HK1177589A - Vaccines comprising cholesterol and cpg as sole adjuvant - carrier molecules - Google Patents

Description

Vaccine comprising cholesterol and CPG as sole adjuvant-carrier molecule

The present invention claims priority from U.S. provisional patent application No. 61/349,244, filed on 28/5/2010, the entire contents of which are incorporated herein by reference.

Technical Field

The present invention relates to vaccines having one or more antigens and cholesterol and uses thereof. The invention also relates to vaccines having one or more antigens and one or more immune modulatory molecules, and cholesterol, and uses thereof.

Background

Cholesterol has been found to enhance the activity of immune modulatory molecules and thus the combination of cholesterol and immune modulatory molecules can be used in the treatment and/or prevention of human and animal disorders. Vaccines comprising one or more antigens and cholesterol are described, as well as vaccines comprising one or more antigens, one or more immune modulatory molecules and cholesterol.

Disclosure of Invention

In certain aspects, the invention relates to vaccines comprising one or more antigens and cholesterol. In certain aspects, the one or more vaccines are each independently a microbial antigen, a self-antigen, a tumor antigen, an allergen, or an addictive substance.

In certain aspects, the invention relates to vaccines comprising one or more antigens and one or more immune modulatory molecules, and cholesterol. In certain aspects, the vaccine further comprises a pharmaceutical carrier. In certain aspects, the one or more antigens are each independently a microbial antigen, a self antigen, a tumor antigen, an allergen, or an addictive substance.

In certain aspects, the invention relates to a method of inducing an antigen-specific immune response in a subject in need thereof, comprising administering a vaccine comprising one or more antigens and cholesterol in an amount effective to induce an antigen-specific immune response in the subject.

In certain aspects, the invention relates to methods of inducing an antigen-specific immune response in a subject in need thereof comprising administering a vaccine comprising one or more antigens and one or more immune modulatory molecules, and cholesterol.

Drawings

FIG. 1: shows antigen-specific cytokine secretion by T cells in the absence of adjuvant or in the presence of CpG or CpG + cholesterol as adjuvant. FIG. 1 a: graph of CD4+ T cells secreting one or two cytokines. FIG. 1 b: map of CD4+ T cells secreting triple cytokines. FIG. 1 c: graph of CD8+ T cells secreting one or two cytokines. FIG. 1 d: map of CD8+ T cells secreting triple cytokines.

FIG. 2: graphs of IL-2 (FIG. 2a) and IFN- γ (FIG. 2b) production in the absence of adjuvant, CpG, and CpG + cholesterol.

FIG. 3: graph of ovalbumin-specific CD8+ T cell response in the absence of adjuvant or in the presence of CpG or CpG + cholesterol as adjuvant. FIGS. 3a to 3 b: cytotoxic T cell response, fig. 3 c-3 d: an antigen-specific CD8+ T cell population.

FIG. 4: graph of ovalbumin specific antibody titers in the absence of adjuvant or in the presence of CpG or CpG + cholesterol as adjuvant. The numbers above the bars indicate the ratio IgG2c/IgG 1.

FIG. 5: transmission electron micrographs of antigen, CpG and cholesterol.

FIG. 6: plots are depicted of the injection site response of calves immunized with pentavalent inactivated virus vaccine BVDV1&2, IBRV, PI3V and BRSV in the presence of CpG + cholesterol (CpG: cholesterol ratio 1:1 or 1:10), Advasure-DEAE/dextran, QCDCR or QCDCR + CpG. Some animals were immunized with commercial vaccines. The placebo animals received sterile saline. Table 1 plots the percentage of calves with clinical disease, fever, leukopenia or viremia after challenge with BVDV-2 after vaccination with the pentavalent inactivated viral vaccine BVDV1&2, IBRV, PI3V and BRSV in the presence of CpG + cholesterol (CpG: cholesterol ratio 1:1 or 1:10), Advasure-DEAE/dextran, QCDCR or QCDCR + CpG. Some animals were immunized with commercial vaccines. The placebo animals received sterile saline.

FIG. 7: graph of antigen-specific antibody responses in pigs immunized with pertactin (p68) formulated with various adjuvants, including CpG + cholesterol.

DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1-CPG79095’TCGTCGTTTTGTCGTTTTGTCGTT3’

SEQ ID NO:2-CpG245555’TCGTCGTTTTTCGGTGCTTTT3’

SEQ ID NO:3-CPG101045’TCGTCGTTTCGTCGTTTTGTCGTT3’

SEQ ID NO:4-CPG101015’TCGTCGTTTTCGGCGGCCGCCG3’

SEQ ID NO:5-CPG101095’TCGTC-GTTTTAC-GGCGCC-GTCCCG3’

SEQ ID NO:6-CpG23407

5’T*C-G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G*T3’

SEQ ID NO:7-CPG21798

5’T*C-G*T*C-G*A*C-G*A*T*C-G*G*C*G*C-G*C*G*C*C*G3’

SEQ ID NO:8-CPG23430

5’T*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G3’

SEQ ID NO:9-CpG24558

5’T*C*G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G*T3’

SEQ ID NO:10-CPG23871

5’JU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G3’

SEQ ID NO:11-CPG23873

5’JU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G*T3’

SEQ ID NO:12-CPG23874

5’*C*G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G*T3’

SEQ ID NO:13-CPG23875

5’EU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G3’

SEQ ID NO:14-CpG23877

5’JU*C-G*T*C*G*A*C*G*A*T*C*G*G*C*G*G*C*C*G*C*C*G*T3’

SEQ ID NO:15-CpG23878

5’JU*C*G*T*C*G*A*C*G*A*T*C*G*G*C*G*G*C*C*G*C*C*G*T3’

SEQ ID NO:16-poly I:C ODN1a5′-ICI CIC ICI CIC ICI CIC ICI CICIC-3′.

SEQ ID NO:17-5’GGGGACGACGTCGTGGGGGGG3’

SEQ ID NO:18-

5’G*G*G_G_A_C_G_A_C_G_T_C_G_T_G_G*G*G*G*G*G3’

SEQ ID NO:19-5’TCGTCGTTTTGTCGTTTTGTCGTT3’

SEQ ID NO:20-5’TCGTCGTTTTGTCGTTTTTTTCGA3’

SEQ ID NO:21-

5’T*C*G*T*C*G*T*T*T*T*T*C*G*G*T*G*C*T*T*T*T3’

SEQ ID NO:22 -

5’T*C*G*T*C*G*T*T*T*T*T*C*G*G*T*C*G*T*T*T*T3’

SEQ ID NO:23 -

5’T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*G*T*C*G*T*T3’

SEQ ID NO:24 -

5’T*C*G*T*C*G*T*T*T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T3’

SEQ ID NO:25 -

5’T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*T*T*T*C*G*A3’

SEQ ID NO:26 - 5’TCGCGTCGTTCGGCGCGCGCCG3’

SEQ ID NO:27 - 5’TCGTCGACGTTCGGCGCGCGCCG3’

SEQ ID NO:28 - 5’TCGGACGTTCGGCGCGCGCCG3’

SEQ ID NO:29 - 5’TCGGACGTTCGGCGCGCCG3’

SEQ ID NO:30 - 5’TCGCGTCGTTCGGCGCGCCG3’

SEQ ID NO:31 - 5’TCGACGTTCGGCGCGCGCCG3’

SEQ ID NO:32 - 5’TCGACGTTCGGCGCGCCG3’

SEQ ID NO:33 - 5’TCGCGTCGTTCGGCGCCG3’

SEQ ID NO:34 - 5’TCGCGACGTTCGGCGCGCGCCG3’

SEQ ID NO:35 - 5’TCGTCGTTTTCGGCGCGCGCCG3’

SEQ ID NO:36 - 5’TCGTCGACGATCGGCGCGCGCCG3’

SEQ ID NO:37 -

5’T*C_G*T*C_G*A*C_G*A*T*C_G*G*C*G*C_G*C*G*C*C*G3’

SEQ ID NO:38 -

5’T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G3’

SEQ ID NO:39 -

5’T*C_G*T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G3’

SEQ ID NO:40 -

5’T*C_G*G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G3’

SEQ ID NO:41 -

5’T*C_G*G*A*C_G*T*T*C_G*G*C*G*C*G*C*C*G3’

SEQ ID NO:42 -

5’T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C*G*C*C*G3’

SEQ ID NO:43 -

5’T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G3’

SEQ ID NO:44 - 5’T*C_G*A*C_G*T*T*C_G*G*C*G*C*G*C*C*G3’

SEQ ID NO:45 - 5’T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C*C*G3’

SEQ ID NO:46-

5’T*C_G*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G3’

SEQ ID NO:47-

5’T*C*G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G3’

SEQ ID NO:48-

5’T*C*G*T*C*G*T*T*T*T*C*G*G*C*G*G*C*C*G*C*C*G3’

SEQ ID NO:49-

5’T*C*G*T*C_G*T*T*T*T*A*C_G*G*C*G*C*C_G*T*G*C*C*G3’

SEQ ID NO:50-

5’T*C_G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G*T3’

(. about.) indicates the presence of stabilized internucleotide linkages, and "_" indicates phosphodiester linkages. J represents an iodo-modified nucleotide and E represents an ethyl-modified nucleotide.

Detailed Description

Aspects of the invention relate to vaccines having one or more antigens and cholesterol, and vaccines having one or more antigens and one or more immunoregulatory molecules and cholesterol. In various aspects of the invention, methods of inducing an antigen-specific immune response in a subject in need thereof by administering the vaccines of the invention are disclosed. Further disclosed is the use of the vaccine in the manufacture of a medicament for the treatment of a condition.

In aspects of the invention, the one or more antigens are each independently a microbial antigen, a self-antigen, a tumor antigen, an allergen, or an addictive substance. In certain aspects, the microbial antigen is derived from a bacterium, virus, or parasite. In certain aspects, the antigen is a peptide, a peptide conjugated to a carrier protein, a polypeptide, a recombinant protein, a purified protein, a whole inactivated pathogen, a live attenuated virus, a live attenuated bacterium, an antigen expressed in a viral or bacterial vector, a polysaccharide conjugated to a carrier protein, a protein conjugated to a virus-like particle, a hapten conjugated to a carrier protein, or a small molecule.

In an aspect of the invention, the antigen is derived from a bacterium. In certain aspects, the bacterial antigen is a whole killed bacterium, an attenuated, or a bacterial or bacterial purified protein.

In aspects of the invention, bacteria include, but are not limited to, the following species and combinations thereof: acinetobacter calcoaceticus (Acinetobacter calcoaceticus), Acetobacter pasteurianus (Acetobacter pasaniana), Actinomyces actinomycetemcomitans (Actinomyces actinometcomomatans), Actinomyces pleuropneumoniae (Actinomyces pleuropneumoniae), Actinomyces israeli (Actinomyces israeli), Actinomyces viscosus (Actinomyces viscosus), Aeromonas hydrophila (Aeromonas hydrophila), Alcaligenes eutrophus (Alcaligenes eutrophus), Thermomyces acidocaldarius (Alicyclobacillus acidocidus), Archaeoglobus fulgidus (Arhaemangelospora), Bacillus species (Bacillus subtilis), Bacillus subtilis (Bacillus subtilis), Bacillus species (Bacillus subtilis), Bacillus species (Bacillus subtilis), Bacillus species (Bacillus subtilis), Bacillus subtilis (Bacillus species (Bacillus subtilis), Bacillus species (Bacillus subtilis), Bacillus species (Bacillus subtilis), Bacillus species (, Burkholderia rhinoceros (Burkholderia glaucae), Brevibacterium (Brachyspira), Brevibacterium dysenteriae (Brachyspira), Brevibacterium Enterobacter (Brachyspira), Campylobacter (Campylobacter) species, Campylobacter coli), Campylobacter fetus (Campylobacter fetus), Campylobacter coli (Campylobacter asiaticus), Campylobacter jejuni (Campylobacter jejuni), Campylobacter psis (Chlamydomonas), Chlamydia trachomatis (Chlamydia trachomatis), Chlamydia Chlamydophila (Chlamydia Chlamydophila), Clostridium chlamydophilum (Clostridium), Clostridium viscosus (Clostridium), Clostridium difficile (Clostridium difficile) species, Enterococcus (Enterococcus), Erysipelothrix rhusiopathiae (Erysipelothrix), Escherichia (Escherichia), Escherichia coli (Escherichia coli), Fusobacterium nucleatum (Fusobacterium subclauum), Haemophilus (Haemophilus influezae), Haemophilus somnus (Haemophilus somnus), Helicobacter (Helicobacter), Helicobacter pylori (Helicobacter pylori), Helicobacter suis (Helicobacter suis), Klebsiella (Klebsiella), Klebsiella pneumoniae (Klebsiella), Lactobacillus pneumoniae (Klebsiella acidophilus), Lactobacillus acidophilus (Lactophilius), Lactobacillus acidophilus (Lactophilus), Leptospira intracellularis (Laterophilus), Leptospira curvata), Leptospira (Leptospira), Leptospira typhii (Leptospira), Leptospira canicola (Leptospira), Leptospira sp), Leptospira canicola (Leptospira, Leptospira sp), Leptospira sp (Leptospira sp), Leptospira sp) Mycobacterium bovis (Halbacco-Parkinsonii hardjo-prajitno), Leptospira interrogans (Leptospira interrogans), Leptospira icterohaemorrhagiae (Leptospira haemolytica), Leptospira pomonensis (Leptospira haemolytica), Leptospira pomona (Leptospira), Leptospira brasiliensis (Leptospira brasiliensis) etc., Listeria species, Listeria monocytogenes (Listinobacterium), Diplococcus meningitidis (Meycococcus), Moraxella species (Moraxella), Mycobacterium species (Mycobacterium), Mycobacterium bovis (Mycobacteria), Mycobacterium tuberculosis (Mycobacterium), Mycobacterium tuberculosis (Mycobacterium, Mycobacterium (Mycobacterium), Mycobacterium pneumoniae (Mycobacterium), Mycobacterium (Mycobacterium pneumoniae (Mycobacterium) and Mycobacterium (Mycobacterium) such as a) or Mycoba, Mycoplasma hyorhinis (Mycoplasma hyopneumoniae), Mycoplasma pneumoniae (Mycoplasma hyopneumoniae), Mycoplasma mycoides (Mycoplasma mycoides) Mycoplasma mycoides (LC subspecies fungoides), Neisseria species (Neisseria), Neisseria gonorrhoeae), Neisseria meningitidis (Neisseria meningitidis), Odorobacter denticola, Pasteurella species (Pasteurella), Pasteurella haemolytica (Mannheimia), Pasteurella multocida (Pasteurella), Pasteurella multocida (Pseudomonas multocida), Pseudomonas paraguas (Pseudomonas paradoxa), Pseudomonas luminescens (Pseudomonas aeruginosa), Pseudomonas aeruginosa (Pseudomonas putida), Pseudomonas aeruginosa strain (Pseudomonas aeruginosa), Pseudomonas aeruginosa (Pseudomonas aeruginosa strain 1), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Pseudomonas aeruginosa strain (Pseudomonas aeruginosa), Pseudomonas aeruginosa (Pseudomonas aeruginosa strain 1), Pseudomonas aeruginosa (Pseudomonas aeruginosa) Pseudomonas fragilis (Pseudomonas fragrans ), Pseudomonas aeruginosa (Pseudomonas luteola), Pseudomonas oleolytica (Pseudomonas oleovans), Pseudomonas sp.B 11-1, Acidophilus quiescens (Pseudomonas immobilis), Rickettsia sp, Rickettsia prohibitiella (Rickettsia prowazekii), Rickettsia rickettsii (Rickettettsia Rickettsia ricksii), Salmonella sp.sp.guillotinensis (Salmonella choleraesuis), Salmonella choleraesuis (Salmonella choleraesuis), Salmonella enterica (Salmonella enterica), Salmonella newport (Salmonella newcastle), Salmonella typhimurium (Salmonella typhimurium), Staphylococcus epidermidis (Staphylococcus epidermidis), Salmonella typhi (Staphylococcus spp., Salmonella typhi), Salmonella typhi (Staphylococcus epidermidis) species (Staphylococcus spp., Staphylococcus epidermidis), Salmonella typhi (Salmonella typhi), Salmonella typhi (Staphylococcus epidermidis) strains (Salmonella typhi), Salmonella typhi (Staphylococcus spp., Staphylococcus epidermidis) and Salmonella typhi (Staphylococcus spp., Staphylococcus, Streptococcus (Streptococcus) species, Staphylococcus suis (Streptococcus monoilformis), beta-hemolytic Streptococcus (beta-hemolytic Streptococcus), Streptococcus pyogenes (Streptococcus pyogenes) (group A Streptococcus), Streptococcus agalactiae (Streptococcus agalactiae) (group B Streptococcus), Streptococcus (Viridans group), Streptococcus faecalis (Streptococcus faecis), Streptococcus bovis (Streptococcus bovis), Streptococcus uberis (Streptococcus lactis), Streptococcus dysgalactiae (Streptococcus dysgalactiae), Streptococcus pneumoniae (anaerobe), Streptococcus pneumoniae (Streptococcus pneumoniae), Streptococcus sobrinus (Streptococcus mutans), Streptococcus sobrinus (Streptococcus sobrinus), Streptococcus faecalis (Streptococcus faecalis), Streptomyces albugineus (Streptococcus faecalis), Streptococcus lactis (Streptococcus lactis), Streptococcus lactis (Streptococcus), Streptococcus lactis), Streptococcus (Streptococcus), Streptococcus lactis), Streptococcus (Streptococcus), Streptococcus lactis), Streptococcus (Streptococcus lactis), Streptococcus (Streptococcus), Streptococcus (Streptococcus), Streptococcus lactis), Streptococcus (Streptococcus), Streptococcus (, Treponema pallidum, Treponema pertenue, Treponema ulcerosa, Treponema bergensis, Treponema inflixima, Treponema inflixiangense, Vibrio species, Vibrio cholerae, Yersinia species.

