FIELD OF THE INVENTION

The present invention relates to a compositions and methods for immunomodulation in cows. In particular, the present invention includes compositions and methods which are effective for increasing first service conception rate in cows.

BACKGROUND OF THE INVENTIO

The highest morbidity and mortality in dairy cattle occurs in the peripartum period. It has been shown that immune function is compromised around calving, ith reduced white cell count and reduced white cell function as demonstrated via myeloperoxidase and phagocytosi s assays. If immune function could be enhanced around calving, then reproductive outcomes, which may be sensitive to immune function, could be improved.

Dairy cattle fertility is declining on an international scale due to multiple factors including increasing herd size, reduced oestrus detection sensitivity and specificity, declining body condition score at calving and increased rate of body condition score loss postpartum. McDougall, J. Reproduction and Development 52, 185-194 (2006). High level s of reproductive performance are necessary to maintain optimum herd health and productivity. There is a need in the art for compositions and methods capable of increasing reproductive abilities of cows and heifers.

SUMMARY OF THE INVENTION

The present invention relates to immunomodulator compositions for increasing conception rates in cows and heifers. In some embodiments, the immunomodulator composition may comprise a cationic liposome delivery vehicle and an isolated bacterially-derived nucleic acid molecule that does not code for an immunogen for increasing conception rate in cows and heifers.

In some embodiments, the nucleic acid molecule comprises at least one

immunostimulatory CpG motif and at least one non-immunostimulatory CpG motif. In further embodiments, the nuclei acid molecule has at least 80% sequence homology with the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In some embodiments, the nucleic acid molecule comprises SEQ ID NO: 1. In other embodiments, the nucleic acid molecule compri ses SEQ ID NO: 2. In other embodiments, the nucleic acid molecule compri ses SEQ ID NO: 3. In other embodiments, the nucleic acid molecule comprises SEQ ID NO: 4. In some embodiments, the liposome delivery vehicle comprises lipids selected from the group consisting of multilamellar vesicle lipids and extruded lipids. In further embodiments, the liposome delivery vehicle compri ses pairs of lipids selected from the group consi sting of N-[l- (2,3-dioleyloxy)propyl]-N,N,N-trimethylanimonium chloride (DOTMA) and cholesterol; N-[l- (2,3-dioleoyloxy )propyl ]N,N, -trimethylammonium chloride (DOTAP) and cholesterol; l-[2- (oleoyloxy)ethyl]-2-oleyl-3 -(2 -hydroxy ethyl)imidazolinium chloride (DOTIM) and cholesterol; and dimethyldioctadecylammonium bromide (DDAB) and cholesterol .

In some embodiments, the i m m unom odul ator composition further comprises a biological agent. In further embodiments, the biological agent is selected from the group consisting of immune enhancer proteins, immunogens, vaccines, antimicrobials or any combination thereof. In some embodiments, the immunomodulator composition further comprises a pharmaceutical ly acceptable carrier.

In some embodiments, the immunomodular composition is for administration and selected from the group consisting of intravenously, intramuscularly, intradermal,

intraperitoneal, subcutaneously, by spray-aerosol, orally, intraocularly, intracheally, intrauterine, intravaginal, and intranasal .

In some embodiments, the conception rate in cows increases relative to the conception rate in a control population.

In some embodiments, the conception rate in non-cycling cows increases relative to the conception rate in a control population.

In some embodiments, the first service conception rate in cows increases relative to the first service conception rate in a control population.

In some embodiments, the first service conception rate in on -cycling cows increases relative to the first service conception rate in a control population .

In some embodiments, the conception rate in cows increases relative to the first service conception rate in a control population as measured by a p-value of < 0.05.

In some embodiments, the conception rate in non-cycling cows increases relative to the conception rate in a control population as measured by a p-value of < 0.05.

In some embodiments, the first service conception rate in cows increases relative to the first serv ice conception rate in a control population as measured by a p-value of < 0.05.

In some embodiments, the first service conception rate in non -cycling cows increases relative to the first serv ice conception rate in a control population as measured by a p-value of < 0.05. The present invention also relates to methods of increasing the conception rate in cows comprising administering to the cows an effective amount of an immunomoduiator composition. In some embodiments, the i m munomodul ator composition may compri se a cationic liposome delivery vehicle and a nucleic acid molecule that does not code for an immunogen.

The present invention also relates to methods of increasing the conception rate in cows comprising administering to the cows an effective amount of an immunomoduiator composition. In some embodiments, the i m munomodul ator composition may compri se a cationic liposome delivery vehicle and an isolated bacteriall -derived nucleic acid molecule that does not code for an immunogen.

The present invention also relates to methods of increasing the conception rate in non- cycling cows comprising administering to the cows an effective amount of an immunomoduiator composition. In some embodiments, the immunomoduiator composition may comprise a cationic liposome delivery vehicle and a nucleic acid molecule that does not code for an immunogen .

The present invention also relates to methods of increasing the conception rate in non- cycling cows comprising administering to the cows an effective amount of an immunomoduiator composition. In some embodiments, the i m m unomod ul ator composition may comprise a cationic liposome delivery vehicle and an i solated bacterially-derived nucleic acid molecule that does not code for an immunogen.

The present invention also relates to methods of increasing the first service conception rate in cows comprising administering to the cows an effective amount of an immunomoduiator composition . In some embodiments, the i m m unom odul ator composition may comprise a cationic liposome delivery vehicle and a nucleic acid molecule that does not code for an immunogen.

The present invention also relates to methods of increasing the first service conception rate in cows comprising administering to the cows an effective amount of an i m m unom odul ator composition . In some embodiments, the immunomoduiator composition may comprise a cationic liposome delivery vehicle and an isolated bacteri ally -deri ved nucleic acid molecule that does not code for an immunogen.

The present invention also relates to methods of increasing the first serv ice concepti on rate in non-cycling cows comprising admini stering to the cows an effective amount of an immunomoduiator composition. In some embodiments, the immunomoduiator composition may compri se a cationic liposome delivery vehicle and a nucleic acid molecule that does not code for an immunogen. The present invention also relates to methods of increasing the first service conception rate in non-cycling cows comprising administering to the cows an effective amount of an i m m unom odul ator composition. In some embodiments, the immunomodulator composition may comprise a cationic liposome delivery vehicle and an isolated bacteri ally -derived nucleic acid molecule that does not code for an immunogen.

