Reference to sequence listing submitted as a compliant xml 1.0 format File (.xml)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (see MPEP § 2442.03(a)), Rule 30 EPC, and § 11 PatV, an electronic sequence listing compliant with WIPO standard ST.26 in the form of an XML 1.0 format file is submitted concurrently with the instant application, and the entire contents of the sequence listing are incorporated herein by reference. For the avoidance of doubt, if discrepancies exist between the sequences mentioned in the specification and the electronic sequence listing, the sequences in the specification shall be deemed to be the correct ones.

Field of the invention

The present invention relates to the field of vaccines for fish.

Background

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.

Although 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). In aquaculture, 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. However, 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).

In Chile, the most recent report on the “Use of antimicrobials in national salmon industry” (Sernapesca, 2021) indicates a significant increase in the use of antimicrobials, with a national “antibiotic consumption rate (ICA)” of 0.047% (470 grams of antimicrobial per ton harvested), 0.012 points higher than values reported between 2018 and 2020. Most of the treatments were carried out in sea farms (98.7% of the national total), mainly for the treatment of Piscirickettsiosis (93.2 % of the total in the sea).

Current strategies to control infection are i) indiscriminate antibiotic use and ii) vaccination. 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.

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.

Several approaches for obtaining attenuated bacteria are available to the skilled person. 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).

Currently 30 vaccines for SRS are registered for use in Chile (27 bacterin, 2 recombinant and one live attenuated bacterial vaccine), administered primarily in water-in-oil emulsions (Servicio Agricola y Ganadero, 2022); nevertheless, regardless of the species of salmon farmed, these vaccines have not been fully protective in reducing total mortality or in delaying the time to onset of the first SRS outbreak under field conditions in Chile (J. Happold, et al., 2020; Rozas-Serri M., 2022). Due to the above, the development of new effective products that contribute to the control of this disease is widely necessary and expected.

Summary of the invention

These and further objects are met with methods and means according to the independent claims of the present invention. The dependent claims are related to specific embodiments.

Brief Description of the Figures

Figure 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.

Figure 3. Gene list of plasmid pPSAl-4

Figure 4. Gene list of the RD1 locus on the chromosome of Piscirickettsia salmonis

Figure 5. TaqMan PCR for plasmid PS Al -4. In blue, 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 .

Figure 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 8. Cytotoxicity of the P102 strain vs. virulent strain (PM15972A1). Measured performed on the supernatant of SHK-1 cells infected at 0.1 MOI for 10 days with each strain.

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.

Figure 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 12. Bacterial counts (CFU/mL) of /< salmonis P102 strain during the growth curve using BMe and BMe supp with AF204. The CFU count 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).

Figure 16. Bacterial count values (CFU/mL) corrected by the dilution factor (groups B, C and D) of the solution. Conditions: A) Wet pellet without stabilizers (bacterial suspension); B) Wet pellet + stabilizers (formula 1); C) Wet pellet + stabilizers (formula 2) and D) Wet pellet + stabilizers (formula 3). daf, days after freezing.

Figure 17. Stability (CFU/mL) of P. salmonis Pl 02 strain with stabilizers cryopreserved (R&D batches C. Bl 19022021 and C.Bl 15032021, Chile).

Figure 18. 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.

Figure 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.

Figure 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.

Figure 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.

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

Figure 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.

Figure 31. Challenge experiment with P. salmonis LF-89 like genogroup. Relative percent survival (RPS) of S. salar vaccinated, post-ip challenge with P. salmonis. DPC, days post challenge. The immunization period was of 470 DD.

Figure 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.

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).

Figure 34. Additional findings during fish necropsy at the end of the immunization period.

The size of the bar is related to the associated frequency in the analyzed samples (10 fish per group/strain).

Figure 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 of the invention

Before the invention is described in detail, it is to be understood that this invention is not limited to the particular component parts or structural features of the devices or compositions described or process steps of the methods described as such devices and methods may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include singular and/or plural referents unless the context clearly dictates otherwise. Further, in the claims, the word “comprising” does not exclude other elements or steps.

It is moreover to be understood that, in case parameter ranges are given which are delimited by numeric values, the ranges are deemed to include these limitation values.

It is further to be understood that 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.

According to one aspect of the invention, a Pisciricketsia salmonis strain is provided 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. The inventors have surprisingly shown that a mutation in this locus, as described hereinbelow, 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.

Furthermore, 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.

In one embodiment, 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.

In one embodiment, 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.

According to one embodiment, the mutation in the RD1 locus is a deletion which has a size of

> 1 kB. In several embodiments the mutation in the RD1 locus is a deletion which has a size of

> 2 kB, > 3 kB, > 4 kB, > 5 kB, > 6 kB, > 7 kB, > 8 kB, > 9 kB, > 10 kB, > 11 kB, > 12 kB, > 13 kB, > 14 kB, > 15 kB, > 16 kB, > 17 kB, and /or > 18kB. According to one embodiment, the mutation in the RD 1 locus, preferably the deletion, is located between the ORFs KW89 30 and KW89 60.

According to one embodiment, 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.

According to one embodiment, the Piscirickettsia salmonis strain comprises, in its genome, at least one further mutation as set forth in Fig. 2

According one other aspect of the invention, a vaccine is provided 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.

According to one embodiment, said vaccine comprises bacteria or subunits or fragments of bacteria, in a concentration of between > 0.05 mg/ml and < 5 mg/ml.

According to another embodiment, said vaccine comprises bacteria in a concentration of between > 1.0 CFU/mL and < 9.9 x 10 ⁹ CFU/ml. As used herein, the term “CFU” relates to colony forming units.

According to one embodiment, 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. According to one embodiment, 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%.

This finding made by the inventors is extremely important, because it has been reported that commercial vaccines do not confer protection against two genetic strains of Piscirickettsia salmonis, LF-89-like and EM-90-like, in Salmon (Figueroa et al., Commercial vaccines do not confer protection against two genetic strains of Piscirickettsia salmonis, LF-89-like and EM- 90-like, in Atlantic salmon. bioRxiv 2021.01.07.424493)

According to one embodiment, said vaccine further comprises one or more adjuvants.

Examples of 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. In a currently preferred embodiment 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. (Immune 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.

In addition, 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. According to one embodiment, 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.

According to one embodiment, 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.

According to one embodiment, 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.

According to one embodiment, 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.

According to one embodiment, said parasitic source is selected from the group consisting of Lepeophtheirus sp., C aligns sp., and Ichthyophthirius sp.

According to one embodiment, said fungal source is selected from the group consisting of Saprolegnia sp., Branchiomyces sanguinis, Branchiomyces demigrans and Icthyophonus hoferi. According to one embodiment, said vaccine is formulated for administration by a route selected from the group consisting of bath, immersion, intraperitoneal injection, intramuscular injection and oral administration.

