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WO2003051305A2 - Vaccins agissant sur le systeme immunitaire inne - Google Patents

Vaccins agissant sur le systeme immunitaire inne Download PDF

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Publication number
WO2003051305A2
WO2003051305A2 PCT/US2002/040046 US0240046W WO03051305A2 WO 2003051305 A2 WO2003051305 A2 WO 2003051305A2 US 0240046 W US0240046 W US 0240046W WO 03051305 A2 WO03051305 A2 WO 03051305A2
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pamp
antigen
ofthe
chaperone
fusion protein
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PCT/US2002/040046
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English (en)
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WO2003051305A3 (fr
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Ruslan Medzhitov
Elizabeth Kopp
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Yale University
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Priority to AU2002361682A priority Critical patent/AU2002361682A1/en
Publication of WO2003051305A2 publication Critical patent/WO2003051305A2/fr
Publication of WO2003051305A3 publication Critical patent/WO2003051305A3/fr

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    • 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
    • A61K39/385—Haptens or antigens, bound to carriers
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6043—Heat shock proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • 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

  • Immunity Multicellular organisms have developed two general systems of immunity to infectious agents.
  • the two systems are innate or natural immunity (also known as “innate immunity”) and adaptive (acquired) or specific immunity.
  • innate immunity also known as "innate immunity”
  • adaptive (acquired) or specific immunity The major difference between the two systems is the mechanism by which they recognize infectious agents.
  • the innate immune system uses a set of germline-encoded receptors for the recognition of conserved molecular patterns present in microorganisms. These molecular patterns occur in certain constituents of microorganisms including: lipopolysaccharides, peptidoglycans, lipoteichoic acids, phosphatidyl cholines, bacteria-specific proteins, including lipoproteins, bacterial DNAs, viral single and double-stranded RNAs, unmethylated CpG-DNAs, mannans and a variety of other bacterial and fungal cell wall components. Such molecular patterns can also occur in other molecules such as plant alkaloids.
  • PAMPs Pathogen Associated Molecular Patterns
  • PRRs Pattern Recognition Receptors
  • Cellular PRRs are expressed on effector cells ofthe innate immune system, including cells that function as professional antigen-presenting cells (APC) in adaptive immunity.
  • effector cells include, but are not limited to, macrophages, dendritic cells, B lymphocytes and surface epithelia.
  • This expression profile allows PRRs to directly induce innate effector mechanisms, and also to alert the host organism to the presence of infectious agents by inducing the expression of a set of endogenous signals, such as inflammatory cytokines and chemokines, as discussed below. This latter function allows efficient mobilization of effector forces to combat the invaders.
  • the adaptive immune system which is found only in vertebrates, uses two types of antigen receptors that are generated by somatic mechanisms during the development of each individual organism.
  • the two types of antigen receptors are the T-cell receptor (TCR) and the immunoglobulin receptor (IgR), which are expressed on two specialized cell types, T-lymphocytes and B- lymphocytes, respectively.
  • TCR T-cell receptor
  • IgR immunoglobulin receptor
  • the specificities of these antigen receptors are generated at random during the maturation of lymphocytes by the processes of somatic gene rearrangement, random pairing of receptor subunits, and by a template-independent addition of nucleotides to the coding regions during the rearrangement.
  • naive CD4 + T-lymphocytes require that both signals, the specific antigen and the B7 molecule, are expressed on the same APC. If a naive CD4 T-cell recognizes the antigen in the absence ofthe B7 signal, the T-cell will die by apoptosis. Expression of B7 molecules on APCs, therefore, controls whether or not the naive CD4 T-lymphocytes will be activated. Since CD4 T-cells control the activation of CD8 T-cells for cytotoxic functions, and the activation of B-cells for antibody production, the expression of B7 molecules determines whether or not an adaptive immune response will be activated.
  • TLRs Toll-like receptors
  • TLRs have been shown to recognize PAMPs such as the bacterial products LPS, peptidoglycan, and lipoprotein.
  • PAMPs such as the bacterial products LPS, peptidoglycan, and lipoprotein.
  • Vaccines have traditionally been used as a means to protect against disease caused by infectious agents. However, with the advancement of vaccine technology, vaccines have been used in additional applications that include, but are not limited to, control of mammalian fertility, modulation of hormone action, and prevention or treatment of tumors.
  • vaccines used to protect against a disease are to induce immunological memory to a particular microorganism. More generally, vaccines are needed to induce an immune response to specific antigens, whether they belong to a microorganism or are expressed by tumor cells or other diseased or abnormal cells. Division and differentiation of B- and T-lymphocytes that have surface receptors specific for the antigen generate both specificity and memory.
  • a vaccine hi order for a vaccine to induce a protective immune response, it must fulfill the following requirements: 1) it must include the specific antigen(s) or fragment(s) thereof that will be the target of protective immunity following vaccination; 2) it must present such antigens in a form that can be recognized by the immune system, e.g., a form resistant to degradation prior to immune recognition; and 3) it must activate APCs to present the antigen to CD4 + T-cells, which in turn induce B-cell differentiation and other immune effector functions.
  • vaccines include, but are not limited to, cowpox virus for inoculating against smallpox, tetanus toxoid to prevent tetanus, whole-inactivated bacteria to prevent whooping cough (pertussis), polysaccharide subunits to prevent streptococcal pneumonia, and recombinant proteins to prevent hepatitis B.
  • Attenuated vaccines are usually immunogenic, their use has been limited because their efficacy generally requires specific, detailed knowledge ofthe molecular determinants of virulence. Moreover, the use of attenuated pathogens in vaccines is associated with a variety of risk factors that in most cases prevent their safe use in humans.
  • An adjuvant is defined as any substance that increases the immunogenicity of admixed antigens. Although chemicals such as alum are often considered to be adjuvants, they are in effect akin to carriers and are likely to act by stabilizing antigens and/or promoting their interaction with antigen-presenting cells. The best adjuvants are those that mimic the ability of microorganisms to activate the innate immune system. Pure antigens do not induce an immune response because they fail to induce the costimulatory signal (e.g., B7.1 or B7.2) necessary for activation of lymphocytes.
  • costimulatory signal e.g., B7.1 or B7.2
  • Immune stimulating complexes are cage-like structures comprising Quil-A, cholesterol, adjuvant active saponin and phospho lipids that induce a wide range of systemic immune responses.
  • ISCOMS Immune stimulating complexes
  • ISCOMS are suitable for repeated administration of different antigens to an individual because these complexes allow the entry of antigen into both MHC I and II processing pathways.
  • ISCOMS have been used with conjugates of modified soluble proteins.
  • the vaccines contemplated by Klein et al. are fusion proteins, in which the component peptides are all selected by virtue of their being antigens (i.e., being recognized by a TCR or IgR) .
  • the vaccines described by Klein et al. are not designed to stimulate the innate immune system. Although many types of vaccines are available, it would be advantageous to have vaccines which provide greater protection.
  • the novel vaccines ofthe present invention comprise one or more isolated PAMPs in combination with one or more antigens.
  • the antigens used in the vaccines ofthe present invention can be any type of antigen (e.g., including but not limited to pathogen-related antigens, tumor-related antigens, allergy-related antigens, neural defect-related antigens, cardiovascular disease antigens, rheumatoid arthritis-related antigens, other disease-related antigens, hormones, pregnancy- related antigens, embryonic antigens and/or fetal antigens and the like). Examples of various types of vaccines, which can be produced by the present invention, are provided in Figure 1.
  • the vaccines are recombinant proteins, or recombinant lipoproteins, or recombinant glycoproteins, which contain a PAMP (e.g., BLP, Flagellin or FimC) and one or more antigens.
  • PAMP e.g., BLP, Flagellin or FimC
  • APCs such as dendritic cells and macrophages. This interaction will have two consequences: First, the PAMP portion ofthe vaccine will interact with a PRR such as a TLR and stimulate a signaling pathway, such as the NF- ⁇ B, JNK and/or p38 pathways.
  • the recombinant vaccine will be taken up into dendritic cells and macrophages by phagocytosis, endocytosis, or macropinocytosis, depending on the cell type, the size ofthe recombinant vaccine, and the identity ofthe PAMP.
  • TLR-induced signaling pathways Activation of TLR-induced signaling pathways will lead to the induction of the expression of cytokines, chemokines, adhesion molecules, and co-stimulatory molecules by dendritic cells and macrophages and, in some cases, B-cells. Uptake ofthe vaccines will lead to the processing ofthe antigen(s) fused to the PAMP and their presentation by the MHC class-I and MHC class-II molecules. This will generate the two signals required for the activation of naive T-cells - a specific antigen signal and the co-stimulatory signal.
  • chemokines induced by the vaccine due to PAMP interaction with TLR
  • TLR cytokines
  • IL-12 cytokines, like IL-12, which will induce T-cell differentiation into Th-1 effector cells.
  • a robust T-cell immune response will be induced, which will in turn activate other aspects ofthe adaptive immune responses, such as activation of antigen-specific B-cells and macrophages.
  • novel vaccines ofthe present invention provide an efficient way to produce an immune response to one or more specific antigens without the adverse side effects normally associated with conventional vaccines.
  • the present invention relates generally to vaccines, methods of making vaccines and methods of using vaccines.
  • the present invention provides vaccines comprising an isolated PAMP, immunostimulatory portion or immunostimulatory derivative thereof and an antigen or an immunogenic portion or immunogenic derivative thereof.
  • An example of a vaccine ofthe present invention is a fusion protein comprising a PAMP, immunostimulatory portion or immunostimulatory derivative thereof and an antigen or an immunogenic portion or immunogenic derivative thereof.
  • the vaccines ofthe present invention can comprise any PAMP peptide or protein, including, but not limited to, the following PAMPs: peptidoglycans, lipoproteins and lipopeptides, Flagellins, chaperones, outer membrane proteins (OMPs), outer surface proteins (OSPs), other protein components ofthe bacterial cell walls, and other PRR. ligands.
  • One PAMP ofthe present invention is BLP, including the BLP encoded by the polypeptide of SEQ ID NO: 2, set forth in Figure 15.
  • BLP BLP encoded by the polypeptide of SEQ ID NO: 2, set forth in Figure 15.
  • Antigens useful in the present invention include, but are not limited to, those that are microorganism-related, and other disease-related antigens, including but not limited to those that are allergen-related and cancer-related.
  • the antigen component ofthe vaccine can be derived from sources that include, but are not limited to, bacteria, viruses, fungi, yeast, protozoa, metazoa, tumors, malignant cells, abnormal neural cells, arthritic lesions, cardiovascular lesions, plants, animals, humans, allergens, hormones and amyloid- ⁇ peptide.
  • the antigens, immunogenic portions or immunogenic derivatives thereof can be composed of peptides, polypeptides, lipoproteins, glycoproteins, mucoproteins and the like.
  • the various vaccines ofthe present invention include, but are not limited to: 1) one or more PAMPs, immunostimulatory portions or immunostimulatory derivatives thereof, conjugated to one or more antigens, immunogenic portions or immunogenic derivatives thereof;
  • a PAMP/antigen fusion protein comprising one or more PAMPs, immunostimulatory portions or immunostimulatory derivatives thereof, and one or more antigens, immunogenic portions or immunogenic derivatives thereof;
  • a modified antigen, immunogenic portion or immunogenic derivative thereof that comprises a leader sequence fused to a lipidation or glycosylation consensus sequence that is further fused to the antigen, or an immunogenic portion or immunogenic derivative thereof.
  • the present invention also encompasses such vaccines further comprising a pharmaceutically acceptable carrier, including, but not limited to, alum. More specifically, the present invention provides fusion proteins comprising one or more PAMPs, immunostimulatory portions or immunostimulatory derivatives thereof, and one or more antigens, immunogenic portions or immunogenic derivatives thereof.
  • the PAMP domains ofthe fusion proteins ofthe present invention can be composed of amino acids, amino acid polymers, peptidoglycans, glycoproteins, and lipoproteins or any other suitable component.
  • One preferred PAMP to use in the fusion proteins ofthe present invention is BLP, including the BLP encoded by the polypeptide of SEQ ID NO: 2.
  • Flagellin is another PAMP to use in the fusion proteins of the present invention, and is provided by (but not limited to) accession numbers P04949 (E, Coli Flagellin) and A24262 (Salmonella Flagellin).
  • Another PAMP to use in the fusion proteins of the present invention is FimC, and is provided by (but not limited to) the amino acid sequence of SEQ ID NO: 14 (accession number AAC77272) and the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 15 (accession number L14598).
  • Useful antigen domain(s) in the fusion proteins ofthe present invention include, but are not limited to, E ⁇ (a peptide antigen derived from mouse MHC class-II I-E), listeriolysin, PSMA, HIV gpl20, Ra5G and TRP-2.
  • the fusion proteins ofthe present invention include a construct comprising the following components: a leader peptide that signals lipidation or glycosylation ofthe consensus sequence, a lipidation and/or glycosylation consensus sequence, and antigen.
  • the fusion proteins ofthe present invention include a construct comprising a leader sequence — CXXN — antigen, wherein the leader peptide is a signal for lipidation ofthe consensus sequence and wherein X is any amino acid, preferably serine.
  • leader peptides useful in the present invention include, but are not limited to, those selected from the peptides of SEQ ID NO: 3 (shown in Figure 15), SEQ ID NO: 4 (shown in Figure 16), SEQ ID NO: 5 (shown in Figure 17), SEQ ID NO: 6 (shown in Figure 18) and SEQ ID NO: 7 (shown in Figure 19).
  • the present invention also provides a fusion protein comprising an isolated PAMP and an antigen, wherein the antigen is a self-antigen.
  • the present invention further provides methods of recombinantly producing the fusion proteins ofthe present invention.
  • the present invention provides recombinant expression vectors comprising a nucleotide sequence encoding the chimeric constructs ofthe present invention as well as host cells transformed with such recombinant expression vectors.
  • Any cell that is capable of expressing the fusion proteins ofthe present invention is suitable for use as a host cell.
  • host cells include, but are not limited to, the cells of bacteria, yeast, insects, plants and animals. More preferably for certain PAMPs such as BLP, the host cell is a bacterial cell. Even more preferably, the host cell is a bacterial cell that can appropriately modify (e.g., lipidation, glycosylation) the PAMP domain ofthe fusion protein when such modification is necessary or desirable.
