US20050043529A1 - Use of nucleic acids containing unmethylated CpG dinucleotide as an adjuvant - Google Patents

Use of nucleic acids containing unmethylated CpG dinucleotide as an adjuvant Download PDF

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US20050043529A1
US20050043529A1 US10/831,775 US83177504A US2005043529A1 US 20050043529 A1 US20050043529 A1 US 20050043529A1 US 83177504 A US83177504 A US 83177504A US 2005043529 A1 US2005043529 A1 US 2005043529A1
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virus
antigen
cpg
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adjuvants
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Heather Davis
Joachim Schorr
Arthur Krieg
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Ottawa Health Research Institute
Coley Pharmaceutical GmbH
University of Iowa Research Foundation UIRF
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Coley Pharmaceutical GmbH
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/39Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/12Viral antigens
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/12Viral antigens
    • A61K39/29Hepatitis virus
    • A61K39/292Serum hepatitis virus, hepatitis B virus, e.g. Australia antigen
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/51Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/523Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/51Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53DNA (RNA) vaccination
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/555Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505Inorganic adjuvants
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/555Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511Organic adjuvants
    • A61K2039/5555Muramyl dipeptides
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/555Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511Organic adjuvants
    • A61K2039/55561CpG containing adjuvants; Oligonucleotide containing adjuvants
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/555Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511Organic adjuvants
    • A61K2039/55566Emulsions, e.g. Freund's adjuvant, MF59
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/555Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511Organic adjuvants
    • A61K2039/55572Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/57Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00Reverse transcribing DNA viruses
    • C12N2730/00011Details
    • C12N2730/10011Hepadnaviridae
    • C12N2730/10111Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10134Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates generally to adjuvants, and in particular to methods and products utilizing a synergistic combination of oligonucleotides having at least one unmethylated CpG dinucleotide (CpG ODN) and a non-nucleic acid adjuvant.
  • CpG ODN unmethylated CpG dinucleotide
  • PBMC peripheral blood mononuclear cells
  • This lymphocyte activation is due to unmethylated CpG dinucleotides, which are present at the expected frequency in bacterial DNA (1/16), but are under-represented (CpG suppression, 1/50 to 1/60) and methylated in vertebrate DNA.
  • Activation may also be triggered by addition of synthetic oligodeoxynucleotides (ODN) that contain an unmethylated CpG dinucleotide in a particular sequence context. It appears likely that the rapid immune activation in response to CpG DNA may have evolved as one component of the innate immune defense mechanisms that recognize structural patterns specific to microbial molecules.
  • ODN synthetic oligodeoxynucleotides
  • CpG-DNA induces proliferation of almost all (>95%) B cells and increases immunoglobulin (Ig) secretion.
  • This B cell activation by CpG DNA is T cell independent and antigen non-specific.
  • B cell activation by low concentrations of CpG DNA has strong synergy with signals delivered through the B cell antigen receptor for both B cell proliferation and Ig secretion (Krieg et al., 1995). This strong synergy between the B cell signaling pathways triggered through the B cell antigen receptor and by CpG DNA promotes antigen specific immune responses.
  • CpG DNA In addition to its direct effects on B cells, CpG DNA also directly activates monocytes, macrophages, and dendritic cells to secrete a variety of cytokines, including high levels of IL-12 (Klinman et al., 1996; Halpern et al., 1996; Cowdery et al., 1996). These cytokines stimulate natural killer (NK) cells to secrete gamma-interferon (IFN- ⁇ -) and have increased lytic activity (Klinman et al., 1996, supra; Cowdery et al., 1996, supra; Yamamoto et al., 1992; Ballas et al., 1996). Overall, CpG DNA induces a Th1 like pattern of cytokine production dominated by IL-12 and IFN- ⁇ with little secretion of Th2 cytokines (Klinman et al., 1996).
  • HBV vaccines are subunit vaccines containing particles of HBV envelope protein(s) which include several B and T cell epitopes known collectively as HBV surface antigen (HBsAg).
  • HBV surface antigen HBsAg particles
  • the HBsAg particles may be purified from the plasma of chronically infected individuals or more commonly are produced as recombinant proteins.
  • These vaccines induce antibodies against HBsAg (anti-HBs), which confer protection if present in titers of at least 10 milli-International Units per milliliter (mIU/ml) (Ellis, 1993).
  • the current subunit vaccines which contain alum (a Th2 adjuvant), are safe and generally efficacious.
  • the present invention relates to methods and products for inducing an immune response.
  • the invention is useful in one aspect as a method of inducing an antigen specific immune response in a subject.
  • the method includes the steps of administering to the subject in order to induce an antigen specific immune response an antigen and a combination of adjuvants, wherein the combination of adjuvants includes at least one oligonucleotide containing at least one unmethylated CpG dinucleotide and at least one non-nucleic acid adjuvant, and wherein the combination of adjuvants is administered in an effective-amount for inducing a synergistic adjuvant response.
  • the subject is an infant.
  • the CpG oligonucleotide and the non-nucleic acid adjuvant may be administered with any or all of the administrations of antigen.
  • the combination of adjuvants may be administered with a priming dose of antigen.
  • the combination of adjuvants is administered with a boost dose of antigen.
  • the subject is administered a priming dose of antigen and oligonucleotide containing at least one. unmethylated CpG dinucleotide before the boost dose.
  • the subject is administered a boost dose of antigen and oligonucleotide containing at least one unmethylated CpG dinucleotide after the priming dose.
  • the antigen may be any type of antigen known in the art.
  • the antigen may be selected from the group consisting of peptides, polypeptides, cells, cell extracts, polysaccharides, polysaccharide conjugates, lipids, glycolipids and carbohydrates.
  • Antigens may be given in a crude, purified or recombinant form and polypeptide/peptide antigens, including peptide mimics of polysaccharides, may also be encoded within nucleic acids.
  • Antigens may be derived from an infectious pathogen such as a virus, bacterium, fungus or parasite, or the antigen may be a tumor antigen, or the antigen may be an allergen.
  • a method of inducing a Th1 immune response in a subject includes the step of administering to the subject in order to induce a Th1 immune response a combination of adjuvants, wherein the combination of adjuvants includes at least one oligonucleotide containing at least one unmethylated CpG dinucleotide and at least one non-nucleic acid adjuvant, and wherein the combination of adjuvants is administered in an effective amount for inducing a Th1 immune response.
  • the combination of adjuvants is administered simultaneously.
  • the combination of adjuvants is administered sequentially.
  • the combination of adjuvants is administered in an effective amount for inducing a synergistic Th1 immune response.
  • the same method is performed but the subject is an infant and the Th1 response can be induced using CpG DNA alone, or CpG DNA in combination with a non-nucleic acid adjuvant at the same or different site, at the same or different time.
  • the invention in other aspects is a composition of a synergistic combination of adjuvants.
  • the composition includes an effective amount for inducing a synergistic adjuvant response of a combination of adjuvants, wherein the combination of adjuvants includes at least one oligonucleotide containing at least one unmethylated CpG dinucleotide and at least one non-nucleic acid adjuvant.
  • the composition may also include at least one antigen, which may be selected from the group consisting of peptides, polypeptides, cells, cell extracts, polysaccharides, polysaccharide conjugates, lipids, glycolipids and carbohydrates.
  • Antigens may be given in a crude, purified or recombinant form and polypeptide/peptide antigens, including peptide mimics of polysaccharides, may also be encoded within nucleic acids.
  • Antigens may be derived from an infectious pathogen such as a virus, bacterium, fungus or parasite, or the antigen may be a tumor antigen, or the antigen may be an allergen.
  • the CpG oligonucleotide may be administered with any or all of the administrations of antigen.
  • the CpG oligonucleotide or the combination of adjuvants may be administered with a priming dose of antigen.
  • the CpG oligonucleotide or the combination of adjuvants is administered with a boost dose of antigen.
  • the subject is administered a priming dose of antigen and oligonucleotide containing at least one unmethylated CpG dinucleotide before the boost dose.
  • the subject is administered a boost dose of antigen and oligonucleotide containing at least one unmethylated CpG dinucleotide after the priming dose.
  • the invention in other aspects include a method of inducing a non-antigen-specific Th1-type immune response, including Th1 cytokines such as IL-12 and IFN- ⁇ , for temporary protection against various pathogens including viruses, bacteria, parasites and fungi.
  • the method involves the steps of administering to a subject at least one non-nucleic acid adjuvant and at least one oligonucleotide containing at least one unmethylated CpG dinucleotide in order to induce a Th1 innate immune response.
  • these adjuvants may be administered more than once.
  • CpG DNA may be used alone at one or more of the administrations.
  • a CpG oligonucleotide is used as an adjuvant.
  • the oligonucleotide in one embodiment contains at least one unmethylated CpG dinucleotide having a sequence including at least the following formula: 5′X 1 X 2 CGX 3 X 4 3′ wherein C and G are unmethylated, wherein X 1 X 2 and X 3 X 4 are nucleotides.
  • the 5′X 1 X 2 CGX 3 X 4 3′ sequence is a non-palindromic sequence.
  • the oligonucleotide may be modified.
  • at least one nucleotide has a phosphate backbone modification.
  • the phosphate backbone modification may be a phosphorothioate or phosphorodithioate modification.
  • the phosphate backbone modification occurs on the 5′ side of the oligonucleotide or the 3′ side of the oligonucleotide.
  • the oligonucleotide may be any size. Preferably the oligonucleotide has 8 to 100 nucleotides. In other embodiments the oligonucleotide is 8 to 40 nucleotides in length.
  • X 1 X 2 are nucleotides selected from the group consisting of: GpT, GpG, GpA, ApA, ApT, ApG, CpT, CpA, CpG, TpA, TpT, and TpG; and X 3 X 4 are nucleotides selected from the group consisting of: TpT, CpT, ApT, TpG, ApG, CpG, TpC, ApC, CpC, TpA, ApA, and CpA.
  • X 1 X 2 are GpA or GpT and X 3 X 4 are TpT.
  • X 1 or X 2 or both are purines and X 3 or X 4 or both are pyrimidines or X 1 X 2 are GpA and X 3 or X 4 or both are pyrimidines.
  • X 2 is a T and X 3 is a pyrimidine.
  • the oligonucleotide may be isolated or synthetic.
  • the invention also includes the use of a non-nucleic acid adjuvant in some aspects.
  • the non-nucleic acid adjuvant in some embodiments is an adjuvant that creates a depo effect, an immune stimulating adjuvant, or an adjuvant that creates a depo effect and stimulates the immune system.
  • the adjuvant that creates a depo effect is selected from the group consisting of alum (e.g., aluminum hydroxide, aluminum phosphate) emulsion based formulations including mineral oil, non-mineral oil, water-in-oil or oil-in-water emulsions, such as the Seppic ISA series of Montanide adjuvants; MF-59; and PROVAX.
  • the immune stimulating adjuvant is selected from the group consiting of saponins purified from the bark of the Q. saponaria tree, such as QS21; poly[di(carboxylatophenoxy)phosphazene (PCPP) derivatives of lipopolysaccharides such as monophosphorlyl lipid (MPL), muramyl dipeptide (MDP) and threonyl muramyl dipeptide (tMDP); OM-174; and Leishmania elongation factor.
  • PCPP poly[di(carboxylatophenoxy)phosphazene
  • MPL monophosphorlyl lipid
  • MDP muramyl dipeptide
  • tMDP threonyl muramyl dipeptide
  • OM-174 Leishmania elongation factor
  • the adjuvant that creates a depo effect and stimulates the immune system is selected from the group consiting of ISCOMS; SB-AS2; SB-AS4; non-ionic block copolymers that form micelles such as CRL 1005; and Syntex Adjuvant Formulation.
  • FIG. 1 has two graphs illustrating humoral and cytotoxic T-lymphocyte (CTL) responses in adult BALB/c mice immunized with 1 ⁇ g recombinant HBsAg protein alone, adsorbed onto alum (25 mg Al 3+ /mg HBsAg), with 100 ⁇ g of immunostimulatory CpG ODN 1826, or with both alum and CpG ODN.
  • CTL cytotoxic T-lymphocyte
  • End-point titers were defined as the highest plasma dilution that resulted in an absorbance value (OD 450) two times greater than that of control non-immune plasma with a cut-off value of 0.05.
  • Each point represents the mean % specific lysis at the indicated effector: target (E:T) cell ratio in a chromium release assay with HBsAg-expressing cells as targets.
  • FIG. 3 is a graph illustrating humoral responses in adult BALB/c mice immunized with 1 ⁇ g recombinant HBsAg protein, with or without alum, and with one of several different oligonucleotides (ODN, 10 ⁇ g).
  • ODN oligonucleotides
  • E:T effector:target
  • FIG. 5 is a graph of humoral responses in BALB/c mice immunized with HBsAg (1 ⁇ g) without adjuvant or with various adjuvants alone or in combination.
  • the adjuvants were: alum (25 mg Al 3+ /mg HBs/Ag), with CpG DNA (10 ⁇ g CpG ODN 1826), monophosphoryl lipid A (MPL, 50 ⁇ g) and Freund's complete adjuvant (mixed 1:1 v/v with HBsAg solution).
  • FIG. 6 is a bar graph depicting the amount of total IgG (end-point ELISA titer) produced at 4 weeks in BALB/c mice immunized with 1 ⁇ g of HBsAg with or without CpG and/or IFA (mineral oil mixed 1:1 v/v) or CFA (complete Freund's adjuvant mixed 1:1 v/v).
  • the numbers above each bar indicate the IgG2a:IgG1 ratio, with a number in excess of 1 indicating a more Th1-like response.
  • FIG. 7 is a bar graph depicting the amount of total IgG produced at 4 weeks in BALB/c mice immunized with 1 ⁇ g of HBsAg with or without CpG and/or MPL (monophosphoryl lipid A, 50 ⁇ g) or alum.
  • the numbers above each bar indicate the IgG2a:IgG1 ratio, with a number in excess of 1 indicating a more Th1-like response.
  • End-point titers were defined as the highest plasma dilution that resulted in an absorbance value (OD 450) two times greater than that of control non-immune plasma with a cut-off value of 0.05.
  • FIG. 10 is a bar graph illustrating humoral responses in neonatal BALB/c mice at 8 weeks after immunization (at 7 days of age) with 1 ⁇ g recombinant HBsAg protein with alum (25 mg Al 3+ /mg HBsAg), with 10 ⁇ g of CpG ODN 1826, or with both alum and CpG ODN.
  • Each point represents the group mean (see FIG. 8 for numbers of animals) for anti-HBs titers (IgG1 and IgG2a isotypes) as determined by end-point dilution ELISA assay.
  • IgG1 antibodies indicate a Th2-biased response whereas IgG2a antibodies are indicative of a Th1-type response.
  • Th1 responses are preferable in some instances since they are associated with IgG2a antibodies that have better neutralization and opsonization capabilities than Th2-type antibodies.
  • Th1 responses are associated with cytotoxic T lymphocytes (CTL) that can attack and kill virus-infected cells.
  • CTL cytotoxic T lymphocytes
  • CpG ODN alone or in combination with alum induced good CTL activity in both adult and neonatal mice.
  • the invention is a method of inducing an antigen specific immune response in a subject.
  • the method includes the step of administering to the subject in order to induce an antigen specific immune response an antigen and a combination of adjuvants, wherein the combination of adjuvants includes at least one oligonucleotide containing at least one unmethylated CpG dinucleotide and at least one non-nucleic acid adjuvant, and wherein the combination of adjuvants is administered in an effective amount for inducing a synergistic adjuvant response.
  • the synergistic combination of adjuvants is particularly useful as a prophylactic vaccine for the treatment of a subject at risk of developing an infection with an infectious organism or a cancer in which a specific cancer antigen has been identified or an allergy where the allergen is known.
  • the combination of adjuvants can also be given without the antigen or allergen for shorter term protection against infection, allergy or cancer, and in this case repeated doses will allow longer term protection.
  • a “subject at risk” as used herein is a subject who has any risk of exposure to an infection causing pathogen or a cancer or an allergen or a risk of developing cancer.
  • a subject at risk may be a subject who is planning to travel to an area where a particular type of infectious agent is found or it may be a subject who through lifestyle or medical procedures is exposed to bodily fluids which may contain infectious organisms or even any subject living in an area that an infectious organism or an allergen has been identified.
  • Subjects at risk of developing infection also include general populations to which a medical agency recommends vaccination with a particular infectious organism antigen. If the antigen is an allergen and the subject develops allergic responses to that particular antigen and the subject is exposed to the antigen, i.e., during pollen season, then that subject is at risk of exposure to the antigen.
  • a prophylactic vaccine that can induce protective immunity against many infectious pathogens more quickly and with fewer doses than traditional vaccines can provide. For instance, fewer than 20% of healthy individuals attain protective levels of anti-hepatitis B (HB) antibodies (10 mIU/ml) after a single dose of subunit hepatitis B Vaccine (HBV) vaccine and only 60-70% reach this level after two doses. Thus, three doses (usually given at 0, 1 and 6 months) are required to seroconvert >90% of vaccinated individuals. The three dose regime is frequently not completed owing to poor patient compliance, and in endemic areas, protective levels may not be induced quickly enough.
  • the methods of the invention are particularly useful as prophylactic treatments because they induce higher levels of antibodies than can be achieved with traditional vaccines and can be administered as fewer total doses.
  • CpG ODN also overcame non-response in mice genetically incapable of providing T-help owing to an absence of class II MHC. Similar results were obtained in orangutans at risk of becoming infected with hepatitis B. It was found that orangutans are hyporesponders to the classical alum-adjuvanted vaccine with less than 10% achieving seroprotection after 2 doses, but that nearly 100% of animals responded with use of CpG oligonucleotides alone or combined with alum. The synergistic response was evident because antibody titers were much higher with CpG ODN plus alum than with CpG ODN alone or alum alone and were more than additive.
  • a subject at risk of developing a cancer is one who is who has a high probability of developing cancer. These subjects include, for instance, subjects having a genetic abnormality, the presence of which has been demonstrated to have a correlative relation to a higher likelihood of developing a cancer and subjects exposed to cancer causing agents such as tobacco, asbestos, or other chemical toxins, or a subject who has previously been treated for cancer and is in apparent remission.
  • a subject at risk of developing a cancer is treated with an antigen specific for the type of cancer to which the subject is at risk of developing and an adjuvant and a CpG oligonucleotide the subject may be able to kill of the cancer cells as they develop. If a tumor begins to form in the subject, the subject will develop a specific immune response against the tumor antigen.
  • the invention also encompasses the use of the combination for the immunotherapeutic treatment of a subject having an infection, an allergy or a cancer.
  • a “subject having an infection” is a subject that has been exposed to an infectious pathogen and has acute or chronic detectable levels of the pathogen in the body.
  • the combination of adjuvants can be used with an antigen to mount an antigen specific immune response that is capable of reducing the level of or eradicating the infectious pathogen.
  • An infectious disease as used herein, is a disease arising from the presence of a foreign microorganism in the body.
  • HBV virus is itself non-pathogenic but with chronic infection the partially developed immune response causes inflammatory changes that eventually leads to cirrhosis and increased risk of hepatocellular carcinoma.
  • Such chronic carriers have circulating HBsAg “e” soluble form of the HBV core antigen (HBeAg) without specific immunity. It is thought that the absence of HBV-specific T-cells, including CTL may contribute to the establishment and maintenance of the chronic carrier state.
  • a “subject having an allergy” is a subject that has or is at risk of developing an allergic reaction in response to an allergen.
  • An “allergy” refers to acquired hypersensitivity to a substance (allergen). Allergic conditions include but are not limited to eczema, allergic rhinitis or coryza, conjunctivitis, hay fever, bronchial asthma, urticaria (hives) and food allergies, and other atopic conditions.
  • allergic diseases are generally treated either symptomatically with antihistimines for example or immunotherapeutically by the injection of small doses of antigen followed by subsequent increasing dosage of antigen.
  • Symptomatic treatment offers only temporary relief.
  • Immunotherapy is believed to induce tolerance to the allergen to prevent further allergic reactions. This approach, however, takes several years to be effective and is associated with the risk of side effects such as anaphylactic response. The methods of the invention avoid these problems.
  • Th1 cytokines that are induced by unmethylated CpG oligonucleotides
  • Th2 immune response are associated with the production of IL-4, IL-5 and IL-10.
  • Th1 responses include both cell-mediated responses (including cytotoxic T-cells) and antibodies, whereas Th2 responses are associated only with antibodies.
  • the antibodies with a Th1 response are of isotypes (e.g. IgG2a) that have better neutralizing and opsonizing capabilities than those of Th2 isotypes (e.g. IgE that mediates allergic responses).
  • IgG2a isotypes
  • an effective dose of a CpG oligonucleotide can be administered to a subject to treat or prevent an allergy.
  • the combination of adjuvants of the present invention will have significant therapeutic utility in the treatment of allergic conditions such as asthma.
  • Such combinations of adjuvants could be used alone or in combination with allergens.
  • melanoma neuroblastomas
  • oral cancer ovarian cancer; pancreas cancer; prostate cancer; rectal cancer; sarcomas; skin cancer; testicular cancer; thyroid cancer; and renal cancer, as well as other carcinomas and sarcomas.
  • a “subject” shall mean a human or vertebrate animal including but not limited to a dog, cat, horse, cow, pig, sheep, goat, chicken, primate, (e.g., monkey), fish (aquaculture species e.g. salmon, trout and other salmonids), rat, and mouse.
  • the CpG oligonucleotides can be double-stranded or single-stranded. Generally, double-stranded molecules are more stable in vivo, while single-stranded molecules have increased immune activity.
  • the CpG oligonucleotides or combination of adjuvants can be used with or without antigen.
  • the terms shall also include polynucleosides (i.e. a polynucleotide minus the phosphate) and any other organic base containing polymer.
  • Nucleic acid molecules can be obtained from existing nucleic acid sources (e.g. genomic or cDNA), but are preferably synthetic (e.g. produced by oligonucleotide synthesis). The entire CpG oligonucleotide can be unmethylated or portions may be unmethylated but at least the C of the 5° CG 3′ must be unmethylated.
  • the invention provides a CpG oligonucleotide represented by at least the formula: 5′N 1 X 1 CGX 2 N 2 3′ wherein at least one nucleotide separates consecutive CpGs; X 1 is adenine, guanine, or thymine; X 2 is cytosine, adenine, or thymine; N is any nucleotide and N 1 and N 2 are nucleic acid sequences composed of from about 0-25 N's each.
  • the invention provides an isolated CpG oligonucleotide represented by at least the formula: 5′N 1 X 1 X 2 CGX 3 X 4 N 2 3′ wherein at least one nucleotide separates consecutive CpGs;
  • X 1 X 2 are nucleotides selected from the group consisting of: GpT, GpG, GpA, ApA, ApT, ApG, CpT, CpA, CpG, TpA, TpT, and TpG;
  • X 3 X 4 are nucleotides selected from the group consisting of: TpT, CpT, ApT, TpG, ApG, CpG, TpC, ApC, CpC, TpA, ApA, and CpA;
  • N is any nucleotide and
  • N 1 and N 2 are nucleic acid sequences composed of from about 0-25 N's each.
  • X 1 X 2 are GpA or GpT and X 3 X 4 are TpT.
  • X 1 or X 2 or both are purines and X 3 or X 4 or both are pyrimidines or X 1 X 2 are GpA and X 3 or X 4 or both are pyrimidines.
  • N 1 and N 2 of the nucleic acid do not contain a CCGG or CGCG quadmer or more than one CCG or CGG trimer. The effect of a a CCGG or CGCG quadmer or more than one CCG or CGG trimer depends in part on the status of the oligonucleotide backbone.
  • the oligonucleotide has a phosphodiester backbone or a chimeric backbone the inclusion of these sequences in the oligonucleotide will only have minimal if any affect on the biological activity of the oligonucleotide. If the backbone is completely phosphorothioate or significantly phosphorothioate then the inclusion of these sequences may have more influence on the biological activity or the kinetics of the biological activity.
  • the CpG oligonucleotide has the sequence 5′TCN 1 TX 1 X 2 CGX 3 X 4 3′.
  • the CpG oligonucleotides of the invention include X 1 X 2 selected from the group consisting of GpT, GpG, GpA and ApA and X 3 X 4 is selected from the group consisting of TpT, CpT and GpT.
  • CpG containing oligonucleotides are preferably in the range of 8 to 30 bases in length.
  • nucleic acids of any size greater than 8 nucleotides are capable of inducing an immune response according to the invention if sufficient immunostimulatory motifs are present, since larger nucleic acids are degraded into oligonucleotides inside of cells.
  • Preferred synthetic oligonucleotides do not include a CCGG or CGCG quadmer or more than one CCG or CGG trimer at or near the 5′ and/or 3′ terminals.
  • Stabilized oligonucleotides where the oligonucleotide incorporates a phosphate backbone modification, as discussed in more detail below are also preferred.
  • the modification may be, for example, a phosphorothioate or phosphorodithioate modification.
  • the phosphate backbone modification occurs at the 5′ end of the nucleic acid for example, at the first two nucleotides of the 5′ end of the oligonucleotide.
  • the phosphate backbone modification may occur at the 3′ end of the nucleic acid for example, at the last five nucleotides of the 3′ end of the nucleic acid.
  • the oligonucleotide may be completely or partially modified.
  • the CpG oligonucleotide is in the range of between 8 and 100 and more preferably between 8 and 30 nucleotides in size.
  • CpG oligonucleotides can be produced on a large scale in plasmids. These may be administered in plasmid form or alternatively they can be degraded into oligonucleotides.
  • nucleic acid/cytokine delivery complex shall mean a nucleic acid molecule and/or cytokine associated with (e.g. ionically or covalently bound to; or encapsulated within) a targeting means (e.g. a molecule that results in higher affinity binding to target cell (e.g. dendritic cell surfaces and/or increased cellular uptake by target cells).
  • a targeting means e.g. a molecule that results in higher affinity binding to target cell (e.g. dendritic cell surfaces and/or increased cellular uptake by target cells.
  • nucleic acid/cytokine delivery complexes include nucleic acids/cytokines associated with: a sterol (e.g.
  • lipid e.g. a cationic lipid, virosome or liposome
  • target cell specific binding agent e.g. a ligand recognized by target cell specific receptor
  • “Palindromic sequence” shall mean an inverted repeat (i.e. a sequence such as ABCDEE′D′C′B′A′ in which A and A′ are bases capable of forming the usual Watson-Crick base pairs. In vivo, such sequences may form double-stranded structures.
  • the CpG oligonucleotide contains a palindromic sequence.
  • a palindromic sequence used in this context refers to a palindrome in which the CpG is part of the palindrome, and preferably is the center of the palindrome. In another embodiment the CpG oligonucleotide is free of a palindrome.
  • a CpG oligonucleotide that is free of a palindrome is one in which the CpG dinucleotide is not part of a palindrome.
  • Such an oligonucleotide may include a palindrome in which the CpG is not part of the palindrome.
  • a “stabilized nucleic acid molecule” shall mean a nucleic acid molecule that is relatively resistant to in vivo degradation (e.g. via an exo- or endo-nuclease). Stabilization can be a function of length or secondary structure. Unmethylated CpG oligonucleotides that are tens to hundreds of kbs long are relatively resistant to in vivo degradation, particularly when in a double-stranded closed-circular form (i.e., a plamid). For shorter CpG oligonucleotides, secondary structure can stabilize and increase their effect.
  • an oligonucleotide For example, if the 3′ end of an oligonucleotide has self-complementarity to an upstream region, so that it can fold back and form a sort of stem loop structure, then the oligonucleotide becomes stabilized and therefore exhibits more activity.
  • Preferred stabilized oligonucleotides of the instant invention have a modified backbone. It has been demonstrated that modification of the oligonucleotide backbone provides enhanced activity of the CpG oligonucleotides when administered in vivo.
  • CpG constructs including at least two phosphorothioate linkages at the 5′ end of the oligonucleotide in multiple phosphorothioate linkages at the 3′ end, preferably 5, provides maximal activity and protected the oligonucleotide from degradation by intracellular exo- and endo-nucleases.
  • modified oligonucleotides include phosphodiester modified oligonucleotide, combinations of phosphodiester and phosphorothioate oligonucleotide, methylphosphonate, methylphosphorothioate, phosphorodithioate, and combinations thereof.
  • phosphodiester modified oligonucleotide combinations of phosphodiester and phosphorothioate oligonucleotide, methylphosphonate, methylphosphorothioate, phosphorodithioate, and combinations thereof.
  • phosphorothioate and phosphodiester oligonucleotides containing CpG motifs are active in immune cells. However, based on the concentration needed to induce CpG specific effects, the nuclease resistant phosphorothioate backbone CpG oligonucleotides are more potent (2 ⁇ g/ml for the phosphorothioate vs. a total of 90 ⁇ g/ml for phosphodiester).
  • oligonucleotides include: nonionic DNA analogs, such as alkyl- and aryl-phosphates (in which the charged phosphonate oxygen is replaced by an alkyl or aryl group), phosphodiester and alkylphosphotriesters, in which the charged oxygen moiety is alkylated. Oligonucleotides which contain diol, such as tetraethyleneglycol or hexaethyleneglycol, at either or both termini have also been shown to be substantially resistant to nuclease degradation.
  • nucleic acid sequences of the invention which are useful as adjuvants are those broadly described above and disclosed in PCT Published Patent Applications claiming priority to U.S. Ser. Nos. 08/738,652 and 08/960,774, filed on Oct. 30, 1996 and Oct. 30, 1997 respectively.
  • Exemplary sequences include but are not limited to those immunostimulatory sequences shown in Table 1.
  • the stimulation index of a particular immunostimulatory CpG DNA can be tested in various immune cell assays.
  • the stimulation index of the CpG oligonucleotide with regard to B cell proliferation is at least about 5, preferably at least about 10, more preferably at least about 15 and most preferably at least about 20 as determined by incorporation of 3 H uridine in a murine B cell culture, which has been contacted with 20 ⁇ M of oligonucleotide for 20 h at 37° C. and has been pulsed with 1 ⁇ Ci of 3 H uridine; and harvested and counted 4 h later as described in detail in copending PCT Published Patent Applications claiming priority to U.S. Ser. Nos.
  • the oligonucleotide containing at least one unmethylated CpG is used in combination with a non-nucleic acid adjuvant and an antigen to activate the immune response.
  • a “non-nucleic acid adjuvant” is any molecule or compound except for the CpG oligonucleotides described herein which can stimulate, the humoral and/or cellular immune response.
  • Non-nucleic acid adjuvants include, for instance, adjuvants that create a depo effect, immune stimulating adjuvants, and adjuvants that create a depo effect and stimulate the immune system.
  • the oligonucleotide containing at least one unmethylated CpG is used alone or in combination with a non-nucleic acid adjuvant and an antigen to activate a cellular immune response.
  • an “adjuvant that creates a depo effect” as used herein is an adjuvant that causes the antigen to be slowly released in the body, thus prolonging the exposure of immune cells to the antigen.
  • This class of adjuvants includes but is not limited to alum (e.g., aluminum hydroxide, aluminum phosphate); or emulsion-based formulations including mineral oil, non-mineral oil, water-in-oil or oil-in-water-in oil emulsion, oil-in-water emulsions such as Seppic ISA series of Montanide adjuvants (e.g., Montanide ISA 720, AirLiquide, Paris, France); MF-59 (a squalene-in-water emulsion stabilized with Span 85 and Tween 80; Chiron Corporation, Emeryville, Calif.; and PROVAX (an oil-in-water emulsion containing a stabilizing detergent and a micelle-forming agent; IDEC, Pharmaceuticals Corporation, San
  • an “immune stimulating adjuvant” is an adjuvant that causes activation of a cell of the immune system. It may, for instance, cause an immune cell to produce and secrete cytokines.
  • This class of adjuvants includes but is not limited to saponins purified from the bark of the Q.
  • saponaria tree such as QS21 (a glycolipid that elutes in the 21 st peak with HPLC fractionation; Aquila Biopharmaceuticals, Inc., Worcester, Mass.); poly[di(carboxylatophenoxy)phosphazene (PCPP polymer; Virus Research Institute, USA); derivatives of lipopolysaccharides such as monophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc., Hamilton, Mont.), muramyl dipeptide (MDP; Ribi) andthreonyl-muramyl dipeptide (t-MDP; Ribi); OM-174 (a glucosamine disaccharide related to lipid A; OM Pharma SA, Meyrin, Switzerland); and Leishmania elongation factor (a purified Leishmania protein; Corixa Corporation, Seattle, Wash.).
  • QS21 a glycolipid that elutes in the 21 st peak with HPLC fractionation; Aquila Biopharmaceuticals, Inc.
  • Adjuvants that create a depo effect and stimulate the immune system are those compounds which have both of the above- identified functions.
  • This class of adjuvants includes but is not limited to ISCOMS (Immunostimulating complexes which contain mixed saponins, lipids and form virus-sized particles with pores that can hold antigen; CSL, Melbourne, Australia); SB-AS2 (SmithKline Beecham adjuvant system #2 which is an oil-in-water emulsion containing MPL and QS21: SmithKline Beecham Biologicals [SBB], Rixensart, Belgium); SB-AS4 (SmithKline Beecham adjuvant system #4 which contains alum and MPL; SBB, Belgium); non-ionic block copolymers that form micelles such as CRL 1005 (these contain a linear chain of hydrophobic polyoxpropylene flanked by chains of polyoxyethylene; Vaxcel, Inc., Norcross, Ga.); and Syntex Adjuvant Formulation (SAF, an oil-in-water e
  • the CpG oligonucleotide containing at least one unmethylated CpG can be administered before, after, and/or simultaneously with the other adjuvant.
  • the combination of adjuvants may be administered with a priming dose of antigen. Either or both of the adjuvants may then be administered with the boost dose.
  • the combination of adjuvants may be administered with a boost dose of antigen. Either or both of the adjuvants may then be administered with the prime dose.
  • a “prime dose” is the first dose of antigen administered to the subject.
  • the prime dose may be the initial exposure of the subject to the infectious microbe and thus the combination of adjuvants is administered to the subject with the boost dose.
  • a “boost dose” is a second or third, etc., dose of antigen administered to a subject that has already been exposed to the antigen.
  • the prime dose administered with the combination of adjuvants is so effective that a boost dose is not required to protect a subject at risk of infection from being infected.
  • CpG oligonucleotides or one of the components in the combination may be given alone for one or more of the administrations.
  • the CpG oligonucleotide containing at least one unmethylated CpG can have an additional efficacy (e.g., antisense) in addition to its ability to enhance antigen-specific immune responses.
  • additional efficacy e.g., antisense
  • an “antigen” as used herein is a molecule capable of provoking an immune response.
  • Antigens include but are not limited to peptides, polypeptides, cells, cell extracts, polysaccharides, polysaccharide conjugates, lipids, glycoliopids and carbohydrates. Antigens may be given in a crude, purified or recombinant form and polypeptide/peptide antigens, including peptide mimics of polysaccharides, may also be encoded within nucleic.
  • the term antigen broadly includes any type of molecule which is recognized by a host immune system as being foreign. Antigens include but are not limited to cancer antigens, microbial antigens, and allergens.
  • cancer antigen is a compound, such as a peptide, associated with a tumor or cancer cell surface and which is capable of provoking an immune response when expressed on the surface of an antigen presenting cell in the context of an MHC molecule.
  • Cancer antigens can be prepared from cancer cells either by preparing crude extracts of cancer cells, for example, as described in Cohen, et al., 1994 , Cancer Research, 54:1055, by partially purifying the antigens, by recombinant technology, or by de novo synthesis of known antigens.
  • Cancer antigens include antigens that are recombinately an immunogenic portion of or a whole tumor or cancer. Such antigens can be isolated or prepared recombinatly or by any other means known in the art.
  • a “microbial antigen” as used herein is an antigen of a microorganism and includes but is not limited to infectious virus, infectious bacteria, infectious parasites and infectious fungi. Such antigens include the intact microorganism as well as natural isolates and fragments or derivatives thereof and also synthetic compounds which are identical to or similar to natural microorganism antigens and induce an immune response specific for that microorganism. A compound is similar to a natural microorganism antigen if it induces an immune response (humoral and/or cellular) to a natural microorganism antigen. Most such antigens are used routinely in the art and are well known to those of ordinary skill in the art. Another example is a peptide mimic of a polysaccharide antigen.
  • Retroviridae e.g. human immunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP; Picornaviridae (e.g. polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g. strains that cause gastroenteritis); Togaviridae (e.g. equine encephalitis viruses, rubella viruses); Flaviridae (e.g.
  • coronoviridae e.g. coronaviruses
  • Rhabdoviradae e.g. vesicular stomatitis viruses, rabies viruses
  • Coronaviridae e.g. coronaviruses
  • Rhabdoviridae e.g. vesicular stomatitis viruses, rabies viruses
  • Filoviridae e.g. ebola viruses
  • Paramyxoviridae e.g. parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus
  • Orthomyxoviridae e.g. influenza viruses
  • Bungaviridae e.g.
  • African swine fever virus African swine fever virus
  • Both gram negative and gram positive bacteria serve as antigens in vertebrate animals.
  • Such gram positive bacteria include, but are not limited to Pasteurella species, Staphylococci species, and Streptococcus species.
  • Gram negative bacteria include, but are not limited to, Escherichia coli, Pseudomonas species, and Salmonella species.
  • infectious bacteria include but are not limited to: Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g. M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M.
  • infectious fungi examples include: Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans .
  • infectious parasites include Plasmodium such as Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale , and Plasmodium vivax.
  • Other infectious organisms i.e. protists
  • Nonhuman vertebrates are also capable of developing infections which can be prevented or treated with the synergistic combination of adjuvants disclosed herein.
  • the methods of the invention are useful for treating infections of animals.
  • the term “treat”, “treated”, or “treating” when used with respect to an infectious disease refers to a prophylactic treatment which increases the resistance of a subject (a subject at risk of infection) to infection with a pathogen or, in other words, decreases the likelihood that the subject will become infected with the pathogen, as well as a treatment after the subject (a subject who: has been infected) has become infected in order to fight the infection, e.g., reduce or eliminate the infection or prevent it from becoming worse.
  • Many vaccines for the treatment of non-human vertebrates are disclosed in Bennett, K. Compendium of Veterinary Products, 3rd ed. North American Compendiums, Inc., 1995.
  • antigens include infectious microbes such as virus, bacteria, parasites and fungi and fragments thereof, derived from natural sources or synthetically.
  • Infectious virus of both human and non-human vertebrates include retroviruses, RNA viruses and DNA viruses.
  • This group of retroviruses includes both simple retroviruses and complex retroviruses.
  • the simple retroviruses include the subgroups of B-type retroviruses, C-type retroviruses and D-type retroviruses.
  • An example of a B-type retrovirus is mouse mammary tumor virus (MMTV).
  • the C-type retroviruses include subgroups C-type group A (including Rous sarcoma virus (RSV), avian leukemia virus (ALV), and avian myeloblastosis virus (AMV)) and C-type group B (including murine leukemia virus (MLV), feline leukemia virus (FeLV), murine sarcoma virus (MSV), gibbon ape leukemia virus (GALV), spleen necrosis virus (SNV), reticuloendotheliosis virus (RV) and simian sarcoma virus (SSV)).
