Composition for prevention, reduction, and / or treatment of sea lice infestation and infection

Abstract

Provided is a composition for prevention, reduction, and / or treatment of sea lice infestation and infection of a host, or for modulation of immune response of a host against sea lice. Also provided is the composition for use in prevention, reduction, and / or treatment of sea lice infestation and infection of a host, or for use in modulation of immune response of a host against sea lice.

Description

Composition for Prevention, Reduction, and / or treatment of Sea Lice Infestation and InfectionTechnical Field

[0001] The present invention relates generally to a composition and a method for prevention, reduction, and / or treatment of sea lice infestation and infection of a host, such as salmonids, or for modulation of immune response of the host against sea lice.Background Art

[0002] Lepeophtheirus salmonis, also known as salmon lice, is a species of copepod in the familyCaligidae. Salmon lice are a type of sea lice living mostly on salmon and have been shown to reduce fish growth and appetite and cause substantial costs to salmon farmers. Salmon lice is currently one of the biggest challenges to the salmon production worldwide. This is mainly due to the increasing occurrence of resistance in salmon lice to the available agents used for treatment. Chitin synthesis inhibitors (Diflubenzuron), organophosphorous compounds (Azamethiphos), and hydrogen peroxide are the chemical agents most frequently used. An increasing problem with resistance against the most commonly used agents has pushed forward interest and research on non-medical treatment and prevention.Technical Solution

[0003] The present invention is based, at least in part, on the discovery that a composition comprising one or more of the following antigen: Vitellogenin-1 and / or a fragment thereof, Vitellogenin-2 and / or a fragment thereof, Vitellogenin-3 and / or a fragment thereof, ovalbumin and / or a fragment thereof, Serpin and / or a fragment thereof, and a combination thereof, is able to prevent, reduce, and / or treat sea lice infestation and infection of a host, such as salmonids, or is able to modulate immune response of the host against sea lice.

[0004] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following embodiments.

[0005] Embodiment 1. A composition for prevention, reduction, and / or treatment of sea lice infestation and infection of a host, or for modulation of immune response of a host against sea lice, comprising one or more antigen selected from:

[0006] Vitellogenin-1 and / or a fragment thereof;

[0007] an isolated polynucleotide encodes the Vitellogenin-1 and / or a fragment thereof;

[0008] Vitellogenin-2 and / or a fragment thereof;

[0009] an isolated polynucleotide encodes the Vitellogenin-2 and / or a fragment thereof;

[0010] Vitellogenin-3 and / or a fragment thereof;

[0011] an isolated polynucleotide encodes the Vitellogenin-3 and / or a fragment thereof;

[0012] Ovalbumin and / or a fragment thereof;

[0013] an isolated polynucleotide encodes the Ovalbumin and / or a fragment thereof;

[0014] Serpin and / or a fragment thereof;

[0015] an isolated polynucleotide encodes the Serpin and / or a fragment thereof; and

[0016] a combination thereof.

[0017] Embodiment 2. The composition according to Embodiment 1, wherein the fragment of the Vitellogenin-1 comprises one or more of the following:

[0018] yolk glycopeptide 42 (YGP42) and / or a fragment thereof;

[0019] Von Willebrand factor type D domain (vWD) and / or a fragment thereof;

[0020] phosvitin and / or a fragment thereof; and

[0021] Pt5 and / or a fragment thereof.

[0022] Embodiment 3. The composition according to Embodiment 1, wherein the fragment of the Vitellogenin-2 comprises one or more of the following:

[0023] yolk glycopeptide 40 (YGP40) and / or a fragment thereof;

[0024] Von Willebrand factor type D domain (vWD) and / or a fragment thereof;

[0025] phosvitin and / or a fragment thereof; and

[0026] Pt5 and / or a fragment thereof.

[0027] Embodiment 4. The composition according to Embodiment 1, wherein the fragment of the Vitellogenin-3 comprises one or more of the following:

[0028] Von Willebrand factor type D domain (vWD) and / or a fragment thereof;

[0029] phosvitin and / or a fragment thereof; and

[0030] Pt5 and / or a fragment thereof.

[0031] Embodiment 5. The composition according to any one of Embodiments 1 to 4, wherein the antigen is an isolated polynucleotide or peptide.

[0032] Embodiment 6. The composition according to Embodiment 5, wherein the isolated polynucleotide is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0033] Embodiment 7. The composition according to any one of Embodiments 1 to 5, wherein the antigen is Vitellogenin-1 and / or a fragment thereof (including, but not limited to, yolk glycopeptide 42 (YGP42) and / or a fragment thereof, Von Willebrand factor type D domain (vWD) and / or a fragment thereof, phosvitin and / or a fragment thereof, and Pt5 and / or a fragment thereof), Vitellogenin-2 and / or a fragment thereof (including, but not limited to, yolk glycopeptide 40 (YGP40) and / or a fragment thereof, Von Willebrand factor type D domain (vWD) and / or a fragment thereof, phosvitin and / or a fragment thereof, and Pt5 and / or a fragment thereof), Vitellogenin-3 and / or a fragment thereof (including, but not limited to, Von Willebrand factor type D domain (vWD) and / or a fragment thereof, phosvitin and / or a fragment thereof, and Pt5 and / or a fragment thereof), Ovalbumin and / or a fragment thereof, phosvitin and / or a fragment thereof, and Pt5 and / or a fragment thereof derived from chicken.

[0034] Embodiment 8. The composition according to Embodiment 5, wherein the polynucleotide encodes Vitellogenin-1 having an amino acid sequence of SEQ ID NO: 1 and / or 2 or an amino acid sequence at least 80% identical to SEQ ID NO: 1 and / or 2, and wherein the peptide is Vitellogenin-1 having an amino acid sequence of SEQ ID NO: 1 and / or 2 or an amino acid sequence at least 80% identical to SEQ ID NO: 1 and / or 2.

[0035] Embodiment 9. The composition according to Embodiment 5, wherein the polynucleotide encodes Vitellogenin-2 having an amino acid sequence of SEQ ID NO: 3 or an amino acid sequence at least 80% identical to SEQ ID NO: 3, and wherein the peptide is Vitellogenin-2 having an amino acid sequence of SEQ ID NO: 3 or an amino acid sequence at least 80% identical to SEQ ID NO: 3.

[0036] Embodiment 10. The composition according to Embodiment 5, wherein the polynucleotide encodes Vitellogenin-3 having an amino acid sequence of SEQ ID NO: 4 or an amino acid sequence at least 80% identical to SEQ ID NO: 4, and wherein the peptide is Vitellogenin-3 having an amino acid sequence of SEQ ID NO: 4 or an amino acid sequence at least 80% identical to SEQ ID NO: 4.

[0037] Embodiment 11. The composition according to Embodiment 5, wherein the polynucleotide encodes Ovalbumin having an amino acid sequence of SEQ ID NO: 5 or an amino acid sequence at least 80% identical to SEQ ID NO: 5, and wherein the peptide is Ovalbumin having an amino acid sequence of SEQ ID NO: 5 or an amino acid sequence at least 80% identical to SEQ ID NO: 5.

[0038] Embodiment 12. The composition according to Embodiment 5, wherein the polynucleotide encodes yolk glycopeptide 42 (YGP42) having an amino acid sequence of SEQ ID NO: 6 or an amino acid sequence at least 80% identical to SEQ ID NO: 6, and wherein the peptide is yolk glycopeptide 42 (YGP42) having an amino acid sequence of SEQ ID NO: 6 or an amino acid sequence at least 80% identical to SEQ ID NO: 6.

