A new panel of epitope mapped monoclonal antibodies recognising the prototypical tetraspanin CD81 [version 1; referees: 1 approved, 2 approved with reservations]

• Main Authors: Joe Grove, Ke Hu, Michelle J. Farquhar, Margaret Goodall, Lucas Walker, Mohammed Jamshad, Heidi E. Drummer, Roslyn M. Bill, Peter Balfe, Jane A. McKeating
• Format: Article
• Language: English
• Published: Wellcome 2017-09-01
• Series: Wellcome Open Research
• Subjects: Antigen Processing & Recognition; Viral Infections (without HIV)
• Online Access: https://wellcomeopenresearch.org/articles/2-82/v1

Background: Tetraspanins are small transmembrane proteins, found in all higher eukaryotes, that compartmentalize cellular membranes through interactions with partner proteins. CD81 is a prototypical tetraspanin and contributes to numerous physiological and pathological processes, including acting as a critical entry receptor for hepatitis C virus (HCV). Antibody engagement of tetraspanins can induce a variety of effects, including actin cytoskeletal rearrangements, activation of MAPK-ERK signaling and cell migration. However, the epitope specificity of most anti-tetraspanin antibodies is not known, limiting mechanistic interpretation of these studies. Methods: We generated a panel of monoclonal antibodies (mAbs) specific for CD81 second extracellular domain (EC2) and performed detailed epitope mapping with a panel of CD81 mutants. All mAbs were screened for their ability to inhibit HCV infection and E2-CD81 association. Nanoscale distribution of cell surface CD81 was investigated by scanning electron microscopy. Results: The antibodies were classified in two epitope groups targeting opposing sides of EC2. We observed a wide range of anti-HCV potencies that were independent of their epitope grouping, but associated with their relative affinity for cell-surface expressed CD81. Scanning electron microscopy identified at least two populations of CD81; monodisperse and higher-order assemblies, consistent with tetraspanin-enriched microdomains. Conclusions: These novel antibodies provide well-characterised tools to investigate CD81 function, including HCV entry, and have the potential to provide insights into tetraspanin biology in general.