  • the D-type retroviruses include Mason-Pfizer monkey virus (MPMV) and simian retrovirus type 1 (SRV-1).
  • the complex retroviruses include the subgroups of lentiviruses, T-cell leukemia viruses and the foamy viruses.
  • Lentiviruses include HIV-1, but also include HIV-2, SIV, Visna virus, feline immunodeficiency virus (FIV), and equine infectious anemia virus (EIAV).
  • the T-cell leukemia viruses include HTLV-1, HTLV-II, simian T-cell leukemia virus (STLV), and bovine leukemia virus (BLV).
  • the foamy viruses include human foamy virus (HFV), simian foamy virus (SFV) and bovine foamy virus (BFV).
  • the family Bunyaviridae including the genus Bunyvirus (Bunyamwera and related viruses, California encephalitis group viruses), the genus Phlebovirus (Sandfly fever Sicilian virus, Rift Valley fever virus), the genus Nairovirus (Crimean-Congo hemorrhagic fever virus, Kenya sheep disease virus), and the genus Uukuvirus (Uukuniemi and related viruses); the family Orthomyxoviridae, including the genus Influenza virus (Influenza virus type A, many human subtype
  • the family Bunyaviridae including the genus Bunyvirus (Bunyamwera and related viruses, California encephalitis group viruses), the genus Phlebovirus (Sandfly fever Sicilian virus, Rift Valley fever virus), the genus Nairovirus (Crimean-Congo hemorrhagic fever virus, Kenya sheep disease virus), and the genus Uukuvirus (Uukuniemi and related viruses); the family Orthomyxoviridae, including the genus Influenza virus (Influenza virus type A, many human subtype
  • Illustrative DNA viruses that are antigens in vertebrate animals include, but are not limited to: the family Poxviridae, including the genus Orthopoxvirus (Variola major, Variola minor, Monkey pox Vaccinia, Cowpox, Buffalopox, Rabbitpox, Ectromelia), the genus Leporipoxvirus (Myxoma, Fibroma), the genus Avipoxvirus (Fowlpox, other avian poxvirus), the genus Capripoxvirus (sheeppox, goatpox), the genus Suipoxvirus (Swinepox), the genus Parapoxvirus (contagious postular dermatitis virus, pseudocowpox, bovine papular stomatitis virus); the family Iridoviridae (African swine fever virus, Frog viruses 2 and 3, Lymphocystis virus of fish); the family Herpesviridae
  • the methods of the preferred embodiments are particularly well suited for treatment of birds such as hens, chickens, turkeys, ducks, geese, quail, and pheasant. Birds are prime targets for many types of infections.
  • Hatching birds are exposed to pathogenic microorganisms shortly after birth. Although these birds are initially protected against pathogens by maternal derived antibodies, this protection is only temporary, and the bird's own immature immune system must begin to protect the bird against the pathogens. It is often desirable to prevent infection in young birds when they are most susceptible. It is also desirable to prevent against infection in older birds, especially when the birds are housed in closed quarters, leading to the rapid spread of disease. Thus, it is desirable to administer the CpG oligonucleotide and the non-nucleic acid adjuvant of the invention to birds to enhance an antigen-specific immune response when antigen is present. The CpG oligonucleotide and the non-nucleic acid adjuvant of the invention could also be administered to birds without antigen to protect against infection of a wide variety of pathogens.
  • CIAV chicken infectious anemia virus
  • CIAV infection results in a clinical disease, characterized by anemia, hemorrhage and immunosuppression, in young susceptible chickens. Atrophy of the thymus and of the bone marrow and consistent lesions of CIAV-infected chickens are also characteristic of CIAV infection. Lymphocyte depletion in the thymus, and occasionally in the bursa of Fabricius, results in immunosuppression and increased susceptibility to secondary viral, bacterial, or fungal infections which then complicate the course of the disease. The immunosuppression may cause aggravated disease after infection with one or more of Marek's disease virus (MDV), infectious bursal disease virus, reticuloendotheliosis virus, adenovirus, or reovirus.
  • MDV Marek's disease virus
  • Vaccination of birds, like other vertebrate animals can be performed at any age. Normally, vaccinations are performed at up to 12 weeks of age for a live microorganism and between 14-18 weeks for an inactivated microorganism or other type of vaccine. For in ovo vaccination, vaccination can be performed in the last quarter of embryo development.
  • the vaccine may be administered subcutaneously, by spray, orally, intraocularly, intratracheally, nasally, in ovo or by other methods described herein.
  • the CpG oligonucleotide and non-nucleic acid adjuvant of the invention can be administered to birds and other non-human vertebrates using routine vaccination schedules and the antigen is administered after an appropriate time period as described herein.
  • Cattle and livestock are also susceptible to infection. Disease which affect these animals can produce severe economic losses, especially amongst cattle.
  • the methods of the invention can be used to protect against infection in livestock, such as cows, horses, pigs, sheep, and goats.
  • the CpG oligonucleotide and the non-nucleic acid adjuvant of the invention could also be administered with antigen for antigen-specific protection of long duration or without antigen for short term protection against a wide variety of diseases, including shipping fever.
  • Bovine viral diarrhea virus (BVDV) is a small enveloped positive-stranded RNA virus and is classified, along with hog cholera virus (HOCV) and sheep border disease virus (BDV), in the pestivirus genus.
  • HOCV hog cholera virus
  • BDV sheep border disease virus
  • Pestiviruses were previously classified in the Togaviridae family, some studies have suggested their reclassification within the Flaviviridae family along with the flavivirus and hepatitis C virus (HCV) groups (Francki, et al., 1991).
  • BVDV which is an important pathogen of cattle can be distinguished, based on cell culture analysis, into cytopathogenic (CP) and noncytopathogenic (NCP) biotypes.
  • CP cytopathogenic
  • NCP noncytopathogenic
  • the NCP biotype is more widespread although both biotypes can be found in cattle. If a pregnant cow becomes infected with an NCP strain, the cow can give birth to a persistently infected and specifically immunotolerant calf that will spread virus during its lifetime. The persistently infected cattle can succumb to mucosal disease and both biotypes can then be isolated from the animal.
  • Clinical manifestations can include abortion, teratogenesis, and respiratory problems, mucosal disease and mild diarrhea.
  • severe thrombocytopenia associated with herd epidemics, that may result in the death of the animal has been described and strains associated with this disease seem more virulent than the classical BVDVs.
  • Equine herpesviruses comprise a group of antigenically distinct biological agents which cause a variety of infections in horses ranging from subclinical to fatal disease. These include Equine herpesvirus-1 (EHV-1), a ubiquitous pathogen in horses. EHV-1 is associated with epidemics of abortion, respiratory tract disease, and central nervous system disorders. Primary infection of upper respiratory tract of young horses results in a febrile illness which lasts for 8 to 10 days. Immunologically experienced mares may be reinfected via the respiratory tract without disease becoming apparent, so that abortion usually occurs without warning. The neurological syndrome is associated with respiratory disease or abortion and can affect animals of either sex at any age, leading to incoordination, weakness and posterior paralysis (Telford, E. A. R.
  • EHV's include EHV-2, or equine cytomegalovirus, EHV-3, equine coital exanthema virus, and EHV-4, previously classified as EHV-1 subtype 2.
  • Sheep and goats can be infected by a variety of dangerous microorganisms including visna-maedi.
  • Cats both domestic and wild, are susceptible to infection with a variety of microorganisms.
  • feline infectious peritonitis is a disease which occurs in both domestic and wild cats, such as lions, leopards, cheetahs, and jaguars.
  • the methods of the invention can be used to vaccinate cats to prevent them against infection.
  • FeLV feline leukemia virus
  • FeSV feline sarcoma virus
  • RD-114 endogenous type C oncornavirus
  • FeSFV feline syncytia-forming virus
  • FeLV is the most significant pathogen, causing diverse symptoms, including lymphoreticular and myeloid neoplasms, anemias, immune mediated disorders, and an immunodeficiency syndrome which is similar to human acquired immune deficiency syndrome (AIDS).
  • AIDS human acquired immune deficiency syndrome
  • FeLV-AIDS a particular replication-defective FeLV mutant, designated FeLV-AIDS, has been more particularly associated with immunosuppressive properties.
  • feline T-lymphotropic lentivirus also referred to as feline immunodeficiency
  • Characteristics of FIV have been reported in Yamamoto et al. (1988) Leukemia, December Supplement 2:204S-215S; Yamamoto et al. (1988) Am. J. Vet. Res. 49:1246-1258; and Ackley et al. (1990) J. Virol. 64:5652-5655. Cloning and sequence analysis of FIV have been reported in Olmsted et al. (1989) Proc. Natl. Acad. Sci. USA 86:2448-2452 and 86:4355-4360.
  • Feline infectious peritonitis is a sporadic disease occurring unpredictably in domestic and wild Felidae. While FIP is primarily a disease of domestic cats, it has been diagnosed in lions, mountain lions, leopards, cheetahs, and the jaguar. Smaller wild cats that have been afflicted with FIP include the lynx and caracal, sand cat, and pallas cat. In domestic cats, the disease occurs predominantly in young animals, although cats of all ages are susceptible. A peak incidence occurs between 6 and 12 months of age. A decline in incidence is noted from 5 to 13 years of age, followed by an increased incidence in cats 14 to 15 years old.
  • the fish immune system has many features similar to the mammalian immune system, such as the presence of B cells, T cells, lymphokines, complement, and immunoglobulins. Fish have lymphocyte subclasses with roles that appear similar in many respects to those of the B and T cells of mammals. Vaccines can be administered orally or by immersion or injection.
  • Aquaculture species include but are not limited to fin-fish, shellfish, and other aquatic animals.
  • Fin-fish include all vertebrate fish, which may be bony or cartilaginous fish, such as, for example, salmonids, carp, catfish, yellowtail, seabream, and seabass.
  • Salmonids are a family of fin-fish which include trout (including rainbow trout), salmon, and Arctic char.
  • shellfish include, but are not limited to, clams, lobster, shrimp, crab, and oysters.
  • Other cultured aquatic animals include, but are not limited to eels, squid, and octopi.
  • Polypeptides of viral aquaculture pathogens include but are not limited to glycoprotein (G) or nucleoprotein (N) of viral hemorrhagic septicemia virus (VHSV); G or N proteins of infectious hematopoietic necrosis virus (IHNV); VP1, VP2, VP3 or N structural proteins of infectious pancreatic necrosis virus (IPNV); G protein of spring viremia of carp (SVC); and a membrane-associated protein, tegumin or capsid protein or glycoprotein of channel catfish virus (CCV).
  • G glycoprotein
  • N nucleoprotein
  • IHNV infectious hematopoietic necrosis virus
  • IPNV infectious pancreatic necrosis virus
  • SVC spring viremia of carp
  • CMV channel catfish virus
  • Polypeptides of bacterial pathogens include but are not limited to an iron-regulated outer membrane protein, (IROMP), an outer membrane protein (OMP), and an A-protein of Aeromonis salmonicida which causes furunculosis, p57 protein of Renibacterium salmoninarum which causes bacterial kidney disease (BKD), major surface associated antigen (msa), a surface expressed cytotoxin (mpr), a surface expressed hemolysin (ish), and a flagellar antigen of Yersiniosis; an extracellular protein (ECP), an iron-regulated outer membrane protein (IROMP), and a structural protein of Pasteurellosis; an OMP and a flagellar protein of Vibrosis anguillarum and V.
  • IROMP iron-regulated outer membrane protein
  • OMP outer membrane protein
  • Vibrosis anguillarum and V.
  • ordalii a flagellar protein, an OMP protein,aroA, and purA of Edwardsiellosis ictaluri and E. tarda; and surface antigen of Ichthyophthirius; and a structural and regulatory protein of Cytophaga columnari; and a structural and regulatory protein of Rickettsia.
  • Polypeptides of a parasitic pathogen include but are not limited to the surface antigens of Ichthyophthirius.
  • allergen refers to a substance (antigen) that can induce an allergic or asthmatic response in a susceptible subject.
  • the list of allergens is enormous and can include pollens, insect venoms, animal dander 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 genuses: Canine ( Canis familiaris ); Dermatophagoides (e.g. Dermatophagoides farinae ); Felis ( Felis domesticus ); Ambrosia ( Ambrosia artemiisfolia; Lolium (e.g.
  • Lolium perenne or Lolium multiflorum Cryptomeria ( Cryptomeria japonica ); Alternaria ( Alternaria alternata ); Alder; Alnus ( Alnus gultinoasa ); Betula ( Betula verrucosa ); Quercus ( Quercus alba ); Olea ( Olea europa ); Artemisia ( Artemisia vulgaris ); Plantago (e.g. Plantago lanceolata ); Parielaria (e.g. Parielaria officinalis or Parietaria judaica ); Blattella (e.g. Blattella germanica ); Apis (e.g. Apis multiflorum ); Cupressus (e.g.
  • Juniperus e.g. Juniperus sabinoides, Juniperus virginiana, Juniperus communis and Juniperus ashei ); Thuya (e.g. Thuya orientalis ); Chamaecyparis (e.g. Chamaecyparis obtusa ); Periplaneta (e.g. Periplaneta americana ); Agropyron (e.g. Agropyron repens ); Secale (e.g. Secale cereale ); Triticum (e.g. Triticum aestivum ); Dactylis (e.g. Juniperus sabinoides, Juniperus virginiana, Juniperus communis and Juniperus ashei ); Thuya (e.g. Thuya orientalis ); Chamaecyparis (e.g. Chamaecyparis obtusa ); Periplaneta (e.g. Periplaneta americana
  • Festuca e.g. Festuca elatior
  • Poa e.g.
  • the antigen is a polypeptide. Minor modifications of the primary amino acid sequences of polypeptide antigens may also result in a polypeptide which has substantially equivalent antigenic activity as compared to the unmodified counterpart polypeptide. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. All of the polypeptides produced by these modifications are included herein as long as antigenicity still exists.
  • the polypeptide may be, for example, a viral polypeptide.
  • One non-limiting example of an antigen useful according to the invention is the hepatitis B surface antigen. Experiments using this antigen are described in the Examples below.
  • substantially purified refers to a polypeptide which is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated.
  • One skilled in the art can purify viral or bacterial polypeptides using standard techniques for protein purification.
  • the substantially pure polypeptide will often yield a single major band on a non-reducing polyacrylamide gel.
  • partially glycosylated polypeptides or those that have several start codons there may be several bands on a non-reducing polyacrylamide gel, but these will form a distinctive pattern for that polypeptide.
  • the purity of the viral or bacterial polypeptide can also be determined by amino-terminal amino acid sequence analysis.
  • the invention also utilizes polynucleotides encoding the antigenic polypeptides.
  • the antigen may be delivered to the subject in a nucleic acid molecule which encodes for the antigen such that the antigen must be expressed in vivo.
  • the nucleic acid encoding the antigen is operatively linked to a gene expression sequence which directs the expression of the antigen nucleic acid within a eukaryotic cell.
  • the “gene expression sequence” is any regulatory nucleotide sequence, such as a promoter sequence or. promoter-enhancer combination, which facilitates the efficient transcription and translation of the antigen nucleic acid to which it is operatively linked.
  • the gene expression sequence may, for example, be a mammalian or viral promoter, such as a constitutive or inducible promoter.
  • Constitutive mammalian promoters include, but are not limited to, the promoters for the following genes: hypoxanthine phosphoribosyl transferase (HPTR), adenosine deaminase, pyruvate kinase, $-actin promoter, muscle creatine kinase promoter, human elongation factor promoter and other constitutive promoters.
  • Exemplary viral promoters which function constitutively in eukaryotic cells include, for example, promoters from the simian virus (e.g., SV40), papilloma virus, adenovirus, human immunodeficiency virus (HIV), rous sarcoma virus, cytomegalovirus (CMV), Rous sarcoma virus (RSV), hepatitis B virus (HBV), the long terminal repeats (LTR) of Moloney leukemia virus and other retroviruses, and the thymidine kinase promoter of herpes simplex virus.
  • simian virus e.g., SV40
  • HAV human immunodeficiency virus
  • CMV cytomegalovirus
  • RSV Rous sarcoma virus
  • HBV hepatitis B virus
  • LTR long terminal repeats
  • Other constitutive promoters are known to those of ordinary skill in the art.
  • the promoters useful as gene expression sequences of the invention also include inducible promoters.
  • Inducible promoters are expressed in the presence of an inducing agent.
  • the metallothionein promoter is induced to promote transcription and translation in the presence of certain metal ions.
  • Other inducible promoters are known to those of ordinary skill in the art.
  • the gene expression sequence shall include, as necessary, 5′ non-transcribing and 5′ non-translating sequences involved with the initiation of transcription and translation, respectively, such as a TATA box, capping sequence, CAAT sequence, and the like.
  • 5′ non-transcribing sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined antigen nucleic acid.
  • the gene expression sequences optionally include enhancer sequences or upstream activator sequences as desired.
  • the antigen nucleic acid is operatively linked to the gene expression sequence.
  • the antigen nucleic acid sequence and the gene expression sequence are said to be “operably linked” when they are covalently linked in such a way as to place the expression or transcription and/or translation of the antigen coding sequence under the influence or control of the gene expression sequence.
  • Two DNA sequences are said to be operably linked if induction of a promoter in the 5′ gene expression sequence results in the transcription of the antigen sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the antigen sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein.
  • a gene expression sequence would be operably linked to an antigen nucleic acid sequence if the gene expression sequence were capable of effecting transcription of that antigen nucleic acid sequence such that the resulting transcript is translated into the desired protein or polypeptide.
  • the antigen nucleic acid sequence may encode a protein, polypeptide, peptide, or peptide mimic of a polysaccharide. It may also cncode more than one antigenic component as a fusion construct. More than one antigen-encoding sequence may be included in the same plasmid vector and these may be linked to the same or different gene expression sequences.
  • the antigen nucleic acid of the invention may be delivered to the immune system alone or in association with a vector.
  • a “vector” is any vehicle capable of facilitating the transfer of the antigen nucleic acid to the cells of the immune system and preferably APCs so that the antigen can be expressed and presented on the surface of an APC.
  • the vector transports the nucleic acid to the immune cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector.
  • the vector optionally includes the above-described gene expression sequence to enhance expression of tne antigen nucleic acid in APCs.
  • the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antigen nucleic acid sequences.
  • Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus.
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus
  • adenovirus adeno
  • Non-cytopathic viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with the gene of interest.
  • Non-cytopathic viruses include retroviruses, the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA.
  • Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle).
  • retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo.
  • adeno-virus and adeno-associated virus which are double-stranded DNA viruses that have already been approved for human use in gene therapy and immunotherapy trials.
  • the adeno-associated virus can be engineered to be replication-deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions.
  • the aueno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection.
  • adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event.
  • the adeno-associated virus can also function in an extrachromosomal fashion.
  • Plasmid vectors have been extensively described in the art and are well-known to those of skill in the art. See e.g., Sanbrook et al., “Molecular Cloning: A Laboratory Manual”, Second Edition, Cold Spring Harbor Laboratory Press, 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen-encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid.
  • Plasmids such as those used for DNA vaccines may be delivered by a variety of parenteral, mucosal and topical routes.
  • the plasmid DNA can be injected by intramuscular, intradermal, subcutaneous or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally.
  • the plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation.
  • gene carrying plasmids can be delivered to the immune system using bacteria.
  • Modified forms of bacteria such as Salmonella can be transfected with the plasmid and used as delivery vehicles.
  • the bacterial delivery vehicles can be administered to a host subject orally or by other administration means.
  • the bacteria deliver the plasmid to immune cells, e.g. dendritic cells, probably by passing through the gut barrier. High levels of immune protection have been established using this methodology.
  • the invention includes a method for immunizing an infant by administering to an infant an antigen and an oligonucleotide containing at least one unmethylated CpG dinucleotide in an effective amount for inducing cell mediated immunity in the infant.
  • the infant is also administered at least one non-nucleic acid adjuvant, as described above.
  • Cell mediated immunity refers to an immune response which involves an antigen specific T cell reaction. The presence of cell mediated immunity can be determined directly by the induction of Th1 cytokines (e.g., IFN- ⁇ , IL-12) and antigen-specific cytotoxic T-cell lymphocytes (CTL).
  • Th1 cytokines e.g., IFN- ⁇ , IL-12
  • CTL antigen-specific cytotoxic T-cell lymphocytes
  • Th1 cytokines include but are not limited to IL-12 and IFN- ⁇ .
  • HBV chronicity results in 10-15% of individuals infected as adolescents or adults, but 90-95% for those infected (either vertically or horizontally) as infants.
  • CpG oligonucleotides may be used, according to the invention, to reduce this further owing to a more rapid appearance and higher titers of anti-HBs antibodies and the induction of HBV-specific CTL, which could help clear virus from the liver of babies infected in utero, and which likely account for most of the failures with infant vaccination.
  • the invention further provides a method of modulating the level of a cytokine.
  • modulate envisions the suppression of expression of a particular cytokine when lower levels are desired, or augmentation of the expression of a particular cytokine when higher levels are desired. Modulation of a particular cytokine can occur locally or systemically.
  • CpG oligonucleotides can directly activate macrophages and dendritic cells to secrete cytokines. No direct activation of proliferation or cytokine secretion by highly purified T cells has been found, although they are induced to secrete cytokines by cytokines secreted from macrophages and may be costimulated through the T cell Receptor. Cytokine profiles determine T cell regulatory and effector functions in immune responses. In general, Th1-type cytokines are induced, thus the immunostimulatory nucleic acids promote a Th1 type antigen-specific immune response including cytotoxic T-cells.
  • Cytokines also play a role in direciing the T cell response.
  • Helper (CD4 + ) T cells orchestrate the immune response of mammals through production of soluble factors that act on other immune system cells, including B and other T cells. Most mature CD4 + T helper cells express one of two cytokine profiles: Th1 or Th2. Th1 cells secrete IL-2, IL-3, IFN- ⁇ , GM-CSF and high levels of TNF- ⁇ . Th2 cells express IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, GM-CSF and low levels of TNF- ⁇ .
  • the Th1 subset promotes both cell-mediated immunity, and humoral immunity that is characterized by immunoglobulin class switching to IgG 2a in mice. Th1 responses may also be associated with delayed-type hypersensitivity and autoimmune disease.
  • the Th2 subset induces primarily humoral immunity and induce class switching to IgG 1 and IgE.
  • the antibody isotypes associated with Th1 responses generally have good neutralizing and opsonizing capabilities whereas those associated with Th2 responses are associated more with allergic responses.
  • IL-12 and IFN- ⁇ are positive Th1 and negative Th2 regulators.
  • IL-12 promotes IFN- ⁇ production, and IFN- ⁇ provides positive feedback for IL-12.
  • IL-4 and IL-10 appear to be required for the establishment of the Th2 cytokine profile and to down-regulate Th1 cytokine production; the effects of IL-4 are in some cases dominant over those of IL-12;
  • IL-13 was shown to inhibit expression of inflammatory cytokines, including IL-12 and TNF- ⁇ by LPS-induced monocytes, in a way similar to IL-4.
  • the IL-12 p40 homodimer binds to the IL-12 receptor and may antagonizes IL-12 biological activity; thus it blocks the pro-Th1 effects of IL-12 in some animals.
  • the invention includes a method of inducing a Th1 immune response in a subject by administering to the subject a combination of adjuvants in an effective amount for inducing a Th1 immune response.
  • the combination of adjuvants includes at least one oligonucleotide containing at least one unmethylated CpG dinucleotide and at least one non-nucleic acid adjuvant. It was not previously known that when CpG was combined with a non-nucleic acid adjuvant, as described above, that the combination would produce an immune response with a Th1 profile to an extent that the individual adjuvants could not produce alone. Preferably the extent of the Th profile produced by the combination of adjuvants is synergistic. Another aspect of the invention is to induce a Th response by using CPG with a non-nucleic acid adjuvant that by itself induces a Th2 response.
  • Th2 profile is characterized by production of IL-4 and IL-10.
  • Non-nucleic acid adjuvants that induce Th2 or weak Th1 responses include but are not limited to alum, saponins, oil-in-water and other emulsion formulations and SB-As4.
  • Adjuvants that induce Th1 responses include but are not limited to MPL, MDP, ISCOMS, IL-12, IFN- ⁇ , and SB-AS2.
  • the addition of CpG oligonucleotide can overcome this Th2 bias and induce a Th1 response that may be even more Th1-like than with CpG alone.
  • the combination of adjuvants may be administered simultaneously or sequentially.
  • the adjuvants When the adjuvants are administered simultaneously they can be administered in the same or separate formulations, and in the latter case at the same or separate sites, but are administered at the same time.
  • the adjuvants are administered sequentially, when the administration of the at least two adjuvants is temporally separated.
  • the separation in time between the administration of the two adjuvants may be a matter of minutes or it may be longer.
  • the separation in time is less than 14 days, and more preferably less than 7 days, and most preferably less than 1 day.
  • the separation in time may also be with one adjuvant at prime and one at boost, or one at prime and the combination at boost, or the combination at prime and one at boost.
  • the nucleic acids can be synthesized de novo using any of a number of procedures well known in the art.
  • the b-cyanoethyl phosphoramidite method eaucage, S. L., and Caruthers, M. H., Tet. Let. 22:1859, 1981
  • nucleoside H-phosphonate method Gagg et al., Tet. Let. 27:4051-4054, 1986; Froehler et al., Nucl. Acid. Res. 14:5399 5407, 1986,; Garegg et al., Tet. Let. 27:4055-4058, 1986, Gaffney et al., Tet. Let.
  • oligonucleotide synthesizers available in the market.
  • CpG dinucleotides can be produced on a large scale in plasmids, (see Sambrook, T., et al., Molecular Cloning: A Laboratory Manual , Cold Spring Harbor laboratory Press, New York, 1989) which after being administered to a subject are degraded into oligonucleotides.
  • plasmids may also encode other genes to be expressed such as an antigen-encoding gene in the case of a DNA vaccine.
  • Oligonucleotides can be prepared from existing nucleic acid sequences (e.g., genomic or cDNA) using known techniques, such as those employing restriction enzymes, exonucleases or endonucleases.
  • nucleic acids are preferably relatively resistant to degradation (e.g., via endo-and exo-nucleases). Secondary structures, such as stem loops, can stabilize nucleic acids against degradation. Alternatively, nucleic acid stabilization can be accomplished via phosphate backbone modifications. A preferred stabilized nucleic acid has at least a partial phosphorothioate modified backbone. Phosphorothioates may be synthesized using automated techniques employing either phosphoramidate or H-phosphonate chemistries. Aryl-and alkyl-phosphonates can be made, e.g., 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. 092,574 can be prepared by automated solid phase synthesis using commercially available reagents. Methods for making other DNA backbone modifications and substitutions have been described (Uhlmann, E. and Peyman, A., Chem. Rev. 90:544, 1990; Goodchild, J., Bioconjugate Chem. 1:165, 1990).
  • nucleic acids may be associated with a molecule that results in higher affinity binding to target cell (e.g., B-cell, monocytic cell and natural killer (NK) cell) surfaces and/or increased cellular uptake by target cells to form a “nucleic acid delivery complex.”
  • target cell e.g., B-cell, monocytic cell and natural killer (NK) cell
  • Nucleic acids can be ionically or covalently associated with appropriate molecules using techniques which are well known in the art.
  • a variety of coupling or cross-linking agents can be used, e.g., protein A, carbodiimide, and N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP).
  • SPDP N-succinimidyl-3-(2-pyridyldithio) propionate
  • Nucleic acids can alternatively be encapsulated in liposomes or virosomes using well-known techniques.
  • Nucleic acids containing an appropriate unmethylated CpG can be effective in any mammal, preferably a human. Different nucleic acids containing an unmethylated CpG can cause optimal immune stimulation depending on the mammalian species. Thus an oligonucleotide causing optimal stimulation in humans may not cause optimal stimulation in a mouse and vice versa.
  • One of skill in the art can identify the optimal oligonucleotides useful for a particular mammalian species of interest using routine assays described herein and/or known in the art.
  • the CpG ODN of the invention stimulate cytokine production (e.g., IL-6, IL-12, IFN- ⁇ , TNF- ⁇ and GM-CSF) and B-cell proliferation in PBMC's taken from a subject such as a human.
  • cytokine production e.g., IL-6, IL-12, IFN- ⁇ , TNF- ⁇ and GM-CSF
  • Preferred CpG ODN can effect at least about 500 pg/ml of TNF- ⁇ , 15 pg/ml IFN- ⁇ , 70 pg/ml of GM-CSF 275 pg/ml of IL-6, 200 pg/ml IL-12, depending on the therapeutic indication.
  • These cytokines can be measured by assays well known in the art.
  • the oligonucleotides listed above or other preferred CpG ODN can effect at least about 10%, more preferably at least about 15% and most preferably at least about 20% YAC-1 cell specific lysis or at least about 30%, more preferably at least about 35%, and most preferably at least about 40% 2C11 cell specific lysis, in assays well known in the art.
  • an effective amount of a CpG oligonucleotide refers to the amount necessary or sufficient to realize a desired biologic effect.
  • an effective amount of an oligonucleotide containing at least one unmethylated CpG and a non-nucleic acid adjuvant for treating an infectious disorder is that amount necessary to cause the development of an antigen specific immune response upon exposure to the microbe, thus causing a reduction in the amount of microbe within the subject and preferably to the eradication of the microbe.
  • the effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular CpG oligonucleotide being administered (e.g.
  • compositions of the invention are administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.
  • an effective amount of the adjuvant combination can be administered to a subject by any mode allowing the oligonucleotide to be taken up by the appropriate target cells.
  • administering the pharmaceutical composition of the present invention may be accomplished by any means known to the skilled artisan.
  • Preferred routes of administration include but are not limited to oral, transdermal (e.g. via a patch), parenteral injection (subcutaneous, intradermal, intravenous, parenteral, intraperitoneal, intrathecal, etc.), or mucosal intranasal, intratracheal, inhalation, and intrarectal, intravaginal etc).
  • An injection may be in a bolus or a continuous infusion.
  • compositions according to the invention are often administered by intramuscular or intradermal injection, or other parenteral means, or by biolistic “gene-gun”application to the epidermis. They may also be administered by intranasal application, inhalation, topically, intravenously, orally, or as implants, and even rectal or vaginal use is possible.
  • Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions for injection or inhalation, microencapsulated, encochleated, coated onto microscopic gold particles, contained in liposomes, nebulized, aerosols, pellets for implantation into the skin, or dried onto a sharp object to be scratched into the skin.
  • the pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above.
  • the pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of present methods for drug delivery, see Langer, Science 249:1527-1533, 1990, which is incorporated herein by reference.
  • the pharmaceutical compositions are preferably prepared and administered in dose units.
  • Liquid dose units are vials or ampoules for injection or other parenteral administration.
  • Solid dose units are tablets, capsules and suppositories.
  • the administration of a given dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units. Multiple administration of doses at specific intervals of weeks or months apart is usual for boosting the antigen-specific responses.
  • the adjuvants and antigens may be administered per se (neat) or in the form of a pharmaceutically acceptable salt.
  • the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof.
  • Such salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric, methane sulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, and benzene sulphonic.
  • such salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.
  • Suitable buffering agents include: acetic acid and a salt (1-2% w/v); citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v); and phosphoric acid and a salt (0.8-2% w/v).
  • Suitable preservatives include benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0:25% w/v) and thimerosal (0.004-0.02% w/v).
  • compositions of the invention contain an effective amount of a combination of adjuvants and antigens optionally included in a pharmaceutically-acceptable carrier.
  • pharmaceutically-acceptable carrier means one or more compatible solid or liquid filler, dilutants or encapsulating substances which are suitable for administration to a human or other vertebrate animal.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • the components of the pharmaceutical compositions also are capable of being comingled with the compounds of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency.
  • compositions suitable for parenteral administration conveniently comprise sterile aqueous preparations, which can be isotonic with the blood of the recipient.
  • acceptable vehicles and solvents are water, Ringer's solution, phosphate buffered saline and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed mineral or non-mineral oil may be employed including synthetic mono-ordi-glycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • Carrier formulations suitable for subcutaneous, intramuscular, intraperitoneal, intravenous, etc. administrations may be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa.
  • the adjuvants or antigens useful in the invention may be delivered in mixtures of more than two adjuvants or antigens.
  • a mixture may consist of several adjuvants in addition to the synergistic combination of adjuvants or several antigens.
  • a variety of administration routes are available. The particular mode selected will depend, of course, upon the particular adjuvants or antigen selected, the age and general health status of the subject, the particular condition being treated and the dosage required for therapeutic efficacy.
  • the methods of this invention may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of an immune response without causing clinically unacceptable adverse effects. Preferred modes of administration are discussed above.
  • compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the compounds into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the compounds into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
  • Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the compounds, increasing convenience to the subject and the physician.
  • Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Pat. No. 5,075,109.
  • Delivery systems also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-di-and tri-glycerides; hydrogel release systems; sylastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like.
  • Specific examples include, but are not limited to: (a) erosional systems in which an agent of the invention is contained in a form within a matrix such as those described in U.S. Pat. Nos. 4,452,775,4,675,189, and 5,736,152, and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Pat. Nos. 3,854,480, 5,133,974 and 5,407,686.
  • pump-based hardware delivery systems can be used, some of which are adapted for implantation.
  • CpG ODN as an adjuvant alone or in combination with other adjuvants was evaluated.
  • the hepatitis B virus surface antigen (HBsAg) given as a recombinant protein or expressed in vivo from a DNA vaccine was used as an exemplary model system in the Examples set forth below.
  • mice were carried out using female BALB/c mice (Charles River, Montreal, QC) at 6-8 weeks of age.
  • mice were obtained through breeding male and female BALB/c mice (Charles River) in the Loeb animal facility (Loeb Health Research Institute, The Ottawa Hospital, Ottawa, ON). Pregnant females were monitored daily to ensure accurate recording of the date of birth. Both male and female neonates were used for immunization.
  • Cynomolgus monkeys (1.5-3 kg) were housed at the Primate Research Center, Bogor, Indonesia.
  • Orantutans (5-20kg) were housed at Wanariset Station for the Orangutan Reintroduction Program of the Indonesian government, Balikpapan, Kalimantan.
  • the subunit vaccine consisted of HBsAg (ay subtype) which had been produced as a recombinant protein in yeast cells (Medix Biotech #ABH0905). This was diluted in saline for use without adjuvant.
  • HBsAg was also formulated with alum and/or CpG ODN as adjuvant.
  • HBsAg protein was mixed with aluminum hydroxide (Alhydrogel 85, [Al 2 O 3 ], Superfos Biosector, Vedbaek, Denmark) in the same ratio of 25 mg Al 3+ per mg protein as used in the commercial vaccines (i.e., 2.5 l 2% Al 2 O 3 per ⁇ g HBsAg).
  • the protein and alum were mixed with a vortex and then left on ice for at least 30 minutes prior to use to allow the protein to adsorb onto the Al 2 O 3 .
  • This solution was mixed again immediately prior to injection by drawing up into the syringe 3-5 times.
  • each animal received a total of 1, 10, 100 or 500 ⁇ g ODN.
  • Newborn mice were immunized within 24 hours of birth or 7 days after birth by bilateral injection of a total of 1 ⁇ g HBsAg into the posterior thigh muscles (2 ⁇ 10 l @ 0.05 mg/ml). All injections were carried out with a 0.3 ml insulin syringe which has a fused 29G needle (Becton Dickenson, Franklin Lakes, N.J.). For injection of adults, the needle was fitted with a collar of polyethylene (PE) tubing to limit penetration of the needle to about 3 mm. All intramuscular injections were carried out through the skin (shaved for adults) and under general anesthesia (Halothane, Halocarbon Laboratories, River Edge, N.J.).
  • Orangutans were immunized by IM injection into the anterior thigh muscle of HBsAg *ay subtype, 20 ⁇ g/ml) combined with alum (25 mg Al3+/mg HBsAg), combined with CpG.
  • CpG ODN 2006 (TCGTCGTTTTGTCGTTTTGTCGTT) (SEQ ID NO 77) was added to the vaccine formulation.
  • Each orangutan received an injection of 1.0 ml containing 20 ⁇ g HBsAg with alum (500 ⁇ g), CpG oligonucleotide (1 mg) or both adjuvants.
  • mice were immunized with HBsAg (1 ⁇ g) alone, with alum (25 ⁇ g Al3+), with one of several different CpG and non-CpG control oligonucleotides of different backbones (10 ⁇ g), or with both alum and an oligonucleotide.
  • mice were immunized as above (except only the 10 ⁇ g dose of CpG ODN was used) and boosted with the identical or a different formulation at 8 weeks, then spleens were removed 2 weeks later for evaluation of CTL activity.
  • mice were immunized with HBsAg (1 ⁇ g) and one of the following non-nucleic acid adjuvants alone or in combination with CpG ODN (10 ⁇ g): monophosphoryl lipid A (MPL, 50 ⁇ g, Ribi); Freund's Complete Adjuvant (CFA; 1:1 v/v); Freund's Incomplete Adjuvant (IFA; 1:1 v/v).
  • MPL monophosphoryl lipid A
  • CFA Complete Adjuvant
  • IFA Incomplete Adjuvant
  • mice Heparinized blood was collected by retrobulbar puncture of lightly anaesthetized mice as described elsewhere (Michel et al., 1995). Plasma was recovered by centrifugation (7 min @ 13,000 rpm). Antibodies specific to HBsAg in plasma were detected and quantified by end-point dilution ELISA assay (in triplicate) on individual samples. Ten-fold serial dilutions of plasma were first added to 96-well microtiter plates with a solid phase consisting of plasma-derived HBsAg particles (100 l/well of HBsAg ay subtype at 1 g/ml, coated overnight at RT) and incubated for 1 hr at 37 C.
  • the bound antibodies were then detected by incubation for 1 hr at 37C with HRP-conjugated goat anti-mouse IgG, IgM, IgG1 or IgG2a (1:4000 in PBS-Tween, 10% FCS; 100 l/well, Southern Biotechnology Inc., Birmingham, Ala.), followed by incubation with OPD solution (100 l/well, Sigma, St. Louis, Mo.) for 30 minutes at RT in the dark. The reaction was stopped by the addition of sulfuric acid (50 l of 4N H 2 SO 4 ). End-point titers were defined as the highest plasma dilution that resulted in an absorbance value (OD 450) two times greater than that of non-immune plasma

