NO343281B1

NO343281B1 - Peptides for the inhibition of trypsin and sea lice infestation.

Info

Publication number: NO343281B1
Application number: NO20162073A
Authority: NO
Inventors: Scott Mckinley
Original assignee: Aqua Health As
Priority date: 2016-12-30
Filing date: 2016-12-30
Publication date: 2019-01-14
Also published as: NO20162073A1

Abstract

Peptides which inhibit trypsin are described. Also described are pharmaceutical compositions comprising such peptides, and the use of such peptides and pharmaceutical compositions for the inhibition of sea lice infestation and for the prevention or treatment of infections in salmonids caused by sea lice.

Description

Field of the invention

The present invention relates to peptides and pharmaceutical compositions comprising such peptides for the inhibition of a trypsin protease from a Caligidae, or for inhibiting the infestation and infection of a caligid copepod in a salmonid, or for the prevention and/or treatment of infections in salmonids caused by sea lice.

Background of the invention

Sea lice present a large economic burden for fish farmers. Sea lice are obligate ectoparasitic copepods on the external surface of marine fish. The term sea lice commonly refer to Lepeophtheirus salmonis and Caligus rogercresseyi.

L. salmonis affects inter alia wild and farmed salmon and the rainbow trout industry in Scotland, Ireland, Norway, Faeroe Islands, the northern Atlantic and Pacific coasts of Canada and the U.S., and the Pacific coast of Japan.

The life cycle of L. salmonis consists of 10 different stages and lasts 7-8 weeks at 10<0>C. Naupilus and copepodid are free swimming and non-parasitic stages, and chalimus, pre adult and adult lice are attached and parasitic stages.

A characteristic feature of attachment and feeding sites of caligid copepods on many of their hosts is that a trypsin-like activity is secreted by L. salmonis onto the salmon skin. It is believed that it is used by the sea lice to feed on the salmon mucus, skin and blood and to protect the sea lice from the salmon immune response.

WO2006010265 describes recombinant vaccines against Caligidae copepods that comprises peptide fragments of L. salmonis trypsin.

NO20141508 describes peptides that induce an immune response of fish against copepods.

EP2623114 describes a vaccine composition containing a peptide of P0 ribosomal protein to control an ectoparasite infestation.

The salmon louse, L. salmonis, is known to secrete a number of proteases upon parasitic infection of its host Salmo salar. A trypsin-like serine protease appears to be the major protease secreted by the louse. This enzyme may provide a potential drug target for treatment of L. salmonis infection in farmed salmon. Based on the modelled 3D structure of the L. salmonis trypsin-1 protein (LsTryp1), 11 peptides have been designed with an amino acid sequence that should allow specific binding to LsTryp1 and inhibition of its protease activity. Here we test the LsTryp1 activity of these peptides against recombinant LsTryp1 that has been expressed in E. coli.

Summary of the invention

A first aspect of the present invention relates to a peptide, wherein said peptide inhibits a trypsin protease protein from a Caligidae, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 10 and SEQ ID NO 11.

In a preferred embodiment comprises the peptide the amino acids of SEQ ID NO 3.

In a preferred embodiment comprises the peptide the amino acids of SEQ ID NO 5.

In a preferred embodiment is said Caligidae selected from the group consisting of Pseudocaligus, Caligus and Lepeophtheirus.

In a preferred embodiment is said Caligidae a Lepeophtheirus salmonis (salmon louse).

In a preferred embodiment is said trypsin the trypsin-1 protein (LsTryp1) from L. salmonis. A second aspect of the present invention relates to a pharmaceutical composition for use against a caligid copepod infection in a salmonid, wherein said pharmaceutical composition comprises a peptide which inhibits a trypsin protease protein, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 10 and SEQ ID NO 11.

In a preferred embodiment comprises the peptide the amino acids of SEQ ID NO 3.

In a preferred embodiment comprises the peptide the amino acids of SEQ ID NO 5.

In a preferred embodiment is said trypsin the trypsin-1 protein (LsTryp1) from L. salmonis.

