Mapping of conserved and species-specific antibody epitopes on the Ebola virus nucleoprotein

Abstract

Filoviruses (viruses in the genus Ebolavirus and Marburgvirus in the family Filoviridae) cause severe haemorrhagic fever in humans and nonhuman primates. Rapid, highly sensitive, and reliable filovirus-specific assays are required for diagnostics and outbreak control. Characterisation of antigenic sites in viral proteins can aid in the development of viral antigen detection assays such immunochromatography-based rapid diagnosis. We generated a panel of mouse monoclonal antibodies (mAbs) to the nucleoprotein (NP) of Ebola virus belonging to the species Zaire ebolavirus. The mAbs were divided into seven groups based on the profiles of their specificity and cross-reactivity to other species in the Ebolavirus genus. Using synthetic peptides corresponding to the Ebola virus NP sequence, the mAb binding sites were mapped to seven antigenic regions in the C-terminal half of the NP, including two highly conserved regions among all five Ebolavirus species currently known. Furthermore, we successfully produced species-specific rabbit antisera to synthetic peptides predicted to represent unique filovirus B-cell epitopes. Our data provide useful information for the development of Ebola virus antigen detection assays.

Keywords: Antibody epitope; BDBV; Bundibugyo virus; EBOV; Ebola virus; GP; MARV; Marburg virus; Monoclonal antibody; NP; Nucleoprotein; RAVV; RESTV; Ravn virus; Reston virus; SUDV; Sudan virus; Synthetic peptide; TAFV; Taï Forest virus; VLP; VP; glycoprotein; mAb; monoclonal antibodies; nucleoprotein; viral protein; virus-like particle.

Fig. 1

Reactivity of mouse mAbs in Western blotting. Vero E6 cells were infected with EBOV (Z), SUDV (S), TAFV (T), BDBV (B), RESTV (R), MARV Angola (A), MARV Musoke (M), MARVOzolin(O), MARV Ci67(C) or RAVV (Ra). Cell culture supernatants containing virus particles were collected, inactivated and subjected to SDS-PAGE under reducing conditions. Mo, mock–infected.

Fig. 2

Reactivities of mAbs to EBOV NP-derived synthetic peptides. Seventy-three overlapping peptide sequences (20 aa in length with a 10 aa overlap) covering the entire amino acid sequence of NPof EBOV Mayinga were coated on ELISA plates at a concentration of 100 μg/ml. Purified mAbs were used as primary antibodies at a concentration of 1 μg/ml. OD measurements were determined at 450 nm.

Fig. 3

Epitope sequences in amino acid sequence alignment and known functional regions NPs. (A) Amino acid sequences of EBOV, SUDV, TAFV, BDBV and RESTV were obtained from GenBank under accession numbers AF272001, AF173836, FJ217162, FJ217161 and AF522874, respectively. Amino acid sequences at positions 421–660 of each virus are shown. EBOV NP peptides recognised by the mAbs are highlighted with solid lines. Corresponding regions of the other NPs to which each mAb showed strong cross-reactivity are underlined (dashed lines). Amino acid sequences used for producing species-specific rabbit antisera are shown in pink. (B) Locations of the identified epitopes are shown in the schematic diagram of NP. Functional domains (Bharat et al., 2012; Noda et al., 2007; Watanabe et al., 2006) are also shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

Fig. 4

Reactivity of rabbit antisera in Western blotting. Rabbit antisera (FS0169, FS0191, FS0046, FS0048, FS0170 and FS0610) were produced using synthetic peptides derived from EBOV, SUDV, TAFV, BDBV, RESTV and MARV, respectively. Experimental conditions were the same as in Fig. 1. EBOV (Z), SUDV (S), TAFV (T), BDBV (B), RESTV (R), MARV Angola (A), MARV Musoke (M), MARV Ozolin (O), MARV Ci67 (C), RAVV (Ra). Mo, mock-infected.