  • the present invention also provides methods of immunizing an animal with the vaccines ofthe present invention, where such methods include, but are not limited to, administering a vaccine parenterally, intravenously, orally, using suppositories, or via the mucosal surfaces.
  • the animal being vaccinated is a human.
  • the immune response can be measured using any suitable method including, but not limited to, direct measurement of peripheral blood lymphocytes, natural killer cell cytotoxicity assays, cell proliferation assays, immunoassays of immune cells and subsets, and skin tests for cell-mediated immunity.
  • the present invention also provides methods of treating a patient susceptible to an allergic response to an allergen by administering a vaccine ofthe present invention and thereby stimulating the TLR-mediated signaling pathway.
  • the present invention also provides methods of treating a patient susceptible to or suffering from Alzheimer's disease by administering a vaccine ofthe present invention in which the target antigen is a peptide or protein associated with Alzheimer's disease, including but not limited to amyloid- ⁇ peptide.
  • the present invention further provides a method of stimulating an innate immune response in an animal and thereby enhancing the adaptive immune response to a foreign or self-antigen which comprises co-administering a PAMP with the foreign or self antigen.
  • the present invention also provides a vaccine which comprises a PAMP conjugated with a foreign or self antigen that stimulates an innate immune response in an animal and thereby enliances the adaptive immune response to a foreign or self-antigen but does not lead to undesirable levels of inflammation.
  • the present invention provides a vaccine which comprises a PAMP conjugated with a foreign or self antigen which, when administered at a therapeutically active dose, stimulates an innate immune response in an animal and thereby enhances the adaptive immune response to a foreign or self- antigen but does not lead to undesirable levels of inflammation.
  • the present invention also provides a method of treatment comprising the steps of administering to an individual a vaccine which comprises a PAMP conjugated with a foreign or self antigen which stimulates an innate immune response in an animal and thereby enhances the adaptive immune response to a foreign or self-antigen but does not lead to undesirable levels of inflammation.
  • Figure 1 shows examples of alternative fusion proteins according to the present invention. Permutations and combinations of these fusion proteins can also be prepared according to the methods ofthe present invention.
  • Figure 2 shows a basic outline for generating different recombinant protein vaccines containing different antigens and a signal to trigger the innate immune response (PAMP). Each antigen is represented by a different shape ofthe central portion ofthe vaccine.
  • Figure 3 shows the BLP/E ⁇ construct.
  • Figure 4 shows that BLP/E ⁇ activates NF- ⁇ B in dose-dependent manner.
  • Figure 5 shows IL-6 production by dendritic cells stimulated with BLP/E ⁇ .
  • Figure 6 shows the induction of dendritic cell activation and vaccine antigen processing and presentation by the MHC class-II pathway.
  • Figure 7 shows the immunostimulatory effect ofthe chimeric construct BLP/E ⁇ on specific T-cells in vitro.
  • Figure 8 shows the effect ofthe chimeric construct, BLP/E ⁇ , on specific T- cell proliferation in vivo.
  • FIG. 9 shows that CpG-induced B-cell activation is dependent upon MyD88.
  • MyD88 "/_ MyD88-deficient cells
  • ICE ' ' ' caspase-1 -deficient cells
  • BlO/ScCr TLR4-deficient cells derived from C57BL/10ScCr mice
  • TLR2 " TLR2-deficient cells.
  • Figure 10 shows that IL-6 production by macrophages during CpG stimulation and CpG-DNA-induced IkB ⁇ degradation is mediated by a signaling pathway dependent on MyD88.
  • Figure 11 shows that wild-type and BlO/ScCr dendritic cells, but not dendritic cells from MyD88 _/" animals produce IL- 12 when stimulated with CpG oligonucleotides.
  • Figure 12 shows activation of NF- ⁇ B by Flagellin fusions.
  • Figure 13 shows induction of NF- ⁇ B in macrophages by Flagellin fusions.
  • Figure 14 shows NF-i B activity in RAW KB cells.
  • Figure 15 shows SEQ ID NO: 3.
  • Figure 16 shows SEQ.ID NO: 4.
  • Figure 17 shows SEQ ID NO: 5.
  • Figure 18 shows SEQ ID NO: 6.
  • Figure 19 shows SEQ ID NO: 7.
  • Figure 20 shows SEQ ID NO: 10.
  • Figure 21 shows SEQ ID NO: 11.
  • Figure 22 shows activation of NF- ⁇ B by FimC.
  • Figure 23 shows that boiling reduces the activity of FimC. DETAILED DESCRIPTION OF THE INVENTION 1.
  • the present invention discloses a novel strategy of vaccine design based on the inventor's recent findings in the field of innate immunity.
  • This approach is not limited to any particular antigen or immunogenic portions or derivatives thereof (e.g., microorganism-related, allergen-related or tumor-related, and the like) nor is it limited to any particular PAMP or immunostimulatory portions or immunostimulatory derivatives thereof.
  • the term "vaccine”, therefore, is used herein in a general sense to refer to any therapeutic or immunogenic or immunostimulatory composition that includes the features of the present invention. A more detailed definition of vaccine is disclosed elsewhere herein.
  • an adaptive immune response requires both the specific antigen or derivative thereof, and a signal (e.g. PAMP) that can induce the expression of B7 on the APCs.
  • PAMP a signal that can induce the expression of B7 on the APCs.
  • the present invention combines, in a single chimeric construct, both signals required for the induction ofthe adaptive immune responses - a signal recognized by the innate immune system (PAMP), and a signal recognized by an antigen receptor (antigen).
  • Fusion of an antigen with a PAMP such as bacterial lipoprotein (BLP) optimizes the stoichiometry ofthe two signals and coordinates their effect on the same APC, thus minimizing the unwanted excessive inflammatory responses that occur when antigens are mixed with adjuvants comprising innate immune stimulants to increase their immunogenicity.
  • the chimeric constructs ofthe present invention will prevent activation of APCs that do not take up the antigen.
  • the chimeric constructs of the present invention exhibit the essential immunological characteristics or properties expected of a conventional vaccine supplemented with an adjuvant, but the chimeric constructs do not induce an excessive inflammatory reaction as is often induced by an adjuvant.
  • the vaccine approach described in the present invention minimizes or eliminates undesired side effects (e.g., excessive inflammatory reaction, autoimmunity) yet induces a very potent and specific immune response, and provides a favorable alternative to vaccines comprising mixtures of antigens and adjuvants.
  • neither the PAMP nor the antigen need consist of a polypeptide.
  • either the PAMP or the antigen, or both may be a peptide or polypeptide.
  • recombinant DNA technology may be utilized in the production of chimeric constructs, for use in vaccines, when both the PAMP, or an immunogenic portion or derivative thereof, and the antigen, or an immunostimulatory portion or derivative thereof, are polypeptides.
  • recombinant teclmiques may also be utilized to produce a protein chimeric construct when a peptide mimetic is used in lieu of a non-protein antigen, such as a polysaccharide or a nucleic acid and the like, and/or a non-protein PAMP, such as a lipopolysaccharide, CpG-DNA, bacterial DNA, single or double- stranded viral RNA, phosphatidyl choline, lipoteichoic acids and the like, for example.
  • a non-protein antigen such as a polysaccharide or a nucleic acid and the like
  • a non-protein PAMP such as a lipopolysaccharide, CpG-DNA, bacterial DNA, single or double- stranded viral RNA, phosphatidyl choline, lipoteichoic acids and the like, for example.
  • the present invention contemplates in one embodiment the use of BLP, the bacterial outer membrane proteins (OMP), the outer surface proteins A (OspA) of bacteria, Flagellins, chaperones including periplasmic chaperones such as FimC and other DNA-encoded PAMPs in the recombinant production of chimeric constructs.
  • OMP bacterial outer membrane proteins
  • OspA outer surface proteins A
  • Flagellins periplasmic chaperones
  • FimC periplasmic chaperones
  • other DNA-encoded PAMPs in the recombinant production of chimeric constructs.
  • BLP has been shown to be recognized by TLRs. (Aliprantis et al. (1999) Science 285: 736-9). The details ofthe approach are described using BLP as the PAMP domain of a PAMP/antigen fusion protein; however any inducers ofthe innate immune system are equally applicable for such purpose in the present invention.
  • one or more PAMP mimetics is substituted in place of a PAMP in a fusion protein.
  • This invention further provides methods for producing chimeric constructs where either the PAMP or an immunostimulatory portion or derivative thereof, or the antigen or an immunogenic portion or derivative thereof, or both the PAMP and the antigen are non-protein.
  • these methods utilize chemical means lo conjugate a PAMP to an antigen thereby producing a non-protein chimeric construct.
  • This invention further provides ways to exploit recombinant DNA technology in the synthesis ofthe peptide vaccines.
  • Many ofthe surface antigens present on the pathogens of interest would not be amenable to encoding by nucleic acids as they are not proteins (e.g., lipopolysaccharides) or possess low protein content (e.g., peptidoglycans).
  • the present invention contemplates the use of peptide mimetics for these surface antigens or an immunogenic protein or derivative thereof, and the use of peptide mimetics in vaccines.
  • the present invention contemplates vaccines comprising chimeric constructs that comprise at least one antigen, or an immunogenic portion or derivative thereof, and at least one PAMP, or an immunogenic portion or derivative thereof.
  • the present invention encompasses vaccines comprising fusion proteins where one or more protein antigens are linked to one or more protein PAMPs or a peptide mimetic of a PAMP.
  • the fusion protein has maximal immunogenicity and induces only a modest inflammatory response.
  • a target antigen, or a domain of a target antigen has a relatively low molecular weight and is not adequately immunogenic because of its small size
  • that antigen or antigen domain can act as a hapten and can be combined with a larger carrier molecule such that the molecular weight ofthe combined molecule will be high enough to evoke a strong immune response against the antigen.
  • the antigen itself serves as the carrier molecule.
  • the PAMP serves as the carrier molecule.
  • a hapten is combined, by either fusion or conjugation, with the PAMP or the antigen domain ofthe vaccine to elicit an antibody response to the hapten.
  • the PAMP and the antigen are combined with a third molecule that serves as the carrier molecule.
  • a carrier molecule can be keyhole, limpet hemocyanin or any of a number of carrier molecules for haptens that are lcnown to the artisan.
  • a fusion protein contains an antigen or antigen domain, a PAMP or a portion of a PAMP or a PAMP mimetic, and a carrier protein or earner peptide.
  • carrier protein can be keyhole limpet hemocyanin or any of a number of carrier proteins or carrier peptides for haptens that are known to the artisan, hicreasing the number of antigens or antigen epitopes, by using multiple antigen proteins and/or multiple domains ofthe same antigen protein or of different antigen proteins and/or some combination ofthe foregoing, are contemplated in this invention. Also contemplated are fusion proteins in which the number of PAMPs or PAMP derivatives or PAMP mimetics is increased. It is within the skill ofthe artisan to determine the optimal ratio of PAMP to antigen domains to maximize immunogenicity and minimize inflammatory response. 2. Definitions
  • Adaptive immunity refers to the adaptive immune system, which involves two types of receptors generated by somatic mechanisms during the development of each individual organism.
  • the "adaptive immune system” refers to both cellular and humoral immunity. Immune recognition by the adaptive immune system is mediated by antigen receptors.
  • Adaptive immune response refers to a response involving the characteristics ofthe “adaptive immune system” described above.
  • Adapter molecule refers to a molecule that can be transiently associated with some TLRs, mediates immunostimulation by molecules ofthe innate immune system, and mediates cytokine-induced signaling.
  • Adapter molecule includes, but is not limited to, myeloid differentiation marker 88 (MyD88).
  • Allergen refers to an antigen, or a portion or derivative of an antigen, that induces an allergic or hypersensitive response.
  • amino acid polymer refers to proteins, or peptides, and other polymers comprising at least two amino acids linked by a peptide bond(s), wherein such polymers contain either no non-peptide bonds or one or more non-peptide bonds.
  • proteins include polypeptides and oligopeptides.
  • Antigen refers to a substance that is specifically recognized by the antigen receptors ofthe adaptive immune system.
  • the term “antigen” includes antigens, derivatives or portions of antigens that are immunogenic and immunogenic molecules derived from antigens.
  • the antigens used in the present invention are isolated antigens.
  • Antigens that are particularly useful in the present invention include, but are not limited to, those that are pathogen-related, allergen-related, or disease-related.
  • Antigenic determinant refers to a region on an antigen at which a given antigen receptor binds.
  • Antigen-presenting cell or “APC” or “professional antigen-presenting cell” or “professional APC” is a cell ofthe immune system that functions in triggering an adaptive immune response by taking up, processing and expressing antigens on its surface.
  • effector cells include, but are not limited to, macrophages, dendritic cells and B cells.
  • Antigen receptors refers to the two types of antigen receptors ofthe adaptive immune system: the T-cell receptor (TCR) and the immunoglobulin receptor (IgR), which are expressed on two specialized cell types, T-lymphocytes and B-lymphocy.es, respectively.
  • TCR T-cell receptor
  • IgR immunoglobulin receptor
  • the secreted form ⁇ l ' lhe imiuunoglul-iiliu receptor is referred to as antibody.
  • the specificities of the antigen receptors are generated at random during the maturation ofthe lymphocytes by the processes of somatic gene rearrangement, random pairing of receptor subunits, and by a template- independent addition of nucleotides to the coding regions during the rearrangement.
  • Chimeric construct refers to a construct comprising an antigen and a
  • PAMP, or PAMP mimetic wherein the antigen and the PAMP are comprised of molecules such as amino acids, amino acid polymers, nucleotides, nucleotide polymers, carbohydrates, carbohydrate polymers, lipids, lipid polymers or other synthetic or naturally occurring chemicals or chemical polymers.
  • a "chimeric construct" refers to constructs wherein the antigen is comprised of one type of molecule in association with a PAMP or PAMP mimetic, which is comprised of either the same type of molecule or a different type of molecule.
  • CpG refers to a dinucleo ⁇ de in which an un ethylated cytosine C) residue occurs immediately 5' to a guanosine (G) residue.
