig. 10a). The same immunization protocol used in our previous vaccine studies100,113,128,153 was applied here, which differed from the longer-interval (6-week) regimen used for wild-type GPΔmuc vaccines (Fig. 9a). Mice were immunized intraperitoneally at weeks 0, 3, 6, and 9, with 3-week intervals between doses. Each injection contained 10 μg of glycan-modified antigen formulated with 100 μl of CpG/AH adjuvant, resulting in a final volume of 200 μl. Serum was collected two week after each vaccination to enable longitudinal comparison of bAb and NAb responses.

We first measured serum bAb responses induced by glycan-modified EBOV GPΔmuc trimers by ELISA using an EBOV GPΔmuc-WL2P4 (1TD0) antigen (Fig. 10b and Fig. S11a, b). Among the three groups, the glycan-trimmed trimer elicited the lowest EC50 titers at all timepoints, while the Kif-treated trimer generated the highest EC50 titers, 212 and 42,360 at weeks 2 and 5, respectively. In terms of EC50 titers, the Kif-treated group was 2.2- and 6.2-fold higher at week 2, and 1.2- and 2.1-fold higher at week 5, compared with the wild-type and glycan-trimmed groups, respectively. All trimer groups reached peak EC50 titers after three doses. Following a similar trend, the Kif-treated EBOV GPΔmuc E2p SApNP group showed the highest bAb titers at all three timepoints (Fig. 10c and Fig. S11a, b). Responses appeared to plateau at week 5, with the Kif-treated E2p SApNP yielding an EC50 titer of 300,502, 1.5- and 3.2-fold higher than its wild-type and glycan-trimmed counterparts, respectively. Wild-type and Kif-treated EBOV GPΔmuc I3-01v9 SApNPs induced comparable bAb responses, both reaching peak EC50 titers after three doses (Fig. 10d and Fig. S11a, b). We next evaluated NAb responses induced by glycan-modified EBOV GPΔmuc vaccines against EBOV-Makona. Overall, the Kif-treated vaccines were the most immunogenic, while the glycan-trimmed vaccines elicited the lowest NAb titers at all three timepoints (Fig. 10e–g and Fig. S11c, d). Specifically, the Kif-treated EBOV GPΔmuc trimer yielded an ID50 titer of 449 at week 5, 1.5- and 1.9-fold higher than its wild-type and glycan-trimmed counterparts, respectively (Fig. 10e). The Kif-treated EBOV GPΔmuc E2p SApNP elicited an ID50 titer of 1,459 at week 5, 2.3- and 4.1-fold higher than those of the wild-type and glycan-trimmed groups, respectively (Fig. 10f). A similar pattern was observed in the I3-01v9b SApNP groups (Fig. 10g). These results demonstrated the beneficial effect of an oligomannose-rich glycan shield on bAb and NAb responses induced by EBOV GPΔmuc vaccines, in marked contrast to the glycan effects observed in our recent HIV-1 Env vaccine study113.

We then used a SUDV GPΔmuc-WL2P4 (1TD0) antigen in ELISA to assess serum bAb responses induced by glycan-modified SUDV GPΔmuc trimers (Fig. 10h and Fig. S11e, f). At all three timepoints, the Kif-treated SUDV GPΔmuc trimer elicited the highest bAb titers, whereas the glycan-trimmed trimer elicited the lowest, consistent with results observed for EBOV. Regardless of glycan modification, all vaccine groups reached peak EC50 titers after three doses. The Kif-treated SUDV GPΔmuc trimer yielded EC50 titers of 425 and 150,326 at weeks 2 and 5, respectively. In terms of EC50 titers, this trimer achieved 6.1- and 13.3-fold increases at week 2, and 8.2- and 27.6-fold increases at week 5, compared with the wild-type and glycan-trimmed counterparts, respectively. Similarly, for both SUDV GPΔmuc E2p and I3-01v9b SApNPs, the Kif-treated groups showed the highest bAb responses at weeks 2 and 5 (Fig. 10i, j and Fig. S11e, f). For both SApNPs, Kif treatment led to plateaued EC50 titers after two doses (week 5), whereas the wild-type and glycan-trimmed groups required three doses to reach their EC50 titers at week 8. The Kif-treated SUDV GPΔmuc E2p and I3-01v9b SApNPs elicited EC50 titers of 329,358 and 271,692 at week 5, respectively, 2.1- and 6.9-fold higher than their wild-type counterparts and 2.3- and 5.7-fold higher than their glycan-trimmed counterparts, respectively. We then evaluated NAb responses induced by glycan-modified SUDV GPΔmuc vaccines against SUDV-Gulu. All three SUDV GPΔmuc trimer groups reached peak ID50 titers after three doses (Fig. 10k and Fig. S11g, h). The Kif-treated SUDV GPΔmuc trimer group showed the highest ID50 titer of 3,652 at week 5, 2.7- and 2.2-fold higher than those of the wild-type and glycan-trimmed groups, respectively (Fig. 10k). While all three E2p SApNPs exhibited comparable ID50 titers (Fig. 10l), the Kif-treated I3-01v9 SApNP elicited the highest ID50 titer of 4,041 at week 8, 2.9- and 3.2-fold higher than its wild-type and glycan-trimmed counterparts, respectively (Fig. 10m). Therefore, oligomannose enrichment conferred a similar advantage to SUDV as to EBOV vaccines (Fig. 10e–g).

We assessed serum bAb responses induced by the wild-type and glycan-trimmed RAVV GPΔmuc trimers by ELISA using a wild-type RAVV GPΔmuc-P2CT (1TD0) antigen (Fig. 10n and Fig. S11i, j). The wild-type group reached a peak EC50 titer of 362,215 after two doses, whereas the glycan-trimmed group required three doses to plateau at 446,121. Overall, no significant differences were observed between the two groups at any timepoint. We also compared NAb responses by testing purified IgG against pseudotyped MARV-Angola and orthoebolaviruses (Fig. 10o and Fig. S10k). Against MARV-Angola, both groups yielded similar IC50 values, ranging from 25.9 to 191.6 μg/ml. Cross-orthoebolavirus neutralization was evaluated using mouse IgG against EBOV-Makona, SUDV-Gulu, and BDBV-Uganda. The wild-type trimer induced stronger cross-genus activity than its glycan-trimmed counterpart, although the longer dosing interval may have contributed to this difference. Neither group showed nonspecific IgG neutralization against lymphocytic choriomeningitis virus pseudoparticles (LCMV-pps), included as a negative control. These findings were largely consistent with serum neutralization data (Fig. 9q).

Sex-dependent antibody responses induced by stabilized SUDV GPΔmuc trimers
We investigated the impact of sex on NAb responses induced by wild-type and Kif-treated SUDV GPΔmuc trimers. Mice were immunized at weeks 0, 3, 6, and 9 by intradermal injection into the four footpads, with 3-week intervals between doses (Fig. S12a). Each dose consisted of 80 μl of antigen/AH adjuvant mix containing 10 μg of trimer antigen and 40 μl of adjuvant. Serum samples collected at week 11, after four injections, were tested against SUDV-Gulu pseudovirus. For the wild-type SUDV GPΔmuc trimer, a significant sex-specific difference was observed: the ID50 titer in female mice (607) was 2.3-fold higher than in male mice (Fig. S12b). For the Kif-treated SUDV GPΔmuc trimer, female mice developed a slightly higher ID50 titer (593) than males, although the difference was not statistically significant (Fig. S12c). In both vaccine groups, female mice mounted stronger NAb responses than males. This result is consistent with our previous study on RSV F trimer vaccines154 and with the broader consensus that females tend to generate stronger humoral immune responses than males following vaccination155,156.

Our analysis of vaccine-induced antibody responses underscores the importance of multivalent display and glycan modification in enhancing filovirus vaccine immunogenicity. Overall, Kif-treated GPΔmuc SApNPs elicited the strongest bAb and NAb responses. However, differences in immunization protocols (e.g., dosing intervals) preclude statistically reliable conclusions from direct comparison of the two studies. Additional in vivo studies are warranted to confirm these findings. Another notable observation is the induction of cross-orthoebolavirus NAbs by the RAVV GPΔmuc-P2CT trimer, detected at both serum and IgG levels (Fig. 9q, right; Fig. 10o). This result suggests that this GP possesses intrinsic immunogenic potential, warranting further validation in animal models, and could be incorporated into a pan-filovirus vaccine. Finally, sex should be considered an important variable in evaluating filovirus vaccine responses.

Methods
Construct design, expression, and purification of filovirus GP immunogens
For orthoebolaviruses, the amino acid sequences of EBOV GP (Mayinga strain, GenBank Accession: NP_066246) with a T42A substitution, SUDV GP (Gulu strain, GenBank Accession: AAU43887), and BDBV GP (R4386L strain, GenBank Accession: AYI50307) were used to design GPΔmuc trimers (Fig. S1a) and SApNPs (Fig. S3h). Notably, an N637D mutation was later introduced to the SUDV GPΔmuc-WL2P4 construct to eliminate a potential N-linked glycosylation site at N637 due to the enzymatic site “AS” between the GP and SApNP backbone. For orthomarburgviruses, the amino acid sequence of RAVV GP (Ravn-87 strain, GenBank Accession: ABE27071) was used to design GPΔTM and various GPΔmuc trimers (Fig. S5a).

Rationally designed EBOV, SUDV, and BDBV GPΔmuc trimers were transiently expressed in HEK293F cells as previously described100. Briefly, HEK293F cells were thawed and suspended in FreeStyle 293 Expression Medium (Life Technologies, Carlsbad, CA) and placed in a shaker incubator at 37 °C, 135 rotations per minute (rpm), with 8% CO2. When the cells reached a density of 2.0 × 106/ml, they were diluted to a concentration of 1.0 × 106/ml in 1 L of FreeStyle 293 Expression Medium for transfection using a polyethyleneimine (PEI) (Polysciences) transfection protocol. In brief, 900 μg of plasmid DNA in 25 ml of Opti-MEM transfection medium (Life Technologies) was mixed with 5 ml of PEI-MAX (1.0 mg/ml) in 25 ml of Opti-MEM and incubated for 30 min at room temperature. Cells were then transfected with the DNA–PEI-MAX mixture and incubated in a shaker incubator at 37 °C, 135 rpm, with 8% CO2. Kifunensine (10 mg/L, Tocris Bioscience) was added at the time of transfection to inhibit α-mannosidase I and promote oligomannose-type glycosylation. Five days later, the supernatants containing the target protein were harvested, clarified by centrifugation at 1126 × g for 22 min, and filtered through a 0.45 μm membrane (Thermo Scientific). Orthoebolavirus GP was purified with an ADI-15878 IAC column, except for EBOV GPΔmuc-WL2P4 used in X-ray crystallography and EBOV GPΔmuc-WL2P4-I3-01v9b/c-T used in nsEM, which were purified with a mAb100 IAC column. Orthoebolavirus GPs were eluted three times with 5 ml of 0.2 M glycine (pH 2.2), neutralized with 0.5 ml of 2 M Tris base (pH 9.0), and buffer-exchanged into phosphate-buffered saline (PBS; pH 7.2). IAC-purified orthoebolavirus GPs were further purified by SEC using a Superdex 200 Increase 10/300 GL column (Cytiva).

RAVV GPΔmuc trimers and all orthoebolavirus GPΔmuc-presenting SApNPs were produced in ExpiCHO cells (Thermo Fisher). Briefly, ExpiCHO cells were thawed and incubated in ExpiCHO Expression Medium (Thermo Fisher) in a shaker incubator at 37 °C, 135 rpm, with 8% CO2. When cell density reached 10 × 106/ml, ExpiCHO Expression Medium was added to adjust the cell density to 6 × 106/ml for transfection. ExpiFectamine CHO–plasmid DNA complexes were prepared for 100-ml transfections according to the manufacturer’s instructions. Kifunensine (10 mg/L) was added at the time of ExpiCHO transfection to inhibit α-mannosidase I. For these constructs, 100 μg of plasmid and 320 μl of ExpiFectamine CHO reagent were mixed in 7.7 ml of cold OptiPRO™ medium (Thermo Fisher). Following the first feed on day 1, cells were cultured in a shaker incubator at 33 °C, 115 rpm, with 8% CO2 using the MaxTiter protocol, with an additional feed on day 5 (Thermo Fisher). Culture supernatants were harvested 13–14 days post-transfection, clarified by centrifugation at 3724 × g for 25 min, and filtered through a 0.45 μm membrane (Thermo Fisher). RAVV GPΔmuc trimers were purified using Ni Sepharose excel resin (Nickel, Cytiva), eluted twice with 15 ml of 0.5 M imidazole, and buffer-exchanged into Tris-buffered saline (TBS; pH 7.2). Orthoebolavirus GPΔmuc-presenting SApNPs were purified using an ADI-15878 IAC column, eluted three times with 5 ml of 0.2 M glycine (pH 2.2), neutralized with 0.5 ml of 2 M Tris base (pH 9.0), and buffer-exchanged into PBS (pH 7.2). Final polishing was performed by SEC using a Superdex 200 Increase 10/300 GL column (Cytiva) for nickel-purified RAVV GPΔmuc trimers and a Superose 6 Increase 10/300 GL column (Cytiva) for IAC-purified orthoebolavirus GPΔmuc-presenting SApNPs. Selected SEC fractions were pooled, aliquoted, and stored in liquid nitrogen or at −80 °C until use.

