PLASMID ENCODING B-CELL ACTIVATING FACTOR RECEPTOR (BAFF-R) AND USES OF SAME IN THE TREATMENT AND PREVENTION OF INFLAMMATORY DISEASES IN FISH

Abstract

The present invention relates to a plasmid that encodes a fusion protein comprising a signal peptide and the extracellular domain of the B-cell activating factor receptor (BAFF-R), and optionally, a fragment of the constant region (Fc) of an immunoglobulin. The invention also relates to compositions comprising said plasmid, and to the use of same in the treatment and/or prevention of inflammatory diseases in fish, more preferably in salmonids.

Claims

1. A fusion protein comprising a first amino acid sequence comprising at least 95%, 96%, 97%, 98%, 99% identity to the interleukin-2 (IL-2) signal peptide of SEQ ID NO: 2 fused to a second amino acid sequence comprising at least 95%, 96%, 97%, 98%, 99% identity to the extracellular domain of the BAFF receptor (BAFF-R) of SEQ ID NO: 4.

2. The fusion protein according to claim 1, comprising at least 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 8.

3. The fusion protein according to claim 1, further comprising an amino acid sequence of an Fc domain of a mouse immunoglobulin.

4. The fusion protein according to claim 3, wherein the immunoglobulin is mouse IgG1 comprising SEQ ID NO: 6.

5. The fusion protein according to claim 1, comprising at least 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 10.

6. A nucleic acid encoding the fusion protein according to claim 1.

7. The nucleic acid according to claim 6, comprising a sequence with at least 95%, 96%, 97%, 98%, 99% identity to any of the sequences selected from the list consisting of SEQ ID NO: 7 or SEQ ID NO: 9.

8. A plasmid comprising a nucleic acid molecule according to claim 6, wherein, optionally: a) the nucleic acid molecule is operably bonded to an expression control sequence suitable for expression in a host cell; and/or, b) the plasmid comprises one or more selectable marker genes.

9. The plasmid according to claim 8, characterized in that it comprises SEQ ID NO: 11.

10. A host cell comprising a nucleic acid molecule according to claim 6.

11. A composition comprising the fusion protein according to claim 1, and at least one excipient and/or carrier.

12. The composition according to claim 11, characterized in that it is a pharmaceutical or veterinary composition.

13. (canceled)

14. A method for the treatment and/or prevention of inflammatory diseases in aquatic animals that comprises the administration of a therapeutically effective amount of the fusion protein according to claim 1.

15. The method according to claim 14 wherein the administration is by intramuscular route.

16. The method according to claim 14, wherein the inflammatory disease is caused by myxozoans of the genus Tetracapsuloides.

17. The method according to claim 14, wherein the inflammatory diseases are Proliferative Kidney Disease and the Red Mark Syndrome.

18. The method according to claim 14, wherein the aquatic animals belong to the salmonid family.

19. The method according to claim 18, wherein the salmonids are selected from the list consisting of: Rainbow trout (Oncorhynchus mykiss); Chinook salmon (Oncorhynchus tshawytscha); Coho salmon (Oncorhynchus kisutch); Atlantic salmon (Salmo salar); Common trout (Salmo trutta); Grayling (Thymallus thymallus); Whitefish (Coregonus spp.); Chum salmon (Oncorhynchus keta); Sockeye salmon (Oncorhynchus nerka); Lake trout (Salvelinus namaycush); Brook trout (Salvelinus fontinalis) and Arctic char (Salvelinus alpinus).

20. The method according to claim 18, wherein the salmonids are Rainbow trout (Oncorhynchus mykiss) and Common trout (Salmo trutta).

21. A composition comprising the nucleic acid according to claim 6, and at least one excipient and/or carrier.

22. A method for the treatment and/or prevention of inflammatory diseases in aquatic animals that comprises the administration of a therapeutically effective amount of the nucleic acid according to claim 6.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0071] FIG. 1. Transcription levels of the gene that encodes the extracellular domain of the Rainbow trout BAFF receptor (BAFF-R) after intramuscular injection of the pBAFF-R plasmid. At day 7 post-injection, the fish were sacrificed to sample the muscle of the dorsal area and determine the levels of gene expression by real-time PCR. The empty plasmid without the gene construct was used as a negative control. The data is shown as the relative expression of each gene compared to the expression of the endogenous control gene EF-1α (mean+SD; n=3-6).

[0072] FIG. 2. Protein corresponding to the extracellular domain of the Rainbow trout BAFF receptor (BAFF-R) detected by the western blot technique using the concentrated supernatant of EPC cells transfected with the pBAFF-R plasmid. The empty plasmid without the gene construct was used as a negative control. M, marker.

