Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
  • A61K39/145—Orthomyxoviridae, e.g. influenza virus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P33/00—Antiparasitic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
  • A61K2039/541—Mucosal route
  • A61K2039/543—Mucosal route intranasal
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55522—Cytokines; Lymphokines; Interferons
  • A61K2039/55527—Interleukins
  • A61K2039/55538—IL-12
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
  • C12N2760/00011—Details
  • C12N2760/16011—Orthomyxoviridae
  • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
  • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• • Y—GENERAL 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
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• Mucosal surfaces are the major portals of entry for bacteria and viruses, and therefore constitute the first line of defense for the host. As such, immunization strategies that enhance mucosal immunity have practical significance for preventing infectious disease. Most parenterally administered vaccines, however, are only partially effective at inducing optimal mucosal immunity. Thus, adjuvants that can enhance mucosal immunity and be delivered in a safe, non-invasive manner are needed.
• the present invention relates to methods of enhancing and/or inducing an immune response (e.g., systemic, mucosal) to a pathogen in a host (e.g., mammalian, including human), which comprises administering intranasally (i.n.) to the host an effective amount of IL-12 and an antigen (e.g., a protein, carbohydrate, lipid, recombinant DNA, whole organism, toxin, organic molecule) of the pathogen.
• an immune response e.g., a protein, carbohydrate, lipid, recombinant DNA, whole organism, toxin, organic molecule
• the immune response can be antigen-specific.
• the immune response can result in enhanced expression of a Thl -type cytokine response (e.g., expression of interferon- ⁇ ) and/or a humoral response (e.g., IgG2a, IgG2b, IgG3).
• a Thl -type cytokine response e.g., expression of interferon- ⁇
• a humoral response e.g., IgG2a, IgG2b, IgG3
• the present invention relates to a method of enhancing an immune response to a pathogen in a host, which comprises administering i.n. to the host an effective amount of IL-12 and an antigen of the pathogen.
• the present invention relates to a method of inducing an immune response to a pathogen in a host, which comprises administering i.n. to the host an effective amount of IL-12 and an antigen of the pathogen.
• the present invention relates to a method of inducing an immune response to a mucosal pathogen in a host, which comprises administering i.n. to the host an effective amount of IL-12 and an antigen of the pathogen.
• Also encompassed by the present invention is a method of inducing a Thl- like immune response to a pathogen in a host, comprising administering i.n. to the host an effective amount of IL-12 and an antigen of the pathogen.
• the present invention also relates to a method of enhancing a mucosal immune response to a pathogen in a host, which comprises administering i.n. to the host an effective amount of IL-12 and an antigen of the pathogen.
• IL-12 administered i.n. is effective for augmenting antigen specific-responses in both mucosal and systemic compartments as described herein, demonstrates that i.n. administration of IL-12 can be used to obtain a potent vaccine adjuvant effect in immunization strategies against pathogens, such as mucosal pathogens.
• Figure 1A-1C are bar graphs showing the effects of IL-12 administered intranasally (i.n.) on respiratory mucosal immune responses; the data are presented as average optical density (O.D.) +/- SEM with four mice per group; bronchoalveolar lavage (BAL) were tested at dilutions corresponding to the linear portions of the titration curve (1 :64 for IgGl and IgA, 1 :8 for IgG2a).
• Figure 2A-2E are graphs of reciprocal serum dilution versus O.D. 405 nm showing the effects of IL-12 administered i.n. on systemic antibody responses; solid symbols represent animals injected with DNP-OVA plus IL-12 and open symbols represent animals injected with DNP-OVA plus phosphate buffered saline (PBS); each line represents binding of antibody from an individual mouse.
• PBS phosphate buffered saline
• Figures 3A-3B are bar graphs showing the effects of IL-12 administered i.n. on total Ig levels; the data are presented as average O.D. +/- SEM with four mice per group; sera were tested at dilutions corresponding to the linear portions of the titration curve (1:6400 for IgGl, 1:200 for IgG2a).
• Figures 4A-4F are bar graphs showing the effects of parenteral (i.p.) and i.n. administration of IL-12 on fecal mucosal responses; the data shown represent day 21 antibody responses for IgA and day 28 responses for IgG isotypes, the peak of each reactive response; the data are presented as average O.D. +/- SEM with 3-4 mice per group.
• Figures 5A-5B are graphs of reciprocal serum dilution versus O.D. 405 nm showing the effects of IL-12 administered i.n. on systemic antibody responses; mice were immunized i.n. on day 0 with purified hemagglutinin and neuraminidase derived from influenza virus (HANA) and treated i.n. with either IL-12 (closed triangles) or phosphate buffered saline (PBS) vehicle (open circles) on days 0, 1, 2 and 3; serum anti-HANA antibody levels on day 14 were determined by isotype- specific ELISA using HANA-coated microtiter plates; each line represents binding of antibody from an individual mouse.
• HANA purified hemagglutinin and neuraminidase derived from influenza virus
• PBS phosphate buffered saline
• Figures 6A-6B are graphs of reciprocal serum dilution versus O.D. 405 nm showing the effects of IL-12 administered i.n. on systemic antibody responses; mice were immunized i.n. on day 0 with HANA and treated i.n. with either IL-12 (closed triangles) or PBS vehicle (open circles) on days 0, 1, 2 and 3 and boosted on day 14; serum anti-HANA antibody levels on day 28 were determined by isotype-specific ELISA using HANA-coated microtiter plates; each line represents binding of antibody from an individual mouse.
• Figure 7A-7B are graphs of reciprocal serum dilution versus O.D. 405 nm showing the effects of IL-12 administered i.n. on respiratory mucosal responses; mice were immunized on day 0 with HANA and treated with either IL-12 or PBS vehicle on days 0, 1, 2 and 3 and boosted on days 14 and 28; on day 28 the mice also received IL-12 or vehicle; mice were sacrificed on day 35, and BAL fluid was assayed for anti-HANA antibody levels by ELISA using HANA coated microtiter plates; each line represents binding of antibody from an individual mouse.
• Figures 8A-8B are graphic representations showing the effects of LL-12 administered i.n. on early systemic antibody responses to the subunit influenza vaccine.
