Title:  DNA based vaccination of fish

United States Patent:  6,180,614

Assignee:  Loeb Health Research Institute at The Ottawa Hospital (Ottawa, CA)

Filed:  July 14, 1998

The present invention relates to methods of immunization of aquaculture species by introducing DNA expression systems into the aquaculture species. Such DNA expression systems preferably include DNA sequences encoding polypeptides of pathogens of species of aquaculture. The present invention also relates to methods of administration of DNA expression systems into aquaculture. Such methods include injection, spray, and immersion techniques. The methods of this invention are useful for prophylactic vaccination or therapeutic immunization of fin-fish, shellfish, or other aquatic animals against infectious diseases.

DESCRIPTION OF THE INVENTION

The present invention provides for methods and compositions for immunizing cultured fin-fish, shellfish, and other aquatic animals against infection by viral, bacterial or parasitic pathogens. In basic outline, DNA encoding a polypeptide component of a pathogen is introduced into an animal, and the polypeptide is expressed in cells of the animal, thus inducing an immune response that confers protection against natural infection by the pathogen or helps overcome an ongoing and possibly chronic infection.

In a preferred embodiment, the present invention provides a method for immunizing cultured fin-fish, shellfish, or other aquatic animals against disease, comprising immersion of the animals in an aqueous solution containing formulated plasmid DNA encoding one or more antigenic determinants of an infectious agent (regardless of codon usage), whereby the DNA enters cells of the animal where it is expressed leading to induction of immune responses. The immunization procedure may be prophylactic to prevent infection from occurring or may be therapeutic to treat pre-existing infections.

Few anti-viral vaccines have been marketed for fish. This is largely due to the difficulty of growing virus in culture for the production of whole killed viral vaccines or safe attenuated strains of virus. Antigen-based vaccines using purified recombinant proteins are difficult and expensive to produce in large scale and may have poor immunogenicity in fish.

DNA-based immunization has several advantages. The antigenic protein is synthesized in vivo giving rise to both humoral and cell-mediated (cytotoxic T lymphocytes) immune responses. However, unlike live attenuated pathogens, which also synthesize protein in vivo, DNA vaccines carry no risk of inadvertent infection. Unlike antigen-based immunization, DNA-based vaccination does not require the use of traditional adjuvants to generate an effective immune response. Furthermore, DNA used in the methods of this invention is inexpensive and easy to manufacture and purify.

DNA-based immunization also allows the host animal to produce foreign antigens within its own tissue thereby resulting in several advantages. One advantage is the efficient presentation of the foreign antigen to the immune system due to the expression of a protein within a self-cell, which could be an antigen-presenting cell. Another advantage is the correct folding, protein modification, and disulfide bonding of a protein expressed in a host cell, especially for viral proteins, which are normally produced in cells of hosts. Recombinant viral proteins synthesized in bacterial or yeast cells may be incorrectly post-translationally modified and are often massed in inclusion bodies, which make the proteins difficult to purify or ineffective if administered in unpurified form.

Immune responses in fish are temperature dependent. Antigen-based vaccines may give rise to sub-optimal immune responses if such vaccines are given at the wrong temperature. DNA-based immunization is advantageous because expression of the antigenic protein could continue over a long period until such time as to stimulate an immune response when the temperature is optimal.

Another advantage of prolonged synthesis of antigen is the induction of immune responses as soon as the immune system is mature. Fish may be unable to induce sufficient immune responses at a young age. For example, trout and halibut may not produce lymphoid cells until as late as ten and thirty days after hatching, respectively, and T-dependent immune responses do not appear until months after hatching. Using the methods of this invention, expression of foreign protein in fish can continue at least four months after transfection indicating that DNA-based immunization may be preferred for vaccination of young fish.

The term "vaccine" herein refers to a material capable of producing an immune response. A vaccine according to this invention would produce immunity against disease in cultured fin-fish, shellfish and other aquatic species. One of skill in the art would readily appreciate that activation of CTL activity resulting from in vivo synthesis of antigen would produce immunity against disease not only prophylactically but also therapeutically (after development of disease in culture).

Aquaculture species treated by methods of this invention will include a diversity of species of cultured fin-fish, shellfish, and other aquatic animals. Fin-fish include all vertebrate fish, which may be bony or cartilaginous fish. A preferred embodiment of this invention is the immunization of fin-fish. These fin-fish include but are not limited to salmonids, carp, catfish, yellowtail, seabream, and seabass. Salmonids are a family of fin-fish which include trout (including rainbow trout), salmon, and Arctic char. Examples of shellfish include, but are not limited to, clams, lobster, shrimp, crab, and oysters. Other cultured aquatic animals include, but are not limited to eels, squid, and octopi.

