Selection of Vaccine Candidates for Fish Pasteurellosis Using Reverse Vaccinology and an In Vitro Screening Approach
The advent of new technologies in recent years has revolutionized the methods by which pathogens are studied and at the same time it has provided new tools to design vaccines against infections for which vaccine development has so far been unsuccessful. T
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Introduction Photobacteriosis or fish pasteurellosis is a septicemia caused by the gram-negative, halophilic bacterium Photobacterium damselae subsp. piscicida (Pdp) [1]. It is considered one of the most threatening diseases in world aquaculture due to high mortality, broad host range, and ubiquitous distribution [2]. Research has been focused on the development of effective vaccines to prevent photobacteriosis and limit antibiotic use in fish farming and consequently to reduce economic losses in aquaculture. Conventional Pdp vaccines, based on inactivated products containing cellular (heat- or formalin-killed bacteria) and soluble antigens (LPS and ribosomal formulations), appeared to be ineffective in protecting against pasteurellosis and the only commercially available vaccine, an ECP-enriched bacterin preparation, gave unreliable results [1, 3]. Recently, recombinant DNA technology has been applied for the development of bivalent subunit vaccine in cobia [4], and a DNA vaccine encoding codon-optimized 7 kDa lipoprotein has been investigated in Japanese flounder [5]. In our laboratory, a biotechnological approach based on reverse vaccinology has been applied to design a vaccine against fish pasteurellosis. Here we describe the selection of antigen vaccine candidates which is accomplished in two steps: (a) in silico methods for selecting surface-exposed or secreted proteins; (b) in vitro screening of the in silico selected vaccine candidates by an adherence inhibition assay. Genomic sequences of Pdp are the starting point for bioinformatic analysis aiming to identify new proteins and predict their localization. Bacterial proteins, when localized on the surface, have the potential to be ideal targets for antibody recognition and
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_12, © Springer Science+Business Media New York 2016
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therefore ideal vaccine antigens [6]. The antigens identified by reverse vaccinology should possibly be further screened by an in vitro assay. This aims to reduce the number of antigen candidates to be tested in the in vivo experiments which evaluate the survival of fish experimentally immunized with those antigens after challenge. The attachment of Pdp to host epithelial cells is a primary prerequisite for infections and a crucial step in pathogenesis. Adherence is a multifactorial process mediated by a number of surface-exposed organelles and secreted toxins, known as adhesins, that are of considerable interest as potential vaccine targets [7]. For this reason, the inhibition of Pdp adherence on fish epithelial cells can be used as a further selection tool to identify proteins with vaccine potential. For the in vitro assays, the selected antigens, produced as recombinant proteins, are used for mice immunization and the immunoglobulins purified from mice immune sera are tested in an adherence inhibition assay with Pdp and fi
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