Method for protecting a progeny of an oviparous animal from a pathogen

Abstract

The present invention is directed to a method for protecting a progeny of an oviparous female from an infection of pathogen or a symptom associated therewith, including administering to the oviparous female a therapeutically effective amount of composition including: (a) a first polypeptide; and (b) an agent capable of specifically binding to the pathogen or a portion thereof. Further provided is composition including: (a) a first polypeptide; and (b) a double stranded RNA (dsRNA) molecule, in a mole per mole ratio (m:m) ranging between 1: 1 and 10: 1.

Description

METHOD FOR PROTECTING A PROGENY OF AN OVIPAROUS ANIMAL FROM A PATHOGENREFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0001] The contents of the electronic sequence listing (NIBN-BGU-P-044-PCT.xml; size: 32,267 bytes; and date of creation: January 15, 2025) is herein incorporated by reference in its entirety.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority of U.S. Provisional Application No. 63 / 622,102, titled "METHOD FOR PROTECTING A PROGENY OF AN OVIPAROUS ANIMAL FROM A PATHOGEN", filed 18 January 2024, the contents of which is incorporated herein by reference in its entirety.FIELD OF INVENTION

[0003] The present invention is in the field of vaccination.BACKGROUND

[0004] Vitellogenin (Vg) is a major lipoprotein (LP) in oviparous animals, being the precursor of the egg-yolk protein vitellin. It is essential for providing the metabolic demands of the developing embryo - and is one of the most abundant LPs in the hemolymph of reproductive (vitellogenic) females. In the giant prawn Macrobrachium rosenbergii which is widely cultured throughout the world (over 450,000 tons annually), vitellogenin (MrVg) is synthesized in the hepatopancreas, secreted to the hemolymph, and taken up by the ovary via receptor-mediated endocytosis.

[0005] LPs and their membrane receptors are conserved throughout evolution, including the human ApoB, several low-density lipoproteins, and the crustacean Vg. The Vg receptor (VgR) belongs to the low-density LP receptor (LDLR) superfamily and contains several conserved domains, including the ligand-binding domains (LBDs), an epidermal growth factor (EGF)-like domain, an O -glycosylation domain, a transmembrane domain,and a short cytosolic tail. Studies of vertebrate and invertebrate LDLR and VgR found that the LBD is comprised of several repeats of about 40 amino acids. Each repeat contains six cysteine residues that form three disulfide bonds. It was found that these six cysteine repeats are important for the LDLR-LDL binding.

[0006] The most extensive study on Vg-VgR interaction was conducted in fish, the blue tilapia, Oreochromis aureus, where it was shown that VgR binds to the lipid binding domain at the N-terminal region of Vg. Accordingly, an 84 amino acid-long fragment from the N-terminal portion of O. aureus Ng was found to be sufficient for VgR binding.

[0007] There is still a great need for a method of trans -generationally vaccinating a progeny by transovarial transmission of a vaccine to the mother of the progeny.SUMMARY

[0008] According to the first aspect, there is provided a method for protecting a progeny of an oviparous female crustacean from an infection of a White Spot Syndrom Virus (WSSV) or a symptom associated therewith, the method comprising administering to the oviparous female crustacean a therapeutically effective amount of composition comprising: (a) a first polypeptide comprising the amino acid: DKX1X2X2X3PX4X5GX6YKYVEAX7X8X9SX10X11X12 (SEQ ID NO: 1), wherein: Xi is selected from the amino acid residues N and K; X2 is selected from the amino acid residues I and V; X3 is selected from the amino acid residues K and R; X4 is selected from the amino acid residues A and S; X5 is selected from the amino acid residues Y and I; X7, is an amino acid residue selected from the group consisting of: S, I, A, and T; X7 is an amino acid residue selected from the group consisting of: H, S, K and E; Xg is selected from the amino acid residues Q and M; X9 is an amino acid residue selected from the group consisting of: E, D, and M; X10 is selected from the amino acid residues V and T; Xu is selected from the amino acid residues L and M; and X12 is selected from the amino acid residues R and K; and (b) a double stranded RNA (dsRNA) having at least 70% complementarity to a transcript of the WSSV.

[0009] According to another aspect, there is provided a progeny obtained from an oviparous female crustacean of the method of the invention.

[0010]

[0011] According to another aspect, there is provided a composition comprising: (a) a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1; and (b) a double stranded RNA (dsRNA) molecule, in a mole per mole ratio (m:m) ranging between 1: 1 and 10: 1.

[0012] According to another aspect, there is provided a composition comprising: (a) a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1; and (b) a dsRNA molecule having at least 80% complementarity to an equal sized transcript of a gene of the WSSV.

[0013] In some embodiments, the transcript encodes a protein being essential to any one of a pathogenic activity, survival, and both, of the WSSV.

[0014] In some embodiments, the crustacean is a decapod crustacean.

[0015] In some embodiments, the transcript encodes a VP28 protein of the WSSV.

[0016] In some embodiments, the dsRNA comprises the nucleic acid sequence set forth in SEQ ID NO: 21.

[0017] In some embodiments, the composition further comprises a second polypeptide comprising a double stranded RNA binding domain (dsRBD).

[0018] In some embodiments, the dsRBD comprises the amino acid sequence as set forth in SEQ ID NO: 2, or a functional analog thereof having at least 75% sequence homology or identity thereto.

[0019] In some embodiments, the first polypeptide and the second polypeptide are bound to one another constituting a chimeric protein comprising both.

[0020] In some embodiments, the method further comprises a step before the administering, comprising determining the female crustacean being suitable for administration with the composition, wherein the determining comprises selecting an early vitellogenic or a vitellogenic female crustacean.

[0021] In some embodiments, the administering is at least once a week administering.

[0022] In some embodiments, the administering is injecting.

[0023] In some embodiments, the injecting is injecting into a muscular tissue of the female.

[0024] In some embodiments, the injecting is into the abdomen of the female.

[0025] In some embodiments, the composition is a vaccine composition.

[0026] In some embodiments, the protecting comprises any one of: increasing survival rate of the progeny, reducing incidence of at least one symptom associated with infection of the pathogen in the progeny, and both.

[0027] In some embodiments, increasing or reducing is compared to a non-treated control.

[0028] In some embodiments, protecting is for a period being at least 14 days longer compared to a non-treated control.

[0029] In some embodiments, the oviparous female is a crustacean, the pathogen is WSSV, and the symptom is appearance of at least one white spot on a carapace of at least one progeny of the oviparous female crustacean.

[0030] In some embodiments, the method further comprises a step after the administering, comprising crossing the administered female with a corresponding male.

[0031] In some embodiments, the composition further comprising a second polypeptide comprising a dsRBD.

[0032] In some embodiments, the dsRNA has at least 80% complementarity to an equal sized transcript of a gene of a pathogen.

[0033] In some embodiments, the pathogen is the white spot syndrom virus (WSSV).

[0034] In some embodiments, the dsRNA molecule comprises the nucleic acid sequence set forth in SEQ ID NO: 21.

[0035] In some embodiments, the composition is formulated for vaccination.

[0036] In some embodiments, vaccination is RNA-based vaccination.

[0037] In some embodiments, the composition is formulated for injection.

[0038] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0039] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE FIGURES

[0040] Fig. 1 includes photographs of A. vanncimei vitellogenic female representing the 5 stages of ovary development, arrows point toward developed ovary.