[0073] FIG. 3. Effect of intramuscular treatment with the pBAFF-R plasmid in Rainbow trout naturally infected with T. bryosalmonae. (A) Degree of posterior kidney inflammation of trout treated with the pBAFF-R plasmid (n=20) or PBS (negative control; n=24). The individual value plot shows the interquartile range and the median of the data. (B) Percentage of trout affected or killed by PKD after intramuscular injection of the pBAFF-R plasmid or PBS (control).

[0074] FIG. 4. Transcription levels of 18S rRNA from T. bryosalmonae after intramuscular injection of the plasmid (0.1 and 1 μg) containing the sequence of the extracellular domain of the BAFF receptor (pBAFF-R) in Rainbow trout naturally infected by T. bryosalmonae. At day 110 post-injection, the fish were sacrificed to sample the posterior kidney and determine the expression levels of the 18S rRNA gene of T. bryosalmonae by real-time PCR. The empty plasmid without the gene construct was used as a negative control. The data is shown as the relative expression of each gene compared to the expression of the endogenous control gene EF-1α (mean+SD; n=5-7).

EXAMPLES

[0075] Next, the invention will be illustrated by means of assays carried out by the inventors that demonstrate the effectiveness of the product of the invention.

Example 1. Obtaining the pBAFF-R Plasmid (SEQ ID NO: 11) Encoding the IL BAFF-R Fusion Protein (SEQ ID NO: 8)

[0076] The nucleotide sequence (SEQ ID NO: 3) that encodes the extracellular domain of BAFF-R (SEQ ID NO: 4) from Rainbow trout was fused to the nucleotide sequence (SEQ ID NO: 1) that encodes the Rainbow trout IL-2 signal peptide (SEQ ID NO: 2) giving rise to the IL-2-BAFF-R construct comprising the nucleotide sequence SEQ ID NO: 7 that encodes the IL-2-BAFF-R fusion protein comprising the amino acid sequence SEQ ID NO: 8.

[0077] To allow further purification of the IL-2-BAFF-R construct, the nucleotide sequence (SEQ ID NO: 5) that encodes the Fc region of the mouse IgG1 immunoglobulin-like domain (SEQ ID NO: 6) was fused thereto, giving rise to the IL-2-BAFF-R-IgG1 construct which comprises the nucleotide sequence SEQ ID NO: 9, which encodes the amino acid sequence SEQ ID NO: 10.

[0078] Thus, the foregoing construct was cloned into the pcDNA3.1+ eukaryotic expression vector (Invitrogen), previously digested by HindIII/XhoI restriction enzymes. Next, said plasmid, called pBAFF-R (SEQ ID NO: 11), was transformed into JM109 E. coli competent cell bacteria (Promega) following the manufacturer's instructions. Transformed colonies were selected on LB agar medium (Invitrogen) supplemented with ampicillin (100 μg/ml) for subsequent plasmid extraction and purification (Invitrogen).

[0079] Subsequently, the nucleotide sequence of the cloned pBAFF-R plasmid was confirmed by sequencing using the T7 primer (SEQ ID NO: 12; 5′-TAATACGACTCACTATAGGG-3′) derived from the vector.

[0080] The empty plasmid without the nucleotide sequence that encodes the IL-2-BAFFR-IgG1 gene construct was used as a negative control.

Example 2. Study of BAFF-R Transcription after Intramuscular Injection of pBAFF-R (SEQ ID NO: 11)

[0081] Rainbow trout (Oncorhynchus mykiss) of 7 cm supplied by the Cifuentes fish farm (Guadalajara, Spain) were used. To that end, the fish were kept at the Animal Health Research Centre (CISA-INIA) at 14° C. and fed daily with a commercial diet (Skretting, Norway). Before starting the experiments, the fish were acclimatised to laboratory conditions for 2 weeks.

[0082] The fish were divided into two groups and injected intramuscularly with 1 μg of pBAFF-R plasmid resuspended in 50 μl of sterile saline solution (0.9% NaCl) or with the same amount of empty plasmid (negative control). The fish were sacrificed 7 days post-injection and a sample was taken from the muscle area of each fish where the injection had been made for RNA extraction.

[0083] The total muscle RNA was extracted using Tri-reagent (Invitrogen), following the manufacturer's instructions and stored at −80° C. until use. The purified RNA was quantified using the Nanodrop 1000 spectrophotometer (Thermo Scientific). Next, 1 μg of RNA was treated with the DNase I enzyme using the RapidOut DNA Removal Kit (Thermo Scientific) to remove traces of genomic DNA and it was used to synthesise cDNA with the RevertAid Reverse Transcriptase enzyme (Thermo Scientific) and oligo(dT)23VN (1.6 μM), as indicated by the manufacturer. The resulting cDNA was diluted to 1:5 with nuclease-free water and stored at −20° C. until use.