• mice were immunized i.n. on day 0 with H1N1 subunit influenza vaccine, and treated i.n. with either IL-12 (closed triangles) or PBS vehicle (open circles) on days 0, 1, 2 and 3.
• Serum anti-HlNl antibody levels on day 14 were determined by isotype-specific ELISA using HLN1 -coated microtiter plates. Each line represents binding of antibody from an individual mouse (4 mice per group). The difference in binding between mice immunized with vaccine and IL-12 and those immunized with vaccine and PBS vehicle was significant at p ⁇ 0.05 for IgG2a.
• FIGS 9A-9E are graphic representations showing the effects of IL-12 administered i.n. on late systemic antibody responses to the subunit influenza vaccine.
• Mice were immunized i.n. on day 0 with H1N1 subunit influenza vaccine, treated i.n. with either IL-12 (closed triangles) or PBS vehicle (open circles) on days 0, 1 , 2 and 3, and boosted with vaccine on days 14 and 28.
• the mice received a second treatment with IL-12 or vehicle.
• Serum anti-HlNl antibody levels on day 35 were determined by isotype-specific ELISA using HINl-coated microtiter plates. Each line represents binding of antibody from an individual mouse (4 mice per group). The differences in binding between mice immunized with vaccine and IL-12 and those immumzed with vaccine and PBS vehicle were significant at p >0.05 for IgG2a, total Ab and total Ig.
• FIGS 10A-10D are graphic representations showing the effects of IL-12 administered i.n. on respiratory mucosal responses.
• Mice were immunized i.n. on day 0 with H1N1 subunit influenza vaccine, treated i.n. with either IL-12 or PBS vehicle on days 0, 1, 2 and 3, and boosted with vaccine on days 14 and 28.
• the mice received a second treatment with IL-12 or vehicle.
• Mice were sacrificed on day 35 and BAL fluid was assayed for anti-HlNl antibody levels by ELISA using H1N1 -coated microtiter plates. Each line represents binding of antibody from an individual mouse (4 mice per group). The differences in binding between mice immunized with vaccine and LL-12 and those immunized with vaccine and PBS vehicle were significant at p ⁇ 0.05 for total Ab, IgGl, IgG2a and IgA.
• FIGS 11 A- 1 ID are graphic representations showing that co-administration of influenza subunit vaccine plus IL-12 protects mice from a subsequent influenza virus infection.
• Mice were immumzed i.n. with H1N1 subunit vaccine plus LL-12 (closed triangles), vaccine plus PBS vehicle (open circles), IL-12 only (open diamonds) or PBS vehicle only (open squares). All mice (8 per group) were then challenged i.n. 4-5 weeks later with 10 3 pfu (A) or 2 x 10 3 pfu (B) of A/PR/8/34 influenza virus. The mice were monitored daily for mortality and weight loss. The differences in survival between mice immunized with vaccine and IL-12 and those immunized with vaccine and PBS were significant atp ⁇ 0.05.
• FIGS 12A-12B are graphic representations showing that LL-12 induced protection against influenza virus infection is mediated by B cells.
• MT mice were immunized i.n. on with HlNl subunit vaccine plus LL-12 (closed triangles), vaccine plus PBS vehicle (open circles) or PBS vehicle only (open diamonds). Wild type (WT) mice were pre-treated with PBS vehicle (open squares). All mice (8 per group) were then challenged i.n. 6-7 weeks later with 10 3 pfu of A/PR/8/34 influenza virus. The mice were monitored daily for mortality and weight loss.
• Figure 13 is a graphic representation showing passive transfer of serum from mice immunized with the subunit influenza vaccine plus IL-12 confers protection against influenza virus challenge.
• FIGS 14A-14B are graphic representations showing passive transfer of BAL fluid i.n. from mice immunized with the subunit influenza vaccine plus LL-12 confers protection against influenza virus challenge.
• BAL fluids were collected from mice immunized with the HlNl subunit influenza vaccine plus LL-12 (closed triangles), vaccine plus PBS (open circles) or PBS vehicle only (open squares). All mice (8 per group) were then inoculated i.n. with pooled BAL fluid and 2 x 10 3 pfu of A/PR/8/34 influenza virus. The differences in survival between mice immunized with vaccine and LL-12 and those immunized with vaccine and PBS were significant atp ⁇ 0.05.
• the present invention relates to methods of enhancing and/or inducing immunity to a pathogen (one or more) in a host, which comprises administering i.n. to the host an effective amount of LL-12 and an antigen of the pathogen (e.g., a mucosal pathogen).
• the methods of the present invention can be used to enhance an immune response to an antigen in a mammalian host, such as a primate (e.g., human), murine, feline, canine, bovine or porcine host.
• the terms “enhance” and/or “enhancing” refer to the strengthening (augmenting) of an existing immune response to a pathogen.
• the term also refers to the initiation of (initiating, inducing) an immune response to a pathogen.
• An antigen (one or more) for use in the methods of the present invention includes (or can be obtained from), but is not limited to, proteins or fragments thereof (e.g., proteolytic fragments), peptides (e.g., synthetic peptides, polypeptides), glycoproteins, carbohydrates (e.g., polysaccharides), lipids, glycolipids, hapten conjugates, recombinant DNA, whole organisms (killed or attenuated) or portions thereof, toxins and toxoids (e.g., tetanus, diphtheria, cholera) and/or organic molecules.
• proteins or fragments thereof e.g., proteolytic fragments
• peptides e.g., synthetic peptides, polypeptides
• glycoproteins e.g., carbohydrates (e.g., polysaccharides), lipids, glycolipids, hapten conjugates, recombinant DNA, whole organisms (killed or attenu
• antigens for use in the present invention include hemagglutinin and neuraminidase obtained or derived from the influenza virus.
• the antigen can be obtained or derived from a variety of pathogens or organisms, such as bacteria (e.g., bacillus, Group B streptococcus, Bordetella, Listeria, Bacillus anthracis, S. pneumoniae, N. meningiditis, H. influenza), viruses (e.g., hepatitis, measles, poliovirus, human immunodeficiency virus, influenza virus, parainfluenza virus, respiratory syncytial virus), mycobacteria (M.