[0041] Figs. 2A-2D include graphs showing oocyte specific delivery (OSDel)-double stranded RNA of the white spot syndrome virus (dsWSSV) or oocyte-specific silencing chimera (OSSCot)fseq -dsWSSV treatments lead to higher survivability upon WSSV challenge. Shrimps (0.8-1 gr) from blank, control, OSDel-dsWSSV (‘OSDel’) and OSSCot-dsWSSV (‘OSSCot’) treated female groups were challenged with WSSV by per os method. For each family, 3 tanks holding 30 shrimps each were monitored daily for 10 days to evaluate the vaccine effect on the shrimp survival. (2A) A Kaplan-Meier survival rate plot of shrimp families. (2B) A histogram represents each shrimp family's average percent survival rate 10 days post-challenge. (2C) A Kaplan-Meier survival rate plot of each treatment group (asterisk indicates p<0.0001). (2D) A histogram representing each treatment group's average percent survival rate 10 days post-challenge, (asterisk indicates p<0.01, Kruskal-Wallis statistical test).

[0042] Fig. 3 includes a violin plot showing that WSSV copy numbers in the OSDel- dsWSSV and OSSCot-dsWSSV treated groups are lower than in the blank and control groups. The violin plot represents the distribution of WSSV copies per gr muscle tissue (3 individuals from each family, 15 individuals per group) — samples collected on the 4th-day post-challenge. There is a higher probability of finding uninfected shrimp (WSSV copies =0) in the OSDel-dsWSSV (OSDel) and OSSCot-dsWSSV (OSSCot) treated groups than in the blank and the control groups.

[0043] Figs. 4A-4E include photograph showing that carapace from the OSDel- dsWSSV and OSSCot-dsWSSV treated shrimp are white spot free. The photographs are of carapaces of individuals taken from (4A) negative control shrimp. (4B-4E) Photographs of carapaces of shrimps following treatments with: blank (4B), control(4C), OSDel-dsWSSV (4D), and OSSCot-dsWSSV (5E) shrimps, collected 10 days post-challenge.

[0044] Figs. 5A-5B include graphs showing that the OSDel-dsWSSV and OSSCot- dsWSSV treatments resulted in a significant delay in the mortality of WSSV-injected shrimp. Thirty individuals (0.8-1.0 gr) from each blank, control, OSDel-dsWSSV (OSDel) and OSSCot-dsWSSV (OSSCot) treated groups were intramuscularly (IM) injected with live WSSV. The shrimps were monitored daily for 10 days to evaluate the treatment effect on the shrimp survival. (5A) A graph representing the Kaplan-Meier survival rate plot of the shrimp families. (5B) A vertical bar graph showing the survival rate of the different treatment groups of shrimps 4 days post WSSV injection. A significant difference in the survival rate, 46±2.2% and 46.67±3.5%, for the OSDel- dsWSSV and OSSCot-dsWSSV treated groups, compared to the blank and control groups, 25.83±2.5% and 28±2.91%, respectively, (p<0.01, according to Kruskal-Wallis test).

[0045] Fig. 6 includes a graphs showing that OSDel-dsVP28 treatment leads to longterm higher survivability upon WSSV challenge. Shrimps (25 gr average weight) from blank, control, OSDel-dsVP28 (OSDel) and OSSCot-dsVP28 (OSSCot) treated female groups were challenged with WSSV by per os method. For each family, tanks holding 40 shrimps were monitored daily for 10 days to evaluate the vaccine effect on the shrimp's survival. A Kaplan-Meier survival rate plot of shrimp families of each treatment group (p<0.001 by Kaplan-Meier pairwise comparison).DETAILED DESCRIPTIONMethods

[0046] According to a first aspect, there is provided a method for protecting a progeny of an oviparous female from an infection of pathogen or a symptom associated therewith.

[0047] In some embodiments, the method comprises administering to the oviparous female a therapeutically effective amount of composition comprising: (a) a first polypeptide; and (b) an agent capable of specifically binding to the pathogen or a portion thereof.

[0048] In some embodiments, the first polypeptide comprising the amino acid: DKX1X2X2X3PX4X5GX6YKYVEAX7X8X9SX10X11X12 (SEQ ID NO: 1), wherein: Xi isselected from the amino acid residues N and K; X2 is selected from the amino acid residues I and V; X3 is selected from the amino acid residues K and R; X4 is selected from the amino acid residues A and S; X5 is selected from the amino acid residues Y and I; X7, is an amino acid residue selected from the group consisting of: S, I, A, and T; X7 is an amino acid residue selected from the group consisting of: H, S, K and E; Xg is selected from the amino acid residues Q and M; X9 is an amino acid residue selected from the group consisting of: E, D, and M; X10 is selected from the amino acid residues V and T; Xu is selected from the amino acid residues L and M; and X12 is selected from the amino acid residues R and K; and

[0049] In some embodiments, a portion of the pathogen comprises a transcript of the pathogen.

[0050] In some embodiments, an agent comprises a polynucleotide. In some embodiments, a polynucleotide comprises a double stranded polynucleotide and / or an antisense polynucleotide. In some embodiments, a polynucleotide is an RNA polynucleotide or a polynucleotide comprising RNA nucleotides.

[0051] In some embodiments, the composition comprises the first polypeptide and the agent in a mole per mole (m:m) ratio ranging 1: 1 and 10: 1, 1: 1 and 100: 1, 10: 1 and 100: 1, 5: 1 and 50: 1, 7: 1 and 40: 1, 6: 1 and 20: 1, and 1: 1 and 20: 1.

[0052] In some embodiments, the agent is administered at a dose of at least 0.1 pg, 1 pg, 3 pg, 5 pg, 10 pg, 15 pg, 20 pg, 50 pg, or 100 pg per 1 g body weight of the oviparous female, or any value and rang therebetween. Each possibility represents a separate embodiment of the invention.

[0053] In some embodiments, the agent comprises a dsRNA molecule.

[0054] In some embodiments, the dsRNA molecule comprises the nucleic acid sequence:least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity or homology thereto, or any range and value therebetween. Each possibility represents a separate embodiment of the invention.

[0055] In some embodiments, the polynucleotide comprises a nucleic acid sequence having complementarity to a transcript of the pathogen.

[0056] In some embodiments, complementarity of a polynucleotide, such as a dsRNA an antisense polynucleotide, for example a dsRNA to a target nucleotide, such as a gene or transcript of a pathogen, is at least 70%, is at least 75%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% complementary, or any range and value therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, complementarity of a polynucleotide, such as a dsRNA an antisense polynucleotide, for example a dsRNA to a target nucleotide, such as a gene or transcript of a pathogen, is 70-85%, 80-90% 92-97%, 95-99%, or 97-100%. Each possibility represents a separate embodiment of the invention.

[0057] In some embodiments, the transcript encodes a protein being essential to a pathogenic activity, survival, or both, of a pathogen. In some embodiments, the transcript encodes an envelope protein. In some embodiments, the transcript encodes VP28 protein ofWSSV.

[0058] In some embodiments, an activity is or comprises infective activity or infection.

[0059] In some embodiments, pathogenic activity comprises escaping or evading an endosome of a cell. In some embodiments, the cell is a host cell. In some embodiments, the host cell is a cell of a progeny / individual being a progeny of an oviparous female, as disclosed herein according to the method of the invention.

[0060] In some embodiments, the pathogen comprises a virus. In some embodiments, the virus comprises a DNA virus or an RNA virus. In some embodiments, the virus comprises a hybrid of DNA and RNA.

[0061] In some embodiments, the vims comprises a single stranded RNA genome (ssRNA). In some embodiments, the vims comprises a double stranded RNA genome (dsRNA). In some embodiments, the vims comprises a single stranded DNA genome (ssDNA). In some embodiments, the vims comprises a double stranded DNA genome (dsDNA).

