HK1190628A - Enhanced immune response in bovine species - Google Patents

Description

Enhanced immune response in bovine species

Technical Field

The present invention relates to a method of immune activation in a member of the bovine species. In particular, the invention includes methods of eliciting systemic, non-specific and antigen-specific immune responses for administration to animals and protection against infectious diseases.

Background

Cattle are the primary target for many types of viral, bacterial and parasitic infections. Modern production practices such as weaning of cattle in the beef and dairy industries, transport of cattle, inclement weather and nutritional requirements can also become risk factors for disease development. Bovine Respiratory Disease (BRD), or as it is commonly referred to as a complex of bovine respiratory disease, occurs in dairy and beef cattle and is one of the leading causes of economic loss in cattle industry worldwide. These losses are due to morbidity, mortality, reduced weight gain, treatment and prevention costs, loss of milk production and negative impact on carcass characteristics (carcas characteristics).

The onset of BRD is thought to result from the various environmental and physiological stressors described above that are accompanied by infectious pathogens. Considered as haemolytic Mannheim (M.), (Mannheimiahaemolytica) (Pasteurella haemolytica: (B.haemolytica))Pasteurellahaemolytica) Pasteurella multocida (B), Pasteurella multocida (B) ((B))Pasteurella multocida) And Hippophaophila somni: (Histophilus somni) (formerly Haemophilus somnus) is part of the normal flora of the upper respiratory tract of cattle. In contrast, the lower respiratory tract is a relatively sterile environment maintained by a number of immunological pathways aimed at preventing the entry of microorganisms. When cattle are subjected to environmental and physiological stressors, the innate and acquired immune functions of the animal are compromised, thereby allowing these aforementioned organisms to proliferate and subsequently colonize the lower respiratory tract. Various bovine viruses are known to have immunosuppressive effects in the lung, such as infectious bovine rhinotracheitis virus (IBRV, IBR or BHV1), Bovine Viral Diarrhea Virus (BVDV), Bovine Respiratory Syncytial Virus (BRSV) and parainfluenza virus type 3 (PI 3). However, to date, mannheimia haemolytica is the most prevalent bacterial pathogen in cases of BRD.

Current prevention and treatment of BRD consists of administration of antibiotics (i.e. metaphalias) to the herd after arrival at the feedlot, antibiotic treatment of sick cattle, and vaccination against BRD virus and bacteria including mannheimia haemolytica.

There are different reasons why current vaccination programs and drug therapies are not optimally suited to control BRD in today's cattle. First, the host defense system plays a major role in combating infectious diseases in cattle. Conventional treatments include administration of antibiotics to treat or control bacterial infections. However, there is no approved drug treatment available for viral infections. For BRD, in most cases there is not only a bacterial infection but also a viral infection. Second, the timing of vaccination is often suboptimal. For respiratory vaccines to be most effective, the product should be administered 2-4 weeks prior to stress or shipping, and this is not generally feasible in commercial cattle production. The vaccine is administered too early or too late to be most effective.

Thus, there is a need for methods of stimulating the immune system and establishing a challenge response to reduce or eliminate disease-causing organisms. It is important that the method be easy to administer, work alone or in combination with vaccines, or help make such vaccines more effective, have a longer duration or do not require increased injections to maximize immunity. The present invention provides methods of eliciting a non-antigen specific immune response in bovine species that is easy to administer, acts alone or in combination with a vaccine, induces a protective response against one or more infectious pathogens.

Brief Description of Drawings

Figure 1.1 illustrates mean rectal temperature data according to the dose of immunomodulator administered as described in example 1.

Figure 1.2 illustrates mean daily weight gain data according to the dose of immunomodulator administered as described in example 1.

Figure 1.3 illustrates model-corrected lung injury scores for doses of immunomodulatory agents administered as described in example 1.

Figure 2.1 illustrates mean rectal temperature data according to the dose of immunomodulator administered as described in example 2.

Figure 2.2 illustrates mean daily weight gain data according to the dose of immunomodulator administered as described in example 2.

Figure 2.3 illustrates model-corrected lung injury scores for doses of immunomodulatory agents administered as described in example 2.

Figure 3.1 illustrates model-corrected lung injury scores for doses of immunomodulatory agents administered as described in example 3.

Figure 3.2 illustrates the lung injury score corrected for the model as described in example 3 for the day of immunomodulator administration.

Figure 4.1 illustrates the percentage of animals protected by the treated group as described in example 4.

Figure 4.2 illustrates the percentage of animals protected by the treated group (<1% lung injury and no lung injury) as described in example 4.

Figure 5.1 illustrates measurements of CD 25 EI expression index (y-axis) in BHV-1 infected cells for all five cell types (x-axis) for each of 6 treatment groups as described in example 5.

Fig. 5.2 illustrates the measurement of CD 25 EI expression index (y-axis) in BRSV infected cells for all five cell types (x-axis) for each of the 6 treatment groups as described in example 5.

Fig. 5.3 illustrates measurements of CD 25 EI expression index (y-axis) in cells infected with BVDV type 1 for all five cell types (x-axis) of each of the 6 treatment groups as described in example 5.

Fig. 5.4 illustrates measurements of CD 25 EI expression index (y-axis) in cells infected with BVDV type 2 for all five cell types (x-axis) of each of the 6 treatment groups as described in example 5.

Figure 5.5 illustrates graphically the measurement of IFN γ expression index (y-axis) in cells infected with BHV-1 for all five cell types (x-axis) of each of the 6 treatment groups as described in example 5.

Figure 5.6 illustrates the measurement of IFN γ expression index (y-axis) in BRSV infected cells for all five cell types (x-axis) of each of the 6 treatment groups as described in example 5.

Figure 5.7 illustrates the measurement of IFN γ expression index (y-axis) in cells infected with BVDV type 1 for all five cell types (x-axis) of each of the 6 treatment groups as described in example 5.

Figure 5.8 illustrates measurements of IFN γ expression index (y-axis) in cells infected with BVDV 2 type for all five cell types (x-axis) of each of 6 treatment groups as described in example 5.

FIG. 5.9 illustrates measurements of IL-4 expression index (y-axis) in BHV-1 infected cells for all five cell types (x-axis) for each of 6 treatment groups as described in example 5.

Fig. 5.10 illustrates the measurement of IL-4 expression index (y-axis) in BRSV infected cells for all five cell types (x-axis) for each of the 6 treatment groups as described in example 5.

FIG. 5.11 illustrates the measurement of IL-4 expression index (y-axis) in cells infected with BVDV1 type for all five cell types (x-axis) for each of 6 treatment groups as described in example 5.

FIG. 5.12 illustrates the measurement of IL-4 expression index (y-axis) in cells infected with BVDV 2 type for all five cell types (x-axis) for each of 6 treatment groups as described in example 5.

Fig. 5.13 illustrates model corrected serum antibody titer estimates (y-axis) in BVDV type 1 infected cells for all five cell types (x-axis) of each of the 6 treatment groups as described in example 5.

Fig. 5.14 illustrates model corrected serum antibody titer estimates (y-axis) in BVDV type 2 infected cells for all five cell types (x-axis) of each of the 6 treatment groups as described in example 5.

Figure 5.15 illustrates model corrected serum antibody titer estimates (y-axis) in BHV-1 infected cells for all five cell types (x-axis) for each of 6 treatment groups as described in example 5.