• bacteria e.g., bacillus, Group B streptococcus, Bordetella, Listeria, Bacillus anthracis, S. pneumoniae, N. meningiditis, H. influenza
• viruses e.g., hepatitis, measles, poliovirus, human immuno
• the antigen of a pathogen can be obtained using skills known in the art.
• the antigen can be isolated (purified, essentially pure) directly from the pathogen, derived using chemical synthesis or obtained using recombinant methodology.
• the antigen can be obtained from commercial sources.
• a suitable antigen for use in the present invention is one that includes at least one B and/or T cell epitope (e.g., T helper cell or cytolytic T cell epitope).
• T cell epitope e.g., T helper cell or cytolytic T cell epitope.
• Other suitable antigens useful in the compositions of the present invention can be determined by those of skill in the art.
• IL-12 is a recently characterized heterodimeric cytokine that has a molecular weight of 75 kDa and is composed of disulfide-bonded 40 kDa and 35 kDa subunits. It is produced by antigen presenting cells such as macrophages and dendritic cells, and binds to receptors on activated T, B and NK cells (Desai, B.B., et al, J. Immunol, - ⁇ 5:3125-3132 (1992); Vogel, L.A., et al, Int. Immunol, 5:1955-1962 (1996)).
• LL-12 has been shown to be an important costimulator of proliferation in Thl clones (Kennedy et al, Eur. J. Immunol. 24:2271-227%, 1994) and leads to increased production of IgG2a antibodies in serum when administered i.p. (Morris, S.C., et al, J.
• interleukin-12 and IL-12 refer to interleukin 12 protein, its individual subunits, multimers of its individual subunits, functional fragments of LL-12, and functional equivalents and or analogues of "interleukin- 12" and "LL-12".
• functional fragments of LL-12 are fragments which, when administered i.n., modulate an immune response against an antigen in a host.
• modified LL-12 protein such that the resulting IL-12 product has activity similar to the LL-12 described herein (e.g., the ability to enhance an immune response when administered i.n.).
• interleukin- 12 also include nucleic acid sequences (e.g., DNA, RNA) and portions thereof, which encode a protein or peptide having the LL-12 function or activity described herein (e.g., the ability to enhance an immune response when administered i.n.).
• nucleic acid sequences e.g., DNA, RNA
• portions thereof which encode a protein or peptide having the LL-12 function or activity described herein (e.g., the ability to enhance an immune response when administered i.n.).
• the term includes a nucleotide sequence which through the degeneracy of the genetic code encodes a similar peptide gene product as LL-12 and has the IL-12 activity described herein.
• a functional equivalent of "interleukin- 12" and "LL-12” includes a nucleotide sequence which contains a "silent" codon substitution (e.g., substitution of one codon encoding an amino acid for another codon encoding the same amino acid) or an amino acid sequence which contains a "silent” amino acid substitution (e.g., substitution of one acidic amino acid for another acidic amino acid).
• IL-12 suitable for use in the methods of the present invention can be obtained from a variety of sources or synthesized using known skills.
• LL-12 can be purified (isolated, essentially pure) from natural sources (e.g., mammalian, such as human sources), produced by chemical synthesis or produced by recombinant DNA techniques.
• natural sources e.g., mammalian, such as human sources
• the LL-12 for use with the present invention can be obtained from commercial sources.
• an effective amount of LL-12 is administered i.n. in the methods of the present invention which is an amount that induces and/or enhances an immune response to an antigen in the host.
• an effective amount of LL-12 is an amount such that when administered i.n. with an antigen to a host, enhances an immune response to the antigen in the host as described herein, relative to the immune response to the antigen in a host when an effective amount of LL-12 is not administered i.n. to the host.
• an "effective amount" of LL-12 is an amount that, when administered i.n. with an antigen, it enhances an immune response to an antigen in a host as described herein, relative to the immune response to the antigen if LL-12 is not administered i.n. to the host.
• the LL-12 and/or the antigen can be administered i.n. as a prophylactic vaccine or a therapeutic vaccine. That is, the LL-12 can be administered either before (to prevent) or after (to treat) the effects of a pathogen which has appeared and/or manifested in a host.
• the LL-12 and/or antigen can be administered to a host who either exhibits the disease state caused by a pathogen from which the antigen is obtained or derived, or does not yet exhibit the disease state caused by a pathogen from which the antigen is obtained or derived.
• the LL-12 and/or antigen can be administered to a host either before or after the disease state is manifested in the host and can result in prevention, amelioration, elimination or a delay in the onset of the disease state caused by the pathogen from which the antigen is obtained or derived.
• the LL-12 and the antigen are administered i.n. to a host.
• Any convenient route of i.n. administration can be used.
• absorption through epithelial or mucocutaneous linings e.g., administering the IL-12 and/or antigen using a nasal mist; administering the IL-12 and/or antigen to the eye using an eye dropper wherein the IL-12 and/or antigen drains into the nasal cavity
• absorption through epithelial or mucocutaneous linings e.g., administering the IL-12 and/or antigen using a nasal mist; administering the IL-12 and/or antigen to the eye using an eye dropper wherein the IL-12 and/or antigen drains into the nasal cavity
• the IL-12 and antigen can be administered together with other components or biologically active agents, such as adjuvants (e.g., alum), pharmaceutically acceptable surfactants (e.g., glycerides), liposomes, excipients (e.g., lactose), carriers, diluents and vehicles.
• adjuvants e.g., alum
• pharmaceutically acceptable surfactants e.g., glycerides
• liposomes e.g., lactose
• excipients e.g., lactose
• carriers e.g., lactose
• diluents and vehicles e.g., diluents and vehicles.
• sweetening, flavoring and/or coloring agents can also be added.
• the LL-12 and or the antigen in the embodiment wherein the antigen is a protein (peptide), can be administered i.n. by in vivo expression of polynucleotides encoding such into a host.
• the IL-12 or the antigen can be administered to a host using a live vector, wherein the live vector containing IL-12 and/or antigen nucleic acid sequences is administered i.n. under conditions in which the LL-12 and/or antigen are expressed in vivo.
• a host can also be injected i.n.
• a host can be injected i.n. with a vector which encodes and expresses LL-12 in vivo in combination with an antigen in peptide or protein form, or in combination with a vector which encodes and expresses an antigen in vivo.
