Field of the invention

The present invention relates to peptides and a pharmaceutical composition comprising such peptides for inhibiting the attachment and infection of a parasite to an animal. Preferably, the parasite is a caligid copepod and the host is a salmonid. The invention provides a vaccine and a feed for the prevention and/or treatment of infections in salmonids caused by a caligid copepod

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, Iceland, Faeroe Islands, the northern Atlantic and Pacific coasts of Chile, Canada and the U.S., and the Pacific coast of Japan.

Objects of the invention

It is an object of the invention to provide a vaccine which effectively inhibits the attachment of a parasite to a host animal. Preferably, it is an object of the invention to inhibit the attachment of a parasite of a long enough period to seriously damage the host animal.

It is a further object of the present invention to provide a vaccine which effectively inhibits an infection caused by a parasite on a host animal.

It is a further object of the invention to identify and provide peptides that inhibits attachment of parasites to host animals, and which also effectively inhibits the infection caused by the parasite on the host animal. It is preferably an object of the invention to provide peptides that inhibit the attachment and infection of caligid copepods on salmonids.

Summary of the invention

A first aspect of the invention relates to a peptide for use in inhibiting the infestation or attachment of a parasite to an animal, or for the prevention and/or treatment of infections of an animal caused by a parasite, wherein said peptide is administered to said animal, and wherein said peptide comprises an amino acid sequence of a parasitic protein transferred to said animal.

In a preferred embodiment is the parasite protein transferred to said animal isolated from a blood sample of said animal infected by said parasite.

In a preferred embodiment comprises the peptide an amino acid sequence from a part of the protein present on the surface of either a 14-3-3 epsilon protein, a peroxiredoxin-2 or Proteasome subunit alpha.

In a preferred embodiment has the peptide a length of 5 to 30 amino acids, more preferably 10-25 amino acids, and more preferably 15-20 amino acids.

In a preferred embodiment is the peptide selected from the group consisting of SEQ ID NO 1 (Ser-Gly-Asn-Ala-Glu-Ala-GIn-Pro-Glu-Ala-Ala-Ala-Ser) and SEQ ID NO 2 (Asn-Lys-Glu- Phe-Lys-Glu-Val-Ser-Lue-Lys-Asp-Tyr-Thr, and variants thereof being at least 70% identical over the entire sequence with the sequences SEQ ID NO 1 , SEQ ID NO 2, SEQ ID NO 3 or SEQ ID NO 4, respectively.

In a preferred embodiment is said animal a fish, preferably wherein the fish is a salmonoid, preferably wherein the salmonoid is selected from the group consisting of Atlantic salmon, coho salmon, Chinook, rainbow trout, and Arctic charr.

In a preferred embodiment is the parasite an ectoparasite, preferably a Caligidae, and more preferably wherein the Caligidae is selected from the group consisting of Pseudocaligus, Caligus and Lepeophtheirus, preferably Lepeophtheirus salmonis.

A second aspect of the present invention relates to a pharmaceutical composition for use in inhibiting the infestation or attachment of a parasite on an animal, or for the prevention and/or treatment of infections of parasite on an animal, wherein said pharmaceutical composition is administered to said parasite and/or animal, wherein said pharmaceutical composition comprises a peptide comprising an amino acid sequence of a parasite protein released to said animal.

In a preferred embodiment is the parasite protein released to said animal isolated from a blood sample of said animal infected by said parasite.

In a preferred embodiment comprises the peptide an amino acid sequence from a part of the protein present on the surface of either the 14-3-3 epsilon protein, peroxiredoxin-2 or Proteasome subunit alpha.

In a preferred embodiment has the peptide a length of 5 to 30 amino acids, more preferably 10-25 amino acids, and more preferably 15-20 amino acids.

In a preferred embodiment is the peptide selected from the group consisting of SEQ ID NO 1 (Ser-Gly-Asn-Ala-Glu-Ala-GIn-Pro-Glu-Ala-Ala-Ala-Ser) and SEQ ID NO 2 (Asn-Lys-Glu- Phe-Lys-Glu-Val-Ser-Lue-Lys-Asp-Tyr-Thr) and variants thereof being at least 70% identical over the entire sequence with the sequences SEQ ID NO 1 and SEQ ID NO 2.

In a preferred embodiment is the animal a fish, preferably wherein the fish is a salmonoid, preferably wherein the salmonoid is selected from the group consisting of Atlantic salmon, coho salmon, Chinook, rainbow trout, and Arctic charr.

In a preferred embodiment is the parasite an ectoparasite, preferably a Caligidae, and more preferably wherein the Caligidae is selected from the group consisting of Pseudocaligus, Caligus and Lepeophtheirus, preferably Lepeophtheirus salmonis.

In a preferred embodiment is the pharmaceutical composition administered to said salmonid by injection.

In a preferred embodiment is the pharmaceutical composition administered to said salmonid orally.

A third aspect of the present invention relates to a vaccine for use in inhibiting the infestation or attachment of a parasite on an animal, or for the prevention and/or treatment of infections of parasite on an animal, wherein said vaccine comprises a pharmaceutical composition according to any of the aspects and embodiments above.

A fourth aspect of the invention relates to a feed composition comprising conventional feed ingredients such as proteins, fat and carbohydrates, wherein said feed composition comprises a peptide comprising an amino acid sequence of a parasite protein released to said animal.

In a preferred embodiment is the parasite protein released to said animal isolated from a blood sample of said animal infected by said parasite.

In a preferred embodiment comprises said peptide an amino acid sequence from a part of the protein present on the surface of either the 14-3-3 epsilon protein, peroxiredoxin-2 or Proteosome subunit alpha.

In a preferred embodiment has the peptide a length of 5 to 30 amino acids, more preferably 10-25 amino acids, and more preferably 15-20 amino acids.

