# Patent

## Title
Nucleic acid and amino acid sequences, and vaccine for the control of ectoparasite infestations in fish

## Abstract

The present invention is related with the isolation and cloning of a new gene, the production of the protein encoded by this gene by using recombinant systems, and the use of this antigen in a vaccine formulation as a purified protein and/or naked DNA, to induce an immune response in aquatic organisms against different ectoparasite species, including the known as sea lice, and pathogens associated with these infestations. The vaccine preparations, administered by oral route, immersion bath or injection, demonstrated its efficacy by producing IgM humoral immune response and reducing the number of parasites per fish in the vaccinated fishes.

## Background
This application is a continuation-in-part of, and claims priority to, U.S. application Ser. No. 12/601,974 filed Nov. 25, 2009, which claims priority to international application PCT/CU2008/000003 filed May 30, 2008, which claims priority to Cuban Application No. 2007-0124 filed May 31, 2007, all of which are incorporated by reference.


FIELD OF THE INVENTION
The present invention relates to aquatic biotechnology, in particular with a gene sequence and the encoded polypeptide, which are protective antigens against ectoparasite infestations in fish, vaccines comprising these antigens and methods for inducing an immune response against ectoparasites in aquatic organisms.
BACKGROUND OF THE INVENTION
Sea lice (Copepoda, Caligidae) are the most widely distributed marine pathogens in salmon industry in the last 30 years. They also spread in the final 15 years to other culture species and wild salmonid populations (Pike, A. W. y Wadsworth, S. L. (2000). Advances in Parasitology 44:233-337, Ragias, V. et al. (2004). Aquaculture 242:727-733). There are three major genera of sea lice: Pseudocaligus, Caligus and Lepeophtheirus. 
Considering salmonid production throughout northern hemisphere, one of these species, Lepeophtheirus salmonis, is the responsible for the main disease outbreaks in salmonid farms. This parasite, just in 2004, was the responsible for direct and indirect losses in worldwide aquaculture, of 100 millions of US dollars (Johnson, S. C., et al. (2004). Zool Studies 43: 8-19). All the sea lice developmental stages in which the parasite is attached to the host, feed on host mucus, skin and blood. The sea lice adhesion and feeding produce lesions that differ in their nature and severity, depending on the sea lice specie, their abundance, the developmental stages present and the host specie (Johnson, S. C et al., “Interactions between sea lice and their hosts”. En: Host-Parasite Interactions. Editors: G. Wiegertjes and G. Flik, Garland Science/Bios Science Publications, 2004, pp. 131-160). In the southern hemisphere, Caligus rogercresseyi is the most important specie affecting chilean salmon industry (González, L. y Carvajal, J. (2003). Aquaculture 220: 101-117). In the case of severe illness, like the one observed in Atlantic salmon (Salmo salar), when the fish are infected by a high number of L. salmonis, extensive damaged and hemorrhagic skin areas are observed in the head and fish back. Also, a distinct area of erosion and sub-epidermal hemorrhage in the perianal region can be seen (Grimnes, A. et al. (1996). J Fish Biol 48: 1179-1194). Sea lice can cause host physiological changes including a stress response, reduction of immune functions, osmorregulation failure and death, if the infection is not treated (Johnson, S. C., et al. (2004). Zool Studies 43: 8-19).
A wide range of chemicals had been used to control sea lice infestations like hydrogen peroxide, organophosphates, ivermectin and other related compounds like emamectin benzoate, molting regulators and pyrethrins (MacKinnon, B. M. (1997). World Aquaculture 28: 5-10; Stone J., et al. (1999). J Fish Dis 22: 261-270). The treatments against sea lice can be apply by immersion baths like organophosphates and pyrethroyds or orally, as ivermectin. These immersion baths are difficult to perform. In addition they are expensive and can have significant effects over fish growth after treatments (MacKinnon, B. M. (1997). World Aquaculture 28: 5-10). Besides, the chemicals commonly used by immersion baths are not effective in all sea lice developmental stages. To date, the use of oral treatments such as SLICE(R) (emamectin benzoate) is predominant in salmon industry. SLICE(R), unlike chemicals administered by immersion, give a short protection against re-infection. This treatment, although is easier to apply compared to immersion baths, is also expensive and requires a period of time before the fish can be destined for human consumption (Stone J., et al. (1999). J Fish Dis 22: 261-270).
There are evidences which suggest the development of resistance against conventional treatments in L salmonis, particularly in populations frequently treated (Denholm, I. (2002). Pest Manag Sci 58: 528-536). This fact together with the necessity of reducing costs and threats to the environment, make imperative the development of new approaches like vaccines to control sea lice infestations in fish. The experience with terrestrial parasites demonstrated that a successful vaccine needs to be comprised for one or more concealed antigens with low or not homology with the host proteins. Sea lice are ectoparasites that feed on host mucus, skin and blood and then, only have limited contact with the host immune system (Boxaspen, K. (2006). ICES Journal of Marine Science 63: 1304-1316). In these cases, it was observed host immune response suppression due to the production of immunomodulatory proteins by the parasite at the adhesion and feeding site (Wikel, S. K., et al., “Arthropod modulation of host immune responses”. En: The Immunology of Host-Ectoparasitic Arthropod Relationships. Editors: Wikel, S. K., CAB Int., 1996, pp. 107-130). These proteins have been investigated for their use as candidate vaccines to control sea lice infestations. They have been patented and have been evaluated in assays performed in vitro to study its effects over the host immune system (Patent No. WO2006010265: RECOMBINANT VACCINES AGAINST CALIGID COPEPODS (SEA LICE) AND ANTIGEN SEQUENCES THEREOF). Tripsins, vitellogenin-like proteins and host adhesion proteins are some of the molecules studied as potential antigens (Johnson, S. C., et al. (2004). Zool Studies 43: 8-19; Boxaspen, K. (2006). ICES Journal of Marine Science 63: 1304-1316).
In general, vaccines are safer than chemical treatments for both, fish and environment. Nevertheless, until now there are not commercial vaccines available against sea lice. Experimental vaccines against L salmonis, which employ animal whole extracts, have been developed. These vaccines were not protective since their administration resulted in minor changes in L salmonis fecundity (Grayson T. H., et al. (1995). J Fish Biol 47: 85-94).
The identification of targets for sea lice prevention and treatment have not been successful as a result of the poor knowledge about the mechanisms involved in the pathology of sea lice infestations in salmons. This handicap makes difficult the progress of researches related to recombinant vaccines development. The reality is that up to the moment an effective vaccine against these ectoparasites has not been developed.
