CA2281913A1 - A vaccine against piscirickettsia salmonis based on a recombinant 17 kd protein - Google Patents

Abstract

A method for the protection against infection of a poikilothermic fish by the bacterial pathogen, Piscirickettsia salmonis comprised of administering either intraperitoneally or orally or by both routes to said animal an immunogenic amount of a pharmaceutical composition consisting essentially of a principal antigen, the OspA protein, its variants or antigenic peptides derived or synthesized therof, with or without an adjuvant.

Description

A VACCINE AGAINST PISCIRICKETTSIA SALMONIS BASED

FIELD OF THE INVENTION
This invention relates to the use of the 17 kD protein (OspA) of Piscirickettsia salmonis, or its homologues, as the basis of, or part thereof, a recombinant vaccine for salmonid rickettsial septicaemia and other rickettsial diseases.
BACKGROUND OF THE INVENTION
The order Rickettsiales historically encompassed any intracellular bacterium and taxonomy was based on only a few phenotypic characteristics {Drancourt, 1994 #59}. More recently, 16S rRNA sequence similarity studies have helped to better define the taxonomy of the order Rickettsiales {Drancourt, 1994 #59}. Rickettsiae cause a variety of medically significant diseases in humans including typhus fever, Rocky Mountain spotted fever, and boutonneuse fever {Vishwanath, 199 #58; Pang, 1994 #62}. Rickettsiae are also agriculturally significant and are the aetiological agents of a variety of veterinary diseases {Rikihisa, 1991 #60}.
The past decade has been a renaissance in the identification of rickettsial and rickettsial-like infections as the aetiological agents of poorly understood diseases and as emerging pathogens {Azad, 1997 # 1; Fryer, 1997 #94; Stenos, 1998 #25; Anderson, 1997 # 142;
Davis, 1998 # 160 } .
Inherent difficulties are associated with rickettsials: it is very difficult to grow large quantities of rickettsiae; rickettsiae have very slow growth rates; and rickettsiae are difficult to separate from host cell material {Higgins, 1998 #136}. Although rickettsiae lack a characterized genetic system for genetic manipulation {Mallavia, 1991 #14}, the advent of recombinant DNA
technology has revolutionized rickettsial research. Characterization of rickettsial pathogenesis and functional analysis of rickettsial proteins has largely relied upon antibody inactivation studies {Seong, 1997 #51; Messick, 1994 #48; Li, 1998 #46}. Recently major rickettsial antigens have been identified and characterized further upon sub-cloning into Escherichia coli {Hahn, 1996 #28; Ching, 1996 #45; Musoke, 1996 #34; Ching, 1992 #26; Anderson, 1990 #42; Carl, 1990 #27; Anderson, 1987 #131 }. Successful transformation of Rickettsia typhi {Troyer, 1999 #159} and Rickettsia prowazekii {Rachek, 1998 #38} have recently raised exciting prospects for the future of rickettsia research.
Antibody studies of rickettsiae have shown that inactivation of specific rickettsial surface proteins can inhibit entry into host cells and establishment of infection {Messick, 1994 #48;
Anacker, 1985 #145; Li, 1998 #46}. Failed attempts at constructing vaccines against human rickettsial diseases have been based on preparations of inactivated whole cells {Summer, 1995 #144}. Although these whole cell vaccines elicit protective responses in animal models, they are only partially effective when used in humans { Summer, 1995 # 144 } . Current vaccine strategies using recombinantly expressed rickettsial proteins identified by antibody studies have been shown to successfully elicit protective immune responses against bacterial challenge {McDonald, 1987 #31; Summer, 1995 #144}.
Piscirickettsia salmonis is the first rickettsiae to be isolated from an aquatic poikilotherm {Fryer, 1990 #3}. P. salmonis is the aetiological agent of salmonid rickettsial septicaemia (SRS), and is an economically significant pathogen of salmonids that is responsible for extensive mortalities in the cold water aquaculture industry. P. salmonis, a gram-negative obligate intracellular bacterium, was first observed in 1989 in a diseased, moribund coho salmon from a saltwater net pen site on the coast of Chile {Bravo S., 1989 #2}. It is now known that P. salmonis is geographically more widespread than was initially suspected, and has recently been observed in Ireland {Rodger, 1993 #14}, Scotland, Norway, and on the Pacific coast of Canada {Brocklebank, 1993 #9}.
P. salmonis has been observed to infect a wide range of salmonid species and causes a systemic infection that targets the kidney, liver, spleen, heart, brain, intestine, ovary, and gills of salmonids { Cvitanich, 1991 # 1 } . Pleomorphic, predominantly coccoid bacteria that range in diameter from 0.5 to 1.5 ~m are found within cytoplasmic vacuoles of cells from infected tissues {Bravo S., 1989 #2}. While initially difficult to culture, P. salmonis was successfully isolated from the kidney of a diseased adult coho salmon on an immortal, Chinook salmon embryo cell line {Fryer, 1990 #3}. Fryer et al. {Fryer, 1992 #7} conducted a 16S rRNA
sequence similarity study which placed P. salmonis in its own genus and species within the order Rickettsiales.
P. salmonis is most closely related to Coxiella burnetii and Wolbachia persica with 87.5% and 86.3% sequence similarity respectively {Fryer, 1992 #7}. P. salmonis appears to belong within the tribe Ehrlichieae because of its morphilogical characteristics {Fryer, 1992 #7}.

