Epitope mapping targeting the K205R protein of African swine fever virus using nanobody as a novel tool

Figures

• 

  Fig 1. Production and identification of Nb-HRP fusion proteins, expressed and secreted, against ASFV K205R protein. The pCAGGS-HA-Nb1-, -Nb14-, -Nb35-, and -Nb82-HRP recombinant plasmids were transfected into HEK293T cells for 60 h to produce Nb-HRP fusion proteins. The HEK293T cells were fixed or collected 60 h after transfection, and the expression of Nb-HRP fusions was analyzed using (A) immunofluorescence assay and (B) western blotting with anti-HA monoclonal antibody as the primary antibody. (C) Cell culture supernatants were collected 60 h after transfection or not, and detection of HRP activity in the Nb-HRP fusions secreted into the cell culture supernatants was conducted using direct ELISA. Data are mean ± SD values of three independent results. *P < 0.05; **P < 0.01; ***P < 0.001;****P < 0.0001. Nb, nanobody; HRP, horseradish peroxidase.

• 

  Fig 2. Four Nbs, namely Nb1, Nb14, Nb35, and Nb82, bind to both prokaryotically and eukaryotically expressed K205R proteins. (A) SDS-PAGE analysis of prokaryotically expressed ASFV-K205R protein. (B) Nb1, Nb14, Nb35, and Nb82’s reaction with ASFV-K205R protein expressed with E. coli was determined using western blotting, with ASFV-DP96R used as a negative control. (C) Four Nbs specifically reacted with the ASFV-K205R protein, as determined by direct ELISA. Data are mean ± SD values of three independent results. ****P < 0.0001. (D) Titration of cell supernatant containing Nb1-, Nb14-, Nb35-, and Nb82-HRP fusion proteins binding to the ASFV-K205R protein. (E) Affinity of Nb1-, Nb14-, Nb35-, and Nb82-His protein toward ASFV-K205R protein. (F) Western blot analysis of eukaryotically expressed ASFV-K205R protein using anti-HA monoclonal antibody for detection. Four Nbs specifically reacted with the eukaryotically expressed ASFV-K205R protein, as determined using (G) western blotting and (H) immunofluorescence assay. Nb, nanobody; ASFV, African swine fever virus.

• 

  Fig 3. Nb1, Nb14, Nb35, and Nb82 can recognize ASFV strains HLJ/18-infected PAMs or Vero cells, according to by IFA and immunoperoxidase monolayer assay. (A) IFA analysis of the reaction of four Nbs with ASFV K205R. PAMs were plated and infected with the HLJ/18 ASFV strain at an MOI of 0.01 for 12 h. Next, PAMs were fixed and used for immunostaining analysis with Nbs-HRP as the primary antibody. (B) Immunoperoxidase monolayer assay identification of the reaction of the four Nbs with ASFV K205R. Vero cells were infected with the HLJ/18 ASFV strain at an MOI of 0.01 for 16 h. Next, the cells were fixed and incubated with Nbs-HRP supernatants at a serial dilution of 1:100, 1:1,000, and 1:2,000. Inactivated anti-ASFV antibody-positive serum was used as the positive control, while anti-ASFV antibody-negative serum was used as the negative control. Nb, nanobody; ASFV, African swine fever virus; IFA, immunofluorescence assay; MOI, multiplicity of infection.

• 

  Fig 4. Location of the linear B-cell epitopes regions of the ASFV-K205R protein. (A) Schematic diagram of different truncated ASFV-K205R protein peptides. (B) Expression and purification of different truncated ASFV-K205R protein peptides using a prokaryotic system. (C) The reaction of Nb1, Nb14, Nb35, and Nb82 with the different truncated versions of ASFV-K205R protein was determined using western blotting. (D) Analysis of the reaction between Nb1, Nb14, Nb35, and Nb82 and different truncated versions of the ASFV-K205R protein using direct ELISA. The full-length ASFV K205R protein served as a positive control, while ASFV DP96R served as a negative control. Data are mean ± SD values of three independent results. ****P < 0.0001; ns, no significant. ASFV, African swine fever virus; Nb, nanobody.

