While approximately half of GP-specific B cell lines obtained from BDBV survivors produced antibodies specific to BDBV GP, 24%–50% of GP-reactive B cell culture supernatants also cross-reacted with EBOV GP ( Figures 1 A and 1D). Similarly, 36% of GP-specific B cell lines obtained from the EBOV survivor cross-reacted with the heterologous BDBV GP ( Figures 1 B and 1D). Despite the apparent presence of B cells encoding cross-reactive antibodies in survivors of BDBV or EBOV infections to GPs from heterologous Ebolavirus species, we detected a very limited cross-reactivity with GPs from MARV, which belongs to a different genus in the family Filoviridae ( Figures 1 A and 1D). In line with this finding, 90% of GP-reactive B cell lines obtained from the MARV survivor reacted with autologous GP, and only 2% of antigen-specific B cell lines produced Ebolavirus cross-reactive Abs ( Figures 1 C and 1D). The limited cross-reactivity of mAbs to GPs from Ebolavirus and Marburgvirus species likely is due in part to low sequence conservation between GPs from two genera (only 27% amino acid identity between BDBV and MARV GP) as well as differences in epitope availability caused by different positions of the mucin-like domains on the GP surface of Ebolavirus and Marburgvirus ().

To generate human cell lines secreting human mAbs to BDBV, we transformed peripheral blood B cells from seven survivors of the 2007 Uganda BDBV outbreak with Epstein-Barr virus, as described in the Experimental Procedures . To determine the breadth of antibody response in survivors of ebolavirus infection, we screened supernatants from EBV-transformed B cell lines for binding to GPs from diverse representatives of filovirus species: BDBV, EBOV, or Marburg virus (MARV) ( Figures 1 A and S1 ). We also used the same GP panel to screen supernatants from transformed B cell lines derived from a survivor of the 2014 EBOV outbreak ( Figure 1 B) or from a donor who survived MARV infection ( Figure 1 C). We color coded GP-reactive supernatants based on the cross-reactivity pattern as follows: species-specific cell lines are highlighted in black; and cross-reactive lines to two or three species are shown in yellow or blue, respectively ( Figures 1 A–1C and S1 ).

Supernatants from EBV-transformed PBMC samples isolated from survivors were screened in ELISA binding assays using BDBV, EBOV or MARV GPs. Height of the bars indicates OD 405 nm values in ELISA binding to full-length extracellular domain of GP of the indicated virus species. Reactive supernates are color-coded based on the cross-reactivity pattern: species-specific cell lines are highlighted in black; cross-reactive lines to 2 or 3 species are shown in yellow or blue, respectively.

(D) Percentages of lines secreting antibodies specific to BDBV, EBOV, or MARV GPs or cross-reactive antibodies to BDBV and EBOV (designated BDBV/EBOV) or BDBV, EBOV, and MARV (designated BDBV/EBOV/MARV) are shown. Increasing intensity of the pink cell fill color corresponds to increasing reactivity for the indicated virus.

(A–C) Supernatants from EBV-transformed PBMC samples isolated from survivors were screened in ELISA-binding assays using BDBV, EBOV, or MARV GPs. Results for four BDBV survivors (A), one EBOV survivor (B), and one MARV survivor (C) are shown. Height of the bars indicates OD 405 nm values in ELISA binding to full-length extracellular domain of GP of the indicated virus species. Reactive supernates are color coded based on the cross-reactivity pattern as follows: species-specific cell lines are highlighted in black; and cross-reactive lines to two or three species are shown in yellow or blue, respectively. Previous work has shown that the amino acid sequence of GP differs between BDBV and EBOV by over 34% and between BDBV and MARV by over 72%.