[0039] Embodiment 13. The composition according to Embodiment 5, wherein the polynucleotide encodes yolk glycopeptide 40 (YGP40) having an amino acid sequence of SEQ ID NO: 7 or an amino acid sequence at least 80% identical to SEQ ID NO: 7, and wherein the peptide is yolk glycopeptide 40 (YGP40) having an amino acid sequence of SEQ ID NO: 7 or an amino acid sequence at least 80% identical to SEQ ID NO: 7.

[0040] Embodiment 14. The composition according to any one of Embodiments 1 to 5, wherein the antigen is Serpin derived from sea lice.

[0041] Embodiment 15. The composition according to Embodiment 14, wherein the polynucleotide encodes Serpin having an amino acid sequence of SEQ ID NO: 8 or an amino acid sequence at least 80% identical to SEQ ID NO: 8, and wherein the peptide is Serpin having an amino acid sequence of SEQ ID NO: 8 or an amino acid sequence at least 80% identical to SEQ ID NO: 8.

[0042] Embodiment 16. The composition according to any one of Embodiments 1 to 15, further comprising Von Willebrand factor type D domain (vWD) derived from sea lice.

[0043] Embodiment 17. The composition according to any one of Embodiments 1 to 16, wherein the polynucleotide encodes Von Willebrand factor type D domain (vWD) having an amino acid sequence of SEQ ID NO: 9, 10, 11, and / or 18 or an amino acid sequence at least 80% identical to SEQ ID NO: 9, 10, 11, and / or 18, and wherein the peptide is Von Willebrand factor type D domain (vWD) having an amino acid sequence of SEQ ID NO: 9, 10, 11, and / or 18 or an amino acid sequence at least 80% identical to SEQ ID NO: 9, 10, 11, and / or 18.

[0044] Embodiment 18. The composition according to Embodiment 5, wherein the polynucleotide encodes phosvitin having an amino acid sequence of SEQ ID NO: 12, 14, and / or 16 or an amino acid sequence at least 80% identical to SEQ ID NO: 12, 14, and / or 16, and wherein the peptide is phosvitin having an amino acid sequence of SEQ ID NO: 12, 14, and / or 16 or an amino acid sequence at least 80% identical to SEQ ID NO: 12, 14, and / or 16.

[0045] Embodiment 19. The composition according to Embodiment 5, wherein the polynucleotide encodes Pt5 having an amino acid sequence of SEQ ID NO: 13, 15, and / or 17 or an amino acid sequence at least 80% identical to SEQ ID NO: 13, 15, and / or 17, and wherein the peptide is Pt5 having an amino acid sequence of SEQ ID NO: 13, 15, and / or 17 or an amino acid sequence at least 80% identical to SEQ ID NO: 13, 15, and / or 17.

[0046] Embodiment 20. The composition according to Embodiment 5, wherein the isolated polynucleotide is constructed in a vector.

[0047] Embodiment 21. The composition according to any one of Embodiments 1 to 20, further comprising a pharmaceutically-acceptable adjuvant, diluent or carrier.

[0048] Embodiment 22. The composition according to any one of Embodiments 1 to 21, wherein the antigen is encapsulated in a nanovesicle.

[0049] Embodiment 23. The composition according to Embodiment 22, wherein the nanovesicle is one or more of liposomes, niosomes, ethosomes, and exosomes.

[0050] Embodiment 24. The composition according to Embodiment 23, wherein the exosomes are derived from an animal cell or a plant cell.

[0051] Embodiment 25. The composition according to Embodiment 24, wherein the animal cell is salmon cell.

[0052] Embodiment 26. The composition according to any one of Embodiments 1 to 25, wherein the sea lice is a Lepeophtheirus or a Caligus.

[0053] Embodiment 27. The composition according to Embodiment 26, wherein the Lepeophtheirus isLepeophtheirus salmonis.

[0054] Embodiment 28. The composition according to Embodiment 26, wherein the Caligus isCaligus rogercresseyi.

[0055] Embodiment 29. The composition according to any one of Embodiments 1 to 28, further comprising one or more antigens of viral origin or bacterial origin.

[0056] Embodiment 30. The composition according to any one of Embodiments 1 to 29, wherein the composition is administrated via oral administration, injection, immersion, bath, spray, or any combination thereof.

[0057] Embodiment 31. The composition according to any one of Embodiments 1 to 30, wherein the composition is a vaccine, immune-stimulant, therapeutic agent, or feed additive.

[0058] Embodiment 32. The composition according to any one of Embodiments 1 to 31, wherein the host is Salmonidae.

[0059] Embodiment 33. A method for prevention, reduction, and / or treatment of sea lice infestation and infection of a host, or for modulation of immune response of a host against sea lice, comprising the step of administering to the host a composition according to any one of Embodiments 1 to 32.

[0060] Embodiment 34. A composition according to any one of Embodiments 1 to 32 for use in prevention, reduction, and / or treatment of sea lice infestation and infection of a host, or for use in modulation of immune response of a host against sea lice.

[0061] Embodiment 35. Use of a composition according to any one of Embodiments 1 to 32 for the manufacture of a medicament for prevention, reduction, and / or treatment of sea lice infestation and infection of a host, or for modulation of immune response of a host against sea lice.Description of Drawings

[0062] Figure 1 shows the fold change in gene expression of interleukin 8 (IL-8), complement factor C3 (C3), cathelicidin (CAMP), and tumor necrosis factor a (TNFa) in salmon cells treated with ovalbumin, relative to untreated cells serving as controls.

[0063] Figure 2 shows the fold change in gene expression of IL-8, C3, CAMP, and TNFa in salmon cells treated with YGP40, relative to untreated cells serving as controls.

[0064] Figure 3 shows the fold change in gene expression of IL-8, C3, CAMP, and TNFa in salmon cells treated with YGP42, relative to untreated cells serving as controls.

[0065] Figure 4 shows the fold change in gene expression of IL-8, C3, CAMP, and TNFa in salmon cells treated with phosvitin, relative to untreated cells serving as controls.Best Mode

[0066] In one aspect, the present invention provides a vaccine composition comprising one or more proteins selected from a group of proteins including Vitellogenin-1, Vitellogenin-2, Vitellogenin-3, Ovalbumin, Serpin, yolk glycopeptide 40 (YGP40), yolk glycopeptide 42 (YGP42), Von Willebrand factor type D domain (vWD), phosvitin, and Pt5, wherein the relative proportions of the individual proteins are not limited to any particular values. The weight percentage of each component may independently range from about 0% to about 100%, and, in certain embodiments, from about 0.1% to about 90%, or any value therebetween.

[0067] Specifically, the present invention provides a composition for prevention, reduction, and / or treatment of sea lice infestation and infection of a host, or for modulation of immune response of a host against sea lice, comprising one or more antigen selected from:

[0068] Vitellogenin-1 and / or a fragment thereof;

[0069] an isolated polynucleotide encodes the Vitellogenin-1 and / or a fragment thereof;

[0070] Vitellogenin-2 and / or a fragment thereof;

[0071] an isolated polynucleotide encodes the Vitellogenin-2 and / or a fragment thereof;

[0072] Vitellogenin-3 and / or a fragment thereof;

[0073] an isolated polynucleotide encodes the Vitellogenin-3 and / or a fragment thereof;

[0074] Ovalbumin and / or a fragment thereof;

[0075] an isolated polynucleotide encodes the Ovalbumin and / or a fragment thereof;

[0076] Serpin and / or a fragment thereof;

[0077] an isolated polynucleotide encodes the Serpin and / or a fragment thereof; and

[0078] a combination thereof.