Polypeptides or polysaccharides of bacterial pathogens include, but are not limited to: iron-regulated outer membrane proteins (IROMP), Outer Membrane Proteins (OMP) and protein a of aeromonas salmonicida causing furunculosis; p57 protein of renal bacterium salmon (renibacterium salmoninarum) causing Bacterial Kidney Disease (BKD); major surface-bound antigens of yersinia's disease (msa), surface-expressed cytotoxins (mpr), surface-expressed hemolysins (ish), and flagellar antigens; extracellular proteins (ECPs), IROMPs and structural proteins of pasteurellosis; OMP and flagellin of vibrio anguillarum (vibris anguillarum) and vibrio ornadelis (v.ordalii); OMP proteins, aroA and purA of Edwardsiella ictaluri (edwards iella ictaluri) and Edwardsiella tarda (e.tarda); surface antigen of Ichthyophthirius; structural and regulatory proteins of the species fibrobacter columni (cytophaga columnifera); and structural and regulatory proteins of rickettsia; IsdA, ClfA, ClfB, Opp3A, HLA and capsular polysaccharide from Staphylococcus aureus.

In aspects of the invention, the antigen is derived from a virus. In certain aspects, the viral antigen is a whole inactivated or inactivated virus, a live attenuated virus, or a virus purification protein or peptide.

In certain aspects, the virus is a virus that infects animals, including but not limited to the following species and combinations thereof: avian herpes virus, avian influenza virus, avian leukemia virus, avian paramyxovirus, border disease virus, bovine coronavirus, bovine epidemic fever virus, bovine herpes virus, bovine immunodeficiency virus, bovine leukemia virus, bovine parainfluenza virus 3, bovine respiratory syncytial virus, Bovine Viral Diarrhea Virus (BVDV), BVDV type I, BVDV type II, canine adenovirus, Canine Coronavirus (CCV), canine distemper virus, canine herpes virus, equine herpes virus, canine influenza virus, canine parainfluenza virus, canine parvovirus, canine respiratory coronavirus, classical swine fever virus, eastern equine encephalitis virus (EEE), equine infectious anemia virus, equine influenza virus, West Nile virus, feline calicivirus, feline immunodeficiency virus, feline infectious peritonitis virus, feline herpes virus, feline influenza virus, feline leukemia virus (FeLV), feline viral rhinotracheitis virus, bovine coronavirus, bovine influenza virus, bovine leukemia virus (FeLV), bovine viral rhinotracheitis virus, bovine herpes virus, bovine immunodeficiency virus, bovine influenza virus, bovine viral, Lentivirus, Marek's disease virus, Newcastle disease virus, ovine herpes virus, ovine parainfluenza type 3 virus, ovine progressive pneumonia virus, ovine lung adenocarcinoma virus, pantoea virus, porcine circovirus type I (PCV), PCV type II, porcine epidemic diarrhea virus, porcine hemagglutinating encephalomyelitis virus, porcine herpes virus, porcine parvovirus, Porcine Reproductive and Respiratory Syndrome (PRRS) virus, pseudorabies virus, rabies virus, provirus, rhinovirus, rinderpest virus, swine influenza virus, infectious gastroenteritis virus, turkey coronavirus, Venezuelan equine encephalitis virus, vesicular stomatitis virus, West Nile virus, and Western equine encephalitis virus.

In certain aspects, the virus is a human-infecting virus, including but not limited to: adenoviridae (most adenoviruses); arenaviridae (hemorrhagic fever virus); astroviridae; bunyaviridae (bunaviridae) (e.g., hantavirus, bunyavirus (bunga virus), phlebovirus, and rhabdovirus); caliciviridae (caliciviridae) (e.g., strains that cause gastroenteritis); coronaviridae (e.g., coronaviruses); filoviridae (e.g., ebola virus); flaviviridae (Flaviridae) (e.g., hepatitis c virus, dengue virus, encephalitis virus, yellow fever virus); hepadnaviridae (Hepadnaviridae) (hepatitis b virus); herpesviridae (herpes simplex virus (HSV)1 and 2, varicella-zoster virus, Cytomegalovirus (CMV), herpes virus); iridoviridae (e.g., african swine fever virus); norwalk virus and related viruses; orthomyxoviridae (e.g., influenza virus); papovaviridae (papillomavirus, polyomavirus); paramyxoviridae (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus); parvoviridae (parvoviruses); picornaviridae (e.g., poliovirus, hepatitis a virus, enterovirus, human coxsackievirus, rhinovirus, echovirus); poxviridae (e.g., smallpox virus, vaccinia virus, poxvirus); reoviridae (reovirus, circovirus, and rotavirus); retroviruses (e.g., human immunodeficiency viruses such as HIV-1 or HIV-2 (also known as HTLV-III, LAV or HTLV-III/LAV) or HIV-III; and other isolates such as HIV-LP); rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus); togaviridae (e.g., equine encephalitis virus, rubella virus); and unclassified viruses (e.g., the etiological agent of spongiform encephalopathy, the etiological agent of hepatitis delta, believed to be a defective satellite of hepatitis b virus (satelite)).

In various aspects of the invention, the antigen may be derived from a parasite. In certain aspects, the parasite is a protein from the Anaplasma genus (anaplama), hookworm (hookworm), ascaris, babesia, coccidia, cryptosporidium, heartworm (dirofilaria immitis), eimeria species, fasciola hepatica (liver fluke), giardia, hammond, isosporococcus (Isopsora), leishmania species, neospora canis, sarcocystis, haemophilus, strongyloides, cestode, toxoplasma gondii, trichinosis species, trichomonas species, or trypanosoma species, and combinations thereof.

In certain aspects, the parasite is an ectoparasite. In certain aspects, ectoparasites include, but are not limited to, classes of ticks including species of hard ticks, rhipicephalus, dermanylus, minus, hyalomma, haemaphysalis, and combinations thereof.

In certain aspects of the invention, the antigen is an autoantigen. In certain aspects, a self antigen is an antigen of a subject's own cells or cell products that elicits an immune response in the subject. In certain aspects, autoantigens include, but are not limited to: a tumor antigen, an antigen associated with alzheimer's disease, an antigen directed against an antibody, or an antigen expressed from a human endogenous retroviral element. The antigen associated with Alzheimer's disease may be tau-amyloid or beta-amyloid. The antigen directed against an antibody may be an antigen directed against a human antibody, for example in certain embodiments the antigen is IgE.

In aspects of the invention, the antigen is a tumor antigen. In certain aspects, the tumor antigen is one or more of: WT1, MUC1, LMP2, HPV E6 or HPV E7, EGFR or variant forms (e.g., EGFRvIII), HER-2/neu, idiotype, MAGE A3, p53 non-mutants, NY-ESO-1, PSMA, GD2, CEA, MelanA/MART1, Ras mutants, gp100, p53 mutants, protease 3(PR1), bcr-abl, tyrosinase, survivin, PSA, hTERT, sarcoma ectopic breakpoint, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG (TMPRSS2ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic, MYCN, RhoC, WM-2, GD3, FusycolGM 7, mesothelin, PSCA, MAGE 1, CYP 72, BO 72, BOB 8672, BO-RG 72, SAC-OB 72, sLe, SALT 72, SAO-OB-sLe, SALT 72, MAG 72, SSX2, XAGE1, B7H3, Legumain (Legumain), Tie2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-beta, MAD-CT-2 or Fos-associated antigen 1. Such tumor antigens have been ranked according to criteria such as, for example, a) therapeutic function, b) immunogenicity, c) role of the antigen in tumorigenicity, d) specificity, expression level and percentage of antigen-positive cells, g) number of epitopes and h) cellular location of antigen expression (see Cheever, m.a. et al, clinical Cancer Research, 9.1.2009, 15(17): 5323-5-5337). In certain embodiments, the tumor antigen is one or more of survivin, Her-2, EFGRvIII, PSA, PAP, or PMSA.

In some aspects of the invention, the antigen is an allergen. An allergen refers to a substance (antigen) that is likely to induce an allergic or asthmatic response in a susceptible subject. The list of allergens is large and may include pollen, insect venom, animal fur dust, fungal spores and drugs (e.g., penicillin). Examples of natural, animal and plant allergens include, but are not limited to, proteins specific to the following species and combinations thereof: agropyron (e.g., creeping wheatgrass), agrostin (e.g., furfur parvifolia), alder (alder), alternaria (alternaria), ragweed (ambrosia americana), citronella (e.g., citronella aureoides), honeybee (e.g., annual bees), avena (e.g., avena sativa), artemisia (wormwood), avena (e.g., oat), betula (birch), cockroach (e.g., blatella germanica), bromus (e.g., bromus formosanus), canine (dog), hinoki (e.japanese cypress), cedar (japanese cedar), cedar (e.g., cypress, green stem, and golden crown), cocksfoot (e.g., cocksfoot), mite (e.g., dust mite), catnip (cat), fescue (e.g., oxtail), chorionic (e.g., chorionic villus), Sabina (e.g., Juniperus sabinoides, sabina pennyensis, juniper, and Juniperus ashei), lolium (e.g., perennial ryegrass and annual ryegrass), elaeagnus (olea europaea), pellitha (e.g., kojic and milfoil), paspalum (e.g., paspalum), periplaneta (e.g., periplaneta americana), phalaris (e.g., phalaris), ramosia (e.g., timothy), plantago (e.g., plantago lanceolata), poachene (e.g., prairia and prairia), quercus (quercus alba), secale (e.g., rye), sorghum (e.g., sorghum halepense), thujaponaria (e.g., Thuya orientalis), and triticale (e.g., wheat).

In some aspects of the invention, the antigen is an addictive substance. An addictive substance is any chemical or biological substance, including synthetic or artificial substances, that causes a subject to develop addiction to the substance. In certain aspects, the addictive substance is nicotine or cocaine. In certain embodiments, the antigen in a vaccine against nicotine addiction is a nicotine or nicotine-like hapten conjugated to a carrier. In certain embodiments, the carrier to which the nicotine or nicotine-like hapten is conjugated is diphtheria toxoid.

In some aspects of the invention, the antigen or hapten is conjugated to a carrier protein. In certain aspects, the carrier protein is a bacterial toxoid or derivative, a pseudomonas exotoxin, KLH, or a virus-like particle. In certain aspects, the bacterial toxoid is diphtheria toxoid or a derivative thereof. In certain aspects, the bacterial toxoid is CRM 197. In certain aspects, the virus-like particle is HBsAg, HBcAg, coliphage Q β, norwalk virus, or influenza HA.

Some aspects of the invention relate to vaccines with cholesterol. The cholesterol is of the formula C₂₇H₄₅White crystalline material of OH. It is a cyclic hydrocarbon alcohol classified as a lipid. Lipids are any group of organic compounds that are insoluble in water but soluble in non-polar organic solvents, greasy to the touch and act as basic structural materials for living cells together with carbohydrates and proteins, including but not limited to fats, oils, waxes, sterols and triglycerides. Cholesterol is insoluble in water but soluble in many organic solvents.

In some aspects of the invention, sterols refer to compounds in animals that are biologically produced from terpene precursors. Which comprises a steroid ring structure having a hydroxyl group (OH). In certain aspects, the hydroxyl group can be attached to carbon-3. The hydrocarbon chains of the fatty acid substituents vary in length. In certain aspects, the hydrocarbon chain can be 16 to 20 carbon atoms. In certain aspects, the hydrocarbon chain can be saturated or unsaturated. Sterols can contain one or more double bonds in the ring structure and can include various substituents attached to the ring. Sterols and their fatty acid esters may be insoluble in water. Fatty acid esters refer to any class of organic compounds corresponding to inorganic salts formed by a condensation reaction in which a molecule of an organic acid is combined with a molecule of an alcohol and one molecule of water is eliminated. In certain aspects, synthetic steroids include, but are not limited to: glucocorticoids (e.g., prednisone, dexamethasone, triamcinolone), mineralocorticoids (e.g., fludrocortisone), vitamin D (e.g., dihydrotachysterol), androgens (e.g., oxandrolone, nandrolone, anabolic steroids), estrogens (e.g., diethylstilbestrol), and progestins (e.g., norethindrone, medroxyprogesterone acetate). In certain aspects, cholates, such as sodium deoxycholate, may be used.

In some aspects of the invention, sterols include, but are not limited to, natural steroids such as beta-sitosterol, stigmasterol, ergosterol, ergocalciferol, and cholesterol. Such sterols are commercially available. For example, cholesterol is disclosed in Merck Index, 12 th edition, page 369.

In some aspects of the invention, sterols may be used as adjuvants. In certain aspects, the amount of sterol can be from about 1 μ g per dose of vaccine to about 5,000 μ g per dose of vaccine. In certain aspects, the amount of sterol can be from about 1 μ g/dose vaccine to about 4,000 μ g/dose vaccine, from about 1 μ g/dose vaccine to about 3,000 μ g/dose vaccine, from about 1 μ g/dose vaccine to about 2,000 μ g/dose vaccine, or from about 1 μ g/dose vaccine to about 1,000 μ g/dose vaccine. In certain aspects, the amount of sterol can be from about 5 μ g/dose of vaccine to about 750 μ g/dose of vaccine, from about 5 μ g/dose of vaccine to about 500 μ g/dose of vaccine, from about 5 μ g/dose of vaccine to about 250 μ g/dose of vaccine, from about 5 μ g/dose of vaccine to about 100 μ g/dose of vaccine, from about 15 μ g/dose of vaccine to about 100 μ g/dose of vaccine, or from about 30 μ g/dose of vaccine to about 75 μ g/dose of vaccine.

In some aspects of the invention, the vaccine has one or more antigens and cholesterol. In certain aspects, the amount of cholesterol is about 0.1-fold to about 50-fold higher by weight compared to the amount of antigen. In certain aspects, the amount of cholesterol is 1-fold to about 10-fold greater than the amount of antigen by weight. In certain aspects, the amount of cholesterol is equal to the antigen by weight.