[0062] In some embodiments, the oviparous female comprises an animal being selected from: an arthropod, a fish, an ave, an amphibian, a reptile, or any combination thereof.

[0063] As used herein, the term “ave” is equivalent to a “bird” and / or “poultry”.

[0064] In some embodiments, an arthropod comprises a crustacean, an insect, or both.

[0065] In some embodiments, a crustacean comprises a decapod cmstacean.

[0066] In some embodiments, a vims is selected from: White Spot Syndrome Vims (WSSV), Yellow head vims (YHV) Taura Syndrom Vims (TSV), Gill-associated Vims (GAV), Mourilyan Vims (MoV), Infectious Myonecrosis Vims (IMNV), Lymphoid Organ Vacuolisation Vims (LOW), Macrobrachium rosenbergii Nodavims / Extra Small Vims (MrNV / XSV), Infectious Pancreatic Necrosis-Like Vims (IPN-like Vims), Infectious Hemoxcytic and Hematopoietic Vims (HPV), Baculoviral Midgut Gland Necrosis Vims (BMNV / PjNOB), Single-Nucleocapsid Polyedrosis Vims (MbNPV), RPS, LSNV, Reo-Pj, Reo-Pm, Reo-Pv, PBRV, SMV, LPV, PHRV, or CcBV.

[0067] In some embodiments, a vims belongs to a vims family selected from: Dicistroviridae, Roniviridae, Bunyaviridcie, Totiviridae, Rhabdoviridcie, Togaviridcie, Nodaviridcie, Birnaviridcie, Reoviridae, Parvoviridae, Iridoviridae, Nonoccluded bacilliform vims, Baculoviridcie, or Nimaviridcie.

[0068] In some embodiments, the composition further comprises a second polypeptide. In some embodiments, the second polypeptide comprises a double stranded RNA binding domain (dsRBD).

[0069] In some embodiments, a dsRBD comprises the amino acid sequence: PVSLLQELCMRRGISPKYDLLQIEGAVHEPTFVYRVTVGEFAANGSGQSKKKA KHAAAKAVLDIIIQGGAASTGGPTTGGPPGAPPELSTQIVSPYDDGIPGNDKNII KPAYGSYKYVEAHQESVLR (SEQ ID NO: 2), or a functional analog thereof having at least 75%, 80%, 90%, 95%, 99% sequence homology or identity thereto, or any valueand range therebetween. Each possibility represents a separate embodiment of the invention.

[0070] In some embodiments, the first polypeptide and the second polypeptide are bound to one another constituting a chimeric protein comprising both. In some embodiments, the first and second polypeptides are bound to one another via a covalent bond. In some embodiments, the covalent bond is or comprises a peptide bond.

[0071] In some embodiments, the method further comprises a step preceding or before the administering (step), comprising determining the female is suitable for administration with the composition as disclosed herein. In some embodiments, determining comprises selecting an early vitellogenic or vitellogenic female.

[0072] Methods and means for determining vitellogenic state of a female are common and would be apparent to one of skill in the art. Non-limiting examples of such methods include, but are not limited to, determining expression levels of vitellogenin in a sample obtained or derived from a female. In some embodiments, the sample comprises hemolymph, blood, or an equivalent bodily fluid. In some embodiments, expression comprises protein levels, transcript levels, or both. In some embodiments, the sample comprises a tissue fragment or biopsy derived from the site of vitellogenin production, e.g., ovary, hepatopancreas, etc. Methods for determining vitellogenin, either protein or transcript, include, but are not limited to PCR, qPCR, western blot, enzyme linked immunosorbent assay (ELISA), and others. In some embodiments, vitellogenic state is determined using a light source, such as would be apparent to one of ordinary skill in the art of crustacean culturing.

[0073] Methods for determining vitellogenic state or stage are common and would be apparent to one of ordinary skill in the art. A non-limiting example include, but is not limited to, quantification of the levels of the vitellogenin protein in the circulation using an immunological assay, for example, enzyme linked immunosorbent assay.

[0074] In some embodiments, vitellogenin levels are compared to a control. In some embodiments, a control comprises a sample obtained or derived from a previtellogenic female, a male, or both.

[0075] In some embodiments, vitellogenin levels of s ample obtained or derived from a male are defined as negative or non- vitellogenic. In some embodiments, early vitellogenic or vitellogenic state is defined as vitellogenin levels being at least 10%,20%, 50%, 100%, 250%, 500%, 750%, or 1,000% greater than control, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0076] In some embodiments, administering comprises at least once a week administering.

[0077] In some embodiments, administering comprises injecting. In some embodiments, the composition is formulated for injection.

[0078] In some embodiments, injecting comprises injecting into a muscular tissue of a female. In some embodiments, injecting is into the abdomen of a female.

[0079] In some embodiments, the composition is or comprises a vaccine composition.

[0080] In some embodiments, protecting comprises: increasing survival rate of a progeny, reducing incidence of at least one symptom associated with infection of a pathogen in a progeny, or both.

[0081] In some embodiments, “increasing” and / or “reducing” is compared to a control. In some embodiments, a control comprises a progeny of an intact female. In some embodiments, a control comprises a progeny of an untreated female. In some embodiments, untreated refers to not treated according to the method of the invention. In some embodiments, a control comprises a progeny of a female being administered with ‘naked’ dsRNA (such as exemplified herein, e.g., without the first polypeptide, the second polypeptide, or both). In some embodiments, a control comprises a progeny of an intact female administered with the first polypeptide, the second polypeptide, or both, is the absence or without dsRNA.

[0082] In some embodiments, the oviparous female comprises a crustacean, the pathogen comprises WSSV, and the symptom comprises appearance of at least one white spot on a carapace of at least one progeny of the oviparous female crustacean.

[0083] In some embodiments, the method further comprises a step proceeding or after the administering, comprising crossing the administered female with a corresponding male, so as or thereby, obtaining a progeny.

[0084] According to another aspect, there is provided a progeny obtained from an oviparous female of the method of the invention.

[0085] In some embodiments, the progeny comprises a larva, a post-larva, or both.

[0086] In some embodiments, the arthropod is a crustacean. In some embodiments, the crustacean is a decapod crustacean. Non-limiting examples of a decapod crustacean include, but are not limited to, a prawn, a shrimp, a lobster, a crab, and a crayfish.

[0087] In some embodiments, administering is injecting. In some embodiments, administering is administering to the ovary. In some embodiments, administering is injecting. In some embodiments, administering is administering to the hemolymph. In some embodiments, administering is administering to the ovary and the hemolymph. In some embodiments, administering is at least once a week, at least twice a week, or at least three times a week, or any range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, administering is once or twice a week, once to three times a week, or twice or three times a week. Each possibility represents a separate embodiment of the invention.

[0088] As used herein, the term “hemolymph” refers to the circulation system.

[0089] In some embodiments, the method further comprises a step of crossing the administered female with a male. In some embodiments, crossing comprises mating with a male. In some embodiments, crossing comprises in vitro inseminating or in vitro fertilization. Methods of in vitro insemination or in vitro fertilization are common and would be apparent to one of ordinary skill in the art. Non-limiting examples include, but are not limited to, sperm or spermatophore collection and incubation with spawned eggs or placement on a receptive female, respectively.Polypeptides

[0090] In some embodiments, the first polypeptide comprises the amino acid sequence: DKXIX2X2X3PX4X5GX6YKYVEA (SEQ ID NO: 3), wherein: Xi is selected from the amino acid residues: N and K; X2 is selected from the amino acid residues: I and V; X3 is selected from the amino acid residues: K and R; X4 is selected from the amino acid residues: A and S; X5 is selected from the amino acid residues: Y and I; and X7, is an amino acid residue selected from: S, I, A and T.

