FIELD OF THE INVENTION

[0001] The present invention generally relates to immunostimuiatory plasmids. The plasmids do not comprise an antibiotic resistance gene. The plasmids can comprise a selectable or screenable marker gene that is not an antibiotic resistance gene (e.g., a LacZ gene). Alternatively, the plasmids can be devoid of any selectable or screenable marker genes.

BACKGROUND OF TH E INVENTION

100021 Unmethyiated CpG moti s occur much more frequently in bacterial DNA than in vertebrate DNA. These motifs activate host defense mechanisms and lead to innate and acquired immune responses. CpG moti fs and their immunostimuiatory effects are rev iewed in Krieg, Ann. Rev. Immunol. 20:709-760 (2002).

[0003] An immunostimuiatory piasmid containing a number of CpG motifs was previously developed and has been shown to be effective for el iciting immune responses when administered to avian and bovine species in an immunomodulator composition comprising the piasmid and a cationic l iposome del iv ery veh icle. See U.S. Patent Application Publications Nos. 2012/0064151 A I ( avian species) and 2013/0295167 A I (bovine species), the contents of both of which are hereby incorporated by reference in their entirety. This piasmid, pMB75.6, is 4242 bp i n length and contains 288 CpG dinucleotides. A map of pMB75.6 is shown in FIG. 1 and the nucleotide sequence of pMB75.6 is prov ided as SEQ I D NO: 2. As described in U.S. Patent Appl ication Publ ication No. 2012/0064151 , the immunomodulator composition containing pMB75.6 elicited a non-antigen-specific immune response that protected chickens from infectious disease when administered in ovo. This non- antigen-specific immune response was further enhanced with administration of at least one biological agent, such as a vaccine. I n addition, the immunomodulator composition containing the pMB75.6 piasmid was found to have an adj uv ant effect and to elicit an increase in the efficacy of v accines. Simi larly, as described in U.S. Patent Application No. 2013/0295167, the immunomodulator composition contain ing pMB75.6 elicited a non- antigen-specific i mmune response in cattle that protected the cattle from infectious disease. 100041 However, as shown in FIG. 1 , the pMB75.6 plasmid contains a kanamycin- resistance gene (Kan ^R ). Antibiotic -based selection and production systems are becoming increasingly disfavored due to concerns about horizontal transfer of the antibiotic resistance gene to bacteria in the environment. Potential horizontal transfer of antibiotic resistance genes is particularly concerning for vectors that are directly administered to a subject (e.g., immunostimulatory plasmids such as pMB75.6 or vectors used for gene therapy or DNA vaccination ). There is therefore a need in the art for immunostimulatory plasmids that arc capable of eliciting an immune response in a subject while also lacking antibiotic resistance genes.

SUMMARY OF TH E INVENTION

[0005] The present invention relates to immunostimulatory plasmids. The immunostimulatory plasmid may comprise a nucleic acid sequence having at least 89% sequence identity with the sequence of SEQ I D NO: 1, SEQ I D NO: 4, or a combination thereof. In some aspects, the immunostimulatory plasmid may comprise a nucleic acid molecule having at least 84% sequence identity with the sequence of SEQ I D NO: 4. I some aspects, the immunosti mulatory plasmid may comprise the sequence of SEQ I D NO: 1. In some aspects, the immunostimulatory plasmid may comprise the sequence of SEQ I D NO: 4.

[0006] In other aspects, the immunostimulatory plasmid may consist of a nucleic acid sequence having at least 89% sequence identity with the sequence of SEQ I D NO: 1, SEQ I D NO: 4, or a combi nation thereof. I some aspects, the immunostimulatory plasm id may consist of a nucleic acid molecule hav ing at least 84% sequence identity with the sequence of SEQ I D NO: 4. I n some aspects, the i mmunostimulatory plasmid may consist of the sequence of SEQ I D NO: 1. I n some aspects, the immunostimulatory plasmid may consist of the sequence of SEQ I D NO: 4.

[0007] In some aspects, the immunostimulatory plasmid preferably does not comprise a nucleic acid sequence encoding a full-length or functional selectable or screenable marker. In other aspects, the immunostimulatory plasm id comprises a nucleic acid sequence encodi ng a selectable or screenable marker that is not an antibiotic resistance gene.

[0008] The present invention also relates to pharmaceutical formulations comprising any of the immunostimulatory plasmids, or DNA sequences, described herein and a pharmaceutically acceptable carrier. [0009] The present invention further relates to immunomodulator compositions comprising a cationic liposome delivery vehicle and any of the immunostimulatory plasmids, or DNA sequences, described herein.

[0010] In some aspects, the present invention relates to methods of using the immunostimulatory plasmids, or DNA sequences, described herein. Suitable methods of use include therapeutic administration to a subject. Such therapeutic administration includes prophylactic treatment, metaphylactic treatment, and post- infection treatment of a subject or subjects.

[0011] The present invention relates to methods of stimulating or eliciting an immune response in a subject. In some aspects, the methods include stimulating an immune response in a subject by administering to the subject an immunomodulator composition described herein. In some aspects, the methods include stimulating an immune response in a subject by administering to the subject an immunostimulatory plasm id, or DNA sequence, described herein.

[0012] Other objects and features will be in part apparent and in part pointed out hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0014] FIG. 1 shows a map of the pMB75.6 piasmid;

[0015] FIG. 2 shows a map of the pGCMB75.6 piasm id;

[0016] FIG. 3 shows a map of the pLacZ75.6 piasmid; and,

[0017] FIG. 4 graphically illustrates that immunomodulator compositions described herein increase the survivability of recipient subjects challenged with a pathogenic virus (FIG. 4A, and FIG. 4B).

DETAILED DESCRIPTION OF THE INVENTION

[0018] In accordance with the present invention, a composition that is capable of eliciting an immune response in a recipient subject, as well as methods of use, have been discovered. In particular, the present invention relates to novel nucleic acid compositions, or immunomodulator compositions, and uses thereof. It has been discovered that such immunomodulator compositions may include DNA sequences described herein with enhanced GC content, CpG motifs, and propagated without including antibiotic resistance genes or coding sequence. The nucleic acid sequences of the invention may be used to stimulate or enhance an immune response in a subject to prevent or treat infectious disease with significantly enhanced safety over other methods of prevention and treatment known in the art. The invention is particularly useful in the treatment and prevention of infectious diseases caused by microorganisms, such as, without limitation, viruses, bacteria, mold, fungus, yeast, parasites and other microbes known in the art. The compositions and methods of using the immunomodulator compositions are discussed in more detail below.

I. Compositions

[0019] Compositions useful in this invention, such as those described herein, are generally able to be used as a prophylactic therapy, metaphylactic therapy, or treatment therapy for infectious diseases. Such compositions are referred to herein as

immunomodulator compositions. The immunomodulator compositions include at least a immunostimulatory plasm id or immunostimulatory DNA sequence, capable of eliciting an immune response in a recipient subject. In some aspects, the immunomodulator compositions may also include a liposome deliv ery v ehicle.

A. Nucleic Acids

100201 In some aspects the present inv ention relates to nucleic acid molecules useful f r the treatment or prevention of infectious disease causing agents. The nucleic acid molecules described herein may be included in an immunostimulatory plasm id, as linear double stranded or single stranded DNA, amino acid sequence, ribonucleic acid (RNA), or combinations thereof. In some aspects, the present invention relates to nucleic acid molecules, vectors, and host cells (in vitro, in vivo, or ex vivo) which contain the immunostimulatory plasmid or immunostimulatory DNA sequence.

[0021] In some aspects, the present invention relates to immunostimulatory plasmids, or DNA sequences, that do not comprise an antibiotic resistance gene. The plasmids may be devoid of any selectable or screenable marker genes. For example, the pGCMB75.6 plasmid described herein does not comprise any full-length or functional selectable or screenable marker genes. The sequence of pGCMB75.6 is prov ided in SEQ I D NO: 1.

[0022] In some aspects, the immunostimulatory plasmids described herein preferably do not comprise a nucleic acid sequence coding for a full-length or functional selectable or screenable marker. In some aspects, the immunostimulatory plasmids do not comprise an antibiotic resistance gene. For example, the plasmids do not comprise a kanamycin resistance gene. In some aspects, the plasmids described herein preferably do not encode an

immunogen.

