title: Cell Attachment Domains of the Porcine Epidemic Diarrhea Virus Spike Protein Are Key Targets of Neutralizing Antibodies.
abstract: Porcine epidemic diarrhea virus (PEDV) causes enteric disease in pigs, resulting in significant economic losses to the swine industry worldwide. Current vaccination approaches against this emerging coronavirus are only partially effective, though natural infection protects pigs against reinfection and provides lactogenic immunity to suckling piglets. The viral spike (S) glycoprotein, responsible for receptor binding and cell entry, is the major target for neutralizing antibodies. However, knowledge of antibody epitopes, their nature and location in the spike structure, and the mechanisms by which the antibodies interfere with infection is scarce. Here we describe the generation and characterization of 10 neutralizing and nonneutralizing mouse monoclonal antibodies raised against the S1 receptor binding subunit of the S protein. By expression of different S1 protein fragments, six antibody epitope classes distributed over the five structural domains of the S1 subunit were identified. Characterization of antibodies for cross-reactivity and cross-neutralization revealed antigenic differences among PEDV strains. The epitopes of potent neutralizing antibodies segregated into two epitope classes and mapped within the N-terminal sialic acid binding domain and in the more C-terminal receptor binding domain. Antibody neutralization escape mutants displayed single amino acid substitutions that impaired antibody binding and neutralization and defined the locations of the epitopes. Our observations picture the antibody epitope landscape of the PEDV S1 subunit and reveal that its cell attachment domains are key targets of neutralizing antibodies.IMPORTANCEPorcine epidemic diarrhea virus (PEDV), an emerging porcine coronavirus, causes an economically important enteric disease in pigs. Effective PEDV vaccines for disease control are currently lacking. The spike (S) glycoprotein on the virion surface is the key player in virus cell entry and, therefore, the main target of neutralizing antibodies. To understand the antigenic landscape of the PEDV spike protein, we developed monoclonal antibodies against the spike protein's S1 receptor binding region and characterized their epitopes, neutralizing activity, and cross-reactivity toward multiple PEDV strains. Epitopes of antibodies segregated into six epitope classes dispersed over the multidomain S1 structure. Monoclonal antibodies revealed antigenic variability in B-cell epitopes between PEDV strains. The epitopes of neutralizing antibodies mapped to two distinct domains in S1 that are involved in binding to carbohydrate and proteinaceous cell surface molecules, respectively, indicating the importance of these cell attachment sites on the PEDV spike protein in eliciting a protective humoral immune response.
authors.affiliation: Institute of Animal Husbandry and Veterinary Science, Shanghai Academy of Agricultural Sciences, Shanghai, People's Republic of China.|Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors.forename: Chunhua
authors.initials: C
authors.lastname: Li
authors.affiliation: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors.forename: Wentao
authors.initials: W
authors.lastname: Li
authors.affiliation: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Lucio de Esesarte
authors.affiliation: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors.forename: Hongbo
authors.initials: H
authors.lastname: Guo
authors.affiliation: MSD Animal Health, Boxmeer, The Netherlands.
authors.forename: Paul
authors.initials: P
authors.lastname: van den Elzen
authors.affiliation: MSD Animal Health, Boxmeer, The Netherlands.
authors.forename: Eduard
authors.initials: E
authors.lastname: Aarts
authors.affiliation: MSD Animal Health, Boxmeer, The Netherlands.
authors.forename: Erwin
authors.initials: E
authors.lastname: van den Born
authors.affiliation: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors.forename: Peter J M
authors.initials: PJM
authors.lastname: Rottier
authors.affiliation: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands b.j.bosch@uu.nl.
authors.forename: Berend-Jan
authors.initials: BJ
authors.lastname: Bosch
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D057134:Antibodies, Neutralizing / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D000915:Antibody Affinity; D018985:Epitopes, B-Lymphocyte / Q000737:chemistry / Q000145:classification / Q000276:immunology; D051379:Mice; D009154:Mutation; D009500:Neutralization Tests; D053485:Porcine epidemic diarrhea virus / Q000737:chemistry* / Q000235:genetics / Q000276:immunology* / Q000502:physiology; D064370:Spike Glycoprotein, Coronavirus / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D013552:Swine
chemical_list: D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D018985:Epitopes, B-Lymphocyte; D064370:Spike Glycoprotein, Coronavirus
keywords: PEDV; coronavirus; monoclonal antibodies; spike protein; virus neutralization