Anti-Ebola virus mAb 3A6 protects highly viremic animals from fatal outcome via binding GP(1,2) in a position elevated from the virion membrane

Hastie, Kathryn M.; Salie, Zhe Li; Ke, Zunlong; Halfmann, Peter J.; DeWald, Lisa Evans; McArdle, Sara; Grinyó, Ariadna; Davidson, Edgar; Schendel, Sharon L.; Hariharan, Chitra; Norris, Michael J.; Yu, Xiaoying; Chennareddy, Chakravarthy; Xiong, Xiaoli; Heinrich, Megan; Holbrook, Michael R.; Doranz, Benjamin; Crozier, Ian; Kawaoka, Yoshihiro; Branco, Luis M.; Kuhn, Jens H.; Briggs, John A. G.; Worwa, Gabriella; Davis, Carl W.; Ahmed, Rafi; Saphire, Erica Ollmann

doi:10.1038/s41467-025-56452-2

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Published: 03 February 2025

Anti-Ebola virus mAb 3A6 protects highly viremic animals from fatal outcome via binding GP_(1,2) in a position elevated from the virion membrane

Nature Communications volume 16, Article number: 1293 (2025) Cite this article

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Abstract

Monoclonal antibodies (mAbs) against Ebola virus (EBOV) glycoprotein (GP_1,2) are the standard of care for Ebola virus disease (EVD). Anti-GP_1,2 mAbs targeting the stalk and membrane proximal external region (MPER) potently neutralize EBOV in vitro and are protective in a mouse model of EVD. However, their neutralization mechanism is poorly understood because they target a GP_1,2 epitope that has evaded structural characterization. Using X-ray crystallography and cryo-electron tomography of mAb 3A6 complexed with its stalk–MPER epitope, we reveal a previously undescribed mechanism in which 3A6 binds to a conformation of GP_1,2 that is lifted from the virion membrane. We further show that in both domestic guinea pig and rhesus monkey EVD models, 3A6 provides therapeutic benefit at high-viremia advanced disease stages and at the lowest dose yet demonstrated for any anti-EBOV mAb-based monotherapy. The findings reported here can guide design of next-generation highly potent anti-EBOV therapeutics and vaccines.

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Introduction

Ebola virus (EBOV; family Filoviridae: species Orthoebolavirus zairense) causes severe and frequently fatal acute human disease in outbreaks that can result in thousands of deaths. Complicating containment efforts, EBOV may persist subclinically in survivors for years and reignite outbreaks. Ebola virus disease (EVD) can be prevented with two licensed vaccines and treated with approved monoclonal antibody (mAb) therapeutics¹. However, even with approved mAbs, outcomes remain poor in patients with high viral loads and/or advanced disease, and the incidence of viral persistence is unknown. As such, identification and optimization of novel mAbs are needed to address these gaps.

All advanced anti-EBOV mAb-based therapeutics and vaccines target protein spikes protruding from virion envelopes². Each spike comprises a single EBOV-encoded glycoprotein (GP_1,2), synthesized by translation of a preproprotein that is cleaved in the Golgi apparatus into GP₁ and GP₂ subunits. A disulfide bond links these two subunits to form heterodimers (Fig. 1a top) that assemble into GP_1,2 trimers^3,4. GP₁ contains a heavily glycosylated mucin-like domain (MLD) that obscures the upper and outer portions of GP_1,2 and a glycan cap domain that shields the virion receptor-binding site in the GP₁ core from the host immune response (Fig. 1a top). Upon virion entry, host cell cathepsins proteolytically process GP_1,2 in the endosome to remove the MLD and glycan cap domain to expose the GP₁ core and binding site for virion receptor NPC intracellular cholesterol transporter 1 (NPC1)^5,6,7. GP₂, a typical class I fusogen, mediates fusion of virion and endosomal membranes to release viral ribonucleocomplexes into the target cell⁸. GP₂ contains an internal fusion loop (IFL), two consecutive heptad repeat regions (HR1 and HR2), a membrane proximal external region (MPER), and a C-terminal transmembrane (TM) domain (Fig. 1a top). HR2 also called the stalk, is largely alpha-helical and connects the GP₂ core to the MPER and TM domain⁷.

**Fig. 1: Structures of human mAb 3A6 in complex with the Ebola virus glycoprotein stalk–MPER peptide.**

Major recognition sites for anti-EBOV mAbs are the GP₁ MLD and glycan cap domain; the GP_1,2 trimer base; and the GP₂ IFL, stalk, and MPER^{9,10,11,12,13,14,15,16,17}. Stalk–MPER is of special interest for therapeutic and vaccine design because of the potent neutralization activity demonstrated by mAbs that bind to it. Indeed, the region has 70% sequence conservation among all six orthoebolaviruses and, for the three viruses that can cause fatal disease, Bundibugyo virus (BDBV), EBOV, and Sudan virus (SUDV), the sequence conservation increases to ≈90%^9,11,13. Despite these important features, mAbs against the stalk–MPER is the least well-characterized of anti-orthoebolavirus mAbs in part because of a comparative lack of structural information about this region; studies to date have used GP_1,2 constructs in which MPER was deleted (for increased solubility and stability). Only one structure, that of the BDBV stalk–MPER targeted by mAb BDBV223, has been examined⁷. Curiously, the angle at which BDBV223 binds to the BDBV GP₂ epitope suggests that, in current models of the EBOV GP_1,2 structure, access would be occluded by the viral or host cell membrane¹⁸. As such, the mechanism by which mAbs that target the stalk–MPER access their epitope remains unclear, further questioning whether access is associated with therapeutic efficacy for patients infected with EBOV or related viruses.

In this work, we use x-ray crystallography to illuminate the discrete binding footprint of mAb 9.6.3A6, a highly potent stalk-MPER-targeted mAb. Using cryo-electron tomographic analysis, we show that this mAb gains access to its occluded epitope by stabilizing EBOV GP_1,2 in a lifted state. Finally, we demonstrate complete mAb 9.6.3A6-mediated post-exposure protection in the stringent guinea pig and rhesus monkey models of EVD.

Results

Structure of mAb 3A6 in complex with the Ebola virus stalk–MPER peptide

mAb 9.6.3A6 (henceforth abbreviated as “3A6 IgG”) was isolated from a human survivor of the 2013–16 Western African EVD outbreak 6 months after hospital discharge¹¹. The predicted linear epitope of 3A6 IgG encompasses GP_1,2 residues 626–640 and extends from the C-terminal end of the stalk to the start of the MPER¹¹ (Fig. 1a, bottom/inset, orange box, outlined in purple in the sequence alignment). To determine the mode of molecular recognition of EBOV by 3A6 IgG, we crystallized the 3A6 antigen-binding fragment (“Fab”, henceforth abbreviated as “3A6”) alone (Supplementary Fig. 1) and in complex with a 14-amino-acid peptide having a sequence corresponding to the EBOV GP_1,2 stalk–MPER epitope (residues 626–640; Fig. 1b–f). Crystals of 3A6 diffracted to 2.5 Å and had an asymmetric unit containing four Fab molecules. Meanwhile, crystals of the 3A6-stalk–MPER peptide complex diffracted to 1.27 Å and had one Fab fragment in the asymmetric unit (Supplementary Table 1). The 3A6 structure was essentially identical in the unbound and peptide-bound states as evidenced by the 0.46 Å root-mean-square deviation (RMSD) over the entirety of the paratope (Supplementary Fig. 1b). Residues I627–G639 of the 3A6-peptide epitope are visible (Fig. 1c–f, Supplementary Fig. 1a–e). The EBOV stalk–MPER peptide is ɑ-helical from its N terminus (I627) to residue T634 and then slightly unravels through the visible terminus at residue G639 (Supplementary Fig. 1).