• a single vector containing the sequences encoding an antigen and the LL-12 protein are also useful in the methods of the present invention.
• Several expression vector systems are available commercially or can be reproduced according to recombinant DNA and cell culture techniques.
• vector systems such as the yeast or vaccinia virus expression systems, or virus vectors can be used in the methods and compositions of the present invention (Kaufman, R.J., A J. ofMeth. in Cell andMolec. Biol, 2:221-236 (1990)).
• Other techniques using naked plasmids or DNA, and cloned genes encapsulated in targeted liposomes or in erythrocyte ghosts, can be used to introduce IL-12 polynucleo tides into the host (Freidman, T., Science, 2 ⁇ :1275-1281 (1991); Rabinovich, N.R., et al, Science, 265: 1401-1404 (1994)).
• the present invention relates to a method of inducing a Thl-like immune response to a pathogen in a host, comprising administering i.n. to the host an effective amount of LL-12 and an antigen of the pathogen.
• the present invention also relates to a method of enhancing a mucosal immune response to a pathogen in a host, comprising administering i.n. to the host an effective amount of LL-12 and an antigen of the pathogen.
• the methods described herein can result in enhanced expression of IFN- ⁇ .
• a humoral response can be induced and/or enhanced in a host, which can result in enhanced expression of IgG2a, IgG2b and/or IgG3 antibody.
• the immune response can be antigen-specific.
• an effective amount of LL-12 is administered i.n. to the host, which is an amount that enhances and/or induces an immune response to the antigen in the host and results in the improved condition of the host (i.e., the disease or disorder caused by the presence of the pathogen from which the antigen is obtained or derived, is prevented, eliminated or diminished).
• the amount of LL-12 used to enhance an immune response to an antigen in a host will vary depending on a variety of factors, including the size, age, body weight, general health, sex and diet of the host, and the time of administration, duration or particular qualities of the disease state.
• Suitable dose ranges of LL-12 are generally about 0.5 ⁇ g to about 150 ⁇ g per kg body weight. In one embodiment, the dose range is from about 2.75 ⁇ g to about 100 ⁇ g per kg body weight. In another embodiment, the dose range is from about 5 ⁇ g to about 50 ⁇ g per kg body weight. Effective dosages may be extrapolated from dose- response curves derived in vitro or in animal model test systems.
• an effective amount of LL-12 is administered i.n. in combination with an antigen. That is, the LL-12 is administered at a time closely related to immunization of the host with an antigen, so that an immune response to the antigen is induced or enhanced in the host relative to the immunization of a host in which IL-12 is not administered.
• the LL-12 can be administered i.n. prior to, preferably just prior to, immunization; at the time of immunization (i.e., simultaneously); or after immunization (subsequently).
• the IL-12 can be administered i.n. prior to immunization with the antigen followed by subsequent administrations of LL-12 after immunization with the antigen.
• IL-12 given i.n. and in a non-invasive manner, redirects the mucosal compartment of the immune system toward Thl type cytokine and antibody profiles.
• delivery of LL-12 modulates the patterns of cytokine and antibody expression in distant systemic compartments of the immune system.
• mice immunized i.n. with DNP-OVA plus LL-12 displayed enhanced levels of IFN- ⁇ mRNA in the lungs after 6 hours with maximal expression noted at 24 hours. There was a similar enhancement of IFN- ⁇ mRNA in the spleen after i.n. administration of LL-12.
• IFN- ⁇ is a potent immunoregulator of Th cell subsets and their effector functions (Trinchieri, G., et al, Res. Immunol, 74(5:423-431 (1995); Trinchieri, G., Immunol Today, 74:335-338 (1993)).
• LFN- ⁇ has been shown to activate macrophages and mediate isotype switching to IgG2a and IgG3 antibody production which is characteristic of Thl -type immune responses (Snapper, CM., et al, Science, 236:944-947 (1987); Snapper, CM., et al, J. Immunol, 140:2121-2127 (1988); Finkelman, F.D., et al, J. immunol, 140:1022- 1027 (1988)).
• an important negative regulator of T-cell responses is interleukin- 10 (LL-10) (Meyaard, L., et al, J. Immunol, 75(5:2776-2782 (1996)).
• LL-10 interleukin- 10
• IL-12 is able to induce human T cells to secrete LL-10 (Meyaard, L., et al, J. Immunol, 755:2776-2782 (1996); Daftarian, P.M., et al, J. Immunol, 757:12-20 (1996); Gerosa, F., et al, J. Exp.
• IL-12 delivered i.n. by a non-invasive route is capable of influencing serum antibody responses in a similar manner.
• Mice that were immunized i.n. with antigen and LL-12 had markedly elevated levels of serum IgG2a, IgG2b and IgG3 compared to animals receiving antigen only.
• the observed increases in IgG2a and IgG3 levels are consistent with the ability of LL-12 to induce IF ⁇ - ⁇ , which is a potent switch factor for both IgG2a and IgG3 antibody responses (Metzger, D.W., et al, Eur. J.
• IL-12 administered i.n. or parenterally resulted in enhancement of fecal IgG2a antibody levels.
• i.n. treatment with LL-12 resulted in reduced IgA expression while parenteral delivery of -14- 99/44635 PC17US99/04678
• IL-12 enhanced IgA levels. These results show an important differential effect of IL-12 given via two different routes of administration. Recently, in contrast to the data described herein, Okada et al. (Okada, E., et al, J. Immunol, 759:3638-3647 (1997)) reported that i.n. immunization with an HIV DNA vaccine in an IL-12 expressing plasmid did not modify fecal IgA antibody levels. Furthermore,
• Thl type immune responses have been shown to be protective against Leishmania (Muller, I., et al, Immunol. Rev., 772:95-113 (1989)) and Listeria (Kratz, S.S., et al, J. Immunol, 747:598-606
• LL-12 is a key cytokine in immune regulation by its ability to direct Th cells towards a Thl phenotype with enhancement of LFN- ⁇ secretion and elevation of IgG2a antibody levels.
• the findings described herein show that the i.n. use of LL-12 as an adjuvant enhances vaccine immunity.
• LL-12 there are no suitable mucosal adjuvants for clinical use at the current time.
• An immediate application for IL-12 given by this route would be for use in conjunction with nasal influenza vaccines currently in clinical trials. Since protection against influenza is mediated by IgG antibody (Palladino, G., et al, J.