Purification of DNA on a large scale may be accomplished by anion exchange chromatography (for example, resins manufactured by Qiagen, U.S. FDA Drug Master File (DMF-6224)).

DNA which is introduced to aquaculture species will encode foreign polypeptides (e.g., those derived from viral, bacterial or parasitic pathogens). Polypeptides of this invention refer to complete proteins or fragments thereof, including peptides which are epitopes (e.g., a CTL epitope) associated with an infectious virus, bacterium or parasite.

DNA sequences encoding a complete or large parts of an antigenic protein are preferred where humoral immunity is desired rather than DNA sequences encoding smaller parts, such as only CTL epitopes, as are preferred where cell-mediated immunity is desired and humoral immunity may be deleterious. In preferred embodiments, the DNA sequences encoding polypeptides of viral pathogens may be selected from the group consisting of glycoprotein (G) or nucleoprotein (N) of viral hemorrhagic septicemia virus (VHSV); G or N proteins of infectious hematopoietic necrosis virus (IHNV); VP1, VP2, VP3 or N structural proteins of infectious pancreatic necrosis virus (IPNV); G protein of spring viremia of carp (SVC); and a membrane-associated protein, tegumin or capsid protein or glycoprotein of channel catfish virus (CCV).

In other preferred embodiments, the DNA sequences encoding polypeptides of bacterial pathogens may be selected from the group consisting of an iron-regulated outer membrane protein, (IROMP), an outer membrane protein (OMP), and an A-protein of Aeromonis salmonicida which causes furunculosis, p57 protein of Renibacterium salmoninarum which causes bacterial kidney disease (BKD), major surface associated antigen (msa), a surface expressed cytotoxin (mpr), a surface expressed hemolysin (ish), and a flagellar antigen of Yersiniosis; an extracellular protein (ECP), an iron-regulated outer membrane protein (IROMP), and a structural protein of Pasteurellosis; an OMP and a flagellar protein of Vibrosis anguillarum and V. ordalli; a flagellar protein, an OMP protein, aroA, and purA of Edwardsiellosis ictaluri and E. tarda; and surface antigen of Ichthyophthirius; and a structural and regulatory protein of Cytophaga columnari; and a structural and regulatory protein of Rickettsia.

In yet another preferred embodiment, the DNA sequences encoding polypeptides of a parasitic pathogen may be selected from one of the surface antigens of Ichthyophthirius.

The methods of this invention could also be used to introduce plasmid vectors encoding polypeptides endogenous to the animal, but which might be normally present in low concentrations (e.g., growth hormones). In this case the expression proteins would serve a physiological role (i.e. enhanced growth) rather than induce an immune response.

Vectors useful in the making of expression plasmids include, but are not limited to, vectors containing constitutive promoters, inducible promoters, tissue-specific promoters, or promoters from the gene of the antigen being expressed. Constitutive promoters may include strong viral promoters, for example, promoter sequences from cytomegalovirus (CMV), Rous sarcoma virus (RSV), simian virus-40 (SV40), or herpes simplex virus (HSV). Tissue-specific promoters may include the muscle beta-actin promoter or the thymidine kinase promoter. An inducible or regulatable promoter, for example, may include a growth hormone regulatable promoter, a promoter under the control of lac operon sequences or an antibiotic inducible promoter or a Zinc-inducible metallothionein promoter.

The vector should include an expression control sequence comprising a promoter (e.g., inducible or constitutive promoters described above) DNA sequence, and may include, but is not limited to, an enhancer element, an RNA processing sequence such as an intronic sequence for splicing of a transcript or a polyadenylation signal (e.g., from simian virus-40 (SV40) or bovine growth hormone (BGH)), a signal sequence for secretion of the expressed protein, or one or more copies of immunostimulatory DNA sequences known as CpG motifs. The vector should also include one or more of the following DNA sequences: bacterial origin of replication sequences, a selectable marker, which may be for antibiotic resistance (e.g., kanamycin) or for non-antibiotic resistance (e.g., .beta.-galactosidase gene).

