Recombinant norovirus-specific scFv inhibit virus-like particle binding to cellular ligands.
BACKGROUND
Noroviruses cause epidemic outbreaks of gastrointestinal illness in all age-groups. The rapid onset and ease of person-to-person transmission suggest that inhibitors of the initial steps of virus binding to susceptible cells have value in limiting spread and outbreak persistence. We previously generated a monoclonal antibody (mAb) 54.6 that blocks binding of recombinant norovirus-like particles (VLP) to Caco-2 intestinal cells and inhibits VLP-mediated hemagglutination. In this study, we engineered the antigen binding domains of mAb 54.6 into a single chain variable fragment (scFv) and tested whether these scFv could function as cell binding inhibitors, similar to the parent mAb.


RESULTS
The scFv54.6 construct was engineered to encode the light (VL) and heavy (VH) variable domains of mAb 54.6 separated by a flexible peptide linker, and this recombinant protein was expressed in Pichia pastoris. Purified scFv54.6 recognized native VLPs by immunoblot, inhibited VLP-mediated hemagglutination, and blocked VLP binding to H carbohydrate antigen expressed on the surface of a CHO cell line stably transfected to express alpha 1,2-fucosyltransferase.


CONCLUSIONS
scFv54.6 retained the functional properties of the parent mAb with respect to inhibiting norovirus particle interactions with cells. With further engineering into a form deliverable to the gut mucosa, norovirus neutralizing antibodies represent a prophylactic strategy that would be valuable in outbreak settings.
Ettayebi
Khalil
K
Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717, USA. kettayeb@montana.edu
Hardy
Michele E
ME
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology; D000918:Antibody Specificity; D000956:Antigens, Viral / Q000276:immunology; D001666:Binding Sites, Antibody / Q000276:immunology; D018938:Caco-2 Cells; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology* / Q000378:metabolism / Q000494:pharmacology; D007143:Immunoglobulin Heavy Chains / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D008024:Ligands; D029322:Norovirus / Q000502:physiology*; D010843:Pichia / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology; D053585:Virus Attachment / Q000187:drug effects
D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D008024:Ligands; D011994:Recombinant Proteins