Suitable formulations are shown herein elsewhere.

According to another aspect of the invention, 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

• being diagnosed for,

• suffering from, or

• being at risk of developing an infectious condition, or for the prevention of such condition.

This language is deemed to encompass both the swiss type claim language accepted ins come countries (in this case, brackets are deemed absent) and EPC2000 language (in this case, brackets and content within the brackets is deemed absent).

According to another aspect of the invention, a method of treating an animal subject (i) being diagnosed for, (ii) suffering from or (iii) being at risk of developing, an infectious condition is provided, which method 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

According to another aspect of the invention, an animal subject is provided 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. According to embodiments of the invention, 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.

According to an embodiment of the invention, 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. Typically, in salmon farming, young salmons are transferred from freshwater basis to outdoor cages at the smolt stage. Typically, vaccination takes place before the transfer.

In early investigations, Piscirickettsia salmonis bacteria failed to grow on a number of artificial bacteriological media and the bacteria were thus considered to be obligate intracellular organisms. It was observed, however, that the bacteria are able to propagate on a number of cell lines of salmon and non-salmonid origin (Cvitanich et al. 1991, Fryer et al. 1990 and 1992). However, relying on culturing the bacteria on suitable cell lines imposes severe limitations for large-scale production, since large-scale culture of P. salmonis in eukaryotic cell lines is laborious and expensive, and as such not profitable for the fish industry.

For these reasons, according to another aspect of the invention, a method of growing or cultivating a bacterium of the Piscirickettsia genus

(i) in the absence of cells of nonbacterial origin, or

(ii) in a substantially extracellular environment, is provided, in which method a growth medium or cultivation medium is used which comprises, inter alia, Eugon Broth.

According to another aspect of the invention, a growth medium or cultivation medium for the cultivation or growth of a bacterium of the Piscirickettsia genus,

(i) in the absence of cells of nonbacterial origin, or

(ii) in a substantially extracellular environment is provided, which medium comprises, inter alia, Eugon Broth.

As used herein, 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. Thus, within the scope of the present invention, 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.

As used herein, the term “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.

In the present context 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 IO ¹⁰ , at least 4 x IO ¹⁰ , or such as at least 5 x IO ¹⁰ bacteria/ml medium in the presence of at the most 10 ⁵ cells of non-bacterial origin/ml culture medium, such as at the most 2 x 10 ⁵ , such as at the most 3 x 10 ⁵ , at the most 4 x 10 ⁵ , at the most 5 x 10 ⁵ , at the most, 6 x 10 ⁵ , at the most 7 x 10 ⁵ , at the most 8 x 10 ⁵ , at the most 9 x 10 ⁵ , or such as in the presence of at the most 1 x 10 ⁶ cells of non-bacterial origin/ml culture medium. 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. For this purpose 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. Alternatively, 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

• the w/w ratio of Eugon Broth : NaCl is in the range of < 2,5 and > 1,4, and/or

• the w/w ratio of Eugon Broth : MgSCU x 7H2O is in the range of <200 and > 50, , and/or

• the w/w ratio of Eugon Broth : Glutamic acid is in the range of < 10 and > 2, and/or

• the w/w ratio of Eugon Broth : L-cysteine is in the range of < 20 and > 50, and/or

• the w/w ratio of Eugon Broth : K2HPO4 is in the range of < 5 and > 1,5

According to one embodiment of the invention, Eugon Broth has the following composition:

Table 1A. Composition of Eugon Broth

Reagent concentration range embodiment (g/L)

(g/L)

Casein Peptone 5 - 10 g/L 7.5 g/L

Meat Peptone 5 - 10 /L 7.5 g/L

Soy Peptone 2.5 - 7.5 g/L 5.0 g/L

Dextrose 2.5 - 7.5 g/L 5.5 g/L

Sodium Chloride 2- 6 g/L 4.0 g/L

L-Cystine 0.5 - 1 g/L 0.7 g/L

Sodium Sulfite 0.05 - 4 g/L 0.2 g/L

According to one embodiment of the method or growth medium, the growth medium has the following composition:

Table IB. Composition of the BMe medium and a prior art medium

„ Concentration range embodiment (g/L)

^Reagent (g/L) ("BMe")

BM4 BMe

Yeast extract 2 - 6 4

Eugon Broth 8 - 15 12,16

NaCl 4 - 10 7,4

MgSO ₄ x 7 H 2O 0,02 - 0,5 0,1

Glutamic acid 0,5 - 4 2

L-cysteine 0,2 - 5 1

K2HPO4 3 - 8 5,09

KH2PO4 0,3 - 3 0,9352

According to further embodiments, the medium is essentially free of at least one of a) mammalian serum, and/or b) mammalian blood or blood extract.

One example for mammalian serum is fetal bovine serum (FBS). One example of 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.

These components are complex and therefore not standardized. Further, they bear the risk of contamination with e.g. prions or mycoplasms. Therefore, leaving them out increases the stability and reproducibility of the production process, and decreases the risk of culture failure due to contamination. Also, it decreases the risk of unwanted infections of the vaccinated fish.

As shown herein, e.g., in Figure 10, a growth medium according to the invention (called BM4* or BMe) 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.

According to another aspect of the invention, a method of determining the stability of mutations in the chromosome of Pisciricketsia salmonis is provided, which method comprises High- Resolution Melting (HRM) analysis.

High Resolution Melt (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. Typically the user will use polymerase chain reaction (PCR) prior to HRM analysis to amplify the DNA region in which their mutation of interest lies. In the sample tube there are now many copies of the DNA region of interest. This region that is amplified is known as the amplicon. After the PCR process 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.

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.

According to one embodiment of that method, 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.

Examples

While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

All amino acid sequences disclosed herein are shown from N-terminus to C-terminus; all nucleic acid sequences disclosed herein are shown 5'->3'.

Materials and methods

Attenuation of P. salmonis P102 strain

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.

Sequence analysis of the 16S gene was used to type the isolated bacteria as an EM90-like strain (see Saavedra et al, 2017 for a modified PCR). The genome sequence of Piscirickettsia salmonis PM15972A1 strain is annotated in the GenBank NCBI database under the accession N° CP012413.

In 2015, a cryopreserved vial of P. salmonis PM15972A1 strain was thawed, seeded on PSA plates and incubated at 18 °C for 9 days until a confluent bacterial growth was observed. For attenuation, on a weekly basis, a loop of confluent growth was re-streaked onto a new PSA plate and incubated at 18 °C up to passage n° 50. At this point, a clone of this passage was selected (P50c7) and genetic studies and in vivo efficacy and safety trials on S. salar were performed to assess the attenuation status of the strain. Due to this isolate did not have the necessary characteristics to be considered as a safety and potential antigen for a live attenuated vaccine, the material from P50c7 was expanded and stored, but also was streaked and used to continue the attenuation until passage n° 100. A sample of the biological material was taken and stored every 10 passages.