[0091] In some embodiments, the first polypeptide comprises the amino acid sequence: DKX1X2X2X3PX4 (SEQ ID NO: 4), wherein: Xi is selected from the amino acid residues: N and K; X2 is selected from the amino acid residues: I and V; X3 is selected from the amino acid residues: K and R; and X4 is selected from the amino acid residues: A and S.

[0092] In some embodiments, the first polypeptide comprises the amino acid sequence: DKX1X2X2X3P (SEQ ID NO: 5), wherein: Xi is selected from the amino acid residues: N and K; X2 is selected from the amino acid residues: I and V; and X3 is selected from the amino acid residues: K and R.

[0093] In some embodiments, the first polypeptide comprises the amino acid sequence: GXgYKYVEA (SEQ ID NO: 6), wherein: Xr, is an amino acid residue selected from: S, I, A and T.

[0094] In one embodiment, the first polypeptide comprises the amino acid sequence: DKNIIKPAYGSYKYVEA (SEQ ID NO: 7).

[0095] In one embodiment, the first polypeptide comprises the amino acid sequence: DKNIIKP (SEQ ID NO: 8).

[0096] In one embodiment, the first polypeptide comprises the amino acid sequence: DKNIVRPA (SEQ ID NO: 9).

[0097] In one embodiment, the first polypeptide comprises the amino acid sequence: GSYKYVEA (SEQ ID NO: 10).

[0098] In one embodiment, the first polypeptide comprises the amino acid sequence: GIYKYVEA (SEQ ID NO: 11).

[0099] In some embodiments, the first polypeptide is further functionalized by the addition of a functional group to the peptide's N' -terminus, C'-terminus, or both. In some embodiments, the functional group is an amino acid. In some embodiments, the functional group is selected from a cysteine residue or a lysine residue.

[0100] The present invention encompasses derivatives of the peptides (first and / or second) of the invention. The term "derivative" or "chemical derivative" includes any chemical derivative of the peptide having one or more residues chemically derivatized by reaction of side chains or functional groups, as long as the derivatized peptide maintains the herein disclosed first polypeptide, i.e., Vg receptor (VgR) binding and subsequent induction of receptor-mediated endocytosis, and / or second polypeptide, i.e., dsRNA binding domain. Such derivatized molecules include, for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatizedto form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine. Also included as chemical derivatives are those peptides, which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acid residues. For example: 4-hydroxyproline may be substituted for proline; 5 -hydroxy lysine may be substituted for lysine; 3- methylhistidine may be substituted for histidine; homoserine may be substituted or serine; and ornithine (O) may be substituted for lysine.

[0101] In addition, a peptide derivative can differ from the natural sequence by chemical modifications including, but are not limited to, terminal-NFE acylation, acetylation, methylation, phosphorylation, pegylation, or thioglycolic acid amidation, and by terminal -carboxlyamidation, e.g., with ammonia, methylamine, and the like. Polypeptides can be either linear, cyclic, or branched and the like, having any conformation, which can be achieved using methods known in the art, as long as the derivatized polypeptide(s) is / are capable of binding to the VgR and subsequently induce receptor mediated endocytosis, bind dsRNA, or both.

[0102] As used herein, the terms “peptide”, “polypeptide” and "protein" are interchangeable, and refer to a polymer of amino acid residues, such as disclosed by SEQ ID Nos: 1 or 2, or fragments thereof, such as for example SEQ ID Nos.: 3-11 in case of SEQ ID NO: 1.

[0103] The term "amino acid" as used herein means an organic compound containing both a basic amino group and an acidic carboxyl group.

[0104] The term "amino acid residue" as used herein refers to the portion of an amino acid that is present in a peptide.

[0105] The term "peptide bond" means a covalent amide linkage formed by loss of a molecule of water between the carboxyl group of one ammo acid and the ammo group of a second ammo acid.

[0106] The terms "polypeptide", "peptide", and "protein" as used herein encompass native peptides, peptidomimetics (typically including non-peptide bonds or other synthetic modifications) and the peptide analogs peptoids and semi-peptoids or any combination thereof. In another embodiment, the terms “peptide”, and "protein" applyto amino acid polymers in which at least one amino acid residue is an artificial chemical analog of a corresponding naturally occurring amino acid.

[0107] One of skill in the art will recognize that individual substitutions, deletions or additions to a peptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a conservatively modified variant where the alteration results in the substitution of an amino acid with a similar charge, size, and / or hydrophobicity characteristics, such as, for example, substitution of a glutamic acid (E) to an aspartic acid (D).

[0108] As used herein, the phrase "conservative substitution" also includes the use of a chemically derivatized residue in place of a non-derivatized residue provided that such peptide displays the requisite function as specified herein.

[0109] Peptide derivatives can also include side chain bond modifications, including but not limited to -CH2-NH-, -CH2-S-, -CH2-S=O, OC-NH-, -CH2-O-, -CH2-CH2-, S=C-NH-, and -CH=CH-, and backbone modifications such as modified peptide bonds. Peptide bonds (-CO-NH-) within the peptide can be substituted, for example, by N- methylated bonds (-N(CH3)-CO-); ester bonds (-C(R)H-C-O-O-C(R)H-N); ketomethylene bonds (-CO-CH2-); a-aza bonds (-NH-N(R)-CO-), wherein R is any alkyl group, e.g., methyl; carba bonds (-CH2-NH-); hydroxyethylene bonds (-CH(OH)-CH2- ); thioamide bonds (-CS-NH); olefinic double bonds (-CH=CH-); and peptide derivatives (-N(R)-CH2-CO-), wherein R is the "normal" side chain, naturally presented on the carbon atom. These modifications can occur at one or more of the bonds along the peptide chain and even at several (e.g., 2-3) at the same time.

[0110] As used herein, the term "chimera" encompasses any conjugate comprising two or more moieties, wherein the two or more moieties are bound to one another either directly or indirectly, and wherein the moieties are either derived from distinct origins or are not naturally bound to one another. In some embodiments, the two or more moieties have: distinct functions, originate or derived from different genes, peptides, genomic regions, or species, distinct chemical classification (e.g., a first polypeptide and a second polypeptide, as exemplified herein).

[0111] In some embodiments, the chimera comprises a first polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 1 and a second polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 2.

[0112] In some embodiments, the chimera comprises the first polypeptide and a linker. In some embodiments, the chimera comprises the first polypeptide, a linker, and dsRNA. In some embodiments, the dsRNA is bound to the linker. In some embodiments, bound is at least partially via electrostatic interactions. In some embodiments, a chimera comprising the first polypeptide, a linker and dsRNA at least partially bound thereto via electrostatic interactions is termed herein “OSDel”.

[0113] In some embodiments, the chimera comprises the first polypeptide and a dsRNA binding domain (dsRBD). In some embodiments, the chimera comprises the first polypeptide, a dsRBD, and dsRNA. In some embodiments, the dsRNA is bound to the dsRBD. In some embodiments, a chimera comprising the first polypeptide, a dsRBD and dsRNA bound thereto is termed herein “OSSCot”. In some embodiments, the chimera further comprises a linker. In some embodiments, OSSCot, further comprises a linker.[001 14] In some embodiments, a chimera comprises a plurality of chimeras. As used herein, the term “plurality” comprises any integer being equal to or greater than 2. In some embodiments, a plurality of chimeras comprises OSDel and OSSCot.