Oligonucleotides having unmethylated CpG dinucleotides have been shown to activate the immune system (A. Krieg, et al., "CpG motifs in Bacterial DNA Trigger Directed B Cell Activation" Nature 374:546-549 (1995)). Depending on the flanking sequences, certain CpG motifs may be more immunostimulatory for B cell or T cell responses, and preferentially stimulate certain species. Copies of CpG motifs in DNA expression vectors act as adjuvants facilitating the induction of an immune response against an expressed protein. A CpG motif, a stretch of DNA containing CpG dinucleotides within a specified sequence, may be as short as 5-40 base pairs in length. Multiple CpG motifs may be inserted into the non-coding region of the expression vector. When a humoral response is desired, preferred CpG motifs will be those that preferentially stimulate a B cell response. When cell-mediated immunity is desired, preferred CpG motifs will be those that stimulate secretion of cytokines known to facilitate a CD8+ T cell response.

Other CpG motifs have be found to inhibit immune responses. In a preferred embodiment of the application, these immunoinhibitory CpG motifs would be removed or mutated in a DNA expression vector used by the methods of this invention, without disrupting the expression of polypeptides therefrom.

An additional preferred embodiment of this invention relates to the administration of a vector containing one or more different DNA sequences, one sequence encoding an antigen and the others encoding polypeptides which may or may not be antigenic. For example, the vector may encode two antigens from the same pathogen. Alternatively, the different antigen(s) may induce an immune response against a different pathogen and thus serve as a multivalent vaccine. Alternatively, the other polypeptides may serve to enhance an immune response against a targeted pathogen (e.g., helper epitopes, cytokines, carrier polypeptides, cholera toxin subunits, or other immunostimulants).

When two or more polypeptide-encoding DNA sequences are present in one vector, the transcription of each antigen-encoding DNA sequence may be directed from its own promoter. Alternatively, one promoter may drive the expression of two or more antigen-encoding DNA sequences joined in frame to each other to express a fusion protein. For example, VP2 and VP3 proteins of infectious pancreatic necrosis virus (IPNV) may be fused. In another embodiment, DNA sequences encoding two or more antigens from different diseases may be joined to form a multivalent vaccine when expressed.

Alternatively, a DNA sequence encoding an antigenic polypeptide may be fused to a DNA sequence encoding a carrier polypeptide. In a preferred embodiment, the carrier polypeptide may contain one or more envelope proteins of the hepatitis B virus, preferably from the human hepatitis B virus. In a more preferred embodiment, the envelope proteins of hepatitis B virus will be the small and major protein (also referred to as surface antigen).

In another embodiment, each polypeptide-encoding DNA sequence in the vector may be under the control of its own promoter for expression of two or more non-fused polypeptides.

Alternatively, the DNA sequences encoding additional antigens may be administered by using a second vector containing such sequences. Such sequences may encode antigens from the same pathogen or different pathogens, or cytokines, cholera toxin subunits, or other immunostimulants. Such a vector may be administered concurrently or sequentially with the first expression vector. A preferred embodiment of this invention is the concurrent administration of expression vectors. One vector may be induced to express protein simultaneously with or after expression of protein from the other vector.

In yet another embodiment of this invention, antigen-expressing vectors may be administered concurrently with an antigen-based vaccine such as a recombinant protein or whole-killed vaccine. In a preferred embodiment, the antigen-expressing vector is administered simultaneously with a protein antigen (i.e. recombinant protein or whole killed pathogen). Another preferred embodiment would be to first administer a DNA vaccine to prime the immune response followed by administration of the protein antigen two to eight weeks later, preferably orally or by immersion, to boost the immune response.

The DNA used in the method of this invention is preferably purified plasmid DNA(s) simply dissolved in an aqueous solution or in a formulation. One of skill in the art would readily appreciate how to formulate DNA used in the methods of this invention with known transfection reagents such as cationic liposomes, fluorocarbon emulsions, cochleates, tubules, gold particles, biodegradable microspheres, or cationic polymers.

Liposomes useful for transfection of DNA of this invention include commercially available liposomes and liposomes containing either cationic lipids or cationic polymers. In a preferred embodiment of this invention, liposomes would include a mixture of a neutral lipid such as dioleoylphosphatidylethanolamine (DOPE) or cholesterol and a cationic lipid.

In a more preferred aspect of the invention, liposomes would include a mixture of cationic polymers and neutral lipids such as DOPE or cholesterol. Such liposomes may be prepared as described herein and in United States Provisional Patent Application entitled, "A Novel Class of Cationic Reagents for High Efficient and Cell-Type-Specific Introduction of Nucleic Acids into Eukaryotic Cells", incorporated by reference herein. Unlike cationic lipids, cationic polymers do not have ester-linkages and have greater stability in vivo as a result. Cationic polymers (also referred to as dendrimers) may be dimeric, cyclic, oligomeric, or polymeric in structure.