Efficacy of antibiotic treatment of SRS is poor because of the intracellular nature of P. salmonis, thereby making management of the disease difficult {Lannan, 1993 #12}. To effectively prevent and control SRS, vaccine development is the only logical alternative.
However, vaccines prepared from whole cell bacterins of mammalian rickettsiae have shown disappointing protection in trials {Hickman, 1991 #7}.
SUMMARY OF THE INVENTION
The present inventors have characterized the surface antigens of the bacterial pathogen P. salmonis and identified and characterized an immunoreactive antigen. The slow growing, rickettsia-like, piscine pathogen, P. salmonis, was grown en mass on Chinook salmon (Oncorhynchus tshawytscha) embyro cell line monolayers (CHSE-214) to purify enough P. salmonis to allow genomic deoxyribonucleic acid (DNA) isolation. A genomic expression library was constructed and screened with high titre anti-P. salmonis rabbit serum identifying immunoreactive clones that encoded a common region of P. salmonis DNA. A 4,983 by insert was excised in E. coli and Exo III/S 1 deletion clones were sequenced. The insert contained 4 intact open reading frames (ORF) encoding a homologue, ospA, of a genus-specific rickettsial 17 kD antigen, a RadA DNA repair enzyme, an insertion sequence-like element encoding a transposase, and a hypothetical BAX protein.. OspA was recognized by both convalescent coho salmon (Oncorhynchus kisutch) serum and rabbit antiserum to both 10 & 20 residue peptides based on predicted protein sequence. The codon usage of the ospA ORF was optimized for expression in E coli by construction of a synthetic version of the ospA gene.
An N-terminal fusion partner was cloned in frame with the ospA gene under control of both T7 and lambda phage promoters to facilitate large scale expression of the protein. The OspA
fusion protein was purified from E. coli as the insoluble fraction of a whole cell lysate.
Suspensions of the insoluble fraction were formulated with an adjuvant and used as a vaccine to immunize coho salmon. Four weeks post-vaccination the salmon were challenged with virulent suspensions of P. salmonis.
The results indicated that the vaccine was protective against virulent challenge.
SEQUENCE LISTING
The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and one letter code for amino acids. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
SEQ ID:1 shows the ospA DNA sequence from P. salmonis SEQ ID:2 shows the amino acid sequence of the precursor (unprocessed) protein OspA
SEQ ID:3 shows the ospA DNA sequence, 17e2, modified for optimal codon usage in E. coli SEQ ID:4 shows the amino acid sequence of the modified for optimal codon usage, in E. coli, precursor (unprocessed) protein OspA (17E2) SEQ ID: 5 shows the DNA sequence, c17e2, of an N-terminal fusion partner with optimized ospA gene SEQ ID: 6 shows the amino acid sequence of an N-terminal fusion partner with optimized OspA (C 17E2) SEQ ID:7 DNA sequence of the forward oligonucleotide used during pTYBI-l7kD
construction SEQ ID:8 DNA sequence of the reverse oligonucleotide used during pTYBI-l7kD
construction SEQ ID:9 oligonucleotide #1 used for construction of optimized ospA gene, 17e2 SEQ ID:10 oligonucleotide #2 used for construction of optimized ospA gene, 17e2 SEQ ID:11 oligonucleotide #3 used for construction of optimized ospA gene, 17e2 SEQ ID:12 oligonucleotide #4 used for construction of optimized ospA gene, 17e2 SEQ ID:13 oligonucleotide #5 used for construction of optimized ospA gene, 17e2 SEQ ID:14 oligonucleotide #6 used for construction of optimized ospA gene, 17e2 SEQ ID:15 amino acid sequence of a 10 residue synthetic polypeptide based on residues 110-119 of OspA
SEQ ID:16 amino acid sequence of a 20 residue synthetic polypeptide based on residues 110-129 of OspA
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions Epitope: An epitope refers to an immunologically active region of an immunogen (protein) that binds to specific membrane receptors for antigen on lymphocytes or to secreted antibodies To generate an immune response to a foreign antigen, lymphocytes and antibodies recognize these specific regions (epitopes) of the antigen rather than the entire molecule.
B cell epitope: The region of an immunogen (protein, polysaccharide, or lipid) which is recognized by B cells when it binds to their membrane bound antibody. The B
cells which recognize that particular region then proliferate and secrete antibody molecules which are specific for that region of the immunogen. B cell epitopes tend to be highly accessible regions on the exposed surface of the immunogen. Stimulation of the immune system by B
cell epitopes results in "humoral" immunity.
T cell epitope: The region (epitope) of an immunogen which is recognized by a receptor on T cells after being processed and presented on the surface of an antigen presenting cell (APC) in the context of a major histocompatability complex (MHC) class I or II
molecule. T cells can be split into two distinct groups, T helper cells (Th) and T cytotoxic cells (T~). T helper cells recognize epitopes bound to MHC class II molecules whereas T cytotoxic cells recognize epitopes bound to MHC class I molecules. T helper cells can be further subdivided into two classes, T,,1 and Th2, Thl being responsible for stimulation of cell-mediated immunity and Th2 cells stimulating the humoral arm of the immune system. When a given T cell recognizes the epitope-MHC complex at the surface of the APC it becomes stimulated and proliferates, leading to the production of a large number of T cells with receptors specific for the stimulating epitope.
Stimulation of the immune system by T cell epitopes normally results in "cell-mediated"
immunity.
Attenuated Bacterial Vaccine: This refers to bacterial strains which have lost their pathogenicity while retaining their capacity for transient growth within an inoculated host.
Because of their capacity for transient growth, such vaccines provide prolonged immune-system exposure to the individual epitopes on the attenuated organisms, resulting in increased immunogenicity and memory-cell production, which sometimes eliminates the need for repeated booster injections. The ability of many attenuated vaccines to replicate within host cells makes them very suitable to induce a cell-mediated immunity. Typically, bacterial strains are made attenuated by introducing multiple defined gene mutations into the chromosome thereby impairing growth in vivo.
Recombinant Vector Vaccine: This refers to the introduction of genes (or pieces of genes) encoding major antigens (or epitopes) from especially virulent pathogens into attenuated viruses or bacteria. The attenuated organism serves as a vector, replicating within the host and expressing the gene product of the pathogen.
Sequence Identity: Identity between two nucleic acid sequences, or two amino acid sequences is expressed in terms of the level of identical residues shared between the sequences.
Sequence identity is typically expressed in terms of percentage identity; the higher the percentage, the more similar the two sequences are.
Sequence Similarity: Similarity between two amino acid sequences is expressed in terms of the level of sequence conservation, including shared identical residues and those residues which differ but which share a similar size, polarity, charge or hydrophobicity. Sequence similarity is typically expressed in terms of percentage similarity; the higher the percentage, the more similar the two sequences are.
Recombinant: A recombinant nucleic acid is one that has a sequence that is not normally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.
Oligonucleotide (oligo): A linear polymer sequence of up to approximately 100 nucleotide bases in length.
Probes and primers: Nucleic acid probes and primers may readily be prepared based on the amino acid and DNA sequence provided by this invention. A probe comprises an isolated nucleic acid attached to a detectable label or reporter molecule. Typical labels incluce radioactive isotopes, ligands, chemiluminescent agents, and enzymes. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed, e.g., in Sambrook et al. .
Primers are short nucleic acids, preferably DNA oligonucleotides 15 nucleotides or more in length. Primers may be annealed to a complementary target DNA strand, and then extended along the target DNA strand by a DNA polymerise enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, e.g., by the polymerise chain reaction (PCR) or other nucleic-acid amplification methods known in the art.
Methods for preparing and using probes and primers are described, for example, in Sambrook, 1989, Ausubel, 1987, and Innis, 1990. PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as DNAStar Lasergene software. One of skill in the art will appreciate that the specificity of a particular probe or primer increases with its length. Thus, for example, a primer comprising 20 consecutive nucleotides will anneal to a target with a higher specificity than a corresponding primer of only 15 nucleotides. Thus, in order to obtain greater specificity, probes and primers may be selected that comprise 20, 25, 30, 35, 40, 50 or more consecutive nucleotides.
Isolated: An "isolated" biological component (such as nucleic acid or protein or organelle) has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods.
The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids. An "isolated" bacterial strain or colony is purified away from other colonies and yields a pure culture without any contaminants upon plating on selective media.
Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements known in the art. A "temperature-sensitive" vector is one which replicates normally at a low growth temperature (i.e., 28°C) and will not replicate at a higher growth temperature (i.e., 42°C) due to mutations at or near the origin of replication. An "imperfectly segregating" vector is one which is not stably inherited by new daughter cells at the time of cell division in the absence of selection pressure due to mutations within the vector sequence.
Host Cell: Refers to those cells capable of growth in culture and capable of expressing OspA protein and/or OspA fusion protein. The host cells of the present invention encompass cells in in vitro culture and include prokaryotic, eukaryotic, and insect cells. A host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers (i.e. temperature, small inducer molecules such as (3-galactosides for controlling expression of T7 or lac promoters or variants thereof). The preferred host cell for the cloning and expression of the OspA protein and OspA-fusion protein is a prokaryotic cell. An example of a prokaryotic cell useful for cloning and expression of the OspA protein of the present invention is E. coli BL21.
Cell Culture: a) Refers to the growth of eukaryotic (non-bacterial) cells in a complex culture medium generally consisting of vitamins, buffers, salts, animal serum, and other nutrients.
b) Refers to the growth of P. salmonis on CHSE-214 and any other cell line that sustains P. salmonis growth.
Fusion Partner: Any DNA sequence cloned in frame to the 5' or 3' end of an ORF
that results in transcription and translation of amino acid sequence added to the N-or C-terminus of the original protein.
Fusion Protein: The term fusion protein used herein refers to the joining together of at least two proteins, an OspA protein and a second protein. In some embodiments of the present invention, the second protein may be fused or joined to a third protein. In the present invention, examples of second proteins include any polypeptide that facilitates the following: expression, secretion, purification, condensation, precipitation, or any property which facilitates concentration or purification.
Variant: Any molecule in which the amino acid sequence, glycosylation, phosphorylation, and/or lipidation pattern, or any other feature of a naturally occurring molecule which has been modified covalently or non-covalently and is intended to include mutants. Some of the variants falling within this invention possess amino acid substitutions, deletions, and/or insertions provided that the final construct possesses the desired ability of OspA. Amino acid substitutions in OspA may be made on a basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. Also included within the definition of variant are those proteins having additional amino acids at one or more of the C-terminal, N-terminal, and within the naturally occurring OspA sequence as long as the variant protein retains the capability to act as an antigen and hence as a vaccine.