In June 2018, one vial was thawed, diluted and cultured to obtain individual clones, which were cryopreserved at -80 °C for further analysis. Colonies belonging to the passage n° 100 were used for in vitro virulence evaluation. After confirming the attenuation of its virulence, the whole genome sequence of these colonies was obtained, and the randomly introduced mutations/deletions were identified and registered. The selected clone, named Pl 02 (clone two from 100th passage) was characterized and used for an in vivo assay. The attenuation of the virulence was also confirmed in vivo.

In vitro virulence attenuation

To determine in vitro virulence, both TCID50 assay (using the CHSE-214 cell model) and a LDH release assay were performed. For LDH release test, SHK-1 cells were cultured without antibiotics in Leibovitz’s L-15 medium supplemented with 10% fetal bovine serum (FBS) at 20 °C in 75-cm2 flasks. Cells were seeded into 24-well plates at a concentration of ~1.0 x 105 cell/well, incubated for 24 h at 20°C and then infected with the selected P. salmonis strain at a multiplicity of infection (MOI) = 0.1. The plates were incubated at 18 °C for 10 days. Cell damage induced by P. salmonis in SHK-1 cell monolayers was determined by the release of the cytosolic enzyme lactate dehydrogenase (LDH) into the medium using a commercial Cytotoxicity LDH Detection Kit (Thermo Scientific). 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.

The in vitro infectivity of P. salmonis was assessed using an endpoint dilution assay (TCID50) as described early, with few modifications (House et al., 1999). 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). As inoculum, 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. Monolayers were infected (8 wells/dilution), incubated for 15 days at 18°C, and then visually inspected for the presence of cytopathic effects. TCID50 were calculated according to the Spearman-Karber algorithm (Hi erholzer and Killington, 1996).

To determine in vitro gene expression, SHK-1 cells were cultured as previously described and then were infected with P. salmonis parent strain or Pl 02 strain at a multiplicity of infection (MOI) = 10. Purified nucleic acid samples were collected from the cell culture 5- and 10-days post infection (dpi).

Genetic characterization

Sequencing of P. salmonis Pl 02 strain was performed at Macrogen, Inc. (Seoul, South Korea) using the Pacific Biosciences (PacBio) SMRT cell 8Pac version 3 and DNA polymerase binding kit P6 version 2 for library preparation. An additional Nextera library was prepared to be run on an Illumina HiSeq 2000 platform. Mapped reads were de novo assembled using SMRT Analysis version 2.3.0.1 (PacBio), and the sequences were checked for errors by mapping Illumina paired-end reads over the genome built with PacBio reads. The assembled data were annotated using Blast2go software. The annotated genome was compared vs. that of the parent strain (PM15972A1, accession n° CP012413) and the P. salmonis ADL-PSA1 strain (p50c7) using Mauve software to look for long polymorphic regions. The primary analysis was combined with variant sequence analysis with the aim of identifying single nucleotide polymorphism or SNP across sequences (CLC Workbench software version 6.5).

Culture medium optimization

Growth media without fetal bovine serum (FBS)

The media selected to evaluate the growth of P. salmonis Pl 02 were taken from the literature. These media were BM4 (Henriquez, 2013), BMJ (WO2017137834A1), MD2 (W02016082050A1), PSB (without FBS) and SF900-III as references. Frozen bacteria were inoculated on PSA plates and incubated for 4 days at 18 °C. Bacterial cells were washed with buffered saline solution (BSS), and adjusted to an optical density (OD) = 1.0 at 600 nm in the same solution. The bacterial suspension was diluted 100 times in 50 ml of each medium until reaching an OD600 of 0.01 (~ 1-3 x 10 ⁵ colony-forming units (CFU) per mL). Growth test was performed in a 125 mL flask at 200 rpm, measuring OD600 at the end of the culture, after 120 hours.

In a second experiment, and based on the previous results, it was decided to perform growth kinetics to evaluate the performance of BM4, BMJ and PSB media. At the same time, an improvement in the media was tested, changing the peptone of the formulation of each of the media by a different protein source. The evaluation of growth kinetics was carried out in a microplate reader EPOCH2 (BioTek Instruments, Inc). The reactivation of the strain as well as the preparation of the bacterial suspension was carried out as described in the previous paragraph. Wells of a flat-bottom 96-well microtiter plate were inoculated with 150 pL (10 replicates/strain). The microplate was incubated at 18 ± 0.1 °C for 3 days, with 1 min of shaking before reading the absorbance at 600 nm each hour.

Antifoam Tolerance 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.

After selecting the appropriate concentration of AF204, the test was repeated in 250 mL flasks. For this, frozen bacteria were inoculated on PSA plates and incubated for 3 days at 18 °C (or until a bacterial lawn was observed). Bacterial cells were washed with BSS and adjusted to OD600 = 1.0 in the same solution. The bacterial suspension was diluted 100 times in 125 mL of BMe and BMe supp with AF204, and each flask was incubated at 18 °C and 150 rpm for 96 h. Each day samples were taken from each culture and the OD600 was recorded. In parallel, bacterial counts were performed on PSA plates (triplicate). The values were recorded and plotted using the statistical program Graphpad Prism 6.0.

Scaling and harvest of the bacteria.

The activation and growth of the bacteria (pre-inoculum) was carried out exactly as described in the previous paragraph, using BMe and BMe supp. with AF204. Once a late exponential / early stationary phase of growth is reached in the AF204-supp. medium, the bacterium is mixed with fresh medium and adjusted to an OD600 = 1.00 (inoculum). 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. The harvest is 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 Purification and Gene Expression Analysis

To evaluate possible differences in gene expression triggered by the infection with P. salmonis wild-type (PM15972A1) and the attenuated P102 strain, 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.

Table 2. List of immune response gene markers selected and description of their function.

Immune Gene Function Description response

Innate illB Cytokine Proinflamatory response.

H8 Cytokine Proinflamatory response, chemotactic activity (neutrophils)

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.

H15 Cytokine Proinflamatory response. Important for the maintenance of T cell memory.

C3 Complement Activation of complement system and effector functions, protein

Adaptive / mhcl APC surface Antigen processing and presentation.

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.

Adaptive / mhcll APC surface Antigen processing and presentation, humoral receptor response cd4 T cell surface T-cell recognition and activation, MHCI presented Ag recognition, receptor cd40* Surface ligand Costimulatory molecule involved in the maturation of CD4+ T helper. cd83* Surface ligand Transmembrane glycoprotein expressed on DCs and leukocytos.