[0115] As used herein, the term “vitellogenin” refers to the precursor protein of the egg yolk protein (e.g., vitellin). In some embodiments, vitellogenin is an arthropod vitellogenin. In some embodiments, vitellogenin is a crustacean vitellogenin. In some embodiments, vitellogenin is the vitellogenin of a crustacean of the Macrobrachium genus. In some embodiments, vitellogenin is a vitellogenin of a crustacean of the family of Penaeidae.

[0116] In some embodiments, the full length vitellogenin is the full length vitellogenin of Macrobrachium rosenbergii (Accession number BAB69831.1).

[0117] In some embodiments, vitellogenin is a fish vitellogenin. In some embodiments, vitellogenin is a reptile vitellogenin. In some embodiments, vitellogenin is an amphibian vitellogenin. In some embodiments, vitellogenin is an ave vitellogenin.

[0118] As used herein, the term "directly" refers to cases wherein the peptide of the invention is bound to the agent in a covalent bond.

[0119] As used herein, the term "indirectly" refers to cases wherein each of the peptide of the invention and the agent are bound to a linker or a spacing element and not directly to one another. In some embodiments, the peptide is covalently bound to the linker. In some embodiments, the agent is either covalently or non-covalently bound to the linker.

[0120] As used herein, the term "covalent bond" refers to any bond which comprises or involves electron sharing. Non-limiting examples of a covalent bond include, but are not limited to: peptide bond, glyosidic bond, ester bond, phosphor diester bond.

[0121] As used herein, the term "non-covalent bond" encompasses any bond or interaction between two or more moieties which do not comprise or do not involve electron sharing. Non-limiting examples of a non-covalent bond or interaction include, but are not limited to, electrostatic, a-cffcct. van der Waals force, hydrogen bonding, and hydrophobic effect. In some embodiments, a non-covalent bond comprises ionic interaction. In some embodiments, the first polypeptide and the dsRNA are bound to one another via ionic interactions.

[0122] In some embodiments, the first polypeptide and the second polypeptide are bound to one another via a linker.

[0123] The term "linker" refers to a molecule or macromolecule serving to connect different moieties of the chimera, that is the first polypeptide and the second polypeptide . In one embodiment, a linker may also facilitate other functions, including, but not limited to, preserving biological activity, maintaining sub-units and domains interactions, and others.

[0124] In another embodiment, a linker may be a monomeric entity such as a single amino acid. In another embodiment, amino acids with small side chains are especially preferred, or a peptide chain, or polymeric entities of several amino acids. In another embodiment, a peptide linker is 2 to 30 amino acids long, 2 to 25 amino acids long, 4 to 23 amino acids long, 4 to 20 amino acids long, 5 to 22 amino acids long, or 2 to 28 amino acids long. Each possibility represents a separate embodiment of the invention. In another embodiment, a peptide linker is at least 6 amino acids long, at least 8 amino acids long, at least 10 amino acids long, at least 12 amino acids long, at least 15 amino acids long, at least 17 amino acids long, at least 20 amino acids long, at least 22 amino acids long, at least 25 amino acids long, at least 27 amino acids long, or at least 30 amino acids long, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In one embodiment, a linker may be a nucleic acid encoding a small peptide chain. In another embodiment, a linker encodes a peptide linker of 6 to 30 amino acids long, 6 to 25 amino acids long, 7 to 23 amino acids long, 8 to 20 amino acids long, 10 to 22 amino acids long, or 12 to 28 amino acids long.Each possibility represents a separate embodiment of the invention. In another embodiment, a linker encodes a peptide linker of at least 6 amino acids long, at least 8 amino acids long, at least 10 amino acids long, at least 12 amino acids long, at least 15 amino acids long, at least 17 amino acids long, at least 20 amino acids long, at least 22 amino acids long, at least 25 amino acids long, at least 27 amino acids long, or at least 30 amino acids long, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0125] In some embodiments, the linker comprises the amino acid sequence I<HI<HI<HI<HI<HI<HI<HI<HI<H (SEQ ID NO: 12). In some embodiments, the linker comprises the amino acid sequence I<HI<HI<HI<HI<HI<HI<HI<H (SEQ ID NO: 13). In some embodiments, the linker comprises the amino acid sequenceIn some embodiments, the linker comprises the amino acid sequence KHKHKHKHKHKH (SEQ ID NO: 15). In some embodiments, the linker comprises the amino acid sequence KHKHKHKHKH (SEQ ID NO: 16). In some embodiments, the linker comprises the amino acid sequence KHKHKHKH (SEQ ID NO: 17). In some embodiments, the linker comprises the amino acid sequence KHKHKH (SEQ ID NO: 18). In some embodiments, the linker comprises the amino acid sequence KHKH (SEQ ID NO: 19). In some embodiments, the linker comprises the amino acid sequence KH. In some embodiments, the linker comprises the amino acid sequence KKKKKKKKK (SEQ ID NO: 20).

[0126] In some embodiments, an agent, that is a polynucleotide is selected from: a single strand RNA, antisense RNA, siRNA, dsRNA, shRNA, guide RNA, micro RNA (miRNA), and DNA. As used herein, DNA refers to any deoxyribonucleic acid polymer, for example, complementary DNA (cDNA), a digested cDNA or genomic DNA (gDNA), a plasmid DNA, and the like. In some embodiments, an agent is any RNA interference (RNAi) inducing polynucleotide.

[0127] An antisense sequence as described herein comprises any one of: antisense oligonucleotide, ribozyme, external guide sequence (EGS) oligonucleotide, siRNA compound, single- or double-stranded RNA interference (RNAi) compound such as siRNA compound, modified bases / locked nucleic acid (LNA), antagomir, peptide nucleic acid (PNAs), or any other oligomeric compound or oligonucleotide mimetic capable of hybridizing to at least a portion of the target nucleic acid, such as a gene or a transcript thereof, and modulate its function. In some embodiments, the antisensesequence comprises an antisense RNA, antisense DNA, chimeric antisense oligonucleotide, antisense oligonucleotide comprising modified linkages, micro interfering RNA (miRNA), and a short hairpin RNA (shRNA).

[0128] As used herein, the term “interfering RNA” refers to any double stranded or single stranded RNA sequence, capable — either directly or indirectly (i.e., upon conversion) — of inhibiting or down regulating gene expression by mediating RNA interference. Interfering RNA includes but is not limited to siRNA and shRNA. RNAi refers to the selective degradation of a sequence-compatible messenger RNA transcript.

[0129] As used herein, the term “shRNA” refers to an RNA molecule comprising an antisense region, a loop portion, and a sense region, wherein the sense region has complementary nucleotides that base pair with the antisense region to form a duplex stem. Following post-transcriptional processing, the small hairpin RNA is converted into a small interfering RNA by a cleavage event mediated by the enzyme Dicer, which is a member of the RNase III family.

[0130] As used herein, the term siRNA refers to any small RNA molecule capable of inhibiting or down regulating gene expression by mediating RNA interference in a sequence specific manner. The small RNA can be, for example, about 18 to 21 nucleotides long.

[0131] As used herein, the term dsRNA refers to any double stranded RNA molecule capable of inhibiting or down regulating gene expression by mediating RNA interference in a sequence specific manner. The dsRNA can be, for example, about 50 to 1,000 nucleotides long, about 50 to 500 nucleotides long, about 150 to 750 nucleotides long, or about 100 to 500 nucleotides long, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0132] In some embodiments, the agent is a peptide. In some embodiments, an agent that is a peptide is an enzyme. In some embodiments, an agent that is a peptide is an apoptosis inducer. The term "apoptosis inducer" encompasses any molecule or compound capable of inducing, or promoting programmed cell death, or any molecule or compound involved in the process of programmed cell death.

