Molecular Mechanism for Antibody-Dependent Enhancement of Coronavirus Entry

Abstract

Antibody-dependent enhancement (ADE) of viral entry has been a major concern for epidemiology, vaccine development, and antibody-based drug therapy. However, the molecular mechanism behind ADE is still elusive. Coronavirus spike protein mediates viral entry into cells by first binding to a receptor on the host cell surface and then fusing viral and host membranes. In this study, we investigated how a neutralizing monoclonal antibody (MAb), which targets the receptor-binding domain (RBD) of Middle East respiratory syndrome (MERS) coronavirus spike, mediates viral entry using pseudovirus entry and biochemical assays. Our results showed that MAb binds to the virus surface spike, allowing it to undergo conformational changes and become prone to proteolytic activation. Meanwhile, MAb binds to cell surface IgG Fc receptor, guiding viral entry through canonical viral-receptor-dependent pathways. Our data suggest that the antibody/Fc-receptor complex functionally mimics viral receptor in mediating viral entry. Moreover, we characterized MAb dosages in viral-receptor-dependent, Fc-receptor-dependent, and both-receptors-dependent viral entry pathways, delineating guidelines on MAb usages in treating viral infections. Our study reveals a novel molecular mechanism for antibody-enhanced viral entry and can guide future vaccination and antiviral strategies.IMPORTANCE Antibody-dependent enhancement (ADE) of viral entry has been observed for many viruses. It was shown that antibodies target one serotype of viruses but only subneutralize another, leading to ADE of the latter viruses. Here we identify a novel mechanism for ADE: a neutralizing antibody binds to the surface spike protein of coronaviruses like a viral receptor, triggers a conformational change of the spike, and mediates viral entry into IgG Fc receptor-expressing cells through canonical viral-receptor-dependent pathways. We further evaluated how antibody dosages impacted viral entry into cells expressing viral receptor, Fc receptor, or both receptors. This study reveals complex roles of antibodies in viral entry and can guide future vaccine design and antibody-based drug therapy.

Keywords: IgG Fc receptor; MERS coronavirus; SARS coronavirus; antibody-dependent enhancement of viral entry; neutralizing antibody; spike protein; viral receptor.

Copyright © 2020 American Society for Microbiology.

Figures

FIG 1

Structural similarity between DPP4 and MAb in binding MERS-CoV spike. (A) Tertiary structure of MERS-CoV RBD in complex with DPP4 (PDB code 4KR0) (30). DPP4 is colored yellow. RBD is colored cyan (core structure) and red (receptor-binding motif). DPP4 binds to the receptor-binding motif of the RBD. (B) Modeled structure of MERS-CoV S-e in complex with DPP4. S-e is a trimer (PDB code 5X5F): one monomeric subunit, whose RBD is in the standing-up conformation, is colored blue, and the other two monomeric subunits, whose RBDs are in the lying-down conformation, are colored gray (18). To generate the structural model of the S-e in complex with DPP4, the RBD in panel A was structurally aligned with the standing-up RBD in the S-e trimer. (C) Tertiary structure of MERS-CoV RBD (PDB code 4L3N) (64). Critical MAb-binding residues were identified through mutagenesis studies (48) and are shown as green sticks.

FIG 2

Interactions between coronavirus spike and RBD-specific MAb. (A) ELISA for detection of the binding between MERS-CoV RBD-specific MAb (i.e., Mersmab1) and MERS-CoV spike ectodomain (S-e). Mersmab1 was precoated on the plate, and recombinant S-e or RBD was added subsequently for ELISA. Binding affinities were characterized as ELISA signal at an optical density (OD) at 450 nm. PBS was used as a negative control. (B) ELISA for detection of the binding between Fab of Mersmab1 and MERS-CoV S-e. Recombinant S-e was used to precoat the plate, and Mersmab1 or Fab was added subsequently for ELISA. (C) Flow cytometry for detection of the binding between MERS-CoV S-e and DPP4 receptor and among S-e, Mersmab1, and CD32A (i.e., Fc receptor). Cells expressing DPP4 or CD32A were incubated with S-e alone, S-e plus Mersmab1, or S-e plus a SARS-CoV RBD-specific MAb (i.e., 33G4). Fluorescence-labeled anti-His6 antibody was added to target the C-terminal His6 tag on S-e. Cells were analyzed using fluorescence-activated cell sorting (FACS). (D) The expression levels of cell-membrane-associated DPP4 and CD32A were characterized using Western blotting targeting their C-terminal C9 tag and then used to normalize the binding affinity as measured in panel C. As an internal control, the expression level of cellular actin was measured using an anti-actin antibody. All of the experiments were repeated at least three times, with similar results, and representative results are shown. Error bars indicate SD (n = 5). Statistical analyses were performed as a one-tailed t test. ***, P < 0.001. Mersmab1 and its Fab both bind to MERS-CoV RBD and S-e.

