GB2522119A - Peptides inducing an immune response against copepods and/or the development of a mucous shield in fish - Google Patents

Peptides inducing an immune response against copepods and/or the development of a mucous shield in fish Download PDF

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GB2522119A
GB2522119A GB1421363.1A GB201421363A GB2522119A GB 2522119 A GB2522119 A GB 2522119A GB 201421363 A GB201421363 A GB 201421363A GB 2522119 A GB2522119 A GB 2522119A
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gin
vaccine
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Diego Latorre
Matias Grosman
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Tecnovax Chile S A
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Tecnovax Chile S A
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1767—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0003—Invertebrate antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/14—Ectoparasiticides, e.g. scabicides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine

Description

PEPTIDES INDUCING AN IMMUNE RESPONSE AGAINST COPEPODS
AND/OR THE DEVELOPMENT OF A MUCOUS SHIELD IN FISH; VACCINES,
USES AND METHODS FOR MODULATING THE FISH IMMUNE RESPONSE
AND/OR FOR INDUCING DEVELOPMENT OF A MUCOUS SHIELD IN FISH
BACKGROUND OF THE INVENTION
Copepods of the Caligidae family, commonly known as sea lice, are the most extensively reported ectoparasites in wild-type and cultured salmon species.
The dominant species that affects salmon farming in Chile is Caligus rogercresseyi, which is present in 99% of salmon farms, infesting both salmonids and native fish and thereby generating high mortality rates.
Global growth of intensive salmonids farming over the last decade has made the control of sea lice one of the main concerns in the industry due to important economic losses and environmental effects generated by these parasites.
Copepodes feed from fish skin, mucus and blood, and there is well-documented literature concerning their taxonomy, life cycles, and parasite-host relationship.
Infestation by sea lice in salmonids produces erosion, epidermis and scale loss, host tissue hemorrhage and osmoregulatory stress which may cause the death of the affected specimens due to their inability to preserve homeostasis.
Stress conditions increase salmon susceptibility to infections because of weakness produced by the attack of sea lice.
As production levels increase in farming centers, the resulting large populations of confined salmons lead to an increasing incidence of ectoparasite pests and associated diseases, a reduction of growth rate of farming fish and lower quality standards resulting from muscle damage. This leads to loss of commercial value, higher production costs necessary to afford treatments for combating the pest and related diseases in salmonids. Further problems are parasite resistance to therapeutic chemicals and toxicity of these products on marine life.
Vaccines using vitelogenin 1 as antigen (EP 2 405 003 and W020071039599), antigens comprising proteins fused to a promiscuous T cell epitope (US 2010/00221271) have been described. However, there remains a need for highly efficient vaccines which are more easily prepared for immunizing fish capable of being infested with copepods. Furthermore, compositions or vaccines promoting the formation in fish of a mucous protective shield against infestations by copepods are also needed.
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°1 Vitellogenin 1 {Lepeophtheirus salmon/s], SEQ ID NO. 2 Vitellogenin 2 {Lepeophtheirus salmonisl, and SEQ ID N°3 Vitellogenin-like protein [Lepeophtheirus saimonis]; Figure 3: Figure 3 is a graph showing the efficacy of vaccine A of the invention expressed as percent reduction of the number of post-challenge parasitic stages in immunized trouts as compared to controls in three stages of challenge: fixation, development of juvenile Chalimus stages (I through IV) and adults (male and fern ale).
Figure 4: S Figure 4 is a graph showing the mean number of male and female adult parasites found per fish and the standard deviation corresponding to 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 of specific antibodies from 4 vaccinated groups (n=5) at various post-vaccination times are represented.
Specific antibody titers were determined by means of an [lisa; Figure 6: Figure 6 shows the amino acid sequence of SEQ ID NO. 1 and identifies the peptides of the invention; Figure 7: Figure 7 is a graph showing the efficacy of the vaccines of the invention expressed as percent reduction of the number of parasitic stages post-challenge in immunized Atlantic salmons as compared to controls, in three stages of challenge: fixation, development of juvenile Chalimus stages (I through IV) and adults (male and female).
Figure 8: Figure 8 is a graph showing the mean number of male and female adult parasites found per fish and the standard deviation of each group (vaccines 1-7 of the invention and controls); Figure 9: Figure 9 is a graph showing induction kinetics of specific antibodies in Atlantic salmon vaccinated with the different vaccines of the invention and their controls, challenged with infective stages of C. rogercresseyi.
Figure 10: S Figure 10 is a graph showing induction kinetics of specific antibodies in mucus of Atlantic salmon vaccinated with the different vaccines of the invention and their controls, challenged with infective stages of C. rogercresseyi.
Figure 11: Figure 11 is a graph showing correlation between reduction percentage of PRI infestation (%) and serology is expressed as inverse log of the titer of specific antibodies for vaccines 1 and A. Figure 12: Figure 12 shows the results of a histological analysis of the epidermis of fish vaccinated with the different vaccines and their controls performed at time points 0, 10, 20, and 30 days post-vaccination. Skin slices stained with PAS-Alcian Blue for identifying mucus-secreting cells are shown.
Figure 13: Figure 13 shows a histological analysis of the epidermis of fish vaccinated with the different vaccines and their controls performed on days 40-50-80 and 120 during immunization and challenge (below). Skin slices stained with PAS for identifying mucus-secreting cells are identified.
Figure 14: Figure 14 is an application scheme timeline showing fish acclimation, immunization, challenge with the parasite and sampling.
SUMMARY 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 to 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, wherein said peptide induces an immune response against copepods in fish. Fish may comprise salmonids, such as Atlantic salmon (Salmo Satar), Rainbow trout (Oncorhynchus mykiss), Coho salmon (Oncorhynchus kisutch), brown trout (Salmo trutta), or Chinook salmon (0.
tshawytscha); and the copepods may comprise Cal/gus rogercresseyi, Caligus absens, Ca//gus acanthopagri, Cal/gus aduncus, Cal/gus aeso pus, Cal/gus aff/nis, Cal/gus furca (us, Cal/gus ala/hi, Cal/gus ala tus, Ca//gus amblygenitalis, Cal/gus angustatus, Cal/gus antennatus, Cal/gus ar// , Cal/gus ar//colus, Cal/gus asper/manus, Ca//gus asymrnetr/cus, Cal/gus atmrnaculatus, Cal/gus balistae Cat/gus belones, Cal/gus bennett4 Ca//gus berych/s, Ca//gus b/aculea (us, Cal/gus b/cycletus, Ca//gus bifurcatus, Ca//gus bifurcus, Cal/gus biser/odenta (us, Ca//gus bock/, Cal/gus bonito, Cal/gus brev/caudatus, Cal/gus brev/caudus, Cal/gus bre v/s3 Caligus buech/erae, Cal/gus callaoens/s, Ca//gus callyodoni, Cal/gus calotomi, Cal/gus carangis, Ca//gus caudatus, Ca//gus centrodont/, Caligus chaenichthy/s, Caligus cheilodactyl/, Cal/gus chelifer, Cat/gus ch/astos, Ca//gus chor/nem/, Cal/gus ch,ysophtys/, Cal/gus clavatus, Cal/gus c/emens/, Ca//gus con fusus, Cal/gus constr/ctus, Cal/gus cookeoli, Cal/gus cord/ventr/s, Cal/gus cordyla, Cal/gus corn utus, Cal/gus coryphaenae, Cal/gus cossack/, Cal/gus costatus, Cal/gus cresseyorum, Cal/gus cr/status, Cal/gus crusmae, Ca//gus cun/copha/us, Cal/gus curtus, Cal/gus cyb//, Cal/gus dacty/opten/ , Cal/gus dacty/us, Cal/gus dakar4 Cal/gus dam p/er, Cal/gus dasyat/cus, Cal/gus debueni, Cal/gus deform/s, Cal/gus d/aphanus, Cal/gus d/centrarchi, Cal/gus dieuze/de/, Ca//gus dig/ta tus, Cal/gus djcdabae, Cal/gus dubius, Ca/igus e/eutheronemi, Caligus elevatus Cal/gus elongatus, Cal/gus engraulidis, Cal/gus enormes, Cal/gus epidemicus Cal/gus opine phe//, Caligus equulae, Caligus eventilis, Cailgus fistulariae, Caligus flex/spina, Cal/gus fortis, Caligus fronsuganinus, Cal/gus fugu, Cal/gus furcisetifer 5, Cal/gus gayl, Caligus germo/, Ca/igus glacial/s3 Caligus gland/for, Cal/gus gracilis, Caligus grand/abdomina/is, Caligus guer/n4 Cal/gus gurnardi, Cal/gus haemulon/s, Cal/gus hamatus, Cal/gus hamruri, Cal/gus hem/con/at!, Cal/gus hobsoni, Cal/gus hoplognathi, Cal/gus hottentotus, Caligus hya//nae, Cal/gus hyal/nus, Ca//gus ignotus, Cal/gus man/s3 Ca//gus infestans, Cal/gus /nopinatus, Cal/gus lu/tans, Cal/gus isonyx, Cal/gus /tacurussens/s, Cal/gus jawahar/ , Caligus kabatae, Cal/gus kahawa/, Cal/gus kala, Cal/gus kalumal, Cal/gus kanagurta, Cal/gus kapuhili, Cailgus k/rU, Cal/gus k/diodes, Cal/gus k/awe!, Cal/gus kurochk/ni, Cal/gus kuwa/tensis, Cal/gus labracis, Cal/gus lacustr/s, Caligus lalandet Cal/gus lat/caudus, Ca//gus lat/genitalis, Caligus latus, Cal/gus lepidopi, Cal/gus lesson/us, Ca//gus lethrin/cola, Cal/gus lich/ae, Caligus ilgatus, Ca//gus 1/gust/cus, Cal/gus littoral/s3 Ca//gus /obodes, Cal/gus /o/l/gunculae, Cal/gus Ion g/abdom/n/s, Cal/gus long/ca uda tus, Cal/gus Ion g/caudus, Caligus Ion g/ceni/c/s, Cal/gus Ion giped/s, Cal/gus Ion g/pennatus, Caligus Ion g/rostr/s, Cal/gus /ong/sp/nosus, Caligus /unatus, Caligus Iutjani, Cal/gus macarovi, Cal/gus macrurus, Ca//gus malabar/cus, Cal/gus mercatorus, Caligus m/n/mus, Cal/gus mordax, Cal/gus mortis, Cal/gus mug/us, Caligus mult/sp/nosus, Cal/gus murrayanus, Cal/gus musa/cus, Cal/gus mutab///s, Cal/gus nanhaiens/s, Cal/gus nenga/, Cal/gus n/beae, Cal/gus nolan/, Cal/gus novocaledon/cus, Cal/gus nuenonnae, Cal/gus obscurus, Calf gus ocul/cola, Cal/gus ocyurus, Cal/gus oligopl/t/si, Cal/gus olsoni, Caligus om/ssus, Cal/gus or/en ta/is) Caligus ov/ceps, Ca//gus pagelli, Cal/gus paget Cal/gus pagr/, Ca//gus pagrosomi, Cal/gus pampi, Cal/gus parv/latus, Ca//gus patulus, Cal/gus paulian/, Cal/gus pect/natus, Caligus pa/a gicus, Cal/gus pa/am yd/s, Cal/gus penr/th Cal/gus ph/psoni, Cal/gus pisc/rius, Caligus placidus, Caligus p/a turus, Ca//gus p/atytars/s, Cal/gus polycanth/, Cal/gus pomacentrus, Cal/gus pomadas Cal/gus praetextus, Cal/gus priacanth/, Cal/gus productos, Cal/gus pseudokaluma/, Caligus pseudo productus, Cal/gus ptero/s, Ca//gus punctatus, Ca//gus quadratus, Cal/gus randaQ Cal/gus ran/caps, Cal/gus rapax, Cal/gus rectus, Caligus regalis, Cal/gus remorae, Ca//gus ren/form/s, Caligus robustus, Caligus rotundigen/tal/s, Cal/gus ruu/maculatus, Cal/gus russell/L Cal/gus salmoneus, Cal/gus sauc/us, Cal/gus savala, Cal/gus schele ge/i, Cal/gus schistonyx, Cal/gus sc/aenops, Caligus sclerot/nosus, Caligus scribae, Caligus sans/ks, Call gus sensor/us, Caligus sepetibensis, Cal/gus ser/o/ae, Cal/gus serratus, Cal/gus s/bogae, Cal/gus s/car/us, Cal/gus s/mi/is, Cal/gus spinosurcu/us, Caligus sp/nosus, Cal/gus stokes, Cal/gus stromate/, Ca/igus suffuscus, Ca/igus tanago, Cal/gus temnodont/s, Ca//gus tenax, Caligus tenu/cauda tus, Cal/gus ten uifurcatus, Cal/gus ten uis, Cal/gus tare s, Cal/gus tetrodontis, Ca//gus thyrs/tae, Ca//gus torped/n/s, Ca//gus trachynoti, Ca//gus tr/abdom/na/is, Cal/gus tr/angular/s, Cal/gus tr/ch/uri, Ca//gus fr/p ada/is, Ca/igus truttae, Cal/gus ty/osur/, Ca//gus undulatus, Ca//gus ungu/dentatus, Cal/gus uranoscop/, Ca//gus va/ic/us, Ca//gus van trosetosus, Cal/gus vexator, Cal/gus w///ungae, Cal/gus wilson/, Ca/igus xystercus, Cal/gus ze/, Ca/igus zylanica, Lepeophthe/rus europaens/s, Lepeophihe/rus grohmann/ Lepeophthe/rus nordmann//, Lepeophthe/rus pectora/es, Lepeophthe/rus salmon/s3 Lepeophthe/rus Thompson, or Tigr/o pus japon/cus.
The peptide may be conjugated to an antigenic protein, for example the peptide may be covalently conjugated to hemocyanin (KLH -keyhole limpet hemocyanin) of Megathura crenu/ata, or others.
A vaccine against infestation of fish with copepods comprising at least one peptide is provided, wherein said peptide has at least 90%, for example 91, 92, 93, 94, 95, 96, 97, 98 and 99 % identity to 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, 01 SEQ ID NO. 28; and excipients and adjuvants. The adjuvant may be any known adjuvant, for example Megathura crenulata hemocyanin (keyhole limpet) (H8283-Sigma), a purified saponine, yeast (1-3) D-glucanes, synthetic or natural microbial derivatives such as monophosphoryl lipid A (MPL), virosomes, polylactic-glycolic acid microparticles, Mycobacter/um ph/el cell wall backbone, amino alkyl glucosaminide phosphate, synthetic acetilated monosaccharides, lipid A derivatives, flagelin, oligodeoxynucleotides containing CpG motifs, genetically bacterial modified toxins, cholera toxin from Vibr/o coleree, heat labile enterotoxin from Escher/chia co/i, human endogenous immunomodulators, cytokine, chemokines, immunopotentiator, double-stranded RNA, small immunopotentiator molecules such as imiquimod, resiquimod.