• 

  Fig 5. Fine mapping of the epitopes in ASFV K205R protein recognized by K205R-Nb1, -Nb14, and -Nb82. (A, D, and G) Schematic diagram of 11 short peptides, namely P1-P12. Reaction of K205R-Nb1, -Nb14, and -Nb82 with seven short peptides (P1-P7), as analyzed by (B and E) dot blot and (C, F, and H) peptide-based ELISA. The full-length ASFV K205R protein served as a positive control, while ASFV DP96R served as a negative control. (I) Reaction of K205R-Nb1, -Nb14, and -Nb82 with P8, P9, P10, P11, and P12 peptides by peptide-based ELISA. Data are mean ± SD values of three independent results. ***P < 0.001;****P < 0.0001; ns, no significant. ASFV, African swine fever virus; Nb, nanobody.

• 

  Fig 6. Fine mapping of the epitopes in the ASFV K205R protein recognized by K205R-Nb35. (A) Schematic diagram of different truncated ASFV K205R protein peptides produced in a eukaryotic expression system. (B) Western blot analysis of the eukaryotically expressed ASFV-K205R protein with anti-HA monoclonal antibody for detection. K205R-Nb35 reacted with different eukaryotically expressed truncated K205R protein peptides, as determined by using (C) western blotting and (D) immunofluorescence assay. (E) Schematic diagram of the synthesis of P13, P14, P15 truncated and mutant K205R peptides. (F) K205R-Nb35 reacted with different truncated and mutant K205R peptides, as revealed by peptide-based ELISA. (G) Schematic diagram of the synthesis of different single amino acid mutant K205R peptides. (H) Peptide-based ELISA detection of K205R-Nb35 reacted with P16, P17, P18, and P19. Data are mean ± SD values of three independent results. ****P < 0.0001; ns, no significant. African swine fever virus; Nb, nanobody.

• 

  Fig 7. Reactivity of identified linear B-cell epitopes with inactivated ASFV-positive serum. (A) Schematic diagram of the synthesis of different truncated and mutant K205R peptides. (B) Reactivity of identified epitope 1MVEPR5 with inactivated ASFV-positive serum using peptide-based ELISA. (C) Reactivity of identified epitope 188RTQF191 with inactivated ASFV-positive serum using peptide-based ELISA. Five inactivated positive pig sera for anti-ASFV antibodies and five ASFV antibody-negative serums were used to test. Data are mean ± SD values of three independent results. ***P < 0.001;****P < 0.0001; ns, no significant.

• 

  Fig 8. Conservation analysis of the two epitopes 1MVEPR5 and 188RTQF191. (A) Secondary structure analysis of ASFV K205R protein using DNASTAR Protean software. Secondary structure analysis of epitopes 1MVEPR5 (B) and 188RTQF191 (C) using DNASTAR Protean software. (D) Conservation analysis of the 1MVEPR5 and 188RTQF191 antigenic regions among ASFV epidemic isolates. The red box indicated the antigenic region included two epitopes 1MVEPR5 and 188RTQF191.

• 

  Fig 9. Predict complex structure using molecular docking. (A) Molecular docking of complex Nb1 and the identified epitope 1MVEPREQFFQ10. (B) Molecular docking of complex Nb14 and the identified epitope 1MVEPREQFFQ10. (C) Molecular docking of complex Nb35 and the identified epitope 188RTQFSDEQKKM198. (D) Molecular docking of complex Nb82 and the identified epitope 1MVEPREQFFQ10. K205R epitope is shown as a cyan stick, the nanobody is represented as a slate cartoon model, and their binding sites are shown as magentas stick structures. The hydrogen bond is depicted by a yellow dashed line.