In other arthropods like ticks, genes involved in different tick's genera reproduction and feeding (Almazán et al. (2003). Vaccine 21:1492-1501) have been identified, employing expression immunization libraries. The results based on RNA interference (de La Fuente et al. (2005). Parasitol Res. 96:137-141) and immunization trials (Almazán et al. (2005). Vaccine 23: 4403-4416) suggest that these genes might be good candidates for vaccine development against different tick species which infest mammals. One of the tick's studied proteins, cement protein, which is produced in the tick salivary glands appear to be a good candidate to confer protection against different tick species (Adama, R. et al. (2005). Vaccine 23: 4329-4341) and consequently, against opportunistic pathogens which use ticks as their hosts (Labuda, M. et al. (2006). PLoS Pathogens 2(4): 251-259).
DESCRIPTION OF THE INVENTION
The current invention solves the problem discussed before, providing new nucleic acid and amino acid sequences as components of a vaccine against ectoparasite infestations in fish. These vaccines comprise an immunogenic amount of a DNA fragment isolated from Caligus rogercresseyi adults and/or a polypeptide encoded by this DNA fragment, formulated in a suitable adjuvant.
New genes which comprises the sequences identified as SEQ ID NOS: 1, 3 and 5, were isolated by using degenerate oligonucleotides. These primers were designed based on sequences similar to the 4D8 protein (or subolesin/akirin) found in different insects and arachnids: Drosophila melanogaster (GeneBank accession number: AAN12062), D. pseudoobscura (GeneBank accession number: EAL30734), Apis mellifera (GeneBank accession number: XP—395252) and the tick Ixodes scapularis (GeneBank accession number: AAV67031). At the DNA level, the isolated fragment only has a short region of homology of 56 nucleotides comprised between nucleotide 476 and nucleotide 532 of the isolated sequence, and nucleotide 321-nucleotide 377 of the 4D8 gene from Rhipicephalus sanguineus. In terms of amino acid sequence, the sequence of the new antigen has low homology (<43%) with the sequences mentioned above and other in the GeneBank database.
The isolated nucleic acids encode for polypeptides, which amino acid sequences are identified in the present invention as SEQ ID NOS: 2, 4 and 6, respectively. It is also an object of the present invention an amino acid sequence in which one or more amino acid residues have been removed, substituted or added to the sequences identified as SEQ ID NOS: 2, 4 and 6, and keeps their potential to induce an immune response against ectoparasites in fish.
Preferably, SEQ ID NOS: 2, 4 and 6 have two, or least two amino acid residues removed, substituted or added and keep their potential to induce an immune response against ectoparasites in fish.
As the main result, it was found that the new genes and/or their corresponding polypeptides expressed by recombinant DNA technology in the yeast Pichia pastoris, produces humoral IgM immune response and important damages in ectoparasite development and reproduction. Moreover, it was observed a significant reduction in the number of parasites per fish in challenge experiments.
SEQ ID NO: 7 is a truncated polypeptide identical to SEQ ID NO: 4, but shortened. SEQ ID NO: 7 maintains the ability to significantly reduce the number of parasites per fish in challenge experiments. See Example 15 below.
The use of these new antigens offers several advantages over the traditional methods employed in salmon industry to treat sea lice infestations described above. The parasite life cycle is interrupted due to the presence of specific antibodies against this protein and the activation of the host cellular immune response. These mechanisms together produce a reduction in the parasite body weight. They also affect the parasite reproduction and consequently, reduce the infestation levels, because of the individual damage which ultimately leads to the reduction of caligid populations. These effects are coupled with both a minimal environmental effect and low risk for human consumption. Besides, as a result of the vaccine administration, the infestation levels of opportunistic pathogens like Piscirickettsia salmonis, an intracellular pathogen that causes large losses in salmon culture, were diminished.
The effects of the use of these new genes and the encoded proteins, are surprising and unexpected, bearing in mind that some authors have considered that host blood is not the main component of the sea lice diet (approximately 15-25% of consumed tissue) and the female does not ingest more blood compared to males as occurs in terrestrial blood-feeding parasites (Bricknell, I. R et al. (2004). Caligus 8: 6; Wagner, G. N. y McKinley, R. S. (2004). J. Fish. Biol. 64:1027-1038). Currently, the knowledge about the molecular mechanisms involved in sea lice digestion is sparse. Thus, it is not obvious that crustacean's physiology is similar to arachnids (Raynard, R. S. et al. (2002). Pest Management Science 58: 569-575).
In one of the embodiment of the present invention, the compositions which comprise the polypeptides defined by the amino acid sequences identified as SEQ ID NOS: 2, 4, 6 and 7 stimulate the IgM immune response in fish, producing damages in the ectoparasites and reducing sea lice infestations levels.
The polypeptide coding sequences were obtained by Polymerase Chain Reaction (PCR) using complementary DNA (cDNA) from adult Caligus spp. as template and degenerate oligonucleotides. Polypeptides which comprise in their polypeptide chain an amino acid sequence at least 50% identical, more preferably about 70% identical, and even more preferably about 80% identical to SEQ. ID NOS: 2, 4, 6 and 7, and produce an immune response against ectoparasite infestations in salmonids and other fish species, are also subject of the present invention.
In a particular embodiment of this invention, the nucleotide sequence coding for the polypeptides mentioned above were cloned into a P. pastoris expression vector pPS7. This vector has the P. pastoris AOX1 promoter (pAOX1), the signal peptide of sucrose invertase 2 (spSUC 2) and the terminator sequence from glyceraldehyde-3-phosphate dehidrogenase, both from S. cerevisiae. It also contains a 3′ AOX P. pastoris chromosomal DNA sequence, necessary for identical recombination with the yeast genome and the selection marker HIS3 of S. cerevisiae. In addition, the vector has a functional replication origin in Escherichia coli and the ampicillin resistance gene as bacterial selection marker. The vectors commonly used to generate recombinant yeast strains are generally integrative. Prior to transformation, the plasmids should be linearized to direct identical recombination by AOX1 region.
The P. pastoris MP36 strain was used for extracellular recombinant protein production. This strain is an auxotrophic his3 mutant obtained from P. pastoris BKM-90 strain (patent EP0438200), which after transformation with the expression vector acquired a His+ phenotype (Yong V., et al. (1992). HIS-3 gene of Saccharomyces cerevisiae complement his− mutation in yeast Pichia pastoris. Biotecnología Aplicada 9: 55-61).