Preferably, the vaccine is an emulsion and the excipierit is a non-mineral oil Montanide ISA 763 Seppic. Fish to be treated may be salmonids, such as Atlantic salmon (Sa/mo Salar), Rainbow trout (Oncorhynchus myk/ss), Coho salmon (Oncorhynchus k/sutch), brown trout (So/mo trutta), or Chinook salmon (0.
tshawytscha); and the copepods may be, without limitation, Ca/igus absens, Caligus acanthopagri, Cal/gus aduncus, Cal/gus aeso pus, Cal/gus aff/nis, Ca//gus furcatus, Ca/igus ala/hi, Caligus a/atus, Caligus amblygen/ta/is, Ca//gus angustatus, Cal/gus antennatus, Caligus ar/i, Cal/gus ar//co/us, Cal/gus asper/manus, Ca/igus asymmetricus, Cal/gus atrnmaculatus, Ca//gus bal/stae Ca//gus be/ones, Ca/igus bennettl, Cal/gus benjch/s, Cal/gus b/acu/eatus, Cal/gus b/cyc/etus, Ca//gus bifurcatus, Ca//gus bifurcus, Ca/igus biser/odentatus, Cal/gus bock/, Gal/gus bonito, Cal/gus brev/caudatus, Cal/gus brev/caudus, Cal/gus bre v/s1 Gal/gus buechlerae, Cal/gus callaoensis, Ga//gus callyodon4 Ga//gus calotom/, Cal/gus carang/s, Cal/gus caudatus, Cal/gus centrodont/, Cal/gus chaen/chthyis, Cal/gus cheilodacty/ Cal/gus chel/fer, Cal/gus ch/astos, Cal/gus chor/nem/, S Cal/gus chtysophtys/, Gal/gus c/a vatus, Gal/gus clemens Call gus con fusus, Cal/gus constrictus, Gal/gus cookeol/, Cal/gus cord/ventr/s, Gal/gus cordyla, Cal/gus corn utus, Cal/gus coryphaenae, Cal/gus cossack/, Gal/gus costatus, Galigus cresseyorum, Cal/gus cr/status, Ca//gus crusmac, Ca//gus cun/cepha/us, Gal/gus curtus, Ca//gus cyb//, Cal/gus dactylopteni, Cal/gus dactylus, Cal/gus dakar/, Cal/gus damp/er/, Cal/gus dasyaticus, Cal/gus debuen Cal/gus deform/s, Cal/gus d/aphanus, Call gus d/centrarchi, Gal/gus d/euze/dei, Gal/gus d/g/tatus, Cal/gus djedabae, Gal/gus dub/us, Gal/gus e/eutheronem/, Gal/gus ale vatus Cal/gus elongatus, Gal/gus engrau//d/s, Gal/gus enormes, Gal/gus ep/demicus Ca//gus ep/nephel/, Cal/gus equulae, Cal/gus event/Ifs, Cal/gus fistular/ae, Gal/gus flex/sp/na, Ga//gus fort/s, Cal/gus fronsuganinus, Cal/gus fugu, Cal/gus furc/set/fer Cal/gus gay/, Cal/gus germo/, Gal/gus glac/alls, Gal/gus gland/far, Gal/gus gra c/I/s, Cal/gus grandiabdom/nal/s, Gal/gus guer/n/, Cal/gus gurnard/, Cal/gus haemulon/s, Cal/gus hamatus, Cal/gus hamrur/, Cal/gus hem/con/at/, Cal/gus hobson Cal/gus hoplognath Cal/gus hottentotus, Cal/gus hyal/nae, Caligus hyal/nus, Cal/gus /gnotus, Ca//gus /nan/s, Cal/gus /nfestans, Cal/gus /nop/natus, Cal/gus /rr/tans, Cal/gus /sonyx, Gal/gus /tacurussens/s, Gal/gus jawahari, Cal/gus kabatae, Gal/gus kahawa/, Ca//gus kala, Cal/gus kalumal, Cal/gus kanagurta, Gal/gus kapuh/l/, Gal/gus k/rt4 Gal/gus k/il/odes, Cal/gus klawel; Cal/gus kurochk/nl; Ca//gus kuwa/tens/s, Cal/gus Iabrac/s, Cal/gus /acustr/s, Cal/gus la/an del; Cal/gus lat/caudus, Ca//gus /at/gen/tal/s, Cal/gus latus, Gal/gus lep/dop Ga//gus lesson/us, Caligus lethr/n/cola, Gal/gus l/ch/ae, Ga//gus 1/gatus, Ga//gus //gusticus, Cal/gus littoralis, Cal/gus /obodes, Cal/gus Iolligunculae, Caligus Ion giabdom/nis, Ca/igus Ion gicaudatus, Caligus Ion g/caudus, Cal/gus Ion g/ceniicis, Ca/igus Ion g/pedis, Cal/gus Ion giponna (us, Caligus Ion g/rostris, Cal/gus Ion gisp/nosus, Call gus lunatus, Caligus lutfan); Caligus macarov); Cal/gus macrurus, Caligus malabar/cus, Ca//gus mercatorus, Caligus m/nimus, Caligus mordax, Caligus mort/s, Cal/gus mu gills, Ca/igus mu/tispinosus, Cal/gus murrayanus, Cal/gus musa/cus, Cal/gus mutab/I/s, Cal/gus nanhaiensis, Cab gus non gal, Cal/gus n/beae, Ca/igus nolan); Caligus novoca/edon/cus, Caligus nuenonnae, Caligus obscurus, Caligus ocul/cola, Caligus ocyurus, Cal/gus ol/go p/it/si, Ca//gus olson); Caligus omissus, Ca//gus orientalis, Cal/gus ov/ceps, Caligus page/l/, Cal/gus pageti, Cal/gus pagri, Cal/gus pagrosomi, Caligus pamp); Caligus parv/latus, Caligus patulus, Ca/igus paul/an); Caligus pect/natus, Caligus pc/a g/cus, Caligus pelarnyd/s, Caligus penrithi, Caligus ph/pson); Cal/gus p/scinus, Cal/gus placidus, Cal/gus platurus, Cal/gus platytars/s, Cal/gus polycanth); Cal/gus pomacentrus, Cal/gus pomadas); IS Cal/gus praetextus, Cal/gus priacanthi, Caligus productos, Cal/gus pseudokaluma/, Caligus psoudoproductus, Cal/gus ptero/s, Cal/gus punctatus, Caligus quadratus, Cal/gus randalli, Ca/igus ran/cops, Caligus rapax, Cal/gus rectus, Cal/gus regal/s5 Ca//gus romorae, Caligus ren/forni/s, Cal/gus robustus, Cal/gus rogercressey); Ca//gus rotund/gon/ta/is, Cal/gus ruf/macula tus, Cal/gus russell!); Caligus salmoneus, Ca//gus saucius, Cal/gus savala, Ca//gus sche/e gel); Cal/gus sch/stonyx, Caligus sciaenops, Cal/gus sclerotinosus, Cal/gus scr/bae, Ca/igus sens///s, Ca//gus sensor/us; Ca/igus sepet/bens/s, Ca//gus ser/olae, Cal/gus serratus, Cal/gus sibogae, Cal/gus s/car/us, Cal/gus s/m/l/s, Cal/gus sp/nosurcu/us, Cal/gus spinosus, Ca//gus stokes, Cal/gus stromate/, Caligus suffuscus, Ca//gus tanago, Ca//gus temnodontis, Ca/igus tenax, Ca//gus ten u/caudatus, Cal/gus tenu/furcatus, Ca//gus ten u/s, Ca//gus teres, Ca//gus tetrodon (is, Ca//gus thyrs/tae, Caligus torpodinis, Caligus trachynot/, Ca/igus triabdomin a/is, Cal/gus triangu/aris, Cal/gus trichiuri, Ca/igus tripedalls, Cal/gus truttac, Caligus ty/osuri, Ca/igus undulatus, Caligus unguidentatus, Caligus uranoscopi, Cal/gus va/ic/us, Cal/gus ventrosetosus, Cal/gus vexator, Cal/gus wi//un gae, Ca/igus wi/soni, Caligus xystercus, Cal/gus zei, Cal/gus zylanica, Lepeophtheirus eurnpaensis Lepeophthe/rus grohmanni,Lepeophtheirus nordmannll, Lepeophthe/rus pectora/is, Lepeophtheirus salmonis, Lepeophtheirus thompsoni, Tigrio pus japon/cus, Paracyclopina nana.
A vaccine against fish infestation by copepods comprising at least one peptide is provided, said peptide having at least 90%, for example 91, 92, 93, 94, 95, 96, 97, 98 and 99 % identity to 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 may be any known adjuvant, for example Megathura crenulata hemocyanin (keyhole limpet) (H8283-Sigma), a purified saponine, yeast p0-3) D-glucan, synthetic or natural microbial derivatives such as monophosphoryl lipid A (MPL), virosomes, polylactic-glycolic acid microparticles, Mycobacterium phlei cell wall backbone, amino alkyl glucosaminide phosphate, synthetic acetylated monosaccharides, lipid A derivatives, flagellin, oligodeoxynucleotides containing CpG motifs, genetically bacterial modified toxins, cholera toxin from Vibr/o colerae, heat labile enterotoxin from Esche rich/a co/i, human endogenous immunomodulators, cytokines, chemokines, immunopotentiator double-stranded RNA, small immunopotentiator molecules such as imiquimod, resiquimod.
The peptides may 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 preparing a vaccine that induces an immune response in fish or for preparing a composition generating the formation of a mucous shield protector in fish.
Further provided is a vaccine against fish infestation by copepods 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 said fish a necessary 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 pg peptide. When the vaccine comprises 4 peptides, 1 to 500 pg of each peptide is administered. The peptide may be conjugated to an antigenic protein, for example KLH.
A method for generating the formation of a mucous shield in fish comprising administering to said fish a necessary amount of a vaccine comprising at least one peptide having at least 90 % identity to 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 pg of the peptide. The fish may be Atlantic salmon (Salmo Salar), Rainbow trout (Oncorhynchus mykiss) and Coho salmon (Oncorhynchus kisutch), brown trout (Salmo trutta) and Chinook salmon (0. tshawytscha) and the copepods may 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 purpose 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 formation of a mucous shield, wherein said shield is a biologic protection against infestation by copepods in fish.
Efficacy assays were carried out with multimeric proteins of the vitellogenin family as candidate immunogens for developinga new vaccine against C. rogercresseyi and other copepod& Candidate soluble proteins as immunogens were separated by electrophoresis on 8% sodium dodecyl sulfate polyacrylamide gels from a suspension of homogenized Caligus rogercresseyi adult parasites.
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) the soluble protein concentrates from the homogenized adult parasites suspension were observed. Under reducing conditions, 4 bands corresponding to proteins of 220 kDa, 173 kDa, 116 kDa, and 97 kDa were observed (Figure 1) Tryptic digestion analysis and Matrix-Assisted Laser Desorptionflonization (Maldi-tof) of bands extracted from the gel showed that the different peptidic fractions were highly homologous (>90%) to amino acid sequences of multimeric phospholipoglyco proteins of the vitellogenin family of copepods, such as vitellogenin 1, vitellogenin 2, and vitellogenin-like of Lepeophtheirus salmonis, Trig/opus 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 of the invention and known proteins of the vitellogenin family (Figure 2).
Table 1 shows that the amino acids sequences of Vitellogenin 1 [Lepeophthoirus sa/monIs], Vitellogenin 2 [Lepeophtheirus salmoni.s] and Vitellogenin-like proteins [Lepeophtheirus satmonis] specifying the homology of the identified peptides to the isolated proteins from Ca/igus rogercresseyi.
Table I __________ __________________________
REFERENCE _______ MASS IDENTIFIED PEPTIDE
22O-kDa SLAVYALK (SEQ ID NO. 4) FYMETIQKV (SEQ ID NO. 5) satmonis] (SEQ ID [Lepeophtheirus KVETTMGVI SPFTKQ (SEQ ID NO. 6) 212 kDa KALVALFQTKM (SEQ ID NO.
Vitellogenin 2 [Lepeophtheirus -7) salmonis] (SEQ ID NO. 2) RYYACGPRS (SEQ ID NO.
_______ _____________ __________ _____ _____ 8) ___________ ______ 173-kDa PLIYGETEIK (SEQ ID NO. 9) QYSHFETDYGLGVSK (SEQ ID NO. 10 VKNSWAFR (SEQ ID NO. 11)
IYGSHFPRNFVIGVNPLKK
Vitellogenin-Iike protein (SEQ ID NO. 12) [Lcpeophtheirus sa/monis] (SEQ IILGHEFTPGYIENR (SEQ ID IDNO.3) NO.13) NAIVSQFQSVM (SEQ ID NO. 14)
SAGSHLDAK (SEQ ID NO. 15)
WGSSYNVYSFLK (SEQ ID ______ _______ ___ ____ NO.16) Peptides obtained from the digestion of isolated proteins of Caligus rogercresseyi of the invention correlated with known proteins belonging to the vitellogenin family. Figure 2 shows the sequences of Vitellogenin [Lepeophtheirus.salmonis], Vitellogenin 2 [Lepeophtheirus salmon/s], and Vitellogenin-like proteins [Lepeophtheirus salmonis]. The peptides which were found to be part of the amino acid sequences of the proteins of the invention are identified.
The isolated proteins of C. rogorcresseyi were used to prepare different vaccines for immunizing fish (trouts), said vaccines comprising: Vaccine A: comprises 1 pg of each of the following proteins: 22OkDa (SEQ ID NO. 1), 2l2kDa (SEQ ID NO. 2), and l73kDa (SEQ ID NO. 3) in an oil emulsion formulated with Montanide ISA 763 Seppic non-mineral oil (70 % v/v oil - 3 0% v/v water), and 10 pg of Megathura crenulata hemocyanin (keyhole limpet) (H8283-Sigma).
Non-specific control composition: Each dose contained 3 pg of BmSS (BmSS recombinant protein from Boophilus microplus gut) in an oil emulsion formulated with Montanide ISA 763 Seppic non-mineral oil (70 % v/v oil -30 % v/v water) and 10 pg of Megathura crenulata hemocyanin (keyhole limpet) (H8283-Sigma).
Adjuvant control compositions: Each dose contained 30 pg of PBS in an oil emulsion formulated with Montanide ISA 763 Seppic non-mineral oil (70 % v/v oil - % v/v water) and 10 pg of Megathura crenulata hemocyanin (keyhole limpet) (H8283-Sigma).
PBS control composition: Each dose contained only 100 p1 of PBS.
After inoculating 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 resulted innocuous and safe. None of the compositions or vaccines modified the behavior or appetite of the fish.
To verify the fixation degree of the copepods a count of parasite specimens was carried out 72hs post-challenge in all the challenged fish. Copepodites were fixed at the expected rate. The degree of protection of the vaccines was expressed as the reduction percent of post-challenge parasitic stages count in trouts immunized with each of the vaccines as compared to controls.
A significant and gradual increase of developing juvenile stages and adults of copepod parasites was observed in control groups. Only vaccine A of the invention resulted highly efficacious at 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 only reached a 25 and 32 %, whereas in ponds with fish treated with adjuvant control composition the protection only reached 19 to 20%, in both cases with respect to PBS control (Fig 3).