[0023] In some aspects, the immunostimulatory plasmids may comprise a nucleic acid sequence coding for a selectable or screenable marker gene that is not an antibiotic resistance gene. For example, the pLacZMB75.6 plasmid described herein comprises a LacZ gene as a screenable marker. A map of pLacZMB75.6 is prov ided in FIG. 3 and the nucleotide sequence of pLacZMB75.6 is prov ided as SEQ ID NO: 4. As shown in FIG. 3, pLacZMB75.6 is sim ilar to pGCMB75.6, but contains a LacZ screenable marker.

[0024] I t will be appreciated that the nucleotide sequences of the pGCMB75.6 or pLacZMB75.6 plasmids may be varied to a certain extent without significantly adversely affecting their immunostimulatory properties. In some aspects, the present invention relates to an immunostimulatory plasmid comprising a nucleic acid sequence having at least 89% sequence identity with the sequence o pG CM 1375.6 ( SEQ I D NO: 1). The

immunostimulatory plasmid preferably comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of pGCMB75.6 ( SEQ ID NO: 1). In some aspects, the immunostimulatory plasmid more preferably comprises the sequence of pGCMB75.6 ( SEQ I D NO: 1).

[0025] In some aspects, the present invention relates to an immunostimulatory plasmid comprising a nucleic acid sequence having at least 84% sequence identity with the sequence of pLacZMB75.6 ( SEQ I D NO: 4). The immunostimulatory plasmid preferably comprises a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of pLacZMB75.6 (SEQ I D NO: 4). In some aspects, the immunosti mulatory plasmid more preferably comprises the sequence of pLacZMB75.6 ( SEQ I D NO: 4). [0026] In some aspects, the present invention relates to an immunostimulatory plasmid consisting of a nucleic acid sequence having at least 89% sequence identity with the sequence of pGCMB75.6 (SEQ I D NO: 1). The immunostimulatory plasmid preferably consists of a nucleic acid sequence hav ing at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of pGCMB75.6 ( SEQ I D NO: 1). I n some aspects, the immunostimulatory plasmid more preferably consists of the sequence of pGCMB75.6 (SEQ I D NO: 1).

[0027] In some aspects, the present invention relates to an immunostimulatory plasmid consisting of a nucleic acid sequence having at least 84% sequence identity with the sequence of pLacZMB75.6 (SEQ I D NO: 4). The immunostimulatory plasmid preferably consists of a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of pLacZMB75.6 (SEQ I D NO: 4). in some aspects, the immunostimulatory plasmid more preferably consists of the sequence of pLacZMB75.6 (SEQ I D NO: 4).

[0028] Another important aspect of this invention provides for immunostimulatory DNA sequences or immunostimulatory pi asm ids capable of stimulating an immune response including nucleic acid sequences that hybridize under high stringency conditions to SEQ ID NO: 1 or SEQ I D NO: 4. Suitable nucleic acid sequences include those that are homologous, substantially similar, or identical to the nucleic acids of the present invention. In some aspects, homologous nucleic acid sequences will have a sequence similarity of at least about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ I D NO: 1 or the respective complementary sequence. In other aspects, homologous nucleic acid sequences will have a sequence si mi larity of at least about 84%, 85%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ I D NO: 4 or the respectiv complementary sequence. Sequence simi larity may be calculated using a number of algori thms known in the art, such as B LAST, described in A ltschul, S. F., et al.. J. Mol. Biol. 215:403- 10, 1990. The nucleic acids may differ in sequence from the above-described nucleic acids due to the degeneracy of the genetic code. In general, a reference sequence will be 18 nucleotides, more usually 0 or more nucleotides, and may comprise the entire nucleic acid sequence of the composition for comparison purposes. 10029| Nucleotide sequences that can hybridize to S EQ I D NO: 1 or SEQ I D NO: 4 are contemplated herein. Stringent hybridization conditions include conditions such as hybridization at 50°C or higher and 0.1X SSC (15 mM sodium ch loride 1 .5 mM sodium citrate). Another example is overnight incubation at 42°C in a solution of 50% formamide, 5X SSC ( 1 0 mM NaCl, 1 5 mM trisodium citrate ), 50 mM sodium phosphate (pH 7.6), 5X Denhardt's solution, 10% dextran sulfate, and 20 iig ml denatured, sheared salmon sperm DNA, followed by washing in 0. 1 X SSC at about 65°C. Exemplary stri ngent hybridization conditions are hybridization conditions that are at least about 80%, 85%, 90%, or 95% as stringent as the above specific conditions. Other stringent hybridization conditions are known in the art and may also be employed to identify homologs of the nucleic acids of the

invention (Current Protocols in Molecular Biology, Unit 6, pub. John Wiley & Sons, N.Y. 1989).

[0030] Mutant nucleotides of the DNA molecules described herein may be used, so long as mutants include nucleic acid sequences maintain the ability to stimulating an immune response as described herein. The DNA sequence of such a mutation will usual ly differ by one or more nucleotides or amino acids. The sequence changes may be substitutions, insertions, deletions, or a combination thereof. Techniques for mutagenesis of cloned genes are known in the art. Methods for site specific mutagenesis may be found in Gustin et al, Biotechn iques 14:22, 1993 ; Barany. Gene 37: 1 1 1-23, 1985; Col icel li et al., Mol. Gen. Genet. 199:537-9, 1985; and Sambrook et al.. Molecular Cloning: A Laboratory anual, CSH Press 1989, pp. 15.3- 15.108 and al l incorporated herein by reference. I n summary, the invent ion relates to nucleic acid sequences capable of stimulating an immune response in a subject and v ariants or mutants thereof. Also, the invention encompasses the i ntermediatary RNAs encoded by the described nucleic acid sequences, as well as any resultant amino acid sequences encoded.

100 11 Where the nucleotide sequence of the immunostimulatory pi as mid varies from the sequences provided in SEQ ID NOs. 1 and 4 the CpG dinucieotides in the piasmid are preferably left intact. Alternatively, if the nucleotide sequence of the piasmid is altered such that a CpG dinucleotide is eliminated, the sequence of the piasmid may be altered at another location such that the total number of CpG dinucieotides in the piasmid remains the same. Further CpG dinucieotides in addition to those already present in the nucleotide sequences of pGCMB75.6 or pLacZMB75.6 may also be introduced into the piasmid. Thus, for example, the immunostimulatory plasmids described herein preferably comprise at least about 200, at least about 220, at least about 240, at least about 260, at least about 270, at least about 275, at least about 280, at least about 283, at least about 285, or at least about 288 CpG

dinucieotides. For example, the immunostimulatory pi asm id can comprise 283 CpG dinucleotides.

[0032] In particular, the present invention relates to pharmaceutical formulations comprising any of the immunostimulatory plasmids or DNA sequences described herein and a pharmaceutically acceptable carrier.

B. Immunomodulator

[0033] Suitable immunomodulator compositions for use with the immunostimulatory plasmids described herein are described in U.S. Patent Application Publications Nos.

2012/0064151 Al (avian species) and 2013/0295167 A 1 (bovine species), the contents of both of wh ich are hereby incorporated by reference in their entirety.

[0034] The immunomodulator composition comprises a liposome delivery vehicle and at least one of the immunostimulatory plasmids, or DNA sequences, described herein.

[0035] A suitable liposome delivery vehicle comprises a lipid composition that is capable of delivering nucleic acid molecules to the tissues of the treated subject. A liposome delivery vehicle is preferably capable of remaining stable in a subject for a sufficient amount of time to deliver a nucleic acid molecule and/or a biological agent. For example, the liposome delivery vehicle is stable in the recipient subject for at least about five minutes, for at least about I hour, or for at least about 24 hours.

