Production and Purification of Recombinant Filamentous Bacteriophages Displaying Immunogenic Heterologous Epitopes
Viruslike particles often combine high physical stability with robust immunogenicity. Furthermore, when such particles are based on bacteriophages, they can be produced in high amounts at minimal cost and typically will require only standard biologically
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Introduction The mammalian adaptive immune system typically responds vigorously to exogenous virus particles. For decades now, this property has been exploited to generate synthetic vaccines that comprise recombinant versions of such particles that are very well defined and noninfectious. Commercially successful examples of such viruslike particles (VLPs) include ENGERIX-B, distributed by GlaxoSmithKline, that is used as a vaccine against hepatitis B virus, and GARDASIL, distributed by Merck, that is used as a vaccine for the prevention of disease caused by human papillomavirus. These recombinant VLPs are produced and purified in the yeast Saccharomyces cerevisiae. Recombinant DNA technology has allowed to introduce heterologous epitopes in the coding information of viral capsomers (the building blocks of virus particles and VLPs) in order to generate hybrid VLPs that display these epitopes on the surface of the resulting VLPs. A useful application of this technology is to dramatically increase the immunogenicity of the heterologous epitopes [1]. Filamentous bacteriophages are frequently used to display foreign epitopes [2]. Filamentous bacteriophages such as E. coli phages M13 and fd belong to the Inoviridae and use the F pilus as a receptor. A remarkable and very practical feature of these phages is that they replicate without killing the infected bacteria, but rather cause a persisting infection with newly assembled phages shedding from the host cells by a budding process [3]. Two viral coat proteins of these filamentous phages are used as fusion
Sunil Thomas (ed.), Vaccine Design: Methods and Protocols, Volume 2: Vaccines for Veterinary Diseases, Methods in Molecular Biology, vol. 1404, DOI 10.1007/978-1-4939-3389-1_31, © Springer Science+Business Media New York 2016
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partners to display foreign epitopes: the minor coat protein pIII that is present in five copies at one end of the virion and the major coat protein pVIII that is assembled in a helical fashion around the circular single-stranded DNA of the phage. Because the pIII protein is essential for F pilus recognition and is present in just five copies per phage, it is not the prime choice for the insertion of heterologous epitopes when increased immunogenicity against these epitopes is aimed for. The major coat protein pVIII is a much better choice for this purpose. However, this protein is just 50 amino acid residues in length [4], and insertion of extra amino acid residues may interfere with the biological function of pVIII, its role being to bind to the viral genome to assemble stable filamentous particles [5]. As a solution to this intrinsic problem, the Smith lab generated the type 88 fd system [6]. This elegant system contains two copies of gene VIII (hence the double “8” in fd88), which allows production of hybrid phages comprising both wild-type pVIII and recombinant pVIII (fused to an epitope of interest) without the need for a helper phage. In the fd-Tet system, a tetracycline resistance gene is inserted in the min
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