Epstein-Barr virus gH/gL has multiple sites of vulnerability for virus neutralization and fusion inhibition

Abstract

Epstein-Barr virus (EBV) is nearly ubiquitous in adults. EBV causes infectious mononucleosis and is associated with B cell lymphomas, epithelial cell malignancies, and multiple sclerosis. The EBV gH/gL glycoprotein complex facilitates fusion of virus membrane with host cells and is a target of neutralizing antibodies. Here, we examined the sites of vulnerability for virus neutralization and fusion inhibition within EBV gH/gL. We developed a panel of human monoclonal antibodies (mAbs) that targeted five distinct antigenic sites on EBV gH/gL and prevented infection of epithelial and B cells. Structural analyses using X-ray crystallography and electron microscopy revealed multiple sites of vulnerability and defined the antigenic landscape of EBV gH/gL. One mAb provided near-complete protection against viremia and lymphoma in a humanized mouse EBV challenge model. Our findings provide structural and antigenic knowledge of the viral fusion machinery, yield a potential therapeutic antibody to prevent EBV disease, and emphasize gH/gL as a target for herpesvirus vaccines and therapeutics.

Keywords: Epstein-Barr virus; antibody therapeutics; fusion machinery; gH/gL; glycoprotein H; herpesvirus; monoclonal antibody; sites of vulnerability; vaccine.

Figure 1.. Isolation and characterization of EBV gH/gL-specific mAbs

(A) EBV gH/gL-specific B cells were isolated from two convalescent donor samples. Immunoglobulin gene assignments, and antibody characteristics are shown for isolated antibodies, and previously described EBV gH/gL-targeting antibodies for reference. (B) Antibody neutralization of EBV infection of Raji B cells and SVKCR2 epithelial cells. (C) Inhibition of B cell and epithelial cell fusion by isolated mAbs. (D) Octet Biolayer interferometry antibody-antigen affinity measurements using EBV gH/gL (left) and gH/gL/gp42 (right) antigens. The association, dissociation rates, and KD values are shown for antibodies. 769B10 values were previously described (Bu et al., 2019). (E) Antibody competition profile using previously described antibodies (mAbs E1D1, CL40, 769B10) as competitors. Antibodies noted in the left column were first bound to gH/gL, followed by binding to a second antibody noted in the top row, with percent competition indicated. See also Figure S1 and S2.

Figure 2.. mAb 769B10 binds to a gH/gL antigenic supersite on EBV gH/gL

(A) Crystal structure of 769B10 Fab in complex with EBV gH/gL/gp42peptide. EBV glycoproteins gH/gL/gp42p are shown in surface representation; gH, gL, and gp42p (colored in light grey, light pink, and pale cyan respectively) bound to mAb 769B10 (cartoon representation, heavy chain and light chain are colored with bright orange and light orange respectively). The integrin tripeptide motif ‘KGD’ is indicated by a red circle. (B) Major antibody loops involved in gH/gL recognition are shown in ribbon representation with gH/gL in surface representation. Antibody residue numbering and CDR loops are designated using the Kabat numbering system. (C) CDR-gH/gL epitope BSA percentage is shown in pie-chart representation. The 769B10 epitope is made up of 73% gH and 27% gL (inner circle), with both heavy and light chain recognition of gH, and predominantly heavy chain recognition of gL (middle), with multiple CDR loops used to recognize the gH/gL (outer). (D) mAb 769B10 contact residues are shown in stick representations based on (1) CDRs H2, and H3; (2) CDR H2; (3) FR H3 antibody contacting regions; (4) CDR L1 and L3. (E) Negative-stain EM maps of 769B10 (top), or 769A7 (bottom) in complex with EBV gH/gL. The EBV gH/gL/gp42p structure is fitted into the electron density for reference. (F) Definition of the antigenic supersite. Structure-based alignment of CL40, AMMO1, and 769B10 (ribbon representation) in complex with EBV gH/gL (surface representation) are shown. (G) Comparison of the 769B10 epitope to CL40 and AMMO1 epitopes. The 769B10 (orange), AMMO1 (green), and CL40 (blue) epitopes are outlined on the gH/gL molecule individually, and overlaid (right panel). The equivalent % BSA between the 769B10 and either AMMO1, or CL40 epitopes are indicated at the base of the two figures, and shown based on total, gH-only, or gL-only overlap. See also Figure S3 and S4, and Table S1.

Figure 3.. mAb 769C2 defines a site of vulnerability with antibody contacts on both gH and gL molecules

(A) Structure of 769C2 Fab (ribbon representation, heavy chain and light chain are colored with sky blue and light blue respectively) in complex with EBV gH/gL/gp42peptide (surface representation; gH, gL, and gp42p are colored in light grey, light pink, and pale cyan respectively) are shown. (B) Major antibody loops involved in gH/gL recognition are shown in ribbon representation with gH/gL in surface representation. (C) CDR-glycoprotein epitope BSA % are shown represented in pie-chart format. The 769C2 epitope is made up of 72% gH and 28% gL (inner circle), with both heavy and light chain recognition of gH and gL (middle), with multiple CDR loops used to recognize the gH/gL molecule (outer). (D) mAb 769C2 contact residues are shown in stick representations based on (1, 2) CDR H1, H2 and H3; (3) CDR L1 and H3; (4) CDR L1 and L3. (E) Negative-stain EM maps of 769C2 (top) and competing mAb 769F8 (bottom) in complex with EBV gH/gL. The EBV gH/gL/gp42p structure is fitted into the electron density for reference. (F) Structural comparison of gH/gL/gp42p (surface representation) in complex with 769C2 or EphA2 (left) or E1D1 (right) respectively. (G) Comparison of the 769C2 epitope (blue, upper left) to mAb E1D1 epitope (purple, lower left) or the EphA2 (bright green, top right) binding site with all shown overlaid (lower right). The % BSA that is identical between the EphA2 binding site (top) and the antibody epitopes is indicated at the base of the figure. See also Figure S3 and S4, and Table S1.