Abstract

The present invention relates generally to adjuvants, and in particular to methods and products utilizing a synergistic combination of immunostimulatory oligonucleotides having at least one unmethylated CpG dinucleotide (CpG ODN) and a non-nucleic acid adjuvant. Such combinations of adjuvants may be used with an antigen or alone. The present invention also relates to methods and products utilizing immunostimulatory oligonucleotides having at least one unmethylated CpG dinucleotide (CpG ODN) for induction of cellular immunity in infants.

Description

    RELATED APPLICATIONS
  • This application is a divisional of U.S. patent application Ser. No. 09/325,193, filed on Jun. 3, 1999, and now pending, which is a continuation in part of U.S. patent application Ser. No. 09/154,614 filed on Sep. 16, 1998, abandoned, which is a National Stage filing of PCT/US98/04703, filed on Mar. 10, 1998, claiming priority to U.S. Provisional Patent Application 60/040,376, filed Mar. 10, 1997, now abandoned.
  • FIELD OF THE INVENTION
  • The present invention relates generally to adjuvants, and in particular to methods and products utilizing a synergistic combination of oligonucleotides having at least one unmethylated CpG dinucleotide (CpG ODN) and a non-nucleic acid adjuvant.
  • BACKGROUND OF THE INVENTION
  • Bacterial DNA, but not vertebrate DNA, has direct immunostimulatory effects on peripheral blood mononuclear cells (PBMC) in vitro (Krieg et al., 1995). This lymphocyte activation is due to unmethylated CpG dinucleotides, which are present at the expected frequency in bacterial DNA (1/16), but are under-represented (CpG suppression, 1/50 to 1/60) and methylated in vertebrate DNA. Activation may also be triggered by addition of synthetic oligodeoxynucleotides (ODN) that contain an unmethylated CpG dinucleotide in a particular sequence context. It appears likely that the rapid immune activation in response to CpG DNA may have evolved as one component of the innate immune defense mechanisms that recognize structural patterns specific to microbial molecules.
  • CpG-DNA induces proliferation of almost all (>95%) B cells and increases immunoglobulin (Ig) secretion. This B cell activation by CpG DNA is T cell independent and antigen non-specific. However, B cell activation by low concentrations of CpG DNA has strong synergy with signals delivered through the B cell antigen receptor for both B cell proliferation and Ig secretion (Krieg et al., 1995). This strong synergy between the B cell signaling pathways triggered through the B cell antigen receptor and by CpG DNA promotes antigen specific immune responses. In addition to its direct effects on B cells, CpG DNA also directly activates monocytes, macrophages, and dendritic cells to secrete a variety of cytokines, including high levels of IL-12 (Klinman et al., 1996; Halpern et al., 1996; Cowdery et al., 1996). These cytokines stimulate natural killer (NK) cells to secrete gamma-interferon (IFN-γ-) and have increased lytic activity (Klinman et al., 1996, supra; Cowdery et al., 1996, supra; Yamamoto et al., 1992; Ballas et al., 1996). Overall, CpG DNA induces a Th1 like pattern of cytokine production dominated by IL-12 and IFN-γ with little secretion of Th2 cytokines (Klinman et al., 1996).
  • Hepatitis B virus (HBV) poses a serious world-wide health problem. The current HBV vaccines are subunit vaccines containing particles of HBV envelope protein(s) which include several B and T cell epitopes known collectively as HBV surface antigen (HBsAg). The HBsAg particles may be purified from the plasma of chronically infected individuals or more commonly are produced as recombinant proteins. These vaccines induce antibodies against HBsAg (anti-HBs), which confer protection if present in titers of at least 10 milli-International Units per milliliter (mIU/ml) (Ellis, 1993). The current subunit vaccines which contain alum (a Th2 adjuvant), are safe and generally efficacious. They, however, fail to meet all current vaccination needs. For example, early vaccination of infants born to chronically infected mothers, as well as others in endemic areas, drastically reduces the rate of infection, but a significant proportion of these babies will still become chronically infected themselves (Lee et al., 1989; Chen et al., 1996). This could possibly be reduced if high titers of anti-HBs antibodies could be induced earlier and if there were HBV-specific CTL. In addition, there are certain individuals who fail to respond (non-responders) or do not attain protective levels of immunity (hypo-responders). Finally, there is an urgent need for an effective treatment for the estimated 350 million chronic carriers of HBV and a therapeutic vaccine could meet this need.
  • SUMMARY OF THE INVENTION
  • The present invention relates to methods and products for inducing an immune response. The invention is useful in one aspect as a method of inducing an antigen specific immune response in a subject. The method includes the steps of administering to the subject in order to induce an antigen specific immune response an antigen and a combination of adjuvants, wherein the combination of adjuvants includes at least one oligonucleotide containing at least one unmethylated CpG dinucleotide and at least one non-nucleic acid adjuvant, and wherein the combination of adjuvants is administered in an effective-amount for inducing a synergistic adjuvant response. In one embodiment the subject is an infant.
  • The CpG oligonucleotide and the non-nucleic acid adjuvant may be administered with any or all of the administrations of antigen. For instance the combination of adjuvants may be administered with a priming dose of antigen. In another embodiment the combination of adjuvants is administered with a boost dose of antigen. In some embodiments the subject is administered a priming dose of antigen and oligonucleotide containing at least one. unmethylated CpG dinucleotide before the boost dose. In yet other embodiments the subject is administered a boost dose of antigen and oligonucleotide containing at least one unmethylated CpG dinucleotide after the priming dose.
  • The antigen may be any type of antigen known in the art. For example, the antigen may be selected from the group consisting of peptides, polypeptides, cells, cell extracts, polysaccharides, polysaccharide conjugates, lipids, glycolipids and carbohydrates. Antigens may be given in a crude, purified or recombinant form and polypeptide/peptide antigens, including peptide mimics of polysaccharides, may also be encoded within nucleic acids. Antigens may be derived from an infectious pathogen such as a virus, bacterium, fungus or parasite, or the antigen may be a tumor antigen, or the antigen may be an allergen.
  • According to another aspect of the invention a method of inducing a Th1 immune response in a subject is provided. The method includes the step of administering to the subject in order to induce a Th1 immune response a combination of adjuvants, wherein the combination of adjuvants includes at least one oligonucleotide containing at least one unmethylated CpG dinucleotide and at least one non-nucleic acid adjuvant, and wherein the combination of adjuvants is administered in an effective amount for inducing a Th1 immune response. In one embodiment the combination of adjuvants is administered simultaneously. In another embodiment the combination of adjuvants is administered sequentially. In some embodiments the combination of adjuvants is administered in an effective amount for inducing a synergistic Th1 immune response. In another aspect, the same method is performed but the subject is an infant and the Th1 response can be induced using CpG DNA alone, or CpG DNA in combination with a non-nucleic acid adjuvant at the same or different site, at the same or different time.
  • The invention in other aspects is a composition of a synergistic combination of adjuvants. The composition includes an effective amount for inducing a synergistic adjuvant response of a combination of adjuvants, wherein the combination of adjuvants includes at least one oligonucleotide containing at least one unmethylated CpG dinucleotide and at least one non-nucleic acid adjuvant. The composition may also include at least one antigen, which may be selected from the group consisting of peptides, polypeptides, cells, cell extracts, polysaccharides, polysaccharide conjugates, lipids, glycolipids and carbohydrates. Antigens may be given in a crude, purified or recombinant form and polypeptide/peptide antigens, including peptide mimics of polysaccharides, may also be encoded within nucleic acids. Antigens may be derived from an infectious pathogen such as a virus, bacterium, fungus or parasite, or the antigen may be a tumor antigen, or the antigen may be an allergen.
  • According to other aspects the invention includes a method for immunizing an infant. The method involves the step of administering to an infant an antigen and an oligonucleotide containing at least one unmethylated CpG dinucleotide and at least one non-nucleic acid adjuvant in an effective amount for inducing cell mediated immunity or Th1-like responses in the infant. The method may also involve the step of administering at least one non-nucleic acid adjuvant.
  • The CpG oligonucleotide may be administered with any or all of the administrations of antigen. For instance the CpG oligonucleotide or the combination of adjuvants may be administered with a priming dose of antigen. In another embodiment the CpG oligonucleotide or the combination of adjuvants is administered with a boost dose of antigen. In some embodiments the subject is administered a priming dose of antigen and oligonucleotide containing at least one unmethylated CpG dinucleotide before the boost dose. In yet other embodiments the subject is administered a boost dose of antigen and oligonucleotide containing at least one unmethylated CpG dinucleotide after the priming dose.
  • The invention in other aspects includes a method of inducing a stronger Th1 immune response in a subject being treated with a non-nucleic acid adjuvant. The method involves the steps of administering to a subject receiving an antigen and at least one non-nucleic acid adjuvant and at least one oligonucleotide containing at least one unmethylated CpG dinucleotide in order to induce a stronger Th1 immune response than either the adjuvant or oligonucleotide produces alone.
  • The invention in other aspects include a method of inducing a non-antigen-specific Th1-type immune response, including Th1 cytokines such as IL-12 and IFN-γ, for temporary protection against various pathogens including viruses, bacteria, parasites and fungi. The method involves the steps of administering to a subject at least one non-nucleic acid adjuvant and at least one oligonucleotide containing at least one unmethylated CpG dinucleotide in order to induce a Th1 innate immune response. For longer term protection, these adjuvants may be administered more than once. In another embodiment, CpG DNA may be used alone at one or more of the administrations.
  • In each of the above described embodiments a CpG oligonucleotide is used as an adjuvant. The oligonucleotide in one embodiment contains at least one unmethylated CpG dinucleotide having a sequence including at least the following formula:
    5′X1 X2CGX3 X4 3′
    wherein C and G are unmethylated, wherein X1X2 and X3X4 are nucleotides. In one embodiment the 5′X1 X2CGX3 X4 3′ sequence is a non-palindromic sequence.
  • The oligonucleotide may be modified. For instance, in some embodiments at least one nucleotide has a phosphate backbone modification. The phosphate backbone modification may be a phosphorothioate or phosphorodithioate modification. In some embodiments the phosphate backbone modification occurs on the 5′ side of the oligonucleotide or the 3′ side of the oligonucleotide.
  • The oligonucleotide may be any size. Preferably the oligonucleotide has 8 to 100 nucleotides. In other embodiments the oligonucleotide is 8 to 40 nucleotides in length.
  • In some embodiments X1X2 are nucleotides selected from the group consisting of: GpT, GpG, GpA, ApA, ApT, ApG, CpT, CpA, CpG, TpA, TpT, and TpG; and X3X4 are nucleotides selected from the group consisting of: TpT, CpT, ApT, TpG, ApG, CpG, TpC, ApC, CpC, TpA, ApA, and CpA. Preferably X1X2 are GpA or GpT and X3X4 are TpT. In other preferred embodiments X1 or X2 or both are purines and X3 or X4 or both are pyrimidines or X1X2 are GpA and X3 or X4 or both are pyrimidines. In one embodiment X2 is a T and X3 is a pyrimidine. The oligonucleotide may be isolated or synthetic.
  • The invention also includes the use of a non-nucleic acid adjuvant in some aspects. The non-nucleic acid adjuvant in some embodiments is an adjuvant that creates a depo effect, an immune stimulating adjuvant, or an adjuvant that creates a depo effect and stimulates the immune system. Preferably the adjuvant that creates a depo effect is selected from the group consisting of alum (e.g., aluminum hydroxide, aluminum phosphate) emulsion based formulations including mineral oil, non-mineral oil, water-in-oil or oil-in-water emulsions, such as the Seppic ISA series of Montanide adjuvants; MF-59; and PROVAX. In some embodiments the immune stimulating adjuvant is selected from the group consiting of saponins purified from the bark of the Q. saponaria tree, such as QS21; poly[di(carboxylatophenoxy)phosphazene (PCPP) derivatives of lipopolysaccharides such as monophosphorlyl lipid (MPL), muramyl dipeptide (MDP) and threonyl muramyl dipeptide (tMDP); OM-174; and Leishmania elongation factor. In one embodiment the adjuvant that creates a depo effect and stimulates the immune system is selected from the group consiting of ISCOMS; SB-AS2; SB-AS4; non-ionic block copolymers that form micelles such as CRL 1005; and Syntex Adjuvant Formulation.
  • Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention.
  • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 has two graphs illustrating humoral and cytotoxic T-lymphocyte (CTL) responses in adult BALB/c mice immunized with 1 μg recombinant HBsAg protein alone, adsorbed onto alum (25 mg Al3+/mg HBsAg), with 100 μg of immunostimulatory CpG ODN 1826, or with both alum and CpG ODN. Left panel: Each point represents the group mean (n=10) for titers of anti-HBs (total IgG) as determined in triplicate by end-point dilution ELISA assay. End-point titers were defined as the highest plasma dilution that resulted in an absorbance value (OD 450) two times greater than that of control non-immune plasma with a cut-off value of 0.05. Right panel: Each point represents the mean % specific lysis at the indicated effector: target (E:T) cell ratio in a chromium release assay with HBsAg-expressing cells as targets.
  • FIG. 2 is a graph illustrating humoral responses in adult BALB/c mice immunized with 1 μg recombinant HBsAg protein, with or without alum, and with 0, 0.1, 1, 10, 100 or 500 μg of CpG ODN 1826 added. Each point represents the group mean (n=10) for anti-HBs titers (total IgG) as determined by end-point dilution ELISA assay.
  • FIG. 3 is a graph illustrating humoral responses in adult BALB/c mice immunized with 1 μg recombinant HBsAg protein, with or without alum, and with one of several different oligonucleotides (ODN, 10 μg). The ODN were made with a natural phosphodiester backbone (O), synthetic phosphorothioate backbone (S) or a chimeric of phosphodiester center regions and phosphorothioate ends (SOS). Most of the ODN contained 1-3 CpG motifs but some of the ODN were non-CpG controls (1911, 1982, 2041). Each point represents the group mean (n=5) for anti-HBs titers (total IgG) as determined by end-point dilution ELISA assay.
  • FIG. 4 is a graph of CTL responses in adult BALB/c mice immunized with 1 μg recombinant HBsAg protein with alum (25 mg Al3+/mg HBs/Ag), with 10 μg of CpG ODN 1826, or with both alum and CpG ODN. Some animals were boosted with the same or a different formulation after 8 weeks. Each point represents the group mean (n=5) for % specific lysis of HBsAg-expressing target cell at various effector:target (E:T) cell ratios.
  • FIG. 5 is a graph of humoral responses in BALB/c mice immunized with HBsAg (1 μg) without adjuvant or with various adjuvants alone or in combination. The adjuvants were: alum (25 mg Al3+/mg HBs/Ag), with CpG DNA (10 μg CpG ODN 1826), monophosphoryl lipid A (MPL, 50 μg) and Freund's complete adjuvant (mixed 1:1 v/v with HBsAg solution). Each point represents the group mean (n=10) for anti-HBS titers (total IgG) as determined by end-point dilution ELISA assay 4 weeks after immunization.
  • FIG. 6 is a bar graph depicting the amount of total IgG (end-point ELISA titer) produced at 4 weeks in BALB/c mice immunized with 1 μg of HBsAg with or without CpG and/or IFA (mineral oil mixed 1:1 v/v) or CFA (complete Freund's adjuvant mixed 1:1 v/v). The numbers above each bar indicate the IgG2a:IgG1 ratio, with a number in excess of 1 indicating a more Th1-like response.
  • FIG. 7 is a bar graph depicting the amount of total IgG produced at 4 weeks in BALB/c mice immunized with 1 μg of HBsAg with or without CpG and/or MPL (monophosphoryl lipid A, 50 μg) or alum. The numbers above each bar indicate the IgG2a:IgG1 ratio, with a number in excess of 1 indicating a more Th1-like response.
  • FIG. 8 is a graph of the percent of young BALB/c mice that seroconverted (end-point dilution titer >100) after immunization at <1, 3, 7 or 14 days of age. Mice were immunized with 10 μg HBsAg-expressing DNA vaccine (pCMV-S), or with recombinant HBsAg (1 μg) with alum (25 mg Al3+/mg HBsAg), CpG ODN 1826 (10 μg) or both alum and CpG ODN. Each point represents the proportion of mice responding, the numbers above the bars show the number of responding over the total number immunized.
  • FIG. 9 has two graphs illustrating humoral and cytotoxic T-lymphocyte (CTL) responses in BALB/c mice immunized at 7 days of age with a DNA vaccine (1 μg pCMV-S), or with 1 μg recombinant HBsAg protein alone, adsorbed onto alum (25 mg Al3+/mg HBsAg), with 100 μg of immunostimulatory CpG ODN 1826, or with both alum and CpG ODN. Upper panel: Each point represents the group mean of animals that seroconverted (see FIG. 8 for numbers of animals) for titers of anti-HBs (total IgG) as determined in triplicate by end-point dilution ELISA assay. End-point titers were defined as the highest plasma dilution that resulted in an absorbance value (OD 450) two times greater than that of control non-immune plasma with a cut-off value of 0.05. Lower panel: Each point represents the mean % specific lysis at the indicated effector: target (E:T) cell ratio in a chromium release assay with HBsAg-expressing cells as targets.
  • FIG. 10 is a bar graph illustrating humoral responses in neonatal BALB/c mice at 8 weeks after immunization (at 7 days of age) with 1 μg recombinant HBsAg protein with alum (25 mg Al3+/mg HBsAg), with 10 μg of CpG ODN 1826, or with both alum and CpG ODN. Each point represents the group mean (see FIG. 8 for numbers of animals) for anti-HBs titers (IgG1 and IgG2a isotypes) as determined by end-point dilution ELISA assay. IgG1 antibodies indicate a Th2-biased response whereas IgG2a antibodies are indicative of a Th1-type response.
  • FIG. 11 is a graph of humoral responses in juvenile Cynomolgus monkeys immunized with Engerix-B vaccine (10 μg recombinant HBsAg protein with alum, SmithKline Beecham biologicals, Rixensart, BE) or with Engerix-B plus 500 μg of CpG ODN 1968. Each point represents the group mean (n=5) for anti-HBs titers in milli-International units/ml (mIU/ml). A titer of 10 mIU/ml is considered protective in humans.
  • FIG. 12 is a bar graph depicting titers of antibodies against HBsAg (anti-HBs) in milliInternational Units per millilitre (mIU/ml) in orangutans immunized with 10 μg HBsAg with alum (like the HBV commercial vaccine), CpG oligonucleotides (CpG ODN 2006, 1 mg) or both alum and CpG ODN. The numbers above the bars show the number of animals with seroconversion (upper numbers, >1 mIU/ml) or with seroprotection (lower numbers, >10 mIU/ml) over the total number of animals immunized. A titer of 10 mIU/ml is considered sufficient to protect humans and great apes against infection.
  • DETAILED DESCRIPTION OF THE INVENTION
  • The invention in one aspect is based on the discovery that formulations containing combinations of immunostimulatory CpG oligonucleotides and non-nucleic acid adjuvants synergistically enhance immune responses to a given antigen. Different non-nucleic acid adjuvants used in combination in the prior art have different affects on immune system activation. Some combinations of adjuvants produce an antigen-specific response that is no better than the best of the individual components and some combinations even produce lower antigen specific responses than with the individual adjuvants used alone. In Gordon et al., for instance, when humans were immunized with C terminal recombinant malaria circumsporozite antigen with alum alone or alum in combination with monophosphoryl lipid A (MPL), the subjects receiving alum alone developed higher antigen specific antibodies at several time points than subjects receiving the combination of adjuvants.
  • It has been discovered according to the invention that the combination of immunostimulatory CpG oligonucleotides and alum, MPL and other adjuvants results in a synergistic immune response. Compared with the recombinant hepatitis B surface antigen (HBsAg) protein vaccine alone, addition of alum increases the level of antibodies in mice against HBsAg (anti-HBs) about 7-fold whereas addition of CpG ODN increases them 32-fold. When CpG ODN and alum are used together, a 500-1000 times higher level of anti-HBs was observed, indicating a strong synergistic response. Additionally, it was found according to the invention that immunization with HBsAg and alum resulted in a strong Th2-type response with almost all IgG being of the IgG1 isotype. CpG ODN induced a high proportion of IgG2a, indicative of a Th1-type response, even in the presence of alum. Furthermore, it was discovered according to the invention that in very young mice (7 day old), immune responses were induced by HBsAg with alum and CpG ODN but not with alum or CpG ODN alone. The antibodies produced with CpG ODN were predominantly of the IgG2a isotype, indicating a strong Th1-type response. This is remarkable considering the strong Th2 bias of the neonatal immune system and the known difficulty in inducing Th1 responses at such a young age. Th1 responses are preferable in some instances since they are associated with IgG2a antibodies that have better neutralization and opsonization capabilities than Th2-type antibodies. As well, Th1 responses are associated with cytotoxic T lymphocytes (CTL) that can attack and kill virus-infected cells. Indeed, CpG ODN, alone or in combination with alum induced good CTL activity in both adult and neonatal mice. These studies demonstrate that the addition of CpG ODN to protein or DNA vaccines in combination with other adjuvants is a valid new adjuvant approach to improve efficacy.
  • Thus in one aspect the invention is a method of inducing an antigen specific immune response in a subject. The method includes the step of administering to the subject in order to induce an antigen specific immune response an antigen and a combination of adjuvants, wherein the combination of adjuvants includes at least one oligonucleotide containing at least one unmethylated CpG dinucleotide and at least one non-nucleic acid adjuvant, and wherein the combination of adjuvants is administered in an effective amount for inducing a synergistic adjuvant response.
  • The synergistic combination of adjuvants is particularly useful as a prophylactic vaccine for the treatment of a subject at risk of developing an infection with an infectious organism or a cancer in which a specific cancer antigen has been identified or an allergy where the allergen is known. The combination of adjuvants can also be given without the antigen or allergen for shorter term protection against infection, allergy or cancer, and in this case repeated doses will allow longer term protection. A “subject at risk” as used herein is a subject who has any risk of exposure to an infection causing pathogen or a cancer or an allergen or a risk of developing cancer. For instance, a subject at risk may be a subject who is planning to travel to an area where a particular type of infectious agent is found or it may be a subject who through lifestyle or medical procedures is exposed to bodily fluids which may contain infectious organisms or even any subject living in an area that an infectious organism or an allergen has been identified. Subjects at risk of developing infection also include general populations to which a medical agency recommends vaccination with a particular infectious organism antigen. If the antigen is an allergen and the subject develops allergic responses to that particular antigen and the subject is exposed to the antigen, i.e., during pollen season, then that subject is at risk of exposure to the antigen.
  • There is a need for a prophylactic vaccine that can induce protective immunity against many infectious pathogens more quickly and with fewer doses than traditional vaccines can provide. For instance, fewer than 20% of healthy individuals attain protective levels of anti-hepatitis B (HB) antibodies (10 mIU/ml) after a single dose of subunit hepatitis B Vaccine (HBV) vaccine and only 60-70% reach this level after two doses. Thus, three doses (usually given at 0, 1 and 6 months) are required to seroconvert >90% of vaccinated individuals. The three dose regime is frequently not completed owing to poor patient compliance, and in endemic areas, protective levels may not be induced quickly enough. The methods of the invention are particularly useful as prophylactic treatments because they induce higher levels of antibodies than can be achieved with traditional vaccines and can be administered as fewer total doses.
  • Additionally between 5 and 10% of individuals are non-responders or hypo-responders to the subunit HBsAg vaccine. This may be MHC-restricted (Kruskall et al., 1992) and is thought to result from a failure to recognize T-helper epitopes. In certain immunocompromised individuals (e.g., kidney dialysis patients, alcoholics) the rate of non-response can approach 50%. As set forth in the Examples below, alum plus CpG ODN gave higher anti-HBs titers than alum alone in a strain of mice which has MHC-restricted hypo-responsiveness to HBsAg, thought to result in a failure to recognize T-helper epitopes. CpG ODN also overcame non-response in mice genetically incapable of providing T-help owing to an absence of class II MHC. Similar results were obtained in orangutans at risk of becoming infected with hepatitis B. It was found that orangutans are hyporesponders to the classical alum-adjuvanted vaccine with less than 10% achieving seroprotection after 2 doses, but that nearly 100% of animals responded with use of CpG oligonucleotides alone or combined with alum. The synergistic response was evident because antibody titers were much higher with CpG ODN plus alum than with CpG ODN alone or alum alone and were more than additive. These results support the proposition that CpG ODN drives the T cell independent activation of B cells. Thus in addition to providing a more effective and easier vaccination protocol the synergistic combination of adjuvants can be used to reduce the percentage of non-responders and hypo-responders.
  • A subject at risk of developing a cancer is one who is who has a high probability of developing cancer. These subjects include, for instance, subjects having a genetic abnormality, the presence of which has been demonstrated to have a correlative relation to a higher likelihood of developing a cancer and subjects exposed to cancer causing agents such as tobacco, asbestos, or other chemical toxins, or a subject who has previously been treated for cancer and is in apparent remission. When a subject at risk of developing a cancer is treated with an antigen specific for the type of cancer to which the subject is at risk of developing and an adjuvant and a CpG oligonucleotide the subject may be able to kill of the cancer cells as they develop. If a tumor begins to form in the subject, the subject will develop a specific immune response against the tumor antigen.
  • In addition to the use of the combination of adjuvants for prophylactic treatment, the invention also encompasses the use of the combination for the immunotherapeutic treatment of a subject having an infection, an allergy or a cancer. A “subject having an infection” is a subject that has been exposed to an infectious pathogen and has acute or chronic detectable levels of the pathogen in the body. The combination of adjuvants can be used with an antigen to mount an antigen specific immune response that is capable of reducing the level of or eradicating the infectious pathogen. An infectious disease, as used herein, is a disease arising from the presence of a foreign microorganism in the body.
  • Many types of infectious pathogens do not have any effective treatments and chronic presence of the pathogen can result in significant damage. For instance, the HBV virus is itself non-pathogenic but with chronic infection the partially developed immune response causes inflammatory changes that eventually leads to cirrhosis and increased risk of hepatocellular carcinoma. An estimated one million people die each year from HBV-related liver disease. Persistent HBV infection of the liver results when acute infection fails to launch an appropriate immune response to clear the virus. Such chronic carriers have circulating HBsAg “e” soluble form of the HBV core antigen (HBeAg) without specific immunity. It is thought that the absence of HBV-specific T-cells, including CTL may contribute to the establishment and maintenance of the chronic carrier state. Indeed, many previously infected individuals, even years after clinical and serological recovery, have traces of HBV in their blood and HBV-specific CTL that express activation markers indicative of recent contact with antigen (Rehermann et al., 1996). These results suggest that sterilizing immunity may not occur after HBV infection and that chronic activation of HBV-specific CD4+ and CD8+ T-cells is responsible for keeping the virus under control.
  • There is currently no cure for the HBV chronic infection. Interferon is used currently but this cures only 10-20% of treated individuals (Niederau et al., 1996). Anti-viral drugs e.g., lamivudine) can reduce circulating virus to undetectable levels, however these return to pretreatment levels if the drug is stopped. Each of these types of treatment is also expensive and has certain undesirable side-effects. Thus the synergistic combination of adjuvants which induces potent Th1 responses, including CTL, is useful for treating a subject having an infection such as HBV.
  • A “subject having an allergy” is a subject that has or is at risk of developing an allergic reaction in response to an allergen. An “allergy” refers to acquired hypersensitivity to a substance (allergen). Allergic conditions include but are not limited to eczema, allergic rhinitis or coryza, conjunctivitis, hay fever, bronchial asthma, urticaria (hives) and food allergies, and other atopic conditions.
  • Currently, allergic diseases are generally treated either symptomatically with antihistimines for example or immunotherapeutically by the injection of small doses of antigen followed by subsequent increasing dosage of antigen. Symptomatic treatment offers only temporary relief. Immunotherapy is believed to induce tolerance to the allergen to prevent further allergic reactions. This approach, however, takes several years to be effective and is associated with the risk of side effects such as anaphylactic response. The methods of the invention avoid these problems.
  • Allergies are generally caused by IgE antibody generation against harmless allergens. The cytokines that are induced by unmethylated CpG oligonucleotides are predominantly of a class called “Th1” which includes IL-12 and IFN-γ. In contrast, Th2 immune response are associated with the production of IL-4, IL-5 and IL-10. Th1 responses include both cell-mediated responses (including cytotoxic T-cells) and antibodies, whereas Th2 responses are associated only with antibodies. The antibodies with a Th1 response are of isotypes (e.g. IgG2a) that have better neutralizing and opsonizing capabilities than those of Th2 isotypes (e.g. IgE that mediates allergic responses). In general, it appears that allergic diseases are mediated by Th2 type immune responses and protective immune responses by Th1 immune response although exaggerated Th1 responses may be also associated with autoimmune diseases.
  • Th2 cytokines, especially IL-4 and IL-5 are elevated in the airways of asthmatic subjects. These cytokines promote important aspects of the asthmatic inflammatory response, including IgE isotype switching, eosinophil chemotaxis and activation and mast cell growth. Th1 cytokines, especially IFN-γ and IL-12, can suppress the formation of Th2 clones and production of Th2 cytokines. “Asthma” refers to a disorder of the respiratory system characterized by inflammation, narrowing of the airways and increased reactivity of the airways to inhaled agents. Asthma is frequently, although not exclusively associated with atopic or allergic symptoms.
  • Based on the ability of the CpG oligonucleotides to shift the immune response in a subject from a Th2 (which is associated with production of IgE antibodies and allergy) to a Th1 response (which is protective against allergic reactions), an effective dose of a CpG oligonucleotide can be administered to a subject to treat or prevent an allergy.
  • Since Th1 responses are even more potent with CpG DNA combined with non-nucleic acid adjuvants, the combination of adjuvants of the present invention will have significant therapeutic utility in the treatment of allergic conditions such as asthma. Such combinations of adjuvants could be used alone or in combination with allergens.
  • A “subject having a cancer” is a subject that has detectable cancerous cells. The cancer may be a malignant or non-malignant cancer. Cancers or tumors include but are not limited to biliary tract cancer; brain cancer; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; intraepithelial neoplasms; lymphomas; liver cancer; lung cancer (e.g. small cell and non-small cell); melanoma; neuroblastomas; oral cancer; ovarian cancer; pancreas cancer; prostate cancer; rectal cancer; sarcomas; skin cancer; testicular cancer; thyroid cancer; and renal cancer, as well as other carcinomas and sarcomas.
  • A “subject” shall mean a human or vertebrate animal including but not limited to a dog, cat, horse, cow, pig, sheep, goat, chicken, primate, (e.g., monkey), fish (aquaculture species e.g. salmon, trout and other salmonids), rat, and mouse.
  • The subject is administered a combination of adjuvants, wherein the combination of adjuvants includes at least one oligonucleotide containing at least one unmethylated CpG dinucleotide and at least one non-nucleic acid adjuvant. An oligonucleotide containing at least one unmethylated C pG dinucleotide is a nucleic acid molecule which contains an unmethylated cytosine-guanine dinucleotide sequence (i.e. “CpG DNA” or DNA containing a 5′ cytosine followed by 3′ guanosine and linked by a phosphate bond) and activates the immune system. The CpG oligonucleotides can be double-stranded or single-stranded. Generally, double-stranded molecules are more stable in vivo, while single-stranded molecules have increased immune activity. The CpG oligonucleotides or combination of adjuvants can be used with or without antigen.
  • The terms “nucleic acid” and “oligonucleotide” are used interchangeably to mean multiple nucleotides (i.e. molecules comprising a sugar (e.g. ribose or deoxyribose) linked to a phosphate group and to an exchangeable organic base, which is either a substituted pyrimidine (e.g. cytosine (C), thymine (T) or uracil (U)) or a substituted purine (e.g. adenine (A) or guanine (G)). As used herein, the terms refer to oligoribonucleotides as well as oligodeoxyribonucleotides. The terms shall also include polynucleosides (i.e. a polynucleotide minus the phosphate) and any other organic base containing polymer. Nucleic acid molecules can be obtained from existing nucleic acid sources (e.g. genomic or cDNA), but are preferably synthetic (e.g. produced by oligonucleotide synthesis). The entire CpG oligonucleotide can be unmethylated or portions may be unmethylated but at least the C of the 5° CG 3′ must be unmethylated.
  • In one preferred embodiment the invention provides a CpG oligonucleotide represented by at least the formula:
    5′N1X1CGX2N23′
    wherein at least one nucleotide separates consecutive CpGs; X1 is adenine, guanine, or thymine; X2 is cytosine, adenine, or thymine; N is any nucleotide and N1 and N2 are nucleic acid sequences composed of from about 0-25 N's each.
  • In another embodiment the invention provides an isolated CpG oligonucleotide represented by at least the formula:
    5′N1X1X2CGX3X4N23′
    wherein at least one nucleotide separates consecutive CpGs; X1X2 are nucleotides selected from the group consisting of: GpT, GpG, GpA, ApA, ApT, ApG, CpT, CpA, CpG, TpA, TpT, and TpG; and X3X4 are nucleotides selected from the group consisting of: TpT, CpT, ApT, TpG, ApG, CpG, TpC, ApC, CpC, TpA, ApA, and CpA; N is any nucleotide and N1 and N2 are nucleic acid sequences composed of from about 0-25 N's each. Preferably X1X2 are GpA or GpT and X3X4 are TpT. In other preferred embodiments X1 or X2 or both are purines and X3 or X4 or both are pyrimidines or X1X2 are GpA and X3 or X4 or both are pyrimidines. In a preferred embodiment N1 and N2 of the nucleic acid do not contain a CCGG or CGCG quadmer or more than one CCG or CGG trimer. The effect of a a CCGG or CGCG quadmer or more than one CCG or CGG trimer depends in part on the status of the oligonucleotide backbone. For instance, if the oligonucleotide has a phosphodiester backbone or a chimeric backbone the inclusion of these sequences in the oligonucleotide will only have minimal if any affect on the biological activity of the oligonucleotide. If the backbone is completely phosphorothioate or significantly phosphorothioate then the inclusion of these sequences may have more influence on the biological activity or the kinetics of the biological activity. In another preferred embodiment the CpG oligonucleotide has the sequence 5′TCN1TX1X2CGX3X43′.
  • Preferably the CpG oligonucleotides of the invention include X1X2 selected from the group consisting of GpT, GpG, GpA and ApA and X3X4 is selected from the group consisting of TpT, CpT and GpT. For facilitating uptake into cells, CpG containing oligonucleotides are preferably in the range of 8 to 30 bases in length. However, nucleic acids of any size greater than 8 nucleotides (even many kb long) are capable of inducing an immune response according to the invention if sufficient immunostimulatory motifs are present, since larger nucleic acids are degraded into oligonucleotides inside of cells. Preferred synthetic oligonucleotides do not include a CCGG or CGCG quadmer or more than one CCG or CGG trimer at or near the 5′ and/or 3′ terminals. Stabilized oligonucleotides, where the oligonucleotide incorporates a phosphate backbone modification, as discussed in more detail below are also preferred. The modification may be, for example, a phosphorothioate or phosphorodithioate modification. Preferably, the phosphate backbone modification occurs at the 5′ end of the nucleic acid for example, at the first two nucleotides of the 5′ end of the oligonucleotide. Further, the phosphate backbone modification may occur at the 3′ end of the nucleic acid for example, at the last five nucleotides of the 3′ end of the nucleic acid. Alternatively the oligonucleotide may be completely or partially modified.
  • Preferably the CpG oligonucleotide is in the range of between 8 and 100 and more preferably between 8 and 30 nucleotides in size. Alternatively, CpG oligonucleotides can be produced on a large scale in plasmids. These may be administered in plasmid form or alternatively they can be degraded into oligonucleotides.
  • The CpG oligonucleotide and immunopotentiating cytokine may be directly administered to the subject or may be administered in conjunction with a nucleic acid delivery complex. A “nucleic acid/cytokine delivery complex” shall mean a nucleic acid molecule and/or cytokine associated with (e.g. ionically or covalently bound to; or encapsulated within) a targeting means (e.g. a molecule that results in higher affinity binding to target cell (e.g. dendritic cell surfaces and/or increased cellular uptake by target cells). Examples of nucleic acid/cytokine delivery complexes include nucleic acids/cytokines associated with: a sterol (e.g. cholesterol), a lipid (e.g. a cationic lipid, virosome or liposome), or a target cell specific binding agent (e.g. a ligand recognized by target cell specific receptor). Preferred complexes should be sufficiently stable in vivo to prevent significant uncoupling prior to internalization by the target cell. However, the complex should be cleavable under appropriate conditions within the cell so that the nucleic acid/cytokine is released in a functional form.
  • “Palindromic sequence” shall mean an inverted repeat (i.e. a sequence such as ABCDEE′D′C′B′A′ in which A and A′ are bases capable of forming the usual Watson-Crick base pairs. In vivo, such sequences may form double-stranded structures. In one embodiment the CpG oligonucleotide contains a palindromic sequence. A palindromic sequence used in this context refers to a palindrome in which the CpG is part of the palindrome, and preferably is the center of the palindrome. In another embodiment the CpG oligonucleotide is free of a palindrome. A CpG oligonucleotide that is free of a palindrome is one in which the CpG dinucleotide is not part of a palindrome. Such an oligonucleotide may include a palindrome in which the CpG is not part of the palindrome.
  • A “stabilized nucleic acid molecule” shall mean a nucleic acid molecule that is relatively resistant to in vivo degradation (e.g. via an exo- or endo-nuclease). Stabilization can be a function of length or secondary structure. Unmethylated CpG oligonucleotides that are tens to hundreds of kbs long are relatively resistant to in vivo degradation, particularly when in a double-stranded closed-circular form (i.e., a plamid). For shorter CpG oligonucleotides, secondary structure can stabilize and increase their effect. For example, if the 3′ end of an oligonucleotide has self-complementarity to an upstream region, so that it can fold back and form a sort of stem loop structure, then the oligonucleotide becomes stabilized and therefore exhibits more activity.
  • Preferred stabilized oligonucleotides of the instant invention have a modified backbone. It has been demonstrated that modification of the oligonucleotide backbone provides enhanced activity of the CpG oligonucleotides when administered in vivo. CpG constructs, including at least two phosphorothioate linkages at the 5′ end of the oligonucleotide in multiple phosphorothioate linkages at the 3′ end, preferably 5, provides maximal activity and protected the oligonucleotide from degradation by intracellular exo- and endo-nucleases. Other modified oligonucleotides include phosphodiester modified oligonucleotide, combinations of phosphodiester and phosphorothioate oligonucleotide, methylphosphonate, methylphosphorothioate, phosphorodithioate, and combinations thereof. Each of these combinations and their particular effects on immune cells is discussed in more detail in copending PCT Published Patent Applications claiming priority to U.S. Ser. Nos. 08/738,652 and 08/960,774, filed on Oct. 30, 1996 and Oct. 30, 1997 respectively, the entire contents of which is hereby incorporated by reference. It is believed that these modified oligonucleotides may show more stimulatory activity due to enhanced nuclease resistance, increased cellular uptake, increased protein binding, and/or altered intracellular localization.
  • Both phosphorothioate and phosphodiester oligonucleotides containing CpG motifs are active in immune cells. However, based on the concentration needed to induce CpG specific effects, the nuclease resistant phosphorothioate backbone CpG oligonucleotides are more potent (2 μg/ml for the phosphorothioate vs. a total of 90 μg/ml for phosphodiester).
  • Other stabilized oligonucleotides include: nonionic DNA analogs, such as alkyl- and aryl-phosphates (in which the charged phosphonate oxygen is replaced by an alkyl or aryl group), phosphodiester and alkylphosphotriesters, in which the charged oxygen moiety is alkylated. Oligonucleotides which contain diol, such as tetraethyleneglycol or hexaethyleneglycol, at either or both termini have also been shown to be substantially resistant to nuclease degradation.
  • The nucleic acid sequences of the invention which are useful as adjuvants are those broadly described above and disclosed in PCT Published Patent Applications claiming priority to U.S. Ser. Nos. 08/738,652 and 08/960,774, filed on Oct. 30, 1996 and Oct. 30, 1997 respectively. Exemplary sequences include but are not limited to those immunostimulatory sequences shown in Table 1.
  • The stimulation index of a particular immunostimulatory CpG DNA can be tested in various immune cell assays. Preferably, the stimulation index of the CpG oligonucleotide with regard to B cell proliferation is at least about 5, preferably at least about 10, more preferably at least about 15 and most preferably at least about 20 as determined by incorporation of 3H uridine in a murine B cell culture, which has been contacted with 20 μM of oligonucleotide for 20 h at 37° C. and has been pulsed with 1 μCi of 3H uridine; and harvested and counted 4 h later as described in detail in copending PCT Published Patent Applications claiming priority to U.S. Ser. Nos. 08/738,652 and 08/960,774, filed on Oct. 30, 1996 and Oct. 30, 1997 respectively. For use in vivo, for example, it is important that the CpG oligonucleotide and adjuvant be capable of effectively inducing activation of Ig expressing B cells. Oligonucleotides which can accomplish this include, for example, but are not limited to those oligonucleotides described in PCT Published Patent Applications claiming priority to U.S. Ser. Nos. 08/738,652 and 08/960,774, filed on Oct. 30, 1996 and Oct. 30, 1997 respectively.
  • The oligonucleotide containing at least one unmethylated CpG is used in combination with a non-nucleic acid adjuvant and an antigen to activate the immune response. A “non-nucleic acid adjuvant” is any molecule or compound except for the CpG oligonucleotides described herein which can stimulate, the humoral and/or cellular immune response. Non-nucleic acid adjuvants include, for instance, adjuvants that create a depo effect, immune stimulating adjuvants, and adjuvants that create a depo effect and stimulate the immune system. In infants, the oligonucleotide containing at least one unmethylated CpG is used alone or in combination with a non-nucleic acid adjuvant and an antigen to activate a cellular immune response.
  • An “adjuvant that creates a depo effect” as used herein is an adjuvant that causes the antigen to be slowly released in the body, thus prolonging the exposure of immune cells to the antigen. This class of adjuvants includes but is not limited to alum (e.g., aluminum hydroxide, aluminum phosphate); or emulsion-based formulations including mineral oil, non-mineral oil, water-in-oil or oil-in-water-in oil emulsion, oil-in-water emulsions such as Seppic ISA series of Montanide adjuvants (e.g., Montanide ISA 720, AirLiquide, Paris, France); MF-59 (a squalene-in-water emulsion stabilized with Span 85 and Tween 80; Chiron Corporation, Emeryville, Calif.; and PROVAX (an oil-in-water emulsion containing a stabilizing detergent and a micelle-forming agent; IDEC, Pharmaceuticals Corporation, San Diego, Calif.).
  • An “immune stimulating adjuvant” is an adjuvant that causes activation of a cell of the immune system. It may, for instance, cause an immune cell to produce and secrete cytokines. This class of adjuvants includes but is not limited to saponins purified from the bark of the Q. saponaria tree, such as QS21 (a glycolipid that elutes in the 21st peak with HPLC fractionation; Aquila Biopharmaceuticals, Inc., Worcester, Mass.); poly[di(carboxylatophenoxy)phosphazene (PCPP polymer; Virus Research Institute, USA); derivatives of lipopolysaccharides such as monophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc., Hamilton, Mont.), muramyl dipeptide (MDP; Ribi) andthreonyl-muramyl dipeptide (t-MDP; Ribi); OM-174 (a glucosamine disaccharide related to lipid A; OM Pharma SA, Meyrin, Switzerland); and Leishmania elongation factor (a purified Leishmania protein; Corixa Corporation, Seattle, Wash.).
  • “Adjuvants that create a depo effect and stimulate the immune system” are those compounds which have both of the above- identified functions. This class of adjuvants includes but is not limited to ISCOMS (Immunostimulating complexes which contain mixed saponins, lipids and form virus-sized particles with pores that can hold antigen; CSL, Melbourne, Australia); SB-AS2 (SmithKline Beecham adjuvant system #2 which is an oil-in-water emulsion containing MPL and QS21: SmithKline Beecham Biologicals [SBB], Rixensart, Belgium); SB-AS4 (SmithKline Beecham adjuvant system #4 which contains alum and MPL; SBB, Belgium); non-ionic block copolymers that form micelles such as CRL 1005 (these contain a linear chain of hydrophobic polyoxpropylene flanked by chains of polyoxyethylene; Vaxcel, Inc., Norcross, Ga.); and Syntex Adjuvant Formulation (SAF, an oil-in-water emulsion containing Tween 80 and a nonionic block copolymer; Syntex Chemicals, Inc., Boulder, Colo.).
  • When the CpG oligonucleotide containing at least one unmethylated CpG is administered in conjunction with another adjuvant, the CpG oligonucleotide can be administered before, after, and/or simultaneously with the other adjuvant. For instance, the combination of adjuvants may be administered with a priming dose of antigen. Either or both of the adjuvants may then be administered with the boost dose. Alternatively, the combination of adjuvants may be administered with a boost dose of antigen. Either or both of the adjuvants may then be administered with the prime dose. A “prime dose” is the first dose of antigen administered to the subject. In the case of a subject that has an infection the prime dose may be the initial exposure of the subject to the infectious microbe and thus the combination of adjuvants is administered to the subject with the boost dose. A “boost dose” is a second or third, etc., dose of antigen administered to a subject that has already been exposed to the antigen. In some cases the prime dose administered with the combination of adjuvants is so effective that a boost dose is not required to protect a subject at risk of infection from being infected. In cases where the combination of adjuvants is given without antigen, with repeated administrations, CpG oligonucleotides or one of the components in the combination may be given alone for one or more of the administrations.
  • The CpG oligonucleotide containing at least one unmethylated CpG can have an additional efficacy (e.g., antisense) in addition to its ability to enhance antigen-specific immune responses.
  • An “antigen” as used herein is a molecule capable of provoking an immune response. Antigens include but are not limited to peptides, polypeptides, cells, cell extracts, polysaccharides, polysaccharide conjugates, lipids, glycoliopids and carbohydrates. Antigens may be given in a crude, purified or recombinant form and polypeptide/peptide antigens, including peptide mimics of polysaccharides, may also be encoded within nucleic. The term antigen broadly includes any type of molecule which is recognized by a host immune system as being foreign. Antigens include but are not limited to cancer antigens, microbial antigens, and allergens.
  • A “cancer antigen” as used herein is a compound, such as a peptide, associated with a tumor or cancer cell surface and which is capable of provoking an immune response when expressed on the surface of an antigen presenting cell in the context of an MHC molecule. Cancer antigens can be prepared from cancer cells either by preparing crude extracts of cancer cells, for example, as described in Cohen, et al., 1994, Cancer Research, 54:1055, by partially purifying the antigens, by recombinant technology, or by de novo synthesis of known antigens. Cancer antigens include antigens that are recombinately an immunogenic portion of or a whole tumor or cancer. Such antigens can be isolated or prepared recombinatly or by any other means known in the art.
  • A “microbial antigen” as used herein is an antigen of a microorganism and includes but is not limited to infectious virus, infectious bacteria, infectious parasites and infectious fungi. Such antigens include the intact microorganism as well as natural isolates and fragments or derivatives thereof and also synthetic compounds which are identical to or similar to natural microorganism antigens and induce an immune response specific for that microorganism. A compound is similar to a natural microorganism antigen if it induces an immune response (humoral and/or cellular) to a natural microorganism antigen. Most such antigens are used routinely in the art and are well known to those of ordinary skill in the art. Another example is a peptide mimic of a polysaccharide antigen.
  • Examples of infectious virus that have been found in humans include but are not limited to: Retroviridae (e.g. human immunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP; Picornaviridae (e.g. polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g. strains that cause gastroenteritis); Togaviridae (e.g. equine encephalitis viruses, rubella viruses); Flaviridae (e.g. dengue viruses, encephalitis viruses, yellow fever viruses); Coronoviridae (e.g. coronaviruses); Rhabdoviradae (e.g. vesicular stomatitis viruses, rabies viruses); Coronaviridae (e.g. coronaviruses); Rhabdoviridae (e.g. vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g. ebola viruses); Paramyxoviridae (e.g. parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g. influenza viruses); Bungaviridae (e.g. Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g. reoviruses, orbiviurses and rotaviruses); Birnaviridae; Hepadndviridae (Hepatitis B virus); Parvovirida (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes virus; Poxviridae (variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g. African swine fever virus); and unclassified viruses (e.g. the etiological agents of Spongiform encephalopathies, the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), the agents of non-A, non-B hepatitis (class 1=internally transmitted; class 2=parenterally transmitted (i.e. Hepatitis C); Norwalk and related viruses, and astroviruses).
  • Both gram negative and gram positive bacteria serve as antigens in vertebrate animals. Such gram positive bacteria include, but are not limited to Pasteurella species, Staphylococci species, and Streptococcus species. Gram negative bacteria include, but are not limited to, Escherichia coli, Pseudomonas species, and Salmonella species. Specific examples of infectious bacteria include but are not limited to: Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g. M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillus antracis, corynebacterium diphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leplospira, Rickettsia, and Actinomyces israelli.
  • Examples of infectious fungi include: Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans. Examples of infectious parasites include Plasmodium such as Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax. Other infectious organisms (i.e. protists) include Toxoplasma gondii.
  • Other medically relevant microorganisms have been descried extensively in the literature, e.g., see C. G. A Thomas, Medical Microbiology, Bailliere Tindall, Great Britain 1983, the entire contents of which is hereby incorporated by reference.
  • Although many of the microbial antigens described above relate to human disorders, the invention is also useful for treating other nonhuman vertebrates. Nonhuman vertebrates are also capable of developing infections which can be prevented or treated with the synergistic combination of adjuvants disclosed herein. For instance, in addition to the treatment of infectious human diseases, the methods of the invention are useful for treating infections of animals.
  • As used herein, the term “treat”, “treated”, or “treating” when used with respect to an infectious disease refers to a prophylactic treatment which increases the resistance of a subject (a subject at risk of infection) to infection with a pathogen or, in other words, decreases the likelihood that the subject will become infected with the pathogen, as well as a treatment after the subject (a subject who: has been infected) has become infected in order to fight the infection, e.g., reduce or eliminate the infection or prevent it from becoming worse. Many vaccines for the treatment of non-human vertebrates are disclosed in Bennett, K. Compendium of Veterinary Products, 3rd ed. North American Compendiums, Inc., 1995.
  • As discussed above, antigens include infectious microbes such as virus, bacteria, parasites and fungi and fragments thereof, derived from natural sources or synthetically. Infectious virus of both human and non-human vertebrates, include retroviruses, RNA viruses and DNA viruses. This group of retroviruses includes both simple retroviruses and complex retroviruses. The simple retroviruses include the subgroups of B-type retroviruses, C-type retroviruses and D-type retroviruses. An example of a B-type retrovirus is mouse mammary tumor virus (MMTV). The C-type retroviruses include subgroups C-type group A (including Rous sarcoma virus (RSV), avian leukemia virus (ALV), and avian myeloblastosis virus (AMV)) and C-type group B (including murine leukemia virus (MLV), feline leukemia virus (FeLV), murine sarcoma virus (MSV), gibbon ape leukemia virus (GALV), spleen necrosis virus (SNV), reticuloendotheliosis virus (RV) and simian sarcoma virus (SSV)). The D-type retroviruses include Mason-Pfizer monkey virus (MPMV) and simian retrovirus type 1 (SRV-1). The complex retroviruses include the subgroups of lentiviruses, T-cell leukemia viruses and the foamy viruses. Lentiviruses include HIV-1, but also include HIV-2, SIV, Visna virus, feline immunodeficiency virus (FIV), and equine infectious anemia virus (EIAV). The T-cell leukemia viruses include HTLV-1, HTLV-II, simian T-cell leukemia virus (STLV), and bovine leukemia virus (BLV). The foamy viruses include human foamy virus (HFV), simian foamy virus (SFV) and bovine foamy virus (BFV).