  • CpG- DNA refers to a synthetic CpG repeat, intact bacterial DNA containing CpG motifs, or a CpG-containing derivative thereof. The immunostimulatory effect of CpG-DNA on B-cells is mediated through a TLR and is dependent upon a "protein adapter molecule".
  • Derivative refers to any molecule or compound that is structurally related to the molecule or compound from which it is derived. As used herein, “derivative” includes peptide mimetics (e.g., PAMP mimetics).
  • Domain refers lo a portion of a protein with its own function. The combination of domains in a single protein determines its overall function.
  • An "antigen domain” comprises an antigen or an immunogenic portion or derivative of an antigen.
  • a “PAMP domain” comprises a PAMP or a PAMP mimetic or an immunostimulatory portion or derivative of a PAMP or a PAMP mimetic.
  • Fusion protein and “chimeric protein” both refer to any protein fusion comprising two or more domains selected from the following group consisting of: proteins, peptides, lipoproteins, lipopeptides, glycoproteins, glycopeptides, mucoproteins, mucopeptides, such that at least two ofthe domains are either from different species or encoded by different genes or such that one ofthe two domains is found in nature and the second domain is not known to be found in nature or such that one ofthe two domains resembles a molecule found in nature and the other does not resemble that same molecule.
  • fusion protein also refers to an antigen or an immunogenic portion or derivative thcrcor which has been modi Hod to contain an amino acid sequence that results in post-translational modification of that amino acid sequence or a portion of that sequence, wherein the post-translationally modified sequence is a ligand for a PRR.
  • the amino acid sequence that results in post- translational modification to form a ligand for a PRR can comprise a consensus sequence, or that amino acid sequence can contain a leader sequence and a consensus sequence.
  • Hapten refers to a small molecule that is not by itself immunogenic but can bind antigen receptors and can combine with a larger carrier molecule to become immunogenic.
  • association with refers to a reversible union between two chemical entities, whether alike or different, to form a more complex substance.
  • In combination with refers to either a reversible or irreversible (e.g. covalent) union between two chemical entities, whether alike or different, to form a more complex substance.
  • Immunoser refers to the ability of a molecule to activate either the adaptive immune system or the innate immune system.
  • antigens are examples of molecules that are capable of stimulating the adaptive immune system
  • PAMPs or PAMP mimetics are examples of molecules that are capable of stimulating the innate immune system.
  • activation of either immune system includes the production of constituents of humoral and/or cellular immune responses that are reactive against the immunostimulatory molecule.
  • Innate immunity refers to the innate immune system, which, unlike the “adaptive immune system", uses a set of germline-encoded receptors for the recognition of conserved molecular patterns present in microorganisms.
  • “Innate immune response” refers to a response involving the characteristics ofthe "innate immune system” described above.
  • Linker refers to any chemical entity that links one chemical moiety to another chemical moiety. Thus, something that chemically or physically connects a PAMP and an antigen is a linker. Examples of linkers include, but are not limited to, complex or simple hydrocarbons, nucleosides, nucleotides, nucleotide phosphates, oligonucleotides, polynucleotides, nucleic acids, amino acids, small peptides, polypeptides, carbohydrates (e.g., monosaccharides, disaccharides, trisaccharides), and lipids.
  • linkers are provided in the Detailed Description Selection included herein. Without limitation, the present invention also contemplates using a peptide bond or an amino acid or a peptide linker to link a polypeptide PAMP and a polypeptide antigen. The present invention further contemplates preparing such a linked molecule by recombinant DNA procedures.
  • a linker can also function as a spacer.
  • Microorganism refers to a living organism too small to be seen with the naked eye. Microorganisms include, but are not limited to bacteria, fungi, protozoans, microscopic algae, and also viruses.
  • “Mimetic” refers to a molecule that closely resembles a second molecule and has a similar effect or function as that ofthe second molecule.
  • “Moiety” refers to one of the component parts of a molecule. While there are normally two moieties in a single molecule, there may be more than two moieties in a single molecule.
  • Molecular pattern refers to a chemical structure or motif that is typically a component of microorganisms, or certain other organisms, but which is not typically produced by normal human cells or by other normal animal cells. Molecular patterns' are found in, or composed of, the following types of molecules: lipopolysaccharides, peptidoglycans, lipoteichoic acids, phosphatidyl cholines, lipoproteins, bacterial DNAs, viral single and double-stranded RNAs, certain viral glycoproteins, unmethylated CpG-DNAs, mannans, and a variety of other bacterial, fungal and yeast cell wall coniponents and the like.
  • Non-protein chimeric construct or “non-protein chimera” refers to a “chimeric construct” wherein either the antigen or the PAMP or the PAMP mimetic, or two or more of them, is not an amino acid polymer.
  • PAMP PAMP
  • PAMP refers to a molecular pattern found in a microorganism but not in humans, which, when it binds a PRR., can trigger an innate immune response.
  • the term “PAMP” includes any such microbial molecular pattern and is not limited to those associated with pathogenic microorganisms or microbes.
  • PAMP includes a PAMP, derivative or portion of a PAMP that is immunostimulatory, and any immunostimulatory molecule derived from any PAMP. These structures, or derivatives thereof, are potential initiators of innate immune responses, and therefore, ligands for PRRs, including Toll receptors and TLRs.
  • PAMPs are immunostimulatory structures that are found in, or composed of molecules including, but not limited to, lipopolysaccharides; phosphatidyl choline; glycans, including peptidoglycans; teichoic acids, including lipoteichoic acids; proteins, including lipoproteins and lipopeptides; outer membrane proteins (OMPs), outer surface proteins (OSPs and other protein components ofthe bacterial cell walls and Flagellins; chaperones including periplasmic chaperones such as FimC; bacterial DNAs; single and double-stranded viral RNAs; unmethylated CpG-DNAs; mannans; mycobacterial membranes; porins; and a variety of other bacterial and fungal cell wall components, including those found in yeast.
  • OMPs outer membrane proteins
  • OSPs outer surface proteins
  • chaperones including periplasmic chaperones such as FimC
  • bacterial DNAs single and double-stranded viral
  • PAMP/antigen conjugate refers to an antigen and a PAMP or PAMP mimetic that are covalently or noncovalently linked.
  • a conjugate may be comprised of a protein PAMP or antigen and a non-protein PAMP or antigen.
  • PAMP/antigen fusion or “PAMP/antigen chimera” refers to any protein fusion formed between a PAMP or PAMP mimetic and an antigen.
  • Passive immunization refers to the administration of antibodies or primed lymphocytes to an individual in order to confer immunity.
  • PAMP mimetic refers to a molecule that, although it does not occur in microorganisms, is analogous to a PAMP in that it can bind to a PRR and such binding can trigger an innate immune response.
  • a PAMP mimetic can be a naturally-occurring molecule or a partially or totally synthetic molecule.
  • certain plant alkaloids, such as taxol are PRR ligands, have an immunostimulatory effect on the innate immune system, and thus behave as PAMP mimetics. (Kawasaki et al. (2000) J. Biol. Chem. 275(4): 2251-2254).
  • PAMP Plasma Recognition Receptor
  • PRR Physical Receptor
  • Cellular PRRs maybe expressed on effector cells ofthe innate immune system, including cells that function as professional APCs in adaptive immunity, and also on cells such as surface epithelia that are the first to encounter pathogens during infection. PRRs may also induce the expression of a set of endogenous signals, such as inflammatory cytokines and chemokines.
  • PRRs useful for the present invention include, but are not limited to, the following: C-type lectins (e.g., humoral, such as coUectins (MBL), and cellular, such as macrophage C-type lectins, macrophage mannose receptors, DEC205); proteins containing leucine-rich repeats (e.g., Toll receptor and TLRs, CD14, RP105); scavenger receptors (e.g., macrophage scavenger receptors, MARCO, WC1); and pentraxins (e.g., C-reactive proteins, serum, amyloid P, LBP, BPIP, CD 1 lb,C and CD 18.
  • C-type lectins e.g., humoral, such as coUectins (MBL), and cellular, such as macrophage C-type lectins, macrophage mannose receptors, DEC205
  • proteins containing leucine-rich repeats
  • Protein mimetic refers to a protein or peptide that closely resembles a non- protein molecule and has a similar effect or function to the non-protein molecule.
  • a peptide mimetic can be a non-protein molecule or non-peptide molecule that closely resembles a peptide or protein and has a similar effect or function to the peptide or protein.
  • “Pharmaceutically acceptable carrier” refers to a carrier that can be tolerated by a recipient animal, typically a mammal.
  • Protein chimeric construct refers to a chimeric construct wherein both the antigen and the PAMP or PAMP mimetic are amino acid polymers.
  • Recombinant refers to genetic material that is produced by splicing genes, gene derivatives or other genetic material. As used herein, “recombinant” also refers to the products produced from recombinant genes (e.g. recombinant protein).
  • Spacer refers to any chemical entity placed between two chemical moieties that serves to physically separate the latter two moieties.
  • a chemical entity placed between a PAMP or PAMP mimetic and an antigen is a spacer.
  • spacers include, but are not limited to, nucleic acids (e.g. untranscribed DNA between two stretches of transcribed DNA), amino acids, carbohydrates (e.g., monosaccharides, disaccharides, trisaccharides), and lipids.
  • “Strong immune response” refers to an immune response, induced by the chimeric construct, that has about the same intensity or greater than the response induced by an antigen mixed with Complete Freund's Adjuvant (CFA).
  • “Therapeutically effective amount” refers to an amount of an agent (e.g., a vaccine) that can produce a measurable positive effect in a patient.
  • TLR Toll-like receptor
  • TLRs refers to any of a family of receptor proteins that are homologous to the Drosophila melanogaster Toll protein. TLRs also refer to type I transmembrane signaling receptor proteins that are characterized by an extracellular leucine-rich repeat domain and an intracellular domain homologous to that ofthe interleukin 1 receptor.
  • the TLR family includes, but is not limited to, mouse TLR2 and TLR4 and their homologues, particularly in other species including humans.
  • This invention also defines Toll receptor proteins and TLRs wherein the nucleic acids encoding such proteins have at least about 70% sequence identity, more preferably, at least about 80% sequence identity, even more preferably, at least about 85% sequence identity, yet more preferably at least about 90% sequence identity, and most preferably at least about 95% sequence identity to the nucleic acid sequence encoding the Toll protein and the TLR proteins TLR2, TLR4 and other members ofthe TLR family.
  • this invention also contemplates Toll receptors and TLRs wherein the amino acid sequences of such Toll receptors and TLRs have at least about 70% sequence identity, more preferably, at least about 80% sequence identity, even more preferably, at least about 85% sequence identity, yet more preferably at least about 90% sequence identity, and most preferably at least about 95%> sequence identity to the amino acid sequences of the Toll protein and the TLRs, TLR2, TLR4 and their homologues.
  • Tumor refers to a mass of proliferating cells lacking, to varying degrees, normal growth control. As used herein, “tumors” include, at one extreme, slowly proliferating “benign” tumors, to, at the other extreme, rapidly proliferating “malignant” tumors that aggressively invade neighboring tissues.
  • Vaccine refers to a composition comprising an antigen, and optionally other ancillary molecules, the purpose of which is to administer such compositions to a subject to stimulate an immune response specifically against the antigen and preferably to engender immunological memory that leads to mounting of an immune response should the subject encounter that antigen at some future time.
  • ancillary molecules are adjuvants, which are non-specific immunostimulatory molecules, and other molecules that improve the pharmacokinetic and/or pharmacodynamic properties ofthe antigen.
  • a vaccine usually consists of the organism that causes a disease (suitably attenuated or killed) or some part ofthe pathogenic organism as the antigen.
  • Attenuated organisms such as attenuated viruses or attenuated bacteria, are manipulated so that they lose some or all of their ability to grow in their natural host.
  • biotechnological approaches used to producing vaccines.
  • the various methods include, but are not limited to, the following:
  • Viral vaccines consisting of genetically altered viruses.
  • the viruses can be engineered so that they are harmless but can still replicate
  • Another approach is to clone the gene for a protein from a pathogenic virus into another, harmless virus, so that that resulting, engineered virus has certain immunologic properties ofthe pathogenic virus but does not cause any disease.
  • Examples ofthe latter method include, but are not limited to, altered vaccinia and adenoviruses used as rabies vaccines for distribution with meat bait, and a vaccinia virus engineered to produce haemagglutinin and fusion proteins of rindepest virus of cattle;
  • E. coli vaccine for pigs, bacterial vaccine for furunculosis in salmon.
  • Biopharmaceutical vaccines consist of proteins, or portions of proteins, that are the same as the proteins in a viral, fungal or bacterial coat or wall, which can be made by recombinant DNA methods;
  • MAPs Multiple antigen peptides
  • Polyprotein vaccines consist of a single protein made by genetic engineering so that the different peptides from the organisms of interest form part of a continuous polypeptide chain
  • Vaccines produced in transgenic plants that can be used as food to provide oral vaccines (e.g., vaccine delivery by eating bananas).
  • the present invention is based in part on the unexpected discovery that vaccines comprising chimeric constructs of a PAMP and an antigen (e.g., the fusion protein BLP/E ⁇ ) exhibit the essential immunological characteristics or properties expected of a conventional vaccine supplemented with an adjuvant.
  • the present invention is based on the finding that BLP/E ⁇ induces activation of NF- ⁇ B and production of IL-6 in macrophages and dendritic cells, respectively, demonstrating that the vaccine is capable of activating the innate immune system.
  • the activity of BLP/E ⁇ is comparable to that of LPS, and is not due to endotoxin contamination, as demonstrated by the lack of inhibition by polymyxin B.
  • the present invention is based on the finding that the BLP/E ⁇ fusion protein induces maturation of dendritic cells, as demonstrated by the induction ofthe cell surface expression ofthe co-stimulatory molecule, B7.2. Additionally, BLP/E ⁇ is appropriately targeted to the antigen processing and presentation pathway, and a functional E ⁇ peptide/MHC class-II complex is generated. This result is demonstrated by FACS analysis using an antibody specific for the E ⁇ peptide complexed with MHC class-II.
  • the present invention is based on the surprising discovery that a recombinant vaccine comprising a BLP/E ⁇ chimeric construct activates antigen- specific T-cell responses in vitro by stimulating dendritic cell activation and generating a specific ligand (E ⁇ /MHC-II) for the T-cell receptor.
