The improved glycoprotein (GP) of EBOV was named GP-ΔM. Alpacas were immunized with this protein, and peripheral blood mononuclear cells (PBMCs) were collected to construct a phage display nanobody library. Six types were identified using this protein for screening, among which Nanosota-EB1 and -EB2 had the highest affinity for this protein. EB1 and EB2 (EB1 His and EB2 His) with HIS tags were expressed and purified in a bacterial expression system, and EB1 and EB2 (EB1 Fc and EB2 Fc) with Fc were expressed and purified in mammals. Figure 1A evaluates the binding affinity of His-labeled nanobodies to GP-ΔM using surface plasmon resonance (SPR), showing high-affinity binding. Figure 1B shows EB1 His and EB2 His binding to the cleaved extracellular domain of GP (GPcl) and the secreted form of GP (sGP), respectively. EB1-His binds to sGP but not to GPcl, while EB2 His does the opposite. Figure 1C shows the entry of EBOV pseudovirus into human cells in the presence of two types of nanobodies. EB1 Fc and EB2 Fc neutralize EBOV pseudovirus entry with moderate and high potency. Figure 1D tests the effect of Fc-labeled nanobodies on true EBOV infection in human cells; EB1 Fc and EB2 Fc neutralize true EBOV infection with low efficiency and high efficacy. 

Fig 1: In Vitro Characterization of Two Novel Anti-EBOV Nanobodies, Nanosota-EB1 and -EB2.

As shown in Figure 2A, the EBOV GP-ΔM (modified GP) trimer consists of three subunits: orange, gray, and green, with two Nanosota-EB1 molecules (blue) bound to it. Figure 2B further analyzes the binding mode: Nanosota-EB1 specifically targets the polysaccharide cap region of EBOV GP (shown in blue), where there are protease cleavage sites (marked by red circles) nearby. Research has shown that EB1 may inhibit the cleavage of host tissue proteases at this site by stabilizing the conformation of the polysaccharide cap, thereby blocking the critical step of virus entry into host cells. The fine structure of the binding site is shown in Figure 2C. Nanosota-EB1 directly interacts with the β 17 chain of the polysaccharide cap while pushing the adjacent β 18 chain away from its original position, inducing it to form a unique cyclic structure. This structural rearrangement may interfere with the subsequent function of viral glycoproteins through steric hindrance effects or conformational locking mechanisms. 

Fig. 2: Structural Basis for the Anti-EBOV Functions of Nanosota-EB1.

A trimer of EBOV binds to three EB2 molecules (Figure 3A), with the binding site (red) being the fourth epitope of GP (HR1, fusion ring, N-terminus of GP2, and β 1/β 2 chains of GP1). The CDR region of EB2 strongly interacts with the N-linked polysaccharide on Asn563 (N563 polysaccharide) of HR1, which is crucial for the stability and membrane fusion function of GP2. 

Fig 3: Structural Basis for the Anti-EBOV Functions of Nanosota-EB2. 

Subsequently, biochemical analysis, efficacy validation, stability evaluation, and other studies showed that EB2 exhibited strong neutralizing activity against EBOV infection in vitro and provided strong protection in a mouse model of EBOV attack, while Nanosota-EB1 provided moderate levels of neutralization and protection, making it a validated effective nanobody and providing potential therapeutic mechanisms for nanobody therapy against EBOV. 

Alpha Lifetech's nanobody development platform provides complete and efficient nanobody production services. We have an animal immunization platform that lays the foundation for the preparation of raw materials for nanobodies. We have a comprehensive phage display library construction and screening system, with multiple phage display systems to provide high-quality conditions for rapid production of nanobodies. We also have an antibody purification platform, affinity determination platform, antibody validation, and characterization platform, which provide more advantages for the later validation of nanobodies. A complete system, fast cycle, and high-quality service help ensure the smooth progress of the project.

Bu F, Ye G, Morsheimer K, Mendoza A, Turner-Hubbard H, et al. (2024) Discovery of Nanosota-EB1 and -EB2 as Novel Nanobody Inhibitors Against Ebola Virus Infection. PLOS Pathogens 20(12): e1012817. https://doi.org/10.1371/journal.ppat.1012817