NO20141508A1 - Peptides that induce an immune response to copepods and / or the development of a mucus shield in fish; vaccines, uses and methods for modulating the immune response of the fish and / or for inducing the development of a mucus shield in fish. - Google Patents

Abstract

Isolated peptides comprising the sequences SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27 or SEQ ID NO. 28, wherein one or more peptides induce an immune response to copepods and / or the formation of a mucus shield in fish. The peptide may be conjugated to an antigenic protein; for example, it may be covalently conjugated to KLH. In addition, vaccines are provided comprising the peptides alone or in combination.

Description

BACKGROUND OF THE INVENTION

Copepods of the Caligidae family, commonly known as fish lice, are the most widely reported ectoparasites of wild and farmed salmon species.

The dominant species attacking salmon farms in Chile is Caligus rogercresseyi, present in 99% of salmon farms, infesting both salmonids and native fish, thereby causing high mortality rates.

Global growth of intensive salmon farming in recent decades has made the control of fish lice one of the main focuses of the industry due to important economic losses and environmental impacts created by these parasites.

Copepods feed on fish skin, slime and blood, and there is well-documented literature regarding their taxonomy, life cycle and parasite-veter relation.

Fish lice infestation in salmonids produces erosion, loss of epidermis and scales, hemorrhage into host tissues, and osmoregulatory stress that may cause death of the infested specimens due to their inability to preserve homeostasis. Stressful conditions increase the salmon's susceptibility to infection due to weakness caused by the fish lice infestation.

As production levels increase in farms, the large populations of captive salmon lead to an increasing incidence of ectoparasitic pests and associated diseases, a reduction in the growth rate of farmed fish and lower quality standards due to muscle damage. This leads to a loss of economic value, higher production costs necessary to afford treatments to control the pests and related diseases of salmonids. Additional problems are parasite resistance to therapeutic chemicals and the toxicity of these products to marine life.

Vaccines using vitellogenin 1 as antigen (EP 2 405 003 and WO2007/039599), antigens comprising proteins fused to a promiscuous T-cell epitope (US 2010/00221271) have been described. However, there is still a need for highly effective vaccines that can be produced more easily, to immunize fish that can be infested with copepods. Furthermore, there is also a need for compositions or vaccines that promote the formation in fish of a mucus shield that protects against infestations with copepods.

DESCRIPTION OF THE DRAWINGS

Figure 1:

Figure 1 shows proteins extracted from C. rogercresseyi separated on 8% sodium dodecyl sulfate polyacrylamide gel under reducing conditions. Lane 1: Molecular weight marker (Fermentas #SM1811). Lane 2: Soluble protein concentrates from C. rogercresseyi;

Figure 2:

Figure 2 shows the amino acid sequences of SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3 and the peptides used for identification; SEQ ID N°l Vitellogenin 1 [Le<p>eo<p>htheims salmonis], SEQ ID NO. 2 Vitellogenin 2 [Le<p>eo<p>htheims salmonis], and SEQ ID N°3 Vitellogenin-like protein [Le<p>eo<p>htheims salmonis] ;

Figure 3:

Figure 3 is a graph showing the effect of vaccine A according to the invention expressed as a percentage reduction of the number of parasitic stages after provocation in immunized trout compared to controls in three provocation stages: fixation, development of juvenile Chalimus stages (I to IV) and adults (males and females).

Figure 4:

Figure 4 is a graph showing the average number of adult male and female parasites found per fish and the standard deviation for each group (vaccine A and controls);

Figure 5:

Figure 5 is a graph showing induced immune response in fish vaccinated with vaccine A and controls; log serum titers for specific antibodies from 4 vaccinated groups (n=5) at different times after vaccination are represented. Specific antibody titers were determined using an Elisa;

Figure 6:

Figure 6 shows the amino acid sequence of SEQ ID NO. 1 and identifies the peptides according to the invention;

Figure 7:

Figure 7 is a graph showing the effect of the vaccines according to the invention expressed as a percentage reduction of the number of parasitic stages after challenge in immunized Atlantic salmon compared to controls, in three challenge stages: fixation, development of juvenile Chalimus stages (I to IV) and adults (males and females).

Figure 8:

Figure 8 is a graph showing the average number of adult male and female parasites found per fish and the standard deviation for each group (vaccine 1-7 according to the invention and controls);

Figure 9:

Figure 9 is a graph showing induction kinetics for specific antibodies in Atlantic salmon vaccinated with the various vaccines according to the invention and their controls, challenged with infective stages of C. rogercresseyi.

Figure 10:

Figure 10 is a graph showing induction kinetics for specific antibodies in mucus from Atlantic salmon vaccinated with the various vaccines according to the invention and their controls, provoked with infectious stages of C. rogercresseyi.

Figure 11:

Figure 11 is a graph showing correlation between percentage of PRI infestation (%) and serology expressed as the inverse log of the titer of specific antibodies for vaccine 1 and

A.

Figure 12:

Figure 12 shows the results of a histological analysis of the epidermis of fish vaccinated with the various vaccines and their controls performed at the times 0, 10, 20 and 30 days after vaccination. Skin strips stained with PAS-Alcian Blue to identify mucus-secreting cells are shown.

Figure 13:

Figure 13 shows a histological analysis of the epidermis of fish vaccinated with the various vaccines and their controls performed on days 40-50-80 and 120 during immunization and challenge (below). Skin strips stained with PAS to identify mucus-secreting cells are identified.

Figure 14:

Figure 14 is an application schedule timeline showing fishermen's acclimatization, immunization, provocation with the parasite and sampling.

