Abstract
BackgroundNoroviruses 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.ResultsThe 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 α 1,2-fucosyltransferase.ConclusionscFv54.6 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.
Highlights
Noroviruses cause epidemic outbreaks of gastrointestinal illness in all age-groups
VL and VH domains of monoclonal antibody (mAb) 54.6 and design of scFv54.6 Anti-rNV mAb 54.6 recognizes non-denatured VP1, inhibits virus-like particles (VLPs)-mediated hemagglutination, and blocks VLP binding to CaCo-2 cells
To determine whether functional activity of the mAb could be reduced to a smaller antigen binding domain, sequences encoding the VL and VH genes of mAb 54.6 were cloned from the hybridoma cells (Figure 1)
Summary
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 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. Noroviruses are non-enveloped positive strand RNA viruses that cause foodborne illness worldwide [1]. Noroviruses are non-enveloped positive strand RNA viruses that cause foodborne illness worldwide [1] They are classified as NIAID Category B priority pathogens because they are transmitted person-to-person and can cause persistent epidemics.
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