The present invention also comprises the sequences identified as SEQ ID NOS: 2, 4, 6, and 7, or fragments obtained from recombinant or natural sources or by chemical synthesis. To obtain the polypeptides of interest, a bacterial expression system and/or yeast different from P. pastoris and/or other systems for recombinant protein production well known in the art can be used.
In another embodiment of the present invention, the target genes were also cloned in pVAX vector (Invitrogen). This vector is specifically designed for use in the development of DNA vaccines. With a minimal sequence this vector provides highest level expression of recombinant proteins while minimizing extraneous genetic elements. The pVAX vector meets the U.S. Food and Drug Administration (FDA) guidelines for DNA vaccine design.
This invention comprises as well a vaccine composition against ectoparasites that affect salmonids and other species, which contains the polypeptides identified as SEQ ID NOS: 2, 4, 6 or 7, or a polypeptide at least 50% identical, preferably about 70% identical, more preferably about 80% identical with SEQ. ID NOS: 2, 4, 6 or 7.
As part of this invention, two or more promiscuous T cell epitopes were incorporated to the polypeptides identified as SEQ ID NOS: 2, 4, 6, or 7. Several studies have established the potential of promiscuous T cell epitopes incorporated as fusion proteins in chimeric peptides and proteins to increase its immunogenicity (El Kasmi K C. et al. (2000). J Gen Virol 81:729-35). These epitopes can be derived from natural immunogens of viral or bacterial origin. These natural epitopes can also be modified by addition, deletion or substitution of one or multiple amino acids to obtain a vaccine candidate that can be tested by its ability to increase the response to a specific antigen. The potential of employing these epitopes in fish vaccines has been demonstrated (Kuzyk M A. et al. (2001) Vaccine 19: 2337-2344).
The effect of these polypeptides over the humoral immune response and sea lice infestation index was evaluated in experiments where the purified polypeptides adjuvated in oil adjuvant were administered to fish by injection. The polypeptides were also effective when administered in the feed or by immersion bath. Humoral IgM immune response was increased in vaccinated animals. Challenge experiments demonstrated that the number of parasites per fish were lower in vaccinated fish.
Another aspect of the present invention is a vaccine composition against ectoparasitic infestations in salmonids and other fish species which comprises a nucleotide sequence containing the sequences identified as SEQ ID NOS: 1, 3 and 5.
As part of this invention, a method to prevent and treat fish infestations by different ectoparasite species was provided. This method is based on the administration of the polypeptides identified as SEQ ID NOS: 2, 4, 6 and 7 or a polypeptide with an amino acid sequence at least 50% identical, prefereably about 70% identical, more preferably about 80% identical, to SEQ ID NOS: 2, 4, 6 or 7 in a vaccine composition to induce an immune response in aquatic organisms (e.g. fish) against different ectoparasites species and/or diminished the damage associated with pathogens and opportunistic diseases associated with these infestations (i.e. reduce the number of parasites in the fish.
The polypeptides identified as SEQ ID NOS: 2, 4, 6, and 7 were obtained as fusion proteins with one or two T cell epitopes. These polypeptides, purified and formulated in oil adjuvant, were administered to fish by injection. The success of vaccination was measured in a challenge experiment as the reduction of the number of parasites per fish, compared to the groups injected with the polypeptide identified as SEQ ID NOS: 2, 4, 6 and 7 and the group injected with PBS.
In a materialization of the present invention, the composition which comprises the polypeptides administered by injection, in doses ranging from 0.1 to 10 μg/g of vaccinated fish body weight. In another aspect of the invention, the vaccine composition is administered in feed formulations, in doses ranging 0.1-300 μg/g of feed, or by immersion baths, in doses between 0.01-1 mg/L of water.
This invention also includes the use of the nucleotide sequences identified as SEQ ID NOS: 1, 3, and 5 alone or in combination with the encoded polypeptide, to make a vaccine composition which comprises naked DNA, to induce an immune response in aquatic organisms (e.g. fish) against different ectoparasite species, and/or diminished the damages related with these pathogens and opportunistic diseases associated with these infestations (i.e. reduce the number of parasites). A naked DNA vaccine containing the cDNA from Caligus spp. and a combination of the purified polypeptides/naked DNA were evaluated. Good results, in terms of IgM immune response and reduction of the number of parasites per fish, were obtained.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Humoral immune response obtained with different doses of the protein named MY32, administered by intraperitoneal injection to 35 g fish (Salmo salar). The graphic shows the geometric mean of the antibody titers (GMT) versus time. The experimental groups are: A. Purified recombinant MY32 protein: 0.5 μg/g of body weight); B. Purified recombinant MY32 protein: 1 μg/g of body weight; Negative control: Phosphate Buffered Saline (PBS) adjuvated in oil adjuvant.
FIG. 2. Humoral immune response obtained with different doses of the protein named MY32, orally administered to 35 g salmons. The graphic shows the geometric mean of the antibody titers (GMT) versus time. The experimental groups are: A. Purified recombinant MY32 protein: 0.5 μg/g of feed; B. Purified recombinant MY32 protein: 1 μg/g of feed; Negative control: Salmonid standard feed without MY32.
FIG. 3. Challenge experiment in Salmo salar immunized with the purified polypeptide MY32, administered at the dose of 1 μg/g of feed. Fish were infested with Lepeophtheirus salmonis. The experimental groups are: A. Feed containing MY32 protein at the dose of 1 μg/g of feed; B. Negative control: Salmonid standard feed without MY32.
FIG. 4. Effect on the number of parasites per fish of the salmon immunization with the DNA coding for the polypeptide MY32 or the combination DNA/purified protein. The fish were immunized by intramuscular injection containing 25 μg of plasmid DNA having the my32 gene from C. rogercresseyi (group A) or with 25 μg of plasmid DNA having the my32 gene from C. rogercresseyi plus the purified polypeptide at a concentration of 0.5 μg/g of body weight (group B). Control fish were immunized with the backbone expression vector (group C).
FIG. 5. Challenge experiment in Salmo salar, previously immunized by immersion bath and infested with C. rogercresseyi. The graphic shows the average number of parasite per fish versus time. The experimental groups are: A: Group immunized with the plasmid containing my32 gene at the dose of 0.01 mg/L of water; B: MY32 polypeptide at the dose of 0.1 mg/L of water; C: Negative control.