Regulation of B cell maturation, homeostasis, and function. socs3 Cytokine Suppressors of cytokine signaling 3. Inhibit the tyrosine activity of

JAK/STAT pathway.

ELFla- Housekeeping Elongation factor 1G3G3 gene

Formulation of vaccine

Stabilizers

Previous studies developed in ADL had suggested that to work with P. salmonis, the theoretical value of the osmotic pressure of the medium must be equal to or greater than 14 atm. Considering the above, different reagents were selected to carry out a first evaluation, among which we find PBS, glucose, sucrose, peptone, skimmed milk, and other formulations developed by the company, in concentrations ranging from 1% to 50%. Of these, 2 solutions were finally selected as preservative agents (confidential). From these two reagents, 3 formulations with different concentrations (formula B-D) were evaluated compared to pure concentrated antigen (A). The bacterial antigen was obtained from a 2 L fermentation batch. Aliquots from each formulation were frozen at -80 °C for 1 and 4 days. Bacterial counts were performed on PSA plates at time zero (post bulk preparation) and after the incubation times. Stability was measured as the maximum recovery of live bacteria (UFC/mL) after one treatment, either cryopreservation or lyophilization.

Stability test

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. For each batch, 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.

In vivo virulence evaluation

Evaluation of the virulence of P. salmonis P102 strain vs. P. salmonis P50c7 strain

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). To differentiate the groups of fish from tanks 1 and 2, 50% of the population was marked through a cut in the adipose fin. 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.

Fish were monitored daily for 37 days (470 DD), and mortalities were removed from the tanks and were recorded and subjected to necropsy. Additionally, samples from liver, head kidney and spleen were collected in ethanol absolute for analysis to confirm the cause of mortality through a specific qPCR for SRS (ADL confidential). On days 7, 14 and 37 (end of trial) 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’-

AGCAACATCTGCACAGACATA-3’), 357_H-L477_Forward (5’-

GTGGCAACACCGATCGG-3’) and 358_H-L477_Reverse (5’-

GCTTGCTATGACGCGTATCT-3’). 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. To carry out this study, strains of different origin were incorporated as reference for dissociation curve analysis.

Evaluation of the virulence of P102 strain in larger fish.

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. salmonis wild-type PM15972A1, P. salmonis P\ 02) or vehicle (BSS). 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.

Reversion to virulence - In vivo and in vitro evaluation

In vivo evaluation

Atlantic salmon smolts were confirmed as “pathogen free” using RT-PCR routine diagnostic methods for IPNV, IS AV, PRV, P. salmonis and R. salmoninarum. After their transfer to the experimental center, fish were tagged using Passive Integrated Transponder tags (PIT tags). The animals were acclimatizing to freshwater for 8 days and fed ad libitum. During the study, 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. On day 0, 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. 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. To obtain the “clarified”, 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. On that day, 5 fish/group were sacrificed to perform necropsy and determine pathological findings associated with SRS. In case animalized colonies were isolated in each passage, expansion on PSA plates was done in order to perform TCID50 to confirm their infective capacity and determine their genetic stability through molecular studies (PCR).

In vitro evaluation

To carry out the in vitro reversion study, P. salmonis Pl 02 was recovered from microplates carrying the TCID50/ml test performed as quality control inoculum in the virulence study already described. For this, after 14 days of incubation, the supernatant from wells of the first dilution column in the 96 wells microplate were recovered. This column is characterized by its clear cytopathic effect. The supernatant was seeded on PSA plates and incubated at 18 °C until colony growth is observed. The grown colonies were recovered and resuspended in BSS, and this suspension was used to make a new TCID50 in CHSE-214 cells as described in the "In vitro virulence attenuation" section. In total, the TCID50 was determined for 5 passages, each assigned with letters from A to E. In parallel to the new infection, PCR-HRM studies were performed in each passage as described elsewhere herein.

In vivo efficacy evaluation

Intraperitoneal (IP) challenge

Homologous challenge (P. salmonis EM-90 like)

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. After their transfer to the experimental center, fish were tagged using Passive Integrated Transponder tags (PIT tags). The animals were acclimated to freshwater for 21 days and fed ad libitum. During the immunization stage, 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). During challenge stage, fish were maintained at 14 ± 1 °C, salinity at 33 ppt and a photoperiod of 24L. At both stages, the water change rate was of 1.2 - 1.5/hour and the specific feeding rate was of 1.5 - 2%. 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).

After immunization, fish were challenged with a virulent P. salmonis EM-90 like strain by intraperitoneal injection in a volume of 0.1 mL/fish. The dose was previously defined from a LD50 trial. The challenge considers duplicate tanks with the two vaccinated groups and the positive control group. The negative control group was kept in a separate tank. 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 for the presence of P. salmonis. At the end of the trial, the relative percentage survival (RPS) of each group was calculated

Heterologous challenge (P. salmonis LF-89 like)

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. During the immunization stage, 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). During 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. 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.

After immunization, fish were challenged with a virulent P. salmonis strain (code PM63907, belonging to the LF89 (B) genogroup) by intraperitoneal injection in a volume of 0.1 mL/fish. The dose was previously defined from a LD50 trial. Fish were monitored daily for 30 days, and mortalities were removed from the tanks. Mortalities were recorded and subjected to necropsy. Additionally, PCR of internal organ samples (head kidney/liver pool) were done to confirm cause of death from SRS. At the end of the trial, the relative percentage survival (RPS) of each group was calculated

Cohabitation challenge

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. 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.

Fish were challenged by cohabitation with shedders injected with 0.1 ml of the challenge inoculum of P. salmonis belonging to the EM90 (A) genogroup. Mortality of study and shedder fish were observed throughout a 60 days period post challenge. At the end of the trial, the relative percentage survival (RPS) of each group was calculated.

During the immunization and challenge period, mortalities were registered daily and assigned to each study group after VIE-tag inspection. 100% of dead fish were analyzed for SRS clinical signs by performing a guided necropsy. Samples of liver and head-kidney from mortalities were taken to confirm the death caused by qPCR-SRS.

Results

Attenuation of P. salmonis Pl 02 strain

As mentioned, 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).

Table 3. Characteristics of the complete genome of the Piscirickettsia salmonis PM15972A1 strain

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

Plsm pPSAl- CP012414 1 39,335 38,3 46 - - - 49 3

1

Plsm pPSAl- CP012415 1 29,784 39,4 39 - - - 43 4

2

Plsm pPSAl- CPO 12416,1 82,130 38,2 86 - - - 88 2

3

Plsm pPSAl- CPO 12417 I 31,951 39,1 41 - - - 45 4

4

Chr, chromosome

Plsm, plasmid

INSDC, International Nucleotide Sequence Database Collaboration

During the attenuation process, from passage n° 50 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.