  • Examples of other RNA viruses that are antigens in vertebrate animals include, but are not limited to, the following: members of the family Reoviridae, including the genus Orthoreovirus (multiple scrotypes of both mammalian and avian retroviruses), the genus Orbivirus (Bluetongue virus, Eugenangee virus, Kemerovo virus, African horse sickness virus, and Colorado Tick Fever virus), the genus Rotavirus (human rotavirus, Nebraska calf diarrhea virus, murine rotavirus, simian rotavirus, bovine or ovine rotavirus, avian rotavirus); the family Picornaviridae, including the genus Enterovirus (poliovirus, Coxsackie virus A and B, enteric cytopathic human orphan (ECHO) viruses, hepatitis A virus, Simian enteroviruses, Murine encephalomyelitis (ME) viruses, Poliovirus muris, Bovine enteroviruses, Porcine enteroviruses, the genus Cardiovirus (Encephalomyocarditis virus (EMC), Mengovirus), the genus Rhinovirus (Human rhinoviruses including at least 113 subtypes; other rhinoviruses), the genus Apthovirus (Foot and Mouth disease (FMDV); the family Calciviridae, including Vesicular exanthema of swine virus, San Miguel sea lion virus, Feline picornavirus and Norwalk virus; the family Togaviridae, including the genus Alphavirus (Eastern equine encephalitis virus, Semliki forest virus, Sindbis virus, Chikungunya virus, O'Nyong-Nyong virus, Ross river virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus), the genus Flavirius (Mosquito borne yellow fever virus, Dengue virus, Japanese encephalitis virus, St. Louis encephalitis virus, Murray Valley encephalitis virus, West Nile virus, Kunjin virus, Central European tick borne virus, Far Eastern tick borne virus, Kyasanur forest virus, Louping III virus, Powassan virus, Omsk hemorrhagic fever virus), the genus Rubivirus (Rubella virus), the genus Pestivirus (Mucosal disease virus, Hog cholera virus, Border disease virus); the family Bunyaviridae, including the genus Bunyvirus (Bunyamwera and related viruses, California encephalitis group viruses), the genus Phlebovirus (Sandfly fever Sicilian virus, Rift Valley fever virus), the genus Nairovirus (Crimean-Congo hemorrhagic fever virus, Nairobi sheep disease virus), and the genus Uukuvirus (Uukuniemi and related viruses); the family Orthomyxoviridae, including the genus Influenza virus (Influenza virus type A, many human subtypes); Swine influenza virus, and Avian and Equine Influenza viruses; influenza type B (many human subtypes), and influenza type C (possible separate genus); the family paramyxoviridae, including the genus Paramyxovirus (Parainfluenza virus type 1, Sendai virus, Hemadsorption virus, Parainfluenza viruses types 2 to 5, Newcastle Disease Virus, Mumps virus), the genus Morbillivirus (Measles virus, subacute sclerosing panencephalitis virus, distemper virus, Rinderpest virus), the genus Pneumovirus (respiratory syncytial virus (RSV), Bovine respiratory syncytial virus and Pneumonia virus of mice); forest virus, Sindbis virus, Chikungunya virus, O'Nyong-Nyong virus, Ross river virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus), the genus Flavirius (Mosquito borne yellow fever virus, Dengue virus, Japanese encephalitis virus, St. Louis encephalitis virus, Murray Valley encephalitis virus, West Nile virus, Kunjin virus, Central European tick borne virus, Far Eastern tick borne virus, Kyasanur forest virus, Louping III virus, Powassan virus, Omsk hemorrhagic fever virus), the genus Rubivirus (Rubella virus), the genus Pestivirus (Mucosal disease virus, Hog cholera virus, Border disease virus); the family Bunyaviridae, including the genus Bunyvirus (Bunyamwera and related viruses, California encephalitis group viruses), the genus Phlebovirus (Sandfly fever Sicilian virus, Rift Valley fever virus), the genus Nairovirus (Crimean-Congo hemorrhagic fever virus, Nairobi sheep disease virus), and the genus Uukuvirus (Uukuniemi and related viruses); the family Orthomyxoviridae, including the genus Influenza virus (Influenza virus type A, many human subtypes); Swine influenza virus, and Avian and Equine Influenza viruses; influenza type B (many human subtypes), and influenza type C (possible separate genus); the family paramyxoviridae, including the genus Paramyxovirus (Parainfluenza virus type 1, Sendai virus, Hemadsorption virus, Parainfluenza viruses types 2 to 5, Newcastle Disease Virus, Mumps virus), the genus Morbillivirus (Measles virus, subacute sclerosing panencephalitis virus, distemper virus, Rinderpest virus), the genus Pneumovirus (respiratory syncytial virus (RSV), Bovine respiratory syncytial virus and Pneumonia virus of mice); the family Rhabdoviridae, including the genus Vesiculovirus (VSV), Chandipura virus, Flanders-Hart Park virus), the genus Lyssavirus (Rabies virus), fish Rhabdoviruses, and two probable Rhabdoviruses (Marburg virus and Ebola virus); the family Arenaviridae, including Lymphocytic choriomeningitis virus (LCM), Tacaribe virus complex, and Lassa virus; the family Coronoaviridae, including Infectious Bronchitis Virus (IBV), Mouse Hepatitis virus, Human enteric corona virus, and Feline infectious peritonitis (Feline coronavirus).
  • Illustrative DNA viruses that are antigens in vertebrate animals include, but are not limited to: the family Poxviridae, including the genus Orthopoxvirus (Variola major, Variola minor, Monkey pox Vaccinia, Cowpox, Buffalopox, Rabbitpox, Ectromelia), the genus Leporipoxvirus (Myxoma, Fibroma), the genus Avipoxvirus (Fowlpox, other avian poxvirus), the genus Capripoxvirus (sheeppox, goatpox), the genus Suipoxvirus (Swinepox), the genus Parapoxvirus (contagious postular dermatitis virus, pseudocowpox, bovine papular stomatitis virus); the family Iridoviridae (African swine fever virus, Frog viruses 2 and 3, Lymphocystis virus of fish); the family Herpesviridae, including the alpha-Herpesviruses (Herpes Simplex Types 1 and 2, Varicella-Zoster, Equine abortion virus, Equine herpes virus 2 and 3, pseudorabies virus, infectious bovine keratoconjunctivitis virus, infectious bovine rhinotracheitis virus, feline rhinotracheitis virus, infectious laryngotracheitis virus) the Beta-herpesviruses (Human cytomegalovirus and cytomegaloviruses of swine, monkeys and rodents); the gamma-herpesviruses (Epstein-Barr virus (EBV), Marek's disease virus, Herpes saimiri, Herpesvirus ateles, Herpesvirus sylvilagus, guinea pig herpes virus, Lucke tumor virus); the family Adenoviridae, including the genus Mastadenovirus (Human subgroups A,B,C,D,E and ungrouped; simian adenoviruses (at least 23 serotypes), infectious canine hepatitis, and adenoviruses of cattle, pigs, sheep, frogs and many other species, the genus Aviadenovirus (Avian adenoviruses); and non-cultivatable adenoviruses; the family Papoviridae, including the genus Papillomavirus (Human papilloma viruses, bovine papilloma viruses, Shope rabbit papilloma virus, and various pathogenic papilloma viruses of other species), the genus Polyomavirus (polyomavirus, Simian vacuolating agent (SV-40), Rabbit vacuolating agent (RKV), K virus, BK virus, JC virus, and other primate polyoma viruses such as Lymphotrophic papilloma virus); the family Parvoviridae including the genus Adeno-associated viruses, the genus Parvovirus (Feline panleukopenia virus, bovine parvovirus, canine parvovirus, Aleutian mink disease virus, etc). Finally, DNA viruses may include viruses which do not fit into the above families such as Kuru and Creutzfeldt-Jacob disease viruses and chronic infectious neuropathic agents (CHINA virus).
  • Each of the foregoing lists is illustrative, and is not intended to be limiting.
  • In addition to the use of the combination of CpG oligonucleotides and non-nucleic acid adjuvants to induce an antigen specific immune response in humans, the methods of the preferred embodiments are particularly well suited for treatment of birds such as hens, chickens, turkeys, ducks, geese, quail, and pheasant. Birds are prime targets for many types of infections.
  • Hatching birds are exposed to pathogenic microorganisms shortly after birth. Although these birds are initially protected against pathogens by maternal derived antibodies, this protection is only temporary, and the bird's own immature immune system must begin to protect the bird against the pathogens. It is often desirable to prevent infection in young birds when they are most susceptible. It is also desirable to prevent against infection in older birds, especially when the birds are housed in closed quarters, leading to the rapid spread of disease. Thus, it is desirable to administer the CpG oligonucleotide and the non-nucleic acid adjuvant of the invention to birds to enhance an antigen-specific immune response when antigen is present. The CpG oligonucleotide and the non-nucleic acid adjuvant of the invention could also be administered to birds without antigen to protect against infection of a wide variety of pathogens.
  • An example of a common infection in chickens is chicken infectious anemia virus (CIAV). CIAV was first isolated in Japan in 1979 during an investigation of a Marek's disease vaccination break (Yuasa et al., 1979, Avian Dis. 23:366-385). Since that time, CIAV has been detected in commercial poultry in all major poultry producing countries (van Bulow et al., 1991, pp. 690-699) in Diseases of Poultry, 9th edition, Iowa State University Press).
  • CIAV infection results in a clinical disease, characterized by anemia, hemorrhage and immunosuppression, in young susceptible chickens. Atrophy of the thymus and of the bone marrow and consistent lesions of CIAV-infected chickens are also characteristic of CIAV infection. Lymphocyte depletion in the thymus, and occasionally in the bursa of Fabricius, results in immunosuppression and increased susceptibility to secondary viral, bacterial, or fungal infections which then complicate the course of the disease. The immunosuppression may cause aggravated disease after infection with one or more of Marek's disease virus (MDV), infectious bursal disease virus, reticuloendotheliosis virus, adenovirus, or reovirus. It has been reported that pathogenesis of MDV is enhanced by CIAV (DeBoer et al., 1989, p. 28 In Proceedings of the 38th Western Poultry Diseases Conference, Tempe, Ariz.). Further, it has been reported that CIAV aggravates the signs of infectious bursal disease (Rosenberger et al., 1989, Avian Dis. 33:707-713). Chickens develop an age resistance to experimentally induced disease due to CAA. This is essentially complete by the age of 2 weeks, but older birds are still susceptible to infection (Yuasa, N. et al., 1979 supra; Yuasa, N. et al., Arian Diseases 24, 202-209, 1980). However, if chickens are dually infected with CAA and an immunosuppressive agent (IBDV, MDV etc.) age resistance against the disease is delayed (Yuasa, N. et al., 1979 and 1980 supra; Bulow von V. et al., J. Veterinary Medicine 33, 93-116, 1986). Characteristics of CIAV that may potentiate disease transmission include high resistance to environmental inactivation and some common disinfectants. The economic impact of CIAV infection on the poultry industry is clear from the fact that 10% to 30% of infected birds in disease outbreaks die.
  • Vaccination of birds, like other vertebrate animals can be performed at any age. Normally, vaccinations are performed at up to 12 weeks of age for a live microorganism and between 14-18 weeks for an inactivated microorganism or other type of vaccine. For in ovo vaccination, vaccination can be performed in the last quarter of embryo development. The vaccine may be administered subcutaneously, by spray, orally, intraocularly, intratracheally, nasally, in ovo or by other methods described herein. Thus, the CpG oligonucleotide and non-nucleic acid adjuvant of the invention can be administered to birds and other non-human vertebrates using routine vaccination schedules and the antigen is administered after an appropriate time period as described herein.
  • Cattle and livestock are also susceptible to infection. Disease which affect these animals can produce severe economic losses, especially amongst cattle. The methods of the invention can be used to protect against infection in livestock, such as cows, horses, pigs, sheep, and goats. The CpG oligonucleotide and the non-nucleic acid adjuvant of the invention could also be administered with antigen for antigen-specific protection of long duration or without antigen for short term protection against a wide variety of diseases, including shipping fever.
  • Cows can be infected by bovine viruses. Bovine viral diarrhea virus (BVDV) is a small enveloped positive-stranded RNA virus and is classified, along with hog cholera virus (HOCV) and sheep border disease virus (BDV), in the pestivirus genus. Although, Pestiviruses were previously classified in the Togaviridae family, some studies have suggested their reclassification within the Flaviviridae family along with the flavivirus and hepatitis C virus (HCV) groups (Francki, et al., 1991).
  • BVDV, which is an important pathogen of cattle can be distinguished, based on cell culture analysis, into cytopathogenic (CP) and noncytopathogenic (NCP) biotypes. The NCP biotype is more widespread although both biotypes can be found in cattle. If a pregnant cow becomes infected with an NCP strain, the cow can give birth to a persistently infected and specifically immunotolerant calf that will spread virus during its lifetime. The persistently infected cattle can succumb to mucosal disease and both biotypes can then be isolated from the animal. Clinical manifestations can include abortion, teratogenesis, and respiratory problems, mucosal disease and mild diarrhea. In addition, severe thrombocytopenia, associated with herd epidemics, that may result in the death of the animal has been described and strains associated with this disease seem more virulent than the classical BVDVs.
  • Equine herpesviruses (EHV) comprise a group of antigenically distinct biological agents which cause a variety of infections in horses ranging from subclinical to fatal disease. These include Equine herpesvirus-1 (EHV-1), a ubiquitous pathogen in horses. EHV-1 is associated with epidemics of abortion, respiratory tract disease, and central nervous system disorders. Primary infection of upper respiratory tract of young horses results in a febrile illness which lasts for 8 to 10 days. Immunologically experienced mares may be reinfected via the respiratory tract without disease becoming apparent, so that abortion usually occurs without warning. The neurological syndrome is associated with respiratory disease or abortion and can affect animals of either sex at any age, leading to incoordination, weakness and posterior paralysis (Telford, E. A. R. et al., Virology 189, 304-316, 1992). Other EHV's include EHV-2, or equine cytomegalovirus, EHV-3, equine coital exanthema virus, and EHV-4, previously classified as EHV-1 subtype 2.
  • Sheep and goats can be infected by a variety of dangerous microorganisms including visna-maedi.
  • Primates such as monkeys, apes and macaques can be infected by simian immunodeficiency virus. Inactivated cell-virus and cell-free whole simian immunodeficiency vaccines have been reported to afford protection in macaques (Stott et al. (1990) Lancet 36:1538-1541; Desrosiers et al. PNAS USA (1989) 86:6353-6357; Murphey-Corb et al. (1989) Science 246:1293-1297; and Carlson et al. (1990) AIDS Res. Human Retroviruses 6:1239-1246). A recombinant HIV gp120 vaccine has been reported to afford protection in chimpanzees (Berman et al. (1990) Nature 345:622-625).
  • Cats, both domestic and wild, are susceptible to infection with a variety of microorganisms. For instance, feline infectious peritonitis is a disease which occurs in both domestic and wild cats, such as lions, leopards, cheetahs, and jaguars. When it is desirable to prevent infection with this and other types of pathogenic organisms in cats, the methods of the invention can be used to vaccinate cats to prevent them against infection.
  • Domestic cats may become infected with several retroviruses, including but not limited to feline leukemia virus (FeLV), feline sarcoma virus (FeSV), endogenous type C oncornavirus (RD-114), and feline syncytia-forming virus (FeSFV). Of these, FeLV is the most significant pathogen, causing diverse symptoms, including lymphoreticular and myeloid neoplasms, anemias, immune mediated disorders, and an immunodeficiency syndrome which is similar to human acquired immune deficiency syndrome (AIDS). Recently, a particular replication-defective FeLV mutant, designated FeLV-AIDS, has been more particularly associated with immunosuppressive properties.
  • The discovery of feline T-lymphotropic lentivirus (also referred to as feline immunodeficiency) was first reported in Pedersen et al. (1987) Science 235:790-793. Characteristics of FIV have been reported in Yamamoto et al. (1988) Leukemia, December Supplement 2:204S-215S; Yamamoto et al. (1988) Am. J. Vet. Res. 49:1246-1258; and Ackley et al. (1990) J. Virol. 64:5652-5655. Cloning and sequence analysis of FIV have been reported in Olmsted et al. (1989) Proc. Natl. Acad. Sci. USA 86:2448-2452 and 86:4355-4360.
  • Feline infectious peritonitis (FIP) is a sporadic disease occurring unpredictably in domestic and wild Felidae. While FIP is primarily a disease of domestic cats, it has been diagnosed in lions, mountain lions, leopards, cheetahs, and the jaguar. Smaller wild cats that have been afflicted with FIP include the lynx and caracal, sand cat, and pallas cat. In domestic cats, the disease occurs predominantly in young animals, although cats of all ages are susceptible. A peak incidence occurs between 6 and 12 months of age. A decline in incidence is noted from 5 to 13 years of age, followed by an increased incidence in cats 14 to 15 years old.
  • Viral, bacterial and parasitic diseases in fin-fish, shellfish or other aquatic life forms pose a serious problem for the aquaculture industry. Owing to the high density of animals in the hatchery tanks or enclosed marine farming areas, infectious diseases may eradicate a large proportion of the stock in, for example, a fin-fish, shellfish, or other aquatic life forms facility. Prevention of disease is a more desired remedy to these threats to fish than intervention once the disease is in progress. Vaccination of fish is the only preventative method which may offer long-term protection through immunity. Fish are currently protected against a variety of bacterial infections with whole killed vaccines with oli adjuvants, but there is only one licensed vaccine for fish against a viral disease. Nucleic acid based vaccinations are described in U.S. Pat. No. 5,780,448 issued to Davis and these have been shown to be protective against at least two different viral diseases.
  • The fish immune system has many features similar to the mammalian immune system, such as the presence of B cells, T cells, lymphokines, complement, and immunoglobulins. Fish have lymphocyte subclasses with roles that appear similar in many respects to those of the B and T cells of mammals. Vaccines can be administered orally or by immersion or injection.
  • Aquaculture species include but are not limited to fin-fish, shellfish, and other aquatic animals. Fin-fish include all vertebrate fish, which may be bony or cartilaginous fish, such as, for example, salmonids, carp, catfish, yellowtail, seabream, and seabass. Salmonids are a family of fin-fish which include trout (including rainbow trout), salmon, and Arctic char. Examples of shellfish include, but are not limited to, clams, lobster, shrimp, crab, and oysters. Other cultured aquatic animals include, but are not limited to eels, squid, and octopi.
  • Polypeptides of viral aquaculture pathogens include but are not limited to glycoprotein (G) or nucleoprotein (N) of viral hemorrhagic septicemia virus (VHSV); G or N proteins of infectious hematopoietic necrosis virus (IHNV); VP1, VP2, VP3 or N structural proteins of infectious pancreatic necrosis virus (IPNV); G protein of spring viremia of carp (SVC); and a membrane-associated protein, tegumin or capsid protein or glycoprotein of channel catfish virus (CCV).
  • Polypeptides of bacterial pathogens include but are not limited to an iron-regulated outer membrane protein, (IROMP), an outer membrane protein (OMP), and an A-protein of Aeromonis salmonicida which causes furunculosis, p57 protein of Renibacterium salmoninarum which causes bacterial kidney disease (BKD), major surface associated antigen (msa), a surface expressed cytotoxin (mpr), a surface expressed hemolysin (ish), and a flagellar antigen of Yersiniosis; an extracellular protein (ECP), an iron-regulated outer membrane protein (IROMP), and a structural protein of Pasteurellosis; an OMP and a flagellar protein of Vibrosis anguillarum and V. ordalii; a flagellar protein, an OMP protein,aroA, and purA of Edwardsiellosis ictaluri and E. tarda; and surface antigen of Ichthyophthirius; and a structural and regulatory protein of Cytophaga columnari; and a structural and regulatory protein of Rickettsia.
  • Polypeptides of a parasitic pathogen include but are not limited to the surface antigens of Ichthyophthirius.
  • An “allergen” refers to a substance (antigen) that can induce an allergic or asthmatic response in a susceptible subject. The list of allergens is enormous and can include pollens, insect venoms, animal dander 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 genuses: Canine (Canis familiaris); Dermatophagoides (e.g. Dermatophagoides farinae); Felis (Felis domesticus); Ambrosia (Ambrosia artemiisfolia; Lolium (e.g. Lolium perenne or Lolium multiflorum); Cryptomeria (Cryptomeria japonica); Alternaria (Alternaria alternata); Alder; Alnus (Alnus gultinoasa); Betula (Betula verrucosa); Quercus (Quercus alba); Olea (Olea europa); Artemisia (Artemisia vulgaris); Plantago (e.g. Plantago lanceolata); Parielaria (e.g. Parielaria officinalis or Parietaria judaica); Blattella (e.g. Blattella germanica); Apis (e.g. Apis multiflorum); Cupressus (e.g. Cupressus sempervirens, Cupressus arizonica and Cupressus macrocarpa); Juniperus (e.g. Juniperus sabinoides, Juniperus virginiana, Juniperus communis and Juniperus ashei); Thuya (e.g. Thuya orientalis); Chamaecyparis (e.g. Chamaecyparis obtusa); Periplaneta (e.g. Periplaneta americana); Agropyron (e.g. Agropyron repens); Secale (e.g. Secale cereale); Triticum (e.g. Triticum aestivum); Dactylis (e.g. Dactylis glomerata); Festuca (e.g. Festuca elatior); Poa (e.g. Poa pratensis or Poa compressa); Avena (e.g. Avena sativa); Holcus (e.g. Holcus lanatus); Anthoxanthum (e.g. Anthoxanthum odoratum); Arrhenatherum (e.g. Arrhenatherum elatius); Agrostis (e.g. Agrostis alba); Phleum (e.g. Phleum pratense); Phalaris (e.g. Phalaris arundinacea); Paspalum (e.g. Paspalum notalum); Sorghum (e.g. Sorghum halepensis); and Bromus (e.g. Bromus inermis).
  • In some aspects of the invention the antigen is a polypeptide. Minor modifications of the primary amino acid sequences of polypeptide antigens may also result in a polypeptide which has substantially equivalent antigenic activity as compared to the unmodified counterpart polypeptide. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. All of the polypeptides produced by these modifications are included herein as long as antigenicity still exists. The polypeptide may be, for example, a viral polypeptide. One non-limiting example of an antigen useful according to the invention is the hepatitis B surface antigen. Experiments using this antigen are described in the Examples below.
  • The term “substantially purified” as used herein refers to a polypeptide which is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. One skilled in the art can purify viral or bacterial polypeptides using standard techniques for protein purification. The substantially pure polypeptide will often yield a single major band on a non-reducing polyacrylamide gel. In the case of partially glycosylated polypeptides or those that have several start codons, there may be several bands on a non-reducing polyacrylamide gel, but these will form a distinctive pattern for that polypeptide. The purity of the viral or bacterial polypeptide can also be determined by amino-terminal amino acid sequence analysis.
  • The invention also utilizes polynucleotides encoding the antigenic polypeptides. It is envisioned that the antigen may be delivered to the subject in a nucleic acid molecule which encodes for the antigen such that the antigen must be expressed in vivo. The nucleic acid encoding the antigen is operatively linked to a gene expression sequence which directs the expression of the antigen nucleic acid within a eukaryotic cell. The “gene expression sequence” is any regulatory nucleotide sequence, such as a promoter sequence or. promoter-enhancer combination, which facilitates the efficient transcription and translation of the antigen nucleic acid to which it is operatively linked. The gene expression sequence may, for example, be a mammalian or viral promoter, such as a constitutive or inducible promoter. Constitutive mammalian promoters include, but are not limited to, the promoters for the following genes: hypoxanthine phosphoribosyl transferase (HPTR), adenosine deaminase, pyruvate kinase, $-actin promoter, muscle creatine kinase promoter, human elongation factor promoter and other constitutive promoters. Exemplary viral promoters which function constitutively in eukaryotic cells include, for example, promoters from the simian virus (e.g., SV40), papilloma virus, adenovirus, human immunodeficiency virus (HIV), rous sarcoma virus, cytomegalovirus (CMV), Rous sarcoma virus (RSV), hepatitis B virus (HBV), the long terminal repeats (LTR) of Moloney leukemia virus and other retroviruses, and the thymidine kinase promoter of herpes simplex virus. Other constitutive promoters are known to those of ordinary skill in the art. The promoters useful as gene expression sequences of the invention also include inducible promoters. Inducible promoters are expressed in the presence of an inducing agent. For example, the metallothionein promoter is induced to promote transcription and translation in the presence of certain metal ions. Other inducible promoters are known to those of ordinary skill in the art.
  • In general, the gene expression sequence shall include, as necessary, 5′ non-transcribing and 5′ non-translating sequences involved with the initiation of transcription and translation, respectively, such as a TATA box, capping sequence, CAAT sequence, and the like. Especially, such 5′ non-transcribing sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined antigen nucleic acid. The gene expression sequences optionally include enhancer sequences or upstream activator sequences as desired.
  • The antigen nucleic acid is operatively linked to the gene expression sequence. As used herein, the antigen nucleic acid sequence and the gene expression sequence are said to be “operably linked” when they are covalently linked in such a way as to place the expression or transcription and/or translation of the antigen coding sequence under the influence or control of the gene expression sequence. Two DNA sequences are said to be operably linked if induction of a promoter in the 5′ gene expression sequence results in the transcription of the antigen sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the antigen sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a gene expression sequence would be operably linked to an antigen nucleic acid sequence if the gene expression sequence were capable of effecting transcription of that antigen nucleic acid sequence such that the resulting transcript is translated into the desired protein or polypeptide.
  • The antigen nucleic acid sequence may encode a protein, polypeptide, peptide, or peptide mimic of a polysaccharide. It may also cncode more than one antigenic component as a fusion construct. More than one antigen-encoding sequence may be included in the same plasmid vector and these may be linked to the same or different gene expression sequences.
  • The antigen nucleic acid of the invention may be delivered to the immune system alone or in association with a vector. In its broadest sense, a “vector” is any vehicle capable of facilitating the transfer of the antigen nucleic acid to the cells of the immune system and preferably APCs so that the antigen can be expressed and presented on the surface of an APC. Preferably, the vector transports the nucleic acid to the immune cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. The vector optionally includes the above-described gene expression sequence to enhance expression of tne antigen nucleic acid in APCs. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antigen nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.
  • Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses, the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in Kriegler, M., “Gene Transfer and Expression, A Laboratory Manual”, W.H. Freeman C.O., New York (1990) and Murry, E. J. Ed. “Methods in Molecular Biology”, vol. 7, Humana Press, Inc., Cliffton, N.J. (1991).
  • Preferred virus for certain applications are the adeno-virus and adeno-associated virus which are double-stranded DNA viruses that have already been approved for human use in gene therapy and immunotherapy trials. The adeno-associated virus can be engineered to be replication-deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the aueno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion.
  • Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well-known to those of skill in the art. See e.g., Sanbrook et al., “Molecular Cloning: A Laboratory Manual”, Second Edition, Cold Spring Harbor Laboratory Press, 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen-encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, and pBlueScript. Other plasmids are well-known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids such as those used for DNA vaccines may be delivered by a variety of parenteral, mucosal and topical routes. For example the plasmid DNA can be injected by intramuscular, intradermal, subcutaneous or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation.
  • It has recently been discovered that gene carrying plasmids can be delivered to the immune system using bacteria. Modified forms of bacteria such as Salmonella can be transfected with the plasmid and used as delivery vehicles. The bacterial delivery vehicles can be administered to a host subject orally or by other administration means. The bacteria deliver the plasmid to immune cells, e.g. dendritic cells, probably by passing through the gut barrier. High levels of immune protection have been established using this methodology.
  • In other aspects the invention includes a method for immunizing an infant by administering to an infant an antigen and an oligonucleotide containing at least one unmethylated CpG dinucleotide in an effective amount for inducing cell mediated immunity in the infant. In some embodiments the infant is also administered at least one non-nucleic acid adjuvant, as described above. Cell mediated immunity, as used herein, refers to an immune response which involves an antigen specific T cell reaction. The presence of cell mediated immunity can be determined directly by the induction of Th1 cytokines (e.g., IFN-γ, IL-12) and antigen-specific cytotoxic T-cell lymphocytes (CTL). The presence of cell mediated immunity is also indicated indirectly by the isotype of antigen-specific antibodies that are induced (e.g. IgG2a>>IgG1 in mice). Thus, if Th1 cytokines or CTL or TH1-like antibodies are induced, cell mediated immunity is induced according to the invention. As discussed above, Th1 cytokines include but are not limited to IL-12 and IFN-γ.
  • Neonates (newborn) and infants (which include humans three months of age and referred to hereinafter as infants) born in HBV endemic areas require particularly rapid induction of strong HBV-specific immunity owing to the high rate of chronicity resulting from infection at a young age. Without immunoprophylaxis, 70-90% of infants born to mothers positive for both HBsAg and the “e” antigen (HBeAg) become infected and almost all of these become chronic carriers (Stevens et al., 1987). Even when vaccinated with a four dose regime of the HBV subunit vaccine commencing on the day of birth, 20% of such infants became chronically infected and this was reduced to only 15% if they were also given HBV-specific immunoglobulin (Chen et al., 1996). HBV chronicity results in 10-15% of individuals infected as adolescents or adults, but 90-95% for those infected (either vertically or horizontally) as infants. CpG oligonucleotides may be used, according to the invention, to reduce this further owing to a more rapid appearance and higher titers of anti-HBs antibodies and the induction of HBV-specific CTL, which could help clear virus from the liver of babies infected in utero, and which likely account for most of the failures with infant vaccination.
  • The invention further provides a method of modulating the level of a cytokine. The term “modulate” envisions the suppression of expression of a particular cytokine when lower levels are desired, or augmentation of the expression of a particular cytokine when higher levels are desired. Modulation of a particular cytokine can occur locally or systemically. CpG oligonucleotides can directly activate macrophages and dendritic cells to secrete cytokines. No direct activation of proliferation or cytokine secretion by highly purified T cells has been found, although they are induced to secrete cytokines by cytokines secreted from macrophages and may be costimulated through the T cell Receptor. Cytokine profiles determine T cell regulatory and effector functions in immune responses. In general, Th1-type cytokines are induced, thus the immunostimulatory nucleic acids promote a Th1 type antigen-specific immune response including cytotoxic T-cells.
  • Cytokines also play a role in direciing the T cell response. Helper (CD4+) T cells orchestrate the immune response of mammals through production of soluble factors that act on other immune system cells, including B and other T cells. Most mature CD4+ T helper cells express one of two cytokine profiles: Th1 or Th2. Th1 cells secrete IL-2, IL-3, IFN-γ, GM-CSF and high levels of TNF-α. Th2 cells express IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, GM-CSF and low levels of TNF-α. The Th1 subset promotes both cell-mediated immunity, and humoral immunity that is characterized by immunoglobulin class switching to IgG2a in mice. Th1 responses may also be associated with delayed-type hypersensitivity and autoimmune disease. The Th2 subset induces primarily humoral immunity and induce class switching to IgG1 and IgE. The antibody isotypes associated with Th1 responses generally have good neutralizing and opsonizing capabilities whereas those associated with Th2 responses are associated more with allergic responses.
  • Several factors have been shown to influence commitment to Th1 or Th2 profiles. The best characterized regulators are cytokines. IL-12 and IFN-γ are positive Th1 and negative Th2 regulators. IL-12 promotes IFN-γ production, and IFN-γ provides positive feedback for IL-12. IL-4 and IL-10 appear to be required for the establishment of the Th2 cytokine profile and to down-regulate Th1 cytokine production; the effects of IL-4 are in some cases dominant over those of IL-12; IL-13 was shown to inhibit expression of inflammatory cytokines, including IL-12 and TNF-α by LPS-induced monocytes, in a way similar to IL-4. The IL-12 p40 homodimer binds to the IL-12 receptor and may antagonizes IL-12 biological activity; thus it blocks the pro-Th1 effects of IL-12 in some animals.
  • In other aspects the invention includes a method of inducing a Th1 immune response in a subject by administering to the subject a combination of adjuvants in an effective amount for inducing a Th1 immune response. The combination of adjuvants includes at least one oligonucleotide containing at least one unmethylated CpG dinucleotide and at least one non-nucleic acid adjuvant. It was not previously known that when CpG was combined with a non-nucleic acid adjuvant, as described above, that the combination would produce an immune response with a Th1 profile to an extent that the individual adjuvants could not produce alone. Preferably the extent of the Th profile produced by the combination of adjuvants is synergistic. Another aspect of the invention is to induce a Th response by using CPG with a non-nucleic acid adjuvant that by itself induces a Th2 response.
  • As described above a Th2 profile is characterized by production of IL-4 and IL-10. Non-nucleic acid adjuvants that induce Th2 or weak Th1 responses include but are not limited to alum, saponins, oil-in-water and other emulsion formulations and SB-As4. Adjuvants that induce Th1 responses include but are not limited to MPL, MDP, ISCOMS, IL-12, IFN-γ, and SB-AS2. When the CpG oligonucleotide is administered with a non-nucleic acid adjuvant the combination of adjuvants causes a commitment to a Th1 profile, that neither the adjuvant nor the CpG oligonucleotide is capable of producing on its own. Furthermore, if the non-nucleic acid adjuvant on its own induces a Th2 response, the addition of CpG oligonucleotide can overcome this Th2 bias and induce a Th1 response that may be even more Th1-like than with CpG alone.
  • The combination of adjuvants may be administered simultaneously or sequentially. When the adjuvants are administered simultaneously they can be administered in the same or separate formulations, and in the latter case at the same or separate sites, but are administered at the same time. The adjuvants are administered sequentially, when the administration of the at least two adjuvants is temporally separated. The separation in time between the administration of the two adjuvants may be a matter of minutes or it may be longer. The separation in time is less than 14 days, and more preferably less than 7 days, and most preferably less than 1 day. The separation in time may also be with one adjuvant at prime and one at boost, or one at prime and the combination at boost, or the combination at prime and one at boost.
  • For use in the instant invention, the nucleic acids can be synthesized de novo using any of a number of procedures well known in the art. For example, the b-cyanoethyl phosphoramidite method (Beaucage, S. L., and Caruthers, M. H., Tet. Let. 22:1859, 1981); nucleoside H-phosphonate method (Garegg et al., Tet. Let. 27:4051-4054, 1986; Froehler et al., Nucl. Acid. Res. 14:5399 5407, 1986,; Garegg et al., Tet. Let. 27:4055-4058, 1986, Gaffney et al., Tet. Let. 29:2619-2622, 1988). These chemistries can be performed by a variety of automated oligonucleotide synthesizers available in the market. Alternatively, CpG dinucleotides can be produced on a large scale in plasmids, (see Sambrook, T., et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor laboratory Press, New York, 1989) which after being administered to a subject are degraded into oligonucleotides. Such plasmids may also encode other genes to be expressed such as an antigen-encoding gene in the case of a DNA vaccine. Oligonucleotides can be prepared from existing nucleic acid sequences (e.g., genomic or cDNA) using known techniques, such as those employing restriction enzymes, exonucleases or endonucleases.
  • For use in vivo, nucleic acids are preferably relatively resistant to degradation (e.g., via endo-and exo-nucleases). Secondary structures, such as stem loops, can stabilize nucleic acids against degradation. Alternatively, nucleic acid stabilization can be accomplished via phosphate backbone modifications. A preferred stabilized nucleic acid has at least a partial phosphorothioate modified backbone. Phosphorothioates may be synthesized using automated techniques employing either phosphoramidate or H-phosphonate chemistries. Aryl-and alkyl-phosphonates can be made, e.g., 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. 092,574) can be prepared by automated solid phase synthesis using commercially available reagents. Methods for making other DNA backbone modifications and substitutions have been described (Uhlmann, E. and Peyman, A., Chem. Rev. 90:544, 1990; Goodchild, J., Bioconjugate Chem. 1:165, 1990).
  • For administration in vivo, nucleic acids may be associated with a molecule that results in higher affinity binding to target cell (e.g., B-cell, monocytic cell and natural killer (NK) cell) surfaces and/or increased cellular uptake by target cells to form a “nucleic acid delivery complex.” Nucleic acids can be ionically or covalently associated with appropriate molecules using techniques which are well known in the art. A variety of coupling or cross-linking agents can be used, e.g., protein A, carbodiimide, and N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP). Nucleic acids can alternatively be encapsulated in liposomes or virosomes using well-known techniques.
  • Nucleic acids containing an appropriate unmethylated CpG can be effective in any mammal, preferably a human. Different nucleic acids containing an unmethylated CpG can cause optimal immune stimulation depending on the mammalian species. Thus an oligonucleotide causing optimal stimulation in humans may not cause optimal stimulation in a mouse and vice versa. One of skill in the art can identify the optimal oligonucleotides useful for a particular mammalian species of interest using routine assays described herein and/or known in the art.
  • The CpG ODN of the invention stimulate cytokine production (e.g., IL-6, IL-12, IFN-γ, TNF-α and GM-CSF) and B-cell proliferation in PBMC's taken from a subject such as a human. Specific, but nonlimiting examples of such sequences include those presented in Table 1 below:
    TABLE 1
    sequences
    GCTAGACGTTAGCGT; (SEQ ID NO: 1)
    GCTAGATGTTAGCGT; (SEQ ID NO: 2)
    GCTAGACGTTAGCGT; (SEQ ID NO: 3)
    GCTAGACGTTAGCGT; (SEQ ID NO: 4)
    GCATGACGTTGAGCT; (SEQ ID NO: 5)
    ATGGAAGGTCCAGCGTTCTC; (SEQ ID NO: 6)
    ATCGACTCTCGAGCGTTCTC; (SEQ ID NO: 7)
    ATCGACTCTCGAGCGTTCTC; (SEQ ID NO: 8)
    ATCGACTCTCGAGCGTTCTC; (SEQ ID NO: 9)
    ATGGAAGGTCCAACGTTCTC; (SEQ ID NO: 10)
    GAGAACGCTGGACCTTCCAT; (SEQ ID NO: 11)
    GAGAACGCTCGACCTTCCAT; (SEQ ID NO: 12)
    GAGAACGCTCGACCTTCGAT; (SEQ ID NO: 13)
    GAGAACGCTGGACCTTCCAT; (SEQ ID NO: 14)
    GAGAACGATGGACCTTCCAT; (SEQ ID NO: 15)
    GAGAACGCTCCAGCACTGAT; (SEQ ID NO: 16)
    TCCATGTCGGTCCTGATGCT; (SEQ ID NO: 17)
    TCCATGTCGGTCCTGATGCT; (SEQ ID NO: 18)
    TCCATGACGTTCCTGATGCT; (SEQ ID NO: 19)
    TCCATGTCGGTCCTGCTGAT; (SEQ ID NO: 20)
    TCAACGTT; (SEQ ID NO: 21)
    TCAGCGCT; (SEQ ID NO: 22)
    TCATCGAT; (SEQ ID NO: 23)
    TCTTCGAA; (SEQ ID NO: 24)
    CAACGTT; (SEQ ID NO: 25)
    CCAACGTT; (SEQ ID NO: 26)
    AACGTTCT; (SEQ ID NO: 27)
    TCAACGTC; (SEQ ID NO: 28)
    ATGGACTCTCCAGCGTTCTC; (SEQ ID NO: 29)
    ATGGAAGGTCCAACGTTCTC; (SEQ ID NO: 30)
    ATCGACTCTCGAGCGTTCTC; (SEQ ID NO: 31)
    ATGGAGGCTCCATCGTTCTC; (SEQ ID NO: 32)
    ATCGACTCTCGAGCGTTCTC; (SEQ ID NO: 33)
    ATCGACTCTCGAGCGTTCTC; (SEQ ID NO: 34)
    TCCATGTCGGTCCTGATGCT; (SEQ ID NO: 35)
    TCCATGCCGGTCCTGATGCT; (SEQ ID NO: 36)
    TCCATGGCGGTCCTGATGCT; (SEQ ID NO: 37)
    TCCATGACGGTCCTGATGCT; (SEQ ID NO: 38)
    TCCATGTCGATCCTGATGCT; (SEQ ID NO: 39)
    TCCATGTCGCTCCTGATGCT; (SEQ ID NO: 40)
    TCCATGTCGTCCCTGATGCT; (SEQ ID NO: 41)
    TCCATGACGTGCCTGATGCT; (SEQ ID NO: 42)
    TCCATAACGTTCCTGATGCT; (SEQ ID NO: 43)
    TCCATGACGTCCCTGATGCT; (SEQ ID NO: 44)
    TCCATCACGTGCCTGATGCT; (SEQ ID NO: 45)
    GGGGTCAACGTTGACGGGG; (SEQ ID NO: 46)
    GGGGTCAGTCGTGACGGGG; (SEQ ID NO: 47)
    GCTAGACGTTAGTGT; (SEQ ID NO: 48)
    TCCATGTCGTTCCTGATGCT; (SEQ ID NO: 49)
    ACCATGGACGATCTGTTTCCCCTC; (SEQ ID NO: 50)
    TCTCCCAGCGTGCGCCAT; (SEQ ID NO: 51)
    ACCATGGACGAACTGTTTCCCCTC; (SEQ ID NO: 52)
    ACCATGGACGAGCTGTTTCCCCTC; (SEQ ID NO: 53)
    ACCATGGACGACCTGTTTCCCCTC; (SEQ ID NO: 54)
    ACCATGGACGTACTGTTTCCCCTC; (SEQ ID NO: 55)
    ACCATGGACGGTCTGTTTCCCCTC; (SEQ ID NO: 56)
    ACCATGGACGTTCTGTTTCCCCTC; (SEQ ID NO: 57)
    CACGTTGAGGGGCAT; (SEQ ID NO: 58)
    TCAGCGTGCGCC; (SEQ ID NO: 59)
    ATGACGTTCCTGACGTT; (SEQ ID NO: 60)
    TCTCCCAGCGGGCGCAT; (SEQ ID NO: 61)
    TCCATGTCGTTCCTGTCGTT; (SEQ ID NO: 62)
    TCCATAGCGTTCCTAGCGTT; (SEQ ID NO: 63)
    TCGTCGCTGTCTCCCCTTCTT; (SEQ ID NO: 64)
    TCCTGACGTTCCTGACGTT; (SEQ ID NO: 65)
    TCCTGTCGTTCCTGTCGTT; (SEQ ID NO: 66)
    TCCATGTCGTTTTTGTCGTT; (SEQ ID NO: 67)
    TCCTGTCGTTCCTTGTCGTT; (SEQ ID NO: 68)
    TCCTTGTCGTTCCTGTCGTT; (SEQ ID NO: 69)
    TCCTGTCGTTTTTTGTCGTT; (SEQ ID NO: 70)
    TCGTCGCTGTCTGCCCTTCTT; (SEQ ID NO: 71)
    TCGTCGCTGTTGTCGTTTCTT; (SEQ ID NO: 72)
    TCCATGCGTGCGTGCGTTTT; (SEQ ID NO: 73)
    TCCATGCGTTGCGTTGCGTT; (SEQ ID NO: 74)
    TCCACGACGTTTTCGACGTT; (SEQ ID NO: 75)
    TCGTCGTTGTCGTTGTCGTT; (SEQ ID NO: 76)
    TCGTCGTTTTGTCGTTTTGTCGTT; (SEQ ID NO: 77)
    TCGTCGTTGTCGTTTTGTCGTT; (SEQ ID NO: 78)
    GCGTGCGTTGTCGTTGTCGTT; (SEQ ID NO: 79)
    TGTCGTTTGTCGTTTGTCGTT; (SEQ ID NO: 80)
    TGTCGTTGTCGTTGTCGTTGTCGTT; (SEQ ID NO: 81)
    TGTCGTTGTCGTTGTCGTT; (SEQ ID NO: 82)
    TCGTCGTCGTCGTT; (SEQ ID NO: 83)
    TGTCGTTGTCGTT; (SEQ ID NO: 84)
    TCCATAGCGTTCCTAGCGTT; (SEQ ID NO: 85)
    TCCATGACGTTCCTGACGTT; (SEQ ID NO: 86)
    GTCGYT; (SEQ ID NO: 87)
    TGTCGYT; (SEQ ID NO: 88)
    AGCTATGACGTTCCAAGG; (SEQ ID NO: 89)
    TCCATGACGTTCCTGACGTT; (SEQ ID NO: 90)
    ATCGACTCTCGAACGTTCTC; (SEQ ID NO: 91)
    TCCATGTCGGTCCTGACGCA; (SEQ ID NO: 92)
    TCTTCGAT; (SEQ ID NO: 93)
    ATAGGAGGTCCAACGTTCTC; (SEQ ID NO: 94)
    GTCGTT (SEQ ID NO: 95)
    GTCGTC (SEQ ID NO: 96)
    TGTCGTT (SEQ ID NO: 97)
    TGTCGCT (SEQ ID NO: 98)
  • Preferred CpG ODN can effect at least about 500 pg/ml of TNF-α, 15 pg/ml IFN-γ, 70 pg/ml of GM-CSF 275 pg/ml of IL-6, 200 pg/ml IL-12, depending on the therapeutic indication. These cytokines can be measured by assays well known in the art. The oligonucleotides listed above or other preferred CpG ODN can effect at least about 10%, more preferably at least about 15% and most preferably at least about 20% YAC-1 cell specific lysis or at least about 30%, more preferably at least about 35%, and most preferably at least about 40% 2C11 cell specific lysis, in assays well known in the art.
  • The term “effective amount” of a CpG oligonucleotide refers to the amount necessary or sufficient to realize a desired biologic effect. For example, an effective amount of an oligonucleotide containing at least one unmethylated CpG and a non-nucleic acid adjuvant for treating an infectious disorder is that amount necessary to cause the development of an antigen specific immune response upon exposure to the microbe, thus causing a reduction in the amount of microbe within the subject and preferably to the eradication of the microbe. The effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular CpG oligonucleotide being administered (e.g. the number of unmethylated CpG motifs or their location in the nucleic acid), 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 adjuvant and antigen without necessitating undue experimentation.
  • The formulations of the invention are administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.
  • For use in therapy, an effective amount of the adjuvant combination can be administered to a subject by any mode allowing the oligonucleotide to be taken up by the appropriate target cells. “Administering” the pharmaceutical composition of the present invention may be accomplished by any means known to the skilled artisan. Preferred routes of administration include but are not limited to oral, transdermal (e.g. via a patch), parenteral injection (subcutaneous, intradermal, intravenous, parenteral, intraperitoneal, intrathecal, etc.), or mucosal intranasal, intratracheal, inhalation, and intrarectal, intravaginal etc). An injection may be in a bolus or a continuous infusion.
  • For example the pharmaceutical compositions according to the invention are often administered by intramuscular or intradermal injection, or other parenteral means, or by biolistic “gene-gun”application to the epidermis. They may also be administered by intranasal application, inhalation, topically, intravenously, orally, or as implants, and even rectal or vaginal use is possible. Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions for injection or inhalation, microencapsulated, encochleated, coated onto microscopic gold particles, contained in liposomes, nebulized, aerosols, pellets for implantation into the skin, or dried onto a sharp object to be scratched into the skin. The pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above. The pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of present methods for drug delivery, see Langer, Science 249:1527-1533, 1990, which is incorporated herein by reference.
  • The pharmaceutical compositions are preferably prepared and administered in dose units. Liquid dose units are vials or ampoules for injection or other parenteral administration. Solid dose units are tablets, capsules and suppositories. For treatment of a patient, depending on activity of the compound, manner of administration, purpose of the immunization (i.e., prophylactic or therapeutic), nature and severity of the disorder, age and body weight of the patient, different doses may be necessary. The administration of a given dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units. Multiple administration of doses at specific intervals of weeks or months apart is usual for boosting the antigen-specific responses.
  • The adjuvants and antigens may be administered per se (neat) or in the form of a pharmaceutically acceptable salt. When used in medicine the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof. Such salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric, methane sulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, and benzene sulphonic. Also, such salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.
  • Suitable buffering agents include: acetic acid and a salt (1-2% w/v); citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v); and phosphoric acid and a salt (0.8-2% w/v). Suitable preservatives include benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9% w/v); parabens (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 a combination of adjuvants and antigens optionally included in a pharmaceutically-acceptable carrier. The term “pharmaceutically-acceptable carrier” means one or more compatible solid or liquid filler, dilutants or encapsulating substances which are suitable for administration to a human or other vertebrate animal. The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being comingled with the compounds of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency.
  • Compositions suitable for parenteral administration conveniently comprise sterile aqueous preparations, which can be isotonic with the blood of the recipient. Among the acceptable vehicles and solvents are water, Ringer's solution, phosphate buffered saline and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed mineral or non-mineral oil may be employed including synthetic mono-ordi-glycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. Carrier formulations suitable for subcutaneous, intramuscular, intraperitoneal, intravenous, etc. administrations may be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa.
  • The adjuvants or antigens useful in the invention may be delivered in mixtures of more than two adjuvants or antigens. A mixture may consist of several adjuvants in addition to the synergistic combination of adjuvants or several antigens.
  • A variety of administration routes are available. The particular mode selected will depend, of course, upon the particular adjuvants or antigen selected, the age and general health status of the subject, the particular condition being treated and the dosage required for therapeutic efficacy. The methods of this invention, generally speaking, may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of an immune response without causing clinically unacceptable adverse effects. Preferred modes of administration are discussed above.
  • The compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the compounds into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the compounds into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
  • Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the compounds, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Pat. No. 5,075,109. Delivery systems also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-di-and tri-glycerides; hydrogel release systems; sylastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to: (a) erosional systems in which an agent of the invention is contained in a form within a matrix such as those described in U.S. Pat. Nos. 4,452,775,4,675,189, and 5,736,152, and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Pat. Nos. 3,854,480, 5,133,974 and 5,407,686. In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation.
  • The present invention is further illustrated by the following Examples, which in no way should be construed as further limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference.
  • EXAMPLES
  • The use of CpG ODN as an adjuvant alone or in combination with other adjuvants was evaluated. The hepatitis B virus surface antigen (HBsAg) given as a recombinant protein or expressed in vivo from a DNA vaccine was used as an exemplary model system in the Examples set forth below.
  • Materials and Methods
  • Animals
  • Experiments on adult mice were carried out using female BALB/c mice (Charles River, Montreal, QC) at 6-8 weeks of age.
  • Newborn mice were obtained through breeding male and female BALB/c mice (Charles River) in the Loeb animal facility (Loeb Health Research Institute, The Ottawa Hospital, Ottawa, ON). Pregnant females were monitored daily to ensure accurate recording of the date of birth. Both male and female neonates were used for immunization.
  • Cynomolgus monkeys (1.5-3 kg) were housed at the Primate Research Center, Bogor, Indonesia.
  • Orantutans (5-20kg) were housed at Wanariset Station for the Orangutan Reintroduction Program of the Indonesian government, Balikpapan, Kalimantan.
  • HBsAg Subunit Vaccination of Mice
  • The subunit vaccine consisted of HBsAg (ay subtype) which had been produced as a recombinant protein in yeast cells (Medix Biotech #ABH0905). This was diluted in saline for use without adjuvant. HBsAg was also formulated with alum and/or CpG ODN as adjuvant. HBsAg protein was mixed with aluminum hydroxide (Alhydrogel 85, [Al2O3], Superfos Biosector, Vedbaek, Denmark) in the same ratio of 25 mg Al3+ per mg protein as used in the commercial vaccines (i.e., 2.5 l 2% Al2O3 per μg HBsAg). The protein and alum were mixed with a vortex and then left on ice for at least 30 minutes prior to use to allow the protein to adsorb onto the Al2O3. This solution was mixed again immediately prior to injection by drawing up into the syringe 3-5 times.
  • For groups treated with CpG ODN, an appropriate volume of synthetic oligodeoxynucleotide (ODN #1826) of the sequence TCCATGACGTTCCTGACGTT (SEQ ID NO. 86) synthesized with a phosphorothioate backbone (Oligos Etc. & Oligo Therapeutics, Wilsonville, Oreg.) was added alone or with alum to HBsAg on the day of injection. Adult mice received a single intramuscular (IM) injection into the left tibialis anterior (TA) muscle of 1 or 2 ug HBsAg, without or with adjuvant (alum and/or CpG ODN), in 50 l vehicle. When CpG DNA was added, each animal received a total of 1, 10, 100 or 500 μg ODN. Newborn mice were immunized within 24 hours of birth or 7 days after birth by bilateral injection of a total of 1 μg HBsAg into the posterior thigh muscles (2×10 l @ 0.05 mg/ml). All injections were carried out with a 0.3 ml insulin syringe which has a fused 29G needle (Becton Dickenson, Franklin Lakes, N.J.). For injection of adults, the needle was fitted with a collar of polyethylene (PE) tubing to limit penetration of the needle to about 3 mm. All intramuscular injections were carried out through the skin (shaved for adults) and under general anesthesia (Halothane, Halocarbon Laboratories, River Edge, N.J.).
  • HBsAg Subunit Vaccination of Monkeys
  • Monkeys were immunized by IM injection into the anterior thigh muscle of Engerix-B® (SmithKline Beecham Biologicals, Rixensart, BE) which comprises HBsAg (ay subtype, 20 μg/ml) adsorbed to alum (25 mg Al3+/mg HbsAg). Each monkey received an injection of 0.5 ml containing 10 μg HbsAg. For some monkeys, 500 μg CpG ODN 1968 (TCGTCGCTGTTGTCGTTTCTT) (SEQ ID NO 72) was added to the vaccine formulation.
  • HBsAg Subunit Vaccination of Orangutans
  • Orangutans were immunized by IM injection into the anterior thigh muscle of HBsAg *ay subtype, 20 μg/ml) combined with alum (25 mg Al3+/mg HBsAg), combined with CpG. CpG ODN 2006 (TCGTCGTTTTGTCGTTTTGTCGTT) (SEQ ID NO 77) was added to the vaccine formulation. Each orangutan received an injection of 1.0 ml containing 20 μg HBsAg with alum (500 μg), CpG oligonucleotide (1 mg) or both adjuvants.
  • Experimental Groups
  • Comparison of CpG ODN and Non-Nucleic Acid Adjuvants with HBsAg Subunit Vaccine
  • Twelve groups of adult BALB/c mice (n=10) were injected with 1 μg HBsAg (i) alone, (ii) mixed with alum, (iii, iv, v, vi, vii) mixed with 0.1, 1, 10, 100 or 500 μg CpG ODN, or (viii, ix, x, xi, xii) mixed with both alum and 0.1, 1, 10, 100 or 500 μg CpG ODN. These mice were bled at 1, 2, 4 and 8 weeks after immunization and the plasma was assayed for anti-HBs. At the end of the study the mice were killed and their spleens removed for assay of CTL activity.
  • Other groups of mice (n=5) were immunized with HBsAg (1 μg) alone, with alum (25 μg Al3+), with one of several different CpG and non-CpG control oligonucleotides of different backbones (10 μg), or with both alum and an oligonucleotide.
  • Other groups of mice (n=5) were immunized as above (except only the 10 μg dose of CpG ODN was used) and boosted with the identical or a different formulation at 8 weeks, then spleens were removed 2 weeks later for evaluation of CTL activity.
  • Other groups of mice were immunized with HBsAg (1 μg) and one of the following non-nucleic acid adjuvants alone or in combination with CpG ODN (10 μg): monophosphoryl lipid A (MPL, 50 μg, Ribi); Freund's Complete Adjuvant (CFA; 1:1 v/v); Freund's Incomplete Adjuvant (IFA; 1:1 v/v).
  • Immunization of Neonates with Subunit or DNA Vaccine
  • Groups of newborn and young BALB/c mice (n=10) aged <24 hours, 3, 7 or 14 days were injected with (i, ii, iii) a total of 1 μg HBsAg with alum, with CpG ODN 1826 (10 μg) or with both alum and CpG ODN, or with (iv) an HBsAg-expressing DNA vaccine (1-μg pCMV-S). Plasma was obtained at 4, 8, 12 and 16 weeks for assay of anti-HBs as total IgG and IgG subtypes (IgG1 and IgG2a). At the end of the study the mice were killed and their spleens removed for assay of CTL activity.
  • Immunization of Cynomolgus Monkeys with HBsAg and Alum or Alum+CpG ODN
  • Two groups of juvenile Cynomolgus monkeys (n=5) were immunized at 0 and 10 weeks with 0.5 ml Engerix-B (HBsAg at 20 mg/ml adsorbed to alum, 25 mg Al3+/mg HBsAg) to which had been added saline (0.1 ml) or CpG ODN 2006 (500 μg in 0.1 ml, SEQ #77). Monkeys were bled at 2, 8, 10, 12 and 14 weeks and plasma was evaluated for anti-HBs titers (mIU/ml).
  • Immunization of Orangutans with HBsAg and Alum or CpG ODN or Alum+CpG ODN
  • Three groups of juvenile orangutans were immunized IM at 0 and 4 weeks with 1 ml of vaccine containing HBsAg (10 μg) plus (i) alum (25 mg Al3+/mg HBsAg)(n=13), (ii) CpG ODN 2006 (SEQ# 77) (m=24) or (iii) alum plus CpG ODN (n=14). Animals were bled at 4.8 and 12 weeks and plasma was evaluated for anti-HBs titers (mIU/ml).
  • Evaluation of Humoral Response to HbsAg
  • Mice: Heparinized blood was collected by retrobulbar puncture of lightly anaesthetized mice as described elsewhere (Michel et al., 1995). Plasma was recovered by centrifugation (7 min @ 13,000 rpm). Antibodies specific to HBsAg in plasma were detected and quantified by end-point dilution ELISA assay (in triplicate) on individual samples. Ten-fold serial dilutions of plasma were first added to 96-well microtiter plates with a solid phase consisting of plasma-derived HBsAg particles (100 l/well of HBsAg ay subtype at 1 g/ml, coated overnight at RT) and incubated for 1 hr at 37 C. The bound antibodies were then detected by incubation for 1 hr at 37C with HRP-conjugated goat anti-mouse IgG, IgM, IgG1 or IgG2a (1:4000 in PBS-Tween, 10% FCS; 100 l/well, Southern Biotechnology Inc., Birmingham, Ala.), followed by incubation with OPD solution (100 l/well, Sigma, St. Louis, Mo.) for 30 minutes at RT in the dark. The reaction was stopped by the addition of sulfuric acid (50 l of 4N H2SO4). End-point titers were defined as the highest plasma dilution that resulted in an absorbance value (OD 450) two times greater than that of non-immune plasma