In some embodiments, the nucleic acid molecule compri ses at least one

i m m unosti m ul atory CpG motif and at least one non-immunostimulatory CpG motif In further embodiments, the nucleic acid molecule has at least 80% sequence homology with the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In some embodiments, the nucleic acid molecule compri ses SEQ ID NO: 1. In other embodiments, the nucleic acid molecule comprises SEQ ID NO: 2. In other embodiments, the nucleic acid molecule comprises SEQ ID NO: 3. In other embodiments, the nucleic acid molecule compri ses SEQ ID NO: 4.

In some embodiments, the liposome delivery vehicle comprises lipids selected from the group consisting of multilamellar vesicle lipids and extruded lipids. In further embodiments, the liposome delivery vehicle compri ses pairs of lipids selected from the group consi sting of N-[l- (2,3-dioleyloxy)propyl]-N,N,N-trimethylanimonium chloride (DOTMA) and cholesterol; N-[l- (2,3-dioleoyloxy )propyl ]N,N,N-trimethylammonium chloride (DOT AP) and cholesterol; l-[2- (ol eoyl oxy )ethy 1 ] -2-ol ey 1 -3 -( 2-hydroxyethyl )i mi dazol i ni um chloride (DOTTM ) and cholesterol; and di methyl di octadecy lam m on i um bromide (DDAB) and cholesterol .

In some embodiments, the i m m unom odul ator composition further comprises a biological agent. In further embodiments, the biological agent is selected from the group consisting of immune enhancer proteins, immunogens, vaccines, antimicrobials or any combination thereof. In some embodiments, the immunomodulator composition further comprises a pharmaceutical ly acceptable carrier.

In some embodiments, the i mm unom odul ar composition i s for administration and selected from the group consisting of intravenously, intramuscularly, intradermal,

intraperitoneal, subcutaneously, by spray-aerosol, orally, intraocularly, intracheally, and intranasal.

BR IEF DESCRIPT IO OF T HE DRAWINGS

FIG. 1 shows a map of the pMB75.6 pi asm id ( SEQ ID NO: 1)

FIG. 2 shows a map of the pGCMB75.6 pi asm id (SEQ ID NO: 2)

FIG. 3. Shows a map of the pLacZMB75.6 pi asm id ( SEQ ID NO: 3) FIG. 4 A shows a chart of the probability (estimated marginal means and standard error of the mean (SEM)) of conception to the first insemination and FIG. 4B shows the probability (estimated marginal means and SEM) of pregnancy within 3 weeks after the planned start of the breeding program for cyclic and non-cyclic cows by treatment groups. The asterisk indicates a difference between treatments within the non -cycling group.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, immunomodulator compositions and methods of use thereof for increasing the conception rate in cows. The immunomodulator composition includes a cationic liposome delivery vehicle and an isolated bacterially-derived nucleic acid molecule that does not code for an immunogen . The compositions and methods of using the immunomodulator compositions are discussed in more detail below.

I. Composition

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 di seases. Such compositions are referred to herein as immunomodulator compositions. The immunomodulator compositions include at least an immunostimulatory plasmid or

immunostimulatory DNA sequence, capable of increasing the conception rate in cows. In some aspects, the immunomodulator compositions may also include a liposome delivery vehicle.

A. Nucleic Acids

In some aspects the present invention relates to nucleic acid molecules useful for increasing the conception rate in cows. The nucleic acid molecules described herein may be included in an immunostimulatory plasmid, 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.

The nucleic acid molecules described herein are enriched in CpG motifs. Such CpG motifs may induce immune stimulation via specific Toll-like receptors, such as TLR9 and

TLR21 . In addition the nucleic acid molecules described herein also contain non-CpG immunostimulatory motifs. In some aspects, the nucleic acid molecules contain about 2-20%

CpG motifs over the frequency of CpG motifs expected in random nucleic acid sequences. In some aspects, the nucleic acid molecules contain about 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15,

16, 17, 18, 1 9, 20, 25, 30, 35, 40%, or more CpG motifs over the frequency of CpG motifs expected in random nucleic acid sequences. In some aspects, the nucleic acid molecules contain about 10% CpG motifs over the frequency of CpG motifs expected in random nucleic acid sequences. In some aspects, compared to vertebrate DNA, an enrichment of CpG motifs of more than 10-fold is observed. In some aspects, the nucleic acid molecules contain about 2 to 50 fold, or more CpG motifs compared to vertebrate DNA. In some aspects, the nucleic acid molecules contain about 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55 fold or more CpG motifs compared to vertebrate DNA.

In some aspects, the present invention relates to immunostimulatory plasmids, or DNA sequences, that do comprise an antibiotic resistance gene. For example, the pMB75.6 plasmid described herein compri ses an the antibiotic resi stance gene kanamycin. The sequence of pMG75.6 is provided in Table 1 as SEQ ID NO: 1.

In some aspects, the present invention relates to immunostimulatory plasmids, or DN A 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 plasmi d described herein does not compri se any full -length or functional selectable or screenable marker genes. The sequence of pGCMB75.6 is provided in Table 1 as SEQ ID NO: 2.

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 i m m unosti m ul atory plasmids do not compri se 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.

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 provided in FIG. 3 and the nucleotide sequence of pLacZMB75.6 is provided in Table 1 as SEQ ID NO: 3. As shown in FIG. 3, pLacZMB75.6 is similar to pGCMB75.6, but contains a LacZ screenable marker.

It will be appreciated that the nucleotide sequences of the pMB75.6, pGCMB75.6 or pLacZMB75.6 plasmids may be varied to a certain extent without significantly adversely affecting their i m m un osti m ul atory properties. In some aspects, the present invention relates to an immunostimulatory plasmid comprising a nucleic acid sequence having at least 80% sequence identity with the sequence of pMB75.6 ( SEQ ID NO: 1). The i m m unosti m ul a tory plasmid preferably comprises a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 pMB75.6 (SEQ ID NO: 1). In some aspects, the immunostimulatory plasmid more preferably comprises the sequence of pMB75.6 (SEQ ID NO: 1).

In some aspects, the present invention relates to an immunostimulatory plasmid comprising a nucleic acid sequence having at least 80% sequence identity with the sequence of pGCMB75.6 (SEQ ID NO: 2). The immunostimulatory plasmid preferably comprises a nucleic acid sequence having at least 80%, at least 81 %), at least 82%, at least 83%>, at least 84%>, at least 85%, at least 86%>, at least 87%o, 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 pGCMB75.6 (SEQ ID NO: 2). In some aspects, the immunostimulatory plasmid more preferably comprises the sequence of pGCMB75.6 (SEQ ID NO: 2).