The paratope of 3A6 can roughly be divided into stalk-binding or MPER-binding areas. The N-terminal/stalk portion of the peptide (residues 627–631) is enveloped by complementarity determining regions (CDRs) H1, H3, and L2 (Fig. 1b, Supplementary Fig. 1). CDR-H3 residues R98, S100, T101, and Y104 form extensive hydrogen bonds to GP₂ residues H628, D629, and D632 (Fig. 1c, e; Supplementary Fig. 1), CDR-H1 residue E31 contacts MPER residue K633, and Y54 of CDR-L2 interacts with surrounding water molecules to contact MPER residue T634 (Fig. 1c; Supplementary Fig. 1). Heavy-chain residues Y32 and Y104 and light-chain residues Y54, L58, and F99 engage in hydrophobic interactions with this region of the peptide (Fig. 1f).

The MPER/C-terminal portion of the peptide is surrounded by CDR-H2, CDR-H1, and CDR-L3. Antibody residues H31 and S32 (CDR-L1), T97 (CDR-L3), and N59 (CDR-H2) engage MPER residues L635, P636, D637, and Q638 (Fig. 1d, e). Residue M33 of the heavy chain and A96, F99, and L101 of the light chain form a hydrophobic pocket around P636 (Fig. 1f). MPER residues D632 and P636 are particularly crucial and form extensive interactions with the Fab (Fig. 1c–f).

Comparison to stalk-binding mAb BDBV223

Only one other high-resolution structure of an orthoebolavirus GP₂ stalk–MPER-targeted mAb is documented. The structure of mAb BDBV223 was determined alone and in complex with its peptide epitope from BDBV¹⁸. Comparison of the mode of recognition of 3A6 and BDBV223 for their cognate targets reveals several differences. BDBV223 binds GP₂ residues 620–634, placing its epitope primarily within HR2 (residues 599–632) (Supplementary Fig. 2a). In addition to an upward shift, the manner in which BDBV223 binds to its epitope is markedly different from that of 3A6. In contrast to 3A6, for which the peptide binds directly in the groove between the heavy and light chains (Fig. 1b), the heavy chain of BDBV223 forms the bulk of interactions with its peptide epitope, with only minor contributions from the light chain of CDR-L3 (Supplementary Fig. 2b). Moreover, while there is no induced fit for 3A6 (Supplementary Fig. 1b), binding of BDBV223 to its peptide epitope results in substantial rearrangements in the heavy-chain CDRs as compared to the unbound Fab. Furthermore, 3A6 binds to EBOV GP_1,2 at an ≈80° angle, while BDBV223 binds at an ≈117° angle (Supplementary Fig. 1c).

Despite these differences, there are curious similarities between 3A6 and BDBV223. Both 3A6 and BDBV223 are escaped by changes at aspartate (D632 and D624, respectively) or proline (P636 or P634, respectively) residues (Fig. 2 and King et al., 2019¹⁸). Although located in different regions of the GP₂ stalk–MPER, each of these residues interacts with the respective mAb in a similar manner. In particular, both D624 and D632 of GP₂ are coordinated by CDR-H3 residues and both P634 and P636 of GP₂ occupy a hydrophobic pocket within CDR-L3 (Fig. 1c–f and Supplementary Fig. 2b).

**Fig. 2: Residues D632 and P636 of the Ebola virus glycoprotein MPER are key for mAb 3A6 binding.**

EBOV GP_1,2 MPER residues D632 and P636 are critical for mAb 3A6 binding

3A6 IgG binds to and neutralizes EBOV but not SUDV in vitro¹¹. In the 3A6 IgG peptide epitope that includes stalk–MPER residues I627–G639, four residues differ substantially between EBOV and SUDV GP₂: K vs. N at 633, T vs. P at 634, D vs. N at 637, and G vs. D at 639 (Fig. 1a, bottom/inset). Using a cell-based antibody-binding assay, we compared binding of 3A6 IgG to full-length (MLD-containing) EBOV GP_1,2 with each of these four residues changed individually or in combination with the corresponding SUDV residues (Fig. 2a and Supplementary Fig. 2a); EBOV GP_1,2 lacking residues 312–462 of the MLD and residues 644–676 of the TM domain (GP_{1,2ΔTM/ΔMLD}) was created. In addition, we used an enzyme-linked immunosorbent assay (ELISA) to evaluate 3A6 binding to recombinant EBOV GP_{1,2ΔTM/ΔMLD} ectodomains containing the same amino acid changes (Fig. 2b). In either case, none of the individual amino-acid residue changes affected 3A6 IgG binding, but binding was completely inhibited when all four residues were changed to the SUDV counterparts (Fig. 2, Supplementary Fig. 3a). Binding of control mAbs that target quaternary epitopes in the fusion domain of GP₂ was not impacted, indicating the overall conformation of GP_1,2 was not altered by any change(s) (Supplementary Fig. 3b, c).

Next, we used alanine-scanning mutagenesis of GP_1,2 to identify individual residues throughout the epitope that are crucial for 3A6 binding. We individually replaced alanine residues (wild-type [WT] Ala to Ser) at positions 627–639 of EBOV GP_1,2 lacking the MLD (GP_1,2ΔMLD; Fig. 1a) and analyzed each resulting EBOV GP_1,2ΔMLD for 3A6 reactivity by flow cytometry (Supplementary Table 2, Supplementary Fig. 4). Notably, both D632A and P636A showed reduced 3A6 IgG binding by more than 80% relative to WT GP_1,2ΔMLD (Fig. 2c). Meanwhile, neither change substantially affected binding of control mAbs KZ52, 1H3, or 4G7 that target conformational epitopes on the base (KZ52 and 4G7) and glycan cap (1H3) of EBOV GP_1,2^7,19,20 (Fig. 2c). These results are consistent with those of previous studies, in which morphologically authentic “biologically contained” EbolaΔVP30 virions²¹, passaged in the presence of 3A6 IgG led to the emergence of glycoproteins bearing P636S and P636Q changes¹¹. Therefore, we next evaluated neutralization of P636S-bearing EbolaΔVP30-eGFP virions by multiple mAbs using a plaque-reduction assay. P636S abolished neutralization activity of both 3A6 and 1E6, another stalk-binding mAb¹¹, but did not affect neutralization of mAbs targeting the glycoprotein core (Supplementary Table 3).

Binding of mAb 3A6 lifts EBOV GP_1,2 relative to the membrane surface

All GP_1,2 structures in which the stalk region can be modeled include a fibritin trimerization motif fused to the GP₂ C terminus^22,23. It is not known whether inclusion of exogenous trimerization domains induces a non-native association of the HR2/MPER helices or if the stalk region adopts a more open and flexible conformation in the context of membrane-bound GP_1,2. Certainly, superimposition of the 3A6–stalk–MPER structure onto a fibritin-stabilized GP_{1,2ΔTM/ΔMLD} trimer (PDB 5JQ7) revealed steric clashes of the bound 3A6 with the other two GP₂ monomers of stalk–MPER (Supplementary Fig. 5a). Hence, the tightly bundled conformation of GP₂ HR2 observed in this crystal structure is incompatible with Fab binding.

