WO2024003035A1 - Vaccins contre la piscirickettsiose (septicémie rickettsienne des salmonidés) - Google Patents

Vaccins contre la piscirickettsiose (septicémie rickettsienne des salmonidés) Download PDF

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WO2024003035A1
WO2024003035A1 PCT/EP2023/067443 EP2023067443W WO2024003035A1 WO 2024003035 A1 WO2024003035 A1 WO 2024003035A1 EP 2023067443 W EP2023067443 W EP 2023067443W WO 2024003035 A1 WO2024003035 A1 WO 2024003035A1
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fish
strain
salmonis
vaccine
piscirickettsia
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Valeska HERRERA
Marcos MANCILLA
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Vaxxinova Norway As
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/0208—Specific bacteria not otherwise provided for
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/29—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Richettsiales (O)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/36—Adaptation or attenuation of cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine

Definitions

  • Vaccines for piscirickettsiosis (salmonid rickettsial septicaemia)
  • the present invention relates to the field of vaccines for fish.
  • SRS is a serious infectious disease in salmon farming caused by the bacterium Piscirickettsia salmonis, a Gram-negative intracellular facultative bacterium. Since its appearance in 1989, it has a major impact on salmon, with a mortality rate of up to 90% in some populations. SRS is predominantly present in Chilean coastal waters, and affects Salmon farms mainly there.
  • P. salmonis is the etiological agent of this disease, its incidence and frequency in farms respond more to a multifactorial model, which includes those associated with the host (species and genetic susceptibility), the pathogen (virulence, antibiotic resistance) the environment (such as water temperature or salinity) and husbandry practices (Rozas-serri M., 2022).
  • the best approaches in controlling infectious diseases are based on dietary supplements, non-specific immunostimulants, vaccine, probiotics, prebiotics, medicinal plant products, improved husbandry practices, movement restrictions, genetically resistant-disease fish, water disinfection and antimicrobial compounds.
  • effective vaccines are probably the most important factors for the growth and success of intensive salmonid farming systems (Maisey et al., 2017; Vargas et al, 2021).
  • the first P. salmonis vaccines were formulated as formalin-inactivated bacterins - i.e. inactivated or killed bacteria (antigens) that are injected to a subject parenterally to produce active immunization, using cell-culture derived organisms grown in CHSE-214 cells, derived from Chinook salmon embryonic cell culture.
  • the first bacterin-vaccination studies showed the potential of formalin-inactivated P. salmonis bacterins as immunogenic agents against SRS. Bacterial challenge in vaccine/control trials yet is complex, with different outcomes depending upon strain type, dose and route of administration.
  • Attenuated bacteria strains Another approach that has been pursued is the development and use of attenuated bacteria strains.
  • An attenuated bacterium has lost pathogenity, but is still infectious and immunogenic, so that subjects immunized therewith become resistant against pathogenic representatives of the bacteria species.
  • An attenuated strain of a bacteria belonging to the Piscirickettsia genus may be generated for instance by passing the bacteria through culture a number of times, or deleting or mutating a gene involved in a biosynthetic pathway.
  • Attenuated Piscirickettsia strains and their use in vaccines are for example disclosed in W02008002152.
  • Such strains can be obtained by providing a sample from a fish, which is infected with bacteria belonging to the Piscirickettsia genus; inoculating into a essentially cell free culture medium bacteria from said sample, and c) selecting a bacterium that propagate freely in the medium.
  • Attenuated strains thus obtained and disclosed in W02008002152 are for example the strains deposited under the Budapest Treaty with the European Collection of Cell Culture (ECACC), Health Protection Agency Porton Down, Salisbury, Wiltshire (UK), SP4 OJG, UK, on June 9, 2006 under the following accession numbers: 06050901, 06050902 and 06050903, or the strain deposited on March 21, 2007 under accession number 07032110 (isolate Al 10014).
  • ECACC European Collection of Cell Culture
  • UK Health Protection Agency Porton Down
  • SP4 OJG UK
  • FIG. 1 Comparison of RD1 locus between parental PM15972A1 and ADL-PSA1/P102 strains. Some predicted genes have been marked to facilitate interpretation of the 18.9 kb deletion found in ADL-PSA1 and derivatives.
  • Figure 2. Optional mutations present in the genome of a Pisciricketsia salmonis strain according to the present invention. *ORF nomenclature related to the annotation of ADL-PSA1 genome (not available in public databases). fDeletion detected in a low complexity zone that does not allow predicting the position with accuracy.
  • ADL-PSAl(also calledP50c7) is a derivative of Al-15972 (also known as PM15972 or PM15972A1), an EM-90-like strain.
  • P101 and Pl 02 are both derivatives of P50c7. + or - indicate the occurrence of the corresponding mutation. Those mutations detected in P50c7, and derivatives were confirmed by Sanger sequencing.
  • FIG. 5 TaqMan PCR for plasmid PS Al -4.
  • the amplification curves for a chromosomal target for several clones selected from P40 and P50c7 are shown.
  • the corresponding signal for a target carried by the 32 kb PS Al -4 plasmid is shown in green.
  • Figure 6 Conventional PCR for 18.9 kb chromosomal deletion.
  • the amplicon can be already observed in material purified from passage 20 th .
  • FIG. 7 Cytopathic effect of two P. salmonis strains in CHSE-214 cell monolayer infection assays (14 days post infection). Arrows indicate the characteristic CPE produced by P. salmonis. MOI, multiplicity of infection; Magnification (40X and 120X). WT (EM 90-like), PM15972A1. Control not infected.
  • Figure 9 Growth kinetics (shaking). The strains under study were inoculated in liquid medium (ADL-PSB) and incubated at 18 °C. The ODeoo was recorded every hour.
  • ADL-PSB liquid medium
  • FIG. 10 Growth kinetics of P. salmonis Pl 02 strain in different culture media (BMJ, BM4, PSB). * indicates media with modifications from the original formula.
  • Figure 11. Growth kinetics of P. salmonis Pl 02 strain in BMe and BMe supp. with AF204. The ODeoo record was extended for up to 100 h.
  • Figure 13 Innate immune response marker gene expression after bacterial infection. Relative expression levels of cytokine genes ifny, illfi, ill 0, H12, H18, H15 and complement protein c3 in in vitro SHK-1 cell model of P. salmonis infection. Values were determined relative to uninfected cells. Differences were statistically significant (*p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.005 and ****p ⁇ 0.001).
  • Figure 14 Adaptive (cellular) immune response marker gene expression after bacterial infection. Relative expression levels of genes mch-I, cd8a, cd8a, tgf/3 and granzyme in in vitro SHK-1 cell model of P. salmonis infection. Values were determined relative to uninfected cells. Differences were statistically significant (*p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.005 and ****p ⁇ 0.001).
  • Figure 15 Adaptive (humoral) immune response marker gene expression after bacterial infection. Relative expression levels of genes mch-II, cd4, cd83 and socs3 in in vitro SHK-1 cell model of P. salmonis infection. Values were determined relative to uninfected cells. Differences were statistically significant (*p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.005 and ****p ⁇ 0.001).
  • FIG. 1 Bacterial count values (CFU/mL) corrected by the dilution factor (groups B, C and D) of the solution.