Cationic polymers in an aqueous solution without neutral lipids are also preferred transfection reagents according to the preferred embodiments of this invention. Cationic polymers have been shown to work well for transfecting fish cells in vitro with plasmids expressing fish pathogen antigens (see Table 1, Example 1).

Cochleates, which are stable phospholipid-calcium precipitates composed of phosphatidylserine, cholesterol and calcium are desirable non-toxic and non-inflammatory transfection reagents that can survive the digestive system. Biodegradable microspheres composed of polymers such as polyester poly(lactide-co-glycolide) have been used to microencapsulate DNA for transfection.

Tubules have been previously described in the literature as lipid-based microcylinders consisting of helically wrapped bilayers of lipid, the edges of which are packed together. DNA may be placed in the hollow center for delivery and controlled release in animals.

With immersion, DNA may enter cells of the epithelium of the skin, the gills or the gut wall. With injection, DNA may enter muscle cells or other cells in muscle tissue (e.g. fibroblasts, immune cells) or cells of viscera within the intraperitoneal cavity. DNA may then be expressed in these transfected cells leading to induction of appropriate immune responses in regional or systemic lymphoid tissue.

The invention provides for pharmaceutical compositions comprising DNA vaccines in an amount effective for the treatment and prevention of diseases caused by pathogens of aquaculture species. According to another embodiment, the pharmaceutical compositions of this invention further comprise a second DNA vaccine, an adjuvant, a recombinant protein, a transfection reagent, or some combination thereof.

Methods of this invention may be useful in the immunization of aquaculture species against many pathogens. Such pathogens include but are not limited to hemmorrhagic septicemia virus, infectious hematopoietic necrosis virus, infectious pancreatic necrosis virus, virus causing spring viremia of carp, channel catfish virus (Herpesvirus ictaluri), grass carp hemorrhagic virus, nodaviridae such as nervous necrosis virus or striped jack nervous necrosis virus, infectious salmon anaemia virus, Aeromonis salmonicida, Renibacterium salmoninarum, Yersinia, Pasteurella (including piscicida), Vibrosis (including anguillarum and ordalii), Edwardsiella (including ictaluri and tarda), Streptococci, and Ichthyophthirius.

In one embodiment of this invention, recombinant plasmid DNA is introduced into animals orally. DNA for oral use may be formulated with biodegradable microspheres, fluorocarbon emulsions, cochleates, or tubules. This is a non-stressful method of immunizing aquaculture species by which DNA may be coated onto or milled into feed in the form of a paste or liquid suspension or incorporated into gelatin capsules and introduced into the environment of the aquaculture species. Preparations of DNA for oral use may include lactose and corn starch. The DNA can be used with or without products to enhance entry into cells of the gut epithelium or more deeply situated cells.

In another embodiment, pure recombinant plasmid DNA is introduced into animals by injection with a needle or a jet-injection system, which does not have a needle. Injection areas of the fin-fish include but are not limited to intraperitoneal, intramuscular, and subcutaneous areas of the fish. In a preferred embodiment, large fin-fish are immunized by injection methods of this invention. Typically, fish are injected with 0.1-0.5 ml of a solution containing DNA. DNA may be injected in a pure form or may be formulated with liposomes, cationic polymers, fluorocarbon emulsions, cochleates, or tubules.

In yet another embodiment of this invention, pure DNA is introduced into a fin-fish by particle bombardment. This method introduces DNA-coated gold particles into the epidermis of a fin-fish using a "gene-gun", which uses compressed helium to shoot the gold particles at high speed into the skin. This method has been shown to be particularly efficient for induction of cell-mediated immune responses with small quantities of DNA in mice.

In another embodiment of this invention, plasmid DNA is introduced to fish by spray. Typically, fish are exposed to spray for at least 2 seconds. Fish may pass through a mist of DNA solution by forcing the vaccine through high-pressure paint-sprayer-type nozzles. Typically, any pressure up to 90 psi is satisfactory. Due to the number of pounds of fish per unit volume that can be vaccinated by spray, it may be more economical to immunize larger fish by this method than by immersion. The DNA can be used with or without products to enhance entry into cells of the skin. For example, the DNA may be associated with liposomes or cationic polymers.