Expression and purification of neutralizing antibodies (NAbs)
Antibodies in the IgG form were transiently expressed in ExpiCHO cells (Thermo Fisher). At 12–14 days post-transfection, cells were centrifuged at 3,724 × g for 25 min, and the supernatants were filtered through a 0.45-μm membrane (Millipore). IgGs were purified using protein A affinity resin (Cytiva) and eluted with 0.3 M citric acid (pH 3.0). The eluate was immediately titrated to neutral pH (7.0) by adding 2 M Tris base (pH 9.0). IgGs were then concentrated and buffer-exchanged into PBS using Amicon 10 kDa filters (Millipore). Concentrations were determined by UV absorption using theoretical extinction coefficients.

Glycan trimming by Endo H treatment and enzyme removal
The protocol for Endo H treatment and removal was described in our previous study113. Briefly, surface glycans of filovirus GPΔmuc trimers and SApNPs were trimmed using endoglycosidase H (Endo Hf), a fusion of Endo H and MBP (NEB, catalog no. P0703L). In each reaction, 1 mg of antigen was mixed with 200 μl of 10× GlycoBuffer 3, 250 μl of Endo Hf, and H2O (if necessary) to a final volume of 2 ml. The mixture was incubated at room temperature (25 °C) for 5 h to allow enzymatic processing of the GP glycans. After incubation, the reaction mixtures were purified by SEC using a Superdex 200 Increase 10/300 GL column (for trimers) or a Superose 6 Increase 10/300 GL column (for SApNPs) to remove the MBP-tagged enzyme. Notably, glycan trimming by Endo H requires expression of filovirus GPΔmuc immunogens in the presence of kifunensine. In our previous study113, amylose resin (NEB, catalog no. E8021S) was used to remove residual Endo Hf from SEC-purified HIV-1 Env trimers and SApNPs. In the present study, this additional step of Endo Hf removal was omitted to preserve vaccine antigens for animal studies. SEC-purified fractions were pooled, aliquoted, and stored in liquid nitrogen or at -80 °C until use.

SDS-PAGE and BN-PAGE
Filovirus GPΔmuc trimers and SApNPs were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blue native-polyacrylamide gel electrophoresis (BN-PAGE). Proteins were mixed with loading dye and loaded onto a 10% Tris-Glycine Gel (Bio-Rad) or a 4–12% Bis-Tris Native PAGE gel (Life Technologies). For SDS-PAGE under reducing conditions, proteins were mixed with 6× Laemmli SDS sample buffer (Thermo Scientific) and boiled for 5 min at 100 °C. SDS-PAGE gels were run at 250 V for 25 min using SDS running buffer (Bio-Rad) and stained with InstantBlue (Abcam). For BN-PAGE, proteins were mixed with 4× native dye and loaded onto a BN-PAGE gel. The gel was run at 150 V for 2–2.5 h with NativePAGE running buffer (Life Technologies), following the manufacturer’s instructions. BN-PAGE gels were stained with Coomassie Brilliant Blue R-250 (Bio-Rad) and destained using a solution of 6% ethanol and 3% glacial acetic acid.

Differential scanning calorimetry (DSC)
The melting temperature (Tm) and other thermal parameters of filovirus GPΔmuc trimers purified by an IAC or nickel column followed by SEC were measured using a MicroCal PEAQ-DSC Man instrument (Malvern). Briefly, purified proteins in PBS buffer were diluted to 0.5–5 μM. Melting was assessed at a scan rate of 60 °C/h from 20 °C to 100 °C. Data processing, including buffer correction, normalization, and baseline subtraction, was conducted using MicroCal PEAQ-DSC software. Gaussian fitting was carried out using GraphPad Prism version 10.3.1.

Site-specific glycan analysis
To generate site-specific glycan profiles, 100 µg aliquots of each antigen were denatured for 1 h in 50 mM Tris/HCl (pH 8.0) containing 6 M urea and 5 mM dithiothreitol (DTT). The denatured antigens were reduced and alkylated by adding 20 mM iodoacetamide (IAA) and incubating for 1 h in the dark, followed by 1 h of incubation with 20 mM DTT to eliminate residual IAA. Alkylated samples were buffer-exchanged into 50 mM Tris/HCl (pH 8.0) using Vivaspin columns (10 kDa cutoff). Three aliquots were then digested separately overnight with trypsin (Mass Spectrometry Grade, Promega), chymotrypsin (Mass Spectrometry Grade, Promega), or alpha lytic protease (Sigma Aldrich) at a ratio of 1:30 (w/w). The next day, peptides were dried and extracted using an Oasis HLB µElution Plate (Waters). Peptides were dried again, resuspended in 0.1% formic acid, and analyzed by nanoLC-electrospray ionization MS using an Ultimate 3000 HPLC (Thermo Fisher Scientific) system coupled to an Orbitrap Eclipse mass spectrometer (Thermo Fisher Scientific). Stepped higher-energy collisional dissociation (HCD) fragmentation was applied. Peptides were separated using an Easy Spray PepMap RSLC C18 column (75 µm × 75 cm). A trapping column (PepMap 100 C18 3 μM 75 μM × 2 cm) was used in line with liquid chromatography (LC) prior to separation with the analytical column. The LC conditions were as follows: 280-min linear gradient consisting of 4-32% acetonitrile in 0.1% formic acid over 260 min followed by 20 mins of alternating 76% acetonitrile in 0.1% formic acid and 4% ACN in 0.1% formic acid, used to ensure all the sample had eluted from the column. The flow rate was set to 300 nl/min. The spray voltage was set to 2.7 kV and the temperature of the heated capillary was set to 40 °C. The ion transfer tube temperature was set to 275 °C. The scan range was 375−1500 m/z. The stepped HCD collision energies were set to 15%, 25%, and 45% and the MS2 for each energy was combined. Precursor and fragment detection were performed using an Orbitrap at a resolution of MS1 = 120,000 and MS2 = 30,000. The AGC target for MS1 was set to standard and injection time set to auto which involves the system setting the two parameters to maximize sensitivity while maintaining cycle time. Full LC and MS methodology can be extracted from the appropriate Raw file using XCalibur FreeStyle software or upon request.

Glycopeptide fragmentation data were extracted from the raw file using Byos 4.6 (Protein Metrics). The glycopeptide fragmentation data were evaluated manually for each glycopeptide; the peptide was scored as true-positive when the correct b and y fragment ions were observed along with oxonium ions corresponding to the glycan identified. The MS data was searched using the Protein Metrics 38 insect N-glycan library. The relative amounts of each glycan at each site as well as the unoccupied proportion were determined by comparing the extracted chromatographic areas for different glycotypes with an identical peptide sequence. All charge states for a single glycopeptide were summed. The precursor mass tolerance was set at 4 ppm and 10 ppm for fragments. A 1% false discovery rate (FDR) was applied. The relative amounts of each glycan at each site as well as the unoccupied proportion were determined by comparing the extracted ion chromatographic areas for different glycopeptides with an identical peptide sequence. Glycans were categorized according to the composition detected.
HexNAc2Hex(9−3) was classified as M9 to M3. Any of these structures containing a fucose were categorized as fucosylated mannose (FM). Complex-type glycans were classified according to the number of HexNAc subunits and the presence or absence of fucose. Core glycans refer to truncated structures smaller than M3. As this fragmentation method does not provide linkage information, compositional isomers are grouped.

Biolayer interferometry
Antigenic profiles of filovirus GPΔmuc trimers and SApNPs were measured on an Octet RED96 (FortéBio, Pall Life Sciences) against a panel of antibodies, including five pan-orthoebolavirus NAbs and two MARV NAbs, all in the IgG form. All assays were performed with agitation set to 1000 rpm in FortéBio 1× kinetic buffer. The final volume for all solutions was 200 μl per well. Experiments were conducted at 30 °C in solid black 96-well plates (Greiner Bio-One). For all antigens, 5 μg/ml antibody in 1× kinetic buffer was loaded onto the surface of anti-human IgG Quantitation (AHQ) Biosensors for 300 s. Next, a 2-fold concentration gradient of antigen, starting at 500 nM for trimers and 10 nM for SApNPs, was used in a 6-point dilution series. A 60-s biosensor baseline step was applied before measuring association of antigen in solution with antibody-loaded biosensors for 200 s. Dissociation of the interaction was monitored for 300 s. The correction of baseline drift was performed by subtracting the mean value of shifts recorded for a sensor loaded with antibody but not incubated with antigen, and for a sensor without antibody but incubated with antigen. Octet data were processed using FortéBio’s data acquisition software (version 8.1). Peak response signals at the highest antigen concentration were summarized in a matrix and color-coded for comparison across constructs. Experimental data for each antigen-antibody pair were fitted to a 1:1 binding model, and the three best-fitting datasets were grouped to calculate KD and KD values. Antibody-antigen binding curves were plotted using GraphPad Prism version 10.3.1. In a supplemental BLI analysis, EBOV and SUDV GPΔmuc-WL2P4 trimers and their E2p and I3-01v9b SApNPs were tested against CA45 and ADI-15878 Fabs using FAB2G Biosensors. The same experimental and data-processing protocols were used.

Enzyme-linked immunosorbent assay (ELISA)
Costar 96-well, high-binding, flat-bottom, half-area plates (Corning) were coated with 50 µl of PBS containing 0.1 μg of the appropriate filovirus GPΔmuc trimer or SApNP antigen. Plates were incubated overnight at 4 °C and then washed five times with PBST wash buffer (PBS containing and 0.05% (v/v) Tween 20). Each well was then blocked for 1 h at room temperature with 150 µl of blocking buffer consisting of 4% (w/v) blotting-grade blocker (Bio-Rad) in PBS. Plates were washed again five times with PBST wash buffer. To evaluate antibody binding to the coating antigens, antibodies were diluted in blocking buffer to a maximum concentration of 10 μg/ml, followed by a 10-fold dilution series. For each antibody dilution, a total volume of 50 µl was added to the appropriate wells. For serum sample analysis, mouse sera were diluted 1:40 in blocking buffer and subjected to a 10-fold dilution series. For each sample dilution, 50 µl of each dilution was added to the wells. Plates were incubated for 1 h at room temperature and then washed five times with PBST wash buffer. For secondary antibody binding, a 1:5000 dilution of goat anti-human IgG antibody (Jackson ImmunoResearch Laboratories) or, for mouse sample analysis, a 1:3000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories) was prepared in PBST wash buffer. Then, 50 µl of the diluted secondary antibody was added to each well. Plates were incubated for 1 h at room temperature and then washed six times with PBST wash buffer. Finally, wells were developed with 50 µl of 3,3’,5,5’-tetramethylbenzidine (Life Sciences) for 3-5 min before the reaction was stopped with 50 µl of 2 N sulfuric acid. Plates were immediately read on a BioTek Synergy plate reader at a wavelength of 450 nm. EC50 values were calculated from full curves using GraphPad Prism version 10.3.1. For samples with OD450 values below 0.5, EC50 values were set to 10 μg/ml in Figs. 1g, 3h, 4c, and 4h to facilitate EC50 plotting and comparison.