In some aspects, the present invention relates to an immunostimulatory plasmid consisting of a nucleic acid sequence having at least 80%> sequence identity with the sequence of pMB75.6 (SEQ ID NO: 1). The immunostimulatory plasmid preferably consists of a nucleic acid sequence having at least 80%>, at least 81 ), at least 82%>, at least 83%>, at least 84%>, at least 85%, at least 86%, at least 87%>, at least 88%. at least 89%, at least 90%>, at least 91%o, 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 p MB 7 .6 (SEQ ID NO: 1). In some aspects, the immunostimulatory plasmid more preferably consists of the sequence of pMB75.6 (SEQ ID NO:

1).

In some aspects, the present invention relates to an immunostimulatory plasmid consisting of a nucleic acid sequence having at least 80%> sequence identity with the sequence of pGCMB75.6 (SEQ ID NO: 2). The immunostimulatory plasmid preferably consists of a nucleic acid sequence having at least 80%>, at least 81 %>, at least 82%>, at least 83%, at least 84%>, at least 85%, at least 86%>, at least 87%o, 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%s sequence identity with the sequence of pGCMB75.6 (SEQ ID NO: 2). In some aspects, the immunostimulatory plasmid more preferably consists of the sequence of pGCMB75.6 (SEQ ID NO: 2).

Another important aspect of this invention provides for immunostimulatory DNA

sequences or immunostimulatory plasmids capable of stimulating an immune response including nucleic acid sequences that hybridize under high stringency conditions to SEQ ID NO: 1 or SEQ

ID NO: 2. 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 75%, 76%, 77%, 78%, 79%, 80% 81%, 82%, 83%,, 84%, 85%, 86%, 87%, 88%, 89%, 90%,, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO: 1 or the respective complementary sequence. In other aspects, homologous nucleic acid sequences will have a sequence simi larity of at least about 75%, 76%, 77%, 78%, 79%, 80% 81%, 82%, 83%, 84%, 85%, 86%, 88%, 89%, 90%, 91%, 92%, 93%,, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO: 2 or the respective complementarv' sequence. Sequence similarity may be calculated using a number of algorithms known in the art, such as BLAST, described in Altschul, S. F., et al., J. Mol. Biol. 2 1 5 :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 1 8 nucleotides, more usually 30 or more nucleotides, and may comprise the enti e nucleic acid sequence of the composition for comparison purposes.

Nucleotide sequences that can hybridize to SEQ ID NO: 1 or SEQ ID NO: 2 are contemplated herein. Stringent hybridization conditions include conditions such as hybridization at 50°C or higher and 0. 1 X SSC (1 5 m M sodium chloride/ 1 .5 niM sodium citrate). Another example is overnight incubation at 42°C in a solution of 50% form amide, 5X SSC ( 1 50 niM NaCl, 1 5 m M tri sodium citrate ), 50 m M sodium phosphate (pH 7.6), 5X Denhardt's solution, 10% dextran sulfate, and 20 g/ml denatured, sheared salmon sperm DNA, followed by washing in 0. 1 X SSC at about 65 C . Exemplary stringent hybridization conditions are hybridization conditions that are at least about 80%>, 85%, 90%, or 95% as stringent as the above specific conditions. Other stringent hybridi ation 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).

Mutant nucleotides of the DNA molecules described herein may be used, so long as mutants include nucleic acid sequences maintain the ability to increase the conception rate in cows as described herein. The DNA sequence of such a mutation will usually 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 ., Biotechniques

14:22, 1993; Barany, Gene 37: 1 1 1 -23, 1985; Colicelli et al ., Mol. Gen. Genet. 199:537-9, 1985; and Sambrook et al .. Molecular Cloning: A Laboratory Manual, CSH Press 1 89, pp. 1 5.3 -

1 5. 108 and all incorporated herein by reference. In summary, the invention relates to nucleic acid sequences capable of activating cytosolic DNA surveillance molecules in a subject and variants or mutants thereof. Also, the invention encompasses the intermediary RNAs encoded by the described nucleic acid sequences, as well as any resultant amino acid sequences encoded.

In some aspects, where the nucleotide sequence of the immunostimulatory plasmid v aries from the sequences provided in SEQ ID NOs 1 and 2, the CpG dinucleotides in the plasmid are preferably left intact. Alternatively, if the nucleotide sequence of the plasmid is altered such that a CpG dinucleotide is eliminated, the sequence of the plasmid may be altered at another location such that the total number of CpG dinucleotides in the plasmid remains the same. Further CpG dinucleotides in addition to those already present in the nucleotide sequence pGCMB75.6 may also be introduced into the plasmid. Thus, for example, the i m m unost mul atory 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 dinucleotides. For example, the immunostimulatory plasmid can compri se 283 CpG dinucleotides.

In some aspects, where the nucleotide sequence of the immunostimulatory plasmid varies from the sequences provided herein, the CpG motif types in the plasmid are varied to modulate the resultant activ ation of the cvtosolic DNA surveil lance molecules. For example, the number of immune stimulatory CpG motifs may be increased to increase the activation of specific cvtosolic DNA surv eillance molecules responsive to a speci ic threshold of immunostimulatory plasmid/ ^' DNA. By way of further example, the number of non-immune stimulatory CpG motifs may be increased to decrease the activation of speci ic cv tosolic DNA surv eillance molecules and/or increase activation of other DNA surveillance molecules.

In particular, the present invention relates to pharmaceutical formulations comprising any of the inmuinosti mul atory plasmids or DNA sequences described herein and a pharmaceutically acceptable carrier.

B. i mmunomodulator

Suitable immunomodulator compositions for use with the i mm u n osti mul atory plasmids described herein are described in U.S. Patent Application Publications Nos. 2012/0064151 A 1 and 20 13/0295 167 A 1 the contents of both of which are hereby incorporated by reference in their entirety.

The immunomodulator composition comprises a liposome delivery vehicle and at least one of the i m m unosti m ul a tory plasmids, or DNA sequences, described herein .

A suitable liposome delivery vehicle comprises a lipid composition that is capable of deliv ering nucleic acid molecules to the tissues of the treated subject. A liposome deliv ery 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 1 hour, or for at least about 24 hours.

A liposome delivery vehicle of the present invention comprises a lipid composition that is capable of facilitating the delivery of 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 picogram (pg) of protein expressed per milligram (mg) of total tissue protein per microgram (μ^) of nucleic acid delivered. 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 ng of nucleic acid delivered; or at least about 50 pg of protein expressed per mg of total tissue protein per g 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 ^ig of nucleic acid delivered, with the above amounts being more preferred.