To assess the stoichiometry of the 3A6-EBOV GP_{1,2ΔTM/ΔMLD} interaction, we used complementary biophysical techniques to evaluate Fab binding to soluble GP_{1,2ΔTM/ΔMLD} ectodomains. Size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) revealed that 3A6-GP_{1,2ΔTM/ΔMLD} complexes elute as a single peak with a molecular mass consistent with that of a fully occupied GP_{1,2ΔTM/ΔMLD} trimer (Supplementary Fig. 5b). Peaks corresponding to binding of one or two 3A6 molecules to GP_{1,2ΔTM/ΔMLD} were not observed. Composition-gradient multi-angle light scattering (CG-MALS) analysis supported this result, revealing that three copies of 3A6 can bind to one GP_{1,2ΔTM/ΔMLD} trimer and do so with equal affinities (K_D = 52.15 [±1.3] nM; Supplementary Fig. 5c). Moreover, negative-stain EM (nsEM) analysis of 3A6–GP_{1,2ΔTM/ΔMLD} complexes also showed binding of three 3A6s to the stalk–MPER (Supplementary Fig. 5d).

These analyses conclusively demonstrated that GP_{1,2ΔTM/ΔMLD} ectodomains can bind to three 3A6 simultaneously. However, the protein used for these studies did not contain an exogenous trimerization domain, a feature that, if it were there, would have constrained access to the epitope. Similarly, the GP₂ C termini in the natural membrane-anchored form would have less freedom of movement than would be available in the GP₂ termini of untethered ectodomains. To image 3A6 bound to GP₂ in its natural transmembrane form, we produced filamentous EBOV-like particles (VLPs) consisting of EBOV matrix protein (VP40) and full-length GP_1,2. VLPs were incubated with 3A6 and analyzed via cryogenic electron tomography (cryo-ET), followed by subtomogram averaging (Fig. 3a, Supplementary Fig. 6, Supplementary Table 4). Tomographic reconstruction of VLP GP_1,2 in the absence of 3A6 was unsuccessful, perhaps due to flexibility of the GP_1,2 on the viral surface. Thus, to represent the state of GP_1,2 in the absence of 3A6, we complexed VLPs with the Fab of the well-characterized mAb KZ52, which binds to the fusion peptide of EBOV GP_1,2⁷. We also imaged a ternary complex of both 3A6 and KZ52 Fab bound to VLP GP_1,2 to determine if binding of one influenced the binding of the other or the position of the GP_1,2 in relation to the VLP membrane.

**Fig. 3: Binding of mAb 3A6 lifts Ebola virus glycoprotein relative to the membrane surface.**

Reconstructions of VLP GP_1,2–3A6 (18 Å) and VLP GP_1,2–KZ52 (8.7 Å) showed the expected chalice-shaped GP_1,2 trimer with extra densities anchored to the stalk and to the base of the chalice, corresponding to 3A6 and KZ52 Fab, respectively (Fig. 3b, c). Likewise, the ternary complex of GP_1,2 with both Fabs confirmed that each binds to discrete non-overlapping sites on GP_1,2 (Fig. 3b, c). The strong Fab density in each reconstruction suggests that the bulk population of GP_1,2 is fully occupied by the bound Fab(s), however, we have not performed a classification aimed at resolving minority populations with fewer numbers of Fabs. Importantly, the KZ52 epitope at the base of GP₁ is sufficiently separated from the stalk–MPER 3A6 binding site, and its binding does not disrupt the native structure of GP_1,2. Although the overall conformation of GP_1,2 was unchanged regardless of the Fab(s) bound to it, we did observe marked differences in the position of GP_1,2 relative to the surface of the VLP. In the presence of 3A6, the GP_1,2 body was displaced vertically away from the VLP membrane by approximately 3 nm as compared to the position of GP_1,2 when bound to KZ52 alone (Fig. 3b). We conclude that 3A6 binding induces this vertical displacement since VLP GP_1,2 complexed with both 3A6 and KZ52 Fabs together resulted in a similarly lifted GP_1,2 (Fig. 3c) with densities that overlapped with VLP GP_1,2–3A6 (Fig. 3d).

To gain insight into the location of the 3A6 epitope in the context of virion-displayed GP_1,2, we used AlphaFold 2^24,25 to model the various states GP_1,2 may sample. Twenty models were generated (five models generated in each of four independent trials) and curated to remove those in which the TM domain was disordered or not positioned in a manner consistent with membrane association (Supplementary Fig. 7a, b). The remaining ten models were sorted into those that adopted a compact MPER structure (n = 6; Supplementary Fig. 7c) and those that adopted an extended MPER (n = 4; Supplementary Fig. 7d). The positional arrangement of the MPER and TM domain in the “compact MPER” models was similar to that of the previously reported solution structure of EBOV MPER/TM⁸ (Supplementary Fig. 7e), in which the soluble and dynamic N-terminal region (residues 632–640) is linked to the well-ordered TM domain (residues 656–676) through a short interfacia l α -helical region and flexible turn (residues 641–655). Further, we found that models with a compact MPER best fit the subtomographic reconstruction of GP_1,2 in complex with KZ52 (Supplementary Fig. 7c, e), while those with an extended MPER fit the reconstruction of GP_1,2 bound to 3A6 (Supplementary Fig. 7d, f).

We next modeled the location of the 3A6 epitope in a representative of each of the compact and extended MPER models and found that, as expected, the epitope is highly occluded in the compact model and exposed in the extended model (Fig. 4). Notably, we find no evidence that 3A6 interacts with membrane alone (Supplementary Fig. 3a), suggesting that 3A6 does not penetrate the membrane to gain access to its epitope. Instead, we propose that 3A6 binds the epitope when GP_1,2 is tilted and/or lifting up and retracting down. Once engaged by 3A6, GP_1,2 is stabilized in a lifted state. The outwardly located epitope is consistent with the requirement that the stalk must be exposed for 3A6 to bind. It remains unclear whether this position is a natural feature of the GP₂ stalk or if it occurs upon 3A6 binding.

**Fig. 4: Vertical breathing of the Ebola virus glycoprotein stalk and MPER may allow 3A6 to bind.**

Low-dose mAb 3A6 monotherapy is protective in stringent models of EVD

Previous in vivo studies demonstrate that prophylactic administration of 3A6 IgG protects laboratory mice from fatal outcomes after exposure to a typically lethal dose of mouse-adapted EBOV (100% protection after a 100-μg dose [≈5 mg/kg] and 50% protection at a 25 μg dose [≈1.25 mg/kg])¹¹. To determine whether 3A6 is also effective in the post-exposure setting, groups of six (three male and three female) guinea pigs were first exposed intraperitoneally (IP) to 1000 PFU of domesticated guinea-pig-adapted EBOV (Day 0). On Day 3, the guinea pigs were either left untreated or treated IP with a single 5 mg dose of anti-EBOV antibodies 3A6 IgG, 1A2 IgG (targets the EBOV GP₂ fusion loop), or 7G7 IgG (targets an unknown epitope on EBOV GP_1,2¹¹) or the anti-influenza A virus (FLUAV) antibody 42-2D2. All animals in the untreated group and those treated with 1A2 or 7G7 succumbed to EBOV infection. All but two of the 42-2D2-treated control animals succumbed to EBOV infection. In contrast, all guinea pigs treated with 3A6 survived and exhibited few or no clinical signs of disease (Fig. 5a, Supplementary Fig. 8a-c).