In some aspects of the invention, the vaccine has one or more antigens and one or more immunomodulatory molecules, and cholesterol. In certain aspects, the vaccine further comprises a pharmaceutical carrier.

In some aspects of the invention, "combination" or "in combination with …" refers to a blend, combination, or proximity of one or more antigens, as well as a blend or combination, or proximity of one or more antigens and one or more immunomodulatory molecules. In some aspects, one or more antigens and/or one or more immunomodulatory molecules may be linked to cholesterol via one or more linkers by physical means. Linkers include, but are not limited to, direct or indirect linkers. In some aspects, one or more antigens and/or one or more immunomodulatory molecules may be encapsulated with cholesterol.

In some aspects of the invention, one or more antigens may be blended with one or more immunomodulatory molecules. In some aspects, one or more antigens may be blended with cholesterol. In some aspects, one or more immunomodulatory molecules can be blended with cholesterol. In some aspects, one or more immunomodulatory molecules can be blended with an antigen and cholesterol. In some aspects, one or more immunomodulatory molecules can be blended with cholesterol, while one or more antigens can be separated. In some aspects, one or more antigens may be blended with cholesterol, while one or more immunomodulatory molecules may be separated. In some aspects, one or more antigens and/or one or more immunomodulatory molecules may be combined with cholesterol.

In some aspects of the invention, the amount of cholesterol relative to the amount of antigen is greater than the amount of antigen. In certain aspects, the amount of cholesterol relative to the amount of antigen is about 0.1-fold to about 50-fold greater by weight compared to the amount of antigen. In certain aspects, the amount of cholesterol relative to the amount of antigen is about 10-fold to about 50-fold, about 20-fold to about 40-fold, about 30-fold to about 35-fold higher by weight compared to the amount of antigen. In certain aspects, the amount of cholesterol relative to the amount of antigen is 1-fold to about 10-fold higher by weight compared to the amount of antigen. In certain aspects, the amount of cholesterol relative to the amount of antigen is equal in weight to the antigen. In certain aspects, the antigen may be one or more antigens and the weight of the antigen is the weight of the one or more antigens.

In some aspects of the invention, the immunomodulatory molecule(s) is a molecule that modulates an immune cell in a subject. The effect may be mediated directly, e.g., through a receptor, or indirectly, e.g., through a cytokine or chemokine released by another immune cell that is directly regulated. Induction of an immune response refers to any increase in the number or activity of immune cells, or an increase in the expression or absolute level of an immune factor, such as a cytokine. Immune cells include, but are not limited to, NK cells, CD4+ T cells, CD8+ T cells, B cells, dendritic cells, macrophages, and other antigen presenting cells. Cytokines include, but are not limited to, interleukins, TNF- α, IFN- β, and IFN- γ. In some aspects, the immunomodulator is one that, when used with an antigen, enhances an antigen-specific humoral (e.g., antibody) immune response and/or cellular (e.g., T cell) immune response.

In certain aspects, the immunomodulatory molecule is a TLR agonist, an antimicrobial peptide, a cytokine, a chemokine, a NOD ligand, or an oligonucleotide. In certain aspects, the TLR agonist is an Oligoribonucleotide (ORN) or a small molecule that activates TLR7 and/or TLR 8. In certain aspects, the TLR agonist is an Oligodeoxynucleotide (ODN) activated by TLR 9. In certain aspects, the TLR9 agonist is an ODN containing an unmethylated CpG motif, a B-class oligodeoxynucleotide, a C-class oligodeoxynucleotide, or a P-class deoxynucleotide. In certain aspects, the TLR9 agonist is IMO-2055, IMO-2125, or IMO-2134(QAX 935). In other aspects, the TLR agonist is a poly (I: C) that activates TLR 3. In certain aspects, the poly (I: C) is ODN1a having the sequence 5 '-ICI CICICI CIC ICI CIC ICI CIC IC-3' (SEQ ID NO: 16).

In some aspects of the invention, oligonucleotides may encompass various chemical modifications and substitutions (as compared to native RNA and DNA) involving phosphodiester internucleoside bridges, β -D-ribose units, and/or native nucleobases (adenine, guanine, cytosine, thymine, uracil). Examples of chemical modifications are known to the skilled worker and are described, for example, by Uhlmann E et al, (1990) ChemRev90:543; "Protocols for Oligonuclotides and antigens" Synthesis and Properties & Synthesis and Analytical Techniques, S.Agrawal, Ed, Humana Press, Totowa, USA1993; crop ST et al, (1996) Annu RevPharmacol toxin 36: 107; 129; and Hunziker J et al, (1995) Mod Synthmethods7: 331-. In certain aspects, an oligonucleotide may have one or more modifications, wherein each modification is at a position of a particular phosphodiester internucleoside bridge and/or a particular β -D-ribose unit and/or a particular natural nucleobase relative to an oligonucleotide of the same sequence consisting of a natural DNA or RNA.

In certain aspects, the oligonucleotide may comprise one or more modifications, and wherein each modification is independently selected from:

a) phosphodiester internucleoside bridges located at the 3 'and/or 5' end of the nucleoside are replaced by modified internucleoside bridges,

b) the phosphodiester bridges located at the 3 'and/or 5' end of the nucleoside are replaced by dephosphorylated bridges,

c) the sugar phosphate unit from the sugar phosphate backbone is replaced by another unit,

d) the beta-D-ribose unit is replaced by a modified sugar unit, and

e) the natural nucleobase is replaced by a modified nucleobase.

In some aspects of the invention, the oligonucleotides may comprise modified internucleotide linkages, such as those described in a) or b) above. These modified linkages may be partially resistant to degradation (i.e., are stabilized). A "stabilized oligonucleotide molecule" is an oligonucleotide resulting from such a modification that is relatively resistant to degradation in vivo (e.g., via an exonuclease or endonuclease). In certain aspects, oligonucleotides with phosphorothioate linkages may provide maximum activity and protect the oligonucleotide from degradation by intracellular exonucleases and endonucleases.

The phosphodiester internucleoside bridge at the 3 'and/or 5' end of the nucleoside may be replaced by a modified internucleoside bridge, wherein the modified internucleoside bridge is for example selected from: thiophosphate, dithiophosphate, NR¹R²Phosphoramidates, borophosphates (boranophosphates), alpha-hydroxybenzylphosphonates, phosphoric acid- (C)₁～C₂₁) -O-alkyl esters, phosphoric acid- [ (C)₆～C₁₂) Aryl radical- (C)₁～C₂₁) -O-alkyl]Ester, (C)₁～C₈) Alkyl phosphonate and/or (C)₆～C₁₂) Aryl phosphate bridging bond, (C)₇～C₁₂) -alpha-hydroxymethylaryl (e.g. as described in WO 95/01363), wherein (C)₆-C₁₂) Aryl group, (C)₆-C₂₀) Aryl and (C)₆-C₁₄) Aryl is optionally substituted with halogen, alkyl, alkoxy, nitro, cyano, and wherein R is¹And R²Each independently hydrogen, (C)₁～C₁₈) Alkyl, (C)₆～C₂₀) Aryl group, (C)₆～C₁₄) Aryl radical- (C)₁～C₈) Alkyl, preferably hydrogen, (C)₁～C₈) Alkyl, preferably (C)₁～C₄) Alkyl and/or methoxyethyl, or R¹And R²Together with the nitrogen atom which carries them, form a five-to six-membered heterocyclic ring which may additionally contain a further heteroatom selected from the group of O, S and N.

Replacement of the phosphodiester bridges at the 3 ' and/or 5 ' end of the nucleoside by dephosphorylated bridges (dephosphorylation) as described, for example, by Uhlmann E and Peyman A in "Methods in molecular biology", volume 20, "Protocols for Oligonucleotides and Analogs", S.Agrawal, Ed., Humana Press, Totowa1993, Chapter 16, p.355, wherein the dephosphorylated bridges are selected, for example, from the group consisting of dephosphorylated bridged acetals (formacetals), 3 ' -thiohydrazinoacetals, methylhydroxylamines, methylenedimethylidines, dimethylsulfoxides and/or silyl groups.

The sugar phosphate units (i.e.the. beta. -D-ribose and phosphodiester internucleoside bridges which together form the sugar phosphate units) from the sugar phosphate backbone (i.e.the sugar phosphate backbone consists of sugar phosphate units) may be substituted by another unit which may, for example, be suitable for the construction of "morpholine derivative" oligomers (e.g.as described in Stirchak EP et al, (1989) Nucleic Acids Res17: 6129-41), i.e.e.by morpholine derivative units; or which are suitable for the construction of polyamide nucleic acids ("PNA"; for example, described in Nielsen PE et al (1994) bioconjugateg Chem5: 3-7), i.e.for example, by PNA backbone units (for example by 2-aminoethylglycine). Oligonucleotides may have other sugar backbone modifications and substitutions, such as peptide nucleic acids with phosphate groups (PHONA), Locked Nucleic Acids (LNA), and oligonucleotides with backbone portions with alkyl or amino linkers. The alkyl linker may be branched or unbranched, substituted or unsubstituted, and chirally pure or racemic mixtures.

The beta-ribose unit or the beta-D-2 ' -deoxyribose unit can be replaced by a modified sugar unit, wherein the modified sugar unit is selected from, for example, beta-D-ribose, alpha-D-2 ' -deoxyribose, L-2 ' -deoxyribose, 2 ' -F-arabinose, 2 ' -O- (C)₁～C₆) Alkylribose (2' -O- (C)₁～C₆) The alkylribose is preferably 2 '-O-methylribose, 2' -O- (C)₂～C₆) Alkenyl ribose, 2' - [ O- (C)₁～C₆) alkyl-O- (C)₁～C₆) Alkyl radical]Ribose), 2' -NH₂-2' -deoxyribose,. beta. -D-xylofuranose,. alpha. -arabinofuranose, 2, 4-dideoxy-. beta. -D-erythropyranose, and also carbocyclic sugar analogs (e.g. as described in Froehler J (1992) Am Chem Soc114: 8320) and/or open-chain sugar analogs (e.g. as described in Vanderdrissche et al, (1993) Tetrahedron49: 7223) and/or bicyclic sugar analogs (e.g. as described in Tarkov M et al, (1993) Helv Chim Acta76: 481).

In certain aspects, for one or both of the nucleotides connected by a phosphodiester or phosphodiester-like internucleoside linkage, the sugar is 2' -O-methyl ribose.

Nucleic acids may also include, but are not limited to, substituted purines and pyrimidines, such as C-5 propynopyrimidine and 7-deaza-7-substituted purine modified bases (Wagner RW et al, (1996) Nat Biotechnol14: 840-4). Purines and pyrimidines include, but are not limited to, adenine, cytosine, guanine and thymine, as well as other naturally and non-naturally occurring nucleobases, substituted and unsubstituted aromatic moieties.

The modified base is in chemistryAny base that differs in nature from the naturally occurring bases typically found in DNA and RNA (e.g., T, C, G, A and U), but which shares a basic chemical structure with the naturally occurring bases described above. The modified nucleobase may for example be selected from hypoxanthine, uracil, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5- (C)₁～C₆) Alkyl uracils, 5- (C)₂～C₆) Alkenyl uracils, 5- (C)₂～C₆) Alkynyl uracil, 5-hydroxymethyl uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5- (C)₁～C₆) Alkyl cytosine, 5- (C)₂～C₆) Alkenyl cytosine, 5- (C)₂～C₆) Alkynyl cytosine, 5-chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N²Dimethylguanine, 2, 4-diaminopurine, 8-azapurine, substituted 7-deazapurine (preferably 7-deaza-7-substituted purine and/or 7-deaza-8-substituted purine), 5-hydroxymethylcytosine, N4-alkylcytosine (e.g., N4-ethylcytosine), 5-hydroxydeoxycytidine, 5-hydroxymethyldeoxycytidine, N4-alkyldeoxycytidine (e.g., N4-ethyldeoxycytidine), 6-thiodeoxyguanosine, deoxyribonucleotide of nitropyrrole, C5-propynylpyrimidine, and diaminopurine (e.g., 2, 6-diaminopurine), inosine, 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine, Hypoxanthine or other modifications of natural nucleobases. This list is illustrative only and should not be considered as limiting.

In some aspects of the invention, for certain formulae described herein, a set of modified bases is defined. For example, the letter Y is used to refer to a nucleotide containing cytosine or modified cytosine. Modified cytosine is a naturally occurring or non-naturally occurring cytosine analog of the pyrimidine base, which can replace the base (cytosine) without compromising the immunostimulatory or immunomodulatory activity of the oligonucleotide. Modified cytosines include, but are not limited to, 5-substituted cytosines (e.g., 5-methylcytosine, 5-fluorocytosine, 5-chlorocytosine, 5-bromocytosine, 5-iodocytosine, 5-hydroxycytosine, 5-hydroxymethylcytosine, 5-difluoromethylcytosine, and unsubstituted or substituted 5-alkynylcytosine), 6-substituted cytosines, N4-substituted cytosines (e.g., N4-ethylcytosine), 5-azacytosine, 2-mercaptocytosine, isocytosine, pseudoisocytosine, cytosine analogs with fused ring systems (e.g., N, N' -propenocyytosine or phenoxazine), and uracils and derivatives thereof (e.g., 5-fluorouracil, 5-bromouracil, 5-bromovinyluracil, uracil, 4-thiouracil, 5-hydroxyuracil, 5-propynyluracil). In certain aspects, cytosines include 5-methylcytosine, 5-fluorocytosine, 5-hydroxycytosine, 5-hydroxymethylcytosine, and N4-ethylcytosine. In certain aspects, the cytosine base is substituted with a universal base (e.g., a 3-nitropyrrole, a P-base), an aromatic ring system (e.g., fluorobenzene or difluorobenzene), or a hydrogen atom (dSpacer).

The letter R is used to refer to guanine or a modified guanine base. Modified guanine is a naturally occurring or non-naturally occurring guanine base analog, which can replace the base (guanine) without compromising the immunostimulatory or immunomodulatory activity of the oligonucleotide. Modified guanines include, but are not limited to, 7-deaza-7-substituted guanines (e.g., 7-deaza-7- (C2-C6) alkynylpurines), 7-deaza-8-substituted guanines, hypoxanthine, N2-substituted guanines (e.g., N2-methylguanine), 5-amino-3-methyl-3H, 6H-Thiazolo [4,5-d ] pyrimidine-2, 7-dione, 2, 6-diaminopurine, 2-aminopurine, purine, indole, adenine, substituted adenine (e.g., N6-methyladenine, 8-oxoadenine), 8-substituted guanine (e.g., 8-hydroxyguanine and 8-bromoguanine), and 6-thioguanine. In certain aspects, the guanine base is substituted with a universal base (e.g., 4-methylindole, 5-nitroindole, and K-base), an aromatic ring system (e.g., benzimidazole or dichlorobenzimidazole, 1-methyl 1H- [1,2,4] triazole-3-carboxamide), or a hydrogen atom (dSpacer).

In certain aspects, other base modifications are also contemplated. For example, the terminal T residue at either end of the oligonucleotide may be replaced by deoxyuridine (U), G of one or more CpG motifs may be replaced by deoxyinosine (I), and the G residue modified to 7-deazadeoxyguanosine. In certain aspects, the 5' terminal T of the oligonucleotide may comprise a halogen substitution. In certain aspects, the halogen substitution is ethyl uridine, bromo uridine, chloro uridine, or iodo uridine.