[0084] The expression levels of the extracellular domain of BAFF-R were analysed by real-time PCR using the LightCycler 96 System instrument (Roche). All amplification reactions were performed in duplicate using the FastStart Essential DNA Green Master reaction mix (Roche) and specific primers (Table 1). The amplification conditions consisted of an initial denaturation step (95° C., 10 minutes), followed by 40 cycles of amplification (95° C. for 10 s, 60° C. for 10 s and 72° C. for 10 s). Moreover, a dissociation curve was obtained by reading the fluorescence signal every degree between the temperatures 60° C. and 95° C. to verify that this signal is due to the amplification of a single product. Negative controls without template DNA and negative reverse transcription (RT-) controls were included in all assays. The expression of the extracellular domain of the BAFF-R receptor was normalised with the expression of the gene that encodes the Rainbow trout elongation factor-1α (EF-1α) (Montero, J., J. et al., J. Virol. 2011; 85: 4046-4056) which is constitutively expressed in all organs to the same extent, using specific primers (Table 1). The expression levels were calculated with the 2-ΔCt method, wherein ΔCt was determined by subtracting the Ct value of EF-1α from the Ct value of the target gene (Livak, K. J., and T. D. Schmittgen. Methods. 2001; 25: 402-408). Statistical analysis was performed using a two-tailed Student's t-test with Welch's correction and the differences were considered statistically significant when p<0.05.

TABLE-US-00001 TABLE 1 List of primers used in transcriptional studies. Forward primer Reverse primer Gene (5′-3′) (5′-3) EF-1α SEQ ID NO: 13 SEQ ID NO: 14 (gatccagaagga (ttacgttcgacc ggtcacca) ttccatcc) BAFF-R SEQ ID NO: 15 SEQ ID NO: 16 (gacaaactgctc (atatccagacag atcacctgtatc) ctggactcactg) 18S rRNA SEQ ID NO: 17 SEQ ID NO: 18 T. (ggacactgcatg (ccatgctagaat bryosalmonae tgctgcatagt) gtccaggcact)

[0085] As can be seen in FIG. 1, after intramuscular injection of the pBAFF-R plasmid, an increase in BAFF-R mRNA expression was detected in the muscle (56 times higher) compared to the expression shown by the trout treated with the empty plasmid (negative control).

Example 3. Obtaining Supernatants with the Extracellular Domain of the Rainbow Trout BAFF Receptor (BAFF-R)

[0086] To verify that the pBAFF-R plasmid of the invention is capable of effectively transcribing and translating the extracellular domain of Rainbow trout BAFF-R, EPC (Epithelioma papulosum cyprinid) cells were transfected with said pBAFF-R plasmid using the 4D-Nucleofector™ kit (Lonza). To that end, the EPC cells maintained in Leibovitz medium (L-15, Life Technologies) supplemented with penicillin (100 units/ml, Life Technologies), streptomycin (100 μg/ml, Life Technologies) and foetal bovine serum (10% FBS, Life Technologies) were trypsinised and transfected with 1 μg of plasmid pBAFF-R using the reagents of the Amaxa P3 Primary cell kit (Lonza). Next, the transfected cells were cultured in 24-well plates at a concentration of 5×10.sup.5 cells/ml. After 24 h of incubation at 20° C., the culture medium was changed using L-15 medium supplemented with antibiotics and 0.1% FBS. After incubating the cells for another 24 h, the supernatants were collected from the wells and concentrated (50×) using 3 kDa molecular-weight cutoff centricons (GE Healthcare Life Sciences). In addition, the culture supernatants of the EPC cells transfected with the empty plasmid (negative control) were also collected. In order to verify the correct transfection of the cells, the pmaxGFP plasmid supplied in the Amaxa kit was used as a positive control for the subsequent visualisation of the green fluorescent protein by fluorescence microscopy (Zeiss Axio Vert.A1).

[0087] The concentrated supernatants of the EPC cells transfected with the pBAFF-R plasmid of the invention and with the empty plasmid were analyzed by polyacrylamide gel electrophoresis (SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis). To that end, the polyacrylamide gel (12%; Bio-Rad) was loaded with 20 μl of concentrated supernatant under reducing conditions and stained with Coomassie blue. Protein transfer was carried out on a polyvinylidene fluoride membrane (PVDF; Bio-Rad) using the Trans-Blot Turbo kit (Bio-Rad) to identify the protein corresponding to the extracellular domain of the Rainbow trout BAFF receptor (BAFF-R) by Western blot. To that end, the membrane was blocked with 5% skim milk in phosphate-buffered saline (PBS) at room temperature for 1 h. Then, the membrane was incubated with a rabbit anti-mouse IgG primary antibody (Sigma-Aldrich) and prepared in the blocking solution at 4° C. for 16 h. The membrane was then washed three times with 0.1% Tween 20 (Sigma-Aldrich) prepared in PBS for 10 min and incubated with a horseradish peroxidase-conjugated donkey anti-rabbit IgG secondary antibody (GE Healthcare Life Sciences) at room temperature for 1 h. After three washes in 0.1% Tween 20-PBS and a final wash in PBS, the membrane was revealed by peroxidase chemiluminescent reaction using the ECL kit (GE Healthcare Life Sciences).