Fig. 5.16 illustrates the average daily increase results for model correction as described in example 5.

Figure 6.1 illustrates BHV1 SNT titers for the treatment groups as described in example 6.

Detailed Description

The methods of the invention for eliciting an immune response in a member of a bovine species comprise administering to a member of a bovine species an effective amount of an immunomodulator composition to elicit an immune response. An immunomodulator composition comprises a liposome delivery vehicle and at least one nucleic acid molecule. In addition, when administered prior to, co-administered with, post-vaccinated with, or mixed with such vaccines, the immunomodulator elicits a non-antigen specific immune response that is effective alone or enhances the effect of at least one biological agent, such as a vaccine.

The methods provide novel therapeutic strategies for protecting bovine species from infectious disease and treating populations with infectious disease. Finally, when the immunomodulator is used in combination with a vaccine, the method of the present invention provides more rapid, longer-lasting and better protection against disease.

1. Composition comprising a metal oxide and a metal oxide

a. Immunomodulator

In one embodiment of the invention, an immunomodulator composition comprises a liposome delivery vehicle and at least one nucleic acid molecule, as described in U.S. patent No. 6,693,086, which is incorporated herein by reference.

Suitable liposome delivery vehicles include lipid compositions capable of delivering nucleic acid molecules to the tissue of the subject being treated. The liposomal delivery vehicle is preferably capable of remaining stable in the subject for a sufficient amount of time to deliver the nucleic acid molecule and/or biological agent. In one embodiment, the liposome delivery vehicle is stable in the recipient subject for at least about 5 minutes. In another embodiment, the liposome delivery vehicle is stable in the recipient subject for at least about 1 hour. In yet another embodiment, the liposome delivery vehicle is stable in the recipient subject for at least about 24 hours.

The liposome delivery vehicles of the present invention include lipid compositions that are capable of fusing with the plasma membrane of a cell to deliver a nucleic acid molecule into the cell. In one embodiment, when delivered, the nucleic acid: liposome complex of the invention is at least about 1 picogram (pg) of expressed protein per milligram (mg) of total tissue protein per microgram (μ g) of delivered nucleic acid. In another embodiment, the transfection efficiency of the nucleic acid: liposome complex is at least about 10 pg expressed protein per mg total tissue protein per μ g delivered nucleic acid; and in yet another embodiment, at least about 50 pg of expressed protein per mg of total tissue protein per μ g of delivered nucleic acid. The transfection efficiency of the complex can be as low as 1 femtograms (fg) of expressed protein per mg of total tissue protein per μ g of delivered nucleic acid, with such amounts being more preferred.

Preferred liposomal delivery vehicles of the invention are those having a diameter of about 100 to 500 nanometers (nm), in another embodiment, about 150 to 450 nm and in yet another embodiment, about 200 to 400 nm.

Suitable liposomes include any liposome, such as those commonly used, for example, in gene delivery methods known to those skilled in the art. Preferred liposome delivery vehicles include Multilamellar Liposome (MLV) lipids and extruded lipids. Methods for preparing MLVs are well known in the art. More preferred liposome delivery vehicles include liposomes with polycationic lipid compositions (i.e., cationic liposomes) and/or liposomes with a cholesterol backbone conjugated with polyethylene glycol. Exemplary cationic liposome compositions include, but are not limited to, N- [1- (2, 3-dioleoyloxy) propyl ] -N, N, N-trimethylammonium chloride (DOTMA) and cholesterol, N- [1- (2, 3-dioleoyloxy) propyl ] -N, N, N-trimethylammonium chloride (DOTAP) and cholesterol, 1- [2- (oleoyloxy) ethyl ] -2-oleyl-3- (2-hydroxyethyl) imidazolinium chloride (DOTIM) and cholesterol, Dimethyl Dioctadecyl Ammonium Bromide (DDAB) and cholesterol, and combinations thereof. The most preferred liposome composition for use as a delivery vehicle includes DOTIM and cholesterol.

Suitable nucleic acid molecules include any nucleic acid sequence, such as coding or non-coding sequences, as well as DNA or RNA. The coding nucleic acid sequence encodes at least a portion of a protein or peptide, while the non-coding sequence does not encode any portion of a protein or peptide. According to the invention, a "non-coding" nucleic acid may comprise a regulatory region of a transcription unit, such as a promoter region. The term "empty vector" is used interchangeably with the term "non-coding" and refers specifically to a nucleic acid sequence in which no protein-coding part is present, e.g., a plasmid vector that does not contain a gene insert. Expression of the protein encoded by the nucleic acid molecule is not required to elicit a non-antigen specific immune response; the nucleic acid molecule does not therefore have to be operably linked to a transcriptional regulatory sequence. However, further advantages (i.e., antigen specificity and enhanced immunity) can be obtained by including nucleic acid sequences (DNA or RNA) encoding the immunogen and/or cytokine in the composition.

Complexation of liposomes with nucleic acid molecules can be achieved using methods standard in the art or described in U.S. patent No. 6,693,086 (incorporated herein by reference). Suitable concentrations of nucleic acid molecules added to the liposomes include concentrations effective to deliver sufficient amounts of nucleic acid molecules into a subject to elicit a systemic immune response. In one embodiment, about 0.1 μ g to about 10 μ g of nucleic acid molecules are combined with about 8 nmol of liposomes, in another embodiment about 0.5 μ g to about 5 μ g of nucleic acid molecules are combined with about 8 nmol of liposomes, and in yet another embodiment about 1.0 μ g of nucleic acid molecules are combined with about 8 nmol of liposomes. In one embodiment, the ratio of nucleic acids to lipids in the composition (μ g nucleic acids: nmol lipids) is at least about 1:1 nucleic acids: lipids by weight (i.e., 1 μ g nucleic acids: 1 nmol lipids), and in another embodiment, at least about 1:5, and in yet another embodiment, at least about 1:10, and in a further embodiment, at least about 1: 20. The proportions expressed herein are based on the amount of cationic lipid in the composition, and not on the total amount of lipid in the composition. In another embodiment, the ratio of nucleic acids to lipids in the compositions of the invention is from about 1:1 to about 1:80 nucleic acids to lipids by weight; and from about 1:2 to about 1:40 nucleic acid: lipid, by weight, in another embodiment; and in further embodiments, from about 1:3 to about 1:30 nucleic acid: lipid; and from about 1:6 to about 1:15 nucleic acid to lipid, by weight, in yet another embodiment.

b. Biological agent

In another embodiment of the invention, the immunomodulator comprises a liposome delivery vector, a nucleic acid molecule and at least one biological agent.

Suitable biological agents are agents effective in the prevention or treatment of bovine disease. Such biologies include immunopotentiating proteins, immunogens, vaccines, antimicrobials, or any combination thereof. Suitable immune enhancing proteins are those proteins known to enhance immunity. By way of non-limiting example, cytokines, including protein families, are a family of proteins known to enhance immunity. Suitable immunogens are proteins that elicit a humoral and/or cellular immune response such that administration of the immunogen to a subject achieves an immunogen-specific immune response against the same or similar proteins encountered within the tissues of the subject. Immunogens may include pathogenic antigens expressed by bacteria, viruses, parasites, or fungi. Preferred antigens include antigens that cause infectious disease in a subject. According to the present invention, the immunogen may be any part of a protein of natural or synthetic origin, which elicits a humoral and/or cellular immune response. Thus, the size of an antigen or immunogen may be as small as about 5-12 amino acids, and as large as the full length protein, including sizes in between. The antigen may be a multimeric protein or a fusion protein. The antigen may be a purified peptide antigen derived from natural or recombinant cells. The nucleic acid sequences of the immunopotentiating protein and the immunogen are operably linked to transcriptional regulatory sequences such that the immunogen is expressed in the tissue of the subject, thereby eliciting an immunogen-specific immune response in the subject in addition to the non-specific immune response.