In a preferred embodiment is the peptide selected from the group consisting of SEQ ID NO 1 (Ser-Gly-Asn-Ala-Glu-Ala-GIn-Pro-Glu-Ala-Ala-Ala-Ser) and SEQ ID NO 2 (Asn-Lys-Glu- Phe-Lys-Glu-Val-Ser-Lue-Lys-Asp-Tyr-Thr), and variants thereof being at least 70% identical over the entire sequence with the sequences SEQ ID NO 1 and SEQ ID NO 2.

In a preferred embodiment is said feed composition fed to a fish, preferably wherein the fish is a salmonoid, preferably wherein the salmonoid is selected from the group consisting of Atlantic salmon, coho salmon, Chinook, rainbow trout, and Arctic charr.

Description of drawings

Figure 1 shows the 8 stages of the life cycle of a parasite salmon louse.

Figure 2 shows the reduction in lice count at 40 days post infection (dpi).

Figure 3 shows how the length of the eggstring of a louse is measured.

Figure 4 shows the results of the common-garden experiment. 4a shows the results for Rainbow trout challenged with Lepeophtheirus salmonis and 4b shows the results for Atlantic salmon challenged with Lepeophtheirus salmonis.

Figure 5 shows the effect of vaccine B on salmon challenged with sea lice

Figure 6 shows the effect of re-challenging of the salmon of figure 5 with sea lice.

Figure 7 shows the effect of vaccine B on total lice count of lice at adult stage. Detailed description of the invention

We have prepared several different peptide sequences and tested their ability to inhibit attachment and infection of a parasite on a salmonid.

Life cycle of the parasite salmon lice consist of 8 stages like, 2 planktonic nauplii, 1 infective copepod, 2 attached chalimus, 2 mobile pre-adult and 1 adult as shown in figure 1 (Illustration: “SLRC Lepeophtheirus salmonis life cycle” by Sea Lice Research Centre).

Chalimus stage is of particular interest in vaccine development because during this stage lice is assumed to have its first interaction with the host through the development of a frontal filament (FF). Studies about FF is incomplete, and its main function is supposed to be as an attachment structure which re-grows during each molting until the parasite reach pre-adult stage. Studies have also mentioned its association with frontal and lateral glands of lice which shed light to its role more than attachment. There is a possibility that lice use its FF to transfer an unknown secretion (e.g. anti-coagulant, immuno-suppressant etc.) into its host which can be the reason for the low/no response of fish to lice infection and can aid in their easy attachment to the host surface. The later argument although not proven has been the rationale for the present invention.

Thus, blood samples collected from salmon during previous vaccination trial (In 2019 a pilot study similar to the current trial was conducted in Matre research station, a facility by Institute of Marine Research, Bergen). The experimental set up consisted of four tanks with 14 fishes (Atlantic salmon post-smolt, 200 g) in each and vaccinated with different vaccine candidates that the present along with the adjuvant as control. Later, the fishes were challenged with salmon lice (L. salmonis) copepods (80 copepods per fish). The vaccine candidates did not show any effect in reducing the lice load per fish and the experiment was closed but the blood sample from a few fishes from the control group was taken and analyzed to study the lice protein present. The sample was subjected to high resolution mass spectrometry (Q- exactive MS) based proteomic analysis to identify lice proteins present in salmon blood. The proteomic data obtained was compared to proteins present in lice-attached-salmon skin and vaccine candidates for current trial was chosen. Blood samples was analyzed using proteomics service of University of Oslo, Oslo (https://www.mn.uio.no/ibv/enqlish/ research/sections/bmb/research-groups/enzymology-and-protein -structure-and- function/proteomics-thiede/proteomics-service/ ) and data was viewed and interpreted using free version of Peak studio (https://www.bioinfor.com/peaks-studio/)), whereas the skin samples was analyzed at MS/Proteomics core facility of Faculty of Chemistry, Biotechnology and Food science, Norwegian University of Life sciences, As. However, not many lice proteins were detected in salmon blood and none of them were directly related to blood clotting or immune suppression. Still, a few proteins were found to be of interest and were looked upon more closely.

We do not know the exact biological action mechanisms which salmon lice use when attacking its host, but our main purpose was to design a vaccine which can effectively eliminate the attached parasite by disrupting its life cycle by targeting the early blood feeding stage of parasite.

Based on this, 12 potential protein candidates with important functions in lice-salmon interactions such as blood feeding, reproduction, parasites life cycle, feeding, digestion, molting, immunity and detoxification were selected from the proteomics data. Selection of vaccine candidates is very crucial among the other criteria’s for studying the overall efficacy of vaccine. Mainly, they should be an antigen which is immune stimulating, non-allergenic, promiscuous, and available at the surface. Thus, 3 lice proteins (1433 epsilon, peroxiredoxin 2 and proteasome subunit alpha) among 12 which was present in salmon blood and were of importance in other blood sucking parasitic species were selected as vaccine candidates. Unique peptide sequences (peptides) of 15-20 amino acids in length, hydrophobic and present on surface of the three selected proteins were selected as vaccine candidates, and these peptides were produced and tested.

Experimental section

Materials and methods

Selection of protein and peptide candidates, preparation of vaccines and observations on fish health and lice qrowth after challenge Selection of protein and peptide candidates

14-3-3 protein epsilon

14-3-3 proteins are ubiquitous regulatory eukaryotic adaptor proteins involved in many cellular processes such as cell-cycle control, signal transduction, protein trafficking, and apoptosis. They exist in different isoforms and are conserved across the species and mainly exist as dimers and by binding to other proteins, they can assist in protein folding, protein localization, and stimulation or inhibition of other protein-protein interactions.