[0088] As can be seen in FIG. 2, the protein corresponding to the extracellular domain of BAFF-R was detected, showing a specific band of 37 kDa.

Example 4. The Administration of the pBAFF-R Plasmid (SEQ ID NO: 11) Reduces the Degree of Posterior Kidney Inflammation and Mortality in Trout Naturally Infected by T. bryosalmonae

[0089] Forty-four 30-40 g trout from the Southampton fish farm (United Kingdom) were used, free from infection by T. bryosalmonae and divided into two groups (20-24 fish per group). The fish of one of the groups were treated with two intramuscular injections (front and back of the dorsal fin) of the pBAFF-R plasmid (10 μg) dissolved in 20 μl of phosphate buffer (PBS) (20 μg of the pBAFF-R plasmid administered in total per fish). The fish of the second group were treated in the same way, but instead of administering the pBAFF-R plasmid, they were only administered the same volume of phosphate buffer (PBS) without plasmid.

[0090] Next, both groups of animals were introduced into a fish farm where an outbreak of PKD had been detected (Test Valley Trout), recording at each moment the number of dead fish. This assay was carried out in the middle of May, when the water temperature increased, reaching values higher than 15° C. At 90 days post-injection, when the temperature was still above 15° C. and the outbreak of PKD persisted on the farm, the trout were sacrificed to analyze the level of posterior kidney inflammation and thus determine the degree of infection by the parasite. The statistical analysis was performed using the Mann-Whitney nonparametric test and the differences were considered statistically significant for p<0.05.

[0091] The results show that the intramuscular administration of the pBAFF-R plasmid in trout produced a decrease in the degree of kidney inflammation caused by PKD infection compared to the degree of kidney inflammation shown by the control group (FIG. 3A).

[0092] The percentage of mortality presented by both groups of animals was also analyzed, showing that said percentage of mortality was lower in the group of trout treated with the pBAFF-R plasmid (4.76%) compared to the percentage shown by the control group (25%) (FIG. 3B). Moreover, as shown in FIG. 3B, the percentage of trout that presented a degree of mortality was greater (52.38%) in the group treated with the pBAFF-R plasmid than in the control group (37.50%).

Example 5. The Administration of the pBAFF-R Plasmid (SEQ ID NO: 11) Reduces the Load of T. bryosalmonae in Trout Naturally Infected by this Parasite

[0093] To determine whether the administration of the pBAFF-R plasmid is capable of modifying the long-term parasite load in trout naturally infected with T. bryosalmonae and that manage to survive the disease, the pBAFF-R plasmid was injected intramuscularly into the fish to carry out transcriptional studies of the 18S rRNA gene of T. bryosalmonae. Due to the marked seasonality of this parasitic disease, the treatment with the pBAFF-R plasmid was carried out during the month of July when an outbreak of PKD was detected, when cases of mortality associated with the characteristic symptoms of this disease appeared and, moreover, the temperature of the water at the fish farm was above 15° C.

[0094] Forty 30-40 g Rainbow trout were used from the Cifuentes fish farm (Guadalajara, Spain) and were divided into four groups (10 fish per group). Each fish was injected intramuscularly with 100 μl of sterile saline solution (0.9% NaCl) containing 0.1 or 1 μg of the pBAFF-R plasmid (two groups), as well as 0.1 or 1 μg of the empty plasmid without the construct in two other groups (negative controls). The fish of each group were sacrificed 110 days post-injection, in the month of November when the water temperature was below 15° C. and the outbreak of PKD had subsided at the fish farm. As expected, inflammation of the kidney was not observed in any case. A posterior kidney sample was taken from each trout for RNA extraction with Tri-reagent and subsequent cDNA synthesis using the methodology explained above in Example 2 was carried out.

[0095] The results show that the administration of a dose of 0.1 μg of the pBAFF-R plasmid did not induce a decrease in the expression of the 18S rRNA gene of T. bryosalmonae in the posterior kidney at 110 days post-injection (FIG. 4). However, when a dose of 1 μg of the pBAFF-R plasmid was administered, a decrease in the parasite load detected in the kidney at the transcriptional level was observed (FIG. 4).

[0096] Thus, these results indicate that the administration of the plasmid of the invention is capable of favouring the specific immune response of the infected organism against the parasite and its elimination from the organism, as well as controlling B-cell mediated inflammation of the kidney tissue, thus favouring the survival and symptoms of the infection caused by T. bryosalmonae, in Rainbow trout.