  • E ⁇ /MHC-II specific ligand for the T-cell receptor.
  • the results of immunization of mice with BLP/E ⁇ and the resultant antigen-specific T- cell responses demonstrate that the recombinant vaccine activates antigen-specific T-cell responses in vivo.
  • mice were immunized with E ⁇ peptide mixed with Complete Freund's Adjuvant (CFA).
  • CFA Complete Freund's Adjuvant
  • the recombinant vaccine ofthe present invention induced an immune response in the mice that is stronger than that produced by E ⁇ peptide mixed with CFA.
  • the present invention is also based on the surprising discovery that immunization with the recombinant vaccines that comprise the chimeric constructs ofthe present invention induce a minimal inflammatory reaction when compared to that induced by an adjuvant.
  • the vaccine unexpectedly induced a strong immune response.
  • the vaccine approach described in the present invention minimizes an undesired side effect (e.g., an excessive inflammatory reaction) yet induces a very potent and specific immune response.
  • the present invention also provides fusion proteins comprising at least one antigen molecule or antigen domain and at least one PAMP or PAMP mimetic for use as vaccines.
  • the fusion protein has maximal immunogenicity and induces only a modest inflammatory response.
  • a fusion system for the production of recombinant polypeptides.
  • heterologous proteins and peptides are often degraded by host proteases; this may be avoided, especially for small peptides, by using a gene fusion expression system.
  • general and efficient purification schemes are established for several fusion partners. The use of a fusion partner as an affinity handle allows rapid isolation ofthe recombinant peptide.
  • the recombinant product may be localized to different compartments or it might be secreted; such strategy could lead to facilitation of purification ofthe fusion partner and/or directed compartmentalization ofthe fusion protein.
  • the present invention also contemplates modified fusion proteins having affinity for metal (metal ion) affinity matrices, whereby one or more specific metal- binding or metal-chelating amino acid residues are introduced, by addition, deletion, or substitution, into the fusion protein sequence as a tag.
  • the fusion partner e g., the antigen or PAMP sequence
  • the antigen or PAMP could also be altered to provide a metal-binding site if such modifications could be achieved without adversely effecting a ligand-binding site, an active site, or other functional sites, and/or destroying important tertiary structural relationships in the protein
  • These metal-binding or metal-chelating residues may be identical or different, and can be selected from the group consisting of cysteine, histidine, aspartate, tyrosine, tryptophan, lysine, and glutamate, and are located so to permit binding or chelation ofthe expressed fusion protein to a metal.
  • Histidine is the preferred metal-binding residue.
  • the metal-binding/chelating residues are situated with reference to the overall tertiary structure ofthe fusion protein to maximize binding/chelation to the metal and to minimize interference with the expression ofthe fusion protein or with the protein's biological activity.
  • a fusion sequence of an antigen, PAMP and a tag may optionally contain a linker peptide.
  • the linker peptide might separate a tag from the antigen sequence or the PAMP sequence. If the linker peptide so used encodes a sequence that is selectively cleavable or digestible by conventional chemical or enzymatic methods, then the tag can be separated from the rest of the fusion protein after purification.
  • the selected cleavage site within the tag may be an enzymatic cleavage site.
  • suitable enzymatic cleavage sites include sites for cleavage by a proteolytic enzyme, such as enterokinase, Factor Xa, trypsin, collagenase, and thrombin.
  • the cleavage site in the linker may be a site capable of cleavage upon exposure to a selected chemical (e.g., cyanogen bromide, hydroxyl amine, or low pH),
  • Cleavage at the selected cleavage site enables separation of the tag from the antigen PAMP fusion protein.
  • the antigen/PAMP fusion protein may then be obtained in purified form, free from any peptide fragment to which it was previously linked for ease of expression or purification,
  • the cleavage site if inserted into a linker useful in the fusion sequences of this invention, does not limit this invention. Any desired cleavage site, of which many are known in the art, may be used for this purpose.
  • the optional linker peptide in a fusion protein ofthe present invention might serve a purpose other than the provision of a cleavage site.
  • the linker peptide might be inserted between the PAMP and the antigen to prevent or alleviate steric hindrance between the two domains.
  • the linker sequence might provide for post-translational modification including, but not limited to, e.g., phosphorylation sites, biotinylation sites, sulfation sites, carboxylation sites, lipidation sites, glycosylation sites and the like.
  • the fusion protein of this invention contains an antigen sequence fused directly at its amino or carboxyl terminal end to the sequence of a PAMP.
  • the fusion protein of this invention comprising an antigen and a PAMP sequence, is fused directly at its amino or carboxyl terminal end to the sequence of a tag.
  • the resulting fusion protein is a soluble cytoplasmic fusion protein.
  • the fusion sequence further comprises a linker sequence interposed between the antigen sequence and a PAMP sequence or sequence of a tag. This fusion protein is also produced as a soluble cytoplasmic protein.
  • an "antigen” is any substance that induces a state of sensitivity and/or immune responsiveness after any latent period (normally, days to weeks in humans) and that reacts in a demonstrable way with antibodies and/or immune cells ofthe sensitized subject in vivo or in vitro.
  • antigens include, but are not limited to, (1) microbial-related antigens, especially antigens of pathogens such as viruses, fungi or bacteria, or immunogenic molecules derived from them; (2) "self antigens, collectively comprising cellular antigens including cells containing normal transplantation antigens and/or tumor-related antigens, RR- Rh antigens and antigens characteristic of, or specific to particular cells or tissues or body fluids; (3) allergen-related antigens such as those associated with environmental allergens (e.g., grasses, pollens, molds, dust, insects and dander), occupational allergens (e.g., latex, dander, urethanes, epoxy resins), food (e.g., shellfish, peanuts, eggs, milk products), drugs (e.g., antibiotics, anesthetics) and (4) vaccines (e.g., flu vaccine).
  • microbial-related antigens especially antigens of pathogens such as viruses, fungi or bacteria
  • the antigen portion used in the vaccines ofthe present invention can contain epitopes or specific domains of interest rather than the entire sequence.
  • the antigenic portions ofthe vaccines ofthe present invention can comprise one or more immunogenic portions or derivatives of the antigen rather than the entire antigen.
  • the vaccine ofthe present invention can contain an entire antigen with intact three-dimensional structure or a portion ofthe antigen that maintains a three-dimensional structure of an antigenic determinant, in order to produce an antibody response by B-lymphocytes against a spatial epitope ofthe antigen.
  • P atho en-Related Anti genes include, but are not limited to, antigens selected from the group consisting of vaccinia, avipox virus, turkey influenza virus, bovine leukemia virus, feline leukemia virus, avian influenza, chicken pneumovirosis virus, canine parvovirus, equine influenza, FHV, Newcastle Disease Virus (NDV),
  • tetani mumps, Morbillivirus, Herpes Simplex Virus type 1, Herpes Simplex Virus type 2, Human cytomegalo virus, Hepatitis A Virus, Hepatitis B Virus, Hepatitis C Virus, Hepatitis E Virus, Respiratory Syncytial Virus, Human Papilloma Virus, Influenza Virus, Salmonella, Neisseria, Borrelia, Chlamydia, Bordetella, and Plasmodium and Toxoplasma, Cryptococcus, Streptococcus, Staphylococcus, Haemophilus, Diptheria, Tetanus, Pertussis, Escherichia, Candida, Aspergillus, Entamoeba, Giardia, and Trypanasoma.
  • Cancer-Related Antigens The methods and compositions ofthe present invention can also be used to produce vaccines directed against tumor-associated protein antigens such as melanoma-associated antigens, mammary cancer-associated antigens, colorectal cancer-associated antigens, prostate cancer-associated antigens and the like.
  • tumor-associated protein antigens such as melanoma-associated antigens, mammary cancer-associated antigens, colorectal cancer-associated antigens, prostate cancer-associated antigens and the like.
  • tumor-related or tissue-specific protein antigens useful in such vaccines include, but are not limited to, antigens selected from the group in the following table.
  • the methods and compositions ofthe present invention can also be used to produce vaccines directed against tumor vascularization.
  • target antigens for such vaccines are vascular endothelial growth factors, vascular endothelial growth factor receptors, fibroblast growth factors and fibroblast growth factor receptors and the like. 3. Allergen-Related Antigens.
  • the methods and compositions ofthe present invention can be used to prevent or treat allergies and asthma.
  • one or more protein allergens can be linked to one or more PAMPs, producing a PAMP/allergen chimeric construct, and administered to subjects that are allergic to that antigen.
  • the methods and compositions ofthe present invention can also be used to construct vaccines that may suppress allergic reactions.
  • the allergen is associated with or combined with a PAMP, including but not limited to BLP, Flagellin or FimC, that can initiate a Thi response upon binding to a TLR.
  • Initiation of innate immunity via a TLR tends to be characterized by production and secretion of cytokines, such as IL-12, that elicit a so-called Thi response in a subject, rather than the typical Th2 response that triggers B-cells to produce immunoglobulin E, the initiator of typical allergic and/or hypersensitive responses.
  • cytokines such as IL-12
  • Thi response in a subject
  • Th2 response the typical Th2 response that triggers B-cells to produce immunoglobulin E
  • IL-12 produced by dendritic cells and macrophages upon PAMP binding to their TLRs will direct T-cell differentiation into Thi effector cells.
  • Cytokines produced by Thi cells such as hiterferon-gamma, will block the differentiation of IL-4 producing Th2 cells and would thus prevent production of antibodies ofthe IgE isotype, which are responsible for allergic responses.
  • allergen-related protein antigens useful in the methods and compositions ofthe present invention include, but are not limited to: allergens derived from pollen, such as those derived from trees such as Japanese cedar (Cryptomeria, Cryptomeriajaponica), grasses (Gramineao), such as orchard-grass (Dactylis, Dactylis glomerata), weeds such as ragweed (Ambrosia, Ambrosia artemisiifolia); specific examples of pollen allergens including the Japanese cedar pollen allergens Cryj 1 (J Allergy Clin. Immunol.
  • allergens derived from fungi Aspergillus, Candida, Alternaria, etc.
  • allergens derived from mites allergens from Dermatophagoides pteronyssinus, Dermatophagoides farinae etc.
  • specific examples of mite allergens including Der p I, Der p II, Der p III, Der p VII, Der f I, Der f II, Der fill, Der f VII etc.
  • house dust allergens derived from animal skin debris, feces and hair (for example, the feline allergen Fel d I); allergens derived from insects (such as scaly hair or scale of moths, butterflies, Chironomidae etc.
  • prophylactic treatment of chronic allergies can be accomplished by the administration of a protein PAMP.
  • the PAMP ofthe prophylactic vaccine is an OMP, more preferably OspA, and most preferably BLP.
  • Flagellin or FimC can be used as the PAMP.
  • amyloid- ⁇ peptide is associated with the pathogenesis of Alzheimer's disease. (Janus et al, Nature (2000) 408: 979-982; Morgan et al, Nature (2000) 408: 982- 985).
  • the chimeric construct used in the vaccines ofthe present invention can include amyloid- ⁇ peptide, or antigenic domains of amyloid- ⁇ peptide, as the antigenic portion ofthe construct, and a PAMP or PAMP mimetic. Examples of other diseases in which vaccines might be generated against self proteins or self peptides are shown in the following table.
  • PAMPs are discrete molecular structures that are shared by a large group of microorganisms, They are conserved products of microbial metabolism, which are not subject to antigenic variability and are distinct from self-antigens. (Medzhitov et al. (1991) Current Opinion in Immunology 9: 4).
  • PAMPs can be composed of or found in, but are not limited to, the following types of molecules: Flagellins, chaperones including periplasmic chaperones such as FimC, lipopolysaccharides (LPS), porins, lipid A-associated proteins (LAP), lipopolysaccharides, fimbrial proteins, unmethylated CpG motifs, bacterial DNAs, double-stranded viral RNAs, mannans, cell wall-associated proteins, heat shock proteins, glycoproteins, lipids, cell surface polysaccharides, glycans (e.g., peptidoglycans), phosphatidyl cholines, teichoic acids (e.g., lipoteichoic acids), mycobacterial cell wall components/membranes, bacterial lipoproteins (BLP), outer membrane proteins (OMP), and outer surface protein A (Osp A).
  • periplasmic chaperones such as FimC, lipopolys
  • the preferred PAMPs ofthe present invention include those that contain a DNA-encoded protein component, such as BLP, Neisseria porin, OMP, Flagellin, FimC and OspA, as these can be used as fusion partners to prepare the preferred embodiment ofthe invention, i.e., fusion proteins comprising a PAMP and an antigen, preferably a self-antigen.
  • BLP DNA-encoded protein component
  • OMP Neisseria porin
  • Flagellin fusion proteins comprising a PAMP and an antigen, preferably a self-antigen.
  • One preferable PAMP for use in the present invention is BLP because BLP is known to induce activation ofthe innate immune response (Henderson et al. (1996) Microbiol.
  • the present invention contemplates derivatives, portions, parts, or peptides of PAMPs that are recognized by the innate immune system for generating vaccines.
  • fragments of PAMPs fragments of PAMPs
  • portions of PAMPs parts of PAMPs
  • peptides of PAMPs all refer to an immunostimulatory part of an entire PAMP molecule.
  • the PAMPs used in the vaccines ofthe present invention can comprise an immunostimulatory portion or derivative ofthe PAMP rather than the entire PAMP, for example E. Coli murein lipoprotein amino acids 1 to 24.
  • the present invention contemplates peptide mimetics of non-protein PAMPs.
  • Peptide mimetics of polysaccharides and peptidoglycans are examples of peptide mimetics which can be used in the present invention.
  • the present invention contemplates using phage selection methods to identify peptide mimetics of these non-protein PAMPs.
  • an antibody raised against a non-protein PAMP can be used to screen a phage library containing randomized short-peptide sequences. Selected sequences are isolated and assayed to determine their usefulness as a protein derivative of a non-protein PAMP in the chimeric constructs ofthe present invention.
  • Such peptide mimetics are useful to produce the recombinant vaccines disclosed herein.
  • the present invention contemplates further examples of PAMP mimics or PAMP mimetics in which analogs of amino acids and/or peptides are substituted for the amino acid and/or peptide residues, respectively, of a peptide-containing PAMP or a protein PAMP.