[0036] A liposome delivery vehicle of the present invention comprises a lipid composition that is capable of fusing with the plasma membrane of a cell to del iver a nucleic acid molecule into a cell. When the nucleic acid molecule encodes one or more proteins, the nucleic acid:liposome complex preferably has a transfection efficiency of at least about 1 pieogram (pg) of protein expressed per milligram (mg) of total tissue protein per microgram ^g) of nucleic acid del ivered. For example, the transfection efficiency of a nucleic acid: liposome complex can be at least about 10 pg of protein expressed per mg of total tissue protein per tig of nucleic acid delivered; or at least about 50 pg of protein expressed per mg of total tissue protein per ^ig of nucleic acid delivered. The transfection efficiency of the complex may be as low as 1 femtogram (fg) of protein expressed per mg of total tissue protein per n of nucleic acid delivered, with the above amounts being more preferred.

[0037] A preferred liposome delivery vehicle of the present invention is between about 100 and 500 nanometers (nm) in diameter. For example, the liposome deliv ery vehicle can be between about 150 and 450 nm or between about 200 and 400 nm in diameter.

[0038] Suitable liposomes include any liposome, such as those commonly used in, for example, gene delivery methods known to those of skill in the art. Preferred liposome delivery vehicles comprise multilamellar vesicle (MLV) lipids and extruded lipids. Methods for preparation of MLVs are wel l known in the art. More preferred liposome del ivery vehicles comprise liposomes having a polycationic lipid composition (i.e., cationic

liposomes) and or liposomes hav ing a cholesterol backbone conj ugated to polyethylene glycol. Exemplary cationic liposome compositions include, but are not l imited to, N-[l -(2,3- dioleyloxy)propyl]-N,N,N-trimethylammonium chloride ( DOT M A ) and cholesterol, N-[ 1 - (2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride ( DOT A P ) and cholesterol, I - [2-(oleoyioxy)ethyi]-2-oleyl-3-(2-hydroxyethyl)-imidazoliniu m chloride (DOTIM) and cholesterol, dimethyidioctadecylammonium bromide (DDAB) and cholesterol, and combinations thereof. A most preferred liposome composition for use as a delivery vehicle includes DOTIM and cholesterol.

[0039] A suitable nucleic acid molecule includes any of the immunostimuiatory piasmids described herein. Coding nucleic acid sequences encode at least a portion of a protein or peptide, while non-coding sequence does not encode any portion of a protein or peptide. According to the present inv ention, "non-coding ^" nucleic acids can include regulatory regions o a transcription unit, such as a promoter region. The term, "empty vector" can be used interchangeably with the term "non-coding, ^" and particularly refers to a nucleic acid sequence in the absence o a protein coding portion, such as a plasmid vector without a gene insert. Expression o a protein encoded by the piasmids described herein is not required for eiicitation of a non-antigen-specific immune response; therefore the piasmids need not contain any coding sequences operatively linked to a transcription control sequence. Howev er, further adv antages may be obtained (i.e., antigen-specific and enhanced immunity ) by including in the composition nucleic acid sequence (DNA or RNA ) which encodes an immunogen and/or a cytokine. Such a nucleic acid sequence encoding an immunogen and/or a cytokine can be included in the immunostimulatory plasmids described herein, or can be included in a separate nucleic acid (e.g., a separate plasmid) in the composition.

[0040] Complexing a liposome with the immunostimulatory plasmids described herein may be achieved using methods standard in the art or as described in U.S. Patent No. 6,693,086, the contents of which arc hereby incorporated by reference in their entirety. A suitable concentration of a plasmid to add to a liposome includes a concentration effective for delivering a sufficient amount of the plasmid into a subject such that a systemic immune response is elicited. For example, from about 0. 1 iig to about 10 iig of plasmid can be combined with about 8 nmoi liposomes, from about 0.5 μg to about 5 iig of plasmid can be combined with about 8 nmol liposomes, or about 1.0 ,ug of plasmid can be combined with about 8 nm l liposomes. The ratio of plasmid to lipid (^i piasmid:nmol lipid) in a composition can be at least about 1 : 1 plasmid:lipid by weight (e.g., 1 ii plasmid: 1 nmol lipid). For example, the ratio of plasmid to lipids can be at least about 1 :5, at least about 1 : 10, or at least about 1 :20. Ratios expressed herein are based on the amount of cationic lipid in the composition, and not on the total amount of lipid in the composition. The ratio of plasmid to lipids in a composition of the invention is suitably from about 1 : 1 to about 1 :80 plasniid: lipid by weight; from about 1 :2 to about 1 :40 plasm id: l ipid by weight; from about 1 :3 to about 1 :30 plasmid: l ipid by weight; or from about 1 :6 to about 1 : 1 5 plasmid: lipid by weight.

C. Biological Agent

[0041] Any of the immunomodulator compositions described herein can further comprise at least one biological agent, in addition to the liposome delivery vehicle and at least one of the plasmids described herein.

100421 Suitable biological agents are agents that are effective in preventing or treating avian or bovine diseases. Such biological agents include immune enhancer proteins, immunogens, vaccines, antimicrobials or any combination thereof. Suitable immune enhancer proteins are those proteins known to enhance immunity. By way of a non-l imiti ng example, a cytokine, which includes a family of proteins, is a known immunity enhancing protein fami ly. Suitable immunogens are proteins which elicit a humoral and/or cel lular immune response such that administration of the immunogen to a subject mounts an immunogen-specific immune response against the same or similar proteins that are encountered within the tissues of the subject. An immunogen may include a pathogenic antigen expressed by a bacterium, a virus, a parasite or a fungus. Preferred antigens include antigens derived from organisms which cause an infectious disease in a subject. According to the present invention, an immunogen may be any portion of a protein, naturally occurring or synthetically derived, which elicits a humoral and/or cellular immune response. As such, the size of an antigen or immunogen may be as small as about 5-12 amino acids and as large as a full length protein, including any sizes in between. The antigen may be a muitimer protein or fusion protein. The antigen may be a purified antigen. Alternatively, the immune enhancer protein or immunogen can be encoded by the immunostimulatory plasmid or by another nucleic acid included in the immunomodulator composition. Where the immune enhancer protein or immunogen is encoded by a nucleic acid molecule in the immunomodulator composition, the nucleic acid sequence encoding the immune enhancer protein or immunogen is opcratively l inked to a transcription control sequence, such that the immunogen is expressed in a tissue of a subject, thereby eliciting an immunogen-specific immune response in the subject, in addition to the non-specific immune response. Techniques to screen for immunogenicity, such as pathogen antigen immunogenicity or cytokine activity are known to those of skill in the art and include a variety of in vitro and in vivo assays.

100431 Where the biological agent is a vaccine, the vaccine may include a live, infectious, viral, bacterial, or parasite vaccine or a killed, inactivated, viral, bacterial, or parasite vaccine. One or more vaccines, live or killed viral vaccines, may be used in combination with the immunomodulator composition of the present invention. Suitable vaccines include those known in the art for avian or bovine species.

[0044] Exemplary vaccines for avian species include, without limitation, those used in the art for protection against Marek's disease virus (MDV), New Castle disease virus (NDV), chick anemia virus (CAV), infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV), turkey herpesvirus (HVT), infectious laryngotracheitis virus (ILTV), avian encephalomyelitis virus (AEV), fowl pox virus (FPV), fowl cholera, av ian influenza virus (AIV), reovirus, avian leucosis virus (ALV), reticuloendotheliosis virus (REV), av ian adenovirus and hemorrhagic enteritis virus (HEV), coccidia, and other diseases know n in the art. In another example, the vaccine may be a vaccine as described by U.S. Pat. Nos.

5,427,791, 6,048,535, and 6,406,702. For example, a vaccine for protection against Marek's disease may be used in combination with the immunomodulator composition of the present inv ention. 100451 Exemplary vaccine for bov ine species include, without limitation, those used in the art for protection against infectious bovine rhinotracheitis (IBR) (Type 1 bovine herpes v irus (BHV1 )). parainfluenza virus type 3 (PI3), bovine respiratory syncytial virus (BRSV), bovine viral diarrhea virus (BVDV Type 1 and 2), Histophilus somni, Mycoplasma bovis, and other diseases known in the art. For example, a vaccine for the protection against

Mannheimia haemolytica may be used in combination with the immunomodulator composition of the present invention.

10041 The biological agent can be an antimicrobial. Suitable antimicrobials include: quinolones, preferably fluoroquinolones, β-lactams, and macrolide-lincosamide- strcptogtamin (MLS) antibiotics.