Original ResidueConservative Substitutions Ala ser Arg lys Asn gln; his Asp glu Gln asn Glu asp Gly pro His asn; gln Ile leu; val Leu ile; val Lys arg; gln; glu Met leu; ile Phe met; leu; tyr Ser thr Thr ser Trp tyr Tyr trp; phe Val ile; leu II. Table 1: More substantial changes in functional or other features may be obtained by selecting substitutions that are less conservative than those in Table 1, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in protein properties will be those in which (a) a hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl or alanyl;
(b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine. Variant peptides having one or more of these more substantial changes may also be employed in the invention, provided that temporin or dermaseptin biological activity is retained.
More extensive amino acid changes may also be engineered into variant dermaseptin or temporin peptides. As noted above however, these variant peptides will typically be characterized by possession of at least 40% sequence identity counted over the full length alignment with the amino acid sequence of their respective naturally occurring sequences using the alignment programs described above. In addition, these variant peptides will retain biological activity.
Confirmation that a dermaseptin or temporin peptide has biological activity may be achieved using the assay systems described above. Following confirmation that the peptide has the desired activity, a nucleic acid molecule encoding the peptide may be readily produced using standard molecular biology techniques. Where appropriate, the selection of the open reading frame will take into account codon usage bias of the plant species in which the peptide is to be expressed.
III. Selection and Creation of Nucleic Acid Sequences Encoding the 17 kD OspA Protein a. Growth & Purification of P. salmonis P. salmonis strains were routinely passaged on Chinook salmon embryo cell line, CHSE-214 (ATCC CRL-1681), at 17°C in Eagle's minimal essential media (MEM) with Earles salts supplemented with 10% newborn calf serum. Type strain P. salmonis LF-89 was obtained from the American Type Culture Collection (ATCC VR-1361) and is herein referred to as P. salmonis.
A protocol for purifying P. salmonis was developed by combining and modifying the protocols of Tamura et al {Tamura, 1982 #19} and Weiss et al {Weiss, 1975 #20}. A 6,320 cm2 Nunc cell factory was seeded with cell line CHSE-214 and infected with 450 ml of cell culture supernatant from fully lysed CHSE-214 monolayers infected with P. salmonis.
Infection was allowed to continue 14-17 days until cytopathic effects obliterated the entire monolayer. Upon destruction of the monolayers cell culture supernatants were collected and pelleted at 10,000 x g for 30 min at 4°C. Pellets were resuspended in MEM and homogenized in a 15 ml Dounce tissue homogenizer. The homogenized suspension was centrifuged at 200 x g for 10 min at 4°C to pellet large host cell debris. The supernatant was filtered twice through glass microfiber and pelleted at 17,600 x g for 15 min at 4°C. Pellets were resuspended in TS-buffer (33 mM
Tris-HCI, 0.25 M sucrose; pH 7.4). Samples were loaded onto Percoll gradients with a final concentration of 40% and centrifuged in a fixed angle rotor (type JA-14) at 20,000 x g for 60 min at 4°C in a Beckman J2-21 centrifuge. Bands were collected by aspiration, diluted with phosphate buffered saline, pH 7.4 {Sambrook, 1989 #2} and pelleted at 20,000 x g for 10 min at 4°C. Pellets were washed twice with PBS. Contents of the bands were negative stained with 0.5% phosphotungstic acid and analyzed by transmission electron microscopy on a Phillips EM
300 at an accelerating voltage of 75 kV.
b. Demonstration of Immunoreactive Molecules In order to characterize the antigenic profile of P. salmonis western blot analysis was carried out using anti-P. salmonis rabbit serum (Fig. 1). Proteinase K
digestion was used to determine if any observed antigens may have been carbohydrate. Six P. salmonis immunoreactive antigens were observed at relative molecular weights of 65, 60, 54, 51, 17, and 11 kD (Fig. 1 ). Proteinase K digestion destroyed all immunoreactive antigens except the 11 kD
antigen (Fig. 1 ).
c. Purification of Genomic DNA & Construction of Library P. salmonis was purified by density gradient centrifugation as previously described {Kuzyk, 1996 #97} from 12,000 cmz of CHSE-214 cells exhibiting full cytopathic effect 14 days after infection with P. salmonis. A single step DNA isolation solution was used to obtain genomic DNA from the purified P. salmonis. Genomic DNA was further purified by equilibrium centrifugation using a CsCI-ethidium bromide gradient to yield 250 ~g of P.
salmonis genomic DNA { Sambrook, 1989 #81 } .
P. salmonis DNA was partially digested using serially diluted EcoR I. Digests containing an average fragment size of 10 kb were chosen for creation of a P. salmonis gene expression library using a lambda ZAP II cloning kit.

