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Recombinant norovirus-specific scFv inhibit virus-like particle binding to cellular ligands.

Ettayebi K, Hardy ME - Virol. J. (2008)

Bottom Line: 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.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717, USA. kettayeb@montana.edu

ABSTRACT

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.

Conclusion: 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.

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scFv54.6 blocks the binding of rNV VLPs to CHO-FTBKE cells. rNV VLPs were incubated with CHO-FTBKE cells in the presence or absence of scFv54.6 and sorted by flow cytometry (A) VLP binding detected by incubation with scFv54.6 followed by anti-c-myc mAb and PE-conjugated goat anti-mouse antibody. (B) VLP binding detected by incubation with anti rNV MAb 72.1 PE-conjugated goat anti-mouse and C) scFv54.6 were pre-incubated with VLPs for one hour prior to addition to the cells. Bound VLPs were detected by MAb 72.1.
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Figure 5: scFv54.6 blocks the binding of rNV VLPs to CHO-FTBKE cells. rNV VLPs were incubated with CHO-FTBKE cells in the presence or absence of scFv54.6 and sorted by flow cytometry (A) VLP binding detected by incubation with scFv54.6 followed by anti-c-myc mAb and PE-conjugated goat anti-mouse antibody. (B) VLP binding detected by incubation with anti rNV MAb 72.1 PE-conjugated goat anti-mouse and C) scFv54.6 were pre-incubated with VLPs for one hour prior to addition to the cells. Bound VLPs were detected by MAb 72.1.

Mentions: CHO cells do not express ABH histoblood group antigens, but CHO cells stably transfected with the rat FTB gene encoding α 1,2-FT confers the ability to bind rNV VLPs to these cells [31]. scFv54.6 was tested for the ability to block binding of rNV VLPs to CHO-FTBKE cells. In the absence of antibody, 48.2% of cells bound VLPs (Figure 5). When VLPs were pre-incubated with scFv54.6, the percentage of positive cells was reduced to 10.2%. No reduction in binding was observed with samples from non-expressing X33 culture subjected to the same purification procedure as the scFv54.6 (see Figure 2A). These results suggest scFv54.6 is able to block binding of rNV VLPs to H type antigen on the cell surface.


Recombinant norovirus-specific scFv inhibit virus-like particle binding to cellular ligands.

Ettayebi K, Hardy ME - Virol. J. (2008)

scFv54.6 blocks the binding of rNV VLPs to CHO-FTBKE cells. rNV VLPs were incubated with CHO-FTBKE cells in the presence or absence of scFv54.6 and sorted by flow cytometry (A) VLP binding detected by incubation with scFv54.6 followed by anti-c-myc mAb and PE-conjugated goat anti-mouse antibody. (B) VLP binding detected by incubation with anti rNV MAb 72.1 PE-conjugated goat anti-mouse and C) scFv54.6 were pre-incubated with VLPs for one hour prior to addition to the cells. Bound VLPs were detected by MAb 72.1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2267775&req=5

Figure 5: scFv54.6 blocks the binding of rNV VLPs to CHO-FTBKE cells. rNV VLPs were incubated with CHO-FTBKE cells in the presence or absence of scFv54.6 and sorted by flow cytometry (A) VLP binding detected by incubation with scFv54.6 followed by anti-c-myc mAb and PE-conjugated goat anti-mouse antibody. (B) VLP binding detected by incubation with anti rNV MAb 72.1 PE-conjugated goat anti-mouse and C) scFv54.6 were pre-incubated with VLPs for one hour prior to addition to the cells. Bound VLPs were detected by MAb 72.1.
Mentions: CHO cells do not express ABH histoblood group antigens, but CHO cells stably transfected with the rat FTB gene encoding α 1,2-FT confers the ability to bind rNV VLPs to these cells [31]. scFv54.6 was tested for the ability to block binding of rNV VLPs to CHO-FTBKE cells. In the absence of antibody, 48.2% of cells bound VLPs (Figure 5). When VLPs were pre-incubated with scFv54.6, the percentage of positive cells was reduced to 10.2%. No reduction in binding was observed with samples from non-expressing X33 culture subjected to the same purification procedure as the scFv54.6 (see Figure 2A). These results suggest scFv54.6 is able to block binding of rNV VLPs to H type antigen on the cell surface.

Bottom Line: 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.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717, USA. kettayeb@montana.edu

ABSTRACT

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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus