The present invention relates to a method to introduce a nucleic acid molecule into a felid by administration of a nucleic acid-cationic lipid complex composition. The method includes the step of administering to the felid, by a parenteral route, a nucleic acid-cationic lipid complex to elicit and/or enhance an immune response. In one embodiment, this method enhances the immune response in a felid compared to a method in which a naked DNA vaccine is administered to a felid. Also provided is a method to deliver a nucleic acid to a felid. This method comprises parenterally administering to the felid a composition that includes a nucleic acid molecule complexed with a cationic lipid.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention relates to a method to elicit an immune response to an antigen in a felid. This method includes the step of parenterally administering to the felid a composition comprising a nucleic acid molecule encoding the antigen in which the nucleic acid molecule is complexed with a cationic lipid. In one embodiment, this method enhances the immune response in a felid compared to a method in which a naked DNA vaccine is administered to a fetid. Also provided is a method to deliver a nucleic acid molecule to a felid. This method comprises parenterally administering to the fetid a composition that includes a nucleic acid molecule complexed with a cationic lipid.

DETAILED DESCRIPTION

The present invention relates to a method to elicit an immune response to an antigen in a fetid. The method includes the step of parenterally administering to the fetid a composition comprising a nucleic acid molecule encoding the antigen in which the nucleic acid molecule is complexed with a cationic lipid. The ability of such a method to elicit an immune response to the antigen encoded by the nucleic acid molecule is new and surprising. Until recently, the general perception of those skilled in the art was that cationic lipids did not enhance the ability of a nucleic acid molecule to elicit an immune response, compared to, for example, delivery of a naked, or unformulated, nucleic acid molecule (i.e., a nucleic acid molecule that is not complexed with, for example, a lipid or other transfection-facilitating agents). Recent studies, cited above, have provided conflicting results: although two studies in mice demonstrated that cationic lipids enhanced the ability of DNA to elicit an immune response, a third study concluded that cationic lipid-complexed DNA was no better than naked DNA at eliciting an immune response. In addition, monkeys administered a nucleic acid molecule-cationic lipid complex did not exhibit seroconversion to the antigen encoded by the nucleic acid molecule. Furthermore, the inventors have demonstrated that while parenteral administration to a felid of a nucleic acid molecule complexed with a cationic lipid results in the felid successfully seroconverting in response to the antigen encoded by the nucleic acid molecule, intranasal administration of such a composition did not result in seroconversion. Thus, the ability to demonstrate seroconversion in cats parenterally administered a nucleic acid molecule complexed with a cationic lipid is completely unpredictable based on previous studies and, as such, is inventive.

One embodiment of the present invention is the use of a composition comprising a nucleic acid molecule encoding an antigen complexed with a cationic lipid to elicit an immune response in a felid. It is to be noted that the term "a" or "an" entity refers to one or more of that entity; for example, a nucleic acid molecule, an antigen, and a cationic lipid refers to one or more nucleic acid molecules, antigens, and cationic lipids, respectively; or to at least one nucleic acid molecule, antigen, and cationic lipid, respectively. As such, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein. It is also to be noted that the terms "comprising", "including", and "having" can be used interchangeably. Furthermore, a member of a group that is "selected from the group consisting of" refers to one or more of members of that group, including combinations thereof.

A nucleic acid molecule of the present invention also referred to herein as a nucleic acid, can be DNA or RNA. In one embodiment, a nucleic acid molecule encodes an antigen that elicits an immune response in a felid. As such, a nucleic acid molecule can simply be a molecule that encodes such an antigen, i.e., a coding region, or the nucleic acid molecule can comprise a coding region operatively linked to a regulatory sequence. As used herein, the phrase operatively linked refers to the joining of a coding region to one or more regulatory sequences such that the coding region is expressed using such regulatory sequence(s) in a felid. Examples of such regulatory sequences include transcription control sequences and translation control sequences that can be recognized by felid cellular mechanisms in order to effect transcription and translation of a coding region. Transcription control sequences are sequences that control the initiation, elongation, and termination of transcription (e.g., promoters, enhancers, introns, polyA sites, terminators). Translation control sequences control the initiation, elongation and termination of translation. Additional regulatory sequences include signal sequences that effect secretion of a protein from a cell and a combination of a signal sequence and a transmembrane sequence (i.e., membrane anchoring domain) that causes a protein to be partially extracellular and partially retained in the membrane and/or cytoplasm. A preferred nucleic acid molecule of the present invention is a plasmid or viral genome that includes a coding region for the desired antigen operatively linked to strong eukaryotic regulatory sequences, including a strong promoter and strong transcription termination/polyadenylation sequences. A preferred plasmid can replicate in bacteria. Procedures by which such a nucleic acid molecule is produced are known to those skilled in the art, and are disclosed, for example, in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press. Appropriate plasmids are known in the art, and may include, but are not limited to, pUC19 and BLUESCRIPT.RTM.. A preferred plasmid is pUC19. Appropriate regulatory sequences are known to those skilled in the art. For example, a suitable promoter includes, but is not limited to the cytomegalovirus immediate early promoter (CMV IE) with or without intron A, a long terminal repeat (LTR) promoter from a retrovirus, or a strong cellular promoter such as .beta.-actin, with CMV IE with intron A being preferred. Similarly, suitable transcription termination sequences include, but are not limited to, bovine growth hormone, SV40 virus or rabbit beta-globin polyadenylation sequences, with a bovine growth hormone sequence being preferred.

A suitable antigen is any antigen that effects an immune response, and as such includes allergens and autoantigens as well as other antigens. An antigen, as used herein, can refer to the full-length antigen or any portion thereof that is capable of eliciting an immune response. Preferred antigens are those that elicit an immune response that protects an animal from disease. Examples of such antigens include, but are not limited to, a protozoan parasite antigen, a helminth parasite antigen, an ectoparasite antigen, a fungal antigen, a bacterial antigen, and a viral antigen. Examples of viral antigens include, but are not limited to, antigens from adenoviruses, caliciviruses, coronaviruses, distemper viruses, hepatitis viruses, herpesviruses, immunodeficiency viruses, infectious peritonitis viruses, leukemia viruses, oncogenic viruses, papilloma viruses, parainfluenza viruses, parvoviruses, rabies viruses, and reoviruses, as well as other cancer-causing or cancer-related viruses. Examples of bacterial antigens include, but are not limited to, antigens from Actinomyces, Bacillus, Bacteroides, Bordetella, Bartonella, Borrelia, Brucella, Campylobacter, Capnocytophaga, Clostridium, Corynebacterium, Coxiella, Dermatophilus, Enterococcus, Ehrlichia, Escherichia, Francisella, Fusobacterium, Haemobartonella, Helicobacter, Klebsiella, L-form bacteria, Leptospira, Listeria, Mycobacteria, Mycoplasma, Neorickettsia, Nocardia, Pasteurella, Peptococcus, Peptostreptococcus, Proteus, Pseudomonas, Rickettsia, Rochalimaea, Salmonella, Shigella, Staphylococcus, Streptococcus, and Yersinia. Examples of fungal antigens include, but are not limited to, antigens from Absidia, Acremonium, Alternaria, Aspergillus, Basidiobolus, Bipolaris, Blastomyces, Candida, Chlamydia, Coccidioides, Conidiobolus, Cryptococcus, Curvalaria, Epidermophyton, Exophiala, Geotrichum, Histoplasma, Madurella, Malassezia, Microsporum, Moniliella, Mortierella, Mucor, Paecilomyces, Penicillium, Phialemonium, Phialophora, Prototheca, Pseudallescheria, Pseudomicrodochium, Pythium, Rhinosporidium, Rhizopus, Scolecobasidium, Sporothrix, Stemphylium, Trichophyton, Trichosporon, and Xylohypha. Example of protozoan and helminth parasite antigens include, but are not limited to, antigens from Babesia, Balantidium, Besnoitia, Cryptosporidium, Eimeria, Encephalitozoon, Entamoeba, Giardia, Hammondia, Hepatozoon, Isospora, Leishmania, Microsporidia, Neospora, Nosema, Pentatrichomonas, Plasmodium, Pneumocystis, Sarcocystis, Schistosoma, Theileria, Toxoplasma, and Trypanosoma, Acanthocheilonema, Aelurostrongylus, Ancylostoma, Angiostrongylus, Ascaris, Brugia, Bunostomum, Capillaria, Chabertia, Cooperia, Crenosoma, Dictyocaulus, Dioctophyme, Dipetalonema, Diphyllobothrium, Diplydium, Dirofilaria, Dracunculus, Enterobius, Filaroides, Haemonchus, Lagochilascaris, Loa, Mansonella, Muellerius, Nanophyetus, Necator, Nematodirus, Oesophagostomum, Onchocerca, Opisthorchis, Ostertagia, Parafilaria, Paragonimus, Parascaris, Physaloptera, Protostrongylus, Setaria, Spirocerca, Spirometra, Stephanofilaria, Strongyloides, Strongylus, Thelazia, Toxascaris, Toxocara, Trichinella, Trichostrongylus, Trichuris. Uncinaria, and Wuchereria. Examples of ectoparasite antigens include, but are not limited to, antigens (including protective antigens as well as allergens) from fleas; ticks, including hard ticks and soft ticks; flies, such as midges, mosquitos, sand flies, black flies, horse flies, horn flies, deer flies, tsetse flies, stable flies, myiasis-causing flies and biting gnats; ants; spiders, lice; mites; and true bugs, such as bed bugs and kissing bugs. Additional examples of suitable allergens include food, grass, weed, tree pollen, other animal and other plant allergens.

Preferred antigens include, but are not limited to, a calicivirus antigen, a coronavirus antigen, a herpesvirus antigen, an immunodeficiency virus antigen, an infectious peritonitis virus antigen, a leukemia virus antigen, a panleukopenia virus antigen, a parvovirus antigen, a rabies virus antigen, a Bartonella antigen, a Yersinia antigen, a Dirofilaria antigen, a Toxoplasma antigen, a tumor antigen, a flea antigen, a flea allergen, a midge antigen, a midge allergen, a mite antigen, a mite allergen, a ragweed allergen, a ryegrass allergen, a cat allergen, a dog allergen, a Bermuda grass allergen, a Johnson grass allergen, or a Japanese cedar pollen allergen. Particularly preferred antigens include a rabies virus glycoprotein G antigen; heartworm PLA2, P39, P4, P22U, Gp29, astacin, cysteine protease, macrophage migration inhibitory factor, venom allergen, TPX-1, TPX-2, transglutaminase, ankyrin, asparaginase, calreticulin, cuticulin, and aromatic amino aid decarboxylase antigens; flea serine protease, cysteine protease, aminopeptidase, serpin, carboxylesterase, juvenile hormone esterase, chitinase, epoxide hydrolase, ecdysone, ecdysone receptor, and ultraspiracle protein antigens; flea salivary antigens; Yersinia F1 and V antigens; and Toxoplasma gondii antigens such as those disclosed in PCT Patent Publication No. WO 99/32633, published Jul. 1, 1999, by Milhausen et al. Additional examples of suitable and preferred allergens are disclosed in U.S. Pat. No. 5,945,294, issued Aug. 31, 1999, by Frank et al. (U.S. Pat. No. 5,945,294); U.S. Pat. No. 5,958,880, issued Sep. 28, 1999, by Frank et al. (U.S. Pat. No. 5,958,880); PCT Patent Publication No. WO 98/45707, published Oct. 15, 1998, by Frank et al. (WO 98/45707); and PCT Patent Publication No. WO 99/38974, published Aug. 5, 1999, by Weber et al. (WO 99/38974).

One embodiment of the present invention is a composition comprising a nucleic acid molecule-cationic lipid complex that further comprises a heterologous nucleic acid molecule encoding an immunomodulator. Such an immunomodulator-encoding nucleic acid molecule can be contained within the same nucleic acid molecule encoding the antigen of the present invention, or can exist as a separate nucleic acid molecule, which can be on the same or separate plasmid or viral genome. The present invention also includes Suitable immunomodulators include compounds that enhance certain immune responses as well as compounds that suppress certain immune responses. Compounds that enhance the immune response include compounds that preferentially enhance humoral immunity as well as compounds that preferentially enhance cell-mediated immunity. Suitable compounds can be selected depending on the desired outcome. Suitable immunomodulators include, but are not limited to, cytokines, chemokines, superantigens, co-stimulatory molecules, adhesion molecules, and other immunomodulators as well as compounds that induce the production of such immunomodulators. Examples of such compounds include, but are not limited to, granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), colony stimulating factor (CSF), erythropoietin (EPO), interleukin 2 (IL-2), interleukin 3 (IL-3), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 10 (IL-10), interleukin 12 (IL-12), interleukin 13 (IL-13), interleukin 18 (IL-18), interferon gamma, interferon gamma inducing factor I (IGIF), transforming growth factor beta (TGF-.beta.), RANTES (regulated upon activation, normal T-cell expressed and presumably secreted), macrophage inflammatory proteins (e.g., MIP-1 alpha and MIP-1 beta), Leishmania elongation initiating factor (LEIF), B7-1, B7-2, CD40, CD40 ligand, ICAM-1, and VCAM.

A composition of the present invention includes a cationic lipid complexed with a nucleic acid molecule encoding an antigen in order to elicit or enhance an immune response to the antigen. As used herein, a cationic lipid is a lipid which has a cationic, or positive, charge at physiologic pH. Cationic lipids can have a variety of forms, including liposomes or micelles. Whether a cationic lipid occurs primarily as a liposome or a micelle can be manipulated by methods known in the art; for example, a freezing and thawing of cationic lipids in aqueous solution will encourage formation of liposomes, rather than micelles. A nucleic acid molecule complexed with a cationic lipid may also be referred to as a nucleic acid molecule-cationic lipid complex, a lipoplex or a complex of the present invention. A complex of the present invention that elicits an immune response is a complex of a nucleic acid molecule which encodes an antigen with a cationic lipid. As used herein, the term complexed with, which is equivalent to complexed to, refers to any method by which a nucleic acid molecule interacts (e.g. binds, comes into contact with a cationic lipid.) Such an interaction can include, but is not limited to encapsulation of a nucleic acid molecule into a cationic liposome, association of a nucleic acid molecule and cationic lipid characterized by non-covalent, ionic charge interactions, and other types of associations between nucleic acid molecules and cationic lipids known by those skilled in the art. It is preferred that cationic lipids have a cationic group, such as a quaternary amine group, and one or more lipophilic groups, such as saturated or unsaturated alkyl groups having from about 6 to 30 carbon atoms. Cationic lipid compositions suitable for use in the present invention include lipid compositions comprised of one type of lipid, or lipid compositions comprised of more than one type of lipid. If there is more than one type of lipid present in a lipid composition, it is necessary that the overall net charge of the lipid composition is cationic, i.e. positive; however, as long as the overall net charge of the lipid composition is cationic, individual lipid types may be neutral or even anionic in charge. A composition of the present invention includes a cationic lipid that is suitable in accordance with the present invention. Cationic lipids suitable for use in the present invention include commercially available cationic lipids, for example DOTMA, available under the trademark name of LIPOFECTIN.RTM., available from Life Technologies Inc., (LTI), Gaithersburg, MD and DDAB, available from Boehringer-Mannheim, Indianapolis, Ind. In addition, suitable cationic lipids can be synthesized as described in the literature; see, for example, Felgner et al., 1987, PNAS 84 7413-7417 regarding the preparation of DOTAP; Douar et al, 1996, Gene Ther 3(9), 789-796 regarding the preparation of Lipid 67; Wheeler et al., 1996, Biochim Biophys Acta 1280(1), 1-11 regarding the preparation of DMRIE; McLean et al., 1997 Am J Physiol 273, H387-404 regarding the preparation of DOTIM; and Hofland et al., 1997, Pharm Res 14(6), 742-749 regarding the preparation of DOSPA. Other suitable cationic lipid compounds are described in the literature. See, for example, Stamatatos et al., 1988, Biochemistry 27, 3917-3925 and Eibl, et al., 1979, Biophysical Chemistry 10, 261-271. Preferred cationic lipids include the class of lipids known as tetramethyltetraalkyl spermine analogs, described by McCluskey et al., (1998), Antisense and Nucleic Acid Drug Development, vol. 8, pp 401-414. Lipids of this type include tetramethyltetralaurylspermine, tetramethyltetramyristylspermine, tetramethyltetrapalmitoylspermine, and tetramethyltetraoleoylspermine. The following lipids, obtained from LTI are of the tetramethyltetraalkyl spermine class, with the alkyl groups containing fatty acid chains of length longer than oleic acid. These lipids are denoted as LTI lipids 4251-781-1, 4251-106-3, 4518-52, D304-200, 4521-52-3, 4251-106-4, 4251-781-2, 4518-53, 4518-31, 4519-30,4519-34, and 2518-111. Preferred cationic lipids include LTI lipid 4251-781-1, LTI lipid 4251-106-3, and LTI lipid 4518-52. In one embodiment, tetramethyltetraalkyl spermine lipids are formulated with a neutral lipid, such as dioleylphosphatidyl-ethanolamine (DOPE).

A nucleic acid molecule-cationic lipid complex can be formed by using techniques known to those skilled in the art, examples of which are described in the Examples section. A complex can be formed, for example, by adding a cationic lipid solution to a nucleic acid molecule, preferably an endotoxin-free nucleic acid molecule, at concentrations appropriate for the present invention, and mixing, for example by pipetting. Preferable nucleic acid molecule-to-cationic lipid ratios are from about 10:1 weight nucleic acid molecule: weight cationic lipid, (e.g. microgram (.mu.g) nucleic acid molecule to .mu.g cationic lipid) to about 1:10 weight nucleic acid molecule: weight cationic lipid. More preferable are ratios from about 1:2 weight of nucleic acid molecule: cationic lipid to about 4:1 weight of nucleic acid molecule: cationic lipid. In a preferred embodiment, the nucleic acid molecule-cationic lipid complex is incubated at room temperature for about 30 minutes before administration. A nucleic acid molecule-cationic lipid complex can be dehydrated and rehydrated using techniques known to those skilled in the art; for example, the complex can be frozen in liquid nitrogen and lyophilized at 150 milliTorr, then reconstituted in solution for injection.

A dose of a nucleic acid molecule-cationic lipid complex to administer to a cat can be reported as the amount of nucleic acid molecule administered to a cat. A preferred dose of a nucleic acid molecule-cationic lipid complex to administer to a cat includes from at least one nanogram (ng) of nucleic acid to about 10 milligram (mg) of nucleic acid molecule. More preferred is a dose range that includes from about 1 .mu.g nucleic acid molecule to about 1 mg of nucleic acid molecule. Particularly preferred is a dose ranging from about 75 .mu.g of a nucleic acid molecule to about 300 .mu.g of a nucleic acid molecule.

A nucleic acid molecule-cationic lipid complex composition of the present invention can be formulated in an excipient that the animal to be treated can tolerate. As such, the present invention includes administration of a composition comprising a nucleic acid molecule-cationic lipid complex, wherein the composition further comprises an excipient. Examples of such excipients include water, saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions. Other useful formulations include suspensions containing viscosity enhancing agents, such as sodium carboxymethylcellulose, sorbitol, mannitol, or dextran. Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability. Examples of buffers include phosphate buffer, bicarbonate buffer and Tris buffer. Standard formulations can either be liquid injectables or solids which can be taken up in a suitable liquid as a suspension or solution for injection. Thus, in a non-liquid formulation, the excipient can comprise dextrose, human serum albumin, preservatives, etc., to which sterile water or saline can be added prior to administration.

In one embodiment of the present invention, the nucleic acid molecule-cationic lipid complex can also include an adjuvant and/or a carrier. One advantage of a nucleic acid molecule-cationic lipid complex is that adjuvants and carriers are not required to produce a composition that administration thereof will elicit an immune response. However, it should be noted that use of adjuvants or carriers is not precluded by the present invention. Adjuvants are typically substances that generally enhance the immune response of an animal to a specific antigen. Suitable adjuvants include, but are not limited to, other bacterial cell wall components; aluminum-based salts; calcium-based salts; silica; polynucleotides; toxins, such as cholera toxin; toxoids, such as cholera toxoid; serum proteins; other viral coat proteins; other bacterial-derived preparations; block copolymer adjuvants, such as Hunter's Titermax.TM. adjuvant (Vaxcel.TM., Inc. Norcross, Ga.); Ribi adjuvants (available from Ribi ImmunoChem Research, Inc., Hamilton, Mont.); and saponins and their derivatives, such as Quil A (available from Superfos Biosector A/S, Denmark). Carriers are typically compounds that increase the half-life of a therapeutic composition in the treated animal. Suitable carriers include, but are not limited to, polymeric controlled release formulations, biodegradable implants, liposomes, bacteria, viruses, oils, esters, and glycols.

An immune response to an antigen includes a humoral, i.e. antibody, response to that antigen and/or a cell-mediated response to that antigen. Methods to measure an immune response are known to those skilled in the art; examples of such methods are disclosed herein. If one or both types of immune response are present, they may protect the felid from disease caused, for example, by the agent from which the antigen was derived. In accordance with the present invention, the ability of an antigen derived from a disease-causing agent to protect an animal from a disease caused by that disease-causing agent or a cross-reactive agent refers to the ability of a nucleic acid molecule-cationic lipid complex of the present invention to treat, ameliorate and/or prevent disease caused by the disease-causing agent or cross-reactive agent, preferably by eliciting an immune response against the antigen derived from the disease-causing agent. It is to be noted that an animal may be protected by a composition of the present invention even without the detection of a humoral or cell-mediated response to the antigen. Protection can be measured by methods known to those skilled in the art, such as by challenging an animal with the agent against which the animal has mounted a putative immune response. In certain cases, the antibody titer of an animal can be used to demonstrate protection. For example, it is known that animals that elicit an antibody response against a rabies glycoprotein G antigen are protected if their sera exhibits a rapid focus fluorescent inhibition test (RFFIT) titer of rabies virus neutralizing antibodies of greater than 1:5. As used herein, an animal that elicits an immune response to an antigen is an animal that has been immunized with that antigen.

The biological mechanism for eliciting and/or enhancing an immune response by the use of a nucleic acid molecule-cationic lipid complex composition of the present invention has not been elucidated, but, without being bound by theory, the inventors believe that the mechanism is likely related to the ability of these compositions to protect DNA from nuclease attack, to facilitate the transfection of both muscle cells and professional antigen presenting cells (APC) in vivo, to increase levels of expression in transfected cells, and/or to distribute DNA to lymphoid organs.

A felid, as used herein, is a member of the family Felidae. Examples of felids include domestic cats, wild cats, and zoo cats. Examples of cats, include, but are not limited to, domestic cats, lions, tigers, leopards, panthers, cougars, bobcats, lynx, jaguars, cheetahs, and servals. A preferred cat to immunize is a domestic cat. The term cat(s) and felid(s) are used interchangeably herein.

As used herein, parenteral administration means administration not through the alimentary canal (e.g. oral administration), but rather by injection through some other route, including but not limited to routes such as subcutaneous, intramuscular (I.M.), intravenous (I.V.), intraperitoneal (I.P.), intradermal (I.D.), intraorbital, intracapsular, intraspinal, and intrasternal. Parenteral administration includes, but is not limited to, administration by any route that includes use of a needle to insert material into the body. Parenteral administration also includes uses of devices other than a syringe and needle to insert material through the skin and or mucosal surfaces into the body, including but not limited to the BIOJECTOR.RTM., POWDERJECT, and MEDIJECT.RTM. needleless injection systems. A preferred route of administration includes intramuscular administration using a needle and syringe.

Acceptable protocols to administer therapeutic compositions in an effective manner include individual dose size, number of doses, and frequency of dose administration. Typically, the first administration of a composition intended to elicit an immune response is called the primary (or prime) administration, also known as the pre-boost. Additional administrations intended to "boost" or increase an immune response to an antigen are termed booster administrations. Determination of a protocol to elicit an immune response in a cat using a nucleic acid molecule-cationic lipid complex of the present invention can be accomplished by those skilled in the art. In one embodiment of the present invention, a nucleic acid molecule encoding a desired antigen complexed with cationic lipid need only be administered once by a route appropriate to the present invention (e.g. parenteral) to stimulate an immune response against the antigen. In a preferred embodiment, such an administration protects the felid from the agent from which the antigen was derived or from an agent against which the immune response is cross-protective.

