Attenuated piscirickettsia salmonis bacterium

专利摘要:
The present invention relates to an attenuated Piscirickettsia salmonis bacterium. The bacterium comprises mutations in the amino acid sequence of each of the rpoD, FecR, ATP-grasp domain protein, and FtsH gene products. The invention also relates to vaccines comprising the attenuated Piscirickettsia salmonis bacterium that are useful for the prevention of microbial pathogenesis. In addition, the invention relates to methods for the preparation of attenuated Piscirickettsia salmonis bacteria, and vaccines comprising such bacteria.
公开号:DK201771015A1
申请号:DKP201771015
申请日:2017-12-22
公开日:2018-01-15
发明作者:Marianne Bordevik；Anja Nygaard；Siv Haugen Tunheim；Marianne Frøystad-Saugen；Are Klevan；Claudia Vargas Maira
申请人:Pharmaq As；
IPC主号:

专利说明:

(19) DANMARK <¹°> DK 2017 71015 A1
<¹²> PATENTANSØGNING
Patent- og
Varemærkestyrelsen (51)
Int.CI.: C 07 K 14/195 (2006.01)
C 12 R 1/01 (2006.01)
C12N 1/36(2006.01)
C 12 N 9/50(2006.01) (21) Ansøgningsnummer: PA 2017 71015 (22) Indleveringsdato: 2017-12-22 (24) Løbedag: 2016-05-25 (41) Aim. tilgængelig: 2017-12-22 (86) International ansøgning nr: PCT/EP2016/061862 (86) International indleveringsdag: 2016-05-25 (85) Videreførelsesdag: 2017-12-22 (30) Prioritet: 2015-05-26 GB 1509004.6 (71) Ansøger: Pharmaq AS, Skogmo Industriområde, N-7863 Overhalla, Norge (72) Opfinder: Marianne Bordevik, P.O. Box 267 Skoyen, c/o Pharmaq AS, 0213 Oslo, Norge Anja Nygaard, P.O. Box 267 Skoyen, c/o Pharmaq AS, 0213 Oslo, Norge
Siv Haugen Tunheim, P.O. Box 267 Skoyen, c/o Pharmaq AS, 0213 Oslo, Norge Marianne Frøystad-Saugen, P.O. Box 267 Skoyen, c/o Pharmaq AS, 0213 Oslo, Norge Are Kievan, P.O. Box 267 Skoyen, c/o Pharmaq AS, 0213 Oslo, Norge
Claudia Vargas Maira, P.O. Box 267 Skoyen, c/o Pharmaq AS, 0213 Oslo, Norge (74) Fuldmægtig: Zacco Denmark A/S, Arne Jacobsens Allé 15, 2300 København S, Danmark (54) Benævnelse: ATTENUATED PISCIRICKETTSIA SALMONIS BACTERIUM (57) Sammendrag:
The present invention relates to an attenuated Piscirickettsia salmonis bacterium. The bacterium comprises mutations in the amino acid sequence of each of the rpoD, FecR, ATP-grasp domain protein, and FtsH gene products. The invention also relates to vaccines comprising the attenuated Piscirickettsia salmonis bacterium that are useful for the prevention of microbial pathogenesis. In addition, the invention relates to methods for the preparation of attenuated Piscirickettsia salmonis bacteria, and vaccines comprising such bacteria.
Fortsættes ...
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SUBSTITUTE SHEET (RULE 26)
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ATTENUATED PlSCIRiCKETTSSA SALMÖNSS BACTERIUM
Field of the Invention in the broadest aspect, the present invention relates to an attenuated bacterium, its 5 preparation, and its use in a live attenuated vaccine,
Bnokgroutid
Salmon Rickettsial Syndrome, SRS, (also known as, Piscirickettsiosis, Coho salmon septicaemia, or Huito disease) is considered to be one of the most important disease .io problems facing, Ihe salmon farming industry. The bacterium Pischickettsia sahnonis is the causative agent of S.RS.
SRS continues to evolve and new outbreaks continually occur which, are increasi ngly insidious and refractory to treatments. New outbreaks frequently show increased {,$ bacterial virulence, clinical and pathological severity and variable presentation under similar conditions of species, age and management measures.
SRS has proven very difficult to control. The use of antibiotics, both piophylactically and during early infection, may inhibit the growth of the pathogen, but failure of eo antibiotic treatment is common, and antibiotic treatments have been largely unsuccessful in stopping disease outbreaks.
Th us, there is a need lor improved methods of controlling P. salmonis,
Vaccines based on live but attenuated micro-organisms (live attenuated vaccines) induce a highly effective type of immune response. Generally, such vaccines induce stronger and more durable immunity than vaccines based on an inaetivaied pathogen as they activate all phases of the immune system. Specifically, once an animal host has been vaccinated with a live attenuated vaccine, entry of the microbial pathogen into the go host induces an accelerated recall of earlier, cell-mediated and/or humoral immunity which is able to control the further growth of the organism before the infection can assume clinically significant proportions, inactivated vaccines (based on killed microorganisms or fragments of micro-organisms) are less likely to be able to achieve the •same magnitude and rapidity of response.
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There is thus a need .for an attenuated strain off. sdrøras, suitable for use in a vaccine. The attenuated vaccine should substantially retain the antigenic capacity of the wild-type strain in order to cause a robust immune response in the host, and thereby provide strong immunity, The vaccine should also Ire sufficiently »virulent to minimise undesirable pathological effects. In addition, the live attenuated vaccine strain should have substantially no likelihood of reversion to a virulent form.
The general approach for attenuating bacteria is the removal of one or more virulence factors. In most cases however, vindene factors are required in order to induce immunity, and delet ion of virulence factors unavoidably impairs the immunogenic capacities ofthe bacterium.
It has now surprisingly been found, that by mutating the I'poD, FacR, ΛΤΡ-grasp domain protein, and Ftsif gene products, an attenuated P. saimonis bacterium can be produced, without impairing the viability or immunogenicity of such bacteria in vivo. By mutating a number of genes in parallel, the likelihood of reversion to a virulent form is minimised. Moreover, these genes were not previously known to relate to virulence factors, and It is therefore surprising that they have now been found to affect bacterial pathogenicity. This also offers the further advantage that. the attenuated bacterium demonstrates substantially the same level of immunogenicity as wild-type strains. The disclosed bacterium has therefore surprisingly been found to be extremely suitable for use in the preparation of live attenuated vaccines.
Sonunary
According to a first aspæl, an attenuated Pisdrickeltsia salmonis bacterium is provided. The bacterium comprises a mutation in the amino acid sapience of each of the rpoP, FecF, ATP-grasp domain protein, and Ftsii gene products.
The attenuated bacterium has a. reduced virulence relative to wild-type P. sannonis. The attenuated bacterium is preferably avirulent and does not induce any symptoms of Salmon Rickettsial Syndrome when administered to fish.
The attenuated bacterium preferably does not revert to a virulent strain after serial passage in fish. For example, the .attenuated .bacterium preferably does not revert to a virulent strain after a, 3.4.5, or 6 passages in fish.
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The attenuated bacterium is preferably capable of inducing immunological protection against Salmon Rickettsial Syndrome when administered to fish, indeed, the attenoated bacterium preferably provides protection to fish against SRS following subsequent challenge with a virulent strain ot P. salmonis. Preferably, when measured in terms of accumulated mortality, the attenuated bacterium provides more than 40%, more than 50%, more than 6o%, or more than 80% protection against SRS. Preferably, when measured io terms of accumulated mortality, the attenuated bacterium provides tOO% protection against SRS.
ro The. mo rations in each of the rpoP, fbcK, ATP-grasp dontaiu protein, and FtsFf genes of the attenuated bacterium, which underlie the mutations in the corresponding g,ene products, may be non-reverting mutations.
The mutations in the amino acid sequence ofthe rpoD, FecR, ATP-grasp domain protein, and FtsPi gene products may be mutations relative, to the sequence ofthe corresponding LF~8o wild-type protein, as derived from the tP-89 genomic sapience that is available under the GenBank accession no. AMFP00000000.2. and as provided as Seq, ID No.s 17,26, 40, and 54, respectively.
so The attenuated bacterium may comprise at least one mutation in 1,2,3, or all 4, ofthe following regions:
a) amino acid residues 462-504 of the rpoP> gene prod»·» r, pros tded as Seq. ID No. 37i
It) amino acid residues 39-1,3/ of the FecR gene piodoct, provided as Seq, ID No.
26;
c) amino acid residues 118-251 ofthe ATP-grasp domain protein gene product, provided as Seq. ID No. 40: and/or,
d) amino acid residues 152-274 of the RsHgene product, provided as Seq. ID No. KA.
i~r»
The attenuated bacterium may comprise i, 2, 3, or all 4, of the following specific mutations:
a) an arginine to cysteine mutation at position 473 ofthe rpoD gene product, provided as Seq. ID No. 17;
b a premature stop codon at the position corresponding to residue 83 of the FecR gene product, provided as Seq. ID No. 26;
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c) a serine to proline mutation at: position 184 of the A.TP-grasp domain protein gene product, provided as Seq. ID No. 40; and/or·,
d) a methionine to isoleucine muration at position 191 of the FtsH gene product, provided as Seq. ID No. 54The amino acid residue numbers given throughout are defined on the basis of the sequences of the corresponding tF~8g wild-type proteins, as shown in Example 2 (and given as Seq. ID No.s it. 26,40. 54k and derived from the LF-89 genomic sequence that is available under the Get iBank accession no. AMFF00000000.2.
The attenuated bacterium maybe the strain PHARMAQ 00.1 deposited with the European Collection of Ceil Cultures, Public health England, Culture Collections, Forton Down, Salisbury 8E4 OJG, United Kingdom, on 09th October 20x4 with, accession number 14100901,
According to a second aspect, the invention provides a live, attenuated vaccine composi tion comprising:
(a) an attenuated Piscirickettsia sahnonis bacterium of the first aspect; and (b a pharmaceutically acceptable carrier or diluent,
The live, attenuated vaccine composition may be in freeze-dried form.
According to a third aspect, the invention provides a method of producing an attenuated bacterium in accordance with the first aspect. The method comprises:
as 1) subjecting an initial population of F. safinoms bacteria to attenuating conditions to produce a putatively attenuated bacterial population;
2) identifying clones of the putatively attenuated bacterial population that have mutations in the amino acid sequences of all of the rpoD, feR, ATP-pmsp domain protein, and PfcH gene products; and then,
3) identifyi ng and selecting clones that have mutations in the am inn acid sequence of all of the rpoD, FccR,ATF-ff’^:fisp dømainpretem, and FfsH gene products and that also exhibit reduced virulence relative ro wild-type bacteria of the genus Piscirickettsia.
According to a fourth aspect. the invention provides a method of raising an immune response in a fish. The method comprises administering to the fish an attenuated /bsciCicteiSM solmonis bacterium of the first aspect.
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According ίο a fifth aspect, the invention provides a method of vaccinating a fish against Salmon Rickettsial Syndrome. The method comprises administering to a fish an imnumo logics lly-effee five amount of a vaccine composition, said vaccine composition comprising an attenuated Piscirickettsia snbnords bacterium of the first aspect.
According to a sixth aspect, the invention provides an attenuated Piseiricke/tsia sa.finonis bacterium of the first aspect, for use in a method of vaccinating a fish.
io According to a seventh aspect, the invention provides an attenuated Ffecirickettsia softnonis bacterium of the first aspect, for use in a method of vaccinating a fish against Salmon Rickettsial Syndrome.
According to an eighth aspect, the invention provides a method of distinguishing the tS PHÄRMAQ oo: strain of Pisciricketisia sadnam's from other strains such as wild-type strains. More specifically, die invention provides a method of distinguishing between wild-type and mutant alleles of a Piseir/ckefteio sa/monis single nucleotide polymorphism (SNP) located ai the position rorrespondingtcc residue number 1417 of Seq. ID No, 1 (in the rpoD gene); so - residue number 247 of Seq. ID No. 4 (In the FecR pene);
residue number 550 of Seq. ID No. 7 (io the A FP-groop domain protein gene); or, residue n umber 573 of Seq. ID No. to (in the fdsif gene),
The method comprises:
i) amplifying by PCR the region of the nucleotide sequence containing the SN P;
it) including in the PCR reaction mix a nucleic acid probe having a sequence complementary to one allele of the SNP, the probe comprising a detectable marker; and ii t) analysing the PCR product for the presence of the marker, wherein the presence of the marker is indicative of the presence of the allele.
The method may further comprise including in the PCR reaction mix a first nucleic acid probe having a sequence complementary to the wild-type alleie of the SNP, and a second nucleic acid probe having a sequence complementary to the mutant allele of the
SNP, the first and second probes comprisi ng different detectable markers.
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-6 The probe or, when two or more probes are used, one or more of the probes, may comprise a to-qo nucleotide subsequence of:
- Seq, iD No. i, the subsequence including residue number 1417;
Seq. iD No. 4, the subsequence including residue number 247;
- Seq. ID No. 7, the subsequence including residue number $50; or,
Seq. ID No. to. the subsequence including residue number 573.
Spoeilicahy. the probe or probes may consist of 0. comprise Seq, ID No. 68.69.72,73, 76, or 77.
u
PCR primer pair may be u ^pd in the method to amplify a region cd'at. least 50 nuf'kotides in length of the subsequence of:
Seq. ID No. t, the subsequence including residue number I4i7;
- Seq, ID No. 4, the xt-bsem -cnee including residue number 24 7;
- Seq. ID No. 7, the subsequence including residue number 55η: or.
Setp ID No. 10, the subsequence including residue number 573.
Specifically, the PCR primer pair may be or comprise Seq. ID No. 67 and 70.71 and 74, or 75 and 78, ££>
Detailed Description
SRS is caused by the gram-negative bacterium. Fiscirickeitsia sabnonis. This was the first rickettsia-like’ bacterium to be recognized as a pathogen offish.
•*w£t
Λ sn/monzs Is generally non-motiie, obligato intracellular bacterium, pleomorphic but predominately coccoid, and 0.5-1.5 urn in diarnet&r. It is currently placed in the class Gammaproteobacteria; order Thiotrichales; and family RiadrickcUsiacaea, and has a closer relationship to, e.g., Legionella and Coxiella, than to members of the genera
Rickettsia. The bacterium replicate by binary fission within membrane bound cytoplasmic vacuoles in fish cell lines and in the ceils of tissues throughout infected fish. The bacteria occur either singularly or in group , giving the. vacuole the appearance of a morula. When R. snhnorris is examined by electron nrie.rosc.opy, the. bacterium displays the typical protoplasmic structure of a prokaryote and the cell wall
5-5 of a gram-negative bacterium.
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The genome of P. salmanis strain LF-89 has been sequenced and published on at least three separate occasions (see, tor example, Eppinger et at. Genome Announe. November/Decentber 201.3 vol 1 no. 6), and is available via DDRl/EMBL/GenBank under the accession no.s AMGCooooooooa, AMFF00000000.2, and
ASSK00000000.2. Unless otherwise indicated, the LF-89 sequence used in the present application is the sequence available under the accession no. AMFF00000000.2, Specifically, unless otherwise indicated, for the purposes of genetic and protein sequence comparison In particular, this LF-89 sequence that is available under the GenBank accession no. AMFFoooooooo.a is considered to represent the sequence of to wiki-type F. sa/monis, and references to the wild-type in this context should be interpreted accordingly. The genomes of P. salmonfe strains EM-90 (NCBI Reference Sequence: rø„„JRHP0000<X>00.l), A1--X5072. (NCBI Reference Sefpience:
NZ„ JRAV00000OOO.2), and 81-32597 (NCBI Reference Sequence:
NZ...JRAD00000000.2} Stave also been published.
The attenuated P. satmords bacterium of the invention is attenuated by means of a mutation in each ofthe rpoD, FecR, AIT-pmsp domain protein, and Fis’rf genes. Specifically, the attenuated bacterium comprises genetic mutations which result in no mutations in the amino add sequence of each ofthe rpoD, FccR, ATP-grosp domain protein, and FtsH gene products, relative to the sequence of the wild-type proteins.
For the purposes of the invention, the term “gene product” is specifically considered to refer to the protein resulting from the expression of a gene.
-C*
For the purposes of the invention, a “mutation” is considered to be any alteration in the gene or protein sequence relative to the wild-type sequence. Genetic mutations that' are of interest are those that result In a mutation (i.e. alteration) in. the resulting amino acid sequence ofthe gene product relative to the wild-type amino acid sequence. Each of the (nutations in the rpoD, PecR, ATP-grasp domain protein, and FtsH genes can be any type of mutation, including an insertion, a deletion, a substitution, or any combination of these, provided that the mutation leads to a change In the amino acid sequence of each of the rpol), FecR, ATP-grasp domain protein, and FisH gene products, relative to the wild-type prote.in seep tence.
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A functional gene product is a protein having the hmctlonal characteristics ofthe wildtype protein. A rpo£>, FecR, A/rF--qrnsp domain protein, or Its i I gene product that is at ieast partially defective in at least one of its functions is considered to bn an attenuated gene product. Any mutation resetting in an attenuated genu product is considered to he an attenuating mutation. The mutations in the amino acid sequence of each ofthe rpoD, FecR, ΛΤΡ-grasp domain protein, anti fdsH gene products, relative to the wildtype proteins, are preferably attenuating mutations.
Attenuating mutations in the rpo.0, FccR. ΛΤΡ-grasp domain protein, and Ftsii gene jo products may knock -out the function of the gene product partially or completely. The partial or total functional knock-out mu be achieved, for exampk', by making a mutation that results in the synthesis of non-functional or partially functional polypeptide. for example, the mutation in the amino acid sequence may comprise the insertion of a stop codon, or may result in the incorporation of an amino acid that is 5 physically or chemically dissimilar to the wild-type residue. Such mutations may result :n the produ-tion o; a truncated protein, a misfolded protein,ora chemically Inactive prorein, lor -wurnple.
The mutaiions iu each oi thr rpoD. FccR, ATPgraspdoPiain protein. and Fu7/ g-mes, j»o w hick result in mutated gen-· products, are prelArahiv non-reverting mutations. These are mutations that duns «•.•ssentiaify no reversion buck to the wild-type when the bacterium is used as a vat cine.
The possibility of reversion of the bact-n'inm to full v indene·¹ is also eliminated b the fact the bacterium contains attenuating mutation,- in four independent genes.
M^lltlifiodCeoes
The gene rpoi) encodes RNA polymerase sigma factor, which is an initiation factor involved in promoting the attachment of RNA polymerase to specific transcription iuitlalion sites. The rpoD gene product is believed to be involved In in the regulation of essential housekeeping genes. For the avoidance of doubt, the wild-type P. salmonis rpoi) gene sequence that is mutated in the present invention is given as Sea. IL' No. ·. and the gene can be identified using the PCR primers of See. ID No.s e. and 3. The amino acid sequence ofthe .full-length wild-type protein is given in Seq. 1T> No,s 14-17.