100471 Suitable quinolones include benofloxacin, binfSoxacin, cinoxacin,

ciprofloxacin, clinafloxacin, danofloxacin, difloxacin. cnoxacin, enro loxacin, fleroxaein. gemifloxacin, ibafloxacin, levofloxacin, lomefloxacin, marbolloxacin, moxifloxacin, norfloxacin, ofloxacin, orbifloxacin, pazufloxacin, pradofloxacin, perfloxaein, sarafloxacin, spartloxacin, temafloxacin, and tosutloxacin. Preferred fluoroquinolones include

ciprofloxacin, danofloxacin, enrofloxacin, moxifloxacin, and pradofloxacin. Suitable naphthyridones include nalidixic acid.

[0048] Suitable β-lactams include penicillins (e.g., amoxicillin, ampiciliin, azlocillin, benzathine penicillin, benzylpcnicillin, carbenicillin, cloxacillin, co-amoxiclav [i.e.

amox ici 11 i n clavii Ian ic acid], dicloxacillin, flucloxacillin, methicillin, mezlocillin, nafcillin, oxacillin, plicnoxymetliylpenicillin, piperacillin, procaine penicillin, temocillin, and ticarcillin); cephalosporins (e.g., cefaclor, cefalonium, cefamandole, cefapririn, cefazolin, cefepime, cefixime, cefotaxime, cefoxitin, cefpirome, cefpodoxime, cefquinome, ceftazidime, ceftiofur, ceftriaxone, cefuroxime, cephalexin, cephalothin, and defotetan); carbapenems and pen ems (e.g., doripenem, crtapenem, faropenem, imipenem, and meropenem): monobactams (e.g., aztreonam, nocardicin A, tabtoxinine-P-lactam, and tigemonam): and β-lactamase inhibitors (e.g., clavulanic acid, sulbactam, and tazobactam). Preferred β-lactams include cephalosporins, in particular, cefazolin.

100491 Suitable MLS antibiotics include clindamycin, lincomycin, pirlimycin, and any macrolide antibiotic. A preferred lincosamide antibiotic is pirlimycin.

[0050] Other antimicrobials include aminoglycosides, clopidol, dimetridazoles, erythromycin, framycctin, furazolidone, halofuginone, 2-pyridoncs, robenidine, sulfonamides, tetracyclines, trimethoprim, various pleuromutil ins (e.g., tiamulin and valnemuiin ), and various streptomycin (e.g., monensin, narasin, and salinomycin).

I I. Methods

100511 An object of the present invention is to provide immunomodulator compositions, immunostimulatory plasmids (or DNA sequence), and methods that elicit protective immunity to uninfected subjects, protective immunity to infected subjects, enhanced immunity to uninfected subjects, enhanced immunity to infected subjects, therapeutic immunity to infected subjects, or combination thereof. As such, the compositions of the invention may be used to prophylactically immunize a subject or be used to treat a subject. The methods described herein include administrating an immunostimulatory plasmid, or DNA sequence, described herein to a subject.

A. Methods of Immune Stimulation

[0052] The present invention is related to methods of eliciting an immune response in a recipient subject. The methods comprise administering to a subject an effective amount of an immunomodulator composition to elicit an immune response. In some aspects, the immunomodulator composition elicits a non-antigen-speci fie immune response that is effective alone. In some aspects, the immunomodulator composition enhances the operation of at least one biological agent such as a vaccine, when administered prior to such a vaccine, co-administered with a vaccine, administered post vaccination, or mixed with the v accine. In some aspects, the methods prov ide new treatment strategies for protecting recipient subjects from infectious diseases and treating populations having infectious disease. In some aspects, the methods prov ide a more rapid, a longer and better protection against a disease when the immunomodulator is used in combination with a vaccine, compared to use of the v accine without the immunomodulator composition.

1005 1 An immune response can be elicited in a recipient subject by administering an effective amount of an immunomodulator composition, which includes any of the liposome delivery vehicles described herein, any of the immunostimulatory plasmids (for DNA sequences ) described herein, and any of the biological agents described herein. It is contemplated that the biological agent may be mixed with or co-administered with the immunomodulator or independently thereof. Independent administration may be prior to or after administration of the immunomodulator. It is also contemplated that more than one administration of the immunomodulator or biological agent may be used to extend enhanced immunity. Furthermore, more than one biological agent may be co-administered with the immunomodulator, administered prior to the immunomodulator, administered after administration of the immunomodulator. or concurrently with the immunomodulator.

100541 An effective amount of any of the immunomodulator compositions described herein may be administered to a subject. The effective amount is sufficient to elicit an immune response in the recipient subject. Such effective amount is any amount that cuases an immune response in a recipient sub ject. Methods of measuring an immune response arc wll known in the art. Also, a skilled artisan will recognize that the effective amount will depend upon age weight, stage of infection, as well as other factors known in the art.

Suitable effective amounts may range from about 0.1 iig to 1,000 ug per subject. In some aspects, the effectiv e amount may range from about 0. 1 ^ig to about 10 Lig, from about 0. 1 ug to about 5 ^ig, from about 0.5 Lig to about 5 iig, from about 0.25 ^ig to about 5 ι«, from about 0.05 ug to about 10 ^ig, from about 5 ug to about 15 iig, from about 10 ^ig to about 15 Lig, from about 10 ug to about 20 ug. from about 20 Lig to about 30 Lig, from about 30 g to about 40 Lig, from about 40 Lig to about 50 ^ig, from about 50 iig to about 70 ug, from about 70 Lig to about 90 Lig, from about 50 ug to about 100 ug, from about 100 ug to about 1 50 ^ig, from about 1 50 ug to about 200 ug, from about 200 ^ig to about 250 ^ig, from about 250 Lig to about 300 ug, from about 300 ug to about 350 Lig, from about 350 ug to about 400 ug, from about 400 Lig to about 450 Lig, from about 450 Lig, to about 500 ug, from about 500 Lig to about 550 ug, from about 550 ^ig to about 600 ug, from about 600 Lig to about 650 ug, from about 650 Lig to about 700 Lig, from about 700 Lig to abou 750 ug, from about 750 ^ig to about 800 g, from about 800 ug to about 850 iig, from about 850 g to about 900 Lig, from about 900 Lig to about 950 Lig, from about 950 g to about 1000 ug. Preferably, in some aspects, the effectiv e amount ranges from about 0.5 Lig to about 10 Lig. Yet, preferably in other aspects the effectiv e amount ranges from about 50 ^ig to about 100 L g. And, preferably in other aspects, the effective amount ranges from about 40 Lig to about 70 Lig.

1005 1 In some aspects, an immune response can be elicited in a member of the avian species by administering an effective amount of any of the immunomodulator compositions described herein to the member of the av ian species. The effective amount is sufficient to elicit an immune response in the member of the avian species. For example, the effective amount of the immunomodulator for an av ian species may be from about 0.05 Mg to about 10 Mg, from about 0. 1 Mg to about 5 Mg, from about 0.5 Mg to about 1.5 Lig, or from about 1 .0 Lig to about 10 iig. By way of example, suitable effective amounts for a subject that is of the avian species may be about 0. 1 ug, 0.2 Mg. 0.3 Mg, 0.4 ,ug, 0.5 Mg. 0.6 Mg. 0.7 ug, 0.8 Mg. 0.9 . g. 1 Lig, 1.2 Lig, 1 .4 Lig, 1 .6 μg, 1.8 Mg, 2.0 Lig. 2.5 μg, 3.0 Lig, 3.5 ug, 4.0 ug. 4.5 Lig, 5.0 .ug, 5.5 ug, 6.0 Lig, 6.5 Lig, 7.0 _M g, 7.5 Lig, 8.0 Lig, 8.5 Lig, 9.0 Mg, 9.5 Mg, 10.0 _M g, 9.5 μ¾ 10.0 μg, 10.5 g, 1 1 .0 Mg, 1 2 Mg, 13 μg, 14 μg, or 15 μg.