In a more preferred embodiment of this invention, a large number of animals can be immunized simultaneously by immersion in a solution containing DNA. In one embodiment, fish are dip-netted into suspensions containing DNA formulations (e.g., DNA formulated with cationic polymers or liposomes) for at least several seconds. The fish are then returned to the holding tanks in which they develop immunity. In another embodiment, fin-fish, shellfish, or other aquatic animals are placed into tanks containing a relatively small volume of water. Concentrated DNA formulations (e.g., DNA formulated with cationic polymers or liposomes) is added to the tank, and animals are left for a period of time up to several hours before the tank is refilled with water to restore the normal aquatic environment. This method of immersion is preferred for the immunization of small fry, which cannot be immunized by direct injection.

The amount of the expression plasmid DNA that may be combined with a carrier material to produce a single dosage form will vary depending upon the host treated, and the particular mode of administration. It should be understood, however, that a specific dosage and treatment regimen for any particular fish will depend upon a variety of factors, including the expression of the particular plasmid DNA employed, the stability and activity of the particular protein or peptide expressed, age, body weight, general health, species of fish, the progress of the disease being treated, and nature of the disease being immunized against or dreaded. The amount of expression plasmid DNA may also depend upon whether other therapeutic or prophylactic agents including additional expression plasmid DNAs and adjuvants, if any, are co-administered with the expression plasmid.

Without being bound by the values listed below, dose ranges for the administration of DNA used in the methods of this invention may be generalized as follows. For immunization of fish via oral routes, 0.1 to 50 .mu.g DNA per fish administered over several consecutive days may be used. For DNA-based immunization by intramuscular or intraperitoneal injection, 0.1 to 10 .mu.g of DNA may be used. For spray immunization, a volume of 1 ml per fish of 0.1 to 10 mg/ml DNA solution may be useful. Fish immunized by immersion methods of this invention may be incubated in a 1 to 100 .mu.g/ml DNA solution at a volume sufficient for fish to survive for a time period necessary for uptake of DNA to produce an immune response by the fish. An effective dosage range for immunization of fish via gene-gun route may be 10 ng to 1 .mu.g.

Adjuvants for immunization are well known in the art and suitable adjuvants can be combined with the DNA sequences described herein by a person skilled in the art to form a pharmaceutical composition. Oil adjuvants are least desirable for the methods of this invention because they create undesirable side-effects such as visceral adhesions (which can restrict growth) and melanized granuloma formations (which can lower the grade of the fish at market) and because they cannot form a homogeneous mixture with DNA preparations. DNA-based immunization does not require oil adjuvants and thus avoids these undesirable effects.

Adjuvants used in immunization with DNA expression plasmids of this invention may include alum or a DNA molecule having unmethylated CpG dinucleotides therein (also referred to as CpG adjuvant). Oligonucleotides having unmethylated CpG dinucleotides have been shown to activate the immune system (A. Krieg, et al., "CpG motifs in Bacterial DNA Trigger Directed B Cell Activation" Nature 374:546-549 (1995)). CpG motifs may be inserted into a plasmid DNA vaccine vector, and replicated in bacteria thereby allowing the CpG motifs to retain their unmethylated form. As such, administration of a CpG adjuvant cloned into plasmid vectors would be simultaneous with the administration of a plasmid DNA vaccine. Alternatively, a CpG adjuvant in the form of free oligonucleotides may be administered before, during or after the administration of a plasmid DNA vaccine.

Oligonucleotides having CpG motifs may be optionally modified at their phosphodiester linkages for stability purposes. Such modifications are well known by those of skill in the art. For example, phosphodiester bonds in an oligonucleotide may be replaced by phosphorothioate linkages.

The present invention also includes pharmaceutical products for all of the uses contemplated in the methods described herein. For example, a pharmaceutical product comprising pure plasmid DNA vector or formulations thereof, operatively coding for an immunogenic polypeptide or peptide, may be prepared in physiologically acceptable administrable form (e.g., saline). The pharmaceutical product may be placed in a container, with a notice associated with the container in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the DNA for veterinary administration. Such notice, for example, may be labeling approved by the Biologics Division of Agriculture and Agri-Food Canada or the United States Department of Agriculture (USDA) or the approved product insert.

Claim 1 of 84 Claims

What is claimed is:

1. A composition for inducing an immune response in an aquaculture species, comprising:

an expression vector having an expression control sequence capable of directing expression in an aquaculture species of at least one immunogenic polypeptide and a polypeptide-encoding DNA sequence encoding at least one immunogenic polypeptide from an aquaculture species pathogen.

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