EXAMPLES
Example 1
Isolation and Cloning of the MY32 Gene
The coding sequence for the MY32 polypeptide was obtained by PCR from cDNA of Caligus rogercresseyi, using degenerate oligonucleotides designed from previously reported sequences for subolesin/akirin in different arthropods.
The oligonucleotides were:








Oligonucleotide that hybridyze with 5′ fragment:



(SEQ ID NO: 8)



ATG GC(T/C) TG(T/C) GC(T/C/G/A) AC(T/C/A/G) (T/C)T(T/C) AA(A/G)



 


(SEQ ID NO: 9)



ATG GC(T/C) TG(T/C) GC(T/C/G/A) AC(T/C/A/G) (T/C)T(A/G) AA(A/G)



 


(SEQ ID NO: 10)



ATG GC(G/A) TG(T/C) GC(T/C/G/A) AC(T/C/A/G) (T/C)T(T/C) AA(A/G)



 


(SEQ ID NO: 11)



ATG GC(G/A) TG(T/C) GC(T/C/G/A) AC(T/C/A/G) (T/C)T(A/G) AA(A/G)



 


Oligonucleotide that hybridyze with 3′ fragment:


(SEQ ID NO: 12)



TT(A/C)AC(A/G)AA(A/C/G/T)G(T/C)(A/G)TC(A/G)TA(C/T)TG(C/T)TC



 


(SEQ ID NO: 13)



