SEA LICE VACCINES

Abstract

Proteins derived from secretory/excretory products of Lepeophtheirus salmonis, including recombinant proteins, DNA encoding the proteins, vaccines and antigens comprising the proteins or the DNA, and uses thereof for the prevention or treatment of sea lice (Lepeophtheirus salmonis or Caligus rogercresseyi) in fish, and related methods of treatment.

Claims

1. A protein comprising the amino acid sequence of SEQ ID NO:1 or 2.

2. A recombinant protein comprising the protein defined in claim 1.

3. A vaccine against caligid copepod infection in fish, the vaccine comprising an immunologically effective amount of the protein according to claim 1, and a pharmaceutically-acceptable adjuvant, diluent or carrier.

4. (canceled)

5. The vaccine according to claim 3, wherein the caligid copepod is Lepeophtheirus salmonis or Caligus rogercresseyi.

6. The vaccine according to claim 3, wherein the fish is salmon.

7. DNA encoding the amino acid sequence of SEQ ID NO:1 or 2.

8. The DNA according to claim 7, wherein the DNA comprises the nucleotide sequence of SEQ ID NO:3 or 4.

9. A vaccine against caligid copepod infection in fish, the vaccine comprising an immunologically effective amount of the DNA according to claim 7 and a pharmaceutically-acceptable adjuvant, diluent or carrier.

10. (canceled)

11. The DNA or vaccine according to claim 9, wherein the caligid copepod is Lepeophtheirus salmonis or Caligus rogercresseyi.

12. The DNA or vaccine according to claim 9, wherein the fish is salmon.

13. An antigen comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, or the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:4.

14. (canceled)

15. (canceled)

16. (canceled)

17. A method of treatment or prevention of caligid copepod infection in fish, comprising administering a therapeutic amount of a protein comprising the amino acid sequence of SEQ ID NO:1 or 2, a recombinant protein comprising the amino acid sequence of SEQ ID NO:1 or 2, a DNA encoding the amino acid sequence of SEQ ID NO:1 or 2, a vaccine against caligid copepod infection in fish, or an antigen comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 or the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:4, optionally with the co-administration of an adjuvant.

18. The method according to claim 17, wherein the caligid copepod is Lepeophtheirus salmonis or Caligus rogercresseyi.

19. The method according to claim 17, wherein the fish is salmon.

Description

[0039] The invention will now be described by way of example with reference to the enclosed Figures, wherein:

[0040] FIG. 1 shows blood serum IgM antibody titer against SEQ ID NO:1 recombinant protein in individual Atlantic salmon parr vaccinated with SEQ ID NO:1 recombinant protein, negative control fluorescent protein or no vaccination control at 602 degree days post-vaccination at 14° C. Individual fish Log.sub.2 titers illustrated with mean±SEM (n=12 fish per group). Data analysed by one-way ANOVA, * signifies significant difference;

[0041] FIG. 2A shows on an SDS-PAGE gel stained with coomassie blue in which one microgram purified SEQ ID NO:4 recombinant protein per well was run;

[0042] FIG. 2B shows a western blot of SEQ ID NO:2 recombinant protein probed with serum from Atlantic salmon vaccinated with SEQ ID NO:1 recombinant protein; and

[0043] FIG. 3 shows the infection intensity of Lepeophtheirus salmonis chalimus on skin of Atlantic salmon smolts 7 days post-infection by immersion with copepodids at 60 copepodids per fish. Fish were challenged at 1508 degree-days post-prime vaccination with 50 μg recombinant protein SEQ ID NO:1, or adjuvant control containing 250 fluorescent protein, intra-dermally/subcutaneously and 1326 degree-days post-boost vaccination with 50 μg recombinant protein antigen with adjuvant Montanide™ ISA 763 A VG. Vaccination with recombinant protein SEQ ID NO:1 significantly reduced the mean sea lice infection intensity on smolts by 33.3% at 7 days post-infection compared to the no vaccination controls (p=0.0294). Data represented as mean number of chalimus settled on the host's skin±SEM. Data combined from 5 replicate cohabitation tanks, total n per treatment=30.

EXAMPLES

Example 1—Maintenance of Atlantic Salmon

[0044] Atlantic salmon parr were maintained in a well water flow through system at 13.5±1.0° C. and a dissolved oxygen (DO) level of 9.0±1.0 mg/L. Fish were fed daily at 1.5% of body weight with a commercial dry pellet, and water parameters monitored daily in each tank (temperature, DO, ammonia, nitrite).

[0045] After the 602 degree-day post-vaccination sampling day, all fish underwent smoltification by exposure to a 24 hour light regime for 2 weeks followed by the gradual introduction of seawater up to a maximum salinity of 32 ppt. Once the addition of seawater was started the system was switched to a full recirculation system at 13.5±1.0° C. and a dissolved oxygen level of 9.0±1.0 mg/L.