[0079] Preferably and in some embodiments, the fragment of the Vitellogenin-1 comprises one or more of the following: yolk glycopeptide 42 (YGP42) and / or a fragment thereof, Von Willebrand factor type D domain (vWD) and / or a fragment thereof, phosvitin and / or a fragment thereof, and Pt5 and / or a fragment thereof. Preferably and in some embodiments, the fragment of the Vitellogenin-2 comprises one or more of the following: yolk glycopeptide 40 (YGP40) and / or a fragment thereof, Von Willebrand factor type D domain (vWD) and / or a fragment thereof, phosvitin and / or a fragment thereof, and Pt5 and / or a fragment thereof. Preferably and in some embodiments, the fragment of the Vitellogenin-3 comprises one or more of the following: Von Willebrand factor type D domain (vWD) and / or a fragment thereof, phosvitin and / or a fragment thereof, and Pt5 and / or a fragment thereof.

[0080] Preferably and in some embodiments, the present invention provides a composition for prevention, reduction, and / or treatment of sea lice infestation and infection of a host, or for modulation of immune response of a host against sea lice, comprising an “effective amount” of one or more antigen as described above.

[0081] Preferably and in some embodiments, the present invention may further comprise a pharmaceutically acceptable carrier, excipient, adjuvant, and / or ingredient.

[0082] As used herein, the term “effective amount” includes “a pharmaceutically effective amount,” “a therapeutically effective amount,” and “an oral effective amount.” As used herein, the term “effective amount” refers to a non-toxic but sufficient amount of the composition according to the present invention to provide the desired biological, immunological, or prophylactic effect. The effective amount is not limited to a specific numerical range, and may vary depending on the antigen, host species, route of administration, and formulation type. The examples provided below are for illustration only and are not intended to limit the scope of the invention.

[0083] By way of non-limiting example, the protein concentration or effective amount of the antigen in the composition may be within a range of approximately 0.1 μg / mL to 10 mg / mL, preferably 1 μg / mL to 1 mg / mL, when formulated for in vitro use, and approximately 0.01 mg / kg to 10 mg / kg body weight, preferably 0.1 mg / kg to 1 mg / kg body weight, when administered in vivo.

[0084] For instance, when the host fish weighs approximately 100 g to 300 g, the total antigen load corresponding to the dosage range of approximately 0.01 mg / kg to 10 mg / kg body weight would be about 1 µg to 3 mg per fish, respectively. For a fish weighing approximately 150 g, the total antigen amount would correspond to approximately 1.5 µg to 1.5 mg per fish, or about 0.01 µg / g to 0.01 mg / g fish body weight. These values are provided by way of non-limiting example, and may vary depending on the specific formulation, antigen composition, and administration route. In certain embodiments, when administered via feed or immersion, the antigen concentration may range from approximately 10 µg / g to 10 mg / kg of feed, depending on feed composition, fish species, and feeding frequency.

[0085] Concentrations outside of these ranges may also be employed, provided that the amount remains non-toxic and capable of eliciting or modulating the desired immune response. The appropriate concentration or dosage can be readily determined and optimized by a person skilled in the art, taking into account factors such as host species, size, immune sensitivity, route of administration, dosing frequency, and combination with other antigens or adjuvants. These examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way.

[0086] In certain embodiments, the composition according to the present invention may comprise one or more proteins or antigens as described herein. In those instances where the composition comprises two or more proteins, the relative ratio between or among the proteins may be adjusted to achieve the desired immunological or prophylactic efficacy. The following examples illustrate representative ratios among two or more antigens that may be included within the composition of the present invention. The total effective amount described in the foregoing sections may be distributed among the individual proteins in accordance with the ratios described herein, such that the cumulative amount of all proteins falls within the effective range defined above.

[0087] Preferably and in some embodiments, when the composition comprises two proteins, and the ratio of the two proteins may be 1:1, 1:2, 1:5, 2:3, 3:7, 1:9, 4:6, 7:3, or any other combination. Preferably and in some embodiments, when the composition comprises three proteins, and the ratio of the three proteins may be 1:1:1, 1:2:3, 2:1:5, 3:5:7, 1:4:8, 5:2:1, 2:3:9, 7:1:3, 7:8:10, 4:1:6, 5:4:2, 10:2:6 or any other combination. Preferably and in some embodiments, when the composition comprises four proteins, and the ratio of the four proteins may be 1:1:1:1, 1:2:1:3, 2:3:5:1, 3:3:9:2, 5:1:1:1, 1:5:2:8, 6:7:8:10, 1:10:2:4, 2:5:8:10, 4:2:1:7, 1:3:6:9, 7:2:3:5, 9:1:4:2, 10:7:7:5, 8:4:2:6, 1:8:4:2, 5:9:1:3, or any other combination. Preferably and in some embodiments, when the composition comprises five proteins, and the ratio of the five proteins may be 1:1:1:1:1, 1:2:3:4:5, 2:3:4:5:6, 1:5:2:3:8, 3:1:5:9:2, 2:4:6:8:10, 5:5:7:2:1, 9:3:2:4:1, or any other combination. Preferably and in some embodiments, when the composition comprises six proteins, and the ratio of the six proteins may be 1:1:1:1:1:1, 1:2:3:4:5:6, 2:2:3:3:4:4, 1:3:5:7:9:11, 2:5:8:10:3:1, 6:4:3:2:1:5, 8:1:2:3:5:7, or any other combination. In certain embodiments, the ratio among the proteins in the composition may also be determined based on their molecular weights, antigenic potency, or relative abundance, and may be adjusted to yield an overall effective amount of the composition. The proteins may be combined in equal proportions (e.g., equimolar or equal mass) or in customized ratios to achieve synergistic or optimized immune responses, without departing from the scope of the invention.

[0088] In certain embodiments, the composition of the present invention may comprise two or more of the proteins or antigens described herein in various combinations. For example, the composition may include combinations of two, three, four, five, six, or more of the following proteins or their fragments: Vitellogenin-1, Vitellogenin-2, Vitellogenin-3, Ovalbumin, Serpin, yolk glycopeptide 40 (YGP40), yolk glycopeptide 42 (YGP42), Von Willebrand factor type D domain (vWD), phosvitin, Pt5, or any other protein or fragment functionally equivalent thereto.

[0089] Representative combinations include, but are not limited to:

[0090] Two-protein combinations such as YGP40 + Ovalbumin, YGP42 + Serpin, Vitellogenin-1 + phosvitin, or YGP42 + Pt5;

[0091] Three-protein combinations such as Ovalbumin + YGP40 + YGP42, Vitellogenin-2 + vWD + Serpin, vWD + YGP40 + YGP42 or Pt5 + YGP40 + YGP42;

[0092] Four-protein combinations such as Vitellogenin-1 + Vitellogenin-2 + Ovalbumin + Serpin, Ovalbumin + YGP40 + YGP42 + phosvitin, vWD + YGP40 + YGP42 + phosvitin, or Vitellogenin-2 + Vitellogenin-3 + YGP42 + phosvitin;

[0093] Five-protein combinations such as Vitellogenin-1 + Vitellogenin-2 + Vitellogenin-3 + Ovalbumin + phosvitin, or Ovalbumin + Serpin + YGP40 + YGP42 + Pt5;

[0094] Six or more protein combinations may also be employed, including any combination of two or more of the aforementioned antigens, their fragments, or derivatives, such as Vitellogenin-derived domains (e.g., vWD, phosvitin, or Pt5) together with one or more of Ovalbumin, Serpin, or other functionally related proteins. The specific combinations may include both full-length proteins and their structural domains, derived from the same or different precursors, provided that the overall composition elicits or modulates an effective immune response against sea lice.