**Fig. 5: Low-dose mAb 3A6 monotherapy protects domesticated guinea pigs and rhesus monkeys against Ebola virus.**

Guinea pigs offer a more stringent model of EVD compared to mice, but the rhesus monkey model better recapitulates key features of EVD and is generally preferred over rodent models for development of EVD medical countermeasures¹. As a proof of concept, we randomized four rhesus monkeys into two groups: administered treatment (n = 3, rhesus monkeys 1–3) and no treatment (n = 1). All monkeys were exposed via intramuscular (IM) injection to a typically lethal 1000-PFU dose of EBOV (Day 0). On Day 4 and Day 7 after exposure, the treatment group monkeys given 25 mg/kg of 3A6 in phosphate-buffered saline (PBS) intravenously, whereas the control monkey was given intravenous PBS only. EBOV replication was confirmed in all monkeys on Day 4 by plaque assay titration and quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR), with 10⁴–10⁶ EBOV PFU per mL and 10⁸–10¹⁰ EBOV glycoprotein (GP) gene equivalents per mL of serum (Supplementary Fig. 8d). Notably, these high viremic levels could still be reversed by 3A6 administration, as evidenced by a decrease in viral load after the first dose on Day 4 and continued reduction to below the limit of detection by Day 21 (Supplementary Fig. 8d). Clinical signs consistent with EVD, observed in monkeys 1 and 3 as early as Day 4, resolved by Day 13 (Supplementary Fig. 8e). Notably, monkey 3 had significantly elevated AST activity and rapidly rising serum creatinine concentrations, suggesting a marked reduction in the glomerular filtration rate (the initial rise was similar to that of the control animal) with pathologic evidence of EBOV-induced liver injury (Supplementary Fig. 9a, b). Nonetheless, this animal still recovered after 3A6 IgG treatment (Fig. 5b, Supplementary Fig. 8e). All three monkeys in the treatment group survived, whereas the control monkey was found dead on Day 8 (Fig. 5b). These preliminary results demonstrate that post-exposure dosing of 3A6 IgG alone reverses the course of EBOV infection and protects against fatal outcomes. Additional studies, including a larger cohort of NHPs and differential dosing (i.e., lower dose of antibody, variation in the number of doses and day of treatment) will further illuminate the therapeutic impact of 3A6 treatment.

Discussion

Vaccines against EVD are approved²⁶ but none are in place for infections caused by other filoviruses. Similarly, the human standards-of-care monotherapy ansuvimab (mAb114) and cocktail atoltivimab/maftivimab/odesivimab (REGN-EB3) are also specific for EBOV and are dosed at 50 mg/kg each (150 mg/kg total for three). As such, antibody therapeutics that can be used at a low dose to reverse advanced disease are urgently needed to treat people with filovirus infections who live in countries with limited resources. The EBOV GP_1,2 stalk–MPER is of interest for therapeutic/vaccine design due to its relatively high amino acid sequence conservation among all orthoebolaviruses, indicating that a single mAb targeting this region could have therapeutic activity against infections by any of these viruses. Moreover, known anti-orthoebolavirus stalk and/or MPER mAbs are highly potent neutralizers in vitro^11,18,27, suggesting that they may be applied in much lower doses compared to mAbs that are currently used in the clinic.

Here we built on previous in vitro and prophylactic laboratory mouse efficacy studies of the EBOV GP_1,2 stalk–MPER-binding mAb 3A6 by demonstrating complete post-exposure protection in stringent models of EVD in domesticated guinea pigs and rhesus monkeys. 3A6 showed unprecedented potency in the rhesus monkey model of disease. In the pilot study performed here, monkeys presenting with severe disease signs associated with fatal outcomes were treated with a single 25-mg/kg dose of 3A6, which reduced Day 4 viral loads of 10⁹–10¹⁰ PFU per mL to undetectable levels by Day 21. Concomitantly, 3A6 reversed clinical signs of advanced disease and decreased elevated liver enzyme activities and serum creatinine concentrations to baseline. The administered dose is half that used for mAb114 monotherapy and one-sixth of that used for the REGN-EB3 mAb cocktail in the same animal model (albeit administered on different days)^10,28. Our data therefore pave the way for development of novel therapeutics that potentially expand the treatment window later in the EVD course for effective intervention in highly viremic patients. Such therapeutics could increase the likelihood of survival for this group of patients seen relative to currently approved mAb therapeutics.

We previously hypothesized that binding of BDBV223, an anti-stalk antibody targeting a similarly occluded epitope in the EBOV-related Bundibugyo virion¹⁸, requires either bending or lifting of GP_1,2. In this study, we experimentally addressed this hypothesis using 3A6, which, at the outset appears to bind to an epitope that is even more occluded than that bound by BDBV223. Our structural and modeling studies suggest that the 3A6-targeted epitope is in close apposition to the membrane and perhaps occluded in some conformations. The inherent flexibility of the stalk region may allow EBOV GP_1,2 to bend or extend, thus enabling 3A6 access and therefore the ability to lift GP_1,2 from the surface of the membrane.

As high-resolution structures of full-length transmembrane GP_1,2 are missing, we do not yet know if the three membrane-proximal stalks are bundled or separated in the natural protein. If bundled, the first 3A6 to bind would likely induce some unbundling and displacement. We also do not know if the first 3A6 to bind would induce a tilt, in which one epitope is exposed, and the second and third 3A6 to bind would support a general lift with all epitopes exposed or if the first to bind would be enough to support a generally lifted and exposed conformation for all three instances of the 3A6 epitope on the trimer.

We hypothesize that by binding to the stalk, 3A6 achieves its potent neutralization activity through the blocking of conformational changes needed to drive fusion of the virion and cell membranes. Human immunodeficiency virus 1 GP160 and influenza virus A (FLUAV) hemagglutinin can also be tilted by binding of anti-MPER antibodies^29,30, indicating that positional flexibility is a common property of class I fusogens.

In conclusion, our studies establish 3A6 as a potent immunotherapeutic against EBOV that achieves complete protection against advanced disease at the lowest dose yet observed for a monotherapy via a previously undescribed mechanism of action. The next desired feature of stalk and MPER-targeted mAbs is breadth: 3A6-like antibodies with pan-orthoebolavirus activity likely exist. Such antibodies could be used at even lower concentrations and at more advanced stages across the filovirus disease spectrum. We have shown that binding of stalk and MPER-targeted antibodies requires a flexible HR2 helical bundle that can accommodate antibody binding. Tethers such as exogenous trimerization domains or presentation of the minimal stalk–MPER peptide on protein scaffolds will likely prevent the elicitation of antibodies that can then recognize native full-length GP_1,2. This study provides new blueprints for the development of stalk–MPER-targeted vaccines that elicit potent and broad immune responses.

Methods

Cell lines

Human embryonic kidney (female) epithelial Expi293F cells (Thermo Fisher Scientific, Waltham, MA, USA) were cultured on orbital shakers in Expi293 expression medium (Thermo Fisher Scientific) at 37 °C in a humidified atmosphere containing 8% carbon dioxide (CO₂). HEK 293 T cells (American Type Culture Collection [ATCC] Manassas, VA, USA; #CRL-3216, human, female) were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) containing L-glutamine (Invitrogen, Carlsbad, CA, USA), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Omega Scientific, Tarzana, CA, USA) and 1% penicillin–streptomycin solution (Thermo Fisher Scientific). Cells were maintained at 37 °C in a humidified atmosphere with 5% CO₂. Drosophila Schneider 2 (S2) cells (Thermo Fisher Scientific) were cultured with Schneider’s Drosophila medium (Thermo Fisher Scientific) in stationary flasks at 27 °C. Stable cell lines were adapted to serum-free conditions and maintained on orbital shakers at 27 °C.

Antibody and antibody fragment expression, purification, crystallization, and visualization

Protein fragment generation, protein and protein fragment purification, crystallization, X-ray structure determination, and negative-stain electron microscopy were performed according to standard protocols.