Protein crystallization and data collection
EBOV GPΔmuc-WL2P4 was transiently expressed in HEK293S cells using the same protocol as for HEK293F cells, followed by IAC and SEC purification. The mAb100/SEC-purified EBOV GPΔmuc-WL2P4 trimer protein at 9.3 mg/ml was subjected to crystallization by sitting-drop vapor diffusion using our automated CrystalMation robotic system (Rigaku) at 20 °C at The Scripps Research Institute166. The reservoir solution consisted of 12% PEG-6000 (v/v) and 0.1 M sodium citrate-citric acid (pH 4.51). Diffraction-quality crystals were obtained after 2 weeks of incubation at 20 °C. Crystals were cryoprotected in well solution containing 25% (v/v) ethylene glycol, mounted on a nylon loop, and flash-cooled in liquid nitrogen. Diffraction data were collected at beamline 12-1 of the Stanford Synchrotron Radiation Lightsource (SSRL) and processed with HKL-2000167. The data were indexed in space group P321.

Structure determination and refinement
The crystal structure of EBOV GPΔmuc-WL2P4 was determined by molecular replacement using Phaser-MR in Phenix (version 1.19.2-4158), with the coordinates of the previously reported EBOV GPΔmuc-WL2P2 structure (PDB ID: 7JPH)100 as the search model. Polypeptide chains were manually adjusted into electron density using Coot168, refined with phenix.refine169, and validated using the wwPDB Validation System170. Carbohydrates were further validated using Privateer in CCP4i171, both before and after refinement. Final data processing and refinement statistics are described in Table S1. All images for crystal structures shown in the figures were generated using PyMOL version 2.3.4. Notably, due to weak electron density, structural models were not built for residues K633-D637 in the GP2 stalk region of EBOV GPΔmuc-WL2P4, or for the C-terminal foldon trimerization motif. These omissions result in apparent gaps in crystal packing when visualizing the GP structure within the electron density map.

Negative-stain electron microscopy (nsEM)
The nsEM analysis of filovirus GPΔmuc trimers, their antibody-bound complexes, and GPΔmuc-presenting SApNPs, was performed by the Core Microscopy Facility at The Scripps Research Institute. Briefly, samples were prepared at concentrations of 0.008 and 0.01 mg/ml, respectively. Carbon-coated copper grids (400 mesh) were glow-discharged, and 8 µl of each sample was adsorbed for 2 min. Excess samples were removed, and grids were negatively stained with 2% uranyl formate for 2 min. Excess stain was wicked away, and the grids were allowed to air-dry. Trimer samples were analyzed at 120 kV with a Talos L120C transmission electron microscope (Thermo Fisher), and images were acquired using a CETA 16M CMOS camera under 73,000× magnification at a resolution of 1.93 Å/pixel and a defocus range of 0.5-2 μm. For SApNP samples, the images were collected under 52,000× magnification. The resulting pixel size at the specimen plane was 2.05 Å, and the defocus was set to −1.50 μm. Computational analysis of the images was performed using the high-performance computing core facility at The Scripps Research Institute. nsEM images were converted to MRC format using EMAN2172 and further processed with CryoSPARC version 4.3.0124. Micrographs were contrast-transfer function (CTF)-corrected by patch CTF estimation. Particles were selected using a blob/template picker and later extracted with a box size of 130 pixels for 2D classification, with 50 and 230 Å used as the minimum and maximum particle sizes for blob picking. 3D models were generated by ab initio reconstruction and optimized using heterogeneous and homogeneous refinement procedures. All nsEM and fitted structure images were generated using UCSF Chimera173 and ChimeraX174–176.

Cryo-electron microscopy (cryo-EM) for SUDV GP-CA45 complexes
The SUDV GPΔmuc-WL2P4 construct was expressed in HEK293F cells and purified using an ADI-15878 column. The eluate was mixed with CA45 Fab at a 1:1.5 molar ratio. The mixture was further purified using a Superose 6 Increase 10/300 GL column, and complex fractions were pooled. Cryo-EM grid preparation and data collection were performed at the UC San Diego Cryo-EM Facility. Vitrification was carried out using a Vitrobot Mark IV (FEI) equilibrated to 4 °C and 100% humidity. Quantifoil Cu 1.2/1.3 300 grids were plasma cleaned using a Solarus II (Gatan) with a mixture of Ar/O2 for 10 s. Following plasma cleaning, the grid was blotted on one side with 3.5 µl of the SUDV GPΔmuc-WL2P4/CA45 Fab complex at a concentration of 0.55 mg/ml for 4 s, and frozen in liquid ethane. The frozen grids were stored in liquid nitrogen until imaging. Cryo-EM data were collected on a Titan Krios G4 (Thermo Fisher) operated at 200 keV, equipped with a Falcon 4 camera and a Selectris-X energy filter (Thermo Fisher). Automated data collection was performed using Smart EPU software (Thermo Fisher). Images were acquired under 130,000× magnification, corresponding to a pixel size of 0.89 Å, with a defocus range of -1 to -2.5 μm. All datasets were processed using CryoSPARC version 3.3. Movie frames were aligned using Patch Motion Correction, and CTF estimation was performed with Patch CTF Estimation (Fig. S2d). Initial particle picking was done with Blob Picker with the particle size set to 100–350 Å, and particles were extracted using a box size of 360 pixels. After 2D classification, selected particle classes were used as input for Template Picking and re-extracted with a box size of 360 pixels. After multiple rounds of 2D classification, 249,139 particles were selected for 3D reconstruction. Three starting reference models were generated using Ab-Initio Reconstruction and used for Heterogeneous Refinement. Particles in the largest cluster (163,924 particles) were refined using Homogeneous Refinement with a focusing mask and C3 symmetry. The refined model was further subjected to 3D Classification, resulting in 10 models. The class associated with the most complete model (41,233 particles) was subjected to another round of Ab-Initio Reconstruction. This intermediate model was further optimized using Non-uniform Refinement and Local Refinement, both using 163,924 particles and a focusing mask. The final GSFSC resolution was 3.13 Å, and the final structural model was built using the crystal structures of EBOV GPΔmuc-WL2P2 (PDB ID: 7JPH)100 and EBOV GPΔmuc-bound CA45 Fab (PDB ID: 6EAY)118 as templates. The structures were fitted into the density map using ChimeraX174–176, followed by real-space refinement in Phenix169, manual model correction in Coot168, and a final round of real-space refinement in Phenix169. Statistics for the cryo-EM reconstruction and model refinement are provided in Table S2. The coordinates and 3D density map have been deposited in Protein Data Bank (PDB) and Electron Microscopy Data Bank (EMDB), respectively.

Rational design of trimer-presenting I3-01v9b/c SApNP scaffolds
We previously redesigned the I3-01v9 scaffold for optimal display of monomeric antigens127. In the resulting I3-01v9a SApNP, the N-terminal helix was extended such that its first residue was positioned just above the NP surface. Here, we further redesigned I3-01v9a to optimize NP display of trimeric antigens with a long coiled-coil stalk (Fig. S3). First, the 11-aa N-terminal helix in I3-01v9a was truncated to 7 aa. Then, a 13-aa helix-turn fragment (9-aa helix + 4-aa turn, all alanine) was fused to this truncated N-terminus. The newly added 9-residue helix was designed to pack within the groove formed by two helices in the I3-01 core. This 9-residue helix was derived from the structure of a human transcription factor (PDB ID: 6G6L). To accommodate this new helix, four mutations were introduced into the I3-01 core helices to remove steric clashes. The helix-turn backbone was first relaxed using iterative modular optimization (IMO)177, followed by structural sampling with the CONCOORD program178 to generate 1000 slightly perturbed backbone conformations. An ensemble-based protein design protocol87,153 was then used to identify the optimal amino acid sequence for the 9-residue N-terminal helix based on Cα and Cβ-based RAPDF179 scoring functions. The 4-residue turn was fixed as “GSGS” during design. The final I3-01v9b design was selected by combining the results from both scoring functions. A second design, I3-01v9c, was derived by mutating the turn sequence from “GSGS” to “GPPS” to increase rigidity. The I3-01v9b structural model was further relaxed using IMO with a minimum backbone perturbation angle. The distance between the N-termini of the I3-01v9b/c trimer model (I3-01v9b/c-T) was 43.9 Å. EBOV GPΔmuc-WL2P4-I3-01v9b/c-T constructs (Fig. S3b) were designed, transiently expressed in HEK293F cells, and structurally validated by nsEM.

Dynamic light scattering (DLS)
Particle size distributions of orthoebolavirus GPΔmuc-presenting SApNPs were measured using a Zetasizer Ultra instrument (Malvern). Briefly, ADI-15878/SEC-purified SApNPs produced from ExpiCHO cells were diluted to 0.2 mg/ml in 1× PBS, and 30 μl of each sample was added to a quartz batch cuvette (Malvern, catalog no. ZEN2112). Measurements were performed at 25 °C in backscatter mode. Data processing was carried out using the Zetasizer particle size analyzer with the Ultra instrument software. The resulting particle size distributions were plotted using GraphPad Prism version 10.3.1.

Mouse immunization and sample collection
Mouse immunization protocols were adapted from our previous studies113,127,129. All animal experiments were conducted in accordance with the guidelines of the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC) and approved by the Institutional Animal Care and Use Committee (IACUC) of The Scripps Research Institute. Female and male BALB/c mice, aged 6–8 weeks, were purchased from The Jackson Laboratory and housed in ventilated cages in environmentally controlled rooms at 20 °C with 50% humidity, under a 12 h/12 h light-dark cycle at an animal biosafety level 2 facility. For mechanistic studies of vaccine transport and immune responses in lymph nodes, mice were intradermally inoculated via four footpads using a 29-gauge insulin needle under 3% isoflurane anesthesia with oxygen. The injection dose was 80 μl of antigen and AP adjuvant mix, containing 40 μg of vaccine antigen per mouse or 10 μg per footpad. In the first immunogenicity study, mice were injected via the intraperitoneal (i.p.) route at weeks 0, 3, 9, and 15. A 6-week interval was maintained between the second and third, and the third and fourth doses. In the second immunogenicity study, our standard 4-dose screening regimen113,127,129,159 was used, with mice injected at weeks 0, 3, 6, and 9 at 3-week intervals. Each injection consisted of 200 μl of antigen/adjuvant mix containing 10 μg of vaccine antigen and 100 μl of adjuvant, including 40 µg/50 µl of CpG (oligonucleotide 1826, a TLR9 agonist from InvivoGen) adsorbed onto 50 µl of AH (InvivoGen). In the sex-dependent antibody response study, mice were injected intradermally via the footpads at weeks 0, 3, 6, and 9, with 3-week intervals between doses. Each mouse received 80 μl of an antigen/AH adjuvant mixture containing 10 μg of immunogen and 40 μl of adjuvant. Blood samples were collected 2 weeks after each injection through the retro-orbital sinus using heparinized capillary tubes. Samples were centrifuged at 13,200 × g for 10 min to separate serum (top layer) and the rest of the whole blood layer. Upon heat inactivation at 56 °C for 30 min, serum was centrifuged again at 13,200 × g for 10 min to remove precipitates. For the last timepoint, the rest of the whole blood layer was diluted with an equal volume of PBS and then overlaid on 4.5 ml of Ficoll in a 15 ml SepMate tube (STEMCELL Technologies) and centrifuged at 300 × g for 10 min at 20 °C to separate peripheral blood mononuclear cells (PBMCs). Cells were washed once in PBS and then resuspended in 1 ml of ACK red blood cell lysis buffer (Lonza). After washing with PBS, PBMCs were resuspended in 1 ml of freezing media (10% dimethyl sulfoxide [DMSO]/90% fetal calf serum [FCS]). Two weeks after the last bleed, spleens were harvested and ground against a 70-μm cell strainer (BD Falcon) to release splenocytes into a cell suspension. Splenocytes were centrifuged, washed in PBS, treated with 5 ml of ACK lysing buffer (Lonza), and frozen with 3 ml of freezing media (10% DMSO/90% FCS). Serum samples from week 11 in the second study were purified using a CaptureSelect™ IgG-Fc (Multispecies) Affinity Matrix (Thermo Scientific) following the manufacturer’s instructions. Purified IgG from individual mice was analyzed in filovirus-pp assays to assess vaccine-induced NAb responses.