[0095] Accordingly, each of these proteins or fragments may be included alone or in combination at any of the ratios described herein, or at other suitable ratios determined by a person skilled in the art. The relative proportions of the antigens may be adjusted and optimized to achieve a desired immune effect, taking into account formulation type, host species, and route of administration. The invention is not limited to any specific antigen combination; rather, any combination capable of eliciting or modulating an effective immune or protective response against sea lice infestation is encompassed within the scope of the present invention.

[0096] The foregoing ratios are provided by way of illustration and are not limiting. The actual proportions may be adjusted based on antigen properties, desired magnitude or quality of the immune response, and / or characteristics of the intended recipient population. Any proportion of the selected proteins that is capable of eliciting an effective immune response is encompassed within the scope of the present invention.

[0097] Preferably and in some embodiments, the antigen is an isolated polynucleotide or peptide. Preferably and in some embodiments, the isolated polynucleotide is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0098] Preferably and in some embodiments, the antigen is Vitellogenin-1 and / or a fragment thereof, Vitellogenin-2 and / or a fragment thereof, Vitellogenin-3 and / or a fragment thereof, Ovalbumin and / or a fragment thereof, phosvitin and / or a fragment thereof, and Pt5 and / or a fragment thereof derived from chicken. Preferably and in some embodiments, the antigen and / or protein may be obtained or produced by artificial extraction from natural sources, by chemical synthesis, or by recombinant expression using molecular biological or genetic engineering techniques.

[0099] Preferably and in some embodiments, the polynucleotide encodes Vitellogenin-1 having an amino acid sequence of SEQ ID NO: 1 and / or 2 or an amino acid sequence at least 80% identical to SEQ ID NO: 1 and / or 2 (e.g., at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO: 1 and / or 2). Preferably and in some embodiments, the peptide is Vitellogenin-1 having an amino acid sequence of SEQ ID NO: 1 and / or 2 or an amino acid sequence at least 80% identical to SEQ ID NO: 1 and / or 2 (e.g., at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO: 1 and / or 2).

[0100] Preferably and in some embodiments, the polynucleotide encodes Vitellogenin-2 having an amino acid sequence of SEQ ID NO: 3 or an amino acid sequence at least 80% identical to SEQ ID NO: 3 (e.g., at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO: 3). Preferably and in some embodiments, the peptide is Vitellogenin-2 having an amino acid sequence of SEQ ID NO: 3 or an amino acid sequence at least 80% identical to SEQ ID NO: 3 (e.g., at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO: 3).

[0101] Preferably and in some embodiments, the polynucleotide encodes Vitellogenin-3 having an amino acid sequence of SEQ ID NO: 4 or an amino acid sequence at least 80% identical to SEQ ID NO: 4 (e.g., at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO: 4). Preferably and in some embodiments, the peptide is Vitellogenin-3 having an amino acid sequence of SEQ ID NO: 4 or an amino acid sequence at least 80% identical to SEQ ID NO: 4 (e.g., at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO: 4).

[0102] Preferably and in some embodiments, the polynucleotide encodes Ovalbumin having an amino acid sequence of SEQ ID NO: 5 or an amino acid sequence at least 80% identical to SEQ ID NO: 5 (e.g., at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO: 5). Preferably and in some embodiments, the peptide is Ovalbumin having an amino acid sequence of SEQ ID NO: 5 or an amino acid sequence at least 80% identical to SEQ ID NO: 5 (e.g., at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO: 5).

[0103] Preferably and in some embodiments, the polynucleotide encodes yolk glycopeptide 42 (YGP42) having an amino acid sequence of SEQ ID NO: 6 or an amino acid sequence at least 80% identical to SEQ ID NO: 6 (e.g., at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO: 6). Preferably and in some embodiments, the peptide is yolk glycopeptide 42 (YGP42) having an amino acid sequence of SEQ ID NO: 6 or an amino acid sequence at least 80% identical to SEQ ID NO: 6 (e.g., at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO: 6).

[0104] Preferably and in some embodiments, the polynucleotide encodes yolk glycopeptide 40 (YGP40) having an amino acid sequence of SEQ ID NO: 7 or an amino acid sequence at least 80% identical to SEQ ID NO: 7 (e.g., at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO: 7). Preferably and in some embodiments, the peptide is yolk glycopeptide 40 (YGP40) having an amino acid sequence of SEQ ID NO: 7 or an amino acid sequence at least 80% identical to SEQ ID NO: 7 (e.g., at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO: 7).

[0105] Preferably and in some embodiments, the antigen is Serpin derived from sea lice. Preferably and in some embodiments, the polynucleotide encodes Serpin having an amino acid sequence of SEQ ID NO: 8 or an amino acid sequence at least 80% identical to SEQ ID NO: 8 (e.g., at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO: 8). Preferably and in some embodiments, the peptide is Serpin having an amino acid sequence of SEQ ID NO: 8 or an amino acid sequence at least 80% identical to SEQ ID NO: 8 (e.g., at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO: 8).

[0106] Preferably and in some embodiments, the antigen is Von Willebrand factor type D domain (vWD) derived from sea lice or chicken. Preferably and in some embodiments, the polynucleotide encodes Von Willebrand factor type D domain (vWD) having an amino acid sequence of SEQ ID NO: 9, 10, 11, and / or 18 or an amino acid sequence at least 80% identical to SEQ ID NO: 9, 10, 11, and / or 18 (e.g., at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO: 9, 10, 11, and / or 18). Preferably and in some embodiments, the peptide is Von Willebrand factor type D domain (vWD) having an amino acid sequence of SEQ ID NO: 9, 10, 11, and / or 18 or an amino acid sequence at least 80% identical to SEQ ID NO: 9, 10, 11, and / or 18 (e.g., at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO: 9, 10, 11, and / or 18).

[0107] Preferably and in some embodiments, the polynucleotide encodes phosvitin having an amino acid sequence of SEQ ID NO: 12, 14, and / or 16 or an amino acid sequence at least 80% identical to SEQ ID NO: 12, 14, and / or 16 (e.g., at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO: 12, 14, and / or 16). Preferably and in some embodiments, the peptide is phosvitin having an amino acid sequence of SEQ ID NO: 12, 14, and / or 16 or an amino acid sequence at least 80% identical to SEQ ID NO: 12, 14, and / or 16 (e.g., at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO: 12, 14, and / or 16).

[0108] Preferably and in some embodiments, the polynucleotide encodes Pt5 having an amino acid sequence of SEQ ID NO: 13, 15, and / or 17 or an amino acid sequence at least 80% identical to SEQ ID NO: 13, 15, and / or 17 (e.g., at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO: 13, 15, and / or 17). Preferably and in some embodiments, the peptide is Pt5 having an amino acid sequence of SEQ ID NO: 13, 15, and / or 17 or an amino acid sequence at least 80% identical to SEQ ID NO: 13, 15, and / or 17 (e.g., at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO: 13, 15, and / or 17).

[0109] Preferably and in some embodiments, the isolated polynucleotide is constructed in a vector. Preferably and in some embodiments, the vector comprises, but is not limited to, an expression vector and a cloning vector.

[0110] Preferably and in some embodiments, the composition of the present invention further comprises a pharmaceutically-acceptable adjuvant, diluent or carrier. Preferably and in some embodiments, the adjuvant comprises, but is not limited to, muramyldipeptides, lipopolysaccharides, lipopolysaccharides, glycans, animal oil, vegetable oil, mineral oil, Montanide®, Carbopol®, mineral oil-in-water emulsion, Freund's adjuvant, and acrylic acid polymers.

