Binding activity of mAb AF-03 to MARV GP and its epitopes.

(A) AF-03 and MARV GP proteins are examined by SDS-PAGE. NR, non-reducing; R, reducing. (B) The binding capacity of AF-03 to MARV GP is determined by ELISA. The absorbance is detected at 450 nm. (C) The binding kinetics of AF-03 to MARV GP is detected by SPR assay. Experiments are independently repeated at least three times, and the data from one representative experiment is shown. (D) The 3-D ribbon structures of the AF-03 Fv fragment. The red ribbon denotes H-CDR1, the light blue denotes H-CDR2, the pink denotes H-CDR3, the orange denotes L-CDR1, the deep blue denotes L-CDR2, the purple denotes L-CDR3. (E) AF-03 and MARV GP complex derived from theoretical modeling. The green ribbon denotes the orientation of the MARV GP fragment, the yellow denotes AF-03 VLCDR, the pink denotes AF-03 VHCDR, the deep blue denotes AF-03 VL and the red ribbon denotes AF-03 VH. (F) By molecular docking analysis of van der Waals interaction, intermolecular hydrogen bonding, polarity interaction and electrostatic interaction, the key amino acid residues of MARV GP are screened.

AF-03 Epitope Identification.

(A) The neutralization activity of AF-03 or MR78 to mutated pseudovirus (Q128S-N129S, Q204A-T205A-Q206A, Y218A, K222A, C226Y) is evaluated in HEK293T cells. The inhibition rate is analyzed. (B) The binding of AF-03 and MR78 to mutant GP (Q128S-N129S or C226Y) is examined by ELISA respectively. (C) Secondary structure of MARV GP and mutants are detected by CD. (D) The epitope overlapping between AF-03 and MR78 is examined by the competitive ELISA.

In vitro and in vivo neutralization of MARV pseudovirus infection by AF-03.

(A) Pseudotypic MARV-Uganda is incubated with AF-03, MR78 or control mAb at 37°C for 1 h before infecting HEK293T cells (left) and Huh7 cells (right) respectively. Luciferase is assayed and inhibition rates are calculated. (B) Pseudotypic MARV-Angola, Musoke and RAVN infect HEK293T cells respectively and neutralization activity of AF-03 to these species is determined. (C) AF-03 (10, 3, 1mg/kg) is administrated at 24 and 4 h before intraperitoneal injection of MARV pseudovirus. On day 4, bioluminescence signals are detected by an IVIS Lumina Series III imaging system. (D) The average radiance value based on the luminescence of (C). *p<0.05, **p<0.01, ***p<0.001.

The neutralization activity of AF-03 to EBOV, SUDV and BDBV harboring cleaved GP.

Pseudotypic EBOV, SUDV, and BDBV are processed with thermolysin at 37°C. Inhibition of these ebola virus entry harboring GP or GPcl by AF-03 is examined by luciferase assay. *p<0.05, **p<0.01, ***p<0.001.

Cellular internalization of AF03-NL.

(A) AF-03, AF03-NL or human IgG1 isotype (MOCK) is incubated with cells at 4L for 1 h to prevent internalization and then at 37L for another 2 h to allow internalization. PE-conjugated secondary antibody is added prior to analysis by flow cytometry. (B,C) pHrodo Red-labelled AF-03 or AF03-NL is incubated with cells at 37□ for 1 h and analyzed by flow cytometry (B) and fluorescence microscopy (C) respectively. The red arrow denotes internalized AF03-NL. Experiments are independently repeated at least three times, and the data from one representative experiment is shown.

Pan-filovirus entry inhibition by AF03-NL.

(A,B) AF-03 or AF03-NL (50-0.0007 μg/ml, 4-fold dilution) is incubated with HEK293T cells at 37°C for 2 h prior to exposure to pseudotypic filovirus species (A) and EBOV mutants (B). Luciferase is assayed and inhibition rates are calculated.

The requirement of CI-MPR for the neutralization activity of AF03-NL.

(A) NPC2 protein is examined by SDS-PAGE. NR, non-reducing; R, reducing. (B) AF03-NL, AF-03, NPC2 alone or equimolar combination of AF-03 and NPC2 is incubated with HEK293T cells at 37°C for 2 h prior to exposure to pseudotypic EBOV. Luciferase is assayed and inhibition rates are calculated. (C) HEK293T cells are treated with siRNA-CI-MPR or negative control vector (NC) respectively and CI-MPR expression is detected by flow cytometry. AF03-NL is incubated with siCI-MPR or NC-treated HEK293T cells at 37°C for 2 h respectively prior to exposure to pseudotypic EBOV. (D) CI-MPR is introduced into Huh7 cells and its expression is detected by flow cytometry. AF03-NL is incubated with CI-MPR or NC-knockin Huh7 cells at 37°C for 2 h respectively prior to exposure to pseudotypic EBOV. Luciferase is assayed and inhibition rates are calculated.