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’9The attenuated bacterium of the invendon »'Όη-prises a mutation in the amine ae id sequence ofthe rpoD gw product, relative to the sequence ofthe wiki-type protein, which is exemplified by the LF-8g sequence, given as Seq. ID No. ty.
The gene FecR encodes an iron di-citrate transport regulator. The Fcr’R gene product Is believed to be involved in regulating a number of genes involved in the uptake of iron and citrate. For the avoidance of doubt, the wild-type P. subnonts PecP gene sequence that is mutated in the present invention is given as Seq. ID No. 4, and the gene can be identified using the PCR primers of Seq. ID No.s 5 and 6. The amino acid sequence of to the full-length wild- type protein is given in Seq. ID No.s 26--29. The attenuated bacterium ofthe invention comprises a mutation in the amino acid sequence of the Fee/ gene product, relative to the sequence ofthe wild-type protein, which Is exemplified by the LF-89 sequence, given as Seq. ID No, 26.
.*5 The ATP-gvasp domain, protein gene encodes a protein with similarity to an alpha-Lglutamate ligase-related protein found In Pseudomonas fGenBank accession No. AP 014655.11. For the avoidance of doubt, the wild-type P. sa/monis ΛΤΡ grasp domain protein gene sequence that is mutated in the present invention is given as Seq. TD No. 7, and fhe gene can be identified using the PCR primers of Seq. ID No.s 8 and 9. The aa amino acid sequence of ihe full-length wild-type protein is given in Seq. fD No.s 40-43. The attenuated bacterium ofthe invention comprises a mutation in the amino acid sequence of the ATP-gra&p domain protein gene product, relative to the sequence of the wild-type protein, which: is exemplified by the LF-89 sequence, given as Seq. ID No,
40.
The gene R.stf encodes an Al P-depend-mt zinc røetnilopn>v-ase, w hieb acN ns a prormshe, ATP-d<-pendent zinc metallopeptidam for both cylopla^mif* and membrane prol-mw Th«* /45// gene piodu-'t -> also behoved to plu a »ole in the quality control of integral membrane proteins. For the avoidane- of doubl, the wdd-tvpe F. satmanis po Ffsli g< »<- < queue» thal is mutated in the present inv< nlion is gbo-u as Seq. ID No, to, and the §<-ne can be uVmtincd nsmgthe PCR printers ofSeq. ID No.s it and t2. Ihe amino acid: sequence of the mb-h-ngth wild-type protein is ghen in Seq. ID No.s 54-5⁷. Tiv· attenuated bacterium of the he, emion comprises a mutation In the amino acid sequence of the Ffs// gene product, relative to the sequence ut th- wild-type protein.
which is cxemplith d by the Lh'-do sequence, givs-m as Seq. 11) No. 54.
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- fi> In sonic embodiments. one or more of the rpoD, Fee/ , APPyp-osp domain protein, and FtsH gene prod «cis are entirely hnocked-ouh with the effect that no functional protein is detectable. Thus, the mutation in the amino acid sequence of the gene product is that there Is no amino acid sequence.
In other embodiments, the mutation may comprise the Introduction of a stop codon, in some embodiments, the genes may be expressed at wild-type levels, but mutated so that the gene products have a different amino acid sequence to that found in. wild-type m strains. The genetic mutation may result in a deletion, an insertion, and/or a substitution of one or more amino acids in the gene product. The genetic mutation may result in foil-length or substantially full-length gene products, or truncated gene products. The mutation may be a point mutation, affecting just one amino acid, or may affect more than one antino add residue, such as, (or example, affecting 2-20 residues,
3-fo residues. 4-12 residues, or 5-10 residues.
For example, in one embodiment of the invention, the rpol) gene Is mutated resulting in the replacement of arginine with cysteine at position 473 in the amino acid sequence of the gene product. As a result of this mutation, the protein encoded by the mutated so rpoD gene has different functional properties to those of the wild-rype protein.
in one embodiment of the invention, the FecR gene is mutated resulting in the insertion of a premature stop codon, for example, in the position of residue 83, and therefore the. production of a truncated gene product, having different functional properties to those of the wild-type protein.
In one embodiment of the invention, the ATP-grasp domain protein gene is mutated resulting in t he replacement of serine with proline at position :84 in the amino acid sequence of the gene product. As a result, the mutated AT -grasp domain protein has :}o different functional properties to those of the wild-type protein.
In one embodiment of the invention, the P/sif gene is mutated resulting in the replacement of methionine with Isoleucine at position tpt in the amino acid sequence of the gene product. As a result, the protein encoded by the mutated Ffs/7 gene has different functional properties to those of the wild-type protein.
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- π In some embodiments, two. throe, or all four, of the specific point imitations described above in the rpofp FecR,ÄTP-grasp domain protein., and FfsH genes may be present in combination. For example, in one embodiment, the attenuated bacterium comprises the specific point mutations described above in three of the rpoi.' FacR, ATP-grasp domain protein, and Ptsll genes, and the fourth gene has a different mutation to that described above, in one embodiment, the attenuated bacterium comprises all tour of the specific, point mutations described above io the rpo/.), FeeR, ATP-grasp domain protein, and FfsH genes, io The bacterium preferably contains only defined mutations, which are fully characterised, it. is less preferred to use a bacterium which has uncharacterised mutations in Its genome as a vaccine, because there would be a risk that the uncharacterised mutations may confer properties on the bacterium that cause undesirable side-effects, m
Frpductmny/fA^
In another aspect of the present invention, the invention provides methods for identifying and/or producing attenuated P. stHmonis clones, .an Tim methods: according to this aspect of the invention include subjecting an initial population of P. soimom's bacteria to atteouating conditions. thereby producing a putatively attenuated bacterial population.
According to this aspect of the invention, the initial population of F. suZmon/s bacteria can he any quantity of P. soZmonis bacteria. The bacteria, in certain embodiments are wild- type F, soZmoms bacteria, A number of strains of P. sn/monis have been isolated following outbreaks of SILS. Any of these isolated strains would potentially be suitable as a starting population for producing a putatively attenuated bacterial population, including the following strains: AL 10 016. AL to 008, AI. 20 218.
Af, 20 219, Al, 20 223, AI. 20 220, AI. 20 470. AL 20 471, AI. 20 455, AL 20 222, Al1597:.7 81-32597, LF-89, EM-90. References co wild-type P. sal moms may refer to any of these strains. Preferably, however, references to wiki-type P. saimords refer to any of strains At-15972, Bt-32597, LF-89, or EM-90, such as in particular, strains Al-15972, LF-89, or EM-90, Unless otherwise indicated, however, tor the specific purposes of genetic and protein sequence comparison, the LF-89 sequence that is av ailable under the GonBauk accession no. AMFFoooooooo.n is considered to represent the sequence
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- 12 ~ of wild-type P, sahncnte, and references to the wild-type in thio context should he interpreted accordingly.
The bacteria used as a starting popttlation for producing a putatively attenuated bacterial population may alterijstively contain one or snore mutations relative to the wild-type, or other strain.
Preferably, the bacteria in the initial population areeionally identical os' substantially rksualiy identical. In other words, the bacteria are preferabh all derived from a single jo parental P. sq/monts bacterial cell and/or have identical or substantially Identical genotypic and/or phenotypic characteristics.
The terns '''attenuating conditions” refers to any condition or combination of condItions which has or have the potential for introducing one or more genetic changes (he., i5 mutations) into the genome of a P. sahnonis bacterin nt. Exemplary, non-limiting, attenuating conditions include, for example, passaging bacteria in culture, transforming bacteria with a genome-insertable genetic clement such as a transposon (e.g., a transposon that random ly inserts into the P. sahnonis genome), exposing bacteria to one or more mutagens (e.g,, chemical mutagens or ultraviolet light), and any so oilier suitable methods.
Indeed, the attenuating mutation» maybe introduced by any suitable method.
A possibility to introduce a mutation at a predetermined site, deliberately rather than randomly, is offered by recombinant DNA-tochnology. Such a mutation may be an «5 insertion, a deletion, a replacement of one or more nucleotides, or any combination of these, with the only proviso that the genetic mutation leads to a mutation in the amino acid sequence of the resulting gene product
For example, one possible method includes cloning the DNA sequence ofthe wild-type .30 gene Into a veelor, such as a plasmid, and inserting a selectable marker into the cloned DMA sequence or deleting a part of the DMA sequence, resulting in its inactivation, A deletion may be introduced by, for example, cutting the DMA sequence using rest riel ion enzymes that cut at two points in or just outsidethe coding sequence and ligating together the two ends in the remaining sequence. A plasmid carrying the inactivated .35 DNA sequence, can be transformed into the bacterium by known techniques such as electroporation and coniugation. It is then possible by suitable selection to identity a
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in some embodiments, one or more further mutations may be introduced into the bacteria to generate strains containing mutations in genes in addition to those in the rpoD, Ft-cR, ATP-grasp domain protein, and Fistf genes.