[0111] Preferably and in some embodiments, the antigen is encapsulated in a nanovesicle to improve the stability, delivery efficiency, and / or penetration of the encapsulated material. Preferably and in some embodiments, the nanovesicle is one or more of liposomes, niosomes, ethosomes, and exosomes. Preferably and in some embodiments, the exosomes are derived from an animal cell or a plant cell. Preferably and in some embodiments, the animal cell is salmon cell.

[0112] Preferably and in some embodiments, the sea lice are a Lepeophtheirus or a Caligus. Preferably and in some embodiments, the Lepeophtheirus isLepeophtheirus salmonis. Preferably and in some embodiments, the Caligus isCaligus rogercresseyi.

[0113] Preferably and in some embodiments, the composition of the present invention further comprises one or more antigens of viral origin or bacterial origin. Preferably and in some embodiments, the antigens of viral origin or bacterial origin may be, but not limited to, one ofMoritella viscosa,Piscirickettsias sp.,Aeromonas sp.,Vibrio sp.,Aliivibrio sp.,Listonella sp.,Tenacibaculum sp.,Pasteurella sp.,Photobacterium sp,Flavobacterium sp.,Yersinia sp.,Renibacterium sp.,Streptococcus sp.,Lactococcus sp.,Leuconostoc sp.,Bifidobacterium sp.,Pediococcus sp.,Brevibacterium sp.,Edwarsiella sp.,Francisella sp.,Pseudomonas sp.,Cytophaga sp.,Nocardia sp.,Mycobacterium sp.,Saprolegnia Sp.,Branchiomyces sanguinis,Branchiomyces demigrans, Viral Hemorrhagic Septicemia Virus (VHSV), Infectious Hematopoietic Necrosis virus (IHNV), Infectious Pancreatic Necrosis Virus (IPNV), Infectious Salmon Anaemia virus (ISAV), Salmon pancreatic disease virus (SPDV), Iridovirus, Nodavirus, Piscine myocarditis virus (PMCV) and Piscine Orthoreovirus (PRV), Spring Viremia of Carp (SVC), Channel Catfish Virus (CCV), Iridovirus, and heart and skeletal muscle inflammation virus (HSMIV) variants or immunogenic fragments thereof.

[0114] Preferably and in some embodiments, the composition is administrated via oral administration, injection, immersion, bath, spray, or any combination thereof. Preferably and in some embodiments, the route of injection includes, but is not limited to, intraperitoneal and intramuscular injection. Preferably and in some embodiments, the composition is administrated via a combination of different routes, for example, but is not limited to, immersion first and then injection, immersion first and then oral administration, immersion first and then spray, injection first and then immersion, injection first and then oral administration, injection first and then bath, injection first and then spray, oral administration first and then injection, oral administration first and then immersion, oral administration first and then injection and immersion, immersion first and then oral administration and injection, and injection first and then spray and oral administration.

[0115] Preferably and in some embodiments, the composition is a vaccine, immune-stimulant, therapeutic agent, or feed additive. Preferably and in some embodiments, the composition is a vaccine. Preferably and in some embodiments, the composition is a DNA vaccine, an RNA vaccine (or mRNA vaccine), or a subunit vaccine. Preferably and in some embodiments, the composition may be an immunomodulator that can regulate or modulate immune responses and the expression of immune-related genes.

[0116] Preferably and in some embodiments, the host may include any organism that can be infested or infected by lice, including humans and non-human animals. In preferred embodiments, the host is an aquatic animal, more preferably a fish species susceptible to sea lice infestation. Preferably and in some embodiments, the host is Salmonidae. Preferably and in some embodiments, the Salmonidae includes, but is not limited to, Atlantic salmon (Salmo salar), coho salmon (Oncorhynchus kisutch), rainbow trout (Oncorhynchus mykiss), sockeye salmon (Oncorhynchus nerka), and Chinook salmon (Oncorhynchus tshawytscha).

[0117] Specifically, the present invention also provides a method for prevention, reduction, and / or treatment of sea lice infestation and infection of a host, such as a Salmonidae, or for modulation of immune response of the host against sea lice. The method comprises the step of administering to the host a composition of the present invention.

[0118] Specifically, the present invention also provides the composition of the present invention for use in prevention, reduction, and / or treatment of sea lice infestation and infection of a host, such as a Salmonidae, or for use in modulation of immune response of the host against sea lice.

[0119] The composition of the present invention has demonstrated remarkable reduces in sea lice number on salmon after the salmon were fed or injected or immersed with the composition.

[0120] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the case of conflict, the present document, including definitions will control.

[0121] As used herein, the term “composition” or “composition of the present invention” refers to a mixture of one or more antigen for prevention, reduction, and / or treatment of sea lice infestation and infection of a host, or for modulation of immune response of a host against sea lice. Examples of the composition of the present invention include, but are not limited to, vaccines, therapeutics, and immuno-stimulants.

[0122] As used herein, the term “modulation of immune response” or “modulate immune response” refers to any action that alters, enhances, suppresses, or regulates components or activity of the immune system, including but not limited to: (i) regulation of immune-related gene expression, such as upregulation or downregulation of immune-related genes; (ii) modulation of innate or adaptive immune responses, including cellular immune responses and humoral immune responses; (iii) alteration of antigen presentation and immune tolerance; (iv) regulation of the complement system; (v) modulation of cytokine or chemokine profiles; (vi) enhancement or suppression of immune cell activation, differentiation, proliferation, or migration; and (vii) indirect modulation through host–microbiota interaction or other biological intermediates; (viii) interference with pathogen or parasite immune evasion mechanisms, including generation of cross-reactive antibodies or effector molecules that recognize parasite-derived antigens.

[0123] As used herein, the term “host” refers to an organism that can be infested or infected by lice, including but not limited to humans, non-human animals, and aquatic species. In preferred embodiments, the host is an aquatic animal, more preferably a fish species susceptible to sea lice infestation. Examples of the host include, but are not limited to, Atlantic salmon (Salmo salar), coho salmon (Oncorhynchus kisutch), rainbow trout (Oncorhynchus mykiss), sockeye salmon (Oncorhynchus nerka), and Chinook salmon (Oncorhynchus tshawytscha). In certain embodiments, the term “host” may also encompass cells or cell lines derived from such organisms that can be stimulated, influenced, or interacted with / by the antigen, protein or composition of the present invention, resulting in modulation or regulation of immune-related gene expression, thereby contributing to or indicative of enhanced defense against pathogens, such as sea lice. As used herein, the term “cell” includes, but is not limited to, skin cells, immune cells, gill cells, hepatocytes, kidney cells, intestinal cells, adipocyte cells and muscle cells. In preferred embodiments, the cell is a skin cell, kidney cell, or immune cell.

[0124] In certain embodiments, the immunostimulatory effects of the antigenic proteins described herein have been observed across multiple salmon cell types, including skin cells, head kidney cells, and spleen cells. Consistent trends in the upregulation of immune-related genes such as interleukin 8 (IL-8), complement factor C3 (C3), cathelicidin (CAMP), and tumor necrosis factor α (TNFα) was observed, indicating that the stimulatory activity of the compositions of the present invention is not restricted to a single cell type, but rather exhibits broad immunomodulatory potential across various salmon immune-associated tissues.

[0125] As used herein, the term “sea lice” refers to a marine ectoparasitic copepod and obligate parasite, feeds on skin, mucus and blood of and lives on the external surface of Salmonidae animals. Examples of sea lice include, but are not limited to, a Lepeophtheirus (such asL. salmonis) or a Caligus (such asC. rogercresseyi).

