Fig 1.
Schematic diagram establishment of MERS-CoV nanobody screening library.
Immunized the alpaca with extracellular domains of MERS-CoV S protein and collected the blood after the final immunization to isolate PBMCs. Extracted RNA to synthesize cDNA via RT-PCR, and established phage screening library. After four rounds of bio-panning, the specific VHH coding sequences were confirmed from the selected positive clones and further assayed using candidate nanobodies. See also S1 Fig.
Fig 2.
Determination and Characterization of MERS-CoV S protein-specific Nbs.
(A). Characterized the affinity of Nbs bound to MERS-CoV S protein by Flow Cytometry. HEK-293T cells, which overexpressed the MERS-CoV S protein, stained by Nb9-Fc, Nb11-Fc, Nb14-Fc, and Nb67-Fc. HEK-293T cells were washed by PBS (Solarbio) and stained by anti-Fc-APC (Abcam). (B). The binding of purified Nbs-Fc with the MERS-CoV S1 subunit was identified using ELISA. Data represented as mean ± SD. (C). The Kinetic binding curves of the MERS-CoV S1 subunit with Nb9-Fc, Nb11-Fc, Nb14-Fc, and Nb67-Fc were detected by BLI. (D). Determination of the MERS-CoV S1 subunit recognition region of Nbs by ELISA. (E, F). Nb9, Nb11, Nb14, and Nb67 could effectively neutralize MERS-CoV pseudovirus in vitro. MERS-CoV pseudovirus was incubated with serially diluted Nbs and detected luciferase activity in Huh-7 cells (E) and Vero E6 cells (F), respectively. The neutralization potency of each Nbs was evaluated in a luciferase assay system. Nb22 was taken as a negative isotype control antibody (NC). Data are represented as mean ± SD from three independent experiments. All experiments were repeated at least twice. (G) Determination of the MERS-CoV S1 subunit recognition region of Nbs by Pull-Down. (H) The neutralization potencies of Nbs. The IC50 and IC90 were labeled orange and cyan, respectively. The intensity of the color indicates the strength of the neutralization activity. ELISA, enzyme-linked immunosorbent assay; BLI, Bio-Layer Interferometry; Nbs, nanobodies; MERS-NTD, N-terminal domain of MERS-CoV Spike glycoprotein; MERS-RBD, receptor-binding domain of MERS-CoV Spike glycoprotein; MERS-CoV, Middle East Respiratory Syndrome Coronavirus.
Fig 3.
Crystal structure and binding interface of Nb9 and Nb14 bound to MERS-CoV RBD.
(A, B). Crystal structures of Nb14 and Nb9 bound to MERS-CoV RBD, respectively. Receptor-binding subdomain, core subdomain, Nb14, and Nb9 are colored in wheat, gray, slate, and cyan, respectively. (C). Schematic illustration of MERS-CoV RBD topology. β strands are drawn as arrows and α helices are drawn as cylinders. (D, E). The interaction region of Nbs and RBD. (CDR1: yellow, CDR2: pink, CDR3: red, and CCL: slate). (F). The structure superimposition of Nb14-RBD and Nb9-RBD. (G, H, and I). Interactions between the residues of RBD and Nb14. The CCL recognized to the β4-β5 loop of RBD (G); The CDR2 of Nb14 was recognized with the β6-β7 loop of RBD (H); CDR3 bound to RBD (I). (J, K, and L). Interaction residues between RBD and Nb9. The CDR1 bound to the β6-β7 loop of RBD (J), the CDR2 bound to the β5-β6 loop of RBD (K), and the CDR3 recognized with β5-β6 loop and β7-β8 loop of RBD (L). CCL, Nb14 complementarity cage loop (FRs of Nb14 aa: G42-L47); CDR, complementarity determining region.
Fig 4.
Confirmation of the neutralizing epitopes.
(A). Diverse epitopes of Nb14 and Nb9. Nb14 epitope is slate and the Nb9 epitope is cyan. (B, C). Epitopes on the RBD recognized by Nb14 and Nb9, respectively. The pseudovirus of MERS-CoV variants identified from the Nb14 and Nb9 epitopes were tested to evaluate the neutralization potency conferred by Nb14 (D) and Nb9 (E), respectively. The names of MERS-CoV variants with an amino acid mutation based on the wild type (WT) of MERS-CoV are indicated. The y-axis shows the ratio of IC50 of indicated variant/IC50 of WT conferred by Nb, and N.D. means not detected. Data are represented as mean ± SD from three independent experiments.
Fig 5.
Neutralizing effects of Nb9 and Nb14 on different MERS-CoV strains.
(A). The binding modes of Nb14 (slate) and Nb9 (cyan) are binding modes to prototype MERS-CoV RBD with the natural mutation sites highlighted in red. (B, C). The epitopes of Nb14 (slate) and Nb9 (cyan) on the surface of MERS-CoV RBD, relative to relevant natural mutation sites highlighted in red. (D, E). Neutralizing potencies of Nb14 and Nb9 against pseudoviruses bearing the natural mutations, respectively. Data are presented as the means ± SD from three independent experiments. (F). Summary of Nb14 and Nb9 neutralizing activities. IC50 neutralization titers for natural mutant variants are presented relative to wild type (WT) of MERS-CoV pseudoviruses.