In some aspects of the invention, oligonucleotides may be synthesized de novo using a number of procedures well known in the art. For example, the b-cyanoethylphosphoramide method (Beaucage, s.l. and carothers, m.h., tet.let).221859,1981), the nucleoside H-phosphonate method (Garegg et al, Tet.27:4051-4054,1986;Froehler et al.,Nucl.Acid.Res.145399-5407,1986, Garegg et al, Tet.274055, 4058,1986, Gaffney et al, Tet.29:2619-2622,1988). These chemical methods can be performed by various automated nucleic acid synthesizers available on the market. These oligonucleotides are referred to as synthetic oligonucleotides. An isolated oligonucleotide generally refers to an oligonucleotide that is separated from components with which it is normally associated in nature. For example, the isolated oligonucleotide may be an oligonucleotide isolated from a cell, nucleus, mitochondria, or chromatin.

In certain aspects of the invention, the internucleotide linkage may be an unstabilized linkage or stabilized linkage (for nucleases), a phosphodiester (unstabilized), a phosphorothioate (stabilized), or another charged backbone or phosphodiester linkage. In certain aspects, if the internucleotide linkage at Y-R is a phosphorothioate, the chirality of the linkage may be random, or, preferably, a phosphorothioate linkage having the Rp configuration.

In some aspects of the invention, modified backbones (e.g., phosphorothioates) can be synthesized using automated techniques employing phosphoramidite or H-phosphate chemistry. Aryl phosphates and alkyl phosphates may be prepared, for example, as described in U.S. Pat. No. 4,469,863; and alkylphosphotriesters in which the charged oxygen moiety is alkylated, as described in U.S. Pat. No. 5,023,243 and european patent No. 0,092,574, can be prepared by automated solid phase synthesis using commercially available reagents. Have already been modified for preparing other DNA backbonesMethods of decorating and substituting are described (e.g., Uhlmann, e. and Peyman, a., chem.rev.90:544,1990;Goodchild,J.,Bioconjugate Chem.1:165,1990). The symbol "indicates the presence of a stabilized internucleotide linkage, and" _ "indicates the presence of a phosphodiester linkage. In some aspects, the one or more immunomodulatory molecules can each independently have a fully natural phosphodiester backbone.

In some aspects of the invention, the one or more immunomodulatory molecules are oligonucleotides comprising at least one unmethylated cpG dinucleotide. An oligonucleotide containing at least one unmethylated cpG dinucleotide is a nucleic acid molecule that contains an unmethylated cytosine-guanine dinucleotide sequence (i.e., a "CpG DNA" or DNA containing a 5 'cytosine followed by a 3' guanine and phosphate bond linkage) and activates the immune system. The entire CpG oligonucleotide may be unmethylated, or certain portions may be unmethylated, but at least 5 'CG 3' must be unmethylated. CpG the term CpG oligonucleotide or CpG nucleic acid as used herein refers to an immunostimulatory CpG oligonucleotide or nucleic acid unless otherwise indicated.

In some aspects of the invention, immunomodulatory molecules include, but are not limited to, oligonucleotides that are class a, class B, class C, class T, class P, or any class with E modifications.

Class a oligonucleotides can potently induce IFN- α and NK cell activation, but are relatively weak at stimulating B cells. Class a oligonucleotides typically have stabilized poly G sequences at the 5 'and 3' ends and palindromic phosphodiester CpG dinucleotide-containing sequences of at least 6 nucleotides and form multimeric structures. Oligonucleotides of class A are described in U.S. Pat. No. 6,949,520 issued on 27/9/2005 and PCT application No. PCT/US00/26527(WO01/22990) published on 5/4/2001. The class A oligonucleotides do not necessarily contain the hexamer palindromic sequence GACGTC, AGCGCT or AACGTT, as described by Yamamoto et al (Yamamoto S et al, J Immunol148:4072-6 (1992)). In some aspects, an "a class" CpG oligonucleotide has the following nucleic acid sequence: 5 'GGGGACGACGTCGTGGGGGGG 3' (SEQ ID NO: 17). In some aspects, class a oligonucleotides include, but are not limited to, 5 'G _ a _ C _ G _ T _ G3' (SEQ id no: 18); wherein ". sup." means phosphorothioate bond, and ". sup." means phosphodiester bond.

Class B oligonucleotides can potently activate B cells, but are relatively weak at inducing IFN- α and NK cell activation. The class B oligonucleotides are monomeric and may be fully stabilized by a complete phosphorothioate backbone. Class B oligonucleotides may also have some natural phosphodiester bonds, for example, between C and G of CpG, in which case they are referred to as semi-soft (semi-soft) oligonucleotides. In certain aspects, the B class CpG oligonucleotides may be represented by at least the following formula: 5' X₁X₂CGX₃X₄3', wherein X₁、X₂、X₃And X₄Is a nucleotide. In some aspects, X₂Is adenosine, guanosine or thymidine. In some aspects, X₃Is cytidine, adenosine or thymidine. In some aspects, the B class CpG oligonucleotides may be represented by at least the following formula: 5' N₁X₁X₂CGX₃X₄N₂3', wherein X₁、X₂、X₃And X₄Is a nucleotide, and N is any nucleotide, and N₁And N₂Are nucleic acid sequences each consisting of about 0 to 25N. In some aspects, X₁X₂Is a dinucleotide selected from the group consisting of GpT, GpG, GpA, ApA, ApT, ApG, CpT, CpA, CpG, TpA, TpT and TpG; x₃X₄Is a dinucleotide selected from the group consisting of TpT, ApT, TpG, ApG, CpG, TpC, ApC, CpC, TpA, ApA and CpA. In certain aspects, X₁X₂Is GpA or GpT and X3X4 is TpT. In some aspects, X₁And/or X₂Is purine and X₃And/or X₄Is a pyrimidine, or X₁X₂Is GpA and X₃And/or X₄Is a pyrimidine. In certain aspects, X₁X₂Is a dinucleotide selected from the group consisting of TpA, ApA, ApC, ApG, and GpG. In certain aspects, X₃X₄Is a dinucleotide selected from the group consisting of TpT, TpA, TpG, ApA, ApG, GpA and CpA. In certain aspects, X₁X₂Is a dinucleotide selected from the group consisting of TpT, TpG, ApT, GpC, CpC, CpT, TpC, GpT and CpG; x₃Is a nucleotide selected from A and T, and X₄Is a nucleotide, but when X₁X₂When it is TpC, GpT or CpG, X₃X₄Is not TpC, ApT or ApC. In some aspects, the CpG oligonucleotide has the sequence 5' TCN₁TX₁X₂CGX₃X₄3'. The CpG oligonucleotides of the present invention may comprise, for example, X selected from the group consisting of GpT, GpG, GpA and ApA₁X₂And X selected from the group consisting of TpT, CpT and TpC₃X₄。

Oligonucleotides of class B have been described in U.S. Pat. Nos. 6,194,388B1 and 6,239,116B1, issued respectively at 27/2001 and 29/5/2001, as well as in PCT application publication No. WO/1996/002555, published at 2/1/1996, and PCT application publication No. WO/1998/018810, published at 7/5/1998. In certain aspects, the class B oligonucleotide is:

CPG79095’TCGTCGTTTTGTCGTTTTGTCGTT3’(SEQ ID NO:1)，

CpG245555’TCGTCGTTTTTCGGTGCTTTT3’(SEQ ID NO:2)，

CPG10104TCGTCGTTTCGTCGTTTTGTCGTT(SEQ ID NO:3)，

5’TCGTCGTTTTGTCGTTTTGTCGTT3’(SEQ ID NO:19),

5’TCGTCGTTTTGTCGTTTTTTTCGA3’(SEQ ID NO:20)，

5’T*C*G*T*C*G*T*T*T*T*T*C*G*G*T*G*C*T*T*T*T3’(SEQ IDNO:21)，

5’T*C*G*T*C*G*T*T*T*T*T*C*G*G*T*C*G*T*T*T*T3’(SEQ IDNO:22)，

5’T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*G*T*C*G*T*T3’(SEQ ID NO:23)，

5’T*C*G*T*C*G*T*T*T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T3’(SEQ ID NO:24)，

or

5’T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*T*T*T*C*G*A3’(SEQ ID NO:25)，

Wherein "-" means a phosphorothioate bond.

C-class oligonucleotides have both traditional "stimulatory" CpG sequences and "GC-rich" or "B-cell neutralizing" motifs. C class CpG oligonucleotides have properties intermediate between A and B classes, and therefore activate B cells and NK cells and induce IFN- α (Krieg AM et al, (1995) Nature374:546-9; Ballas ZK et al, (1996) J Immunol157:1840-5; Yamamoto S et al, (1992) J Immunol148: 4072-6). Class C oligonucleotides contain a single palindromic sequence, such that they can form secondary structures such as stem-loop structures or tertiary structures such as dimers. The backbone of the class C oligonucleotide may have a fully stabilized, chimeric or semi-soft backbone. Class C oligonucleotides comprise a class B type sequence and a GC-rich palindromic or near-palindromic sequence. Such is described in U.S. application publication No. 20030148976 published at 8/7/2003 and PCT application publication No. WO2008/068638 published at 12/6/2008. In certain aspects, the class C oligonucleotide is:

CPG101015’TCGTCGTTTTCGGCGGCCGCCG3’(SEQ ID NO:4)，

CPG101095 'TCGTC-GTTTTAC-GGCGCC-GTCCCG 3' (SEQ ID NO:5, wherein the short horizontal line represents a semi-soft phosphodiester linkage),

CpG234075 'TC-GTCGTTTTCGGCGCGCGCCGT 3' (SEQ ID NO:6, wherein the short horizontal line represents a semi-soft phosphodiester bond),

5’TCGCGTCGTTCGGCGCGCGCCG3’(SEQ ID NO:26)，

5’TCGTCGACGTTCGGCGCGCGCCG3’(SEQ ID NO:27)，

5’TCGGACGTTCGGCGCGCGCCG3’(SEQ ID NO:28),

5’TCGGACGTTCGGCGCGCCG3’(SEQ ID NO:29)，

5’TCGCGTCGTTCGGCGCGCCG3’(SEQ ID NO:30),

5’TCGACGTTCGGCGCGCGCCG3’(SEQ ID NO:31)，

5’TCGACGTTCGGCGCGCCG3’(SEQ ID NO:32)，

5’TCGCGTCGTTCGGCGCCG3’(SEQ ID NO:33),

5 'TCGCGACGTTCGGCGCGCGCCG 3' (SEQ ID NO:34), or

5’TCGTCGTTTTCGGCGCGCGCCG3’(SEQ ID NO:35)。

In some aspects, the C-class CpG oligonucleotide is:

5’T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G3’(SEQID NO:38)，

5’T*C_G*T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G3’(SEQ ID NO:39),

5’T*C_G*G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G3’(SEQ IDNO:40),

5’T*C_G*G*A*C_G*T*T*C_G*G*C*G*C*G*C*C*G3’(SEQ IDNO:41),

5’T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C*G*C*C*G3’(SEQ IDNO:42),

5’T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G3’(SEQ IDNO:43),

5’T*C_G*A*C_G*T*T*C_G*G*C*G*C*G*C*C*G3’(SEQ ID NO:44),

5’T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C*C*G3’(SEQ ID NO:45),

5’T*C_G*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G3’(SEQID NO:46),

5’T*C*G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G3’(SEQID NO:47),

5’T*C*G*T*C*G*T*T*T*T*C*G*G*C*G*G*C*C*G*C*C*G3’(SEQID NO:48),

5 'T C G T G C G C G T G C G3' (SEQ ID NO:49) or

5’T*C_G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G*T3’(SEQ ID NO:50)，

Wherein ". sup." means phosphorothioate bond, and ". sup." means phosphodiester bond. In any of these sequences, ethyl uridine or halo may be substituted for 5' T; examples of halogen substitutions include, but are not limited to, bromouridine or iodouridine substitutions.

The P-class oligonucleotides are in some cases capable of inducing much higher levels of IFN- α secretion than the C-class oligonucleotides. The P-type oligonucleotides are capable of self-assembly into concatamers (concatamers) spontaneously in vitro and/or in vivo. The P-type oligonucleotides are further disclosed in PCT application No. WO2008/068638 published on 12.6.2008. In certain aspects, the P-type oligonucleotide is:

CpG21798

5’T*C-G*T*C-G*A*C-G*A*T*C-G*G*C*G*C-G*C*G*C*C*G3’(SEQID NO:7)，

CpG23430

5’T*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G3’(SEQ ID NO:8)，

CpG24558

5’T*C*G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G*T3’(SEQ ID NO:9)，

CpG23871

5’JU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G3’(SEQ ID NO:10)，

CpG23873

5’JU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G*T3’(SEQ ID NO:11)，

CpG23874

5’JU*C*G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G*T3’(SEQ ID NO:12)，

CpG23875

5’EU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G3’(SEQ ID NO:13)，

CpG23877

5’JU*C-G*T*C*G*A*C*G*A*T*C*G*G*C*G*G*C*C*G*C*C*G*T3’(SEQ ID NO:14)，

CpG23878

5 'JU C G T C G C G C G T3' (SEQ ID NO:15) or

5’T*C_G*T*C_G*A*C_G*A*T*C_G*G*C*G*C_G*C*G*C*C*G3’(SEQ ID NO:37)。

Compared to class B or C oligonucleotides, class T oligonucleotides induce lower levels of IFN- α and IFN-related cytokine and chemokine secretion, while maintaining the ability to induce IL-10 levels similar to class B oligonucleotides. The T-type oligonucleotides are further disclosed in PCT application No. WO2008/068638 published on 12.6.2008.

E modifications can be made on any class of CpG oligonucleotides. This is an oligonucleotide with lipophilic substituted nucleotide analogs outside the CpG motif and which has an enhanced ability to stimulate interferon alpha (IFN- α) production and induce TLR9 activation. The E-modified oligonucleotide is further disclosed in PCT application No. WO2008/068638 published on 12.6.2008.

In some aspects of the invention, the one or more immunomodulatory molecules are in an effective amount to induce or enhance an antigen-specific immune response. In certain aspects, the enhanced antigen-specific immune response is a Th1 immune response. In certain aspects, the Th1 immune response results in antigen-specific induction of IFN- γ, or induction of multifunctional T cells secreting two or more cytokines. In certain aspects, such cytokines include, but are not limited to, IL-2 and IFN- γ or IFN- γ, TNF- α, and IL-2.

In some aspects of the invention, the amount of immunomodulatory molecule ranges from about 1 μ g per dose of vaccine to about 5mg per dose of vaccine. In certain aspects, the amount of immunomodulatory molecule is from about 1 μ g/dose of vaccine to about 4 mg/dose of vaccine, from about 1 μ g/dose of vaccine to about 3 mg/dose of vaccine, from about 1 μ g/dose of vaccine to about 2 mg/dose of vaccine, or from about 1 μ g/dose of vaccine to about 1 mg/dose of vaccine. In certain aspects, the amount of immunomodulatory molecule is from about 10 μ g/dose of vaccine to about 750 μ g/dose of vaccine, from about 10 μ g/dose of vaccine to about 500 μ g/dose of vaccine, from about 10 μ g/dose of vaccine to about 250 μ g/dose of vaccine, from about 10 μ g/dose of vaccine to about 100 μ g/dose of vaccine, from about 20 μ g/dose of vaccine to about 100 μ g/dose of vaccine, or from about 30 μ g/dose of vaccine to about 100 μ g/dose of vaccine. In certain aspects, the amount of immunomodulatory molecule is about 500 μ g per dose of vaccine. In certain aspects, the amount of immunomodulatory molecule is about 250 μ g per dose of vaccine.

In some aspects of the invention, the amount of immunomodulatory molecule relative to the amount of cholesterol is greater than the amount of cholesterol. In some aspects of the invention, the ratio of the amount of immunomodulatory molecule to the amount of cholesterol is about 100:1, or about 75:1, or about 50:1, or about 25:1, or about 15:1, or about 10:1, or about 5:1 by weight. In some aspects of the invention, the amount of immunomodulatory molecule relative to the amount of cholesterol is about the same as the amount of cholesterol. That is, the ratio of the amount of immunomodulatory molecule to the amount of cholesterol is 1:1 by weight. In some aspects of the invention, the amount of immunomodulatory molecule relative to the amount of cholesterol is less than the amount of cholesterol. In some aspects of the invention, the ratio of the amount of immunomodulatory molecule to the amount of cholesterol is about 1:100, or about 1:75, or about 1:50, or about 1:25, or about 1:15, or about 1:10, or about 1:5 by weight. In one aspect, the ratio of the amount of immunomodulatory molecule to the amount of cholesterol is 1:10 by weight. Those skilled in the art will recognize that the proportions given may be as shown above or substantially as shown above.