[0126] As used herein, the term “isolated” means that the referenced material is removed from the environment in which it is normally found. Thus, an isolated biological material can be free of cellular components, i.e., components of the cells in which the material is found or produced. In the case of an isolated peptide or protein it may be associated with other proteins or nucleic acids, or both, with which it associates in the cell or with cellular membranes if it is a membrane-associated protein. An isolated organelle, cell, or tissue is removed from the anatomical site in which it is found in an organism. An isolated material may be, but need not be, purified. As used herein, the terms “isolated polynucleotide” and “isolated peptide” refers to polynucleotide and peptide that are not naturally occurring substances.

[0127] The terms “nucleic acid”, “polynucleotide”, “nucleic acid molecule” and the like may be used interchangeably herein and refer to a series of nucleotide bases (also called “nucleotides”) in DNA and RNA. The nucleic acid may contain deoxyribonucleotides, ribonucleotides, and / or their analogs. The term “nucleic acid” includes, for example, single-stranded and double-stranded molecules. A nucleic acid can be, for example, a gene or gene fragment, exons, introns, a DNA molecule (e.g., cDNA), an RNA molecule (e.g., mRNA), recombinant nucleic acids, plasmids, and other vectors, primers and probes. Both 5′ to 3′ (sense) and 3′ to 5′ (antisense) polynucleotides are included. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A poly-nucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs.

[0128] The terms “polypeptide”, “oligopeptide”, “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, un-natural amino acids, etc.), as well as other modifications known in the art. It is understood that, because the polypeptides of this invention are based upon an antibody, the polypeptides can occur as single chains or associated chains.

[0129] As used herein, the terms “derived from chicken” refers to that a material has been isolated, extracted, synthesized or produced from chicken tissue, cells, or genetic material; this can include a material naturally present in chicken meat, eggs, or cells, or a material that is produced using chicken DNA or cell lines in a laboratory or industrial setting, or by chemical synthesis or recombinant expression based on chicken-derived sequences. Examples of chicken sources include, but are not limited to, chicken meat or muscle tissue, chicken eggs, chicken blood, chicken cell culture, and genetically engineered organisms using chicken genes.

[0130] As used herein, the terms “derived from sea lice” refers to that a material has been isolated, extracted, synthesized or produced from any development stage of sea lice, including, but not limited to, copepodids / chalimus, pre-adult, and adult sea lice with egg-strings. This can include a material naturally present in sea lice, or a material that is produced using sea lice DNA or cell lines in a laboratory or industrial setting or by chemical synthesis or recombinant expression based on sea lice-derived sequences. Examples of sea lice include, but are not limited to a Lepeophtheirus (such asLepeophtheirus salmonis) or a Caligus (such asCaligus rogercresseyi).

[0131] As used herein, the terms “vector” refers to DNA molecules used as vehicles to carry foreign genetic material into a host cell. As used herein, the terms “expression vector” refers to vectors used for transcription and translation of an inserted gene, leading to protein production. Key features of an expression vector include, but are not limited to, a promoter, a ribosome binding site, terminator sequences, tags for purification. Example of expression vectors include, but are not limited to, pET series, pcDNA3, pGEX, and pCMV vectors. As used herein, the terms “cloning vector” refers to vectors used for replicating and propagating foreign DNA in a host cell. Key features of a cloning vector include, but are not limited to, original of replication, selectable marker (e.g., antibiotic resistance), and multiple cloning site. Examples of cloning vectors include, but are not limited to, pUC19, pBR322, and pBluescript.

[0132] The composition of the present invention may further comprise a suitable pharmaceutical carrier. The appropriate carrier is evident to those skilled in the art and will depend in large part upon the route of administration. Additional components that may be present in this invention are adjuvants, preservatives, surface active agents, chemical stabilizers, suspending or dispersing agents. Typically, stabilizers, adjuvants and preservatives are optimized to determine the best formulation for efficacy in the target subject.

[0133] As used herein, the term “treat,” “treating,” or “treatment” encompasses alleviation of one or more symptom thereof, a reduction in the severity thereof, or inhibition of the progression thereof. Treatment does not necessarily mean that the disease, disorder, or condition is totally cured. To be an effective treatment, a useful composition herein needs only to reduce the severity of a disease, disorder, or condition, reduce the severity of symptoms associated therewith, or provide improvement to a patient or subject’s quality of life.

[0134] As used herein, the term “reduce,” “reducing,” or “reduction” refers to lowering the number of infected subjects in a group, lowering the number of sea lice in an infected subject, lowering or eliminating the number of subjects exhibiting clinical signs of infection, or lowering the severity of any clinical signs that are present in one or more subjects, in comparison to infection without treatment of the composition of the present invention. Preferably, the number of infected subjects, the number of sea lice in an infected subject, the number of subjects exhibiting clinical signs of infection, and / or the severity of any clinical signs are reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90, or 100%, in comparison to subjects infected by sea lice and not being treated with the composition of the present invention.

[0135] As used herein, the term “prevent,” “preventing,” or “prevention” refers to being able to substantially preclude, avert, obviate, forestall, stop, hinder, or a combination thereof, any aspect of a disease, condition, or combination thereof from happening, especially by advance action.

[0136] As used herein, the terms “about,” “around,” or “approximately,” when used in connection with a numeric value or range, refer to a value within experimental or measurement uncertainty, manufacturing tolerances, or a relative or absolute deviation sufficient to achieve the intended technical effect in the relevant context. Unless otherwise specified, such terms encompass ±10% of the referenced value; in some embodiments they encompass ±5%, ±1%, ±15%, ±20%, depending on the magnitude of the value, the precision of the method, and the biological system (e.g., in vitro versus in vivo, single antigen versus multi-antigen compositions).

[0137] Numerical quantities provided herein are approximate; accordingly, the modifiers “about,” “around,” or “approximately” may be inferred even if not expressly stated. Unless stated otherwise, all ranges include their endpoints and all intermediate values and sub-ranges. The described values may be expressed in any appropriate units (e.g., mass, volume, molarity, weight / volume %, particle counts, copy numbers, dose per body weight), and routine optimization by one of ordinary skill in the art may adjust such values to account for species, size, route of administration, dosing regimen, and co-administration in combination products, without departing from the scope of the invention.

[0138] The phrase “comprising” as used herein is open-ended, indicating that such embodiments may include additional elements. In contrast, the phrase “consisting of” is closed, indicating that such embodiments do not include additional elements (except for trace impurities). The phrase “consisting essentially of” is partially closed, indicating that such embodiments may further comprise elements that do not materially change the basic characteristics of such embodiments.

[0139] Where applicants have defined an invention or a portion thereof with an open-ended term such as “comprising,” it should be readily understood that (unless otherwise stated) the description should be interpreted to also describe such an invention using the terms “consisting essentially of” or “consisting of.”

[0140] It is noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a polynucleotide” includes a plurality of such polynucleotides and reference to “the polypeptide” includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements or use of a “negative” limitation.

[0141] In those instances where a convention analogous to “one or more of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “a system having one or more of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0142] The present invention is further illustrated by the following examples, which are provided for the purpose of demonstration rather than limitation. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0143] EXAMPLE

[0144] Example 1In vitro Analysis of Immune Stimulation

[0145] Salmon cells were treated with ovalbumin (SEQ ID NO: 5), YGP40 (SEQ ID NO: 7), or YGP42 (SEQ ID NO: 6), or phosvitin (SEQ ID NO: 14), respectively, for 24 hours. Untreated cells served as the negative control. After stimulation, cells were collected for RNA extraction. RNA was extracted using a Direct-zol RNA Miniprep Kit (Zymo Research, Irvine, CA, USA) in accordance with the manufacturer’s instructions. Reverse transcription and real-time PCR analyses for gene expression of interleukin 8 (IL-8), complement factor C3 (C3), cathelicidin (CAMP), and tumor necrosis factor a (TNFa) were conducted with ReverTra AceTMqPCR RT Kit & Master Mix (TOYOBO Co., Ltd., Osaka, Japan) along with the PowerTrack SYBR Green Master Mix (ThermoFisher Scientific, Waltham, MA, USA) on a QuantStudio™ 5 Real-Time PCR System (Applied Biosystems, USA).