• LL-12 i.n. would be a means to augment both mucosal and systemic antibody responses towards influenza.
• i.n. administration of a subunit influenza vaccine plus LL-12 markedly enhances systemic and respiratory IgG2a levels.
• a non-invasive i.n. delivery system was used to evaluate the ability of LL-12 to modulate both mucosal and systemic components of the immune system. Mice immunized i.n.
• DNP-OVA DNP conjugated to OVA
• IL-12 treatment can effectively modulate antigen-specific immune responses and enhance immunization strategies for mucosal vaccines.
• the results clearly demonstrate the effectiveness of LL-12 administered i.n. for augmenting antigen specific-responses in both mucosal and systemic compartments.
• the findings show that LL-12 can be used as a potent vaccine adjuvant for immunization strategies against mucosal pathogens.
• the methods and described herein can be used to treat and/or prevent a disease or condition associated with a pathogen having one or more antigens in a host.
• the methods described herein can utilize an effective amount of LL-12 in combination with a single antigen or multiple antigens which can be derived from the same pathogen, from different strains of a pathogen or from different pathogens.
• LL-12 and one or more antigens can be used to prevent and/or treat one or more disease or condition associated with the pathogen(s) from which the antigen(s) is derived.
• EXAMPLE 1 MODULATION OF MUCOSAL AND SYSTEMIC IMMUNITY BY LNTRANASAL INTERLEUKIN 12 DELIVERY
• mice Six to eight week-old female BALB/c mice were obtained from the National Cancer Institute (Bethesda, MD). Mice were housed in the animal facility at the Medical College of Ohio, and provided food and water ad libitum. Animal care and experimental procedures were in compliance with the Institutional Animal Care and Use Committee (I ACUC) of the Medical College of Ohio .
• I ACUC Institutional Animal Care and Use Committee
• mice were immunized i.n. with 50 ⁇ l of sterile phosphate-buffered saline (PBS) containing 100 ⁇ g of dinitrophenyl hapten conjugated to ovalbumin (DNP-OVA; Biosearch Technologies, San Raphael, CA) and 10 ⁇ g cholera toxin B-subunit (CTB; Sigma, St. Louis, MO). This was followed on days 0, 1, 2 and 3 with intranasal i.n. of 1 ⁇ g of recombinant murine IL-12 in PBS containing 1% normal
• mice BALB/c mouse serum (PBS-NMS) or, in the case of control mice, with PBS-NMS only. Mice were boosted i.n. with the same amount of DNP-OVA and CTB on days 14 and 28. On day 28, the mice also received 1 ⁇ g of LL-12 in PBS-NMS or PBS- NMS only. For i.p. inoculations, mice were immunized with 100 ⁇ g of DNP-OVA in complete Freund's adjuvant (CFA; Life Technologies, Gaithersburg, MD) on day 0, followed by injection of 1 ⁇ g of LL-12 in PBS-NMS on days 0, 1, 2 and 3. Control mice received antigen and PBS-NMS only.
• CFA complete Freund's adjuvant
• mice were boosted by the same route on days 14 and 28 with DNP-OVA in incomplete Freund's adjuvant (IF A; Life Technologies). On day 28, the mice were also injected i.p. with LL-12 in PBS-NMS or PBS-NMS only. Sera were prepared by bleeding mice from the orbital plexus. -17- O 99/44635 PCT/US99/04678
• RNA isolation from snap frozen spleens and lungs was performed with Trizol reagent (Gibco-BRL Gaithersburg, MA) according to the manufacturer's instructions. Briefly, the frozen tissues were homogenized with a mortar and pestle, and immediately transferred into polystyrene tubes containing 2.0 ml of Trizol reagent.
• the homogenized samples were incubated for 5 minutes at room temperature to allow dissociation of the nucleoprotein complexes and centrifuged at 12,000g for 10 minutes at 4°C
• the supernatant fluids were mixed for 15 seconds with 0.4 ml of chloroform, incubated for 15 minutes on ice, and centrifuged at 12,000g for 15 minutes at 4°C Following centrifugation, the RNA in the aqueous phase was precipitated at -20 °C for one hour by the addition of 1.0 ml isopropanol.
• the samples were centrifuged for 15 minutes at 12,000g and the RNA pellet was washed twice with 1.0 ml of 75% ethanol.
• the pellet was air-dried for 2-5 minutes, solubilized in DEPC-treated water, and stored at -80 °C
• the final preparation of total RNA yielded a 260/280 ratio of 1.7-2.0.
• First strand cDNA synthesis was performed following the manufacturer's instructions (Gibco-BRL). Briefly, 1 ⁇ g of oligo(dT), 3 ⁇ g of total RNA, and sterile DEPC-treated water were added to a sterile eppendorf tube to a final volume of 11 ⁇ l. The mixture was incubated at 70°C for 10 minutes and then chilled on ice. Subsequently, the following components were added in order: 4.0 ⁇ l of 5X first strand buffer, 2 ⁇ l of 0.1 M DDT, and 1 ⁇ l of dNTP mixture (10 mM each of dATP, dGTP, dCTP and dTTP).
• the contents of the tube were mixed gently and incubated at 42 °C for 2 minutes, followed by the addition of 1 ⁇ l (200 U) of Superscript U reverse transcriptase (RT).
• RT Superscript U reverse transcriptase
• the tubes were placed into the wells of the Perkin Elmer Thermal Cycler 480 (Perkin Elmer Cetus, Norwalk, CT), incubated at 95°C for 5 minutes and then subjected to the following amplification profile: 1 minute at 95 °C, 1 minute at 56°C and 1 minute at 72°C for a duration of 35 cycles. This followed by an incubation at 72°C for 10 minutes followed by a soak cycle at 4°C.
• the PCR products were separated on a 2.5% agarose gel and stained with ethidium bromide. The bands were visualized and photographed using UV transillumination.
• HPRT Hypoxanthine phosphoribosyl transferase
• mice were sacrificed and their tracheas were exposed and intubated using a 0.58 mm OD polyethylene catheter (Becton Dickinson, Sparks, MD).
• the lungs were lavaged two to three times with PBS containing 5 mM EDTA.