Mean abundance was calculated as the average of adult male and female is parasites per fish, considering the total number of fish of each group (Fig 4). The average number of male and female adult 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 of the invention. Fish immunized with vaccine A showed less difference in abundance between male and female.
A comparison between the average number of male and female adult 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% decrease in the infestation with C. rogercresseyi, and furthermore that this composition or vaccine was found to be safe, innocuous and efficacious Figure 5 shows specific antibody titers detected by ELISA in serum from animals treated with the different vaccines. Only vaccine A turned out to be immunogenic, showing a significant difference with respect to antibody titers of fish inoculated with non-specific, adjuvant and PBS controls.
In order to determine the subsistence of antibody titers in the groups of fish treated according to figure 5, fish were bled at different times, and sera were analyzed by ELISA as described in the examples. As can be seen in figure 5, only vaccine A of 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 preventing and/or activating a humoral immune response in salmonids. The peptides, or a combination thereof, increase mucus density, the number and diameter of secretory cells and epithelial thickness, thereby generating a biologic shield or mucous against infestation by pathogens, for example by sea lice C. rngercresseyi, thus reducing by 80% (with respect to the control) the number of lice in juvenile stages and adults after challenge with the parasite in the treated salmonids.
As mentioned previously, the proteins of the invention identified by mass spectroscopy having molecular weights of 220 kDa (SEQ ID NO. 1), 212k0a (SEQ ID NO. 2), and l73kDa (SEQ ID NO. 3) belong to multimers of the vitellogenin family.
To find immunogenic peptides within the protein sequence, an analysis for predicting linear B epitopes with BepiPred 1.Ob (Technical University of Denmark) in the 220 koa protein (Vitellogenin 1 [Lepeophtheirus salmonh.s] (SEQ ID NO. 1) was carried out. Twelve peptides were selected-Their sequence is that shown in figure 6. The selected peptides were also conjugated to a hemocyanin extracted from the mollusk designated keyhole limpet (KLH -Keyhole limpet hemocyanin [La pa cafiforniana]).
Peptide sequences used for manufacturing the vaccines were as follows: Peptide 1: GYSPSYYGWAPSKEYVYEFE (SEQ ID NO. 17). MW: 2525.75 Cystein was added to COOHt, and it was conjugated to KLH.
Peptide 2: ESLFVEKDEPVVVTNWKKALL (SEQ ID NO. 18). MW: 2548.01 Cystein was added to COOHt, and it was conjugated to KLH.
Peptide 3: SQKEIHEVMEESGRACTGKQ (SEQ ID NO. 19) MW: 2282.70 It was conjugated to KLH at the cysteine of the sequence.
Peptide 4: STVSHQIPKPKTPKTVGNLF (SEQ ID NO. 20) MW: 2282.70.
IS Cystein was added to COOHt, and it was conjugated to KLH.
Peptide 5: KTLKAKSPQLYYVSTVSFSD (SEQ ID NO. 21) MW: 2282,70 Cystein was added to 000Ht, and it was conjugated to KLH.
Peptide 6: QKITQKLQITPRTLQEPELS (SEQ ID NO. 22) MW: 2282.70 Cystein was added to COOHt, and it was conjugated to KLH.
Peptide 7: HGLPFKYTKTRNFVDVQSVAPTASGFPVRIQ (SEQ ID NO. 23) MW: 2282.70 Cystein was added to COOHt, and it was conjugated to KLH.
Peptide 8: CSQSSTNTVNPNTCEEKERS (SEQ ID NO. 24) MW: 2282.70 It was conjugated to KLH at the cysteine of the sequence.
Peptide 9: PVNESSGSSTPPSSTPGPLL (SEQ ID NO. 25) MW: 2282.70 Cystein was added to COOHt, and it was conjugated to KLH.
Peptide 10: SCQGIPTPEEKTKFEKESHE (SEQ ID NO. 26) MW: 2282.70 It was conjugated to KLH at the cysteine of the sequence.
Peptide ii: PTTYNRMIEEASNCQSSSSSGSGMGGGS (SEQ ID NO. 27) MW: 2282.70 It was conjugated to KLH at the cysteine of the sequence.
Peptide 12: SSPSSSDSSSHHAQPSTGRFQ (SEQ ID NO. 28) MW: 2282.70 Cystein was added to COOHt, and it was conjugated to KU-I.
Peptides ito 4 correspond to the amino terminal sequence, peptides 5 to 8, correspond to the medium region and peptides 9 to 12 correspond to the carboxyl terminal region of the vitellogenin 1 protein (SEQ ID NO. 1) of LepeophtheIrus saimonis.
During the immunization, challenge and monitoring periods of 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 of the invention comprises 50 pg of each of 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 of the invention comprises 50 pg of each of 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 of the invention comprises 50 pg of each of 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 pg of hemocyanin keyhole limpet from Megathura crenulata (H8283-Sigma), to a final volume of 0.05m1.
Vaccine 5 of the invention comprises 50 pg of each of 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 of the invention comprises 50 pg of each of 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 of the invention comprises 50 pg of each of 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 pg of hemocyanin keyhole limpet from Megathura crenulata (H8283-Sigma), to a final volume of 0.05m1.
Composition 8 is the adjuvant control composition: Each dose contained 30 pg of 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 pg of Megathura crenulata hemocyanin (keyhole limpet) (H8283-Sigma).
Composition 9 corresponds to the PBS control composition: Each dose of the PBS control vaccine contained only 0.05 p1 of PBS.
The vaccines were all innocuous and safe. None of the compositions or vaccines modified the behavior or the appetite of the fish.
A count of parasite specimens was carried out 72hs post-challenge in all the fish challenged to measure fixation. Copepodites were fixed at the expected rate. The degree of protection of the vaccines was expressed as percent reduction of parasitic stages post-challenge in salmons immunized with each of the vaccines as compared to controls.
Vaccines 1 and A resulted to be the most effective. Vaccine 1 showed a reduction of 81% and 77.7% and vaccine A of 72 and 68.5 % in the number of juvenile and adult specimens, respectively. Vaccines 2, 3, 4, 5, 6, and 7 showed less reduction in the number of specimens (Fig 7).
Counts carried out in ponds with fish immunized with vaccines 2 and 3 showed 11-24.2 % and 25-35.4% efficacy as 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 (vaccines 5-6-7), whereas the adjuvant control composition showed 18-20% less than the PBS control (Fig 7).
In addition, mean abundance per fish was determined. Mean abundance corresponds to the amount of male and female adult parasites found in each fish as compared to the total number in all fish of each group. The number of male and female adult 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 that comprised peptides 1-4 conjugated with KLH, the latter vaccine showing the least difference in mean abundance between male and female parasites (Fig 8).
A comparison of mean 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 differences observed were statistically significant.
No significant differences were observed between the number of male and female adult parasites in the PBS control group and the adjuvant control groups and vaccines 5, 6 and 7. However, there was a relatively lower abundance in the group treated with vaccines 2 and 3 as compared to the PBS control.
It was observed that through immunization with peptides 1-4 of the amino-terminal portion of the C rogercresseyi protein of 220 kDa (SEQ ID NO. 1) conjugated with KLH a 78% reduction of infestation induced through challenge with infestation of C rogercresseyi was achieved, and in addition this vaccine is safe, innocuous and efficacious.
It was further observed that the efficacy of vaccine 1 is somewhat superior to that seen in fish immunized with vaccine A comprising complete proteins.
Moreover, the synthesis of such small peptides, for example by solid phase techniques, is an automated process and is readily carried out. Conjugation with transporter proteins, for example KLH, induces an improved immunological response and an adequate protection of salmons infested with C rogercresseyi.
Sera were titered in 2-fold serial dilutions from the 1:4 dilutions. The variation coefficient of positive and negative sera was calculated in 12 determinations as an intra-assay repeatability rate, resulting in values from 4 to 19% The results show that immunization of Atlantic salmon with a dose of 200ug of peptides 1-4 (vaccine 1) induces high titers of specific serum antibodies, detected by ELISA (Fig 9). A single immunization in vaccinated fish was enough to induce serum titers higher than 1.5 and 2 log between 20 and 40 dpv, which increased to 3 log during the immunization course. Serum titers obtained from fish treated with vaccine I were correlated with an increase of 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 vaccines 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 assayed without dilution. The results show, for example, that a single immunization of Atlantic salmon with 200ug of peptides 1-4 formulated with hemocyanin in non-mineral oil (vaccine 1) induced production of specific antibodies in mucus which increased during the immunization period. These levels of antibodies obtained by immunization of fish vaccine 1 were correlated with the increase in specific antibody levels found in mucus of 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 response in mucus from untreated control fish (Fig 10).
It will be obvious for a person skilled in the art that the peptides and vaccines of the invention may be used for immunization against any type of copepod, given that the peptides used therefor are comprised in vitellogenin I of, for example, Lepeophtheirus salmonis or other known copepods.
The presence of specific antibodies in serum and mucus of fish vaccinated with vaccines 1 and A is correlated with a significant reduction in post-challenge infestation percentage of fish with C. rogercresseyI. The vaccine formulated with peptides 1-4 showed the best results. These results strongly suggest that detecting serum-mucous antibodies is an essential tool for demonstrating potency of a vaccine, 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 carried out analyzing three fields per slice. A statistical treatment of the results was performed according to the Kruskal Wallis test for differences in the medians. Similarly, correlations were made between reduction of infestation by challenge with C rogercresseyi and the SHIELD (shield) effect produced by variation in the number and diameter of mucus producing cells, as well variation of wall thickness. (Tables 2 to 9) Table 2. Thickness, number and diameter of mucus-secreting cells at time 0 (pre-vaccination) for different vaccines and control groups. ____ Vaccine Thickn S. E.E. Vaccine NJof E.E. Vaccine Dianiete E.E.
Vaccine 1 2792 7.02 1.81 Vaccine 13.47 3,66 095 Vaccine 12.93 1.69 0,44 Vaccine 2 29.06 6.92 1.79 Vaccine 12.93 518 1.34 14.34 -1.47 0.38 Vaccine 3 27.64 6.25 1.61 13.33 6.59 1.7 Vaccine 13.64 1.69 0.44 Vaccine 4 29.29 2.87 11.73 3.01 0.78 cine 12.5 2.27 0.59 VaccineS 31.72 7.09 1.83 12.53 3.18 0.82 rcat 13.44 1.75 0.45 VaccineS 25.17 844 2.18 cine 13.8 5.31 1.37 accine 14.73 1.83 0.47 Vaccine 7 29,15 11.3 2.92 cine 13.8 5.31 1.37 Vaccine 1296 1.9 0.49 Adjuvant 33.42 9.38 2.42 Adjuvan 12.6 2.26 0.58 Adjuvan 13.44 1.75 0.45 control _____ ______ t control ______ ______ t control __________ _____ ______ Control 31.73 8.42 2.17 itrdl 12.93 3.51 0.91 Control 12.94 1.31 0.34 Table 3. Thickness, number and diameter of mucus-secreting cells 10 days post-vaccination for differont vaccines and control groups. ______ ____ Vaccine ckn E.E. Vaccine LEE Vaccine Diamete g* ±L Vaccine 1 28.14 9.02 2.33 cme 13.8 5.31 1.37 vaccine 15.16 1.9 0.49 Vaccine 2 27.67 3.56 Vaccine 12.8 0.58 Vaccine 14.45 1.56 Vaccine 3 32.29 7.18 1.85 Vaccine 11,2 2.51 0.65 Vaccine 13.62 1.72 Vaccine 4 28.83 7.05 1.82 11.4 2.41 0.62 Vaccine 15.57 1.4 0.36 VaccineS 30.76 517 1.49 Vaccine -12.6 2,26 0,58 Vaccine 13.74 2.23 0.34 VaccineS 28.35 6.93 1.79 Vaccine 12.93 3.51 0.91 ace 14.68 2.01 0.26 Vaccine 28.84 7.38 191 -Vaccine 10.67 3.29 0.85 Vaccine 12.52 1.43 o.
Adjuvant 2844 5.82 1.5 11.47 3.36 0.87 Adjuvan 13.28 1.47 0.38 control ______ ______ t control ______ ______ t control ______ ______ control 32.21 7.02 1.81 rti'dl 11.68 4.38 1.57 ti'ol. 13.03 1.31 034 Table 4. Thickness, number and diameter of mucus-secreting cells 20 days post-vaccination for different vaccines and control_groups. _______ Thickn S. . No. of S. . Diamete S. Vaccine E.E. Vaccine E.E. Vaccine E.E.
ess D. _____ ________ cells D. r D. ____ Vaccine 1 28.14 9.02 2.33 13.8 5.31 1.37 15.16 1.9 0.49 Vaccine 2 767 3.56 Vaccine 12.8 2.24 cLs8 rcdre 14.45 1.56 0.28 Vaccine 3 32.29 7.18 1.85 Vaccine 11.2 2.51 0.65 Vaccine 13.62 1.72 ft31 Vaccine 4 28.83 7.05 1.82 Vaccine 11.4 2.41 0.62 Vaccine 15.57 1.4 0.36 Vaccine 5 30.76 -5.77 1.49 cc 12.6 2.26 0.58 ne 13.74 23 0.34 Vaccine 6 28.35 6.93 1.79 12.93 3.51 0.91 Vaccine 14.68 2.01 0.26 Vaccine 7 28.84 7.38 1.91 amne 10.67 3.29 085 Vaccine 12.52 1.43 0.37 Adjuvant 28.44 5.82 1.5 11.47 3.36 087 Adjuvan 13.28 1.47 a38 control ______ t control ______ ______ t control __________ ______ Control 32.21 7.02 1.81 11.68 4,38 1.57 Control 13.03 1.31 0.34 Table 5. Thickness, number and diameterof mucus-secreting cells 30 days post-vaccination for different vaccines and control groups. ____ ____ Vaccine Thickn E.E. Vaccine No. of S. E.E. Vaccine Diamete E.E.