Protein expression and purification

Immunoglobulins were expressed with an Expi293 Expression System (Thermo Fisher Scientific). Light and heavy chain-encoding plasmids were prepared using an endotoxin-free kit (NucleoBond Xtra Midi Plus EF; Takara Bio, San Jose, CA, USA) and used to transfect Expi293 cells at a 2:1 ratio of light chain to heavy chain with Expifectamine 293 transfection reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. mAbs containing supernatants from transfected cells were clarified by centrifugation and then incubated with protein A agarose resin (GenScript, Piscataway, NJ, USA) in batch format overnight, followed by washing, elution, and buffer exchange into Dulbecco’s PBS (DPBS; Thermo Fisher Scientific) as previously described by Smith et al., 2009³¹. Antibodies used in vivo were verified to be endotoxin-free using a commercial detection kit (Thermo Fisher Scientific). All antibodies produced in this study were expressed as human IgG1.

Fab fragments were generated from purified IgG1s through digestion with 3% immobilized papain (Thermo Fisher Scientific) for 2 h, followed by purification with a Mono Q anion exchange chromatography column (GE Healthcare, Chicago, Illinois, USA) and size-exclusion chromatography with a Superdex 75 Increase 10/300 GL column (GE Healthcare) in 1X tris(hydroxymethyl)aminomethane-buffered saline (tris-buffered saline [TBS]; Thermo Fisher Scientific). Fractions with pure Fab were concentrated using Ultra Centrifugal Filter Units (Amicon, Miami, FL, USA). Epitope peptide representing GP_1,2 residues 626–640 was chemically custom-synthesized by Thermo Fisher Scientific and purified via high-performance liquid chromatography (HPLC).

Recombinant WT and Ebola virus/H.sapiens-tc/COD/1976/Yambuku-Mayinga GP_1,2 ectodomain variants, the latter lacking residues 312–462 of the MLD and residues 644–676 of the transmembrane domain (GP_{1,2ΔTM/ΔMLD}), were expressed in S2 cells. All constructs carried a C-terminal double-strep tag for affinity purification. Stably transfected cells were selected with 6 μg/mL puromycin (InvivoGen, San Diego, CA, USA). The resulting strep-tagged proteins were purified using a 5-mL StrepTrap column (Cytiva, Marlborough, MA, USA) according to the manufacturer’s protocol and then further purified with size-exclusion chromatography (SEC) using a Superdex 200 column (Cytiva) in 1X TBS.

Protein crystallization

3A6 was crystallized in 28% polyethylene glycol (PEG) 400, 0.1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) + sodium hydroxide (NaOH) pH 7.5 buffer, and 0.2 M calcium chloride (CaCl₂) (all constituents from Hampton Research, Viejo, CA, USA) at 20 °C. To form the 3A6–peptide complex, purified 3A6 was concentrated to 5 mg/mL, combined with a five-fold excess of peptide, and incubated at 4 °C for 18 h. The Fab–peptide complex was crystallized in 30% PEG 3000, 0.1 M tris pH 7.0, and 0.2 M sodium chloride (NaCl) (all constituents from Hampton Research) at 20 °C. Crystals were flash-cooled in liquid nitrogen, with 15% ethylene glycol (Hampton Research) as a cryoprotectant.

X-ray data collection and protein structure determination

X-ray diffraction data of Fab–peptide complexes were collected on beamline 12–2 at the Stanford Synchrotron Radiation Lightsource, and Fab diffraction data were collected on beamline 23ID-B at the Advanced Photon Source^32,33. One dataset for the Fab crystal was used, and two datasets from separate Fab–peptide complex crystals were merged for processing using autoPROC with XDS^34,35 for indexing and integration, followed by POINTLESS³⁶ and AIMLESS³⁷ programs for data reduction, scaling, merging, and calculation of structure factor amplitudes and intensity statistics. One Fab–peptide complex per asymmetric unit was present in space group P1 21 1 (a = 52.3 Å, b = 66.4 Å, c = 68 Å, α = γ = 90°, β = 104.2°), and four Fabs were present in the asymmetric unit of the Fab structure in space group P1 (a = 53.7 Å, b = 65.7 Å, c = 125.6 Å, α = 98.7°, β = 91.4°, γ = 96.0°). Crystal structures were determined by molecular replacement using PHASER³⁸ within the CCP4 package³⁹, using a homology model predicted with the SWISS-MODEL server⁴⁰. Iterative manual model rebuilding was performed using Coot⁴¹ and refined with PHENIX⁴². The peptide was built into different Fourier maps and calculated prior to inclusion of the respective structural elements. Final atomic coordinates and structure factors of the Fab–peptide complex and apo–Fab structures were deposited in the Protein Data Bank (PDB) under identification numbers (IDs) 7RPU and 7RPT, respectively. Figures were created in PyMOL (http://www.pymol.org/) and Chimera X.

Size-exclusion chromatography coupled to multi-angle light scattering

Size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) experiments were performed using a Superdex 200 Increase 10/300 column (Cytiva), an ÄKTA fast protein liquid chromatography (FPLC) purifier (Cytiva) in line with a miniDAWN MALS detector (Wyatt Technology, Santa Barbara, CA. USA), and an Optilab digital refractive index detector (Wyatt Technology). All experiments were performed in 1X TBS. ASTRA VI software was used to combine these measurements and enable the absolute molar mass and extinction coefficient of the eluting GP, Fab, or GP–Fab complex to be determined^43,44.

Composition gradient multi-angle light scattering

Composition gradient multi-angle light scattering (CG-MALS) experiments were performed with a Calypso II composition gradient system (Wyatt Technology) to prepare different compositions of buffer, GP, and antibody and deliver to the miniDAWN detector and an online ultraviolet (UV) detector (Cytiva). The extinction coefficient obtained from the SEC-MALS experiment was used to measure the concentration of the GP during CG-MALS experiments. Polycarbonate filter membranes with 0.1 µM pore size (Millipore Sigma, Burlington, MA, USA) were installed in the Calypso system for sample and buffer filtration. GP was diluted to a stock concentration of 40–60 µg/mL in TBS. Fab was diluted to a stock concentration of 50–60 µg/mL in TBS. The automated Calypso method consisted of a dual-component “crossover” gradient to assess hetero-association between the GP and Fab. For each composition, a volume of 0.7 mL of protein solution was injected into the UV and MALS detectors until an equilibrium was reached within the MALS flow cell and the flow stopped for 300–800 s. Data were collected, and analyses were performed with CALYPSO software. GP₂–3A6 association was measured in triplicate with two different preparations of GP and Fab.

Protein assays

ELISA and a cell-based antibody-binding assay were performed with WT virus GPs or variants created via alanine scanning performed in accordance with standard protocols.

Cell-based antibody binding assay

To evaluate binding of mAbs to GP variants, HEK 293 T cells expressing full-length GP_1,2 or variants thereof were incubated with unlabeled mAbs at 10 μg/mL, followed by staining with DyLight 488 anti-human IgG and detection of fluorescence by microscopy. The binding of a control conformational mAb (ADI-15878)^9,45,46 was used as a control for GP expression levels. Secondary antibody binding alone was used as a negative control to assess background binding. In detail, HEK 293 T cells were plated at ≈1 × 10⁵ cells per well in 24-well plates treated with Poly-L-lysine solution (Millipore Sigma) 1 d prior to transfection. Cells were transiently transfected with 0.5 μg DNA per well using TransIT-LT1 transfection reagent (Mirus Bio, Madison, WI, USA). At 48 h post-transfection, cells were fixed with 4% paraformaldehyde (PFA; Electron Microscopy Sciences, Hatfield, PA, USA) in DPBS for 20 min. Cells were then incubated for 1 h at room temperature with 10 μg/mL primary mAbs in DPBS supplemented with 1% bovine serum albumin (BSA; Millipore Sigma). Cells were subsequently incubated at room temperature for 1 h with DyLight 488 anti-human IgG secondary antibody (SA5-10126; Thermo Fisher Scientific) and Hoechst 33342 (Invitrogen) in DPBS supplemented with 1% BSA. Finally, cells were imaged on a widefield fluorescence Axiovert 200 M Marianas microscope with a 10x/0.3 dry objective (ZEISS, Feasterville, PA, USA) or a confocal LSM780 microscope with a 10x/0.3 dry objective (ZEISS). Images were analyzed in QuPath⁴⁷. Nuclei were segmented using the Hoechst image, and the objects were expanded by 5 μm to locate approximate cell boundaries. Anti-human IgG positive and -negative cells were counted using a trained object classifier. The classifier was optimized for the widefield and confocal images separately. Then, all data from three biological replicates were combined, and the 3A6-positive cell percentage was normalized against that obtained with ADI-15878.