[0056] In some aspects, an immune response can be elicited in a member of the bovine species by administering an effective amount of any of the immunomodulator compositions described herein to the member of the bovine species. The effective amount is sufficient to elicit an immune response in the member of the bovine species. For example, the effective amount of the immunomodulator for a bovine species can be from about 1 μg to about 1000 μg per animal, from about 5 μg to about 500 μg per animal, from about 10 Mg to about 100 μg per animal, from about 10 Mg to about 50 μg per animal, or from about 40 μg to about 60 Lig per animal. By way of example, suitable effectiv e amounts for a subject that is of the bov ine species may be about 30 Lig, 35 μ , 40 Lig. 45 Mg, 50 Mg, 55 M , 60 Mg, 65 Mg, 70 Mg, 75 Mg, 80 Mg. 85 μg. 90 Mg, 95 Mg, 100 μ . 1 10 μg, 120 μg, 130 _M g. 140 Mg, 150 Mg, 160 Mg, 170 Mg, 80 Mg, 190 Mg, 200 Mg or up to 500 Mg, or up to 1000 Mg.

B. Conditions for Use

100571 The methods of the invention elicit an immune response in a subject such that the subject is protected from a disease that is amenable to eiicitation of an immune response. As used herein, the phrase "protected from a disease ^" refers to reducing the symptoms of the disease; reducing the occurrence of the disease; reducing the clinical or pathologic severity of the disease; or reducing shedding of a pathogen causing a disease. Protecting a subject can refer to the ability of a therapeutic composition of the present inv ention, when administered to a subject, to prevent a disease from occurring, cure, and/or alleviate or reduce disease symptoms, clinical signs, pathology, or causes. For example, without l imitation, clinical signs of Bovine Respiratory Disease ( BRD ) include lung lesions, increased temperature, depression (e.g. anorexia, reduced responsiveness to external stimuli, droopy ears), nasal discharge, and respiratory character (e.g. respiratory rate, respiratory effort ). The immunomodulator compositions described herein may be administered to cattle that are suspected of being exposed to BRD to prevent or reduce the severity of the above-described clinical signs of BRD. By way of further example, without l imitat ion, cl inical signs of Marek's disease i n avian subjects includes reduced hatchabiiity and bird survivability. The immunomodulator compositions described herein may be administered to avian subjects that are suspected of being exposed to Marek's disease virus to prevent or reduce the severity of the above- described clinical signs of Marek's disease.

[0058] As such, protecting a subject from a disease encompasses both preventing disease occurrence (prophylactic treatment) and treating a subject that has a disease (therapeutic treatment). In particular, protecting a subject from a disease is accomplished by eliciting an immune response in the subject by inducing a beneficial or protective immune response which may, in some instances, additionally suppress, reduce, inhibit, or block an overactive or harmful immune response. The term "disease" refers to any deviation from the normal health of a subject and includes a state when disease symptoms are present, as well as conditions in which a dev iation ( e.g., infection, gene mutation, genetic defect, etc.) has occurred, but symptoms are not yet manifested.

[0059] Methods of the invention may be used for the prevention of disease, stimulation of effector cell immunity against disease, elimination of disease, alleviation of disease, and prev ention of a secondary disease resulting from the occurrence of a primary disease.

[0060] In some aspects, methods described herein may be used to improve the acquired immune response of the subject when co-administered with a vaccine versus administration of the vaccine by itself. Generally a vaccine once administered does not immediately protect the subject as it takes time to stimulate acquired immunity. The term "improve ^" refers, in the present inv ention, to elicitation of an innate immune response in the subject until the vaccine starts to protect the subject and/or to prolong the period of

protection, v ia acquired immunity, given by the vaccine.

[0061] In some aspects, methods of the invention include administering the composition to protect against infection of a wide variety of pathogens. The composition administered may or may not include a speci ic antigen to elicit a specific response. It is contemplated that the methods of the invention will protect the recipient subject from disease resulting from infectious microbial agents including, without limitation, viruses, bacteria, fungi, and parasites. A skilled artisan will recognize and appreciate that a immunomodulator composition, as described herein, is effective against numerous infectious agents, which are too numerous to list. The infectious agents prov ided herein are provided for exemplary purposes and are provided without limitation of the scope of use.

[0062] Exemplary viral infectious diseases in avian species include, without limitation, those resulting from in fection with chicken in fectious anemia virus (CIAV), Marek's disease virus (MDV), herpesv irus chicken (HCV), herpesvirus turkey (HTV), infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious laryngotracheitis v irus (ILTV), paramyxovirus type 3, av ian

encephalomyelitis (AEV), fowl pox v irus (FPV), fowl cholera, avian influenza vi rus (AIV), rcovirus, av ian leucosis virus (ALV), reticuloendotheliosis virus (REV), avian adenovirus, hemorrhagic enteritis virus (HEV), pneumov irus, pigeon pox virus, recombinants thereof, and other viruses known in the art.

[0063] Exemplary bacterial infections in avian species include, without limitation, those resulting from infection with gram positive bacteria, gram negative bacteria, or fungi such as Bordetella spp., Campylobacter jejuni, Clostridium botulinum, Clostridium colinum, Clostridium perfringens, Erysipelothrix insidiosa, Escherichia coli, Hemophilus gallinarum, Mycoplasma gallisepticum, Mycoplasma meleagridis, Mycoplasma synoviae, Pasteurella multocida, Riemerella anatipestifer, Salmonella spp. , Salmonella enteritidis, Salmonella gallinarum, Salmonella pullorum, and other bacteria known in the art.

[0064] Exemplary fungi or moid infections in avian species include, without limitation, those resulting from infection with Aspergillus flavus, Aspergillus fumigates, Candida albicans, and other infectious fungi or mold known in the art. Exemplary disease condit ions include, without l imitation, those resulting from toxins from gram positive bacteria, gram negative bacteria, or fungi such as Clostridium botulinum toxin, Clostridium perfringens toxins, Escherichia coli enterotoxin, Fusarium mycotoxins Pasteurella leukotoxin, Staphylococcus toxins, and other toxins known in the art.

[0065] Exemplary parasites in avian species include, without limitation, Ascaridia galli, Capillaria annulata, Capillaria contorta, Capillaria obsignata, coccidia spp. , Eimeria meleagridis, Heterakis gallinae, Syngamus trachea, and other parasites known in the art.

[0066] Exemplary viral infectious diseases in bov ine species include, without l imitation, those resulting from infection with bluetongue virus, bovine adenov irus, bov ine cal iciv irus, bov ine coronavirus (BCV), bovine enterovirus, bov ine herpesv irus Type 1 (BHV1), bovine herpesvirus Type 4 (BHV4), bov ine leukemia virus, bov ine parvovirus, bovine reoviras, bovine respiratory syncytial virus (BRSV), bovine rhinovirus, bovine viral diarrhea v irus Type 1 (BVDV1), bovine v iral diarrhea v irus Type 2 (BVDV2 ), in fectious bov ine rhinotrac Ileitis ( I BR ), malignant catarrhal fever virus, parain fluenza virus type 3 (PIV3), rabies virus, vesicular stomatitis virus (VSV), recombinants thereof, and other viruses known in the art.

100671 Exemplary bacterial infections in bov ine species include, without l im itation, those resulting from in fection with gram positive bacteria, gram negative bacteria, or mycobacteria such as Arcanobacterium pyogenes, Bacillus anthracis, Bacillus anthrax, Brucella abortus, Campylobacter fetus, Campylobacter jejuni, Clostridium botulinum, Clostridium chauveoi, Clostridium colinum, Clostridium hemolyticum, Clostridium novyi, Clostridium perfringens, Clostridium septicum, Clostridium tetani, Corynebacterium, Escherichia coli, Fusobacterium necrophorum, Fusobacterium spp., Histophilus somni, Histophilus spp., Leptospira spp., Mannheimia haemolytica, Moraxella spp., Muellerius spp., Mycobacterium paratuberculosis, Mycobacterium spp., Mycoplasma bovirhinis, Mycoplasma bovis, Mycoplasma dispar, Mycoplasma spp., Pasteurella multocida, Salmonella spp., Treponema spp., Ureaplasma diversum, and other bacteria known in the art.

[0068] Exemplary fungi or mold infections in bov ine species includes, without l imitat ion, those resulting from i nfection with Actinobacterim spp., Aspergillus spp., and Histomonas spp., and other infectious fungi or mold known in the art.