__________ ess____ D. _____ ______ cells D. ________ r D. ______ Vaccine 1 37.37 * 5.21 1.86 Vaccine 15.4 * 3.36 0.87 Vaccine 17.68 * 1.01 0.41 Vaccine 2 3a33 5.71 2.25 Vaccine 11.68 2.38 1.57 Vaccine 19 1.33 0.37 Vaccine 3 32.4 6.5 2.19 ccine 13.13 2.18 0.82 13.67 1.28 0.44 Vaccine4 38.65 * 1.99 Vaccine 4.79 1.5 Vaccine 16.61 * 1.33 0.36 Vaccine 5 34.32 6.46 1.48 Vaccine 10.67 3.29 0.85 Vaccine 14.26 1.39 0.28 Vaccine 6 33.41 7.84 3.57 Vaccine 11.47 3.36 0.87 Vaccine 14.53 1.28 0.31 Vaccine 7 34.88 4.64 2.52 Vaccine 12.01 3.88 1.49 rcci3.4l 1.62 a34 Adjuvant 32.8 5.21 1.89 Adjuvan 11.89 0.79 Adjuvan 13.65 1.19 0.3 control ______ ______ ____ t control ______.. _____ t control ______ _____ Control 3119 602 233 Control 1127 246 089 itroI 1434 1 28 039 Table 6. Thickness, number and diameter of mucus-secreting cells 40 days_post-vaccination for different vaccines and control groups. ____ ____ Thickn S. . No. of S. . Diamete S. Vaccine FE. Vaccine E.E. Vaccine E.E.
ess D. cells D. ________ r D. _____ Vaccine 1 39Q5* 5.65 2.19 Vaccine 16.83 1.67 0.89 Vaccine 18.45 * 1.47 0.38 Vaccine 2 38.11 9.15 1.99 Vaccine 13.01 3.88 1.49 Vaccine 13.53 1.13 o Vaccine 3 35.02 7.13 3.48 Vaccine 13.2 2.89 0.93 Vaccine 14.17 1.16 0.3 Vaccine 4 40.18 * 5.7 ss Vaccine 15.21 2.47 1.56 Vaccine 17.82* 1.4 0.36 -VaccineS 33.28 7.41 3.52 Vaccine 12.89 3.58 0.79 Vaccine 13.44 1.9 0.49 Vaccine 6 34.79 7.83 2.89 Vaccine 11.62 2.46 0.89 Vaccine 14.69 1.07 0.28 Vaccine 7 31.32 5.07 2.33 acc 12.27 3.14 1.12 rcci 13.53 1.19 0.34 Adjuvant 36,58 6.35 3.56 Adjuvan 12.23 3.03 1.23 Adjuvan 15.65 0.29 control _______ ______ ______ t control _______ ______ t control ________ ______ Control 34.94 7,12 1.85 11.45 2.84 0:92 trol 14.77 1.04 0.33 Table 7. Thickness, number and diameter of mucus-secreting cells 50 days post-vaccination for different vaccines and control groups. ______ ____ -Vaccine -ickn E.E. Vaccine JeisOf FE. Vacctne iamete E.E.
Vaccine 1 46,2 * 8.42 2.17 Vaccine 17.23 2.46 0.75 Vaccine 19.45 * 1.69 1.44 Vaccine2 39.75 774 ss 13.67 4.31 1.33 Vaccine 14.41 147 1.38 Vaccine 3 36.89 6.51 1.68 Vaccine 13.2 2.89 0.93 Vaccine 13.44 1.69 0.44 Vaccine 4 48,77 * 6.8 1.76 Vaccine 16.76 2.86 1.44 cd 17.29 * 1.9 0.49 VaccineS 39.59 8.77 1.23 Vaccine 11.89 3.58 0.79 ccine 13.58 1.4 0.36 Vaccine 6 37.32 9.52 2.2 Vaccine 11.27 2.46 0.89 Vaccine 15.47 1.9 0.49 Vaccine 7 36.26 4.05 Vaccine 12.01 3.14 1.12 rccmne 43.31 -iT 0.49 Adjuvant 34.16 8.17 4.43 Adjuvan 12,84 3.82 0.82 Adjuvan 15,23 2.27 0.59 control _________ ______ t control ____ -______ ______ t control __________ _____ ______ Control 36.96 9.03 2.85 Control 12.04 3.62 1.06 Control 15.57 1.75 0.45 Table 8. Thickness, number and diameter of mucus-secreting cells 80 days post-vaccination for different vaccines and control groups. ____ Thickn S. No. of S. . Diamete S. Vaccine E.E. Vaccine E.E. Vaccine E.E.
________ -ess D. _____ ________ cells D. _____ ________ r D. _____ Vaccine 1 49.62 * 9.13 3.84 Vaccine 17.68 3.28 1.79 cci 19.72 * 2.13 0.13 Vaccine 2 39.21 6.18 2.16 rci 13.22 3.05 0.94 Vaccine 14.7 1.68 0.68 Vaccine 3 37.5 6.3 Vaccine 1381 2.65 0.72 Vaccine 13.38 -1.15 0.15 Vaccine 4 52.25 * 11.2 3.69 Vaccine 16.85 3,83 1.39 Vaccine 18.68 * 1.31 0,31 VaccineS 38.89 8.16 1.31 Vaccine 11.89 1.78 0.65 13.51 1.33 0.25 Vaccine 6 38.88 7.63 2.1 11.27 2.67 0.97 14.73 1.3 0.65 Vaccine 7 34.76 8.96 1.18 12.92 1.32 0.88 13.65 1.23 0.74 Adjuvant, 35.8 7.31 2.24 13.14 2.18 0.91 15.44 -1.62 0.68 Control 33.19 5.22 control 12.04 3.62 1.06 Control 14.84 1.38 Table 9. Thickness, number and diameter of mucus-secreting cells 120 days post-vaccination for different vaccines and control groups. ______ ____ ____ Vaccine Thickn E.E. Vaccine of FE. Vaccine Diamete FE.
Vaccine 1 52.28 * 9.09 3.61 Vaccine 16.49 3.28 1.79 ace 20.17 * 1.3 0.28 Vaccine 2 42.14 5.96 345 Vaccine 12.07 2.51 1.17 Vaccine 16.62 1.44 0.24 Vaccine 3 4136 7.19 a65 13.32 1.32 0.88 Vaccine 15.49 1.26 0.3 Vaccine 4 53.72 * 9.92 2.42 Vaccine 17.51 3.83 1,39 Vaccine 19.16 * 1.22 0.3 VaccineS 39.45 7.96 2.15 cci 12.23 2.52 1.05 14.42 1.34 0.41 Vaccine6 36.99 5.78 3.71 Vaccine 13.68 2.23 1.12 15.22 1.32 0.35 Vaccine 7 39.06 6.32 2.16 13.3 3.68 0.92 Vaccine 14.65 1.27 0.33 Adjuvant 37.46' 5.66 2.26 " 13.58 3.47 1.48 Adjuvan 14.37 1.38 0.47 control _______ ______________ t control _______ ______ t control __________ Control 34.89 -7.12 1.76 Control 2.66 0.86 Control 13.87 1.43 0.27 At time 0, histological samples taken from the abdominal and lateral zone of the fish were stained with the same intensity both for FAS as for PAS-Alcian Blue staining, with prevalence of neutral mucopolysaccharides. Between days 10 and post-vaccination the mucus of the fish immunized with Vaccine 1 and Vaccine A turned more dense and more acidic (Figures 12 and 13) The thickness of the epithelium and the number of PAS+ cells did not show S significant differences between day 0 and day 20. These observations are described in tables 2 through 9 and are also shown in figures 12 and 13.
By 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 vaccines 1 and A as compared to day 0 and as compared with the controls (PBS and adjuvant). In the case of vaccines 2-3-5- 6, and 7, the values were lower than those observed for vaccines 1 and A. Immunogenic stimulation also caused hyperplasia of goblet cells At day 40 post-vaccination, activation of macrophages and an increase of lymphocytic infiltration were observed, and they were accompanied by an increase of specific serum antibody titers. At day 50 post-vaccination, the immune response was potentiated, further increasing epithelia thickness, the number of PAS+ cells and the diameter thereof, mainly, but not exclusively, in fish treated with vaccines 1 and A. From day 80 to day 120 post-vaccination, a significant increase in the number and diameter of cells was observed, keeping the same values observed during challenge. A relatively significant increase of epithelium thickness was recorded as compared to data from day 50.
There were no statistically significant differences in epithelium thickness, number of FAS+ cells and their diameter in control fish. There were however minor variations in fish treated with vaccines 2-3-5-6, and 7.
It would be obvious for a person of skill in the art that the peptides and vaccines of the invention may be used as compositions useful for generating a mucous shield, arid, as has been shown, the mucous shield decreases infestation by copepods 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 of skill without departing from the spirit of the present invention and/or the scope of the appended claims.
Examples
Example 1: Isolation, processing, analysis and identification of proteins and peptides Proteins were isolated from a suspension of 0.5g 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 2mL tubes containing ceramic and glass beads (prefilled 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 mm in a centrifuge (Eppendorf Refrigerated Microcentrifuge Model 5417 R-USA). The supernatant was collected and then concentrated with CentriPlus YM5O (cut off>50 kDa) (Millipore-Fisher Sci) at 2500 rpm in a Sorvall centrifuge with 5534 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 digestion followed by mass spectrometry and Maldi-tof (Matrix-Assisted Laser Desorptiontlonization-time of flight).
Bands extracted from a 8% sodium dodecyl sulfate polyacrylamide gel were destained using methanol-acetic (20:7), diluted in 0.1M of ammonium carbonate p1-I 8.0 and then reduced with 10 mM Dithiotreitol for 30 minutes at room temperature, followed by alkylation with 50mM iodoacetamide. Tryptic digestion and bidimentional separation were performed according to Cordwell et al 1999. A Thermo Electron LTQ-FT spectrometer with a Protana nanospray system as ion source was used for mass spectrometry. Phenomenex Jupiter 1O/C18 reverse phase columns were used as interphase. The samples subjected to tryptic digestion were injected in the column and peptides were eluted with 0.1M acetic acid -100% acetonitrile with a gradient flow of 0.4p1/min during 2 hs. The nanospray source was operated at 2.5 kV. Analysis by tryptic digestion and mass spectrometry Maldi-tof of the gel-extracted bands revealed that different peptidic fractions had been obtained which were analyzed using the Sequest algorithm and the NCBI NR -2006 data base.
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-unprotection-washing. The free amino terminal end of a peptide bound to a solid phase was coupled to a single N-protected amino acid unit. This unit was then unprotected, thereby showing a new amino terminal end which could bind to another amino acid.
The conjugation to KLH was performed through the free cysteine group or by addition using the MBS method (m-maleimidobenzoyl-N-hydroxysuccinimide Ester or activated maileimide) which is preferred for coupling amino acids according to Hermanson. The peptides were sequenced using the method
described by Merrifield
Example 3: Preparation of the vaccines Vaccine A: Three soluble proteins of high molecular weight were selected from the parasite homogenate. Protein quantification was carried out by densitometry in a densitometer UV-P System using BSA (V-Sigma fraction) as reference. Each dose of vaccine A contained 1 pg 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 an oil emulsion formulated with Montanide ISA 763 Seppic non-mineral oil (70 % v/v oil -30 % v/v water), and 10 pg of hemocyanin keyhole limpet from Megathura crenulata (H8283-Sigma).
Non-specific control composition: BmSS recombinant protein from Boophilus microplus intestine was used, which is highly immunogenic and protective in bovine infestation with native ticks. Each dose of vaccine contained 3 pg of BmSS in an oil emulsion formulated with Montanide ISA 763 Seppic non-IS mineral oil (70 % v/v oil -30 % v/v water) and 10 pg of Megathura crenulata hemocyanin (keyhole limpet) (H8283-Sigma).
Adjuvant control compositions: Each dose of vaccine adjuvant controls contained 30 pg of PBS in an oil emulsion formulated with Montanide ISA 763 Seppic non-mineral oil (70 % v/v oil -30 % v/v water) and 10 pg of Megathura crenulata hemocyanin (keyhole limpet) (H8283-Sigma).
PBS control: Each dose of PBS control vaccine contained only 100 p1 of PBS.
Once the peptides were obtained, part of the same were 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 pg of each of 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 pg of each of 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 pg of each of 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 oH (70 % v/v oil -3Q % v/v water) and 10 pg of hemocyanin keyhole limpet from Megathura crenufata (H8283-Sigma), to a final volume of 0.05ml.
Vaccine 5 comprised 50 pg of each of non-conjugated peptides 1-4; Vaccine 6 comprised 50 pg of each of non-conjugated peptides 5-8; Vaccine 7 comprised 50 pg of each of non-conjugated peptides 9-12.
Each of them was prepared as an emulsion in Montanide ISA 763 Seppic non-mineral oil (70 % v/v oil -30 % v/v water) and 10 pg of hemocyanin keyhole limpet from Megathura crenulata (H8283-Sigma), to a final volume of 0.05m1.
Composition 8, adjuvant controls: Each dose of vaccine adjuvant controls contained 30 pg of 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 pg of Megathura crenulata hemocyanin (keyhole limpet) (H8283-Sigma).
Composition 9 PBS control: Each dose of PBS control vaccine contained only 0,05 ml of PBS.
Example 4: Assays in fish and challenge: The assay included the most sensitive species to C. rogercresseyi: rainbow trouts (Oncorhynchus mykiss) of 30g free from infestation (n = 50/group) without previous vaccination and without antibiotic treatment before commencement 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 only with PBS.
The assays were carried out at the Aquaculture Unit of the city of Mercedes [Unidad do Acuicultura do Ia ciudad do Mercedes](Province of Buenos Aires, Argentina). The animals were acclimated during 4 days in 200 L fresh water ponds at a temperature of 17-20 °C, with 5 mg/I oxygen (minimum), with a turnover rate of IL/hour 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 niL/fish) intraperitoneally in the ventral midline using a 1 mL syringe and 25G x 5/811 needle. The control group was vaccinated with 0.1 mI/fish of sterile PBS and marked with a cut made in the adipose fin for its later identification. No reactions at the injection site Itissue damage/ survival were observed. After the vaccination, the fish were placed in identified ponds where they remained without stressing conditions until their immunization period was completed. At 450-500 UTA, fish were moved to ponds with sea water (2sppt), before the challenge with infecting stages of C. rogercressoyt Temperature was monitored daily throughout this period,.
During the days of adaptation and during the immunization period, the fish were fed with a commercial diet at 3.5% body weight every day. Immunization schedule and sampling are shown in figure 14.
Serum and mucus samples were taken prior to vaccination, at the time of the challenge and every 10 days after the same to determine the immuno response to the vaccine. Average parasitic load of challenged fish was determined.
Challenge using C. rocjercresseyi For the culture of C rogercresseyi, specifically the small copepod stage, ovigerous females were collected weekly from a filter at a processing farm for Atlantic salmon using tipped tweezers. Samples were sent to the laboratory transported in plastic containers containing sea water with constant aeration system. After spawning, naupliar stages were withdrawn and placed in beakers containing 600 ml of filtered and sterilized sea water under constant aeration. They were kept in a Hotcold-S culture chamber at an average temperature of 13°C, until copepodites emerged. Once infective stages were obtained, counting was performed in a Neubauer chamber, and concentrating the specimens at 4000 copepodites/600 mL of filtered water.
Challenge took place when the fish reached 600UTA, then introducing 4000 copepodites (in the specified quantity of filtered sea water) into each 50 fish! pond, and expecting a 50%.fixation rate. The amount of water was reduced to 50%, oxygen bubbling and water flow were stopped for 6 hours after infection (static flow), after which they were resumed and water flow rate was kept at 0.5 liters! hour so as to avoid affecting fixation of C rogercresseyi.
Post-challenge parasitic load and reduction efficacy 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 I, II, Ill) and upon development of chalimus IV, female and male adults.
Treatment with ijeptide vaccines The treatment was carried out in the most commercialized species, Atlantic salmon (Salmo salar), using 30g specimens free from infestation (n = 50/group) without prior vaccination, without a history of recent condition and without antibiotic treatments previous to commencement of the assay. The assays were carried out at the Unit of Aquaculture of the city of Mercedes [Unidad de Acuicu/tura do Ia ciudad do Mercedesl(Frovince 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 I mL syringe and 25G x 518" needle. The control group was vaccinated with 0.1 mI/fish of sterile PBS and marked with a cut practiced in the adipose fin for its later identification. Any reactions at the injection site /tissue damage/ survival were recorded. Challenge was performed when the Atlantic salmons reached a weight of 80g, at 600UTA.