BRIEF DESCRIPTION OF THE INVENTION

An object of the present invention is to provide an isolated peptide comprising an amino acid sequence having at least 90%, for example 91, 92, 93, 94, 95, 96, 97, 98 and 99% identity with the following sequences: SEQ ID NO . 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27 or SEQ ID NO. 28, where the peptide induces an immune response against copepods in fish. Fish may include salmonids, such as Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), Coho salmon (Oncorhynchus kisutch), brown trout (Salmo trutta), or Chinook salmon (O. tshawytschd); and the copepods may include Caligus rogercresseyi, Caligus absens, Caligus acanthopagri, Caligus aduncus, Caligus aesopus, Caligus affinis, Caligus furcatus, Caligus alaihi, Caligus alatus, Caligus amblygenitalis, Caligus angustatus, Caligus antennatus, Caligus arii, Caligus ariicolus, Caligus asperimanus, Caligus asymmetricus, Caligus atromaculatus, Caligus balistae, Caligus belänes, Caligus bennetti, Caligus berychis, Caligus biaculeatus, Caligus bicycletus, Caligus bifurcatus, Caligus bifurcus, Caligus biseriodentatus, Caligus bocki, Caligus bonito, Caligus brevicaudatus, Caligus brevicaudus, Caligus brevis, Caligus buechlerae, Caligus callaoensis, Caligus callyodoni, Caligus calotomi, Caligus carangis, Caligus caudatus, Caligus centrodonti, Caligus chaenichthyis, Caligus cheilodactyli, Caligus chelifer, Caligus chiastos, Caligus chorinemi, Caligus chrysophrysi, Caligus clavatus, Caligus clemensi, Caligus confusus, Caligus constrictus, Caligus cookeoli , Caligus cordiventris, Caligus c ordyla, Caligus cornutus, Caligus coryphaenae, Caligus cossacki, Caligus costatus, Caligus cresseyorum, Caligus cristatus, Caligus crusmae, Caligus cunicephalus, Caligus curtus, Caligus cybii, Caligus dactylopteni, Caligus dactylus, Caligus dakari, Caligus dampieri, Caligus dasyaticus, Caligus debueni, Caligus deformis, Caligus diaphanus, Caligus dicentrarchi, Caligus dieuzeidei, Caligus digitatus, Caligus djedabae, Caligus dubius, Caligus eleutheronemi, Caligus elevatus, Caligus elongatus, Caligus engraulidis, Caligus enormes, Caligus epidemicus, Caligus epinepheli, Caligus equulae, Caligus eventilis, Caligus fistulariae . Caligus hobsoni, Caligus hoplognathi, Caligus hottent otus, Caligus hyalinae, Caligus hyalinus, Caligus ignotus, Caligus inanis, Caligus infestans, Caligus inopinatus, Caligus irritans, Caligus isonyx, Caligus itacurussensis, Caligus jawahari, Caligus kabatae, Caligus kahawai, Caligus kala, Caligus kalumai, Caligus kanagurta, Caligus kapuhili, Caligus kirti, Caligus kirtiodes, Caligus klawei, Caligus kurochkini, Caligus kuwaitensis, Caligus labracis, Caligus lacustris, Caligus lalandei, Caligus laticaudus, Caligus latigenitalis, Caligus lotus, Caligus lepidopi, Caligus lessonius, Caligus lethrinicola, Caligus lichiae, Caligus ligatus, Caligus ligusticus , Caligus littoralis, Caligus lobodes, Caligus lolligunculae, Caligus longiabdominis, Caligus longicaudatus, Caligus longicaudus, Caligus longicervicis, Caligus longipedis, Caligus longipennatus, Caligus longirostris, Caligus longispinosus, Caligus lunatus, Caligus lutjani, Caligus macarovi, Caligus macrurus, Caligus malabaricus, Caligus mercatorus, Caligus minimus, Caligus mordax, Caligus mortis, Caligus mugilis, Caligus multispinosus, Caligus murrayanus, Caligus musaicus, Caligus mutabilis, Caligus nanhaiensis, Caligus nengai, Caligus nibeae, Caligus nolani, Caligus novocaledonicus, Caligus nuenonnae, Caligus obscurus, Caligus oculicola, Caligus ocyurus, Caligus oligoplitisi, Caligus olsoni, Caligus omissus, Caligus orientalis, Caligus oviceps, Caligus pageli, Caligus pageti, Caligus pagri, Caligus pagrosomi, Caligus pampi, Caligus parvilatus, Caligus patulus, Caligus pauliani, Caligus pectinatus, Caligus pelagicus, Caligus pelamydis, Caligus penrithi, Caligus phipsoni, Caligus piscinus , Caligus placidus, Caligus platurus, Caligus platytarsis, Caligus polycanthi, Caligus pomacentrus, Caligus pomadasi, Caligus praetextus, Caligus priacanthi, Caligus productos, Caligus pseudokalumai, Caligus pseudoproductus, Caligus pterois, Caligus punctatus, Caligus quadratus, Caligus randalli, Caligus raniceps, Caligus rapax, Caligus rectus, Caligus regalis, Caligus remorae, Caligus reniformis, Caligus robustus, Caligus rotundigenitalis, Caligus rufimaculatus, Caligus russella, Caligus salmoneus, Caligus saucius, Caligus savala, Caligus schelegeli, Caligus schistonyx, Caligus sciaenops, Caligus selerotinosus, Caligus scribae, Caligus sensilis, Caligus sensorius, Caligus sepetibensis, Caligus seriolae, Caligus serratus, Caligus sibogae, Caligus sicarius, Caligus similis, Caligus spinosurculus, Caligus spinosus, Caligus stokes, Caligus stromatei, Caligus suffuscus, Caligus tanago, Caligus temnodontis, Caligus tenax, Caligus tenuicaudatus, Caligus tenuifurcatus, Caligus tenuis, Caligus teres, Caligus tetrodontis , Caligus thyrsitae, Caligus torpedinis, Caligus trachynoti, Caligus triabdominalis, Caligus triangularis, Caligus trichiuri, Caligus tripedalis, Caligus truttae, Caligus tylosuri, Caligus undulatus, Caligus unguidentatus, Caligus uranoscopi, Caligus validus, Caligus ventrosetosus, Caligus vexator, Caligus willungae, Caligus wilsoni, Caligus xystercus, Caligus zei, Caligus zylanica, Lepeophtheims europaensis, Lepeophtheims grohmanni, Lepeophtheims nordmannii, Lepeophtheims pectorales, Lepeophtheims salmonis, Lepeophtheims Thompson, or Tigriopus japonicus.

The peptide can be conjugated to an antigenic protein, for example the peptide can be covalently conjugated to hemocyanin (KLH - keyhole limpet hemocyanin) from Megathura crenulata, or others.

A vaccine against infestation of fish with copepods comprising at least one peptide is provided, wherein the peptide has at least 90%, for example 91, 92, 93, 94, 95, 96, 97, 98 and 99% identity with the sequences of SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27 or SEQ ID NO. 28; and excipients and adjuvants. The adjuvant can be any known adjuvant, for example Megathura crenulata hemocyanin (keyhole snail) (H8283-Sigma), a purified saponin, yeast (3(1-3) D-glucans, synthetic or natural microbial derivatives such as monophosphoryl lipid A (MPL), virosomes, polylactide glycolic acid microparticles, Mycobacterium phlei cell wall skeleton, aminoalkylglucosaminide phosphate, synthetic acetylated monosaccharides, lipid A derivatives, flagellin, oligodeoxynucleotides containing CpG motifs, genetically modified bacterial toxins, cholera toxin from Vibrio colerae, heat-labile enterotoxin from Escherichia coli, human endogenous immunomodulators, cytokine, chemokines, immunopotentiator, double-stranded RNA, small immunopotentiator molecules such as imiquimod, resikimod.