Claims (8)

1-98. (Canceled).
99. A method for maintaining suppression of a Th2 immune response in a subject, the method comprising: administering to a subject a first dose of an immunostimulatory nucleic acid; and administering to the subject a second dose of an immunostimulatory nucleic acid, wherein the immunostimulatory nucleic acid comprises a nucleotide sequence comprising 5′-CG-3′, and wherein the second dose is administered from about 10 days to about 8 weeks after the first dose.
100. The method of claim 99, wherein the second dose is administered about 4 weeks after the first dose.
101. The method of claim 99, wherein the subject is a human.
102. The method of claim 99, wherein the first and the second doses are administered by inhalation.
103. A method for maintaining stimulation of a Th1 immune response in a subject, the method comprising: administering to a subject a first dose of an immunostimulatory nucleic acid; and administering to the subject a second dose of an immunostimulatory nucleic acid, wherein the immunostimulatory nucleic acid comprises a nucleotide sequence comprising 5′-CG-3′, and wherein the second dose is administered from about 10 days to about 8 weeks after the first dose.
104. The method of claim 103, wherein the second dose is administered about 4 weeks after the first dose.
105. The method of claim 103, wherein the subject is a human.
US10/831,775 1997-03-10 2004-04-23 Use of nucleic acids containing unmethylated CpG dinucleotide as an adjuvant Abandoned US20050043529A1 (en)

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Cited By (63)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20010044416A1 (en) * 2000-01-20 2001-11-22 Mccluskie Michael J. Immunostimulatory nucleic acids for inducing a Th2 immune response
US20020164341A1 (en) * 1997-03-10 2002-11-07 Loeb Health Research Institute At The Ottawa Hospital Use of nucleic acids containing unmethylated CpG dinucleotide as an adjuvant
US20030026801A1 (en) * 2000-06-22 2003-02-06 George Weiner Methods for enhancing antibody-induced cell lysis and treating cancer
US20030091593A1 (en) * 2001-09-14 2003-05-15 Cytos Biotechnology Ag In vivo activation of antigen presenting cells for enhancement of immune responses induced by virus like particles
US20030099668A1 (en) * 2001-09-14 2003-05-29 Cytos Biotechnology Ag Packaging of immunostimulatory substances into virus-like particles: method of preparation and use
US20030139364A1 (en) * 2001-10-12 2003-07-24 University Of Iowa Research Foundation Methods and products for enhancing immune responses using imidazoquinoline compounds
US20030148976A1 (en) * 2001-08-17 2003-08-07 Krieg Arthur M. Combination motif immune stimulatory oligonucleotides with improved activity
US20030191079A1 (en) * 1994-07-15 2003-10-09 University Of Iowa Research Foundation Methods for treating and preventing infectious disease
US20030212026A1 (en) * 1999-09-25 2003-11-13 University Of Iowa Research Foundation Immunostimulatory nucleic acids
US20030232074A1 (en) * 2002-04-04 2003-12-18 Coley Pharmaceutical Gmbh Immunostimulatory G, U-containing oligoribonucleotides
US20040005338A1 (en) * 2002-06-20 2004-01-08 Cytos Biotechnology Ag Packaged virus-like particles for use as adjuvants: method of preparation and use
US20040067905A1 (en) * 2002-07-03 2004-04-08 Coley Pharmaceutical Group, Inc. Nucleic acid compositions for stimulating immune responses
US20040092472A1 (en) * 2002-07-03 2004-05-13 Coley Pharmaceutical Group, Inc. Nucleic acid compositions for stimulating immune responses
US20040131628A1 (en) * 2000-03-08 2004-07-08 Bratzler Robert L. Nucleic acids for the treatment of disorders associated with microorganisms
US20040132685A1 (en) * 1994-07-15 2004-07-08 The University Of Iowa Research Foundation Immunostimulatory nucleic acid
US20040142469A1 (en) * 1994-07-15 2004-07-22 University Of Iowa Research Foundation Immunomodulatory oligonucleotides
US20040152649A1 (en) * 2002-07-03 2004-08-05 Coley Pharmaceutical Group, Inc. Nucleic acid compositions for stimulating immune responses
US20040157791A1 (en) * 1998-06-25 2004-08-12 Dow Steven W. Systemic immune activation method using nucleic acid-lipid complexes
US20040171571A1 (en) * 2002-12-11 2004-09-02 Coley Pharmaceutical Group, Inc. 5' CpG nucleic acids and methods of use
US20040198680A1 (en) * 2002-07-03 2004-10-07 Coley Pharmaceutical Group, Inc. Nucleic acid compositions for stimulating immune responses
US20040235777A1 (en) * 1998-05-14 2004-11-25 Coley Pharmaceutical Gmbh Methods for regulating hematopoiesis using CpG-oligonucleotides
US20040235774A1 (en) * 2000-02-03 2004-11-25 Bratzler Robert L. Immunostimulatory nucleic acids for the treatment of asthma and allergy
US20050013812A1 (en) * 2003-07-14 2005-01-20 Dow Steven W. Vaccines using pattern recognition receptor-ligand:lipid complexes
US20050054601A1 (en) * 1997-01-23 2005-03-10 Coley Pharmaceutical Gmbh Pharmaceutical composition comprising a polynucleotide and optionally an antigen especially for vaccination
US20050059619A1 (en) * 2002-08-19 2005-03-17 Coley Pharmaceutical Group, Inc. Immunostimulatory nucleic acids
US20050075302A1 (en) * 1994-03-25 2005-04-07 Coley Pharmaceutical Group, Inc. Immune stimulation by phosphorothioate oligonucleotide analogs
US20050100983A1 (en) * 2003-11-06 2005-05-12 Coley Pharmaceutical Gmbh Cell-free methods for identifying compounds that affect toll-like receptor 9 (TLR9) signaling
US20050101557A1 (en) * 1994-07-15 2005-05-12 The University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20050119273A1 (en) * 2003-06-20 2005-06-02 Coley Pharmaceutical Gmbh Small molecule toll-like receptor (TLR) antagonists
US20050130911A1 (en) * 2003-09-25 2005-06-16 Coley Pharmaceutical Group, Inc. Nucleic acid-lipophilic conjugates
US20050181422A1 (en) * 2000-09-15 2005-08-18 Coley Pharmaceutical Gmbh Process for high throughput screening of CpG-based immuno-agonist/antagonist
US20050197314A1 (en) * 1998-04-03 2005-09-08 University Of Iowa Research Foundation Methods and products for stimulating the immune system using immunotherapeutic oligonucleotides and cytokines
US20050239734A1 (en) * 2003-10-30 2005-10-27 Coley Pharmaceutical Gmbh C-class oligonucleotide analogs with enhanced immunostimulatory potency
US20050239733A1 (en) * 2003-10-31 2005-10-27 Coley Pharmaceutical Gmbh Sequence requirements for inhibitory oligonucleotides
US20050256073A1 (en) * 2004-02-19 2005-11-17 Coley Pharmaceutical Group, Inc. Immunostimulatory viral RNA oligonucleotides
US20060003962A1 (en) * 2002-10-29 2006-01-05 Coley Pharmaceutical Group, Ltd. Methods and products related to treatment and prevention of hepatitis C virus infection
US20060140875A1 (en) * 2004-10-20 2006-06-29 Coley Pharmaceutical Group, Inc. Semi-soft C-class immunostimulatory oligonucleotides
US20060211644A1 (en) * 2005-02-24 2006-09-21 Coley Pharmaceutical Group, Inc. Immunostimulatory oligonucleotides
US20060229271A1 (en) * 2005-04-08 2006-10-12 Coley Pharmaceutical Group, Inc. Methods for treating infectious disease exacerbated asthma
US20060241076A1 (en) * 2005-04-26 2006-10-26 Coley Pharmaceutical Gmbh Modified oligoribonucleotide analogs with enhanced immunostimulatory activity
US20060251623A1 (en) * 2003-07-10 2006-11-09 Caytos Biotechnology Ag Packaged virus-like particles
US20060286070A1 (en) * 1999-09-27 2006-12-21 Coley Pharmaceutical Gmbh Methods related to immunostimulatory nucleic acid-induced interferon
US20070009710A1 (en) * 2000-08-04 2007-01-11 Toyo Boseki Kabushiki Kaisha Flexible metal-clad laminate and method for producing the same
US20070065467A1 (en) * 1994-07-15 2007-03-22 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules for activating dendritic cells
US20080026986A1 (en) * 2006-06-05 2008-01-31 Rong-Fu Wang Reversal of the suppressive function of specific t cells via toll-like receptor 8 signaling
US20080045473A1 (en) * 2006-02-15 2008-02-21 Coley Pharmaceutical Gmbh Compositions and methods for oligonucleotide formulations
US20080226649A1 (en) * 2000-12-08 2008-09-18 Coley Pharmaceutical Gmbh CPG-like nucleic acids and methods of use thereof
US20090117132A1 (en) * 2005-07-07 2009-05-07 Pfizer, Inc. Anti-Ctla-4 Antibody and Cpg-Motif-Containing Synthetic Oligodeoxynucleotide Combination Therapy for Cancer Treatment
US20090142362A1 (en) * 2006-11-06 2009-06-04 Avant Immunotherapeutics, Inc. Peptide-based vaccine compositions to endogenous cholesteryl ester transfer protein (CETP)
US20090214578A1 (en) * 2005-09-16 2009-08-27 Coley Pharmaceutical Gmbh Immunostimulatory Single-Stranded Ribonucleic Acid with Phosphodiester Backbone
US20090306177A1 (en) * 2005-09-16 2009-12-10 Coley Pharmaceutical Gmbh Modulation of Immunostimulatory Properties of Short Interfering Ribonucleic Acid (Sirna) by Nucleotide Modification
US20090311277A1 (en) * 2002-07-03 2009-12-17 Coley Pharmaceutical Group, Inc. Nucleic acid compositions for stimulating immune responses
US20100010193A1 (en) * 1999-02-17 2010-01-14 Csl Limited Immunogenic complexes and methods relating thereto
US7662949B2 (en) 2005-11-25 2010-02-16 Coley Pharmaceutical Gmbh Immunostimulatory oligoribonucleotides
US20100098722A1 (en) * 2003-03-26 2010-04-22 Cytos Biotechnology Ag Packaging of Immunostimulatory Substances Into Virus-Like Particles: Method of Preparation and Use
US7741300B2 (en) 1998-06-25 2010-06-22 National Jewish Medical And Research Center Methods of using nucleic acid vector-lipid complexes
US8574564B2 (en) 2005-12-14 2013-11-05 Cytos Biotechnology Ag Immunostimulatory nucleic acid packaged particles for the treatment of hypersensitivity
US8574599B1 (en) 1998-05-22 2013-11-05 Ottawa Hospital Research Institute Methods and products for inducing mucosal immunity
US8580268B2 (en) 2006-09-27 2013-11-12 Coley Pharmaceutical Gmbh CpG oligonucleotide analogs containing hydrophobic T analogs with enhanced immunostimulatory activity
US8883174B2 (en) 2009-03-25 2014-11-11 The Board Of Regents, The University Of Texas System Compositions for stimulation of mammalian innate immune resistance to pathogens
US9260517B2 (en) 2009-11-17 2016-02-16 Musc Foundation For Research Development Human monoclonal antibodies to human nucleolin
US9404126B2 (en) 2006-06-12 2016-08-02 Kuros Biosciences Ag Processes for packaging aggregated oligonucleotides into virus-like particles of RNA bacteriophages
US10286065B2 (en) 2014-09-19 2019-05-14 Board Of Regents, The University Of Texas System Compositions and methods for treating viral infections through stimulated innate immunity in combination with antiviral compounds