FIG 3

Antibody-dependent enhancement of coronavirus entry. (A) Antibody-mediated MERS-CoV pseudovirus entry into human cells. The human cells included HEK293T cells exogenously expressing DPP4, HEK293T cells exogenously expressing one of the Fc receptors (CD16A, CD32A, or CD64A), and macrophages (induced from THP-1 monocytes) endogenously expressing a mixture of Fc receptors. The antibody was Mersmab1. An anti-SARS MAb (i.e., 33G4) was used as a negative control. Efficiency of pseudovirus entry was characterized by luciferase activities accompanying entry. HEK293T cells not expressing any viral receptor or Fc receptor were used as a control. (B) Fc- or Fab-mediated MERS-CoV pseudovirus entry into human cells. The Fc or the Fab portion of Mersmab1 was used in MERS-CoV pseudovirus entry performed as for panel A. (C) Expression levels of DPP4 receptor in different cell lines. Total RNA was extracted from three different cell lines: HEK293T, MRC5, and HeLa. Then qRT-PCR was performed on the total RNAs from each cell line. The expression level of DPP4 in each cell line is defined as the ratio between the RNA of DPP4 and the RNA of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (D) Antibody-mediated MERS-CoV pseudovirus entry into HeLa cells that do not express DPP4 receptor. The experiments were performed in the same way as for panel A, except that HeLa cells replaced HEK293T cells. (E) Antibody-mediated SARS-CoV pseudovirus entry into human cells. DPP4 and Mersmab1 were replaced by ACE2 and 33G4, respectively. Mersmab1 was used as a negative control. All of the experiments were repeated at least three times, with similar results, and representative results are shown here. Error bars indicate SD (n = 4). Statistical analyses were performed as a one-tailed t test. ***, P < 0.001. RBD-specific MAbs mediate ADE of coronavirus entry while blocking viral-receptor-dependent coronavirus entry.

FIG 4

Antibody-induced conformational changes of coronavirus spike. (A) Purified MERS-CoV pseudoviruses were incubated with recombinant DPP4, MAb, or PBS and then treated with trypsin. Samples were subjected to Western blotting. MERS-CoV spike and its cleaved fragments (all of which contained a C-terminal C9 tag) were detected using an anti-C9 tag monoclonal antibody. Both DPP4 and Mersmab1 triggered conformational changes of MERS-CoV spike, allowing it to cleaved at the S2′ site by trypsin. (B) Negative-stain electron microscopic analysis of MERS-CoV S-e in complex with the Fab of Mersmab1. Both a field of particles and windows of individual particles are shown. Black arrows indicate S-e-bound Fabs. According to previous studies (18, 20, 21), the Fab-binding site on the trimeric S-e is accessible only when the RBD is in the standing-up position.

FIG 5

Pathways for antibody-dependent enhancement of coronavirus entry. (A) Impact of proprotein convertases on ADE of MERS-CoV entry. During packaging of MERS-CoV pseudoviruses, HEK293T cells were treated with proprotein convertase inhibitor (PPCi). The MERS-CoV pseudoviruses packaged in the presence of PPCi were then subjected to MERS-CoV pseudovirus entry into HEK293T cells expressing either DPP4 receptor or CD32A receptor. (B) Western blot of MERS-CoV pseudoviruses packaged in the presence or absence of PPCi. MERS-CoV spike protein was detected using anti-C9 antibody targeting its C-terminal C9 tag. As an internal control, another viral protein, p24, was detected using an anti-p24 antibody. (C) Impact of cell surface proteases on ADE of MERS-CoV entry. HEK293T cells exogenously expressing TMPRSS2 (which is a common cell surface protease) were subjected to MERS-CoV pseudovirus entry. TMPRSS2 enhanced both the DPP4-dependent and antibody-dependent entry pathways. (D) Impact of lysosomal proteases on ADE of MERS-CoV entry. HEK293T cells exogenously expressing DPP4 or CD32A were pretreated with one of the lysosomal protease inhibitors E64d and Baf-A1 and then subjected to MERS-CoV pseudovirus entry. Lysosomal protease inhibitors blocked both the DPP4-dependent and antibody-dependent entry pathways. HEK293T cells not expressing DPP4 or CD32A were used as a negative control. All of the experiments were repeated at least three times, with similar results, and representative results are shown. Error bars indicate SD (n = 4). Statistical analyses were performed as a one-tailed t test. ***, P < 0.001; *, P < 0.05. Antibody-dependent and DPP4-dependent viral entries share the same pathways.

FIG 6

Antibody dosages for antibody-dependent enhancement of coronavirus entry. (A) Impact of antibody dosages on MERS-CoV pseudovirus entry into HEK293T cells exogenously expressing either DPP4 or CD32A. MAb blocks the DPP4-dependent entry pathway; it enhances the antibody-dependent entry pathway at lower concentrations and blocks it at higher concentrations. (B) Impact of antibody dosages on MERS-CoV pseudovirus entry into HEK293T cells exogenously expressing both DPP4 and CD32A. In the presence of both DPP4 and CD32A, MAb blocks viral entry at low concentrations, enhances viral entry at intermediate concentrations, and blocks viral entry at high concentrations. (C) Same experiment as in panel A, except that MRC5 cells replaced HEK293T cells. Here MRC5 cells express DPP4 receptor endogenously. (D) Same experiment as in panel B, except that MRC5 cells replaced HEK293T cells. Here MRC5 cells endogenously express DPP4 and exogenously express CD32A. Please refer to the text for more detailed explanations. All of the experiments were repeated at least three times, with similar results, and representative results are shown. Error bars indicate SD (n = 4).

FIG 7

Two previously published structures of coronavirus spike proteins complexed with antibody. (A) SARS-CoV S-e complexed with S230 MAb (PDB code 6NB7). The antibody binds to the side of the RBD, away from the viral-receptor-binding site, stabilizes the RBD in the lying-down state, and hence does not trigger conformational changes of SARS-CoV S-e. (B) MERS-CoV S-e complexed with LCA60 MAb (PDB code 6NB4). The antibody binds to the viral-receptor-binding site in the RBD, stabilizes the RBD in the standing-up state, and hence triggers conformational changes of MERS-CoV S-e.