In one embodiment, administration of a complex of the present invention to a felid in order to elicit an immune response actually enhances the immune response generated by the felid as compared to the immune response generated upon administration of a naked DNA vaccine to a felid, wherein the naked DNA vaccine consists essentially of a naked DNA molecule; i.e., a DNA molecule that is not complexed with lipids. Finding that a complex of the present invention enhances an immune response is surprising both in view of the conflicting studies known to those skilled in the art as described herein as well as in view of the studies described in more detail in the Examples, in which administration of naked DNA vaccines to cats elicited immune responses in only some cats within each group, or population, tested, whereas administration of a complex of the present invention could result in up to 100% seroconversion of all cats in a population tested. As used herein, enhancement of the immune response can include increasing the amount, or titer, of antibody elicited by a complex of the present invention that encodes an antigen to the desired antigen and/or agent from which the antigen was derived as compared to the titer of antibody generated by a naked DNA vaccine that encodes the same antigen. In one embodiment, such an enhancement can be induction of no antibody titer with a naked DNA vaccine to induction of a protective antibody titer with a complex of the present invention. Enhancement of an immune response can also refer to augmentation of the cell-mediated response to the antigen and/or agent encoded by a complex of the present invention as compared to the response generated by a naked DNA vaccine encoding the same antigen. Enhancement of immune response can also include conferring or augmenting protection from disease by a complex of the present invention compared to the protection, if any, conferred by a naked DNA vaccine encoding the same antigen. In one embodiment, enhancement of the immune response includes increasing the likelihood of a cat seroconverting in response to antigen encoded by a complex of the present invention in comparison to the likelihood of the cat responding to the same antigen encoded by a naked DNA vaccine. In other words, in a group of cats being vaccinated with a complex of the present invention, a greater number of cats will seroconvert in response to antigen encoded by the complex rather than to the same antigen encoded by a naked DNA vaccine. Preferably, the likelihood that a cat will seroconvert when administered a single dose of a complex of the present invention that encodes an antigen is at least about 50%, preferably at least about 75%, more preferably at least about 90% and even more preferably at least about 100%. In the case where a primary and booster administration of the complex is administered, the likelihood that a cat will seroconvert is preferably at least about 75%, more preferably at least about 90%, and even more preferably at least about 100%.

The present invention includes a method to administer a nucleic acid molecule to a felid. The method includes the step of parenterally administering a composition comprising said nucleic acid molecule complexed with a cationic lipid. Such a nucleic acid molecule can encode either a protein or a RNA molecule. In one embodiment, the nucleic acid molecule encodes a protein or RNA molecule that, when expressed at an appropriate level, has a protective effect upon the cat. As used herein, a protein refers to a full-length protein or any portion thereof that is at least about 5 amino acids in length and has a useful function, including, but not limited to, ability to elicit an immune response, elicit an immunomodulatory effect (e.g., an immunomodulator that stimulates or reduces the immune response), effect gene therapy, effect enzyme activity, or otherwise effect cell division, differentiation, development and cell death. As used herein, a RNA molecule refers to any RNA species that can be encoded by a nucleic acid molecule, including, but not limited to antisense RNA, a molecule capable of triple helix formation, a ribozyme, or other nucleic acid-based drug compound. As such, any protein or RNA molecule that can be expressed at an appropriate level in a cat, which protects a cat from disease, would be included in this invention. Diseases from which to protect a felid include, but are not limited to, infectious diseases, genetic diseases, oncological diseases, and other metabolic diseases, including diseases that lead to abnormal cell growth, degenerative processes, and immunological defects. Compositions of the present invention can protect animals from a variety of diseases including, but not limited to, allergies, arthritic diseases, autoimmune diseases, cancers, cardiovascular diseases, graft rejection, hematopoietic disorders, immunodeficiency diseases, immunoproliferative diseases, immunosuppressive disorders, infectious diseases, inflammatory diseases, jaundice, septic shock, and other immunological defects, as well as other genetic or metabolic defects. Methods to produce and use a composition comprising any nucleic acid molecule of the present invention complexed with any cationic lipid of the present invention are as described herein.
 

Claim 1 of 2 Claims

1. A method to vaccinate a felid against an infectious disease, said method comprising parenterally administering to said felid a composition comprising a nucleic acid molecule complexed with a cationic lipid, wherein said nucleic acid molecule encodes a rabies virus antigen capable of protecting said felid from disease caused by rabies virus.

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