Families Citing this family (454)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5849719A (en) * 1993-08-26 1998-12-15 The Regents Of The University Of California Method for treating allergic lung disease
US20030109469A1 (en) * 1993-08-26 2003-06-12 Carson Dennis A. Recombinant gene expression vectors and methods for use of same to enhance the immune response of a host to an antigen
US8956621B2 (en) 1994-11-08 2015-02-17 The Trustees Of The University Of Pennsylvania Compositions and methods for treatment of cervical dysplasia
US8791237B2 (en) 1994-11-08 2014-07-29 The Trustees Of The University Of Pennsylvania Compositions and methods for treatment of non-hodgkins lymphoma
US8114414B2 (en) * 1994-11-08 2012-02-14 The Trustees Of The University Of Pennsylvania Compositions and methods for treatment of cervical cancer
US5981501A (en) * 1995-06-07 1999-11-09 Inex Pharmaceuticals Corp. Methods for encapsulating plasmids in lipid bilayers
US7422902B1 (en) * 1995-06-07 2008-09-09 The University Of British Columbia Lipid-nucleic acid particles prepared via a hydrophobic lipid-nucleic acid complex intermediate and use for gene transfer
US20030078223A1 (en) * 1996-01-30 2003-04-24 Eyal Raz Compositions and methods for modulating an immune response
WO1997028259A1 (en) * 1996-01-30 1997-08-07 The Regents Of The University Of California Gene expression vectors which generate an antigen specific immune response and methods of using the same
US20060002949A1 (en) * 1996-11-14 2006-01-05 Army Govt. Of The Usa, As Rep. By Secretary Of The Office Of The Command Judge Advocate, Hq Usamrmc. Transcutaneous immunization without heterologous adjuvant
US5980898A (en) * 1996-11-14 1999-11-09 The United States Of America As Represented By The U.S. Army Medical Research & Material Command Adjuvant for transcutaneous immunization
US20060002959A1 (en) * 1996-11-14 2006-01-05 Government Of The United States Skin-sctive adjuvants for transcutaneous immuization
US6797276B1 (en) 1996-11-14 2004-09-28 The United States Of America As Represented By The Secretary Of The Army Use of penetration enhancers and barrier disruption agents to enhance the transcutaneous immune response
DE69841002D1 (en) 1997-05-14 2009-09-03 Univ British Columbia Highly effective encapsulation of nucleic acids in lipid vesicles
US20040006034A1 (en) * 1998-06-05 2004-01-08 Eyal Raz Immunostimulatory oligonucleotides, compositions thereof and methods of use thereof
US20040258703A1 (en) * 1997-11-14 2004-12-23 The Government Of The Us, As Represented By The Secretary Of The Army Skin-active adjuvants for transcutaneous immunization
FR2776928B1 (en) * 1998-04-03 2000-06-23 Merial Sas ADJUVATED DNA VACCINES
US20040247662A1 (en) * 1998-06-25 2004-12-09 Dow Steven W. Systemic immune activation method using nucleic acid-lipid complexes
US20010034330A1 (en) * 1998-08-10 2001-10-25 Charlotte Kensil Innate immunity-stimulating compositions of CpG and saponin and methods thereof
ATE315405T1 (en) 1998-08-10 2006-02-15 Antigenics Inc CPG COMPOSITIONS, SAPONIN ADJUVANTS AND METHODS OF USE THEREOF
US6558951B1 (en) * 1999-02-11 2003-05-06 3M Innovative Properties Company Maturation of dendritic cells with immune response modifying compounds
GB9909077D0 (en) * 1999-04-20 1999-06-16 Smithkline Beecham Biolog Novel compositions
WO2000061151A2 (en) 1999-04-12 2000-10-19 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Oligodeoxynucleotide and its use to induce an immune response
US6977245B2 (en) 1999-04-12 2005-12-20 The United States Of America As Represented By The Department Of Health And Human Services Oligodeoxynucleotide and its use to induce an immune response
HU229255B1 (en) * 1999-04-19 2013-10-28 Smithkline Beecham Biolog Vaccines
US6558670B1 (en) 1999-04-19 2003-05-06 Smithkline Beechman Biologicals S.A. Vaccine adjuvants
US20050002958A1 (en) * 1999-06-29 2005-01-06 Smithkline Beecham Biologicals Sa Vaccines
US6514948B1 (en) * 1999-07-02 2003-02-04 The Regents Of The University Of California Method for enhancing an immune response
US20050226890A1 (en) * 1999-08-12 2005-10-13 Cohen David I Tat-based vaccine compositions and methods of making and using same
WO2001037869A1 (en) * 1999-11-19 2001-05-31 Csl Limited Vaccine compositions
ATE378348T1 (en) * 2000-01-14 2007-11-15 Us Health OLIGODEOXYNUCLEOTIDES AND THEIR USE FOR INDUCING AN IMMUNE RESPONSE
US20020098199A1 (en) * 2000-03-10 2002-07-25 Gary Van Nest Methods of suppressing hepatitis virus infection using immunomodulatory polynucleotide sequences
US7157437B2 (en) * 2000-03-10 2007-01-02 Dynavax Technologies Corporation Methods of ameliorating symptoms of herpes infection using immunomodulatory polynucleotide sequences
US20020107212A1 (en) * 2000-03-10 2002-08-08 Nest Gary Van Methods of reducing papillomavirus infection using immunomodulatory polynucleotide sequences
US20010046967A1 (en) * 2000-03-10 2001-11-29 Gary Van Nest Methods of preventing and treating respiratory viral infection using immunomodulatory polynucleotide
US20020028784A1 (en) * 2000-03-10 2002-03-07 Nest Gary Van Methods of preventing and treating viral infections using immunomodulatory polynucleotide sequences
CA2404041A1 (en) * 2000-03-28 2001-10-04 The Regents Of The University Of California Methods for increasing a cytotoxic t lymphocyte response in vivo
US20080044435A1 (en) * 2004-03-16 2008-02-21 Cohen David I Tat-Based Tolerogen Compositions and Methods of Making and Using Same
JP2005503320A (en) * 2000-08-25 2005-02-03 イエダ・リサーチ・アンド・デベロツプメント・カンパニー・リミテツド Methods for treating or preventing autoimmune diseases with CpG-containing polynucleotides
ES2377077T3 (en) * 2000-10-18 2012-03-22 Glaxosmithkline Biologicals S.A. Vaccines comprising the MAGE antigen bound to a protein D fragment
WO2006091720A2 (en) * 2000-12-08 2006-08-31 3M Innovative Properties Company Compositions and methods for targeted delivery of immune response modifiers
US20030008000A1 (en) * 2001-03-08 2003-01-09 Wong Jonathan P. DNA vaccine using liposome-encapsulated plasmid DNA encoding for hemagglutinin protein of influenza virus
US8771702B2 (en) 2001-03-26 2014-07-08 The Trustees Of The University Of Pennsylvania Non-hemolytic LLO fusion proteins and methods of utilizing same
US7700344B2 (en) 2001-03-26 2010-04-20 The Trustees Of The University Of Pennsylvania Compositions and methods for enhancing the immunogenicity of antigens
US20030050268A1 (en) * 2001-03-29 2003-03-13 Krieg Arthur M. Immunostimulatory nucleic acid for treatment of non-allergic inflammatory diseases
EP1857122B1 (en) 2001-06-05 2010-12-01 CureVac GmbH Stabilised mRNA with increased G/C content, coding for a viral antigen
US6818787B2 (en) * 2001-06-11 2004-11-16 Xenoport, Inc. Prodrugs of GABA analogs, compositions and uses thereof
US7785610B2 (en) * 2001-06-21 2010-08-31 Dynavax Technologies Corporation Chimeric immunomodulatory compounds and methods of using the same—III
ES2487645T3 (en) * 2001-06-21 2014-08-22 Dynavax Technologies Corporation Chimeric immunomodulatory compounds and methods of use thereof
US20030138458A1 (en) * 2001-06-29 2003-07-24 Michael Houghton HCV E1E2 vaccine compositions
US7666674B2 (en) 2001-07-27 2010-02-23 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Use of sterically stabilized cationic liposomes to efficiently deliver CPG oligonucleotides in vivo
US20030148316A1 (en) * 2001-08-01 2003-08-07 Lipford Grayson B. Methods and compositions relating to plasmacytoid dendritic cells
EP1420829A4 (en) * 2001-08-07 2006-05-17 Dynavax Tech Corp Immunomodulatory compositions, formulations, and methods for use thereof
WO2003015716A2 (en) * 2001-08-13 2003-02-27 Ige Therapeutics, Inc. Immunoglobulin e vaccines and methods of use thereof
WO2003020884A2 (en) * 2001-08-14 2003-03-13 The Government Of The United States Of America As Represented By The Secretary Of Health And Human Services Method for rapid generation of mature dendritic cells
WO2003020889A2 (en) * 2001-08-30 2003-03-13 3M Innovative Properties Company Methods of maturing plasmacytoid dendritic cells using immune response modifier molecules
JP2005501917A (en) * 2001-09-07 2005-01-20 ザ トラスティーズ オブ ボストン ユニバーシティ Methods and compositions for treating immune complex related diseases
EP1451581A4 (en) * 2001-10-05 2006-01-11 Coley Pharm Gmbh Toll-like receptor 3 signaling agonists and antagonists
US20050238660A1 (en) * 2001-10-06 2005-10-27 Babiuk Lorne A Cpg formulations and related methods
WO2003039595A2 (en) * 2001-11-07 2003-05-15 Inex Pharmaceuticals Corporation Mucosal adjuvants comprising an oligonucleotide and a cationic lipid
US20040014779A1 (en) * 2001-11-16 2004-01-22 3M Innovative Properties Company Methods and compositions related to IRM compounds and toll-like recptor pathways
WO2003050135A2 (en) 2001-12-07 2003-06-19 Board Of Regents The University Of Texas System Use a parapox b2l protein to modify immune responses to administered antigens
DE10162480A1 (en) 2001-12-19 2003-08-07 Ingmar Hoerr The application of mRNA for use as a therapeutic agent against tumor diseases
US7615227B2 (en) * 2001-12-20 2009-11-10 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Use of CpG oligodeoxynucleotides to induce angiogenesis
CA2365732A1 (en) * 2001-12-20 2003-06-20 Ibm Canada Limited-Ibm Canada Limitee Testing measurements
US8466116B2 (en) 2001-12-20 2013-06-18 The Unites States Of America As Represented By The Secretary Of The Department Of Health And Human Services Use of CpG oligodeoxynucleotides to induce epithelial cell growth
US8088388B2 (en) * 2002-02-14 2012-01-03 United Biomedical, Inc. Stabilized synthetic immunogen delivery system
PT1478327E (en) * 2002-02-22 2015-08-04 Meda Ab Method of reducing and treating uvb-induced immunosuppression
CA2479187A1 (en) * 2002-03-15 2003-09-25 Astral, Inc. Immunostimulatory double stranded rna and methods of inducing, enhancing or modulating the immune response
EP2258712A3 (en) 2002-03-15 2011-05-04 Multicell Immunotherapeutics, Inc. Compositions and Methods to Initiate or Enhance Antibody and Major-histocompatibility Class I or Class II-restricted T Cell Responses by Using Immunomodulatory, Non-coding RNA Motifs
US20070037769A1 (en) * 2003-03-14 2007-02-15 Multicell Immunotherapeutics, Inc. Compositions and methods to treat and control tumors by loading antigen presenting cells
GB0210128D0 (en) * 2002-05-02 2002-06-12 Chiron Spa Nucleic acids and proteins from streptococcus groups A & B
US20040013649A1 (en) * 2002-05-10 2004-01-22 Inex Pharmaceuticals Corporation Cancer vaccines and methods of using the same
US20040009944A1 (en) * 2002-05-10 2004-01-15 Inex Pharmaceuticals Corporation Methylated immunostimulatory oligonucleotides and methods of using the same
KR100456681B1 (en) * 2002-05-22 2004-11-10 주식회사 대웅 Immnune-stimulating and controlling Composition comprising bacterial chromosomal DNA fragments and detoxified lipopolysaccharides
CA2388049A1 (en) 2002-05-30 2003-11-30 Immunotech S.A. Immunostimulatory oligonucleotides and uses thereof
GB0212666D0 (en) * 2002-05-31 2002-07-10 Secr Defence Immunogenic sequences
US20040009949A1 (en) * 2002-06-05 2004-01-15 Coley Pharmaceutical Group, Inc. Method for treating autoimmune or inflammatory diseases with combinations of inhibitory oligonucleotides and small molecule antagonists of immunostimulatory CpG nucleic acids
DE10229872A1 (en) 2002-07-03 2004-01-29 Curevac Gmbh Immune stimulation through chemically modified RNA
AU2003255969A1 (en) * 2002-07-17 2004-02-02 Coley Pharmaceutical Gmbh Use of cpg nucleic acids in prion-disease
EP1551376A4 (en) * 2002-08-12 2010-10-06 Dynavax Tech Corp Immunomodulatory compositions, methods of making, and methods of use thereof
DK1545597T3 (en) 2002-08-15 2011-01-31 3M Innovative Properties Co Immune Stimulating Compositions and Methods for Stimulating an Immune Response
EP1537208A1 (en) * 2002-09-13 2005-06-08 Replicor, Inc. Non-sequence complementary antiviral oligonucleotides
US8263091B2 (en) * 2002-09-18 2012-09-11 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Method of treating and preventing infections in immunocompromised subjects with immunostimulatory CpG oligonucleotides
CA2503457A1 (en) * 2002-10-25 2004-05-06 University Of Connecticut Health Center Apparatus and method for immunotherapy of a cancer through controlled cell lysis
AU2003304107B2 (en) * 2002-11-01 2008-05-15 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Method of preventing infections from bioterrorism agents with immunostimulatory CpG oligonucleotides
EP2298360A3 (en) * 2002-11-21 2011-07-13 Bayhill Therapeutics, Inc. Methods and immune modulatory nucleic acid compositions for preventing and treating disease
WO2004053057A2 (en) * 2002-12-11 2004-06-24 3M Innovative Properties Company Gene expression systems and recombinant cell lines
WO2004052293A2 (en) * 2002-12-11 2004-06-24 Hawaii Biotech, Inc. Recombinant vaccine against flavivirus infection
AU2003287316A1 (en) * 2002-12-11 2004-06-30 3M Innovative Properties Company Assays relating to toll-like receptor activity
ATE544772T1 (en) 2002-12-23 2012-02-15 Dynavax Tech Corp IMMUNO-STIMULATING SEQUENCE OLIGONUCLEOTIDES AND METHOD OF USE THEREOF
US8158768B2 (en) 2002-12-23 2012-04-17 Dynavax Technologies Corporation Immunostimulatory sequence oligonucleotides and methods of using the same
EP2572714A1 (en) 2002-12-30 2013-03-27 3M Innovative Properties Company Immunostimulatory Combinations
US7067155B2 (en) * 2003-01-03 2006-06-27 Tbni, Inc. Anti-inflammatory humate compositions and methods of use thereof
CA2513655C (en) 2003-01-21 2011-11-22 Chiron Corporation Use of tryptanthrin compounds for immune potentiation
EP1592302A4 (en) * 2003-02-13 2007-04-25 3M Innovative Properties Co Methods and compositions related to irm compounds and toll-like receptor 8
ATE491463T1 (en) 2003-02-20 2011-01-15 Univ Connecticut Health Ct METHOD FOR PRODUCING ALPHA (2) MACROGLOBULIN-ANTIGEN MOLECULE COMPLEXES
WO2004075865A2 (en) * 2003-02-27 2004-09-10 3M Innovative Properties Company Selective modulation of tlr-mediated biological activity
EP1601365A4 (en) 2003-03-04 2009-11-11 3M Innovative Properties Co Prophylactic treatment of uv-induced epidermal neoplasia
WO2004080292A2 (en) 2003-03-13 2004-09-23 3M Innovative Properties Company Method of tattoo removal
ATE556711T1 (en) * 2003-03-13 2012-05-15 3M Innovative Properties Co METHOD FOR IMPROVING SKIN QUALITY
US20040192585A1 (en) 2003-03-25 2004-09-30 3M Innovative Properties Company Treatment for basal cell carcinoma
CA2520181A1 (en) * 2003-03-26 2004-10-14 Astral Inc. Selected rna motifs to include cell death and/or apoptosis
WO2004087153A2 (en) 2003-03-28 2004-10-14 Chiron Corporation Use of organic compounds for immunopotentiation
AU2004226605A1 (en) * 2003-04-02 2004-10-14 Coley Pharmaceutical Group, Ltd. Immunostimulatory nucleic acid oil-in-water formulations for topical application
US20050013855A1 (en) * 2003-04-09 2005-01-20 Biodelivery Sciences International, Inc. Cochleate compositions directed against expression of proteins
US20040265351A1 (en) * 2003-04-10 2004-12-30 Miller Richard L. Methods and compositions for enhancing immune response
EP1617871A4 (en) * 2003-04-10 2010-10-06 3M Innovative Properties Co Delivery of immune response modifier compounds using metal-containing particulate support materials
WO2004100965A1 (en) * 2003-05-15 2004-11-25 Japan Science And Technology Agency Immunostimulant
US8080642B2 (en) * 2003-05-16 2011-12-20 Vical Incorporated Severe acute respiratory syndrome DNA compositions and methods of use
MXPA05012421A (en) * 2003-05-16 2006-02-22 Hybridon Inc Synergistic treatment of cancer using immunomers in conjunction with chemotherapeutic agents.
US7803386B2 (en) 2003-06-05 2010-09-28 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Poly-gamma-glutamic conjugates for eliciting immune responses directed against bacilli
US7320795B2 (en) * 2003-07-30 2008-01-22 Vaccine Research Institute Of San Diego Rodent hepatitis B virus core proteins as vaccine platforms and methods of use thereof
US7144712B2 (en) * 2003-07-30 2006-12-05 Vaccine Research Institute Of San Diego Human hepatitis B virus core proteins as vaccine platforms and methods of use thereof
CA2534060C (en) * 2003-07-30 2012-06-12 Vaccine Research Institute Of San Diego Hepatitis virus core proteins as vaccine platforms and methods of use thereof
US20080039533A1 (en) * 2003-07-31 2008-02-14 3M Innovative Properties Company Bioactive Compositions Comprising Triazines
CA2534313C (en) * 2003-08-05 2013-03-19 3M Innovative Properties Company Formulations containing an immune response modifier
JP2007502288A (en) 2003-08-12 2007-02-08 スリーエム イノベイティブ プロパティズ カンパニー Oxime-substituted imidazo-containing compounds
WO2005018574A2 (en) * 2003-08-25 2005-03-03 3M Innovative Properties Company Immunostimulatory combinations and treatments
WO2005020912A2 (en) * 2003-08-25 2005-03-10 3M Innovative Properties Company Delivery of immune response modifier compounds
CA2536819A1 (en) * 2003-08-26 2005-03-03 The Council Of The Queensland Institute Of Medical Research Immunogenic agent and pharmaceutical composition for use against homologous and heterologous pathogens including plasmodium spp
AU2004268625B2 (en) 2003-08-27 2011-03-31 3M Innovative Properties Company Aryloxy and arylalkyleneoxy substituted imidazoquinolines
EP1663222A4 (en) * 2003-09-02 2008-05-21 3M Innovative Properties Co Methods related to the treatment of mucosal associated conditions
JP2007504269A (en) 2003-09-05 2007-03-01 スリーエム イノベイティブ プロパティズ カンパニー Method for treating CD5 + B cell lymphoma
US8541002B2 (en) 2003-09-12 2013-09-24 Agenus Inc. Vaccine for treatment and prevention of herpes simplex virus infection
ES2544477T3 (en) 2003-10-03 2015-08-31 3M Innovative Properties Company Alkoxy substituted imidazoquinolines
US7544697B2 (en) 2003-10-03 2009-06-09 Coley Pharmaceutical Group, Inc. Pyrazolopyridines and analogs thereof
WO2005034979A2 (en) * 2003-10-11 2005-04-21 Inex Pharmaceuticals Corporation Methods and compositions for enhancing innate immunity and antibody dependent cellular cytotoxicity
WO2005041891A2 (en) * 2003-10-31 2005-05-12 3M Innovative Properties Company Neutrophil activation by immune response modifier compounds
EP1682544A4 (en) 2003-11-14 2009-05-06 3M Innovative Properties Co Hydroxylamine substituted imidazo ring compounds
CA2545774A1 (en) 2003-11-14 2005-06-02 3M Innovative Properties Company Oxime substituted imidazo ring compounds
NZ547467A (en) 2003-11-25 2010-06-25 3M Innovative Properties Co Substituted imidazo ring system and methods
US20050277127A1 (en) * 2003-11-26 2005-12-15 Epitomics, Inc. High-throughput method of DNA immunogen preparation and immunization
US20050287118A1 (en) * 2003-11-26 2005-12-29 Epitomics, Inc. Bacterial plasmid with immunological adjuvant function and uses thereof
WO2005055932A2 (en) * 2003-12-02 2005-06-23 3M Innovative Properties Company Therapeutic combinations and methods including irm compounds
US20050226878A1 (en) * 2003-12-02 2005-10-13 3M Innovative Properties Company Therapeutic combinations and methods including IRM compounds
US9090673B2 (en) * 2003-12-12 2015-07-28 City Of Hope Synthetic conjugate of CpG DNA and T-help/CTL peptide
EP1550458A1 (en) * 2003-12-23 2005-07-06 Vectron Therapeutics AG Synergistic liposomal adjuvants
US20050175630A1 (en) * 2003-12-23 2005-08-11 Eyal Raz Immunogenic compositions and methods of use thereof
JP4817599B2 (en) * 2003-12-25 2011-11-16 独立行政法人科学技術振興機構 Immune activity enhancer and method for enhancing immune activity using the same
JP2007517035A (en) 2003-12-29 2007-06-28 スリーエム イノベイティブ プロパティズ カンパニー Arylalkenyl and arylalkynyl substituted imidazoquinolines
EP1699398A4 (en) * 2003-12-30 2007-10-17 3M Innovative Properties Co Enhancement of immune responses
EP1699788A2 (en) 2003-12-30 2006-09-13 3M Innovative Properties Company Imidazoquinolinyl, imidazopyridinyl and imidazonaphthyridinyl sulfonamides
US7973016B2 (en) * 2004-01-23 2011-07-05 Joslin Diebetes Center Methods of treating, reducing, or preventing autoimmune conditions
US20050181035A1 (en) * 2004-02-17 2005-08-18 Dow Steven W. Systemic immune activation method using non CpG nucleic acids
AU2005216075B2 (en) * 2004-02-20 2011-03-10 Idera Pharmaceuticals, Inc. Potent mucosal immune response induced by modified immunomodulatory oligonucleotides
WO2005090968A1 (en) * 2004-03-16 2005-09-29 Inist Inc. Tat-based immunomodulatory compositions and methods of their discovery and use
US8697873B2 (en) 2004-03-24 2014-04-15 3M Innovative Properties Company Amide substituted imidazopyridines, imidazoquinolines, and imidazonaphthyridines
TWI235440B (en) * 2004-03-31 2005-07-01 Advanced Semiconductor Eng Method for making leadless semiconductor package
US20070166384A1 (en) * 2004-04-09 2007-07-19 Zarraga Isidro Angelo E Methods , composition and preparations for delivery of immune response modifiers
CN101426524A (en) * 2004-04-28 2009-05-06 3M创新有限公司 Compositions and methods for mucosal vaccination
EP1766094A4 (en) * 2004-05-18 2009-11-25 Vical Inc Influenza virus vaccine composition and method of use
US20050267145A1 (en) * 2004-05-28 2005-12-01 Merrill Bryon A Treatment for lung cancer
DK1765310T3 (en) 2004-05-28 2016-01-11 Oryxe MIXING for transdermal delivery of LAV AND HØJMOLEKYLVÆGTFORBINDELSER
WO2005123080A2 (en) 2004-06-15 2005-12-29 3M Innovative Properties Company Nitrogen-containing heterocyclyl substituted imidazoquinolines and imidazonaphthyridines
TW200613554A (en) 2004-06-17 2006-05-01 Wyeth Corp Plasmid having three complete transcriptional units and immunogenic compositions for inducing an immune response to HIV
WO2006065280A2 (en) 2004-06-18 2006-06-22 3M Innovative Properties Company Isoxazole, dihydroisoxazole, and oxadiazole substituted imidazo ring compounds and methods
US8026366B2 (en) 2004-06-18 2011-09-27 3M Innovative Properties Company Aryloxy and arylalkyleneoxy substituted thiazoloquinolines and thiazolonaphthyridines
WO2006038923A2 (en) 2004-06-18 2006-04-13 3M Innovative Properties Company Aryl substituted imidazonaphthyridines
US20110104186A1 (en) 2004-06-24 2011-05-05 Nicholas Valiante Small molecule immunopotentiators and assays for their detection
CA2571421A1 (en) 2004-06-24 2006-01-05 Nicholas Valiante Compounds for immunopotentiation
JP2008506789A (en) * 2004-07-18 2008-03-06 シーエスエル、リミテッド Immunostimulatory complex and oligonucleotide formulation for inducing enhanced interferon-gamma response
US20060045886A1 (en) * 2004-08-27 2006-03-02 Kedl Ross M HIV immunostimulatory compositions
DE102004042546A1 (en) * 2004-09-02 2006-03-09 Curevac Gmbh Combination therapy for immune stimulation
AU2005294129B2 (en) 2004-10-08 2010-12-23 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services, Centers For Disease Control And Prevention Modulation of replicative fitness by using less frequently used synonymous codons
US20070243215A1 (en) * 2004-10-08 2007-10-18 Miller Richard L Adjuvant for Dna Vaccines
ATE521621T1 (en) * 2004-11-29 2011-09-15 Changchun Huapu Biotechnology Co Ltd CPG SINGLE STRAND DESOXYNUCLEOTIDES FOR USE AS ADJUVANTS
WO2006065751A2 (en) * 2004-12-13 2006-06-22 Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Cpg oligonucleotide prodrugs, compositions thereof and associated therapeutic methods
WO2006071997A2 (en) 2004-12-30 2006-07-06 3M Innovative Properties Company Treatment for cutaneous metastases
ES2392647T3 (en) 2004-12-30 2012-12-12 3M Innovative Properties Company Chiral tetracyclic compounds that induce interferon biosynthesis
US8034938B2 (en) 2004-12-30 2011-10-11 3M Innovative Properties Company Substituted chiral fused [1,2]imidazo[4,5-c] ring compounds
JP4836145B2 (en) * 2005-01-28 2011-12-14 クウォン,ヒュン−ジョ Oligonucleotide derived from mycobacteria for immune function stimulation, immune disease treatment, atopic dermatitis treatment and / or normal immune cell protection
WO2006084251A2 (en) 2005-02-04 2006-08-10 Coley Pharmaceutical Group, Inc. Aqueous gel formulations containing immune reponse modifiers
US7968563B2 (en) 2005-02-11 2011-06-28 3M Innovative Properties Company Oxime and hydroxylamine substituted imidazo[4,5-c] ring compounds and methods
JP2008531722A (en) 2005-03-04 2008-08-14 ダイナバックス テクノロジーズ コーポレイション Composition comprising a structurally stable conjugate molecule
EP1869043A2 (en) 2005-04-01 2007-12-26 Coley Pharmaceutical Group, Inc. Pyrazolopyridine-1,4-diamines and analogs thereof
AU2006232375A1 (en) 2005-04-01 2006-10-12 Coley Pharmaceutical Group, Inc. 1-substituted pyrazolo (3,4-c) ring compounds as modulators of cytokine biosynthesis for the treatment of viral infections and neoplastic diseases
US20080193474A1 (en) * 2005-04-25 2008-08-14 Griesgraber George W Immunostimulatory Compositions
PL2179737T3 (en) 2005-07-01 2014-01-31 Index Pharmaceuticals Ab Modulating responsiveness to steroids
ES2450593T3 (en) 2005-07-01 2014-03-25 Index Pharmaceuticals Ab Immunostimulant method
WO2007008904A2 (en) * 2005-07-08 2007-01-18 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Targeting poly-gamma-glutamic acid to treat staphylococcus epidermidis and related infections
CA2615658A1 (en) * 2005-07-19 2007-01-25 Dow Global Technolgies Inc. Recombinant flu vaccines
US20090087456A1 (en) * 2005-09-07 2009-04-02 James Edward Eyles Adjuvanted vaccine
US20100130425A1 (en) 2005-09-09 2010-05-27 Oregon Health & Science University Use of toll-like receptor ligands in treating excitotoxic injury, ischemia and/or hypoxia
WO2007041190A2 (en) * 2005-09-30 2007-04-12 The University Of Iowa Research Foundation Polymer-based delivery system for immunotherapy of cancer
CA2625969A1 (en) * 2005-10-28 2007-05-03 Index Pharmaceuticals Ab Composition and method for the prevention, treatment and/or alleviation of an inflammatory disease
AU2007239095B2 (en) 2006-01-09 2012-05-03 The Regents Of The University Of California Immunostimulatory combinations for vaccine adjuvants
DE102006007433A1 (en) * 2006-02-17 2007-08-23 Curevac Gmbh Immunostimulant adjuvant useful in vaccines against cancer or infectious diseases comprises a lipid-modified nucleic acid
EP1988896A4 (en) * 2006-02-22 2011-07-27 3M Innovative Properties Co Immune response modifier conjugates
BRPI0708912B8 (en) 2006-03-14 2021-07-27 Univ Oregon Health & Science in vitro methods for detecting mycobacterium tuberculosis and cd8 expressing t cells that specifically recognize seq id no: 11 in an individual
WO2008008432A2 (en) 2006-07-12 2008-01-17 Coley Pharmaceutical Group, Inc. Substituted chiral fused( 1,2) imidazo (4,5-c) ring compounds and methods
US8323664B2 (en) * 2006-07-25 2012-12-04 The Secretary Of State For Defence Live vaccine strains of Francisella
AU2007280690C1 (en) 2006-07-31 2012-08-23 Curevac Gmbh Nucleic acid of formula (I): GIXmGn, or (II): CIXmCn, in particular as an immune-stimulating agent/adjuvant
DE102006035618A1 (en) * 2006-07-31 2008-02-07 Curevac Gmbh New nucleic acid useful as immuno-stimulating adjuvant for manufacture of a composition for treatment of cancer diseases e.g. colon carcinomas and infectious diseases e.g. influenza and malaria
US8865185B2 (en) 2006-09-08 2014-10-21 The Trustees Of The University Of Pennsylvania Methods of use for HSV-1 and HSV-2 vaccines
CA2663109A1 (en) * 2006-09-08 2008-03-13 The Trustees Of The University Of Pennsylvania Hsv-1 and hsv-2 vaccines and methods of use thereof
US20080166377A1 (en) * 2006-10-25 2008-07-10 Peter Hotez Human Hookworm Vaccine
EP3564674B1 (en) 2006-11-01 2021-08-25 Ventana Medical Systems, Inc. Haptens, hapten conjugates, compositions thereof and method for their preparation and use
US20080149123A1 (en) 2006-12-22 2008-06-26 Mckay William D Particulate material dispensing hairbrush with combination bristles
EP2591800A1 (en) * 2006-12-28 2013-05-15 The Trustees of The University of Pennsylvania Herpes simplex virus combined subunit vaccines and methods of use thereof
US10478490B2 (en) 2006-12-28 2019-11-19 The Trustees Of The University Of Pennsylvania Herpes simplex virus combined subunit vaccines and methods of use thereof
US20130028925A1 (en) * 2006-12-28 2013-01-31 Harvey Friedman Herpes simplex virus combined subunit vaccines and methods of use thereof
JP5596980B2 (en) 2007-02-28 2014-10-01 アメリカ合衆国 Brachyury polypeptides and methods of use
US20090004213A1 (en) 2007-03-26 2009-01-01 Immatics Biotechnologies Gmbh Combination therapy using active immunotherapy
US8518903B2 (en) 2007-04-19 2013-08-27 University of Pittsburgh—of the Commonwealth System of Higher Education Use of toll-like receptor-9 agonists
AU2008279584A1 (en) * 2007-04-27 2009-01-29 Dow Global Technologies Inc. Improved production and in vivo assembly of soluble recombinant icosahedral virus-like particles
DK2170353T3 (en) * 2007-05-18 2015-07-27 Adiutide Pharmaceuticals Gmbh The phosphate-modified oligonucleotide analogs with immunostimulatory activity
CA2687178C (en) * 2007-05-23 2014-02-04 Ventana Medical Systems, Inc. Polymeric carriers for immunohistochemistry and in situ hybridization
KR101313915B1 (en) 2007-07-27 2013-10-01 이매틱스 바이오테크놀로지스 게엠베하 Novel immunogenic epitopes for immunotherapy
DK2660248T3 (en) 2007-07-27 2015-09-07 Immatics Biotechnologies Gmbh Novel immunotherapy against brain tumors
SI2567707T1 (en) 2007-07-27 2017-01-31 Immatics Biotechnologies Gmbh Composition of tumour-associated peptides and related anti-cancer vaccine
US7879812B2 (en) 2007-08-06 2011-02-01 University Of Iowa Research Foundation Immunomodulatory oligonucleotides and methods of use therefor
WO2009030254A1 (en) 2007-09-04 2009-03-12 Curevac Gmbh Complexes of rna and cationic peptides for transfection and for immunostimulation
WO2010008411A1 (en) 2007-11-09 2010-01-21 The Salk Institute For Biological Studies Use of tam receptor inhibitors as immunoenhancers and tam activators as immunosuppressors
EP3100718B1 (en) 2008-01-02 2019-11-27 Arbutus Biopharma Corporation Improved compositions and methods for the delivery of nucleic acids
DK2176408T5 (en) 2008-01-31 2015-12-14 Curevac Gmbh Nucleic acids comprising FORMULA (NuGiXmGnNv) a AND DERIVATIVES AS IMMUNE STIMULATING AGENTS / ADJUVANTS.
ME02267B (en) 2008-03-27 2016-04-28 Immatics Biotechnologies Gmbh Novel immunotherapy against neuronal and brain tumors
WO2009127060A1 (en) 2008-04-15 2009-10-22 Protiva Biotherapeutics, Inc. Novel lipid formulations for nucleic acid delivery
SI2113253T1 (en) 2008-04-30 2010-06-30 Immatics Biotechnologies Gmbh Novel formulations of tumour-associated peptides binding to human leukocyte antigen (HLA) class I or II molecules for vaccines
PL2119726T5 (en) 2008-05-14 2018-04-30 Immatics Biotechnologies Gmbh Novel and powerful MHC-class II peptides derived from survivin and neurocan
US8703490B2 (en) 2008-06-05 2014-04-22 Ventana Medical Systems, Inc. Compositions comprising nanomaterials and method for using such compositions for histochemical processes
CA2960734C (en) 2008-06-27 2021-08-24 Zoetis Services Llc Adjuvants containing saponins, sterols and quaternary amines
TWI351288B (en) * 2008-07-04 2011-11-01 Univ Nat Pingtung Sci & Tech Cpg dna adjuvant in avian vaccines
EP2385371B1 (en) 2008-09-22 2014-10-22 Oregon Health and Science University Methods for detecting a mycobacterium tuberculosis infection
WO2010037408A1 (en) 2008-09-30 2010-04-08 Curevac Gmbh Composition comprising a complexed (m)rna and a naked mrna for providing or enhancing an immunostimulatory response in a mammal and uses thereof
SI2172211T1 (en) 2008-10-01 2015-03-31 Immatics Biotechnologies Gmbh Composition of tumor-associated peptides and related anti-cancer vaccine for the treatment of glioblastoma (GBM) and other cancers
EP2350043B9 (en) 2008-10-09 2014-08-20 TEKMIRA Pharmaceuticals Corporation Improved amino lipids and methods for the delivery of nucleic acids
US8552165B2 (en) * 2008-12-09 2013-10-08 Heather Davis Immunostimulatory oligonucleotides
CN102333538B (en) 2008-12-09 2014-01-15 科勒制药集团有限公司 Immunostimulatory oligonucleotides
CA3036963A1 (en) 2009-01-29 2010-08-05 Arbutus Biopharma Corporation Lipid formulations comprising cationic lipid and a targeting lipid comprising n-acetyl galactosamine for delivery of nucleic acid
WO2010099472A2 (en) 2009-02-27 2010-09-02 The U.S.A. Of America, As Represented By The Secretary, Department Of Health And Human Services Spanx-b polypeptides and their use
WO2010099322A1 (en) 2009-02-27 2010-09-02 Government Of The U.S.A. As Represented By The Secretary, Department Of Health And Human Services Francisella tularensis variants: compositions and methods of use
WO2010111292A1 (en) 2009-03-23 2010-09-30 Nanirx, Inc. Treatment of cancer with immunostimulatory hiv tat derivative polypeptides
WO2010115118A2 (en) 2009-04-03 2010-10-07 Antigenics, Inc. Methods for preparing and using multichaperone-antigen complexes
GB0906234D0 (en) 2009-04-14 2009-05-20 Secr Defence Vaccine
CA3151387A1 (en) 2009-05-05 2010-11-11 Arbutus Biopharma Corporation Lipid compositions for the delivery of therapeutic agents
WO2010129687A1 (en) 2009-05-05 2010-11-11 Alnylam Pharmaceuticals, Inc Methods of delivering oligonucleotides to immune cells
NZ622843A (en) 2009-06-10 2015-10-30 Tekmira Pharmaceuticals Corp Improved lipid formulation
CA2767127A1 (en) 2009-07-01 2011-01-06 Protiva Biotherapeutics, Inc. Novel lipid formulations for delivery of therapeutic agents to solid tumors
US20110033515A1 (en) * 2009-08-04 2011-02-10 Rst Implanted Cell Technology Tissue contacting material
WO2011026111A1 (en) 2009-08-31 2011-03-03 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Oral delivery of a vaccine to the large intestine to induce mucosal immunity
ES2443952T3 (en) 2009-09-02 2014-02-21 Novartis Ag Immunogenic compositions that include modulators of TLR activity
US20110053829A1 (en) 2009-09-03 2011-03-03 Curevac Gmbh Disulfide-linked polyethyleneglycol/peptide conjugates for the transfection of nucleic acids
CN102782138A (en) 2009-10-07 2012-11-14 Uvic工业合伙公司 Vaccines comprising heat-sensitive transgenes
WO2011047340A1 (en) 2009-10-16 2011-04-21 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Insertion of foreign genes in rubella virus and their stable expression in a live, attenuated viral vaccine
EP3360566B1 (en) 2009-11-20 2019-12-25 Oregon Health&Science University Methods for detecting a mycobacterium tuberculosis infection
WO2011073215A2 (en) 2009-12-14 2011-06-23 Immatics Biotechnologies Gmbh Hla-binding peptides derived from prostate-associated antigenic molecules and methods of use thereof
WO2011112599A2 (en) 2010-03-12 2011-09-15 The United States Of America, As Represented By The Secretary. Department Of Health & Human Services Immunogenic pote peptides and methods of use
GB201004551D0 (en) 2010-03-19 2010-05-05 Immatics Biotechnologies Gmbh NOvel immunotherapy against several tumors including gastrointestinal and gastric cancer
GB201004575D0 (en) 2010-03-19 2010-05-05 Immatics Biotechnologies Gmbh Composition of tumor associated peptides and related anti cancer vaccine for the treatment of gastric cancer and other cancers
GB201019331D0 (en) 2010-03-19 2010-12-29 Immatics Biotechnologies Gmbh Methods for the diagnosis and treatment of cancer based on AVL9
CA3197245A1 (en) 2010-05-14 2011-11-17 The General Hospital Corporation Compositions and methods of identifying tumor specific neoantigens
PT2772265T (en) 2010-05-14 2018-04-20 Univ Oregon Health & Science Recombinant hcmv and rhcmv vectors and uses thereof
TWI455722B (en) * 2010-06-04 2014-10-11 Pfizer Vaccines Llc Conjugates for the prevention or treatment of nicotine addiction
CN103025876A (en) 2010-07-30 2013-04-03 库瑞瓦格有限责任公司 Complexation of nucleic acids with disulfide-crosslinked cationic components for transfection and immunostimulation
EP2600901B1 (en) 2010-08-06 2019-03-27 ModernaTX, Inc. A pharmaceutical formulation comprising engineered nucleic acids and medical use thereof
ES2458355T3 (en) 2010-09-01 2014-05-05 Novartis Ag Adsorption of immunopotentiators on insoluble metal salts
EP3431100B1 (en) 2010-09-14 2021-09-08 University of Pittsburgh- Of the Commonwealth System of Higher Education Computationally optimized broadly reactive antigens for influenza
US10668092B2 (en) 2010-09-24 2020-06-02 The John Hopkins University Compositions and methods for treatment of inflammatory disorders
US9072760B2 (en) 2010-09-24 2015-07-07 University of Pittsburgh—of the Commonwealth System of Higher Education TLR4 inhibitors for the treatment of human infectious and inflammatory disorders
CN104531671A (en) 2010-10-01 2015-04-22 现代治疗公司 Engineered nucleic acids and methods of use thereof
NZ609916A (en) 2010-12-14 2015-03-27 Immatics Biotechnologies Gmbh Hla-binding peptides derived from prostate-associated antigenic molecules and methods of use thereof
WO2012088425A2 (en) 2010-12-22 2012-06-28 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Gap junction-enhancing agents for treatment of necrotizing enterocolitis and inflammatory bowel disease
BR112013022397A2 (en) * 2011-03-02 2017-09-26 Derek O’Hagan vaccines combined with lower doses of antigen and / or adjuvant
CA2831613A1 (en) 2011-03-31 2012-10-04 Moderna Therapeutics, Inc. Delivery and formulation of engineered nucleic acids
FR2975600B1 (en) 2011-05-24 2013-07-05 Assist Publ Hopitaux De Paris AGENTS FOR THE TREATMENT OF TUMORS
MX355623B (en) 2011-06-03 2018-04-25 3M Innovative Properties Co Hydrazino 1h-imidazoquinolin-4-amines and conjugates made therefrom.
US9475804B2 (en) 2011-06-03 2016-10-25 3M Innovative Properties Company Heterobifunctional linkers with polyethylene glycol segments and immune response modifier conjugates made therefrom
US9580475B2 (en) 2011-06-20 2017-02-28 University of Pittsburgh—of the Commonwealth System of Higher Education Computationally optimized broadly reactive antigens for H1N1 influenza
US20130023736A1 (en) 2011-07-21 2013-01-24 Stanley Dale Harpstead Systems for drug delivery and monitoring
US20140348865A1 (en) 2011-09-12 2014-11-27 The United States Of America, As Represented By The Secretary, Department Of Health And Human Ser. Immunogens based on an hiv-1 v1v2 site-of-vulnerability
US9464124B2 (en) 2011-09-12 2016-10-11 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
WO2013049535A2 (en) 2011-09-30 2013-04-04 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Influenza vaccine
EP2763701B1 (en) 2011-10-03 2018-12-19 Moderna Therapeutics, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US20140286983A1 (en) 2011-10-20 2014-09-25 The Government of the USA as represented by the Secretaryof the Dept. of Health and Human Services Dengue virus e-glycoprotein polypeptides containing mutations that eliminate immunodominant cross-reactive epitopes
EP2596806A1 (en) 2011-11-25 2013-05-29 Index Pharmaceuticals AB Method for prevention of colectomy
JP2015501844A (en) 2011-12-16 2015-01-19 モデルナ セラピューティクス インコーポレイテッドModerna Therapeutics,Inc. Modified nucleosides, nucleotides and nucleic acid compositions
US9901631B2 (en) 2012-01-10 2018-02-27 The Broad Institute, Inc. Method and cells for the production of viral vaccines
US20140086849A1 (en) 2012-09-21 2014-03-27 Elizabeth McKenna Naturally-Occurring CpG Oligonucleotide Compositions and Therapeutic Applications Thereof
EA201491081A1 (en) 2012-01-16 2016-11-30 Элизабет Маккенна COMPOSITIONS AND METHODS FOR THE TREATMENT OF DISEASES AND LIVER DISORDERS
WO2013113326A1 (en) 2012-01-31 2013-08-08 Curevac Gmbh Pharmaceutical composition comprising a polymeric carrier cargo complex and at least one protein or peptide antigen
CN104136456A (en) 2012-02-07 2014-11-05 高等教育联邦系统-匹兹堡大学 Computationally optimized broadly reactive antigens for h3n2, h2n2, and b influenza viruses
IN2014DN05695A (en) 2012-02-13 2015-05-15 Univ Pittsburgh
JP6170077B2 (en) 2012-02-16 2017-07-26 エータイアー ファーマ, インコーポレイテッド Histidyl tRNA synthetase for treating autoimmune and inflammatory diseases
RS58077B1 (en) 2012-02-24 2019-02-28 Arbutus Biopharma Corp Trialkyl cationic lipids and methods of use thereof
JP6345603B2 (en) 2012-03-08 2018-06-20 ノバルティス アーゲー Adjuvanted formulation of booster vaccine
WO2013142808A1 (en) 2012-03-23 2013-09-26 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Pathogenic phlebovirus isolates and compositions and methods of use
DE18200782T1 (en) 2012-04-02 2021-10-21 Modernatx, Inc. MODIFIED POLYNUCLEOTIDES FOR THE PRODUCTION OF PROTEINS ASSOCIATED WITH DISEASES IN HUMANS
US10501512B2 (en) 2012-04-02 2019-12-10 Modernatx, Inc. Modified polynucleotides
US9572897B2 (en) 2012-04-02 2017-02-21 Modernatx, Inc. Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins
US9283287B2 (en) 2012-04-02 2016-03-15 Moderna Therapeutics, Inc. Modified polynucleotides for the production of nuclear proteins
US9409956B2 (en) 2012-05-23 2016-08-09 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Salmonella typhi Ty21a expressing Yersinia pestis F1-V fusion protein and uses thereof
CN104428008B (en) 2012-05-24 2020-10-09 美国政府(由卫生和人类服务部的部长所代表) Multivalent meningococcal conjugates and methods of making conjugates
US9687542B2 (en) 2012-06-19 2017-06-27 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Rift valley fever virus replicon particles and use thereof
EP3511425A1 (en) 2012-07-12 2019-07-17 Persimmune, Inc. Personalized cancer vaccines and adoptive immune cell therapies
WO2014043518A1 (en) 2012-09-14 2014-03-20 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Brachyury protein, non-poxvirus non-yeast vectors encoding brachyury protein, and their use
US9562066B2 (en) * 2012-09-25 2017-02-07 University Of Pittsburgh-Of The Commonwealth System Of Higher Education Oral therapy of necrotizing enterocolitis
HRP20220607T1 (en) 2012-11-26 2022-06-24 Modernatx, Inc. Terminally modified rna
EP2948469A4 (en) 2013-01-23 2016-11-02 Univ Leland Stanford Junior Stabilized hepatitis b core polypeptide
ES2921174T3 (en) * 2013-03-15 2022-08-19 Corium Inc Set of microstructures for the delivery of active agents, method of manufacturing the set of microstructures and liquid formulation thereof
US8980864B2 (en) 2013-03-15 2015-03-17 Moderna Therapeutics, Inc. Compositions and methods of altering cholesterol levels
CN105377292A (en) 2013-04-07 2016-03-02 博德研究所 Compositions and methods for personalized neoplasia vaccines
EP2986717A1 (en) 2013-04-19 2016-02-24 The Regents of The University of California Lone star virus
BR112015030989A2 (en) * 2013-06-14 2019-09-24 Intervet Int Bv pharmaceutical composition and use of a pharmaceutical composition
CA2919268C (en) 2013-07-25 2023-09-05 Exicure, Inc. Spherical nucleic acid-based constructs as immunostimulatory agents for prophylactic and therapeutic use
EP3632458A1 (en) 2013-07-26 2020-04-08 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods and pharmaceutical compositions for the treatment of bacterial infections
TWI819228B (en) 2013-08-05 2023-10-21 德商伊瑪提克斯生物科技有限公司 Novel peptides, cells, and their use against several tumors, methods for production thereof and pharmaceutical composition comprising the same
WO2015022401A1 (en) * 2013-08-14 2015-02-19 INSERM (Institut National de la Santé et de la Recherche Médicale) Immunoadjuvant compositions and uses thereof
SG11201510746WA (en) 2013-08-21 2016-03-30 Curevac Ag Respiratory syncytial virus (rsv) vaccine
CA3005608C (en) 2013-09-19 2020-06-30 Zoetis Services Llc Water-in-oil emulsions comprising immunostimulatory oligonucleotides
US10010606B2 (en) 2013-09-30 2018-07-03 Los Alamos National Security, Llc Mosaic conserved region HIV immunogenic polypeptides
US10023626B2 (en) 2013-09-30 2018-07-17 Modernatx, Inc. Polynucleotides encoding immune modulating polypeptides
EA201690675A1 (en) 2013-10-03 2016-08-31 Модерна Терапьютикс, Инк. POLYNUCLEOTES ENCODING THE RECEPTOR OF LOW DENSITY LIPOPROTEINS
EP3052127A1 (en) 2013-10-04 2016-08-10 PIN Pharma, Inc. Immunostimulatory hiv tat derivative polypeptides for use in cancer treatment
CA2925021A1 (en) 2013-11-01 2015-05-07 Curevac Ag Modified rna with decreased immunostimulatory properties
GB201319446D0 (en) 2013-11-04 2013-12-18 Immatics Biotechnologies Gmbh Personalized immunotherapy against several neuronal and brain tumors
EP3091074B1 (en) 2013-11-21 2019-08-07 Repertoire Genesis Incorporation T cell receptor and b cell receptor repertoire analysis system, and use of same in treatment and diagnosis
WO2015077717A1 (en) 2013-11-25 2015-05-28 The Broad Institute Inc. Compositions and methods for diagnosing, evaluating and treating cancer by means of the dna methylation status
CN112107693B (en) 2013-12-03 2023-05-26 西北大学 Liposome particles, method for preparing said liposome particles and use thereof
WO2015085147A1 (en) 2013-12-05 2015-06-11 The Broad Institute Inc. Polymorphic gene typing and somatic change detection using sequencing data
JP6758185B2 (en) 2013-12-13 2020-09-23 ザ ユナイテッド ステイツ オブ アメリカ, アズ リプレゼンテッド バイ ザ セクレタリー, デパートメント オブ ヘルス アンド ヒューマン サービシーズ Multiepitope TARP peptide vaccine and its use
KR20230076867A (en) 2013-12-20 2023-05-31 더 브로드 인스티튜트, 인코퍼레이티드 Combination therapy with neoantigen vaccine
CA2936286A1 (en) 2014-04-01 2015-10-08 Curevac Ag Polymeric carrier cargo complex for use as an immunostimulating agent or as an adjuvant
GB201408255D0 (en) 2014-05-09 2014-06-25 Immatics Biotechnologies Gmbh Novel immunotherapy against several tumours of the blood, such as acute myeloid leukemia (AML)
CA2986096A1 (en) 2014-05-30 2015-12-03 Sanofi Pasteur Inc. Expression and conformational analysis of engineered influenza hemagglutinin
PL3164113T3 (en) 2014-06-04 2019-09-30 Exicure, Inc. Multivalent delivery of immune modulators by liposomal spherical nucleic acids for prophylactic or therapeutic applications
GB201411037D0 (en) 2014-06-20 2014-08-06 Immatics Biotechnologies Gmbh Novel immunotherapy against several tumors of the blood, in particular chronic lymphoid leukemai (CLL)
MX2017004448A (en) 2014-10-06 2017-10-23 Exicure Inc Anti-tnf compounds.
CA2968531A1 (en) 2014-11-21 2016-05-26 Northwestern University The sequence-specific cellular uptake of spherical nucleic acid nanoparticle conjugates
EP3234193B1 (en) 2014-12-19 2020-07-15 Massachusetts Institute of Technology Molecular biomarkers for cancer immunotherapy
WO2016100977A1 (en) 2014-12-19 2016-06-23 The Broad Institute Inc. Methods for profiling the t-cel- receptor repertoire
GB201501017D0 (en) 2014-12-23 2015-03-04 Immatics Biotechnologies Gmbh Novel peptides and combination of peptides for use in immunotherapy against hepatocellular carcinoma (HCC) and other cancers
KR102382773B1 (en) 2015-01-16 2022-04-04 조에티스 서비시즈 엘엘씨 Foot-and-mouth disease vaccine
CA3014427A1 (en) 2015-02-13 2016-08-18 Icahn School Of Medicine At Mount Sinai Rna containing compositions and methods of their use
BR112017020491A2 (en) 2015-03-25 2018-07-17 The Regents Of The University Of Michigan compositions and methods for delivery of biomacromolecule agents.
GB201505585D0 (en) 2015-03-31 2015-05-13 Immatics Biotechnologies Gmbh Novel peptides and combination of peptides and scaffolds for use in immunotherapy against renal cell carinoma (RCC) and other cancers
GB201507030D0 (en) 2015-04-24 2015-06-10 Immatics Biotechnologies Gmbh Immunotherapy against lung cancers, in particular NSCLC
GB201507719D0 (en) 2015-05-06 2015-06-17 Immatics Biotechnologies Gmbh Novel peptides and combination of peptides and scaffolds thereof for use in immunotherapy against colorectal carcinoma (CRC) and other cancers
WO2016180852A1 (en) 2015-05-12 2016-11-17 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for preparing antigen-specific t cells from an umbilical cord blood sample
EP3294324A1 (en) 2015-05-13 2018-03-21 Agenus Inc. Vaccines for treatment and prevention of cancer
JP6913032B2 (en) 2015-05-20 2021-08-04 ザ・ブロード・インスティテュート・インコーポレイテッド Common neoantigen
AU2016270979B2 (en) 2015-06-02 2020-11-12 Sanofi Pasteur Inc. Engineered influenza antigenic polypeptides and immunogenic compositions thereof
TWI750122B (en) 2015-06-09 2021-12-21 美商博德研究所有限公司 Formulations for neoplasia vaccines and methods of preparing thereof
JP6886410B2 (en) 2015-06-10 2021-06-16 ザ ユナイテッド ステイツ オブ アメリカ, アズ リプレゼンテッド バイ ザ セクレタリー, デパートメント オブ ヘルス アンド ヒューマン サービシーズ Process for preparation and purification of nucleic acid-containing compositions
EP3919507A3 (en) 2015-07-01 2022-01-12 Immatics Biotechnologies GmbH Novel peptides and combination of peptides for use in immunotherapy against ovarian cancer and other cancers
GB201511546D0 (en) 2015-07-01 2015-08-12 Immatics Biotechnologies Gmbh Novel peptides and combination of peptides for use in immunotherapy against ovarian cancer and other cancers
CN113880938A (en) 2015-07-06 2022-01-04 伊玛提克斯生物技术有限公司 Novel peptides and peptide compositions for immunotherapy of esophageal and other cancers
GB201513921D0 (en) 2015-08-05 2015-09-23 Immatics Biotechnologies Gmbh Novel peptides and combination of peptides for use in immunotherapy against prostate cancer and other cancers
CN108024914A (en) 2015-09-17 2018-05-11 Jrx生物技术有限公司 Improve the method for the aquation or wetting action of skin
WO2017059280A1 (en) 2015-10-02 2017-04-06 The University Of North Carolina At Chapel Hill Novel pan-tam inhibitors and mer/axl dual inhibitors
US10548970B2 (en) 2015-10-05 2020-02-04 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Human rotavirus G9P[6] strain and use as a vaccine
WO2017075570A1 (en) 2015-10-30 2017-05-04 The United States Of America, As Represented By Secretary, Department Of Health And Human Sevices Compositions and methods for the treatment of her2-expressing solid tumors
CA3006779A1 (en) 2015-12-09 2017-06-15 Admedus Vaccines Pty Ltd Immunomodulating composition for treatment
GB201522667D0 (en) 2015-12-22 2016-02-03 Immatics Biotechnologies Gmbh Novel peptides and combination of peptides for use in immunotherapy against breast cancer and other cancers
WO2017143076A1 (en) 2016-02-16 2017-08-24 Dana-Farber Cancer Institute, Inc. Chimeric antigen receptors and methods of use thereof
GB201602918D0 (en) 2016-02-19 2016-04-06 Immatics Biotechnologies Gmbh Novel peptides and combination of peptides for use in immunotherapy against NHL and other cancers
CN116375797A (en) 2016-03-01 2023-07-04 伊玛提克斯生物技术有限公司 Peptides, peptide compositions and cell-based drugs for immunotherapy of bladder cancer and other cancers
MX2018012152A (en) 2016-04-06 2019-02-07 Immatics Biotechnologies Gmbh Novel peptides and combination of peptides for use in immunotherapy against aml and other cancers.
WO2017184590A1 (en) 2016-04-18 2017-10-26 The Broad Institute Inc. Improved hla epitope prediction
WO2017189448A1 (en) 2016-04-25 2017-11-02 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Bivalent immunogenic conjugate for malaria and typhoid
CA3026096A1 (en) 2016-06-02 2017-12-07 Sanofi Pasteur Inc. Engineered influenza antigenic polypeptides and immunogenic compositions thereof
MA45159A (en) 2016-06-03 2019-04-10 Sanofi Pasteur Inc MODIFIED INFLUENZA HEMAGGLUTININ POLYPEPTIDES
US10947277B2 (en) 2016-06-13 2021-03-16 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Nucleic acids encoding zika virus-like particles and their use in zika virus vaccines and diagnostic assays
WO2018009604A1 (en) 2016-07-08 2018-01-11 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Chimeric dengue/zika viruses live-attenuated zika virus vaccines
WO2018009603A1 (en) 2016-07-08 2018-01-11 The United State of America, as represented by the Secretary, Department of Health and Human Service Chimeric west nile/zika viruses and methods of use
US11850279B2 (en) 2016-07-13 2023-12-26 Ohio State Innovation Foundation Platforms and methods for optimizing host antigen presentation and host antitumor and antipathogen immunity
WO2018027123A1 (en) 2016-08-05 2018-02-08 Sanofi Pasteur, Inc. Multivalent pneumococcal polysaccharide-protein conjugate composition
US11241489B2 (en) 2016-08-05 2022-02-08 Sanofi Pasteur Inc. Multivalent pneumococcal polysaccharide-protein conjugate composition
US11364304B2 (en) 2016-08-25 2022-06-21 Northwestern University Crosslinked micellar spherical nucleic acids
TWI796299B (en) 2016-08-26 2023-03-21 德商英麥提克生物技術股份有限公司 Novel peptides and scaffolds for use in immunotherapy against head and neck squamous cell carcinoma and other cancers
US10172933B2 (en) 2016-10-31 2019-01-08 The United States Of America, As Represented By The Secretary Of Agriculture Mosaic vaccines for serotype a foot-and-mouth disease virus
EP3574116A1 (en) 2017-01-24 2019-12-04 The Broad Institute, Inc. Compositions and methods for detecting a mutant variant of a polynucleotide
TWI796314B (en) 2017-01-27 2023-03-21 德商英麥提克生物技術股份有限公司 Novel peptides and combination of peptides for use in immunotherapy against ovarian cancer and other cancers
AU2018214451B2 (en) 2017-02-06 2022-02-03 Meat & Livestock Australia Limited Immunostimulating compositions and uses therefore
CN111093691A (en) 2017-04-03 2020-05-01 内恩疗法公司 Protein antigens and uses thereof
CN111499716A (en) 2017-04-10 2020-08-07 伊玛提克斯生物技术有限公司 Peptides and peptide compositions thereof for cancer immunotherapy
EA201992416A1 (en) 2017-04-10 2020-02-25 Имматикс Байотекнолоджиз Гмбх PEPTIDES AND COMBINATIONS OF PEPTIDES FOR APPLICATION IN IMMUNOTHERAPY OF LEUKOSIS AND OTHER TYPES OF CANCER
TW201841934A (en) 2017-04-10 2018-12-01 德商英麥提克生物技術股份有限公司 Novel peptides and combination thereof for use in the immunotherapy against cancers
WO2018201090A1 (en) 2017-04-28 2018-11-01 Exicure, Inc. Synthesis of spherical nucleic acids using lipophilic moieties
WO2018213803A1 (en) 2017-05-19 2018-11-22 Neon Therapeutics, Inc. Immunogenic neoantigen identification
US11642405B2 (en) 2017-06-19 2023-05-09 Washington University Nanoemulsion and methods of use thereof
EP3641789A4 (en) 2017-06-22 2021-08-11 Board Of Regents, The University Of Texas System Methods for producing regulatory immune cells and uses thereof
CR20200059A (en) 2017-07-07 2020-06-01 Immatics Biotechnologies Gmbh Novel peptides and combination of peptides for use in immunotherapy against lung cancer, including nsclc, sclc and other cancers
US10800823B2 (en) 2017-07-07 2020-10-13 Immatics Biotechnologies Gmbh Peptides and combination of peptides for use in immunotherapy against lung cancer, including NSCLC, SCLC and other cancers
JP2020526558A (en) 2017-07-13 2020-08-31 ノースウェスタン ユニバーシティ Common and direct methods for preparing oligonucleotide functionalized metal-organic framework nanoparticles
BR112020001255A2 (en) 2017-07-21 2020-07-28 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services immunogenic compositions of neisseria meningitidis
US11419928B2 (en) 2017-10-25 2022-08-23 The Johns Hopkins University Methods and compositions for treating cancer
US11235046B2 (en) 2017-11-04 2022-02-01 Nevada Research & Innovation Corporation Immunogenic conjugates and methods of use thereof
BR112020008927A2 (en) 2017-11-08 2020-10-20 Neon Therapeutics, Inc. t cell compositions and methods
WO2019126197A1 (en) 2017-12-18 2019-06-27 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Bacterial polysaccharide-conjugated carrier proteins and use thereof
CN111741767A (en) 2017-12-19 2020-10-02 麻省理工学院 Antigen-adjuvant coupling agents and methods of use
CN111629751A (en) 2018-01-22 2020-09-04 美国政府(由卫生和人类服务部的部长所代表) Broad spectrum protective inactivated influenza virus vaccines
DE102018107224A1 (en) 2018-02-21 2019-08-22 Immatics Biotechnologies Gmbh Peptides and combinations of peptides of non-canonical origin for use in immunotherapy against various cancers
CN112292137A (en) 2018-02-21 2021-01-29 得克萨斯大学体系董事会 Universal antigen presenting cells and uses thereof
CA3034912A1 (en) 2018-02-28 2019-08-28 Pfizer Inc. Il-15 variants and uses thereof
JP7329529B2 (en) 2018-03-28 2023-08-18 サノフィ パスツール インコーポレイテッド Methods of Producing Broadly Protective Vaccine Compositions Containing Hemagglutinin
CN112584858A (en) 2018-04-03 2021-03-30 赛诺菲 Antigenic OSPA polypeptides
EP3773698A1 (en) 2018-04-03 2021-02-17 Sanofi Ferritin proteins
CN112512565A (en) 2018-04-03 2021-03-16 赛诺菲 Antigenic influenza-ferritin polypeptides
MX2020010199A (en) 2018-04-03 2021-01-08 Sanofi Sa Antigenic respiratory syncytial virus polypeptides.
CN112512566A (en) 2018-04-03 2021-03-16 赛诺菲 Antigenic epstein-barr virus polypeptides
TW202016131A (en) 2018-05-16 2020-05-01 德商英麥提克生物技術股份有限公司 Peptides for use in immunotherapy against cancers
MX2020012607A (en) 2018-05-23 2021-01-29 Pfizer Antibodies specific for gucy2c and uses thereof.
US11434292B2 (en) 2018-05-23 2022-09-06 Pfizer Inc. Antibodies specific for CD3 and uses thereof
US10925947B2 (en) 2018-06-29 2021-02-23 Immatics Biotechnologies Gmbh A*03 restricted peptides for use in immunotherapy against cancers and related methods
TW202019955A (en) 2018-07-31 2020-06-01 德商英麥提克生物技術股份有限公司 Immunotherapy with b*07 restricted peptides and combination of peptides against cancers and related methods
US20200087363A1 (en) 2018-09-17 2020-03-19 Immatics Biotechnologies Gmbh B*44 restricted peptides for use in immunotherapy against cancers and related methods
TW202024121A (en) 2018-09-18 2020-07-01 德商英麥提克生物技術股份有限公司 Immunotherapy with a*01 restricted peptides and combination of peptides against cancers and related methods
US20210382068A1 (en) 2018-10-02 2021-12-09 Dana-Farber Cancer Institute, Inc. Hla single allele lines
KR20210124205A (en) 2018-12-04 2021-10-14 더 락커펠러 유니버시티 HIV Vaccine Immunogen
EP3894547A1 (en) 2018-12-12 2021-10-20 The United States of America, as represented by The Secretary, Department of Health and Human Services Recombinant mumps virus vaccine expressing genotype g fusion and hemagglutinin-neuraminidase proteins
US20220023410A1 (en) 2018-12-14 2022-01-27 University Of Georgia Research Foundation, Inc. Crimean-congo hemorrhagic fever virus replicon particles and use thereof
WO2020131586A2 (en) 2018-12-17 2020-06-25 The Broad Institute, Inc. Methods for identifying neoantigens
TW202039535A (en) 2018-12-18 2020-11-01 德商英麥提克生物技術股份有限公司 Immunotherapy with b*08 restricted peptides and combination of peptides against cancers and related methods
US20220370606A1 (en) 2018-12-21 2022-11-24 Pfizer Inc. Combination Treatments Of Cancer Comprising A TLR Agonist
EP3946446A1 (en) 2019-04-02 2022-02-09 Sanofi Antigenic multimeric respiratory syncytial virus polypeptides
KR20220029584A (en) 2019-05-27 2022-03-08 이매틱스 유에스 인코포레이티드 Viral vectors and their use in adoptive cell therapy
CN114096280A (en) 2019-07-12 2022-02-25 俄勒冈健康与科学大学 Therapeutic constructs for co-delivery of mitotic kinase inhibitors and immune checkpoint inhibitors
US20220249389A1 (en) 2019-07-12 2022-08-11 Oregon Health & Science University Immunotherapeutic constructs and methods of their use
BR112022010228A2 (en) 2019-12-17 2022-09-06 Us Health ATTENUATED LIVE LEISHMANIA PARASITE VACCINES WITH INCREASED SAFETY CHARACTERISTICS
BR112022009723A2 (en) 2019-12-17 2022-08-09 Pfizer SPECIFIC ANTIBODIES TO CD47, PD-L1, AND USES THEREOF
WO2021144020A1 (en) 2020-01-15 2021-07-22 Immatics Biotechnologies Gmbh Antigen binding proteins specifically binding prame
JP2023515829A (en) 2020-02-28 2023-04-14 サノフィ パスツール インコーポレイテッド High-dose influenza vaccine for pediatric subjects
US11213482B1 (en) 2020-03-05 2022-01-04 University of Pittsburgh—Of the Commonwealth System of Higher Educat SARS-CoV-2 subunit vaccine and microneedle array delivery system
DE102020111571A1 (en) 2020-03-11 2021-09-16 Immatics US, Inc. WPRE MUTANT CONSTRUCTS, COMPOSITIONS, AND RELATED PROCEDURES
DE102020106710A1 (en) 2020-03-11 2021-09-16 Immatics US, Inc. WPRE MUTANT CONSTRUCTS, COMPOSITIONS, AND RELATED PROCEDURES
CN116323668A (en) 2020-07-17 2023-06-23 辉瑞公司 Therapeutic antibodies and uses thereof
AU2021325081A1 (en) 2020-08-13 2023-04-13 Biontech Us Inc. T cell manufacturing compositions and methods
DE102020125465A1 (en) 2020-09-29 2022-03-31 Immatics Biotechnologies Gmbh Amidated peptides and their deamidated counterparts presented by non-HLA-A*02 molecules for use in immunotherapy against various types of cancer
TW202229312A (en) 2020-09-29 2022-08-01 德商英麥提克生物技術股份有限公司 Amidated peptides and their deamidated counterparts displayed by non-hla-a*02 for use in immunotherapy against different types of cancers
DE102020125457A1 (en) 2020-09-29 2022-03-31 Immatics Biotechnologies Gmbh Amidated peptides and their deamidated counterparts presented by HLA-A*02 molecules for use in immunotherapy against various types of cancer
KR20230142704A (en) 2020-12-14 2023-10-11 바이오엔테크 유에스 인크. Tissue-specific antigens for cancer immunotherapy
CR20230295A (en) 2020-12-31 2023-07-27 Immatics Us Inc CD8 POLYPEPTIDES, COMPOSITIONS AND METHODS OF USE OF THESE
DE102021100038A1 (en) 2020-12-31 2022-06-30 Immatics US, Inc. MODIFIED CD8 POLYPEPTIDES, COMPOSITIONS AND METHODS OF USE THEREOF
TW202241925A (en) 2021-01-15 2022-11-01 德商英麥提克生物技術股份有限公司 Peptides displayed by hla for use in immunotherapy against different types of cancers
WO2023288263A1 (en) 2021-07-16 2023-01-19 The Board Of Trustees Of The University Of Illinois Universal vaccine for influenza virus based on tetrameric m2 protein incorporated into nanodiscs
WO2023018817A1 (en) 2021-08-11 2023-02-16 Sanofi Pasteur Inc. Truncated influenza neuraminidase and methods of using the same
US20230190805A1 (en) 2021-10-06 2023-06-22 Immatics Biotechnologies Gmbh Methods of identifying metastatic lesions in a patient and treating thereof
WO2023059857A1 (en) 2021-10-08 2023-04-13 Sanofi Pasteur Inc. Multivalent influenza vaccines
WO2023079113A1 (en) 2021-11-05 2023-05-11 Sanofi Hybrid multivalent influenza vaccines comprising hemagglutinin and neuraminidase and methods of using the same
WO2023079507A1 (en) 2021-11-05 2023-05-11 Sanofi Respiratory syncytial virus rna vaccine
WO2023081798A1 (en) 2021-11-05 2023-05-11 Sanofi Pasteur Inc. Multivalent influenza vaccines comprising recombinant hemagglutinin and neuraminidase and methods of using the same
WO2023081925A1 (en) 2021-11-08 2023-05-11 Immatics Biotechnologies Gmbh Adoptive cell therapy combination treatment and compositions thereof
US20230310571A1 (en) 2021-11-30 2023-10-05 Sanofi Pasteur Inc. Human metapneumovirus vaccines
WO2023102388A1 (en) 2021-11-30 2023-06-08 Sanofi Pasteur Inc. Human metapneumovirus viral vector-based vaccines
WO2023111262A1 (en) 2021-12-17 2023-06-22 Sanofi Lyme disease rna vaccine
WO2023144206A1 (en) 2022-01-27 2023-08-03 Sanofi Pasteur Modified vero cells and methods of using the same for virus production
WO2023144798A1 (en) 2022-01-31 2023-08-03 Genevant Sciences Gmbh Ionizable cationic lipids for lipid nanoparticles
WO2023177579A1 (en) 2022-03-14 2023-09-21 Sanofi Pasteur Inc. Machine-learning techniques in protein design for vaccine generation
WO2023212655A1 (en) 2022-04-28 2023-11-02 Immatics US, Inc. Il-12 polypeptides, il-15 polypeptides, il-18 polypeptides, cd8 polypeptides, compositions, and methods of using thereof
US20230348561A1 (en) 2022-04-28 2023-11-02 Immatics US, Inc. Dominant negative tgfbeta receptor polypeptides, cd8 polypeptides, cells, compositions, and methods of using thereof
US20230348548A1 (en) 2022-04-28 2023-11-02 Immatics US, Inc. Membrane-bound il-15, cd8 polypeptides, cells, compositions, and methods of using thereof
WO2023215825A1 (en) 2022-05-05 2023-11-09 Immatics US, Inc. Methods for improving t cell efficacy
WO2023214082A2 (en) 2022-05-06 2023-11-09 Sanofi Signal sequences for nucleic acid vaccines