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

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 M LVs are well know n in the art. More preferred liposome delivery vehicles compri se liposomes hav ing a polycationic lipid composition (i .e., cationic liposomes) and/or liposomes having a cholesterol backbone conj ugated to polyethylene glycol . Exemplary cationic liposome compositions include, but are not limited to, N-[l-(2,3- dioleyloxy)propyl]-N,N,N- tri methyl am m oni um chloride (DOT MA ) and cholesterol, N-[ 1 - (2,3-di ol eoyloxy Jpropyl ]-N,N, N- tri methyl am m oni um chloride (DOTAP) and cholesterol, I - [ 2-( ol eoyl oxy )eth\ ]-2-ol ev -3 -( 2- hydroxy ethyl )-i mi dazol i ni um chloride (DOTIM ) and cholesterol,

dimethyldioctadecylammonium bromide (DDAB) and cholesterol, and combinations thereof. A most preferred liposome composition for use as a delivery vehicle includes DOTIM and cholesterol .

A suitable nucleic acid molecule includes any of the immunostimulatory pi asm ids 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 invention, "non-coding ^" nucleic acids can include regulatory regions of 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 of a protein coding portion, such as a plasmid vector without a gene insert. Expression of a protein encoded by the plasmi ds described herein is not required for activation of cytoso!ic DNA surveillance molecules; therefore the plasmids need not contain any coding sequences operatively linked to a transcription control sequence. However, further advantages 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 may be included in the i m m unost i m ul atory plasmids described herein, or can be included in a separate nucleic acid (e.g., a separate plasmid) in the composition.

Complexing a liposome w ith the i m m un osti m ul atory 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 are 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 tig to about 10 ,ug of plasmid can be combined with about 8 nmol liposomes, from about 0.5 ng to about 5 Lig of plasmid can be combined with about 8 nmol liposomes, or about 1.0 ng of plasmid can be combined with about 8 nmol liposomes. The ratio of plasmid to lipid ( g plasmid :nmol lipid) in a composition can be at least about 1 : 1

plasmid:lipid by weight (e.g., I itg plasmid: I 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 plasmid.lipid by weight; from about 1 :2 to about 1 :40 plasmid:lipid by weight; from about 1 :3 to about 1 :30 plasmid: lipid by weight; or from about 1 :6 to about 1 : 1 5 plasmid:lipid by weight.

C. Biological Agent

Any of the i mmunomodul ator compositions described herein can further comprise at least one biological agent, in addition to the liposome deliv ery vehicle and at least one of the plasmids described herein.

Suitable biological agents are agents that are effectiv e in prev enting or treating 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-limiting example, a cytokine, which includes a family of proteins, is a known immunity enhancing protein family. Suitable immunogens are proteins which elicit a humoral and/or cellular immune response such that administration of the immunogen to a subject mounts an i m m un ogen -sped fi c 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 1 2 amino acids and as large as a full length protein, including any sizes in between. The antigen may be a multimer protein or fusion protein. The antigen may be a purified antigen. Alternatively, the immune enhancer protein or immunogen can be encoded by the immunostimulatory pi asm id or by another nucleic acid included in the i m m unom odul ator 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 operatively linked to a transcription control sequence, such that the immunogen is expressed in a tissue of a subject, thereby eliciting an i m m unogen -sped fi c 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.

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 kil led viral vaccines, may be used in combination with the

i m m unom odul ator composition of the present invention. Suitable vaccines include those known in the art for avian or bovine species.

The biological agent can be an antimicrobial . Suitable antimicrobials include: qui nol ones, preferably fluoroquinolones, β-lactams, and macrolide-lincosamidestreptogramin (MLS) antibiotics.

Suitable quinolones include benofloxacin, binfloxacin, cinoxacin, ciprofloxacin, clinafloxacin, danofloxacin, difloxacin, enoxacin, enrofloxacin, fleroxacin, gemifloxacin, ibafloxacin, levofloxacin, !om ofloxacin, marbofloxacin, moxifloxacin, norfloxacin, ofloxacin, orbifloxacin, pazufl oxacin, pradofloxacin, peril oxacin, sarafloxacin, sparfloxacin, temafloxacin, and tosufl oxacin . Preferred fluoroquinolones include ciprofloxacin, danofloxacin, enrofloxacin, moxifloxacin, and pradofloxacin. Suitable naphthyridones include nalidixic acid. Suitable β- lactams include penicillins (e.g., amoxicillin, ampicillin, azlocillin, benzathine penicillin, benzylpenicillin, carbenicillin, cl oxacillin, co-amoxiclav [i .e. am oxi cil 1 i n/cl avul ani c acid], dicloxacil lin, flucloxacillin, methicillin, mezlocillin, nafcillin, oxacillin,

phenoxymethylpeni ci 11 i n, 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 penems (e.g., doripenem, ertapenem, 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.

Suitable MLS antibiotics include cli ndamycin, li neomycin, pirlimycin, and any macrolide antibiotic. A preferred lincosamide antibiotic is pirlimycin.

Other antimicrobials include aminoglycosides, clopidol, dimetridazoles, erythromycin, framycetin, furazolidone, halofuginone, 2-pyridones, robenidine, sulfonamides, tetracyclines, trimethoprim, various pleuromutilins (e.g., tiamulin and valnemulin ), and various streptomycin (e.g., monensin, narasin, and salinomycin).

II. Methods

A. Methods of Immune Stimulation

In one embodim ent of the invention, an i mmune response is elicited in a fem ale member of the bovine species by administering an effective amount of an immunomodulator composition to the female member of the bovine species. The effective amount is sufficient to elicit an immune response in the female member of the bovine species. The i m m unom odul ator includes a liposome deliver}' vehicle and a nucleic acid molecule.

In one embodiment, the effective amount of the i m m unom odul ator is from about 1 micrograms to about 1000 micrograms per animal. In another embodiment, the effective amount of the immunomodulator is from about 5 micrograms to about 500 micrograms per animal. In yet another embodiment, the effective amount of the immunomodulator is from about 10

micrograms to about 100 micrograms per animal. In a further embodiment, the effective amount of the immunomodulator is from about 10 micrograms to about 50 micro grams per animal.

In another embodiment of the invention, an immune response is elicited in a female member of the bovine species by administering an effective amount of an immunomodulator, which includes a liposome delivery vehicle, an isolated nucleic acid molecule, and a biological agent. It is contemplated that the biological agent may be mixed with or coadministered with the immunomoduiator or independently thereof. Independent administration may be prior to or after administration of the immunomoduiator. It is also contemplated that more than one

administration of the immunomoduiator or biological agent may be used to extend enhanced immunity. Furthermore, more than one biological agent may be co-administered with the immunomoduiator, administered prior to the immunomoduiator, administered after

administration of the immunomoduiator, or concurrently.