When bacterial cells are attenuate!.! by passaging In vitro, the ceils may be passaged any ro number of times, such as for example, at least io, 20,40,60,80,100,120, or more times in vitro.
The initial population of P. sedmonds, after being subjected to attenuating conditions, is referred to as a putatively attenuated bacterial population, individual clones of the putatively attenuated bacterial population can be obtained by standard microbiological techniques including, for example, serially diluting the cells and plating out individual cells on appropriate media.
Once obtained, the individual clones of the putatively attenuated bacterial population no are assayed for mutations in each of the rpoD, PecK, ATP-grnsp domain protein, and
FisH genes. The mutated gene sequences are then analysed to determine whether the resulting amino acid sequences have any mutations. Mutations in the amino acid sequences of the gene products are considered to be any differences In the amino acid sequences compared to the wild-type P. sohnonis sequence.
Any suitable method may be used to determine whether a putatively attenuated P. sahnomis bacterium exhibits mutations lit each of the rpoD, FecR, ATP-grasp domain protein, and FYsH genes, and consequently arty mutations in the amino acid sequence of any of the rpoD, FecR. ΛΡΡ-grasp domain protein., and R$F< gene products.
Se
One method by which mutations in these genes may be identified is by amplifying and sequencing portions of the genes. Any suitable PCR method may be used to amplify portions of the genes and pairs of PCR primers suitable for amplifying specific portions of the rpoD, Fecß, ATP-grasp domain protein, and FtsH genes are given in Seq, ID
No.s: 2 and 3; 5 and ό; 8 and 9; and u and 12. respectively. The amino acid sequence of
DK 2017 71015 A1
WO 2ίί16.'Ί89!ί(»7
- i4 the gone product can be determined from the genetic sequence using any suitable eonijn hat tonal tool.
Fort.be avoidancoofdoubt, the primers given In Seq. ID Nas: a «and 3; 5 and 6; 8 and a: and π and 12, may also be used to identify the genes referred to as rpoD, FpcR. ATPgrasp damoin protein., and F'isH respectively.
The portions of the genes ampihied using the PCR primers of Seq. ID No.s: 2 and 3; 5 and 6: 8 and 9; and 1 and 12 are regions of the genes, which when mutated, have been found to be particularly associated with the attenuation ofthe bacteria. Specifically, differences between the annuo acid sequences of these portions of the rpoD, FecP, Tl'T-prasp domain protein, and F/s 7 gene products, and the wji<| type sequences, may la-, indicative of attenuating mutations in the gene products. Therefore, in some einbixiintents, referer tees to mutations in the arnino acid sequence of the rpaD, Fee/ ,
s.$ A TP-grasp domain protein, and Pt$H gene products preferably refer to the presence of initiations in the antino acid sequence of those portions of the gene products corresponding to the regions that may be amplified by the PCR primers of Seq. ID No# 2 anti 3; 5 and 6; 8 and 9; and ft and 12.
ό Differences in fhe amino acid sequences of the rpoP), FecR, ATP-grasp domain protein, and FisHgene products between the putatively attenuated and wiki-rype F, so;n on s bacteria may in some cases not be accompanied by reduced function of the gene product, For the purposes of the invention, such mutations arc not considered to be attenuating mutations, Non-.attenttating mutations may be found in the portions of the gene products corresponding to the regions amplified using the PCR primers of 8 err ID No.s: 2. and 3; 5 and 6; S and u; and n and ,·2. or elsewhere in the genes.
fn some embodiments, some (such as i, 2, or 3) of the rpoD, tecR, ATP-ffrasp domain protein, and Fistf gene products may include attenuating mutations, while the o remainder of the rpaD, FeeR, ATP-grasp domain protein, and FisH gene products contain non-attenuating mutations.
The clones that, have been identified as having mutations in the amino acid sequence of each of the rpoD, FecR, ΛΤΡ-grasp domain protein, and FZs/·/ gene products, relative to the wiki Type sequence, are then tested for virulence.
DK 2017 71015 A1
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individual cloned thai are identified as having mutations in the amine acid sequence of cadi ofthe rpo£>, Feck, Al’P-prosp domain protein, and Rs/7 gene products can he tested for virulence by any suitable method. For example, the attenuated bacteria may be administered to an animal that is susceptible to infection by the wild-type version of the bacterium, and the presence and severity of disease determ ined.
In the present context, an animal that is susceptible to infection by a wild-type P. salmonis bacterium·' is an animal that shows at least one clinical symptom after being challenged with a wild-type P. salmonis bacterium. Such symptoms are known to to persons of ordinary skill in the art. For example, in the ease of a putatively attenuated P. salmonis strain that exhibits mutations in the amino acid sequence of each ofthe rpoi), FecR, ATP-grasp domain protein, and FtsPl gene products, the strain can be administered to, for example, salmon (which are normally susceptible to infection by wild-type P. salmonis}. Clinical symptoms of SRS in salmon are known to the skilled tg person, in some embodiments, the symptoms investigated may include the accumulated mortality of a population. If the accumulated mortality is lower in animals challenged with the putatively attenuated JP, salmonis strähn compared to dsh that have been so infected with a wild-type P. salmonis strain, then the putatively attenuated P. salmonis strain is deemed to have reduced virulence.
In some embodiments, the symptoms investigated may’ include the presence or accumulation or P. salmonis genomes in tissue samples taken from animals challenged with the putatively attenuated P. salmonis strain. If the presence of P. salmonis DNA is reduced compared to fish that have been infected with a wild-type P. salmonis strain, then the putatively attenuated P. salmonis strain is deemed to have reduced virulence.
Thus, if the putatively attenuated P. salmonis strain, when administered to salmon, jo results In fewer and/or less severe symptoms when compared to nsh that have been infected with a wild-type P. salmonis strain, then the putatively attenuated P. salmonis strain is deemed to have reduced virulence''. Any degree of reduction in any relevant symptoms will identify the putatively attenuated strain as having reduced virulence.
DK 2017 71015 A1
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- 56 For the purposes ofthe invention, gny strain that is found ίο have a reduced virulence is considered to be an attenuated strain. In preferred embodiments, the putatively atten*.rated strain is avirulent.
Clone that exhibit snutatkms in the amino acid sequence of each of 5he rpoD, FecR, .i'PP-grasp domain protein, and CisH gene pnxhuds, artd that also exhibit redu< cd virulence relative to wild-type P. salmonis are identified as attenuated P. salmonis clones of the present invention..
jo An exemplary, live, attenuated P. salmonis done of the present ifivention, which exhibits non-reverting genetic mutations resulting in inntatioiis iit the amino acid sequence of eiich ofthe rpof>, FecR, Λ'ΓΡ-grasp domafr. protein, and CfsH gene products is the strain designated PHARMAQ OOt.
Specifically. relative to wild-type P. salmonis, PHARMAQ oo.i has been found to have musatiOiJS located at post tlons corresponding to:
- residue number .1417 of Seq. ID No. : fin the rpalJ gene);
- residue number 247 of Seq, 10 No, 4 fin the Peek gonep residue number 550 of Seq. ID No. fin the /EfP-grøsp domain protein gene); no and,
- residue number 573 of Seq. ID No. io (in the FtsH genet.
For the purpose of the present disclosure, the .‘nutations found in the P. salmonis strähn PHARMAQ oot are considered to represent single nucleotide polymorphisms (SNP).
Various methods of distinguishing between SNP alleles are known to the skilled person, and can be used to determine whether a given strain is PHARMAQ oot, hi particular, various different snethods have been developed for the -detention of specific alleles, or DNA sequence variants, at the same locus by polymerase chain reaction. For example, suitable methods may be based on the use of PCR priniers with a 3) end specific for one of the allelic variants, or on the use of nucleic acid probes having a sequence complementary to the stiqnence of one particular individual allelic variant.
PHARMAQ 00 i has been deposited with the European Collection of Cell Cultures, Public health England, Culture Collection«, Porton Down, Salisbury- SP4 OJG, United
Kingdom, on oo^ih October 2014 and was assigned accession number 14500901,
DK 2017 71015 A1 wo 28tøW7
- ί/··
Vaccine
A vaccine comprising the disclosed attenuated bacterium maybe formulated using known techniques.
it has now surprisingly been found that an attenuated P. sahnonis bacterium having a combi oat ton of mutations io the toe amino acid sequences of the rpoZX EeeR, ATPgrasp domain protein, and Ptsfi gene products gives a vaccine having superior properties for at least two reasons.
io Firstly, due to the presence of multiple mutations io four independent genes there is a significantly reduced chance of reversion of attenuation of the bacterium. Therefore, the bacterium can survive in the vaccinated host fora longtime and at high levels, resulting in better protection.
Secondly, the disclosed bacterium does not cause reduced humtmogenicity compared to wild type strains because antigens important for immunogenicity are still expressed.
The vaccine composition preferably comprises a live, attenuated P. sohnanis bacterium and a pharmaceutically acceptable carrier, ao