Citations (63)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4185090A (en) * 1972-02-02 1980-01-22 Abbott Laboratories Chemically modified endotoxin immunizing agent
US4188375A (en) * 1976-09-25 1980-02-12 Bayer Aktiengesellschaft Process for the preparation of vaccines
US4578269A (en) * 1982-10-18 1986-03-25 Bror Morein Immunogenic protein or peptide complex, method or producing said complex and the use thereof as an immune stimulant and as a vaccine
US4981684A (en) * 1989-10-24 1991-01-01 Coopers Animal Health Limited Formation of adjuvant complexes
US5100662A (en) * 1989-08-23 1992-03-31 The Liposome Company, Inc. Steroidal liposomes exhibiting enhanced stability
US5178860A (en) * 1989-09-01 1993-01-12 Coopers Animal Health Limited Adjuvant complexes and vaccine made therefrom
US5723130A (en) * 1993-05-25 1998-03-03 Hancock; Gerald E. Adjuvants for vaccines against respiratory syncytial virus
US5723335A (en) * 1994-03-25 1998-03-03 Isis Pharmaceuticals, Inc. Immune stimulation by phosphorothioate oligonucleotide analogs
US5728518A (en) * 1994-01-12 1998-03-17 The Immune Response Corporation Antiviral poly-and oligonucleotides
US6027732A (en) * 1996-02-21 2000-02-22 Morein; Bror Iscom or iscom-matrix comprising hydrophobic receptor molecules for antigenic substances
US6174872B1 (en) * 1996-10-04 2001-01-16 The Regents Of The University Of California Method for treating allergic lung disease
US6194388B1 (en) * 1994-07-15 2001-02-27 The University Of Iowa Research Foundation Immunomodulatory oligonucleotides
US6207646B1 (en) * 1994-07-15 2001-03-27 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US6214806B1 (en) * 1997-02-28 2001-04-10 University Of Iowa Research Foundation Use of nucleic acids containing unmethylated CPC dinucleotide in the treatment of LPS-associated disorders
US6218371B1 (en) * 1998-04-03 2001-04-17 University Of Iowa Research Foundation Methods and products for stimulating the immune system using immunotherapeutic oligonucleotides and cytokines
US6221882B1 (en) * 1997-07-03 2001-04-24 University Of Iowa Research Foundation Methods for inhibiting immunostimulatory DNA associated responses
US6339068B1 (en) * 1997-05-20 2002-01-15 University Of Iowa Research Foundation Vectors and methods for immunization or therapeutic protocols
US20020028784A1 (en) * 2000-03-10 2002-03-07 Nest Gary Van Methods of preventing and treating viral infections using immunomodulatory polynucleotide sequences
US20020042387A1 (en) * 2000-02-23 2002-04-11 Eyal Raz Method for treating inflammatory bowel disease and other forms of gastrointestinal inflammation
US6503533B1 (en) * 1994-07-28 2003-01-07 Georgetown University Antisense ogligonucleotides against Hepatitis B viral replication
US6506386B1 (en) * 1998-08-05 2003-01-14 Smithkline Beecham Biologicals, S.A. Vaccine comprising an iscom consisting of sterol and saponin which is free of additional detergent
US20030022852A1 (en) * 2000-03-10 2003-01-30 Nest Gary Van Biodegradable immunomodulatory formulations and methods for use thereof
US6514948B1 (en) * 1999-07-02 2003-02-04 The Regents Of The University Of California Method for enhancing an immune response
US20030026782A1 (en) * 1995-02-07 2003-02-06 Arthur M. Krieg Immunomodulatory oligonucleotides
US20030026801A1 (en) * 2000-06-22 2003-02-06 George Weiner Methods for enhancing antibody-induced cell lysis and treating cancer
US20030050268A1 (en) * 2001-03-29 2003-03-13 Krieg Arthur M. Immunostimulatory nucleic acid for treatment of non-allergic inflammatory diseases
US20030049266A1 (en) * 2000-12-27 2003-03-13 Fearon Karen L. Immunomodulatory polynucleotides and methods of using the same
US20030050263A1 (en) * 1994-07-15 2003-03-13 The University Of Iowa Research Foundation Methods and products for treating HIV infection
US6534062B2 (en) * 2000-03-28 2003-03-18 The Regents Of The University Of California Methods for increasing a cytotoxic T lymphocyte response in vivo
US20030055014A1 (en) * 2000-12-14 2003-03-20 Bratzler Robert L. Inhibition of angiogenesis by nucleic acids
US20030059773A1 (en) * 2000-03-10 2003-03-27 Gary Van Nest Immunomodulatory formulations and methods for use thereof
US20030064064A1 (en) * 1998-09-18 2003-04-03 Dino Dina Methods of treating IgE-associated disorders and compositions for use therein
US6544518B1 (en) * 1999-04-19 2003-04-08 Smithkline Beecham Biologicals S.A. Vaccines
US6552006B2 (en) * 2000-01-31 2003-04-22 The Regents Of The University Of California Immunomodulatory polynucleotides in treatment of an infection by an intracellular pathogen
US20030078223A1 (en) * 1996-01-30 2003-04-24 Eyal Raz Compositions and methods for modulating an immune response
US20040006010A1 (en) * 1996-10-11 2004-01-08 Carson Dennis A. Immunostimulatory polynucleotide/immunomodulatory molecule conjugates
US20040006034A1 (en) * 1998-06-05 2004-01-08 Eyal Raz Immunostimulatory oligonucleotides, compositions thereof and methods of use thereof
US20040009942A1 (en) * 2000-03-10 2004-01-15 Gary Van Nest Methods of preventing and treating respiratory viral infection using immunomodulatory polynucleotide sequences
US20040009944A1 (en) * 2002-05-10 2004-01-15 Inex Pharmaceuticals Corporation Methylated immunostimulatory oligonucleotides and methods of using the same
US20040009949A1 (en) * 2002-06-05 2004-01-15 Coley Pharmaceutical Group, Inc. Method for treating autoimmune or inflammatory diseases with combinations of inhibitory oligonucleotides and small molecule antagonists of immunostimulatory CpG nucleic acids
US20040030118A1 (en) * 1998-05-14 2004-02-12 Hermann Wagner Methods for regulating hematopoiesis using CpG-oligonucleotides
US6693086B1 (en) * 1998-06-25 2004-02-17 National Jewish Medical And Research Center Systemic immune activation method using nucleic acid-lipid complexes
US20040038922A1 (en) * 2000-10-06 2004-02-26 Jean Haensler Vaccine composition
US20040053880A1 (en) * 2002-07-03 2004-03-18 Coley Pharmaceutical Group, Inc. Nucleic acid compositions for stimulating immune responses
US20040067902A9 (en) * 2000-02-03 2004-04-08 Bratzler Robert L. Immunostimulatory nucleic acids for the treatment of asthma and allergy
US20040067905A1 (en) * 2002-07-03 2004-04-08 Coley Pharmaceutical Group, Inc. Nucleic acid compositions for stimulating immune responses
US6727230B1 (en) * 1994-03-25 2004-04-27 Coley Pharmaceutical Group, Inc. Immune stimulation by phosphorothioate oligonucleotide analogs
US20050013812A1 (en) * 2003-07-14 2005-01-20 Dow Steven W. Vaccines using pattern recognition receptor-ligand:lipid complexes
US20050019340A1 (en) * 2000-10-18 2005-01-27 Nathalie Garcon Vaccines
US6852705B2 (en) * 2000-01-21 2005-02-08 Merial DNA vaccines for farm animals, in particular bovines and porcines
US20050054601A1 (en) * 1997-01-23 2005-03-10 Coley Pharmaceutical Gmbh Pharmaceutical composition comprising a polynucleotide and optionally an antigen especially for vaccination
US20050059619A1 (en) * 2002-08-19 2005-03-17 Coley Pharmaceutical Group, Inc. Immunostimulatory nucleic acids
US20060003962A1 (en) * 2002-10-29 2006-01-05 Coley Pharmaceutical Group, Ltd. Methods and products related to treatment and prevention of hepatitis C virus infection
US20060019916A1 (en) * 2004-04-02 2006-01-26 Coley Pharmaceutical Group, Inc. Immunostimulatory nucleic acids for inducing IL-10 responses
US20060019923A1 (en) * 2004-07-18 2006-01-26 Coley Pharmaceutical Group, Ltd. Methods and compositions for inducing innate immune responses
US20060058251A1 (en) * 1994-07-15 2006-03-16 University Of Iowa Research Foundation Methods for treating and preventing infectious disease
US20070041998A1 (en) * 2003-03-24 2007-02-22 Intercell Ag Use of alum and a th1 immune response inducing adjuvant for enhancing immune responses
US20070066554A1 (en) * 1999-09-25 2007-03-22 Coley Pharmaceutical Gmbh Immunostimulatory nucleic acids
US20070065467A1 (en) * 1994-07-15 2007-03-22 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules for activating dendritic cells
US20080009455A9 (en) * 2005-02-24 2008-01-10 Coley Pharmaceutical Group, Inc. Immunostimulatory oligonucleotides
US20080045473A1 (en) * 2006-02-15 2008-02-21 Coley Pharmaceutical Gmbh Compositions and methods for oligonucleotide formulations
US7488490B2 (en) * 1997-03-10 2009-02-10 University Of Iowa Research Foundation Method of inducing an antigen-specific immune response by administering a synergistic combination of adjuvants comprising unmethylated CpG-containing nucleic acids and a non-nucleic acid adjuvant
US20110033421A1 (en) * 1999-09-27 2011-02-10 Coley Pharmaceutical Gmbh Methods related to immunostimulatory nucleic acid-induced interferon

Family Cites Families (159)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3627874A (en) 1969-07-16 1971-12-14 Merck & Co Inc Vaccine preparation
US3761585A (en) 1971-04-05 1973-09-25 Beecham Group Ltd Vaccines containing modified allergenic material
US3906092A (en) 1971-11-26 1975-09-16 Merck & Co Inc Stimulation of antibody response
US5023243A (en) 1981-10-23 1991-06-11 Molecular Biosystems, Inc. Oligonucleotide therapeutic agent and method of making same
US5766920A (en) 1982-08-11 1998-06-16 Cellcor, Inc. Ex vivo activation of immune cells
US5075109A (en) 1986-10-24 1991-12-24 Southern Research Institute Method of potentiating an immune response
CA1339596C (en) 1987-08-07 1997-12-23 New England Medical Center Hospitals, Inc. Viral expression inhibitors
NZ230747A (en) 1988-09-30 1992-05-26 Bror Morein Immunomodulating matrix comprising a complex of at least one lipid and at least one saponin; certain glycosylated triterpenoid saponins derived from quillaja saponaria molina
DE68927679T2 (en) 1988-10-27 1997-06-26 Univ Minnesota Immunosuppressants from liposomes containing lymphokine IL -2
US5786189A (en) 1989-11-29 1998-07-28 Smithkline Beecham Biologicals (S.A.) Vaccine
US5248670A (en) 1990-02-26 1993-09-28 Isis Pharmaceuticals, Inc. Antisense oligonucleotides for inhibiting herpesviruses
US5514577A (en) 1990-02-26 1996-05-07 Isis Pharmaceuticals, Inc. Oligonucleotide therapies for modulating the effects of herpes viruses
EP0468520A3 (en) 1990-07-27 1992-07-01 Mitsui Toatsu Chemicals, Inc. Immunostimulatory remedies containing palindromic dna sequences
ATE154947T1 (en) 1990-08-16 1997-07-15 Isis Pharmaceuticals Inc OLIGONUCLEOTIDES FOR MODULATING THE EFFECTS OF CYTOMEGALOVIRUS INFECTIONS
US6042838A (en) * 1991-02-15 2000-03-28 Uab Research Foundation immunogenic compositions for mucosal administration of pneumococcal surface protein A (PspA)
US5683985A (en) 1991-04-18 1997-11-04 The Salk Institute For Biological Studies Oligonucleotide decoys and methods relating thereto
CA2110040A1 (en) 1991-05-31 1992-12-10 Lyle J. Arnold, Jr. Compositions and delivery systems for transdermal administration of neutral oligomers
US6030954A (en) * 1991-09-05 2000-02-29 University Of Connecticut Targeted delivery of poly- or oligonucleotides to cells
US5576302A (en) 1991-10-15 1996-11-19 Isis Pharmaceuticals, Inc. Oligonucleotides for modulating hepatitis C virus having phosphorothioate linkages of high chiral purity
US5599797A (en) * 1991-10-15 1997-02-04 Isis Pharmaceuticals, Inc. Oligonucleotides having phosphorothioate linkages of high chiral purity
DK0761231T3 (en) 1992-06-25 2000-05-08 Smithkline Beecham Biolog Vaccine containing adjuvants
JPH07504683A (en) 1992-07-08 1995-05-25 シェリング・コーポレーション Use of GM-CSF as a vaccine adjuvant
US5585479A (en) 1992-07-24 1996-12-17 The United States Of America As Represented By The Secretary Of The Navy Antisense oligonucleotides directed against human ELAM-I RNA
US6107062A (en) 1992-07-30 2000-08-22 Inpax, Inc. Antisense viruses and antisense-ribozyme viruses
US6004534A (en) 1993-07-23 1999-12-21 Massachusetts Institute Of Technology Targeted polymerized liposomes for improved drug delivery
US5679647A (en) 1993-08-26 1997-10-21 The Regents Of The University Of California Methods and devices for immunizing a host against tumor-associated antigens through administration of naked polynucleotides which encode tumor-associated antigenic peptides
AU705889B2 (en) 1993-08-26 1999-06-03 Regents Of The University Of California, The Method, compositions and devices for administration of naked polynucleotides which encode antigens and immunostimulatory peptides
US20030109469A1 (en) 1993-08-26 2003-06-12 Carson Dennis A. Recombinant gene expression vectors and methods for use of same to enhance the immune response of a host to an antigen
US5874085A (en) 1993-11-10 1999-02-23 Henry M. Jackson Foundation For The Advancement Of Military Medicine Vaccine for enhanced production of IgA antibodies
US5595756A (en) * 1993-12-22 1997-01-21 Inex Pharmaceuticals Corporation Liposomal compositions for enhanced retention of bioactive agents
US5543152A (en) 1994-06-20 1996-08-06 Inex Pharmaceuticals Corporation Sphingosomes for enhanced drug delivery
US5741516A (en) 1994-06-20 1998-04-21 Inex Pharmaceuticals Corporation Sphingosomes for enhanced drug delivery
US7935675B1 (en) * 1994-07-15 2011-05-03 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US5753613A (en) * 1994-09-30 1998-05-19 Inex Pharmaceuticals Corporation Compositions for the introduction of polyanionic materials into cells
US6027726A (en) * 1994-09-30 2000-02-22 Inex Phamaceuticals Corp. Glycosylated protein-liposome conjugates and methods for their preparation
US6168787B1 (en) 1995-04-24 2001-01-02 John Wayne Cancer Institute Pluripotent vaccine against enveloped viruses
UA56132C2 (en) 1995-04-25 2003-05-15 Смітклайн Бічем Байолоджікалс С.А. Vaccine composition (variants), method for stabilizing qs21 providing resistance against hydrolysis (variants), method for manufacturing vaccine
CA2220433C (en) 1995-05-11 2010-02-16 Applied Research Systems Ars Holding N.V. Il-6 activity inhibitor
EP1489184A1 (en) 1995-06-07 2004-12-22 Inex Pharmaceutical Corp. Lipid-nucleic acid particles prepared via a hydrophobic lipid-nucleic acid complex intermediate and use for gene transfer
US5705385A (en) * 1995-06-07 1998-01-06 Inex Pharmaceuticals Corporation Lipid-nucleic acid particles prepared via a hydrophobic lipid-nucleic acid complex intermediate and use for gene transfer
US5981501A (en) 1995-06-07 1999-11-09 Inex Pharmaceuticals Corp. Methods for encapsulating plasmids in lipid bilayers
GB9513261D0 (en) * 1995-06-29 1995-09-06 Smithkline Beecham Biolog Vaccines
US5985662A (en) 1995-07-13 1999-11-16 Isis Pharmaceuticals Inc. Antisense inhibition of hepatitis B virus replication
US6248720B1 (en) 1996-07-03 2001-06-19 Brown University Research Foundation Method for gene therapy using nucleic acid loaded polymeric microparticles
US5736152A (en) * 1995-10-27 1998-04-07 Atrix Laboratories, Inc. Non-polymeric sustained release delivery system
US5780448A (en) 1995-11-07 1998-07-14 Ottawa Civic Hospital Loeb Research DNA-based vaccination of fish
WO1997028259A1 (en) 1996-01-30 1997-08-07 The Regents Of The University Of California Gene expression vectors which generate an antigen specific immune response and methods of using the same
SE9600647D0 (en) 1996-02-21 1996-02-21 Bror Morein New use
US5843770A (en) 1996-03-11 1998-12-01 The Immune Response Corporation Antisense constructs directed against viral post-transcriptional regulatory sequences
US6620805B1 (en) 1996-03-14 2003-09-16 Yale University Delivery of nucleic acids by porphyrins
DE69737231T2 (en) 1996-04-19 2007-10-11 Merial Ltd. VACCINATION WITH NUCLEIC ACIDS AGAINST PARVOVIRUS INFECTIONS
CA2283557A1 (en) 1997-03-10 1998-09-17 Heather L. Davis Use of nucleic acids containing unmethylated cpg dinucleotide as an adjuvant
US5965542A (en) 1997-03-18 1999-10-12 Inex Pharmaceuticals Corp. Use of temperature to control the size of cationic liposome/plasmid DNA complexes
US6835395B1 (en) 1997-05-14 2004-12-28 The University Of British Columbia Composition containing small multilamellar oligodeoxynucleotide-containing lipid vesicles
US20030104044A1 (en) 1997-05-14 2003-06-05 Semple Sean C. Compositions for stimulating cytokine secretion and inducing an immune response
JP2001526688A (en) 1997-05-19 2001-12-18 メルク エンド カンパニー インコーポレーテッド Oligonucleotide adjuvant
US6589940B1 (en) 1997-06-06 2003-07-08 Dynavax Technologies Corporation Immunostimulatory oligonucleotides, compositions thereof and methods of use thereof
DE69840850D1 (en) 1997-06-06 2009-07-09 Dynavax Tech Corp INHIBITORS OF IMMUNSTIMULATORY DNA SEQUENCE ACTIVITY
AU8428998A (en) 1997-07-24 1999-02-16 Inex Pharmaceuticals Corporation Preparation of lipid-nucleic acid particles using a solvent extraction and direct hydration method
AU757175B2 (en) 1997-09-05 2003-02-06 Regents Of The University Of California, The Use of immunostimulatory oligonucleotides for preventing or reducing antigen-stimulated, granulocyte-mediated inflammation
GB9727262D0 (en) 1997-12-24 1998-02-25 Smithkline Beecham Biolog Vaccine
JPH11209289A (en) 1998-01-22 1999-08-03 Taisho Pharmaceut Co Ltd Mucosal immunity inducer
NZ506603A (en) 1998-04-09 2002-10-25 Smithkline Beecham Biolog S Adjuvant compositions comprising polyoxyethylene ether or polyoxyethylene ester
JP2002513763A (en) 1998-05-06 2002-05-14 ユニバーシティ オブ アイオワ リサーチ ファウンデーション Methods for preventing and treating parasitic infections and related diseases using CPG oligonucleotides
DK1077722T3 (en) 1998-05-22 2006-11-27 Ottawa Health Research Inst Methods and products for the induction of mucosa immunity
US6562798B1 (en) 1998-06-05 2003-05-13 Dynavax Technologies Corp. Immunostimulatory oligonucleotides with modified bases and methods of use thereof
WO1999066947A1 (en) 1998-06-23 1999-12-29 The Board Of Trustees Of The Leland Stanford Junior University Adjuvant therapy
US20030022854A1 (en) 1998-06-25 2003-01-30 Dow Steven W. Vaccines using nucleic acid-lipid complexes
US20040247662A1 (en) 1998-06-25 2004-12-09 Dow Steven W. Systemic immune activation method using nucleic acid-lipid complexes
EP1100807A1 (en) 1998-07-27 2001-05-23 University Of Iowa Research Foundation STEREOISOMERS OF CpG OLIGONUCLEOTIDES AND RELATED METHODS
ATE315405T1 (en) 1998-08-10 2006-02-15 Antigenics Inc CPG COMPOSITIONS, SAPONIN ADJUVANTS AND METHODS OF USE THEREOF
US20010034330A1 (en) 1998-08-10 2001-10-25 Charlotte Kensil Innate immunity-stimulating compositions of CpG and saponin and methods thereof
EP1108017A2 (en) 1998-09-03 2001-06-20 Coley Pharmaceutical GmbH G-motif oligonucleotides and uses thereof
FR2783170B1 (en) 1998-09-11 2004-07-16 Pasteur Merieux Serums Vacc IMMUNOSTIMULATING EMULSION
WO2000020039A1 (en) 1998-10-05 2000-04-13 The Regents Of The University Of California Methods and adjuvants for stimulating mucosal immunity
AU6425999A (en) 1998-10-09 2000-05-01 Dynavax Technologies Corporation Anti hiv compositions comprising immunostimulatory polynucleotides and hiv antigens
DE69935606T9 (en) 1998-10-16 2021-03-11 Glaxosmithkline Biologicals S.A. ADJUVANCE SYSTEMS AND VACCINE
CO5210925A1 (en) 1998-11-17 2002-10-30 Novartis Ag TETRASUSTITUID DIAMINUM NITROGUANIDINE DERIVATIVES
AUPP807399A0 (en) 1999-01-08 1999-02-04 Csl Limited Improved immunogenic lhrh composition and methods relating thereto
EP1165773A4 (en) 1999-02-02 2005-01-05 Biocache Pharmaceuticals Inc Advanced antigen presentation platform
US6887464B1 (en) 1999-02-02 2005-05-03 Biocache Pharmaceuticals, Inc. Advanced antigen presentation platform
WO2000045849A2 (en) 1999-02-05 2000-08-10 Genzyme Corporation Use of cationic lipids to generate anti-tumor immunity
US6977245B2 (en) 1999-04-12 2005-12-20 The United States Of America As Represented By The Department Of Health And Human Services Oligodeoxynucleotide and its use to induce an immune response
AU4642600A (en) 1999-04-15 2000-11-02 Regents Of The University Of California, The Methods and compositions for use in potentiating antigen presentation by antigenpresenting cells
US6558670B1 (en) * 1999-04-19 2003-05-06 Smithkline Beechman Biologicals S.A. Vaccine adjuvants
WO2000067023A1 (en) 1999-04-29 2000-11-09 Coley Pharmaceutical Gmbh Screening for immunostimulatory dna functional modifyers
US6737066B1 (en) 1999-05-06 2004-05-18 The Immune Response Corporation HIV immunogenic compositions and methods
CA2412345A1 (en) 1999-06-16 2000-12-21 University Of Iowa Research Foundation Antagonism of immunostimulatory cpg-oligonucleotides by 4-aminoquinolines and other weak bases
US20050002958A1 (en) 1999-06-29 2005-01-06 Smithkline Beecham Biologicals Sa Vaccines
ATE419869T1 (en) 1999-08-19 2009-01-15 Dynavax Tech Corp METHOD FOR MODULATING AN IMMUNE RESPONSE USING IMMUNSTIMULATIVE SEQUENCES AND COMPOSITIONS THEREOF
US20050249794A1 (en) 1999-08-27 2005-11-10 Semple Sean C Compositions for stimulating cytokine secretion and inducing an immune response
US7223398B1 (en) 1999-11-15 2007-05-29 Dynavax Technologies Corporation Immunomodulatory compositions containing an immunostimulatory sequence linked to antigen and methods of use thereof
WO2001051083A2 (en) 2000-01-13 2001-07-19 Antigenics Inc. Innate immunity-stimulating compositions of cpg and saponin and methods thereof
AU3108001A (en) 2000-01-20 2001-12-24 Coley Pharmaceutical Group, Inc. Immunostimulatory nucleic acids for inducing a th2 immune response
US20030130217A1 (en) 2000-02-23 2003-07-10 Eyal Raz Method for treating inflammatory bowel disease and other forms of gastrointestinal inflammation
US20020156033A1 (en) 2000-03-03 2002-10-24 Bratzler Robert L. Immunostimulatory nucleic acids and cancer medicament combination therapy for the treatment of cancer
US20040131628A1 (en) 2000-03-08 2004-07-08 Bratzler Robert L. Nucleic acids for the treatment of disorders associated with microorganisms
US20020107212A1 (en) 2000-03-10 2002-08-08 Nest Gary Van Methods of reducing papillomavirus infection using immunomodulatory polynucleotide sequences
US20020098199A1 (en) 2000-03-10 2002-07-25 Gary Van Nest Methods of suppressing hepatitis virus infection using immunomodulatory polynucleotide sequences
US7157437B2 (en) 2000-03-10 2007-01-02 Dynavax Technologies Corporation Methods of ameliorating symptoms of herpes infection using immunomodulatory polynucleotide sequences
US20020142978A1 (en) 2000-04-07 2002-10-03 Eyal Raz Synergistic improvements to polynucleotide vaccines
US6893821B2 (en) 2000-05-05 2005-05-17 The Regents Of The University Of California Agents that modulate DNA-PK activity and methods of use thereof
EP1280521B1 (en) * 2000-05-12 2005-07-20 Pharmacia & Upjohn Company LLC Vaccine composition, method of preparing the same, and method of vaccinating vertebrates
US6339630B1 (en) * 2000-05-18 2002-01-15 The United States Of America As Represented By The United States Department Of Energy Sealed drive screw operator
US20020165178A1 (en) 2000-06-28 2002-11-07 Christian Schetter Immunostimulatory nucleic acids for the treatment of anemia, thrombocytopenia, and neutropenia
US20020198165A1 (en) 2000-08-01 2002-12-26 Bratzler Robert L. Nucleic acids for the prevention and treatment of gastric ulcers
WO2002018631A2 (en) * 2000-09-01 2002-03-07 Epigenomics Ag Diagnosis of illnesses or predisposition to certain illnesses
US20020091097A1 (en) 2000-09-07 2002-07-11 Bratzler Robert L. Nucleic acids for the prevention and treatment of sexually transmitted diseases
EP1366077B1 (en) 2000-09-15 2011-05-25 Coley Pharmaceutical GmbH PROCESS FOR HIGH THROUGHPUT SCREENING OF CpG-BASED IMMUNO-AGONIST/ANTAGONIST
GB0025577D0 (en) * 2000-10-18 2000-12-06 Smithkline Beecham Biolog Vaccine
ES2307568T3 (en) 2000-12-08 2008-12-01 Coley Pharmaceutical Gmbh CPG TYPE NUCLEIC ACIDS AND SAME USE METHODS.
US7244438B2 (en) 2001-01-05 2007-07-17 Intercell Ag Uses for polycationic compounds
US6780448B1 (en) * 2001-02-06 2004-08-24 David Howard Pasteurization of food products
US7713942B2 (en) 2001-04-04 2010-05-11 Nordic Vaccine Technology A/S Cage-like microparticle complexes comprising sterols and saponins for delivery of polynucleotides
ES2487645T3 (en) 2001-06-21 2014-08-22 Dynavax Technologies Corporation Chimeric immunomodulatory compounds and methods of use thereof
US20030148316A1 (en) 2001-08-01 2003-08-07 Lipford Grayson B. Methods and compositions relating to plasmacytoid dendritic cells
EP1420829A4 (en) 2001-08-07 2006-05-17 Dynavax Tech Corp Immunomodulatory compositions, formulations, and methods for use thereof
DE60229422D1 (en) 2001-08-17 2008-11-27 Coley Pharm Gmbh COMBINATION MOTIF IMMUNOSTIMULATING OLIGONUCLEOTIDES WITH IMPROVED EFFECT
US20030119774A1 (en) 2001-09-25 2003-06-26 Marianna Foldvari Compositions and methods for stimulating an immune response
EP1451581A4 (en) 2001-10-05 2006-01-11 Coley Pharm Gmbh Toll-like receptor 3 signaling agonists and antagonists
US20050238660A1 (en) 2001-10-06 2005-10-27 Babiuk Lorne A Cpg formulations and related methods
WO2003094836A2 (en) 2001-10-12 2003-11-20 University Of Iowa Research Foundation Methods and products for enhancing immune responses using imidazoquinoline compounds
WO2003086280A2 (en) 2002-04-04 2003-10-23 Coley Pharmaceutical Gmbh Immunostimulatory g,u-containing oligoribonucleotides
US7807803B2 (en) 2002-07-03 2010-10-05 Coley Pharmaceutical Group, Inc. Nucleic acid compositions for stimulating immune responses
AU2003267986A1 (en) * 2002-07-03 2004-01-23 Depuy Mitek, Inc. Vaccines to induce mucosal immunity
US7605138B2 (en) 2002-07-03 2009-10-20 Coley Pharmaceutical Group, Inc. Nucleic acid compositions for stimulating immune responses
US7576066B2 (en) 2002-07-03 2009-08-18 Coley Pharmaceutical Group, Inc. Nucleic acid compositions for stimulating immune responses
AU2003255969A1 (en) 2002-07-17 2004-02-02 Coley Pharmaceutical Gmbh Use of cpg nucleic acids in prion-disease
WO2004026888A2 (en) 2002-09-19 2004-04-01 Coley Pharmaceutical Gmbh Toll-like receptor 9 (tlr9) from various mammalian species
JP2006512927A (en) 2002-12-11 2006-04-20 コーリー ファーマシューティカル グループ,インコーポレイテッド 5 'CPG nucleic acids and methods of use thereof
AU2004226605A1 (en) 2003-04-02 2004-10-14 Coley Pharmaceutical Group, Ltd. Immunostimulatory nucleic acid oil-in-water formulations for topical application
WO2004094671A2 (en) 2003-04-22 2004-11-04 Coley Pharmaceutical Gmbh Methods and products for identification and assessment of tlr ligands
US7410975B2 (en) 2003-06-20 2008-08-12 Coley Pharmaceutical Group, Inc. Small molecule toll-like receptor (TLR) antagonists
US7077098B2 (en) * 2003-08-26 2006-07-18 Shuba Yaroslav M Vane-type piston, four-cycle multi-chamber rotary internal combustion engine
CA2536139A1 (en) 2003-09-25 2005-04-07 Coley Pharmaceutical Group, Inc. Nucleic acid-lipophilic conjugates
US20050215501A1 (en) 2003-10-24 2005-09-29 Coley Pharmaceutical Group, Inc. Methods and products for enhancing epitope spreading
UA88457C2 (en) 2003-10-30 2009-10-26 Коли Фармасьютикал Гмбх Immunostimulatory nucleic acid with enhanced immunostimulatory potency
US20050239733A1 (en) 2003-10-31 2005-10-27 Coley Pharmaceutical Gmbh Sequence requirements for inhibitory oligonucleotides
US20050100983A1 (en) 2003-11-06 2005-05-12 Coley Pharmaceutical Gmbh Cell-free methods for identifying compounds that affect toll-like receptor 9 (TLR9) signaling
TWI247371B (en) * 2004-02-06 2006-01-11 Advanced Semiconductor Eng Semiconductor package and method for manufacturing the same
TW200533750A (en) 2004-02-19 2005-10-16 Coley Pharm Group Inc Immunostimulatory viral RNA oligonucleotides
AU2005326144A1 (en) 2004-06-08 2006-08-03 Coley Pharmaceutical Gmbh Abasic oligonucleotide as carrier platform for antigen and immunostimulatory agonist and antagonist
JP2008506789A (en) 2004-07-18 2008-03-06 シーエスエル、リミテッド Immunostimulatory complex and oligonucleotide formulation for inducing enhanced interferon-gamma response
MY159370A (en) 2004-10-20 2016-12-30 Coley Pharm Group Inc Semi-soft-class immunostimulatory oligonucleotides
SG161260A1 (en) 2005-04-08 2010-05-27 Coley Pharm Group Inc Methods for treating infectious disease exacerbated asthma
US20060241076A1 (en) 2005-04-26 2006-10-26 Coley Pharmaceutical Gmbh Modified oligoribonucleotide analogs with enhanced immunostimulatory activity
US20090117132A1 (en) 2005-07-07 2009-05-07 Pfizer, Inc. Anti-Ctla-4 Antibody and Cpg-Motif-Containing Synthetic Oligodeoxynucleotide Combination Therapy for Cancer Treatment
KR20080047463A (en) 2005-09-16 2008-05-28 콜리 파마슈티칼 게엠베하 Modulation of immunostimulatory properties of short interfering ribonucleic acid (sirna) by nucleotide modification
BRPI0616235A2 (en) 2005-09-16 2011-06-14 Coley Pharm Gmbh phosphodiester main chain immunostimulatory monofilamentated ribonucleic acid
WO2007038720A2 (en) 2005-09-27 2007-04-05 Coley Pharmaceutical Gmbh Modulation of tlr-mediated immune responses using adaptor oligonucleotides
AU2006318464B2 (en) 2005-11-25 2011-02-17 Zoetis Belgium Sa Immunostimulatory oligoribonucleotides
US8027888B2 (en) 2006-08-31 2011-09-27 Experian Interactive Innovation Center, Llc Online credit card prescreen systems and methods
WO2008033432A2 (en) 2006-09-12 2008-03-20 Coley Pharmaceutical Group, Inc. Immune modulation by chemically modified ribonucleosides and oligoribonucleotides
PT2078080E (en) 2006-09-27 2015-09-18 Coley Pharm Gmbh Cpg oligonucleotide analogs containing hydrophobic t analogs with enhanced immunostimulatory activity
EP2068912A2 (en) 2006-09-27 2009-06-17 Coley Pharmaceutical Group, Inc. Compositions of tlr ligands and antivirals
US20100144846A1 (en) 2006-10-26 2010-06-10 Coley Pharmaceutical Gmbh Oligoribonucleotides and uses thereof
WO2008057529A2 (en) 2006-11-06 2008-05-15 Coley Pharmaceutical Group, Inc. Peptide-based vaccine compositions to endogenous cholesteryl ester transfer protein (cetp)
MX2009012482A (en) 2007-05-17 2009-12-02 Coley Pharm Group Inc Class a oligonucleotides with immunostimulatory potency.
CN101820908A (en) 2007-10-09 2010-09-01 科利制药公司 The immune stimulatory oligonucleotide analogs that comprises modified sugar moieties