B. Conception rates

The methods of the invention are useful for increasing conception rates in cows. In preferred embodiments, the conception rate in cows increases relative to the conception rate in a control population. In some preferred embodiments, the conception rate in cows increases relative to the conception rate in a control population as measured by a p-value of < 0.05.

The methods of the invention are useful for increasing first service conception rates in cows. As used herein, first service conception rate refers to the proportion of cows bred to artificial insemination that conceived to the first insemination. In preferred embodiments, the first service conception rate in cows increases relative to the first service conception rate in a control population . In some preferred embodiments, the first service conception rate in cows increases relative to the first service conception rate in a control population as measured by a p- value of < 0.05.

The methods of the invention are useful for increasing conception rates in non-cycling cows. As used herein, "non-cycling ^" refers to cows not detected in oestrus by observation or by removal of tail paint approximately 30 days prior to the planned start of breeding. In preferred embodiments, the conception rate in non-cycling cows increases relative to the conception rate in a control population. In some preferred embodiments, the conception rate in non-cycling cows increases relative to the conception rate in a control population as measured by a p-value of < 0.05.

The methods of the invention are useful for increasing first-service conception rates in non-cycling cows. In preferred embodiments, the first service conception rate in non-cycling cows increases relative to the first service conception rate in a control population. In some preferred embodiments, the first service conception rate in non-cycling cows increases relative to the first service conception rate in a control population as measured by a p-value of < 0.05. C. Administration

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.

Vaccination of the bovine species can be performed at any age. The v accine may be administered intravenously, intramuscularly, intradermal, intraperitoneal, subcutaneously, by spray/aerosol, orally, intraocularly, intratracheally, intranasal, or by other methods known in the art. Further, it is contemplated that the methods of the invention may be used based on routine vaccination schedules. The immunomodulator may also be administered intravenously, intramuscularly, subcutaneously, by spray, orally, intraocularly, intratracheal ly, nasally, 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 admini stered by itself to the animal prior to parturition . In another embodiment, the immunomodulator is admini stered by itself to the animal post parturition. In yet another embodiment, the immunomodulator is administered by itself to the animal at the same time as parturition. In still another embodiment, the

immunomodulator is admini stered by itself to the animal both prior to parturition and at the same time as parturition. In a further embodiment, the immunomodulator composition i s coadministered at the same time as the v accination prior to parturition. In yet a further

embodiment, the immunomodulator composition is co-administered at the same time as the vaccination at the same time as parturition . The co-administration may include admini stering the vaccine and immunomodulator in the same general location on the animal at two different sites next to each other (i.e., injections next to each other at the neck of the animal ), on opposing sides of the animal at the same general location (i .e., one on each side of the neck), or on different locations of the same animal. In another embodiment, the immunomodulator composition i s administered prior to vaccination and parturition. In a further embodiment, the

i m m unom odul ator composition is admini stered after vaccination but prior to parturition . In a further embodiment, the immunomodulator composition is administered after parturition to an animal that has been vaccinated prior to parturition. A skilled artisan will recognize that administration routes may vary depending upon the subject and the health or state of the subject.

In one embodiment, the immunomodulator is admini stered by itself to the animal prior to breeding. In another embodiment, the i mm unom odul ator is admini stered by itself to the animal post breeding. In yet another embodiment, the immunomodulator is administered by itself to the animal at the same time as breeding. In still another embodiment, the immunomodulator is administered by itself to the animal both prior to breeding and at the same time as breeding. In a further embodiment, the i m m unom odul ator composition i s co-administered at the same time as the vaccination prior to breeding. In yet a further embodiment, the immunomodulator composition is co-administered at the same time as the vaccination at the same time as breeding. The co-admini stration may include administering the vaccine and immunomodulator in the same general location on the animal at two different sites next to each other (i.e., injections next to each other at the neck of the animal ), on opposing sides of the animal at the same general location (i .e., one on each side of the neck), or on different locations of the same animal . In another embodiment, the immunomodulator composition is administered prior to vaccination and breeding. In a further embodiment, the i m m unomodul ator composition is admini stered after vaccination but prior to breeding. In a further embodiment, the immunomodulator composition is admini stered after breeding to an animal that has been vaccinated prior to breeding. A ski lled artisan will recognize that administration routes may vary depending upon the subject and the health or state of the subject.

In one embodiment, the immunomodulator is administered from about 1 to about 14 days prior to challenge or from about I to about 14 days post challenge. In another embodiment, the immunomodulator is admini stered from about I to about 7 days prior to challenge or from about I to about 7 days post challenge. In yet another embodiment, the immunomodulator i s administered 1 , 2, 3, 4, 5, 6, 7 days prior to challenge or 1, 2, 3, 4, 5, 6, 7 days post challenge. In a preferred embodiment, the imm nomodulator is administered 3 days prior to challenge or 3 days post challenge.

Other delivery 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 delivery systems are available and known to those of ordinary skill in the art. They include polymer based systems such as pol y ( 1 acti de-gl y col i de ), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U. S. Pat. No. 5,075, 109. 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 triglycerides; hydrogel release systems; sylastic systems; peptide based systems; wax coatings; compressed tablets using convention binders and excipients; partially fused implants; and the like. Speci ic examples include, but are not limited to erosional systems in which an agent of the invention is contained in a form within a matrix such as those described in U.S. Pat. Nos.

4,452,775, 4,675, 189 and 5,736,152, and diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Pat. Nos. 3,854,480, 5,133,974 and 5,407,686. In addition, pump-based hardware deliveiy systems can be used, some of which are adapted for implantation.

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

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 preventing 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 to 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 of ordinary skill in the art can empirically determine the effecti ve amount of immunomodulator without necessitating undue experimentation.

The term "cow" as used herein refers to any member of the bovine species that is capable of bearing offspring, including without limitation, female bovine, cows, and heifers.

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 activation of cells, particularly phagocytic cells). Exemplary cytokines include, without limitation, iiiterleukin-2 (IL-2), interleukin-12 (IL 12), interleukin- 15 (IL- 15), interleukin-18 (IL- 1 8), interferon-a (IFNa), and interferon-γ (IFNy). The term "elicit ^" can be used interchangeably with the terms activate, sti mulate, generate or upregulate.

The term "eliciting an immune response ^" in a subject refers to specifically control ling 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).

The term "operatively 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 initiation, 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 w hich function in avian, fi sh, mammalian, bacteria, plant, and insect cell s. While any transcriptional control sequences may be used with the invention, the sequences may include naturally occurring transcription control sequences naturally associated with a sequence encoding an immunogen or immune stimulating protein.