Additionally, an in vivo study was carried out on S. salar to determine the level of protection conferred by the strain. However, even though the efficacy results were optimal (RPS >80 %), its safety was not appropriate, since the fish immunized with this antigen still manifested moderate histopathological findings derived from the virulence of the isolate (data not shown). Due to the above, the strain continued to be passaged until passage number 100. At this point, 2 clones were isolated: P100-1 (P101) and P100-2 (P102). Both clones were stored at -80 °C, sequenced and subjected to in vitro infectivity studies to determine if there were differences with respect to clone P50c7 (ADL-PSA1). In vitro virulence attenuation

To determine the infectivity of the strains, 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.

As shown in Table 4, the Pl 02 strain produced a lower infectivity compared to the P101 clone, P50 and the PM15972A1 strains. Although 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).

Table 4. Summary of main mutations found in different Piscirickettsia salmonis attenuated strains.

Gene ORF* Predicted function Relative Mutation P50c7 P101 P102 position marC P500 440 Hypothetical protein, 505 G > C, A169P - - +

MarC family regulator mcp02 P500 1250 Secreted f Deletion A, fs + + + metalloprotease Mcp02 amt P500 2098 Aminotransferase 717 Insertion G, fs class-V family protein ADIVIHSLS239GG

HCYSFTE at C- terminal domain rasGEF P500 2193 rasGEF domain protein 791 T > G, M264R + + + hflC P500 2928 Protease modulator 187 G > A, V63I + + +

HflC

RD1 KW89 35 to cyo operon, a MFS 18.9 kb deletion + + +

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.

In order to give robustness to the in vitro attenuation studies, a cytotoxicity assay was performed. The release of lactate dehydrogenase (LDH), a cytosolic enzyme, present in many different cell types and that is released into the medium once the cell is lysed, is an indicator of cytotoxicity. As seen in Figure 8, the cytotoxicity percentage produced by the Pl 02 strain in SHK-1 cells 10 days post infection is lower than that produced by the virulent strain.

Both the infectivity and cytotoxicity studies were also performed with the P50c7 and P101 strains, however, both showed higher values of each parameter compared to the Pl 02 strain (data not shown).

Genetic characterization

The hybrid assembly strategy, using combined short-read and long-read sequencing datasets, it has been recommended to generate fully resolved and accurate bacterial genome assemblies. Long reads provide information regarding the structure of the genome and short reads facilitate detailed assembly at local scales and can be used to correct errors in long reads (Rhoads A. and Au KF, 2015; De Maio, N. et al., 2019).

Comparative studies of NGS sequencing of P. salmonis Pl 02 strain, revealed that, as detected in P50, the loss of a fragment of 18.9 kb in the chromosome, called RD1, is maintained. This region contains genes for aerobic metabolism & putative virulence factors (Figure 2, Table 5).

Furthermore, 5 SNPs have been identified in different genes across the chromosome and have only detected in the attenuated strains: marC, mcp02, amt, rasGEF and hflC. Of these, the SNP in marC is the only one found exclusively in the Pl 02 strain, which makes this locus of great interest to develop traceability genetic tools. Additionally, and as in previous passages, the plasmid PS Al -4 (32 Kb) is absent from Pl 02 background.

Table 5. Summary with the genes identified in the 18,9 kb segment (RD 1 ) .

Gene Function

IS30 Transposase

_ hyp- _

Msf Transporter MFS l cyoA Citocrome c oxidase Subunit _ cyoB _ Citocrome c oxidase Subunit _ cyoC Citocrome c oxidase Subunit III cyol) Citocrome c oxidase Subunit IV cyoE _ Protohem IX Famesystransferase _ _

IS30 _ Transposase _

IS630 _ Transposase _ _ _ _ dde _ DDE superhendonuclease family _

IS30 _ Transposase _ GCN5- related acetyltransferase

FAD binding protein ydjX Membrane protein, SNARE-associated domain hyp-

IS630 Transposase dde DDE superhendonuclease family mhpC Adhydrolase 1 superfamily hyp.

IS982 Transposase

Growth media without fetal bovine serum (FBS)

Before selecting and improving the culture media, it had already been described that there were no significant differences in the growth kinetics of the attenuated strains P50c7, P101 and Pl 02 with respect to the virulent PM15972A1 strain (Figure 9).

This result, in addition to the results observed in the infectivity studies and other basic microbiological data (Table 6), were decisive to discard the P101 strain from all subsequent analyses, because it did not present major in vitro differences with the P50c7 strain.

Table 6. Summary of the main in vitro characteristics observed in /'. PM I 572I A l (parent), P50c7, P101 and P102 strains.

P. salmonis strains

Charasteristic / „

Parent P50c7 P101 P102 parameter in vitro passages 0 50 100 100

, Gram Gram Gram Gram

Morfology , ’ . , ’ . , ’ . , ’ . coccobacilh coccobacuh coccobacuh coccobacilh

Growth in ADL-PSA (solid media) ^{yes yes yes yes}

Growth in ADL-PSB

(liquid media) ^{yeS yeS yeS yeS}

Growth kinetics _{16.18 h} 16-18 h 16-18 h 16-18 h

(generation time)

Plasmid content 4 3 3 3

Infectivity in vitro

(TCID50/mL)

Regarding the bacterial growth kinetics, the results of the first test using reference media showed that BM4, PSB and MD2 media were the most suitable for growing the attenuated Pl 02 strain (Table 7).

Table 7. ODeoo values after 120 h of incubation at 18 °C and 200 rpm.

Time (h) BM4 BMJ MD2 SF900-III PSB*

120 E6 072 002 002 uT

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. After 120 h, 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). On the other hand, 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*.

Due to the above, and because the study of new nutritive media for the production of live antigen was focused: (i) to allow optimal growth of the strains under study; (ii) simplification of the medium, using components of low/medium commercial value in the formulation and that do not represent a difficulty for the scaling of the culture, the BMJ and BM4 media were selected in addition to the PSB medium for a subsequent study.

The growth curves observed in the study in microplates with the BM4, BMJ and PSB media and their proposed variants show that the incorporation of the different protein source generated an improvement in the performance of the BM4 and PSB media, but not in BMJ (Figure 10).

As seen in Figure 10, the BM4* (also called BMe) and PSB* medium were those that provided the best results. However, 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.