To evaluate the inhibitory activity of isolated mAbs, we tested mAbs in a BDBV neutralization assay. Of the 90 BDBV GP-reactive mAbs, 31 had half-maximal inhibitory concentration (IC) values <10 μg/ml, and we defined these as neutralizing antibodies (nAbs) ( Figures 2 B, where nAb names are highlighted in red, and S3 ). Several nAbs displayed an extremely high neutralizing potency, with ICvalues below 1 ng/ml ( Figure 2 B). Also, 18 of 31 nAbs bound only to BDBV GP in ELISA, six nAbs recognized BDBV and EBOV GPs, and the remaining seven nAbs bound to GPs from representatives of three Ebolavirus species, BDBV, EBOV, and SUDV. These results suggested that cross-reactive mAbs in our panel might possess neutralizing activity to multiple ebolaviruses. To test this hypothesis, we screened BDBV425 (a group 2A nAb) in an EBOV neutralization assay as the nAb with the lowest half-maximal effective concentration (EC) value to the heterologous EBOV GP, and we determined that BDBV425 neutralized the heterologous EBOV. Encouraged by this result, we tested nAbs from groups 3A and 3B in EBOV or SUDV neutralization assays to determine whether cross-reactive nAbs can neutralize three Ebolavirus species. We found two cross-reactive nAbs from group 3A (BDBV43 and BDBV324) that neutralized all three ebolaviruses BDBV, EBOV, and SUDV ( Figure 2 D, BDBV43). The remaining five nAbs from groups 3A and 3B neutralized BDBV and EBOV, but failed to neutralize SUDV ( Figure 2 D, BDBV289). Analysis of the Ab heavy-chain variable domain sequences for 26 nAbs revealed that all BDBV-specific and cross-reactive nAbs were encoded by unique Ab genes ( Table S1 ).

Red circles represent percent neutralization relative to control at different antibody concentrations. Logistic curves are indicated by solid lines, and 95% confidence intervals are indicated by dashed lines.

We further characterized the binding of species-specific or cross-reactive mAbs to recombinant GPs by performing a binding assay with the recombinant form of GP that is secreted from the cell to the extracellular space during natural infection (sGP, secreted GP) (). While the Ebolavirus GP is a trimer, sGP forms dimers in which each protomer shares only the amino-terminal 295 amino acids with GP. The majority of mAbs recognized epitopes shared between BDBV GP and BDBV sGP (designated groups 1A, 2A, or 3A) ( Figures 2 A and 2C). We also identified antibodies that bound to BDBV GP, but failed to bind BDBV sGP in ELISA (designated groups 1B, 2B, or 3B) ( Figures 2 A and 2C). Antibodies from groups 1B, 2B, or 3B also bound the recombinant GP form that lacks highly glycosylated mucin-like domains (BDBV GPΔmuc), suggesting that mAbs from these three groups target epitopes outside of mucin-like domains ( Figure S2 ).

The virion glycoproteins of Ebola viruses are encoded in two reading frames and are expressed through transcriptional editing.

We fused transformed cells from B cell lines producing BDBV GP-reactive Abs with myeloma cells and generated 90 cloned hybridomas secreting BDBV GP-reactive human mAbs. To determine the breadth of mAb binding, we screened the mAbs in ELISA-binding assays using recombinant GPs from multiple filoviruses: BDBV, EBOV, SUDV, or MARV GPs. While 33 Abs recognized only the autologous BDBV GP (designated groups 1A and 1B), 20 Abs recognized both BDBV and EBOV GPs (groups 2A and 2B), and 37 Abs recognized all three GPs from BDBV, EBOV, and SUDV (groups 3A and 3B) ( Figures 2 A and 2C ; Data S1 ). The relative proportions of antibodies that recognize GPs from 1, 2, or 3 Ebolavirus species did not correlate fully with the B cell line frequencies in the initial screen, which can be explained by our prioritization on recovery of a high number of cross-reactive mAbs. We were not able to isolate Abs that bind to the heterologous MARV GP ( Figures 2 A and 2C; Data S1 ).

(D) Neutralization activity of representative neutralizing mAbs from three binding groups against BDBV, EBOV, or SUDV. Error bars represent the SE of the experiment, performed in triplicate.

(B) Heatmap showing the neutralization potency of BDBV GP-specific mAbs against BDBV. The IC 50 value for each virus-mAb combination is shown. IC 50 values greater than 10,000 ng/ml are indicated (>). Neutralization assays were performed in triplicate.