Example 5: Serum and Histological Assays 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 post challenge until 120 days post-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 at a concentration of 5OpgImL. As a secondary antibody, an anti 1gM of coho salmon (Oncorhyncus kisutch) (IgGi monoclonal fraction) (Grupo Bios-Bios, Chile) was employed and as a conjugated antibody a mouse anti-lgG marked with peroxidase (goat anti- mouse, Dako, Denmark) and ABTS (2,2'azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)as a substrate.
For the histological analysis samples of fish epidermis were taken using a scalpel, making cuts on the abdominal and lateral zones of the fish on days 0-10- 20-30-40-50, 80, and 120. Cuts were embedded in 4% formol buffer and they were stained using PAS (periodic acid-Shift) and PAS-Alcian Blue dyes.
Mucus was obtained by scraping the surface of the fish with a scalpel. The extracted material was placed in 15 mL tubes with 2 mL of PBS+ protease inhibitor coctail (Promega 06521 50X) thereby achieving a dense suspension. It was centrifuged at 3000g for 10 minutes and the supernatant was collected and kept at -20°C. Samples were assayed non-diluted and in duplicate. Five 5 serum and mucus samples were per group and per sampling 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-Poijak A, Gooley AA, Duncan M, Williams KL, Humphery-Smith I., 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 lmmuriopathology Vet Immunol Immunopathol. 2008 Dec 15;126(3-4):171-98.
Epub 2008 Aug 3.
Tadiso TM, Krasnov A, Skugor 5, Afanasyev 5, Hordvik I, Nilsen F. Gene expression analyses of immune responses in Atlantic salmon during early stages of infection by salmon louse (Lepeophtheirus salmonis) revealed 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
SEQUENCE LISTING
<110> Tecnovax de Chile S.A <120> PEPTIDES INDUCING AN IMMUNE RESPONSE AGAINST COPEPODS AND/OR A MUCOUS SHIELD IN FISH, VACCTNES, uSES AND METHODS THEREFOR <130> 2001 <160> 26 <170> PatenLln version 3.5 <210> 1 <211> 1965 <212> EDRT <213> LepeophLheirvs sairnonis <400> 1 Met Lys Leu Vol Leo Per Leu Leu Leu Phe Ala Gly Vai Ala Leu Gly 1 5 10 15 Tyr Per Pro 5cr Tyr Tyr Gly Trp Ala Pro Ser Lys Glu Tyr Vol Tyr 25 30 Glu Phe G1.u Thr Gin Met leo Thr Gly lie Pro Gb Tie Arg 3cr Gln 40 45 Tyr 3cr Sly Leu Lys Leo Ser 3cr Lys Vol Arg Tic Gin 5cr Phe Pro 55 60 Asp Tyr 5cr I,eu Arg Vol Gin PLc Vol Gin Pro Lys PLc Vol Thr Val 70 75 80 Asn Gin Gb Ile Pro 3cr Tie Asp Giy Arq Leo Tyr Val Pro 3cr Thr 85 90 95 His 5cr Gb Gin Leo Pro Lys Al a lie Tyr Gly Pro Leu Vol Thr Pro 105 110 PLc Glu Va L his Phe Lys Arg sly Vol lie Gb 3cr Ten Phe Vol Glu 123 125 Lys Asp mu Pro Val Val Vai Thr Asn Trp Lys Lys Ala Ten Leo 5cr 130 135 140 Gin lie Gin Thr Asp Lou Her Gly Se] Thrg Gin G-ly His Vat Gin Lys 150 155 160 Leo Asn His Gb Vol PLc Pro Leo Vat Ala Lys Asp Her Gin Glu Met 170 175 Lys Asn Vol 5cr PLc Phe His Thr Met Giu Arg Thr Leu His Gi y Asp 185 190 Cys Gb Thr TIn Tyr Thr Lou His Pro Leo Pro Len Tyr Gin Ala his 200 205 Glu Leo Gb Giu Gin Trp Arq Asn His Lys Tie Lys Val PLc Gb Thr 210 215 220 Val Pro Glu Tyr PIe Gin Her lie Arg 3cr Gin Lys Gin lie His Gin 225 230 235 240 Val Met Glu Gin 5cr Giy Arq Ala ys Thr Giy Lys Gin Tyr Pho Gin 245 250 255 Vat Thr Lys Thr Ills Asn PLc Asp Asn Cys Arg Gin Arg Pro Val PIe 260 265 270 8cr Ala Trp 5cr Giy lie Lys 5cr Asn Gys Asp Val TSr Arg Her Giy 275 280 285 Cys Asp Asp Ala PLc Ass Her lie Val Her Thr Arq Tyr lie Tie Gys 290 295 300 Gly Thr Pro Asp Lou Phe Val lie Arg Lys Aia Thr TSr Glu ftsn lie 305 310 315 320 lie Her Lou 8cr Pro TSr Giy Phe Asn TIn Pro Glu Lys Leo Thr 3cr 325 330 335 Phe 3cr TSr Val Thr Lou Giu Leo Arq Thr Tie Len Her Thr Val 3cr 340 345 350 lbs Gin lie Pro Lys Pro lys TSr Pro Lys Thr Va]. lily Asn Len Phe 355 360 365 Met Gin Tyr Pro Gin His Gi. 5cr Phe Asn 3cr GJn Per lie 5cr Gin 370 3/5 380 Gin Trp Thr Lys Gly 5cr lie Tic 3cr Pro Thr Asn lie 3cr Giy Phe 385 390 395 400 Thr Gly Phe Lye 5cr Len 5cr dy Phe Tyr Pro lie Ills Pro MeL Pro 405 410 415 TSr Met Asp Thr Aia Pro TSr Len Lou lyr Pro lie 3cr Ten 5cr Lys 420 425 430 Pro Gin Len Tie Arg Asp Vat Gin Gin Met Met 3cr Lys lie Vol Arg 435 440 445 Gin Thr Tyr Gin Vai Pro Gin 5cr Gys 5cr 3cr 3cr 3cr Asp Len Aia 450 455 460 Giy Tyr Tic Va]. 5cr lie Ala Gin Ala Len Arg Pro Len 5cr Ten Thr 465 470 475 480 Gin Len Lys Giu Len Asp Thr Gin Vat ills Arg She MeL Gin Vol Arg 485 490 495 Asp Lys Gin Ala lie Len Thr Per GIn Tyr Leo She Tyr Asp Tie Lcu 500 505 510 Ala Met Vai Giy TSr Asn Pro 5cr lie 3cr Tyr lie Jys Gin Leo Tie 515 520 525 Giy 5cr Asp Lys Tie Pro lie Gin Tyr Aic Pre Asp Vai Len Gin 3cr 530 535 540 so Ala Len Arg Asn lie Lys Thr Pro TSr Pro Gin Leu Phe Asn Leo Vol 545 550 555 560 Phe Thr Met Vai Lys Thr Leo Lys Aia Lye 5cr Pro Gin Leu Tyr Tyr 565 570 575 Val 3cr TSr Val 3cr PLc Her Asp Len Lou His Arq Ala Cys lie Asn 580 585 590 Pro 3cr 3cr Met Val Ala Gin PLc Pro Val His Vai Tyr Gly Asn PLo 595 600 605 Cys Asn Pro Gb TSr Pro PLc lie lys Asp Gin Tyr Tie Thr PLc Leo 610 615 620 Giu 3cr Gin lie Gin Giy Gly 3cr Gin Gly Her Lys 3cr Gin Lys Val 625 630 635 640 Val Lou lie Asn Ala Len Gly Lys Leo Gly his Tyr Lys Ala VaT 3cr 645 650 655 TSr Len Val Lys PLc lie Gin Gly Lys Vai 3cr Gin Sb Pro Met lie 660 665 670 Arg 3cr Leo Ala Val Tyr Ala Len Lys Arg TSr Ala MeL Gin Tyr Pro 675 680 685 Ala Lys Vai Lys Pro lie Len Met 3cr lie lie Asn Asn Pro Gly Giu 690 695 700 His Pro Giu Vai Arg ITo Ala Ala Vol 3cr Va! Len Pro Phe Sor Her 705 /10 715 720 Pro 3cr Thr TSr Giu Len Gb Lys lie Ala Leo Arg Thr Trp Phc Glu 725 730 735 Pro 3cr Lys Gin Val Thr 3cr PLc lie Tyr 3cr Thr Len Lys 3cr Len /40 745 750 Arg Thr Thr Gin Val Pro (flu Leo Met Gin Phe Arg Asn Lys Vat Lys 755 760 765 3cr Val Tie Pro Met Vol Arg Arg TSr His 3cr Gly lie Gin Pho 3cr 770 775 780 His Asn Tie His lie 3cr TSr PLc Lou Asp Tyr Len Lys lie Val Ala 785 790 795 800 Asn Asn Lye Leo Giu Tie Vat Asn Thr Pro G1 9cr Met Leo Pro Ala 805 810 815 Lye lie 3cr Phe 9cr Glu Asp Trp lie Aen Arg 9cr Met Arg lie Lye 820 825 830 Gly Leu 3cr PLc 3cr Lou Tyr Ser GIn Gly Not Asp Tyr Val Phe Gb 835 040 845 Lye Met Met Thr His Leo Gly lou Lye Gb 3cr Pro 9cr Pro lie Va] 850 855 860 Thi. 9cr Giu Leo Gin Lye lie Thr GIn Lye Leo Gin Tic Thr Pro Arg 865 870 875 880 Thr leo Gin Glu Pro Gin Leo 3cr Lou lye lie Lye Phe Met Giy Leo 885 090 895 Gin Arg lie Phc 3cr Leu Asp 3cr Lye Phe Tyr Met Gb Thr lie Gin 933 905 91 0 Lye Vai Thr Giu lye Lou Arq 3cr 3cr Pro Gbn lie Leu 9cr His dy 915 920 925 Leu Pro Phe Lye Tyr Thr Lye Thr Arg Aen Phe Val Asp Va] Gin 3cr 930 935 940 Val Am Pro Thr Ala 3cr Gly Phe Pro Vol Arg lie Gin 3cr Vol Thr 945 950 955 960 Pro Met Vat Tyr 3cr Vai lye Giy Tyr Thr 3cr Gly Aen PLc 3cr 9cr 965 970 975 Len Vat Pro 3cr Val Gin His Giu Val 5cr Phe Len His 5cr Ala Arg 980 985 990 Vol Lye Tic Thr Pro lie Leo His Ala Lye V-al Gb Thr Thr Met Gly 995 1000 1005 Val lie 3cr Pro Phc Thr Lye Gin Tyr TIc Giy 3cr Gly PLc Glu 1010 1015 1020 Met: Gly Leo His 3cr 3cr Tbr Pro Ten Asp Val Lye Val Her Val 1025 1030 1035 Asn Her Leu Gly Gin Len Lye Leo Thr Met lye 3cr Pro Gb Gin 1040 1045 1050 Vai Gin Asn Gin Val GIn Ten Ala His Val Tyr Thr Lye Pro PLc 1055 1060 1065 TSr Phe Lye Lye 3cr Tyr do TAr Tic Val Pro Ala Her Arg Her 1070 1075 1080 Pro Gly Aen Lye Met lie Leo 3cr Gly Thr lie Leu Lye Lye Phe 1035 1090 1095 Thr Phe Ass Pro Thr Lys 3cr Thr Val Gly lie Asp Ala Pro Len 1100 1105 1110 Lye lie Her Thr Asp Tyr Pro Val Met Asp Leo Ala Val Ala Tyr 1115 1120 1125 Lye Lye Phe Her Aen Ala Pro Asn Pro Met Ala lie Leu Lye Ala 1130 1135 1140 Met Thr lie Pro 3cr Thr Leo Arg lyr Val 3cr Phe Aen Leo Lye 1145 1150 1155 PLc Aen Pro Thr TAr Her Thr Thy Lye Gb Leo Arq Thr Arq PLc 1160 1165 11.70 Her Len Ala 3cr Gly Tyr Lys Pro Ala Pro Her Gb Phe lie Arg 1175 1180 1165 Tyr MeL Leo Pro Arg oIly Tyr TSr Gin Glu TAr (flu lie Met Lye 1190 1195 1200 Met: dye Arq Gin His Arq Pro file Asp lie TAr Gly Cys lie Gin 1205 1210 1215 Her Gin TAr Lye 3cr Ler His Ala Ala Hor Gin Vai Her Aen Gin 1220 1225 1230 Ala Arg 5cr Leu Cys Ten Gin His Vai Arg lie Asn Met Phe Pro 1235 1240 1245 l.J.e Lye 5cr Gin 3cr Tie Pho Asp Gin Gin Met Gin Asn Cys Tie 1250 1255 1260 Lye 5cr Val Lye lie Gys Glu 5cr Vai Arg Phe Vai Cys Per Gin 1265 1270 1275 3cr 5cr Thr Asn Thr Vai Asn Pro Len Thr Cys Ciii Olu Lye Gin 1260 1285 1290 Arq 5cr Cys lie 7yr Arg Gin lie Asn Lou lie Lye Leo Asn Thr 1295 1300 1305 Vai Leo His Len Leo Gin Thr Giy TSr Gly Val 5cr Val Thr Val 1310 1315 1320 Asp Aia 5cr Lou Asn 3cr Pro Tyr Gin Met Arg Thr Tyr 3cr Thr 1325 1330 1335 lie Len Ala Len Giy Aia 5cr Thr Oar Gin His His Gin lie Arg 1340 1345 1350 Gly Tyr Vai Asn Ala Gin lie Lye 5cr His Gin Len Pro Ala His 1355 1360 1365 vol Len lie Aia Asp 5cr Lye Per Arg Leo Pro 5cr Tie 5cr 5cr 1370 1375 1360 Arg Trp Aen Leu Giu Gin Met Tie Asn Asp Giu lie Thr Met Giu 1385 1390 1395 His Asp Met 1/al lie Tyr Tyr Ciy Lye Arq Thr 5cr Pro Arg Asp 1400 1405 1410 Gl.y Met His Lys lie lie Ten Ciii Ala Phe Ala Thr Lys Per Asn 1415 1420 1425 Gi,y Leo Arg Lye Per Ito Vol Gin Per Pro Gin Tyr lie Too Gys 1430 1435 1440 Asn Lys Gin lie Gly Gin Gly Arg Thr Len Ala Pro Vai Cys Gin 1445 1450 1455 Lys Len Arg His Len 3cr Ala 3cr Val Asp Thr PLc Lys lie Arg 1460 1465 1470 TAr Lys Phe Pro Met Arg Gin Thy 3cr 5cr TAr Tyr Ala Tw° 5cr 1475 1480 1485 Tyr lie Arg Asn Val Leu Gin Val Phe Phe Pro Tyr Len TAr 1490 1495 1500 Gin Arq Tyr Phe Asp Ala Thy 3cr Ala Val Gin dy lie Lys Lys 1505 1510 1515 Asp Glu Thr Lou Len Gin Ala Pho VaTh 3cr Arg Gin Giy Asp Len 1520 1525 1530 Ala Gin Tie Lys Tyr Lys Gin His Gly The Gin Trp Asp Vai 3cr 1535 1540 1545 Am lie Arg i1eu Pro Lys TAr Leu Thr Gin His Ile Lou Pro Len 1550 1555 1560 3cr Phe Arq Asn His Gin Len Thy Thr Giy 3cr Arq Phe Tie Gin 1565 1570 1575 Lys PLc TAr 3cr Gin Gin 5cr Pro Ala 3cr Cys Thr Val Glu Pro 1580 1585 1590 Ama PLc Va] TAr TAr Phe Asp Asn Lys Thr Tyr Pro Tyr TSr en isys 1600 1605 Asn Asp Cys Gin His Len lie Vai Tys Asp Cys 5cr Gly Len Trp 1610 1615 1620 Pro Met Ala Vai Thr Ala Arg Lys Ala Gly 3cr Gln Met Gin Val 1625 1630 1635 Lys Met Tie Val Giy Lys His Val Vai Val Met 3cr Pro Len Gin 1640 1645 1650 3cr Her Val lie Asu Asn lie