  • the chimeric construct is a construct comprising
  • CpG or CpG-DNA and an antigen.
  • the CpG or CpG-DNA can be conjugated to a protein or non-protein antigen.
  • peptide mimetics of CpG or CpG-DNA that mimic the structural, functional, antigenic or immunogenic properties of CpG, can be produced and used to generate an antigen-PAMP (where PAMP is a CpG peptide mimetic) protein chimeric construct.
  • This chimeric construct can be produced by recombinant DNA techniques and the expressed fusion protein can be used in the compositions and methods ofthe present invention.
  • D. Peptide Mimetics This invention also includes a mimetic ofthe three-dimensional structure of a PAMP or antigen.
  • this invention also includes peptides that closely resemble the three-dimensional structure of non-peptide PAMPs and antigens.
  • Such peptides provide alternatives to non-polypeptide PAMPs or antigens, respectively, by providing the advantages of, for example: more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, and/or altered specificity (e.g., a broad-spectrum of biological activities), and other advantages.
  • analogs of PAMP and/or antigen proteins can be synthesized such that one or both consists partially or entirely of amino acid and /or peptide analogs.
  • Such analogs can contain non-naturally-occurring amino acids, or naturally-occurring amino acids that do not commonly occur in proteins, including but not limited to, D-amino acids or amino acids such as ⁇ -alanine, ornithine or canavanine, and the like, many of which are known in the art.
  • analogs of PAMPs and/or antigens can be synthesized such that one or both consists partially or entirely of peptide analogs containing non-peptide bonds, many examples of which are known in the art.
  • Such analogs may provide greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.) and or altered specificity (e.g., a broad-spectrum of biological activities) when compared with the naturally-occurring PAMP and/or antigen as well as other advantages.
  • the contemplated molecular structures are peptide-containing molecules that mimic elements of protein secondary structure, (see, for example, Johnson et al. (1993) Peptide Turn Mimetics, in Biotechnology and Pharmacy,
  • analogs of peptides are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of a subject peptide.
  • non-peptide compounds are also referred to as "peptide mimetics” or “peptidomimetics” (Fauchere (1986) Adv. Drug Res.15, 29-69; Veber et al. (1985) Trends Neurosci. 8: 392-396; Evans et al. (1987) J. Med. Chem. 30: 1229-1239) and are usually developed with the aid of computerized molecular modeling.
  • Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent therapeutic or prophylactic effect.
  • Labeling of peptide mimetics usually involves covalent attachment of one or more labels, directly or through a spacer (e.g., an amide group), to non-interfering position(s) on the peptide mimetic that are predicted by quantitative structure- activity data and molecular modeling.
  • Such non-interfering positions generally are positions that do not form direct contacts with the macromolecule(s) (e.g., in the present example they are not contact points in PAMP -PRR complexes) to which the peptide mimetic binds to produce the therapeutic effect.
  • Derivitization (e.g., labeling) of peptide mimetics should not substantially interfere with the desired biological or pharmacological activity ofthe peptide mimetic.
  • PAMP peptide mimetics can be constructed by structure-based drug design through replacement of amino acids by organic moieties. (Hughes (1980) Philos. Trans. R. Soc. Lond. 290: 387-394; Hodgson (1991) Biotechnol. 9: 19-21; Suckling
  • peptide mimetics can be aided by identifying amino acid mutations that increase or decrease binding of PAMP to its PRR.
  • Approaches that can be used include the yeast two-hybrid method (Chien et al. (1991) Proc. Natl. Acad. Sci. USA 88: 9578-9582) and using the phage display method.
  • the two- hybrid method detects protein-protein interactions in yeast. (Fields et al. (1989) Nature 340: 245-246).
  • the phage display method detects the interaction between an immobilized protein and a protein that is expressed on the surface of phages such as lambda and M13. (Amberg et al. (1993) Strategies 6: 2-4; Hogrefe et al. (1993) Gene 128: 119-126). These methods allow positive and negative selection for protein-protein interactions and the identification ofthe sequences that determine these interactions.
  • Flagellin Bacterial flagella are made up ofthe structural protein Flagellin, which induces expression of chemokine IL-8 and activation of NF- ⁇ B in human and mouse cells. Additionally Flagellin activates mammalian cells via a Toll-Like Receptor, TLR5. These findings, as well as the fact that Flagellin proteins are extremely conserved in bacteria, indicate that Flagellin is a pathogen-associated molecular pattern (PAMP) that would be recognized by the innate immune system. Because Flagellin is a protein and a PAMP, it is also suitable for the generation of recombinant fusion vaccines. As described in the Examples section below, a series of fusion constructs were tested for their ability to activate the mammalian innate immune system.
  • PAMP pathogen-associated molecular pattern
  • NF- ⁇ B Activation of NF- ⁇ B was used as a read-out in the experiments because it is a critical pathway indicative ofthe triggering ofthe Toll-Like Receptors, and has been demonstrated to be a property ofthe recombinant fusion vaccines.
  • Type I pili are made up of several proteins encoded by the fim genes. The assembly of Type I pili and their extrusion through the outer bacterial membrane is highly regulated and requires the presence of a periplasmic protein, FimC.
  • FimC is a conserved periplasmic chaperone ofthe PapD superfamily. It consists of two immunoglobulin-like domains and participates in the folding and assembly of each pilus subunit in a process called donor strand complementation, which prevents pilus protein aggregation in the periplasm. Because FimC is conserved and absolutely required for pilus assembly, we cloned and expressed it to see if it could induce an innate immune response and potentially be used as an adjuvant for a vaccine.
  • FimC activated NF- ⁇ B in mouse cell lines which express the innate immune system receptors TLR2 and TLR4, suggesting that FimC is a ligand for one of these, or perhaps another Toll-like receptor. Ligation of TLRs is necessary for the induction of an immune response; therefore, FimC is likely to be a good candidate for a vaccine adjuvant. Because FimC is a single gene product, it can be used as a fusion protein with an antigen of interest for the generation of recombinant vaccines.
  • the present invention also contemplates conservative variants of naturally- occurring protein PAMPs, peptides of PAMPs, and peptide mimetics of PAMPs that recognize the corresponding PRRs. Such variants are examples of PAMP mimetics.
  • the conservative variations include mutations that substitute one amino acid for another within one ofthe following groups:
  • Aromatic residues Phe, Tyr and Trp.
  • the types of substitutions selected may be based on the analysis ofthe frequencies of amino acid substitutions among the PAMPs of different species (Schulz et al. Principles of Protein Structure, Springer-Verlag, 1978, pp. 14-16) on the analyses of structure- forming potentials developed by Chou and Fasman (Chou et al. (1974) Biochemistry 13: 211; Schulz et al. (1978) Principles in Protein Structure, Springer- Verlag, pp. 108-130), and on the analysis of hydrophobicity patterns in proteins developed by Kyte and Doolittle (Kyte et al. (1982) J Mol. Biol. 157: 105-132).
  • the present invention also contemplates conservative variants that do not affect the ability ofthe PAMP to bind to its PRR.
  • the present invention includes PAMPs with altered overall charge, structure, hydrophobicity/hydrophilicity properties produced by amino acid substitution, insertion, or deletion that retain and/or improve the ability to bind to their receptor.
  • the mutated PAMP has at least about 70% sequence identity, more preferably at least about 80% sequence identity, even more preferably, at least about 85% sequence identity, yet more preferably at least about 90% sequence identity, and most preferably at least about 95% sequence identity to its corresponding wild-type PAMP.
  • the present invention provides methods of treating subjects comprising passively immunizing an individual by administering antibodies or activated immune cells to a subject to confer immunity, and administering a vaccine comprising a fusion protein ofthe present invention, preferably wherein the administered antibody or activated immune cells are directed against the same antigen ofthe fusion protein ofthe vaccine.
  • Such treatments can be sequential, in either order or simultaneous.
  • This combination therapy contemplates the use of either monoclonal or polyclonal antibodies that are directed against the antigen of the PAMP/antigen fusion.
  • the present invention provides methods of treating subjects comprising passively immunizing an individual by administering antibodies or activated immune cells to a subject to confer immunity, and administering a vaccine comprising a chimeric construct ofthe present invention, wherein the administered antibody or activated immune cells are preferably directed against the same antigen ofthe chimeric construct.
  • Such treatments can be sequential, in either order, or simultaneous.
  • This combination therapy contemplates the use of either monoclonal or polyclonal antibodies that are directed against the antigen ofthe PAMP/antigen chimeric construct.
  • the present invention also contemplates the use of a vaccine comprising a chimeric construct ofthe present invention in combination with a second treatment where such second treatment is not an immune-directed therapy.
  • a non-limiting example of such combination therapy is the combination of a vaccine comprising a fusion protein ofthe present invention in combination with a chemotherapeutic agent, such as an anti-cancer chemotherapeutic agent.
  • a further non-limiting example of such combination therapy is the combination of a vaccine comprising a fusion protein construct ofthe present invention in combination with an anti- angiogenic agent.
  • a further non-limiting example of such combination therapy is the combination of a vaccine comprising a fusion protein ofthe present invention in combination with radiation therapy, such as an anti-cancer radiation therapy.
  • Yet a further non-limiting example of combination therapy is the combination of a vaccine comprising a fusion protein ofthe present invention in combination with surgery, such as surgery to remove or reduce vascular blockage.
  • a combination of more than one other therapeutic with a vaccine contemplated in this invention is a combination of more than one other therapeutic with a vaccine contemplated in this invention.
  • a non-limiting example is a combination of a vaccine contemplated in this invention in combination with passive immunotherapy treatment and chemotherapy treatment.
  • treatments can be sequential or simultaneous.
  • the PAMP domain can comprise the entire PAMP or an ii nunostimulatory portion ofthe PAMP.
  • the fusion protein has maximal immunogenicity and induces minimal inflammatory response.
  • Such desirable properties might be achieved, for example, by using two or more different antigens, and/or portions of different antigens, and/or by using more than one copy ofthe same antigen or portions ofthe same antigen, and/or by a combination of both.
  • two or more different PAMPs, or portions of different PAMPs, and/or two or more copies ofthe same PAMP, or portions ofthe same PAMP, and/or a combination of both can be used.
  • a further embodiment contemplates fusion proteins containing multiple antigens, and/or portions of antigens, together with multiple PAMPs, and/or portions of PAMPs. It is within the skill ofthe artisan to determine the desirable ratio of PAMP to antigen domains to maximize immunogenicity and minimize inflammatory response.
  • a fusion system for the production of recombinant polypeptides.
  • heterologous proteins and peptides are often degraded by host proteases; this may be avoided, especially for small peptides, by using a gene fusion expression system.
  • general and efficient purification schemes are established for several fusion partners. The use of a fusion partner as an affinity handle allows rapid isolation and purification ofthe recombinant peptide.
  • the recombinant product may be localized to different compartments or it might be secreted; such strategy could lead to facilitation of purification ofthe fusion partner and/or directed compartmentalization ofthe fusion protein.
  • fusion proteins include: the Staphylococcal protein A fusion system and the synthetic ZZ variant, both of which have IgG affinity and have been used for the generation of antibodies against short peptides; the glutathione S-transferase fusion system (Smith et al. (1988) Gene 60); the ⁇ -galactosidase fusion system; and the trpE fusion system (Yansura (1990) Methods Enzym. 185: 61).
  • the present invention also contemplates modified fusion proteins having affinity for metal ion affinity matrices, whereby one or more specific metal-binding or metal-chelating amino acid residues are introduced, by addition, deletion, or substitution, into the fusion protein sequence as a tag.
  • a fusion partner either an antigen or a PAMP domain, is modified to contain an added metal- chelating amino acid tag.
  • metal-binding or metal-chelating residues may be identical or different, and can be selected from the group consisting of cysteine, histidine, aspartate, tyrosine, tryptophan, lysine, and glutamate, and are located so to permit binding or chelation ofthe expressed fusion protein to a metal. Histidine is the preferred metal-binding residue.
  • the metal-binding/chelating residues are situated with reference to the overall tertiary structure ofthe fusion protein to maximize binding/chelation to the metal and to minimize interference with the expression of the fusion protein its biological activity.
  • a fusion sequence of an antigen, PAMP and a tag may optionally contain a linker peptide.
  • the linker peptide might separate a tag from the antigen sequence or the PAMP sequence. If the linker peptide so used encodes a sequence that is selectively cleavable or digestible by conventional chemical or enzymatic methods, then the tag can be separated from the rest ofthe fusion protein after purification.
  • the selected cleavage site within the tag may be an enzymatic cleavage site.
  • suitable enzymatic cleavage sites include sites for cleavage by a proteolytic enzyme, such as enterokinase, Factor Xa, trypsin, collagenase, thrombin and the like.
  • the cleavage site in the linker may be a site capable of cleavage upon exposure to a selected chemical or condition, e.g., cyanogen bromide, hydroxylamine, or low pH, or other chemicals or conditions known in the art.
  • a selected chemical or condition e.g., cyanogen bromide, hydroxylamine, or low pH, or other chemicals or conditions known in the art.
  • Cleavage at the selected cleavage site enables separation ofthe tag from the antigen PAMP fusion protein.
  • the antigen/PAMP fusion protein may then be obtained in purified form, free from any peptide derivative to which it was previously linked for ease of expression or purification.
  • the cleavage site if inserted into a linker useful in the fusion sequences of this invention, does not limit this invention. Any desired cleavage site, of which many are known in the art, may be used for this purpose.
  • linker peptides might be to direct cleavage ofthe antigen in intracellular processing so as to facilitate peptide presentation on the surface of the APC.
  • Appropriate cleavage sites might be inserted via linkers such that the fusion protein is not cleaved until it is internalized by the APC. Under such circumstances, such a peptide cleavage site can be introduced via a linker between the PAMP and the antigen to generate intracellular antigen free of PAMP.
  • Such directed cleavage could also be used particularly to facilitate production within the APC of specific peptides that have been identified as interacting with particular HLA haplotypes.
  • different domains from a single antigen or from more than one antigen might be separated by linkers containing cleavage sites so that these epitopes could be appropriately processed for presentation on the surface ofthe APC.
  • the optional linker peptide in a fusion protein ofthe present invention might serve a purpose other than the provision of a cleavage site.