A third aspect of the invention relates to a process for inhibiting the infestation and infection of a caligid copepod on a salmonid, or for the prevention and/or treatment of infections in salmonids caused by sea lice, wherein a peptide or a pharmaceutical composition comprising said peptide is administered to said caligid copepod and/or said salmonid, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 10 and SEQ ID NO 11.

In a preferred embodiment comprises the peptide the amino acids of SEQ ID NO 3.

In a preferred embodiment comprises the peptide the amino acids of SEQ ID NO 5.

The sequences referred to above and in the claims are given in table 1.

Description of the drawings

Figure 1 shows a plasmid map of pET40-LsTryp1.

Figure 2 shows a portion of the sequencing data for LsTryp1 aligned to the expected sequence of pET40-LsTryp1. It shows the P159A mutation, which was the only amino acid change from expected based on published genomic L. salmonis sequence.

Figure 3 shows a Western blot for His-tag proteins isolated from BL21 with pET40-LsTryp1 (lane 1), pET-40b lacking the LsTryp1 gene (lane 2), pET40-LsTryp1 without induction of protein expression (lane 3) and protein size ladder (Fermentas; lane 4).

Figure 4 shows the analysis of LsTryp1 purification. A) Coomassie stained SDS-PAGE gel. B) Western blot for His-tagged proteins (same samples as in "A"). Lane 1: flow through of IMAC column, Lanes 2-4: Sequential elution from IMAC column, Lane 5: Protein ladder (NEB)

Figure 5 shows the effect on LsTryp1 activity by various peptides. Potent serine protease inhibitor, PMSF is included as positive control of trypsin inhibition.

Figure 6 shows gelatin zymograph of protease containing extracts from various sources. A) No inhibitor present. B) In the presence of a mixture of SEQ ID NO 3 (P3) and SEQ ID NO 5 (P5) at a concentration of 50µg/mL each.

Figure 7 shows the protease activity of rLsTrp after incubation in S. salar serum over 3 days as a measure of peptide stability in salmon blood. ∗denotes a statistically significant difference compared to serum without peptide collected at the same time point (p < 0:05).

Figure 8 shows a graph of a number of lice found on control vs. peptide treated salmon from in vivo experiment 1 (high peptide concentration). Observations made 10 days and 21 days post lice exposure. Error bars represent standard deviation.

Figure 9 shows a graph of a number of lice found and area of those lice on control vs. peptide treated salmon from in vivo experiment #2 (low peptide concentration). Observations made seven days post lice exposure and 17 days post administration of peptide. Error bars (`size' column only) represent standard error of the mean. ∗ indicates a statistically significant difference (P < 0:01).

Detailed description of the invention

We have prepared a number of different peptide sequences and tested their ability to inhibit a trypsin from sea lice.

Experimental section

Materials and methods

Strains and culture conditions

E. coli DH10B was used for cloning and production of plasmid DNA. Cultures were grown in ZYM-505 medium at 37°. DNA manipulations were performed using standard procedures. Plasmid DNA was purified using QIAprep Spin Miniprep Kit (Qiagen). E. coli BL21(DE3)pLysS (referred to as BL21 from here on) was used as a protein expression host for LsTryp1. BL21 was routine culture of BL21 was in MDAG-135 and protein expression was performed in auto-induction medium, MDA-5052. Antibiotics kanamycin (Km) and chloramphenicol (Cm) were used at 100 and 33 µg/mL, where appropriate.