In another embodiment of the invention, the biological agent is a vaccine. Vaccines may include live, infectious, viralA bacterial or parasitic vaccine or a killed, inactivated, viral, bacterial or parasitic vaccine. In one embodiment, 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 bovine species. Exemplary vaccines include, without limitation, those used in the art against Infectious Bovine Rhinotracheitis (IBR) (bovine herpes virus type 1 (BHV1)), parainfluenza virus type 3 (PI3), Bovine Respiratory Syncytial Virus (BRSV), bovine viral diarrhea virus (BVDV types 1 and 2), histophilus somni, mycoplasma bovis (c.) (bha (r)), (BHV (r)), (r) (b) f (r)), and (c) (b) f (r) (bMycoplasma bovis) And other diseases known in the art. In an exemplary embodiment, a vaccine for protection against mannheimia haemolytica may be used in combination with an immunomodulator composition of the present invention.

In yet another embodiment of the present invention, the biologic is an antimicrobial agent. Suitable antimicrobial agents include: quinolones, preferably fluoroquinolones, beta-lactams and macrolide-streptogramin-lincosamides (MLS) antibiotics.

Suitable quinolones include dinoxacin, binofloxacin, cinoxacin, ciprofloxacin, clinafloxacin, danofloxacin, difloxacin, enoxacin, enrofloxacin, fleroxacin, gemifloxacin, ebafloxacin, levofloxacin, lomefloxacin, marbofloxacin, moxifloxacin, norfloxacin, ofloxacin, orbifloxacin, pazufloxacin, pradofloxacin, pefloxacin, temafloxacin, tosufloxacin, sarafloxacin, gemifloxacin and sparfloxacin. Preferred fluoroquinolones include ciprofloxacin, enrofloxacin, moxifloxacin, danofloxacin and pridoxacin. Suitable naphthyridinones include naphthyridonic acid.

Suitable β -lactams include penicillins, such as benzathine, benzylpenicillin (penicillin G), phenoxymethylpenicillin (penicillin V), procaine, methicillin, oxacillin, nafcillin, cloxacillin, dicloxacillin, flucloxacillin, temocillin, amoxicillin, ampicillin, amoxicillin complex amoxicillin (amoxicillin and clavulanic acid), azlocillin, carbenicillin, ticarcillin, mezlocillin, piperacillin; cephalosporins, such as cefalonium, cephalexin, cefazolin, cefapirin, cefquinome, ceftiofur, cephalothin, cefaclor, cefuroxime, cefamandole, defotetan, cefoxitin, ceftriaxone, cefotaxime, cefpodoxime, cefixime, ceftazidime, cefepime, cefpirome; carbapenems and penems, such as imipenem, meropenem, ertapenem, faropenem, doripenem, monobactams, such as aztreonam (bacterikem), tigemonam, nocardicidin A, tabtoxinine-B-lactam; and beta-lactamase inhibitors such as clavulanic acid, tazobactam and sulbactam. Preferred β -lactams include cephalosporins, in particular cefazolin.

Preferred MLS antibiotics include any macrolide, lincomycin, clindamycin, pirlimycin. The preferred lincosamide is pirlimycin.

Other antimicrobial agents include 2-pyridinones, tetracyclines, sulfonamides, aminoglycosides, trimethoprim, dimetridazole, erythromycin, neomycin B, furazolidone, various pleuromutilins such as thiamine, valnemulin, various streptomycins, clopidol, salinomycin, monensin, halofuginone, narasin, robenidine, and the like.

2. Method of producing a composite material

a. Method of immunostimulation

In one embodiment of the invention, an immune response is elicited in a member of a bovine species by administering to the member of the bovine species an effective amount of an immunomodulator composition. The effective amount is sufficient to elicit an immune response in a member of the bovine species. Immunomodulators include liposome delivery vehicles and nucleic acid molecules.

In one embodiment, the effective amount of an immunomodulator is from about 1 microgram to about 1000 microgram per animal. In another embodiment, the effective amount of an immunomodulator is from about 5 micrograms to about 500 micrograms per animal. In yet another embodiment, the effective amount of an immunomodulator is from about 10 micrograms to about 100 micrograms per animal. In a further embodiment, the effective amount of an immunomodulator is from about 10 micrograms to about 50 micrograms per animal.

In another embodiment of the invention, an immune response is elicited in a member of the bovine species by administering an effective amount of an immunomodulator, including a liposome delivery vector, an isolated nucleic acid molecule and a biological agent. It is contemplated that the biological agent may be mixed with or co-administered with the immunomodulator, or it may be administered separately. The separate administration may be before or after the administration of the immunomodulator. It is also contemplated that more than one administration of an immunomodulator or biologic may be used to amplify the enhanced immunity. In addition, more than one biological agent may be co-administered with the immunomodulator, administered before the immunomodulator, administered after the immunomodulator, or administered simultaneously.

b. Disease and disorder

The methods of the invention elicit an immune response in a subject, thereby protecting the subject from a disease susceptible to treatment by eliciting an immune response. As used herein, the phrase "protecting against a disease" refers to alleviating the symptoms of a disease; reduce the incidence of disease and lessen the clinical or pathological severity of the disease, or reduce shedding of the pathogen responsible for the disease. Protecting a subject may refer to the ability of a therapeutic composition of the invention, when administered to a subject, to prevent the onset of disease, treat and/or alleviate or reduce symptoms, clinical signs, pathology or cause of disease. Examples of clinical signs of BRD include lung injury, elevated body temperature, depression (e.g., anorexia, reduced response to external stimuli, drooping ears), runny nose, and respiratory characteristics (e.g., respiratory rate, respiratory effort). Thus, protection of a member of a bovine species from disease includes prevention of disease development (prophylactic treatment) and treatment of a member of a bovine species having disease (therapeutic treatment). In particular, protection of a subject from disease is accomplished by eliciting an immune response in a member of the bovine species that induces a beneficial or protective immune response that may also, in some cases, suppress, reduce, inhibit or block an overactive or harmful immune response. The term "disease" refers to any deviation from normal health of a member of the bovine species and includes the state when disease symptoms are present and conditions in which a deviation (e.g., infection, genetic mutation, genetic defect, etc.) has occurred but the symptoms have not yet manifested.

The methods of the invention can be used to prevent disease, stimulate effector cell immunity to disease, eliminate disease, slow disease, and prevent secondary disease resulting from the development of primary disease.

The invention can also improve the adaptive immune response of an animal when co-administered with a vaccine, as compared to administration of the vaccine alone. Since it takes time to stimulate adaptive immunity, it is not usual that a single administration of the vaccine will immediately protect the animal. The term "improve" in the present invention refers to eliciting an innate immune response in an animal until the vaccine begins to protect the animal and/or extends the period of protection through adaptive immunity given by the vaccine.