We compared the 14-3-3 epsilon sequence of lice (L. salmonis) with salmon (S.salar) to select unique part of sequence, i.e. lice sequences that were not present in the salmon variant. We also looked for sequences that potentially were on the surface of the protein, and that also were hydrophobic. The sequence Ser-Gly-Asn-Ala-Glu-Ala-GIn-Pro-Glu-Ala-Ala- Ala-Ser) (SEQ ID NO 1 ) was identified and selected as a possible good candidate peptide.

Peroxiredoxin-2

Peroxiredoxins (Prxs) are highly abundant thiol-specific proteins that mainly function against hydrogen peroxide (H2O2), organic hydroperoxides, and peroxynitrite within living organisms. Additionally, Prxs function as redox sensors, cell signaling pathway and inflammation modulators, and barriers for cancer cell formation. The production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the body of organisms can be enhanced via in vivo metabolic reactions, such as aerobic respiration, which results in oxidative stress. The imbalance of ROS/RNS levels may lead to establishment of oxidative stress conditions. The overproduction of ROS is counteracted by a well-defined antioxidant system, which comprises Prxs, superoxide dismutase, catalase, and thioredoxin (Yu, B.P. 1994). The catalytic mechanism and structural arrangement of the Prxs are divided into six different classes based on sequence (Prx1-6). These classes are further divided into two main groups in accordance with the number and position of cysteine (Cys) residues at the active site: 2-Cys Prxs and 1 -Cys Prxs (Prx6). Selected proteins sequences of lice were matched to salmon using NCBI-blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi ) and area (15-20 Amino acid in length) that was unique to lice was selected using MUSCLE multiple sequence alignment tool (https://www.ebi.ac.uk/Tools/msa/muscle/). Furthermore, its position in the 3D structure was identified using homology modelling using bioinformatic tools like Roberta (http://robetta.bakerlab.org/ ) and SWISS-MODEL (https://swissmodel.expasy.org/). If a protein is available with a match identity of 60% or more then it is selected as a reference to model lice protein structure because structures of lice proteins are not available yet) and that part of protein present on the surface was selected as the candidate antigen. Moreover, its hydrophobicity was checked using the total hydrophobic-hydrophilic amino acids present in the sequence.

Based on this investigation, we identified and selected two peptide sequence candidates from peroxiredoxin-2.

Asn-Lys-Glu-Phe-Lys-Glu-Val-Ser-Lue-Lys-Asp-Tyr-Thr (SEQ ID NO 2)

Ser-Tyr-Lue-Ala-Asp-Ala-Glu-GIn-Ser-Lys-Lys (SEQ ID NO 3)

Proteasome subunit

The proteasome is a multicatalytic proteinase complex, which is characterized by its ability to cleave peptides with Arg, Phe, Tyr, Leu, and Glu adjacent to the leaving group at neutral or slightly basic pH.

Based on the assessment of unique lice sequences, hydrophobicity, etc., one sequence was identified and selected as a vaccine candidate.

Arg-Lys-Ala-Arg-Lys-Met-Ser-Glu-Glu-Tyr-Thr-Met-lle (SEQ ID NO 4) These four peptide sequences SEQ ID NO 1 to SEQ ID NO 4 were tested in vaccines against parasites (see below).

Preparation of vaccines

The candidate peptides were produced and delivered as lyophilized powder by Proimmune (www.thinkpeptides.com) and emulsified (using SEPPIC's protocol) into vaccines using a suitable adjuvant for salmon, Montanide ™ ISA 763 A VG from SEPPIC, France (https://www.seppic.com/en/montanide-isa-w-o). A total of 6 vaccines were formulated namely, the peptide identified from the 14-3-3 protein (SEQ ID NO 1 ) (vaccine A), the peptides from peroxiredoxin (vaccine B (SEQ ID NO 2) and vaccine C (SEQ ID NO 3)), and the peptide from PSA (SEQ ID NO 4)(vaccine D), and a combination of vaccine A (SEQ ID NO 1) and B (SEQ ID NO 2) (named as vaccine E) and combination of A, B, C, D (named as vaccine F), and adjuvant alone was used as control as given in table 1 below.

The emulsified vaccines were kept in refrigerator (4 °C) until use and will be stored in the same temperature up to one year. Tanks contained 14 fish each, and the vaccine dosage was 1.5 pg protein for 1 gm fish approximately. Therefore, 300 pg (100 μl) w) as injected in each fish which weighed approximately 215 g average. Fishes were starved 2 days before and 2 days after vaccination.

Table 1

Challenge trial was conducted in Matre research station, a facility by Institute of Marine Research, Bergen. Post-smolt salmon (one week after transferred from fresh water to sea water) for the study was maintained at 12 °C and 20-25 ppt salinity initially. During vaccination, they were carefully netted from the tank into 10 I bucket and were anesthetized using Finquel (1 g in 10 I of sea water for 1 min) and vaccinated using 9 mm needle syringe. Needle was changed between each tank to avoid cross contamination of vaccines.

One week after vaccination, salinity was raised to 34 ppt, and water level maintained at 400 I, 24 hr light and 24 hr feeding. Feed was purchased from Skretting and was of standard size 3 mm (Nutra Olympic 3.0, Skretting, Norway) and using an automatic feed dispenser was continuously fed to fishes without ration. No mortality was observed after vaccination. After 28 days (13.5 x 28 = 378 degree days), water level was reduced to 15 cm and water current reduced to half, and lice copepods (approximately 80 per fish) was distributed evenly to all tanks. After 10 min, water level and circulation was brought back to normal. It is to be noted that, the copepods used does not belong to wild lice and is a lab bred strain which was inbred for 50-60 generations in Institute of Marine Research lice facility.