TT(G/T)AC(A/G)AA(A/C/G/T)G(T/C)(A/G)TC(A/G)TA(C/T)TG(C/T)TC







The amplified DNA was cloned into a commercial pGEM Teasy vector (Promega) and it was sequenced with Thermo Sequenase Premixed cycle Sequencer Kit (Amersham Pharmacia) according to manufacturer's instructions.
Example 2
Construction of Pichia pastoris Expression Vector Containing the Coding Sequence for MY32 Polypeptide, Transformation of MP36 Strain and Protein Expression
The my32 gene was amplified by PCR with specific oligonucleotides. These specific oligonucleotides incorporated a histidine tail to facilitate the protein immunodetection and purification. The PCR product was treated with T4 polynucleotide kinase enzyme to phosphorylate the gene ends and facilitate its cloning into the expression vector. The expression vector pPS7 was enzymatically digested with Nco I restriction endonuclease, treated with nuclease S1 and phosphatase alkaline to blunt and desphosphorylate the ends. The recombinant plasmid obtained was named pPS7-my32.
Prior to transformation, the plasmids were linearized with Pvu II restriction enzyme. The P. pastoris strain MP36 was transformed by electroporation with the recombinant expression vector. This strain is an auxotrophic his3 mutant which acquired the His+ phenotype after transformation.
The transformants identified by Dot Blot were analyzed by Southern Blot to determinate in which the integration event occurs. The gene my32 is under the regulation of AOX1 promoter. This promoter is inducible by methanol. The protein was obtained at high levels in the periplasm. It was solubilized and purified by affinity chromatography to metal ions employing Ni-NTA (Quiagen) and obtained 95% pure.
Example 3
Construction of MY32 DNA Expression Vector in Eukaryotic Cells for Naked DNA Vaccine
The gene my32 was excised from pGEM T easy (Promega) by digestion with EcoR I. The insert was purified by QiaQuick kit (Qiagen, Hamburg, Germany) and ligated to pVAX vector (Invitrogen) previously digested with EcoR I. Recombinant DNA employed in immunization experiments was purified by Endo Free Plasmid Mega kit (Qiagen, Hamburg, Germany).
Example 4
Humoral Immune Response to Different Doses of Purified Recombinant Protein MY32 Administered by Intraperitoneal Injection
Three experimental groups of Atlantic salmon (Salmo salar) of 35 g mean weight were intraperitoneally injected with the purified polypeptide in oil adjuvant. The experimental groups (of 50 animals each) were:

    
    
        A. Purified recombinant MY32 protein: 0.5 μg/g of body weight.
        B. Purified recombinant MY32 protein: 1 μg/g of body weight.
        C. Negative control: PBS in oil adjuvant.
    
    


The dynamic of the IgM humoral immune response was measured by ELISA in a 90 days period. High IgM titers were demonstrated in the two doses assayed (FIG. 1).
Example 5
Challenge Experiment: Artificial Caligus rogercresseyi Infestations in Salmo salar Previously Immunized by Intraperitoneal Injection
Two hundred salmon of 35 g of average body weight were distributed into two experimental groups, 100 fish per group. Salmons from one group were injected with the purified protein at the dose of 1 μg/g of body weight adjuvated in oil adjuvant Montanide 888 at week 0. Negative control group received a PBS injection adjuvated in Montanide 888.
Two weeks later, salmon were extensively infested with an average of 100 parasites per fish, at copepodid developmental stage. At day 60, the number of parasites per fish was counted, finding a reduction in more than 80% (Table 1) in the group injected with the purified polypeptide, as compared to negative control group. Fish sera were collected at weeks 0, 4 and 8 and they were evaluated by ELISA for MY32 IgM specific response. High antibody titers were found at weeks 4 and 8 (Data not shown).







TABLE 1







Effect of the intraperitoneal injection with purified MY32 protein on the


number of parasites per fish at days 28 and 60 after the injection.









Number of



parasites/fish









Experimental groups
28 days
60 days





Purified MY32 protein adjuvated in oil adjuvant
45 ± 5
15 ± 3


(1 μg/g of body weight)


Negative control: PBS in oil adjuvant
92 ± 7
90 ± 8









Example 6
Humoral Immune Response in Fish Vaccinated with Different Doses of Purified MY32, Administered by the Oral Route
Three groups of juvenile Atlantic salmon (Salmo salar), mean body weight of 35 g, were handled. An oral formulation was prepared including the MY32 polypeptide in the salmon basal diet. Fishes belonging to negative control group were fed with basal diet alone. The fishes were fed once a day for five days. The experimental groups (of 50 fishes each) were:

    
    
        A. Feed containing purified MY32 protein at the dose of 0.5 μg/g of feed
        B. Feed containing purified MY32 protein at the dose of 1 μg/g of feed
        C. Negative control: Commercial feed
    
    


By using ELISA, the time course of the IgM humoral immune response was evaluated in a 90 days period, which showed high antibody titers at the doses assayed (FIG. 2).
Example 7
Challenge Experiment in Salmo salar Previously Immunized with the Purified Polypeptide MY32 and Infested with Lepeophtheirus salmonis 
Two hundred salmons with an average body weight of 35 g were distributed into two groups of 100 fishes each one. An oral formulation was prepared mixing the purified MY32 polypeptide with salmon basal diet, at the dose of 1 μg/g of body weight. Negative control group was fed of basal diet alone. Fishes were fed once a day for 5 days with:

    
    
        A. Feed containing purified MY32 protein at the dose of 0.5 μg/g of feed
        B. Feed containing purified MY32 protein at the dose of 1 μg/g of feed
        C. Negative control: Commercial feed
    
    