The methods of the invention comprise administering a composition to provide protection against infection by a wide variety of pathogens. The composition administered may or may not include a specific antigen that elicits a specific response. It is contemplated that the methods of the invention will protect recipient subjects from diseases caused by infectious microbial pathogens, including, without limitation, viruses, bacteria, fungi, and parasites. Exemplary viral infections include, without limitation, those caused by Infectious Bovine Rhinotracheitis (IBR) (bovine herpes virus type 1 (BHV1)), parainfluenza virus type 3 (PI3), Bovine Respiratory Syncytial Virus (BRSV), bovine viral diarrhea virus (BVDV types 1 and 2), bovine adenovirus, Bovine Coronavirus (BCV), bovine calicivirus, bovine parvovirus, BHV4, bovine reovirus, bovine enterovirus, bovine rhinovirus, malignant catarrhal fever virus, bovine leukemia virus, rabies virus, Vesicular Stomatitis Virus (VSV), bluetongue (circovirus), recombinants thereof, and other viral infections known in the artViral infections. Exemplary bacterial infections include, without limitation, those consisting of gram-positive or gram-negative bacteria and mycobacteria such as Escherichia coli (E.coli: (B.coli))Escherichia coli) Pasteurella multocida (B), (C)Pasteurella multocida) Clostridium perfringens (A), (B), (C)Clostridium perfringens) Clostridium quail (C.), (Clostridium colinum) Campylobacter jejuni: (A), (B)Campylobacter jejuni) Clostridium botulinum (1)Clostridium botulinum) Clostridium novyi (C.nokokii) ((C.nokokii))Clostridium novyi) Shore D Clostridium (Clostridium chauveoi) Clostridium (ii) and (ii)Clostridium septicum) Clostridium hemolyticus (Clostridium hemolyticum) Clostridium tetani (Clostridium tetani)Clostridium tetani) Mannheim hemolyticus bacterium (Mannheimia haemolytica) Differential ureaplasma (M.urealyticum)Ureaplasma diversum) Mycoplasma distinguishii (A), (B), (C), (Mycoplasma dispar) Mycoplasma bovis (I)Mycoplasma bovis) Mycoplasma bovis (M.bovis) ((Mycoplasma bovirhinis) Hippophakia somnifera (A), (B), (C), (B), (C)Histophilus somni) Campylobacter fetus: (A), (B), (C)Campylobacter fetus) Leptospira species (Leptospira spp.) Cryptobacterium pyogenes: (A)Arcanobacterium pyogenes) Bacillus anthracis (B.anthracis) (B.anthracis)Bacillus anthrax) Fusobacterium necrophorum (A), (B), (C)Fusobacterium necrophorum) Clostridium species (A), (B), (C)Fusobacterium spp.) Treponema species (A)Treponema spp.) Corynebacterium (I) and (II)Corynebacterium) Brucella abortus: (Brucella abortus) Mycobacterium paratuberculosis: (Mycobacterium paratuberculosis) Mycobacterium species (A), (B), (C), (Mycobacterium spp.) Histophilus species (Histophilus spp.) Moraxella species (A), (B), (C)Moraxella spp.) Species of the genus Muller (Mueller: (Mr.)Muellerius spp.) Mycoplasma species (A), (B), (C), (Mycoplasma spp.) Salmonella species (II)Salmonella spp.) Bacillus anthracis (B.anthracis) (B.anthracis)Bacillus anthracis) And bacterial infections caused by other bacterial infections known in the art. Exemplary fungal or mold infections include, without limitation, infections caused by actinomycete species (A)Actinobacterim spp.) Aspergillus species (A), (B), (C)Aspergillus spp.) And tissue of the genus Trichomonas (Histomonas spp.) And fungal or fungal infections caused by other infectious fungal or fungal infections known in the artAnd (6) dyeing. Exemplary parasites include, without limitation, neospora species (Neospora spp.) Nematodiasis (D)Trichostrongylus), Cupressus spp (Cooperia) The anaplasma genus (Anaplasma spp) Babesia species (b)Babesia spp) Genus Zymophyta (A. sp.), (BChorioptes spp) Species of the genus cysticercus (cysticercus) (II)Cysticercus spp) Genus Peptophilus species (a)Dermatophilus spp) Cow hair louse: (Damalinia bovis)、Dictylocaulus sppSpecies of Eimeria (Eimeria)Eimeria spp) Eperythrozoon species (A)Eperythrozoon spp) Haemonchus species (II)Haemonchus spp) Species of the genus tick fly: (Melophagus spp) Species of mullerian nematode (A), (B), (C), (B), (Muellerius spp) Species of Microcoleptoterus (C.tenuis) (C.tenuis)Nematodirus spp) (ii) species of the genus lyssodexis: (Oestrus spp) Ostertagia species (A), (B), (C), (Ostertagia spp) Genus Prurites species (a)Psoroptes spp) Scabies genus species (Sarcoptes spp) Serpentis species (A)Serpens spp) Species of strongyloides (A), (B), (C), (Strongyloides spp) The toxoplasma belongs to the species (Toxoplasma spp) And species of Trichuris (S) ((S))Trichuris spp) The genus Trichophyton (A), (B), (C)Trichophyton spp) And Trichomonas species (Tritrichomas spp) Genus fasciolopsis (a)Fascioloides spp) An edge amorphous body (Anaplasma marginale) And other parasites known in the art.

c. Test subject

The methods of the invention may be administered to any subject or member of the bovine species, whether domesticated or wild. In particular, it can be administered to those subjects that are commercially bred for breeding, meat or dairy production. Suitable bovine subjects include, without limitation, antelope, buffalo, yak, cow, and bison. In one embodiment, the member of the bovine species is bovine. Bovine species include, without limitation, cows, bulls, beef bulls, heifers, castrated bulls, beef cattle, or dairy cattle. The skilled person will appreciate that the method of the invention will be largely advantageous for rearing cattle for breeding, meat or dairy production, as they are particularly susceptible to environmental exposure to infectious pathogens.

d. Administration of

A variety of routes of administration are available. The particular mode selected will, of course, depend on the particular biological agent selected, the age and general health of the subject, the particular condition to be treated, and the dosage required for therapeutic efficacy. The methods of the invention may be practiced using any mode of administration that produces effective levels of immune response without causing clinically unacceptable side 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.

The inoculation of bovine species can be performed at any age. The vaccine may be administered intravenously, intramuscularly, intradermally, intraperitoneally, subcutaneously, by spray/aerosol, orally, intraocularly, intratracheally, intranasally, or by other methods known in the art. Furthermore, it is contemplated that the methods of the present invention may be used based on conventional vaccination schedules. The immunomodulator may also be administered intravenously, intramuscularly, subcutaneously, by spraying, orally, intraocularly, intratracheally, intranasally or by other methods known in the art. In one embodiment, the immunomodulator is administered subcutaneously. In another embodiment, the immunomodulator is administered intramuscularly. In yet another embodiment, the immunomodulator is administered as a spray. In a further embodiment, the immunomodulator is administered orally.