Preferably the vaccine is an emulsion and the excipient is a non-mineral oil Montanide ISA 763 Seppic. Fish to be treated may be salmonids, such as Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), Coho salmon (Oncorhynchus kisutch), brown trout (Salmo truttd), or Chinook salmon (O. tshawytscha); and the copepods may be, without limitation Caligus absens, Caligus acanthopagri, Caligus aduncus, Caligus aesopus, Caligus affinis, Caligus furcatus, Caligus alaihi, Caligus alatus, Caligus amblygenitalis, Caligus angustatus, Caligus antennatus, Caligus arii, Caligus ariicolus, Caligus asperimanus, Caligus asymmetricus, Caligus atromaculatus, Caligus balistae, Caligus belänes, Caligus bennetti, Caligus berychis, Caligus biaculeatus, Caligus bicycletus, Caligus bifurcatus, Caligus bifurcus, Caligus biseriodentatus, Caligus bocki, Caligus bonito, Caligus brevicaudatus, Caligus brevicaudus, Caligus brevis, Caligus buechlerae, Caligus callaænsis, Caligus callyodoni, Caligus calotomi, Caligus carangis, Caligus caudatus, Caligus centrodonti, Caligus chaenichthyis, Caligus cheilodactyli, Caligus chelifer, Caligus chiastos, Caligus chorinemi, Caligus chrysophrysi, Caligus clavatus, Caligus clemensi, Caligus confusus, Caligus constrictus, Caligus cookeoli , Caligus cordiventris, Caligus cordyla, C aligus cornutus, Caligus coryphaenae, Caligus cossacki, Caligus costatus, Caligus cresseyorum, Caligus cristatus, Caligus crusmae, Caligus cunicephalus, Caligus curtus, Caligus cybii, Caligus dactylopteni, Caligus dactylus, Caligus dakari, Caligus dampieri, Caligus dasyaticus, Caligus debueni, Caligus deformis , Caligus diaphanus, Caligus dicentrarchi, Caligus dieuzeidei, Caligus digitatus, Caligus djedabae, Caligus dubius, Caligus eleutheronemi, Caligus elevatus, Caligus elongatus, Caligus engraulidis, Caligus enormes, Caligus epidemicus, Caligus epinepheli, Caligus equulae, Caligus eventilis, Caligus fistulariae, Caligus flexispina, Caligus fortis, Caligus fronsuganinus, Caligus fugu, Caligus furcisetifer, Caligus gayi, Caligus germoi, Caligus glacialis, Caligus glandifer, Caligus gracilis, Caligus grandiabdominalis, Caligus guerini, Caligus gurnardi, Caligus haemulonis, Caligus hamatus, Caligus hamruri, Caligus hemiconiati, Caligus hobsoni, Caligus hoplognathi, Caligus hottentotus, C aligus hyalinae, Caligus hyalinus, Caligus ignotus, Caligus inanis, Caligus infestans, Caligus inopinatus, Caligus irritans, Caligus isonyx, Caligus itacurussensis, Caligus jawahari, Caligus kabatae, Caligus kahawai, Caligus kala, Caligus kalumai, Caligus kanagurta, Caligus kapuhili, Caligus kirti , Caligus kirtiodes, Caligus klawei, Caligus kurochkini, Caligus kuwaitensis, Caligus labracis, Caligus lacustris, Caligus lalandei, Caligus laticaudus, Caligus latigenitalis, Caligus lotus, Caligus lepidopi, Caligus lessonius, Caligus lethrinicola, Caligus lichiae, Caligus ligatus, Caligus ligusticus, Caligus littoralis, Caligus lobodes, Caligus lolligunculae, Caligus longiabdominis, Caligus longicaudatus, Caligus longicaudus, Caligus longicervicis, Caligus longipedis, Caligus longipennatus, Caligus longirostris, Caligus longispinosus, Caligus lunatus, Caligus lutjani, Caligus macarovi, Caligus macrurus, Caligus malabaricus, Caligus mercatorus, Caligus minimus, Caligus mordax, Caligus mortis, Caligus mugilis, Caligus multispinosus, Caligus murrayanus, Caligus musaicus, Caligus mutabilis, Caligus nanhaiensis, Caligus nengai, Caligus nibeae, Caligus nolani, Caligus novocaledonicus, Caligus nuenonnae, Caligus obscurus, Caligus oculicola, Caligus ocyurus, Caligus oligoplitisi, Caligus olsoni, Caligus omissus Caligus Orientalis placidus, Caligus platurus, Caligus platytarsis, Caligus polycanthi, Caligus pomacentrus, Caligus pomadasi, Caligus praetextus, Caligus priacanthi, Caligus productos, Caligus pseudokalumai, Caligus pseudoproductus, Caligus pterois, Caligus punctatus, Caligus quadratus, Caligus randalli, Caligus raniceps, Caligus rapax, Caligus rectus, Caligus regalis, Caligus remorae, Caligus renifor mis, Caligus robustus, Caligus rogercresseyi, Caligus rotundigenitalis, Caligus rufimaculatus, Caligus russella, Caligus salmoneus, Caligus saucius, Caligus savala, Caligus schelegeli, Caligus schistonyx, Caligus sciaenops, Caligus scleroanosus, Caligus scribae, Caligus sensilis, Caligus sensorius, Caligus sepeabensis, Caligus seriolae, Caligus serratus, Caligus sibogae, Caligus sicarius, Caligus similis, Caligus spinosurculus, Caligus spinosus, Caligus stokes, Caligus stromatei, Caligus suffuscus, Caligus tanago, Caligus temnodontis, Caligus tenax, Caligus tenuicaudatus, Caligus tenuifurcatus, Caligus tenuis, Caligus teres . willungae, Caligus wilsoni, Caligus xyst ercus, Caligus zei, Caligus zylanica, Lepeophtheims europaensis, Lepeophtheims grohmanni, Lepeophtheims nordmannii, Lepeophtheims pectoralis, Lepeophtheims salmonis, Lepeophtheims thompsoni, Tigriopus japonicus, Paracyclopina nona.

A copepod fish infestation vaccine comprising at least one peptide is provided, wherein the peptide has at least 90%, for example 91, 92, 93, 94, 95, 96, 97, 98 and 99% identity with the sequences of SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27 or SEQ ID NO. 28; and excipients and adjuvants. The adjuvant can be any known adjuvant, for example Megathura crenulata hemocyanin (keyhole snail) (H8283-Sigma), a purified saponin, yeast (3(1-3) D-glucan, synthetic or natural microbial derivatives such as monophosphoryl lipid A (MPL), virosomes, polylactide glycolic acid microparticles, Mycobacterium phlei cell wall skeleton, aminoalkyl glucosaminide phosphate, synthetic acetyl pea monosaccharides, lipid A derivatives, flagellin, oligodeoxynucleotides containing CpG motifs, genetically modified bacterial toxins, cholera toxin from Vibrio colerae, heat labile enterotoxin from Escherichia coli, human endogenous immunomodulators, cytokines, chemokines, immunopotentiator double-stranded RNA, small immunopotentiator molecules such as imiquimod, resikimod.

The peptides can be conjugated to an antigenic protein, for example KLH or to any

other known antigenic protein.

Further provided is the use of the peptide for the preparation of a vaccine which induces an immune response in fish, or for the preparation of a composition which generates the formation of a mucus shield protection in fish.

Further provided is a copepod fish infestation vaccine comprising the proteins of SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3; excipients and adjuvants.

A method for modulating an immune response in fish comprising administering to the fish a required amount of a vaccine comprising at least one peptide selected from the group consisting of SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28; and excipients. The method comprises administering from 1 to 500 µg of peptide. When the vaccine comprises 4 peptides, 1 to 500 µg of each peptide is administered. The peptide may be conjugated to an antigenic protein, for example KLH.

A method of generating the formation of a mucus shield in fish comprising administering to the fish a necessary amount of a vaccine comprising at least one peptide having at least 90% identity with a sequence selected from the group consisting of SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28 and combinations thereof; and excipients. For example, administering from 1 to 500 µg of the peptide. The fish can be Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss) and Coho salmon (Oncorhynchus kisutch), brown trout (Salmo trutta) and Chinook salmon (O. tshawytscha) and the copepods can belong to the Caligidae family. The peptide may be conjugated to an antigenic protein, for example Keyhole Limpet Hemocyanin from Megathura crenulata.

DETAILED DESCRIPTION OF THE INVENTION

For the purposes of the present invention, the term "vaccine" refers to a composition that induces an immune response in an animal, for example in fish, and it also refers to a composition that induces the formation of a mucus shield, the shield being a biological protection against copepod infestation in fish. Efficacy analyzes were performed with multimeric proteins from the vitellogenin family as candidate immunogens to develop a new vaccine against C. rogercresseyi and other copepods.

Candidate soluble proteins on which immunogens were separated by electrophoresis

8% sodium dodecyl sulfate polyacrylamide gels from a suspension of homogenized adult Caligus rogercresseyi paiasites.

When subjected to electrophoresis on sodium dodecyl sulfate polyacrylamide gels under non-reducing conditions, 3 prominent bands of 220 kDa (SEQ ID NO. 1), 212 kDa (SEQ ID NO. 2) and 173 kDa (SEQ ID NO. 3) from the soluble the protein concentrates from the homogenized adult parasite suspension observed. Under reducing conditions, 4 bands corresponding to proteins of 220 kDa, 173 kDa, 116 kDa and 97 kDa were observed (Figure 1).