[0146] As shown in FIG. 1 to FIG. 4, treatment with ovalbumin (FIG. 1), YGP40 (FIG. 2), YGP42 (FIG. 3), and phosvitin (FIG. 4) individually resulted in upregulation of mRNA expression of all of IL-8, complement factor C3, CAMP, and TNFa in salmon cells. The results suggest that the components of the composition of the present invention effectively regulates gene expression in salmon cells and induce the activation of multiple immune pathways in salmon cells, such as the complement system, the promotion of inflammatory cytokines, and antimicrobial peptide expression, thereby enhancing the fish’s ability to combat sea lice infections.

[0147] Furthermore, in addition to the proteins described above (ovalbumin, YGP40, YGP42, and phosvitin), other individual proteins disclosed in the present invention also exhibited similar trends in the regulation of the immune-related genes.

[0148] Example 2In vivoStudy of Immune Stimulation

[0149] Sixteen Atlantic salmon were divided randomly and equally into two groups; one group administered of the present composition (for example, a recombinant protein mixture comprising ovalbumin + YGP42 + YGP40) at a dose of about 1 µg per gram of body weight, in a total volume of 0.1 mL and the other group received the same volume of phosphate buffered saline (PBS) as control. A dose was applied to the nasal ridge. Tissues were collected 5 days post-administration for gene expression analysis.

[0150] In order to assess gene expression in tissue samples, fish tissues were first homogenized in TRIzol (Invitrogen, Waltham, MA, USA) by a tissue homogenizer (Precellys Evolution Touch, Bertin Technologies, France). The resulting homogenate was subjected to centrifugation to eliminate tissue debris, and the supernatant underwent RNA extraction as described in Example 1. Reverse transcription and real-time PCR analyses for gene expression of IL-8, C3, CAMP, TNFa, and toll-like receptor 5 (TLR5) were conducted with ReverTra AceTMqPCR RT Kit & Master Mix (TOYOBO Co., Ltd., Osaka, Japan) along with the PowerTrack SYBR Green Master Mix (ThermoFisher Scientific, Waltham, MA, USA) on a QuantStudio™ 5 Real-Time PCR System (Applied Biosystems, USA).

[0151] As shown in Table 1, treatment with a combination of ovalbumin, YGP40, and YGP42 resulted in upregulation of mRNA expression of IL-8, complement factor C3, CAMP, TNFa, and TLR5 in salmon skin tissue. The results suggests that the composition of the present invention effectively regulates gene expression in salmon cells and induce the activation of multiple immune pathways in salmon cells, such as the complement system, the promotion of inflammatory cytokines, antimicrobial peptide expression, and Toll-like receptor signaling pathway, thereby enhancing the fish’s ability to combat sea lice infections.

[0152] Furthermore, in addition to the combination of proteins described above (ovalbumin, YGP40, YGP42), other combinations of proteins disclosed in the present invention (for example, ovalbumin + YGP40 + YGP42 + phosvitin, YGP40 + YGP42 + Pt5, Serpin + YGP40 + YGP42 etc.), as well as administration of individual proteins disclosed herein (for example, ovalbumin, YGP40, YGP42, phosvitin, Pt5, Serpin, or others), also exhibited similar trends in the regulation of immune-related genes.

[0153] The results of Examples 1 and 2 suggest that the composition and its components of the present invention can activate immune responses and enhance the host’s ability to defend against parasitic infestation.

[0154] Table 1

[0155]

[0156] Example 3 Sea Lice Challenge Study

[0157] Atlantic salmon (Salmo salarL.) were kept in seawater at 10°C ± 1°C, 32 ‰ average salinity, and fed daily with automatic feeders at 0.8-1 % body weight throughout the challenge study. Fifty fish were randomly and equally divided into 2 groups. The test group was administered a composition comprising ovalbumin, YGP40, YGP42, and phosvitin at a dosage of about 0.2–0.3 µg per gram of body weight via intraperitoneal route. The control group was administered with the same volume of PBS via the same route. Seven days post-vaccination, each fish was challenged with 20 copepodid-stage sea lice. Sea lice counting was performed 23 days post-challenged. For example, for a fish weighing approximately 100 g, the total administered dose corresponds to about 20–30 µg of protein per fish.

[0158] The results showed that Fish treated with the composition (test group) carried markedly fewer sea lice, approximately 5–12 sea lice per fish, a reduction of about 40–75% compared with the control group, which carried 13–18 sea lice per fish.

[0159] Furthermore, in addition to the foregoing protein combination (ovalbumin, YGP40, YGP42, and phosvitin), administration of other protein combinations disclosed in the present invention (for example, YGP40 + YGP42 + Pt5, Serpin + YGP40 + YGP42 etc.) and administration of individual protein disclosed in the present invention (for example, Vitellogenin-1, Vitellogenin-2, Vitellogenin-3, Ovalbumin, Serpin, YGP40, YGP42, phosvitin, Pt5, or Von Willebrand factor type D domain (vWD) alone) also exhibited similar trends in reducing the number of sea lice infesting fish.

[0160] In addition, the protein combination used serves merely as an example. Similar trends in reducing sea lice infestation were observed when the constituent proteins were formulated in various suitable ratios, such as equal proportions (by weight, mole, or volume), ratios adjusted according to their respective molecular weights, or ratios determined based on practical factors including production efficiency, purification yield, solubility, mixing compatibility, and cost considerations.

[0161] These results indicate that administration of the composition provides a protective immune response sufficient to prevent, reduce, and inhibit infestation and / or attachment of sea lice to fish. Therefore, the composition of the present invention can be used as a prophylactic and / or curative measure against sea lice infection. The composition of the present invention provides a highly biosafe and sustainable control strategy for the aquaculture industry, effectively reducing reliance on chemical agents and ecological impacts, and reducing growth stagnation, increased mortality, and yield losses caused by sea lice infestation and infection, thus possessing significant industrial application value and economic benefits.

[0162] It should be understood that the selection of proteins, the administration amount (or effective amount), and the ratios among the proteins described in the above example are merely illustrative. These specific examples are provided to facilitate understanding of the concepts disclosed herein by those skilled in the art, and should not be construed as limiting the scope of the invention or its possible embodiments.