Afterwards, fishes were infested with an average of 90-95 sea lice per fish, approximately. The parasites were counted weekly for 6 weeks. It was observed a reduction in a number of parasites per fish in oral vaccinated groups as compared to negative control group (FIG. 3). At weeks 4 and 8, sera were collected and evaluated by ELISA for IgM against MY32. High antibody titers were found.
Example 8
Salmon Immunization with Naked DNA of the Sequence Coding for MY32 Gene and Evaluation of Infestation by Caligus rogercresseyi 
Hundred fifty salmons (Salmo coho) of 35 g in average weight were distributed into three groups of 50 fishes each one. Fishes were immunized with an intramuscular injection of 25 μg of the plasmid DNA containing the gene my32 from C. rogercresseyi (group A) or 25 μg of the plasmid DNA containing the gene my32 combined with the encoded polypeptide at a concentration of 0.5 μg/g of body weight (group B). Negative control fishes were immunized with the plasmid backbone vector (group C). Fishes from groups A and B showed higher IgM titer as compared to the negative control group, at weeks 4 and 7 post vaccination. After 4 weeks, fishes infested with an average of 100 sea lice per fish were incorporated to the tanks. The experiment was followed by counting the number of parasites for 12 weeks. The results showed that groups A and B had lower number of parasites as compared to group C. The reduction in the infestation levels was 70% for group A (naked DNA) and 95% for group B (naked DNA+polypeptide) as compared to negative control group (FIG. 4).
Example 9
Challenge Experiment in Salmons Salmo salar Previously Immunized by Immersion Bath and Infested with C. rogercresseyi 
Hundred fifty salmons of 30 g as average weight were distributed into three groups of 50 fishes each one. For immersion bath, the water level in the tanks was decreased. The plasmid pVAXmy32 was administered to one group. Another group received the purified protein and a control group received PBS. The immersion bath treatment was done for 90 min. The experimental groups were:

    
    
        A: Plasmid pVAXmy32 at the dose of 0.01 mg/L of water.
        B: Purified MY32 protein at the dose of 0.1 mg/L of water
        C: Negative control group: PBS
    
    


After three weeks post vaccination, salmon were extensively infested with an average of 90-95 sea lice (C. rogercresseyi) per fish, approximately. The experiment was conducted for 12 weeks and showed that fishes immunized with my32 gene or MY32 polypeptide had a lower number of parasites, as compared to negative control group (FIG. 5).
Example 10
Challenge Experiment, with Infestation by Caligus rogercresseyi of Salmo salar Previously Immunized by Intraperitoneal Injection with Purified MY32 Polypeptide and the MY32-Promiscuous T Cell Epitopes Fusion Proteins
Five hundred salmons of 35 g of weight were distributed into 5 groups of 100 fishes per group. Fishes were intraperitoneally injected at week 0. The experimental groups were:
Group 1: MY32 protein adjuvated in Montanide 888, at the dose of 1 μg/g of body weight. Group 2: Purified MY32-tetanus toxoid ttP2 promiscuous T cell epitope (830-844QYIKANSKFIGITEL (SEQ ID NO: 14); GenBank X04436) protein adjuvated in oil adjuvant Montanide 888 (1 μg/ g of body weight). Group 3: Purified MY32-measles virus promiscuous T cell epitope (288-302 LSEIKGVIVHRLEGV (SEQ ID NO: 15); GenBank M81903) protein adjuvated in oil adjuvant Montanide 888 (1 μg/g of body weight). Group 4: Purified MY32-ttP2-measles virus promiscuous T cell epitopes protein adjuvated in oil adjuvant Montanide 888(1 μg/g of body weight). Group 5: Negative control group was injected with PBS in oil adjuvant.
Two weeks later, the fishes were infested with an average of 100 sea lice per fish at copepodid stage. On day 60, it was found a reduction of the number of parasites per fish in more than 95% (Table 2) in the group injected with the purified MY32-ttP2-measles virus promiscuous T cell epitopes as compared to negative control group. Sera from the animals were collected at weeks 0, 4 and 8 and evaluated for IgM against MY32 by ELISA. High antibody titers were found at weeks 4 and 8 (data not shown) in all the groups immunized with the protein or the protein fused to promiscuous T cell epitopes. The highest titer was found in the group vaccinated with MY32-ttP2-measles virus promiscuous T cell epitopes.







TABLE 2







Effect of the intraperitoneal injection of purified MY32 protein and the


fusion protein MY32-promiscuous T cell epitopes, over the number of


parasites per fish, at days 28 and 60 after injection.










Average number




of parasites per fish









Experimental group
Day 28
Day 60





Group 1
50 ± 5
20 ± 3 


Group 2
30 ± 3
8 ± 2


Group 3
32 ± 6
9 ± 3


Group 4
15 ± 2
3 ± 1


Group 5
92 ± 7
90 ± 8 









Example 11
Isolation and Cloning of the MY32 Gene from Lepeophtheirus salmonis 
The coding sequence for the MY32 polypeptide was obtained by PCR from cDNA of Lepeophtheirus salmonis (my32-Ls) (samples obtained from two different geographical regions), using degenerate oligonucleotides designed from previously reported sequences for subolesin/akirin in different arthropods.
The oligonucleotides were:








Oligonucleotide that hybridyze with 5′ fragment:



(SEQ ID NO: 8)



ATG GC(T/C) TG(T/C) GC(T/C/G/A) AC(T/C/A/G) (T/C)T(T/C) AA(A/G)



 


(SEQ ID NO: 9)



ATG GC(T/C) TG(T/C) GC(T/C/G/A) AC(T/C/A/G) (T/C)T(A/G) AA(A/G)



 


(SEQ ID NO: 10)



ATG GC(G/A) TG(T/C) GC(T/C/G/A) AC(T/C/A/G) (T/C)T(T/C) AA(A/G)



 


(SEQ ID NO: 11)



ATG GC(G/A) TG(T/C) GC(T/C/G/A) AC(T/C/A/G) (T/C)T(A/G) AA(A/G)



 


Oligonucleotide that hybridyze with 3′ fragment:


(SEQ ID NO: 12)



TT(A/C)AC(A/G)AA(A/C/G/T)G(T/C)(A/G)TC(A/G)TA(C/T)TG(C/T)TC



 