Tryptic cleavage analysis and matrix-assisted laser desorption/ionization (Maldi-tof) of bands extracted from the gel showed that the various peptide fractions were highly homologous (>90%) to amino acid sequences of multimeric phospholipoglycoproteins from the vitellogenin family of copepods, such as vitellogenin 1, vitellogenin 2 and vitellogenin equations of Lepeophtheims salmonis, Trigiopus japonicus, and Paracyclopina nana, respectively.

Sequences of the peptidic fractions were analyzed using the information available in GenBank. The results showed that there was a correlation between the isolated proteins according to the invention and known proteins from the vitellogenin family (figure 2).

Table 1 shows that the amino acid sequences of Vitellogenin 1 [Le<p>eo<p>htheims salmonis], Vitellogenin 2 [Le<p>eo<p>htheims salmonis] and Vitellogenin-like proteins [Le<p>eo<p>htheims salmonis] specifies the homology of the identified peptides with the isolated proteins from Caligus rogercresseyi.

Peptides obtained from the cleavage of isolated proteins from Caligus rogercresseyi according to the invention correlated with known proteins belonging to the vitellogenin family. Figure 2 shows the sequences of Vitellogenin 1 [Le<p>eo<p>htheims salmonis'], Vitellogenin 2 [Le<p>eo<p>htheims salmonis] and Vitellogenin-like proteins [Le<p>eo<p>htheims salmonis]. The peptides which proved to be part of the amino acid sequences of the proteins according to the invention have been identified.

The isolated proteins from C. rogercresseyi were used to prepare various vaccines for immunization of fish (trout), where the vaccines comprise: Vaccine A: comprises 1 µg of each of the following proteins: 220 kDa (SEQ ID NO. 1), 212 kDa (SEQ ID NO. 2) and 173 kDa (SEQ ID NO. 3) in an oil emulsion formulated with

Montanide ISA 763 Seppic non-mineral oil (70% v/v oil - 30% v/v water), and 10 ug Megathura crenulata hemocyanin (keyhole snail)

(H8283-Sigma).

Non-specific control composition: Each dose contained 3 µg BmSS (BmSS recombinant protein from Boophilus microplus gutj in an oil emulsion formulated with Montanide ISA 763 Seppic non-mineral oil (70% v/v oil - 30% v/v water) and 10 u<g>Megathura crenulata hemocyanin (keyhole snail)

(H8283-Sigma).

Adjuvant control compositions: Each dose contained 30 µg PBS in an oil emulsion formulated with Montanide ISA 763 Seppic non-mineral oil (70% v/v oil - 30% v/v water) and 10 µg Megathura crenulata hemocyanin (keyhole snail) (H8283-Sigma) .

PBS control composition: Each dose contained only 100 µl of PBS.

After inoculation of fish with the vaccine or their controls, the fish were challenged with an infection with C. rogercresseyi

During fish immunization, challenge and monitoring periods, it was observed that the vaccines did not cause local, systemic, inflammatory and/or granulomatous reactions at the site of administration. The vaccines proved harmless and safe. None of the compounds or vaccines modified the fish's behavior or appetite.

To verify the degree of fixation of the copepods, a count of parasite specimens was carried out 72 hours after provocation on all the provoked fish. Copepodites were fixed at the expected rate. The degree of protection of the vaccines was expressed as the reduction percentage of the number of parasitic stages after provocation in trout immunized with each of the vaccines compared to controls.

A significant and gradual increase of development of juvenile stages and adults of copepod parasites was observed in the control groups. Only vaccine A according to the invention proved highly effective in reducing the number of juvenile and adult specimens. This vaccine showed a protection of from 70 to 75% depending on the stage. In ponds with fish treated with a non-specific control composition, protection reached only 25 and 32%, while in ponds with fish treated with an adjuvant control composition, protection reached only 19 to 20%, in both cases relative to the PBS control (Fig. 3) .

Average abundance was calculated as the average of adult male and female parasites per fish, taking into account the total number of fish in each group (Fig. 4). The average number of adult male and female parasites per fish in the PBS control group was from 4.1 to 4.6 times higher than the number detected in fish treated with vaccine A according to the invention. Fish immunized with vaccine A showed less difference in abundance between males and females.

A comparison between the mean number of adult male and female parasites found in the PBS control group and in the groups treated with non-specific control compositions and adjuvant control provided no significant differences.

It was observed that immunization with vaccine A and subsequent challenge of the vaccinated fish caused a 75% reduction in infestation with C. rogercresseyi, and further that this composition or vaccine proved to be safe, harmless and effective.

Figure 5 shows specific antibody titers detected by ELISA in serum from animals treated with the various vaccines. Only vaccine A was found to be immunogenic, showing a significant difference in antibody titers for fish inoculated with non-specific, adjuvant and PBS controls.

To determine the existence of antibody titers in the group of fish treated according to Figure 5, the fish were bled at various times, and sera were analyzed by ELISA as described in the examples. As can be seen in figure 5, only vaccine A according to the invention induced a specific immune response which lasted for at least 120 days and was significantly higher than that induced by the control compositions.

The present invention also relates to peptides and combinations thereof, conjugated or not, in an oily composition or vaccine for the prevention and/or activation of a humoral immune response in salmonids. The peptides, or a combination thereof, increase mucus thickness, the number and diameter of secretory cells and epithelial thickness, thereby generating a biological shield or mucus against infestations with pathogens, for example with fish louse C. rogercresseyi, thus reducing by 80%

(compared to the control) the number of fish lice in juvenile stages and adults after provocation

with the parasite in the treated salmonids.

As previously mentioned, the proteins according to the invention identified by mass spectroscopy with molecular weights of 220 kDa (SEQ ID NO. 1), 212 kDa (SEQ ID NO. 2) and 173 kDa (SEQ ID NO. 3) belong to multimers in the vitellogenin family.

To find immunogenic peptides in the protein sequence, an analysis was performed to predict linear B epitopes with BepiPred 1.0b (Technical University of Denmark) in the 220 kDa protein (Vitellogenin 1 [Le<p>eo<p>htheims salmonis] (SEQ ID NO. 1). Twelve peptides were selected. Their sequence is shown in Figure 6. The selected peptides were also conjugated to a hemocyanin extracted from the mollusk called keyhole limpet hemocyanin (KLH - Keyhole limpet hemocyanin [ Lapa californiana]).

Peptide sequences used for the preparation of the vaccines were as follows:

Peptide 1: GYSPSYYGWAPSKEYVYEFE (SEQ ID NO. 17). MV: 2525.75 D

Cysteine was added to COOHt, and it was conjugated to KLH.

Peptide 2: ESLFVEKDEPVWTNWKKALL (SEQ ID NO. 18). MV: 2548.01

Cysteine was added to COOHt, and it was conjugated to KLH.

Peptide 3: SQKEIHEVMEESGRACTGKQ (SEQ ID NO. 19) MV: 2282.70

It was conjugated to KLH at the cysteine of the sequence.

Peptide 4: STVSHQIPKPKTPKTVGNLF (SEQ ID NO. 20) MV: 2282.70.

Cysteine was added to COOHt, and it was conjugated to KLH.

Peptide 5: KTLKAKSPQLYYVSTVSFSD (SEQ ID NO. 21) MV: 2282.70

Cysteine was added to COOHt, and it was conjugated to KLH.

Peptide 6: QKITQKLQITPRTLQEPELS (SEQ ID NO. 22) MV: 2282.70

Cysteine was added to COOHt, and it was conjugated to KLH.