1006 1 Exemplary parasites in bovine species include, without l imitation, Anaplasma spp., Anaplasma marginale, Babesia spp., Chorioptes spp., Cooperia, Cysticercus spp., Damalinia bovis, Dermatophilus spp., Dictylocaulus spp., Eimeria spp., Eperythrozoon spp., Fascioloides spp., Haemonchus spp., Melophagus spp., Muellerius spp., Nematodirus spp., Neospora spp., Oestrus spp., Ostertagia spp., Psoroptes spp., Sarcoptes spp., Serpens spp., Strongyloides spp., Toxoplasma spp., Trichophyton spp., Trichostrongylus, Trichuris spp., and Tritrichomonas spp., and other parasites known in the art.

100701 Exemplary infectious disease agents in bovine species also include those agents causing mastitis, metritis, cryptosporidiosis, and any other infectious disease the bov ine species is suscept ible to. C. Administration

[0071] A variety of administration routes are available. The particular mode selected will depend, of course, upon the particular biological agents selected, the age and general health status of the subject, the particular condition being treated and the dosage required for therapeutic efficacy. The methods of this invention may be practiced using any mode of administration that produces effective levels of an immune response without causing clinically unacceptable adverse effects. The compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art.

[0072] The immunomodulator composition may be administered intravenously, intramuscularly, intradermally, intraperitoncally, subcutaneously, by spray, in ovo by feather follicle method, orally, intraocuiarly, intratracheaily, intranasal ly, or by other methods known in the art. In one aspect, the immunomodulator is administered subcutaneously. In another aspect, the immunomodulator may be administered intramuscularly. In another aspect, the immunomodulator is administered as a spray. In another aspect, the immunomodulator may be administered orally.

[0073] In one respect, the immunomodulator may be administered by itself to the subject prior to challenge (or infection ). In another aspect, the immunomodulator may be administered by itself to the subject post challen e (or infection ). In another aspect, the immunomodulator may be administered by itself to the subject at the same time as challenge (or infection).

[0074] In some aspects, the immunomodulator composition may be co-administered at the same time as the vaccination prior to challenge. In some aspects, the immunomodulator composition may be co-administered at the same time as the vaccination at the same time as challenge (or infection). In some aspects, the co-administration may include administering the vaccine and immunomodulator in the same general location on the subject at two different sites next to each other (i.e., injections next to each other at the neck of the subject), on opposing sides of the subject at the same general location (i.e., one on each side of the neck), or on different locations of the same subject. In some aspects, the immunomodulator composition can be administered prior to vaccination and challenge. In some aspects, the immunomodulator composition may be administered after vaccination but prior to challenge. The immunomodulator composition can be administered after challenge to a subject that has been vaccinated prior to challenge (or infection ). 1007 1 A skilled artisan will recognize that administration routes may vary depending upon the subject and the health or state of the subject. The administration routes provided for avian and bov ine species are for exemplary purposes and arc provided without limitation.

[0076] Vaccination of avian species may be performed at any age. Vaccinations may be administered to 18 day old embryos (in ovo) and above for a live microorganism and 3 weeks and older for an inactivated microorganism or other type of vaccine. For in ovo vaccination, vaccination may be administered in the last quarter of development. The vaccine may be administered subcutaneous ly, by the feather follicle method, by spray, orally, intraocularly, intratracheally, intranasally, in ovo, or by other methods know in the art. Oral vaccines may be administered in drinking water. Further, it is contemplated that the methods of the invention may be used based on routine vaccination schedules.

[0077] The immunomodulator composition may also be administered to an avian species subcutaneously, by the feather follicle method, by spray, intraocularly,

intratracheally. intranasally, in ovo, or by other methods known in the art. For example, the immunomodulator composition can be administered in ovo. Alternatively, the

immunomodulator composition can be administered as a spray.

[0078] The immunomodulator composition can be administered in ovo to an avian embryo in the last quarter of its development. For example, the immunomodulator composition can be administered in ovo to a 18-day-old or 1 -day-old embryo. The administration to the egg may be prior to challenge (or infection) or post challenge.

[007 1 The immunomodulator can be administered to an animal of the avian or bov ine species from about 1 to about 14 days prior to challenge or from about 1 to about 14 days post challenge. For example, the immunomodulator can be administered from about 1 to about 7 days prior to challenge or from about 1 to about 7 days post challenge. The immunomodulator is suitably administered 1, 2, 3, 4, 5, 6, 7 days prior to chal lenge or 1, 2, 3, 4, 5, 6, 7 days post challenge.

[ 00801 Vaccination of bov ine species may be performed at any age. The v accine may be administered intravenously, intramuscularly, intradermally, intrapcritoneally,

subcutaneously, by spray, orally, intraocularly, intratracheally, intranasally, or by other methods known in the art. Further, it is contemplated that the methods described herein may be used based on routine vaccination schedules. 100811 Other del ivery systems may include time-release, delayed release, or sustained release delivery systems. Such systems can avoid repeated administrations of the

compositions therefore increasing convenience. Many types of release deliv ery systems are available and known to those of ordinary skill in the art. They include polymer based systems such as poly(lactide-giycoiide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, poiyhydroxybutyric acid, and polyanhydrides. M icrocapsules of the foregoing polymers containing drugs are described in, for example, U.S. Patent No.

5,075,109.

[0082] Delivery systems also include non-polymer systems that are lipids including sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-, di- , and tri-glycerides; hydrogei release systems; silastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to, erosionai systems in which an agent of the inv ention is contained in a form within a matrix such as those described in U.S. Patent Nos. 4,452,775, 4,675,189, and 5,736, 152, and diffusional systems in which an activ e component permeates at a controlled rate from a polymer such as described in U.S. Patent Nos. 3, 854,480, 5, 133,974, and 5,407,686. In addition, pump-based hardware delivciy systems can be used, some of which are adapted for implantation.

[0083] As various changes could be made in the above composition, products, and methods without departing from the scope of the invention, it is intended that all matter contained in the above description and in the examples given below shall be interpreted as illustrative and not in a limiting sense.

DEFINITIONS

[0084] The term "effective amount" refers to the amount necessary or sufficient to realize a desired biologic effect. For example, an effective amount of immunomodulator for treating or prev enting an infectious disease is that amount necessary to cause the

development of an immune response upon exposure to the microbe, thus causing a reduction in the amount of microbe within the subject and preferably the eradication of the microbe. The effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the size of the subject, or the severity of the disease or condition. One f ordinary skill in the art can empirical ly determine the effective amount of immunomoduiator without necessitating undue experimentation.

[0085] The term "cytokine" refers to an immune enhancing protein family. The cytokine family includes hematopoietic growth factor, interleukins, interferons,

immunoglobulin superfamily molecules, tumor necrosis factor family molecules and chemokines (i.e. proteins that regulate the migration and activat ion of cells, particularly phagocytic cells). Exemplary cytokines include, without l imi tation, intcrleuki n-2 (IL-2), interleukin-12 (IL- 12), interleukin- 1 5 (IL- 15), interleukin-18 (IL- 18), interferon-a (IFNa), and i nterferon -y (IFNy).

[0086] The term "elicit" can be used interchangeably with the terms activate, stimulate, generate or upregulate.

[0087] The term "eliciting an immune response" in a subject refers to specifically control l ing or influencing the activity of the immune response, and can include activating an immune response, upregulating an immune response, enhancing an immune response and. or altering an immune response (such as by eliciting a type of immune response which in turn changes the prevalent type of immune response in a subject from one which is harmful or ineffective to one which is beneficial or protective).

[0088] The term "operativeiy linked" refers to linking a nucleic acid molecule to a transcription control sequence in a manner such that the molecule is able to be expressed when transfected (i.e., transformed, transduced or transfected) into a host cell. Transcriptional control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription in itiation, such as promoter, enhancer, operator and repressor sequences. A variety of such transcription control sequences are known to those skilled in the art. Preferred transcription control sequences include those which function in avian, fish, mammalian, bacteria, viral, plant, and insect cells. While any transcriptional control sequences may be used with the invention, the sequences may include natural ly occurring transcription control sequences natural ly associated with a sequence encoding an immunogen or immune stimulating protein.