• Approximately 1.5 ml of lavage fluid was obtained per mouse and blood contamination was monitored using Hemastix (Bayer Corporation, Elkhart, IN).
• the recovered BAL fluid was centrifuged at 12,000g for 5 minutes at 4°C and the supernatant was stored at -70°C until use.
• Fecal extracts were prepared by the method of deVos and Dick (deVos, T., et al, J. Immunol.
• Anti-DNP and anti-OVA antibody levels were determined by ELISA as described (Buchanan, J.M., et al, Int. Immunol, 7:1519-1528 (1995); Metzger, D.W., Eur. J. Immunol, 27:1958-1965 (1997)). Briefly, microtiter plates (Nalge Nunc International, Rochester, NY) were coated overnight with 10 ⁇ g/ml DNP- bovine serum albumin (BSA) or 100 ⁇ g/ml of OVA in PBS. The plates were washed with PBS containing 0.1% (w/v) gelatin and 0.05% (v/v) Tween 20.
• BSA DNP- bovine serum albumin
• mice were immunized with DNP- OVA and CTB +/- IL-12, and levels of cytokine mRNA in the lungs of individual animals were analyzed by RT-PCR after 6 and 24 hours. There was found to be a sha ⁇ increase in the expression of IFN- ⁇ mRNA in mice 6 hours after treatment with LL-12 and this expression remained elevated for at least 24 hours compared to immunized mice not exposed to LL-12.
• IFN- ⁇ mRNA has been found to downregulate Th2 type cytokines such as IL-5 (Mosmann, T.R., et al,
• Cytokine expression in the lungs was compared to that in spleens after i.n. inoculation of antigen plus LL-12. There was an enhancement of splenic IFN- ⁇ mRNA expression 6 hours after treatment with IL-12. This increase was still pronounced at 24 hours whereas untreated mice had nearly undetectable levels of IFN- ⁇ mRNA at this time point. Increases of LL-10 mRNA levels were also detected in the spleens of LL-12 treated mice, with maximal expression at 24 hours compared to untreated controls. The ability of LL-12 given i.p. to induce systemic
• Intranasal IL-12 administration modulates respiratory antibody responses
• Intranasal IL-12 administration modulates serum antibody responses
• mice that received antigen and LL-12 by either route had significantly higher levels (p ⁇ 0.05) of fecal IgG2a anti-DNP antibody levels compared to immunized mice not exposed to LL-12 ( Figures 4A-4F).
• mice that received only antigen parenterally had no detectable IgG2a in fecal extracts.
• parenteral treatment with antigen and LL-12 also resulted in enhancement of fecal IgA levels (p ⁇ 0.05), i.n. delivery of LL-12 resulted in a decrease (p ⁇ 0.05) of IgA antibody levels.
• mice were immunized i.n. on day 0 with HANA and treated i.n. with either IL-12 or PBS vehicle on days 0, 1, 2 and 3. Serum anti-HANA antibody levels on day 14 were determined by isotype-specific ELISA using HAHA-coated microtiter plates. See Figures 5A-5B.
• mice were immunized i.n. on day 0 with HANA and treated i.n. with either LL-12 or PBS vehicle on days 0, 1, 2 and 3 and boosted on day 14.
• Serum anti-HANA antibody levels on day 28 were determined by isotype-specific ELISA using HAHA-coated microtiter plates. See Figures 6A-6B.
• mice were immunized on day 0 with HANA and treated with either LL- 12 or PBS vehicle on days 0, 1, 2 and 3 and boosted on days 14 and 28; on day 28 the mice also received LL-12 or vehicle. Mice were sacrificed on day 35, and BAL fluid was assayed for anti-HANA antibody levels by ELISA using HANA coated microtiter plates. See Figures 7A-7B.
• mice Six-to eight-week old female BALB/c mice were obtained from The National Cancer Institute (Bethesda, MD). C57BL/6 IgM deficient ( ⁇ MT) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Mice were housed in the animal facility at the Medical College of Ohio and provided food and water ad libitum. All animal care and experimental procedures were performed in compliance with the Institutional Animal Care and Use Committee (IACUC) guidelines. -24- O 99/44635 PCT/US99/04678
• mice were immunized i.n. on day 0 with 25 ⁇ l of sterile PBS containing 1 ⁇ g of subunit influenza vaccine which consisted of soluble hemagglutinin subtype 1 (HI) and neuraminidase subtype 1 (Nl) purified from influenza virus A/PR8/34 (provided by Dr. Doris Bucher, New York Medical College, New York, NY). This was followed on days 0, 1, 2 and 3 with i.n.
• Ketamine HCL Fort Dodge Laboratories, Fort Dodge, IO
• Xylazine Bayer Corporation, Shawnee Mission, KA
• Mice were immunized i.n. on day 0 with 25 ⁇ l of sterile PBS containing 1 ⁇ g of subunit influenza vaccine which consisted of soluble hemagglutinin subtype 1 (HI) and neuraminidase subtype 1 (Nl) purified from influenza virus A/PR8/34 (provided by Dr. Doris Bucher
• RNA isolation from snap frozen spleens and lungs was performed with the Ambion Total RNA Isolation Kit (Austin, TX) according to the manufacturer's instructions. Briefly, the frozen tissues were homogenized with a mortar and pestle and immediately transferred into tubes containing 1.0 ml of denaturation solution. Following phenol-chloroform extraction, the homogenized samples were centrifuged at 10,000 x g for 10 minutes at 4°C The supernatants were subjected to another round of phenol-chloroform extraction and the resulting RNA was precipitated with isopropanol, washed twice with 75% ethanol and solubilized in DEPC-treated water.
• RNA concentration was determined by spectrophotometric analysis at 260 nm. Three micrograms of total RNA were reverse transcribed into cDNA using a reverse transcription kit (Life Technologies, Gaithersburg, MD) utilizing oligo (dT) 16.lg primers. The resulting cDNA was amplified using specific primers for LFN- ⁇ and LL-10 with hypoxanthine phosphoribosyl transferase (HPRT) primers as a control. The sense and antisense primers utilized had the following sequences: IFN- ⁇
• PCR amplification was performed by mixing 2 ⁇ l of cDNA, 0.25 mM dNTPs (Invitrogen Corporation, San Diego, CA), 0.8 ⁇ M primer and 2.5 U of Tag DNA Polymerase (Life Technologies) in a final volume of 50 ⁇ l in 60 mM Tris-HCl (pH 8.5), 15 mM (NH 4 ) 2 SO 4 , 0.4 mM MgCl 2 .
