Characterization of monoclonal antibodies against feline coronavirus accessory protein 7b

• Published in: Veterinary microbiology Vol. 184; pp. 11 - 19
• Main Authors: Lemmermeyer, Tanja, Lamp, Benjamin, Schneider, Rainer, Ziebuhr, John, Tekes, Gergely, Thiel, Heinz-Jürgen
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 29.02.2016
• Subjects: Animals; Antibodies, Monoclonal - genetics; Antibodies, Monoclonal - metabolism; Antibodies, Viral - genetics; Antibodies, Viral - metabolism; Binding Sites; Coronavirus, Feline - immunology; Escherichia coli; Escherichia coli - genetics; Feline coronavirus; Glycosylation; Immune Evasion; Monoclonal antibodies; N-glycosylation; ORF 7b; Recombinant Proteins - genetics; Recombinant Proteins - immunology; Viral Proteins - genetics; Viral Proteins - immunology; Feline coronavirus; Monoclonal antibodies; ORF 7b; N-glycosylation
• ISSN: 0378-1135; 1873-2542
• DOI: 10.1016/j.vetmic.2015.12.009

•Generation of monoclonal antibodies against accessory protein 7b of feline coronavirus.•Identification of reactive epitopes.•Relevance of glycosylation site for antigenicity. Feline coronaviruses (FCoVs) encode five accessory proteins termed 3a, 3b, 3c, 7a and 7b of unknown function. These proteins are dispensable for viral replication in vitro but are supposed to play a role in virulence. In the current study, we produced and characterized 7b-specific monoclonal antibodies (mAbs). A recombinant form of the 7b protein was expressed as a fusion protein in Escherichia coli, purified by immobilized metal affinity chromatography and used as immunogen. Two hybridoma lines, 5B6 and 14D8, were isolated that expressed mAbs that recognized 7b proteins of both FCoV serotypes. Using an extensive set of N- and C-terminally truncated 7b proteins expressed in E. coli and a synthetic peptide, the binding sites of mAbs 5B6 and 14D8 were mapped to an 18-residue region that comprises the only potential N-glycosylation site of the FCoV 7b protein. The two mAbs were suitable to detect a 24-kDa protein, which represents the nonglycosylated form of 7b in FCoV-infected cells. We speculate that glycosylation of 7b is part of the viral evasion strategy to prevent an immune response against this antigenic site.