Example 2—Vaccination of Atlantic Salmon

[0046] Atlantic salmon parr were acclimatized in the experimental system for 25 days prior to vaccination. Purified recombinant protein consisting of SEQ ID NO:1 was delivered as a prime intra-dermal vaccination at a dose of 50 μg protein per fish in a total volume of 30 μl in sterile phosphate buffered saline (PBS; 3 injections of 10 μl per fish; n=48 fish; duplicate tanks of 24 fish per group). All fish were tagged under the jaw in order to identify treatment groups. Two hundred and ten degree-days post-prime vaccination, the fish received a booster vaccination containing the corresponding recombinant protein treatment delivered intra-peritoneal at a dose of 50 μg each recombinant protein in a total volume of 100 μl adjuvanted with Montanide ISA 763A VG (Seppic; following manufacturers' instructions). Fish were anaesthetized by immersion in MS-222 at a concentration of 100 mg/L in system seawater buffered with 100 mg/L sodium bicarbonate prior to vaccinations and tagging. Controls included: 1) no vaccination control and 2) control HN-tagged recombinant fluorescent protein at a dose of 250 μg recombinant protein per fish with Montanide ISA 763A VG adjuvant.

[0047] At 602 degree-days post-prime vaccination 12 Atlantic salmon were euthanized with 250 mg/L MS-222 buffered with 100 mg/L sodium bicarbonate. Blood sera samples were taken from the caudal vein. The blood was allowed to clot for one hour followed by clot retraction at 4° C. overnight and centrifugation at 3000×g for 7 minutes, and the serum frozen at −80° C. Serum samples were utilized to determine specific antibody titres via ELISA at 602 days post-vaccination. Western blot was performed in order to determine whether or not blood serum from Atlantic salmon immunized with SEQ ID NO:1 recombinant protein cross-reacted to SEQ ID NO:2.

Elisa

[0048] Elisa plates were coated with 100 μl per well 4 μg/ml SEQ ID NO:1 recombinant protein in carbonate:bicarbonate coating buffer (Sigma) overnight at 4° C. Plates were washed three times with low salt wash buffer (LSWB), blocked with 250 μl per well 3% (w/v) casein in PBS overnight at 4° C. Plates were washed again three times with LSWB and 100 μl fish sera diluted in PBS in doubling serial dilutions starting at 1/25 in duplicates were applied to wells, and incubated overnight at 4° C. Plates were subsequently washed five times with high salt wash buffer (HSWB) with a 5 minute incubation on the last wash. One hundred μl primary mouse IgG anti-salmonid IgM antibody (Biorad cat #MCA2182) at 1/500 in PBS was applied to the wells for 1 hour at 22° C., followed by five washes with HSWB and 100 μl of the conjugated secondary goat anti-mouse IgG—HRP (Sigma cat #A4416) at 1/1000 in 1% (w/v) BSA in 1×LSWB for 1 hour at 22° C. The plates were washed again five times with HSWB prior to the addition of 100 μl per well TMB substrate for 10 minutes. The reaction was stopped by the addition of 50 μl 2 M sulphuric acid, and the plates read for absorbance at 450 nm. Each plate contained relevant controls: 1) pooled positive serum, 2) pooled negative plasma, and 3) no serum controls. The titer was calculated as the reciprocal of the dilution that was above the cut-off i.e. above negative control mean A450 nm plus 3×SD. The coefficient of variation of the 450 nm absorbance of sample replicates within a plate, and pooled positive serum between plates was always ≤15%.

SDS-PAGE

[0049] One microgram of SEQ ID NO:2 recombinant protein was loaded onto a Bio-rad mini-protean TGX 4-15% precast gradient gel under reducing conditions with 2-B-mercaptoethanol sample buffer (Bio-rad). Samples were incubated at 95° C. for 5 min in reducing sample buffer. Gels were run at 200 V for 40 minutes followed by staining for 1 hour in 0.25% coomassie blue. Gels were de-stained in 40% methanol, 10% acetic acid until the desired contrast was observed.

Western Blot

[0050] One microgram of SEQ ID NO:2 recombinant protein was loaded onto a Bio-rad mini-protean TGX 4-15% precast gradient gel under reducing conditions with 2-B-mercaptoethanol sample buffer (Bio-rad). Samples were incubated at 95° C. for 5 min in reducing sample buffer. Gels were run at 200 V for 40 min, and then transferred to nitrocellulose membrane (Bio-Rad). Transfers were run at 30 V overnight at room temperature.