Observations on fish health and lice growth after challenge

On the day of challenge (day1 ), the majority of the fishes were pale white in color without any prominent dark spots, but after 24 hrs of infection, one fish from group A started developing dark black and blue spots whereas others remained pale white. Whereas, in group B and C, majority had developed dark black spots when compared to the E, F, D and control group which had faint black spots. In general, all fishes in all vaccine groups looked healthy and were swimming well. D group always stayed near the water inlet until the final days of experiment. Development of dark spots after copepod infection suggests a host response in early stages in salmon.

On 3 dpi (day post infection), group C developed intense dark black spots whereas group A and B had more of visible blue spots. In previous studies in the same lab, it has been observed that the blue spots disappear quickly when fishes are transferred to fresh water. Group A, B and C had more blue spots in general when compared to the rest. It can be some reaction in host due to parasite development but no correlation between spots and early lice stages has been stated so far. Fishes were actively swimming on this day.

On 4 dpi, in all groups on average 30 - 40 spots were prominent. Group C had more dark spots and developed slight pink coloration whereas A, D, E still were white and pale. In F, one fish had developed many blue spots than the rest (7-10). In control, spots were less with respect to majority of vaccinated tanks. Feces consistency also had changed slightly in vaccinated tanks. The vaccinated fishes were compared to non-vaccinated fishes maintained in the same facility to get an unbiased opinion of spots and coloration.

On 5 dpi, lice counting was conducted for 1 fish per vaccine group to identify chalimus stage. It is difficult at this stage to differentiate between chalimus 1 and 2 therefore, a total chalimus count is taken. On this day, fishes seem to be rubbing and banging their body against tank walls and filter. They had also lost their shoaling behavior when compared to their unchallenged counterparts. Group C and F was different from the rest at this stage with more prominent dark spots.

For counting, one fish per group was chosen and immersed in an anesthetic (Aquacalm (methomidal), 1 ml :10 I in sea water) which will sedate the fish but will not affect lice. The generally used anesthetic (Finquel) is not good for lice therefore an alternative was used. Location of lice was observed, and the majority was found to be on the dorsal area and around the operculum and none on the blue spots. We assume the blue spot was the area where copepods started infecting on day 1 . Lice on the gills was avoided from counting in all cases. All lice were in chalimus stage and the numbers are as given in table 2 below,

Table 2, 5 dpi Lice was counted by expert who was well trained for counting but still, these numbers cannot be considered accurate due to its small size and difficulty in identification. Although care was taken to replace the detached lice back into its assigned tank after netting, there is a possibility that some has gone unnoticed and can affect the number at later stages. Some reduction can also be due to loss of some lice during the cleaning of tank bottom which was done once a week.

On 6 dpi, all fishes were swimming well and there was no increase in the number of blue spots. One fish in group F has developed curved caudal peduncle (like in scoliosis) and hereafter was seen laying at the bottom with very slight movement.

On 7 dpi, it appeared that the blue spots were slowly starting to fade. This again suggests the possibility of blue spot related to early lice stage attachment. There is a high chance that it has some connection with frontal filament attachment or cementing secretions of Chalimus stage, (note: Salmon can turn completely blue when stressed. Blue pigments are cyanophores; one among the 4 color pigments). On this day chalimus has become visible on fish, mainly on the dorsal, ventral and adipose fin.

On 8 dpi, blue spots disappeared from majority of the groups especially control, but fishes showed irritation on the attachment spots and were rubbed their body on the tank sides to avoid lice.

On 9 dpi, the fishes were swimming, but they were not very active and was still rubbing the body against the tank wall. They always took turns among each other to position themselves in front of the water inlet to get rid of attached lice. Group C and F were promising at this stage due to its low lice occurrence when viewed externally. However, group C turned to be the most infected vaccine group at later stages (group C is an example which shows the difficulty in lice detection in early stages). Fish behavior and coloration in each vaccine groups were different suggesting the vivid effect of vaccine.

On 10 dpi, counting was done in one fish from each group as explained previously. Gills were excluded from counting. Here, we should note that there can be always a chance of few lice which are in the transition phase from chalimus 2 to pre-adult 1 and it is very difficult to assign them to correct stage so a few which were identified in chalimus 2 state could actually be a pre-adult 1 lice.

Table 3, 10 dpi

Majority of lice were attached to dorsal fin, dorsal side, ventral fin, pectoral and caudal fin and some were seen distributed throughout the body surface. There were slight scale loss and redness in group C.

On 11 dpi, large sized lice (when compared to the rest) was seen in group A along the dorsal line. Lice in this group seemed to be fast growers when compared to group E which was slow. In group B, fishes were consistently banging against the tank. One fish in group C had developed a minor lesion at the dorsal fin. In general, base of the fins was red in majority of the fishes. Lice load in group F was very less when compared to others.

On 12 dpi, lice were growing and becoming more visible due to its dark coloration, but many transparent lice was also spotted. Some lice were transparent and free floating in water while still attached to host body. It looked like dead lice or sometimes an exoskeleton.

On 13 dpi, lice are expected to transform into the pre-adult stage, the blood feeding motile stage and as expected, lice in the group A, B, C, E and control had blood in their gut suggesting its shift from chalimus 2 to pre adult 1 . In group C, lice load was very high still but not all are drinking blood. From this day onwards lice were seen on the tank sides detached from the salmon body in all tanks and more in group D (which had more water flow at the inlet and could have displaced lice from salmon). In summary, group C was the most infected one and control was the least with lice observed and groups A and E had the lice with higher growth rate.

Vaccine B and C especially is designed to act after this stage of lice life cycle.

On 14 dpi, Lice is seen to be moving in fish body and in group E, blood in the lice guts were less compared to others.

On 15 dpi, no change to be reported.