Histology, immunostaining, and imaging for lymph node tissues
To study vaccine distribution, trafficking, retention, cellular interaction, and GC reactions in sentinel lymph nodes, SUDV GPΔmuc trimer and SApNPs (E2p and I3-01v9b) adjuvanted with AP were intradermally injected into four footpads of mice. A similar protocol was described in our previous studies113,127,129. Briefly, each injection consisted of 80 μl of antigen/adjuvant mix containing 40 μg of filovirus antigen (10 μg/footpad; 40 μg/mouse). Mice were euthanized at timepoints ranging from 2 h to 8 weeks after a single-dose injection. Fresh sentinel lymph nodes were collected and embedded in frozen section compound (VWR International, catalog no. 95057-838). Sample molds were immersed in liquid nitrogen and stored at -80 °C before shipping to The Centre for Phenogenomics (Toronto, Canada). Lymph node tissues were sectioned at 8 μm thickness using a cryostat (Cryostar NX70). Primary antibodies were applied to lymph node sections and incubated overnight at 4 °C. After washing with TBS containing 0.1% Tween-20 (TBST), biotin- or fluorophore-conjugated secondary antibodies were added, and sections were incubated at 25 °C for 1 h. Lymph node sections were stained with NAbs ADI-15878116, ADI-15946119, CA45118, or mAb100134 (1:100), followed by biotinylated goat anti-human secondary antibody (Abcam, catalog no. ab7152, 1:30), streptavidin-horseradish peroxidase (HRP) reagent (Vectastain Elite ABC-HRP Kit, Vector, catalog no. PK-6100), and ImmPACT diaminobenzidine (DAB; Vector, catalog no. SK-4105).
To study the interaction between SUDV GPΔmuc immunogens with lymph node cell populations, FDCs were labeled using anti-CD21 primary antibody (Abcam, catalog no. ab75985, 1:1800), followed by anti-rabbit secondary antibody conjugated with Alexa Fluor 555 (Thermo Fisher, catalog no. A21428, 1:200). FDCs were also co-stained using anti-FDC-M1 primary antibody (BD Biosciences, catalog no. 551320, 1:20), followed by anti-rat secondary antibody conjugated with Alexa Fluor 488 (Abcam, catalog no. ab150165, 1:200). Subcapsular sinus macrophages were labeled with anti-sialoadhesin (CD169) antibody (Abcam, catalog no. ab53443, 1:600), followed by anti-rat secondary antibody conjugated with Alexa Fluor 488 (Abcam, catalog no. ab150165, 1:200). B cells were labeled with anti-B220 antibody (eBioscience, catalog no. 14-0452-82, 1:100), followed by anti-rat secondary antibody conjugated with Alexa Fluor 647 (Thermo Fisher, catalog no. A21247, 1:200). GC responses were assessed by immunohistology. GC B cells were stained with FITC-conjugated rat anti-GL7 antibody (BioLegend, catalog no. 144604, 1:250). Tfh cells were stained with anti-CD4 antibody (BioLegend, catalog no. 100402, 1:100), followed by anti-rat secondary antibody conjugated with Alexa Fluor 488 (Abcam, catalog no. ab150165, 1:1000). GC cells were stained with Bcl6 antibody (Abcam, catalog no. ab220092, 1:300), followed by anti-rabbit secondary antibody conjugated with Alexa Fluor 555 (Thermo Fisher, catalog no. A21428, 1:1000). Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, catalog no. D9542, 100 ng/ml).
Stained lymph node tissues were scanned using an Olympus VS-120 slide scanner equipped with a Hamamatsu ORCA-R2 C10600 digital camera or an Akoya Vectra Polaris imaging system with a 20×/0.45 NA objective. The transport of SUDV GPΔmuc trimer and SApNP immunogens, as well as the GCs they induced in lymph nodes, were quantified from bright-field and fluorescent images using ImageJ software180.

Electron microscopy analysis of protein nanoparticles in lymph node tissues
To assess interactions between SUDV GPΔmuc immunogens and stromal and immune cells in lymph nodes, TEM analysis was performed by the Core Microscopy Facility at The Scripps Research Institute (TSRI). The protocol for lymph node isolation, processing, and analysis followed our previous studies113,129. To visualize AP-adjuvanted SUDV GPΔmuc SApNPs (E2p and I3-01v9b) interacting with FDCs and phagocytic cells, mice were injected with 200 μl of antigen/adjuvant mix containing 100 μg of SUDV immunogen and 100 μl of AP adjuvant, administered into two hind footpads (50 μg/footpad). Fresh popliteal lymph nodes were collected at 2, 12, and 48 h after a single-dose injection. Lymph node tissues were bisected and immersed in oxygenated fixative containing 2.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) overnight at 4 °C. Lymph node samples were then fixed and stained with 0.5% uranyl acetate overnight at 4 °C, infiltrated with LX-112 (Ladd) epoxy resin, and polymerized at 60 °C. Ultrathin tissue sections (70 nm) were cut and mounted on copper grids for imaging. TEM was performed at 80 kV using a Talos L120C transmission electron microscope (Thermo Fisher), and images were acquired with a CETA 16M CMOS camera for further analysis.

Lymph node disaggregation, cell staining, and flow cytometry
To assess vaccine-induced humoral immune responses, the frequency and number of GC B cells (GL7+B220+) and Tfh cells (CD3+CD4+CXCR5+PD-1+) were measured by flow cytometry to quantify GC reactions (Fig. S9). The protocol for lymph node tissue collection, processing, and analysis followed our previous studies113,127,129. Briefly, mice were administered with 80 μl of antigen/adjuvant mix containing 40 μg of vaccine immunogen (10 μg/injection, 40 μg/mouse) into four footpads. Mice were euthanized at 2, 5, and 8 weeks after a single-dose injection, or at 2 and 5 weeks after the boost. Fresh sentinel lymph nodes were isolated and immediately processed by mechanical disaggregation. Lymph node tissues were then incubated in enzyme digestion solution at 37 °C for 30 min on a rotator, with the resulting cells filtered through a 70 μm cell strainer to obtain a single-cell suspension. Anti-CD16/32 antibody (BioLegend, catalog no. 101302, 1:50) was added to the cell samples to block the nonspecific binding of Fc receptors and the samples were kept on ice for 30 min. Next, cells were transferred to 96-well V-shaped-bottom microplates with a cocktail of antibodies and stains, including: Zombie NIR live/dead stain (BioLegend, catalog no. 423106, 1:100), Brilliant Violet 510 anti-mouse/human CD45R/B220 antibody (BioLegend, catalog no. 103247, 1:300), FITC anti-mouse CD3 antibody (BioLegend, catalog no. 100204, 1:300), Alexa Fluor 700 anti-mouse CD4 antibody (BioLegend, catalog no. 100536, 1:300), PE anti-mouse/human GL7 antibody (BioLegend, catalog no. 144608, 1:500), Brilliant Violet 605 anti-mouse CD95 (Fas) antibody (BioLegend, catalog no. 152612, 1:500), Brilliant Violet 421 anti-mouse CD185 (CXCR5) antibody (BioLegend, catalog no. 145511, 1:500), and PE/Cyanine7 anti-mouse CD279 (PD-1) antibody (BioLegend, catalog no. 135216, 1:500). Cells were incubated with the antibody cocktail on ice for 30 min and centrifuged at 400 × g for 10 min. They were then fixed with 1.6% paraformaldehyde (Thermo Fisher Scientific, catalog no. 28906) in Hank’s Balanced Salt Solution (HBSS) on ice for 30 min, centrifuged again at 400 × g for 10 min, resuspended in HBSS blocking solution at 4 °C. Sample acquisition was performed on a 5-laser AZE5 flow cytometer (Yeti, Bio-Rad) with Everest software at the Core Facility of The Scripps Research Institute. Data analysis was conducted with FlowJo version 10.

Pseudovirus production and neutralization assays
A filovirus-pseudoparticle (pp) neutralization assay100 was performed to assess the neutralizing activity of vaccine-induced mouse sera and purified mouse IgG. Filovirus-pps were generated by co-transfecting HEK293T cells with the envelope-deficient HIV-1 pNL4-3.lucR−E− plasmid (NIH AIDS Reagent Program: https://www.aidsreagent.org/) and an expression plasmid encoding the GP gene from EBOV Makona (GenBank accession no. KJ660346), SUDV Gulu (GenBank accession no. AY729654), BDBV Uganda (GenBank accession no. KR063673), or MARV Angola (GenBank accession no. DQ447653) at a 4:1 ratio using the Lipofectamine 3000 transfection protocol (Thermo Fisher). After 72 h, pseudoviruses were harvested from the supernatant by centrifugation at 3,724 × g for 10 min, aliquoted, and stored at -80 °C until use. For pseudovirus neutralization assays, mouse sera (starting dilution 1:100) or purified IgG (starting at 300 μg/ml) were serially diluted 3-fold and incubated with pseudoviruses at 37 °C for 1 h in white solid-bottom 96-half-well plates (Corning). Then, 0.8 × 104 Huh-7 cells were added to each well, and the plates were incubated at 37 °C for 60-72 h. After incubation, the supernatant was removed, and cells were lysed. Luciferase reporter gene expression was quantified by adding Bright-Glo Luciferase substrate (Promega) according to the manufacturer’s instructions. Luciferase activity (in relative light units, RLU) was measured on a BioTek microplate reader using Gen5 software. Values from experimental wells were compared to virus-only wells, with background luminescence from a series of uninfected wells subtracted from both. Dose-response neutralization curves were fit by nonlinear regression (0-100% constraints) in GraphPad Prism version 10.3.1, from which IC50 and ID50 were calculated. The same protocols were used to produce and assay pseudoparticles displaying the envelope glycoprotein of lymphocytic choriomeningitis virus (LCMV-pps) as a negative control for IgG neutralization.