  • CFU/mL Bacterial count values corrected by the dilution factor (groups B, C and D) of the solution.
  • FIG. Stability (CFU/mL) of P. salmonis Pl 02 strain with stabilizers cryopreserved (R&D batches C. Bl 19022021 and C.Bl 15032021, Chile).
  • FIG. 1 Stability (CFU mL-1) of P. salmonis Pl 02 strain cryopreserved in LN2 (liquid phase). Values obtained from 3 vials per time, each titrated in triplicate.
  • Figure 19 In vivo virulence of the attenuated strains (P. salmonis P50c7 and Pl 02) and wildtype strain. Each group consisted of 40 fish.
  • Figure 20 Average weight and length obtained at the end of the in vivo virulence study in each evaluated group. Differences in size were statistically significant for the strain P50c7 dose lOOx (p ⁇ 0.05).
  • Figure 21 Histological findings in samples of fish vaccinated with attenuated strains (lx dose). The size of the bar is related to the associated frequency in the analyzed samples (5 fish per group, kidney and liver). Samplings were performed at 7, 14 and 37 days post immunization. Upper panel: fish immunized with P50c7 strain; lower panel, fish immunized with Pl 02 strain.
  • FIG 22 Relative expression of a selection of innate and adaptive immune response markers in head kidney samples obtained after immunization with P. salmonis P50c7 strain (white) and Pl 02 strain (grey). Black bars correspond to the non-immunized control. Relative expression levels between immunized and control groups were compared using a one-way ANOVA with GraphPad Prism 6.0 software. Differences were statistically significant (*p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.005 and ****p ⁇ 0.001).
  • Figure 23 In vivo virulence of the attenuated strains (P. salmonis P50 and Pl 02) and wild-type strain. Results obtained from two tanks, each containing two treatment and one control group each consisting of 30 fish.
  • Figure 24 Innate immune response marker gene expression after bacterial injection. Relative expression levels between immunized fish and control groups were compared using t-test with GraphPad Prism 6.0 software. Differences were statistically significant (*p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.005 and ****p ⁇ 0.001).
  • Figure 25 Innate and adaptive immune response marker gene expression after bacterial injection. Relative expression levels between immunized fish and control groups were compared using t-test with GraphPad Prism 6.0 software. Differences were statistically significant (**p ⁇ 0.01, ***p ⁇ 0.005 and ****p ⁇ 0.001). Values were determined relative to uninfected cells
  • Figure 26 Dissociation curves for locus 440 (marC). The red lines correspond to dissociation curves produced by strains PM15972A1 (P0), P50c7 and other P50c7 strains that were also subjected to the in vitro reversion to virulence study. The green ones represent the Pl 02 strain and the isolates from the 5 in vitro passages. The single blue line is the pattern exhibited by the LF-89 type strain.
  • FIG. 27 Dissociation curves for locus 477 (methyltransferase).
  • the red lines correspond to strains PM15972A1 (P0).
  • the green lines represent the P50c7 and Pl 02 strains and their corresponding isolates recovered after 5 in vitro passages.
  • the blue line is the LF-89 type strain.
  • FIG. 28 Challenge experiment with P. salmonis EM-90 like genogroup. Relative percent survival (RPS) of S. salar vaccinated with the two formulations, post-ip challenge with P. salmonis. DPC, days post challenge.
  • RPS Relative percent survival
  • Figure 29 Average weight and length obtained at the beginning (0 DD) and the end (630 DD) of the immunization stage in each group. There were no statistically significant differences between the vaccinated groups vs. the control
  • FIG. 30 Histological findings in samples of fish vaccinated with attenuated Pl 02 strain (fresh and freeze dried, lx dose). The size of the bar is related to the associated frequency in the analyzed samples (5 fish per group/strain). Upper panel: histological findings in kidney; lower panel, histological findings in liver.
  • FIG 31 Challenge experiment with P. salmonis LF-89 like genogroup.
  • the immunization period was of 470 DD.
  • FIG 32 Cohabitation challenge with P. salmonis EM-90 like genogroup. Relative percent survival (RPS) of S. salar vaccinated with Pl 02 strain and a commercial competitor vaccine, post cohabitation challenge with P. salmonis. DPC, days post challenge.
  • RPS Relative percent survival
  • Figure 33 Pathological findings in samples of fish vaccinated with attenuated P102 strain and a vaccine competitor (inactivated, pentavalent vaccine) after immunization period (655 DD).
  • the size of the bar is related to the associated frequency in the analyzed samples (10 fish per group/strain).
  • the size of the bar is related to the associated frequency in the analyzed samples (10 fish per group/strain).
  • FIG. 35 IgM concentration at the end of the immunization period (655 DD).
  • ELISA assay against SRS developed by ADL).
  • Control fish not immunized.
  • Ps/Vxx fish vaccinated with Pl 02 strain.
  • Competitor fish vaccinated with inactivated vaccine.
  • embodiments disclosed herein are not meant to be understood as individual embodiments which would not relate to one another.
  • Features discussed with one embodiment are meant to be disclosed also in connection with other embodiments shown herein. If, in one case, a specific feature is not disclosed with one embodiment, but with another, the skilled person would understand that does not necessarily mean that said feature is not meant to be disclosed with said other embodiment. The skilled person would understand that it is the gist of this application to disclose said feature also for the other embodiment, but that just for purposes of clarity and to keep the specification in a manageable volume this has not been done.
  • a Pisciricketsia salmonis strain which is characterized in that it comprises, in its genome, at least a mutation in the RD1 locus and/or lacks the plasmid pPSAl-4
  • the RD 1 locus in Piscirickettsia salmonis strains encodes, inter alia, a cyo operon (cytochrome o operon), a MFS transporter (Major facilitator superfamily), a SNARE-associated membrane protein (soluble N-ethylmaleimide-sensitive-factor attachment receptor), a peptidase and several transposases.
  • a mutation in this locus is suitable to inactivate at least one of these genes and thus contribute to the attenuation of the pathogen, making it a suitable vaccine candidate.
  • wildtype Piscirickettsia salmonis strains comprise four plasmids, pPSAl-1 (46 genes), pPSAl-2 (39 genes), pPSAl-3 (86 genes) and pPSAl-4 (41 genes).
  • pPSA4-l has 41 genes and encompasses the gene locus tags KW89 RS16315-KW89 RS16315. The inventors have surprisingly shown that the loss of plasmid pPSAl-4 is suitable to contribute to the attenuation of the pathogen, making it a suitable vaccine candidate.
  • the Piscirickettsia salmonis strain according to the invention preferably comprises, in its genome, at least a mutation in the RD1 locus, while it optionally lacks the plasmid pPSAl-4.
  • the Piscirickettsia salmonis strain according to the invention preferably lacks the plasmid pPSAl-4, while it optionally comprises, in its genome, at least a mutation in the RD1 locus.
  • the mutation in the RD1 locus is a deletion which has a size of
  • the mutation in the RD1 locus is a deletion which has a size of
  • the mutation in the RD 1 locus is located between the ORFs KW89 30 and KW89 60.