  • the linker peptide might be inserted between a PAMP domain and an antigen domain to prevent or alleviate steric hindrance between the two domains.
  • the linker sequence might provide for post-translational modification including, but not limited to, e.g., phosphorylation sites, biotinylation sites, sulfation sites, carboxylation sites, glycosylation sites, lipidation sites, and the like.
  • the fusion protein of this invention contains a domain of an antigen or an immunogenic portion of an antigen fused directly at its amino or carboxyl terminal end to the domain of a PAMP or an immunostimulatory portion of a PAMP.
  • the fusion protein of this invention contains a domain of a PAMP, or an immunostimulatory portion of a PAMP, or a sequence that can be post-translationally modified to produce a PAMP, inserted within the domain of an antigen, or an immunogenic portion of an antigen, h yet another embodiment, the fusion protein of this invention contains a domain of an antigen, or an immunogenic portion of an antigen, inserted within the domain of a PAMP, or an immunostimulatory portion of a PAMP, or a sequence that can be post- translationally modified to produce a PAMP.
  • the fusion protein of this invention comprising an antigen and a PAMP sequence, is fused directly at its amino or carboxyl terminal end to the sequence of a tag.
  • the resulting fusion protein is a soluble cytoplasmic fusion protein.
  • the fusion sequence further comprises a linker sequence interposed between the antigen sequence and a PAMP sequence or sequence of a tag. This fusion protein is also produced as a soluble cytoplasmic protein.
  • Protein PAMPs, protein antigens, and derivatives thereof can be generated using standard peptide synthesis technology. Alternatively, recombinant methods can be used to generate nucleic acid molecules that encode protein PAMPs, protein antigens and derivatives thereof.
  • Nucleic acids encoding PAMP/antigen fusions can easily be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci, et al. ((1981) J. Am. Chem. Soc. 103: 3185- 3191) or using automated synthesis methods.
  • larger nucleic acids can readily be prepared by well known methods, such as synthesis of a group of oligonucleotides that define various modular segments ofthe nucleic acid encoding the PAMP/antigen fusion, followed by ligation of oligonucleotides to build the complete nucleic acid molecule.
  • the present invention further provides recombinant nucleic acid molecules that encode PAMP/antigen fusion proteins.
  • a "recombinant nucleic acid molecule” refers to a nucleic acid molecule that has been subjected to molecular manipulation in vitro. Methods for generating recombinant nucleic acid molecules are well lcnown in the art. (Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press).
  • a nucleotide sequence that encodes a PAMP/antigen fusion is operably linked to one or more expression control sequences and/or vector sequences.
  • a vector contemplated by the present invention is at least capable of directing the replication or insertion into the host chromosome, and preferably also expression, of a nucleotide sequence encoding a PAMP/antigen fusion.
  • Expression control elements that are used for regulating the expression of an operably linked protein encoding sequence are known in the art and include, but are not limited to, inducible promoters, constitutive promoters, secretion signals, enhancers, transcription terminators and other regulatory elements.
  • inducible promoters that is readily controlled, such as being responsive to a nutrient in the medium, is used.
  • the vector containing a nucleic acid molecule encoding a PAMP/antigen fusion will include a prokaryotic rephcon, e.g., a nucleotide sequence having the ability to direct autonomous replication and maintenance ofthe recombinant nucleic acid molecule intrachromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith.
  • a prokaryotic rephcon e.g., a nucleotide sequence having the ability to direct autonomous replication and maintenance ofthe recombinant nucleic acid molecule intrachromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith.
  • a prokaryotic rephcon e.g., a nucleotide sequence having the ability to direct autonomous replication and maintenance ofthe recombinant nucleic acid molecule intrachromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith.
  • Typical bacterial drug resistance genes are those that confer resistance to ampicillin (Amp) or tetracycline (Tet).
  • Vectors that include a prokaryotic rephcon can further include a prokaryotic or viral promoter capable of directing the expression (transcription and translation) ofthe PAMP/antigen fusion in a bacterial host cell, such as E. coli.
  • a promoter is an expression control element formed by a nucleic acid sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a nucleic acid segment ofthe present invention.
  • Typical of such vector plasmids are pUC8, pUC9, pBR322 and pBR329 available from Biorad Laboratories (Richmond, CA), pPL and pKK-223 available from Amersham Pharmacia Biotech, Piscataway, NJ.
  • Expression vectors compatible with eukaryotic cells can also be used to express nucleic acid molecules that contain a nucleotide sequence that encodes a PAMP/antigen fusion.
  • Eukaryotic cell expression vectors are well known in the art and are available from several commercial sources. Typically, such vectors provide convenient restriction sites for insertion ofthe desired DNA segment. Typical of such vectors are pSVL and pKSV-10 (Amersham Pharmacia Biotech), pBPV-l/pML2d (International Biotechnologies, Inc.), pTDTl (ATCC, #31255), the vector pCDM8 described herein, and other like eukaryotic expression vectors.
  • Eukaryotic cell expression vectors used to construct the recombinant molecules ofthe present invention may further include a selectable marker that is effective in a eukaryotic cell, preferably a drug resistance selection marker.
  • a preferred drug resistance marker is the gene whose expression results in neomycin resistance, e.g. , the neomycin phosphotransferase (neo) gene. (Southern et al. (1982) J. Mol. Anal. Genet. 1:327-341).
  • the selectable marker can be present on a separate plasmid, and the two vectors are introduced by cotransfection ofthe host cell, and selected by culturing in the presence ofthe appropriate drug for the selectable marker.
  • the present invention further provides host cells transformed with a nucleic acid molecule that encodes a PAMP/antigen fusion protein ofthe present invention.
  • the host cell can be either prokaryotic or eukaryotic.
  • Eukaryotic cells useful for expression of a PAMP/antigen fusion protein are not limited, so long as the cell line is compatible with cell culture methods and compatible with the propagation ofthe expression vector and expression ofthe fusion protein.
  • Preferred eukaryotic host cells include, but are not limited to, yeast, insect and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human fibroblastic cell line. Any prokaryotic host can be used to express a recombinant nucleic acid molecule.
  • the preferred prokaryotic host is E. coli. In embodiments where the
  • PAMP is a lipoprotein
  • expression ofthe PAMP/antigen fusion protein in a bacterial cell is preferred.
  • Expression ofthe nucleic acid in a bacterial cell line is desirable to ensure proper post-translational modification of the protein portion of the lipoprotein.
  • the host cells selected for expression ofthe PAMP/antigen fusion e.g. lipoprotein/antigen fusion
  • the host cells selected for expression ofthe PAMP/antigen fusion is the cell that natively produces the lipoprotein ofthe lipoprotein/antigen fusion.
  • Transformation of appropriate cell hosts with nucleic acid molecules encoding a PAMP/antigen fusion ofthe present invention is accomplished by well known methods that typically depend on the type of vector and host system employed. With regard to transformation of prokaryotic host cells, electroporation and salt treatment methods are typically employed. (See e.g., Cohen et al. (1972) Pro.c Natl. Acad. Sci. USA 69:2110; Maniatis et /., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  • cells resulting from the introduction of a nucleic acid molecule encoding the PAMP/antigen fusions ofthe present invention can be cloned to produce single colonies.
  • Cells from those colonies can be harvested, lysed and their nucleic acids content examined for the presence ofthe recombinant molecule using a method such as that described by Southern (1975) (J. Mo!. Bio/. 98: 503), or Berent el al. ( 1985) (Biotech. 3: 208) or the proteins produced from the cell assayed via an immunological method.
  • the present invention further provides methods for producing a PAMP/antigen fusion protein that uses one ofthe nucleic acid molecules herein described.
  • the production of a recombinant protein typically involves the following steps. First, a nucleic acid molecule is obtained that encodes a PAMP/antigen fusion protein. Said nucleic acid molecule is then preferably placed in an operable linkage with suitable control sequences, as described above. The expression unit is used to transform a suitable host and the transformed host is cultured under conditions that allow the production ofthe PAMP/antigen fusion protein.
  • the fusion protein is isolated from the medium or from the cells; recovery and purification ofthe fusion protein may not be necessary in some instances where some impurities may be tolerated.
  • the desired coding sequences may be obtained from genomic fragments and used directly in an appropriate host.
  • the construction of expression vectors that are operable in a variety of hosts is accomplished using an appropriate combination of replicons and control sequences.
  • the control sequences, expression vectors, and transformation methods are dependent on the type of host cell used to express the gene and were discussed in detail earlier.
  • a skilled artisan can readily adapt any host/expression system lcnown in the art for use with the nucleotide sequences described herein to produce a PAMP/antigen fusion protein.
  • Endonucleases are nucleases that are able to break internal phosphodiester bonds within a nucleic acid molecule.
  • nucleases include, but are not limited to, SI endonuclease from the fungus Aspergillus oryzae, deoxyribonuclease (DNase I), and restriction endonucleases.
  • the cutting and joining processes that underlie DNA manipulation are carried out by enzymes called restriction endonucleases (for cutting) and ligases (for joining).
  • Suitable restriction endonuclease cleavage sites can, if not normally available, be added to the ends of the coding sequence so as to provide an excisable nucleic acid sequence to insert into these vectors.
  • restriction endonuclease cleavage sites may also be inserted in the nucleic acid sequence encoding the PAMP/antigen fusion protein.
  • these cleavage sites are engineered between nucleotide sequences encoding identical or different PAMPs; between identical or different antigens, or between nucleotide sequences encoding PAMP and antigen.
  • Appropriate cleavage sites well know to those skilled in the art include, but are not limited to, the following: EcoRI, BamHI, -3g//II, Pvul, Ev-.II, Hm-ffll, Hinf , Sau3A, AM, Taql, Hael ⁇ l and Notl. (T. A. Brown (1996) Gene Cloning: An Introduction, Second Edition, Chapman & Hall, Chapter 4:49-83).
  • vaccines ofthe present invention include PAMP/antigen conjugates such as, but not limited to, the following: protein/nucleic acid conjugates, nucleic acid/protein conjugates, nucleic acid/nucleic acid conjugates, peptide- mimetic/nucleic acid conjugates, nucleic acid/peptide mimetic conjugates, peptide mimetic/peptide mimetic conjugates, lipopolysaccharide/protein conjugates, lipoprotein/protein conjugates, R ⁇ A/protein conjugates, CpG-D ⁇ A/protein conjugates, nucleic acid analog/protein conjugates, and mannan protein conjugates.
  • PAMPs identified in the future are comprised of yet other chemical classes, conjugates containing such chemicals in combination with antigen domains can also be contemplated.
  • ⁇ on-protein PAMPs such as CpG or CpG-D ⁇ A, and lipopolysaccharides may be conjugated to protein or non-protein antigens by conventional techniques.
  • PAMP/antigen conjugates may be linked through polymers such as PEG, poly-D-lysine, polyvinyl alcohol, polyvinylpyrollidone, immunoglobulins, and copolymers of D-lysine and D-glutamic acid. Conjugation ofthe PAMP and antigen to the polymer linker may be achieved in any number of ways, typically involving one or more crosslinking agents and functional groups on the PAMP and antigen.
  • Polypeptide PAMPs and antigens will contain amino acid side chains such as amino, carbonyl, or sulfhydryl groups that will serve as sites for linking the PAMP and antigen to each other. Residues that have such functional groups may be added to either the PAMP or antigen. Such residues may be incorporated by solid phase synthesis techniques or recombinant techniques, both of which are well known in the peptide synthesis arts.
  • functional amino and sulfhydryl groups may be incorporated therein by conventional chemistry.
  • primary amino groups may be incorporated by reaction with ethylenediamine in the presence of sodium cyanoborohydride and sulfhydryls may be introduced by reaction of cysteamine dihydrochloride followed by reduction with a standard disulfide reducing agent, hi a similar fashion the polymer linker may also be derivatized to contain functional groups if it does not already possess appropriate functional groups.
  • Heterobifunctional crosslinlcers such as sulfosuccinimidyl(4- iodoacetyl) aminobenzoate, which link the .epsilon.
  • the vaccines ofthe present invention contain one or more PAMPs, immunostimulatory portions, or immunostimulatory derivatives thereof (e.g., a domain recognized by the innate immune system), and one or more antigens, immunogenic portions, or immunogenic derivatives thereof (e.g., a domain recognized by the adaptive immune system). Since a PAMP mimetic, by definition, has the ability to bind PRRs and initiate an innate immune response, vaccine formulations contemplated by this invention include PAMP mimetics in place of PAMPs. Thus, the present invention contemplates vaccines comprising chimeric constructs including at least one antigen domain and at least one PAMP domain. In one specific embodiment, the vaccines ofthe present invention comprise a BLP/E ⁇ fusion protein.
  • the vaccines comprising the chimeric constructs ofthe present invention, can be formulated according to lcnown methods for preparing pharmaceutically useful compositions, whereby the chimeric constructs are combined in a mixture with a pharmaceutically acceptable carrier.
  • a composition is said to be a
  • pharmaceutically acceptable carrier if its administration can be tolerated by the recipient and if that composition renders the active ingredient(s) accessible at the site where the action is required.
  • Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier,
  • suitable carriers are well-known to those in the art. (Ansel et al, Pharmaceutical Dosage Forms and Drug Delivery Systems, 5 th Edition (Lea & Febiger 1990); Gennaro (ed.), Remington's Pharmaceutical Sciences 18th Edition (Mack Publishing Company 1990)).
  • excipients examples include, water, dextrose, glycerol, ethanol, and combinations thereof.
  • the vaccines ofthe present invention may further contain auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, stabilizers or other carriers that include, but are not limited to, agents such as aluminum hydroxide or phosphate (alum), commonly used as a 0.05 to 0.1 percent solution in phosphate buffered saline, to enhance the effectiveness thereof.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers or other carriers that include, but are not limited to, agents such as aluminum hydroxide or phosphate (alum), commonly used as a 0.05 to 0.1 percent solution in phosphate buffered saline, to enhance the effectiveness thereof.
  • the chimeric constructs ofthe present invention can be used as vaccines by conjugating to soluble immunogenic carrier molecules.
  • Suitable carrier molecules include protein, including keyhole limpet hemocyanin, which is a preferred carrier protein.