Cloning, expression and purification of LsTryp1

Approximately 15 adult L. salmonis were harvested from infected salmon and placed in 10 mL RNAlater reagent (Qiagen). Total RNA was purified using the RNeasy Mini Kit (Qiagen). The RNA preparation was cleared of any genomic DNA carry-over by DNaseI digestion (Fermentas) followed by heat inactivation of the DNaseI enzyme. First-strand cDNA synthesis was performed using 1 µg L. salmonis RNA and M-MulV reverse transcriptase (200 U) primed with an oligo-dT primer in the presence of RNase inhibitor, by manufacturer’s instructions (NEB). PCR amplification of the LsTryp1 gene was performed with PrimeSTAR GXL DNA Polymerase (Clonetech) using primers F-LsTryp1_3 (5’-gttccccctcagatcaaatactctgag-3’) and R-LsTryp1_1 (5’-ctattggtgttcagcaatccagtcaatg-3’; IDT) with an annealing temperature of 60°C and 35 cycles. PCR product of the expected size was (672 bps) was confirmed by agarose gel electrophoresis and this product was purified by gel extraction using the NucleoSpin Gel and PCR Clean-up kit (MACHEREY-NAGEL GmbH & Co). PCR product of the LsTryp1 gene was ligated into pET-40b expression vector (Novagen) which had been digested with restriction enzyme, ScaI (NEB) using T4 DNA ligase (NEB). The product of this ligation was used to transform E. coli DH10B. Km resistant tranformants were screened by PCR for the presence of plasmid containing the LsTryp1 gene in the correct orientation. Plasmid DNA was extracted from a positive clone and sequence of the correct LsTryp1 gene was confirmed by DNA sequencing (Eurofins MWG Operon). This plasmid (pET40-LsTryp1) was introduced into E. coli BL21 for protein expression.5 mL culture was grown for 24 h in auto-induction medium at 30°C. Cells were then collected by centrifugation and lysed in BugBuster Protein Extraction Reagent (Novagen) supplemented with benzonase and lysozyme and purified over a Ni-NTA His•Bind Resin (Novagen) column. Presence of LsTryp1 was monitored by SDS-PAGE gel electrophoresis and western blot, performed by standard methods. SDS-PAGE gels were stained with coomassie blue dye. For western blotting, protein from SDS-PAGE was transferred to nitrocellulose membranes and probed with rabbit α-His-tag antibody and IR800 conjugated α-rabbit IgG antibody (Rockland Immunochemicals Inc.) visualized on an Odyssey CLx IR scanner (LI-COR Biosciences).

Protease inhibition assay

Protease activity of recombinant LsTryp1 protein was assayed using the Pierce Fluorescent Protease Assay Kit by manufactures instructions (Thermo-Fisher Scientific) in 384-well microtitre plate (White, low protein binding assay plate; Greiner Bio-One). Activity of LsTryp1 was measured in the presence or absence of each of the peptides of interest.

Lyophilized peptides were reconstituted in assay buffer (10 mM tris, pH 7.4) to make stock solutions with a final concentration of 1 mM. Peptides were added to the FITC-casein substrate to a final concentration of 100 µM. Recombinant LsTryp extracted from E. coli BL21 was drop dialyzed on a 0.025 µm, mixed cellulose esters membrane filter against assay buffer for 2 hours then diluted 1:2 in assay buffer.35 µL of this LsTryp1 solution was added to 35 µL of substrate with or without peptide for a final assay volume of 70 µL. Lysate extracted from of a culture of BL21 lacking the recombinant LsTryp1 gene, but otherwise processed in the same manor to the LsTryp1 extraction, was used as baseline to control for possible carryover of endogenous E. coli proteases. Serine protease inhibitor, phenylmethanesulfonyl fluoride (PMSF) was included as a positive control for trypsin inhibition. All test samples were assayed in quadruplicate. Protease digestion was allowed to proceed for 30 minutes after LsTryp1 addition before reading output on a SpectraMax M plate reader (Molecular Devices) with an excitation wavelength of 494 nm and emission wavelength of 521 nm.

Results

Cloning of the LsTryp1 gene and expression of active LsTryp1

PCR of the LsTryp1 gene with the primer sequences presented above produced a DNA fragment of consistent with the size expected for the PCR using L. salmonis cDNA as a template (672 base pairs). The PCR amplified fragment includes only the enzyme domain and lacks the signal peptide and activation peptide of the natural full-length transcript to avoid problems of improper processing to mature protease in the E. coli host.