  • the mutation in the RD1 locus comprises a deletion of ORFs KW89_35 to KW89_55
  • Exemplary Piscirickettsia salmonis strains developed by the inventors that comprise this mutation are called ADL-PSA-1 (P50c7, “passage No 50c7”), P101 (“passage no 100 clone 1”) and Pl 02 (“passage no 100 clone 2”) herein.
  • Exemplary Piscirickettsia salmonis strains developed by the inventors that lack the plasmid pPSAl-4 are called ADL-PSA-1 (P50c7, “passage No 50c7”), P101 (“passage no 100 clone 1”) and Pl 02 (“passage no 100 clone 2”) herein.
  • the Piscirickettsia salmonis strain comprises, in its genome, at least one further mutation as set forth in Fig. 2
  • a vaccine which comprises, as an immunogenic antigen, a bacterium from a strain according to any one of the aforementioned claims, or a subunit or fragment of said bacterium.
  • said vaccine comprises bacteria or subunits or fragments of bacteria, in a concentration of between > 0.05 mg/ml and ⁇ 5 mg/ml.
  • said vaccine comprises bacteria in a concentration of between > 1.0 CFU/mL and ⁇ 9.9 x 10 9 CFU/ml.
  • CFU relates to colony forming units.
  • the vaccine is being suitable and/or formulated for administration to a fin fish, preferably a Telostei, more preferably a Salmonid, most preferably a Salmon.
  • the teleostei further include, but are not limited to, basses, tuna, bonito, breams, cods, snappers, flatfish, catfish, yellowtails and tilapias.
  • the vaccine protects a fin fish against infection with Pisciricketsia salmonis strain types EM-90 like and LF-89 like, preferably with a relative percent survival of > 70%.
  • said vaccine further comprises one or more adjuvants.
  • adjuvants suitable for being used in aquaculture are muramyldipeptides, lipopolysaccharides, several glucans and glycans, mineral oil and Carbopol®.
  • An extensive overview of adjuvants suitable for fish and shellfish vaccines is given in the review paper by Jan Raa (1996), the content of which is incorporated herein by reference in its entirety for enablement purposes.
  • the vaccine of the invention may further comprise a suitable pharmaceutical carrier.
  • the vaccine is formulated as an emulsion of water in oil.
  • the vaccine may also comprise a so-called "vehicle".
  • a vehicle is a device to which the antigen adheres, without being covalently bound to it.
  • Such vehicles are i.a. biodegradable nano/micro- particles or -capsules of PLGA (poly-lactide-co-gly colic acid), alginate or chitosan, liposomes, niosomes, micelles, multiple emulsions and macrosols, all known in the art.
  • a special form of such a vehicle, in which the antigen is partially embedded in the vehicle, is the so-called ISCOM.
  • Immuno stimulating complexes which are spherical open cage-like structures (typically 40 nm in diameter) that are spontaneously formed when mixing together cholesterol, phospholipids and Quillaja saponins under a specific stoichiometry.
  • the complex displays immune stimulating properties.
  • the vaccine may comprise one or more suitable surface-active compounds or emulsifiers, e.g. Cremophore, Tween® and Span®. Also adjuvants such as interleukin, CpG and glycoproteins may be used.
  • said vaccine further comprises at least a) one further antigen from a bacterial source other than a bacterium of the Piscirickettsia genus, and/or b) an antigenic material obtained from a viral source, an antigenic material obtained from a parasitical source, and/or an antigenic material obtained from a fungal source.
  • said antigen from a bacterial source is selected from the group consisting of live, attenuated or killed bacteria of the species but not limiting to Aeromonas sp., Vibrio sp., Listonella sp., Moritella viscosa, Photobacterium damsela, Flavobacterium sp..
  • Yersinia sp. Renibacterium sp., Streptococcus sp., Lactococcus sp., Leuconostoc sp., Bifidobacterium sp., Pediococcus sp., Brevibacterium sp., Edwarsiella sp., Francisella sp., Pseudomonas sp., Cytophaga sp., Nocardia sp., Mycobacerium sp., or subunits or fragments of these bacteria, and any combination hereof.
  • said antigenic material obtained from a viral source is selected from the group consisting of Glycoprotein of Viral Hemorrhagic Septicemia Virus (VHSV); nucleoprotein of Viral Hemorrhagic Septicemia Virus (VHSV); glycoprotein of Infectious Hematopoietic Necrosis virus (IHNV); inactivated Pancreatc Necrosis Virus; VP1, VP2, VP3 or nucleoprotein, structural proteins of Infectious Pancreatic Necrosis Virus (IPNV); G protein of Spring Viremia of Carp (SVC); and a membrane-associated protein, tegumin or capsid protein or glycoprotein of Channel Catfish Virus (CCV); antigenic material obtained from ISA virus.
  • VHSV Glycoprotein of Viral Hemorrhagic Septicemia Virus
  • VHSV nucleoprotein of Viral Hemorrhagic Septicemia Virus
  • IHNV Infectious Hematopoietic Necrosis
  • said viral source is selected from the group consisting of pancreatic disease virus (SPDV), Iridovirus, Infectious Salmon Anaemia virus (ISAV) and heart and skeletal muscle inflammation virus.
  • SPDV pancreatic disease virus
  • Iridovirus Iridovirus
  • ISAV Infectious Salmon Anaemia virus
  • heart and skeletal muscle inflammation virus a group consisting of pancreatic disease virus (SPDV), Iridovirus, Infectious Salmon Anaemia virus (ISAV) and heart and skeletal muscle inflammation virus.
  • said parasitic source is selected from the group consisting of Lepeophtheirus sp., C aligns sp., and Ichthyophthirius sp.
  • said fungal source is selected from the group consisting of Saprolegnia sp., Branchiomyces sanguinis, Branchiomyces demigrans and Icthyophonus hoferi.
  • said vaccine is formulated for administration by a route selected from the group consisting of bath, immersion, intraperitoneal injection, intramuscular injection and oral administration.
  • the use of the Piscirickettsia salmonis strain according to the above description, or a subunit or fragment thereof, or the use of the vaccine according to the above description is provided (for the manufacture of a medicament) in the treatment of an animal subject
  • a method of treating an animal subject (i) being diagnosed for, (ii) suffering from or (iii) being at risk of developing, an infectious condition comprises administration of the Piscirickettsia salmonis strain according to the above description, or a subunit or fragment thereof, or the vaccine according to the above description, in a sufficient dose
  • an animal subject that has been vaccinated with the Piscirickettsia salmonis strain according to the above description, or a subunit or fragment thereof, or with the vaccine according to the above description, or with a method according to the above description.
  • the animal subject is a fin fish, preferably a Telostei, more preferably a Salmonid, most preferably a Salmon.
  • the Teleostei further include, but are not limited to, basses, tuna, bonito, breams, cods, snappers, flatfish, catfish, yellowtails and tilapias.
  • the fish is a pre-smolt or smolt.
  • Smolt is a young salmon at the stage when it migrates from fresh water to the sea.
  • young salmons are transferred from freshwater basis to outdoor cages at the smolt stage.
  • vaccination takes place before the transfer.
  • a method of growing or cultivating a bacterium of the Piscirickettsia genus for these reasons, according to another aspect of the invention, a method of growing or cultivating a bacterium of the Piscirickettsia genus
  • a growth medium or cultivation medium which comprises, inter alia, Eugon Broth.