ELISA

Microtiter plate wells were coated with purified recombinant WT or mutant GP_{1,2ΔTM/ΔMLD} and incubated at room temperature for 1 h before blocking with 3% BSA (Millipore Sigma) in DPBS containing 0.05% TWEEN-20 (Thermo Fisher Scientific) for 1 h. Serial dilutions of mAb were applied to the wells and incubated for 1 h at room temperature. The bound antibodies were detected using Jackson Immuno Research Labs peroxidase-conjugated goat anti-human IgG (#109036006; Thermo Fisher Scientific) with horseradish peroxidase (diluted 1:4000) and 3,3’,5,5’-tetramethylbenzidine (TMB) substrate (Thermo Fisher Scientific) before 50 µL of 1 N sulfuric acid solution (Thermo Fisher Scientific) was added to stop the reaction. Absorbance at 450 nm was then measured using a Spark microplate reader (Tecan, Männedorf, Switzerland). Half-maximal response (EC₅₀) values for mAb binding were determined using Prism 7 (GraphPad Software, Boston, MA, USA) after log-transformation of antibody concentrations using EC₅₀ shift nonlinear regression analysis.

Plaque reduction assay using biologically contained EBOV

A biologically contained EBOV, EbolaΔVP30 virus²¹, was used to assess the impact of a P636S change on 3A6-mediated neutralization as previously described by Davis et al.¹¹. Briefly, Ebola-GP-P636SΔVP30-eGFP virus was incubated with 10 μg/mL of mAb at 37 °C for 60 min. The virus/mAb mixture was then inoculated onto Vero VP30 cells, seeded the previous day in 12-well plates. After 60 min of incubation, cells were washed to remove any unbound virus and overlaid with 1.25% methylcellulose media to allow for plaque formation. Seven days after infection, the number of plaques was quantified after immunochemistry staining with an antibody against the VP40 protein.

Negative-stain electron microscopy

GP₂–3A6 complexes were obtained by incubating GP_{1,2ΔTM/ΔMLD} with a three-fold molar excess of 3A6 overnight followed by purification using a Superdex 6 Increase 10/300 GL SEC column. The complexes were diluted to 0.01 mg/mL, and a volume of 4 μL of the complex solutions was applied to each freshly plasma-cleaned carbon-coated 400-mesh copper grid (Electron Microscopy Sciences) for 1 min. The solutions were blotted from the grids, followed by staining with 1% uranyl formate (Electron Microscopy Sciences) for 1 min. The stain was blotted from the grids, which were then air-dried before imaging. Images were collected on an FEI Titan Halo 300 kV electron microscope (Thermo Fisher Scientific) at a magnification of ×57,000 with a Falcon II camera. Contrast transfer function (CTF) correction, particle picking, 2-dimensional class averaging, and 3-dimensional reconstruction and refinement were all performed using cryoSPARC⁴⁸.

Virion-like particle preparation, purification, and visualization

EBOV VLPs were prepared from HEK 293 T cells by co-expression of full-length GP_1,2 and VP40 essentially as previously described by Wan et al.⁴⁹, except that Gibco PBS (Thermo Fischer Scientific) was used instead of tris + NaCl + ethylenediaminetetraacetic acid (EDTA) (TNE) buffer. Clarification of supernatants from four 150 mm dishes was performed at 3000 × g for 15 min at 4 °C. After density gradient purification, pellets were resuspended in 200 µL Gibco PBS.

Cryogenic electron tomography

Sample preparation, data collection, and tomogram reconstruction

Fabs (1 mg/mL) were mixed with purified VLPs and 10 nm colloidal gold and incubated for 30–60 min at 4 °C. Different combinations of Fabs were prepared, imaged, and processed in parallel. The different mixtures (4 µL) were added to C-Flat 2/2 EM grids (Protochips, Morrisville, NC, USA) and vitrified by back-side blotting (4-s blotting time) using a LeicaGP cryo plunger (Leica, Deerfield, IL, USA) and stored in liquid nitrogen until imaging.

Cryogenic electron tomography data collection was performed essentially as previously described by Ke et al.⁵⁰ on a Titan Krios electron microscope equipped with Gatan Bioquantum energy filter and K3 direct electron camera (Thermo Fisher Scientific). The nominal magnification was 64,000×, giving a pixel size of 1.386 Å on the specimens. The defocus range was −2.0 to −4.5 μm, with a 0.25 µm step size (Supplementary Table 4).

Subtomogram averaging

Tomograms were reconstructed using IMOD⁵¹, and the initial steps of subtomogram alignment and averaging were implemented using MATLAB (MathWorks) scripts and subTOM package (https://github.com/DustinMorado/subTOM/releases/tag/v1.1.0), which were derived from the TOM⁵² and AV3⁵³ packages as previously described by Ke et al.⁵⁰.

To generate an initial starting model for each structure, 50–100 copies of GP were manually identified from VLP filaments that were down-scaled by 6× binning of the voxels and subjected to reference-free subtomogram alignment (Supplementary Fig. 5A). To identify the positions of all the particles on the surface of the VLP filaments, markers were placed manually along the filaments within the tube using the Volume Tracer function in UCSF Chimera (v.1.13.1)⁵⁴. The radii of the filaments were determined centered at the membrane using the Pick Particle Chimera Plugin⁵⁵. An oversampled cylindrical grid of points was generated on the filament surfaces with ≈8 nm of spacing, and subtomograms were extracted for all grid points with a box size of 64 pixels (≈50 nm) centered at a radius 10 nm above these grid positions. Initial Euler angles were assigned to each subtomogram based on the orientation of the normal vectors relative to the cylinder surface. The oversampled positions were further aligned in the subTOM package. Duplicated particles and poorly aligned particles were removed from further analysis.

Subsequent processing was performed in RELION⁵⁶, as previously described by Ke et al.⁵⁰. Our reported averages are the primary average structures for each sample. We used a tight mask to obtain a higher resolution of the GP structure, while a cylindrical mask including the membrane-proximal region to obtain a better overview map including the membrane region where the Fab fragment is located (Supplementary Fig. 5B). The VLP GP_1,2–3A6 structure was refined to 17.7 Å with 9602 particles from 26 tomograms; the VLP GP_1,2–KZ52 structure was refined to 8.9 Å (tight mask) and 12.7 Å (cylinder mask) with 13,520 particles from 18 tomograms; and the VLP GP_1,2–KZ52/3A6 structure was refined to 7.4 Å (tight mask) and 9.3 Å (cylinder mask) with 40,072 particles from 42 tomograms (Supplementary Fig. 5C, Supplementary Table 4). Contours shown in Figs. 3 and 4 were normalized by eye so that the GP_1,2 densities are as equivalent as possible between different maps.