The Val Asn Gly lie His ho Pro 1655 1660 1665 Len Val Gin lIly Sly Lou Tyr Lys Tyr lie Gin Pro Val Aso Gin 1670 1675 1680 Her 3cr Gly 5cr 3cr Thr Pro Pro 3cr 3cr Thr Pro Sly Pro Lou 1685 1690 1695 Leu 3cr 3cr Leu Sly Pro lie 3cr TSr Gin Sly Arg TSr Val Tie 1700 1705 1710 lys Phe Trp Her Tyr Len Asp Sly TSr Val, Val Val Lys His Tie 1715 1720 1725 Lys Thr Sly Leu lie Val Tie Phe Asp Sly Glu Arg lie Glu Val 1/30 1735 1740 Asn Pro Pro Ala Phe Leu 5cr 3cr Lys Ala Cys Sly 110 Cys Sly 1745 1750 1755 Asp Met Asn dy Gin 3cr Her Ala Asp Len Ala 3cr Pro Lys Met 1760 1765 1770 Cys lie The Slu Gin Pro Arg Met Ala Ala Tyr 5cr Tyr Met lie 1775 1783 1785 Lys Slu 5cr Cys Gin Gly lie Pro TSr Pro Gin Gin Lys TSr Lys 1790 1795 1800 Phe Gin Lys Gin 3cr His Gin Cys Val Leu Lys Lys lie 3cr Vol 1805 1810 1815 Ihr Pro Len Gin Asp Len lie Thr Arq Leo Tic Lys Val Arg 5cr 1820 1825:1830 Sly Pro Len Sly lie lie 3cr lys His Len lie Glu Tyr Arg Ala 1835 1840 Tt845 Asn SLy Asri Gin lie Cys Phe 3cr Gin Arq Vol Lou Asp lie Cys 1850 1855 1860 Gly Sly Arg Her Val Pro Vol Tie (fly His Met ALa Her Thr Pro 1865 1870 1875 Phe Thr Gys Len Gin Her Tyr Her His Len Ala Lys His Ten Lys 1833 1385 1893 Gin Arq Val Val Ala Asn Gin Gin lie Pro Gin Len Met Lys Tyr 1895 1900 19C5 Pro Thr Thr Tyr Asn Arg Met lb Gin Gin Ala Her Asn Cys Gin 1910 1915 1920 Her Her Her Her Her Gly Her Gly Met Gly Gly Gly Her Len Pro 1925 1930 1935 3cr 3cr Pro Her 3cr 3cr Asp 3cr Her 3cr His His Ala Gb Pro 1940 1945 1950 3cr Thr Gly Arg Phe Gin Pro Gin Ile Tyr Asn Tyr 1955 960 1965 <210> 2 <211> 1900 <212> PRT <213> Lepeophtheirus sairnonis <400> 2 Met Lys Leu Phe Ala Phe Leu Cys Phe lIe Gly Len Ala Thr Ala Thr 1 5 10 15 Her Pro Her Trp Gin Trp G1r Ala Sly Lys Gin His lie Phe His Tyr 25 30 Thr Sly Arg Len Len 3cr Sly lie Pro Gly Len Arg Pro His Phe Her 40 45 Gly Ile Sly lie GI.u Thr Gln Vol His Len Gin Vai Lys 3cr Len Gin 55 60 Asp lie Arg Len Aso Len Arg Gin Vol Asn Tyr Thr Gln Vol Asn GJ y 65 73 75 80 Pro Len 3cr Pro Gly Leu Pro His Val Thr 3cr Per Tyr Giu Gly 3cr 90 95 Aso Trp Arg Tyr Val Len Lou Pro Gin Phe TSr Gin A1.a Pro lie Asp 105 110 Ile Lys Lys Len Len Lys Val Pro lie Thr Phe Ala lie His Asp Gly 120 125 GIn Tie lys Thr lie Thr Val Per Gly Thr Glu Gin Gin Trp 3cr Len 130 135 140 Phe Lys I]S Ala Len Val Ala Len Pho Gin Thr Lys Met Gin TSr 3cr iSO 155 160 TSr Len Asp Ten Gin Met Asn TSr Tie Val Lys Asp 3cr Asp 3cr TSr 1/0 175 Lys Asn Tyr Trp Lys Val 3cr Gin Gin Thr lie Gin Giy Val Cys Asp 185 190 Val lie Tyr Gin Vai Asn Gb Len Pro Gin Tyr lie Val Lys GThj Arq 200 205 Ala His Tyr Phe Pro His Lee Gin Gys Thr 3cr Lys Lys Phe Tyr Gin 210 215 220 Tie Thr Lys TSr Lys Gin Tie Asp 3cr Cys Lys Lys 3cr Ala Val Tyr 225 230 235 240 TSr Phe Met Lys Pro Ala Va1 Lys Ala Gin 3cr Cys Asn Her Phe Lys 245 250 255 Cys Ten Her Asn The The Gly 5cr Ala Per See Not Thr Arg Tyr Tyr 260 265 270 Ala Cys Giy Pro Arg Her Asn Trp Tie Len Gin TSr lie Val Asn Gin 275 280 285 Cl y lie Val Gin Arg Pro Vai Gly Val Lys 3cr Gin TSr lie TSr TSr 290 295 300 Gly Thr Arg Gin Vol Leu Lys Leo Arg Thr lie Gin Pro lie 5cr 3cr 305 310 315 320
S
Glu Val Pro Lys Pro Pro Gb Pro Arg Thr TSr Glu 5cr Tie Met Tyr 325 330 335 Giu Tyr lie Asn Ala Gly Gin Val 3cr Arg Gin Gin lie Giy Tie Tie 340 345 350 Pro Lys lie Pro Gin 3cr Gb Leu Lys 5cr Gly Glu lie Tyr Lys Tyr 355 360 365 Leu Pro Arg His Phe Asn Pro Ala Pro 3cr 5cr Thr Glu 3cr Lys Gin 370 375 380 His Leu 3cr Ala TEr Lys lie Lys Ala Gb Len Lys 5cr Tyr lie Tie 385 390 395 400 3cr Tic lie Asp Asp Lou 5cr 3cr Val, Glu Glu Leu Ala Gin Lys Glu 405 410 415 I1 Pro Leu Arq Lou TSr Thr Phe lie Arg Giy Met Thr Leu Len Lys 420 425 430 Vol Gb Asp lie lys 5cr Len Tyr Thr Asp Len Lys 5cr Thr Val Tyr 435 440 445 3cr Pro Ala His 3cr Asn Gin Gin Lys lb 5cr Met Phe His Asn lie 450 155 460 PLc Phe Asp Ala Vab Met Val 5cr Gly Thr TSr Pro Ala VaT leo Phe 465 470 475 480 Len Lys Asp Met lie Lys 5cr Lys Gb lie Pro Thr Tyr Gin Ala Thr 485 490 495 SO Tyr Lou Leu MeL Leo Lou Pro His His Tie lie TSr Pro TSr lys Giu 500 505 510 Val Phe TSr Set Leu Leo Glu lie lie Gin 5cr Glu ho Val. Tie 5cr 515 520 525 Asn Thr lie Len Tyr Asp Thr Ala lie Len 5cr Met Set Asn Len Val 530 535 540 Gin Lys Tbr Cys Leu Asp Lys 5cr Arg Gin Val Per Tyr Pro TSr Ala 545 550 555 560 Val. Phe Gly Gin Phe Gys Asp Ala Gin 5cr Gin lie Val TSr Glu Lys 565 570 575 Trp lie Pro Tyr Len TSr Lys Ai.a Ye] Gin Thr Ala Pro Thr Ala Asp 580 505 590 Arq Arg Asn ALa lie lie Met Aia Len Gly Aia Len Lys Hs Lys Asp 595 600 605 lie lie Pro Ala Leo Leu Pro Leo Vai Glu Giy His Giy Pro Il! e Glu 610 615 620 Gin Gly Set dy Vai Aia Phe Pro Asn lie 9cr Arg TSr Leu 3cr ILe 625 630 635 640 Tyr Ala lie Gly Asp Val Arg Val His His Pro Gin Len Vol leo Pro 645 650 655 lie lie Leu 3cr Va] Tyr 9cr Asp Pro Ala Giu Asn TSr Gin Leo Arq 660 665 670 lie Ala Aia She Asn Met Len Va] Asp Met Asp Pro Pro Met Asn lie 675 680 685 lie Gln Lys Tie Aia Aia Met TSr Trp 5cr Gin Lys Asu TSr Glu Vai 690 695 700 lou Lys Lys TSr Tyr TSr GI y Phe Tyr Thr Len 9cr Arg Sot Vai Asp 705 710 715 720 ho Per Asn Len Gin Asp TSr Per Pro Giu Per Thr Leu Ala Lys Lys 725 730 735 TSr Gin Leo Val Val Pro Lou Met Arg lys Thr 5cr Gly Val Leu Gin 740 745 750 Per Thr 3cr Thr Leu Tyr (flu 3cr 3cr His Len Pro Leo Leu Sir Ala 755 760 765 Sly tyr Met Asn lie Phe 3cr Trp lie 5cr Asn Tyr Ary 3cr Val lie 770 775 780 Pro Per Asp Val Pho Met Lys ILe tie Asn Ear Ile Sly Glu Phe Asn 785 790 795 BOO Tyr 3cr Pro Phe Glu Sly Ala Phe Lys Lou Tyr Sly Gly Ass Asp Phe 805 810 815 Met (flu Lys Leo 3cr Asn Phe Lye Asp Met 5cr Gin Per Gin Gin Lye 820 525 830 Ala Ala Phe Gb Leo Phe 5cr Lye Pro Sly 5cr Pro Thr (flu Sly Gin 835 840 845 Val Tyr Sly Sly Arg Tyr Thr 5cr flu 5cr Arg Arg Gin 3cr Met Met 850 855 860 Gin His Glu Thr Gln Tyr Leu Met Lye Thr Gly Thr Pro Sly Len Leo 865 8/0 875 880 Giu 5cr Thr tie Gin 5cr Val Lye 5cr Gin Lou His Ala Sir Trp Met 885 890 895 lye Val Val Sin Gin Len Lye Tic Gin Thr Arg Gin Asp Sly Pro Lou 900 905 910 Ala Thr Len Val Tyr Len hen Phe Phe Asp Asp Ala Plir Phe Phe Thr 915 920 925 Per Tb 3cr (flu Val TIir Val Thr Ala Len Aeg Sin Lye Tie Teu Pro 930 935 940 Tyr Len Lye Asp Ala Lye 3cr Aen Asn Trp Thr Gin Tlir lie Gin Lye 945 950 955 960 Val rye 5cr Thr lle Gys Glu Tys Gin Len Pro Val Aen Sly Gin lye 965 970 575 Met Vai Asn Len Gly 3cr Ala Gin Phe Lee lie Pro Her Asp Met Gly 980 985 990 Pho Pro lie Val lie Gin Gin Asn Met Pro Giy Leu Vol 5cr lie Arg 995 1000 1005 Sly Her Met Gin Met Asn Cys Asn Val Gin Thy Pro Thr lie Lys 1010 1015 1020 Phe Gin Ala Lou Pro Met Len 3cr Val His His His Val His Vol 1025 1030 1035 Gly Thr Tyr 5cr Pro Phe Thr Lys Lys Leu Va! Va! Thr Sly Len 1040 1045 1050 Lys Gin Asp Leu Thr Val Asn lie Pro Thr Lys Thy His Val Len 1055 1060 1065 Tyr Lys Vai 3cr Thr Asn Gin Vol Lys lie lie Len Lys Pro Val 1070 10/5 1080 Gin Met Lys Lys Pro Thr Asp Len Len Tyr Phe 5cr Thr Lys Pro 1085 1090 1095 Phe Thr Thr Ala Gin 3cr Tyr Phe Her Leu Met Pro Arg Cys His 1100 1105 1110 3cr 5cr Asp Len Lys Tyr lie Lys 5cr Glu Val Pro Met Arg Ala 1115 1120 1125 Vol 5cr lie Pro Her Giy Sly 3cr Val Lee Sly Len 5cr Lou Her i130 1135 1140 Ala Thr lie Gin Thr Gin Thr Pro PLc Lee Asp Lee Pro Tyr Val 1145 1150 1155 Met Gin MeL Aia Lys Met Tyr Asn Tyr Asn Pro Val Asn Met lie 1160 1165 1170 Arg Phe Ten Pus Ala Pro His Her Vol Thr Gin Asn Sly Gin Pro 1175 1180 1185 3cr Tb Arg Tyr His TSr Phe Arg Va] lys lyr Asp Ala Leo 5cr 1190 1195 1200 5cr 3cr TSr Lys Glu Ala Glu Phe Thr She Lou Pro Giy Cys Ala 1205 1210 1215 Vai Lys Lys Met Giy Gin Tys Gin Pro Len Tie Met 3cr lie Ala 1220 1225 1230 Pro Lys Thr 3cr Gly Gin Giy Gly 3cr TSr leo Trp 3cr Trp Met 1235 1240 1245 Pro Tyr Giy Vol Gin Val His Pro lie Per TSr Lys Vai Lou Pro 1250 1255 1260 Vol Vai Gin Glu TSr Leo Lys 3cr Tic MeL Met Gb Lys Thr His 1265 1270 1275 Met 3cr His Gly She A1.a Ala Vol Lou Arg Leo Giu Ala lys Phc 1280 1283 1290 Asn Gly Giy Gin Arg Pro Arg Len Phe Thr Tyr Gln Ala 3cr lie 1295 1300 1305 Gly Arg Giy Lys Asp Lou Ala Asn Vol Thr Arg Trp Hs Giy TSr 1310 1315 1320 Trp Gb TSr Gb Pro Asn 3cr Pro 3cr Met Scr Gin Lye lie Cys 1325 1330 1335 Vab Asp Gly His Vai TSr Lou Pro Thr Vol Pro Thr Trp Asp lIe 1340 1345 1350 Gly Met Thr 7\rg Asil MeL Ass Met Asn Leo Leo lie Lys Aon TSr 1355 1360 1365 lie Gly Phe Gly Arg TSr Cys Asn Gb 3cr Gin fle Lys TSr Phe 1370 1375 1380 Val TSr 3cr Arg Val 3cr Gin Lys Gin Len Gin Trp 3cr Arg @10 1385 1390 1395 Her Pro Val Ala Lys lie Cys Glu Lys Pho lie Glu Arg Arg Vai 1400 1405 1410 Pro Giy Ala Met Her Thr Pro Gb Cys Gin Glu Thr His Trp Leo 1415 1420 1425 Ala Arg Met Tyr Asp Gin Val Asp lie Lys Phe Gin Her lie Asn 1430 1435 1440 Vai Pro Thr TSr lie Lys Asn TSr Val Gly I5 Vab Thr Her Her 1445 1450 1455 Lou Gin Arg Leo Trp Trp Pro Tyr Vab Thr Gb Asn His Her Pile 1460 1465 1470 Thr His Val TSr Gly Ills Tyr Gin Pro Lys Arg Glu Her 9cr lie 1475 1460 1485 His Leo Her Phe Cys lys 9cr Her Glu Thr Vai Her Met Thr Leu 1490 1495 1500 Lys TSr Pro Gin Arg His Val Lys Pho Her Asn lie Arg lie Pro 1505 1510 1515 Tyr Vai Phe Arg Pro Phe Leu Pro Phe Val Ala Gly Glu Her Asn 1520 1525 1530 Leu Met Lys Ala Val Gin lie Thr Her Gly 5cr Arg Met Leu Pro 1535 1540 1545 Thr Cys Arg lie Glu Lys Asp Trp Leo Arg Thr Phe Asp Aso Lys 1550 1555 1-560 3cr Leo Pro Leo HIs Met Asp Asp Cys The His Vai lie Ala Gly 1565 1570 1.575 Asp Cys Her Thr Thr Met Gin Plie Gly lIe Lou Ala Arg Vol. Viii 1580 1585 1590 Pro His TSr Val Ala Lys Glu lie Lys Val Tyr Met Gin Lys Thr 1595 1600 1605 Glu Vol Lys Leu Tic Pro Thr Pro 5cr Tyr Her Arg Gly Asn Arg 1610 1615 1620 Asp Vai lys lie Gin Tie Asn Gly Her Gin Pho Va1 lIe Pro Arg 1625 1630 1635 Glu lie TSr Lys Thr Phe Pro Val Gly 3cr Vol Thr Pro Vol Val 1640 1645 1650 Gin lie Tyr Arg 5cr Tie Asp Asp Vol Tyr His Len Lys Gly lie 1655 1660 1665 Thr Thr Ply lie TSr lie Lys TSr Asn Gly Gin Arq lie 3cr VaT 1670 1675 1660 3cr Pro Her Len Per Met Lys Gly Arg Ten Cys fly lie Gys OiLy 1685 1690 1695 Asp Met Asn Asp Gin Ala Len Ala Asp Tic 3cr Giy Pro Thr Arg 1700 1705 1710 Cys Va] Tyr 3cr Per Pro Per Val Gin Vol Ala Ala Tyr Arq Val 1715 1720 1725 3cr Thr Pro Gin Cys 3cr Pro Met Asp Thr Lys lie Lys Gin lIe 1730 1735 1740 Leu Asp Len Gin Thr Asn Asn Cys Ala Arq Phe Gin Gin Met Pro 1745 1750 1755 Thr Gin Vol lie Lys TSr Tyr Ala 3cr lie Ala Ply Lys Cys Thr 1760 1765 1770 Arg Gin Gin His MeL Tie Len Gin Arg Gly Her Glu TSr Cys Pho 1775 1780 1785 Her TSr TSr Pro Vol TSr Gin Cys Gly Ala Gin Gys Per Pro Lys 1790 1795]B00 Pro Lys Glri Len Aia His Lys Lys Vol Gly Phe His Cys Met Lys 1805 11810 1815 5cr Gly Arq I1eu Tlr Giu Leu Tyr Ang Gin Lys Vai lie Gin GI y 1820 1825 1831)