  • a growth medium or cultivation medium for the cultivation or growth of a bacterium of the Piscirickettsia genus is provided.
  • the term “in the absence of cells of nonbacterial origin” comprise absence of eukaryotic cells, in particular vertebrate cells or mammalian cells. Such cells that would otherwise be suitable as host cells for a bacterium belonging to the Piscirickettsia genus.
  • a method and growth medium is provided wherein a bacterium belonging to the Piscirickettsia genus is, or can be, cultured without the use of host cells.
  • substantially extracellular environment refers to a culture of bacteria, wherein at least 10%, such as at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98, 99, 99.5 or 100% of the bacteria propagate freely in the medium.
  • the terms "substantially extracellular environment” and "absence of cells of non-bacterial origin” define a culture of bacteria, wherein bacteria are cultured to a TCID50 titre of at least 1 x 10 4 bacteria/ml medium, or at least 5 x 10 4 , at least 1 x 10 5 , at least 5 x 10 5 , at least 1 x 10 6 , at least 5 x 10 6 , at least 1 x 10 7 , at least 5 x 10 7 , at least 1 x 10 8 , at least 5 x 10 8 , at least 1 x 10 9 , at least 2 x 10 9 , at least 3 x 10 9 , at least 4 x 10 9 , at least 5 x 10 9 , at least 6 x 10 9 , at least 7 x 10 9 , at least 8 x 10 9 , at least 9 x 10 9 , at least 1 x 10 10 , at least 2 x 10 10 , at least 3 x 10 9 , at least 4 x 10 9
  • the culture may be established by inoculating a culture with a volume of a stock solution corresponding preferably to 0.25 to 4%, more preferably 0.5 to 2%, most preferably 1% of the of the final volume of medium in said culture and having an optical density of between 2.5 and 3.5, such as an optical density of 2.8 or 2.9.
  • the bacteria may be cultured in ventilated spinner flasks, preferably at 30-200 RPM and a temperature of 18.5 to 19.5°C for a period of from 1-10 days, such as from 1 to 8 days, such as from 2 to 5 days, such as from 2 to 4 days, such as from 2 to 3 days.
  • Piscirickettsia salmonis can be grown in shaker flasks or in static cultures.
  • Eugon Broth is a medium used for the cultivation of microorganisms
  • the formula name, Eugon Broth was used to describe the eugonic growth of fastidious microorganisms in such medium. According to different embodiments of the method or growth medium according to the above description
  • the w/w ratio of Eugon Broth : yeast extract is in the range of ⁇ 4 and > 2, and/or
  • Eugon Broth has the following composition:
  • the growth medium has the following composition:
  • the medium is essentially free of at least one of a) mammalian serum, and/or b) mammalian blood or blood extract.
  • mammalian serum is fetal bovine serum (FBS).
  • FBS fetal bovine serum
  • mammalian blood or blood extract is sheep blood as e.g. used in a cell free cultivation method for Piscirickettsia salmonis as disclosed in W02008002152.
  • BM4* or BMe a growth medium according to the invention which is suitable for cell free cultivation of Pisciricketsia salmonis, and comprises inter alia Eugon Broth, yet is devoid of mammalian serum, mammalian blood or blood extract, has shown advantageous in terms of performance and costs.
  • a method of determining the stability of mutations in the chromosome of Pisciricketsia salmonis comprises High- Resolution Melting (HRM) analysis.
  • HRM High- Resolution Melting
  • HRM analysis is a powerful technique in molecular biology for the detection of mutations, polymorphisms and epigenetic differences in double-stranded DNA samples.
  • HRM analysis is performed on double stranded DNA samples.
  • PCR polymerase chain reaction
  • the HRM analysis begins. The process is simply a precise warming of the amplicon DNA from around 50 °C up to around 95 °C. At some point during this process, the melting temperature of the amplicon is reached and the two strands of DNA separate or "melt" apart.
  • HRM Details of HRM are e.g. disclosed in Reed et al (2007), the content of which is incorporated herein by reference for enablement purposes.
  • This approach allows, inter alia to identify and determine the stability of 2 specific mutations in the chromosome of P. salmonis, namely, a) locus 440 - marC gene - so as to differentiate Pl 02 from other P. salmonis strains; and b) locus 477 - methyltransferase -so as to differentiate P50c7 and Pl 02 strains from other P. salmonis strains.
  • At least one primer is used which comprises a nucleic acid sequence selected from the group consisting of any of SEQ ID NOs: 40 - 43.
  • Piscirickettsia salmonis Pl 02 derives from a virulent P. salmonis isolate PM- 15972 (also known as PM15972A1). This isolate was recovered from a diseased specimen of Atlantic salmon, Salmo salar in 2010, Puerto Montt, Chile. Infected kidney was streaked onto a PSA plate and incubated at 18 °C. Bacterial colonies obtained after 14 days of incubation were isolated and seeded onto new PSA plates and incubated for seven days at 18 °C. The colonies were confirmed as Piscirickettsia salmonis using a PCR method (modified by ADL, based on Kuzyk et. al. 2001) and stored at -80 °C 2010 using PSB + 20% DMSO.
  • PSB is a broth medium designed for primary isolation of P. salmonis, its recipe is fully described in Henriquez et al, 2016. PSA is a solid derivative of PSB + 1.5% agar.
  • TCID50 assay using the CHSE-214 cell model
  • FBS fetal bovine serum
  • LDH activity levels were analyzed in 50 pL aliquots of cell-free supernatant obtained from each well. Additionally, the supernatant of cells lysed with L-15 medium containing 1% Triton X-100 was used as a total lysis control (maximum LDH liberation) and the supernatant of uninfected cells was used as a negative control (basal LDH liberation). Optical densities measured at 490 nm and 680 provided the basis for calculations as recommended by the manufacturer.
  • CHSE-214 cells were cultured in 96-well flat-bottom microplates in antibiotic-free L-15 Medium supplemented with 10% FBS and incubated at 20 °C until they reached confluence ( ⁇ 5.0 x 10 4 cells/well).
  • bacterial suspensions ⁇ 5.0 x 10 7 cfu/mL were prepared in L-15 supplemented with 2% FBS, and log 10 dilutions were made up to the 10th dilution.
  • TCID50 were calculated according to the Spearman-Karber algorithm (Hi erholzer and Killington, 1996).
  • BSS buffered saline solution
  • OD optical density
  • the antifoam selected for was “Antifoam 204” (Sigma Aldrich), an aqueous emulsion for bacterial and mammalian systems.
  • the supplier recommends the use of AF204 in a concentration range between 0.01% and 0.005%; however, there was no evidence in the literature on the tolerance of P. salmonis to this range of antifoams. For this reason, a MIC for AF204 was performed in a 96-well microplate as described by Henriquez et al, 2016, withsome slight modifications. The range to be evaluated was established between 0.32% toO.000625% using BMe medium instead of ADL-PSB. The microplate was incubated at 18 ⁇ 0.1 °C for 3 days and the absorbance at 580 nm for each well was measured using themicroplate reader EPOCH2. The lowest concentration where bacterial growth was notdetected corresponded to the MIC value.