(SEQ ID NO: 13)



TT(G/T)AC(A/G)AA(A/C/G/T)G(T/C)(A/G)TC(A/G)TA(C/T)TG(C/T)TC







The characterization of 5′ and 3′ ends of my32-Ls was carried out by RACE.
The amplified DNA was cloned into a comercial pGEM Teasy vector (Promega) and sequenced.
As a result, we obtained two cDNA sequences (SEQ ID No: 3 and SEQ ID No: 5). They share 99% of the amino acid sequence. There is a change between the sequences at amino acid 132 (C instead Y) produced as a consequence of a nucleotide change at position 395 (G instead A), corroborated by different PCR reactions.
Example 12
Construction of E. coli Expression Vector Containing the Coding Sequence for MY32-Ls Polypeptide and Protein Expression
For the expression of my32-Ls cDNA in E. coli, the coding regions of SEQ ID No: 3 and SEQ ID No: 5 were amplified from plasmid DNA by PCR using specific primers. These primers contain Nco I and Hind III restriction sites, to insert the amplified fragments into the corresponding cloning sites in the pET28a expression vector (Novagen). Additionally, to obtain a polypeptide with a deletion in the last 21 amino acids in the C-terminal end, a PCR was performed using SEQ ID No: 3 as template. The amplified band lacks the last 63 bp.
Another construction was made to fuse promiscuous T cell epitopes ttP2 (tetanus toxoid, 830-844QYIKANSKFIGITEL (SEQ ID NO: 14); GenBank X04436) and MV (measles virus, 288-302 LSEIKGVIVHRLEGV (SEQ ID NO: 15); GenBank M81903) to SEQ ID No: 3. The T cell epitopes were amplified by PCR from a synthetic gene with specific primers containing Nco I-Bam H I restriction sites and cloned into the corresponding sites in the pET28a expression vector. The SEQ ID No: 3 was amplified with primers containing Bam H I- Hind III restrictions sites into the same restriction sites of pET28a.
The final vectors (pET28a-my32-Ls) were tested by restriction endonuclease site analysis and DNA sequencing. In the construct, the inserted gene was under the control of the inducible T7 promoter and yielded the polypeptide with a C-terminal fusion His tail. For expression of the recombinant polypeptides, the expression plasmids were transformed into E. coli BL21(DE3). Single clones of BL21(DE3) transformed with pET28a-my32 and pET28a plasmids, respectively, were grown overnight at 37° C. in Luria Bertani medium containing 50 μg/mL of kanamycin. Cultures were then diluted (1:20) in fresh LB medium and grown at 37° C. until the optical density at 600 nm reached approximately 0.5. The expression of recombinant proteins was initiated by the addition of IPTG to a final concentration of 0.5 mM and incubation continued during 5 h for induction of recombinant protein expression. After induction, the bacterial cells were harvested by centrifugation at 10,000×g for 10 min at 4° C. The inclusion bodies were solubilized and the protein purified by affinity chromatography to metal ions employing Ni-NTA (Quiagen) and obtained 95% pure. As a result, we obtained four different polypeptides: my32-Ls1 (SEQ ID No: 4 fused to His tail), my32-Ls2 (SEQ ID No: 6 fused to His tail), my32-Ls3 (SEQ ID No: 7 fused to His tail) and my32-Ls4 (SEQ ID No: 4 fused to T cell epitopes ttP2-MV at the N-terminal.
Example 13
Construction of MY32-Ls DNA Expression Vector in Eukaryotic Cells for Naked DNA Vaccine
The gene my32-Ls cDNA (SEQ ID No: 3) was excised from pGEM T easy (Promega) by digestion with EcoR I. The insert was purified by QiaQuick kit (Qiagen, Hamburg, Germany) and ligated to pVAX vector (Invitrogen), previously digested with EcoR I. The recombinant DNA employed in immunization experiments was purified by Endo Free Plasmid Mega kit (Qiagen, Hamburg, Germany).
Example 14
Use of MY32 from L. salmonis in a Vaccination/Challenge Experiment in Salmo salar 
Sixty salmons with an average weight of 80 g were distributed in 2 groups of 30 fish each. The vaccine group was injected intraperitoneally with my32-Ls1 (SEQ ID No: 4) at a dose of 3 μg/g of body weight of salmon, formulated in an oil adjuvant. The negative control group was immunized the vehicle buffer adjuvated. After 500 arbitrary thermal units, the salmons were adapted to seawater in a single tank and they were infested with 2000±200 copepodids. The challenge was carried out under dark conditions, constant aeration, support oxygenation, temperature (15-17° C.) and salinity (approx. 30 ppm) control. In order to avoid loss of copepodids and facilitate their attachment, the flow of water in the pond was closed and the replacement was done manually every 48 hours. In addition, 220μ sieves were installed in the sewer. After copepodids settlement, the fish were distributed in two replicates tanks per groups of 15 fish each tank. At day 40, the fishes were sacrificed by an anesthesia overdose, and the evaluation of the results proceeded by parasites counting. The results in the Table 3 show a significant decrease in the number of parasites per fish in the groups vaccinated compared to negative controls.







TABLE 3







Parasite count results at the end of the challenge experiment.