Peptide 7: HGLPFKYTKTRNFVDVQSVAPTASGFPVRIQ (SEQ ID NO. 23) MV: 2282.70

Cysteine was added to COOHt, and it was conjugated to KLH.

Peptide 8: CSQSSTNTVNPNTCEEKERS (SEQ ID NO. 24) MV: 2282.70

It was conjugated to KLH at the cysteine of the sequence.

Peptide 9: PVNESSGSSTPPSSTPGPLL (SEQ ID NO. 25) MV: 2282.70

Cysteine was added to COOHt, and it was conjugated to KLH.

Peptide 10: SCQGIPTPEEKTKFEKESHE (SEQ ID NO. 26) MV: 2282.70

It was conjugated to KLH at the cysteine of the sequence.

Peptide 11: PTTYNRMIEEASNCQSSSSSGSGMGGGS (SEQ ID NO. 27) MV: 2282.70. It was conjugated to KLH at the cysteine of the sequence.

Peptide 12: SSPSSSDSSSHAQPSTGRFQ (SEQ ID NO. 28) MV: 2282.70

Cysteine was added to COOHt, and it was conjugated to KLH.

Peptides 1 to 4 correspond to the amino-terminal sequence, peptides 5 to 8 correspond to the intermediate region and peptides 9 to 12 correspond to the carboxyl-terminal region of the vitellogenin 1 protein (SEQ ID NO. 1) of Lepeophtheim salmonis.

During the immunization, provocation and monitoring periods for the fish, it was observed that the vaccines did not cause local, systemic, inflammatory and/or granulomatous reactions at the site of administration.

Vaccine 1 according to the invention comprises 50 ug of each of the conjugated peptides 1-4 (peptides of SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, and SEQ ID NO. 20);

Vaccine 2 according to the invention comprises 50 ug of each of the conjugated peptides 5-8 (peptides of SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, and SEQ ID NO. 24);

Vaccine 3 according to the invention comprises 50 µg of each of the conjugated peptides 9-12 (peptides of SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, and SEQ ID NO. 28);

Each of the vaccines was prepared as an emulsion in Montanide ISA 763 Seppic non-mineral oil (70% v/v oil - 30% v/v water) and 10 µg hemocyanin keyhole limpet ^ Megathura crenulata (H8283-Sigma) to a final volume of 0.05 ml.

Vaccine 5 according to the invention comprises 50 µg of each of the non-conjugated peptides 1-4 (peptides of SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, and SEQ ID NO. 20);

Vaccine 6 according to the invention comprises 50 µg of each of the non-conjugated peptides 5-8 (peptides of SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, and SEQ ID NO. 24);

Vaccine 7 according to the invention comprises 50 µg of each of the non-conjugated peptides 9-12 (peptides of SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, and SEQ ID NO. 28).

Each of the vaccines was prepared as an emulsion in Montanide ISA 763 Seppic non-mineral oil (70% v/v oil - 30% v/v water) and 10 µg hemocyanin keyhole limpet ^ Megathura crenulata (H8283-Sigma) to a final volume of 0.05 ml.

Composition 8 is the adjuvant control composition: Each dose contained 30 µg PBS in an oil emulsion formulated with Montanide ISA 763 Seppic non-mineral oil (70% v/v oil - 30% v/v water) and plus 10 µg Megathura crenulata hemocyanin (keyhole snail) ( H8283-Sigma).

Composition 9 corresponds to the PBS control composition: each dose of the PBS control vaccine contained only 0.05 µl of PBS.

The vaccines were all harmless and safe. None of the compounds or vaccines modified the fish's behavior or appetite.

A count of parasite specimens was performed 72 hours after challenge in all the challenged fish to measure fixation. Copepodites were fixed at the expected rate. The degree of protection of the vaccines was expressed as the percentage reduction of parasitic stages after challenge in salmon immunized with each of the vaccines compared to controls.

Vaccine 1 and A proved to be the most effective. Vaccine 1 showed a reduction of 81% and 77.7% respectively, and vaccine A of 72 and 68.5% in the number of juvenile and adult specimens. Vaccine 2, 3, 4, 5, 6 and 7 showed a smaller reduction in the number

specimens (Fig. 7).

Counts carried out in ponds with fish immunized with vaccine 2 and 3 showed 11-24.2% and 25-35.4% efficacy compared to the control with PBS (Fig. 7). A protection of 18-28%, 22-27% and 28-24%, respectively, was observed in controls formulated with non-conjugated peptides (vaccine 5-6-7), while the adjuvant control composition showed 18-20% less than the PBS control ( Fig. 7).

In addition, the average abundance per fish was determined. Average abundance corresponds to the amount of adult male and female parasites found in each fish compared to the total number in all fish in each group. The number of adult male and female parasites found per fish in the PBS control group was 5.5 and 3.88 times more than that detected in fish treated with vaccine 1 comprising peptide 1-4 conjugated with KLH, where the last vaccine showed the the smallest difference in average abundance between male and female parasites (Fig. 8).

A comparison of the average abundance in the PBS control group with that of vaccine A shows an abundance 3.6 and 2.84 times higher than the latter. The observed differences were statistically significant.

No significant differences were observed between the number of adult male and female parasites in the PBS control group and the adjuvant control groups and vaccine 5, 6 and 7. However, there was a relatively lower abundance in the group treated with vaccine 2 and 3 compared to the PBS control.

It was observed that through immunization with peptide 1-4 of the amino-terminal part of the C rogercresseyi <p>rotem of 220 kDa (SEQ ID NO. 1) conjugated with KLH, a 78% reduction of infestation induced through challenge with infestation was achieved of C rogercresseyi, and in addition this vaccine is safe, harmless and effective.

It was further observed that the effect of vaccine 1 is somewhat superior to that seen in fish immunized with vaccine A comprising complete proteins. Furthermore, the synthesis of such small peptides, for example by solid phase techniques, is an automated process and is easily performed. Conjugation with transport proteins, for example KLH, induces an improved immunological response and an adequate protection for salmon infested with C rogercresseyi.

Sera were titrated in 2-fold serial dilutions from the 1:4 dilutions. The coefficient of variation for positive and negative sera was calculated in 12 determinations as an intra-assay repeatability rate, resulting in values ranging from 4 to 19%.

The results show that immunization of Atlantic salmon with a dose of 200 µg of peptide 1-4 (vaccine 1) induces high titers of specific serum antibodies, detected by ELISA (Fig. 9). A single immunization in vaccinated fish was sufficient to induce serum titers higher than 1.5 and 2 log between 20 and 40 dpv, which increased to 3 log during the course of immunization. Serum titers obtained from fish treated with vaccine 1 were correlated with an increase in specific antibody levels found in the group of fish vaccinated with vaccine A. Vaccines 2-3-5-6 and 7 induced similar and lower serum titers than those observed with vaccine 1 and A. Controls with PBS and adjuvant did not induce a specific antibody response in vaccinated fish (Fig. 9).

The level of specific antibodies was determined by measuring absorbance at 405 nm in mucus extracted from vaccinated fish (Fig. 10). Samples were analyzed without dilution. The results show, for example, that a single immunization of Atlantic salmon with 200 µg of peptide A formulated with hemocyanin in non-mineral oil (vaccine 1) induced the production of specific antibodies in mucus which increased during the immunization period. These levels of antibodies obtained by immunization of fish with vaccine 1 were correlated with the increase in specific antibody levels found in mucus from the group of fish vaccinated with vaccine A. Vaccines 2-3-5-6 and 7 induced lower levels of antibodies. Controls with PBS and adjuvant did not induce specific antibody responses in mucus from untreated control fish (Fig. 10).