  • the activation and growth of the bacteria was carried out exactly as described in the previous paragraph, using BMe and BMe supp. with AF204.
  • Scaling was performed in a 2 L bioreactor (Biostat - A, Sartorius), diluting the bacteria 100-fold in 2 L of BMe medium supp. with AF204.
  • the fermentation parameters correspond to 18 ⁇ 1 °C, aeration rate at 2000 ccm and stirring at 200 rpm, and were maintained for 48 - 52 h.
  • RNA Purification and Gene Expression Analysis are performed by a mixed strategy using ultra flow filtration (dialysis filters + peristaltic pump) and centrifugation at 2,600 x g for 1 h. During the fermentation process, aliquots were taken to measure the OD600 and determine the CFU/mL. The same criteria apply to the stage of harvesting, bulk formulation and packaging of the final product.
  • RNA extraction and cDNA synthesis were performed as described by Mancilla et al. (2018).
  • RNA was purified using the EZNA Total RNA Kit I (Omega Bio-Tek, Norcross, GA, USA). After DNAse I digestion, purified RNA was stored at -70°C. Reverse transcription was performed using the M-MLV RT Kit (Promega, USA) and synthesized cDNA was stored at -20°C.
  • Gene expression was analyzed by qPCR using the Maxima SYBR® Green qPCR Master Mix (Thermo Scientific) in a StepOne PCR machine (Applied Biosystems, Waltham, MA, USA), whereby each reaction was performed using 250 nM of primers (see sequence listing below) and 1.0 pL of 1 :2 diluted cDNA as a template.
  • the thermal profile included an initial step of 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C.
  • the fold change of gene expression was calculated according to the 2-AACt method (Pfaffl, 2001), using the expression of the elongation factor la (efl la) as a normalizer and the expression of all markers under control conditions as a calibrator. In total, 17 immune response genes were evaluated, which are described in Table 2. All primers used in this study are listed in the sequence listing below. PCR efficiencies were determined by linear regression analysis of sample data using LinReg- PCR.
  • H12 Cytokine Promotes differentiation of immune cells into Thl-type cells and IFNy secretion ifny Cytokine Increase respiratory burst / phagocytosis by macrophages. T cells proliferation and maturation. illO Cytokine Th2 marker, inhibits synthesis of cytokines, immunosuppressive function.
  • MHCI Cell receptor mediated cd8a T cell surface T-cell recognition and activation
  • MHCI presented Ag recognition.
  • response receptor cd86 T cell surface T-cell recognition and activation MHCI presented Ag recognition, receptor tgf6 Cytokine Regulatory Thl/Th2 cell proliferation, promoting Treg cell generation.
  • granzyme Serine Enzyme that enters the target cells and cleave to host proteins to induce protease apoptosis.
  • the stability was evaluated in a product prototype with a frozen format (2 batches: C.B1 19022021 and C.B1 15032021), and the storage temperatures selected were -20 ⁇ 1 °C, -70 ⁇ 1 °C and -80 ⁇ 1 °C.
  • a P. salmonis P102 cryovial was seeded on 2 PSA plates and incubated at 18 °C for 3 days.
  • the strain was expanded in BMe plates and incubated at 18 °C for 4 days, until an homogeneous bacterial lawn was observed.
  • the biomass was collected, washed twice, and cryopreserving solutions were added.
  • 72 amber serum vials were aliquoted (filled with 2 mL each), and 24 units were stored per condition. The samplings were carried out at the following times: day 0, 3, 7, 14, 21, month 1, 3, 6, 9, 12, 15 (so far), and at each time bacterial count is performed in 2 vials/batch using PSA plates.
  • Atlantic salmon smolts were confirmed as “pathogen free” using RT-PCR routine diagnostic methods for IPNV, IS AV, PRV, P. salmonis and Renibacterium salmoninarum.
  • the animals were acclimated to seawater for 14 days and fed ad libitum. During the study, they were maintained at 12 ⁇ 1°C, with a continuous exchange of seawater flow rate of ⁇ 4.0 L/min, a photoperiod of 24 light hours, and a specific feeding rate of 1.5 - 2%.
  • Prior to any handling and marking fish were anesthetized with benzocaine. Finally, euthanasia was performed using an overdose of anesthetic. All efforts were made to provide best growth conditions and minimize suffering.
  • the in vivo challenge was carried out on 240 fish with an approximate weight of 30 - 40 g. These fish were allocated to four 0.35 m3 tanks, yielding initial densities of 30 kg/m.
  • the fish were distributed as follows: tank 1, 80 fish for P50 and P102 strains (low dose, IX); tank 2, 80 fish for P50c7 and P102 strains (high dose, lOOx); tank 3, 40 fish for PM15972A1 strain (positive control, 100X dose) and tank 4, 40 fish inoculates with BSS (negative control).
  • Each fish was injected intraperitoneally with a dose of 0.1 ml with its respective treatment and dose.
  • the inoculation dose of the treatments that included bacterial suspensions was confirmed by bacteriological counts on PSA plates.
  • liver and head kidney samples were taken from 5 fish/group, to perform histopathology analysis, gene expression analysis (ifny, H8, illO, H12, cd8a and mhc-II immune gene markers), and bacteriological isolation studies by swabbing samples from each tissue on ADL-PSA plates. The plates were incubated for up to 30 days, until colonies of P. salmonis are observed. The colonies obtained were subjected to PCR-HRM studies.
  • the High-Resolution Melting (HRM) analysis a post-PCR analysis method, was used to identify and determine the stability of 2 specific mutations in the chromosome: locus 440 - marC gene - which allows Pl 02 to be differentiated from the rest of the strains; and locus 477 - methyltransferase - which allows differentiation of the P50c7 and Pl 02 strains from the rest of the P. salmonis strains.
  • DNA extraction was performed with the GeneJet DNA purification extraction kit (ThermoFisher).
  • the material was adjusted to 5 ng/pl, and the PCR-HRM was performed with the Melt Doctor HRM kit (Applied Biosystems) in a real-time thermal cycler (Step One, Applied Biosystems) using the following primer sets: 361_H-L440_Forward (5’- GCCTTTATTGCAGCGCTTAC-3’), 362_H-L440_Reverse(5’-
  • the PCR program included 1 cycle of an initial denaturation at 95 °C for 10 min, 40 cycles of denaturation at 95 ° C for 15 sec and annealing/extension at 60 °C for 1 min, and a continuous melt curve that has a setting of 15 sec at 95 ° C, 1 min at 60 °C and 15 sec at 95 °C.
  • strains of different origin were incorporated as reference for dissociation curve analysis.
  • the virulence of strain Pl 02 was again evaluated in an in vivo trial, however, this time larger fish were used.
  • the in vivo challenge was carried out on 270 fish with an approximate weight of 90 - 100 g. During the study, they were maintained at 14 ⁇ 1 °C, with a continuous exchange of seawater flow rate of ⁇ 1.2 - 1.5/h, a photoperiod of 24 light hours, salinity at 30 - 33 ppt and a specific feeding rate of 1.5 - 2%.
  • Fish were uniformly allocated to three 0.5 m3 tanks, yielding initial densities of 25 - 30 kg/m 3 and 90 fish/tank. 30 fish of each tank comprised a single treatment group that received 0.1 mL of bacterial suspension (either P.
