Nanobody-Based Bispecific Antibody Delivers Broad-Spectrum Protection Against Multiple Ebolavirus Species
核心洞察
Researchers identified two camelid-derived nanobodies, 1A10 (搜索) and BA2 (搜索), that potently neutralize EBOV, SUDV, and BDBV, with each conferring 100% protection against EBOV in murine challenge models.
An engineered bispecific antibody, BA2-1A10 (搜索), achieved IC50 values of 1.9 nM, 2.21 nM, 0.81 nM, and 1.68 nM against EBOV, BDBV, SUDV, and TAFV, respectively, with a 20-fold potency gain against SUDV.
Cryo-EM structures revealed that 1A10 (搜索) and BA2 (搜索) bind adjacent, non-overlapping epitopes within the conserved internal fusion loop, enabling synergistic neutralization and a higher genetic barrier to viral escape.
A team of researchers has engineered a nanobody-based bispecific antibody that provides broad-spectrum protection against multiple species of Ebolavirus, addressing a critical unmet need in the treatment of Ebola virus disease (搜索) (EVD). The work, published in Nature Communications, identifies two naturally occurring single-domain antibodies—1A10 (搜索) and BA2 (搜索)—that potently neutralize Zaire ebolavirus (搜索) (EBOV), Sudan ebolavirus (搜索) (SUDV), and Bundibugyo ebolavirus (搜索) (BDBV), and demonstrates that a bispecific construct combining the two offers enhanced potency and a higher genetic barrier to viral escape.
The Ebola virus glycoprotein (搜索) (GP), the sole viral protein exposed on the virion surface, serves as the principal mediator of viral entry and a key determinant of pathogenicity. GP biosynthesis yields two functionally distinct subunits—the receptor-binding GP1 and the fusion-mediating GP2—that assemble into metastable trimeric spike complexes. Within the endosomal compartment, host cathepsins B and L cleave GP1 to unmask the receptor-binding site, which then engages the endosomal receptor Niemann–Pick C1 (NPC1), triggering membrane fusion.
A persistent threat with limited therapeutic options
Ebolavirus has caused recurrent outbreaks of viral hemorrhagic fever with high case fatality rates, with EBOV associated with case fatality rates approaching 90% in the absence of supportive care. In 2022, the Democratic Republic of the Congo experienced its 14th EVD outbreak, while Uganda reported an outbreak driven by SUDV that resulted in 164 cases (142 confirmed and 22 probable) and 55 deaths across nine districts.
Two antibody-based therapeutics—Ebanga (mAb114) and Inmazeb (REGN-EB3)—have received regulatory approval from the U.S. Food and Drug Administration. However, both are primarily efficacious against EBOV and show limited or no activity against SUDV and BDBV, species that also retain epidemic potential. Moreover, conventional full-length IgG antibodies (~150 kDa) are constrained by poor permeability across the blood–brain barrier, which hampers efficacy during late-stage infection when viral dissemination to immune-privileged sites is common. Consequently, even with antibody therapy, case fatality rates can remain as high as 30%.
Isolation of broad-spectrum nanobodies
To generate natural broad-spectrum nanobodies, the researchers expressed and purified the extracellular domains of glycoproteins from SUDV, BDBV, and EBOV, each with a truncated mucin-like domain (dMLD-GP). Camels were immunized with these antigens, and two independent phage display libraries (each with a complexity of ~10⁸ CFU) were constructed. An iterative biopanning approach alternating antigen exposure among the three GPs yielded 102 unique nanobody clones with confirmed GP reactivity.
Initial characterization using pseudotyped recombinant vesicular stomatitis viruses expressing ebolavirus glycoproteins revealed five cross-reactive nanobodies (1A10 (搜索), BA2 (搜索), BC2, ZC2, ZH7). Notably, 1A10-Fc and BA2-Fc exhibited the broadest neutralization profiles, with activity against all three pseudotyped viruses (IC50 = 1.5–30 nM). Among all antibodies, only BA2-Fc exhibited potent neutralization against Reston ebolavirus (RESTV), with an IC50 of 2.3 nM. These findings were validated using authentic viruses in plaque reduction neutralization tests (PRNT) conducted in biosafety level 4 (BSL-4) facilities, where both assay platforms consistently identified 1A10 and BA2 as the most broadly neutralizing nanobodies.
Mechanisms of neutralization
Surface plasmon resonance (SPR) analysis revealed distinct binding behaviors. 1A10 (搜索)-Fc bound to uncleaved GP-dTM with nanomolar affinity (KD = 1.62 nM) but exhibited picomolar affinity for cleaved GP-CL (KD = 7.51 pM), reflecting an approximately 200-fold enhancement. In contrast, BA2 (搜索)-Fc maintained consistent nanomolar affinities for GP-dTM, GP-dMLD, and GP-CL (KD = 2.80 nM, 2.00 nM, and 1.48 nM, respectively), indicating its ability to recognize both pre- and post-cleavage states of GP.