Insertion of the LsTryp1 PCR product into the ScaI site of pET-40b results in an in-frame translational fusion of LsTryp1 to the upstream DsbC gene, separated by a 6x histidine tag (His-tag) and a thrombin cleavage site. The DsbC gene encodes a periplasm localized disulfide bond isomerase enzyme, which, fused to the LsTryp1 protein, should cause it to be excreted to the periplasmic space of E. coli. The periplasm provides a more favorable environment for the proper folding of disulfide bond-rich proteins (there are four disulfide bonds in LsTryp1) as does the enzyme activity of the DsbC fusion partner. The His-tag allows the protein to be purified by immobilized metal ion affinity chromatography (IMAC). A map of the plasmid is shown in Figure 1, with important elements annotated.

The Dsb-LsTryp1 fusion gene in pET40-LsTryp1 is downstream of a T7 polymerase promoter and relies on the presence of the T7 RNA polymerase protein to be present in the host cell for transcription. The BL21(DE3)pLysS expression host contains the gene for T7 RNA polymerase, but this gene is under control of the Lac repression (LacI), which is in a repressed state unless lactose is present and glucose is not. This allows controlled expression of our protease until which time these conditions are met. To limit potential toxic effects of LsTryp1 on the host cells, expression is delayed until late in the growth phase. This was achieved through the use of auto-induction medium, which contains both glucose and lactose in a specific ratio so that glucose is present and used as the main energy source while the culture is growing to sufficient density. At the point at which glucose (which is used by the bacteria preferentially to lactose) is used up by the host cells, lactose repression of the T7 RNA polymerase is lifted and high-level expression of the DsbC-LsTryp1 fusion begins. After a culture period of a length appropriate to allow for induction of T7 RNA polymerase expression and accumulation of the recombinant fusion protein, cells were harvested and a soluble protein extract was isolated.

Protein extracts were analyzed for presence of the DsbC-LsTryp1 fusion by western immune blot probed with an antibody specific to the 6x His tag epitope located in the linker region between the DsbC and LsTryp1. Figure 3 shows multiple reactive bands corresponding the expected sizes of the fusion protein and trypsin cleavage products of the full-length fusion protein. The full-length product is expected to be a protein of 49.2 kDa. The linker region between DsbC and LsTryp1 contains a trypsin cleavage site just a few amino acids up from the start of LsTryp1. Cleave by a protease with trypsin-like specificity would result in a 25.4 kDa fragment composed of DsbC and the 6x His-tag. The blot in Figure 3 clearly shows protein bands of these approximate sizes. A 27.1 kD fragment corresponding to the LsTryp1 protein should also be produced, but this would not be expected to react with the -His-tag antibody. A third band observed likely corresponds to a second trypsin cleavage fragment. These results show that the DsbC-LsTryp1 is expressed in the E. coli BL21. The presence of smaller fragments of the full-length protein of a size congruent with trypsin cleavage at an exposed trypsin cleavage site is consistent with expression of an active trypsin protease. However, it is possible that the cleavage observed is due to activity of endogenous periplasmic proteases, such as the trypsin-like serine protease DegP. Major involvement of DegP in cleavage of the DsbC-LsTryp1 fusion is unlikely as DegP is a heat shock protein and is not highly expressed at 30°, the temperature at which these cells were cultured.

Purification of LsTryp1

The presence of the 6x His-tag motif in the linker region between LsTryp1 and its fusion expression partner, DsbC, could allow purification of the full-length fusion protein through specific binding to a IMAC resin. However, as this fusion protein was found to have already been processed into its cleaved state, releasing the LsTryp1 portion from the 6x His affinity tag, purification over a Ni-NTA resin will likely result in major loss of the recombinant LsTrypsin enzyme. Such a purification was attempted anyway to verify this conclusion. When the soluble total protein fraction extracted from BL21 was subjected to IMAC purification, a band corresponding to the size of the free LsTryp1 cleavage product (27.1 kDa) did not bind to the resin, but was instead found in the column flow through (Figure 4A, lane 1), and not in significant amount in any of the fractions eluted from the column with imidazole. Western blots of these same fractions with an α-His tag antibody did strongly detect the presence of a the His-tag epitope in the flow-through fraction (Figure 4B), further supporting the conclusion that the 27.1 kDa band in this fraction is indeed recombinant LsTryp1.