Vaccinated






group
Vaccinated group


Parameters
1-my32Ls1
2-my32Ls1
Control 1
Control 2





# of
12 ± 6a
10 ± 6a
40 ± 9b
37 ± 10b


parasites/fish


% Infestation
69
74
—
—


inhibition





Different letters indicate significant differences (p < 0.05)






Example 15
Use of Three Different MY32-Ls Protein Sequences from L. salmonis in a Vaccination/Challenge Experiment in Salmo salar 
Hundred twenty salmons with an average weight of 80 g were distributed in 4 groups of 30 fish each. The experimental groups were:

    Group 1: Injected intraperitoneally with my32-Ls1 (SEQ ID No: 4 fused to His tail) at a dose of 3 μg/g body weight of salmon, formulated in an oil adjuvant.
    Group 2: Injected intraperitoneally with my32-Ls2 (SEQ ID No: 6 fused to His tail) at a dose of 3 μg/g body weight of salmon, formulated in an oil adjuvant.
    Group 3: Injected intraperitoneally with my32-Ls3 (SEQ ID No: 7 fused to His tail) at a dose of 3 μg/g body weight of salmon, formulated in an oil adjuvant.
    Group 4: Negative control group, immunized with the vehicle buffer adjuvated in an oil adjuvant.


After 500 arbitrary thermal units, the salmons were adapted to seawater in a single tank and they were infested with 2000±200 copepodids. The challenge was carried out as described above, but without replicate tanks. At day 40, the fishes were sacrificed by an anesthesia overdose, and the evaluation of the results proceeded by parasites counting. The results in the Table 4 show a significant decrease (67-77%) in the number of parasites per fish in all the vaccinated groups compared to negative control.







TABLE 4







Parasite count results at the end of the challenge experiment.












Vaccinated
Vaccinated
Vaccinated



Parameters
group 1
group 2
group 3
Control





# of parasites/fish
7 ± 3a
10 ± 5a
8 ± 2a
30 ± 9b


% Infestation
77%
67%
73%
—


inhibition





Different letters indicate significant differences (p < 0.05)






Example 16
Salmon Immunizations with Naked DNA of the Sequence Coding for MY32-Ls Gene and Evaluation of Infestation by L. salmonis 
Salmo salar of 35 g in average weight were distributed into three groups of 60 fishes each one. Fishes were immunized with an intramuscular injection of 25 μg of the plasmid DNA containing the gene my32 from L. salmonis (group 1) or 25 μg of the plasmid DNA containing the gene my32-Ls combined with the encoded polypeptide at a concentration of 0.5 μg/g of body weight (group 2). Negative control fishes were immunized with the plasmid backbone vector (group 3). The challenge was performed as described in Example 4. The experiment was followed for 12 weeks. The results showed that groups 1 and 2 had lower number of parasites as compared to group 3. The reduction in the infestation levels was 67% for group 1 and 86% for group 2 as compare to negative control group (Table 5).







TABLE 5







Parasite count results at the end of the challenge experiment.












Parameters
Group 1
Group 2
Group 3







# of parasites/fish
12 ± 4a
5 ± 2a
36 ± 8b



% Infestation
67
86
—



inhibition







Different letters indicate significant differences (p < 0.05)






Example 17
Challenge Experiment, with Infestation by L. salmonis of Salmo salar Previously Immunized by Intraperitoneal Injection with Purified MY32 Polypeptide and the MY32-Promiscuous T Cell Epitopes Fusion Proteins
Two hundred fifty salmons of 50 g of weight were distributed into 5 groups of 50 fish per group. Fishes were intraperitoneally injected at week 0.
The experimental groups were:

    Group 1: MY32-Ls1 adjuvated in Montanide 888, at the dose of 3 μg/g of body weight.
    Group 2: MY32-Ls4 adjuvated in oil adjuvant Montanide 888 (3 μg/g of body weight).
    Group 3: Negative control group was injected with PBS in oil adjuvant.


The challenge was performed as described in Example 14. On day 60 after challenge, it was found a reduction of 92% in the number of parasites per fish (Table 6) in the group injected with the purified MY32-ttP2-measles virus promiscuous T cell epitopes as compared to negative control group.







TABLE 6







Effect of the intraperitoneal injection of purified MY32-Ls and the fusion


protein MY32-Ls-promiscuous T cell epitopes, over the number of


parasites per fish, at day 60 after challenge.












Parameters
Group 1
Group 2
Group 3







# of parasites/fish
16 ± 6a
4 ± 2a
50 ± 8b



% Infestation
68
92
—



inhibition







Different letters indicate significant differences (p < 0.05)

## Claims

1. An immunogenic composition comprising the polypeptide identified as SEQ ID NO: 4 formulated in a suitable adjuvant.

2. A method for inducing an immune response in a salmonid against sea lice, and/or reducing the number of sea lice in the salmonid, said method comprising administering an effective amount of a composition comprising the polypeptide identified as SEQ ID NO: 4 to the salmonid.

3. The method according to claim 2, wherein the composition is administered by injection at doses ranging between 0.1-10 μg/g of body weight of the salmonid.

4. The method according to claim 2, wherein the composition is administered in feed formulations at doses ranging between 0.1-300 μg/g of feed.

5. The method of claim 2, wherein the composition is administered by immersion baths at doses ranging between 0.01-1 mg/l of water.

6. A fusion polypeptide comprising an isolated polypeptide consisting of the amino acid sequence identified as SEQ ID NO: 4 and a peptide consisting of a promiscuous T cell epitope of viral or bacterial origin.

7. The fusion polypeptide of claim 6, wherein said promiscuous T cell epitope is SEQ ID NO: 14 or SEQ ID NO: 15 or a combination thereof.