S
Leu Va1 Len Pro Glu Ten Gin Set Arq Pro lie Thr Plie Set Thr 1835 1840 1845 Ala Tie Len Val Pro Gin Her Gys 3cr 5cr lie Val 3cr Gly His 1850 1855 1860 Pro lie Leu 8cr Len lie Her 8cr 3cr Asn 5cr Asn Giy lIe Met 1865 1870 1875 Asn Thr lie Asp MeL Gly Gi,y 3cr 5cr Gly 3cr Asn Lily 3cr lie 1880 1885 1890 Giy 3cr Giy 3cr 3cr Arq Len 1895 1900 <210> 3 <211> 1520 <212> PAT <213> .TepeophLheirus salmonis <400> 3 Met Her Pro Leu lie Lys Met Arg Val lie Ala lie Len Phe Gys Phe 1 5 10 15 Val Ala TSr 3cr 3cr GAy 5cr lie The Gin Asp Gly Thr Giu Tyr Thr 25 30 Phe Glu TSr Gin TSr 3cr Ala Va]. Val Giy TSr Met Asp His lie Pro 40 45 Ills 3cr 3cr Gly Phc 3cr Tyr Lys Tyr Met TSr Gin Met Gin lie His 50 55 60 Gly Asn 3cr lie Lys Val Lys Len Her Asp Phe Lys Ten Her (lie Phe 70 75 80 Asn Sly Lys His Gin Sly Gly Gin Tyr Pro Phe Asp His TSr Asa Phe 90 95 lie Ala TSr Asn Arq Asp Val Pro Pro Phe Glu Vai Lys Then Asp Her l0[) 105 110 His Giy Leu Phe Her Her Leu Tys Val Her Pro Lys Len Thr lie PLc 115 123 125 Gin Arg Asn Met lie Lys Giy Trp Val Gin Lee Gin Len Asn MeL Asp 135 140 Lys lie Asn His His Gin His Glu Phe His Her Gin Gin Gin 8cr lie 150 155 160 PLc Giy Asp Cys Asp Thr Lee Tyr Thr \Tai Asn Asp His Lys lie Val 170 175 Lys 3cr Vai Thr His Thr Lys Asp Cys Lys Asn Arq Vel His Vai Leu 185 190 lie Asp Asp Trp Arg Gly His Arg Cys Asp Len Giu Asp Pro Asp His 195 200 205 Pro Gin 3cr Arg Gin Asn Pro Asri Giy Len Tyr 3cr Ala Her Asn Thr 210 215 223 Tle Tyr Val Val Asp Lys Lys Gly Asp His Phe His Pro iys Ala lie 225 230 235 240 lie dy 3cr Her Her Vai Va] Ala Gin PLc Tyr Gin 3cr Gin Giy lie 245 250 255 3cr Phe Va] Ala His Her Aso Her 3cr Her Tie Len Lys 3cr 3cr Gly 263 265 270 Am lie 3cr Gin Gin lie Thr Val Vai Giy Vol Asp Len Asp 8cr Len 275 280 285 His Tyr G.m PLo Gin Asp Her Gin Tyr Thr Trp Lys Her Gin Arg Asp 290 295 303 Len Lys Aia Arg Gin Giy Tyr T1eu Ala Thr Giy Gin Phc PLc Gin Asp 305 310 315 320 Asp MeL Pro Tim lie Her Lys Tyr Val lys Gin Lys Leu Ala Lys TSr 325 330 335 His Asp Tie Met Asn Lys Met Her ?hr Glu Ala 3cr Thr lie Gin Tys 340 345 350 Aid His Met Tyr Gly lie Asn Her lie Tyr Pro Ala Met Lou Ala Met 355 360 365 Asp Tyr 3cr Ala Lea Lys Gin Lou Her Gin Gin Len His Her Asp Lys 370 375 380 8cr Ala Gin C] y Vol Tyr Arg Tyr Ann Lea PLc Asn Glu Lou Len Gly 385 390 395 400 8cr Ten Gly Thr 3cr Ala Her Ala lie Lou Val Arg Asp Met Tie Ala 405 410 415 Gin Ann Lys Phe Asp Asn Phe Arg Asp Ala Val Arg TSr Lou Thr Ala 420 425 430 Vai Pro PLc His Tie Arg His Pro Her Lys Gin Lou Len Lys Gly PLc 435 440 445 Gin Thr Leu Tyr 3cr Tyr Gin Giy His Gin Phe Ile Lys Asp TSr lie 450 455 460 Pro lie Vol Leu Gly His Thu Ala Arg Vai Thr Gys Gin Arg Ala Gly 465 470 475 480 Vai Met His 8cr Pro Ala 3cr Gin Glu Gys Phe His Her Vai Val Asp 485 490 495 Gly Tyr Ala Asp Lys Thr Tie Gl,u Lys fle Met Giy 3cr Her Asp His 500 505 510 Thr mu Gin lie Lys Lou Ten Gly Met 3cr Phe Ann Len Arg Lou G]y 5T5 523 525 Asn Vai Ala Gin Lys Len Lys Pro Len Tie Tyr Giy Gin TSr Gin Tic 530 535 540 Lys 5cr Giy His Lou Arg Thr Lou Ala Val Pro Ala Ala Ala PLc Gly 545 550 555 560 Ala lie Asn 5cr Gly Lys Gly Ala Gin His Leu Len Pro lie Phe Val 565 570 575 Glu Thr Gin Asri Asp His Gin Len Arg Len Thr Ala iou 5cr Tyr Len 580 585 590 Met Asp Ala His Pro Thr 5cr TSr His Phe Asn TSr lie Val Ala Vol 595 600 605 l5 Len Tyr Arg Gin Lys Asp Tyr Glu Va1 lie Ass' Tyr Ala Phe Thr Len 610 615 620 Phe Gin iys Tyr Ala Arg Ass' lie Ass' Pro Cys Lys Lys Per Val 5cr 625 630 635 640 Val Len Ala Lys Tyr Phe Len Lys Tyr leo Lys Gin Tyr Per Ills Phe 645 650 655 GIn Thr Asp Tyr Gly Ten Gly Val 3cr Tys TSr Tyr 3cr Arg Gin Phe 660 665 670 Gin Gin Pro Lys Tyr Gly Cys Gly dy Gb Tyr 5cr Tyr Trp Val lie 675 680 685 Giy Sex His Arg 5cr Thr Len Pro Leu Sex Val Ala lie 3cr Met Asp 690 695 700 3cr TSr Met Phe Gly Giy Tyr Thr Aia Ass' Gly Met Cys Vai Gln Teu 705 710 715 720 Arq lie Glu Giy Len 5cr Lys Ala Ten lie Arg Lys PLc Lys Thr lie 725 730 735 5cr Pro Asp Tie Trp Lys Par Gin Gin Len Lys Asn lie Len Met dy 740 745 750 Asp Met His lie Lys Gin Arg Pro Asp Gin Pro lie Ass' Vai Gb Vai 755 760 765 Lou Len Phc Vab Lys Asn Sex Vol Vs'i Ala Phe Arq Gin Tyr Asp Gin 770 775 700 Asp 3cr lie Lys Glu Gly Gly 5cr Leo lye G1. Tie Phe Asp Gin Lou 785 790 795 800 Gin Gly Lou Gly Asp Thr Tyr 3cr Met Asn His Gin Arg Ala Met Arq 805 810 815 PLc Gly 5cr Leu Leu Tyr Gin Gin Pro Len Gin lie Gly Ala Pro Val 820 025 830 Is Ala Tyr Met Asn 5cr Phe Thr Gly Val Phe Asp Tie Gin Ala Thr Vol 835 840 845 Lys Lye Gly Aen Ala Arg Gly Len Met Phe Arg Asp Val Lys Tyr Thr 055 860 Met Asn lie Phe GLy Gl.n Gly 5cr Arg lie Met Met Val Gin Asn Leu 865 870 875 880 Gin Thr Lye Asn Ala Tyr Her Val 5cr GIn Asp Arg lie Tyr Gly 3cr 885 890 895 His Phe Pro Arg Asn Phe Val lie Gly Val Asn Pro Leu Lys Lye Gin 900 905 910 Phe Lys Len 5cr Vai Glu Arg Pro Pro Tyr Gin Asp Pro Len Met Tie 915 920 925 Met Met His 5cr Gin Thr Asn Vai Vai TSr Arg 5cr Gin 5cr Tie Asn 930 935 940 Ace Tys Gin Asp lie 5cr Ala Asn Cys Ala Gin Cys Lye TSr Tie Thr 945 950 955 960 Pro Tie Her Tyr Gly Pro Asp Ala Ala Lys Thr Arg Vol TIe Vol Asp 965 970 975 Arg Gin Gys Asp Asn Thr Gly 5cr Tyr TIe His Gly Gin Tyr She Asp 980 985 990 Gys 0Th Met Glu 3cr Asn Arg Giy Lye Vai Len Tyr Ills Len Trp Arq 995 1000 1005 Ala Met Thr Pro Tyr His Lys Asn Pro Lys Thr lie Giy Asn 3cr 1010 1315 1020 lie Arg Met Gly lie Arg Gin Tie Arg Ala Tyr Pho Val Phe Phe 1025 1030 1035 Pro Arg Ala Glu Lys Cys Gly Ala Met Leo Arg Trp 5cr Gin Sor 1340 1045 1050 Is Lys Gin Asn Pro Val Tys Gin Tie Glu lie 8cr Len Arg Phe Asn 1055 1060 1065 Thr Set Pro Asri Gly Gin Arg Leu Tyr Phe Arg Cly Arg Lys ftp 1070 1075 1080 Ala Leu Thr Gly lie Va! Lys Ala Lys Gly Glu Pro Gin Asp Arg 1085 1090 1095 Val Tyr Lye lie lie Lea Gly His Glu Phe Thr Pro Oily Tyr lie 1100 1105 1110 Gin Asn Arq Lou Lys Phe Arg Met Gin Arg Val Ala Val Pro Gly illS 1120 1125 Leu Lou 5cr Asp Tyr 3cr ho Cys Phe Asn Met Gin Asn Lys Tyr 1130 1135 1140 Pro Asp Plie Giy Gin Gin Phe Met Thr Tyr Asp Lys 8cr Thr Gin 1145 1150 1155 Leu Lye Met 3cr Gly Asn Aia Arg Lou Gin Tyr Giy Aia Ala Ala 1160 1165 1170 Asp Cys Asp 5cr Thr Pro Gly Gin MeL Lys Lou 3cr Phe Gin His 1175 1130 1185 Gin Thr Thr Gin Glu Ala Arg Gin Ala Met Jys His Thr Trp Tyr 1190 1195 1200 Tyr Gin Lys Cys Met Gin Gin lye Gin His Pro Giu Trp Ala Her 1205 1210 1215 Avg Giy Asp Arg ieu Pro Phe Thr Glu Ala Gys His Met Thr Thr 1220 1225 1230 Tip Asp Ala Thr Thy Ala Arg Lys Tyr Thr Trp Lys Met Asn Phe 1235 1240 1245 Val Lys Met Thc Asp Arg Met Asn Ala lie \Tai 3cr Gin Phe Gin 1250 1255 1260 3cr Vol Met Lys Thr Gly Lou Leu Pro Tyr Trp Asp lie Asp Pro 1265 1270 1275 Giu lie Tie Pro Ala Thr Soy Ala Asp Pro His Met Asn lie Glu 1280 1285 1290 Ala Thr Tee Lys Asn His Asp Lys han Val Asp Tic Tyr MeL Glu 1295 1300 1305 TAr 3cr Gin Gly Giy Gin His Phe Asn Asp lIe Pro Leu 3cr Lee 1310 1315 1320 Asn Trp Arg Pro Met Lee Avg Asn Leu Lys Phe TAr 3cr Asn Thr 1325 1330 1335 Ary Arg Lee Met Gin Tyr Lys lie lie His Giy Gys TAr Ala Thr 1340 1345 1350 Tie Asp His Va] Tyr Thr Leu Aso Asn Val TAr Tyr Pro Tyr TAr 1355 1360 1365 Pro Thr 5cr Cys Trp Thr Leu Ala 3cr Gly ills Cys 3cr Pro His 1375 1380 Pro TAr Tyr Ale Vol Pho Val lys Lys 5cr Ala Cly Her His Lee 1385 390 1395 Asp Ala Lys Tie Tyr Leu Gly Giy His 3cr lie (lie Phe Gin Ala 1400 1405 1410 Her Giy Pro Lys iys lie Aso Val Lou lie han Giy G]n Ala lie 1415 1420 1.425 Gin Va]. Gly Giu Lys Gin His Val His Glu G],n Asp Gly Gin Glu 1430 1435 1440 lie She Lys Val Len Lys Trp Gly 5cr 3cr Tyr Asn Vai Tyr 8cr 1445 1450 1455 I0 She Leo Lys lie Trp Vol Val Tyr Asp Gly His Ala Val 3cr Len 1460 1465 1470 lie Pro Ala Pro 5cr Vol Thr Gly Gin His Cys G] y Leo Cys dy 1475 1480 1485 Asn She Asn Arg Asn Gin Tyr Asp G],u She Gin 5cr Lys Asp Ala 1490 1495 1500 ills G1n Leu Lys Thr 8cr Gin G1.u Lou Val Gb Asp Tyr Lys Trp 1505 1510 1515 Lys Cys <210> 4 <211> 8 <212> PRT <213> Artificial. Sequence <220> <223> identified peptide 1 <400> 4 3cr Len Ala Vol Tyr Ala Leo Tys 1 5 <210> 5 <211> 9 <212> SRI' <21,3> Artificial Sequence <220> <223> identified peptide 2 <400> 5 Pbs Tyr Met Gin TSr lie Gin Lys Val 1. 5 <210> 6 <211> 15 <212> PRT <213> Artificial Sequence <223> <223> identifjed peptide 3 <403> 6 Lys Val Giu Thr Tbr Met Gly Vai lie Per Pro Phe Thr iys Gin 1 5 10 15 <210> 7 <211> ii <212> PRT <213> Artificial Sequence <220> <223> identified peptide 4 <430> 7 Lys Ala Leu Val Ala Leu Phe Gin Thr Lye Met 1 5 10 <210> 0 <211> 9 <212> PRT <213> Artificial Sequence <223> <223> identified peptide 5 <400> 8 Arg Tyr Tyr Ala Cys Sly Pro Arg Ocr 1 5 <210> 9 <211> 10 <212> PRI <213> Artificial Sequence <220> <223> idenLified poptide 6 <430> 9 Pro Teu lie Tyr Ci y Glu Thr Glu lie lye 1 5 10 <210> 10 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> identified peptide 7 <400> 13 Gin Tyr 3cr His Phe 0Th Thr Asp yr Sly Icu Sly Vai 3cr Tys 1 5 10 15 <210> 11 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> identified peptide 9 <400> 11 Val Lys Asn Sen Val Val Ala Phe Arg 1 5 <210> 12 <211> 19 <212> PRT <213> ArtificIal Sequence <220> <223> identified peptido 9 <400> 12 ile Tyr Sly 5cr His She Pro Arq Asn Phe Vol lie Sly Vol Asn Pro 1 5 10 15 Ieu Lys Tys <210> 13 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> identified peptide 10 <400> 13 lie lie Lee Sly His Glu She Thr Pro Sly Tyr Ile Gin Asn Arg 1 5 10 15 <210> 14 <211> 11 <212> PR? <213> Artificial Sequence <220> <223> identified peptide 11 <400> 14 Asn Ala Tie Val 3cr Gin Phe Gin 3cr Vol Met 1 5 10 <210> 15 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> identified peptide 12 <400> 15 5cr Ala Sly 5cr His 1eu Asp Mn lys 1 5 <210> 16 <211> 12 <212> PRT <213> ArLificial Sequence <220> <223> identified peptide 13 <400> 16 Trp Sly 5cr Ser Tyr Asn Val Tyr Her She Lou Lys 1 5 10 <210> 17 <211> 20 <212> PR? <213> Artificial Sequence <220> <223> peptide 1 of SEQ IF) NO. 1 <400> 17 Giy Tyr 5cr Pro 5cr Tyr Tyr Sly Trp Ala Pro Her Lys Gin Tyr Vai 1 5 10 15 Tyr [flu Phe Gin 20 <210> 18 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> peptide 2 of SEQ ID NO.