Despite the finding that IMAC purification was not successful due to cleavage of LsTryp1 from the affinity tag before the chromatography step, the protein lysate flow through from the IMAC column was greatly enriched for the LsTryp1 band (Figure 4). This is likely due to trypsin cleavage of contaminating proteins during the IMAC process. The large excess of other soluble protein from the bacterial lysates (Figure 4A, lane 5) is reduced to the point that the LsTryp1 band is the major protein component after the lysate is subjected to the IMAC process, despite no specific purification of LsTryp1 having occurred. This observation again further strengthens the conclusion of the successful recombinant production of an active protease. Furthermore, the apparent digestion of the majority of contaminating host proteins by the protease activity of recombinant LsTryp1 provides a fortuitous purification method: auto-purification by protease digestion of host proteins.

Assay for inhibition by putative LsTryp1 inhibiting peptides

Protease activity of rLsTrp1 protein was assayed using the Pierce Fluorescent Protease Assay Kit by manufactures instructions (Thermo-Fisher Scientic) in a white, low protein binding, 384-well microtitre assay plate (Greiner Bio-One). Activity of rLsTrp1 was measured in the presence or absence of each of the 11 peptides of interest. Lyophilized peptides were reconstituted in assay buffer (10 mM tris, pH 7.4) to make stock solutions with a final concentration of 1 mM. Peptides were added to the FITC-casein substrate to a final concentration of 100 µM. rLsTrp extracted from E. coli BL21 was drop dialyzed on a 0.025 µm, mixed cellulose esters membrane filter against assay buffer for 2 h then diluted 1:2 in assay buffer. 35 µL of this LsTrp1 solution was added to 35 µL of substrate with or without peptide for a final assay volume of 70 µL. All test samples were assayed in quadruplicate. Protease digestion was allowed to proceed for 30 minutes after rLsTrp1 addition before reading output on a SpectraMax M plate reader (Molecular Devices) with an excitation wavelength of 494 nm and emission wavelength of 521 nm.

Protease activity of recombinant LsTryp1 was measured in the presence of 11 potential inhibitory peptides as given in table 1. Two of the 11 peptides (SEQ ID NO 3 (P3) and SEQ ID NO 5 (P5)) displayed an inhibitory activity indistinguishable in magnitude from that of the potent serine protease inhibitor, PMSF, at the same molar concentration. This data(figure 5) suggests that these two peptides are highly potent inhibitors of LsTryp1 activity, with activity comparable to small molecule inhibitors, but with the potential for much great target specificity. Proteases, such as LsTryp1, are important effectors of L. salmonis infection of salmon.

Table 1

Amino acid sequences for the peptides tested for inhibiting of LsTryp1

SEQ ID NO 1 VPEPVVQSPITTTT

SEQ ID NO 2 VRCGRGRMLLLIERGQRFAT

SEQ ID NO 3 VLGEEWHMEGCM

SEQ ID NO 4 LMSFRKDAVMAIM

SEQ ID NO 5 KLTINDASLC

SEQ ID NO 6 TTTLLLHFTFDFNRVGV

SEQ ID NO 7 DVVQVGYQMDHII

SEQ ID NO 8 LNELETEMV

SEQ ID NO 9 LKFMLPRRWCTGC

SEQ ID NO 10 KFLHLDFNNGL

SEQ ID NO 11 TPYYTTVNPTSPYTYDV

Effect of LsTrp inhibitory peptides on trypsins of salmon, lobster and mammalian origin

Zymography, a technique to detect activity of individual proteases in a complex sample, was used to determine the effect of the SEQ ID NO 3 (P3) and SEQ ID NO 5 (P5) mixture on trypsins for other organisms. A lysate of the E. coli strain used to express rLsTrp was used as the LsTrp source, and homogenates of intestines from S. salar and lobster were used as samples containing trypsin from these organisms. Purified bovine trypsin was available as a source of mammalian trypsin. Zymography analysis was done by standard procedures.