It will be obvious to the person skilled in the art that the peptides and vaccines according to the invention can be used for immunization against any type of copepod, given that the peptides used for this are included in vitellogenin 1 from, for example, Lepeophtheims salmonis or other known copepods.

The presence of specific antibodies in serum and mucus from fish vaccinated with vaccine 1 and A is correlated with a significant reduction in infestation percentage after provocation of fish with C. rogercresseyi. The vaccine formulated with peptide 1-4 showed the best results. These results strongly suggest that the detection of serum-mucus antibodies is a crucial tool for demonstrating a vaccine's potency, and that there is a correlation between effective protection (as percent reduction of infestation) and immune response (Fig. 11).

Histological studies evaluating the number of mucus-secreting cells, their diameter, and thickness of epithelium were also performed by analyzing three fields per strip. A statistical treatment of the results was carried out according to the Kruskal Wallis test for differences in the medians. In a similar way, correlations were made between the reduction of infestation by provocation with C rogercresseyi and the SHIELD (shield) effect formed by variation in the number and diameter of mucus-producing cells, as well as variation in wall thickness. (Tables 2 to 9)

At time 0, histological samples taken from the abdominal and lateral zone of the fish were stained with the same intensity for both PAS and PAS-Alcian Blue staining, with a prevalence of neutral mucopolysaccharides. Between days 10 and 50 after vaccination, the mucus of the fish immunized with Vaccine 1 and Vaccine A became thicker and more acidic (figures 12 and 13)

The thickness of the epithelium and the number of PAS+ cells did not show significant differences between day 0 and day 20. These observations are described in Tables 2 to 9 and are also shown in Figures 12 and 13.

At day 30 after vaccination, a significant thickening of the epithelium was observed, as well as an increase in the number and diameter of mucus-secreting cells PAS+ in the groups treated with vaccine 1 and A compared to day 0 and compared to the controls (PBS and adjuvant). In the case of vaccine 2-3-5-6 and 7, the values were lower than those observed for vaccine 1 and A. Immunogenic stimulation also caused goblet cell hyperplasia.

At day 40 post-vaccination, activation of macrophages and an increase in lymphocytic infiltration were observed, and they were accompanied by an increase in specific serum antibody titers. At day 50 post-vaccination, the immune response was potentiated, further increasing the epithelial thickness, the number of PAS+ cells and their diameter, mainly, but not exclusively, in fish treated with vaccine 1 and A.

From day 80 to day 120 after vaccination, a significant increase in the number and diameter of cells was observed, maintaining the same values observed during challenge. A relatively significant increase in epithelial thickness was recorded compared to data from day 50.

There were no statistically significant differences in epithelial thickness, number of PAS+ cells and their diameter in control fish. However, there were small variations in fish treated with vaccine 2-3-5-6 and 7.

It will be apparent to those skilled in the art that the peptides and vaccines of the invention can be used as compositions useful in generating a slime shield and, as shown, the slime shield reduces copepod infestation in treated fish.

This invention is better illustrated in the following examples, which should not be construed as limiting its scope. On the contrary, it should be clearly understood that other embodiments, modifications and equivalents thereof may be possible after reading the present description, which may be suggested by a person skilled in the art without deviating from the purpose of the present invention and/or the scope of the accompanying claims.

Examples

Example 1: Isolation, processing, analysis and identification of proteins and peptides

Proteins were isolated from a suspension of 0.5 g of adult specimens of C. rogercresseyi in PBS-Tween 0.05%. Samples were frozen and homogenized using a Precellys 24 tissue homogenizer (Bertin Technologies-France) using 2 ml tubes containing ceramic beads and glass beads (pre-filled bead tubes cat. no. 03119.200.RD000 Precellys-France). Two cycles of 50 sec at 6000 rpm were performed, and then at 5000 rpm for 15 min in a centrifuge (Eppendorf Refrigerated Microcentrifuge Model 5417 R-USA). The supernatant was collected and then concentrated with CentriPlus YM50 (cutoff > 50 kDa) (Millipore-Fisher Sei) at 2500 rpm in a Sorvall centrifuge with SS34 rotor.

The collected soluble proteins were separated by electrophoresis on 8% sodium dodecyl sulfate polyacrylamide gels under reducing and non-reducing conditions and stained with Coomassie Brilliant Blue G-250 as described by Laemmli et al 1970.

Tryptic cleavage followed by mass spectrometry and Maldi-tof (Matrix-Assisted Laser Desorption/Ionization- time of flight).

Bands extracted from an 8% sodium dodecyl sulfate polyacrylamide gel were destained using methanol-acetic acid (20:7), diluted in 0.1 M ammonium carbonate pH 8.0, and then reduced with 10 mM dithiothreitol for 30 min at room temperature, followed by alkylation with 50 mM iodoacetamide. Tryptic cleavage and two-dimensional separation were performed according to Cordwell et al 1999. A Thermo Electron LTQ-FT spectrometer with a Protana nanospray ion source was used for mass spectrometry. Phenomenex Jupiter 10/C18 reverse phase columns were used as interphase. The samples subjected to tryptic digestion were injected into the column and peptides were eluted with 0.1 M acetic acid - 100% acetonitrile with a gradient flow of 0.4 µl/min over 2 hours. The nanospray source was operated at 2.5 kV. Analysis by tryptic cleavage and Maldi-tof mass spectrometry of the gel-extracted bands revealed that different peptide fractions were obtained which were analyzed using the Sequest algorithm and the NCBI NR-2006 database.

Example 2: Preparation of conjugated peptides

Conjugated peptides were obtained using solid phase chemical synthesis (SPPS) techniques. SPPS follows a general pattern of repetitive cycles of coupling-washing-deprotection-washing. The free amino terminal end of a peptide bound to a solid phase was linked to a single N-protected amino acid unit. This unit was then deprotected, thereby showing a new amino terminal end that can bind to another amino acid.

The conjugation to KLH was carried out through the free cysteine group or by addition using the MBS method (m-maleimidobenzoyl-N-hydroxysuccinimide Ester or activated maleimide) which is preferred for linking amino acids according to Hermanson. The peptides were sequenced using the method described by Merrifield

Example 3: Production of the vaccines

Vaccine A: Three high molecular weight soluble proteins were selected from the parasite homogenate. Protein quantification was performed by densitometry in a densitometer UV-P system with BSA (V-Sigma fraction) as reference. Each dose of vaccine A contained 1 µg of each of the proteins of 220465.60 Da (SEQ ID NO. 1), 212947.00 Da (SEQ ID NO. 2) and 173132.50 Da (SEQ ID NO. 3) in a oil emulsion formulated with Montanide ISA 763 Seppic non-mineral oil (70% v/v oil - 30% v/v water), and 10 µg hemocyanin keyhole limpet from Megathura crenulata (H8283-Sigma).

Non-specific control composition: BmSS recombinant protein from Boophilus microplus- taxm was used, which is highly immunogenic and protective in bovine infestation with resident ticks. Each dose of vaccine contained 3 µg BmSS in an oil emulsion formulated with Montanide ISA 763 Seppic non-mineral oil (70% v/v oil - 30% v/v water) and 10 µg Megathura crenulata hemocyanin (keyhole snail) (H8283-Sigma) .

Adjuvant Control Compositions: Each dose of vaccine adjuvant controls contained 30 µg PBS in an oil emulsion formulated with Montanide ISA 763 Seppic non-mineral oil (70% v/v oil - 30% v/v water) and 10 µg Megathura crenulata hemocyanin (keyhole snail) (H8283- Sigma).

PBS control: Each dose of PBS control vaccine contained only 100 µl of PBS.