[0089] The terms "nucleic acid molecule" and "nucleic acid sequence" can be used interchangeably and i nclude DNA, RNA, or derivativ es of either DNA or RNA. The terms also include oligonucleotides and larger sequences such as plasmids, such as the immunostimulatory plasmids described herein, and including both nucleic acid molecules that encode a protein or a fragment thereof, and nucleic acid molecules that comprise regulatory regions, introns, or other non-coding DNA or RNA. Typically, an oligonucleotide has a nucleic acid sequence from about I to about 500 nucleotides, and more typically, is at least about 5 nucleotides in length. The nucleic acid molecule can be derived from any source, including mammalian, fish, bacterial, insect, viral, plant, synthetic sources or combinations thereof. A nucleic acid molecule can be produced by methods commonly known in the art such as recombinant DNA technology (e.g., polymerase chain reaction (PGR), amplification, cloning) or chemical synthesis. Nucleic acid molecules include natural nucleic acid molecules and homologues thereof, including, but not limited to, natural allelic variants and modified nucleic acid molecules in which nucleotides have been inserted, deleted, substituted, or inverted in such a manner that such modifications do not substantially interfere with the nucleic acid molecule's ability to elicit an immune response useful in the methods of the present inv ention. A nucleic acid homologue may be produced using a number of methods known to those skilled in the art (see, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, 1989), which is incorporated herein by reference.

100901 The terms "selectable marker ^" and "selectable marker gene ^" refer to a gene that encodes a product that protects the organism in which the gene is expressed from a selective agent (e.g., an antibiotic) or a condition that would normally kill the organism or inhibit its growth. Selectable marker genes are most commonly antibiotic resistance genes (e.g., kanamycin resistance genes, ampiciliin resistance genes, chloramphenicol resistance genes, tetracycline resistance genes, etc.). Thus, for example, when E. coli cells are subjected to a transformation procedure to introduce a piasmid encoding a kanamycin resistance gene and then grown on or in media containing kanamycin, only the E. coli cells that have successfully taken up the piasmid and expressed the kanamycin resistance gene will survive. The terms "selectable marker" and "selectable marker gene" also include genes that code for enzymes involved in the synthesis of a compound that is essential for the growth of an organism. When introduced into an auxotrophic organism that is unable to synthesize the essential compound, such genes allow the organism to grow in a medium that has been supplemented with the essential compound. For example, bacterial celis that are auxotrophic for the amino acid lysine due to a mutation in or the absence of an enzyme involved in lysine biosynthesis normally are unable to grown on media that has not been supplemented with lysine. When such bacteria arc subjected to a transformation procedure to introduce a pi asm id encoding the enzyme involved in lysine biosynthesis, the bacteria that have successfully taken up the plasmid and expressed the enzyme will survive when grown on media that has not been supplemented with lysine. The terms "selectable marker" and "selectable marker gene" further include genes that allow for poison/antidote selection. For example, the ccdB gene encodes a protein that binds to DNA gyrase, an essential enzyme for cell division. Upon binding to DNA gyrase, the ccdB gene product impairs gene replication and induces cell death. Thus, bacterial expressing the ccdB gene product cannot survive. The ccdA gene encodes a protein (the "antidote") that acts as a natural inhibitor of the ccdB gene product. Thus, when bacteria hav ing the ccdB gene in their bacterial genome are subjected to a transformation procedure to introduce a plasmid encoding the ccdA gene product, only the cells that successfully take up the plasmid and express the ccdA gene will survive.

[0091] The terms "screenable marker" and "screenable marker gene" refer to a gene that encodes a product that allows an observer to distinguish between cells expressing the screenable marker gene and ceils that are not expressing the screenable marker gene.

Screenable marker gene systems are well known in the art and include, for example, lacZ genes and genes encoding fluorescent proteins such as green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein ( RFP ), blue fluorescent protein (BFP), or cyan fluorescent protein (CFP).

[0092] As used herein, the term "subject" refers to a living organism having a central nervous system. In particular, subjects include, but are not limited to, human subjects or patients and companion animals. Exemplary companion animals may include domesticated mammals (e.g., dogs, cats, horses), mammals with significant commercial value (e.g., avian species, bovine species, daily cows, beef cattle, sporting animals), mammals with significant scientific values (e.g., captive or free specimens of endangered species), or mammals which otherwise have value. Suitable subjects also include: mice, rats, dogs, cats, ungulates such as cattle, swine, sheep, horses, and goats, lagomorphs such as rabbits and hares, other rodents, and primates such as monkeys, chimps, and apes. Subjects may be any member of the avian species, whether domestic or wild, and may be commercially reared for breeding, meat or egg production. Exemplary avian species include, without limitation, chickens, turkeys, geese, ducks, pheasants, quail, pigeons, ostriches, caged birds, birds in zoological collections and aviaries and the l ike. Subjects may be any member of the bovine species, whether domestic or wild, and may be commercially reared for breeding, meat or mi l production. Exemplary bovine species include, without limitation, antelopes, buffalos, yaks, cattle, bison, and the like. Species of cattle include, without limitation, cows, bulls, steers, heifer, ox, beef cattle, daily cattle, and the like. Subjects may be any member of an aquaculture species, including without li mitation, any species of fish, crustaceans, molluscs, liv ing in freshwater or saltwater. In some aspects, subjects may be diagnosed with an infectious disease, may be at risk for an infectious disease, or may be experiencing an in fect ious disease. Subjects may be of any age including in utcro, new born, adolescence, adult, middle age, or elderly.

EXAMPLES

[0093] The following non-limiting examples are provided to further illustrate the present invention.

Example 1 : Preparation of the pGCMB75.6 plasmid.

100941 A map of pGCMB75.6 is shown in FIG. 2. In pGCMB75.6, the kanamycin- resistanec gene of pMB75.6 (see F IG. 1) has been replaced by non-coding sequence from E. coli K- 12. To generate the pGCMB75.6 plasmid, an AscI single cutter restriction site was introduced into pMB75.6 at a site 5' of the kanamycin resistance gene to generate pM B75.6 AscI (SEQ I D NO: 3). To introduce the Asc I restriction site, an adenine present in the sequence of pGCMB75.6 was mutated to a guan ine, thereby changing the sequence AGCGCGCC in the pGCMB75.6 plasmid to GGCGCGCC. This modi fication was accomplished using a mutagenesis-based approach by ligation during amplification. A single primer was used, which carried the Asc I restriction site in its middle.

100951 A 1779 nt long AscI ( GGCGCGCC ) / Xhol ( CTCGAG ) fragment was synthesized by Life Technologies GmbH ( Darmstadt, Germany ). This fragment contained fiv e regions comprising non-coding sequence from E. coli K- 1 2 (exchanges 1-5, abbreviated as "exc. 1 ," "exc. 2, ^" etc. in FIG 2), an F l origin of replication, and a truncated lacZ gene (see FIG. 2). In the case of exchange 4, the E. coli sequence was manually changed at several positions in order to increase the GC-content of the plasmid. In addition, in exchange 2, a single nucleotide was changed to delete a Dral restriction site, so that there would only be a single Dral site in the plasmid. 1009 1 As shown in FIG. 2, pGCMB75.6 contains several regulatory elements (an F l origin of replication, a CMV promoter, and a pUC origin of replication) and a multiple cloning site. pGCMB75.6 also contains a truncated lacZ gene. However, pGCMB75.6 does not comprise any full-length or functional selectable or screenable marker genes.

pGCMB75.6 is 4242 bp in length and contains 283 CpG dinucleotides.

100971 One positive clone, carrying the new introduced AscI site (p B75. AscI ) was digested with AscI ( GGCGCGCC) / Xhol (CTCGAG ) to generate a 2463 fragment containing the CMV promoter, multiple cloning site, and pUC origin of replication (see FIG. 2). The 1779 nt AscI ( GGCGCGCC) / Xhol (CTCGAG ) synthesized fragment was digested with AscI / Xhol as well.