Proteins of these samples were separated by SDS-PAGE under nonreducing conditions and without first boiling the samples in gels co-polymerized with 0.1% gelatin, which is used as the protease substrate. After electrophoresis SDS was removed from gels so that proteins could renature into their active state by incubation in renaturing buffer in the presence and absence of a peptide mixture of SEQ ID NO 3 (P3) and SEQ ID NO 5 (P5) at 100 µmol each for 2 hours. The gels were then fixed stained and destined as described previously for SDS-PAGE. Due to the gelatin present in the gel, the entire gel will stain positive for protein, except for regions were the gelatin has been removed through the action of proteases present within the gel. The zymograph shown in Figure 6 shows light bands of protein clearing in the lane corresponding to rLsTrp in the control gel not exposed peptide, but this clearing was absent in the gel incubated with the inhibitory peptide mixture. The other samples containing proteases from other organisms all showed bands of clearing despite the presence of SEQ ID NO 3 (P3) and SEQ ID NO 5 (P5), suggesting that these peptides do not display inhibitory activity against these proteases.

Stability of LsTrp inhibitory peptides in salmon serum

The stability of peptides SEQ ID NO 3 (P3) and SEQ ID NO 5 (P5) in salmon blood was indirectly assayed by incubating the peptides in the serum fraction of freshly collected salmon blood for various times up to 3 days then analysing the protease activity of rLsTrp in the presence of serum plus peptide mixture. Peptides were reconstituted fresh from lyophilized aliquots at 10 mg/mL in 10 mM tris, pH 7.4 an added to the serum fraction of fresh salmon blood to a final concentration of 1 mg/mL each. Serum needed to be diluted 1:1 with the same tris solution used to reconstitute the peptides to avoid precipitation of serum proteins upon addition of peptides. The mixture of serum and peptides was incubated at room temperature with samples removed immediately after addition of peptide then again 1 day, 2 day and 3 days after addition of peptides. Removed portion was passed through a spin column membrane filtration device with a molecular weight cut-off of 10 kDa, which would allow the peptides to pass through (MW of 2.3 and 1.6 kDa for SEQ ID NO 3 (P3) and SEQ ID NO 5 (P5), respectively, while retaining proteins and larger peptides from the serum which should remove any serum peptidases. Salmon serum without peptides added (but also diluted in 10 mM tris and treated identically all other ways) was incubated and collected alongside the peptide and serum mixture, for use as a negative control. Samples were stored at -80°C until the protease assay was performed. Protease assay was performed as described above. Protease activity of rLsTrp in the presence of peptides mixed with salmon serum still show strong inhibitory activity when collected immediately after combining with the serum. Peptides that had been incubated in salmon serum for 1, 2 and 3 days showed a reduced ability to inhibit protease activity of the rLsTrp enzyme at the longer time points. This suggests that the peptides are present in their active form in the serum, but begin to loose potency (likely due to degradation by serum proteases) over a time range relevant to the 3 day period observed here. The fact that the a reduction in activity could still be measured going out three days for the peptides in fresh serum is a strong indication that they possess sufficient stability for in vivo activity to be possible. The results are shown in figure 7.

Effects of the various peptides in protecting live salmon from sea lice infection

High peptide concentration

Salmon of the treatment group were anesthetized in 50 mg/L tricaine methanesulfonate (TMS) were injected with a mixture of 100 mg each peptide P3 (SEQ ID NO3) and P5 (SEQ ID NO 5) suspended in 0.1 mL sesame oil. A control group of an equal number of salmon received sesame oil alone. Each group contained 12 fish for 24 in total. After a two hour period to allow recovery from the anesthetic, both treatment and control fish were infected with L. salmonis at 650 lice per fish in a single infection tank then and split into separate tanks after an hour of exposure to the copepodites. Three fish from each group were sacrificed and examined for lice presence on the gills and body after 3 days, 10 days and the remaining from each group were observed 21 days post lice exposure. At 3 days lice were very and accurate counts were not possible, so the first time point with usable data was 10 days. At the 10 day time point about two thirds of the lice were still present on the gills and few had matured to the stage at which they would be found on the body. At this time point and average of 24 lice were observed on the control fish, but only a single louse could be found on the peptide treated salmon combined. At the 21 day time point all fish of the treatment group were all healthy and completely free of lice. The control group, however, were thoroughly infested (Figure 8) and morbidly ill.