The terms "nucleic acid molecule ^" and "nucleic acid sequence ^" can be used

interchangeably and include DNA. RNA, or derivatives of either DNA or RNA. The terms also include oligonucleotides and larger sequences, including both nucleic acid molecules that encode a protein or a fragment thereof, and nucleic acid molecules that compri se regulatory regions, introns, or other non-coding DNA or RNA. Ty pically, an oligonucleotide has a nucleic acid sequence from about 1 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, i nsect, viral, plant, or synthetic sources. 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 substantial ly interfere with the nucleic acid molecule's ability to encode an immunogen or immune stimulating protein useful in the methods of the present invention. 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. 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.

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, ampicilli n 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 plasmid 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 plasmid and expressed the kanamycin resistance gene will sur ive. The terms "selectable marker" and "selectable marker gene" also include genes that code for enzymes involved in the synthesis of a compound that i s 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 cells that are auxotrophic for the amino acid lysine due to a mutation in or the absence of an enzyme involved in lysine biosynthesi s normally are unable to grown on media that has not been supplemented with lysine. When such bacteria are subjected to a transformation procedure to introduce a plasmid 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 poi son/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 cel l 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 having 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.

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 cells 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).

Tabic 1 : Plasmi d DNA sequences

Plasmid SEQ SEQUENCE

I D

NO.

pMB75.6 1 ctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctc attttttaaccaata ggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgt tgttcca gtttggaacaagagtccactattaaagaacgtggactccaacgtcaaagggcgaaaaacc gtctatc agggcgatggcccactacgtgaaccatcaccctaatcaagttttttggggtcgaggtgcc gtaaagc actaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaa cgtg gcgagaaaggaagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcg gt cacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcgtccca ttcgc cattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgcc agctgg cgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcac gacgtt gtaaaacgacggccagtgagcgcgcgtaatacgactcactatagggcgaattgggtaccg ggccc cccctcgagcaggatctatacattgaatcaatattggcaattagccatattagtcattgg ttatatagcat aaatcaatattggctattggccattgcatacgttgtatctatatcataatatgtacattt atattggctcatg tccaatatgaccgccatgttgacattgattattgactagttattaatagtaatcaattac ggggtcattagt tcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctg accgccca acgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaataggga ctttccat tgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtat catatgcca agtccgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtac atgacctt acgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgat gcggttttgg cagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccacccc attgacgt caatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactc cgccccatt gacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagt gaaccg tcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccg atccag cctcccctcgaagccgatctgataacggtaccgataagctggcggccgattaagctacag aagttg gtcgtgaggcactgggcaggtaagtatcaaggttacaagacaggtttaaggagaccaata gaaact gggcttgtcgagacagagaagactcttgcgtttctgataggcacctattggtcttactga catccacttt gcctttctctccacaggtgtccactcccaggttcaattacagctcttaagcagccgcaag cttgatatc gaattcctgcagcccgggggatccactagttctagagcggccgccaccgcggtggagctc gaatt atcagatcgattaataactatgctcaaaaattgtgtacctttagctttttaatttgtaaa ggggttaataag gaatatttgatgtatagtgccttgactagagatcataatcagccataccacatttgtaga ggttttacttg ctttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttg ttgttaactt gtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataa agcatttttttc actgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggatcat cagatctgccgg tctccctatagtgagtcgtattaatttcgataagccaggttaacctgcattaatgaatcg gccaacgcg cggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgc gctcggt cgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacaga atcagg ggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaa ggc cgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacg ctcaagt cagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctcc ctcgt gcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcggg aagcgtgg cgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagc tgggctgtgt gcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtc caacccg gtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgagg tatgta ggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagta tttggtat ctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaa acaaacc accgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaagga tctcaaga agatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagg gattttggtc atgagcgcgcctaggcttttgcaaagatcgatcaagagacaggatgaggatcgtttcgca tgattga acaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatga ctgggc acaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgccc ggttct ttttgtcaagaccgacctgtccggtgccctgaatgaactgcaagacgaggcagcgcggct atcgtg gctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaag ggact ggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccg agaaagt atccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccatt cgaccac caagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcag gatgat ctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgagc atgc ccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtgg aaaatg gccgcttttctggattcatcgactgtggccggctgggtgiggcggaccgctatcaggaca tagcgtt ggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgct ttacggta tcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgag cgggactct ggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccac cgccg ccttctatgaaaggttgggcttcggaatcgttttccgggacgccggctggatgatcctcc agcgcgg ggatctcatgctggagttcttcgcccaccctaggcgcgctcatgagcggatacatatttg aatgtattt agaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccac

pGCMB75.6 2 tgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacg ccaatag ggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtac atcaagtgt atcatatgccaagtccgccccctattgacgtcaatgacggtaaatggcccgcctggcatt atgccca gtacatgaccttacgggactttcctacttggcagtacatctacgtattagtcatcgctat taccatggtg atgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttcca agtctcca ccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatg tcgtaacaa ctccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcag agctcg tttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaag acaccgg gaccgatccagcctcccctcgaagccgatctgataacggtaccgataagctggcggccga ttaagc tacagaagttggtcgtgaggcactgggcaggtaagtatcaaggttacaagacaggtttaa ggagac caatagaaactgggcttgtcgagacagagaagactcttgcgtttctgataggcacctatt ggtcttact gacatccactttgcctttctctccacaggtgtccactcccaggttcaattacagctctta agcagccgc aagcttgatatcgaattcctgcagcccgggggatccactagttctagagcggccgccacc gcggtg gagctcgaattatcagatcgattaataactatgctcaaaaattgtgtacctttagctttt taatttgtaaag gggttaataaggaatatttgatgtatagtgccttgactagagatcataatcagccatacc acatttgtag aggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacataaaatga atgcaattgt tgttgttaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaa tttcacaaataa agcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatca tgtctggatcatca gatctgccggtctccctatagtgagtcgtattaatttcgataagccaggttaacctgcat taatgaatcg gccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactg actcgct gcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggtt atccac agaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaa ccg taaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaa aaatcga cgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccct ggaag ctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttct cccttcggg aagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcg ctccaagct gggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcg tcttgag tccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagc agagc gaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactag aagaaca gtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctct tgatccggc aaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcaga aaaaaag gatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaact cacgttaag ggattttggtcatgggcgcgcctaggcttttgcaaagatcgatcaagagacaggatgagg atcgtttc gcagcttttcattctgactgcaacgggcaataagtctctgtgtggattaaaaaaagagtg tctgatagc agcttctgaactggttacctgccgtgagtaaattaaaattttattgacttaggtcactaa ggcgccttgc gctgaggttgcgtcgtgatatcatcagggcagaccggttacatccccctaacaagctgta taaagag aaatactatctcattggcgttgcccgcacctgacagtgcgacgttgggctgcgtccgtcg accaacg gtaccgaggtaacagcccaatctatccatgatctcggccaggccgggtcggccgttatgc agcccg gctcgggtatgaagccattaaggagccgacccagcgcgaccgggcggccggtcacgctgc ctct gctgaagcctgcctgtcactccctgcgcggcgtacccgccgttctcatcgagtaggctcc ggatcg cgaccccggacgggccctgggcccaggagcggcctatgacaaatgccgggtagcgatccg gca ttcagcattgactgcgcacggatccagtccttgcaggagccttatgccgaccgtagcaaa aaatgag cccgagccgatcgcgagttgtgatccggtcccgccgattgccggtcgcgatgacggtcct gtgtaa gcgttatcgttaccaattgtttaagaagtatatacgctacgaggtacttgataacttctg cgiagcatac atgaggttttgtataaaaatggcgggcgatatcaacgcagtgtcagaaatccgaaacagt ctgcggg actctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatttcgat tccacc gccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccggctggatgatc ctccagc gcggggatctcatgctggagttcttcgcccaccctaggcgcgctcatgagcggatacata tttgaat gtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccaccta aattgtaa gcgttaatattttgttaaaattcgcgtt

ggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtt tggaacaa gagtccactattaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcaggg cgatg gcccactacgtgaaccatcaccctaatcaagttttttggggtcgaggtgccgtaaagcac taaatcgg aaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgaga aag gaagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctg cg cgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcgtcccattcgccatt caggct gcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaa agggg gatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgta aaacgac ggccagtgagcgcgcgtaatacgactcactatagggcgaattgggtaccgggccccccct cgagc aggatctatacattgaatcaatattggcaattagccatattagtcattggttatatagca taaatcaatatt ggctattggccattgcatacgttgtatctatatcataatatgtacatttatattggctca tgtccaatatgac cgccatgttgacattgattattgactagttattaatagtaatcaattacggggtcattag ttcatagcccat atatggagttccgcgttacataacttacggtaaatggcccgcctggc pLacZMB75.6 3 tgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacg ccaatag ggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtac atcaagtgt atcatatgccaagtccgccccctattgacgtcaatgacggtaaatggcccgcctggcatt atgccca gtacatgaccttacgggactttcctacttggcagtacatctacgtattagtcatcgctat taccatggtg atgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttcca agtctcca ccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatg tcgtaacaa ctccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcag agctcg tttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaag acaccgg gaccgatccagcctcccctcgaagccgatctgataacggtaccgataagctggcggccga ttaagc tacagaagttggtcgtgaggcactgggcaggtaagtatcaaggttacaagacaggtttaa ggagac caatagaaactgggcttgtcgagacagagaagactcttgcgtttctgataggcacctatt ggtcttact gacatccactttgcctttctctccacaggtgtccactcccaggttcaattacagctctta agcagccgc caaaacaaaattcctcaaaaatcatcatcgaatgaatggtgaaataatttccctgaataa ctgtagtgtt ttcagggcgcggcataataattaactatgctcaaaaattgtgtacctttagctttttaat ttgtaaagggg ttaataaggaatatttgatgiatagtgccttgactagagatcataatcagccataccaca tttgtagagg ttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatg caattgttgtt gttaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttc acaaataaagc atttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgt ctggatcatcagat ctgccggtctccctatagtgagtcgtattaatttcgataagccaggttaacctgcattaa tgaatcggcc aacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgact cgctgcg ctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatc cacaga atcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccg taa aaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaa tcgacg ctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctgg aagctc cctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctccc ttcgggaag cgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctc caagctggg ctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtct tgagtcca acccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagag cgagg tatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaga acagtatt tggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatc cggcaaac aaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaa aaggatct caagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgt taagggatt ttggtcatgggcgcgcctaggcttttgcaaagatcgatcaagagacaggatgaggatcgt ttcgcag cttttcattctgactgcaacgggcaataagtctctgtgtggattaaaaaaagagtgtctg atagcagctt ctgaactggttacctgccgtgagtaaattaaaattttattgacttaggtcactaaggcgc cttgcgctga ggttgcgtcgtgatatcatcagggcagaccggttacatccccctaacaagctgtataaag agaaata ctatctcattggcgttgcccgcacctgacagtgcgacgttgggctgcgtccgtcgaccaa cggtacc gaggtaacagcccaatctatccatgatctcggccaggccgggtcggccgttatgcagccc ggctcg ggtatgaagccattaaggagccgacccagcgcgaccgggcggccggtcacgctgcctctg ctga agcctgcctgtcactccctgcgcggcgtacccgccgttctcatcgagtaggctccggatc gcgacc ccggacgggccctgggcccaggagcggcctatgacaaatgccgggtagcgatccggcatt cagc attgactgcgcacggatccagtccttgcaggagccttatgccgaccgtagcaaaaaatga gcccga gccgatcgcgagttgtgatccggtcccgccgattgccggtcgcgatgacggtcctgtgta agcgtta tcgttaccaattgtttaagaagtatatacgctacgaggtacttgataacttctgcgtagc atacatgagg ttttgtataaaaatggcgggcgatatcaacgcagtgtcagaaatccgaaacagtctgcgg gactctg gggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccacc gccgc cttctatgaaaggttgggcttcggaatcgttttccgggacgccggctggatgatcctcca gcgcggg gatctcatgctggagttcttcgcccaccctaggcgcgctcatgagcggatacatatttga atgtattta gaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctaaattgta agcgtta atattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaatagg ccgaaatcggcaa aatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaa caagagtc cactattaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatg gccca ctacgtgaaccatcaccctaatcaagttttttggggtcgaggtgccgtaaagcactaaat cggaaccc taaagggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaagga agg gaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgt aac caccacacccgccgcgcttaatgcgccgctacagggcgcgtcccattcgccattcaggct gcgca actgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggg gatgt gctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacg acggcc agtgagcgcgcgtaatacgactcactatagggcgaattgggtaccgggccccccctcgag gtcga cggtatcgataagcttgatatcgaattcctgcagcccgggggatccactagttctagagc ggccgcc accgcggtggagctccagcttttgttccctttagtgagggttaattgcgcgcttggcgta atcatggtc atagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccgg aagcataaa

gtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgc

EXAMPLE S

The following example illustrates various embodiments of the invention.