Table 8. Composition of the BMe medium and a prior art medium Final concentration Final concentration (g/L) (g/L)

Yeast extract 4 4

Eugon Broth 12,16

NaCl 7,4 9

MgSO ₄ x 7H ₂ O 0,1 0,1

Glutamic acid 2 2

L-cysteine 1 1

K2HPO4 5,09 6,3 KH2PO4 0,9352

Peptone from 0 meat 2O - 0,08 - 0,02

Antifoam Tolerance

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 growth curves of the Pl 02 strain and the bacterial counts obtained are shown in Figure 11 and 12, respectively. The use of AF204 at the suggested dose considerably delays the beginning of the curve (the lag phase almost doubles in time), nonetheless, once it reaches the early exponential phase, its behavior is quite similar to that observed in BMe medium without antifoam. In terms of bacterial counts, no significant differences are observed once at ODeoo close to 2.00 (late exponential) is reached.

Gene Expression Analysis

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.

In fish, as well as in other vertebrates, 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. These 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. salmonis, a minimal or no activation of U12 an upregulation of illO has been described in in vitro models, which might constitute a virulence mechanism used to promote intracellular bacterial replication in salmonid macrophages. This mechanism optimized the cellular environment for the pathogenic microorganism and down regulated some antimicrobial effectors. (Alvarez CA., et al 2016).

As seen in Figures 13, 14 and 15, there is a clear differentiated pattern between the relative expression levels manifested by cells infected with a virulent strain versus an attenuated one, especially in some markers associated with innate immune response (ifny, illO, H12 and U8) and cell mediated response mhc-II, CD4) after 10 days of infection (prolonged infection). In the case of the parent strain (PM15972A1), the exacerbated expression of certain genes encoding proinflammatory and immunomodulatory cytokines, such ifny/U8 and U10/U12 respectively, as well as the immunomodulation observed at the level of cell response markers mhc-II, CD4, CD83 and socs3) has been previously described in cellular and in vivo models (Alvarez CA et al, 2016; Tandberg JI et al, 2016., Rozas-serri et al., 2018, Xue X et al, 2021}.

Formulation of vaccine

Stabilizers

The addition of stabilizers had a positive effect on the stability of the strain after deep freezing storage at -80 °C for short time. The 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, meanwhile, does not offer as much stability as the two previous conditions, however, it is still superior to the control group (Figure 16).

Table 9. Final concentration of each stabilizer (cryoprotectant) per condition.

Condition Stabilizer 1 Stabilizer 2

A 5.71% 0%

D 8.21% 10%

Stability test The stability results with the two experimental batches show that, if the vials are kept at -70 ° / -80 °C, their titers are slightly affected (Figure 17). The results confirms that storage at temperatures below -70 °C using the cryopreservatives already described allows the strain to remain stable for at least 9 -12 months. The results also indicate that storage at higher temperatures (-20 °C) is not optimal for preserving the strain, and that after one month the recovery of the bacteria is significantly lower.

The results obtained are currently being validated in experimental batches, which have shown a stability quite similar to that obtained in Figure 17 (data not shown). Similarly, stability was evaluated on vaccine (cryovials) stored for up to 4 months in liquid nitrogen (liquid phase, approx. -196 °C). In this study, a drop in bacterial titer like that observed in those samples stored at -70 / -80 °C was observed. From month 1, the viability of the strain (measured in CFU/mL) remained stable until the end of the test (Figure 18).

In vivo virulence evaluation

Evaluation of the virulence of P. salmonis P102 strain vs. P. salmonis P50 strain

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). At the end of the trial (day 37), 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). No mortality was recorded in the group injected with the vehicle (saline solution), P50c7 lx dose and Pl 02 lx dose. All registered mortalities were confirmed as positive for SRS by necropsy assessment of clinical signs of the disease and by PCR (data not shown).

The negative effect of the P50 strain at high concentrations was reflected in the fish growth at the end of the study. Indeed, fish immunized with an overdose of the P50 strain showed statistically significant differences in length and weight (Figure 20). Kidney and liver samples from 5 fish per time point derived for histopathological analysis revealed that alterations in the parenchyma of both types of tissue are evident for surviving fish immunized with lOOx doses of strain P50c7, and to a lesser degree for those immunized with strain Pl 02 (results not shown). In contrast, in the fish immunized with a lx dose, no major alterations were observed between both groups, with the exception of histoneutrophilic hepatitis in fish vaccinated with the P50c7 strain. (Figure 21). The development of hepatitis in the late stage of immunization may be related to the lower survival of this group during bacterial challenge.

At final time (37 days post immunization), head kidney and liver samples from the control group were also analyzed, however, some histopathological findings were also found, such as biliary hyperplasia, nephritis and circulating leukocytes at a frequency of 40%, 20% and 60% respectively (data not graphed). The detection of findings in the control group, which was completely isolated from fish inoculated with P. salmonis, would reflect an intrinsic physiological state of the fish or the effect of environmental conditions to which they were subjected, and would support the fact that the Pl 02 strain is safe, and that a big part of the observed findings could have an external origin.

The evaluation of the immune response was carried out in fish immunized intraperitoneally with the P50c7 and P102 strain. Head kidney and liver extracted at 7, 14 and 37 days were analyzed using RT-PCR (representative results in Figure 22). Since there were no significant differences in immune response markers evaluated from liver of fish samples immunized with attenuated strains versus the control group, these results are not presented in this report.

In contrast to what was observed for virulent strains of P. salmonis. immunization with the attenuated strains does not seem to induce the classic proinflammatory response. In fact, no induction of ifny and H8 was observed in the early stages of infection in the head kidney. However, H12 showed repression in its expression, even in the analysis after 37 days of immunization, which is similar to that observed with the virulent strain. It is interesting that illO overexpression does not occur with the attenuated strain, on the contrary, there is a strong repression in late stages, showing a deficiency in immunomodulatory activity, which could account for the attenuated phenotype. Similarly, mhc-II shows an opposite tendency (overexpression) to the virulent strain (repression) (Rozas-serri et al., 2018). Regarding 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.

Sampling day (post Strain Dose N° of positive fish (P. salmonis immunization) colonies isolation) n= 5

7 Control - 0

P50c7 lx 0

P102 lx 0

P50c7 lOOx 4

P102 lOOx 4

14 Control - 0

P50c7 lx 0

P102 lx 0

P50c7 lOOx 5

P102 lOOx 5

37 Control - 0

From the colonies obtained, PCR-HRM studies were performed to determine the genetic stability of two molecular markers (SNPs found in marC and methyltransferase genes). In general, 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).

Evaluation of the virulence of P102 strain in larger fish.

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).

For this experiment, head kidney samples were taken from 5 fish/group at short times (5, 12 and 20 days post-inoculation) to carry out a comparative study of gene expression between the immune response triggered by a virulent strain (PM15972A1) and that of an attenuated one (Pl 02) at an equivalent dose (Figure 24 and 25).