  • the chimeric construct can be conjugated to the carrier molecule using standard methods. (Hancock et al, "Synthesis of Peptides for Use as Immunogens," in Methods in Molecular Biology: hnmunochemical Protocols, Manson (ed.), pages 23-32 (Humana Press 1992)).
  • the present invention contemplates a vaccine composition comprising a pharmaceutically acceptable injectable vehicle.
  • the vaccines ofthe present invention may be administered in conventional vehicles with or without other standard carriers, in the form of injectable solutions or suspensions.
  • the added carriers might be selected from agents that elevate total immune response in the course ofthe immunization procedure.
  • Liposomes have been suggested as suitable carriers.
  • the insoluble salts of aluminum that is aluminum phosphate or aluminum hydroxide, have been utilized as carriers in routine clinical applications in humans, Polynucleotides and polyelectrolytes and water soluble carriers such as muramyl dipeptides have been used.
  • Preparation of injectable vaccines ofthe present invention includes mixing the chimeric construct with muramyl dipeptides or other carriers.
  • the resultant mixture may be emulsified in a mannide monooleate/squalene or squalane vehicle.
  • Four parts by volume of squalene and/or squalane are used per part by volume of mannide monooleate.
  • Methods of formulating vaccine compositions are well-known to those of ordinary skill in the art. (Rola, Immunizing Agents and Diagnostic Skin Antigens. In: Remington's Pharmaceutical Sciences, 18th Edition, Gennaro (ed.), (Mack Publishing Company 1990) pages 1389-1404).
  • Control release preparations can be prepared through the use of polymers to complex or adsorb chimeric construct.
  • biocompatible polymers include matrices of poly(ethylene-co- vinyl acetate) and matrices of a polyanhydride copolymer of a stearic acid dimer and sebacic acid.
  • the rate of release ofthe chimeric construct from such a matrix depends upon the molecular weight of the construct, the amount ofthe construct within the matrix, and the size of dispersed particles. (Saltzman et al. (1989) Biophys. J.
  • the chimeric construct can also be conjugated to polyethylene glycol (PEG) to improve stability and extend bioavailability times (e. g. , Katre et al. ; U. S . Patent 4,766, 106) .
  • PEG polyethylene glycol
  • the vaccines of this invention may be administered parenterally.
  • the usual modes of administration ofthe vaccine are intramuscular, sub-cutaneous, and intra- peritoneal injections. Moreover, the administration may be by continuous infusion or by single or multiple boluses.
  • the gene gun has also been used to successfully deliver plasmid DNA for inducing immunity against an intracellular pathogen for which protection primarily depends on type 1 CD8.sup. + T-cells. (Kaufmann et al. (1999) J. Immun. 163(8): 4510-4518).
  • compositions ofthe present invention are applicable to the treatment of both noninfectious and infectious diseases and noninfectious diseases, including but not limited to genetic disorders, using such vaccination methods.
  • Eck et al. (1996) Gene-Based Therapy, h : Goodman & Gilman's The Pharmacological Basis of Therapeutics, Ninth Edition, Chapter 5, McGraw Hill).
  • the vaccine ofthe present invention may be formulated and delivered in a manner designed to evoke an immune response at a mucosal surface.
  • the vaccine compositions may be administered to mucosal surfaces by, for example, nasal or oral (intragastric) routes.
  • Other modes of administration include suppositories and oral forniulations.
  • binders and carriers may include polyalkalene glycols or triglycerides.
  • Oral formulations may include normally employed incipients such as pharmaceutical grades of saccharine, cellulose and magnesium carbonate.
  • compositions can take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain about 1 to 95% ofthe chimeric construct.
  • the vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective, protective and immunogenic dosages.
  • the quantity of vaccine employed will of course vary depending upon the patient's age, weight, height, sex, general medical condition, previous medical history, the condition being treated and its severity, and the capacity ofthe individual's immune system to synthesize antibodies, and produce a cell-mediated immune response.
  • a dosage of the chimeric construct which is in the range of from about 1 :g agent /kg body weight of patient to 100 mg agent/kg body weight of patient, although a lower or higher dosage may also be administered.
  • Precise quantities ofthe active ingredient depend on the judgment ofthe practitioner.
  • Suitable dosage ranges are readily determinable by one skilled in the art and may be on the order of nanograms ofthe chimeric construct to grams ofthe chimeric construct, depending on the particular construct.
  • the dosage range ofthe active ingredient is nanograms to micrograms; more preferably nanograms to milligrams; and most preferably micrograms to milligrams.
  • Suitable regimes for initial administration and booster doses are also variable, but may include an initial administration followed by subsequent administrations.
  • the dosage may depend on the route of administration and will vary according to the size ofthe subject.
  • the present invention encompasses vaccines containing antigen and PAMPs from a single organism, such as from a specific pathogen.
  • the present invention also encompasses vaccines that contain antigenic material from several different sources and or PAMP material isolated from several different sources.
  • Such combined vaccines contain, for example, antigen and PAMPs from various microorganisms or from various strains ofthe same microorganism, or from combinations of various microorganisms.
  • the antigen/PAMP fusion proteins are administered to a mammal in a therapeutically effective amount.
  • a vaccine preparation is said to be administered in a "therapeutically effective amount” if the amount administered is can produce a measurable positive effect in a recipient.
  • a vaccine preparation of the present invention produces a positive effect in a recipient if it invokes a measurable humoral and/or cellular immune response in the recipient.
  • this invention contemplates a desirable therapeutically effective amount as one in which the vaccine invoices in the recipient a measurable humoral and/or cellular immune response versus the target antigen but causes neither excessive nonspecific inflammation nor an autoimmune response versus non-target antigen(s).
  • the term "treatment” refers to both therapeutic treatment and prophylactic or preventative treatment.
  • the present invention contemplates using the disclosed vaccines to treat patients in need thereof.
  • the patients may be suffering from diseases such as, but not limited to, cancer, allergy, infectious disease, autoimmune disease, neurological disease, cardiovascular disease, or a disease associated with an allergic reaction.
  • the present invention contemplates administering the disclosed vaccines to passively immunize patients against diseases such as but not limited to, cancer, allergy, infectious disease, autoimmune disease, neurological disease, cardiovascular disease, or disease associated with an allergic reaction.
  • the present invention contemplates administering the disclosed vaccines to immunize patients against diseases in addition to those cited in the previous sentence in which the objective is to rid the body of specific molecules or specific cells.
  • a non-limiting example might be the removal or prevention of deposition of plaque in cardiovascular disease.
  • the vaccines ofthe present invention can be used to enhance the immunity of animals, more specifically mammals, and even more specifically humans (e.g., patients) in need thereof. Enhancement of immunity is a desirable goal in the treatment of patients diagnosed with, for example, cancer, immune deficiency syndrome, certain topical and systemic infections, leprosy, tuberculosis, shingles, warts, herpes, malaria, gingivitis, and atherosclerosis.
  • the advantages ofthe vaccines ofthe present invention are that they induce a strong immune response against the target antigen with minimal undesired inflammatory reaction, as well as minimal instances of autoimmune disease.
  • Such a reduced side effect profile has a distinct advantage over other vaccine approaches, particularly with respect to targeting of self antigens, because with many other vaccine strategies, in order to elicit a robust response against the self antigen, strong adjuvants are used and they result in excessive inflammation and can increase the risk of autoimmune disease.
  • immunoenhancement refers to any increase in an organism's capacity to respond to foreign antigens or other targeted antigens, such as those associated with cancer, which includes an increased number of immune cells, increased activity and increased ability to detect and destroy such antigens, in those cells primed to attack such antigens.
  • the strength of an immune response can be measured by standard tests including, but not limited to, the following: direct measurement of peripheral blood lymphocytes by means known to the art; natural killer cell cytotoxicity assays (Provinciali et al. (1992) J Immunol. Meth. 155: 19-24), cell proliferation assays (Vollenweider et al. (1992) J Immunol. Meth. 149: 133-135), immunoassays of immune cells and subsets (Loeffler et al. (1992) Cytom. 13: 169-174; Rivoltini et al. (1992) Can. Immunol. Immunother. 34: 241 -251); and skin tests for cell- mediated immunity (Chang et al.
  • Enhanced immune response is also characterized by healing of systemic and local infections, and reduction of symptoms in disease, e.g. decrease in tumor size, alleviation of symptoms of leprosy, tuberculosis, malaria, naphthous ulcers, herpetic and papillomatous warts, gingivitis, atherosclerosis, the concomitants of AIDS such as Kaposi's sarcoma, bronchial infections, and the like.
  • the procedures ofthe present invention can be used to generate a chimeric construct comprising one or more antigens of interest and one or more PAMPs.
  • a small, non-immunogenic epitope tag (such as a His tag) can be added to facilitate the purification of fusion protein expressed in bacteria.
  • the combination of antigen with a PAMP such as BLP, Flagellin or FimC provides signals necessary for the activation ofthe antigen-specific adaptive and innate immune responses.
  • a large number of differing fusion proteins comprising different combinations of antigens and PAMPs can be readily generated using recombinant DNA technology or conjugation chemistry that is well lcnown in the art. Virtually any antigen can be used to generate a vaccine by this approach using the same technology.
  • recombinant vaccine product can be generated using a bacterial expression system.
  • the product can be purified from bacterial cultures using standard techniques. The approach is thus extremely economical and cost efficient.
  • recombinant vaccine product can be produced and purified from cultures of yeast or other eukaryotic cells including, without limitation, insect cells or mammalian cells.
  • Conjugated non-protein vaccine product can also be produced chemically in relatively large amounts. This is particularly the case if the PAMP and the antigen can both be obtained by relatively straightforward purification procedures and then conjugated together with relatively simple and efficient conjugation chemistry.
  • a chimeric construct containing a protein component and a non-protein component can be conveniently obtained by preparing the protein component by recombinant means and the non-protein component by chemical means and then linking the two components with linker chemistry well lcnown in the art, some of which is described herein.
  • linker chemistry well lcnown in the art, some of which is described herein.
  • the antigens and PAMPs contemplated in this invention can be naturally occurring, they can be purified from their natural sources and then linked together chemically. Both T-cell and B-cell antigens can be used to generate vaccines by this approach.
  • Fusion of an antigen with a PAMP such as BLP, Flagellin or FimC optimizes the stoichiometry ofthe two signals thus minimizing the unwanted excessive inflammatory responses (which occur, for example, when antigens are mixed with adjuvants to increase their immunogenicity). Fusion of an antigen with a PAMP such as BLP increases the likelihood that
  • APCs activated in response to the vaccine productively trigger the desired adaptive immune response. Activation of such APCs in the absence of uptake and presentation ofthe antigen can lead to the induction of autoimmune responses, which, again, is one ofthe problems with commonly used adjuvants that prevents or limits their use in humans.
  • the fusion proteins ofthe present invention comprise an antigen or an immunogenic portion thereof which has been modified to contain an amino acid sequence comprising a leader sequence and a consensus sequence, that results in the post-translational modification ofthe consensus sequence or a portion of that sequence, wherein the post-translationally modified sequence is a ligand for a PRR.
  • the modified antigens include, but are not limited to, antigens that contain the bacterial lipidation consensus sequence CXXN (SEQ ID NO: 1), wherein X is any amino acid, but preferably serine.
  • leader sequences are well known in the art, but a preferred leader sequence is described by the first 20 amino acids of SEQ ID NO: 2, wherein the first 20 amino acids of SEQ ID NO: 2 are set forth in set forth in SEQ ID NO: 3. Examples of additional suitable leader sequences are described in the Sequence Listing as SEQ ID NO: 4-7.
  • a preferred chimeric construct comprises a leader sequence fused, in frame, to a sequence comprising the bacterial lipidation consensus sequence of SEQ ID NO: 1 further fused to an antigen (e.g. leader sequence — CXXN — antigen).
  • this modification ofthe antigen can be referred to as a fusion
  • this modification can be achieved without fusing DNA, but rather by introducing, by mutagenesis, a leader sequence followed by the CXX sequence into DNA encoding any antigen of interest.
  • the resultant product is a chimeric construct or fusion protein that is a ligand for a PRR and is capable of stimulating both the innate and adaptive immune systems.
  • this chimeric construct or fusion protein comprises additional polar or charged amino acids to increase the hydrophilicity of the chimeric construct or fusion protein without altering the immunogenic or immunostimulatory properties ofthe construct.
  • PAMP pathogen-associated molecular pattern
  • the protein sequence ofthe bacterial lipoprotein (BLP) used in the vaccine cassette for fusion with an antigen of interest is as follows: MKATKLVLGAVILGSTLLAGCSSNAKIDQLSSDVQTLNAKVDQLSNDVNA MRSDVQAAKDDAARANQRLDNMATKYRK (SEQ ID NO: 2).
  • the leader sequence includes amino acid number 1 through amino acid number 20 of SEQ ID NO: 2.
  • the first cysteine (amino acid number 21 of SEQ ID NO: 2) is lipidated in bacteria. This lipidation, which can only occur in bacteria, is essential for BLP recognition by Toll and TLRs.
  • the C-terminal lysine (amino acid number 78 of SEQ ED NO: 2) was mutated to increase the yield of a recombinant vaccine, because this lysine can form a covalent bond with the peptidoglycan.
  • the fusion protein was expressed in bacteria and induced with IPTG.
  • the protein was purified by lysis and sonication in 8 M Urea, 20 mM Tris, 20 mM NaCl, 2% Triton-X-100, pH 8.0.
  • the lysate was passed over a 100 ml Q-Sepharose ion exchange column in the same buffer and washed with 5 column volumes of 8 M Urea, 20 mM Tris, 20 mM NaCl, 0.2% Triton-X-100, pH 8.0.
  • the protein was eluted by salt gradient (20 mM NaCl to 800 mM NaCl). Positive fractions were identified by immunoblotting using an antibody to the Histidine tag.
  • Example 2 Stimulation of NF-icB by BLP/E ⁇ model antigen in RAW cells
  • Example 3 BLP/E ⁇ Model Vaccine Induces the Production of IL-6 by Dendritic Cells In Vitro
  • An effective vaccine must be able to stimulate dendritic cells (DC)to mature and present antigen.