<400> 18 so Glu 5cr Leu The Va1 Glu Lys Asp Glu Pro Val Vnl Val Thr Asn Trp 1 5 10 15 Lys Lys Ala Leu Len <210> 19 <211> 20 <212> PAT <213> Artificial Sequence <220> <223> peptidc 3 of SEQ ID NO. 1 <400> 19 5cr Gin Lye Glu lie His Glu Vol Met Gb Gb Ser Gly Arq AT a Cys 1 5 10 15 Thr Gly Lys GIn <213> 20 <211> 20 <212> PAT <213> Artificial Sequence <223> <223> peptide 4 of SEQ ID NO. 1 <400> 20 Ser Thr Val 5cr His Gin lie Pro Lys Pro Lye TAr Pro Lye Thr Vol 1 5 13 15 Gly Asn Leu Phe <210> 21 <211> 20 <212> PRT <213> Artificial, Sequence <220> <223> peptide 5 of SEQ ID NO. 1 <430> 21 Lye TAr Leu lye Ala Lye 5cr Pro Gin 1]cu Tyr Tyr Vol 8cr Thr Val ii 5 10 15 Her Phe Her Asp <210> 22 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> peptide 6 of SEQ ID NO. 1 <400> 22 Gin Lys lie Thr Gin Lys leu Gin lie Thr Pro Arg Thr Lea GIn Gin 1 5 10 15 Pro Gin Leu Scr <210> 23 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> peptide 7 of SEQ ID NO. 1 <400> 23 His Giy Leu Pro Phe lys Tyr Thr Lys Thr Arg Asn The Val Asp Val 1 5 10 15 Gin 5cr Val Ala Pro Thr Ala 5cr Gly Phe Pro Val Arq lie Gin 20 25 30 <210> 24 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> peptide B of SEQ ID NO. i <400> 24 Cys 3cr Gin 3cr 5cr Thr l'csn Thr Vai Asn Pro Asn Thr Cys Gin Gin 1 3 10 15 lys Gin Arg 5cr <210> 25 <211> 20 <212> PRI <213> ArLLficiai Sequence <220> <223> poptide S of SEQ ID NO. 1 <400> 25 Pro Val Asn Gin 3cr 3cr G1.y 3cr 3cr Thr Pro Pro 5cr 3cr Thr Pro 1 5 10 15 Giy Pro Len Leu <210> 26 <211> 20 <212> PRF <213> Artificial Sequence <220> <223> peptide 10 of SEQ ID NO. I <400> 26 Per Cys Gin Gly Tie Pro Thr Pro Gin Gin Lys Thr Lys Phe Gin Lys 1 5 10 15 Gin 5cr His Gin <210> 27 <211> 28 <212> PRT <2i3> ArGificiai Sequence <220> <223> peptide 11 of SEQ ID NO. I <400> 27 Pro Thr TSr Tyr Asn Arg Met Tie Gin Giu Ala 3cr Asn Cys Gin 3cr 1 5 10 15 Ser 3cr 3cr 5cr Gly Per Gly Met Giy G1y Giy 3cr 25 <210> 28 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> peptide 12 of SEQ ID NO. 1 <400> 28 3cr Per Pro Per 3cr 3cr Asp Per Her 3cr His Ills Ala Gin Pro 3cr 1 5 10 15 Thr Giy Arg Phe Gin 20

Claims (40)

  1. CLAIMSHaving thus specifically described and determined the nature and the best mode for carrying out the present invention, the inventors claim ownership and of exclusive right on: 1. An isolated peptide, characterized by comprising an amino acid sequence having at least 90 % identity to 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, said peptide inducing an immune response against copepods and/or generating a mucous shield in fish.
  2. 2. The peptide according to claim 1, characterized in that the fish are selected from the group consisting of Atlantic salmon (.Salmo Salar), Rainbow trout (Oncorhynchus mykiss), Coho salmon (Oncorhynchus k/stitch), brown trout (Salmo trutta), and Chinook salmon (0. tshawytscha) and the copepods belong to the Caligidae family.
  3. 3. The peptide according to claim 1, characterized by comprising an antigenic protein conjugated to said peptide.
  4. 4. The peptide according to claim 3, characterized in that the antigenic protein is hemocyanin (KLH -keyhole limpet hemocyanin) from Mogathura crenulata.
  5. 5. A vaccine which induces an immune response against copepods and/or a mucous shield in fish, characterized by comprising at least one peptide, wherein said peptide has an amino acid sequence showing at least 90 % identity to 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. 6. The vaccine according to claim 5, characterized in that the fish are selected from the group consisting of Atlantic salmon (Sa/mo Salar), Rainbow trout (Oncorhynchus mykiss), Coho salmon (Oncorhynchus kisutch), brown trout (Salmo trutta), and Chinook salmon (0. tshawytscha) and the copepods belong to the Caligidae family.
  7. 7. The vaccine according to claim 5, characterized in that it is in the form of an emulsion.
  8. 8. The vaccine according to claim 5, characterized in that the excipient is a non-mineral oil.
  9. 9. The vaccine according to claim 5, characterized in that the peptide comprises an antigenic protein conjugated to said peptide.
  10. 10. The vaccine according to claim 9, characterized in that the antigenic protein is hemocyanin from the keyhole limpet Iviogathura crenulata.
  11. 11. A vaccine that induces an immune response against copepods and/or the development of a mucous shield in fish, characterized by comprising peptides as set forth in SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, and SEQ ID NO. 20; and excipients.
  12. 12. The vaccine according to claim 11, characterized in that at least one of the peptides is conjugated to an antigenic protein.
  13. 13. The vaccine according to claim 11, characterized in that the four peptides are conjugated to an antigenic protein.
  14. 14. A vaccine that induces an immune response against copepods and/or a mucous shield in fish, characterized by comprising peptides as set forth in SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, and SEQ ID NO. 24; and excipients.
  15. 15. The vaccine according to claim 14, characterized in that at least one of the peptides is conjugated to an antigenic protein.
  16. 16. The vaccine according to claim 14, characterized in that the four peptides are conjugated to an antigenic protein.
  17. 17. A vaccine that induces an immune response against copepods and/or the development of a mucous shield in fish, characterized by comprising peptides as set forth in SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, and SEQ ID NO. 28; and excipients.
  18. 18. The vaccine according to claim 17, characterized in that at least one of the peptides is conjugated to an antigenic protein.
  19. 19. The vaccine according to claim 17, characterized in that the four peptides are conjugated to an antigenic protein.
  20. 20. The vaccine according to any of claims 11 14, and 17, characterized in that the copepod belong to the Caligidae family.
  21. 21. The vaccine according to any of claims 11, 14 and 17, characterized in that the excipient is a non-mineral oil.
  22. 22. The vaccine according to any of claims 11, 14, and 17, characterized in that the fish are selected from the group consisting of Atlantic salmon (Salmo Salar), Rainbow trout (Oncorhynchus mykiss), Coho salmon (Oncorhynchu.s k/stitch), brown trout (Salmo trutta), and Chinook salmon (0. tshawytscha) and the copepods belong to the Caligidae family.
  23. 23. The vaccine according to any of claims 11, 14, and 17, characterized in that it is in the form of an emulsion.
  24. 24. Use of the peptides of claim 1 for preparing a vaccine.
  25. 25. Use of the peptides of Ia claim 1 for preparing a composition that induces development cia mucous shield in fish.
  26. 26. A vaccine against copepods infesting fish, characterized by comprising the proteins of SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3; excipients and adjuvants.
  27. 27. The vaccine according to claim 26, characterized in that the excipient is a non-mineral oil.
  28. 28. A method for modulating an immune response in fish, characterized by comprising administering to said fish the necessary amount of a vaccine comprising at least one peptide having at least 90 % identity to 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. 29. The method according to claim 28, characterized in that the peptide is administered in an amount of from 1 and 500 pg.
  30. 30. The method according to claim 28, characterized in that the fish are 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 (0. tshawytscha) and the copepods belong to the Caligidae family.
  31. 31. The method according to claim 28, characterized in that the peptide comprises an antigenic protein conjugated to said peptide.
  32. 32. The method according to claim 31, characterized in that the antigenic protein is hemocyanin from the keyhole limpet Megathura crenu/ata.
  33. 33. A method for modulating immune response in fish, characterized by comprising administering to said fish a necessary amount of a vaccine comprising the proteins set forth in SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3; excipients and adjuvants.
  34. 34. The method according to claim 33, characterized in that each protein is administered in an amount of from 1 and 10 pg.
  35. 35. The method according to claim 33, characterized in that the fish are selected from the group comprising Atlantic salmon (Se/mo Safer), Rainbow trout (Oncorhynchus mykiss) and Coho salmon (Oncorhynchus kisutch), brown trout (Salmo trutta) and Chinook salmon (0. tshawytscha) and the copepods belong to the Caligidae family.
  36. 36. A method for generating the development of a mucous shield in fish, characterized by comprising administering to said fish a necessary amount of a vaccine comprising at least one peptide having at least 90 % identity to 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. 37. The method according to claim 36, characterized in that the peptide is administered an amount of from 1 and 500 pg.
  38. 38. The method according to claim 36, characterized in that the fish are selected from the group comprising Atlantic salmon (Se/mo Safer), Rainbow trout (Oncorhynchus mykiss) and Coho salmon (Oncorhynchus kisutch), brown trout (Salmo trutta) and Chinook salmon (0. tshawytscha) and the copepods belong to the Caligidae family.
  39. 39. The method according to claim 36, characterized in that the peptide comprises an antigenic protein conjugated to said peptide.
  40. 40. The method according to claim 39, characterized in that the antigenic protein is hemocyariin from the keyhole limpet Megathura crenu/ata.
GB1421363.1A 2012-05-17 2012-05-17 Peptides inducing an immune response against copepods and/or the development of a mucous shield in fish Withdrawn GB2522119A (en)

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