100981 Both fragments (the 2463 nt vector fragment and the 1779 nt insert fragment) were gel el u ted from a 1% agarose gel by using the QIAquick Gel Extraction Kit from Qiagen. A ligation was performed using 600 ng of the 1779 nt fragment and 400 ng of the 2463 nt pM B75.6 AscI vector fragment in a total volume of 20 μΐ, overnight at 16°C. The 20 μΙ was then dialyzed for 2h against 20 ml H2O on ice, and then mixed with 5 μΐ electro- competent DH5a E. coli cells and 40 μ I Η.Ό ( Invitrogen F-f801acZAM15 Δ ( lacZYA-argF) U169 recA 1 endA 1 hsdR 1 7 ( rk-, mk+) gal- phoA supE44 λ- tlii- 1 gyrA96 re I A 1 ; Lot no. 1376481, Part no. 44-0097 ). Following transformation and 1 h regeneration in SOC media, the cells were plated on LB plates without kanamycin at a 10 dilution. This dilution allowed for isolation of individual clones.

[0099] Colony PGR was performed on individual clones using the following primers listed in Table 1.

Table 1. PGR primers.

1001001 The locations of the Ai l- 13 rev and AH-22 for primers arc shown in FIG. 2. A premix containing Taq polymerase was used. PGR reactions were loaded onto a 1% agarose gel containing ethidium bromide. Positive clones which carried the correct plasmid (pGCMB75.6) showed a product of 606 bp. [00101 1 A total of more than 10,000 clones were screened to identify three positive clones. The three positive clones were immediately transferred to fresh LB plates and were used to create 3 Research Cell Banks (RGB; Sys 3733, Sys 3734 and Sys 3735).

[00102] In parallel, medium was inoculated with these three clones and plasmid DNA was generated. Complete sequence certification of each of the three subclones was done using the following primers listed in Table 2.

Table 2. PGR for sequence certification.

Example 2 : Generation of the p LacZ.M B75.6 plasmid.

[001031 To generate the pLacZMB75.6 plasmid (FIG. 3 ; SEQ I D NO: 4), a 1307 nt Xhol ( CTCGAG ) / Dral (TTTAAA ) fragment was synthesized by Life Technologies GmbH ( Darmstadt, Germany ). This 1307 nt fragment contained a portion of the lacZ gene (265 nt). Thus, when l igated into pGCMB75.6, this fragment elongates the truncated LacZ gene located upstream of the Xhol restriction site and allows the LacZ gene to be expressed (compare FIG. 2 and FIG. 3). In addition 9 1 nt of the multiple cloning site were exchanged with E. coli non coding sequence, in order to el iminate sequence homology with the newly introduced LacZ gene region and avoid recombination. I n addition, the 5 ' region of the CMV promoter was deleted ( 265 nt) in order to generate a plasmid of the same size as plasmid pGCMB75.6. [00104] pGCMB75.6 and the 13 1 1 nt synthesized fragment were both digested with Xhol and Dral. Both fragments were gel e In ted from a 1% agarose gel by using the QIAquiek Gel Extraction Kit from Qiagen.

[00105] A ligation was performed using 240 ng of the 13 1 1 nt fragment and 240 ng of the vector fragment in a total volume of 20 μΐ, overnight at 16°C. 3 μ Ι were mixed with 10 μΐ electro-competent DH5a E. coli cells (Invitrogen F-f801acZAM15 Δ ( lacZYA-argF ) U169 recA 1 endA I hsdR17 ( rk-, mk+) gal- phoA supE44 λ- tli i- 1 gyrA96 relA l ; Lot no. 1376481, Part no. 44-0097 ) and 40 μ I 10% Glycerin solution. Fol lowing transformation and 1 h regeneration in SOC media, the cells were plated on LB X-Gal I PTG plates without kanamycin at a 10 ' and 10 ¹ dilution. This dilution allowed for isolation of individual clones.

[00106] Identification of colonies with plasmid was performed via blue/white selection and colony PGR was performed to confirm using the following primers listed in Table 3.

Table 3. PGR primers for colony confirmation.

[00107] The locations of the A H- 15 and AH-39 primers are shown in FIG. 3. A premix containing Taq polymerase was used. PGR reactions were loaded onto a 1% agarose gel containing ethidium bromide. Positive clones which carried the correct plasmid

(pLacZMB75.6) showed a product of 777 bp.

[00108] Four positive clones were identified and immediately transferred to fresh LB plates and were used to create Research Cell Banks (RCB; Sys 3736, Sys 3737, Sys 3738 and Sys 3739).

[00109] In parallel, medium was inoculated with these four clones and plasmid DNA was generated. Complete sequence certification of each of the four subclones was done using the same pri mers as for pGCMB75.6 but instead of using primer AH- 16 (SEQ I D NO: 10) pri mer A H -24 was used ( See Table 4).

Table 4. PGR primer for A H-24.

A H-24 CGCGTAATACGACTCACTATAG (SEQ I D NO. 17) Example 3: Immunomodulator composition

[00110] The immunomodulator is a composition comprising a cationic lipid and non-coding DNA sequence described herein. The synthetic immunomodulator lipid components [l-[2-[9-(Z)-octadecenoyloxy]]-2-[8](Z)-heptadecenyi]-3- [hydroxyethy I ] i m idazol i n i urn chloride (DOTIM) and a synthetic neutral lipid cholesterol arc formulated to produce liposomes approximately 200 nm in diameter ( See, U.S. Patent 6,693,086). The DNA component is pGCMB75.6 or pLacZMB75.6. Being negatively charged, the plasmid DNA associates with the pos i t i ve ly-charged (cationic) liposomes (See, U.S. Patent 6,693,086).

Example 4: Immunomodulator composition administration to mammal model increased survivability in virus challenge.

[001 1 1 1 The efficacy of the immunomodulator compositions described herein was evaluated in a mammal model challenged with pathogen. M ice were admi nistered the immunomodulator composition, containing pGCMB75.6 DNA and cationic liposomes, via intraperitoneal injection. The immunomodulator composition was administered at 0.01 iig. 0.02 Lig, 0.04 ng, and 1 .00 ug concentrations. Control m ice were administered 0.9% NaCl solution via intraperitoneal in jection. Twenty-four hours post-immunomodulator

admi nistration, all animals were chal lenged with vi ral appl ication v ia intraperitoneal injection (0.2 ml 10 ^{3 4} K I t) (i m l ). The challenge virus was Pseudorabies ( PR ). The survivabi lity rates are depicted in FIG. 4. M ice receiving an immunomodulator composition described herein had higher dose dependent survivabi lity rates than cont rol mice.

Example 5: Immunomodulator composition administration to avian model increased hatchability and survivability in pathogen challenge.

[00112] The efficacy of the immunomodulator compositions described herein was ev aluated in an avian model chal lenged with pathogen by spray on day El 9 (embryonic day 19). Chicken eggs were administered an immunomodulator composition, containing pGCMB75.6 DNA and cationic liposomes, via in ovo administration on day E18 ( embryonic day 18). Control eggs received a placebo control di l uent (D5W) (in ovo). Bil l broth (spray) was used as mock challenge (T l). The details of the study treatment groups are provided in Table 5 below. Table 5. Avian I in in u n o in od u lat o r composition treatment descriptions.

¹ Day 18 of egg incubation; ² 16 eggs per flat; ³ 64 eggs per sub-tray (partitioned to accommodate two groups per tray); ⁴ The chicks hatched from each section of the tray were transferred to one pen; ³ Avian pathogenic Escherichia coli (APEC).

1001 1 1 The DNA component of the immunomodulator composition included one of three clones isolated of pGCMB75.6 DNA (i.e. Jv77, X5872, or X5928). As can be observed from the results summarized in Table 6 below, both the average hatchability and average survivabi l ity were improved when an immunomodulator composition described herein was administered compared to eggs receiving control treatment.

Table 6. Avian hatchability and survivability rates.*

*A11 val idation tests passed ( comparison between two control groups ). Significant difference compared with the Control Chall group. [00114] When introducing elements of the present invention or the preferred embodiments(s) thereof, the articles "a", "an", "the" and "said" are intended to mean that there are one or more of the elements. The terms "comprising", "including" and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[00115] In view of the above, it will be seen that the several objects of the invention are achieved and other advantageous results attained.

[00116] As various changes could be made in the above products, compositions, and methods without departing from the scope of the inv ention, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as i l l ustrative and not in a l imiting sense.