In one embodiment, the immunomodulator is administered separately to the animal prior to challenge (or infection). In another embodiment, the immunomodulator is administered separately to the animal after challenge (or infection). In yet another embodiment, the immunomodulator is administered separately to the animal at the same time as the challenge (or infection). In further embodiments, the immunomodulator composition is co-administered simultaneously with vaccination prior to challenge. In still further embodiments, the immunomodulator composition is co-administered at the same time as the vaccination at the same time as the challenge (or infection). Co-administration may include administering the vaccine and immunomodulator at two different sites adjacent to each other on the same general site in the animal (i.e., injecting adjacent to each other on the neck of the animal), on the same general site on opposite sides of the animal (i.e., once on each side of the neck), or on different sites in the same animal. In another embodiment, the immunomodulator composition is administered prior to vaccination and challenge. In further embodiments, the immunomodulator composition is administered after vaccination but prior to challenge. In further embodiments, the immunomodulator composition is administered after challenge to an animal that has been vaccinated prior to challenge (or infection).

In one embodiment, the immunomodulator is administered from about 1 day to about 14 days before challenge or from about 1 day to about 14 days after challenge. In another embodiment, the immunomodulator is administered from about 1 day to about 7 days before challenge or from about 1 day to about 7 days after challenge. In yet another embodiment, the immunomodulator is administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days prior to challenge or 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days after challenge.

Other delivery systems may include timed release, delayed release or slow release delivery systems. Such systems can avoid repeated application of the composition, thereby increasing convenience. Many types of delivery systems are available and known to those of ordinary skill in the art. They include polymer-based systems such as poly (lactide-co-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules containing the above polymers of drugs are described, for example, in U.S. Pat. No. 5,075,109. The delivery system also includes non-polymeric systems, which are lipids, including sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-di-and triglycerides; a hydrogel release system; silicone rubber (silastic) systems; a peptide-based system; a wax coating; compressed tablets using conventional binders and excipients; a partially fused implant; and so on. Specific examples include, but are not limited to, eroding systems (where the formulations of the present invention are contained in an intramatrix form, such as those described in U.S. Pat. nos. 4,452,775, 4,675,189, and 5,736,152) and dispersing systems (where the active ingredient permeates from the polymer at a controlled rate, such as described in U.S. Pat. nos. 3,854,480, 5,133,974, and 5,407,686). In addition, pump-based hardware delivery systems may be used, some of which are adapted for implantation.

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

Definition of

The term "effective amount" refers to an amount necessary or sufficient to achieve a desired biological effect. For example, an effective amount of an immunomodulator for use in the treatment or prevention of an infectious disease is the amount necessary to elicit the onset of an immune response upon exposure to a microorganism, thereby resulting in a reduction in the amount of the microorganism in the subject and preferably eradication of the microorganism. An effective amount for any particular application may vary depending on factors such as the disease or condition to be treated, the size of the subject, or the severity of the disease or condition. Effective amounts of immunomodulators can be determined empirically by one of ordinary skill in the art without undue experimentation.

The term "cytokine" refers to a family of proteins that are immunopotentiating. The cytokine family includes hematopoietic growth factors, interleukins, interferons, immunoglobulin superfamily molecules, tumor necrosis factor family molecules, and chemokines (i.e., proteins that regulate the migration and activation of cells, particularly phagocytes). Exemplary cytokines include, without limitation, interleukin-2 (IL-2), interleukin-12 (IL12), interleukin-15 (IL-15), interleukin-18 (IL-18), interferon-alpha (IFN α), and interferon-gamma (IFN γ).

The term "trigger" may be used interchangeably with the terms activate, stimulate, produce, or upregulate.

The term "eliciting an immune response" in a subject refers to an activity that specifically controls or affects an immune response, and may include activating an immune response, up-regulating an immune response, enhancing an immune response, and/or altering an immune response (e.g., by eliciting a type of immune response that in turn changes the predominant type of immune response in a subject from a deleterious or ineffective type to a beneficial or protective type).

The term "operably linked" refers to the linkage of a nucleic acid molecule to a transcriptional regulatory sequence in such a way that the molecule is capable of being expressed when transfected (i.e., transformed, transduced or transfected) into a host cell. Transcriptional regulatory sequences are sequences that control the initiation, extension, and termination of transcription. Particularly important transcriptional regulatory sequences are those which control the initiation of transcription, such as promoter, enhancer, operator and repressor sequences. A variety of such transcriptional regulatory sequences are known to those of skill in the art. Preferred transcriptional regulatory sequences include those that function in avian, fish, mammalian, bacterial, plant and insect cells. Although any transcriptional regulatory sequence may be used in the present invention, the sequence may include a naturally occurring transcriptional regulatory sequence that is naturally associated with a sequence encoding an immunogen or an immunostimulatory protein.

The terms "nucleic acid molecule" and "nucleic acid sequence" are used interchangeably and include DNA, RNA, or derivatives of DNA or RNA. The term also includes oligonucleotides and larger sequences, including nucleic acid molecules that encode proteins or fragments thereof, as well as nucleic acid molecules that comprise regulatory regions, introns, or other non-coding DNA or RNA. Typically, oligonucleotides have nucleic acid sequences of about 1 to about 500 nucleotides in length, and more typically, at least about 5 nucleotides in length. The nucleic acid molecule may be derived from any source, including mammalian, fish, bacterial, insect, viral, plant or synthetic sources. Nucleic acid molecules can be produced by methods generally known in the art, such as recombinant DNA techniques (e.g., Polymerase Chain Reaction (PCR), amplification, cloning) or chemical synthesis. Nucleic acid molecules include natural nucleic acid molecules and homologs 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 ability of the nucleic acid molecule to encode an immunogen or an immunostimulatory protein useful in the methods of the invention. Nucleic acid homologs can be generated using a number of methods known to those of skill in the art (see, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, 1989), which is incorporated herein by reference. Techniques for screening for immunogenicity, e.g., pathogen antigen immunogenicity or cytokine activity, are known to those skilled in the art and include a variety of in vitro and in vivo assays.

Examples

The following examples illustrate various embodiments of the present invention.

Example 1 evaluation of cattle receiving DNA immunomodulators before and after the onset of natural bovine respiratory disease.

The objective of this study was to determine the efficacy of DNA immunomodulators administered to calves before and after the natural occurrence of BRD.

Immunomodulator

The immunomodulator used in this study was a composition comprising a cationic lipid and non-coding DNA. Synthetic immunomodulator lipid components [1- [2- [9- (Z) -octadecenoyloxy ] ] -2- [8] (Z) -heptadecenyl ] -3- [ hydroxyethyl ] imidazolinium chloride (DOTIM) and synthetic neutral lipid cholesterol were formulated to produce liposomes of approximately 200 nm diameter (see, U.S. Pat. No. 6,693,086). The DNA component is a 4242 base pair non-coding DNA plasmid produced in e.coli, which is negatively charged and binds to positively charged (cationic) liposomes (see, U.S. patent 6,693,086).

Research animals

84 weaning-aged Holstein castrated calves were selected from a herd without a current history of respiratory disease. Each individual calf was initially evaluated and determined to be in good health. 84 calves were divided into 7 treatment groups of 12 calves each. Only animals that were not vaccinated against mannheimia haemolytica were included in this study. Animals did not receive an antimicrobial agent for 30 days prior to administration of the DNA immunomodulator.

As shown in table 1.1 below, different doses of the above-described DNA immunomodulators were administered to the treatment groups on the day of treatment. The dilution scheme for the DNA immunomodulator is provided in table 1.2. The DNA immunomodulator is administered from the intramuscular and cranium of calves to the left shoulder, the abdomen to the nuchal ligament, and the caudal dorsum to the jugular sulcus.