Claims

1.A composition for prevention, reduction, and / or treatment of sea lice infestation and infection of a host, or for modulation of immune response of a host against sea lice, comprising one or more antigen selected from: Vitellogenin-1 and / or a fragment thereof; an isolated polynucleotide encodes the Vitellogenin-1 and / or a fragment thereof;Vitellogenin-2 and / or a fragment thereof; an isolated polynucleotide encodes the Vitellogenin-2 and / or a fragment thereof;Vitellogenin-3 and / or a fragment thereof; an isolated polynucleotide encodes the Vitellogenin-3 and / or a fragment thereof;Ovalbumin and / or a fragment thereof;an isolated polynucleotide encodes the Ovalbumin and / or a fragment thereof;Serpin and / or a fragment thereof; an isolated polynucleotide encodes the Serpin and / or a fragment thereof; anda combination thereof.2.The composition according to claim 1, wherein the fragment of the Vitellogenin-1 comprises one or more of the following: yolk glycopeptide 42 (YGP42) and / or a fragment thereof;Von Willebrand factor type D domain (vWD) and / or a fragment thereof;phosvitin and / or a fragment thereof; and Pt5 and / or a fragment thereof.3.The composition according to claim 1, wherein the fragment of the Vitellogenin-2 comprises one or more of the following: yolk glycopeptide 40 (YGP40) and / or a fragment thereof;Von Willebrand factor type D domain (vWD) and / or a fragment thereof;phosvitin and / or a fragment thereof; and Pt5 and / or a fragment thereof.4.The composition according to claim 1, wherein the fragment of the Vitellogenin-3 comprises one or more of the following:Von Willebrand factor type D domain (vWD) and / or a fragment thereof;phosvitin and / or a fragment thereof; and Pt5 and / or a fragment thereof.5.The composition according to any one of claims 1 to 4, wherein the antigen is an isolated polynucleotide or peptide. 6.The composition according to claim 5, wherein the isolated polynucleotide is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). 7.The composition according to any one of claims 1 to 5, wherein the antigen is Vitellogenin-1 and / or a fragment thereof, Vitellogenin-2 and / or a fragment thereof, Vitellogenin-3 and / or a fragment thereof, Ovalbumin and / or a fragment thereof, phosvitin and / or a fragment thereof, and Pt5 and / or a fragment thereof derived from chicken. 8.The composition according to claim 5, wherein the polynucleotide encodes Vitellogenin-1 having an amino acid sequence of SEQ ID NO: 1 and / or 2 or an amino acid sequence at least 80% identical to SEQ ID NO: 1 and / or 2, and wherein the peptide is Vitellogenin-1 having an amino acid sequence of SEQ ID NO: 1 and / or 2 or an amino acid sequence at least 80% identical to SEQ ID NO: 1 and / or 2.9.The composition according to claim 5, wherein the polynucleotide encodes Vitellogenin-2 having an amino acid sequence of SEQ ID NO: 3 or an amino acid sequence at least 80% identical to SEQ ID NO: 3, and wherein the peptide is Vitellogenin-2 having an amino acid sequence of SEQ ID NO: 3 or an amino acid sequence at least 80% identical to SEQ ID NO: 3.10.The composition according to claim 5, wherein the polynucleotide encodes Vitellogenin-3 having an amino acid sequence of SEQ ID NO: 4 or an amino acid sequence at least 80% identical to SEQ ID NO: 4, and wherein the peptide is Vitellogenin-3 having an amino acid sequence of SEQ ID NO: 4 or an amino acid sequence at least 80% identical to SEQ ID NO: 4.11.The composition according to claim 5, wherein the polynucleotide encodes Ovalbumin having an amino acid sequence of SEQ ID NO: 5 or an amino acid sequence at least 80% identical to SEQ ID NO: 5, and wherein the peptide is Ovalbumin having an amino acid sequence of SEQ ID NO: 5 or an amino acid sequence at least 80% identical to SEQ ID NO: 5.12.The composition according to claim 5, wherein the polynucleotide encodes yolk glycopeptide 42 (YGP42) having an amino acid sequence of SEQ ID NO: 6 or an amino acid sequence at least 80% identical to SEQ ID NO: 6, and wherein the peptide is yolk glycopeptide 42 (YGP42) having an amino acid sequence of SEQ ID NO: 6 or an amino acid sequence at least 80% identical to SEQ ID NO: 6.13.The composition according to claim 5, wherein the polynucleotide encodes yolk glycopeptide 40 (YGP40) having an amino acid sequence of SEQ ID NO: 7 or an amino acid sequence at least 80% identical to SEQ ID NO: 7, and wherein the peptide is yolk glycopeptide 40 (YGP40) having an amino acid sequence of SEQ ID NO: 7 or an amino acid sequence at least 80% identical to SEQ ID NO: 7.14.The composition according to any one of claims 1 to 5, wherein the antigen is Serpin derived from sea lice. 15.The composition according to claim 14, wherein the polynucleotide encodes Serpin having an amino acid sequence of SEQ ID NO: 8 or an amino acid sequence at least 80% identical to SEQ ID NO: 8, and wherein the peptide is Serpin having an amino acid sequence of SEQ ID NO: 8 or an amino acid sequence at least 80% identical to SEQ ID NO: 8.16.The composition according to any one of claims 1 to 15, further comprising Von Willebrand factor type D domain (vWD) derived from sea lice.17.The composition according to any one of claims 1 to 16, wherein the polynucleotide encodes Von Willebrand factor type D domain (vWD) having an amino acid sequence of SEQ ID NO: 9, 10, 11, and / or 18 or an amino acid sequence at least 80% identical to SEQ ID NO: 9, 10, 11, and / or 18, and wherein the peptide is Von Willebrand factor type D domain (vWD) having an amino acid sequence of SEQ ID NO: 9, 10, 11, and / or 18 or an amino acid sequence at least 80% identical to SEQ ID NO: 9, 10, 11, and / or 18.18.The composition according to claim 5, wherein the polynucleotide encodes phosvitin having an amino acid sequence of SEQ ID NO: 12, 14, and / or 16 or an amino acid sequence at least 80% identical to SEQ ID NO: 12, 14, and / or 16, and wherein the peptide is phosvitin having an amino acid sequence of SEQ ID NO: 12, 14, and / or 16 or an amino acid sequence at least 80% identical to SEQ ID NO: 12, 14, and / or 16.19.The composition according to claim 5, wherein the polynucleotide encodes Pt5 having an amino acid sequence of SEQ ID NO: 13, 15, and / or 17 or an amino acid sequence at least 80% identical to SEQ ID NO: 13, 15, and / or 17, and wherein the peptide is Pt5 having an amino acid sequence of SEQ ID NO: 13, 15, and / or 17 or an amino acid sequence at least 80% identical to SEQ ID NO: 13, 15, and / or 17.20.The composition according to claim 5, wherein the isolated polynucleotide is constructed in a vector. 21.The composition according to any one of claims 1 to 20, further comprising a pharmaceutically-acceptable adjuvant, diluent or carrier. 22.The composition according to any one of claims 1 to 21, wherein the antigen is encapsulated in a nanovesicle. 23.The composition according to claim 22, wherein the nanovesicle is one or more of liposomes, niosomes, ethosomes, and exosomes. 24.The composition according to claim 23, wherein the exosomes are derived from an animal cell or a plant cell.25.The composition according to claim 24, wherein the animal cell is salmon cell.26.The composition according to any one of claims 1 to 25, wherein the sea lice is a Lepeophtheirus or a Caligus.27.The composition according to claim 26, wherein the Lepeophtheirus isLepeophtheirus salmonis.28.The composition according to claim 26, wherein the Caligus isCaligus rogercresseyi.29.The composition according to any one of claims 1 to 28, further comprising one or more antigens of viral origin or bacterial origin. 30.The composition according to any one of claims 1 to 29, wherein the composition is administrated via oral administration, injection, immersion, bath, spray, or any combination thereof.31.The composition according to any one of claims 1 to 30, wherein the composition is a vaccine, immune-stimulant, therapeutic agent, or feed additive.32.The composition according to any one of claims 1 to 31, wherein the host is Salmonidae.33.A method for prevention, reduction, and / or treatment of sea lice infestation and infection of a host, or for modulation of immune response of a host against sea lice, comprising the step of administering to the host a composition according to any one of claims 1 to 32.34.A composition according to any one of claims 1 to 32 for use in prevention, reduction, and / or treatment of sea lice infestation and infection of a host, or for use in modulation of immune response of a host against sea lice.35.Use of a composition according to any one of claims 1 to 32 for the manufacture of a medicament for prevention, reduction, and / or treatment of sea lice infestation and infection of a host, or for modulation of immune response of a host against sea lice.

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