Compared to that observed in the study using a lx dose, the fish immunized with a lOOx dose show a much more evident and differential gene expression profile between both phenotypes. It has been described that Piscirickettsia salmonis induces illO overexpression and reduces ill 2 expression in vitro and in vivo, which could be a strategy to promote intracellular survival and replication. Different expression patterns of immune response-related genes in Atlantic salmon infected with P. salmonis suggest strong innate response stimulation, but an inhibition of the adaptive response (Rozas-Serri et al., 2019). In this way, 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. On the other hand, 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. Regarding the effect over the adaptive immune response, 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. At day 12, we can observe an increased expression level of the mhc-II gene. 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. After 20 days, 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.

Reversion to virulence - In vivo and in vitro evaluation

In vivo evaluation

The in vivo study of reversion to virulence showed that the methodology used does not affect the welfare of the fish, and confirms that the dose proposed in group 1, as well as the cleared from infected fish (injectable fraction) obtained in each passage, does not produce internal injuries in fish or findings associated with SRS infection. Nonetheless, a doubt arises because the bacteriological and qPCR results demonstrated the absence of the bacterium or its genetic material even at the time of passage on day 3 post inoculation. This observation allows us to infer that the persistence of the live bacteria or the genetic material of the pathogen is much faster than expected (Table 11).

Table 11. Mean Ct values of sampled fish, supernatant (injectable fraction) or organ tissue sampled at 3, 7 and _ 11 post inoculation. _

Ct passage n° 1 Ct passage n° 2 Ct passage n° 3

Sample ELF1 SRS ELF2 SRS ELF3 SRS

Head 17,88 No Ct 17,72 No Ct 18,52 No Ct kidney

Liver 17,84 No Ct 16,7 No Ct 17,18 No Ct

Spleen 19,58 No Ct 18,84 No Ct 20,00 No Ct

Injectable 16,72 No Ct 16 No Ct 14,7 No Ct fraction

Pellet tissue 23,22 No Ct 16,1 No Ct 14,5 38,9

(HK+L+S)

*The detection limit of P. salmonis was set to a Ct value of 35; all simples with a Ct value lower or equal to 35.0 were considered positive. ELF1 (elongation factor 1, housekeeping gene). The values of each tissue were obtained from an average of Ct of 5 fish. The detail of the Ct obtained by each fish can be found in table 2 of the annex.

As mentioned, the bacteriological studies of isolation and recovery of the bacteria were not successful even when plates were incubated at 18 °C for up to 30 days (data not shown). To define if the lack of bacterial growth was due to the detection limit of the technique, qPCR analyzes were performed to detect SRS in the same samples used for bacteriology procedures, however, the results were consistent with what had been observed by molecular analyses (Table H).

In vitro evaluation

The values for the TCID50/mL and bacterial counts obtained in each passage are summarized in Table 12. As can be seen, there is no increase in the infective capacity (virulence) of the isolates as the in vitro passages progress, even when the bacterial counts remain in a close order of magnitude.

Table 12. Values for TCID50/mL and bacterial counts of each in vitro passage (A-E).

P102 strains (isolated colonies from each in vitro cell passage, CHSE-214 cells)

A B C D E

TCID50/ml 8.89E+04 2.81E+04 2.81E+03 8.89E+03 2.11E+04

CFU/mL 1.00E+07 3.67E+07 8.33E+06 6.33E+07 1.60E+07

CFU, colony forming unit

Similarly, the results of the PCR-HRM show that genetic stability is maintained in each passage (Table 13). This factor is particularly important when we refer to the marC marker, since it is the only chromosomal marker validated to distinguish the Pl 02 strain vs P. salmonis p50c7 or other P. salmonis, including field strains. Below, it is shown representatives dissociation curves of a PCR-HRM analysis (Figure 26 and 27).

Table 13. Summary of the results obtained by PCR-HRM throughout the course of the study.

Passage A Passage B Passage C Passage D Passage E

Locus/Name 440- 477- 440- 477- 440- 477- 440- 477- 440- 477 - marC tmet* marC tmet* marC tmet* marC tmet* marC tmet*

PM15972A1 1 1 1 1 1 1 1 1 1 1

PM63907 (LF-89) 3 3 3 3 3 3 3 3 3 3

P50c7 (Original clone) 1 2 1 2 1 2 1 2 1 2

P50c7 (passage A) 1 2 1 2 1 2 1 2 1 2

P50c7 (passage B) 1 2 1 2 1 2 1 2 1 2

P50c7 (passage C) 1 2 1 2 1 2 1 2 1 2

P50c7 (passage D) 1 2 1 2 1 2 1 2 1 2

P50c7 (passage E) 1 2 1 2 1 2 1 2 1 2

1, red dissociation curves

2, green dissociation curves

3, blue dissociation curves In vivo efficacy evaluation

IP challenge

Homologous challenge (P. salmonis EM-90 like)

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.

To evaluate the protective capacity of the antigen (fresh Pl 02 strain) and Pl 02 “freeze dried” format, fish were given lethal intraperitoneal injections of P. salmonis PM15972A1 after a 630 DD immunization period. Fish mortalities were monitored daily. The final relative percent survival (RPS) of the group 1 (fresh Pl 02 strain) was slightly higher than group vaccinated with reconstitute freeze dried P102 strain (93,8% vs 91,8%, respectively) (Figure 28). The control group (fish injected with the vehicle during immunization, not challenged) had no mortality, while the positive control group (fish injected with BSS during immunization and then challenged) achieved a mortality of 81.1% after 28 days post challenge (data not plotted). In the case of the positive control group, mortalities began on day 13 post-challenge, reaching a cumulative mortality of approx. 80% at 20 DPC.

Regarding the effect on fish growth ascribed to the type of antigen, we can observe that at a target dose there are no significant variations in the length and weight of the fish immunized with the fresh strain or reconstituted from a lyophilized vial after 630 DD (Figure 29).

On the other hand, gene expression studies at the end of immunization carried out in S. salar head kidney samples, showed that the immune response markers ifny, H8, ill 0, H12, mhc-II and cd8a are similarly expressed in all groups, with only some slight significant differences between Pl 02 strain vaccinated vs the non-vaccinated groups. Indeed, it was possible to see slight differences in terms of proinflammatory response markers such as H8 and ifny, and in the adaptive immune response markers cd8a and mhcll. Nonetheless, due to the low magnitude observed and sample size (5 fish), this significance could be questioned (not shown).

The results for quantification of specific IgM against SRS by ELISA using the OspA protein as capture antigen, showed that at the end of immunization there are no significant differences in the antibody titer observed in sera from vaccinated fish versus the group control (data not shown). This observation can be interpreted as the protection in the challenge stage can be ascribed to mechanisms associated with cellular immunity over antibody response.