[0133] As used herein, the term "enzyme" encompasses any peptide capable of specifically catalyzing a reaction, i.e., an enzymatic procedure. As used herein, “enzymatic procedure” is any procedure catalyzed or performed by an enzyme, to namea few, nucleic acid molecule(s) ligation, reverse transcription, amplification, digestion, dephosphorylation, and others. An outcome of an enzymatic procedure comprises a desired product and by-products.Composition

[0134] According to another aspect, there is provided a composition comprising: (a) a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1; and (b) a double stranded RNA (dsRNA) molecule, in a mole per mole ratio (m:m) ranging between 1: 1 and 10: 1, 1 : 1 and 100: 1, 10: 1 and 100: 1, 5: 1 and 50: 1, 7: 1 and 40: 1, 6: 1 and 20: 1, and 1: 1 and 20: 1. Each possibility represents a separate embodiment of the invention.

[0135] According to another aspect, there is provided a composition comprising: (a) a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1; and (b) a dsRNA molecule having at least 70%, 80%, 90%, 95%, or 99% complementarity to an equal sized transcript of a gene of the WSSV, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the dsRNA molecule has 70%-100%, 80-100%, 90-100%, or 95-100% complementarity to an equal sized transcript of a gene of the WSSV. Each possibility represents a separate embodiment of the invention.

[0136] In some embodiments, the pathogen and / or virus gene encodes a VP28 protein of the WSSV.

[0137] In some embodiments, the dsRNA molecule comprises the nucleic acid sequence set forth in SEQ ID NO: 21.

[0138] In some embodiments, the pathogen is or comprises the white spot syndrom virus (WSSV).

[0139] In some embodiments, the composition further comprises a second polypeptide comprising a dsRBD. In some embodiments, the dsRBD comprises the amino acid sequence as set forth in SEQ ID NO: 2, or a functional analog thereof having at least 75% sequence homology or identity thereto.

[0140] In some embodiments, the first polypeptide and the second polypeptide are covalently bound, thereby constituting a chimera.

[0141] In some embodiments, the composition is a vaccine composition.

[0142] In some embodiments, the composition is formulated for vaccination.

[0143] In some embodiments, the composition of is formulated for injection.

[0144] As used herein, the term "vaccine" includes all prophylactic and therapeutic vaccines. The vaccine compositions described herein are suitable for administration to subjects in a biologically compatible form in vivo. The expression "biologically compatible form suitable for administration in vivo" as used herein means a form of the substance to be administered in which any toxic effects are outweighed by therapeutic effects. The substances may be administered to any animal. In some embodiments, a vaccine as described herein is administered to: arthropod, amphibian, reptile, ave, to elicit degradation, breakdown, knockdown, silencing, or any combination thereof, of at least one RNA molecule obtained, derived, produced, transcribed, expressed, or any combination thereof, of a pathogen.

[0145] In some embodiments, a vaccine refers to a composition being administered to a mother, which induces the degradation, breakdown, knockdown, silencing, or any combination thereof, of at least one RNA molecule obtained, derived, produced, transcribed, expressed, or any combination thereof, of a pathogen in a progeny of the female.

[0146] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0147] As used herein, the term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates. Antibacterialagents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; and agents for the adjustment of tonicity such as sodium chloride or dextrose are also envisioned. The carrier may comprise, in total, from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.

[0148] As used herein, the term "pharmaceutically acceptable" means suitable for administration to a subject, e.g., a human. For example, the term "pharmaceutically acceptable" can mean approved by a regulatory agency of the Federal or a state government or listed in the U. S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0149] In some embodiments, the composition comprises an active agent / ingredient and a carrier. In some embodiments, the active agent / ingredient consists essentially of (a) the first polypeptide or the first polypeptide and the second polypeptide; and (b) the dsRNA molecule.

[0150] As used herein, the term “consists essentially of’ denotes that a given compound or substance constitutes the vast majority of the active agent / ingredient’ s portion or fraction of the composition.

[0151] In some embodiments, consists essentially of means that: (a) the first polypeptide or the first polypeptide and the second polypeptide; and (b) the dsRNA molecule, constitute at least 95%, at least 98%, at least 99%, or at least 99.9% by weight, of the active agent / ingredient(s) of the composition, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.General

[0152] In the discussion unless otherwise stated, adjectives such as “substantially” and “about” modifying a condition or relationship characteristic of a feature or features of an embodiment of the invention, are understood to mean that the condition or characteristic is defined to within tolerances that are acceptable for operation of the embodiment for an application for which it is intended. Unless otherwise indicated, the word “or” in the specification and claims is considered to be the inclusive “or” rather than the exclusive or, and indicates at least one of, or any combination of items it conjoins.

[0153] It should be understood that the terms “a” and “an” as used above and elsewhere herein refer to “one or more” of the enumerated components. It will be clear to one of ordinary skill in the art that the use of the singular includes the plural unless specifically stated otherwise. Therefore, the terms “a”, “an” and “at least one” are used interchangeably in this application.

[0154] For purposes of better understanding the present teachings and in no way limiting the scope of the teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0155] In the description and claims of the present application, each of the verbs, “comprise”, “include” and “have” and conjugates thereof, are used to indicate that the object or objects of the verb are not necessarily a complete listing of components, elements or parts of the subject or subjects of the verb.

[0156] Other terms as used herein are meant to be defined by their well-known meanings in the art.

[0157] Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive.

[0158] Throughout this specification and claims, the word “comprise” or variations such as “comprises” or “comprising,” indicate the inclusion of any recited integer or group of integers but not the exclusion of any other integer or group of integers.

[0159] As used herein, the term “consists essentially of’, or variations such as “consist essentially of’ or “consisting essentially of’ as used throughout the specification and claims, indicate the inclusion of any recited integer or group of integers, and the optional inclusion of any recited integer or group of integers that do not materially change the basic or novel properties of the specified method, structure or composition.

[0160] As used herein, the terms "comprises", "comprising", "containing", "having" and the like can mean "includes", "including", and the like; "consisting essentially of or"consists essentially" likewise has the meaning ascribed in U.S. patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments. In one embodiment, the terms "comprises", "comprising", "having" are / is interchangeable with "consisting".

[0161] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.EXAMPLES

[0162] Generally, the nomenclature used herein, and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds.) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley- Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.Materials and MethodsVaccine preparation

[0163] According to Cohen et al., (2023; Front Mar Sci 10) and Ilouz et al., (2024; Aquaculture 580, 740321)) the vaccine formulations were prepared shortly before injection. Briefly, the first treatment group was injected with a solution containing (KH)9-VgP linked to dsVP28 (20 pg dsVP28 / g body weight, dsVP28-peptide molar ratio 1: 10). The second treatment group was injected with a solution containing OSSCot piggybacking dsVP28 (20 pg dsVP28 / g body weight, dsVP28:OSSCot molar ratio 1: 10). The control group (n=5) was injected with free dsVP28. Chloroquine (20 mM final concentration) was incorporated into the injection mixtures as an endosomal escape reagent.In vivo vaccination trial

[0164] Female L. vannamei shrimp were observed, and their vitellogenic state was assessed (Fig. 1). Twenty (20) females in the late ovary stage II, with an average weight of 51.79 ± 1.12 g (Table 3), underwent meticulous weighing, tagging with eye identifiers, and subsequent allocation into four distinct groups. The first group is blank, in which females are intact. The second group is a control group where each female was injected with naked dsRNA encoding P28, the WSSV envelop protein (dsWSSV, 20 pg per gram of body weight) . The females of the third group were inj ected with the dsWS S V connected to OSDel, as described by Cohen et al. The females of the fourth group were injected with dsWSSV connected to OSSCot, as described by Ilouz et al. The progress of ovary development in the experimental females was monitored daily, with nearly daily vaccine injections until the ovary reached stage IV, totaling three consecutive injections (Table 3). Upon reaching stage V, females were paired with males for mating. After mating, females were transferred to a spawning tank, and observations were made on embryo hatching rates, clutch size, and health (Table 4).Table 3. Females daily monitoring summary table. The weight, ovary development status, and vaccination dates are described for each femaleTable 4. Egg production, fertilization and hatching rates of the femalesNursery Phase