AlphaFold 2 modeling

We used ColabFold²⁵ to generate complete structures of the EBOV GP using the AlphaFold 2 multimer protocol²⁴. Four independent analyses were performed to generate a total of 20 models. Two experiments used previously solved structures of EBOV GP (PDB 6VKM or 5JQ3) as a template for modeling and two experiments used the PDB100 as templates for modeling.

Alanine scanning and antibody binding test

Alanine scanning was performed by introducing single alanine residues (alanines were changed to serines) into GP_1,2ΔMLD region 627–639 via site-directed mutagenesis of GP_1,2ΔMLD-encoding plasmid. The plasmid clones were individually arrayed into 384-well plates and transfected into HEK 293T cells. Protein variants were cell-surface-expressed for 22 h²⁰. The indicated mAbs were incubated with the cells for 1 h before an Alexa Fluor 488-conjugated secondary antibody (Thermo Fisher Scientific) was added. Antibody binding was assessed by detection of cellular fluorescence with a high-throughput flow cytometer (Intellicyt, Albuquerque, NM, USA). Background fluorescence was measured in vector-transfected control cells and mAb reactivity against the variants was calculated with respect to reactivity with GP_1,2ΔMLD by subtracting the signal from mock-transfected controls and normalized to signals from WT GP_1,2ΔMLD-transfected controls. Residues predicted to be involved in the epitope were identified when mAb and variant did not react. But, when reactivity of other control mAbs was observed, GP variants that were misfolded or were expressed at low levels were excluded.

Animal studies

Animal exposure and treatment experiments using infectious EBOV were performed in the biosafety level 4 (BSL-4) laboratory at the Integrated Research Facility at Fort Detrick (IRF-Frederick), Division of Clinical Research (DCR), National Institutes for Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH) under accreditation (000777) by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC), Laboratory Animal Welfare approval (D16-00602) by the Public Health Service (PHS), and United States Department of Agriculture (USDA) registration (51-F-0016). Animal experiments were approved by the NIAID DCR Animal Care and Use Committee (ACUC) and followed the recommendations provided in the Guide for the Care and Use of Laboratory Animals.

Domesticated guinea pigs

Hartley strain domesticated guinea pigs (Cavia porcellus (Linnaeus, 1758)) of both sexes, ages 6–8 weeks (Charles River Laboratories, Wilmington, MA, USA), were assigned to five groups of six animals (three males and three females) each. All animals were exposed IP to 1000 PFU of domesticated guinea pig-adapted Ebola virus/UTMB/C.porcellus-lab/COD/1976/Yambuku-Mayinga-GPA on Day 0. Animals in each group were injected IP on Day 3 with 5 mg of 3A6, 1A2, 7G7, or 42-2D2 anti-FLUAV IgG in 3 mL of DPBS or administered no treatment. Animals were observed daily for clinical signs of disease and were assigned a clinical score of 0–3 (0=none; 1=mild; 2=moderate; 3=severe). Animals reaching endpoint criteria (score of 3) were humanely euthanized. Weight was recorded daily starting 1 d before exposure until all animals recovered from disease (Day 15), then twice weekly until the study endpoint on Day 28. Blood was collected twice, on Day 6 after exposure and at the time of euthanasia.

Rhesus monkeys

Four rhesus monkeys (Macaca mulatta (Zimmermann, 1780)) of both sexes, aged 4–5 years (WorldWide Primates, Miami, FL, USA) were single-housed and acclimated to BSL-4 conditions prior to virus exposure. On Day 0, monkeys were sedated using IM injection of 15 mg/kg of Ketamine HCl (KetaThesia; Henry Schein, Melville, NY, USA) and exposed IM with a target dose of 1000 PFU of Ebola virus/H.sapiens-tc/COD/1995/Kikwit-9510621 (NR-50306, Lot 9510621, ≥95% 7U abundance at the GP editing site; BEI Resources, Manassas, VA, USA); the same dose and lot of this virus previously resulted in fatal outcome at 5–8 d post-exposure in 12 out of 12 untreated rhesus monkeys (“historical controls”⁵⁷). On Day 4 and Day 7 after exposure, monkeys 1–3 received 25 mg/kg of 3A6 in DPBS (provided by Chakravarthy Reddyvia) by intravenous infusion, and the control monkey received an equivalent volume of DPBS. All monkeys were observed for the development of clinical signs of EBOV infection and scored daily according to a four-point scoring scale. Physical examination and blood collection were conducted on the monkeys twice prior to exposure (Day -14 and Day -7) and at 4, 7, 9, 12, 21, and 28 d after exposure. Complete blood counts with reticulocytes and differential was analyzed on a Sysmex XT-2000iV hematology instrument (Sysmex America, New York, NY, USA). Sera were obtained after separation at room temperature and centrifugation for 15 min at 1500 × g followed by analysis using the Piccolo general chemistry 13 panel on a Piccolo Xpress analyzer (Abaxis, NJ, USA). Prothrombin and activated partial thromboplastin times were measured on a CS-2500 system automated coagulation analyzer (Sysmex America). Infectious titers were determined in sera using an Avicel-based crystal violet stain plaque assay on Vero E6 cell culture monolayers (BEI Resources) as previously described by Shurtleff et al, 2012⁵⁸. Sera were inactivated in TRIzol LS (Thermo Fisher Scientific) according to the manufacturer’s instructions and nucleic acid extracted using the QIAamp Viral RNA Mini Kit (Qiagen, Germantown, MD, USA). The Critical Reagents Program (CRP) EZ1 RT-PCR kit assay (BEI Resources) was used in accordance with manufacturer’s instructions³³ on an Applied Biosystems 7500 FastDx Real-Time PCR instrument (Thermo Fisher Scientific) to quantify EBOV nucleic acids in sera and to transform results into log₁₀ genome equivalents (GE) per mL of sample. The control monkey succumbed on Day 8 whereas the treated monkeys underwent elective euthanasia approximately 3 mo after virus exposure.

Statistical analysis

Statistical details of experiments, including numbers of replicates and measures of precision (standard deviation, SD), can be found in the figure legends, figures, results, and methods. Dose-response ELISA curves were fit to a EC₅₀ shift by nonlinear regression analysis. All analyses were performed with Prism 7.

Resource availability

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Erica Ollmann Saphire (erica@lji.org).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

Structure factors and associated model coordinates generated in this study have been deposited in the Protein Data Bank (PDB; http://www.rcsb.org) under PDB accession codes 7RPU (3A6–stalk–MPER, https://doi.org/10.2210/pdb7RPU/pdb) and 7RPT (apo3A6, https://doi.org/10.2210/pdb7RPt/pdb). Tomographic reconstructions have been deposited in the Electron Microscopy Data Bank (EMDB; https://www.ebi.ac.uk/emdb/) under accession codes EMD-45833, EMD-45834, EMD-45835, EMD-45836 https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-45836), and EMD-45837.