  • DC dendritic cells
  • BLP/E ⁇ could induce DC function, we tested the ability of bone marrow-derived DC to produce IL-6 after stimulation in vitro. Bone marrow dendritic cells were isolated and grown for 5 days in culture in the presence of 1% GM-CSF.
  • Example 4 BLP/E ⁇ Stimulates Maturation of Immature Dendritic Cells To determine whether BLP/E ⁇ vaccine can be processed and presented by dendritic cells, we stimulated dendritic cells with the vaccine and tested them for the surface expression of B7.2 and E ⁇ peptide bound to MHC Class II. Cultured bone marrow-derived dendritic cells (5 days) were stimulated with E ⁇ peptide or BLP/E ⁇ and were stained with an antibody to the B7.2 costimulatory molecule and/or with Yae antibody which recognizes E ⁇ peptide bound to MHC Class II. Analysis was performed by FACS ( Figure 6).
  • Example 5 BLP/E ⁇ Model Vaccine Stimulates Specific T-Cells In Vitro.
  • Bone marrow derived mouse DC were isolated and plated into medium containing 1% GM-CSF at 750,000 cells/well. Cells were cultured for 6 days and then the DC were collected, washed, and counted then replated in 96-well dishes at 250,000 cells per well. Cells were stimulated with the above indicated antigens and left three days to mature. After 3 days, the DC were resuspended and plated in a 96-well dish at either 5,000 or 10,000 cells/well.
  • T-cells from lymph nodes from a 1H3.1 TCR transgenic mouse (1H3.1 TCR is specific for the E ⁇ peptide)were plated on the DC at 100,000 cells/well. Cells were left for 3 days in culture then "pulsed" with 0.5:Ci/well of 3 H- thymidine. The cells were harvested 24 hours later and incorporation of thymidine (T-cell proliferation) was measured in cpm ( Figure 7).
  • Example 6 BLP/E ⁇ Activates Specific T-cells In Vivo To assess the ability ofthe vaccine to generate a specific T-cell response in vivo, we injected the fusion protein into a mouse. Three mice were injected as follows:
  • mice #1 and #3 appeared normal.
  • T-cell proliferation assay T-cells were plated in a 96-well plate at 400,000 cells/well and were restimulated with either E ⁇ peptide or with BLP/E ⁇ at the indicated doses. Cells were left 48 hours to begin proliferation, pulsed with 0.5:Ci/well of 3 H-Thymidine in medium and harvested 16 hours later. Thymidine incorporation was measured by counting in a beta-plate reader ( Figure 8).
  • Example 7 Model Vaccine Cassette with an Allergen-Related Antigen Using the procedures set forth above for the production ofthe BLP/E ⁇ model antigen, a vaccine cassette with an allergen-related antigen is produced using the pollen allergen Ra5G from the giant ragweed (Ambrosia trifida).
  • the amino acid sequence of Ra5G is as follows: MKNIFMLTLF ILIITSTIKA IGSTNEVDEI KQEDDGLCYE GTNCGKVGKY CCSPIGKYCVCYDSKAICNKNCT (SEQ ID NO: 9).
  • the amino acid sequence of this allergen can be fused with the BLP amino acid sequence (SEQ ID NO: 1) to generate the BLP/Ra5G fusion protein.
  • the resultant recombinant vaccine places the allergen in the context of an IL-12 inducing signal, where the PAMP in this case is BLP).
  • this vaccine When introduced into a subject, this vaccine will generate allei en-speci lic T-cell responses that will be differentiated into Thi responses due to the induction of IL-12 by BLP in dendritic cells and macrophages.
  • Example 8 Model Vaccine Cassette with a Tumor-Related Antigen Using the procedures set forth above for the production of the BLP/E ⁇ model antigen, a vaccine cassette with a tumor-related antigen is produced using the model tumor antigen, Tyrosinase-Related Protein 2 (TRP-2).
  • TRP-2 Tyrosinase-Related Protein 2
  • the nucleic acid sequence and corresponding amino acid sequence of TRP-2 is provided in SEQ ID NO: 10 (shown in Figure 20) and SEQ ID NO: 11 (shown in Figure 21), respectively.
  • the region used for BLP fusion includes nucleic acid number 840 through nucleic acid number 1040 of SEQ ID NO: 10.
  • the T-cell epitope includes nucleic acid number 945 through nucleic acid number 968 of SEQ ID NO: 10.
  • TRP-2 A region ofthe TRP-2 that can be used for the vaccine construction is shown below: LDLAKKSIHPDYVITTQHWLGLLGPNGTQPQIANCSVYDFFVWLHYY
  • a T-cell epitope of SEQ ID NO: 12 is VYDFFVWL (SEQ ID NO: 13).
  • TLRs The family of TLRs has recently been identified as an essential component of innate immune recognition in both Drosophila and mammalian organisms (Hoffmann et al. (1999) Science 284:1313-1318; hnler et ⁇ /. (2000) Curr. Opin. Microbiol 3:16-22). Drosophila Toll is required for the detection of fungal infection and the induction ofthe antifungal peptide drosomycin (Lemaitre et al. (1996) Cell 86:973-983). In the mouse, TLR2 and TLR4 were shown to mediate recognition of bacterial PGN and LPS, respectively (Takeuchi et al. (1999) Immunity 11 :443-451). The functions ofthe other members ofthe Drosophila and mammalian Toll families are currently unknown, although it is expected that at least some of them are involved in innate immune recognition as well.
  • CpG-DNA is recognized by a Toll receptor other than TLR2 and TLR4.
  • TLR2 and TLR4 Cell lines that express endogenous or transfected TLR1 through TLR6 did not respond to CpG-DNA (data not shown), suggesting that some other member of the Toll family may mediate CpG-DNA recognition.
  • B-cells from the indicated mouse strains were purified from spleen by complement kill of CD4 + , CD8 + and macrophages.
  • Non-adherent cells were cultured in the presence or absence of different amounts of stimulating CpG-DNA (5'-TCCATGACGTTCCTGACGTT-3' (SEQ ID NO. 8), phosphorothioate modified) at 1 x 10 6 cells/ml. After 48 h, the cells were pulsed with [ 3 H]thymidine (0.5 ⁇ Ci per well, NEN) for 16 h and processed for beta counting.
  • CD86 and MHC class-II molecules on B-cells were tested to determine whether these processes are mediated by the MyD88 signaling pathway.
  • CD86 and MHC class -II cell surface expression were analyzed by FACS.
  • B-cells were prepared as above and cultured at 3 x 10 ⁇ cells/ml with or without 10 mM CpG for 12 h. After the stimulation, the surface expression of CD86 and MHC class II were analyzed by flow cytometry. Results, shown in Figure 9B, represent gated B-cells. The shaded area represents stimulated cells, whereas the unshaded area represents untreated controls. As shown in Figure 9B, CpG-DNA strongly induced expression of CD86 and MHC class-II on B-cells from wild-type and TLR4-deficient mice. By contrast, this induction was completely abrogated in MyD88 deficient B-lymphocytes.
  • Example 12 Cloning of Salmonella Tymphimurium Flagellin and E. coli
  • Flagellin Full-length Salmonella typhimurium Flagellin and E coli Flagellin were cloned from the respective genomic DNAs and expressed as recombinant proteins in E coli . Flagellin was expressed alone, or as a fusion protein with antigenic epitopes from ovalbumin (SIINF ⁇ KL), tyrosinase-2 protein (TRP2) cloned from murine B16 cells, or the C-terminal fragment of I- ⁇ protein, which contains the ⁇ epitope. In addition, all ofthe recombinant proteins contained a C-terminal 6x-histidine repeat to aid in purification.
  • IINF ⁇ KL ovalbumin
  • TRP2 tyrosinase-2 protein
  • Fusion proteins were purified by passing filtered lysates over a Niclcel- NTA agarose column followed by extensive washes in several buffers containing imidazole. Purified proteins were eluted in 250mM imidazole, passed twice over a Polymyxin B column to remove contaminating lipopolysaccharide and then dialyzed extensively overnight in PBS at 4°C. The resulting purified proteins were very stable and retain activity at 4°C for at least a month.
  • the human 293 cell line and the murine RAW cell line were stably transfected with a reporter gene containing two copies ofthe Igic NF- ⁇ B site driving transcription of luciferase (this construct is referred to as "pBIIxluc").
  • the resulting cell lines (293LUC and RAWkb) were plated in 24-well dishes and treated 24 hours later with Flagellin fusion proteins or a control protein (lacZ) that was made in the same vector and purified exactly the same way as the Flagellin proteins.
  • Cell lysates were made after 5 hours of treatment and were tested for luciferase activity to indicate induction of NF- ⁇ B.
  • Flagellin proteins significantly induced NF- ⁇ B in this assay, particularly in 293 cells whereas the control protein had no effect, as shown in Figures 12 and 13. It is believed that this induction was not due to contamination by LPS since polymyxin B did not inhibit the activation in RAWKB cells, and 293LUC cells do not respond to LPS but do respond to Flagellin, as indicated by Figures 12 and 14.
  • the results ofthe In vitro assays demonstrate that Flagellin fusion proteins retain their ability to stimulate Toll-Like Receptors and can therefore be used for the generation of recombinant Flagellin- Antigen fusion proteins for the purpose of vaccination.
  • Flagellin- Antigen fusion proteins In Flagellin- Antigen fusion proteins, Flagellin is believed to stimulate the innate immune system by triggering Toll-Like Receptors, whereas the antigen fused to Flagellin provides epitopes for recognition by T and B lymphocytes.
  • Example 14 CpG and IL-6 Production in Macrophages Adherent thioglycollate-elicited peritoneal exudate cells (PECs) from the indicated mouse strains were treated with different stimuli for 24 h. The release of IL-6 into the supernatant was analyzed by specific enzyme-linked immunosorbent assay (ELISA) using anti-mouse IL-6 monoclonal antibodies.
  • ELISA enzyme-linked immunosorbent assay
  • CpG-DNA is also lcnown to have a pronounced stimulatory effect on macrophages (Stacey et al. (2000) Curr. Top. Microbiol. Immunol. 247: 41-58; Lipford et al. (1998) Trends Microhiol. 6: 496-500; Stacey et al. (1996) J. Immunol. 157: 2116-2122), CpG- induced expression of IL-6 by wild-type and MyD88 was examined in deficient macrophages. Cells derived from caspase-1 knock-out mice were used as a control for IL-1 -mediated induction of IL-6.
  • Example 15 CpG-DNA-Induced I ⁇ B ⁇ Degradation
  • Peritoneal macrophages were stimulated with CpG-DNA, or LPS as a control, for 0, 10, 20, 60, and 90 minutes and lysed thereafter.
  • 30 mg total protein was processed for SDS-PAGE and analyzed by immunoblotting for I ⁇ B ⁇ protein.
  • Figure 10B In wild-type cells, both LPS and CpG-DNA induced NF- ⁇ B activation, as evidenced by the degradation of I ⁇ B protein ( Figure 10B).
  • CpG-DNA has been shown to be a potent inducer of DC activation (Sparigan et al. (1998) Eur. J. Immunol. 28: 2045-2054).
  • DC play a pivotal role in the initiation ofthe adaptive immune responses (Banchereau et al. (1998) Nature 392: 245-252).
  • PAMPs microbe-derived products
  • DC undergo developmental changes collectively referred to as maturation (Banchereau et al. (1998) Nature 392: 245-252).
  • the hallmark of DC maturation is the induction of cell surface expression of CD80 and CD86 molecules, as well as migration into lymphoid tissues and production of cytokines such as IL- 12 (Banchereau et al.
  • MyD88 -/- animals produce IL-12 when stimulated with CpG oligonucleotides.
  • Wild-type, BlO/ScCr, and MyD88 -/- bone marrow DC were prepared from bone marrow suspensions cultured for 5 days in DC Growth Medium (RPMI 5% FC + 1% GM-CSF) and stimulated with 10 mm CpG or 10 mm GpC oligonucleotides or left untreated. Supernatants were taken 24 h and 48 h after stimulation and analyzed for IL-12 by ELISA using specific capture and detection antibodies.
  • CpG/ E ⁇ Chimeric Construct A non-protein PAMP, CpG, was conjugated to the characterized mouse antigen, E ⁇ , through a PEG polymer linker and/or copolymers of D-lysine and D- glutamate, according to the methods described in U.S. Pat. No. 6,06,0056.
  • a CpG- DNA derivative, comprising CpG 0 was used as the non-protein PAMP.
  • E. coli FimC Full-length E. coli FimC was cloned from the genomic DNA and expressed as a recombinant protein in E. coli.
  • the recombinant FimC protein contained a C- terminal 6x-histidine repeat to aid in purification.
  • FimC was expressed in E. coli and induced with IPTG.
  • the protein was purified by lysis and sonication in buffer containing PBS, 0.5%. Triton X-100,
  • the murine RAW macrophage and NIH 3T3 cells lines were stably transfected with a reporter gene containing an NF-icB-dependent firefly luciferase gene. Stimulation of these cells with activators of NF-icB leads to production of luciferase which is measured in cell lysates by use of a luminometer. Cells were stimulated with the indicated amounts of FimC, left 5 hours and harvested for luciferase measurement, As a control, RAW macrophage and NIH 3T3 cells were stimulated with LPS in the presence and absence of polymyxin B
  • PmB inactivates endotoxin and, as expected, the activation of NF- ⁇ B activity in the LPS+PmB sample is diminished by a statistically significant degree.
  • Treatment with PmB does not prevent FimC from activating NF- B activity to a statistically significant degree ( Figure 22).
  • Figure 22 shows that the activation of NF- ⁇ B seen with FimC is not due to contamination ofthe preparation with endotoxin.
  • boiling FimC eliminates FimC activity ( Figure 23), indicating that the activity depends on an intact conforaiation of FimC and is not due to LPS contamination.

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Abstract

L'invention concerne de nouveaux vaccins, ainsi que des procédés de production et d'utilisation desdits vaccins. Les nouveaux vaccins selon l'invention associent les deux signaux nécessaires à l'activation des lymphocytes T natifs, à savoir un antigène spécifique et le signal co-stimulant, ce qui provoque une réponse immunitaire spécifique et vigoureuse des lymphocytes T.
PCT/US2002/040046 2001-12-14 2002-12-13 Vaccins agissant sur le systeme immunitaire inne WO2003051305A2 (fr)

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