  • Fresh bacterial suspensions (lOOx dose) were used to prepare the inocula to be administered to the fish via intraperitoneal injection. In parallel, a sample of each inoculum was used to perform bacterial counts at the time of inj ection. Fish were monitored daily for 30 days, and mortalities were removed from the tanks. As for tanks 1 and 2, mortalities were recorded and subjected to necropsy. Additionally, PCR of internal organ samples (head kidney /liver pool) were done to assess the presence of P. salmonis. The third tank was intervened on days 5, 12 and 20 post infection, removing three live fish/group to collect head kidney tissue samples for gene expression analysis.
  • the innate immune response markers evaluated were the following: In the case of innate immune response markers, ifny, H8, ill fl, illO, H12 and tnfa, were evaluated, (tnfa is a cytokine that participates in early infection response and has a key role in regulating inflammation). In the case of the adaptive immune response, cd8a and mhc-H, markers of cellular and humoral immune response, respectively, were selected.
  • Atlantic salmon smolts were confirmed as “pathogen free” using RT-PCR routine diagnostic methods for IPNV, IS AV, PRV, P. salmonis and R. salmoninarum.
  • fish were tagged using Passive Integrated Transponder tags (PIT tags).
  • PIT tags Passive Integrated Transponder tags
  • the animals were acclimatizing to freshwater for 8 days and fed ad libitum.
  • fish were maintained at 12 ⁇ 1 °C in fresh water, with a water change rate of 1.2 - 1.5/hour, a photoperiod of 8L-16D and a specific feeding rate of 1.5 - 2%.
  • Prior to any handling and marking fish were anesthetized with Isoeugenol. Euthanasia was performed using an overdose of anesthetic. All efforts were made to provide best growth conditions and minimize suffering.
  • the in vivo study was carried out on 100 fish with an approximate weight of 30 - 40 g.
  • the experimental design has only two tanks.
  • Tank 1 is only a fish reservoir and tank 2 is where the fish from tank 1 are received.
  • twenty fish (group 1) were injected intraperitoneally (IP) with 0.1 mL of P. salmonis strain Pl 02 at a lx dose and were transferred to tank 2.
  • IP intraperitoneally
  • Pl 02 0.1 mL of P. salmonis strain
  • After 3 days, 5 fish from group 1 were sacrificed, and their liver, head-kidney and spleen were removed.
  • Each tissue was divided into 2 fractions: 1 minor to perform SRS persistence studies by qPCR and bacteriological isolation on PSA plates, and a major fraction to prepare the "injectable fraction", a suspension that should contain live animalized bacteria.
  • the 3 organs were carefully macerated under sterile conditions, and the mixture was 1 centrifuged at 1,200 x g for 5 min. The supernatant (injectable fraction) was recovered and used to inject a new group of 20 fish from tank 1 at a dose of 0.1 ml/fish. This process was repeated on days 7 and 11, until a total of 4 groups of fish were obtained. The conformation of each new group was made from samples of fish from the previous group, for example, to form group 3, five fish from group 2 were sacrificed. Each group were differentiated by pit tag and by adipose and/or caudal fin cut. Monitoring was extended until day 25.
  • IP Intraperitoneal
  • Atlantic salmon pre-smolt were confirmed as “pathogen free” using RT-PCR routine diagnostic methods for IPNV, IS AV, PRV, P. salmonis and R. salmoninarum.
  • fish were tagged using Passive Integrated Transponder tags (PIT tags). The animals were acclimated to freshwater for 21 days and fed ad libitum.
  • PIT tags Passive Integrated Transponder tags
  • the animals were acclimated to freshwater for 21 days and fed ad libitum.
  • fish were maintained at 12 ⁇ 1 °C, salinity at 3-33 ppt and with a photoperiod of 6 weeks winter (12L-12D) plus 4 weeks summer (24L).
  • challenge stage fish were maintained at 14 ⁇ 1 °C, salinity at 33 ppt and a photoperiod of 24L.
  • the water change rate was of 1.2 - 1.5/hour and the specific feeding rate was of 1.5 - 2%.
  • fish Prior to any handling and marking, fish were anesthetized with benzocaine. Euthanasia was performed using an overdose of anesthetic. All efforts were made to provide best growth conditions and minimize suffering.
  • the in vivo study was carried out on 280 fish with an approximate weight of 35 - 45 g. Fish were uniformly allocated to two 1 m 3 tanks, yielding initial densities of 10 - 15 kg/m 3 and 140 fish/tank. 35 fish of each tank comprised a single treatment group that received 0.1 mL of bacterial suspension (either fresh P. salmonis Pl 02 or freeze dried Pl 02 strain, dose lx) or vehicle (BSS). The inocula were administered to the fish via intraperitoneal injection. Fish were monitored daily for 50 days (630 DD), and mortalities were removed from the tanks. One day before the challenge, samples of 3 fish per group/tank were taken to perform serological studies (ELISA IgM), histopathology analysis (kidney, liver and spleen) and gene expression (head kidney and liver).
  • ELISA IgM histopathology analysis
  • gene expression head kidney and liver.
  • Atlantic salmon pre-smolt were confirmed as “pathogen free” using the same RT-PCR routine diagnostic methods described.
  • Fish were tagged using Passive Integrated Transponder tags (PIT tags). The animals were acclimatizing to freshwater for 21 days and fed ad libitum.
  • PIT tags Passive Integrated Transponder tags
  • the animals were acclimatizing to freshwater for 21 days and fed ad libitum.
  • fish were maintained at 12 ⁇ 1°C, salinity at 3-32 ppt and with a photoperiod of at least 2 weeks winter (6L-18D) plus 4 weeks summer (24L).
  • challenge stage fish were maintained at 15 ⁇ 1°C, salinity at 32 ppt and a photoperiod of24L.
  • Tanks were supplied with seawater pumped directly from the shoreline at a continuous seawater exchange rate of ⁇ 4.0 L/min, equivalent to a water renewal rate of 0.8 - 1 time per hour.
  • seawater exchange rate ⁇ 4.0 L/min, equivalent to a water renewal rate of 0.8 - 1 time per hour.
  • fish Prior to any handling and marking, fish were anesthetized with Isoeugenol. Finally, euthanasia was performed using an overdose of anesthetic. All efforts were made to provide best growth conditions and minimize suffering.
  • the immunization was carried out on 160 fish with an approximate weight of 30 - 40 g.
  • Fish were uniformly allocated to four 0.35 m 3 tanks.
  • 40 fish of each tank comprised a single treatment group that received 0.1 mL of bacterial suspension (P. salmonis P102, dose lx, duplicate) or BSS vehicle (for negative and positive control groups), at an approximate density of 30 kg/m 3 .
  • the inoculum was administered to the fish via intraperitoneal injection. Fish were monitored daily for 36 days (473 DD), and mortalities were removed from the tanks. A natural temperature was maintained during this stage and gradually increased during the challenge to 15.0 ⁇ 1 °C.