As used herein, the terms "disorder," "condition," and "disease" are used interchangeably.

In some aspects of the invention, the vaccine can be used as a prophylactic vaccine for preventing an infection (e.g., an infectious disease), a condition associated with a self-antigen, or a condition associated with an addictive substance. Preferably, prophylactic vaccination is used in subjects who have not yet been diagnosed with the condition against which the vaccine is directed, and more preferably, in subjects considered to be at risk of developing one of these conditions. For example, the subject may be a subject at risk for developing an infection with an infectious organism, a condition susceptible to a condition associated with a self-antigen, or a condition associated with an addictive substance.

As used herein, a "subject at risk" is a subject at risk of any exposure to an infectious pathogen, a subject at risk of developing a chronic or treatment-resistant infectious disease, a subject at risk of developing cancer, a subject at risk of developing allergy, a subject at risk of developing asthma, a subject at risk of developing a condition associated with an addictive substance, a subject at risk of developing a condition associated with aberrant protein folding, or a subject at risk of developing an autoimmune condition of origin. Subjects at risk also include subjects with a predisposition to develop such a disorder. Certain susceptibilities may be genetic (and thus identified by genetic analysis or by family history). Certain susceptibilities are environmental (e.g., prior exposure to infectious agents, self-antigens, or addictive substances). For subjects at risk of developing an infection, one example of such subjects is subjects living in an area where a particular type of infectious agent is found or is expected to travel to that area, or may be subjects exposed to the organism (by direct or indirect contact with a body fluid that may contain an infectious organism) by lifestyle or medical procedures. Subjects at risk of developing an infection also include the general population for which a medical institution recommends vaccination against a particular infectious organism.

The subject is a human or a non-human animal treated with a veterinary drug. Non-human animal subjects include, but are not limited to, dogs, cats, birds, horses, cows, pigs, sheep, goats, chickens, non-human primates (e.g., monkeys, orangutans), and fish (aquatic species, e.g., salmon).

As used herein, an "infectious disease" is a disease that results from the presence of a foreign microorganism (e.g., a bacterium, virus, parasite, or fungus) within the body.

In some aspects of the invention, bacteria include, but are not limited to, the following species and combinations thereof: acinetobacter calcoaceticus (Acinetobacter calcoaceticus), Acetobacter pasteurianus (Acetobacter pasaniana), Actinomyces actinomycetemcomitans (Actinomyces actinometcomomatans), Actinomyces pleuropneumoniae (Actinomyces pleuropneumoniae), Actinomyces israeli (Actinomyces israeli), Actinomyces viscosus (Actinomyces viscosus), Aeromonas hydrophila (Aeromonas hydrophila), Alcaligenes eutrophus (Alcaligenes eutrophus), Thermomyces acidocaldarius (Alicyclobacillus acidocidus), Archaeoglobus fulgidus (Arhaemangelospora), Bacillus species (Bacillus subtilis), Bacillus subtilis (Bacillus subtilis), Bacillus species (Bacillus subtilis), Bacillus species (Bacillus subtilis), Bacillus species (Bacillus subtilis), Bacillus subtilis (Bacillus species (Bacillus subtilis), Bacillus species (Bacillus subtilis), Bacillus species (Bacillus subtilis), Bacillus species (, Burkholderia rhinoceros (Burkholderia glaucae), Brevibacterium (Brachyspira), Brevibacterium dysenteriae (Brachyspira), Brevibacterium Enterobacter (Brachyspira), Campylobacter (Campylobacter) species, Campylobacter coli), Campylobacter fetus (Campylobacter fetus), Campylobacter coli (Campylobacter asiaticus), Campylobacter jejuni (Campylobacter jejuni), Campylobacter psis (Chlamydomonas), Chlamydia trachomatis (Chlamydia trachomatis), Chlamydia Chlamydophila (Chlamydia Chlamydophila), Clostridium chlamydophilum (Clostridium), Clostridium viscosus (Clostridium), Clostridium difficile (Clostridium difficile) species, Enterococcus (Enterococcus), Erysipelothrix rhusiopathiae (Erysipelothrix), Escherichia (Escherichia), Escherichia coli (Escherichia coli), Fusobacterium nucleatum (Fusobacterium subclauum), Haemophilus (Haemophilus influezae), Haemophilus somnus (Haemophilus somnus), Helicobacter (Helicobacter), Helicobacter pylori (Helicobacter pylori), Helicobacter suis (Helicobacter suis), Klebsiella (Klebsiella), Klebsiella pneumoniae (Klebsiella), Lactobacillus pneumoniae (Klebsiella acidophilus), Lactobacillus acidophilus (Lactophilius), Lactobacillus acidophilus (Lactophilus), Leptospira intracellularis (Laterophilus), Leptospira curvata), Leptospira (Leptospira), Leptospira typhii (Leptospira), Leptospira canicola (Leptospira), Leptospira sp), Leptospira canicola (Leptospira, Leptospira sp), Leptospira sp (Leptospira sp), Leptospira sp) Mycobacterium bovis (Halbacco-Parkinsonii hardjo-prajitno), Leptospira interrogans (Leptospira interrogans), Leptospira icterohaemorrhagiae (Leptospira haemolytica), Leptospira pomonensis (Leptospira haemolytica), Leptospira pomona (Leptospira), Leptospira brasiliensis (Leptospira brasiliensis) etc., Listeria species, Listeria monocytogenes (Listinobacterium), Diplococcus meningitidis (Meycococcus), Moraxella species (Moraxella), Mycobacterium species (Mycobacterium), Mycobacterium bovis (Mycobacteria), Mycobacterium tuberculosis (Mycobacterium), Mycobacterium tuberculosis (Mycobacterium, Mycobacterium (Mycobacterium), Mycobacterium pneumoniae (Mycobacterium), Mycobacterium (Mycobacterium pneumoniae (Mycobacterium) and Mycobacterium (Mycobacterium) such as a) or Mycoba, Mycoplasma hyorhinis (Mycoplasma hyopneumoniae), Mycoplasma pneumoniae (Mycoplasma hyopneumoniae), Mycoplasma mycoides (Mycoplasma mycoides) Mycoplasma mycoides (LC subspecies fungoides), Neisseria species (Neisseria), Neisseria gonorrhoeae), Neisseria meningitidis (Neisseria meningitidis), Odorobacter denticola, Pasteurella species (Pasteurella), Pasteurella haemolytica (Mannheimia), Pasteurella multocida (Pasteurella), Pasteurella multocida (Pseudomonas multocida), Pseudomonas paraguas (Pseudomonas paradoxa), Pseudomonas luminescens (Pseudomonas aeruginosa), Pseudomonas aeruginosa (Pseudomonas putida), Pseudomonas aeruginosa strain (Pseudomonas aeruginosa), Pseudomonas aeruginosa (Pseudomonas aeruginosa strain 1), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Pseudomonas aeruginosa strain (Pseudomonas aeruginosa), Pseudomonas aeruginosa (Pseudomonas aeruginosa strain 1), Pseudomonas aeruginosa (Pseudomonas aeruginosa) Pseudomonas fragilis (Pseudomonas fragrans ), Pseudomonas aeruginosa (Pseudomonas luteola), Pseudomonas oleolytica (Pseudomonas oleovans), Pseudomonas sp.B 11-1, Acidophilus quiescens (Pseudomonas immobilis), Rickettsia sp, Rickettsia prohibitiella (Rickettsia prowazekii), Rickettsia rickettsii (Rickettettsia Rickettsia ricksii), Salmonella sp.sp.guillotinensis (Salmonella choleraesuis), Salmonella choleraesuis (Salmonella choleraesuis), Salmonella enterica (Salmonella enterica), Salmonella newport (Salmonella newcastle), Salmonella typhimurium (Salmonella typhimurium), Staphylococcus epidermidis (Staphylococcus epidermidis), Salmonella typhi (Staphylococcus spp., Salmonella typhi), Salmonella typhi (Staphylococcus epidermidis) species (Staphylococcus spp., Staphylococcus epidermidis), Salmonella typhi (Salmonella typhi), Salmonella typhi (Staphylococcus epidermidis) strains (Salmonella typhi), Salmonella typhi (Staphylococcus spp., Staphylococcus epidermidis) and Salmonella typhi (Staphylococcus spp., Staphylococcus, Streptococcus (Streptococcus) species, Staphylococcus suis (Streptococcus monoilformis), beta-hemolytic Streptococcus (beta-hemolytic Streptococcus), Streptococcus pyogenes (Streptococcus pyogenes) (group A Streptococcus), Streptococcus agalactiae (Streptococcus agalactiae) (group B Streptococcus), Streptococcus (Viridans group), Streptococcus faecalis (Streptococcus faecis), Streptococcus bovis (Streptococcus bovis), Streptococcus uberis (Streptococcus lactis), Streptococcus dysgalactiae (Streptococcus dysgalactiae), Streptococcus pneumoniae (anaerobe), Streptococcus pneumoniae (Streptococcus pneumoniae), Streptococcus sobrinus (Streptococcus mutans), Streptococcus sobrinus (Streptococcus sobrinus), Streptococcus faecalis (Streptococcus faecalis), Streptomyces albugineus (Streptococcus faecalis), Streptococcus lactis (Streptococcus lactis), Streptococcus lactis (Streptococcus), Streptococcus lactis), Streptococcus (Streptococcus), Streptococcus lactis), Streptococcus (Streptococcus), Streptococcus lactis), Streptococcus (Streptococcus lactis), Streptococcus (Streptococcus), Streptococcus (Streptococcus), Streptococcus lactis), Streptococcus (Streptococcus), Streptococcus (, Treponema pallidum, Treponema pertenue, Treponema ulcerosa, Treponema bergensis, Treponema inflixima, Treponema inflixiangense, Vibrio species, Vibrio cholerae, Yersinia species.

In some aspects of the invention, viruses include, but are not limited to, the following species and combinations thereof: avian herpes virus, avian influenza virus, avian leukemia virus, avian paramyxovirus, border disease virus, bovine coronavirus, bovine epidemic fever virus, bovine herpes virus, bovine immunodeficiency virus, bovine leukemia virus, bovine parainfluenza virus 3, bovine respiratory syncytial virus, Bovine Viral Diarrhea Virus (BVDV), BVDV type I, BVDV type II, canine adenovirus, Canine Coronavirus (CCV), canine distemper virus, canine herpes virus, equine herpes virus, canine influenza virus, canine parainfluenza virus, canine parvovirus, canine respiratory coronavirus, classical swine fever virus, eastern equine encephalitis virus (EEE), equine infectious anemia virus, equine influenza virus, West Nile virus, feline calicivirus, feline immunodeficiency virus, feline infectious peritonitis virus, feline herpes virus, feline influenza virus, feline leukemia virus (FeLV), feline viral rhinotracheitis virus, bovine coronavirus, bovine influenza virus, bovine leukemia virus (FeLV), bovine viral rhinotracheitis virus, bovine herpes virus, bovine immunodeficiency virus, bovine influenza virus, bovine viral, Lentivirus, Marek's disease virus, Newcastle disease virus, ovine herpes virus, ovine parainfluenza type 3 virus, ovine progressive pneumonia virus, ovine lung adenocarcinoma virus, pantoea virus, porcine circovirus type I (PCV), PCV type II, porcine epidemic diarrhea virus, porcine hemagglutinating encephalomyelitis virus, porcine herpes virus, porcine parvovirus, Porcine Reproductive and Respiratory Syndrome (PRRS) virus, pseudorabies virus, rabies virus, provirus, rhinovirus, rinderpest virus, swine influenza virus, infectious gastroenteritis virus, turkey coronavirus, Venezuelan equine encephalitis virus, vesicular stomatitis virus, West Nile virus, and Western equine encephalitis virus.

In some aspects of the invention, parasites include, but are not limited to, the following species and combinations thereof: proteins from the genus Anaplasma (Anaplama), Fasciola hepatica (liver fluke), coccidia, Eimeria species, neospora caninum, Toxoplasma gondii, Giardia, Dirofilaria (Dirofilaria immitis), hookworm (hookworm), Trypanosoma species, Leishmania species, Trichomonas species, Cryptosporidium parvum, Babesia, Hematospora, Taenia, strongyloides, ascaris, Trichinella, Sarcocystis, Hammond, Isosporus (Isosporia). In certain aspects, the parasite is an ectoparasite. In certain aspects, ectoparasites include, but are not limited to, classes of ticks including species of hard ticks, rhipicephalus, dermanylus, minus, hyalomma, haemaphysalis, and combinations thereof.

In some aspects of the invention, fungi include, but are not limited to, spores, molds, and yeasts (e.g., candida species).

As used herein, a chronic or treatment-resistant infectious disease is a disease with an extended period of infection (sometimes lasting weeks, months, or even life-long) or an infection that is resistant to other treatments that are generally successful. In certain aspects, chronic or treatment resistant viral infections include, but are not limited to, HBV, HCV, HIV, HPV, HSV-1 or HSV-2.

In some aspects of the invention, a subject having cancer may be a subject having detectable cancerous cells. The cancer may be a malignant or non-malignant cancer. Cancers or tumors include, but are not limited to: biliary cancer, bladder cancer, brain cancer, breast cancer, cervical cancer, choriocarcinoma, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, glioblastoma, intraepithelial neoplasia, lymphoma (e.g., follicular lymphoma), liver cancer, lung cancer (e.g., small cell and non-small cell lung cancers), leukemia (e.g., hair cell leukemia, chronic myelogenous leukemia, cutaneous T cell leukemia), melanoma (e.g., malignant melanoma), multiple myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer, kidney cancer, and other malignant tumors and sarcomas (e.g., squamous cell tumor, renal cell tumor, prostate tumor, bladder tumor, or colon tumor).

In some aspects of the invention, a subject having an allergy is a subject having or at risk of developing an allergic reaction corresponding to an allergen. An allergen refers to acquired hypersensitivity to a substance (allergen). Allergic conditions include, but are not limited to, eczema, allergic rhinitis or rhinitis, pollinosis, conjunctivitis, bronchial asthma, urticaria (urticaria) and food allergies, as well as other atopic conditions.

At present, allergic diseases are often treated by injection of a small dose of antigen followed by a subsequent increasing dose of antigen. It is believed that this procedure induces tolerance to the allergen to prevent further allergic reactions. However, these methods will take years to be effective and also involve the risk of side effects, such as anaphylactic shock.

Allergy is usually caused by the production of IgE antibodies against harmless allergens. The predominant cytokines induced by systemic or mucosal administration of immunostimulatory nucleic acids are a class of cytokines called Th1 (examples of which are IL-12 and IFN- γ), and they induce both humoral and cellular immune responses. The type of antibody associated with Th1 responses is generally more protective because of its high neutralizing and opsonizing capacity. Another major type of immune response, which is associated with the production of IL-4, IL-5 and IL-10 cytokines, is the Th2 immune response. Th2 responses are primarily directed to antibodies and have a lower protective effect against infection, and certain Th2 isoforms (e.g., IgE) are associated with allergy. In general, allergic diseases appear to be mediated by a Th 2-type immune response, whereas a Th1 response provides the best protection against infection, but an excessive Th1 response is associated with autoimmune diseases. The ability of administration of one or more immunomodulatory molecules to shift the immune response in a subject from Th2 (which is associated with IgE antibody production and allergy) to a Th1 response (which may protect against allergic reactions) can be administered to the subject in an amount effective to induce the immune response of the immunomodulatory molecule to treat or prevent allergy.