d. Immunological Screening of Library Approximately 10,000 plaques of P. salmonis lambda expression library were screened per round with a desired density of 1,000 plaques per 80 mm petri dish. Plaques were lifted in duplicate using 80 mm nitrocellulose discs impregnated with 10 mM IPTG.
Screening followed the protocol of Sambrook et al. (1989) using anti-P. salmonis rabbit serum.
Immunoreactive plaques were picked and rescreened until pure cultures were obtained. Lambda clones were then amplified and the pBluescript phagemid excised into E. coli.
Screening of the P. salmonis expression library with high titre anti-P.
salmonis rabbit serum identified several strongly immunoreactive plaques. These plaques were picked and rescreened until pure and were confirmed to contain inserts. Initial attempts to excise the clones into E. coli from the lambda clones were unsuccessful which suggested the clones may encode products toxic to E. coli. Restriction fragment length analysis using frequently cutting enzymes suggested that all clones contained a common region of DNA. The clones contained a 5 kb insert (Example 1 ).
Genomic DNA from all the lambda clones, P. salmonis, CHSE-214, and vector plasmid DNA was analyzed by DNA dot blotting using insert DNA from one clone (Clone pB
12) as the probe. Hybridization revealed that the pB 12 insert was of P. salmonis origin.
The pB 12 insert also hybridized with all other immunoreactive lambda clone samples indicating that all the inserts encoded an overlapping fragment of P. salmonis DNA.
e. DNA Sequence Analysis of Clone pBl2 DNA sequence analysis of clone pB 12 (Example 1 ) identified 4 complete ORFs within the 4,983 by insert and 1 partial ORF (Example 1 ). The predicted amino acid sequences of these ORFs was subjected to homology searches using database tools (eg. BLAST2 and FASTA3). No significant matches were found when searching for DNA sequence homology to the pB 12 insert.
The 499 by ' alr ORF (Example 1 ) was predicted to encode a 176 residue (res.) protein fused to the N-terminus of LacZ. The predicted molecular weight (m.w.) of the LacZ-'Alr fusion is 22.2 kD. The predicted 'Alr ORF amino acid sequence shares 44% identity and 63% similarity with C-terminal portions of known alanine racemase enzymes from Klebsiella aerogenes (GenBank AAC38140), Salmonella typhimurium (GenBank A29519), and E. coli (GenBank BAA36048).
A 732 by ORF (bax; Example 1 ) was predicted to encode a 243 res., 27.6 kD
protein. Both FASTA3 and BLAST2 only identified low scoring similarity (33% identical, 49%
similar) between the central 187 amino acid region of the bax ORF and a 274 res.
uncharacterized, hypothetical protein in E. coli K12 (BAX; GenBank AAB18547).
A 1368 by ORF (radA; Example 1 ) was predicted to encode a 456 res., 49.4 kD
protein. A
high degree of amino acid homology was found over the entire length of the radA ORF and RadA DNA repair enzymes from a variety of bacteria. P. salmonis RadA is most homologous to RadA of Pseudomonas aeruginosa (SwissProt P96963) with 62% identity and 77%
similarity.
P. salmonis RadA also exhibits 59% identity and 75% similarity to E. coli RadA
(SwissProt P24554).
A 486 by ORF (ospA; Example 1 ), immediately following radA, was predicted to encode a 162 res., 17.7 kD protein with amino acids 21-162 having substantial sequence similarity with the mature chain of the rickettsial 17 kD genus common antigen. The predicted 17 kD antigen was up to 41 % identical and 62% similar to the 17 kD protein antigens of R.
prowazekii (SwissProt G112704), Rickettsia japonica (SwissProt Q52764), Rickettsia rickettsii (SwissProt P05372), and Rickettsia typhi (SwissProt P22882). The 17 kD protein of rickettsiae is translated as a precursor protein containing a 20 amino acid signal peptide. During processing the signal peptide is removed and the N-terminal cysteine residue is lipid-modified to form the mature protein. The first 21 amino acids of the P. salmonis OspA protein are predicted to be a signal peptide and contain a bacterial lipidation pattern as well.
The final 717 by ORF (tnpA; Example 1) was predicted to encode a 239 res., 27.7 kD
protein. This ORF is flanked by a perfect 288 by direct repeat. Amino acid similarity searches returned strong matches between the tnpA ORF and a variety of transposases.
The closest match was a transposase (GenBank U83995) in a Porphyromonas gingivalis insertion element, IS 195, with 47% identity and 65% similarity {Lewis, 1998 #116}.
f. Identification of the ospA ORF as the 17 kD Antigen Rabbit antibodies raised against 10-mer and 20-mer synthetic peptides of this region reacted with an immunoreactive product in P. salmonis around the 17 kD predicted mass of the ospA

