Identifying epitopes responsible for neutralizing antibody and DC-SIGN binding on the spike glycoprotein of the severe acute respiratory syndrome coronavirus.
The severe acute respiratory syndrome-associated coronavirus (SARS-CoV) uses dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN) to facilitate cell entry via cellular receptor-angiotensin-converting enzyme 2. For this project, we used recombinant baculoviruses expressing different lengths of SARS-CoV spike (S) protein in a capture assay to deduce the minimal DC-SIGN binding region. Our results identified the region location between amino acid (aa) residues 324 to 386 of the S protein. We then generated nine monoclonal antibodies (MAbs) against the S protein to map the DC-SIGN-binding domain using capture assays with pseudotyped viruses and observed that MAb SIa5 significantly blocked S protein-DC-SIGN interaction. An enhancement assay using the HKU39849 SARS-CoV strain and human immature dendritic cells confirmed our observation. Data from a pepscan analysis and M13 phage peptide display library system mapped the reactive MAb SIa5 epitope to aa residues 363 to 368 of the S protein. Results from a capture assay testing three pseudotyped viruses with mutated N-linked glycosylation sites of the S protein indicate that only two pseudotyped viruses (N330Q and N357Q, both of which lost glycosylation sites near the SIa5 epitope) had diminished DC-SIGN-binding capacity. We also noted that MAb SIb4 exerted a neutralizing effect against HKU39849; its reactive epitope was mapped to aa residues 435 to 439 of the S protein. We offer the data to facilitate the development of therapeutic agents and preventive vaccines against SARS-CoV infection.
Shih
Yi-Ping
YP
AIDS Prevention and Research Center, National Yang-Ming University, Taipei 111, Taiwan, Republic of China.
Chen
Chia-Yen
CY
Liu
Shih-Jen
SJ
Chen
Kuan-Hsuan
KH
Lee
Yuan-Ming
YM
Chao
Yu-Chan
YC
Chen
Yi-Ming Arthur
YM
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral / Q000235:genetics; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D004279:DNA, Viral / Q000235:genetics; D003713:Dendritic Cells / Q000276:immunology / Q000821:virology; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D066298:In Vitro Techniques; D037181:Lectins, C-Type / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009500:Neutralization Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics / Q000276:immunology*; D045169:Severe Acute Respiratory Syndrome / Q000276:immunology / Q000821:virology; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D000099215:DC-Specific ICAM-3 Grabbing Nonintegrin
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral; D015815:Cell Adhesion Molecules; D004279:DNA, Viral; D000939:Epitopes; D037181:Lectins, C-Type; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; D000099215:DC-Specific ICAM-3 Grabbing Nonintegrin; C578557:spike glycoprotein, SARS-CoV; C578553:spike protein, mouse hepatitis virus