Figure 4.. mAb 770F7 defines a novel epitope on EBV gH D-IV

(A) Structure of 770F7 Fab in complex with EBV gH/gL/gp42peptide (surface representation; gH, gL, and gp42p are colored in light grey, light pink, and pale cyan respectively) are shown bound to mAb 770F7 (ribbon representation, heavy chain and light chain are colored with forest green and pale green respectively). (B) CDR-glycoprotein epitope BSA % are shown represented in pie-chart format. The 770F7 epitope is on the gH molecule (inner circle), with both heavy and light chain recognition of gH (middle), with predominant recognition by the CDR H2 and H3 loops (outer). (C) Major antibody loops involved in gH/gL recognition are shown in ribbon representation with gH/gL in surface representation. (D) mAb 770F7 contact residues are shown in stick representations based on (1) CDR H2, and H3; (2) CDR H3; (3) CDR L2; (4) CDR H3 and CDR L1. (E) Structure model of 770F7 and CL59 in complex with gH/gL/gp42p (surface representation); 770F7 Fab is shown in ribbon representation colored as in A, and CL59 Fab is shown as both cartoon and surface representation in salmon color. See also Figure S3 and S4, and Table S1.

Figure 5.. Antigenic sites of vulnerability on the EBV gH/gL and gH/gL/gp42 fusion machinery

Structure-based composite model of EBV gH/gL and gH/gL/gp42 with neutralizing antibodies. EBV gH domains are indicated by dotted lines for (A-D). Antibody names that are underlined indicate that the structural information is provided by negative-stain EM maps and antibody docking. Antibodies and gH/gL molecules are shown in surface representation. (A) mAbs 769C2 (blue), 770F8 (rose), and E1D1 (purple) targeting the gH/gL D-I region. (B) mAbs 769B10 (orange), 769A7 (gold), AMMO1 (periwinkle blue), and CL40 (blue) targeting gH/gL D-I and D-II. (C) mAbs 1D8 (teal), and 769C5 (brown) targeting the gH/gL D-II and D-III region. (D) mAbs CL59 (salmon), and 770F7 (green), targeting the gH/gL D-IV region. (E) EBV glycoproteins gH/gL/gp42 (surface representation) are shown bound to eleven mAbs. The antibody-antigen model is shown in two orientations to allow visibility of the antibodies. (F) Antibody and gp42 interfaces are shown on the gH/gL/gp42p molecules (surface representation). See also Figures S4-S5.

Figure 6.. Comparison of herpesvirus antigenic sites of vulnerability on the gH/gL fusion machinery

(A) Comparisons of the 796B10 epitope to VZV RC (purple), EBV CL40 (blue), and HCMV 13H11 (yellow) antibody epitopes are mapped on the respective viral gH/gL molecules, shown in surface representation. The EBV gH/gL structural overlap (left) with VZV and HCMV gH/gL molecules indicates the different orientation or tilt of the gH D-I/gL domains in each molecule relative to each other. The approximate location of the gH DI-DIV domains are indicated by horizontal dotted lines. (B) Overlay of HCMV and EBV gH/gL molecules with HCMV mAbs 3G16, 13H11 and EBV mAbs 770F7, CL40, and CL59 shown in surface representation with two orientations. (C) Comparison of mAb 796C2-gH/gL recognition to HCMV gH/gL-targeting mAb 1–32 (green) shown in ribbon representation, with herpesvirus gH/gL molecules shown in surface representation. The approximate location of the gH DI-DIV domains are indicated by horizontal dotted lines. See also Figure S5.

Figure 7.. Passive transfer of gH/gL mAbs in humanized mice and challenge with EBV

(A) Mice were given six doses of gH/gL mAbs 769C2, 770F7, 769B10, or PBS intraperitoneally and challenged intravenously with EBV after the second dose of mAb. (B) Survival rate for each group of mice after challenge. (C) EBV DNA copies in the blood was quantified by real-time qPCR. Each dot represents one mouse and the dotted line indicates the detection limit of the assay. Whiskers indicates minimum and maximum data points and box represents upper and lower quartiles; the horizontal line in the box is the median. Median EBV DNA copies in the blood were calculated. (D) Number and percent of animals in each treatment group with macroscopic lesions on the surface of liver, kidney, spleen, or lung, and with microscopic lesions in these organs showing lymphoma, atypical B cell infiltrates, or lymphoproliferative lesions that do not meet the criteria for lymphoma. (E) Representative images of lymphoma in the kidneys, spleen, and lungs of the mice. Treatment groups are indicated. In situ hybridization was performed for EBV EBER1 with eosin counterstain (upper panel) and CD20 staining for human B cells using 3,3’ diaminobenzidine as a chromogen (lower panel). (F) Heatmap of EBER1 expression in lung, liver, spleen, and kidney in mouse. A score of 0 indicates no EBER1 expression, 1 represents scattered EBER⁺ cells, 2 indicates a moderate number of EBER+ cells, 3 represents intense infiltration of the tissue with EBER+ cells. See also Figures S6-S7.