In some aspects of the invention, an allergen refers to a substance (e.g., an antigen) that induces an allergic or asthmatic response in a susceptible subject. Allergens include, but are not limited to, pollen, insect venom, animal dander, fungal spores, and drugs (e.g., penicillin). Examples of natural, animal and plant allergens include, but are not limited to, proteins specific to the following species: canis (canis), dermatophagoides (e.g., dermatophagoides farinae), felis (felis domestica), ragweed (ambrosia americana), lolium (e.g., perennial ryegrass or annual ryegrass), salix (japanese cedar), alternaria (alternaria alternata), alder (alder), betula (verrucosa), quercus (quercus americana), oliv (olea), artemisia (artemisia argyi), plantago (e.g., plantago lanceolata), pellitory (e.g., medicinal wall (Parietaria officinalis) or achilleta), cockroaches (e.g., german cockroach), honeybee (e.g., uniflora), cedar (e.g., cypress, sabina virginiana and sabina), sabina chinensis (e.g., Juniperus sabina, pencilaria, oerioides or Juniperus), thuja (e.g., thuja), thuja (e (eastern), thuja (e, such as hinoki), japanese cypress), periplaneta (e.g., american cockroach), agropyron (e.g., alpine serra), secale (e.g., rye), triticale (e.g., wheat), dactylus (e.g., dactylis glomerata), festuca (e.g., oxtail), poa (e.g., poa pratensis or poa canadensis), avena (e.g., oat), eriodictyonia (e.g., chorion), citronella (e.g., citronella), avena (e.g., avena sativa), agrostis (e.g., furfurescence grass), ramaria (e.g., timothy grass), phalaris (e.g., phalaris), brome (e.g., bacon), sorghum (e.g., sorghum halepense), and brome (e.g., bromus formononensis).

In some aspects of the invention, asthma refers to a respiratory condition characterized by inflammation, airway narrowing, and increased airway responsiveness to inhalants. Th2 cytokines (e.g., IL-4 and IL-5) are elevated in the airways of asthma subjects. These cytokines contribute to important aspects of the inflammatory response of asthma, including IgE isotype switching, eosinophil chemotaxis and activation, and mast cell growth. Th1 cytokines, particularly IFN-gamma and IL-12, were able to inhibit the formation of Th2 clone null and the production of Th2 cytokine. Asthma is often associated with atopic or allergic symptoms, although not exclusively.

In some aspects of the invention, the disorder associated with aberrant protein folding is a disorder caused by misfolding (misfolding) or a protein associated with an error in the subject's DNA resulting in incorrect folding of the protein. In some aspects, the disorder associated with abnormal protein folding is an amyloidosis disorder (e.g., alzheimer's disease, MS) or a prion disorder (e.g., a Transmissible Spongiform Encephalopathy (TSE) including, but not limited to, bovine spongiform encephalopathy (BSE, mad cow disease) and Creutzfeld Jakob Disease (CJD)) in humans. In some aspects, conditions involving DNA errors in a subject that result in incorrect folding of the protein include, but are not limited to, cystic fibrosis and cancers associated with the p53 protein.

In some aspects of the invention, an autoimmune disorder is any disorder that involves an overactive immune response of a subject's body to substances and tissues (e.g., autoantigens) that are typically present in the subject. In certain aspects, the autoimmune disorder is Rheumatoid Arthritis (RA), lupus erythematosus, or crohn's disease.

In some aspects of the invention, a condition associated with a self-antigen is any condition caused by an antigen of a subject's own cells or cellular products that leads to an immune response in the subject. For example, in certain embodiments, the autoantigen is a tumor antigen, an antigen associated with alzheimer's disease, an antigen directed against an antibody, or an antigen expressed by a human endogenous retroviral element. In certain aspects, the tumor antigen is one or more of: WT1, MUC1, LMP2, HPV E6 or HPV E7, EGFR or variant forms thereof (e.g., EGFRvIII), HER-2/neu, idiotype, MAGE A3, p5 non-mutants, NY-ESO-1, PSMA, GD2, CEA, MelanA/MART1, Ras mutants, gp100, p53 mutants, protease 3(PR1), bcr-abl, tyrosinase, survivin, PSA, hTERT, sarcoma ectopic breakpoint, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG (TMPRSS2ETS fusion protein), NA 56, PAX3, ALK, androgen receptor, cyclin B1, poly-mer, MYCN, RhoC, TRP-2, GD3, Fucosyl GM1, mesothelin, PSCA, MAGE 8258, SAGE 8258, BCE 8672, BOB 8672, BOOB-72, SAOB-RG-72, SALT 72, sLe, SAOB-S sLe, SALT 72, SAO-S sLe, AML sLe, SAC sLe, MAG-III, TMAS-III, MAG-III, SAC sLe, AKAP-4, SSX2, XAGE1, B7H3, legumin (Legumain), Tie2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-beta, MAD-CT-2 or Fos-associated antigen 1. The antigen associated with Alzheimer's disease may be tau-amyloid or beta-amyloid. The antigen directed against an antibody may be an antigen directed against a human antibody, for example in certain embodiments the antigen is IgE.

In some aspects of the invention, the vaccine can be used to prevent respiratory viral infection in an animal. In certain aspects, the respiratory viral infection is BVDV1, BVDV2, IBRV, P13V, or BRSV.

In some aspects of the invention, a condition associated with an addictive substance is any condition in a subject that is involved in developing addiction to an addictive chemical or biological substance. For example, in certain embodiments, the addictive substance may be nicotine or cocaine. In certain embodiments, the vaccine for preventing or treating addiction comprises nicotine or a nicotine-like hapten conjugated to a carrier. In certain embodiments, the carrier to which the nicotine or nicotine-like hapten is conjugated is a bacterial toxoid or derivative, pseudomonas exotoxin, KLH, or virus-like particle. In certain embodiments, the bacterial toxoid is diphtheria toxoid or a derivative thereof, e.g., CRM₁₉₇. In certain aspects, the virus-like particle is HBsAg, HBcAg, coliphage Q β, norwalk virus, or influenza HA.

As used herein, the terms "treatment," "treated" or "treating" when used with respect to an infectious disease refer to prophylactic treatment that increases resistance to, or in other words reduces the likelihood of, infection by a pathogen in a subject (a subject at risk of infection), as well as treatment to combat infection (e.g., reduce or eliminate infection or avoid exacerbations thereof) after a subject (an infected subject) has been infected.

The terms "treat," "treated" or "treating" when used with respect to cancer refer to prophylactic treatment that increases resistance to cancer or reduces the likelihood that a subject will develop cancer (a subject at risk of developing cancer), as well as treatment that reduces the effect of (e.g., reduces or eliminates or avoids worsening of) a condition after the subject (a subject that has or has just been diagnosed with cancer) has developed such a condition or has begun to develop signs or symptoms of such a condition.

The terms "treat," "treated" or "treating" when used with respect to asthma or allergy refer to prophylactic treatment that increases resistance to, or reduces the likelihood that, a subject (a subject at risk of developing asthma or allergy) will develop such a disorder, as well as treatment that reduces the effect of (e.g., reduces or eliminates or avoids worsening of) a disorder after a subject (a subject that has or has just been diagnosed with asthma or allergy) has developed such a disorder or has begun to develop signs or symptoms of such a disorder.

The terms "treat," "treated," or "treating," when used with respect to a condition associated with an addictive substance, refer to prophylactic treatment that increases resistance to developing such a condition in a subject (a subject at risk of developing a condition associated with an addictive substance) or reduces the likelihood that a subject will develop a condition associated with an addictive substance, and treatment that reduces the effect of (e.g., reduces or eliminates or avoids worsening of) a condition associated with an addictive substance after a subject (a subject that has or has just been diagnosed with) has developed such a condition or has begun to develop signs or symptoms of such a condition.

The terms "treat," "treated," or "treating," when used with respect to a disorder associated with abnormal protein folding, refer to prophylactic treatment that increases resistance to development of such a disorder in a subject (a subject at risk of developing a disorder associated with abnormal protein folding) or reduces the likelihood that a subject will develop a disorder associated with abnormal protein folding, as well as treatment that reduces the effect of (e.g., reduces or eliminates or avoids worsening of) a disorder after a subject (a subject that has or has just been diagnosed with a disorder associated with abnormal protein folding) has developed such a disorder or begins to develop signs or symptoms of developing such a disorder.

The terms "treat," "treated" or "treating" when used with respect to an autoimmune disorder refer to prophylactic treatment that increases resistance to development of such a disorder in a subject (a subject at risk of developing an autoimmune disorder) or reduces the likelihood that a subject will develop an autoimmune disorder, as well as treatment that reduces the effect of (e.g., reduces or eliminates or avoids worsening of) an autoimmune disorder after a subject (a subject that has or has just been diagnosed with an autoimmune disorder) has developed such a disorder or has begun to develop signs or symptoms of such a disorder.

The terms "treat," "treated," or "treating," when used with respect to a disorder associated with an autoantigen, refer to prophylactic treatment that increases resistance to development of such a disorder in a subject (a subject at risk of developing a disorder associated with an autoantigen) or reduces the likelihood that a subject will develop a disorder associated with an autoantigen, as well as treatment that reduces the effect of (e.g., reduces or eliminates or avoids worsening of) a disorder after a subject (a subject that has or has just been diagnosed with) has developed such a disorder or has begun to develop signs or symptoms of such a disorder.

Treatment of a subject with the vaccines described herein results in a reduction in infection or a complete elimination of infection, a reduction in signs/symptoms associated with a condition associated with an autoantigen or a complete elimination of the condition, or a reduction in signs/symptoms associated with a condition associated with an addictive substance or a complete elimination of the condition. A subject is considered treated if symptoms associated with an infectious disease, cancer, allergy, asthma, a disorder associated with abnormal protein folding, an autoimmune disorder, a disorder associated with an autoantigen, or a disorder associated with an addictive substance are reduced, controlled, or eliminated as a result of the treatment. For infectious diseases, the treatment also encompasses a reduction in the amount of infectious agent present in the subject (e.g., the amount can be measured by standard assays known to those of ordinary skill in the art, such as ELISA). For cancer, the treatment also encompasses a reduction in cancerous cells or tissues and/or a reduction in signs/symptoms associated with the cancer. For allergy, the treatment also encompasses a reduction in the signs/symptoms associated with the allergy. For asthma, the treatment also encompasses a reduction in the signs/symptoms associated with the asthma. For autoimmune disorders, the treatment also encompasses a reduction in the immune response to the autoimmune disorder and/or a reduction in the signs/symptoms associated with the disorder. For disorders associated with aberrant protein folding, the treatment also encompasses a reduction in the amount of aberrant protein and/or a reduction or reversal of the signs/symptoms associated with the disorder. For conditions associated with an autoantigen, the treatment also encompasses a reduction in the amount of autoantigen present in the subject or a reduction in the immune response induced by the autoantigen. For disorders associated with addictive substances, the treatment also encompasses a reduction in signs/symptoms associated with addiction to the addictive substance.

The dosage forms of the invention are administered in pharmaceutically acceptable solutions which conventionally contain pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, adjuvants and optionally other therapeutic ingredients. For certain vaccine formulations using cholesterol, the ethanol may be replaced with a pharmaceutically acceptable surfactant and an aqueous solution to solvate the cholesterol into an aqueous formulation.

For use in therapy, an effective amount of one or more immunomodulatory molecules can be administered to a subject by any mode of delivery of the immunomodulatory molecules to a desired surface. Administration of the pharmaceutical compositions of the present invention may be accomplished by any means known to those skilled in the art. Preferred routes of administration include, but are not limited to, parenteral (e.g., intramuscular, subcutaneous, intradermal, intravenous injection), topical (e.g., transdermal) to the skin, or mucosal (e.g., oral, intranasal, intravaginal, intrarectal, buccal, intraocular, or sublingual). In the case of cancer treatment, it may comprise intratumoral administration.

In some aspects of the invention, an "effective amount" of an immunomodulatory molecule refers to an amount necessary or sufficient to achieve a desired biological effect. For example, an effective amount of an immunomodulatory molecule for treating a disorder can be an amount necessary to eradicate a microbial infection or tumor. An effective amount for use as a vaccine adjuvant may be an amount useful to boost the immune response of a subject to a vaccine. An "effective amount" for treating an infectious disease, cancer, allergy, asthma, an autoimmune disorder, a disorder associated with aberrant protein folding, a disorder associated with an autoantigen, or a disorder associated with an addictive substance may be an amount useful for inducing an antigen-specific immune response. For any particular application, the effective amount may vary depending on the following parameters: the disease or condition to be treated, the particular immunomodulatory molecule administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular immunomodulatory molecule without undue experimentation.

Subject doses of the compounds described herein for topical delivery are typically from about 0.1 μ g/administration to about 50 mg/administration, which may be given daily, weekly or monthly and any other amount of time in between depending on the application. More generally, the topical dose is about 10 μ g/application to about 10 mg/application, and optionally about 100 μ g to about 1mg and 2-4 times daily or weekly. More typically, the immunostimulant dose is from about 1 μ g/administration to about 10 mg/administration, and most typically from about 10 μ g to about 1mg, administered daily or weekly. The subject dose of a compound described herein for parenteral administration for the purpose of inducing an antigen-specific immune response, wherein the compound is delivered with the antigen but not with other therapeutic agents, is typically about 5-fold to about 10,000-fold higher, and more typically about 10-fold to about 1,000-fold, and most typically about 20-fold to about 100-fold higher than the effective topical dose for vaccine adjuvant or immunostimulant administration. The compounds described herein for parenteral delivery are administered when one or more immunomodulatory molecules are administered in combination with other therapeutic agents or in a specialized delivery vehicle, e.g., for inducing innate immune responses, for increasing ADCC, for inducing antigen-specific immune responses, typically at a dose of about 0.1 μ g/administration to about 10 mg/administration, which may be given daily, weekly or monthly and any other amount of time in between, depending on the application. More generally, the parenteral dose for such purposes is from about 10 μ g/administration to about 5 mg/administration, and most typically from about 100 μ g to about 1mg and 2-4 times daily or weekly. However, in certain embodiments, parenteral dosages for the above purposes may be used in the range of about 5-fold to about 10,000-fold higher than the typical dosages described above.

For any of the compounds described herein, a therapeutically effective amount can initially be determined from an animal model. For immunomodulatory molecules that have been tested in humans and for compounds known to exhibit similar pharmacological activity, such as other adjuvants (e.g., LT) and other antigens used for vaccination purposes, therapeutically effective doses can also be determined from human data. Higher doses may be required for parenteral administration. The dosage applied may be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dosage to achieve maximum efficacy based on the above and other methods is well known in the art and is well within the ability of one of ordinary skill in the art.

One or more immunomodulatory molecules may be administered alone or in combination with one or more antigens, cholesterol, or other therapeutic agents via any of the routes described herein.

When systemic delivery of one or more immunomodulatory molecules is desired, it can be formulated for parenteral administration by injection (e.g., bolus injection or continuous infusion). Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the immunomodulatory molecules in water-soluble form. Additionally, suspensions of immunomodulatory molecules can be prepared as suitable oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils (e.g. sesame oil) or synthetic fatty acid esters (e.g. ethyl oleate or triglycerides), or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextrans. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the immunomodulatory molecules to allow for the preparation of highly concentrated solutions.

The therapeutic agent may be diluted or increased in volume with an inert material. These diluents may include sugars, in particular mannitol, a-lactose, anhydrous lactose, cellulose, dextrans and/or starch. Certain inorganic salts may also be used as fillers, including calcium triphosphate, magnesium carbonate and/or sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx1500, Emcompress and Avicell.