As referred to below, day-1 of treatment refers to the start date of the study after the initial selection where the calves were evaluated and determined to be suitable for the study. Day 0 of treatment is the day following day-1, and so on.

TABLE 1.1 administration schedule of immunomodulators

Most calves were observed to experience different levels of BRD in the morning on day 0. By day 5, it was observed that all calves remaining in the study population had met the case definition of BRD incidence. Cattle were removed from the study population only when euthanized because severe BRD indicated use. No other infectious/non-infectious diseases were observed and thus need to be removed in this study.

Evaluation of

On days 1-5 of the study, calves were evaluated for various health indicators. For example, rectal temperature and average daily body weight of each calf was measured daily during the study period. Animals were evaluated approximately at the same time (+/-3 hours) each day from day 1 to day 5. Figures 1.1 and 1.2 present the mean values of rectal temperature and the mean daily weight gain according to the dose of immunomodulator administered.

On day 5, all calves were euthanized and autopsied. The lung injury score (based on the degree of lung consolidation assessed by visual inspection and manual palpation) was determined for each individual calf at necropsy.

Figure 1.3 presents lung injury scores for doses of administered immunomodulators. The overall lung injury score for each day of administration was about 11% and 14% at day-1 and day 0, respectively. For 500, 200, 50 and negative control groups, lung injury scores of 11.2%, 9.0%, 10.8% and 19.9% were shown, respectively. The greatest difference between the control and treated groups (200 μ g) was a reduction of about 11%.

The estimates of model corrections in fig. 1.3 reflect the raw mean values that correct for all statistical model covariates (i.e. dose, day and dose x days) and the pen in which the calves were housed throughout the study. Thus, the model-corrected estimate may exhibit a difference compared to the original average.

Subsequent bacteriology (lung cultures) and virology (nasal swabs) were also performed. Of the remaining calves (69) euthanized on day 5, 11.6% of the calves were found to shed herpes virus type 1 (BHV-1) in nasal secretions. For lung cultures from all study animals, 41% were Mh positive, 31.3% were culture positive for pasteurella multocida (Pm), 10.8% were culture positive for both Mh and Pm, and histophilus somni was not isolated throughout the study population. In this study, no culture of M.bovis was performed.

Results

In this study, doses of DNA immunomodulator (i.e., 500 μ g, 200 μ g, and 50 μ g) achieved a significant reduction in lung injury score compared to negative controls (P = 0.1284; see fig. 1.3). However, the day of DNA immunomodulator administration (i.e., day-1 or day 0) was not significantly correlated with the lung injury score. No statistical differences in lung injury scores were observed between the dose groups of DNA immunomodulators. Rectal temperature tends to be significantly correlated with the dose of DNA immunomodulator (P =0.1190), but not with the day of administration. No significant difference was observed between the doses of DNA immunomodulator and negative control with respect to average daily weight gain.

There is a strong tendency for DNA immunomodulators to reduce lung injury compared to negative controls, thus providing evidence that the product has the potential to protect lung tissue during a BRD outbreak. In this study, the day of treatment administration was not associated with lung injury, thus suggesting that it is not important whether the cattle received the DNA immunomodulator the day before or on the same day as the clinical signs associated with BRD. This result is important because the timing of exposure to BRD pathogens is generally unknown in typical production systems and is further complicated by the effects of multiple stressors experienced by cattle throughout the production chain. Therefore, it would be extremely valuable in the beef and dairy industries to provide producers with products that provide flexibility in the timing of administration associated with the onset of BRD.

Example 2 evaluation of cattle receiving DNA immunomodulator at the same time or one day after challenge with the Mannheim haemolytica test

The aim of this study was to determine the efficacy of DNA immunomodulators administered to calves simultaneously with or one day after experimental challenge with mannheimia haemolytica.

Immunomodulator

The immunomodulator used in this study was the composition described in example 1 above.

Research animals

From a herd without a current history of respiratory disease, 84 holstein castrated calves of weaning age and weighing an average of about 300 pounds (136 kg) were selected. Each individual calf was initially evaluated and determined to be in good health. 84 calves were divided into 7 treatment groups of 12 calves each. Only animals that were not vaccinated against mannheimia haemolytica were included in this study. Animals did not receive an antimicrobial agent for 30 days prior to administration of the DNA immunomodulator. As shown in table 2.1 below, different doses of DNA immunomodulators were administered to the treatment groups on the day of treatment. The dilution scheme for the DNA immunomodulator is provided in table 2.2. The DNA immunomodulator is administered from the intramuscular and cranium of calves to the left shoulder, the abdomen to the nuchal ligament, and the caudal dorsum to the jugular sulcus.

As referred to below, treatment day 0 refers to the start date of the study after the initial selection in which calves were evaluated and determined to be in good health. Day 1 of treatment is the day following day 0, and so on.

TABLE 2.1 application schedules of immunomodulators and Mh challenge

Experimental attack

On day 0, the total amount used was 3.12X 10⁷Individual Colony Forming Units (CFU) of mannheimia haemolytica challenge calves. The inoculum was administered via the respiratory tract. By day 3, it was observed that all calves in the study population had met the case definition of BRD incidence. The median day of onset is one day.

Evaluation of

As in the previous examples, on days 1-5 of the study, calves were evaluated for various health indicators. Rectal temperature and average daily body weight of each calf was measured daily during the study period. Animals were evaluated at approximately the same time each day. Figures 2.1 and 2.2 present the mean values of rectal temperature and mean daily weight gain in relation to the dose administered with the immunomodulator.

On day 5, all calves were euthanized and autopsied. The lung injury score was determined for each individual calf at necropsy according to the protocol described in example 1.

Figure 2.3 presents model-corrected lung injury scores associated with doses of administered immunomodulators.

Results

In this study, doses of DNA immunomodulator (i.e., 500 μ g, 200 μ g, and 50 μ g) significantly reduced lung injury scores compared to negative controls. However, lower doses (200 μ g and 50 μ g) are better at reducing lung injury than the 500 μ g dose. The day of DNA immunomodulator administration (i.e., day 0 or day 1) was not significantly correlated with the lung injury score. No statistical differences in lung injury scores were observed between the DNA immunomodulator dose groups. Rectal temperature was significantly reduced in calves administered with DNA immunomodulators compared to negative controls, but not dose-related. No significant difference was observed between the doses of DNA immunomodulator and negative control with respect to average daily weight gain.

There is a strong tendency for DNA immunomodulators to reduce lung injury compared to negative controls, thus providing evidence that the product has the potential to protect lung tissue during a BRD outbreak. In this study, the day of treatment administration was not associated with lung injury, thus suggesting that it is not important whether the cattle received the DNA immunomodulator the day before or on the same day as the clinical signs associated with BRD. This result is important because the timing of exposure to BRD pathogens is generally unknown in typical production systems and is further complicated by the effects of multiple stressors experienced by cattle throughout the production chain. Therefore, it would be extremely valuable in the beef and dairy industries to provide producers with products that provide flexibility in the timing of administration associated with the onset of BRD.

Example 3 evaluation of cattle receiving DNA immunomodulator two days before or simultaneously with the Experimental challenge with Mannheim haemolytica

The objective of this study was to determine the efficacy of DNA immunomodulators administered to calves two days prior to or simultaneously with experimental challenge with mannheimia haemolytica.