Competitive ELISA assays showed that 1A10 (搜索), but not BA2 (搜索), partially competed with NPC1 for GP-CL binding, suggesting that 1A10 may inhibit viral entry by blocking receptor engagement. Additionally, thermolysin cleavage assays demonstrated that 1A10 delayed GP proteolytic processing in a time-dependent manner, whereas BA2 had negligible impact on proteolysis. Collectively, these findings indicate that 1A10 acts through a multifaceted mechanism—interfering with cell attachment, GP proteolytic processing, blocking NPC1 binding, and potentially disrupting membrane fusion—while BA2 primarily neutralizes through inhibition of cellular adhesion and membrane fusion.
Structural basis for broad neutralization
Cryo-EM structures of EBOV GP-dMLD in complex with 1A10 (搜索) and BA2 (搜索) were determined at 2.56 Å and 2.96 Å resolution, respectively. In the GP-dMLD/1A10 complex, three 1A10 copies bound simultaneously to the GP trimer, with binding sites centered at the internal fusion loop (IFL) base and spanning both GP1 and GP2. 1A10 buried approximately 456 Ų of surface area on GP1 and approximately 396 Ų on GP2.
In the GP-dMLD/BA2 (搜索) structure, three copies of BA2 bound to the GP trimer at the highly conserved hydrophobic fusion peptide of the IFL, with the footprint spanning two adjacent GP monomers. Each BA2 buried approximately 536 Ų of surface area on one GP protomer and approximately 164 Ų on another.
The structures revealed that 1A10 (搜索) and BA2 (搜索) recognize distinct yet overlapping conserved epitopes within the IFL (residues 510–551), a critical functional domain for membrane fusion. The key interacting residues are highly conserved across the genus of ebolavirus, providing a structural explanation for the broad neutralization potency of both nanobodies.
Engineering a bispecific antibody
To enhance neutralization potency and broaden activity, the researchers engineered a bispecific antibody by genetically linking the variable domains of BA2 (搜索) and 1A10 (搜索) in tandem with a flexible glycine-serine linker, followed by an IgG-Fc domain (BA2-1A10 (搜索)-Fc). PRNT assays demonstrated that the engineered bispecific antibody achieved IC50 values of 1.9 nM, 2.21 nM, 0.81 nM, and 1.68 nM against EBOV, BDBV, SUDV, and Tai Forest ebolavirus (TAFV), respectively. Compared to 1A10-Fc alone, the bispecific antibody showed approximately 3.6-fold, 3-fold, 38-fold, and 5.7-fold improvements in neutralization efficacy against these viruses.
Viral escape assays revealed a notable advantage of the bispecific format. In VSV-SUDV infections, the monospecific antibodies 1A10 (搜索)-Fc and BA2 (搜索)-Fc each acquired two mutations (E106K/L122F and K155Q/L122F, respectively), with E106K and K155Q located within the respective antibody binding sites. In contrast, no mutations were detected in viruses passaged under bispecific antibody pressure, suggesting that simultaneous targeting of two conserved GP epitopes imposes a higher genetic barrier to viral escape.
In vivo protective efficacy
In a mouse model challenged with a lethal dose of mouse-adapted Ebola virus (MA-EBOV), a single administration of either BA2 (搜索)-Fc or 1A10 (搜索)-Fc (5 mg/kg, intraperitoneally) at 1 day post-infection conferred 100% protection from death, with no signs of weight loss. In contrast, PBS-treated mice exhibited only 17% survival. When treatment was delayed to 2 days post-infection, BA2-Fc and 1A10-Fc still achieved 100% protection, while PBS-treated animals exhibited 100% mortality.
In a Syrian hamster model of SUDV infection, administration of either 1A10 (搜索)-Fc or BA2-1A10 (搜索)-Fc (5 mg/kg at 1 day post-infection) provided 100% protection against mortality and weight loss, with animals treated with the bispecific antibody exhibiting continuous weight gain indicative of rapid recovery. In IFNAR⁻/⁻ mice challenged with recombinant VSV expressing BDBV glycoprotein, a single dose of BA2 (搜索)-Fc, 1A10-Fc, or BA2-1A10-Fc (25 mg/kg) administered at 1 day post-infection conferred 100% protection from death.
The authors note that while non-human primate models are the most predictive system for EVD, practical and biosafety constraints precluded their use in the present study. The robust protection conferred across EBOV, SUDV, and BDBV challenge systems nonetheless provides strong justification for subsequent evaluation in non-human primates, which will be essential to fully assess pharmacokinetics, dosing strategies, and therapeutic window in a translational context.
The researchers conclude that the biparatopic nanobody combines high expression yields (20 mg/L in HEK293F cells), excellent solubility, and robust protective efficacy, solidifying its potential as a leading broad-spectrum therapeutic candidate. T.C.T., M.H.W., S.C., and E.T.L. are named inventors on two patents covering the sequences of the 1A10 (搜索) and BA2 (搜索) nanobodies described in the study.