An efficacious recombinant subunit vaccine against the salmonid rickettsial pathogen Piscirickettsia salmonis
Introduction
Rickettsiae cause a number of significant human and veterinary diseases. Traditionally, rickettsial vaccines have been based on inactivated whole cell preparations. While simple bacterin vaccines are available for a number of rickettsial diseases, incomplete and variable protection has limited their usage to only high risk individuals and livestock [1], [2]. Currently, no recombinant vaccines are available against any rickettsial diseases, although several protective antigens of rickettsiae have been identified as potential candidates for recombinant vaccines [3], [4], [5], [6], [7], [8], [9].
Piscirickettsia salmonis is the aetiological agent of salmonid rickettsial septicaemia (SRS), a devastating disease of farmed salmonid fish. P. salmonis was first isolated in 1989 from a moribund coho salmon from a saltwater net pen site in Chile where cumulative mortalities from SRS had peaked between 60 and 90% [10]. SRS responds poorly to antibiotic treatment and annual cumulative mortalities still commonly reach 40% in problem regions. The Chilean aquaculture industry attributes annual losses of US$150 million to SRS [11]. Sporadic outbreaks of SRS have since emerged among farm-raised salmon in Scotland, Ireland, Norway, and Canada, although so far cumulative mortalities have not been as high as those in Chile [12], [13], [14], [15].
P. salmonis is a gram negative, obligate intracellular bacteria and 16S rRNA phylogenetic analysis placed it within the gamma subdivision of Proteobacteria most closely related to another rickettsiae, Coxiella burnetii [16], [17]. P. salmonis infections are septicaemic and initially target monocytes followed by infection of the endothelium of the kidney, liver, spleen, heart, brain, intestine, ovary, and gills of salmonids [18]. SRS occurs during the saltwater stage of the salmonid life cycle, and is horizontally transmitted [19]. Natural reservoirs of P. salmonis and a vector for its transmission have not been identified.
Vaccine trials using inactivated whole cell preparations of the salmonid pathogen P. salmonis have yielded disappointing results in field trials [11]. No evidence of a significant protective effect was observed in coho salmon (Oncorhynchus kisutch) vaccinated with a P. salmonis bacterin, and disease was even exacerbated in some vaccinated groups suggesting the presence of immunosuppressive antigens [11]. Traditional control of mammalian rickettsial diseases has primarily relied upon chemoprophylaxis and control of insect and rodent vectors [20].
Several putative major surface antigens of P. salmonis have been recently revealed [21], [22], but only a single 17 kDa putative outer surface protein, OspA, has been cloned and characterized [23]. OspA is recognized by coho salmon convalescent sera [23] and thus is considered to be a good candidate for a recombinant vaccine against P. salmonis.
Although many aspects of the fish immune system remain uncharacterized, sufficient information is available to conclude that fish have the basic mechanisms possessed by immune systems of higher vertebrates [24]. A lack of isogenic strains of fish and monoclonal antibodies against T cell specific markers has caused research of the cellular arm of the fish immune system to lag behind that of the humoral response. Nevertheless, the teleost adaptive immune system is orchestrated by lymphocytes and is capable of specificity and immunological memory [24]. Genes encoding various components of the immune system have also been characterized in some teleosts: immunoglobulin IgM [25], [26], major histocompatibility complex (MHC) class I and II molecules [24], [27], [28], β2-microglobulin, T cell receptors [29], and Tap and Lmp proteases [30], [31].
During the past decade, numerous studies have established the potential for promiscuous T cell epitopes (TCE's) incorporated into chimeric peptides and proteins to enhance the immunogenicity of other epitopes within the chimeric peptide or protein in mammalian immune systems [32], [33], [34], [35], [36], [37]. Promiscuous TCE's from measles virus fusion protein (MVF) (288–302) [38] and Clostridium tetani tetanus toxin (tt) P2 epitope (830–844) [39] were used here to construct chimeric fusion proteins with OspA to determine if these mammalian TCE's could enhance the immunogenicity of OspA within the salmonid immune system.
We report that recombinantly produced OspA forms an effective subunit vaccine for P. salmonis in coho salmon. Furthermore, the addition of xenobiotic TCE's to OspA fusion protein constructs significantly improved the immunogenicity of OspA which was reflected by a markedly increased protection of vaccinated salmon.
Section snippets
Bacterial strains and media
P. salmonis and CHSE-214 samples were obtained from the American Type Culture Collection (ATCC). Type strain P. salmonis LF-89 (ATCC VR-1361), herein referred to as P. salmonis, was routinely grown on monolayers of a chinook salmon embryo cell line, CHSE-214 (ATCC CRL-1681) and purified using Percoll (Amersham Pharmacia) density gradient centrifugation as previously described [21].
Escherichia coli XL1-Blue (Stratagene) and BL21 (Pharmacia) were used for general cloning and expression,
Inactivated whole cell P. salmonis protects coho salmon
Control coho salmon injected with adjuvant reached a cumulative mortality of 66.7%, 48 days after challenge with P. salmonis (Fig. 2). Coho salmon vaccinated with neat adjuvanated P. salmonis bacterin were protected with a 35% RPS (Fig. 2). Salmon vaccinated with P. salmonis bacterin (dil. 1:5) exhibited only a mild protective response (17.5% RPS) and salmon vaccinated with bacterin (dil. 1:25) experienced 86.7% cumulative mortality significantly higher than that of the unvaccinated controls (
Discussion
Prior to the development and testing of a candidate recombinant vaccine for P. salmonis, we investigated whether or not it was possible to elicit a protective immune response in salmon against P. salmonis using a simple bacterin vaccine. Although the real efficacy of bacterin vaccines against other rickettsiae is highly suspect [1], [2], [47], several are still commercially available against a variety of rickettsiae: Ehrlichia risticii, Cowdria ruminantium, and Coxiella burnetii.
When a P.
Acknowledgements
We thank S. Balfry for care of the experimental animals and R. Roper and J. Drennan for informative discussions. M. Kuzyk was supported by funding from the National Science and Engineering Research Council and the Science Council of British Columbia with PGS A and GREAT awards, respectively. This work was also supported by funding from the Canadian Bacterial Diseases Network.
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