• the mixtures were incubated at 95 °C for 5 minutes and then subjected to the following amplification profile: 1 minute at 95°C, 1 minute at 56°C and 1 minute at 72°C for a duration of 35 cycles. This was followed by a final extension for 10 minutes at 72 °C
• the PCR products were separated on a 2.5% agarose gel, stained with ethidium bromide and visualized by UV transillumination.
• RNA levels were determined utilizing the RiboQuant multi-probe ribonuclease protection assay system (Pharmingen, San Diego, CA) according to the manufacturer's instructions. Briefly, 10 ⁇ g of total RNA was hybridized to a 32 P labeled RNA probe overnight at 56 °C The single-stranded nucleic acid was digested with ribonuclease for 45 minutes at 30 °C, subjected to phenol-chloroform extraction, and resolved on a 6% denaturing polyacrylamide gel. Transcript levels were quantified on a Storm 840 Phosphorlmager (Molecular Dynamics, Sunnyvale, CA). Total RNA was normalized to the housekeeping gene glyceraldehyde 3- phosphate dehydrogenase and relative cytokine mRNA levels were expressed as arbitrary values. Collection of Bronchoalveolar Lavage Fluid
• mice For collection of BAL fluid, the mice were sacrificed and their tracheas intubated using a 0.58 mm OD polyethylene catheter (Becton Dickinson, Sparks, MD). The lungs were then lavaged two to three times with PBS containing 5 mM EDTA. The recovered BAL fluid was centrifuged at 12,000 x g for 5 minutes at 4°C and the supernatant was stored at -70 °C until use.
• Anti-HlNl levels in serum and BAL were determined by ELISA essentially as described (Buchanan, J.M., et al, Int. Immunol, 7:1519-1528 (1995); Buchanan, R.M.,et al/. J. Immunol, 757:5525-5533 (1998)) with minor modifications. Briefly, microtiter plates (Nalge Nunc International, Rochester, NY) were coated overnight with 1 ⁇ g/ml of HlNl in PBS. The plates were washed with PBS containing 0.3% Brij-35 (Sigma, St.
• the plates were incubated with goat anti-mouse IgA conjugated to biotin (Sigma), then washed and incubated with alkaline phosphatase- conjugated streptavadin (BIO RAD, Richmond, CA). Total immunoglobulins were measured in the same fashion except that the plates were coated with 10 ⁇ g/ml affinity-purified goat anti-mouse Ig (Southern Biotechnology Associates)
• mice were immunized i.n. on day 0 with 25 ⁇ l of PBS containing 1 ⁇ g of HlNl subunit influenza vaccine. This was followed on days 0, 1, 2 and 3 with i.n. inoculation of 1 ⁇ g of LL-12 in PBS-NMS or with PBS-NMS only. Some mice received only LL-12 in PBS-NMS or only PBS-NMS (no HlNl subunit vaccine). Approximately 4-5 weeks after primary immunization, viral challenge was performed using infectious A/PR8/34 influenza virus (provided by Dr. Doris Bucher) administered i.n. to anesthetized mice in 40 ⁇ l of sterile PBS. The mice were weighed daily and monitored for morbidity and mortality.
• mice were injected i.p. with 100 ⁇ l of a 1:10 dilution of pooled serum and challenged 5 hours later with infectious influenza virus i.n.
• LL-12 given parenterally has profound regulatory effects on the immune system through its ability to preferentially activate Thl and NK cells, and induce LFN- ⁇ production (Trinchieri, G., et al, Res. Immunol, 146:423-431 (1995); Gately, M.K., et al, Annu. Rev. Immunol, 75/495-521 (1998)).
• the effects of i.n. administration of LL-12 on respiratory cytokine gene expression have now been examined. Analysis of cytokine mRNA expression in the lungs of individual mice (3 mice per group) after a single i.n. inoculation of LL-12 or PBS vehicle and HlNl subunit influenza vaccine.
• mice were sacrificed 24 hours or 48 hours after treatment, and total lung RNA was assayed for the expression of the indicated cytokines by RT-PCR: LL-10 (455 bp), IFN- ⁇ (459 bp) and HPRT (162 bp). It was found the i.n. treatment of mice with HlNl subunit influenza vaccine 28 O 99/44635 " " PCT/US99/04678
• LL-12 had an enhancing effect on expression of lung IFN- ⁇ mRNA levels within 24 hours compared to immunization with vaccine only. This increase in LFN- ⁇ mRNA levels was still evident 48 hours after IL-12 inoculation.
• cytokine mRNA patterns in the spleens of immunized mice were examined. Analysis of cytokine mRNA expression in the spleens of individual mice (3 mice per group) after a single i.n. inoculation of LL-12 or PBS vehicle and HlNl subunit influenza vaccine. Mice were sacrificed 24 hours or 48 hours after treatment, and total splenic RNA was assayed for the expression of the indicated cytokines by RT-PCR: LL-10 (455 bp), IFN- ⁇ (459 bp), and HPRT (162 bp).
• HlNl subunit vaccine plus LL-12 resulted in a substantial increase in splenic LFN- ⁇ mRNA expression within 24 hours compared to mice that received vaccine alone. Elevated levels of IFN- ⁇ were still evident at 48 hours in IL-12 treated mice. Splenic LL-10 mRNA levels remained elevated at both 24 hours and 48 hours after IL-12 treatment. Finally, no IL-5 mRNA was detected in the spleens of either IL-12 treated or control animals. Simultaneous amplification of HPRT mRNA confirmed that equal amounts of RNA were utilized in all RT-PCR reactions. To further quantify the levels of cytokine mRNA transcripts observed after i.n.
• cytokine mRNA levels in the lungs and spleens were analyzed by ribonuclease protection assay. It was found that LFN- ⁇ mRNA levels were increased 2-fold in the lungs of animals 24 hours and 48 hours after treatment with HlNl plus LL-12 compared to mice that received vaccine alone (the Table). Furthermore, IL-10 mRNA expression was -29- 99/44635 PCT/US99/04678
• LFN- ⁇ mRNA was elevated 5-fold at 24 hours and 2-fold at 48 hours after LL-12 treatment.