  • Atlantic salmon pre-smolt were confirmed as “pathogen free” using the same RT-PCR routine diagnostic methods for the following pathogens: P. salmonis, R. salmoninarum, F. psychrophylum, PRV and IPNV. All fish in the study have never been treated with antibiotics and were clinically healthy. Animals were acclimatizing to freshwater for 14 days and fed ad libitum. During the immunization stage, fish were maintained at 12 ⁇ 1°C, salinity between 0- 10% and flow at 1.0 - 1.6 tanks turnover/h. During challenge stage, fish were maintained at 15 ⁇ 1°C, salinity at 29-35% and flow at 1.0 - 1.2 tanks turnover/h.
  • the photoperiod light:dark was of 16:8 h for 2 ’A weeks post vaccination and 24: Oh during smoltification and challenge periods.
  • fish Prior to any handling and marking, fish were anesthetized with benzocaine. Euthanasia was performed using an overdose of anesthetic.
  • the in vivo study was carried out on 360 fish with an approximate weight of 35 - 45 g. All fish within each tank/group were marked using VIE-tag one week before vaccination. Fish were uniformly allocated to two 1 m 3 tanks, yielding initial densities of 10 kg/m 3 and 180 fish/tank at vaccination day. Each tank contained 60 fish belonging to one of two experimental/vaccination groups (Pl 02 strain or commercial inactivated vaccine) and one control group (BSS). The vaccines were administered to the fish via intraperitoneal injection. After fulfilling the smoltification process, all fish were acclimated to seawater. Fish were monitored daily for 55 days (650 DD), and mortalities were removed from the tanks. After immunization period, 10 fish were sampled for blood (for serological studies by ELISA) and weight was registered. One week before challenge, fish were acclimated to 15 °C water.
  • the genome sequence of P. salmonis PM15972A1 strain is annotated in the GenBank NCBI database under the accession N° CP012413. From a genetic point of view, the strain has a chromosome and 4 plasmids (Table 3).
  • Type Name INSDC Size (pb) % CG Protei rRNA tRN Other Gene Pseudo n A RNA gene Chr - CPO 1241.3,1 3.062,470 39.8 2931 18 56 4 3.139 130
  • a PSA plate was seeded with a cryopreserved vial diluted to isolate individual colonies. Eight individual clones were selected and screened for plasmid detection by qPCR, using specific primers designed by ADL (unpublished data), and one of the 8 identified colonies (P50c7), which did not carry a 32 kb plasmid pPSAl-4, was selected and stored at -80 °
  • the colony was sequenced by using two NGS technologies (Illumina HiSeq and PacBio), and its genome was annotated (data not available in public databases) and compared with the genome of the parent strain (PM15972A1).
  • the results showed the detection of some SNPs in the chromosome, together with the deletion of a fragment of approx. 18.9 kb, named “RD1”.
  • This deleted region which encodes several metabolic and putative virulence genes, can be detected by conventional PCR targeting the scar left by the recombination of flanking ends, which is shown in Figure 6.
  • a 1,300 bp amplicon appears when chromosomes are lacking the 18.9 kb region.
  • An increased signal intensity of the band can be interpreted as the sample enrichment by deletion-carrying chromosomes over the passages.
  • infection assays were performed on CHSE-214 ceils to calculate TCID50 with the method of Spearman & Karber. After 14 days of incubation at 18 °C, a microscopic observation allows to determine the number of wells with cytopathic effect in the monolayer (positive), which is then used to calculate the TCED50 or titer.
  • the Pl 02 strain produced a lower infectivity compared to the P101 clone, P50 and the PM15972A1 strains.
  • the TCID50 values do not seem to be different (Table 6)
  • the microscopic observation of cells infected at the same multiplicity of infection (MOI) for a defined time allows to clearly observing a limited monolayer destruction caused by the Pl 02 strain in comparison with a wild-type P. salmonis strain (Figure 7).
  • KW89 55 transporter a SNARE- comprising 20 ORFs associated membrane protein, a peptidase and several transposases
  • P50 is a derivative of Al-15972 (also known as PM15972 or PM15972A1), an EM-90-like strain.
  • P101 and Pl 02 are both derivatives of P50c7. + or - indicate the occurrence of the corresponding mutation. Those mutations detected in P50c7, and derivatives were confirmed by Sanger sequencing.
  • LDH lactate dehydrogenase
  • PSB* formula without FBS
  • the strain was not able to grow in MD2 and SF900-III media, so it was decided to adapt the strain prior to kinetic tests using T25 cell culture bottles and static growth conditions.
  • the strain Pl 02 was able to grow in SF900-III medium, reaching an ODeoo of 1.56; however, adaptation to MD2 medium took more than two weeks, reaching only an ODeoo of 0.12.
  • the strain adapted to MD2 was transferred into a new flask, but again did not reach optimal growth (data not shown).
  • the SF900-III medium considered as the reference medium for the fermentation process, was also discarded due to the poor growth performance compared to other fetal bovine serum-free media, such as PSB*.
  • BM4* also called BMe
  • PSB* medium were those that provided the best results.
  • BMe offers commercial advantages in terms of costs .
  • the suitability of BMe medium for the growth of Pl 02 strain was supported by other flask growth kinetics (data not shown).
  • Medium composition is found in Table 8.
  • the MIC result for the antifoam AF204 was 0.005%, which indicates that the P. salmonis P102 strain is not capable of tolerating the range recommended by the supplier (data not shown). This test was repeated in 125 mL flasks, obtaining the same result: P. salmonis grows up to 0.0025% of AF204. Due to the above, it was determined that the concentration of AF204 that can be used for supplementing the BMe medium for a fermentation stage should be lower than the manufacturer instruction.
  • the in vitro gene expression study was performed using 17 immune response gene markers, including genes representative of an innate immune response and an adaptive immune response (humoral and cellular).
  • the relative expression results of each marker in SHK-1 cells infected at MOI 10 for 5 and 10 days post infection are described in Figure 13, 14 and 15.
  • the first line of defense against pathogen invasion is the innate immune system, a system in which macrophages play an essential role in triggering immune responses. Macrophages primarily act as antigen-presenting cells, but these cells are also responsible for most phagocytic activity, in addition to regulating the immune system cascade triggered by the secretion of proinflammatory cytokines.
  • proinflammatory cytokines include interleukin (IL)- 12 and TFNy, which are key components for the efficient performance of phagocytes in teleost fish. In the case of P.
  • cryoprotectant formula that provide more stability correspond to B and C, with a decrease in the bacterial titer of less than one logarithm on average after freezing process at -80 °C.
  • Condition D does not offer as much stability as the two previous conditions, however, it is still superior to the control group ( Figure 16).
  • the in vivo assay was carried out infecting S. salar smolt with two different doses of P. salmonis strains via intraperitoneal injection: a lethal dose of lOOx according to information obtained in previous studies developed by ADL Diagnostics (data not shown) and a target dose of lx (immunization dose).
  • a cumulative mortality of 100% was obtained for the group of fish inoculated with the virulent strain PM15972, 40% for the fish vaccinated with the P50c7 strain lOOx dose and 2.5% for the fish vaccinated with Pl 02 strain at lOOx dose, demonstrating a significant reduction in virulence in vivo in this new candidate strain (Figure 19).
  • mhc-II shows an opposite tendency (overexpression) to the virulent strain (repression) (Rozas-serri et al., 2018).
