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The use of a rigid silica-based packing material with large particle and pore size, 37–55 μm and 500 A pore, for affinity chromatography makes it possible to combine high selectivity with short analysis times. Both large and small molecules have been covalently bonded to the Protein-Pak TM Affinity Epoxy-Activated bulk packing for purification of glycoproteins, immunoglobulins, enzymes, lectins and other proteins. Recombinant protein A, GammaBind TM G, heparin, Cibacron Blue F3G-A, sulfanilamide, N-acetyl- D -glucosamine, concanavalin A and aminophenylboronic acid were covalently attached to the affinity packing for selective purification of proteins.

Objective: To use our knowledge of the three-dimensional structure and self-assembly mechanism of RNA bacteriophage capsids to develop novel virus-like particles (VLPs) for drug delivery and epitope presentation. Methods: Site-directed mutagenesis of a recombinant MS2 coat protein expression construct has been used to generate translational fusions encompassing short epitope sequences. These chimeric proteins still self-assemble in vivo into T = 3 shells with the foreign epitope in an accessible location. Covalent conjugation has also been used to generate RNA stem-loops attached to the toxin, ricin A chain, or to nucleotide-based drugs, that are still capable of stimulating self-assembly of the capsid in vitro. These packaged drugs can then be directed to specific cells in culture by further covalent decoration of the capsids with targeting molecules. Results: Chimeric VLPs are strongly immunogenic when carrying either B or T cell epitopes, the latter generating cytokine profiles consistent with memory responses. Immune responses to the underlying phage epitopes appear to be proportional to the area of the phage surface accessible. Phage shells effectively protect nucleic acid-based drugs and, for the toxin construct, make cell-specific delivery systems with LD50 values in culture sub-nanomolar. Conclusion: VLP technology has potential for therapeutic and prophylactic intervention in disease.

Publisher Summary The use of RNA bacteriophage, particularly MS2, for presenting epitopes at the surface of a large nucleic acid-flee virus-like particle is described in this chapter. The production of chimeric virus-like particles (VLPs) depends on finding the sites of insertion for the epitopes of interest that do not disrupt the processing and assembly of the particle and present the new sequence in a defined structural context. For some systems, N- or C-terminal extensions are the preferred location; however, such locations suggest that the epitope might not be constrained at one end. In RNA bacteriophages, the preferred site of insertion is at the top of the N-terminal β -hairpin. Appropriate modification of the recombinant coat protein gene allows insertion to occur between residues G14 and T15, allowing presentation in the context of a surface-accessible constrained loop on every subunit of the VLP. The immunoaffinity chromatography experiments described in the chapter suggest that from the three-dimensional structure of the phage particle, insertion of peptide epitopes within the β hairpin blocks access of the anti- MS2 IgG molecules to their epitopes on the wild-type virus. Because it is important to consider secondary immune responses against the epitope carrier in a vaccine, this result is important because it suggests that with enhanced formulations it would be possible to mask B-cell antigenic carrier sites.

We have produced a plasmid expression vector for the coat protein of RNA bacteriophage MS2. The vector has been modified to introduce a unique KpnI restriction site within the coat protein gene at a site corresponding to the most radially distant feature of the bacteriophage capsid, namely the top of the N-terminal beta-hairpin (between residues 15 and 16). Insertion of DNA oligonucleotides at this site allows the production of chimeric MS2 coat proteins having foreign peptide sequences expressed as the central part of the hairpin. We have produced chimeras with a number of different peptide sequences (up to 24 amino acids in length) chosen because of their known antigenic properties. The chimeric coat proteins self-assemble into largely RNA-free phage-like capsids in Escherichia coli and can be easily disassembled and reassembled in vitro. Such peptide-presenting particles may have a number of biotechnological applications, including use as a cost-effective, synthetic vaccine. We have tested the antigenicity of one such construct in vivo in mice and have shown that these particles are immunogenic and that antibody titres against the inserted peptide epitope can be obtained.