Statistical analysis
Data were collected from n = 8 mice per group in the immunization studies and n = 3-5 mice per group in the mechanistic study. All ELISA and pseudovirus neutralization assays were performed in duplicate. Different vaccine constructs and adjuvant-formulated filovirus vaccine antigens were compared using one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison post hoc test. Two-tailed unpaired t-tests were used to compare two independent vaccine groups. Statistical significance is indicated in the figures as follows: ns (not significant), *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Graphs were generated and statistical analyses were performed using GraphPad Prism version 10.3.1.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Filoviruses, notably Ebola and Marburg viruses, continue to challenge global health systems with frequent, high mortality outbreaks. While two vector based vaccines exist for Ebola, a pan filovirus vaccine capable of protecting against diverse viral is needed. The current manuscript reports a comprehensive strategy that combines structural biology and in silico protein nanoparticle engineering to address this unmet gap. Specifically, the authors describe the stabilization of glycoprotein (GP) trimers from Ebola, Sudan, Bundibugyo ebolaviruses, and Ravn marburgvirus, supported by a 3.2 Å crystal structure of redesigned Ebola GP and cryo EM models of GP–antibody complexes. They further engineer a self assembling protein nanoparticle (SApNP) scaffold to display these stabilized GPs, demonstrating markedly improved lymph node retention, follicular dendritic cell presentation, and germinal center responses in a murine model. Finally, the study evaluates the breadth and potency of antibody responses elicited by both soluble GP trimers and GP displaying nanoparticles. These findings present a promising foundation for next generation, broadly protective filovirus vaccines. While this is generally a comprehensive study, several issues exist.\nMajor:\n1 - A key concern is the poor quality and pronounced anisotropy of the reported SUDV GP-CA45 mAb cryo EM map, which substantially undermines confidence in the atomic model’s side chain placements. Because the EM volume quality varies directionally, interpreting precise amino acid interactions at the antibody–GP interface becomes highly speculative. This uncertainty calls into question the validity of mechanistic conclusions drawn from those modeled contacts.\n2- A second significant issue lies in the identification of follicular dendritic cells (FDCs). In immunostaining experiments, the authors rely on anti-CD21 antibodies to mark FDCs, but CD21 is also expressed on B cells, confounding interpretation of antigen retention on true FDC networks. Use of CD35, which is more specific to FDCs, would have been more appropriate.\n3 – The authors report size-exclusion chromatography peaks that indicate aggregated GP trimers. Could the aggregation have been caused by acid elution from the affinity resin?\nMinor:\n1 – The SEC profiles for the third set of trimers (SUDV) seem to have been truncated, as the baseline UV traces are not visible.\n2 – Should the Y axes in Fig. 9n say ED50, as the concentration is not known, only the dilution?", "reviewer_2": "Lee, Zhang and co-workers evaluate a general strategy for stabilizing glycoprotein structures of filoviruses. They then placed these constructs on self-assembling protein nanoparticle (SApNP). Lymph node dynamics of the SApNPs were investigated in a mouse model and showed longer retention in lymph node follicles, greater presentation on follicular dendritic cell dendrites, and stronger germinal center reactions. Functional antibody responses induced by filovirus GP trimers and SApNPs bearing wildtype and modified glycans are assessed in mice. Our study provides a foundation for next-generation filovirus vaccine development. The manuscript is well written and presented. The results are important for moving forward a promising vaccine platform.\nComments:\n1. Line 48. The current ICTV Taxonomy lists 9 genuses in the Filoviridae. They have also changed the names to orthoebolavirus and orthomarburgvirus. The authors may wish to consider using the new genus names throughout. I don’t think that the new species names should be used.\n2. Line 91. The Moderna and Pfizer SARS-CpV-2b mRNA vaccines also express stabilized constructs. It seems like that should also be mentioned for completeness.\n3. Line 128 – sentence is not clear.\n4. Line 335. It might be helpful to define Kifunensine, as an inhibitor of class I α-mannosidases that prevents α-mannosidases I from trimming mannose residues on glycoproteins, thus resulting in oligomannose-type glycans.\n5. Paragraphs at 441 and 459 – the hypotheses are not clearly stated. I would rewrite these paragraphs to not be test of hypotheses or explicitly state what the hypotheses are.\n6. Line 758 – would rewrite this to say that future studies are needed to determine whether improved in vitro antigenicity correlate with improved in vivo immunogenicity as well as protection from disease. Might be better to move this sentence down to the paragraph at line 784.\n7. Line 778. The term “Kif-treated, oligomannose-rich immunogens” doesn’t seem accurate [I get the jargon]. The cells used to produce the immunogen were Kif-treated.\n8. Line 782 – Consider another word besides “discrepancy.” I would mention these results in the abstract. It’s important to point out that” there may not be a general glycan strategy that fits all viral GP vaccines.”", "reviewer_3": "This manuscript presents a promising strategy for developing next-generation filovirus vaccines by stabilizing GP glycoprotein structures, optimizing their display on self-assembling nanoparticles, and exploring the effects of glycan modifications. The study includes high-resolution structural analyses of GPs from multiple filoviruses, though these structural data are not fully integrated into the immunogen design process. In addition, the authors investigate and compare the lymph node dynamics of GP antigens in a mouse model, providing insights into how antigen presentation differs between soluble and nanoparticle-displayed formats. This multi-faceted approach offers valuable data for vaccine development. However, several aspects require clarification or further validation, particularly regarding the rationale connecting structural and immunological components, methodological choices in antigenicity assessment, and the interpretation of immunization results. The manuscript would benefit from clearer explanations, stronger statistical support, and more direct evidence to support some of the conclusions.\nDetailed Comments:\nMajor concerns:\n1. The manuscript asserts that structural data supported immunogen design, but it does not convincingly establish this connection. If the structures did not directly inform the design of vaccine constructs, then separating the structural and vaccine design components into two focused manuscripts should be considered. This would help sharpen the manuscript’s message and clarify the individual contributions of each part.\n2. Although the study provides data on antigen retention and germinal center responses, the comparison of functional antibody responses—particularly neutralizing and cross-neutralizing antibody titers—between different vaccine constructs is not clearly presented or discussed. This makes it difficult to determine whether nanoparticle display confers an actual benefit in protective immune responses beyond improved antigen presentation.\n3. Relatedly, while the nanoparticle-based vaccines elicit stronger markers of immune activation, such as germinal center formation and lymph node retention, this enhancement does not appear to translate into consistently stronger neutralization. This disconnect should be explicitly addressed in the discussion, as it raises questions about the practical advantage of nanoparticle presentation in the absence of clear improvements in functional antibody responses.\nMinor concerns:\n1.The cryo-EM reported appear technically sound, but the structure validation would benefit from the inclusion of correlation coefficients and EMRinger scores in the refinement statistics. These are widely used metrics that improve the transparency and reliability of structural assessments and would strengthen the claims about model quality.\n2.The redesign of the I3-01v9a nanoparticle to optimize its N-terminal geometry for GP stalk compatibility is a notable feature, but the manuscript does not present comparative data to demonstrate the necessity or functional benefit of this modification. Including a comparison of structural or biochemical outcomes before and after the redesign would better support the rationale for this engineering effort.\n3.Several RMSD-based structural comparisons presented in the results rely on a limited subset of atoms, which may not reflect the full extent of structural similarity or divergence. Broader atom selections, such as inclusion of entire domains or full protomers, would yield more robust comparisons and improve the reliability of the conclusions drawn from these analyses.\n4.The rationale behind the use of different immunization intervals for the initial versus glycan-modified groups is not explained. This difference in scheduling could influence immune outcomes and complicates interpretation of comparative results, so it is important that the authors clarify their reasoning and address how this may affect their conclusions.\n5.The use of IgG-format antibodies in BLI assays to assess GP antigenicity is not ideal due to avidity effects, which obscure true affinity differences and reduce the assay’s sensitivity to detect variation between constructs. The use of Fab fragments would provide a more accurate 1:1 binding model and allow for a clearer comparison of antigen binding properties between trimeric and nanoparticle-displayed immunogens.\n6.The data on RAVV GP trimer immunization lack statistical support for the claim that the wildtype trimer is more effective than the glycan-trimmed versions. Additionally, the conclusion that RAVV GP is “more immunogenic” or holds promise as a pan-filovirus immunogen is currently speculative and should be supported with comparative data or a more cautious interpretation."}, "author_response": "Response to Reviewer Comments\nReviewer #1 (Remarks to the Author):\nGeneral comments: Filoviruses, notably Ebola and Marburg viruses, continue to challenge global health\nsystems with frequent, high mortality outbreaks. While two vector based vaccines exist for Ebola, a pan\nfilovirus vaccine capable of protecting against diverse viral is needed. The current manuscript reports a\ncomprehensive strategy that combines structural biology and in silico protein nanoparticle engineering to\naddress this unmet gap. Specifically, the authors describe the stabilization of glycoprotein (GP) trimers\nfrom Ebola, Sudan, Bundibugyo ebolaviruses, and Ravn marburgvirus, supported by a 3.2 Å\ncrystal structure of redesigned Ebola GP and cryo EM models of GP–antibody complexes. They further engineer\na self-assembling protein nanoparticle (SApNP) scaffold to display these stabilized GPs, demonstrating\nmarkedly improved lymph node retention, follicular dendritic cell presentation, and germinal center\nresponses in a murine model. Finally, the study evaluates the breadth and potency of antibody responses\nelicited by both soluble GP trimers and GP displaying nanoparticles. These findings present a promising\nfoundation for next generation, broadly protective filovirus vaccines.\nWhile this is generally a comprehensive study, several issues exist.\nResponse: We thank Reviewer #1 for their thoughtful and constructive evaluation of our manuscript. We\nappreciate the reviewer’s recognition of the significance and timeliness of our work toward developing a\nbroadly protective filovirus vaccine, and for highlighting the strengths of our multidisciplinary approach\nthat integrates structural biology, nanoparticle engineering, and immunological assessment. We are grateful\nfor the insightful comments, which have helped us further refine the manuscript and clarify key aspects of\nour methodology and conclusions. In the revised version, we have included additional immunohistology\nand animal data. We have also made edits throughout the manuscript to improve clarity and flow. A detailed\npoint-by-point response shown below, with the changes highlighted in the main text.\nMajor:\n1. A key concern is the poor quality and pronounced anisotropy of the reported SUDV GP-CA45 mAb\ncryo EM map, which substantially undermines confidence in the atomic model’s side chain placements.\nBecause the EM volume quality varies directionally, interpreting precise amino acid interactions at the\nantibody–GP interface becomes highly speculative. This uncertainty calls into question the validity of\nmechanistic conclusions drawn from those modeled contacts.\nResponse: We thank the reviewer for this important observation. We fully acknowledge that anisotropy\nrepresents a limitation in our cryo-EM analysis of the SUDV GP–CA45 Fab complex. Throughout this\nproject, we exercised considerable care during model building and refinement to ensure that only regions\nsupported by high-quality density were retained in the final atomic model. Specifically, we removed regions\nin GP1 (A187–L218), GP2 (E522–N525), and the C-terminal stalk (P612–N637), even though initial\nbackbone traces had been built, because they did not meet our predefined criteria for density continuity and\ninterpretability. To further address the reviewer’s concern, we compiled high-resolution snapshots of the\ncryo-EM density at the GP–CA45 interface, presented fragment by fragment, to illustrate side-chain fits\nthat support the modeled interactions (see Fig. S2i and S2j). These images demonstrate that key interface\nresidues are supported by well-defined density. Additionally, we compared our SUDV GP–CA45 structure\nto several published cryo-EM structures of EBOV and BDBV GPs in complex with broadly neutralizing\nantibodies (bNAbs), including EBOV GP with ADI-15878 (EMD-8935), EBOV GP with EBOV-515\n(EMD-23719), and EBOV GP with EBOV-520 (EMD-20301). As shown in Fig. S2k-S2m, the density\nquality and model interpretability of our structure are comparable to those peer-reviewed examples. We are\nhappy to provide the full cryo-EM map upon request for independent assessment. Finally, we have revised\nthe manuscript to describe how we addressed anisotropy in the density at the GP–CA45 interface in our\nstructural modeling and interpretation (see page 11, lines 244-245 and page 12-13, lines 267-273).\n1\n2. A second significant issue lies in the identification of follicular dendritic cells (FDCs). In\nimmunostaining experiments, the authors rely on anti-CD21 antibodies to mark FDCs, but CD21 is\nalso expressed on B cells, confounding interpretation of antigen retention on true FDC networks.\nUse of CD35, which is more specific to FDCs, would have been more appropriate.\nResponse: We thank the reviewer for this important comment regarding FDC identification. FDCs are\nknown to express high levels of complement receptor 1 (CR1; CD35) and complement receptor 2 (CR2;\nCD21). While both CD21 and CD35 can be used as FDC markers, they are also expressed on B cells and,\nto a lesser extent, on other immune cells such as mast cells, macrophages, and activated granulocytes. To\naddress this concern, we performed an additional mouse immunization followed by immunohistological\nanalysis using the FDC-specific marker FDC-M1 (Clone FDC-M1, BD Biosciences, Cat. #551320). The\nresults confirmed that SUDV I3-01v9b SApNPs colocalized with FDC networks labeled by both CD21⁺\nand FDC-M1⁺ signals at 48 hours post-injection, supporting our original interpretation. These new data\nhave been incorporated into the revised manuscript (see page 22, lines 484–488, and Fig. S6h). We also\ntested an anti-CD35 antibody (Clone 8C12, Abcam, Cat. #ab245683); however, this antibody did not yield\nusable staining under our experimental conditions.\n3. The authors report size-exclusion chromatography peaks that indicate aggregated GP trimers.\nCould\nthe aggregation have been caused by acid elution from the affinity resin?\nResponse: We thank the reviewer for this thoughtful suggestion. In our previous study of stabilized RSV\nfusion (F) constructs [Lee et al., Nat Commun. 15, 9939 (2024)], we observed that acid elution during\naffinity purification can contribute to protein aggregation. However, in the current study, we found that\ntrimers purified using Nickel affinity columns showed even greater levels of aggregation compared to those\npurified with the ADI-15878 antibody column (Fig. S1b). This suggests that the aggregation observed after\nADI-15878 purification is unlikely to be due to acid elution alone. Notably, ADI-15878 affinity purification\nsubstantially improves the purity of the trimer preparation.\nMinor:\n1. The SEC profiles for the third set of trimers (SUDV) seem to have been truncated, as the baseline UV\ntraces are not visible.\nResponse: We thank the reviewer for this comment. We would like to clarify that the third panel of SEC\nprofiles in Fig. 1b corresponds to BDBV R4368L GPΔmuc-WL2P2 and WL2P4, not the SUDV trimers. All\nsix trimers shown in Fig. 1b have full baseline traces visible in the high-resolution images. The SEC profiles\npresented reflect raw chromatographic data obtained directly from the instrument. Baseline variation can\noccur between runs due to sample-specific factors and instrument sensitivity. In some cases, the baseline\nmay fall slightly below the plotted y-axis range, but the full UV trace was captured and analyzed and will\nbe provided in the Source Data file accompanying the publication.\n2. Should the Y axes in Fig. 9n say ED50, as the concentration is not known, only the dilution?\nResponse: We thank the reviewer for this thoughtful question. In the context of serum binding assays, EC\n50\n(half-maximal effective concentration) is the more appropriate term than ED (median effective dose).\n50\nthe serum dilution at which 50% of the maximal binding to antigen is observed. In our ELISA experiments,\nwe serially diluted serum samples and measured their binding to the antigen. The resulting titration curves\nwere used to calculate EC titers based on half-maximal signal. By contrast, ED is typically used in\n50 50\npharmacological or in vivo efficacy studies to indicate the dose required to produce a defined effect in 50%\nof a test population. Therefore, we have retained the EC label in Fig. 9n. The use of the term “EC titer”\n50 50\nshould make it clear to readers that it represents a dilution-based measurement—analogous to how\n“neutralizing titer” refers to a dilution, not an absolute concentration, in vaccine studies like this one.\nGeneral: Lee, Zhang and co-workers evaluate a general strategy for stabilizing glycoprotein structures of\nfiloviruses. They then placed these constructs on self-assembling protein nanoparticle (SApNP). Lymph\nnode dynamics of the SApNPs were investigated in a mouse model and showed longer retention in lymph\nnode follicles, greater presentation on follicular dendritic cell dendrites, and stronger germinal center\nreactions. Functional antibody responses induced by filovirus GP trimers and SApNPs bearing wildtype\nand modified glycans are assessed in mice. Their study provides a foundation for next-generation filovirus\nvaccine development. The manuscript is well written and presented. The results are important for moving\nforward a promising vaccine platform.\nResponse: We thank the reviewer for their careful evaluation and positive comments on the design and\npresentation of our study. We appreciate the recognition of our integrated approach combining structural\nstabilization, nanoparticle display, and immunological analysis and its potential to advance next-generation\nfilovirus vaccine development. The reviewer’s feedback was valuable in guiding our revisions. In the\nrevised manuscript, we have included new experimental data from immunohistological analysis and a\nmouse immunization study addressing sex as a biological variable. We have also made edits throughout the\nmanuscript to improve clarity and flow. All modifications are highlighted in the revised manuscript.\n1. Line 48. The current ICTV Taxonomy lists 9 genuses in the Filoviridae. They have also changed the\nnames to orthoebolavirus and orthomarburgvirus. The authors may wish to consider using the new\ngenus names throughout. I don’t think that the new species names should be used.\nResponse: We thank the reviewer for this helpful comment. In accordance with the latest ICTV taxonomy,\nwe have revised the manuscript to state that the family Filoviridae comprises nine recognized genera. We\nhave updated the genus names to Orthoebolavirus and Orthomarburgvirus and now refer to EBOV, SUDV,\nBDBV, and RAVV as members of these respective genera throughout the manuscript. We have retained\ncommonly used species names to ensure clarity for readers.\n2. Line 91. The Moderna and Pfizer SARS-CpV-2b mRNA vaccines also express stabilized constructs. It\nseems like that should also be mentioned for completeness.\nResponse: To provide a more complete context, we have included two references highlighting the use of\nprefusion-stabilized SARS-CoV-2 spike constructs in mRNA vaccines. Please see page 5, lines 92–94.\n3. Line 128 – sentence is not clear.\nResponse: We thank the reviewer for pointing this out. The sentence has been revised for clarity as follows:\n“While the EBOV and SUDV GPΔmuc-WL2P4 trimer and SApNP vaccines elicited serum neutralization\nlimited to orthoebolaviruses, the RAVV GPΔmuc-P2CT trimer induced detectable cross-genus NAb\nresponses.” Please see page 6, lines 128-130.\n4. Line 335. It might be helpful to define Kifunensine, as an inhibitor of class I α-mannosidases that\nprevents α-mannosidases I from trimming mannose residues on glycoproteins, thus resulting in\noligomannose-type glycans.\nResponse: As the reviewer suggested, we have added a definition of Kifunensine at its first mention in the\ntext. The revised sentence is as follows: “Supplementing ExpiCHO cultures with kifunensine (Kif) — a\nclass I α-mannosidase inhibitor that blocks ER glycan processing and leads to accumulation of high\nmannose — did not affect neutralizing antibody (NAb) responses.” Please see page 15, lines 337-339.\n5. Paragraphs at 441 and 459 – the hypotheses are not clearly stated. I would rewrite these paragraphs\nto not be test of hypotheses or explicitly state what the hypotheses are.\nResponse: We thank the reviewer for this suggestion. We have revised the relevant paragraphs to remove\nexplicit references to a hypothesis and instead directly describe the experiments and the immunological\n3\nparameters being investigated. These changes clarify the intent of the experiments without framing them as\nformal hypothesis tests. Please see page 20, lines 436–438, as well as paragraphs on pages 21-23.\n6. Line 758 – would rewrite this to say that future studies are needed to determine whether improved in\nvitro antigenicity correlate with improved in vivo immunogenicity as well as protection from disease.\nMight be better to move this sentence down to the paragraph at line 784.\nResponse: We thank the reviewer for this helpful suggestion. As recommended, we removed the original\nsentence from line 758 and incorporated a revised version into the paragraph into the future directions\nsection. After outlining the first objective on antibody epitope mapping for wildtype and glycan-modified\nGP vaccine constructs, we added the following sentence: “Such analyses may explain why improved\nepitope exposure following glycan trimming did not translate into stronger NAb responses in vivo” This\nrevision more clearly underscores the need for future studies to determine whether enhanced in vitro\nantigenicity correlates with in vivo immunogenicity and protection. Please see page 37, lines 821–822.\n7. Line 778. The term “Kif-treated, oligomannose-rich immunogens” doesn’t seem accurate [I get the\njargon]. The cells used to produce the immunogen were Kif-treated.\nResponse: We thank the reviewer for this comment. We revised this sentence and now it reads: while\noligomannose-rich immunogens – produced in the presence of kifunensine (Kif) – induced the strongest\nbAb and NAb responses at most timepoints.” Please see page 36, lines 811-812.\n8. Line 782 – Consider another word besides “discrepancy.” I would mention these results in the abstract.\nIt’s important to point out that” there may not be a general glycan strategy that fits all viral GP vaccines.”\nResponse: We thank the reviewer for this suggestion. We have replaced the word “discrepancy” with\n“differences” and revise the sentence (see page 36, lines 814-816). While we agree with the reviewer that\nthis finding is important, we are unable to include it in the Abstract due to strict 200-word limit. To\nemphasize its significance, we have added a sentence to the final paragraph of the Introduction: “Although\nno universal glycan modification strategy may apply to all viral GPs, enriching the filovirus GP glycan\nshield with oligomannose-type glycans improved vaccine-induced NAb responses in mice.” (see pages 6-\n7, lines 131-133).\nReviewer #3 (Remarks to the Author):\nThis manuscript presents a promising strategy for developing next-generation filovirus vaccines by\nstabilizing GP glycoprotein structures, optimizing their display on self-assembling nanoparticles, and\nexploring the effects of glycan modifications. The study includes high-resolution structural analyses of GPs\nfrom multiple filoviruses, though these structural data are not fully integrated into the immunogen design\nprocess. In addition, the authors investigate and compare the lymph node dynamics of GP antigens in a\nmouse model, providing insights into how antigen presentation differs between soluble and nanoparticle-\ndisplayed formats. This multi-faceted approach offers valuable data for vaccine development. However,\nseveral aspects require clarification or further validation, particularly regarding the rationale connecting\nstructural and immunological components, methodological choices in antigenicity assessment, and the\ninterpretation of immunization results. The manuscript would benefit from clearer explanations, stronger\nstatistical support, and more direct evidence to support some of the conclusions.\nResponse: We thank the reviewer for their thoughtful evaluation and for recognizing the potential of our\nmulti-faceted approach to inform next-generation filovirus vaccine development. We also acknowledge the\nconstructive concerns regarding the integration of structural data into immunogen design, interpretation of\nimmune responses, and presentation of supporting data. In response, we have clarified the rationale linking\nour structural findings to vaccine design, expanded methodological explanations, and strengthened the\ninterpretation of key immunological findings. We hope that these revisions address the reviewer’s concerns\nand help to more clearly convey the rigor of our work. Based on the reviewers’ comments, we have included\n4\nnew data from immunohistological analysis and mouse immunization. We have also made edits throughout\nthe manuscript to improve clarity and flow. All changes are highlighted in the revised manuscript.\nMajor concerns:\n1. The manuscript asserts that structural data supported immunogen design, but it does not\nconvincingly establish this connection. If the structures did not directly inform the design of vaccine\nconstructs, then separating the structural and vaccine design components into two focused\nmanuscripts should be considered. This would help sharpen the manuscript’s message and clarify\nthe individual contributions of each part.\nResponse: We thank the reviewer for this thoughtful comment. Our structural analyses, combined with in\nvitro characterization, form the foundation of our immunogen design strategy. The structural data confirmed\nthat the stabilizing mutations preserved the native-like prefusion conformation of GP and maintained key\nneutralizing epitopes, which is an essential validation step prior to nanoparticle display and immunological\nevaluation. In our previous study [He et al., Nat. Commun. 12, 2633 (2021)], we introduced mutations in\nthe HR1c bend and HR2 stalk of EBOV GP, yielding two lead designs: GPΔmuc-WL2P2 and GPΔmuc-\nWL2P4. While both GP constructs were functional, WL2P2 exhibited a biphasic thermal denaturation profile\nindicative of structural heterogeneity, while WL2P4 showed a more favorable, single-peak DSC profile. In\nthis study, we extended both designs to SUDV and BDBV and found that WL2P4 consistently showed\nsuperior biochemical and biophysical properties across all three orthoebolavirus backbones. To validate the\nstructural fidelity of this design, we determined the crystal structure of EBOV GPΔmuc-WL2P4 and\ncompared it with previously solved WL2 and WL2P2 structures, establishing a clear structural rationale for\nselecting WL2P4 as the optimal platform. We further determined the cryo-EM structure of SUDV GPΔmuc-\nWL2P4 in complex with CA45, confirming the structural integrity of WL2P4 in a different orthoebolavirus\nbackbone and resolving local epitope features at a conserved bNAb site. In summary, the crystallographic\nand cryo-EM data were critical for demonstrating that our rationally designed immunogens adopt native-\nlike conformations and preserve key neutralizing epitopes. Presenting the structural and immunogen design\ncomponents together reflects the iterative nature of the approach and underscores the rationale linking\nstructural validation to downstream immunogenicity. We believe a unified presentation most effectively\nconveys the significance and translational potential of our findings.