ORF product (Example 2). Expression of the 17 kD antigen was induced in clone pBCKS-l7kD
and was recognized by rabbit serum against the synthetic peptides (Example 2).
Serum from coho salmon fry that had survived a challenge with P. salmonis also recognized the induced 17 kD product (Example 2). These data confirm that the ospA ORF encodes the immunoreactive 17 kD OspA antigen.
g. Optimization of the ospA ORF for E. coli Expression The coding sequence of ospA was optimized using codons used frequently by E.
coli (Example 3). Six overlapping oligonucleotides representing the optimized ospA
gene were synthesized using standard phosphoamidite method. The gene was assembled using 2 successive PCR reactions with the oligonucleotides and the full length product was cloned into an appropriate cloning vector. DNA sequence of the optimized ospA gene was verified by sequence analysis using an automated sequencer. Production of the OspA protein from the optimized ospA
gene was confirmed upon subcloning the optimized ospA gene to the pET21 (+) (Novagene) expression vector and inducing expression using the T7 promoter (Example 3).
h. Description of the Fusion Protein Construct The level of OspA production from the optimized ospA gene was still relatively low. It is well known to persons skilled in the art that fusion partners can aid in increasing the level of production of proteins. We constructed both N- and C-terminal fusions (Examples 4 & 5) with the ospA gene. In our examples we show that some fusions resulted in increased production of the OspA-fusion with the N-terminal fusion partner being more favourable than the C-terminal fusion partner. It is possible that presence of a signal peptide on the N-terminus of OspA may hamper high level production of OspA. Therefore, the N-terminal fusion partner may increase OspA production by masking the signal peptide. Similar increases in OspA
production may be obtained from deletion of the region of the ospA gene that encodes the signal peptide.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1. Western blot analysis of P. salmonis. A whole cell lysate of P.
salmonis was analyzed by 12% polyacrylamide SDS-PAGE and reacted with anti-P. salmonis rabbit serum followed by immunochemical staining. Note the immunoreactive protein migrating at 17 kD.
Molecular weight standards are shown in kD.
Figure 2. A. Schematic of spatial relationships of ORFs in P. salmonis clone pB 12, 4,983 bp. The Xba I and Hind III sites were used to subclone the ospA ORF into pBCKS(+) (Example 2). B. DNA sequence of the P. salmonis ospA ORF and amino acid sequence of the OspA protein translated from the ospA ORF. C. Pairwise sequence alignment of the P. salmonis 17 kD antigen, OspA, and the R. prowazekii 17 kD antigen (SwissProt G112704).
The pairwise alignment was generated using the FASTA3 algorithm. The P. salmonis 17 kD
antigen shares 41 % identity (black background) and 62% similarity (black box) with the 17 kD
antigen of R. prowazekii. Synthetic peptides (SEQ ID:15, SEQ ID:16) representing the region from residues 110-129 of the P. salmonis 17 kD antigen were used to generate rabbit polyclonal serum.
Figure 3. A. Map of pBCKS-l7kD, the pBCKS(+) plasmid encoding the subcloned ospA
ORF (Xba IlHind III fragment of clone pB 12). Cm is chloramphenicol resistance, T7 is T7 promoter. B. Analysis of OspA expression. Whole cell lysates of E. coli clones and P. salmonis were analyzed by SDS-PAGE (12% polyacrylamide). P. salmonis whole cell lysate was reacted with rabbit polyclonal serum generated against a 10 residue peptide (SEQ
ID:15) of OspA
recognizing a strongly immunoreactive product in the 17 kD region of P.
salmonis. Expression of the OspA by clone pBCKS-l7kD was induced at 42 C and is visible stained by Coomassie blue. Rabbit polyclonal serum generated against a 20 residue peptide (SEQ
ID:16) of OspA
recognized the expressed 17 kD protein in induced pBCKS-l7kD samples.
Convalescent serum from coho salmon also recognized the induced 17 kD protein in pBCKS-l7kD.
Arrows identify the expressed 17 kD antigen. Molecular weight standards are shown in kD.
Figure 4. A. Schematic representation of the strategy employed during the synthesis of the E. coli codon optimized ospA gene, 17e2. B. DNA sequence of the 6 overlapping oligonucleotides used. C. DNA sequence of the E. coli codon optimized ospA
gene, 17e2.
Figure 5. Amino acid sequence of the OspA protein, 17E2, expressed from the optimized ospA gene, 17e2. DNA sequence of the N-terminal ospA gene fusion construct, c 17e2. Amino acid sequence of the OspA-fusion protein, C 17E2, containing an N-terminal fusion.
Figure 6. A. Maps of the expression vectors encoding the optimized ospA fusion construct under the control of T7, pETC-17E2, and lambda promoters, pKLPR-C17E2. Ap is ampicillin resistance, Km is kanamycin resistance, T7 P is the T7 promoter, PLR is lambda right promoter.