Fig 6.
Mechanism of Nb14 and Nb9 with different neutralizing epitopes.
(A). Division of diverse class epitope regions of MERS-CoV RBD, and model structures of hDPP4 (PDB, 4L72; yellow), Nb14 (slate), and Nb9 (cyan) bound to MERS-CoV RBD. (The epitope of class 1: green; class 2: red; and class 3: black; see also in S9 Fig). (B). Competitive bindings of monomeric Nb14 and Nb9 with RBD to hDPP4 were measured by Huh-7 cell surface staining. Huh-7 cells were incubated with RBD and Nb14 or Nb9, followed by staining with anti-His-APC (RBD) and analyzed by FACS. The cell staining was repeated, and one representative result was shown. (C, D, and E). Neutralization mechanism of Nb14. Structural superimposition demonstrates that the epitope of Nb14 is distinct from the hDPP4 binding site, but clashes with the N229 glycan of hDPP4 recognized to the RBD (C). The N229 glycan shows the sticks with green (PDB, 4L72). (D). The CDR2 of Nb14 connected to RBD with a tight and wide range interface and formed a cavity that adapted to the W535 of RBD. (E). The conformation changes of W535 and D539 in different state RBD. MERS-CoV RBD (cyan), Nb14-RBD (wheat), and hDPP4-RBD (yellow). (F, G, and H). Structure definition of the neutralizing mechanism of Nb9. Comparison structure superimposition shows conformational change of the RBD β5-β6 loop in the Nb9-Bound State (F). Nb9 clashes with hDPP4 (red labeled), and the β5-β6 loop exhibits an inward curved conformation to prevent the approach of the hDPP4 short helix when bound to RBD. MERS-CoV RBD (cyan), Nb9-RBD (wheat), MERS-4V2 scFv (green), and hDPP4-RBD (RBD, Magenta; hDPP4, yellow; PDB, 4L72). (G) Zoom-in view of the aligned RBD β5-β6 loops in unbound (4KQZ: cyan; 4L3N: orange) and hDPP4-bound (4L72: Magenta; 4KR0: yellow) with the Nb9 bound (wheat) state. (H). The residues of L506, D510, and E513 from the RBD β5-β6 loop exhibit conformational changes when bound to Nb9, interrupting the interaction between hDPP4 and RBD. In hDPP4-RBD (PBD, 4L72), the residues show with yellow; and in Nb9-RBD, the residues show with wheat.
Fig 7.
Structural superimpositions of the hDPP4-RBD, Nb14-RBD, and Nb9-RBD crystal structures onto the MERS-CoV S trimers glycoprotein in Receptor-Binding inactivated and activated states.
(A). MERS-CoV S trimers in an inactivated state with all RBD in the “down” positions (PDB: 5w9j). One S protomer is shown as a cartoon (RBD in red, NTD in green, and S2 subunit in orange). (B, C, and D). Structure superimpositions of the hDPP4-RBD (B), Nb14-RBD (C), and Nb9-RBD (D), onto inactivated MERS-CoV S trimers, respectively. RBD bind to Nb14 in the inactivated state without a steric clash, but not hDPP4, and Nb9. (E). MERS-CoV S trimers in activated state with one RBD in the “up” positions (PDB: 5w9h). (F, G, and H). Structure superimpositions of the hDPP4-RBD (F), Nb14-RBD (G), and Nb9-RBD (H), onto activated MERS-CoV S trimers, respectively.
Fig 8.
Synergistic neutralization effects of nanobodies Nb9 and Nb14.
(A). The assembly of ternary complexes by Nb9, Nb14, and RBD is purified by size exclusion chromatography (SEC). (B). FACS analysis of Nb14 cooperated with Nb9 to inhibit RBD bound to Huh-7 cells. Huh-7 cells were incubated with RBD and then incubated with Nb9 or Nb9+Nb14, followed by staining with anti-His-APC (RBD) and analyzed by FACS. The combination rate and inhibition rate of RBD to Huh-7 cells significant statistical differences were analyzed by GraphPad Prism 9. Data are represented as mean ± SD from three independent experiments. (C). Neutralizing effects of Nb14 combined with Nb9 against pseudotyped MERS-CoV. Nbs were diluted 3-fold and tested alone or in combination to calculate percent neutralization and then combined Nbs at constant ratios. The constant ratios of the combined Nbs were their IC50s, and the x-axis means a dose of 1 was at the IC50 concentration. Fractional effect (FA) plots were generated by the CompuSyn program and showed a combination of dosage versus effect. Median effect plot of calculated CI values (logarithmic) versus FA values, in which a log CI of <0 is synergism and a log CI of >0 is antagonism. The data shown are average values from three independent experiments.