On 16 dpi, group E has lice with reduced size and group F has lowest number of lice per fish.

Table 4, 17 dpi On 18 dpi, size difference is observed among the lice attached in various groups. Lice is not growing in the same phase, but it was difficult to conclude the reason of the variation between the groups and within the groups.

On 19 dpi, previously dispersed lice are now seen segregated as clumps. Majority is seen as pairs which suggests they are mating. This is mainly seen in groups A, B, C and D. However, lice number in group A was much less than the rest. This parasite has a very systematic pattern of infection in salmon and the lack of studies in this area is a clear limitation for selecting suitable vaccine candidates. More thorough studies to understand its behavioral biology should be done to aid in selection of ideal vaccine candidate against them.

On 20, 21 dpi, no change to be reported.

On 22 dpi, lice in all tanks were seen in pairs of male and female. Males which was smaller in size than females were seen on top of females. It was also noted during counting that adult males were also seen paired up with pre-adult females.

Water quality was also found deteriorating after this day. Water was found oily and with a foul smell in group A and control especially. The reason for this in just group A and control is unknown. Therefore, multiple water exchanges were done on the same day in all tanks.

On 23 dpi, blood was found in the gut of lice in all the tanks.

Three fishes from each vaccine groups were selected randomly for counting. Tanks were also selected random to avoid any sort of bias by the counting personnel.

On 24 dpi, sampling day. Three fish was anesthetized in Finquel (1 g in 10 I seawater for 1 min) and blood drawn from caudal vein using heparinized 5 ml syringes. Thereafter, the fishes were euthanized in overdose of the same anesthetic and dissected. Blood, spleen, head kidney, muscle (with skin) and lice (male and females) were immediately frozen in dry ice and stored at -80 °C. All samples except lice were sampled individually whereas, lice from 3 fishes were pooled to one tube and stored. The samples were also carefully checked for inflammation, granulation, pigmentation (at the site of injection), wound, and other secondary infections. None of the fishes had any of the above indications, suggesting that our vaccination was good and that the vaccines were absorbed well into the body. Only unusual symptoms noted was for, one fish in group F which had a spleen with complete granules and one fish in control group had split spleen (or spleen seen as two parts). However, slight black distorted pigments were present around spleen alone in all the fishes in all groups in varying amounts.

Fish weight was taken on day 1 for three groups and on the sampling day for all groups and is as given below,

Figure 2 shows the percentage reduction in the number of lice in each vaccine group (A, B, C, D, E, F) when compared to the control group. So, when the lice numbers at each dpi is compared to the control, the highest reduction is for A, B and F (where F is a combination vaccine of all 4 peptides).

Table 6, fish weight

On 26 dpi, fishes in group C were dead due to handling error by the lab. This vaccine group was important so the female lice from dead fish were soon transferred to healthy non- vaccinated fish to observe the egg strings. As the males was not transferred at the time, the chance of females producing egg strings is also low. Still, during final sampling we could observe that few males were present on fish by chance therefore, we assume females produced egg strings in normal way. Although this group is excluded from the experiment, the eggstring length measurement was taken and are noted in the table below.

From this day onwards monitoring of fish health was done by Lab manager of Matre station.

Fishes were healthy until 26 dpi but showed signs of emaciation and sore formation after this day.

On 32 dpi, female lice started to produce egg strings in all groups. The egg strings might be shed in a day or two so the females without egg strings counted on the sampling day might belong to an intermediate stage of shedding 1 ^st eggstring and production of 2 ^nd eggstring. The earlier stages like chalimus were seen mainly in the operculum, head and tail end but adult stages are mainly distributed behind dorsal fin and adipose fin and few throughout the body surface. All fishes were swimming normal but wounds (soft, without scales, red sores but not deep) were seen at the attachment area. Group a had majority of females when compared to males. Group 2 had very high number of males when examined visually. At this stage, control fishes had very high number of males and female lice and 4-5 salmon had signs of sore formation. Still, control group remained healthy, and swimming and no mortality was observed. Fishes (11 in number) were left till 40 dpi to observe the eggstring formation and length among the vaccinated groups.

On 40 dpi, 3 fish from each group was sedated in Aquacalm and were counted individually for adult males, adult females with egg string and adult female without egg strings. Egg string lengths from few female lice was also measured from these three individual fishes. Rest 8 fishes from the group was euthanized by overdose of Aquacalm which will also detach the lice from fish body. Therefore, total adult females with eggstring were counted from these 8 fishes together and noted. We didn’t observe any lice attached on the belly of the fish for any experimental groups and fishes remained clean but with mild sores on the dorsal area. Whereas severe scale loss was observed in group D.

Eggstring of lice was measure by placing them on a transparent scale and the length was noted as indicated in figure 3 (modified from https://en.wikipedia.org/wiki/Salmon louse). Table 7, 40 dpi lice counts and eggstring measurements

Table 8, summary of total lice counting: Conclusion

From the current trial, it is very clear that there is a reduction in total lice count in fish group A (14-3-3) (SEQ ID NO 1 ) and B (pxrs2) (SEQ ID NO 2) when compared to control. Vaccine group A (1433) has significant reduction in total lice number and also number of females. The reduction in F could be due to the effect of A or B or the collective interaction of all four candidates. Although the results were good in A and B, similar reduction in lice was not observed in the combination groups E (which is A+B) and we assume that this may be due to the low amount of individual proteins used to make the combination vaccine. A non-linear effect in antibody response is also possible. In the individual vaccines, each peptide weighed up to 300 ug per dose whereas the combination vaccine was prepared such that the final concentration amounted to 300 ug. So the actual amount of each peptide contained was half (vaccine E) or a quarter (vaccine F) of that present in the individual vaccines. Moreover, the egg string length does not change among the control and vaccine groups.