\n2. Although the study provides data on antigen retention and germinal center responses, the\ncomparison of functional antibody responses—particularly neutralizing and cross-neutralizing\nantibody titers—between different vaccine constructs is not clearly presented or discussed.\nThis\nmakes it difficult to determine whether nanoparticle display confers an actual benefit in protective\nimmune responses beyond improved antigen presentation.\nResponse: We thank the reviewer for this comment. In this study, we examined lymph node trafficking\nand immune responses elicited by SUDV GPΔmuc-WL2P4 in both trimeric and SApNP formats. Compared\nto soluble trimers, SApNPs exhibited prolonged retention in lymph node follicles, greater association with\nfollicular dendritic cells (FDCs), and more robust and sustained germinal center (GC) reactions. To assess\nvaccine-induced functional antibody responses, we measured both binding antibody (bAb) and neutralizing\nantibody (NAb) titers. E2p and I3-01v9b SApNPs consistently elicited higher EC titers at all time points\n50\ncompared to the trimer. For NAb responses, both SApNPs also produced the highest ID titers after two\n50\ndoses, although the differences from the trimer group were not statistically significant. These data suggest\nthat nanoparticle display may accelerate the development of functional antibody responses, consistent with\nenhanced antigen delivery and GC activation. We note that the current immunization schedule was designed\nto provide an initial readout of immunogenicity rather than optimize the response. It is possible that the full\nbenefit of SApNPs will become more apparent with extended dosing intervals, which will be explored in\nfuture studies. Additional discussion can be found in our response to Comment #3.\n3. Relatedly, while the nanoparticle-based vaccines elicit stronger markers of immune activation,\nsuch as germinal center formation and lymph node retention, this enhancement does not appear to\n5\ntranslate into consistently stronger neutralization. This disconnect should be explicitly addressed\nin the discussion, as it raises questions about the practical advantage of nanoparticle presentation\nin the absence of clear improvements in functional antibody responses.\nResponse: We thank the reviewer for this insightful comment. As detailed in the manuscript, multivalent\ndisplay of GPΔmuc trimers on SApNPs enhances antigen retention in lymph node follicles, increases\nassociation with follicular dendritic cells (FDCs), and promotes more robust and durable germinal center\n(GC) reactions compared to soluble trimers. After two doses, SApNPs elicited the highest EC and ID\n50 50\ntiters, consistent with the stronger GC responses observed in the lymph node analysis. However, following\nadditional immunizations, the soluble trimers generated NAb titers comparable to those of the SApNPs,\nlikely reflecting a plateau in the SApNP-induced responses by week 8. These findings suggest that while\nSApNPs accelerate the development of functional antibody responses, repeated dosing can enable soluble\ntrimers to achieve similar outcomes. Thus, a key advantage of nanoparticle vaccines lies in their ability to\ninduce potent responses with fewer doses. As noted in our response to Comment #2, the current regimen\nwas designed to obtain an initial immunogenicity readout rather than optimized for maximal response, and\nextended dosing intervals may further enhance the immunogenicity of SApNPs. Lastly, to support broader\napplicability, we included results from male mouse immunizations alongside previously reported female\ncohorts (see pages 31-32, lines 705–717).\nMinor concerns:\n1. The cryo-EM report appears technically sound, but the structure validation would benefit from the\ninclusion of correlation coefficients and EMRinger scores in the refinement statistics. These are\nwidely used metrics that improve the transparency and reliability of structural assessments and\nwould strengthen the claims about model quality.\nResponse: We thank the reviewer for this helpful suggestion. As recommended, we performed EMRinger\nvalidation for the cryo-EM structure, which yielded a score of 2.60. This value is consistent with good side-\nchain placement and overall model quality. We have included this information in the revised manuscript\n(see page 11, lines 242).\n2. The redesign of the I3-01v9a nanoparticle to optimize its N-terminal geometry for GP stalk\ncompatibility is a notable feature, but the manuscript does not present comparative data to\ndemonstrate the necessity or functional benefit of this modification. Including a comparison of\nstructural or biochemical outcomes before and after the redesign would better support the rationale\nfor this engineering effort.\nResponse: We thank the reviewer for this comment. In our previous study [He et al., Nat. Commun. 12,\n2633 (2021)], we reported I3-01v9 SApNPs displaying EBOV GPΔmuc-WL2P2 trimers, in which a 10GS\nlinker was used to anchor the GPΔmuc trimer to the wider N-termini of the I3-01v9 subunits arranged\naround each threefold axis of the particle surface. The SEC profile of these non-optimized I3-01v9 SApNPs\n(Fig. 4f in that publication), obtained using a Superose 6 Increase column, showed a noticeable peak\ncorresponding to unassembled species. In the current study, the SEC profile of EBOV GPΔmuc-presenting\nI3-01v9b SApNPs, with optimized N-terminal geometry and no flexible linker between the GP and NP\nscaffold, exhibited a reduced trimer peak and higher NP yield compared to the earlier I3-01v9 construct\n(Fig. 3d, left). Moreover, when I3-01v9b was used to present SUDV and BDBV GPΔmuc-WL2P4 trimers,\nwe observed comparable or improved SEC profiles, with negligible amounts of unassembled trimer species\n(Fig. 3d, middle and right). Together, these results demonstrate that the computationally redesigned I3-\n01v9b significantly improves both the biochemical properties and assembly efficiency of the GPΔmuc-\npresenting SApNPs. We have revised the manuscript to clarify the rationale for this redesign in the Results\n(page 13, lines 275-281) and further context in the Discussion section (pages 34-35, lines 770-792) to\nemphasize its broader utility as an NP carrier for diverse viral GPs containing a narrow stalk.\n6\n3. Several RMSD-based structural comparisons presented in the results rely on a limited subset of\natoms, which may not reflect the full extent of structural similarity or divergence. Broader atom\nselections, such as inclusion of entire domains or full protomers, would yield more robust\ncomparisons and improve the reliability of the conclusions drawn from these analyses.\nResponse: We thank the reviewer for this helpful suggestion. We would like to clarify that all resolved\nbackbone Cα atoms in the GPΔmuc protomer—the broader atom selection referenced by the reviewer—\nwere included in the comparison between the crystal structures of EBOV GPΔmuc-WL2P2 and -WL2P4.\nIn\naddition, a focused comparison of the HR1c bend was performed to assess local conformational differences\nin this critical region for GP stabilization (see pages 10-11, lines 224–227). For the cryo-EM structure of\nSUDV GPΔmuc-WL2P4 in complex with CA45 Fab, the map resolution (3.1–3.4 Å) limited confident\nbackbone modeling in certain regions (see our response to reviewer #1, question 1, and Fig. S2f). As a\nresult, the number of Cα atoms included in RMSD-based calculations for the cryo-EM model was smaller\nthan for the crystal structures (154 vs. 243 atoms). Moreover, due to <80% sequence identity among EBOV,\nSUDV, and BDBV GPs, some residues were excluded due to alignment gaps or divergence in non-\nconserved structural regions. The structural comparisons presented in the manuscript were carefully\ndesigned to maximize the inclusion of well-resolved, structurally aligned backbone atoms while avoiding\npotential artifacts from poorly modeled or non-homologous regions. We believe this approach provides a\nrobust and interpretable evaluation of structural similarity and divergence across the filovirus GPs analyzed.\n4. The rationale behind the use of different immunization intervals for the initial versus glycan-\nmodified groups is not explained. This difference in scheduling could influence immune outcomes\nand complicates interpretation of comparative results, so it is important that the authors clarify\ntheir reasoning and address how this may affect their conclusions.\nResponse: We thank the reviewer for this suggestion. The mouse immunization data shown in Figs. 9 and\n10 were generated from two separate studies conducted at different stages of our research, each with its\nown experimental rationale and design. In our first study, we aimed to assess antibody responses induced\nby rationally designed filovirus constructs. The decision to use longer intervals was based on our lymph\nnode analysis, which demonstrated that GPmuc-presenting SApNPs are retained in lymph node follicles\nfor over eight weeks (Fig. 6) and elicit robust, long-lived GC reactions at weeks 5 and 8 after a single-dose\ninjection (Fig. 8). Based on these findings, we adopted an immunization schedule with 6-week intervals\nbetween the second and third, and third and fourth doses. NAb responses were detectable at weeks 5, 11,\nand 17, and plateaued by week 11 after three doses. These results suggested that extended intervals and\nmore than three doses may not be necessary to achieve a potent NAb response. Around the same time, we\nconducted a separate study on RSV F trimer vaccines, recently reported in Nature Communications [Lee et\nal., Nat. Commun. 15, 9939 (2024)]. In that study, we found that a shorter interval was sufficient to elicit\nstrong NAb responses. Based on these insights, our second filovirus vaccine study, which was focused on\nglycan-modified constructs, used a shorter 3-week interval between doses. This shorter regimen is also\nconsistent with our standard “screening” approach in other proof-of-concept vaccine studies.\nWe agree that the two studies do not represent a direct head-to-head comparison of wildtype versus glycan\nmodified antigens. However, both studies used the same 3-week interval between the first and second doses,\nallowing for a direct comparison of early-stage NAb responses. In the submitted manuscript, statistical\nanalyses were performed only for serum samples collected at week 5 (following the second dose), based on\nID values. For samples collected after the third immunization, ID titers were plotted but not subjected\n50 50\nto statistical analysis due to differences in immunization schedules. To clarify this limitation, we included\nthe following statement in the last paragraph of the Results section: “However, due to differences in\nimmunization protocols (e.g., dosing intervals), additional in vivo studies are needed to confirm these\nresults.” (see page 32, lines 720–722).\n5. The use of IgG-format antibodies in BLI assays to assess GP antigenicity is not ideal due to avidity\neffects, which obscure true affinity differences and reduce the assay’s sensitivity to detect variation\n7\nbetween constructs. The use of Fab fragments would provide a more accurate 1:1 binding model\nand allow for a clearer comparison of antigen binding properties between trimeric and\nnanoparticle-displayed immunogens.\nResponse: We thank the reviewer for this thoughtful comment. In general, we agree that Fabs offer a more\naccurate 1:1 binding model in BLI assays, particularly for monomeric or single-epitope antigens. However,\nthis approach faces challenges when applied to GPΔmuc trimers, which display three identical binding sites\nper trimer, and even greater challenges when these trimers are multivalently displayed on SApNPs.\nAn additional constraint arises from the biosensor configuration itself. The commonly used anti-human Fc\nCapture (AHC) biosensor is not suitable for large particles with dense surface antigen display. During the\nearly development of our BLI assays in 2017, we collaborated with FortéBio’s R&D scientists to evaluate\na panel of biosensors on various SApNPs (24-mer FR vs. 60-mer E2p/I3-01). These studies showed that\nanti-human IgG Quantitation (AHQ) biosensors offered the most reliable and reproducible signals for large,\nvirus-sized particles. In contrast, AHC biosensors often yielded artificially low or negative signals, likely\ndue to steric hindrance or signal dampening caused by densely packed surface antigens. AHQ biosensors\nare designed to quantify analyte density in solution and are therefore better suited for detecting antigens\npresented on large protein NPs. While this setup does incorporate avidity effects, particularly with IgGs,\nit\nremains the only configuration that produces measurable BLI responses for both trimers and SApNPs.\nTo further evaluate the reviewer’s suggestion and demonstrate the limitations of Fab-based assays, we\nproduced CA45 and ADI-15878 Fabs and performed BLI assays using FAB2G biosensors. These assays\nyielded consistent profiles for EBOV and SUDV GPΔmuc-WL2P4 trimers. However, the corresponding\nSApNPs generated minimal or undetectable signals using FAB2G biosensors, similar to what we observed\nwith AHC biosensors. These findings reinforce our conclusion that using IgGs paired with AHQ sensors\nprovides the most practical and interpretable method for antigenic comparison between trimers and SApNPs.\nFor these reasons, we continue to use IgGs with AHQ biosensors in the BLI assays presented in this study.\n8\n6. The data on RAVV GP trimer immunization lack statistical support for the claim that the wildtype\ntrimer is more effective than the glycan-trimmed versions. Additionally, the conclusion that RAVV\nGP is “more immunogenic” or holds promise as a pan-filovirus immunogen is currently\nspeculative and should be supported with comparative data or a more cautious interpretation.\nResponse: We thank the reviewer for this helpful comment. We agree that the current data do not support\na statistically robust comparison between wildtype and glycan-trimmed RAVV GPΔmuc trimers, as the\ntwo groups were immunized using different schedules (week 17 vs. week 11). As such, these neutralization\ndata are presented as observational rather than comparative. Nonetheless, the similar trends observed for\nEBOV and SUDV GPΔmuc immunogens, with and without glycan trimming, provide contextual support\nfor the RAVV findings. To assess the NAb responses elicited by RAVV GPΔmuc trimers, we purified total\nIgG from mouse sera to eliminate nonspecific background in pseudovirus assays. Control experiments using\nLCMV pseudoparticles confirmed that the purified IgG did not exhibit nonspecific neutralization. We then\ntested these IgG samples against EBOV-Makona, SUDV-Gulu, and BDBV-Uganda pseudoviruses. Both\nwildtype and glycan-trimmed RAVV GPΔmuc trimers elicited detectable cross-neutralizing activity against\nthe tested orthoebolaviruses. This finding suggests that RAVV GP may possess intrinsic immunogenic\npotential. However, we agree that the current mouse data are limited in scope and do not support a definitive\nconclusion. We have revised the manuscript accordingly to present these findings more cautiously and to\nhighlight the need for additional in vivo studies evaluating the inclusion of RAVV GP as a component of a\npan-filovirus vaccine. Please see pages 28 and 31, as well as page 32, lines 724-725, in the Results section,\nand page 37, lines 827-830, in the Discussion section.