B. 12% polyacrylamide SDS-PAGE analysis of C17E2 expression. Samples from the lambda promoter expression represent the insoluble fraction (i.f.) of whole cells lysates. Whole cell (w.c.) samples from T7 expression are loaded along with a sample of the insoluble fraction Note the abundant expression of the OspA-fusion product at 28.5 kD in the induced samples.
Molecular weight standards are shown in kD.
Figure 7. Map of pTYBl-l7kD. An ospA-fusion construct encoding a C-terminal fusion partner was placed under the control of T7 promoter. The C-terminal fusion partner contained a self cleaving spacer region and chitin binding domain.
Figure 8. Antibody titres of coho salmon groups against the OspA-fusion candidate vaccine. Salmon were immunized with either the vaccine candidate, adjuvant, or no immunization control (naive). Data was determined by ELISA and absorbance values were plotted vs. serum dilution.
Figure 9. Plot of cumulative coho salmon mortalities vs. time. Mortalities were recorded for 37 days following P. salmonis challenge. Note the protective effect of the OspA-fusion vaccine candidate. Each group contained a total of 50 fish.
EXAMPLES
1. Sequence Analysis of P. salmonis Insert Producing Immunoreactive Material A directional deletion library of P. salmonis clone pB 12 was constructed to facilitate sequence analysis. Exo III and S 1 nuclease were used to construct double-stranded nested deletions in the direction of lacZ. Restriction endonucleases EcoR I and Sac I
were used to generate opposing overhangs protecting the vector from Exo III digestion. Upon ligation and screening, 32 deletion clones were selected that represented the entire insert and differed in size by 100-500 bp.
Double stranded plasmid DNA samples were sequenced using a combination of dye primer and dye termination. Sequencing reactions were analyzed using an automated DNA
sequencer.
Sequence data were assembled and analyzed using commercially available computer software packages.
DNA sequencing of pB 12 Exo III/S 1 nuclease deletion clones revealed that the insert was 4,983 bp. Coding predictions identified 4 intact ORFs and 1 partial ORF
creating a fusion in frame with LacZ (Fig. 2). The predicted ORFs were subjected to BLAST2 {Altschul, 1997 #149} and FASTA3 {Pearson, 1998 #148} analysis to determine if any similar sequences were known (Fig. 2).
2. Identification of the ospA ORF as the Source of OspA
Residues 110-129 of the 17 kD antigen encoded by the predicted ospA ORF were predicted to be a B cell epitope by the Jameson-Wolf method {Jameson, 1988 #140}.
Antibodies were generated in New Zealand white rabbits against 10 and 20 amino acid synthetic peptides (SEQ
ID:15; SEQ ID:16) representing amino acids 110-129 of the predicted OspA amino acid sequence (SEQ ID:2). Peptides were glutaraldehyde conjugated to for 1 h at 4°C in a 10 ml reaction volume with 500 ~g/ml keyhole limpet hemocyanin and 1%
glutaraldehyde. For the primary immunization, rabbits received 250 ~g of conjugated peptide mixed 1:1 with Freund's complete adjuvant. Each rabbit was boosted three times at 2 week intervals with 250 ~,g of conjugated peptide per boost mixed 1:1 with Freund's incomplete adjuvant.
Table 2: Synthetic polypeptides used to generate polyclonal rabbit antibodies against OspA.
Peptide Sequence mer (SEQ ID:15) pro-Val-Arg-Thr-Tyr-Gln-Arg-Tyr-Asn-Lys mer pro-Val-Arg-T'hr-Tyr-Gln-Arg-Tyr-Asn-Lys-Gln-Glu-Arg-Arg-Gln-Gln-Tyr-Cys-Arg-Glu (SEQ ID:16) The 17 kD antigen ospA ORF was subcloned into pBCKS(+) under control of the T7 promoter. The Xba IlHind III fragment of clone pB 12 was ligated with Xba IlHind III digested pBCKS(+) to generate clone pBCKS-l7kD. Induction of the T7 promoter by shifting growth temperature to 42°C resulted in expression of a 17 kD protein observed by Coomassie staining of whole cell lysates of induced clone pBCKS-l7kD SDS-PAGE samples (Fig. 3).
Western blot analysis of whole cell lysates of P. salmonis and pBCKS-l7kD with rabbit antibodies generated against synthetic peptides of OspA reacted with a 17 kD protein in both P.
salmonis and the induced sample of pBCKS-l7kD confirming the ospA ORF as the source of then translated OspA protein (Fig. 3).