Once the peptides were obtained, a portion of the same was conjugated with the antigenic protein KLH using a liquid phase conjugation method such as that described in the examples. The following vaccines were prepared: Vaccine 1 comprised 50 µg of each of the conjugated peptides 1-4 (peptides of SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, and SEQ ID NO. 20);

Vaccine 2 comprised 50 µg of each of the conjugated peptides 5-8 (peptides of SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, and SEQ ID NO. 24);

Vaccine 3 comprised 50 µg of each of the conjugated peptides 9-12 (peptides of SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, and SEQ ID NO. 28);

Each vaccine was prepared as an emulsion in Montanide ISA 763 Seppic non-mineral oil (70% v/v oil - 30% v/v water) and 10 µg hemocyanin keyhole limpet from Megathura crenulata (H8283-Sigma) to a final volume of 0 .05 ml.

Vaccine 5 comprised 50 µg of each of the non-conjugated peptides 1-4;

Vaccine 6 comprised 50 µg of each of the non-conjugated peptides 5-8;

Vaccine 7 comprised 50 µg of each of the non-conjugated peptides 9-12.

Each was prepared as an emulsion in Montanide ISA 763 Seppic non-mineral oil (70% v/v oil - 30% v/v water) and 10 µg hemocyanin keyhole limpet from Megathura crenulata (H8283-Sigma) to a final volume of 0.05 ml.

Composition 8, Adjuvant Controls: Each dose of vaccine adjuvant controls contained 30 µg PBS in an oil emulsion formulated with Montanide ISA 763 Seppic non-mineral oil (70% v/v oil - 30% v/v water) plus 10 µg Megathura crenulata hemocyanin (keyhole snail ) (H8283-Sigma).

Composition 9, PBS control: Each dose of PBS control vaccine contained only 0.05 ml of PBS.

Example 4: Analyzes in fish and provocation:

The analysis included the species most sensitive to C. rogercresseyi: rainbow trout (Oncorhynchus mykiss) of 30 g free from infestation (n = 50/group) without previous vaccination and without antibiotic treatment before the start of the study.

Group 1 was vaccinated with vaccine A, group 2 with the non-specific control composition, group 3 with the adjuvant control composition and group 4 with PBS only.

The analyzes were carried out at the aquaculture unit of the city of Mercedes [Unidadde Acuicultura de la ciudad de Me/*cefifes] (Province of Buenos Aires, Argentina). The animals were acclimatized during 4 days in ponds of 200 l fresh water at a temperature of 17-20 °C, with 5 mg/l oxygen (minimum), with a turnover rate of 1 l/h and with a density of up to 20 kg/ m3.

Fish were anesthetized with 20% benzocaine at a dose of 50 ppm. A single immunization was performed (0.1 ml/fish) intraperitoneally in the ventral midline using a 1 ml syringe and 25G x 5/8" needle. The control group was vaccinated with 0.1 ml/fish of sterile PBS and labeled with a cut in the adipose fin for later identification. No injection site reactions/tissue damage/survival were observed. After vaccination, fish were placed in identified ponds where they remained without stressful conditions until their immunization period was complete. At 450-500 UTA, fish were moved to ponds with seawater (25 ppt), before the challenge with infective stages of C. rogercresseyi The temperature was monitored daily throughout this period.

During the days of adaptation and during the immunization period, the fish were fed a commercial diet at 3.5% body weight each day. The immunization plan and sampling are shown in figure 14.

Serum and mucus samples were taken before vaccination, at the time of challenge and every 10 days thereafter to determine the immune response to the vaccine. The average parasite load for challenged fish was determined.

Provocation with C . rose cresseyi

For the culture of C rogercresseyi, specifically the small copepod stage, egg-bearing females were collected weekly from a filter at an Atlantic salmon farm using pointed tweezers. Samples were sent to the laboratory transported in plastic containers containing seawater, with a constant aeration system. After hatching, the larval stages were pulled out and placed in beakers containing 600 ml of filtered and sterilized seawater under constant aeration. They were kept in a Hotcold-S culture chamber at an average temperature of 13 °C until copepodites emerged. As soon as infective stages were achieved, counting was carried out in a Neubauer chamber, and concentration of the specimens at 4000 copepodites / 600 ml of filtered water.

Provocation took place when the fish reached 600UTA, so introducing 4000 copepodites (in the specified amount of filtered seawater) into each 50 fish/pond, expecting a 50% fixation rate. The amount of water was reduced to 50%, oxygen bubbling and water flow were stopped for 6 h after infection (static flow), after which they were resumed and the water flow rate was kept at 0.5 L/h to avoid affecting fixation of C rogercresseyi.

Post-challenge parasite load and reduction effect of C rogercresseyi loads were determined in vaccinated versus control groups at the time of fixation, at the time of development of juvenile stages (Chalimus L II, IO) and at development of chalimus IV, adult females and males.

Treatment with peptide vaccines

The treatment was carried out in the most commercialized species, Atlantic salmon (Salmo salar), using 30 g specimens free of infestation (n = 50/group) without previous vaccination, without a history of recent condition and without antibiotic treatments before starting the analysis. The analyzes were carried out at the aquaculture unit of the city of Mercedes [ Unidad de Acuicultura de la ciudad de Mercedes] (province of Buenos Aires, Argentina). Fish were anesthetized with 20% benzocaine at a dose of 50 ppm. A single immunization was performed (0.1 ml/fish) intraperitoneally in the ventral midline using a 1 ml syringe and 25G x 5/8" needle. The control group was vaccinated with 0.1 ml/fish of sterile PBS and labeled with a cut made in the adipose fin for later identification No injection site reactions / tissue damage / survival observed Provocation was performed when the Atlantic salmon reached a weight of 80g, at 600UTA.

Example 5: Serum and histological analyses

Serum and mucus samples were collected to test specific antibody titers using an ELISA from day 0 or pre-immune, up to 10, 20, 30, 40 days, at the time of challenge (50 days) and every 10 days after challenge until 120 days after vaccination (dpv).

Proteins of 220 kDa (SEQ ID NO. 1), 212 kDa (SEQ ID NO. 2) and 173 kDa (SEQ ID NO. 3) were used as capture antigens (eng.: capture antigens) in concentrations of 50 ug/ml. As a secondary antibody, an anti-IgM from coho salmon (Oncorhyncus kisutch) (IgGl monoclonal fraction) (Grupo Bios-Bios, Chile) was used, and as a conjugated antibody an anti-murine IgG labeled with peroxidase (goat anti-murine, Dako, Denmark) and ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) as a substrate.

For the histological analysis, samples of fish epidermis were taken with a scalpel, with cuts in the abdominal and lateral zones of the fish on days 0-10-20-30-40-50, 80 and 120. The cuts were encapsulated in 4% formalin buffer, and they were stained with PAS (periodic acid-Shiff) and PAS-Alcian Blue stains.

Slime was obtained by scraping the fish's surface with a scalpel. The extracted material was placed in 15 ml tubes with 2 ml of PBS+ protease inhibitor cocktail (Promega G6521 50X) to thereby obtain a dense suspension. It was centrifuged at 3000 g for 10 minutes and the supernatant was collected and kept at -20°C. Samples were analyzed undiluted and in duplicate. 5 serum and mucus samples were taken per group and per sample time for the serum analysis, and 3 samples were used for the histological analysis.

References:

Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 1970 227 (5259): 680-685.

Cordwell SJ, Wilkins MR, Cerpa-Poljak A, Gooley AA, Duncan M, Williams KL, Humphery-Smith L, Cross-species identification of proteins separated by two-dimensional gel electrophoresis using matrix-assisted laser desorption ionisation/time-of- flight mass spectrometry and amino acid composition. Electrophoresis. 1995 Mar;16(3):438-43.