Examples of pharmaceutically acceptable carriers or diluents useful in the presen t invention include water, a preservative, culture medium, stabilisers such os SEGA, carbohydrates (e.g. sorbitol, mannitol, starch, sucrose, glucose, dextran), proteins such as albumin or casein, protein containing agents such as bovine serum or skimmed milk, gy and buffers (e.g, phosphate buffer.).
The vaccine may or may nor comprise an adjuvant. Adjuvants are non-specific stimulators of the immune system. They enhance the. immune response of the host to the vaccine. Examples of adjuvants known in the art are Freunds Complete and yo incomplete adjuvant, vitamin E, non-ionic block polymers, mnramyldipeptides,
TSCOMs (immune stimulating complexes), Saponins, mineral oil, vegetable oil, and Carbo}X>l.
Vaccine formulations comprising the disclosed attenuated bacterium can be prepared in the torn· of a suspension, or in a lyophilized form or, alternatively, in a frozen form. If
DK 2017 71015 A1
WO 2ÖPPi 89867 the formulation fe to be frozen« glycerol or other similar agents may be included in the formulation to enhance the stability ofthe bacterium when frozen.
Reconstitution is advantageousiy effected in a buffer at a suitable pH to ensure the 5 viability of the bacteria.
The combined administration of several vaccines is desirable, in order to save time, effort, and money. Preferably, vaccine formulations comprising the disclosed attenuated bacterium may be used together with other vaccines, such as, for example, ία an inactivated, oil adjuvanted vaccine. The vaccines may be administered together, for example. In a single composition, or separately.
The animal to which the vaccine comprising the disclosed attenuated bacterium is 15 administered is preferably a fish. The vaccine may be administered to any species of fish that is susceptible to SRS infection.
Of particular note, the vaccine is suitable for treating fish ofthe order Samoniformea. Per example, the claimed formulation may he used to treat salnmn sech as Atlantic and co Pacific salmon, such as Coho salmon, and trout such as rainbow trout and brown trout.
fite present invention includes methods of vaccinating fish against P, sedmonfe infection, such as 3RS.
The methods according to this aspect of the invention comp-rise administering io a fish an inununologically-effective amount of a vaccine composition comprising a live, attenuated P. s«/nwnis bacterium of the Invention. The expression “immunologioallyeffective amount” means the amount of vaccine composition required to invoke the po production of pjolective levels of immunity in a host upon vaccination.
The vaccine composition may be administered to the host in any manner known in the art. in particular, the vaccine formulation may be suitable for parenteral administration, sue.h as by intruperitoneal injection.
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An infection caused by a uucmorgfuilsm, especialiy is pathogen, may therefore be prevented by administering an effective dose of a vaccine prepared according to the invention.
The dosage of the vaccine employed will be dependent on various factors including the size anti weight of the host, the type of vaccine formulated. and the formulation.
For «sample, a dosage for Atlantic salmon, Coho salmon or rainbow trout, with an average weight of 2$-;jo grams, may comprise the administration of from mo- to io mo·“, mo-^! to txwr*, or mo·* to ixio³ TCID_S«per fish, such as from mo* to άοο⁷
TCIDy. per fish. As the skilled person will appreciate, the preferred dosage may depend on the age, weight and type of fish to he vaccinated, and the mode of administration. For example, dosages may need to be increased for larger, more robust fish, and decreased for smaller, more delicate fish.
in
Examples
The invention wifi now be explained in further detail in the following Examples, which demonstrate the development of the claimed live, attenuated bacterium, and its use in a vaccine.
The P. sabnom’s strain used as the starting bacterial population for the production of an attenuated bacterium was a strain originally isolated from an outbreak of SRS in Atlantic salmon in the X region in Chile.
2$
The isolate was for the six first passages cultivated. in the presence of eukaryotic cells.
For the next passages until passage 104, the P. sahnonis isolate was cultivated in ceil free insect cell medium at acCC. To secure a homogenous culture, the passage {04 go culture was serially di luted In insect cell medium and seeded into 96 well cell culture plates. Bacteria grown in chosen vrefis were further passed Into new wells at an early stage when it was most likely that the growth originated from a single bacterium.
After a total of τι i passages, one clone from the weds was inoculated into a spinner flask and cultivated in insect ceil medium. This passage was used as the origin of the
DK 2017 71015 A1
- no putatively attenuated bacterial population, and the isolate was named “PHARMAQ oof, which corresponds to passage .113,
Tbs bacterial isolate was verified Io be P. so'moms using a commercial kil SRB Fluorotest Directo” from Bios Chile, Chile.
FHÅRMAQ 001 ha$ been deposited with the European Collection of Cell Cultures, Public health England, Culture Collections, Porten Down. Salisbury S.P4 OJG. United Kingdom, on oo^,!l October 2014 and was assign«.! accession number i4tooqoi.
The genomes of PHARMAQ om and the virulent starting strain were sequenced and the sequences were compared.
Genetic differences between the two strains, i.e. mutations accumulated by the PHARMAQ 00· strain during its production, were identified.
Because these genetic differences underpin the observed differences in the virulence of PHARMAQ oot and the starting strain, mutations were identified in PHARMAQ 001 that resulted in a significant change in the amino acid sequence of the encoded protein. Specifically, four significant mutations in PHaRMAQ oot relative to the starting strain were identified. The identity· of the four genes was determined, and the genes were found to be rpoZX ATF-grasp domain protein, and Et'sH (all annotated by the fGS Prokaryotic Annotation Pipeline), and Fee/ (GenBank reference: KGB03484..Ü
PCR primers were designed to allow the specific region of each of the ipoD.AT’P-grasp domain profihn, F'tsH, and Pec/ genes containing the identified mutation to be amplified. In each of the gene sapiences shown below, the mutation is shown in bold ami underlined, and the primer binding sites are highlighted in grey and underlined.
PHARMAQ 001 cpnD gene cequestcc (Seq, ID No. 1):
ccc0AiCC'i'G.mtÄ00tTncr<;Ä'roÄÄT0ÄGC.ArGCCA3Ci'C;.oC;cöZiÄ0A~GA'rscr0Ä0 dhPriricdfysebssGsyqdhyhhhdpydhhWiffhhdsdddriridhnGfSoodnotiedd dhhAbOsdfiiOQdddWiittnityrisAOAhsss-yflftshddriithPriridddhyydBIdhit
DK 2017 71015 A1
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- 2i 2722's:2®62222£ä££OlÄ7226022227£ä22:2li22/7622222266700Ä:02o&0262260/ 26226002 ooooooooooogoooooxoooo&oooo/OoooooooooOoooooosooooo'OO: :027277020622666602222062222606020622266202666727022022277272
7P /6206720200626660266622220672626666K2262226O62222262022622020· TCTArs.a.GrÄ.rTÄÄroÄCÄr’n'CGCCT.ÄÄÄGqc'CÄATÄTGrxrcCÄTÄÄÄÄTCÄiXKÄC'rAC /2262222222222202666262222221626176:266622622222266621:22222222: :62262200:66666722222202266:2622:2222662227226:666222:222662206227: /62202621652226262626222222620666266667/67726:26262266662:6262766: r> ^ο2θθοοοοθοοοθ2 ;|εοοθ7θοοοοοθοοθθδ££2θθοθ7θοοοοδθίΐοκθο0θ2θοθθθ/ /ΟΟΟΟΟΟΟΟΟΟΟΟΟΟΟο^ίΟΟΟΟΟίκΙοΟΟοΟοΟοΟΟοΟοΟΟΟΟΙοΟΟΟΟΟίΟΟοΟίΟΟΟΟΟ: 02£77£777222262002£:£i£7£-’'7227:££220 7700076£7-£7££^:, ££ .:.0 Ο ΑΓΤ0·16266 ίΐοίοίοίοοοίοοοοοοοοοίοοίΐοοοοοοίοοοοίοοοοίοοίοοοίοοίοοοοίίοοί
0777047700740l200l77004|707:0£5d0020£070704S770:“OS0007Ä7770004
2GG700627002l|lO|lO|llillllll22062022C622222222.£.1227CGGG672
0770760072726Ϊθ660Α26Λ72666220θΐ7660 700270762667066670672222 :05560/ 1. Ο/Οθ00 5.:25.:6.IG;·/,.-;·55, ' 60-.0/2.056O£;O,5;i>5/i':A/:5.:5^:./5./5:-55550: OG A/l/OGr/G/O,,·, 5:- 5.,5.-.15 0-5:50¹0:67-:
6266.200022266226722662.700CT66626220222222£:|ll|Ollllll|:
:/6Ϊ702226222726206:θ776222θ22227672θ6/ΐ£θ6260222622261102ΐ22222:1 /£01:2011006200226226662266602662226601G2612622022G16626626600 iOfBolollooooloOooooOoAolGoOGGGooOoooOoOfSAoioGllolooOoooAoAoo: :e2:20000:00777SOOOSGOÄ772G00770072d£i004£5GG7Ä770SG7:OOG72G70GOO· 7.7:40777777077777 77777770007777' £77777.777.77777070-177 77770777770 /707707707700::::
PCR Primers-.
Forward rpoiDSeq. ID No, 2); CCiVG« klVVfFiVlVCGCrCiU Re er$, ,ρ^ίρ [Srq. ID No. 3h rCGCCÆVrCGGTG'lTrCCA'l
FHÅRMÅQ 001 ®:Ä gene sequence (Seq. ID No. 4):
.£62G0C200626622220225562266766226ll0|i|l|^: 7 £26262iO72O7O£02666££600000666667£6662600 ö o.:A ; ΑΛ ; 0 k>0, ä To Ä i i-.Ao 0 a^G'G.<o Λ AA v'XzDd1>n> Io
220202207 72< . 5 .0
2220222222222222
0220:1222222220200
20222200700022725
227272270077677200007077222707777220:7227770 ''.......^v ·' ’' ’
222270222:2222222272/722222077727777722272020 2222£22202S22220200202&22222222202227272222
Primers:
Forward FecÄ (Seq. ID No. 5I: TGGTCAAGTGA'FGACAAGCTAC 07 Reverse FeeR. (Seq. ID No. 6): ACACAGTACl’^,rcCGGATACCTl'
4<>
0222:220222222607 22222202222-2222 £
PHARMAQ 001 ATP-grasp domainprotein gene sequence (Seq. ID No. 7):
/5 26i5.2:1,6655 0:0:5/:5..2/1 .5:5. ,1.2ί-..0>£:127/7.667: 22022222222226222222722222227: 21022662222222222060222772222:
2702702000727222262027270202222 :220007222:0672227222222220667222.
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DK 2017 71015 A1
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Sv Sv ©A©©©X©©GAXGÄAS©©©©©©GGXGqGGG©©©©©GXGAG:©i©A©©XXA©'i'gAGG©G©SS© '' - ··^f rtm.w ..... '. . · t
CATCÄCÄTTTCTÄÄTSwTTA^XSÖQOTATXC&GTCT'rOOTOßCCAM/GTGS.rCÄCGCC $i Ai®®TTiiBTbdisd/BhdaAvvvasTddh2TA7Yiie|liyd|iy|yghl|i5ys 7<i^: /©£'gsgxgg7©©©axgggggx®gg©aa©©©©©gxggg©g©g©©©©©gxggg©©©©©gaggg ,SGG;t©©©:i:G©£GSAG©©©AAAS©©GAGGGr'GA^:'::©AG©GG'GG©qGG©©G©X©©©G:G7'^:3'©S©;
£©xBsG©A©A©©©AXG©GGG©©©i©©G©©xxxGGAG©©©x©|GGG©AGG©iri©AA£5©©GGG /GA;rA©X©SGG®GGGi7©®A©©A©©©G;i:AGAGA:i::i:©iiXGAt':t;©i3©A'r©Gg©XGGr©©S©©'r©^: <>10/ /ss/2:GqAq®s|y|q|Aqqyyaf/Yf'6sskhss7YAvy7®sSTSTQQSTAiTWfii:yxiBf^: : :&ÄÄ©&© II^OieiliiieilildCTTAa'rGCGCGTCCTGGT’rTKSAi&TTCAAA'r'r g· ··' ........ g - ’ i: .',' . :'. ' ·. s - ' -- - .·..-.