Exam le 1: Efficacy of imnuinomodtilator composition in increasing conception rates in non-cycling cows

The purpose of this study was to assess the effect of modulating immune function at calving on reproductive outcomes of the treated cows.

Immunomodulator

The immunomodulatory used in this study was the composition described above in Example 1.

Study Animals

875 Trie si an, Jersey or cross-bred cows aged > 2 years were obtained from two commercial dairy herds Farm A and B in the Waikato district of New Zealand. The cows were selected based on being newly calved and lactating. Cows were excluded if they were treated with antibiotics, non-steroidal anti-inflammatories or corticosteroids in the 30 days preceding calving or if they exhibited gross evidence of any disease at calving. The remaining animals were blocked by age (heifers versus cows) and assigned within sequential pairs of animals presented from pre-prepared randomization lists to the treatment or placebo groups.

Treatment

Treatment was randomized and the treatment allocation was not included on any of the post treatment sampling sheets and the milk samples were assigned a unique number at accession which was used during subsequent laboratory analysis. On the day of calving (day 0) and following physical examination and enrollment, the appropriate treatment was administered by intramuscular injection into the right gluteal muscle. The injection site was swabbed with a cotton bail moistened in 70% methylated spirits prior to injection. Treatment was repeated in the evening of day 3 or the morning of day 4 and again at day 7 postpartum with injection in the right gluteal muscles.

Breeding Management Tail paint (a heat detection aid) was applied approximately 30 days before the planned start of the breeding program. Those cows not detected in oestrus by observation or removal of the tail paint were treated with a combination of progesterone, GnRH, and prostaglandin F _2a and bred to set time artificial insemination. For the first 37 davs at the Farm A site and 47 davs at the Farm B site, those cows detected in standing oestais were bred by artificial insemination.

Thereafter intact bull s were run with the herd. The total length of the mating period was 77 and 80 days for Farm A and B, respectively. Cows were examined by trans-rectal ultrasonography at 83 and 1 13 days after the start of the breeding program (Farm A) and 90 and 120 days after this start of the breeding program (Farm B). Those cows detected pregnant, had the stage of gestation estimated. Where the estimated stage of gestation was within 7 days of recorded breeding, either artificial insemination or natural mating, the recorded date was accepted as the day of conception.

Results

The three-week submission rate (the proportion of cows detected and inseminated within three weeks at the start of the breeding program ), the first service conception rate (the proportion of cows bred to artificial insemination that conceived to the first insemination ) and the 3 and 6 week in calf rate (the proportion of cows confirmed pregnant within the first three and six weeks of the breeding program ), and the final pregnancy rates were calculated.

The binomial reproductive outcomes were initially analyzed in bivariate (chi squared ) analysis. Multivariate logistic regression models were then undertaken including the explanatory v ariables of treatment, herd, age (2, 3 and >3 years old), days in milk at the start of the breeding program (categorized as 50 to 71 days and >72 days) and breed (Friesian versus other breeds). Additional ly , the planned start of mating to concepti on interv al was calculated for each cow and Kaplan-Meier survival analysi s was used to calculate the median days to conception.

The treatment groups were balanced for breed code and age and p-v alues were calculated using a chi -squared test, as depicted in Table 1.

Table 1 : Number of cows in treated and control groups by breed code and age

Treated Control P-value

Age Group

2 years 79 73 0.62

3 years 91 82 0.48

> 4 years 267 279 0.33

Total 437 434 ___ Breed

Fresian 245 258 0.29

Jersey 6 6 1.00*

Crossbred 186 169 0.29

Total 437 433 ___

P-value calculated using Fisher's exact test.

Additionally, the mean and median time from calving to planned start of mating was the same between the treatment and control groups, as depicted in Table 2. The P-value comparing median days from calving to planned start of breeding for the treatment and control groups was calculated using a Mann-Whitney test.

Table 2: Mean, standard error ( SE), and median days from calving to start of breeding program for cows in the treated and control groups

n Mean SE Median P-value

Treated 437 72.4 0.43 75

0.92

Control 434 72.4 0.43 75

As shown in Table 3, there were no differences between treatment and control groups for the probability of submi ssion to artificial insemination in 2 1 days after planned start of mating (PSM) ( submission in 3 weeks), probability of conception to the first insemination after PSM (conception to first service), probability of pregnancy by 2 1 and 42 days (pregnant in 3 weeks and pregnant in 6 weeks, respectively) after PSM and overal 1 pregnancy at the bivariate level . P- values were calculated using a Chi-square test.

Table 3: Number (%) of cows by treatment groups

Treated (%) Control (%) P-value

Treated as a non-cycler 75/437 (17.2) 91 /434 (21 .0) 0.15

Submission in 3 weeks 383/432 (88.7) 394/430 (91.6) 0.14

Conception to 1 ^st service 263/383 (68.7) 254/394 (64.5) 0.21

Pregnant in 3 weeks 273/432 ( 63.2) 260/430 (60.5) 0.41

Pregnant in 6 weeks 344/432 (79.6) 327/430 (76.0) 0.21

Overall pregnant 384/432 (88.9) 383/430 (89.1) 0.93 In a multivariable regression analysis that controlled for potential effect modifiers including breed (Hoi tein, Jersey and crossbred), age (2, 3 and 4+ years), herd (1 and 2), ovarian cyclic activity (cycling and not cycling based on the insertion of a CIDR device) and time from calving to PSM (50 to 71 days and > 72 days), there was a higher probability of conception to first service and pregnant in 3 weeks for non-cyclic cows in the treated group compared to their counterparts in the control group. FIG. 4A shows a chart of the probability (estimated marginal means and standard error of the mean (SEM)) of conception to the first insemination and FIG. 4B shows the probability (estimated marginal means and SEM) of pregnancy within 3 weeks after the planned start of the breeding program for cyclic and non-cyclic cows by treatment groups. The asterisk indicates a difference between treatments within the non-cycling group. The median time from PSM to pregnancy was 15 and 16 days for the treated and control groups respectively (P = 0.27).

Numerically more of the treated cows conceived to first service (69% versus 65%), were pregnant by three (63%) versus 61%) and six weeks (80%> versus 76%) than the untreated controls. There was a treatment by non-cycling status interaction whereby amongst those cows diagnosed as not detected in oestrus and treated for this condition, the treated cows had a higher first service conception rate and a higher proportion pregnant by three weeks into the breeding program.