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Acknowledgements

We are grateful to Dustin Morado, Kun Qu, and Joaquin Oton (Medical Research Council Laboratory of Molecular Biology) for support in collecting and/or processing cryogenic electron tomography data and to Stanford Synchrotron Light Source (SSRL) and Advanced Photon Source (APS) for assistance in collection of X-ray data. We thank Dawid Zyla (La Jolla Institute for Immunology) for assistance in the generation of AlphaFold models. We thank Anya Crane (National Institutes of Health [NIH], National Institute of Allergy and Infectious Diseases [NIAID], Integrated Research Facility at Fort Detrick, Frederick, MD, USA [IRF-Frederick]) for critically editing the manuscript and Jiro Wada (NIH NIAID IRF-Frederick) for creating figures. We gratefully acknowledge Peter B. Jahrling and Lisa E. Hensley (NIH NIAID IRF-Frederick) for supporting the studies involving animals. The work for this study was supported by NIH NIAID U19 AI142790 (E.O.S. and C.W.D.), DARPA contract W31P4Q-14-1-0010 (C.W.D. and R.A.), NIH NIAID contract HHSN272201400058C to B.J.D., and UK Medical Research Council (MC_UP_1201/16), the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (ERC-CoG-648432 MEMBRANEFUSION), and the Max Planck Society (J.B.). This research was further supported through the NIH/NIAID prime contract with Battelle Memorial Institute (Contract No. HHSN272200700016I) and subsequently with Laulima Government Solutions (Contract No. HHSN272201800013C). G.W. and M.R.H. performed this work as employees under these contracts. J.H.K. performed this work partly as an employee under the Battelle Memorial Institute contract and partly as an employee of Tunnell Government Services, formerly a subcontractor of Battelle Memorial Institute and now of Laulima Government Solutions. This work has been further funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261201500003I, Task Order No. HHSN26100043 and Contract No. 75N91019D00024, Task Order No. 75N91019F00130 (I.C.). S.M. was funded by an Imaging Scientist grant (2019‐198153) from the Chan Zuckerberg Initiative. Use of the SSRL SLAC National Accelerator Laboratory is supported by the US Department of Energy (DOE) Office of Science Basic Energy Sciences (BES) program under Contract No. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Biological and Environmental Research (BER) program and by the NIH National Institute of General Medical Sciences (NIGMS; P41GM103393). This research also used resources of the Advanced Photon Source (APS), a DOE Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory (ANL) under Contract No. DE-AC02-06CH11357. The General Medical Sciences and Cancer Institute Structural Biology Facility at the Advanced Photon Source (GM/CA) project has been funded in whole or in part with Federal funds from the National Cancer Institute (ACB-12002) and the NIGMS (AGM-12006). The Eiger 16M detector at the ANL X-ray Science Division (XSD) was funded by NIH grant S10 OD012289. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the U.S. Army. The funding sources were not involved in study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication. The views and conclusions contained in this document are those of the authors and should not be interpreted as necessarily representing the official policies, either expressed or implied, of the U.S. Department of Health and Human Services or of the institutions and companies affiliated with the authors, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

Funding

Open access funding provided by the National Institutes of Health.

Author information

Zunlong Ke
Present address: Department of Molecular Biosciences, the University of Texas at Austin, Austin, TX, USA
Xiaoli Xiong
Present address: Guangzhou Regenerative Medicine and Health—Guangdong Laboratory, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Science Park, Guangzhou, Guangdong Province, China
Xiaoying Yu
Present address: Arcturus Therapeutics, San Diego, CA, USA
Zhe Li Salie
Present address: Eli Lilly, San Diego, CA, USA
Michael J. Norris
Present address: Department of Biochemistry, University of Toronto, Toronto, ON, Canada
Ariadna Grinyó
Present address: Vall d’Hebron Institute of Oncology, Hospital del Mar Research Institute, Barcelona, Spain
These authors contributed equally: Zhe Li Salie, Zunlong Ke.

Authors and Affiliations

Center for Vaccine Innovation, La Jolla Institute for Immunology, La Jolla, CA, USA
Kathryn M. Hastie, Zhe Li Salie, Sharon L. Schendel, Chitra Hariharan, Michael J. Norris, Xiaoying Yu & Erica Ollmann Saphire
Division of Structural Studies, Medical Research Council Laboratory of Molecular Biology, Cambridge, UK
Zunlong Ke, Xiaoli Xiong & John A. G. Briggs
Department of Cell and Virus Structure, Max Planck Institute of Biochemistry, Martinsried, Munich, Germany
Zunlong Ke & John A. G. Briggs
Influenza Research Institute, Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI, USA
Peter J. Halfmann & Yoshihiro Kawaoka
Integrated Research Facility at Fort Detrick, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Fort Detrick, Frederick, MD, USA
Lisa Evans DeWald, Michael R. Holbrook, Jens H. Kuhn & Gabriella Worwa
Microscopy Core, La Jolla Institute for Immunology, La Jolla, La Jolla, CA, USA
Sara McArdle
Integral Molecular, Philadelphia, PA, USA
Ariadna Grinyó, Edgar Davidson & Benjamin Doranz
Department of Microbiology and Immunology, Emory Vaccine Center, Atlanta, GA, USA
Chakravarthy Chennareddy, Carl W. Davis & Rafi Ahmed
Zalgen Labs LLC, Frederick, MD, USA
Megan Heinrich & Luis M. Branco
Clinical Monitoring Research Program Directorate, Frederick National Laboratory for Cancer Research, Frederick, MD, USA
Ian Crozier
Division of Virology, Institute of Medical Science, University of Tokyo, Tokyo, Japan
Yoshihiro Kawaoka
The Research Center for Global Viral Diseases, National Center for Global Health and Medicine Research Institute, Tokyo, Japan
Yoshihiro Kawaoka
Pandemic Preparedness, Infection and Advanced Research Center (UTOPIA), University of Tokyo, Tokyo, Japan
Yoshihiro Kawaoka
Department of Medicine, University of California San Diego, La Jolla, CA, USA
Erica Ollmann Saphire

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Kathryn M. Hastie
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Zhe Li Salie
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Zunlong Ke
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Contributions

Conceptualization, K.M.H., Z.L.S., J.A.G.B., L.E.D., G.W., M.H.H., C.W.D., R.A., and E.O.S. Methodology, K.M.H., Z.L.S., Z.K., E.D., B.D., and E.O.S. Investigation, K.M.H., Z.L.S., Z.K., P.J.H., S.A. E.D., L.E.D., A.G., C.C., C.H., S.M., M.J.N., and X.X. Formal analysis, K.M.H., Z.L.S., X.Y., Z.K., J.A.G.B., L.E.D., G.W., E.O.S., Writing—Original Draft, Z.L.S. and E.O.S. Writing—Review & Editing, K.M.H., Z.L.S., Z.K., S.L.S., X.Y., I.C., J.H.K., J.A.G.B., G.W., R.A., and E.O.S. Visualization, K.M.H., Z.L.S, Z.K., X.Y., S.M. Supervision, B.D., Y.K., J.A.G.B., and E.O.S. Resources, B.D., M.H., L.M.B., I.C., and J.H.K. Funding Acquisition, J.A.G.B., C.W.D., R.A., and E.O.S.

Corresponding authors

Correspondence to Jens H. Kuhn, John A. G. Briggs, Gabriella Worwa, Carl W. Davis, Rafi Ahmed or Erica Ollmann Saphire.

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Competing interests

A.G., E.D., and B.J.D. are employees of Integral Molecular, and B.J.D. is a shareholder in that company. The remaining authors declare no competing interests.

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Animal exposure and treatment experiments using infectious EBOV were performed in registered an ethics accredited biosafety level 4 (BSL-4) laboratories with appropriate approvals.

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Hastie, K.M., Salie, Z.L., Ke, Z. et al. Anti-Ebola virus mAb 3A6 protects highly viremic animals from fatal outcome via binding GP_(1,2) in a position elevated from the virion membrane. Nat Commun 16, 1293 (2025). https://doi.org/10.1038/s41467-025-56452-2

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Received: 19 December 2023
Accepted: 17 January 2025
Published: 03 February 2025
DOI: https://doi.org/10.1038/s41467-025-56452-2