Phage display is a powerful technology for selection of novel binding functions from large populations of peptide or antibody fragments. From a sufficiently complex library, phage bearing peptides or antibodies with practically any desired binding activity can be physically isolated by affinity selection, and, since each particle carries in its genome the genetic information for its own replication, the selectants can be amplified in bacteria. Existing display systems are based mostly on the filamentous phage M13. It is a powerful technology, but it has limitations for certain applications. Our work has concentrated on development of a new display platform based on virus-like particles (VLPs) of bacteriophage MS2, which addresses some of these limitations. The work presented here has two main foci - the display of single-chain antibody fragments (scFvs) on the MS2 VLP surface and the affinity selection of epitopes and epitope mimic from random sequence peptide libraries using monoclonal antibodies as targets. Here we demonstrate the display of several different scFvs via genetic fusion at the C-terminus of the MS2 coat protein single-chain dimer, including the following: M18 (scFv against anthrax protective antigen), AF-20 (scFv against AF-20 antigen, found on hepatocellular carcinoma cells), and scFv26/scFv66 (scFvs against Nipah virus G and F protein, respectively). Work with M18 demonstrates successful scFv incorporation into the MS2 VLP and intact antibody structure and function. VLPs displaying scFv AF-20 were used in both confocal microscopy and FACS experiments to demonstrate that these scFv-bearing VLPs can be specifically targeted to cells expressing the receptor. The functionality of VLPs displaying scFv26 or scFv66 was demonstrated in neutralization assays with NiV G protein-pseudotyped vesicular stomatitis virus (VSV). We also demonstrate the affinity selection of epitopes and epitope mimics from libraries of random sequence peptide displayed within the AB-loop of the MS2 VLP. Several different antibodies recognizing both protein and carbohydrate antigens were used as the selection targets. They included the following: the 2C7 and 2-1-L8 (monoclonal antibodies directed to lipooligosaccharide of Neisseria gonorrhoeae), MDVP-55A and GTX29202 (antibodies against a discontinuous epitope of the envelope protein of Dengue virus); MCA5792 (an antibody against the peptidoglycan of Staphylococcus aureus); and 2H1 (an antibody to the capsular glucuronoxylomannan (GXM) of the fungus Cryptococcus neoformans, and SYA/J6 (which recognizes the lipopolysaccharide of Shigella flexneri). From these selections, families of potential mimotopes were developed, and tested for activity with the selecting antibody and (in some cases) were used in mouse immunizations to attempt to promote an immune response against the original antigen. In the cases where immunizations were attempted, high-titer antibodies were generated that bound to the original antigen, suggesting the suitability of the MS2 VLP platform for identification of mimotopes for diverse epitope types.
VLP, MS2, scFv, Bacteriophage, Affinity Selection, Random Libraries
Level of Degree
Biomedical Sciences Graduate Program
Peabody, David S.
First Committee Member (Chair)
Second Committee Member
Third Committee Member
Lino, Christopher Andrew. "DISPLAY OF PEPTIDES AND SINGLE-CHAIN ANTIBODIES ON BACTERIOPHAGE MS2 VIRUS-LIKE PARTICLES." (2013). http://digitalrepository.unm.edu/biom_etds/135