(A) Heatmap showing the binding of BDBV mAbs to a panel of filovirus GPs. The EC 50 value for each GP-mAb combination is shown, with dark red, orange, yellow, or white shading indicating high, intermediate, low, or no detectable binding, respectively. EC 50 values greater than 10,000 ng/ml are indicated (>). NAb names are highlighted in red.

To determine whether Abs from distinct binding groups targeted different antigenic regions on the BDBV GP surface, we performed a quantitative competition-binding assay using a real-time biosensor. We tested four BDBV nAbs from binding group 1A, five nAbs from binding group 1B, four nAbs from group 3A, and three nAbs from group 3B in a tandem blocking assay, in which BDBV GP was attached to the biosensor. We also tested five non-neutralizing antibodies from group 1A to determine whether non-neutralizing antibodies target a unique epitope on GP surface. Non-neutralizing and neutralizing mAbs from group 1A and nAbs from group 3A blocked binding of each other to the GP antigen and segregated into a single competition-binding group ( Figure 3 ). These results suggest that mAbs from groups 1A and 3A target a single antigenic region that contains epitopes shared between GP and sGP ( Figure 2 A). The nAbs from group 3B that did not recognize sGP in ELISA ( Figure 2 A) segregated into a separate competition-binding group. Group 1B antibodies were interesting in that two nAbs in this group competed for binding with group 3B nAbs, while three nAbs from the group competed for binding with antibodies from group 3A ( Figure 3 ). These findings suggested that there are at least two major antigenic regions recognized by human BDBV nAbs, based on competition-binding studies. The first major antigenic region contains epitopes that both sGP and GP share (recognized by mAbs from groups 1A and 3A) as well as epitopes that are present only on the GP surface (recognized by three mAbs from group 1B). The second major antigenic region contains only epitopes that are present on the GP surface, but not sGP (recognized by two mAbs from group 1B and three mAbs from group 3B).

Data from competition-binding assays using non-neutralizing mAbs from binding group 1A (white background) and neutralizing mAbs from binding groups 1A, 1B, 3A, or 3B (pink background). Numbers indicate the percentage binding of second mAb in the presence of the first mAb compared to binding of un-competed second mAb. MAbs were judged to compete for the same site if maximum binding of the second mAb was reduced to <30% of its un-competed binding (black boxes with white numbers). The mAbs were considered non-competing if maximum binding of the second mAb was >70% of its un-competed binding (white boxes with red numbers). Gray boxes with black numbers indicate an intermediate phenotype (competition resulted in between 30% and 70% of un-competed binding). Blue, purple, and green dashed lines indicate what appear to be major competition groups; the blue and purple groups overlap substantially, but not completely.

Diverse Patterns of Molecular Recognition Defined by Negative-Stain EM

To determine the location of the two major antigenic regions targeted by human BDBV nAbs, we performed negative-stain single-particle EM studies using antibodies from groups 1A and 1B. The EM class averages and reconstructions showed clearly that the two major antigenic regions, defined in competition-binding experiments, corresponded to two distinct sites on the GP surface: the glycan cap and the GP base.

Bale et al., 2012 Bale S.

Dias J.M.

Fusco M.L.

Hashiguchi T.

Wong A.C.

Liu T.

Keuhne A.I.

Li S.

Woods Jr., V.L.

Chandran K.

et al. Structural basis for differential neutralization of ebolaviruses. Figure 4 BDBV-Neutralizing Antibodies Bind to the Glycan Cap or Base Region of GP Show full caption (A) Shown are negative-stain EM reference-free 2D class averages of group 1A antibodies that bind both the glycan cap of GP and sGP, and group 1B antibodies that bind the glycan cap of GP, but not sGP. BDBV GP or GPΔmuc was used to generate complexes. (B) 3D reconstructions of glycan cap binders from groups 1A and 1B reveal that these antibodies bind the glycan cap at overlapping but distinct epitopes. Top (left) and side (right) views of the complexes are shown. (C) Reference-free 2D class averages of group 1B antibodies (left) reveal that these antibodies bind an epitope below the base of GP that is flexible. In the middle image, GP is colored yellow and each Fab is colored green. The righthand panel illustrates a superimposition of crystal structures of SUDV GPΔmuc (PDB: 3VE0 ) and Fabs (PDB: 3CSY ) to demonstrate how Fabs may bind to GP. (D) The composite model delineates the epitopes of the glycan cap mAbs in group 1A or 1B. Side (above) and top (below) views are shown. Bale et al., 2012 Bale S.

Dias J.M.

Fusco M.L.

Hashiguchi T.

Wong A.C.

Liu T.

Keuhne A.I.

Li S.

Woods Jr., V.L.

Chandran K.

et al. Structural basis for differential neutralization of ebolaviruses. (E) Docking a crystal structure of SUDV GPΔmuc (PDB: 3VE0 ) (), which contains a more complete model of the glycan cap region targeted by group 1A/B mAbs, reveals how group 1A/B mAbs target a broad region in the GP1 centered on the glycan cap, near the beginning of the mucin-like domains. Group 1B mAbs that target the base likely bind to a loop near the membrane proximal external region (MPER) that is flexible and has not yet been resolved at high resolution. TM, transmembrane region; CT, cytoplasmic tail. See also Figure S4 Figure S4 Raw Data and Validation of EM Models, Related to Figure 4 Show full caption (A) Raw EM micrograph (far left), 2D reference-free class averages (middle left), and an FSC curve with resolution indicated (far right) of BDBV41 in complex with BDBV GPΔmuc. (B) As in (A) but of BDBV335 in complex with BDBV GPΔmuc. (C) As in (A), but of BDBV432 in complex with BDBV GPΔmuc. (D) As in (A) but of BDBV353 in complex with BDBV GP. (E) As in (A), but of BDBV289 in complex with BDBV GP. Refinement package used to generate each reconstruction is indicated on the far left. Scale bar indicates 200 nm. Figure S5 Generation of Escape Mutant Viruses for BDBV41, Related to Figure 5 Show full caption (A) Neutralization activity of BDBV41 against wild-type VSV/BDBV-GP (circles, straight curves), VSV/BDBV-GP#7 (squares, dashed curves), or VSV/BDBV-GP#15 (triangles, dotted curves) escape mutant viruses. (B) Amino acid changes in BDBV41 escape mutant viruses. Comparison of the structures of glycan cap-directed mAbs from group 1A with those in group 1B revealed that the antibodies have partially overlapping epitopes, but approach the glycan cap at distinct angles ( Figures 4 A, 4B, and S4 ). We fitted a previously determined atomic resolution structure of SUDV GPΔmuc (), which reveals more residues of the glycan cap region than the equivalent EBOV structure, into the envelope of GP from the EM reconstructions, and we determined the regions targeted by each mAb ( Figures 4 D and 4E). BDBV335, which binds GP and sGP equally well, mainly targets a region between residues 274 and 282. This region appears well defined in the BDBV335 EM map, indicated by the large lobe on the outside of the glycan cap that closely resembles that region in the GP crystal structure. When viewed along the 3-fold axis of GP, BDBV41 binds to the right of BDBV335, further up on the glycan cap, close to a loop that extends from residue 266 to 277. Consistent with this position, we passaged a chimeric VSV in which the G protein was replaced with BDBV GP as a sole surface protein (VSV/BDBV-GP) in the presence of mAb BDBV41 to generate a neutralization escape mutant virus that was completely resistant to the antibody and that possessed two amino acid substitutions, G271R and T272S ( Figure S5 ). The mutation at the 272 position likely explains why BDBV41 is a group 1 antibody, i.e., only recognizes BDBV (with T272), but not EBOV or SUDV (which have the alternate residue K272). BDBV41 also may make contacts with a loop that extends toward the mucin-like domains, from residue 309 to 312 or further in regions that were unresolved in the GP crystal structure. BDBV432 binds to the left of BDBV335, at the top of a helix loop at residues 259–266, and likely makes extensive contacts with a loop from residues around 302–312. Despite a lack of binding to sGP, BDBV432, as well as BDBV353, binds in the glycan cap region, suggesting that these mAbs make contacts with residues that are exclusive to GP.