;fo:iSGGSSGGAGGSGAGGSGGGGGS7GAOGGGGGG7GGGG:AAA6G2AiÄif i5 Primers':
Forward ΑΤΈ-ζfi’osp domain protein (Seq. ID No. 3): GGIGAGCGn I PCGCAAAGI Reverse A7P-gra$p domain protein (Seq. ID No. 9): TCXV.)CAT<IAGT(JGGCCrn'
PHÅRMAQ 002-f'isHgene.sequence (Seq. ID No. 1©):
©©; AGqbGGqqÄÄGSGGGAGGGGÄGGGGGGÖGGGG'GGGGGGGG'rGGGGGGGAqiqiöGkGAGG/ /AfS£/X©©©©GGG©©©X©Ä©©©©©©©GXG©q©G©©G2/GG©|©|©©©A© /qx©®GX©®ÄA©©©£
BGGSGG.S©©©©©G©©GGG©©©A3G©©7'G©GAiS:GÄ©©«©©XGOGG7A©©G©©©;i©©©SG /i©AA©©SGG:|GGGG©l©©©GGGGijlGA©XG©©©©G©SA©|©©©©©S©GG©iXG©S®©©© <AGGG®©©G©GG©S©©©©©A©A©©©GG©GG7©AA©©©GGGG©GGä©©G©AG©©©GGG©ä©© <25; ^;;W:eGGiäA£GÄSGWSSGGGG©7©©:Ä©©GÄGGASW©©©G©©©SGA:BGA©©©©GG©
Α|©©Α©|©®©ίΒΒ©©©Χ©ίΒδ|Β©:β©0©©©Ιθΐ©©Ι©®®|β|θΒ ©^©XGCIIG©T©AGGGXCAAA7'TAAA0TXAAGTrTG©3'©AXGArøCrGGCTOTSS0©G© /GGGAAAS©©GG©G7AAAA©a©SGGGl|Gh:f;f©|©X5GG©©l©©©S':GeBTiS/foAhS /XX:SGG©©G©AAAAX-XGeGeAAG©©GX©XXGGxAq©XG©GGGSGGGGqAA©AGGXASGGGG /·:<> ;©::AAXæAGGXhAA©G©©XXGGGGGX©A©S©GAAiGXS©©©XXGXXXXGXA©XX©AG©©XGX ;GO4AX©XX©Al&XG:|y©©X|©©X©XGGGX©©AX©SGöSGXS©S©©AXAX©XXXGAXGA© /GGAAAG©©AGGG©GA©GGXGiAXiAX©XX:XAXGaAG©A©AXXG©XGG©GXGG©©©©G©AG /G©X©S©XGAGSX©X:@fO5GSSGGGGGA0NBä©WqiNShG©©©A©©XGAASXiB©AÄ©©XG ©TGGAGA7©©AXGGTrrT0Å©|i|i|iii|i|GATTGCC©C©ACGGGTCGT :©©©GAXGX5:XXG©A©©GGGG&XG©X©G©©X©©©GGG©«XX'£©^;S©GGGGGGGGXGA©X©XG: foSGGX©©3©©©GSXG|AA©©©G©'GG©GG©GAl©©XiS55SGGGAGAGS©©XAAGGGGGGX' /GG©GG®©GAG©XXi©SGGGX©AGX©AXGSGGGG’£©©XA©GGG:XGGÖG©©GGAG©GGGG /OGGG©©G©AAGX3::©©X©AAXSAAGGGGGA©XGXXXGeGG©©©©XAAAGAX.SAKA©©©X©^: /©©GGXAGGGGXGsGXXXSAGGA-XGGGAAAGGXAAäAXXXSGGXGGÖGäGXGASGGGGGX
-/O :.©OA©wä.-AxX<©A©/©AAGAGG.AAA.AA©©7GXAAGs.-GG©G<GX©AX©AGGGGGG'3©A©©©© /Ax:X0G©©q©X©X’i©GG©A©©GGix©3GG©X©©©©XSXGXA|A©X©G©GAXX©XGGGG©©© /©GXGGxGGG©©AGGXGXGS©GAG©XAXGXGGGXGAA©AGGGX1SGG55S0©gaxxGAGGG /GAG©©GG'7©GAGS©©g©AAXG©GSGGXAG©XAX@GGGGA©©XÄGG©©X©AAGGA©GAöXG^ ;+;+;i G6:XSS:X©AAG<G©©yXAAGGA©G©©G:+.-.AXGSjAA©©AG.Aj, λ a.GOAAAGGGGGAGssaG XG ©5 Aa/©©©©AAX.5XG©X©AGAAAGXGGGG©©'©£X©X6GGG©©G:X:A©G©©GG.AXA XXAXAXGGiA /e5GGAA©©GGGXGA©G©©©X5©©GOXGGXG©©©GXlS.SGGGA©©©©GGAAXX©XeAGA·/: BAAAGGXGXGGX©©XAXXGAÄGSG©AAG7G©SXGA©AX©AXXGixGSXAAGGAXAGtSSG ;GGX©A©A«GAXX®AAAX©AAXAAXGGSGAGAXXGXG6AX©G©AG©©GGGAXSGG®G©AX©; :©XGXGXGSGA©©AXXGSXGGG©A©©AASXGGGXGAXGXAAiGGGGGG©©©©G©©GXGAA® ;©©A©©gAAGS5X©©©©AXGAGGggXGG©AX©AGGSGG©©G©AX©AGGSXGXGqXiaAXGAG· AXAGAAG©5©XXGAXGGXAAGAX©AA©AGGGA©ÄXXAAXG|GlG'XäA©A©G©GXG5XGAA: ^©A©©©©GGGGAGS©GffoGGGG7BGG©l;3O»eA®S|G|lS©g©©gSS©©GAG£;S2Ag:Ci /'β
DK 2017 71015 A1
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Primers;
Forward Ptsii (Seq. ID No. h): TGGTTCCAGTAAGCtCACGTC Reverse Ftsff (Sen. ID No, 12): ACAATCACCCCl^vrCG( iTCC
Thu mutations in the rpoD, FecR, ΑΊΡ-prospdomain protein, and FteHgenes observed in PHARMAQ ooi have been found to be unique by comparison with DNA sequences from other strains of P. sabnonis. For the purpose of the present disclosure, the mutations found in the P, satmonis strain PHARMAQ OOt ('shown in bold and underlined in the sequences above) are considered to represent single nucleotide to polymorphisms and are located at positions corresponding to:
residue number tai of'Seq. it) No. t (in the rpoD gene):
residue number 247 of Seq. ID No, 4 (in the Feed peuc);
residue (number 550 of Seq. ID No. 7 fin the ATP-grasp domain protein gene);
and, 5 -- residue number 573 of Seq. I’D No. to (in the Ftsff gene).
I able f shows the occurrence of the specific mutations In the rpoiJ, Fec.R, ATP-grasp domain protein, and FtsH genes observed in PTIARMAQ 001 (described In Example 2) in to different wiid-tvpe virulent strains of P. sabnoms.
Table 1
Si rainOriginGroupGenerpoOK>cKAfi'-gtipt-OHPHnRMÜJoo!å11 ·ίϊΐiic s&liftøft«Oh ft £BM -yo likex........XXXAtDtek: ssibws, .Chile·ÉM-go tikeAft................................ft..ft.Λ· -,><·· m2Atlantic sslmo-i, 'ChitsEM -90 Amft......Ai 20Λtϊ:Λ:Ϊs i: ’ riff:tOOif, CL I A't.C-Ay tikeftuAL 20 220Fmw. CivicLF-by like/ft........ftftftA (,20- ΛΌTU>:3t. CbfSot.F -K9 i: k>-'nft. ftAL 20 222tmax. CkOt;i.r-Sy ftftftftA3-15772Attener sahnoa, ChileBM -yo likeAft................ftftft1ύ-725<>νCebu riiii:'Sy:3, 'Nvi)..ti-sy iib'/ft',',','ftft'.'iihnf-.ilDriX:, C'hriifi.r-Sy..ftft.ft.ft 1f’M-'.joAtisaer salsaon, ChileBM-yOiiftftAftA/^::''^:::hft
In Table 1, X” indicates that tire specific mutation found in PHARMAQ 001 (described in Example 2) in each of the rpoD, A TP~gra$p domain protein, FfsH. or Feed genes is present, and n indicates that the mutation is not present. As shown in Tabic t. none of
DK 2017 71015 A1
- 24 the strains of P, saimonis that were examiner· were found to possess any of the mutations described in Exampie 2 in the rpoD, FecR,ATF~grasp domain protein, and F/sD genes.
Thus, only PHARMAQ 001, and no other strains of P. saimonis, possesses the described mutations in the rpa.D, FecR, AF'P-grasp domain protein, and FtsH genes.
The presence of these mutations in the rpoD, PecR, ΛΤΡ-grasp domain protein, and Ftsfi genes therefore provides a means of differentiating and distinguishing the o PHARMAQ 001 strain from other Piscirickettsfa saimonis strains,
A method of identifying the PHARMAQ not strain involves analysing the DNA sequence of the specific portions of each of the rpa/d, FeeR, ATP~arasp domain protein, and FtsH genes containing the identified mutation. The specific portions of the genes 5 may be amplified by polymerase chain reaction (PCR) using specific DNA primers (shown above) followed by DNA sequencing using standard methods. When compared to the sequences of LE-89 (and other wild type strains) the sequences of each of the specific amplified portions of the rpaD, PecR,.ATP-g rasp domain protein, and FtsH genes harbors a DNA point mutation which is specific and unique for PHARMAQ 00i.
Mutations in the rpoD, FecR. ATP-prosp domain protein, and FtsH genes were identified due to the fact that they are the four mutations in PHARMAQ no: which result in a significant change in the amino acid sequence of a protein.
as Amino acid sequence alignments between a number of virulent wild-type strains of F, saimonis and the PHARMAQ oot attenuated strain for each of the τροΐλ ΡαοΡ,ΛΤΡ' grasp domain protein, and FtsH gene products were investigated and are shown below.
The amino acid sequences ofF. sabnonis strains LP-89, EM-90, At-t5972, and Br30 32507 were obtai ued from the published genome sequences of these strains (LF- ifo.
DDBJ/EMBI,/GenBank accession no, AMFP'00000000,2; EM-90. GenBank accession no.: JRHP00000000-1; Αχ*·5972, GenBank accession no.; JRAVoooooooo.2; and Bi32597, GenBank accession no.: JRAD00000000.2). The amino acid sequences for the other strains listed below were obtained frøen the relevant virulent wild-ty pe strain by means of standard PCR and sequencing methods using the PCR primer pairs described above (Seq. ID No,s 2 and 3,5 and 6,8 and 9, it and :a).
DK 2017 71015 A1
WO 20.1ft/189Öö
røøP amino acid seouence alignment:
PHARK&0...001 MDossKHSQF tasLxvRGKQß (ssuæs'tcvn» hbrcs-sssss sivs £ vwsx s mexewsTA
JO gBOßS-iXR—QS' ii ,JVRGRQQ
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PHÄSMAQ._001 PDPDSLLMKE KASSSSXæ&n SAV&AU3SÖA äF'XRRRCRVR äiiMFOiSSVS LRX’RiRXSXßß
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DK 2017 71015 A1
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The rpoD amino acid sequence alignment reveals that there are natural polymorphisms ofthe rpoPgenc product between wild-type strains. However, all ofthe wild-type strains are viru lent and therefore none of the differences between the wild-type yo sequences can be considered to affect virulence. PHARMAQ oot has an arginine to cysteine mutation at position 473 of the amino acid sequence of the cpof) gene product and this mutation is not seen in any of the wild-type strains investigated, such as At15972, 81-32597, EM-90, and/or Li-89. The protein sequence of the rpoOgene product in PHARMAQ 00.1 is otherwise identical to that of wild-type strains including
p.5 Ar -15972. EM 90, and LF- 89. The sequences of it wild- type strains were examined in the region of amino acid, residues 462-504 ofthe rpoD gene product. All ofthe sequences were found to be identical in this region, but different, to that of PHARMAQ 001,
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Thx¹ F'ec/ί amino acid sequence alignment reveals that there are natural polymorphisms of the FecR. gene product between wiid-type strains. However, all of the wild-type strains are virulent and therefore none of the differences between the wi id-type sequences can be considered to affect virulence. PHÄRMAQ 001 has a premature stop codon introduced at position 83 of the amino acid, and this mutation is not seen in any of the wild-type strains investigated, such as A;-/5972, ßi-32597, EM-90, and/or LF89. The protein sequence ofthe FecR gene product in PHARMAQ 00· is otherwise identical m that of wild-type strains Including Ai-15972, EM-90, and LF-Sp. The
DK 2017 71015 A1
WO .ttrU/Wki
• 29seqneuces of 13 wild-type strains were examined in the region of amino acid residues 39 137 of'the F*cR gene product. None of the wild-type sequences were found to be prematurely truncated, whereas in contrast, the FecR gene product of PHARMAQ 001 is terminated by a stop codon in position 83.
ATP-gru$p domain protein amino acid sequence alignment:
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DK 2017 71015 A1
WO 2016/1(0067
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All ofthe wild-type sequences of the A'/'F-protsp· domain protein· gei w product that were investigated were found to be identical. However, PHARMAQ ooi has a serine to proline mutation at position 184 ofthe amino acid sequence of the/VfP-c/rosp domain protein gene product which is not seen in the wiid-type sequence. The sequences of .13 wild-type strains were examined in the region of amino acid residue:; 118-251 of the. ATP-grasp domain protein gene product. /Ml ofthe sequences were found to he identical in this region, hut different to that of FRARMAQ 001.
RsH amino acid sequence alignment:
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DK 2017 71015 A1
WO 201 {i/s8«067 ,χχ.χχχχα aiaasaa MAAaAA la xaaaa ALSiiAxA AL 'SA- L_: äXAAXAA !5