14-3-3 vaccinated groups (SEQ ID NO 1 ) showed a reduction at chalimus stage itself, which indicates that the vaccine acts in the initial stage of the parasitic development agreeing to the earlier studies which suggested the tentative action of 1433 in early detection of infection in certain parasites. Therefore, vaccine A is also a promising vaccine in our lice trial. The 14-3- 3 protein involves in a multitude of functions by binding with hundreds of ligands, and also perform a multitude of regulatory functions, including molecular interactions, subcellular localization, scaffolding and stability. As a result, 14-3-3 proteins can participate in multiple cellular biological functions, including the cell cycle, apoptosis, autophagy, cell signal transduction and other cellular activities. However, stage-specific nature of 14-3-3 is important in this trial. This specific protein was identified from 20 dpi salmon blood from the previous trial (2019), and it is seen effective in the initial stages of the current experiment suggesting that the younger parasite might have more of this protein than the adult stages. It is only through a blood analysis from salmon at 5, 10 and 40 dpi, we can conclude the stage specific effect. Finally, it is the stage-specific expression of the 14-3-3 proteins in parasites, in addition to their cell-cycle key molecule characteristics, singled them out as potential vaccine candidates against respective infections or disease and it was once again evident from our study. Example 2

Vaccine effect on Atlantic salmon and Rainbow trout challenged with salmon lice

In the vaccine trial conducted in 2021 at Matre research station (example 1 , above), we used 14 fish per tank, 200 g, 1 tank per treatment, 80 copepods per fish (1120 copepods per tank approximately), 378 degree days before challenge, 13.5 degree water temperature and the result of total lice count (reference is made to table 8 and figure 2) for different counting points are as follows:

(A= 1433, B= pxrs2 and E= A+B (half dose each)

We found that A was the best performing vaccine and B had the potential to reduce lice in long term. Therefore, both vaccines A and B and their combination, i.e. vaccine E was as a logic principle chosen for further trial. Vaccine B was also selected for vaccination against Caligus rogercresseyi (in Chile).

The selected candidates in the part 2 phase of the trial in Matre, was challenged against salmon lice in Atlantic salmon and Rainbow trout through a common-garden experiment. Experimental set up was similar to the previous trial. Atlantic salmon, 30 each, 100g was kept in 2 tanks. Each tank had equal number of vaccinated and unvaccinated fish (15 each). Likewise, 40 rainbow trout each was distributed into 2 tanks with equal number of vaccinated and unvaccinated fish (15 each). Vaccine E, i.e. the A+B combination was tested in these tanks in full dose (150+150= 300 ug per fish) and half dose (75+75= 150 ug per fish). Lice load was 15 copepods per fish and challenged after 481 degree days.

At 51 dpi, in full dose vaccine, in Atlantic salmon, wounds developed in half of the fishes in a mild manner. There was no mortality during the challenge. Total lice count at 51 dpi was 127 out of 450 copepods dispersed initially. This is equal to 4.2 lice per fish. However, not much difference was observed among vaccinated and unvaccinated fishes which was kept together in the same tank. This we assume could be due to jumping of lice from host to host within the tank.

In the tank which received half concentration, at 51 dpi, total lice count was 100 out of 450 dispersed in the tank, which calculates to 3.3 lice per fish. Wounds were mild but lower in number than full dosed vaccine group fishes. However, we couldn’t find any remarkable difference among vaccinated and unvaccinated fishes in the same tank as in half dose vaccine group. The response of salmon toward challenge with vaccine E, i.e. the A+B vaccine was in general similar in full concentration and half concentration vaccines (controls were not available to compare).

In half dose and full dose rainbow trout, vaccine showed a great reduction in the lice load at the end of 51 dpi. Copepods dispersed initially was 600 each (15*40 fish) in both vaccine groups. One fish was dead at the end of the study in half dose group (insignificant). There was almost 0 lice in full dose vaccine and average 1 lice per fish in half dose vaccine group. Mucus consistency seem to be a little difference from salmon and could be a factor in the reduction observed. Fishes were healthy and without wounds.

Common garden experiment shows us the differential response of selected peptides on salmon and trout. However, the idea of keeping vaccinated and unvaccinated fishes in the same tank made it difficult to correlate the effect of vaccines and reduction of lice.

Meanwhile in Chile, Vaccine B gave promising results after challenging fishes (salmon) with Caligus spp. Fishes weighing 250 g was injected full dose (250 pig) vaccine B emulsified in adjuvant, Montanide ISA 761 at 600 degree days and a laboratory strain of lice was infected (20 copepods per fish) and thereby the trial followed upto 897 degree days and counting done at chalimus, juvenile and adult stages. Total lice count at 51 dpi shows a reduction of 92% lice when compared to the controls. The results are given in figure 5.

These vaccinated fishes were then re-challenged again with copepods of wild strain (which is said to be a resistant variety toward pesticides and other chemicals). However, during the re- infestation, vaccine efficacy is slightly reduced (54.8%) and this we assume could be due to resistant lice variety along with a compromised immunity of host between first and second challenge. The result of total lice count is as follows. The results are given in figure 6.

Furthermore, no significant adverse side effects or residues were observed for the vaccine B.

The results so far have led to a positive conclusion that vaccine B has the potential to be tested as a vaccine candidate against salmon lice. Thus, a new trial was set up in Matre, Norway and in Chile in ideal conditions. Challenge in Chile- 2022

Fishes (salmon) (100 g) was distributed into 8 tanks, 614 degree days, 40 fish per tank with 30 copepods per fish. The salmons were challenged with 30 lice copepods of field strain per fish. A microdose was used, 100 ug in 0,05 mL/IP x 100 grams fish of vaccine B emulsified in Montanide ISA 761 adjuvant. There is a reduction in total lice count at adult stage but the average reduction from 3 replicate of vaccine B when compared to control amounts only 35%. This is lower compared to 92% reduction observed in the previous trial in Chile. The results are shown in figure 7 and table 9.