Immunomodulator

The immunomodulator used in this study was the composition described in example 1 above.

Research animals

96 Holstein castrated calves weighing an average of about 800-. Each individual calf was initially evaluated and determined to be in good health. The 96 calves were divided into 8 treatment groups of 12 calves each. Only animals that were not vaccinated against mannheimia haemolytica were included in this study. Animals did not receive an antimicrobial agent for 30 days prior to administration of the DNA immunomodulator. As shown in table 3.1 below, different doses of DNA immunomodulators were administered to the treatment groups on the day of treatment. The dilution scheme for the DNA immunomodulator is provided in table 3.2. The DNA immunomodulator is administered from the intramuscular and cranium of calves to the left shoulder, the abdomen to the nuchal ligament, and the caudal dorsum to the jugular sulcus.

As indicated below, treatment day-2 points to the start date of the study when treatment groups 1-3 were administered the immunomodulator. Day 0 of treatment is two days after day-2, and so on.

TABLE 3.1 application schedules of immunomodulators and Mh challenge

Experimental attack

On day 0, the total amount used was 1.9X10¹⁰Individual CFUs attack the calves. The inoculum was administered via the respiratory tract.

Evaluation of

As in the previous examples, on days 1-5 of the study, calves were evaluated for various health indicators. On day 5, all calves were euthanized and autopsied. The lung injury score was determined for each individual calf at necropsy.

Figure 3.1 presents model-corrected lung injury scores associated with doses of administered immunomodulators. Figure 3.2 presents model-corrected lung injury scores associated with the day of immunomodulator administration.

Results

In this study, doses of DNA immunomodulator (i.e., 200 μ g, 50 μ g, and 25 μ g) significantly reduced lung injury scores compared to negative controls. However, no statistical differences in lung injury scores were observed between the dose groups of DNA immunomodulators. The days of DNA immunomodulator administration (i.e., days-2 and 0) were not significantly correlated with lung injury scores. A significant reduction in lung injury was observed at day 0 when the immunomodulator was administered compared to day-2.

Example 4 Co-administration of immunomodulators with Mh challenge of inactivated Mh vaccine

The aim of this study was to determine the efficacy of DNA immunomodulators co-administered with inactivated Mh vaccines to calves subjected to experimental challenge with mannheimia haemolytica.

Immunomodulator

The immunomodulator used in this study was the composition described in example 1 above.

Research animals

81 holstein bulls of 12 weeks of age were selected from a herd without a current history of respiratory disease. Each calf individual was evaluated and determined to be in good health. Only animals that were not vaccinated against mannheimia haemolytica were included in this study. Animals did not receive antimicrobial agent for 30 days prior to administration of the inoculum.

Experimental infection and challenge

Challenge or experimental infection involves exposure to an inoculum of mannheimia haemolytica. For the first inoculum, at 1.7X10⁸Concentration per animal organisms were used, and for the second inoculum, 2.4X10¹⁰The organisms were used at a concentration per animal. The animals were also challenged with a spray via another respiratory route. The concentration of organisms in the spray inoculum was 1.9X10¹⁰Per animal.

The efficacy of the immunomodulators as described above administered to calves subsequently exposed to mannheimia haemolytica was determined by the 12 treatment groups detailed in table 3.

TABLE 4.3 study treatment groups

Oil MH = haemolytica Mannheim vaccine (Pulmo-Guard PHM)

Water MH = haemolytic Mannheim vaccine (One Shot)

NC = no mixing and no spray attack (for background gross pathology)

CC = contact and spray challenged

SE = used as a vaccination attack (intratracheal attack)

All animals, except SE and NC, were challenged with the spray.

SC = subcutaneous route of injection

IM = intramuscular route of injection

NA = inapplicable

Animals in the T8 group will be treated after intranasal challenge.

On study day 0, all animals in the T1, T2, and T12 groups were administered an immunomodulator subcutaneously. On day 7, immunomodulators were administered subcutaneously to the T3, T4 and T5 groups. On day 13, the immunomodulators were administered subcutaneously to group T6 and intramuscularly to group T7. On day 15, immunomodulators were administered subcutaneously to the T8 group.

All animals receiving the vaccine were vaccinated according to the label instructions. Immunomodulators and vaccines were administered close together near the lymph nodes (neck) -two injections (one vaccine injection, another immunomodulator injection). All animals receiving subcutaneous injections were injected near the lymph nodes in the lower scapular region.

On day 10 of the study, all T11 calves were transported away from the site in a freight trailer for approximately 24 hours to stress the calves. On study day 11, all T11 animals were administered 20 mL of inoculum containing mannheimia haemolytica transtracheally and 4 hours later were administered 25 mL of inoculum. On study day 14, all groups were mixed and transported away from the site in a freight trailer for approximately 24 hours, except for T9, to stress the calves. On study day 14, all animals except the NC group were mixed into a large pen for 12 to 16 hours and then returned to their respective pens (each animal owns a respective pen). On day 15 of the study, 20 mL of mannheimia haemolytica was administered to all groups except T9 and T11 via another respiratory route. Animals were observed daily for clinical abnormalities and mortality during the study. All animals were negative or had low titers when screened prior to purchase of the animals. The animals had high titers prior to treatment, which indicates that the animals were serologically transformed to mannheimia haemolytica prior to receiving treatment.

Results

Animals in the T8 group had significantly lower lung injury.

This study suggested that early protection (day 7) occurred with or without vaccine (T4 and T5 compared to T3). See fig. 4.1 and 4.2.

Example 5 evaluation of adaptive immunity in cattle vaccinated with a commercial live vaccine when co-administered with a DNA immunomodulator

The objective of this study was to determine whether co-administration of DNA immunomodulators increased the adaptive immunity provided by the Modified Live Virus (MLV) vaccine.

Immunomodulator

The immunomodulator used in this study was the composition described in example 1 above.

Research animals

72 weaning-aged Holstein castrated calves were selected from a herd without the current history of respiratory disease. The 72 calves were divided into 6 treatment groups of 12 calves each. Each calf individual was evaluated and determined to be in good health. All calves had no serum antibodies against BHV-1, BVDV1 and 2 types, and BRSV. Furthermore, all calves were found to be seronegative for PI-3 antibodies. The calves were then determined by immunohistochemistry to be negative for persistent infection with the bovine viral diarrhea virus.

The vaccine and different doses of DNA immunomodulator were administered intramuscularly to the treatment group on the day of treatment as shown in table 5.1 below. The dilution scheme for the DNA immunomodulator is provided in table 5.2. On study day 0, all animals in groups T1-T4 were administered the immunomodulator. All animals receiving the vaccine were vaccinated according to the labeling instructions. Immunomodulators and vaccines were administered close together from cranium to anterior shoulder-two injections (one vaccine injection, another immunomodulator injection).

TABLE 5.1 administration schedule for immunomodulators and vaccines

Group of	Targeted dosage	Day of administration of vaccine and/or immunomodulator	Number of animals
				T1	MLV + immunomodulator (500 μ g) IM	0	12
T2	MLV + immunomodulator (200 μ g) IM	0	12
				T3	MLV + immunomodulator (100 μ g) IM	0	12
T4	MLV + immunomodulator (50 μ g) IM	0	12
				T5	MLV	0	12
T6	Untreated	NA	12

MLV = haemolytica Mannheim vaccine (Bovi-shield:) modified live 4 weight (4-way) viral respiratory vaccine

IM = intramuscular route of injection.