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The FtsH amino acid sequence alignment reveals that there are natural polymorphisms ofthe RfsH gene product between wild-type strains. However, all ofthe wild-type strains art- v indent and therefore none of the difference between the wiId-type sequences can be considered to affect virulence. PHARMAQ 001 ha a methionine io isoleucine mutation at position jqt of the amino acid scrjuencc winch is not seen in any rto of the wild-type steal ns hr. esligaied, such as A - /5070, $1-32597, EM-90, a nd/or LFDK 2017 71015 A1
WO 2016/187067
- 34 89. The protein sequence ofthe Fis tf gene product in PHARMAQ 001 is otherwise identical to that of wild-type strains including Λι-ί;>072, EM-Ofj, and LF-Sq. The sequences of 53 wdld-type strains were examined in the region of amino acid residues 152-274 of the Fgs/f gene product Ah of the sequences were found to be identical in this
S region, but different to that of PHARMAQ oot.
ft is clear from the alignments that .P. scrfmotiis gene products are very highly conserved in ail ofthe strains investigated. As would be expected, there are natural polymorphisms in the genes wherein some, ofthe wild-type strains have a sequence that jo is different front that of other wild-type strains. However, since all the strains except PHAR.MAQ 001 are virulent, these differences cannot contribute to the loss of virulence and consequent attenuated phenotype observed in PHARMAQ oot.
PHARMAQ ooi has mutations in the amino acid sequence ofthe rpoD, FccR, ΛΤΡ
J5 grasp domain profein, and Ftsil gene products relative to the wild-type sequence, and also has an attenuated phenotype. These imitations are the only mutations observed in PHARMAQ oot which lean to a significant alteration in the amino acid sequence of a protein, arid they are not observed In any of the virulent strains investigated.
no in a P. saknonir, strain with an attenuated phenotype, if a mutation is observed in one of the rpoi), Fee.R, ATP-grasp domain protein, or FisFfgenes which is also present in a virulent wild-type strain, then the mutation cannot be responsible for the attenuated phenotype. Mutations, and in particular attenuating mutations, in the rpoi), Fecit,
ATP-grasp domain protein, or Ft$H genes are therefore only significant if thev lead to a difference in the amino acid sequence of the gene product relat ive to the protein sequence of one of the virulent wild-type strains, such as At-15972,81-32597. EM-90, and/or LF-Sq.
b>ainRkAl.fh0'.te qc To elassif> the PisciricRettsia sabnonis isolates in Tabb· i, a simplified MLSA-scheme was used employing the genetic information within the arginine N-sucdnyltransferase fast*. giutamate-i-semhddehyde aminotransferase s’heml. , L-sorme dehydratase fsdhL}, and UDP-gfocosfM-epimerasv fgalE) genes for phylogenetic predictions fas annotated iu the genome, of A >-35972). Sequence information was obtained by standard
PCR and sequencing DNA sequences were assembled. quality checked .and trimmed using vector ΝΊΤ& software. Sequences for the strains At-15072.. Rt-32507,1.F-Sp and
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EM-90 were retrieved from (5enBank. Sequence information from the four genes for each strain were trimmed and concatenated before alignment.
Sequence alignments and. phylogenetic predictions were performed in MEGA$, as ,5 described in Tamura et ai. (Mol Bio! Evol. 20n Oct; 28(10): 2731-9, incorporated herein by reference), with the following parameters:
Staitsffca! Method'. Maximum Likelihood (iOO Bootstrap Replications)
Substitution Model·. Tamura-Nei model
Rotes: Gamma distributed with invariant sites (16 Discrete Gamma Categories) .10
Upon phylogenetic analysis of DNA sequences from the tour genes above, two distinct and separate genotypes were identified. The two genotypes are here referred to as the LF-89 group and the EM -90 group. PHARMAQ 001 was found to group together with the EM-00 group. The LF-89 sequence used in the preparation of the phylogenetic tree was the genomic sequence deposited under GenBank accession no. AMGCoooooooo.r.
LF-89 group
EM-90 group
Ö.Ö'i no yggeipg·
PHAKMAQ 001 was cultivated in Insect cell medium to an (»D^vnm ~ 3.0 before the addition of a eryoproteetant and storage at -80 'C. The bacterial content was determined to be 2,6 x io^sTCiD_SQ/mI b> end-point titration.
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An ampoule of frozen isolate PHARMAQ oo: was thawed at room temperature and a curol dose containing 1,3 x 10'TCiDw/ ml was injected mtraperitoneaUy into Atlantic salmon with an average weight of 25-30 grants,
The fish were held at tfTC for 44 clays in freshwater. No fish died or showed any clinical signs, of SRS.
Tissue samples (from head kidney and. spleen'» from vaccinated fish were taken every week for three weeks after vaccination. Tin· presence of bacterial genomz-s in the tissue to samples was analyzed by real timequantitative-PCR. The results are shown in Table 2.
iy found to
As shown in Table a, the presence of PHÅRMAQ 001 genomes was gener peak at 7 days post vaccination with Ct values around 28.
This experiment demonstrates that PHARMAQ 001 is avirulent and does not induce any symptoms of SRS. Therefore, PHARMAQ 001 is safe and suitable for use as an attenuated live vaccine, hwlaæ
To examine the efficacy ofthe P. salmonis isolate RK.ARMAQ 001 as a vaccine, against SRS, Rainbow trout (Oncorhynchus mykiss) with an average weight of 30 grams were injected with different doses of the vaccine isolate. The vaccine isolate was either preserved as a frozen vaccine and then diluted in PBS for use at. a concentration of 2xio^r>Τ€ΠΧ,.>. or as a lyophilized vaccine and then diluted in PBS for use at a concentration of 5.2x10* TCiD.sc- per fish.
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After an immunization period in fresh water for 504 degree days, the trout were challenged with a highly virulent wild-type strain of P. salmonis. The virulent wild-type .strain was injected intraperitonealiy arid the fish were observed for 34 days, as shown in Figure 1.
,5
Figure t shores the accumulated mortality following challenge with a virulent P. salmonis strain.
The attenuated F. salmonis strain PHARMAQ 00.1 was found to work wet! as a live 10 attenuated vaccine, and provided 100% protection against SRS.
in addition, the study shows that PHARMAQ 001 can he either frozen or lyophilized prior to being used as a vaccine.
.s.tfotyggQh^
To investigate the potential for reversion to virulence, the isolate PHxARMAQ oot was serially passaged through Atlantic, salmon. The trial was performed in fresh water at t5^yC- Atlantic salmon were Injected with isolate PHARMAQ oot. Homogenates were no prepared from head kidneys 7 days after injection, and new Atlantic salmon were injected with the homogenate.
SUBSTITUTE SHEET (RULE 26)
DK 2017 71015 A1 wo
-38After a further 7 days, homogenates were prepared from the head kidneys of fhe second fish, and further Atlantic salmon were injected with the homogenate.
No mortality or clinical signs of SRS wore observed in any fish during the trial.
The liver from fish from each passage was homogenized. and homogenates were tested lor ihe presence of live P, sajmonis by plating onto CHAB agar plates. Bacteria were only detected after the first passage.
to The bacterial loads in liver and spleen were investigated by measuring the presence of P. sa/monis genomes by real time quantitative PCR one week after injection of PH ARMAQ oot or homogenate. The results (shown in Table 3) demonstrate that the bacterial loads io spleen and liver were reduced when head kidney homogenates were passaged from the first injected fish into passages 2 and 3 offish.
Table 3
Initial injectionPassage 2Passage 3Liver2S.9nclndSpleen28.232,8nd
(od- not detected)
This experiment shows that PHARMAQ 001 docs not revert to a virulent strain after co serial, passage in Atlantic salmon. PHARMAQ oot Is therefore suitable for use as a live atten oated vaccine.
Bacterial cultures were grown in ΕχΟΊΙ ’riter High medium from Sigma with no 25 supplements. Thu cultures were incubated in ventilated spioner Husks at 75 rpm cud
20^vC, 2 passages after thawing. The growth was monitored by OH««™»» measurement.
Table 4
stepMediapHVenUiatioaSUrrUigTt'Kip jVoS.ofI swish«!)'fimeEndOiVSjAwviriViSk‘7 iiiirK i^htw ................eoBtToised<A:psex; Ii %:·. iSsys3.»SpinorWtBTiaa-iUghNU ............iS..,,...........j.·> 5’p;Ra days4,4
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The results shown in Table 4 demonstrate the cultivation of P. sabnaw's' in spinner flasks as the OD«-, reached 3-4 after 3-4 days of incubation.
P. salmonis strain PHARMAQ oot was grown tit spinner flasks in $f-ooo medium.
PHARMAQ øOFwas harvested from the spinner flask and 20% skimmed milk in water (made from dry skimmed tniik and heated for 15 minutes at 80°O was «tided i;t to the re P. salmonis etilture to a final concentration of ίθ ό skimmed milk. 2ml of this mixture (P. salmonis and to% skimmed milk) was placed into toml glass vials and then freeze dried in a f^bconeo FreeZone Triad freeze dryer.
ϊ5 Freezing: 3 hours hold at -s’fr'
Segment 1: ramping rale: o.p’C/min; holding time: t hour; shelf temperature: -4O°C Segment2: ramping rate: o.r’C/min; holding time; 8 hours; shelf temperature: -25%’ Segments: ramping rate: o.ft’C/mln; holding time: 24 hours; shelf temperature: -to°C Segment 4: ramping rale: o.i^aC/min; holding lime: indefinite: shelf temperature: 4°C so Vacuum was set to opBar in step 1-4
The vials were sealed under vacuum and tk<· process eras stopped. The freeze dried materials were stored at 2--8 °C,
To determine the viability of dm samples, titration (TCffh_(;/ntlt was performed on the culture at ban esi and after fre-ze drying. The freeze dried cake was rehydrated with end PBS before titration.
Titer (TCIDs»/ml) at harvest: 8.7 x 10^ cells/ml 30 Titer (TCiD_So/ml) after freeze drying: 3.2 x to⁷ cells/ml
Thus, after freeze drying, the samples are sufficiently viable for use as a ‘vaccine.