To aid dissolution of the therapeutic agent in an aqueous environment, a surfactant may be added as a wetting agent. The surfactant may comprise an anionic detergent including sodium lauryl sulphate, sodium dioctyl sulphosuccinate and/or sodium dioctyl sulphonate. Cationic detergents may be used, which may include benzalkonium chloride or benzethonium chloride. A list of potential nonionic detergents that can be included in the formulation is: lauromacrogol 400, macrogol 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and/or 60, glycerol monostearate, polysorbate 40, 60, 65 and/or 80, sucrose fatty acid ester, methylcellulose and carboxymethylcellulose. In some aspects, nonionic detergents include, but are not limited to: octoxynol (e.g., t-octylphenoxy polyethoxyethanol (TRITON X-100)^TM) Polyoxyethylene esters (e.g., polyoxyethylene polysorbitol monooleate (TWEEN 80)^TM) Cholate, and cholic acid derivatives (e.g., sodium deoxycholate or taurodeoxycholate). In some aspects, the formulation may comprise 3D-MPL, Laureth9, TRITONX-100^TM、TWEEN80^TMAnd deoxidationSodium cholate. These surfactants may be present in the preparation of the immunomodulatory molecules alone or as a mixture in varying proportions.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the immunomodulatory molecules in water-soluble form. Additionally, suspensions of immunomodulatory molecules can be prepared as suitable oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils (e.g. sesame oil) or synthetic fatty acid esters (e.g. ethyl oleate or triglycerides), or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextrans. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the immunomodulatory molecules to allow for the preparation of highly concentrated solutions.

Alternatively, the immunomodulatory molecule can be in powder form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to use.

For oral administration, the compound (e.g., the immunomodulatory molecule alone, or in combination with one or more antigens, cholesterol, and/or therapeutic agents) can be conveniently formulated by mixing the immunomodulatory molecule with a pharmaceutically acceptable carrier as is well known in the art. Such carriers enable the immunomodulatory molecules of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions and the like, for oral ingestion by a subject to be treated. Pharmaceutical preparations for oral use can be obtained as solid excipients, optionally by processing the mixture of granules after grinding the resulting mixture and, if necessary, adding suitable auxiliaries, to obtain tablets or dragee cores. Suitable excipients are in particular: fillers, for example sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations, for example, corn starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose; and/or polyvinylpyrrolidone (PVP). If necessary, disintegrating agents such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof (such as sodium alginate) may be added. Alternatively, the oral formulation may also be formulated in saline or buffer (i.e., EDTA) in order to neutralize acidic conditions in vivo, or may be administered without any carrier.

The invention also contemplates oral dosage forms of the above agents or formulations. The agent or formulation may be chemically modified to allow efficient oral delivery of the derivative. Typically, the chemical modification contemplated is the attachment of at least one moiety to the agent or formulation itself, wherein the moiety allows (a) inhibition of proteolysis; and (b) is taken from the stomach or small intestine into the bloodstream. There is also a need to increase the overall stability of the agent or formulation and increase the circulation time in vivo. Examples of such moieties include: polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethylcellulose, dextrane dextran, polyvinyl alcohol, polyvinylpyrrolidone and polyproline (Abuchowski and Davis,1981, "simple Polymer-Enzyme additives", Hounenberg and Roberts, Wiley-Interscience, New York, N.Y., p. 367 383; Newmark et al, 1982, J.apple. biochem.4: 185. sup. 189). Other polymers which may be used are poly-1, 3-dioxolane and poly-1, 3, 6-trioxane. As indicated above, preferred for pharmaceutical use are polyethylene glycol moieties.

Intranasal delivery of the pharmaceutical compositions of the present invention is also contemplated. Intranasal delivery allows the pharmaceutical compositions of the invention to be delivered directly to the bloodstream, without the need for deposition of the product in the lungs, following administration of the therapeutic product to the nose. Formulations for intranasal delivery include those employing dextrans or cyclodextrins. In some aspects, formulations for intranasal delivery (or mucosal delivery) may comprise 3D-MPL, Laureth9, TRITON X-100^TM、TWEEN80^TMAnd deoxycholate. In some aspects, such formulations can be combined with an antigen (e.g., an influenza virus antigen).

For intranasal administration, useful are small hard vials with attached metered dose nebulizers. In some aspects, the metered dose is delivered by inhalation of a pharmaceutical composition of a solution of the present invention into a volume-defined chamber having pores of a size that can aerosolize an aerosol formulation by forming a spray upon compression of the liquid within the chamber. The chamber is compressed to administer the pharmaceutical composition of the invention. In some aspects, the chamber is a piston arrangement. Such devices are commercially available.

Alternatively, a plastic squeeze bottle may be used having apertures or openings of a size that can aerosolize the aerosol formulation by forming a spray when the bottle is squeezed. The opening is typically found in the top of the bottle, which is typically adapted to partially fit within the nasal passage for effective administration of the aerosol formulation. In some aspects, the nasal inhaler provides metered doses of the aerosol formulation for administration of metered doses of the medicament.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

The compounds may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas (e.g., containing conventional suppository bases such as cocoa butter or other glycerides).

In addition to the formulations, the compounds may also be formulated as depot preparations. Such long-acting formulations may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt).

The pharmaceutical compositions may also contain suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include, but are not limited to, sodium carbonate, sodium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycol.

Suitable liquid or solid pharmaceutical preparation forms are, for example: an aqueous or saline solution for inhalation; or microencapsulated, spiral encapsulated (encocholated) or coated on fine gold particles; is contained in a liposome; is atomized by gas; aerosol; granules for implantation into the skin; or on a sharp object to be deflected into the skin. Pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro) capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations of the active compounds with extended release, in which excipients and additives and/or auxiliaries, such as disintegrants, binders, coatings, swelling agents, lubricants, taste enhancers, sweeteners or solubilizers and the like, are used as described above in accordance with the customary practice. The pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of drug delivery methods see Langer, Science249: 1527-.

The immunomodulatory molecule and optionally other therapeutic agents and/or antigen source (neat) may be administered or in the form of a pharmaceutically acceptable salt, which when used in medicine should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may be conveniently used to prepare pharmaceutically acceptable salts thereof. Such salts include, but are not limited to, salts prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluenesulfonic, tartaric, citric, methanesulfonic, formic, malonic, succinic, naphthalene-2-sulfonic, and benzenesulfonic acids. Furthermore, such salts may also be prepared as alkali metal or alkaline earth metal salts, for example, sodium, potassium or calcium salts of the carboxylic acid group.

In some aspects of the invention, the formulation may further comprise cholic acid or a derivative thereof. In some aspects, it may be in the form of a salt. In some aspects, derivatives include, but are not limited to, derivatives of cholic acid and salts thereof. In some aspects, sodium salts of cholic acid or cholic acid derivatives are contemplated. In some aspects, cholic acids and derivatives thereof include, but are not limited to: cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid, cholanic acid, hyodeoxycholic acid and derivatives, for example, glycine, bovine sulfonic acid, amidopropyl-1-propanesulfonic acid, amidopropyl-2-hydroxy-1-propanesulfonic acid derivatives, or N, N-bis (3D-glucamidopropyl) deoxycholic acid amide of the above-mentioned cholic acid. In some aspects, sodium deoxycholate (NaDOC) may be present in the vaccines of the invention.

Suitable buffers include: acetic acid and salt (1% to 2%, weight/volume ratio); citric acid and salt (1-3%, weight/volume ratio); boric acid and salts (0.5% -2.5%, weight/volume ratio); and phosphoric acid and salts (0.8% to 2%, weight/volume ratio). Suitable preservatives include benzalkonium chloride (0.003% to 0.03%, weight/volume); chlorobutanol (0.3-0.9%, weight/volume ratio); p-hydroxybenzoate (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).

The pharmaceutical compositions of the invention contain an effective amount of one or more immunomodulatory molecules, and optionally one or more antigens, cholesterol, and/or other therapeutic agents, optionally contained in a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to humans or other vertebrates. The term "carrier" refers to a natural or synthetic organic or inorganic ingredient with which the active ingredient is mixed to facilitate administration. In addition, the components of the pharmaceutical composition may also be admixed together, or with each other, with the compounds of the present invention in such a way that there is no interaction that would significantly impair the desired pharmaceutical efficacy.

The invention is further illustrated by the following examples, which should not be construed as further limiting. All references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are specifically incorporated herein by reference in their entirety.

Examples

Example 1 immunogenicity and potency data for cholesterol as delivery vehicle for CpG ODN

The use of liposomes containing cationic lipids with cholesterol showed an enhanced CpG ODN potency. As an adjuvant to enhance cellular immunity, the use of cholesterol microspheres (without addition of other lipids) was tested. C57BI/6 mice (n =5 per group) were immunized intramuscularly on days 0, 14 and 21 with ovalbumin (10 μ g), CpG only (CpG2455, 10 μ g) and CpG (CpG 245510 μ g) + cholesterol (1 μ g). Antigen-specific T cells (CD4+ and CD8+) that secreted single, multiple, or triple cytokines (IL-2, IFN-. gamma., and TNF-. alpha.) were determined on day 28 using flow cytometry.

Results and discussion

CpG + cholesterol increased the population of multifunctional CD8+ cells compared to CpG alone. Only CpG and CpG + cholesterol produced CD4+ cells that produced both single and double cytokines (fig. 1 a). CpG + cholesterol produced CD4+ cells that produced triple cytokines (fig. 1 b). CpG and CpG + cholesterol alone produced CD8+ cells that produced single, double and triple cytokines (fig. 1c and 1 d).

CpG + cholesterol showed enhanced secretion of antigen-specific IL-2 (FIG. 2a) and IFN- γ (FIG. 2b), a Th1 biased cytokine. No enhancement of pro-inflammatory or Th 2-biased cytokines was shown.

Cytotoxic T lymphocytes were assayed for response to CpG only and CpG + cholesterol. CpG + cholesterol enhanced ovalbumin-specific cytotoxic T cell responses (fig. 3a and 3b) and increased antigen-specific CD8T cell populations compared to CpG alone (fig. 3c and 3 d). Cholesterol alone showed similar levels to the non-adjuvanted control.

Humoral responses to CpG only and CpG + cholesterol were determined. CpG + cholesterol showed increased ovalbumin-specific antibody titers and a Th1 bias compared to CpG alone (figure 4). The numbers above each bar represent the ratio IgG2c/IgG 1. In mice, higher IgG2a or 2c indicated a shift towards Th1 immune responses, while higher IgG1 titers indicated a shift towards Th2 immune responses. For CpG and CpG + cholesterol, the amount of IgG2c was higher than that of IgG1 (2.04 and 4.66, respectively), indicating a bias towards Th1 immune response.

In either case, cholesterol alone showed no significant adjuvant activity.

Co-delivery of antigen with CpG and antigen with CpG + cholesterol did not show a slow mobility of CpG in electrophoresis. Compared to free CpG, the same amount of CpG was observed in the supernatant of CpG + cholesterol when quantified with UV. This indicates that there is no strong binding between CpG and cholesterol.

Subcutaneous immunization is not as effective as intramuscular injection and thus does not provide any evidence of co-delivery. However, co-delivery appears to play a role in co-formulation of the antigen and CpG that exhibit the strongest response.

Without wishing to be bound by a particular theory, Transmission Electron Microscopy (TEM) indicates that cholesterol forms insoluble helical micelles (micells) that may interact with the cell membrane to allow CpG delivery (fig. 5).

Example 2 quinquevalent (IBR, BRSV, PI3, BVDV1&2) inactivated vaccines elicit immunogenicity, safety and efficacy against BVDV-2 in calves

Injection site reactions of calves vaccinated with pentavalent inactivated virus vaccines (bovine viral diarrhoea (BVDV1&2), Infectious Bovine Rhinobronchitis (IBRV), parainfluenza virus type 3 (PI3V) and Bovine Respiratory Syncytial Virus (BRSV)) (15% of each viral antigen in a 2ml dose) in the presence of adjuvant were determined. The adjuvants CpG + cholesterol (CpG: cholesterol ratio 1:1 or 1:10), Advasure-DEAE/dextran, QCDCR (saponin carrier complex) or QCDCR + CpG are administered. Some animals were immunized with a commercial vaccine. The placebo animals received sterile saline. Calves (7/group; 9 to 12 months of age) were vaccinated subcutaneously with inactivated 2ml BVDV1&2, IBRV, PI3V and BRSV on days 0 and 22 and challenged intranasally with 4ml BVDV-2 (non-cytopathic type 2 bovine viral diarrhea Virus 2; strain 2455) on day 42.

The adjuvants used in the vaccines of each treatment group were as follows: treatment group T01 received sterile saline (without adjuvant). Treatment group T02 received a vaccine in which the adjuvant was an oil emulsion contained in a commercial vaccine.Treatment group T03 received a vaccine in which the adjuvant was CpG-23877(250 μ g)/cholesterol (250 μ g), thereby providing a ratio of CpG to cholesterol of 1: 1. Treatment group T04 received a vaccine in which the adjuvant was CpG-23877(250 μ g)/cholesterol (2500 μ g), thereby providing a ratio of CpG to cholesterol of 1: 10. Treatment group T05 received a vaccine in which adjuvant was AdvaSure. Treatment group T06 received adjuvants which were Quil A (250. mu.g), cholesterol (250. mu.g), dimethyldioctadecylammonium bromide (DDA; 100. mu.g), Carbopol(0.0375. mu.g) and N- (2-deoxy-2-L-leumindo-b-D-glucopyranosyl) -N-octadecyldodecanoic acid hydrochloride (also under the tradename Bay R1005)Commensurable) (1,000. mu.g) vaccine. This combination of components is referred to herein as QCDCR. Treatment group T07 received a vaccine in which the adjuvants were QCDCR (in the amounts given in T06) and CpG23877(250 μ g).

Blood samples were collected on days 0, 22, 42 and 56 and analyzed for IgG antibody titers using ELISA. BVDV ELISA was developed and optimized at PAH using p67H fragment of BVDV as antigen. Briefly, NUNC Maxisorp plates were coated with 0.2ug/ml of a carbonate-bicarbonate buffer (pH9.6) solution of recombinant p67H fragment BVDV antigen and incubated overnight at 4 ℃. The coating antigen was then discarded and the plate was soaked at 37 ℃ and blocked for 1 hour (300. mu.l/well) using a 1% ovalbumin in PBS-Tween solution. The blocking buffer was then removed, diluted serum samples (seven 5-fold serial dilutions; starting at 1: 50) were added and the plates were incubated for 1 hour at 37 ℃. The plate was washed 4 times in PBS-T (0.05% Tween20) after which 100. mu.l of sheep anti-bovine IgG-h + l-HRP conjugate in blocking buffer (1:4000) was added and incubated for 1 hour at room temperature in the dark. The plate was again washed in PBS-T as described above and TMB substrate (100. mu.l/well) was added. After 5 to 10 minutes of incubation, the reaction was stopped with 2N sulfuric acid (50. mu.l/well) and the Optical Density (OD) at 450nM was determined. Results are expressed as geometric mean titers.

Results and discussion

BVDV-2 challenge is symptomatic of fever, leukopenia (WBC count about 40% less than the mean WBC count prior to challenge), and thrombocytopenia, respiratory distress, depression, reproductive disorders (abortion), and diarrhea 3 to 12 days after challenge and immune modulation. Protective immunity against BVDV is a Th1 type immune response. Cell-mediated immunity is mediated by CD4+ T cells. CD8+ T cells are crucial for the clarity of the viral and memory responses. IFN-alpha and IFN-gamma are protective against BVDV. The vaccine should generally induce CMI and humoral immunity against BVDV. Table 1 describes the percentage of calves with clinical disease, fever, leukopenia or viremia after challenge with BVDV-2 after vaccination with the pentavalent inactivated viral vaccines BVDV1&2, IBRV, PI3V and BRSV in the presence of CpG + cholesterol (CpG: cholesterol ratio 1:1(T03) or 1:10(T04)), Advasure-DEAE/Dextran (T05), QCDCR (T06), QCDCR + CpG (T07), commercial vaccine (T02) or sterile saline (T01).