[0165] Clutches with a hatching yield of 30,000 nauplii were chosen to continue with the experiment. The development of nauplii, zoea, mysis, and post-larvae were carefully observed. Post-larvae aged 10 days from each selected clutch (800 individuals) were cultured in the Shrimpvet hatchery, while additional 800 post-larvae were sent toShrimpvet Aquamekong farms and raised until reaching a weight of 0.8-1.0 gram. Shrimps of appropriate weight from the hatchery and the farm were then transported to the Shrimpvet laboratory in Ho Chi Minh City for the challenge trial.WSSV challenge trial

[0166] Shrimps from the same family were mixed, and 120 individuals from each family were acclimated in 4 different 90 L tanks (30 individuals / tank) before undergoing White Spot Syndrome Virus (WSSV) challenge.

[0167] Two WSSV challenge experiments were conducted: (i) via oral administration (per os , and (ii) intramuscular injection (IM). For the per os challenge, the shrimp were fed with 1 meal of shrimp muscle tissue mixture made of 1 :3 weight per WSSV-positive inoculum tissue and specific pathogen free (SPF) shrimp tissue (4.4 lx 108WSSV copies per gr tissue). The meal makes up 5% of the shrimp's body weight. For the injection challenge, each shrimp was injected with 40 pl saline containing 31 virus copies (7.73x l07 / ml WSSV stock diluted 105). The WSSV dosage for each experiment was determined by calibration trial (data not shown).Infection evaluation during 0.8-1 gr shrimp WSSV challenge qPCR analysis

[0168] Four (4) days post-challenge 3 shrimps per family, 1 shrimp per tank, were collected for WSSV viral load evaluation by qPCR in SHRIMPVET lab.Histology

[0169] Four (4) days post-challenge, 2 moribund shrimp per family were fixed with Davidson's AFA for the histological examination for EHP damages in the hepatopancreas caused by general health assessment. All shrimp sampled for histopathology purposes were injected with AFA Davidson's fixative, processed, and stained with hematoxylin and eosine (H&E) using routine histological methods (Bell & Lightner, 1988; Lightner, 1996). The histological sections were analyzed in the SHRIMPVET lab by light microscopy for the damages in shrimp hepatopancreas of AHPND (Tran et al., 2013).EXAMPLE 1Offspring of OSDel-dsWSSV- and OSSCot-dsWSSV-treated females possess higher survival rates compared to control

[0170] Offspring shrimps from each family were divided into 3 tanks (30 individuals / tank) and fed with a mixture of 1 :3 WSSV-positive inoculum tissue and SPF shrimp tissue. Shrimp mortality was recorded daily for 10 days post-challenge, and a Kaplan-Meier survival plot was generated (Fig. 2A). Families with higher survival rates were: OSDell, OSDel2, OSDel4, and OSSCot4 (Fig. 2A). Among 5 families, three of the control groups were utterly dead 10 days after the challenge (Fig. 2B). The Kaplan- Meier survival rate of each group (5 families, 90 shrimp each / group; 540 individuals / group) clearly represents the significant difference in the survival rate (Fig. 2C). A significantly higher survival rate was observed in the OSDel-dsWSSV and OSSCot-dsWSSV treated female groups compared to the blank and the control groups (p<0.0001). The survival rate for the OSDel-dsWSSV and OSSCot-dsWSSV treated female groups were 35.63±8.95% and 30.8±8.71%, respectively, while the survival rate for the control and blank groups were as low as 10.57±5.48% and 12.93±6.4%, respectively (Fig. 2D). Therefore, the offspring of the OSDel-dsWSSV- and OSSCot- dsWSSV-treated female groups acquired an immune response to the virus. Bayesian analysis was performed to calculate the survival probability after the treatment.EXAMPLE 2Survival probability after viral challenge significantly increases in offspring of OSDel-dsWSSV- or OSSCot-dsWSSV-treated females

[0171] Bayesian analysis was performed to calculate the probability of survival after WSSV per os experiment. The Bayesian factor 1.839 in the OSDel-dsWSSV treated clutches (Table 1) and the Bayesian factor 1.708 in the OSSCot-dsWSSV treated clutch (Table 2) support the alternative hypothesis. The obtained BFs imply that the likelihood of shrimp survival after treatment is nearly twice as high as without treatment.Table 1. Bayesian analysis for OSDel-dsWSSV group after WSSV per os experiment

[0172] The Bayesian factor denotes the probability of treatedand survived shrimps among all survivors, while p(D / —H ) denotes the probability of the treated un-survived among all un-survived shrimps, H denotes survived, -H denotes un-survived, D denotes treatment and -D denotes no treatment or control, BF=1.839.Table 2. Bayesian analysis for OSSCot-dsWSSV group after WSSV per os experiment

[0173] The Bayesian factor denotes the probability of treatedand survived shrimps among all survivors, while p(D / —H ) denotes the probability of the treated unsurvived among all unsurvived shrimps, H denotes survived, -H denotes unsurvived, D denotes treatment and -D denotes no treatment or control, BF=1.708.EXAMPLE 3Reduced WSSV infection was observed in OSDel-dsWSSV- and OSSCot- dsWSSV-treated groups

[0174] Four days post-challenge, 3 shrimps from each family were randomly sampled for quantitative PCR (qPCR) analysis to assess the WSSV copy number. The violin plot represents the copy number of WSSV (per gr muscle tissue) in treated and control groups (Fig. 3). The width of the violin represents the distribution of the WSSV copy numbers in each family. A narrow violin indicates that few individuals harbor a specific WSSV copy number, whereas a wide violin indicates that many individuals contain the specific WSSV copy numbers. Herein, more individuals possessing zero amount of WSSV copies were observed in the OSDel-dsWSSV- and OSSCot-dsWSSV-treated groups compared to the blank and control groups, as a comprehensive wide violin line up with the zero of the Y-axis (Fig. 3, dashed line). These results indicate that the probability offinding uninfected healthy shrimp is greater in the OSDel-dsWSSV- and OSSCot- dsWSSV-treated groups.EXAMPLE 4Shrimps from OSDel-dsWSSV- and OSSCot-dsWSSV-treated groups are white spots free

[0175] White spot appearance on the shrimp carapace is one of the shrimp WSSV- infection symptoms. At the end of the challenge experiment, on the 10thday, shrimps were sampled for carapace observation. No white spots appeared on the carapace of individuals ofthe OSDel-dsWSSV- and OSSCot-dsWSSV-treated groups (Figs. 4C-4D, respectively), similarly to the clean carapace of carapace of individuals from the negative control group (Fig. 4A). In sharp contrast, small white spots were highly evident on the carapace of shrimp ofthe blank and control groups (Figs. 4B-4C, respectively).EXAMPLE 5OSDel-dsWSSV and OSSCot-dsWSSV treatments delays WSSV-related mortality