3. Synthesis & Cloning of Optimized ospA Gene A nucleic acid molecule was designed to encode the OspA protein precursor (OspA
including signal peptide). This nucleic acid was constructed by PCR using 6 overlapping oligonucleotides (SEQ ID:9, SEQ ID:10, SEQ ID:11, SEQ ID:12, SEQ ID:13, and SEQ ID:14).
Synthesis of ospA gene was done by three subsequent PCR using the six synthetic overlapping oligonucleotides (Fig. 4A & Fig. 4B). PCR-1 involved overlapping oligonucleotides SEQ ID:11, SEQ ID:12 (0.05 pmol/~l each) and SEQ ID:10, SEQ ID:13 (0.25 pmol/~1 each).
Product of PCR-1 (1 ~1) was used as a template in PCR-2 using oligonucleotides SEQ ID:9 and SEQ ID:14 as primers (0.25 pmol/~1). Both PCR were performed using Taq I polymerase (Boehringer), supplied buffer and deoxynucleotide triphosphates (dNTP) (Amersham Pharmacia).
Temperature cycling was as follows: PCR-1 & 2: 92°C 30 sec., 55°C 30 sec., 72°C 30 sec., 1 cycle 92°C 30 sec., 70°C 30 sec., 72°C 30 sec., 29 cycles Product of PCR2 (Fig. 4C) was cloned into plasmid vector pBCKS(+) as a BamH I -Hind III fragment resulting to pBCKS-17E2. DNA sequence of the insert was verified by DNA
sequencing using methods known to those skilled in the art. The DNA fragment of pBCKA-17E2 carrying optimized ospA gene was than cloned to pET21 (+) as a Nde I -Hind III DNA
fragment resulting to pET-17E2.
4. Expression of Optimized OspA Antigen With N-Terminal Fusion Partner A. Expression using T7 promoter system DNA fragment of pBCKA-17E2 carrying optimised ospA gene was cloned, using methods known to one skilled in the art, to pETC (Microtek International) resulting to pETC-17E2 as a BamHI-HindIII fragment carrying ospA fused to a desired fusion partner under control of T7 promoter (Fig. 5, Fig. 6A).
Strain E. coli BL21 [E.coli B, F-, ompT, hsdS (rs; ms), gal, dcm] (Pharmacia) carried the recombinant expression plasmid pETC-17E2 and helper plasmid pGPl-2 ~.
Expression experiment was performed in 4 L flask. During the growth phase, the culture was grown in TFB with agitation 0300 RPM) at 28-30°C to late log phase.
Then cells were diluted with an equal volume of fresh TFB media and growth continued at 42°C 3-6 hours. Product was accumulated inside cells as insoluble aggregates of protein. Cells from 1 ml of culture were sedimented in a microcentrifuge, washed with water, resuspended in 1 ml of water and disrupted by sonication. Insoluble material was sedimented, washed with water and analysed by 15% SDS-PAGE as is known to one skilled in the art (Fig. 6B).
B.Expression usin;~ lambda promoter system DNA fragment of pETC-17E2 carrying fused optimised ospA gene was recloned, using methods known to one skilled in art, to pKLPR-8 (Microtek International 1998 Ltd.) resulting in pKLPR-C 17E2 as a Xba I - Kpn I fragment carrying the ospA fusion under control of phage lambda promoter. Plasmid also carries repressor gene CI875 of the lambda promoter (Fig. 5).
Strain E. coli BL21 [E.coli B, F-, ompT, hsdS (rs, ms), gal, dcm] (Pharmacia) carried the recombinant expression plasmid pKLPR-C 17E2 (Fig. 6A). During the growth phase, the culture was grown in TFB with agitation (300 RPM) at 28-30 °C to late log phase. Then cells were diluted with an equal volume of fresh TFB media and growth continued at 42°C 3-6 hours.
Product was accumulated inside cells as insoluble aggregates of protein. Cells from 1 ml of culture were sedimented in a microcentrifuge, washed with water, resuspended in 1 ml of water and disrupted by sonication. Insoluble material was sedimented, washed with water and analysed by 15% SDS-PAGE as is known to one skilled in the art (Fig. 6B).

5. Expression of Optimized OspA Antigen With C-Terminal Fusion Partner The P. salmonis ospA ORF was subcloned into the Impact CN Expression System (New England Biolabs) to add a C-terminal fusion partner containing a self cleaving spacer region and chitin binding domain to aid in purification and antibody generation of OspA
(Fig. 7).
The ospA ORF was PCR amplified from clone pB 12 using custom primers (Table 3) designed to incorporate Nde I and Sap I restriction enzyme cleavage sites onto the 5' and 3' ends of the ospA ORF. The ospA PCR product was digested with Nde I and Sap I
restriction enzymes and ligated with the pTYB 1 vector (NEB) of the Impact CN system digested with Nde I and Sap I to create the OspA fusion construct, pTYBI-l7kD (Fig. 7). Positive clones were identified by screening Kpn I and Nde I digests of plasmid preps from potential positive clones by agarose gel electrophoresis. Positive clones were confirmed to contain the ospA ORF in frame with the chitin binding domain by DNA sequence analysis.