TABLE 1 percentage of calves with clinical disease, fever, leukopenia or viremia

Vaccines administered to calves in the T02 (commercial vaccine), T03(CpG: cholesterol 1:1), T04(CpG: cholesterol 1:10), T05 (Advasure-DEAE/dextran dextro-dex), T06(QCDCR) and T07(QCDCR + CpG) groups were superior in suppressing fever compared to saline controls. Vaccines administered to calves in the T03(CpG: cholesterol 1:1), T04(CpG: cholesterol 1:10), T05 (Advasure-DEAE/dextran dextro-dextran), T06(QCDCR) and T07(QCDCR + CpG) groups were superior in suppressing viremia compared to the commercial vaccine (T02) and the saline group (T01). T04 and T07 completely inhibited viremia, whereas the calves of the commercial vaccine (T02) group developed viremia in a shorter time than the controls. Although leukopenia was not completely avoided in any of the vaccinated groups, vaccine effects were observed on multiple days in T03(CpG: cholesterol 1:1), T04(CpG: cholesterol 1:10), T05 (Advasure-DEAE/D-dextran), T06(QCDCR) and T07(QCDCR + CpG) compared to the control and commercial vaccine (T02) groups. Calves of the T03 and T04 groups experienced less clinical disease than the other groups. Taken together, the data indicate that vaccines containing CpG (e.g. E modified P class CpG) (T03, T04 and T07) display enhanced potency and that these vaccines are more potent than commercial vaccines.

The injection site response developed after adjuvant administration is shown in figure 6 a. The commercial vaccine (T02) and the Advasure-DEAE/dextran (T05) vaccines are more reactive than the other vaccines tested. Vaccines containing QCDCR + CpG (T07) and CpG + cholesterol (T03 and T04) are the safest. In calves immunized with CpG + cholesterol, all symptoms elicited by BVDV-2 were reduced compared to unvaccinated control animals.

The first dose of vaccine administered induced low levels of serum neutralizing antibody titers. By day 42, all tested vaccines induced 100% seroconversion to BVDV1 and the IBRV antigen. By day 42, all of the vaccines tested, except the Advasure-DEAE/dextran (T05) vaccine induced 100% seroconversion to BVDV 2. After challenge, BVDV1 and BVDV2 antibody responses were boosted to significantly higher levels in all groups (table 2).

TABLE 2, part A

TABLE 2, part B

SN-serum neutralization

Each primary vaccination (T02-T07) elicited a BVDV-specific IgG antigen response that was enhanced by the booster vaccination and BVDV2 challenge. There were no significant differences between IgG titers between groups.

All vaccine formulations were immunogenic and induced serum neutralization (BVDV1 and BVDV2) as well as IgG BVDV-specific antibodies boosted by re-vaccination and challenge. Protection against BVDV challenge is achieved by cell-mediated immunity (CMI; IFN γ secretion and activation of BVDV-specific CD4+ and CD8+ T cells), but antibodies neutralize free virus and thus can protect against challenge if present at high levels (e.g., in the colostrum fed to calves at birth). CMI (Th-1 type) responses are detected by IFN γ cytokines in the secretory family in vitro, whereas humoral (Th-2 type) responses can be determined by detection of IL-4.

BVDV1 and BVDV2 antigens induced low levels of IFN γ response and were not significantly different (P >0.1) between treatment groups (data not shown). The BSRV antigen induced IFN γ responses in all groups except T01 (saline) and T03(CpG: cholesterol 1: 1). The IBR antigen induced the strongest IFN γ responses in T05 (Advasure-DEAE/dextran) and T06(QCDCR) on all days post vaccination, and weak but positive responses in T02 (commercial vaccine), T04(CpG: cholesterol 1:10) and T07(QCDCR + CpG). PI3 antigens were found in T02 (commercial vaccine), T05 (Advasure-DEAE/dextran), T06(QCDCR) and T07(QCDCR + CpG).

Example 3 immunogenicity of subunit (pertactin) Bordetella bronchiseptica vaccine formulated with different adjuvants in pigs

The antigen-specific immune response of pigs immunized with pertactin (p68) formulated with various adjuvants, including CpG + cholesterol, was evaluated.

Institute of researchThe veterinary products for research (IVP) used were as follows: the vaccine was administered at a dose of 1-mL. Treatment group T01 received 20mM phosphate buffered saline. Treatment group T02 received adjuvants which were Quil A (250. mu.g), cholesterol (250. mu.g), dimethyldioctadecylammonium bromide (DDA; 100. mu.g), Carbopol(0.075%) and N- (2-deoxy-2-L-leu-mino-b-D-glucopyranosyl) -N-octadecyldodecanoic acid hydrochloride salt (also under the tradename Bay R1005)Commensurable) (1,000. mu.g) vaccine. This combination of components is referred to herein as QCDCR. The composition also contained CpG23878(250 μ g) and pertactin (10 μ g). Treatment group T03 received a vaccine containing cholesterol (2,500. mu.g), CpG23877 (250. mu.g) and pertactin (10. mu.g). Treatment group T04 received a vaccine containing cholesterol (2,500. mu.g), CpG23878 (250. mu.g) and pertactin (10. mu.g). Treatment group T05 received a composition containing 6% Al (OH)₃Aluminum and pertactin (10. mu.g).

Sixty four (64) clinically healthy high health status pigs of only two sexes were used in the study. The swine or its enclosure (dam) has no history of vaccination or contact with bordetella bronchiseptica (b. No pigs had a positive pertactin titer (> 200 defined) from sera collected at the source farm or on day 1.

On day 0, pigs were inoculated with a 1.0mL dose of vaccine administered by Intravenous (IM) injection in the left neck. On day 21, pigs were re-vaccinated with the same IVP and dose as before and administered to the right neck. Within one hour of each vaccination, immediate adverse events associated with the vaccination were observed by the investigator or qualified technician.

The primary outcome variable was serum pertactin antibody titer (total IgG). Serum samples were tested for pertactin antibodies using ELISA. Nunc Maxisorp plates were coated with 50 ng/well of pertactin in carbonate buffer (pH 9.1). Plates were washed and blocked with 1x PBS with 0.05% Tween20 and 1% skim milk powder (1 hour, room temperature). Serum samples diluted in blocking buffer were added to the plates, incubated (1 hour, room temperature), washed and incubated (1 hour, room temperature) with HRP conjugate (Bethyl goat anti-porcine IgG (h + l)) diluted 1:1250 in blocking buffer. After the final wash, ABTS (KPL50-62-00) substrate was added and OD values were read after 12 min incubation at room temperature. Titers were calculated based on a cut-off of 20% of the OD value of the 1:1000 dilution of the positive control serum pool.

Serum samples from T01, T03, T04, T05 and T08 were also tested for pertactin-specific IgG1 and IgG2 antibodies using ELISA. NuncMaxisorp plates were coated with 50 ng/well of pertactin in carbonate buffer (pH 9.1). Plates were washed and blocked with 1x PBS with 0.05% Tween20 and 1% skim milk powder (1 hour, room temperature). Serum samples diluted in blocking buffer were added to the plates, incubated (1 hour, room temperature), washed and incubated (1 hour, room temperature) with monoclonal antibodies (IgG1-Serotec MCA635 or IgG2-Sertec MCA636) diluted 1:100 in blocking buffer. After the final wash, ABTS (KPL50-62-00) substrate was added and OD values were read after 20 min incubation at room temperature. IgG1 titers were calculated based on a 50% cutoff of the OD values of the 1:1000 dilutions of the positive control serum pool. IgG2 titers were calculated based on a cutoff value of OD of 0.2.

Antigen-specific IFN- γ production was tested on PBMCs collected from heparin blood samples. IFN- γ response (Th1) was tested by ELISPOT to determine the frequency of INF- γ secreting cells/million cells SFC/10 from PBMC⁶(after subtraction of the background of the media control). In addition, IFN- γ responses were modulated based on the Stimulation Index (SI) of pertussis adhesin-stimulated cells compared to media controls. The stimulation index for the sample must be at least 2X in order to be considered positive.

Results and discussion

Pigs were vaccinated on day 0 and day 21 and the vaccines are shown in table 3.

TABLE 3

Vaccine group	Adjuvant (dose)	Carrier
			T01	Is free of	Is free of
T02	CpG23878(250μg)	QCDCR
			T03	CpG23877(1:10)	Cholesterol
T04	CpG23878(1:10)	Cholesterol
			T05	Aluminum glue	Is free of

Blood and serum samples from PBMC isolates were taken and analyzed on days 1, 7, 20, 28 and 35. Total IgG, IgG1 and IgG2 antibodies were tested on serum samples using ELISA against purified LPS-free recombinant pertactin. Isotype antibodies were obtained from Bethyyl Labs or AbD Serotec.

The Bordetella pertussis adhesin-specific IgG levels were increased in the T02(CpG + QCDCR), T03(CpG23877+ cholesterol; 1:10) and T04(CpG23878+ cholesterol; 1:10) groups compared to the other vaccines tested.

No post-vaccination adverse events were reported within the observation time immediately after vaccination. No observation of the response due to inoculation was recorded. Pigs in treatment group T01 remained negative for pertactin ELISA antibody throughout the study.

All pigs had a negative pertactin specific ELISA titer (< 200) on day 1. The percentage of pigs that had undergone seroconversion after vaccination was: t01 was 0%, and T02, T03, T04 and T08 were 100%. The treatment group with CpG #23878 adjuvant and QCDCR vehicle (T02) had GMTs of 906.0 and 24728.5 on day 20 and day 35, respectively; the GMT was significantly higher (P.ltoreq.10) for these two days than for all other treatment groups (tables 4 and 5). The negative control T01 had significantly lower mean values at day 20 and day 35 than the other groups. At both post-vaccination time points, the GMT of T08 (formulated with aluminium hydroxide) was significantly lower than that of bordetella pertussis adhesin vaccines formulated with CpG (T02, T03, T04) using QCDCR or cholesterol vehicle. A graph of antigen-specific antibody responses in pigs immunized with pertactin (p68) formulated with various adjuvants including CpG + cholesterol is presented in fig. 7.

TABLE 4 pertactin specific Total IgG ELISA titers (day 20)

Mean geometric minimum variance, standard error and range of antibody titers from swine after day 20 of IVP administration

^a,b,c,dThe geometric mean values with different superscripts are significantly different (P.ltoreq.0.10)

TABLE 5 pertactin specific Total IgG ELISA titers (day 35)

Mean geometric minimum variance, standard error and range of antibody titers from swine 35 days after IVP administration

^a,b,c,d,eThe geometric mean values with different superscripts are significantly different (P.ltoreq.0.10)

Only samples from the selected treatment groups were tested for isotype specific pertactin serum antibody titers (table 6). T03(CpG23878, QCDCR formulated) had an IgG2/IgG1 ratio of 0.40, (CpG23878, cholesterol formulated) of 2.64.

TABLE 6 isotype-specific pertactin serum antibody titers

By Stimulation Index (SI) and by plaque forming cells (SFC/10)⁶) The average pertactin-specific IFN- γ response was determined. There was comparable variability of pertactin-specific IFN- γ responses in pigs within different groups and between time points, in part because there were only 8 subjects per group. At all times including before inoculation (day 1)There were significant differences between the treatments (P.ltoreq.0.10). The mean SI for T02 was significantly higher than T01 and T03 for all post-vaccination time points (days 7, 20, 28 and 35). In contrast, the SI of T05 was not different from T01 at any post-vaccination time point. The SI of T04 was significantly higher than T01 on days 20 and 35. SFC/10 of T02 in 3 of 4 post-inoculation time points⁶The average is significantly higher than T01 and T05. IFN- γ at T03 and T08 (SI and SFC/10) at any post-vaccination time point⁶) The response was not different from T01.

Those skilled in the art will recognize, or be confident in view of, many equivalents to the specific embodiments of the aspects described herein that may be obtained using no more than routine experimentation. Such equivalents are intended to be encompassed by the present claims.

Claims (20)

1. A vaccine comprising one or more antigens, one or more isolated immunostimulatory oligonucleotides, and cholesterol.

2. The vaccine of claim 1, wherein the one or more antigens are each independently a microbial antigen, a self antigen, a tumor antigen, an allergen, or an addictive substance.

3. The vaccine of claim 2, wherein the one or more antigens are each independently a peptide, a peptide conjugated to a carrier protein, a peptide conjugated to a virus-like particle, a polypeptide, a recombinant protein, a purified protein, a whole inactivated pathogen, a live attenuated virus or antigen expressing viral vector, a live attenuated bacterium or antigen expressing bacterial vector, a polysaccharide conjugated to a carrier protein, a hapten conjugated to a carrier protein, or a small molecule.

4. The vaccine of claim 3, wherein the antigen is derived from a bacterium, virus, or parasite.

5. The vaccine of claim 4, wherein

a) The bacterial antigen is a whole killed bacterium, a live attenuated bacterium, or a bacterial purified protein; or

b) The viral antigen is a whole inactivated virus, a live attenuated virus, or a virus purification protein.

6. The vaccine of claim 3, wherein the carrier protein is a bacterial toxoid or derivative, a Pseudomonas exotoxin, KLH, or a virus-like particle.

7. The vaccine of claim 6, wherein

a) The bacterial toxoid is diphtheria toxoid or a derivative thereof; or

b) The virus-like particle is HBsAg, HBcAg, coliphage Q beta, Norwalk virus or influenza HA.

8. The vaccine of claim 2, wherein

a) The addictive substance is nicotine or a nicotine-like molecule; or

b) The tumor antigen is one or more of survivin, Her-2, EFGRvIII, PSA, PAP or PMSA.

9. The vaccine of claim 3, wherein the hapten conjugated to a carrier protein is a nicotine or nicotine-like molecule conjugated to diphtheria toxoid or derivatives thereof.

10. The vaccine of claim 1, wherein the amount of cholesterol is 0.1-fold to 50-fold, 1-fold to 10-fold, or equal to the amount of antigen by weight relative to the amount of antigen.

11. A vaccine comprising one or more antigens and one or more immune modulatory molecules and cholesterol.

12. The vaccine of claim 11, further comprising a pharmaceutical carrier.

13. The vaccine of claim 11, wherein the one or more antigens are each independently a microbial antigen, a self antigen, a tumor antigen, an allergen, or an addictive substance.

14. The vaccine of claim 11, wherein the one or more antigens are each independently a peptide, a polypeptide, a recombinant protein, a purified protein, a whole inactivated pathogen, a live attenuated virus or virus vector expressing an antigen, a live attenuated bacterium or bacterium vector expressing an antigen, a polysaccharide conjugated to a carrier protein, a hapten conjugated to a carrier protein, or a small molecule.

15. The vaccine of claim 14, wherein the carrier protein is a bacterial toxoid or derivative thereof, a pseudomonas exotoxin, KLH, or a virus-like particle.

16. The vaccine of claim 15, wherein

a) The bacterial toxoid is diphtheria toxoid or a derivative thereof; or

b) The virus-like particle is HBsAg, HBcAg, coliphage Q beta, Norwalk virus or influenza HA.

17. The vaccine of claim 11, wherein the amount of cholesterol is 0.1-fold to 50-fold, 1-fold to 10-fold, or equivalent to the antigen, by weight relative to the amount of antigen.

18. The vaccine of claim 11, wherein the one or more immune modulatory molecules are each independently a TLR agonist, an antimicrobial peptide, a cytokine, a chemokine, or a NOD ligand.

19. The vaccine of claim 18, wherein the TLR agonists are each independently an Oligoribonucleotide (ORN), a small molecule that activates TLR7 and/or TLR8, or an Oligodeoxynucleotide (ODN) activated via TLR 9.

20. A method of inducing an antigen-specific immune response in a subject in need thereof, the method comprising administering a vaccine comprising one or more antigens and one or more isolated CpG oligonucleotides and cholesterol in an amount effective to induce an antigen-specific immune response in the subject.