  • the bacteriological analyses it is important to highlight that the bacterium was able to be isolated only from samples immunized with a lOOx dose or from one fish immunized with the P50c7 strain at a lx dose (Table 10).
  • Table 10 Colony isolation from immunized fish using different doses.
  • PCR-HRM studies were performed to determine the genetic stability of two molecular markers (SNPs found in marC and methyltransferase genes).
  • SNPs found in marC and methyltransferase genes.
  • 2 animalized colonies of each strain were analyzed per time (depending on availability and regardless of the isolation matrix, whether it was head kidney or liver).
  • the HRM analyzes showed that all the colonies maintained the two mutations, and that there was no reversion or alteration in these genes (data not shown).
  • the in vivo assay was carried out infecting S. salar smolt cohorts with similar amounts of P. salmonis strains via intraperitoneal injection. According to our experience with the challenge strain, the horizontal transmission of P. salmonis PM15972A1 is negligible in the period set for the experiment, and any mortality occurring within that time frame may be interpreted as an effect of the injected material.
  • the results of this study demonstrated that there were no mortalities recorded in the group of fish immunized with a lOOx dose of strain Pl 02. Meanwhile, the group of fish immunized with the parent strain registered the first mortalities on day 8 post immunization, which increased exponentially up to 86.7% 11 days post immunization. This percentage remained stable until the end of the trial (Figure 23). Necropsies conducted on dead fish revealed pathognomonic signs of piscirickettsiosis in internal organs, regardless of the challenge strain. This information was complemented with the results of specific qPCR for SRS (not shown).
  • our virulent strain has a gene expression profile as described in the literature, stimulating a strong inflammatory response in early stages through U8, ifny, tnfa and il 1 fl genes and generating an imbalance between the illO and H12 cytokines.
  • This immunomodulatory effect extends to the cellular immune response gene marker cd8a, which is strongly down-regulated 5 days after treatment. The inflammatory response decreases progressively. On day 20, we were unable to obtain surviving fish from this group, so it was not possible to carry out the analysis at that point.
  • the attenuated strain is also capable of inducing a proinflammatory response in the kidney, however, this effect was weak and seems to be delayed compared to what was observed in fish inoculated with a virulent strain.
  • the Pl 02 strain is also capable of negatively regulating cd8a gene marker 5 days post injection, however, this response turns negligible as the infection progresses.
  • the increase in the expression levels of mhc- II in the antigen-presenting cells allows us to infer that there is an activation of the CD4+ T cells, and therefore an activation of the adaptive humoral immune response.
  • the inflammatory response is minimal, and it seems that the slight imbalance observed in previous days is completely reversed. In general terms, we can conclude that strain Pl 02 does not trigger an inflammatory response or interfere immune response.
  • ELF1 elongation factor 1, housekeeping gene
  • the attenuated antigen (P. salmonis Pl 02 strain) was prepared on the day of vaccination in the laboratory and kept refrigerated until use. Regarding the lyophilized format, a vial of the lyophilized strain was also reconstituted in a saline solution prior to vaccination, diluting the content of the cake in a previously calculated volume. In both cases, a quality control (bacterial viability and infective capacity (TCID50/ml)) was carried out. Both suspensions maintained similar values in terms of CFU/ml (lx dose) and infective capacity in cell culture.
  • hyperplasia and leukocytes could mean a compensatory response against a greater cellular demand following antigenic exposure (vaccines) or a systemic or focal infection, so that none of the observed findings represent a negative effect produced by the live attenuated antigen.
  • the control group fish injected with the vehicle during immunization, not challenged
  • the positive control group fish injected with BSS during immunization and then challenged
  • Mortalities in the control group began after 9 days post-challenge, reaching a cumulative mortality of approx. 60% at 11 dpc. Since the mortality record was quite similar even between both tanks, data were averaged and plotted on a single graph ( Figure 31).
  • the attenuated strain is able to yield cross-protection.
  • the strain is able to provide a protection of approx. 90% in vaccinated fish, however, if we calculate the RPSeo or RPS70 of the group of fish immunized, the protection conferred by the attenuated strain reached 100%.
  • VIE Visible Implant Elastomer
  • the necropsy revealed only some pathological findings (splenomegaly and pale focal nodes in liver) in those fish immunized with the commercial inactivated vaccine (competitor). Regarding the description of other relevant findings, presence of visceral melanosis and adhesions, and loss of appetite (lower content of feces in the intestine) were also observed. On the other hand, fish vaccinated with the Pl 02 strain do not show unwanted findings or side effects (Figure 34).
  • adhesions and melanosis presented in vaccinated fish are well-known side effects derived from oil adjuvant present in the vaccines (Meza et al., 2019).
  • the signal peptides may be encompassed in the reproduced sequences. In such case, the sequences shall be deemed disclosed with and without signal peptides.
  • a readily available tool to identify signal peptides in a given protein sequence is SignalP - 6.0 provided by Dansk Technical University under https://services.healthtech.dtu.dk/service.php7SignalP
  • the open reading frame (ORF) of each gene is shown according to the PM15972A1 genome annotation (SEQ ID NO: 39). Orientation is given by the arrangement of the start codons (typically, ATG or TTGin sense direction, or CAT in antisense direction, which results in ATG as the reverse complement) and stop codons (typically, TAG, TAA, TGA or TCT in sense direction, or TCA, CTA or TTA in antisense direction, which result in TGA, TAG or TAA as the reverse complement.
  • start codons typically, ATG or TTGin sense direction, or CAT in antisense direction, which results in ATG as the reverse complement
  • stop codons typically, TAG, TAA, TGA or TCT in sense direction, or TCA, CTA or TTA in antisense direction, which result in TGA, TAG or TAA as the reverse complement.
  • the respective genes within the ORF are marked in underline. Neighboring genes can be distinguished by straight and wavy underline. The startcodons and stopcodons are marked in bold.

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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

La présente invention concerne des souches atténuées de Piscirickettsia salmonis et leur utilisation en tant que vaccin.
PCT/EP2023/067443 2022-06-27 2023-06-27 Vaccins contre la piscirickettsiose (septicémie rickettsienne des salmonidés) WO2024003035A1 (fr)

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WO2016082050A1 (fr) 2014-11-24 2016-06-02 Pontificia Universidad Católica Del Valparaíso Milieu de culture permettant la croissance de la bactérie piscirickettsia salmonis
WO2017137834A1 (fr) 2016-02-08 2017-08-17 University Of Oslo Milieux de culture bactérienne
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US10857218B2 (en) * 2015-05-26 2020-12-08 Pharmaq As Attenuated Piscirickettsia salmonis bacterium

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WO2008002152A2 (fr) 2006-06-29 2008-01-03 Pharmaq As Procédé de culture de bactéries du genre piscirickettsia
CA2656032A1 (fr) * 2006-06-29 2008-01-03 Pharmaq As Procede de culture de bacteries du genrepiscirickettsia
WO2016082050A1 (fr) 2014-11-24 2016-06-02 Pontificia Universidad Católica Del Valparaíso Milieu de culture permettant la croissance de la bactérie piscirickettsia salmonis
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WO2017137834A1 (fr) 2016-02-08 2017-08-17 University Of Oslo Milieux de culture bactérienne
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