Patent Citations (98)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4185090A (en) * 1972-02-02 1980-01-22 Abbott Laboratories Chemically modified endotoxin immunizing agent
US4188375A (en) * 1976-09-25 1980-02-12 Bayer Aktiengesellschaft Process for the preparation of vaccines
US4578269A (en) * 1982-10-18 1986-03-25 Bror Morein Immunogenic protein or peptide complex, method or producing said complex and the use thereof as an immune stimulant and as a vaccine
US5100662A (en) * 1989-08-23 1992-03-31 The Liposome Company, Inc. Steroidal liposomes exhibiting enhanced stability
US5178860A (en) * 1989-09-01 1993-01-12 Coopers Animal Health Limited Adjuvant complexes and vaccine made therefrom
US4981684A (en) * 1989-10-24 1991-01-01 Coopers Animal Health Limited Formation of adjuvant complexes
US5723130A (en) * 1993-05-25 1998-03-03 Hancock; Gerald E. Adjuvants for vaccines against respiratory syncytial virus
US20030027782A1 (en) * 1993-08-26 2003-02-06 Carson Dennis A. Method for treating allergic lung disease
US5728518A (en) * 1994-01-12 1998-03-17 The Immune Response Corporation Antiviral poly-and oligonucleotides
US5723335A (en) * 1994-03-25 1998-03-03 Isis Pharmaceuticals, Inc. Immune stimulation by phosphorothioate oligonucleotide analogs
US20050075302A1 (en) * 1994-03-25 2005-04-07 Coley Pharmaceutical Group, Inc. Immune stimulation by phosphorothioate oligonucleotide analogs
US6727230B1 (en) * 1994-03-25 2004-04-27 Coley Pharmaceutical Group, Inc. Immune stimulation by phosphorothioate oligonucleotide analogs
US6194388B1 (en) * 1994-07-15 2001-02-27 The University Of Iowa Research Foundation Immunomodulatory oligonucleotides
US20060058251A1 (en) * 1994-07-15 2006-03-16 University Of Iowa Research Foundation Methods for treating and preventing infectious disease
US7879810B2 (en) * 1994-07-15 2011-02-01 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US7674777B2 (en) * 1994-07-15 2010-03-09 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US7517861B2 (en) * 1994-07-15 2009-04-14 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20080031936A1 (en) * 1994-07-15 2008-02-07 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20080026011A1 (en) * 1994-07-15 2008-01-31 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20050004061A1 (en) * 1994-07-15 2005-01-06 The University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20070078104A1 (en) * 1994-07-15 2007-04-05 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20070065467A1 (en) * 1994-07-15 2007-03-22 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules for activating dendritic cells
US20070066553A1 (en) * 1994-07-15 2007-03-22 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20050004062A1 (en) * 1994-07-15 2005-01-06 University Of Iowa Research Foundation Immunomodulatory oligonucleotides
US20070010470A9 (en) * 1994-07-15 2007-01-11 University Of Iowa Research Foundation Immunomodulatory oligonucleotides
US6207646B1 (en) * 1994-07-15 2001-03-27 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20070009482A9 (en) * 1994-07-15 2007-01-11 University Of Iowa Research Foundation Immunomodulatory oligonucleotides
US20030050261A1 (en) * 1994-07-15 2003-03-13 Krieg Arthur M. Immunostimulatory nucleic acid molecules
US20060089326A1 (en) * 1994-07-15 2006-04-27 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US7888327B2 (en) * 1994-07-15 2011-02-15 University Of Iowa Research Foundation Methods of using immunostimulatory nucleic acid molecules to treat allergic conditions
US20030050263A1 (en) * 1994-07-15 2003-03-13 The University Of Iowa Research Foundation Methods and products for treating HIV infection
US20050009774A1 (en) * 1994-07-15 2005-01-13 University Of Iowa Research Foundation Immunomodulatory oligonucleotides
US20060003955A1 (en) * 1994-07-15 2006-01-05 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20050032736A1 (en) * 1994-07-15 2005-02-10 The University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20050070491A1 (en) * 1994-07-15 2005-03-31 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20050059625A1 (en) * 1994-07-15 2005-03-17 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20050054602A1 (en) * 1994-07-15 2005-03-10 The University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20050037403A1 (en) * 1994-07-15 2005-02-17 University Of Iowa Research Foundation Immunomodulatory oligonucleotides
US20050037985A1 (en) * 1994-07-15 2005-02-17 Krieg Arthur M. Methods and products for treating HIV infection
US20050049216A1 (en) * 1994-07-15 2005-03-03 The University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20050049215A1 (en) * 1994-07-15 2005-03-03 The University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US6503533B1 (en) * 1994-07-28 2003-01-07 Georgetown University Antisense ogligonucleotides against Hepatitis B viral replication
US20030026782A1 (en) * 1995-02-07 2003-02-06 Arthur M. Krieg Immunomodulatory oligonucleotides
US20030078223A1 (en) * 1996-01-30 2003-04-24 Eyal Raz Compositions and methods for modulating an immune response
US6027732A (en) * 1996-02-21 2000-02-22 Morein; Bror Iscom or iscom-matrix comprising hydrophobic receptor molecules for antigenic substances
US6174872B1 (en) * 1996-10-04 2001-01-16 The Regents Of The University Of California Method for treating allergic lung disease
US20040006010A1 (en) * 1996-10-11 2004-01-08 Carson Dennis A. Immunostimulatory polynucleotide/immunomodulatory molecule conjugates
US7001890B1 (en) * 1997-01-23 2006-02-21 Coley Pharmaceutical Gmbh Pharmaceutical compositions comprising a polynucleotide and optionally an antigen especially for vaccination
US20050054601A1 (en) * 1997-01-23 2005-03-10 Coley Pharmaceutical Gmbh Pharmaceutical composition comprising a polynucleotide and optionally an antigen especially for vaccination
US20090060927A1 (en) * 1997-01-23 2009-03-05 Coley Pharmaceutical Gmbh Pharmaceutical compositions comprising a polynucleotide and optionally an antigen especially for vaccination
US6214806B1 (en) * 1997-02-28 2001-04-10 University Of Iowa Research Foundation Use of nucleic acids containing unmethylated CPC dinucleotide in the treatment of LPS-associated disorders
US7488490B2 (en) * 1997-03-10 2009-02-10 University Of Iowa Research Foundation Method of inducing an antigen-specific immune response by administering a synergistic combination of adjuvants comprising unmethylated CpG-containing nucleic acids and a non-nucleic acid adjuvant
US20050032734A1 (en) * 1997-05-20 2005-02-10 Krieg Arthur M. Vectors and methods for immunization or therapeutic protocols
US6339068B1 (en) * 1997-05-20 2002-01-15 University Of Iowa Research Foundation Vectors and methods for immunization or therapeutic protocols
US20070027098A1 (en) * 1997-06-06 2007-02-01 Eyal Raz Immunostimulatory oligonucleotides, compositions thereof and methods of use thereof
US6221882B1 (en) * 1997-07-03 2001-04-24 University Of Iowa Research Foundation Methods for inhibiting immunostimulatory DNA associated responses
US7354711B2 (en) * 1997-07-03 2008-04-08 University Of Iowa Research Foundation Methods for inhibiting immunostimulatory DNA associated responses
US6521637B2 (en) * 1997-07-03 2003-02-18 University Of Iowa Research Foundation Methods for inhibiting immunostimulatory DNA associated responses
US6218371B1 (en) * 1998-04-03 2001-04-17 University Of Iowa Research Foundation Methods and products for stimulating the immune system using immunotherapeutic oligonucleotides and cytokines
US20040030118A1 (en) * 1998-05-14 2004-02-12 Hermann Wagner Methods for regulating hematopoiesis using CpG-oligonucleotides
US20040006034A1 (en) * 1998-06-05 2004-01-08 Eyal Raz Immunostimulatory oligonucleotides, compositions thereof and methods of use thereof
US6693086B1 (en) * 1998-06-25 2004-02-17 National Jewish Medical And Research Center Systemic immune activation method using nucleic acid-lipid complexes
US6506386B1 (en) * 1998-08-05 2003-01-14 Smithkline Beecham Biologicals, S.A. Vaccine comprising an iscom consisting of sterol and saponin which is free of additional detergent
US20030064064A1 (en) * 1998-09-18 2003-04-03 Dino Dina Methods of treating IgE-associated disorders and compositions for use therein
US6544518B1 (en) * 1999-04-19 2003-04-08 Smithkline Beecham Biologicals S.A. Vaccines
US6514948B1 (en) * 1999-07-02 2003-02-04 The Regents Of The University Of California Method for enhancing an immune response
US20070066554A1 (en) * 1999-09-25 2007-03-22 Coley Pharmaceutical Gmbh Immunostimulatory nucleic acids
US20110033421A1 (en) * 1999-09-27 2011-02-10 Coley Pharmaceutical Gmbh Methods related to immunostimulatory nucleic acid-induced interferon
US6852705B2 (en) * 2000-01-21 2005-02-08 Merial DNA vaccines for farm animals, in particular bovines and porcines
US6552006B2 (en) * 2000-01-31 2003-04-22 The Regents Of The University Of California Immunomodulatory polynucleotides in treatment of an infection by an intracellular pathogen
US20070037767A1 (en) * 2000-02-03 2007-02-15 Coley Pharmaceutical Group, Inc. Immunostimulatory nucleic acids for the treatment of asthma and allergy
US20040067902A9 (en) * 2000-02-03 2004-04-08 Bratzler Robert L. Immunostimulatory nucleic acids for the treatment of asthma and allergy
US20020042387A1 (en) * 2000-02-23 2002-04-11 Eyal Raz Method for treating inflammatory bowel disease and other forms of gastrointestinal inflammation
US20030022852A1 (en) * 2000-03-10 2003-01-30 Nest Gary Van Biodegradable immunomodulatory formulations and methods for use thereof
US20030059773A1 (en) * 2000-03-10 2003-03-27 Gary Van Nest Immunomodulatory formulations and methods for use thereof
US20040009942A1 (en) * 2000-03-10 2004-01-15 Gary Van Nest Methods of preventing and treating respiratory viral infection using immunomodulatory polynucleotide sequences
US20020028784A1 (en) * 2000-03-10 2002-03-07 Nest Gary Van Methods of preventing and treating viral infections using immunomodulatory polynucleotide sequences
US7183111B2 (en) * 2000-03-10 2007-02-27 Dynavax Technologies Corporation Immunomodulatory formulations and methods for use thereof
US6534062B2 (en) * 2000-03-28 2003-03-18 The Regents Of The University Of California Methods for increasing a cytotoxic T lymphocyte response in vivo
US20030026801A1 (en) * 2000-06-22 2003-02-06 George Weiner Methods for enhancing antibody-induced cell lysis and treating cancer
US20040038922A1 (en) * 2000-10-06 2004-02-26 Jean Haensler Vaccine composition
US20050019340A1 (en) * 2000-10-18 2005-01-27 Nathalie Garcon Vaccines
US20030055014A1 (en) * 2000-12-14 2003-03-20 Bratzler Robert L. Inhibition of angiogenesis by nucleic acids
US20030049266A1 (en) * 2000-12-27 2003-03-13 Fearon Karen L. Immunomodulatory polynucleotides and methods of using the same
US20030050268A1 (en) * 2001-03-29 2003-03-13 Krieg Arthur M. Immunostimulatory nucleic acid for treatment of non-allergic inflammatory diseases
US20040009944A1 (en) * 2002-05-10 2004-01-15 Inex Pharmaceuticals Corporation Methylated immunostimulatory oligonucleotides and methods of using the same
US20040009949A1 (en) * 2002-06-05 2004-01-15 Coley Pharmaceutical Group, Inc. Method for treating autoimmune or inflammatory diseases with combinations of inhibitory oligonucleotides and small molecule antagonists of immunostimulatory CpG nucleic acids
US20040067905A1 (en) * 2002-07-03 2004-04-08 Coley Pharmaceutical Group, Inc. Nucleic acid compositions for stimulating immune responses
US20040053880A1 (en) * 2002-07-03 2004-03-18 Coley Pharmaceutical Group, Inc. Nucleic acid compositions for stimulating immune responses
US20050059619A1 (en) * 2002-08-19 2005-03-17 Coley Pharmaceutical Group, Inc. Immunostimulatory nucleic acids
US20060003962A1 (en) * 2002-10-29 2006-01-05 Coley Pharmaceutical Group, Ltd. Methods and products related to treatment and prevention of hepatitis C virus infection
US20070041998A1 (en) * 2003-03-24 2007-02-22 Intercell Ag Use of alum and a th1 immune response inducing adjuvant for enhancing immune responses
US20050013812A1 (en) * 2003-07-14 2005-01-20 Dow Steven W. Vaccines using pattern recognition receptor-ligand:lipid complexes
US20060019916A1 (en) * 2004-04-02 2006-01-26 Coley Pharmaceutical Group, Inc. Immunostimulatory nucleic acids for inducing IL-10 responses
US20090017021A1 (en) * 2004-07-18 2009-01-15 Coley Pharmaceutical Group, Ltd. Methods and compositions for inducing innate immune responses
US20060019923A1 (en) * 2004-07-18 2006-01-26 Coley Pharmaceutical Group, Ltd. Methods and compositions for inducing innate immune responses
US20080009455A9 (en) * 2005-02-24 2008-01-10 Coley Pharmaceutical Group, Inc. Immunostimulatory oligonucleotides
US20080045473A1 (en) * 2006-02-15 2008-02-21 Coley Pharmaceutical Gmbh Compositions and methods for oligonucleotide formulations

Cited By (159)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050075302A1 (en) * 1994-03-25 2005-04-07 Coley Pharmaceutical Group, Inc. Immune stimulation by phosphorothioate oligonucleotide analogs
US7935675B1 (en) 1994-07-15 2011-05-03 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20050239732A1 (en) * 1994-07-15 2005-10-27 Krieg Arthur M Immunostimulatory nucleic acid molecules
US8309527B2 (en) 1994-07-15 2012-11-13 University Of Iowa Research Foundation Immunomodulatory oligonucleotides
US20070202128A1 (en) * 1994-07-15 2007-08-30 Coley Pharmaceutical Group, Inc Immunomodulatory oligonucleotides
US8258106B2 (en) 1994-07-15 2012-09-04 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US8158592B2 (en) 1994-07-15 2012-04-17 Coley Pharmaceutical Group, Inc. Immunostimulatory nucleic acid molecules
US8148340B2 (en) 1994-07-15 2012-04-03 The United States Of America As Represented By The Department Of Health And Human Services Immunomodulatory oligonucleotides
US20030191079A1 (en) * 1994-07-15 2003-10-09 University Of Iowa Research Foundation Methods for treating and preventing infectious disease
US8129351B2 (en) 1994-07-15 2012-03-06 The University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US8114848B2 (en) 1994-07-15 2012-02-14 The United States Of America As Represented By The Department Of Health And Human Services Immunomodulatory oligonucleotides
US8058249B2 (en) 1994-07-15 2011-11-15 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US8008266B2 (en) 1994-07-15 2011-08-30 University Of Iowa Foundation Methods of treating cancer using immunostimulatory oligonucleotides
US7888327B2 (en) 1994-07-15 2011-02-15 University Of Iowa Research Foundation Methods of using immunostimulatory nucleic acid molecules to treat allergic conditions
US20040087538A1 (en) * 1994-07-15 2004-05-06 University Of Iowa Research Foundation Methods of treating cancer using immunostimulatory oligonucleotides
US7879810B2 (en) 1994-07-15 2011-02-01 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20040106568A1 (en) * 1994-07-15 2004-06-03 University Of Iowa Research Foundation Methods for treating and preventing infectious disease
US7723500B2 (en) 1994-07-15 2010-05-25 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20040132685A1 (en) * 1994-07-15 2004-07-08 The University Of Iowa Research Foundation Immunostimulatory nucleic acid
US20040142469A1 (en) * 1994-07-15 2004-07-22 University Of Iowa Research Foundation Immunomodulatory oligonucleotides
US20040147468A1 (en) * 1994-07-15 2004-07-29 Krieg Arthur M Immunostimulatory nucleic acid molecules
US7723022B2 (en) 1994-07-15 2010-05-25 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20070065467A1 (en) * 1994-07-15 2007-03-22 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules for activating dendritic cells
US20100125101A1 (en) * 1994-07-15 2010-05-20 Krieg Arthur M Immunostimulatory nucleic acid molecules
US20040167089A1 (en) * 1994-07-15 2004-08-26 The University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US7713529B2 (en) 1994-07-15 2010-05-11 University Of Iowa Research Foundation Methods for treating and preventing infectious disease
US20040171150A1 (en) * 1994-07-15 2004-09-02 University Of Iowa Research Foundation Immunomodulatory oligonucleotides
US20040181045A1 (en) * 1994-07-15 2004-09-16 University Of Iowa Research Foundation Immunomodulatory oligonucleotides
US7674777B2 (en) 1994-07-15 2010-03-09 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20040229835A1 (en) * 1994-07-15 2004-11-18 The University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20080031936A1 (en) * 1994-07-15 2008-02-07 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20080026011A1 (en) * 1994-07-15 2008-01-31 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20040152657A1 (en) * 1994-07-15 2004-08-05 University Of Iowa Research Foundation Immunomodulatory oligonucleotides
US20050004061A1 (en) * 1994-07-15 2005-01-06 The University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20050004062A1 (en) * 1994-07-15 2005-01-06 University Of Iowa Research Foundation Immunomodulatory oligonucleotides
US20050009774A1 (en) * 1994-07-15 2005-01-13 University Of Iowa Research Foundation Immunomodulatory oligonucleotides
US20070010470A9 (en) * 1994-07-15 2007-01-11 University Of Iowa Research Foundation Immunomodulatory oligonucleotides
US20050032736A1 (en) * 1994-07-15 2005-02-10 The University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20050049215A1 (en) * 1994-07-15 2005-03-03 The University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20050049216A1 (en) * 1994-07-15 2005-03-03 The University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20050054602A1 (en) * 1994-07-15 2005-03-10 The University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20070078104A1 (en) * 1994-07-15 2007-04-05 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20050070491A1 (en) * 1994-07-15 2005-03-31 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20060094683A1 (en) * 1994-07-15 2006-05-04 University Of Iowa Research Foundation Immunomodulatory oligonucleotides
US20060003955A1 (en) * 1994-07-15 2006-01-05 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20050101557A1 (en) * 1994-07-15 2005-05-12 The University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20050101554A1 (en) * 1994-07-15 2005-05-12 University Of Iowa Research Foundation Methods for treating and preventing infectious disease
US20050277609A1 (en) * 1994-07-15 2005-12-15 The University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20050277604A1 (en) * 1994-07-15 2005-12-15 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20050148537A1 (en) * 1994-07-15 2005-07-07 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20050171047A1 (en) * 1994-07-15 2005-08-04 The University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20050267057A1 (en) * 1994-07-15 2005-12-01 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20050244379A1 (en) * 1994-07-15 2005-11-03 University Of Iowa Research Foundation Immunomodulatory oligonucleotides
US20050233999A1 (en) * 1994-07-15 2005-10-20 Krieg Arthur M Immunostimulatory nucleic acid molecules
US20050245477A1 (en) * 1994-07-15 2005-11-03 University Of Iowa Research Foundation Immunomodulatory oligonucleotides
US20090060927A1 (en) * 1997-01-23 2009-03-05 Coley Pharmaceutical Gmbh Pharmaceutical compositions comprising a polynucleotide and optionally an antigen especially for vaccination
US20050054601A1 (en) * 1997-01-23 2005-03-10 Coley Pharmaceutical Gmbh Pharmaceutical composition comprising a polynucleotide and optionally an antigen especially for vaccination
US20030091599A1 (en) * 1997-03-10 2003-05-15 Coley Pharmaceutical Gmbh Use of nucleic acids containing unmethylated CpG dinucleotide as an adjuvant
US8202688B2 (en) 1997-03-10 2012-06-19 University Of Iowa Research Foundation Use of nucleic acids containing unmethylated CpG dinucleotide as an adjuvant
US20030224010A1 (en) * 1997-03-10 2003-12-04 Coley Pharmaceutical Gmbh Use of nucleic acids containing unmethylated CpG dinucleotide as an adjuvant
US20020164341A1 (en) * 1997-03-10 2002-11-07 Loeb Health Research Institute At The Ottawa Hospital Use of nucleic acids containing unmethylated CpG dinucleotide as an adjuvant
US20110081366A1 (en) * 1997-10-30 2011-04-07 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20050197314A1 (en) * 1998-04-03 2005-09-08 University Of Iowa Research Foundation Methods and products for stimulating the immune system using immunotherapeutic oligonucleotides and cytokines
US20040235777A1 (en) * 1998-05-14 2004-11-25 Coley Pharmaceutical Gmbh Methods for regulating hematopoiesis using CpG-oligonucleotides
US20040235778A1 (en) * 1998-05-14 2004-11-25 Coley Pharmaceutical Gmbh Methods for regulating hematopoiesis using CpG-oligonucleotides
US20040234512A1 (en) * 1998-05-14 2004-11-25 Coley Pharmaceutical Gmbh Methods for regualting hematopoiesis using CpG-oligonucleotides
US8574599B1 (en) 1998-05-22 2013-11-05 Ottawa Hospital Research Institute Methods and products for inducing mucosal immunity
US20040157791A1 (en) * 1998-06-25 2004-08-12 Dow Steven W. Systemic immune activation method using nucleic acid-lipid complexes
US7741300B2 (en) 1998-06-25 2010-06-22 National Jewish Medical And Research Center Methods of using nucleic acid vector-lipid complexes
US8173141B2 (en) 1999-02-17 2012-05-08 Csl Limited Immunogenic complexes and methods relating thereto
US7776343B1 (en) 1999-02-17 2010-08-17 Csl Limited Immunogenic complexes and methods relating thereto
US20100010193A1 (en) * 1999-02-17 2010-01-14 Csl Limited Immunogenic complexes and methods relating thereto
US7271156B2 (en) 1999-09-25 2007-09-18 University Of Iowa Research Foundation Immunostimulatory nucleic acids
US20030212026A1 (en) * 1999-09-25 2003-11-13 University Of Iowa Research Foundation Immunostimulatory nucleic acids
US7776344B2 (en) 1999-09-27 2010-08-17 University Of Iowa Research Foundation Methods related to immunostimulatory nucleic acid-induced interferon
US20060286070A1 (en) * 1999-09-27 2006-12-21 Coley Pharmaceutical Gmbh Methods related to immunostimulatory nucleic acid-induced interferon
US20110033421A1 (en) * 1999-09-27 2011-02-10 Coley Pharmaceutical Gmbh Methods related to immunostimulatory nucleic acid-induced interferon
US20010044416A1 (en) * 2000-01-20 2001-11-22 Mccluskie Michael J. Immunostimulatory nucleic acids for inducing a Th2 immune response
US20040235774A1 (en) * 2000-02-03 2004-11-25 Bratzler Robert L. Immunostimulatory nucleic acids for the treatment of asthma and allergy
US20070037767A1 (en) * 2000-02-03 2007-02-15 Coley Pharmaceutical Group, Inc. Immunostimulatory nucleic acids for the treatment of asthma and allergy
US20060154890A1 (en) * 2000-02-03 2006-07-13 Coley Pharmaceutical Group, Inc. Immunostimulatory nucleic acids for the treatment of asthma and allergy
US20040131628A1 (en) * 2000-03-08 2004-07-08 Bratzler Robert L. Nucleic acids for the treatment of disorders associated with microorganisms
US7534772B2 (en) 2000-06-22 2009-05-19 University Of Iowa Research Foundation Methods for enhancing antibody-induced cell lysis and treating cancer
US20030026801A1 (en) * 2000-06-22 2003-02-06 George Weiner Methods for enhancing antibody-induced cell lysis and treating cancer
US20070009710A1 (en) * 2000-08-04 2007-01-11 Toyo Boseki Kabushiki Kaisha Flexible metal-clad laminate and method for producing the same
US20050181422A1 (en) * 2000-09-15 2005-08-18 Coley Pharmaceutical Gmbh Process for high throughput screening of CpG-based immuno-agonist/antagonist
US7820379B2 (en) 2000-09-15 2010-10-26 Coley Pharmaceutical Gmbh Process for high throughput screening of CpG-based immuno-agonist/antagonist
US20080226649A1 (en) * 2000-12-08 2008-09-18 Coley Pharmaceutical Gmbh CPG-like nucleic acids and methods of use thereof
US20030148976A1 (en) * 2001-08-17 2003-08-07 Krieg Arthur M. Combination motif immune stimulatory oligonucleotides with improved activity
US8834900B2 (en) 2001-08-17 2014-09-16 University Of Iowa Research Foundation Combination motif immune stimulatory oligonucleotides with improved activity
US20030099668A1 (en) * 2001-09-14 2003-05-29 Cytos Biotechnology Ag Packaging of immunostimulatory substances into virus-like particles: method of preparation and use
US9950055B2 (en) 2001-09-14 2018-04-24 Kuros Biosciences Ag Packaging of immunostimulatory substances into virus-like particles: method of preparation and use
US8691209B2 (en) 2001-09-14 2014-04-08 Cytos Biotechnology Ag Packaging of immunostimulatory substances into virus-like particles: method of preparation and use
US20030091593A1 (en) * 2001-09-14 2003-05-15 Cytos Biotechnology Ag In vivo activation of antigen presenting cells for enhancement of immune responses induced by virus like particles
US20030139364A1 (en) * 2001-10-12 2003-07-24 University Of Iowa Research Foundation Methods and products for enhancing immune responses using imidazoquinoline compounds
US9428536B2 (en) 2002-04-04 2016-08-30 Zoetis Belgium Sa Immunostimulatory G, U-containing oligoribonucleotides
US8658607B2 (en) 2002-04-04 2014-02-25 Zoetis Belgium Immunostimulatory G, U-containing oligoribonucleotides
US8153141B2 (en) 2002-04-04 2012-04-10 Coley Pharmaceutical Gmbh Immunostimulatory G, U-containing oligoribonucleotides
US20030232074A1 (en) * 2002-04-04 2003-12-18 Coley Pharmaceutical Gmbh Immunostimulatory G, U-containing oligoribonucleotides
US20040005338A1 (en) * 2002-06-20 2004-01-08 Cytos Biotechnology Ag Packaged virus-like particles for use as adjuvants: method of preparation and use
US8114419B2 (en) 2002-07-03 2012-02-14 Coley Pharmaceutical Group, Inc. Nucleic acid compositions for stimulating immune responses
US20040198680A1 (en) * 2002-07-03 2004-10-07 Coley Pharmaceutical Group, Inc. Nucleic acid compositions for stimulating immune responses
US20090311277A1 (en) * 2002-07-03 2009-12-17 Coley Pharmaceutical Group, Inc. Nucleic acid compositions for stimulating immune responses
US20040152649A1 (en) * 2002-07-03 2004-08-05 Coley Pharmaceutical Group, Inc. Nucleic acid compositions for stimulating immune responses
US20040067905A1 (en) * 2002-07-03 2004-04-08 Coley Pharmaceutical Group, Inc. Nucleic acid compositions for stimulating immune responses
US20040092472A1 (en) * 2002-07-03 2004-05-13 Coley Pharmaceutical Group, Inc. Nucleic acid compositions for stimulating immune responses
US7807803B2 (en) 2002-07-03 2010-10-05 Coley Pharmaceutical Group, Inc. Nucleic acid compositions for stimulating immune responses
US8283328B2 (en) 2002-08-19 2012-10-09 Coley Pharmaceutical Group, Inc. Immunostimulatory nucleic acids
US20050059619A1 (en) * 2002-08-19 2005-03-17 Coley Pharmaceutical Group, Inc. Immunostimulatory nucleic acids
US8304396B2 (en) 2002-08-19 2012-11-06 Coley Pharmaceutical Group, Inc. Immunostimulatory nucleic acids
US7998492B2 (en) 2002-10-29 2011-08-16 Coley Pharmaceutical Group, Inc. Methods and products related to treatment and prevention of hepatitis C virus infection
US20060003962A1 (en) * 2002-10-29 2006-01-05 Coley Pharmaceutical Group, Ltd. Methods and products related to treatment and prevention of hepatitis C virus infection
US20060246035A1 (en) * 2002-10-29 2006-11-02 Coley Pharmaceutical Gmbh Methods and products related to treatment and prevention of hepatitis c virus infection
US7956043B2 (en) 2002-12-11 2011-06-07 Coley Pharmaceutical Group, Inc. 5′ CpG nucleic acids and methods of use
US20040171571A1 (en) * 2002-12-11 2004-09-02 Coley Pharmaceutical Group, Inc. 5' CpG nucleic acids and methods of use
US20100098722A1 (en) * 2003-03-26 2010-04-22 Cytos Biotechnology Ag Packaging of Immunostimulatory Substances Into Virus-Like Particles: Method of Preparation and Use
US20050119273A1 (en) * 2003-06-20 2005-06-02 Coley Pharmaceutical Gmbh Small molecule toll-like receptor (TLR) antagonists
US7410975B2 (en) 2003-06-20 2008-08-12 Coley Pharmaceutical Group, Inc. Small molecule toll-like receptor (TLR) antagonists
US20060251623A1 (en) * 2003-07-10 2006-11-09 Caytos Biotechnology Ag Packaged virus-like particles
US20050013812A1 (en) * 2003-07-14 2005-01-20 Dow Steven W. Vaccines using pattern recognition receptor-ligand:lipid complexes
US20050130911A1 (en) * 2003-09-25 2005-06-16 Coley Pharmaceutical Group, Inc. Nucleic acid-lipophilic conjugates
US20100183639A1 (en) * 2003-09-25 2010-07-22 Coley Pharmaceutical Group, Inc. Nucleic acid-lipophilic conjugates
US8188254B2 (en) 2003-10-30 2012-05-29 Coley Pharmaceutical Gmbh C-class oligonucleotide analogs with enhanced immunostimulatory potency
US20050239734A1 (en) * 2003-10-30 2005-10-27 Coley Pharmaceutical Gmbh C-class oligonucleotide analogs with enhanced immunostimulatory potency
US20050239733A1 (en) * 2003-10-31 2005-10-27 Coley Pharmaceutical Gmbh Sequence requirements for inhibitory oligonucleotides
US20050100983A1 (en) * 2003-11-06 2005-05-12 Coley Pharmaceutical Gmbh Cell-free methods for identifying compounds that affect toll-like receptor 9 (TLR9) signaling
US20050256073A1 (en) * 2004-02-19 2005-11-17 Coley Pharmaceutical Group, Inc. Immunostimulatory viral RNA oligonucleotides
US7795235B2 (en) 2004-10-20 2010-09-14 Coley Pharmaceutical Gmbh Semi-soft c-class immunostimulatory oligonucleotides
US20060140875A1 (en) * 2004-10-20 2006-06-29 Coley Pharmaceutical Group, Inc. Semi-soft C-class immunostimulatory oligonucleotides
US20090137519A1 (en) * 2004-10-20 2009-05-28 Coley Pharmaceutical Group, Inc. Semi-soft c-class immunostimulatory oligonucleotides
US20110201672A1 (en) * 2004-10-20 2011-08-18 Krieg Arthur M Semi-soft c-class immunostimulatory oligonucleotides
US20080009455A9 (en) * 2005-02-24 2008-01-10 Coley Pharmaceutical Group, Inc. Immunostimulatory oligonucleotides
US20060211644A1 (en) * 2005-02-24 2006-09-21 Coley Pharmaceutical Group, Inc. Immunostimulatory oligonucleotides
US20060229271A1 (en) * 2005-04-08 2006-10-12 Coley Pharmaceutical Group, Inc. Methods for treating infectious disease exacerbated asthma
US20060241076A1 (en) * 2005-04-26 2006-10-26 Coley Pharmaceutical Gmbh Modified oligoribonucleotide analogs with enhanced immunostimulatory activity
US20090117132A1 (en) * 2005-07-07 2009-05-07 Pfizer, Inc. Anti-Ctla-4 Antibody and Cpg-Motif-Containing Synthetic Oligodeoxynucleotide Combination Therapy for Cancer Treatment
US20090214578A1 (en) * 2005-09-16 2009-08-27 Coley Pharmaceutical Gmbh Immunostimulatory Single-Stranded Ribonucleic Acid with Phosphodiester Backbone
US20090306177A1 (en) * 2005-09-16 2009-12-10 Coley Pharmaceutical Gmbh Modulation of Immunostimulatory Properties of Short Interfering Ribonucleic Acid (Sirna) by Nucleotide Modification
US20100316659A1 (en) * 2005-11-25 2010-12-16 Coley Pharmaceutical Gmbh Immunostimulatory oligoribonucleotides
US20110206719A1 (en) * 2005-11-25 2011-08-25 Coley Pharmaceutical Gmbh Immunostimulatory oligoribonucleotides
US8354522B2 (en) 2005-11-25 2013-01-15 Coley Pharmaceutical Gmbh Immunostimulatory oligoribonucleotides
US7662949B2 (en) 2005-11-25 2010-02-16 Coley Pharmaceutical Gmbh Immunostimulatory oligoribonucleotides
US8574564B2 (en) 2005-12-14 2013-11-05 Cytos Biotechnology Ag Immunostimulatory nucleic acid packaged particles for the treatment of hypersensitivity
US20080045473A1 (en) * 2006-02-15 2008-02-21 Coley Pharmaceutical Gmbh Compositions and methods for oligonucleotide formulations
US20080026986A1 (en) * 2006-06-05 2008-01-31 Rong-Fu Wang Reversal of the suppressive function of specific t cells via toll-like receptor 8 signaling
US9404126B2 (en) 2006-06-12 2016-08-02 Kuros Biosciences Ag Processes for packaging aggregated oligonucleotides into virus-like particles of RNA bacteriophages
US10358656B2 (en) 2006-06-12 2019-07-23 Kuros Biosciences Ag Oligonucleotides packaged into virus-like particles of RNA bacteriophages
US9902972B2 (en) 2006-06-12 2018-02-27 Kuros Biosciences Ag Processes for packaging oligonucleotides into virus-like particles of RNA bacteriophages
US8580268B2 (en) 2006-09-27 2013-11-12 Coley Pharmaceutical Gmbh CpG oligonucleotide analogs containing hydrophobic T analogs with enhanced immunostimulatory activity
US9382545B2 (en) 2006-09-27 2016-07-05 Coley Pharmaceutical Gmbh CpG oligonucleotide analogs containing hydrophobic T analogs with enhanced immunostimulatory activity
US10260071B2 (en) 2006-09-27 2019-04-16 Coley Pharmaceutical Gmbh CpG oligonucleotide analogs containing hydrophobic T analogs with enhanced immunostimulatory activity
US20090142362A1 (en) * 2006-11-06 2009-06-04 Avant Immunotherapeutics, Inc. Peptide-based vaccine compositions to endogenous cholesteryl ester transfer protein (CETP)
US9504742B2 (en) 2009-03-25 2016-11-29 The Board Of Regents, The University Of Texas System Compositions for stimulation of mammalian innate immune resistance to pathogens
US9186400B2 (en) 2009-03-25 2015-11-17 The Board Of Regents, The University Of Texas System Compositions for stimulation of mammalian innate immune resistance to pathogens
US8883174B2 (en) 2009-03-25 2014-11-11 The Board Of Regents, The University Of Texas System Compositions for stimulation of mammalian innate immune resistance to pathogens
US10722573B2 (en) 2009-03-25 2020-07-28 The Board Of Regents, The University Of Texas System Compositions for stimulation of mammalian innate immune resistance to pathogens
US9260517B2 (en) 2009-11-17 2016-02-16 Musc Foundation For Research Development Human monoclonal antibodies to human nucleolin
US10385128B2 (en) 2009-11-17 2019-08-20 Musc Foundation For Research Development Nucleolin antibodies
US10286065B2 (en) 2014-09-19 2019-05-14 Board Of Regents, The University Of Texas System Compositions and methods for treating viral infections through stimulated innate immunity in combination with antiviral compounds

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