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-40 The attenuated live strain of P. salntonis (rpoD/ FecRf ATF-grasp domain protein/ Ρίχ./ϊ) has been shown -o be well tolerated in healthy fish hosts and to colonise the host In a manner consistent with its utility as an effective vaccine to protect against SRS. it has also been demonstrated to elicit a specific immune response. The PHARMAQ ooi live attenuated P. sobnoms strain has been found to be particularly effective for Uns purpose.
An assay was developed for identifying and detecting the PHARMAQ goj strain of P. safmonis. The genome of the PHARMAQ oot strain contains single nucleotide io polymorphisms (SNP1 in each of the rpoD. FecfL ATP-grasp domain protein, and PfsfZ genes, each of which results in a chemically significant alteration of the amino acid sequence of the resulting gene product. The assay involves typing each of these single nucleotide polymorphisms, and determining whether the wild-type or mutant allele is present.
Specifically, the assay involves using target-specific PCR primers to amplify the nucleic acid sequence in t he region of the SNP. Separate probes specific for each of the wildtype and mutant, alleles are included Lu the reaction mix. Each probe is labelled with ri different detectable marker such as a fluorescent dye. In the example described below, no the probe specific for the wild-type sequence was labelled with the PAM fiuorophore, and the probe specific for the mutant sequence was labelled with the VIC fluorescent dye. An overview of the primers and probes used for each SNR analysis is shown in Table 5. In the present example, the SN'P analysis was performed using the ABI RRISMuL 7900 HT Sequence Detection System,
Table 5
Targtrt OuePsUnu'e/PixibeSeqnex»«»Seq. to X«. 1rpoPfg,jp SA f·' /..yiiwUcc · S'.Vif’tt ft-ίΡη iGG.yC,AHX<0GA'ihtq₍'_:··: vpyiy·]·67 IVProoex-VJCMuiantAlleieÄCÄÄ«XnTAAQ£GCGTCTC68...................Irpni^} it/>;>: ; ' 6. ti / i '! :;js_:· •S.'p.'e·ÄÄrjeriAÄÖCGCcrc’tc6»>.yw.O SAT tViwe'twKewse Erasergc< c- c rrcTi'GG s cca rrrcAC70
DK 2017 71015 A1
ÅTP-grasp domain proteinA7P-Gro«p /..<>·>» <;!« Frost· ·! ,WP Ancet«)«: fpi'icöid Pr&wefTGGtGATGGTGCGTCiAC I f I.'V/'P-Gso.·.;¹ Ansjos'«: Protein WToéei WC .M«£ön 'z5i'fefeGGVKAGföGAGCGiAC; 72.: 4 TF-Groep Domen; Protei« JÖWWOT IVRd-Ts/pe 4/lefe;.·< (: v'AG O2'G VG73 ίA'f. P-G'i'i.’Si.l /.VwtMi« Protei« AVF tteirr/'s.’.n Äewrse PrwwCGCC6ATs'GCäC0Fs'GaT<I4AGr-iFisHNs«· Suppeterrin« Pevw&rd PriiiK!’CCAAAAGi TAGGCGQCAAAAT FCC75 .11FiaH VR'ßii««· -WC Λίηίϊϊΐη .·!.%·{ ·TGÖGCCAA<TAT€A76 1X ·£;:; ü/PsoR’l·'- Rat Wt&M «pe 4 SefeAmoXYAAGCAliA” 1NN SDP OrmAf')! Prim« GCl'AATAGCGI cn 'ACCrCTi'CCA76 1
RNA was isolated front cultures of P, sn/monis, including PHARMAQ oot and wild type strains including the virulent starting strain. The samples were prepared as shown in Table 6. Ail tests were performed using QuantiTect Probe RT-PCR kit (Qiagen).
Table 6
ReagentFinal «tucentratfonaX Master mix(Recomtnended by bit supplier, eontauts cäNTPs, MgCfø {ftnal røneenirai ion ψη.M), HotStertTsoDÄA Polymerase, &od passive reference dye (ROX)tXf-'orø&rd primer;« ; nW (o ο si ()f >.o μΜ ;«>ί·.ηί·>μ)Reverse Prisserpoo PM {o.$ pi of i,n pM solution)VProbe-Vtnapo fib· (< »·. id of i.v apt «>1 n bop)MPfiibe-EXMv 5 «Μ { ,,r-j μ) _υ: j,o tAi soke toriET- bnzymentix tQnasPTeeO0,1 ,sl‘Tes's pistet pg· to s op per -n’artionOH _:tOTo io pi
DK 2017 71015 A1
Samples were analysed in triplicates on 384-well plates. Each plate was subjected to a pre-read, for determination of background fluorescence in each well prior to the realtime RT-PCR step. The real-time RT-FCR was performed using standard enzymes and buffers, with the parameters shown in Table 7.
Table 7
StepTemperaturelaueCyvtes ·(} Ri-ves-sr ί -ψΗναSON 30 taifixtea1 1s! ΡΝΛ ix »i vs !< tx se iu:· is at ίο: ι95^tfC15 minutesi is) ne-ißtUfa-t:·;. A:U;-tai:Sg i:!5ÜνΥΤ1extension ^zk> 16<rC• irtireü'...... Ϊ
All primers and probes were optimized to allow annealing and extension at 6oX This to temperature is also believed to be significant for the competition between the two probes in the SNP assay, as it leads to binding and cleavage of the correct probe as well as destabilization of the incorrect probe, depending on the SNP at the probe site.
After the real-time RT-PCR reaction had been performed, the plate was subjected to an /5 end-point analysis, by perform: ng a post- read of the fluorescence in each well, a nd comparing the result to the data stored from the pre-read. The results are shown in Table $ (in which a pins sign indicates a cycle threshold of less than or equal to 30 and a minus sign indicates no detectable fluorescent signal), co Table 8
rpoi)ATP <k:<sn;· i«proteinITT: VPi’öheiViC.iPfefe' 1VPr<,0<-; V7TAiiefeΛί/Όϊ/χχ -RW-TvpHASeteyPfwheji ;CAfti&fflf ,4 SeteÅ/Probes -,%WPNcteTt pe,4;te« PtiA.RMAQ on i. iin.cnt:;:Ad vnecuR' st-xin4’}<<Vinjieat starting strain 4bi4-<y - S >iWild type sireinÄ*y:4—..... ::l·Wlkl type P.saiiminis strain Β<1*1T*<^:: y·^·4*
DK 2017 71015 A1
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The assay clearly identified each of the mutant alleles in the PHARMAQ oox strain, and also identified the presence of the wild-type allele in all wild type strains tested. For all tests, the discrimination between the two allelic variants was very good. The assay permits clear distinction between wild type and PHARMAQ oo.t P. saimonis strains.
in order to address arious Issues and advance the art, the entirety of this disclosure shows by way of illustration various embodiments In which the claimed invention may he practiced and provide foran attenuated P> salmonis bacterium and an improved P.
m so/monix vaccine. The advantages and features ofthe disclosure are of a representative sample of embodiments only, and arc not exhaustive and/or exclusive. They are presented only to assist in understanding and teach the claimed features, it is to be understood that advantages, embodiments, examples, fianctlons. feat ures, and/or other aspects of the disclosure, arc not to be considered limitations on the disclosure as 5 defined by the claims or limitat ions on equb alents to the claims, and that other embodiments may be utilised and modifications may be made without departing from the scope and/or spirit of the disclosure. Various embodiments may suitably comprise, consist of, or consist essentially of. various combinations ofthe disclosed elements, components, features, parts, steps, means, etc. In addition, the disclosure Includes no other inventions not presently claimed, hut which may br· claimed in future.
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This document certifies that:
Bacteria Piscirickettsia salmonss
Deposit Reference 14100901
Has been accepted as a patent deposit, in accordance with
The Budapest Treaty of 1977
With the European Collection of Ceil Cultures on
October 2014
Bryan Bolton
Head of Business Development and External Communications
Catone Coitecfesns. Pusfc HsatSf £> yar<5, Porian Oowr>. Sa>>stiu>y. SP4 OJO, US 7-+44(8} '3806'25A F: >44(0) 3980 611315 £: cuSiafäcoiietSions@p:)e.govufc ’Λύ'.wx.pre-callDrec.'iÜsdicns.org.uK
ES
NCTC
NCPV h>7:x;f<7p:;;o$5--..:kTi V %. } .,
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权利要求:
Claims (13)
[1] 1. Aa attenuated RvcMdærtsæ saimonis bacterium compi o.mg imitation in the amino acid sequence of each of the rpoD, FeeR, ATP-grasp domain protein, and I id! gene products; wherein said Piscirick&tsia salmonis bacterium is avirulent and does not induce symptoms of Salmon Rickettsial Syndrome when administered to fish
[2] 2. The attenuated bacterium as claimed in claim 1, which does not revert, io a virulent strain after serial passage in fish.
[3] 3. The attenuated bacterium as claimed in claim 1 or 2., wherein the mutations in the amino acid sequence ofthe rpoD, FecR, ATP-grasp domain protein, and FtsH gene products are mutations relati ve to the sequence of the corresponding
Lf-89 wild-type protein, as derived from the Lf-89 genomic sequence that is available under the GenBank accession no. AMFFoooooooo.2, and as provided as Seq. ID No.s
17,26,40, and $4, respectively.
[4] 4. The attenuated bacterium as claimed in any one ofthe previous claims, comprising a mutation in the region of:
a) amino acid residues 462-504 of the rpo.D gene product, provided as Seq, ID No,
17;
b) amino acid residues 39-137 of the FecR gene product, provided as Seq. ID No.
26;
c) amino acid residues 118-251 of the ATP-grasp domain protein gene product, provided as Seq. ID No. 40; and/or,
d) amino acid residues 152-274 ofthe FlsH eene product, provided as Seq. ID No.
54.
[5] 5, The attenuated bacterium as claimed in any one ofthe previous claims, comprising:
a) an arginine to cysteine mutation at position 473 of the rpoD gene product, provided as Seq. ID No. 1.7;
b) a premature stop codon at the position corresponding to residue 83 of the FecR gene product, provided as Seq. ID No. 26;
c) a serine to praline mutation at position 184 of the AFP-grasp domain protein gene product, provided as Seq. ID No, 40; and/or,
d) a methionine to isoleueine mutation at position 191 of the FtsH gene product, provided as Seq. I'D No. 54,
[6] 6, The attenuated bacterium as claimed in anv one of the proi tous claims, whuh is the strain PHARMAQ 001 deposited with the European Collection of Cell Cultures. Public health England, Culture Collections, Forton Down, Salisbury SP4 OJG, United Kingdom, on
09th October 2014 with accession number 14100901.
[7] 7, A live, attenuated vaccine composition comprising;
(a) an attenuated Piscirickettsia salmonis bacterium as claimed in any one of the previous claims; and {b) a pharmaceutically acceptable carrier or diluent
[8] 8, The live, attenuated vaccine composition as claimed in claim 7, in freeze-dried form.
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[9] 9, The live attenuated vaccine composition as Claimed in eiaini 7 -or 8 for use in vaccination ofa fish against Salmon Rickettsial Syndrome, said vaccine composition comprised in the vaccine composition in immunologicaily effective amount,
[10] 10, A method of distinguishing between wild-type and mutant alleles ofa
Piseiricfaffsi'a sa/monis single nucleotide polymorphism (SNP) located at fee position corresponding to:
residue number .1417 of Seq. ID No. 1 (in the rpoD gene);
residue number 247 of Seq. ID No. 4 (in the Fee/t gene);
residue number 550 of Seq, ID No. 7 (in theN/Y^-groxg domain preterit gene);
or, residue number 573 of Seq. fD No. 10 (in fee F/sH gene), wherein the method comprises;
i) amplifying by PCR the region of the nucleotide sequence containing the SNP;
ii) including in the PCR. reaction mix a nucleic acid probe having a sequence complementary to one allele of fee SNP, the probe comprising a detectable marker; and iii) analysing fee 'PCR product for the presence of the marker, wherein the presence of the marker is indicative ofthe presence ofthe allele.
[11] 11, The method as claimed in claim 10, wherein fee method further comprises including in the PCR reaction mix a first nucleic acid probe having a sequence complementary to fhe wild-type allele of the SNP, and a second nucleic acid probe having a sequence complementary to the mutant, allele of the SNP, the first and second probes comprising different detectable markers.
[12] 12, A nucleic acid probe for use in fee method of claim 1(), wherein the probe comprises a 10-40 nucleotide subsequence of:
Seq. ID No. 1, fee subsequence including residue number 1417;
Seq. ID No. 4, the subsequence including residue number 247;
Seq. ID No. 7, the subsequence including residue number 550; or,
Seq. ID No, 10, the subsequence including residue number 573.
[13] 13, A PCR primer pair for use in the method of claim 10, wherein the PCR primers may he used to amplify a region of at least 50 nucleotides in length of the subsequence of;
Seq. ID No. 1, the subsequence including residue number 1417;
Seq. ID No. 4, the subsequence including residue number 247;
Seq. ID No. 7, the subsequence including residue number 550; or,
Seq. ID No. 10, the subsequence Including residue number 573.
wherein the primer pairs comprise Seq, iD No. 67 and 70, 71 and 74, or 75 and 78.
DK 2017 71015 A1
WO 201.6/189()67
-1/1 -

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WO2016189067A1|2016-12-01|

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US20190091317A1|2019-03-28|

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DK180223B1|2020-08-21|

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CL2019003825A1|2020-05-15|

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US10857218B2|2020-12-08|

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GB201509004D0|2015-07-08|

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EP3303371A1|2018-04-11|

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CA2986834A1|2016-12-01|

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CL2017002982A1|2018-05-25|

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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WO2014198913A2|2013-06-14|2014-12-18|Cermaq As|Novel piscirickettsia salmonis isolate|

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法律状态:
2020-08-21| PME| Patent granted|Effective date: 20200821 |

优先权:

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申请号 | 申请日 | 专利标题

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GBGB1509004.6A|GB201509004D0|2015-05-26|2015-05-26|Attenuated bacterium|

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PCT/EP2016/061862|WO2016189067A1|2015-05-26|2016-05-25|Attenuated piscirickettsia salmonis bacterium|

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