Evaluation of

Immunoassays were performed on samples of suitable blood samples taken from calves on days 0, 13, 28, 27, 34 and 41. Cell-mediated immunity (CMI) assays were performed on each sample. The pathogens of interest for this study are BHV-1, BVDV1 and 2, and BRSV. Where appropriate, the laboratory uses standardized procedures and methods for the target pathogen as previously detailed.

Results

Model correction data for CMI results for each day of sample collection in all treatment groups were determined. When comparing the DNA immunomodulator treatment group-MLV vaccine combination with cattle receiving the MLV vaccine alone, no statistical differences were detected between all treatment groups, cell types and antigens (P >0.10) (see fig. 5.1-5.12). Specifically, fig. 5.1-5.4 present the measurement of CD 25 EI expression index (y-axis) for all five cell types (x-axis) for each of the six treatment groups. Figures 5.5-5.8 present the measurement of IFN γ expression index (y-axis) for all five cell types (x-axis) for each of the six treatment groups. FIGS. 5.9-5.12 present measurements of IL-4 expression index (y-axis) for all five cell types (x-axis) for each of the six treatment groups. Estimates were generated for each of the 4 BRD viral pathogens represented in their respective graphs. For these statistical evaluations, all comparisons were made with the "MLV only" treatment group.

Statistically significant (P <0.10) treatment x-day interactions of BVDV1 (day 28 and day 35) and BVDV 2 (day 42) were examined. No significant findings were detected for BHV-1 at any of the time points listed (P > 0.10). Graphical representations of these findings are presented on fig. 5.13-5.15. BRSV data due to antibody seroconversion observed in the negative control treated group were removed from the analysis. Note that for all statistical evaluations, all comparisons were made with the "MLV only" treatment group.

Individual animal body weights were also collected during the course of the study. A graphical presentation of the model-corrected average daily weight gain results is presented in fig. 5.16. When compared to the MLV only group, no significant findings were detected between treatment groups (P > 0.10).

In summary, the DNA immunomodulator does not enhance CMI when co-administered with an MLV vaccine, compared to administration of an MLV vaccine alone. However, 500. mu.g of DNA immunomodulator can improve humoral immunity when co-administered with MLV vaccines (particularly BVDV). It should be noted, however, that co-administration of DNA immunomodulators at doses of 500. mu.g, 200. mu.g, 100. mu.g and 50. mu.g did not impair the positive immune effect induced by the MLV vaccine, despite the lack of sustained improvement in acquired immunity. In addition, performance (e.g., ADG) is not negatively affected by administration of DNA immunomodulators.

Example 6 evaluation of adaptive immunity in cattle vaccinated with commercial vaccines when co-administered with DNA immunomodulators

The objective of this study was to determine whether co-administration of DNA immunomodulators increased the adaptive immunity provided by vaccines containing inactivated antigens.

Immunomodulator

The immunomodulator used in this study was the composition described in example 1 above.

Research animals

From a herd of cattle with no current history of respiratory disease, 48 holstein cows, 3-5 months old, were selected. The 48 cattle were divided into 6 treatment groups of 8 cattle each. Each individual animal was evaluated and determined to be in good health. All animals had no serum antibodies against BHV-1, BVDV type 1 and type 2. The animals were also determined by PCR to be negative for persistent infection with bovine viral diarrhea virus. Animals were not selected for SNT titers against BRS virus and PI3 virus.

The vaccine and different doses of DNA immunomodulator were administered intramuscularly to the treatment group on the day of treatment as shown in table 5.1 below. The vaccines comprise BHV1 and BVDV types 1 and 2 as inactivated antigens, as well as modified live PI3 virus and BRS virus. The immunomodulator and vaccine are administered separately from the cranium to the anterior shoulder on the same side of the animal, separately in the same area on the opposite side of the animal, or mixed in a single syringe. The dilution scheme for the DNA immunomodulator is provided in table 5.2.

TABLE 6.1 administration schedule of immunomodulators and vaccines

Vaccine = combined (inactivated and modified live) 4-virus respiratory vaccine (Rispoval-

IM = intramuscular route of injection.

Evaluation of

Immunoassays were performed on samples of appropriate blood samples taken from cattle on days 0, 3, 5,7, 9, 11, 14, 17, 20, 23, and 27. The pathogens of interest for this study were BHV-1, BVDV1 and 2. Antibody titer information was also determined against BRS virus and PI3 virus. For the target pathogens specified previously, the laboratory used standardized Serum Neutralization Tests (SNT) as a procedure.

Results

Statistically significant (P <0.010) treatment x-day interactions of BHV1 (day 27) were tested. No significant findings were detected for BHV1 at all other time points, and BVDV type 1 and type 2 at any of the time points listed (P > 0.10). Since the animals were not seronegative at the start of the study, the results of BRSV and PI3 titers were not further evaluated. The effect of the treatment cannot be verified. A graphical representation of these findings is presented on fig. 6.1. Note that for all statistical evaluations, all comparisons were made with the "vaccine and 5% glucose" treatment group.

Claims (11)

1. An immunomodulator composition, wherein the immunomodulator composition comprises:

a. a cationic liposome delivery vehicle; and

b. nucleic acid molecules

It is used for the treatment of bovine respiratory disease in cattle.

2. The composition of claim 1, wherein the liposome delivery vehicle comprises a lipid selected from the group consisting of multilamellar liposome lipids and extruded lipids.

3. The composition of claims 1 and 2, wherein the liposome delivery vehicle comprises a lipid delivery vehicle selected from the group consisting of DTMA and cholesterol; DOTAP and cholesterol; DOTIM and cholesterol; and lipid pairs of DDAB and cholesterol.

4. The composition of claims 1-3, wherein the nucleic acid molecule is an isolated gene insert-free nucleic acid vector of bacterial origin, or a fragment thereof.

5. The composition of claims 1-4 for administration selected from intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, by spray/aerosol, oral, intraocular, intratracheal, and intranasal.

6. The composition of claims 1-5, wherein the biological agent is selected from an immunopotentiating protein, an immunogen, a vaccine, an antimicrobial agent, or any combination thereof.

7. The composition of claim 1, wherein the bovine respiratory disease is caused by a viral infection and/or a bacterial infection.

8. The composition of any one of claims 1 to 7 for use in reducing clinical signs in cattle caused by Mannheim haemolytica and comprising an immunomodulator composition, wherein the immunomodulator composition comprises:

a. DOTIM in combination with cholesterol lipids; and the combination of (a) and (b),

b. a nucleic acid molecule which is an isolated gene insert-free nucleic acid vector of bacterial origin, or a fragment thereof.

9. The composition of claim 8, further comprising a biologic.

10. The composition of any one of claims 1-9 for use in improving an acquired immune response in an animal administered a vaccine, the composition comprising an immunomodulator composition, wherein the immunomodulator composition comprises:

a. DOTIM in combination with cholesterol lipids; and the combination of (a) and (b),

b. a nucleic acid molecule which is an isolated gene insert-free nucleic acid vector of bacterial origin, or a fragment thereof.

11. The composition of claim 10, wherein the immunomodulator composition is co-administered with the vaccine, or is administered after the vaccine, before the vaccine, or is administered in admixture with the vaccine.