• splenic IL-10 mRNA levels were increased 8-fold at 24 hours and 5-fold at 48 hours after IL-12 treatment.
• LL- 12 delivered i.n. modulates both mucosal and systemic immunity to the DNP hapten.
• IL-12 delivered i.n. has similar effects on antibody responses to HlNl influenza vaccine.
• IgG2a anti-HlNl antibody levels were markedly enhanced after IL-12 treatment compared to mice that received vaccine alone. Therefore, i.n. IL-12 treatment resulted in early activation of serum IgG2a antibody responses.
• mice Similar analysis were performed on day 35 sera to determine the long-term effects of i.n. LL-12 treatment. At this time point, IL-12-treated mice had 6-fold higher levels of total anti-HlNl serum antibody than mice immunized with the vaccine alone ( Figures 9A-9E). Moreover, there was an increase in total (nonspecific) Ig after i.n. LL-12 treatment. IgG2a antibody levels were still dramatically enhanced in mice that received IL-12. Furthermore, IgGl anti-HlNl antibodies, evident in both experimental and control groups, were moderately elevated in LL-12 treated mice compared to mice receiving only vaccine, an observation which is consistent with our previous findings (Buchanan, J.M., et al, Int.
• mice that were immunized and treated with LL-12 displayed elevated BAL fluid IgA anti-HlNl antibody levels compared to animals not exposed to LL-12. This result is in stark contrast to the absence of detectable IgA in the circulation of these mice. It was also found that levels of both IgGl and IgG2a anti- H1N1 antibodies were dramatically enhanced in BAL fluid after IL-12 administration compared to mice that received vaccine alone. These results firmly establish the influence of LL-12 delivered i.n. in augmenting respiratory antibody expression.
• mice were immunized i.n. with HlNl vaccine on day 0 and treated with 1 ⁇ g of LL-12 or PBS vehicle on days 0, 1, 2 and 3. Some mice received only LL-12 or PBS vehicle. Four to five weeks after immunization, the mice were inoculated i.n. with infectious A/PR8/34 influenza virus and monitored daily for morbidity and mortality. In the first experiment, a dose of virus was used that allowed 50% survival of mice after exposure to just vaccine ( Figures 11 A-l IB).
• mice inoculated i.n. with vaccine and IL-12 were then challenged with A/PR8/34 influenza virus 5 hours later.
• mice inoculated with vaccine or PBS-NMS only, all succumbed to infection ( Figure 13).
• animals that received serum from mice immunized with the vaccine plus IL-12 exhibited 50% survival after viral challenge.
• mice that received BAL fluid from mice treated with HlNl plus LL-12 were protected against virus infection. These mice exhibited no transient weight loss over the course of the infection while both of the other treatment groups displayed progressive weight loss leading to death. Furthermore, mice that received BAL fluid from animals immunized with vaccine alone had viral lung titers of 10 3 pfu on day 4 after infection while mice that received BAL fluid from animals treated with vaccine plus LL-12 had viral lung titers of ⁇ 100 pfu. Finally, the overall health of virus-challenged animals that received BAL fluid from -33- O 99/44635 PCT/US99/04678
• mice vaccinated with HlNl plus LL-12 remained noticeably better than mice which received BAL fluid from animals vaccinated with HlNl alone.
• passive transfer of BAL fluid i.n. from mice immunized with HlNl subunit vaccine plus LL- 12 provided dramatic protection against influenza virus challenge.
• LL-12 delivered i.n. with an influenza subunit vaccine serves as a potent mucosal adjuvant and confers increased protection against subsequent viral infection.
• Use of B cell deficient mice and passive transfer of serum or BAL fluid demonstrated that the protection induced by LL-12 is mediated by antibody.
• LFN- ⁇ has a variety of immunoregulatory functions, which include induction of the Thl cell differentiation and activation of NK cells (Boehm, U., et al, Annu. Rev. Immunol, 75:749-795 (1998)).
• IFN- ⁇ enhances the production of opsonizing murine antibodies such as IgG2a (Buchanan, J.M., et al, Int. Immunol, 7:1519-1528 (1995); Metzger, D.W., et al, Eur. J.
• LL-10 mRNA expression was also induced in lungs and spleens by i.n. treatment with IL-12.
• LL- 10 is mainly produced by T cells and monocytes, and has been shown to inhibit Thl cell differentiation (Fiorentino, D.F., et al, J. Immunol, 745:3444-3451 (1991); Ding, L., et al, J.
• Example 2 the effects of i.n. IL-12 on responses to a clinically relevant influenza subunit vaccine was examined.
• IL- 12 treatment was found to have a dramatic effect on the early onset of the humoral response, as reflected by significant enhancement of IgG2a anti-HlNl antibody levels.
• animals that received vaccine alone did not develop early IgG2a responses.
• There was little detectable IgGl antibody during the early phase of the immune response in animals that received vaccine alone or vaccine and IL-12.
• IgG2a levels were still enhanced in LL-12 treated mice and IgGl levels were also somewhat elevated, an observation that is in agreement with previous findings in the lysozyme system (Buchanan, J.M., et al, Int.
• LL-12 i.n. administration also resulted in significant increases in respiratory antibody levels, including IgG and IgA anti-HlNl antibody levels.
• IgA is the predominant antibody in mucosal secretions, and is thought to play a major role in preventing attachment of pathogens to mucosal epithelial surfaces (Lamm, M.E., Annu. Rev. Immunol, 57:311-340 (1997)).
• Adjuvants that have been used to enhance mucosal immune responses include microbial products such as CT and LT, which have been utilized in a variety of delivery systems (Staats, H.F., et al, Curr. Opin. Immunol, 5:572-583 (1994); Elson, CO., In Mechanisms in the Pathogenesis of Enteric Disease, Paul, P.S., et al, eds., (NY:Plenum Press), pages 373-385 (1997)).
• CT is a potent inducer of the Th2-type responses
• LT elicits a mixed Thl and Th2 response (Marinaro, M., et al, J. Exp.

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