Low peptide concentration

Fish were injected peritoneally with a mixture of 50 mg each of peptide P3 (SEQ ID NO 3) and P5 (SEQ ID NO 5) in 0.1 mL sesame oil (treatment group), with an equal number of fish receiving 0.1 mL sesame oil alone (control group). Ten days following administration of peptide fish were exposed to L. salmonis at 650 lice per fish. Seven days later fish were sacrificed by lethal dose of TMS immediately before observing fish for level of lice infestation.

As the fish were removed from the tanks it was noticed that the control fish were obviously more lethargic than fish from the peptide-treated group, as determined by a clear lack of ght upon transfer from the experimental tank. After sacrifice, salmon were photographed to document external physical injuries. Individuals from both groups displayed injuries consistent with L. salmonis infection, but severity of lesions found on control fish (C1-3) were striking in comparison to the treatment group (T1-3). Unfortunately, pictures taken to document fish in this experiment were not of sufficient quality to distinguish lice on the fish or other details such as damage to salmon surface. Fish C3 showed extensive damage to the base of the dorsal fin. Other individuals of both groups also exhibited lesions in this region, but not to the gruesome extent seen with C3. This same fish was barely responsive during transfer from the experimental tank, and appeared near death at the time of sacrifice. The treatment group individuals did not display significant injuries skin consistent with L. salmonis parasitism, but to an extent qualitatively less severe than that of the control group that did not receive the peptide treatment.

Lice were removed from the surface of each fish and counted. Many lice had become detached from the fish and could be recovered from the TMS solution in which the fish were sacrificed for both treatment groups. Control fish were associated with a total lice count of 52, compared to 30 for the treatment group (a 42.3% reduction; Figure 9).

Claims

1. A peptide, wherein said peptide inhibits a trypsin protease protein from a Caligidae, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 10 and SEQ ID NO 11.

2. A peptide in accordance with claim 1, wherein the peptide comprises the amino acids of SEQ ID NO 3.

3. A peptide in accordance with claim 1, wherein the peptide comprises the amino acids of SEQ ID NO 5.

4. A peptide in accordance with claim 1, wherein said Caligidae is selected from the group consisting of Pseudocaligus, Caligus and Lepeophtheirus.

5. A peptide in accordance with claim 4, wherein said Caligidae is Lepeophtheirus salmonis.

6. A peptide in accordance with any of the preceding claims, wherein said trypsin is L. salmonis trypsin-1 protein.

7. A pharmaceutical composition for use against a caligid copepod infection in a salmonid, wherein said pharmaceutical composition comprises a peptide which inhibits a trypsin protease protein from a Caligidae, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 10 and SEQ ID NO 11.

8. A pharmaceutical composition in accordance with claim 7, wherein the peptide comprises the amino acids of SEQ ID NO 3.

9. A pharmaceutical composition in accordance with claim 7, wherein the peptide comprises the amino acids of SEQ ID NO 5.

10. A pharmaceutical composition in accordance with claim 7, wherein said Caligidae is selected from the group consisting of Pseudocaligus, Caligus and Lepeophtheirus.

11. A pharmaceutical composition in accordance with claim 7, wherein said Caligidae is salmon louse Lepeophtheirus salmonis.

12. A pharmaceutical composition for use in inhibiting the infestation and infection of a caligid copepod on a salmonid, or for the prevention and/or treatment of infections in salmonids caused by sea lice, wherein said peptide or pharmaceutical composition comprising said peptide is administered to said caligid copepod and/or said salmonid, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 10 and SEQ ID NO 11.

13. Pharmaceutical composition in accordance with claim 12, wherein the peptide or pharmaceutical composition is administered to said salmonid by injection.

14 Pharmaceutical composition in accordance with claim 12, wherein the peptide or pharmaceutical composition is administered to said salmonid orally.

15. Pharmaceutical composition in accordance with claim 14, wherein said peptide or pharmaceutical composition is comprised in a feed composition.