Table 9

Effect of vaccine B on salmon challenged with salmon lice

The reason is quite unclear but the fishes received all treatments including additional vaccines in a similar way. Also the infestation pressure was a bit higher than the last time but not exceeding the upper limits that a fish can tolerate in such a study. However, infestation load can be reason for reduction in efficacy in both trials.

Thus, considering the initial trials in Norway and Chile where vaccine B was found to be effective over controls, this peptide was selected to further testing and validation of its efficacy in Matre, Norway in comparison with a higher purity vaccine of the same peptide. Lice challenge in Matre, Norway - 2022.

Experimental set up consist of 8 tanks with 40 fishes (salmon) each and with vaccine B (97% purity) 150 ug , vaccine B (80% purity) 150 ug, duplicates of vaccine E (combination A+B (150+150= 300 ug), control with adjuvant (Montanide ISA 763) (abbreviated in the table 10 as ADJ) and a control without vaccination (abbreviated in the table as NIL). All fishes were pre- vaccinated against other diseases as a usual vaccination protocol at the research station. Fishes were kept at 13 degree and infected with 20 lice per fish.

Fishes were healthy in majority groups but some welfare related changes were observed among the treatment groups at the end of last time point. Thus, a welfare scoring was done at the end of the experiment (51 dpi and 52 dpi on 10 fish each). Total count of lice and lice per fish is calculated and given in table 10, below:

Table 10

Effect of vaccine B, vaccine E

Control fishes were all euthanized at the end of 24 dpi due to deteriorated health conditions. Therefore, beyond this point, reduction is calculated with adjuvant alone tank taken as a reference. However, tanks administered with adjuvant alone did not have any mortality throughout the experiment. All tanks with B peptide seem to be similar in behavior. However, vaccine B with low percent of purity was found better than higher purity (98%) in reducing the lice load. Percentage reduction is calculated based on number of lice per fish in each vaccine group to that of reference group. (-) mark indicates the reduction. Values are as shown below, in table 11 .

Table 11

Reduction in sea lice number

If these values are color coded based on the rank-color system provided, then the first 2 best vaccines with high reduction of lice load B peptide> A+B combination.

If these values are color coded (shades of gray) based on the rank-color system provided, then the first 2 best vaccines with high reduction of lice load B peptide> A+B combination combination, as shown in table 12. Table 12

Fish welfare scoring

Welfare scoring is done to check the effect and side effects of the vaccine on a visual examination based on modified Spielberg scoring system. Where, values are given ranging from lowest 0 to highest 2 - 6, based on the health parameter analyzed. The lowest value 0 meaning ‘no reaction’ and highest meaning ‘very severe’. For this, 10 fish are taken from each tank and checked in regard to cranial deformities, fin condition, reduced growth (K-factor), cataract (0-3), gill cover (0-3), fins (0-3), wound (0-3), scale loss (0-3), presence of vaccine residues on injection site (0-3), degree of adherence of organs (0-6), melanin deposition on the organs and peritoneum (0-3), lice count, digesta consistency in the gut (0-1 ), inflammation internally (0-3), organ deformities (0-1 ), tapeworm infection (0: no content, 1 : residue of faeces) . Average score per tank in calculated and based on the score received for above parameters. All vaccinated fish groups in this experiment didn’t have any vaccine residues or melanisation or adherence. They received lowest scores and all scores in the vaccinated groups were below the control group (Table 13). Therefore, all treatment groups were concluded to be in good health condition. Table 13, Fish welfare scoring Definitions

The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y- carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) as well as pyrrolysine, pyrroline-carboxy-lysine, and selenocysteine.

Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the amino acid sequence or polynucleotide sequence in the comparison window may comprise additions or deletions (i.e. , gaps) as compared to the reference sequence (e.g., a polypeptide of the invention), which does not comprise additions or deletions, for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

The term "variant" as used herein includes modifications, substitutions, additions, derivatives, analogs, fragments or chemical equivalents of the amino acid sequences disclosed herein that perform substantially the same function as the peptide inhibitors disclosed herein in substantially the same way. Variants of the peptide inhibitors disclosed herein also include, without limitation, conservative amino acid substitutions. A "conservative amino acid substitution" as used herein, is one in which one amino acid residue is replaced with another amino acid residue without abolishing the desired function or activity of the peptide inhibitors disclosed herein. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, lysine, arginine; and phenylalanine, tyrosine. Conserved amino acid substitutions involve replacing one or more amino acids of the polypeptides of the disclosure with amino acids of similar charge, size, and/or hydrophobicity characteristics. When only conserved substitutions are made the resulting variant should be functionally equivalent. Changes, which result in production of a chemically equivalent or chemically similar amino acid sequence, are included within the scope of the disclosure. If the peptide inhibitors of the present disclosure are made using recombinant DNA technology, variants of the peptide inhibitors may be made by using polypeptide engineering techniques such as site directed mutagenesis, which are well known in the art for substitution of amino acids. For example a hydrophobic residue, such as glycine can be substituted for another hydrophobic residue such as alanine. An alanine residue may be substituted with a more hydrophobic residue such as leucine, valine or isoleucine. A negatively charged amino acid such as aspartic acid may be substituted for glutamic acid. A positively charged amino acid such as lysine may be substituted for another positively charged amino acid such as arginine. The phrase "conservative substitution" also includes the use of a chemically derived residue in place of a non-derivatized residue provided that such polypeptide displays the requisite activity. In one embodiment, the peptides described herein comprise, consist essentially of, or consist of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 and have 1 ,2 or 3 conservative amino acid substitutions. Variants of the peptides of the present invention also include additions and deletions to the amino acid sequences disclosed herein.