Table 3: Oligonucleotide primers used during construction of pTYBl-l7kD. Bold nucleotides are not homologous to the template ospA ORF.
Primer Sequence Forward (SEQ ID:7) 5' - GAG AGA ACA TAT GAA CAG AGG ATG TTT GCA AGG - 3' Reverse (SEQ ID:B) 5' - GCC ATA AGC TCT TCC GCA TTT TTC TGT TGA AAT GAC TTG C -3' 6. Salmonid Antibody Response to OspA-fusion Vaccine Coho salmon antibody response to the OspA with N-terminal fusion partner vaccine candidate (Example 4) was assayed enzyme linked immunosorbant assay (ELISA).
Coho salmon fry (125 per group; ~15 g mean weight) were each injected intraperitoneally (IP) 0.2 ml of an formalin inactivated (1 ml/L) adjuvanated (MicrogenTM) vaccine (5:1 vaccine:adjuvant) containing 50 ~g of total protein purified as the insoluble fraction from E
coli BL21 expressing the ospA fusion construct pET-C 17E2 (Example 4). A control group of fish received 0.2 ml of adjuvant diluted with saline 5:1. A second control group was comprised of non-vaccinated salmon.
Four weeks post-immunization, 5 fish from each group were bled from the caudal vein, kept on ice, blood was pooled for each group and. Serum was collected by centrifugation of pooled blood at 5,000 rpm for 20 min in a clinical centrifuge. ELISA plates were coated with ~g of C17E2 protein in 100 ~1 of coating buffer (Tris buffered saline (TBS), pH 7.5, 0.5%
Tween-20). Plates were covered with parafilm and incubated at 4°C
overnight. Coating solution was removed and wells were blocked with 200 ~1 of Tween-TBS with 3% bovine serum. Plates were washed 3 times with Tween-TBS. Fish serum from each group was serially diluted in Tween-TBS with 3% bovine serum and added to wells. Plates were then incubated at 15°C for 1 h and then washed 3 times with Tween-TBS. Second antibody, a mixture of 2 monoclonal antibodies (mAb) against salmon immunoglobulin, IPA2C7 (dil. 1/100) and Beecroft (dil.
1/500), were diluted in Tween-TBS with 3% bovine serum, added to plates and incubated at room temperature for 1 h. Plates were washed 3 times with TBS-Tween. Third antibody, alkaline phosphatase conjugated goat anti-mouse IgGI (dil. 1/2000), was added to plates and incubated at room temperature for 1 h. Plates were washed 3 times. The ELISA was developed with 100 ~l of 1 mg/ml para-nitrophenyl phosphate in alkaline phosphatase buffer and incubated at room temperated overnight and absorbance at 405 nm was measured spectrophotometrically.

The results indicate that naive (~) and adjuvant controls (~) had no antibody activity against the vaccine protein whereas there was significant reactivity in serum (~) from the vaccine immunized coho (Fig. 8).

7. Protection of Immunized Salmonids Against P. salmonis Challenge All groups of salmon in Example 6 were challenged four weeks post-immunization with a 0.1 ml IP injection of P. salmonis with a titre of 1055 50% tissue culture infectious dose (TCIDSO) per ml. Mortalities were recorded for 30 days post-challenge.
Mortalities were attributed to P. salmonis by PCR analysis of kidney using P. salmonis 16S rRNA
specific primers {Fryer, 1992 #7}.
The results indicate that naive (~) and adjuvant controls (~) had severe mortalities (>50%) and the vaccinates (~) were significantly protected with only 5% mortality (Fig. 9).

Claims (11)

1. A method for the protection against infection of a poikilothermic fish by the bacterial pathogen, Piscirickettsia salmonis comprised of administering either intraperitoneally or orally or by both routes to said animal an immunogenic amount of a pharmaceutical composition consisting essentially of a principal antigen, the OspA protein, its variants or antigenic peptides derived or synthesized therof, with or without an adjuvant.

2. A method as in claim 1 where the OspA antigen or a variants therof are fused to another protein or protein fragment either at the N or C terminus where that protein may be either used to facilitate expression and/or the formation of insoluble intracellular aggregates.

3. A method as in claim 1 where the OspA antigen or a variants therof have been fused to other proteins or protein fragments where those proteins or protein fragments are T
and/or B cell epitopes.

4. A method as in claims 1-3, where the said vaccine or variants therof are encapsulated in or adsorbed to an insoluble polymeric matrix.

5. A method as in claims 1-4, where the principle OspA antigen, its variants, fragments or synthetic peptides therof are sequence homologues of the OspA protein, such as the OspA
homologues from other rickettsial bacterial pathogens within the order Rickettsiales of human and non-human animals and where the protection against infection is of a mammal (human or non-human animal) and where the route of administration is either oral, intramuscular or intradermal.

6. A method as in claims 1-5, where DNA of a sequence corresponding to that of ospA, fragments or synthetic oligonucleotides therof or of DNA sequence homologues of ospA, fragments or synthetic oligonucleotides derived therof are used as a vaccine.

7. A method as in claims 1-3, where the principle antigen is either that corresponding to OspA
or a OspA homologue where the sequence has been optimized for expression in a suitable expression host microorganism such as an E. coli bacterial strain.

8. A method as in claims 1-3 where the expression of the OspA protein, its variants or antigenic peptides derived or synthesized therof in E. coli is effected by lambda or phage T7promoter DNA sequences.

9. A method as in claims 1-5 where the principle antigen is a lipoprotein derivative of the OspA
protein, its variants, a fragment or synthesized peptide s therof.

10. An immunological method for the detection of humoral antibody to protein OspA or to P.
salmonis in sera of poikilothermic fishes where either the OspA protein, a fragment or synthesized peptide therof or OspA and its fusion partner as described in claims 2 & 3 are used to adsorb or bind to fish immunoglobulin from fish sera.

11. A method whereby the plasmid based expression vectors, pBCKS-17kD, pETC-17E2 or pKLPR-C17E2 are used to express the OspA protein, its variants, a fragment or synthesized peptide therof, a fusion partner-OspA protein therof or where OspA proteins or protein fragments are expressed with T and/or B cell epitopes.

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