\n9"}, {"round": 2, "reviewer_comments": {"reviewer_1": "no further comments", "reviewer_2": "The authors have carefully addressed each of my concerns. I have nmo further comments.", "reviewer_3": "The authors have made substantial improvements, and the manuscript is much clearer and more cohesive. Several earlier concerns have been addressed with helpful additions, while a few areas could still benefit from further clarification or discussion.\nMajor:\n1. The authors’ explanation connecting structural validation to the selection of WL2P4 is convincing.\n2. No revision was provided. The lack of statistical significance in neutralizing antibody responses between different vaccine constructs needs to be clearly described and discussed.\n3. Adding data from male cohorts is a welcome expansion. The “accelerated kinetics” interpretation is now better framed, though still based on non-harmonized datasets. Quantitative metrics such as area-under-the-curve or time-to-threshold analyses could provide a more robust basis for this point.\nMinor:\n1. The inclusion of the EMRinger score is appreciated. However, the model–map correlation coefficients was not provided as suggested.\n2. I3-01v9b redesign comment was well addressed.\n3. The explanation for atom selection is reasonable, and the current analyses are acceptable.\n4. The explanation for the differing schedules is now clear and limitations are acknowledged. To avoid potential misinterpretation, visual contrasts between non-equivalent regimens might be better placed in the Supplement.\n5. The additional experiments with Fabs help to illustrate the technical challenges, and it would be helpful to include these results, even if limited, in the Supplement. Stating explicitly that the reported IgG/AHQ values represent apparent rather than intrinsic affinities would make the interpretation more transparent.\n6. The moderated language on the RAVV GP immunogenicity and contextualization of the findings make this section appropriate.\nAdditional comments regarding the cryo-EM structure:\nSpecifically, the Ramachandran favored score is 92.79%, which is slightly low for a 3.1 Å structure, and there are CaBLAM outliers at 5.38%, indicating backbone geometry concerns. The correlation coefficients are mixed: CC(mask) = 0.73 and CC(volume) = 0.72 are reasonable, but CC(box) = 0.60, CC(peaks) = 0.51, and CC(ligand) = 0.53 are noticeably low. These values suggest suboptimal model-to-map fit, with evidence of overbuilding and unexplained map density in certain regions.\nFrom a biological perspective, this structure is central to validating the authors’ rational antigen design strategy, demonstrating that the engineered GP can present a native-like conformation. The antibody epitope region appears relatively well-resolved and interpretable, which is essential for supporting their mechanistic conclusions. However, other regions of the GP trimer are less well-resolved, reducing overall confidence in the global model.\nIf the authors are unable to further refine and improve the map and model, I strongly recommend that they report the limitations transparently. This should include a clear statement in the text, as well as complete validation data as mentioned above, with local resolution maps, and representative density figures for different regions. Clear reporting will allow readers to appropriately interpret and utilize the structure."}, "author_response": "Response to Reviewer Comments\nReviewer #1 (Remarks to the Author):\nNo further comments\nReviewer #2 (Remarks to the Author):\nThe authors have carefully addressed each of my concerns. I have no further comments.\nReviewer #3 (Remarks to the Author):\nGeneral comments: The authors have made substantial improvements, and the manuscript is much clearer\nand more cohesive. Several earlier concerns have been addressed with helpful additions, while a few areas\ncould still benefit from further clarification or discussion.\nResponse: We thank Reviewer #3 for the thoughtful and constructive evaluation of our manuscript. This\nis the second round of revision, and we are pleased that most of the reviewer’s earlier concerns have been\naddressed. The other two reviewers have expressed satisfaction with the revised manuscript, and we have\ncarefully considered the additional points raised here. We are grateful for these insightful comments, which\nhave helped us further refine the text and clarify key aspects of the study. We have also made focused edits\nto improve clarity and flow, all of which are highlighted in the revised version.\n1. No revision was provided. The lack of statistical significance in neutralizing antibody responses\nbetween different vaccine constructs needs to be clearly described and discussed.\nResponse: We thank the reviewer for this comment. In the first mouse study (Fig. 9), we assessed antibody\nresponses to rationally designed filovirus GPmuc vaccine constructs in trimer and SApNP (E2p and I3-\n01v9b) forms, whereas in the second mouse study (Fig. 10), we focused on different glycan modifications\nof the same constructs. We agree with the reviewer that the NAb responses (ID titers) did not show\n50\nsignificant differences between trimers and SApNPs (Fig. 9), although Kif-treated groups reached statistical\nsignificance in several cases (Fig. 10).\nSeveral factors may explain the lack of statistical significance between vaccine constructs. First, epitope\nexposure differs between soluble trimers (fully exposed) and NP-displayed forms, where IFL and stalk\nepitopes are less accessible. As a result, the proportion of functional NAbs targeting specific epitopes likely\ndiffers. Increasing NP size and tuning antigen spacing may improve epitope exposure and enhance NAb\nresponses (Nguyen et al., NPJ Vaccines 2021, 6:70; Ellis et al., Cell Rep. 2023, 42:113552). Indeed, we are\nactively optimizing the SApNP platform to generate size-adjustable NPs. Second, longitudinal analysis\nshowed that NAb responses (ID titers) tracked well with binding antibody (bAb) responses measured by\n50 50\nID titers were more variable. Similar differences between trimer and NP immunogens have been reported\n50\nfor EBOV GP (Powell et al., Front. Immunol. 2022, 13:942897; Xu et al, Proc. Natl. Acad. Sci. U.S.A.\n2024, 121:e2316960121), likely depending on construct design and immunization regimen. Finally, animal\nmodels may contribute to the weaker significance in NAb responses. Mice are known to produce antibodies\nwith shorter HCDR3 loops, which limit access to IFL and stalk epitopes on NPs. These epitopes may be\nmore effectively targeted in rabbits, non-human primates, or humans.\nBased on the reviewer’s suggestion, we modified the Results for Fig. 9 (page 27, lines 598-599; page 28,\nline 625) to clearly state the lack of statistical significance with a brief explanation (page 29, lines 645-651).\nWe also modified the Discussion to address possible causes and solutions (page 37, lines 837-843).\n1\n2. Adding data from male cohorts is a welcome expansion. The “accelerated kinetics” interpretation\nis now better framed, though still based on non-harmonized datasets. Quantitative metrics such as\narea-under-the-curve or time-to-threshold analyses could provide a more robust basis for this point.\nResponse: We thank the reviewer for recognizing the inclusion of new experimental data from a mouse\nimmunization study addressing sex as a biological variable. In response to this comment, we added a section\nin the Discussion describing the possible accelerated development of functional antibody responses induced\nby SApNP display (page 37, lines 823-829). Because vaccine injections and endpoint serum collections\nwere performed at different time points, area under the curve (AUC) and time-to-threshold (TTT) analyses\ncould not be applied consistently across all conditions. To avoid potential misinterpretation, additional in\nvivo experiments will be required to verify this conclusion. We have revised the manuscript accordingly to\nhighlight the need for further in vivo investigation (page 33, lines 735-738).\n3. The inclusion of the EMRinger score is appreciated. However, the model–map correlation\ncoefficients was not provided as suggested.\nResponse: We thank the reviewer for this comment. We have included map–model correlation coefficients\nand other key validation parameters for our cryo-EM structure, along with 27 reported orthoebolavirus GP–\nantibody complexes from the EMDB, in Fig. S2f. The Results section was revised to reference these data\nand to note the less optimal coefficients (page 11, lines 241-243).\n4. The explanation for the differing schedules is now clear and limitations are acknowledged. To\navoid potential misinterpretation, visual contrasts between non-equivalent regimens might be\nbetter placed in the Supplement.\nResponse: We thank the reviewer for this comment. Based on the first mouse study of antibody responses\nto rationally designed filovirus vaccine constructs (Fig. 9), EC titers in both E2p and I3-01v9b SApNP\n50\ngroups plateaued after three doses. Therefore, in our second study, we calculated and plotted longitudinal\nEC and ID titers on one graph to provide a more complete view of the datasets (Fig. 10). As expected,\n50 50\nmost peak titers were observed after three immunizations, either at week 11 or week 8. To avoid potential\nmisinterpretation, we have clarified this point in the revised manuscript (page 33, lines 735-738) and added\na statement in the Fig. 10 legend (page 77, lines 1932-1935).\n5. The additional experiments with Fabs help to illustrate the technical challenges, and it would be\nhelpful to include these results, even if limited, in the Supplement. Stating explicitly that the\nreported IgG/AHQ values represent apparent rather than intrinsic affinities would make the\ninterpretation more transparent.\nResponse: We thank the reviewer for this suggestion. We have included additional BLI data on Fabs in the\nSupplementary Materials (Fig. S3l) and reported it in the Results section (pages 15-16, lines 336-341). We\nalso clarified that the reported IgG/AHQ values represent apparent rather than intrinsic affinities.\n6. Additional comments regarding the cryo-EM structure:Specifically, the Ramachandran favored\nscore is 92.79%, which is slightly low for a 3.1 Å structure, and there are CaBLAM outliers at\n5.38%, indicating backbone geometry concerns. The correlation coefficients are mixed: CC(mask)\n= 0.73 and CC(volume) = 0.72 are reasonable, but CC(box) = 0.60, CC(peaks) = 0.51, and\nCC(ligand) = 0.53 are noticeably low. These values suggest suboptimal model-to-map fit, with\nevidence of overbuilding and unexplained map density in certain regions. From a biological\nperspective, this structure is central to validating the authors’ rational antigen design strategy,\ndemonstrating that the engineered GP can present a native-like conformation. The antibody epitope\nregion appears relatively well-resolved and interpretable, which is essential for supporting their\nmechanistic conclusions. However, other regions of the GP trimer are less well-resolved, reducing\noverall confidence in the global model. If the authors are unable to further refine and improve the\nmap and model, I strongly recommend that they report the limitations transparently. This should\ninclude a clear statement in the text, as well as complete validation data as mentioned above, with\n2\nlocal resolution maps, and representative density figures for different regions. Clear reporting will\nallow readers to appropriately interpret and utilize the structure.\nResponse: We thank the reviewer for this insightful comment. The Ramachandran favored percentage\n(92.79%) is slightly below the ideal (>95%), but with 0% in the disallowed region, the overall model\nremains within acceptable standards. We also acknowledge that CC(box), CC(peaks), and CC(ligand) are\ntoward the lower end and may reflect suboptimal map-model fit. The lower CC(box) likely reflects missing\nflexible regions in the density map, particularly the CA45 constant domains (heavy: S113-T211; light:\nT109-S215), the GP glycan cap, and the C-terminal GP2 stalk (C601-E611). The reduced CC(peaks) is\nattributable to weak side-chain density, while the lower CC(ligand) values reflect the intrinsic flexibility of\nglycans, which are generally poorly resolved in cryo-EM maps. Importantly, the GP-CA45 interface was\nwell resolved and interpretable, providing key evidence for our rational design of GP presenting native-\nlike bNAb epitopes. Thus, excluding less resolved regions does not compromise the structural quality or\nbiological interpretation of the antigen–antibody interface.\nTo further address the reviewer’s concern about model quality, we surveyed 27 cryo-EM structures of\northoebolavirus GP-antibody complexes in the EMDB and compared map-model correlation coefficients\nand other key parameters with those of our structure. Our values fall within the reported range, though some\nare toward the lower end. In the GP trimer, three regions—GP1 (A187–L218), GP2 (E522–N525), and the\nC-terminal stalk (P612–N637)—were excluded from the final model due to limited local resolution. The\nCA45 constant domains were also excluded because their density was poorly resolved owing to intrinsic\nflexibility. Similar exclusions or partial modeling of these regions are common among reported GP–NAb\nstructures in the EMDB, suggesting that their absence does not compromise biological interpretation.\nFinally, we agree that the limitations of our cryo-EM analysis should be reported with transparency. We\nhave added a model validation plot and summary to the Supplemental Information (Fig. S2f), revised the\nResults section to describe the suboptimal map–model fit (pages 11, lines 241–243; pages 11–12, lines\n247–250), and included a statement in the Discussion (page 35, lines 777–779) to highlight these limitations\nand reference the detailed model evaluation (Fig. S2f).\n3"}, {"round": 3, "reviewer_comments": {"reviewer_3": "The authors have substantially improved the manuscript in response to earlier feedback.\nThese thoughtful revisions convincingly address prior concerns and clarify the importance of the structural perspective within the study. The manuscript is now well-balanced and coherent. I support publication in its current form."}, "author_response": ""}]

Decision letter