The invention also features a pharmaceutical composition comprising a peptide combination, defined herein. The pharmaceutical composition may be a vaccine or a feed.

A pharmaceutical composition comprises in addition to the peptide or peptide combination, therapeutically inactive ingredients, such as a pharmaceutically acceptable or physiologically acceptable excipient, carrier and/or adjuvants, which are well-known to the person skilled in the art and may include, but are not limited to, solvents, emulsifiers, wetting agents, plasticizers, solubilizers (e.g. solubility enhancing agents) coloring substances, fillers, preservatives, anti-oxidants, anti-microbial agents, viscosity adjusting agents, buffering agents, pH adjusting agents, isotonicity adjusting agents, mucoadhesive substances, and the like. Examples of formulation strategies are well-known to the person skilled in the art.

In some embodiments, the peptide may be formulated (e.g. mixed together) with immune- modifying agents like adjuvants. The adjuvant may be any conventional adjuvant, including but not limited to oxygen-containing metal salts, e.g. aluminium hydroxide, chitosan, heat- labile enterotoxin (LT), cholera toxin (CT), cholera toxin B subunit (CTB), polymerized liposomes, mutant toxins, e.g. LTK63 and LTR72, microcapsules, interleukins (e.g. IL-1 BETA, IL-2, IL-7, IL-12, IN FG AMMA), G -CSF, MDF derivatives, CpG oligonucleotides, LPS, MPL, PL-derivatives, phosphophazenes, Adju-Phos(R), glucan, antigen formulation, liposomes, DDE, DHEA, DMPC, DMPG, DOC/Alum Complex, Freund's incomplete adjuvant, ISCOMs(R), LT Oral Adjuvant, muramyl dipeptide, monophosphoryl lipid A, muramyl peptide, and phospatidylethanolamine. Additional examples of adjuvants are described, for example, in "Vaccine Design — the subunit and adjuvant approach" (Edited by Powell, M. F. and Newman, M. J. ; 1995, Pharmaceutical Biotechnology (Plenum Press, New York and London, ISBN 0-306-44867-X) entitled "Compendium of vaccine adjuvants and excipients" by Powell, M. F. and Newman .

In some embodiments, the pharmaceutical composition may be formulated for parenteral administration, such as formulated for injection, e.g. subcutaneous and/or intradermal injection. Therefore, in some embodiments, the pharmaceutical composition may be a liquid (i.e. formulated as a liquid), including a solution, a suspension, a dispersion, and a gelled liquid. For example, a liquid pharmaceutical composition may be formed by dissolving a powder, granulate or lyophilizate of a peptide combination described herein in a suitable solvent and then administering to a subject. Suitable solvents may be any solvent having physiologically acceptable properties and able to dissolve the peptide combination in desired concentrations. A desired concentration may depend on the aliquot to be administered (i.e. to be injected) and the desired single dose.

A freeze-dried composition may also be formulated into a solid dosage form that is administered for example by the oral route such as by oral mucosa. Thus, in some embodiments, the pharmaceutical composition may be formulated for oral administration, for example for sublingual administration. Therefore, the pharmaceutical composition may be a solid dosage form, such as a freeze-dried solid dosage form, typically a tablet, a capsule or sachet, which optionally may be formulated for fast disintegration.

Pharmaceutical formulations and delivery systems appropriate for the compositions, methods and uses of the invention are known in the art (see, e.g., Remington: The Science and Practice of Pharmacy (2003) 20th ed., Mack Publishing Co., Easton, PA; Remington's Pharmaceutical Sciences (1990) 18th ed., Mack Publishing Co., Easton, PA; The Merck

Index (1996) 12th ed., Merck Publishing Group, Whitehouse, NJ;

Pharmaceutical Principles of Solid Dosage Forms (1993), Technomic Publishing Co., Inc., Lancaster, Pa.; Ansel ad Soklosa, Pharmaceutical Calculations (2001 ) 11th ed., Lippincott Williams & Wilkins, Baltimore, MD; and Poznansky et aL, Drug Delivery Systems (1980), R. L. Juliano, ed., Oxford, N.Y., pp. 253-315).

As mentioned, pharmaceutical compositions can be formulated to be compatible with a particular route of administration, such as by intradermal or by sublingual administration. Thus, pharmaceutical compositions may include carriers, diluents, or excipients suitable for administration by various routes. Exemplary routes of administration for contact or in vivo delivery for which a composition can optionally be formulated include inhalation, intranasal, oral, buccal, sublingual, subcutaneous, intradermal, epicutaneous, rectal, transdermal, or intralymphatic.

For oral, buccal or sublingual administration, a composition may take the form of, for example, tablets or capsules, optionally formulated as fast-integrating tablets/capsules or slow-release tablets/capsules. In some embodiments, the tablet is freeze-dried, optionally a fast-disintegrating tablet or capsule suitable for being administered under the tongue.

The pharmaceutical composition may also be formulated into a "unit dosage form", which used herein refers to physically discrete units, wherein each unit contains a predetermined quantity of a peptide or peptide combination, optionally in association with a pharmaceutical carrier (excipient, diluent, vehicle or filling agent) which, when administered in one or more doses, may produce a desired effect. Unit dosage forms also include, for example, ampules and vials, which may include a composition in a freeze-dried or lyophilized state (a lyophilizate) or a sterile liquid carrier, for example that can be added prior to administration or delivery in vivo. Unit dosage forms additionally include, for example, ampules and vials with liquid compositions disposed therein. Application Project

<120> Title : Peptides for the inhibition of parasite infection

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<141> CurrentFilingDate : 2021-11-08

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