Finally, regarding the histopathological findings, we have found that, as in the virulence study, the control fish (non-immunized) presented an average frequency of a wide variety of findings in both kidney and liver (data not shown). However, if we discard the redundant findings of the control group, we only found mild vacuolar degeneration in the liver in one fish immunized with the freeze-dried antigen. This finding is usually associated with the type of diet administered to fish (Figure 30 lower panel). In the case of the kidney findings, tubular degeneration, hyperplasia in hematopoietic tissue and circulating leukocytes were observed at low frequency and mild intensity (Figure 30, upper panel). Tubular degeneration has been associated mainly with fish exposed to suboptimal water quality conditions (waste metabolic, etc.). This finding does not represent a functional detriment of the organ. Thus, 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.

Heterologous challenge (P. salmonis EM-90 like)

Compared to the in vivo studies already described, efficacy of the attenuated strain using an LF- 89-like isolate for challenge was tested. The rational of this experiment was determining the degree of protection conferred by Pl 02 antigen against a lethal heterologous challenge, beyond describing the response of the fish during the immunization stage from a histological, relative gene expression or serological point of view. The final relative percent survival (RPS) of the tank 1 (vaccinated fish with Pl 02 strain, lx) was 90%, and its replicate (tank 2) showed a RPS of 89.7 %. The control group (fish injected with the vehicle during immunization, not challenged) had no mortality, while the positive control group (fish injected with BSS during immunization and then challenged) achieved a mortality of 100% after 18 days post challenge (dpc). 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 results obtained reflected that the attenuated strain is able to yield cross-protection. As already mentioned, even in an aggressive challenge model such the one presented in Figure 27, 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%.

Cohabitation challenge

Since it is well known that P. salmonis is capable of horizontal transmission to other individuals, we decided to evaluate the efficacy of the Pl 02 antigen in a controlled model of cohabitation challenge, to mimic what occurs during natural infection conditions.

One week after Visible Implant Elastomer (VIE) tagging, fish were weighed and intraperitoneally (i.p.) injected with the vaccines (P102 strain, inactivated commercial vaccine) or control substances and then distributed in two tanks. After immunization period of 655 DD (in which no mortalities were recorded), fish maintained in seawater were challenged with P. salmonis by cohabitation with shedders. The challenge carriers (shedders) were from the same batch of fish and were i.p. injected with P. salmonis, before to be transferred to the challenge tanks. Three study groups were challenged at 15 °C in two replicated tanks, and mortality was observed throughout 60 days.

At the end of challenge, accumulated mortality had reached 100% in control group on both tank replicates. On tank 1, RPSeo values for group 1 (vaccinated with Pl 02 strain) were 98% and 100% for tanks 1 and 2 respectively, while RPSeo values for vaccine 2 (inactivated competitor, pentavalent) were 84% and 92% for tanks 1 and 2 respectively. RPSfmai (end of challenge) of both immunized groups showed that the protection conferred by the attenuated strain was 98%, while the commercial vaccine only conferred 18 and 28%. Because in general, the behavior of the curves was quite similar in both tanks, Figure 32 shows the average accumulated mortality values of the two tanks over time.

At the end of the immunization period, 10 fish from each group were sampled, in order to evaluate visible secondary effects in the organs by necropsy, and to take blood samples for serological studies by specific ELISA for SRS.

As shown in Figure 33, 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).

The detection of pale focal nodes in the liver and ascites in immunized but non-challenged fish has already been reported. This could be related to individual differences between fish, iatrogenic harm caused by vaccination or necropsy, or artifacts during preparation of tissue for analysis. Regarding to splenomegaly, it has been described that the application of intraperitoneal vaccines produces an inflammatory reaction that surrounds the pyloric caeca, pancreas, spleen, intestine and liver, depending on the severity of the response. The described inflammatory response has been complemented by histopathological findings and could explain the detection of two individuals with mild splenomegaly. On the other hand, the presence of adhesion and melanosis in vaccinated fish might also be due to side effects of vaccine components. Adhesions and melanosis presented in vaccinated fish are well-known side effects derived from oil adjuvant present in the vaccines (Meza et al., 2019).

Finally, it is important to mention that, as already reported, there are no significant differences in the IgM levels of the fish vaccinated with the Pl 02 strain compared to the non-vaccinated group (control) at the end of immunization period. This result would reinforce the idea that the protection produced by the Pl 02 strain could be explained by an activation of the cellular-type immune response over the one mediated by antibodies (humoral response).

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Below are the genes found in the 18,9 kb fragment (deleted in the P50c7 and its derived strains). Two PCR reactions were performed to test the deletion.

Table 14. Summary with the genes identified in the 18.9 kb segment (RD1).

Gene Function

IS30 Transposase hyp-

Msf Transporter MFS_1 cyoA Citocrome c oxidase Subunit II cyoB Citocrome c oxidase Subunit I cyoC Citocrome c oxidase Subunit III cyoD Citocrome c oxidase Subunit IV cyoE Protohem IX Farnesystransferase

IS30 Transposase

IS630 Transposase dde DDE superhendonuclease family

IS30 Transposase

GCN5- related acetyltransferase

FAD binding protein ydjx Membrane protein, SNARE-associated domain hyp-

15630 Transposase dde DDE superhendonuclease family mhpC Adhydrolase_l superfamily hyp-

15982 Transposase

Sequence Listing

The following sequences form part of the disclosure of the present application. A WIPO ST 26 compatible electronic sequence listing is provided with this application, too. For the avoidance of doubt, if discrepancies exist between the sequences in the following table and the electronic sequence listing, the sequences in this table shall be deemed to be the correct ones.

In some cases, 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

Table 15. Sequence Listing

Annotated sequence

In the following table, 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.

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.

The following numerical abbreviations are being used:

1 KW89_35, integrase (antisense)

2 KW89 36, HP (antisense)

3 KW89 37, MFS (antisense)

4 KW89 38, cyoA (sense)

5 KW89 39, cyoB (sense)

6 KW89_40, cyoC (sense) KW89_41, cyoD (sense) KW89 42, Protoheme IX (sense) KW89_43, integrase (sense) 0 KW89_44, transposase (sense) 1 KW89 45, DDE family protein (sense) 2 KW89_46, integrase (sense) 3 KW89_47, GCN5-family acetyltransferase (antisense) 4 KW89 48, FAD-binding protein (antisense) 5 KW89 49, membrane protein (antisense) 6 KW89 50, membrane protein (antisense) 7 KW89 51, transposase (sense) 8 KW89_52, DDE family protein (sense) 9 KW89 53, peptidase (sense) 0 KW89_54, tautom erase (sense) 1 KW89_55, transposase (antisense) 2 KW89_56, integrase (antisense)