[0051] Post-blotting, membranes were blocked for 60 minutes in 1% (w/v) BSA in PBS. The membranes were then cut and probed with pooled polyclonal Atlantic salmon serum at 1/100, that was pooled from Atlantic salmon vaccinated with SEQ ID NO:1 recombinant protein, overnight at 4° C. The membranes were subsequently washed 3× with PBS-Tween 20 (0.1%; PBS-T) and then incubated with a monoclonal antibody specific for salmonid IgM at dilutions of 1/500 for 1 hour in 1% BSA in PBS-T. The membranes were subsequently washed three times with PBS-T and incubated with HRP-conjugated goat anti-mouse IgG at 1/3000 in 1% BSA in PBS-T, followed by detection with Opti-4CN™ substrate kit (Bio-rad) following manufacturers' instructions.

Sea Lice Cultivation

[0052] Filtered natural seawater which had undergone reverse osmosis to ensure a salinity of 32 ppt±2 ppt for the sea lice hatchery was obtained from the CCAR facility in Franklin, Me. and added to a 400-litre recirculating system. The system consists of 20 hatching chambers with a fine mesh screen bottom to allow aeration and to hold eggs/larval stages. The hatching chambers were held in 2 litre glass beakers submerged in the system water with an air stone underneath the hatching chamber. Seawater was maintained at a salinity of 32 ppt±2 ppt. The temperature was maintained at 11±1° C. Water quality was monitored and recorded daily for salinity temperature and dissolved oxygen content. The seawater in the beakers underwent a 50% change every other day to ensure good water quality.

[0053] Sea lice were collected from a commercial farm harvest. Egg strings from gravid females were removed and placed into the hatching chambers. Egg strings were sorted by coloration to house eggs of similar development together (darker eggs are more developed). Hatching chambers were checked daily to assess larval stages of the sea lice and unhatched egg strings moved to new hatching chambers daily to ensure larvae of similar ages were housed together. To assess larval development, the volume of the water in the hatching chamber was reduced to concentrate the sea lice then duplicate 1 mL samples were taken per hatching chamber. The samples were placed into a petri dish and larval stages were staged and viability assessed using a microscope.

[0054] Once the percentage of copepodids reached over 80%, the sea lice were used for infestation. Only copepodids which were 4 days old or less were used for infestations i.e. molted into copepodid ≤4 days ago.

Sea Lice Infestation

[0055] At 602 days post—prime vaccination all Atlantic salmon from the treatment groups were co-housed in six replicate 100 gallon tanks for sea lice challenge (n=6 fish per treatment per tank i.e. 36 fish per treatment total combined). Atlantic salmon were challenged with L. salmonis copepodids at 1508 degree-days post-prime vaccination using a bath challenge method to achieve a final infection level on the no vaccination controls of 10 to 20 lice per fish.

[0056] Briefly, the tank volume was lowered to ⅓rd original volume and the copepodids were added to each of the replicate tanks at three different locations to ensure sea lice were distributed throughout the tank to give 60 copepodids per fish. The dissolved oxygen was monitored continuously throughout the 1 hour bath infection to maintain DO at 8.5±0.5 mg/L. After an hour, the water flow was re-instated in order to fill the tank back to its original volume but then left static for a further 1.5 hours prior to re-instating water flow.

Evaluation of Sea Lice Infection

[0057] Seven days post-challenge (once the sea lice reach the late chalimus stage) the Atlantic salmon were euthanized by immersion in 250 mg/L MS-222 in system water until at least 10 minutes post-cessation of opercular movements. The number and stage of sea lice on the skin and gills was counted on each fish using a stereo-microscope. The number of sea lice in each developmental stage was tallied and recorded on the skin and gills separately. The treatment group that the fish belonged to were recorded, i.e. tag colour on left and/or right jaw line, as well as the weight and length of the fish. Skin samples with underlying muscle tissue were taken and fixed in 10% neutral buffered formalin for wax embedding, sectioning and haematoxylin and eosin staining, in order to evaluate the histopathology of the louse attachment site between treatment groups.

Results

[0058] Systemic specific IgM response:

[0059] Fish vaccinated with SEQ ID NO:1 recombinant protein were found to possess significantly higher specific IgM antibody titers against SEQ ID NO:1 recombinant protein in the blood serum at 602 degree-days post-vaccination than the fish vaccinated with the fluorescent protein control (p=0.049) or the no vaccination control group (p=0.0257; FIG. 1). [0060] Cross-reaction of serum specific IgM from Atlantic salmon vaccinated with SEQ ID NO:1 recombinant protein with SEQ ID NO:2 recombinant protein:

[0061] The blood serum from fish vaccinated with SEQ ID NO:1 recombinant protein contained IgM antibodies at 602 degree-days post-vaccination which recognized SEQ ID NO:2 recombinant protein via Western blot (FIG. 2). [0062] Efficacy:

[0063] Vaccination of Atlantic salmon parr with protein SEQ ID NO:1 significantly reduced the mean sea lice infection intensity on the skin of smolts at 7 days post-infection by 33.3% compared to the no vaccination controls (p=0.0294; FIG. 3).