Raynard RS; Bricknell IR, Billingsley PF, Nisbet AJ, Vigneau A, Sommerville C Development of vaccines against sea lice. Pest Manag Sci 58:569-575.

Kollner B , Wasserrab B , Kotterba G, Fischer U Evaluation of immune functions of rainbow trout (Oncorhynchus mykiss)—how can environmental influences be detected? Toxicology Letters 131 (2002) 83-95.

Alvarez-Pellitero P. Fish immunity and parasite infections: from innate immunity to immunoprophylactic prospects. Veterinary Immunology and Immunopathology Vet Immunol Immunopathol. 2008 Dec 15;126(3-4):171-98. Epub 2008 Aug 3.

Tadiso TM, Krasnov A, Skugor S, Afanasyev S, Hordvik I, Nilsen F. Gene expression analyzes of immune responses in Atlantic salmon during early stages of infection by salmon louse (Le<p>eo<p>htheims salmonis) reveal ed bi -phasic responses coinciding with the copepod-chalimus transition. BMC Genomics 2011, 12:141

Bravo S. The reproductive output of sea lice Caligus rogercresseyi under controlled conditions. Experimental Parasitology 125 (2010) 51-54

Hermanson, G.T. (2008). Bioconjugate Techniques. 2nd edition, Academic Press, New York. (Part No. 20036). Chapter 19 discusses carrier protein uses and the maleimide-activation chemistry.

Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide Merrifield B. Journal of the American Chemical Society 1963 85 (14): 2149

Claims (26)

Having thus specifically described and determined the nature of and the best way of carrying out the present invention, the inventors claim ownership and exclusive rights to: 1. Isolated peptide, characterized by comprising an amino acid sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28 and combinations thereof, where the peptide induces an immune response against copepods and/or generates a mucus shield in fish. 2. The peptide according to claim 1, characterized in that the fish is selected from the group consisting of Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), Coho salmon (Oncorhynchus kisutch), brown trout (Salmo trutta) and Chinook salmon (O. tshawytscha) and the copepods belong to the Caligidae family. 3. The peptide according to claim 1, characterized by comprising an antigenic protein conjugated to the peptide. 4. The peptide according to claim 3, characterized in that the antigenic protein is hemocyanin (KLH - Keyhole Limpet Hemocyanin) from. Megathura crenulata. 5. Vaccine that induces an immune response against copepods and/or a mucus shield in fish, characterized by comprising at least one peptide, where the peptide has an amino acid sequence that shows at least 90% identity with a sequence selected from the group consisting of SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28 and combinations thereof; excipients and adjuvants. 6. The vaccine according to claim 5, characterized in that the fish is selected from the group consisting of Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), Coho salmon (Oncorhynchus kisutch), brown trout (Salmo trutta) and Chinook salmon (O. tshawytscha) and the copepods belong to the Caligidae family. 7. The vaccine according to claim 5, characterized in that it is in the form of an emulsion. 8. The vaccine according to claim 5, characterized in that the excipient is a non-mineral oil. 9. The vaccine according to claim 5, characterized in that the peptide comprises an antigenic protein conjugated to the peptide. 10. The vaccine according to claim 9, characterized in that the antigenic protein is hemocyanin from a keyhole snail Megathura crenulata. 11. Vaccine that induces an immune response against copepods and/or the development of a mucus shield in fish, characterized by comprising peptides as shown in SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, and SEQ ID NO. 20; and excipients. 12. The vaccine according to claim 11, characterized in that at least one of the peptides is conjugated to an antigenic protein. 13. The vaccine according to claim 11, characterized in that the four peptides are conjugated to an antigenic protein. 14. Vaccine that induces an immune response against copepods and/or a mucus shield in fish, characterized by comprising peptides as shown in SEQ JD NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, and SEQ ID NO. 24; and excipients. 15. The vaccine according to claim 14, characterized in that at least one of the peptides is conjugated to an antigenic protein. 16. The vaccine according to claim 14, characterized in that the four peptides are conjugated to an antigenic protein. 17. Vaccine that induces an immune response against copepods and/or the development of a mucus shield in fish, characterized by comprising peptides as shown in SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, and SEQ ID NO. 28; and excipients. 18. The vaccine according to claim 17, characterized in that at least one of the peptides is conjugated to an antigenic protein. 19. The vaccine according to claim 17, characterized in that the four peptides are conjugated to an antigenic protein. 20. The vaccine according to any one of claims 11, 14 and 17, characterized in that the copepod belongs to the Caligjdae family. 21. The vaccine according to any one of claims 11, 14 and 17, characterized in that the excipient is either a non-mineral oil. 22. The vaccine according to any one of claims 11, 14 and 17, characterized in that the fish is selected from the group consisting of Atlantic salmon (Salmo Salar), rainbow trout (Oncorhynchus mykiss), Coho salmon (Oncorhynchus kisutch), brown trout (Salmo trutta) and Chinook salmon (O. tshawytscha) and the copepods belong to the Caligjdae family. 23. The vaccine according to any one of claims 11, 14 and 17, characterized in that it is in the form of an emulsion. 24. Use of the peptides according to claim 1 for the production of a vaccine. 25. Use of the peptides according to claim 1 for the preparation of a composition which induces the development of a mucus shield in fish. 26. Vaccine against copepods that infect fish, characterized by including the proteins of SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3; excipients and adjuvants. 27. The vaccine according to claim 26, characterized in that the excipient is a non-mineral oil. 28. Method for modulating an immune response in fish, characterized by comprising administering to the fish the required amount of a vaccine comprising at least one peptide having at least 90% identity with a sequence selected from the group consisting of SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28 and combinations thereof; and excipients. 29. The method according to claim 28, characterized in that the peptide is administered in an amount of from 1 to 500 µg. 30. The method according to claim 28, characterized in that the fish is selected from the group comprising Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss) and Coho salmon (Oncorhynchus kisutch), brown trout (Salmo trutta) and Chinook salmon (O. tshawytscha) and the copepods belong to the Caligjdae family. 31. The method according to claim 28, characterized in that the peptide comprises an antigenic protein conjugated to the peptide. 32. The method according to claim 31, characterized in that the antigenic protein is hemocyanin from a keyhole snail Megathura crenulata. 33. Method for modulating immune response in fish, characterized by comprising administering to the fish a necessary amount of a vaccine comprising the proteins as shown in SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3; excipients and adjuvants. 34. The method according to claim 33, characterized in that each protein is administered in an amount of from 1 to 10 µg. 35. The method according to claim 33, characterized in that the fish is selected from the group comprising Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss) and Coho salmon (Oncorhynchus kisutch), brown trout (Salmo trutta) and Chinook salmon (O. tshawytscha) and the copepods belong to the Caligjdae family. 36. Method for generating the development of a mucus shield in fish, characterized by comprising administering to the fish a necessary amount of a vaccine comprising at least one peptide having at least 90% identity with a sequence selected from the group consisting of SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27 and SEQ ID NO. 28; and excipients. 37. The method according to claim 36, characterized in that the peptide is administered in an amount of between 1 and 500 µg. 38. The method according to claim 36, characterized in that the fish is selected from the group comprising Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss) and Coho salmon (Oncorhynchus kisutch), brown trout (Salmo trutta) and Chinook salmon (O. tshawytscha) and the copepods belong to the Caligjdae family. 39. The method according to claim 36, characterized in that the peptide comprises an antigenic protein conjugated to the peptide. 40. The method according to claim 39, characterized in that the antigenic protein is hemocyanin from a keyhole snail Megathura crenulata.