[0176] A WSSV injection challenge experiment was conducted in parallel to the per os challenge experiment. Thirty shrimps weighing 0.8-1 gr from the blank, control, OSDel-dsWSSV- and OSSCot-dsWSSV-treated female groups were challenged by intramuscular (IM) injection of live WSSV. Shrimp mortality was recorded daily for 10 days post-challenge, and a survival curve for each group is presented as a Kaplan-Meier plot (Fig. 5A). A delay in the mortality in the OSDel-dsWSSV- and OSSCot-dsWSSV- treated groups was clearly evident. Mortality of injected shrimps was observed 2 days post-challenge (Fig. 5A). Lower mortality rates were evident for OSDel-dsWSSV- and OSSCot-dsWSSV-treated individuals starting from the 2ndto the 8thday, compared to individuals of the blank and control groups, where 100% mortality was observed already on day six post-challenge. Plotting the survival rate of day 4 post-challenge reveals a significantly higher survival rate in the OSDel-dsWSSV and OSSCot-dsWSSV-treated groups, compared to the blank and control groups (Fig. 5B, p<0.01).EXAMPLE 6Adult offspring of OSDel-dsWSSV-treated females possess higher survival rates compared to control

[0177] Fl shrimp (0.8 gr) families of each treatment were mixed and cultured in Shrimpvet farm until they reached an average 25 gr weight. Forty (40) Fl -grown shrimps from each treatment were challenged with White Spot Syndrome Virus (WSSV) by feeding pellets submerged with the WSSV inoculum. Four (4) consecutive contaminated meals were applied, and survival rates were monitored for over 10 days. Over the 10- day experiment period, OSDel treatment exhibited significantly higher survival rates than the control group. The higher survival rate of OSDel-treated offspring is noticeable 62 hours post-challenge until the experiment termination (230 hours), indicating significantly increased resistance to the virus (Figs. 6A-6B). The OSSCot-treated offspring possessed no survival difference from the control groups. On experiment termination day, a 25% survival rate was observed in the OSDel-treated offspring compared to zero survivors from the control and OSSCot groups. These results suggest that offspring from the OSDel-treated group acquired a protective immune response against WSSV that lasted 4 months, a period usually sufficient to reach the market size.

[0178] Bayesian analysis provided statistically robust evidence that OSDel -dsVP28 treatments significantly enhanced shrimp survival 10 days following WSSV infection. The Bayesian factor 3.7 (OSDel-dsVP28) strongly supports this conclusion (Table 5). This value implies a nearly four-fold increased survival probability in the OSDel- dsVP28 -treated group compared to controls, highlighting the potential of these treatments for improving shrimp disease resistance.Table 5. Bayesian analysis

[0179] While the present invention has been particularly described, persons skilled in the art will appreciate that many variations and modifications can be made. Therefore, the invention is not to be construed as restricted to the particularly describedembodiments, and the scope and concept of the invention will be more readily understood by reference to the claims, which follow.

Claims

CLAIMSWhat is claimed is:

1. A method for protecting a progeny of an oviparous female crustacean from an infection of a White Spot Syndrome Virus (WSSV) or a symptom associated therewith, the method comprising administering to said oviparous female crustacean a therapeutically effective amount of composition comprising: a. a first polypeptide comprising the amino acid: DKX1X2X2X3PX4X5GX6YKYVEAX7X8X9SX10X11X12 (SEQ ID NO: 1), wherein: Xi is selected from the amino acid residues N and K; X2 is selected from the amino acid residues I and V; X3 is selected from the amino acid residues K and R; X4 is selected from the amino acid residues A and S; X5 is selected from the amino acid residues Y and I; Xr, is an amino acid residue selected from the group consisting of: S, I, A, and T; X7 is an amino acid residue selected from the group consisting of: H, S, K and E; Xx is selected from the amino acid residues Q and M; X9 is an amino acid residue selected from the group consisting of: E, D, and M; X10 is selected from the amino acid residues V and T; Xu is selected from the amino acid residues L and M; and X12 is selected from the amino acid residues R and K; and b. a double stranded RNA (dsRNA) having at least 70% complementarity to a transcript of said WSSV.

2. The method of claim 1, wherein said transcript encodes a protein being essential to any one of a pathogenic activity, survival, and both, of said WSSV.

3. The method of claim 1 or 2, wherein said crustacean is a decapod crustacean.

4. The method of any one of claims 1 to 3, wherein said transcript encodes a VP28 protein of said WSSV.

5. The method of any one of claims 1 to 4, wherein said dsRNA comprises the nucleic acid sequence set forth in SEQ ID NO: 21.

6. The method of any one of claims 1 to 5, wherein said composition further comprises a second polypeptide comprising a double stranded RNA binding domain (dsRBD).

7. The method of claim 6, wherein said dsRBD comprises the amino acid sequence as set forth in SEQ ID NO: 2, or a functional analog thereof having at least 75% sequence homology or identity thereto.

8. The method of claim 6 or 7, wherein said first polypeptide and said second polypeptide are bound to one another, thereby constituting a chimeric protein comprising both.

9. The method of any one of claims 1 to 8, further comprising a step before said administering, comprising determining said female crustacean being suitable for administration with said composition, wherein said determining comprises selecting an early vitellogenic or a vitellogenic female crustacean.

10. The method of any one of claims 1 to 9, wherein said administering is at least once a week administering.

11. The method of any one of claims 1 to 10, wherein said administering is injecting.

12. The method of claim 11, wherein said injecting is injecting into a muscular tissue of said female.

13. The method of claim 11 or 12, wherein said injecting into the abdomen of said female.

14. The method of any one of claims 1 to 13, wherein said composition being a vaccine composition.

15. The method of any one of claims 1 to 14, wherein said protecting comprises any one of: increasing survival rate of said progeny, reducing incidence of at least one symptom associated with infection of said pathogen in said progeny, and both.

16. The method of claim 15, wherein said increasing or reducing is compared to a non-treated control.

17. The method of claim 15 or 16, wherein said protecting is for a period being at least 14 days longer compared to a non-treated control.

18. The method of any one of claims 1 to 17, wherein said symptom is appearance of at least one white spot on a carapace of at least one progeny of said oviparous female crustacean.

19. The method of any one of claims 1 to 18, further comprising a step after said administering, comprising crossing said administered female with a corresponding male.

20. A progeny obtained from an oviparous female crustacean of the method of any one of claims 1 to 19.

21. A composition comprising: a. a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1; and b. a double stranded RNA (dsRNA) molecule,in a mole per mole ratio (m:m) ranging between 1: 1 and 10: 1.

22. The composition of claim 21, further comprising a second polypeptide comprising a dsRBD.

23. The composition of claim 22, wherein said dsRBD comprises the amino acid sequence as set forth in SEQ ID NO: 2, or a functional analog thereof having at least 75% sequence homology or identity thereto.

24. The composition of claim 22 or 23, wherein said first polypeptide and said second polypeptide are covalently bound, thereby constituting a chimera.

25. The composition of any one of claims 22 to 24, wherein said dsRNA has at least 80% complementarity to an equal sized transcript of a gene of a pathogen.

26. The composition of claim 25, wherein said pathogen is the white spot syndrom virus (WSSV).

27. The composition of claim 26, wherein said gene encodes a VP28 protein of said WSSV.

28. A composition comprising: a. a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1; and b. a dsRNA molecule having at least 80% complementarity to an equal sized transcript of a gene of the WSSV.

29. The composition of claim 28, wherein said gene encodes a VP28 protein of said WSSV.

30. The composition of any one of claims 21 to 29, wherein said dsRNA molecule comprises the nucleic acid sequence set forth in SEQ ID NO: 21.

31. The composition of any one of claims 21 to 30, being a vaccine composition.

32. The composition of any one of claims 21 to 31, being formulated for vaccination.

33. The composition of claim 32, wherein said vaccination is RNA-based vaccination.

34. The composition of any one of claims 21 to 33, being formulated for injection.

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