WO2013134656A1 - Induction de réponse immunitaire de th17 - Google Patents

Induction de réponse immunitaire de th17 Download PDF

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WO2013134656A1
WO2013134656A1 PCT/US2013/029907 US2013029907W WO2013134656A1 WO 2013134656 A1 WO2013134656 A1 WO 2013134656A1 US 2013029907 W US2013029907 W US 2013029907W WO 2013134656 A1 WO2013134656 A1 WO 2013134656A1
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seq
polypeptide
fusion protein
composition
lipidated
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PCT/US2013/029907
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Todd Gierahn
Richard Malley
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Genocea Biosciences, Inc.
Children's Medical Center Corporation
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Publication of WO2013134656A1 publication Critical patent/WO2013134656A1/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
    • A61K39/02—Bacterial antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/085—Staphylococcus
    • 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
    • A61K39/02—Bacterial antigens
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A61K39/092—Streptococcus
    • 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
    • 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
    • C07K14/295—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Chlamydiales (O)
    • 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
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • 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
    • A61K2039/6018—Lipids, e.g. in lipopeptides
    • 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
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/90—Fusion polypeptide containing a motif for post-translational modification

Definitions

  • Streptococcus pneumoniae causes approximately 11% of mortality worldwide in children under five. Pneumococcus causes invasive disease including sepsis, meningitis, and, most frequently, pneumonia; it is also a common cause of primarily mucosal diseases such as acute otitis media and sinusitis. Since the introduction of conjugated polysaccharide vaccines (PCVs), rates of pediatric invasive pneumococcal disease due to strains covered by these vaccines have dropped significantly. However, the high manufacturing complexity and associated cost of PCVs limit their use in the developing world, where morbidity and mortality from pneumococcal disease are highest. Additionally, there are over 90 identified pneumococcal serotypes, and regional distribution of these serotypes is highly variable.
  • PCVs conjugated polysaccharide vaccines
  • the mammalian immune system is intricately regulated, allowing pathogen- specific immunity to be rapidly induced in response to infection.
  • Humoral immunity is mediated by B-lymphocytes and marked by the production of antibodies in response to antigens.
  • Cellular immunity is mediated by T-lymphocytes and involves production of cytotoxic T-lymphocytes, activated macrophages, activated Natural Killer cells, and secretion of cytokines.
  • T- lymphocytes CD4+ T cells have the ability to proliferate and differentiate into T helper cells responsive to specific antigens. Sub-populations of T helper cells are classified by the cytokines they produce.
  • T H 17 cells notably produce interleukin-17 (IL-17A, IL-17F), in addition to IL-21 and IL-22.
  • Interleukin-17 in turn stimulates stromal cells to release inflammatory cytokines such as IL-8 and IL-6, which function as chemo-attractants for neutrophils and macrophages.
  • T H 17-mediated immune response has been noted against extracellular bacterial and fungal pathogens such as Klebsiella pneumoniae, Candida albicans, and Citrobacter rodentium, and against intracellular bacteria such as Listeria monocytogenes, Mycobacterium tuberculosis, Salmonella enterica, and others (Curtis, M.M, and Way, S.S. (2009), Interleukin-17 in Host Defense against Bacterial Mycobacterial and Fungal Pathogens, Immunology 126: pp. 177-185). Accordingly, compositions capable of inducing T H 17-mediated immune responses against Streptococcus pneumonia are desirable.
  • the fusion protein is lipidated.
  • the first polypeptide is fused to the N-terminus of the second polypeptide.
  • the first polypeptide comprises an exogenous signal sequence.
  • the first polypeptide is membrane-associated.
  • the second polypeptide does not include a lipobox motif.
  • the present disclosure provides a lipidated fusion protein comprising a first polypeptide that includes a lipidated N-terminal cysteine and second polypeptide from an organism susceptible to host T H 17 immune response, wherein the fusion protein elicits a host T R 17 immune response to said organism or pathogen.
  • the present disclosure describes compositions comprising fusion proteins (e.g., a population of fusion proteins) described herein. In some embodiments, at least 0.1 % of the fusion proteins within the composition contain a lipid moiety.
  • the compositions described herein further comprise a second adjuvant, such as alum, cholera toxin or its nontoxic mutant derivatives, or E. coli heat-labile toxin or its nontoxic mutant derivatives.
  • the composition further comprises a peptide or polypeptide from the same organism or pathogen as the second polypeptide.
  • the adjuvant composition induces one or more of a mucosal immune response, a dermal immune response, and a systemic immune response. In some embodiments, the adjuvant composition inhibits colonization. In some embodiments, the adjuvant composition induces IL-17A. In some embodiments, the adjuvant composition induces IL-22.
  • the present disclosure provides a method of eliciting or increasing a host T H 17 immune response, comprising administering to a subject in need thereof a fusion protein described herein, thereby eliciting or increasing a T R 17 immune response.
  • the present disclosure provides methods of making a fusion protein described herein.
  • the method comprises expressing in a host cell a nucleic acid encoding a fusion protein described herein.
  • the method further comprises isolating or purifying the fusion protein.
  • the method comprises producing the fusion protein synthetically (e.g., chemically).
  • the method comprising providing a first polypeptide and a second polypeptide described herein and conjugating, fusing, and/or joining the first and second polypeptides, thereby producing the fusion protein.
  • the present disclosure provides fusion proteins produced by a method described herein. Brief Description of the Drawing
  • FIG. 1A-B depicts exemplary results of T H 17 response and colonization assays, following intranasal immunization with lipidated and non-lipidated forms of SP2108 or SP0148 in the presence of cholera toxin (CT) adjuvant.
  • CT cholera toxin
  • FIG. 1A Systemic whole pneumococcus (WCB)-specific IL-17A values from immunized animals are shown in FIG. 1A. Each symbol represents the response from a single animal, and lines represent the median value for the group.
  • Colony forming units (CFU) of bacteria per nasal lavage are shown in FIG. IB. Lines represent median values and p- values were calculated using Mann- Whitney test compared to CT immunized controls. Immunization with lipidated antigens resulted in 1-2 log higher IL-17A secretion compared with non-lipidated mutants, correlating with more significant protection from colonization.
  • CFU Colony forming units
  • FIG. 2 depicts exemplary representative results of a T H 17 immune response assay following subcutaneous immunization with lipidated and non-lipidated forms of SP2108 adsorbed on alum.
  • Systemic IL-17A values from immunized animals are shown. Each symbol represents the response from a single animal, and lines represent the median value for the group.
  • immunization with lipidated SP2108 resulted in 5 to 10-fold higher IL-17A secretion than immunization with non-lipidated SP2108.
  • FIG. 3A-C depicts exemplary results of T H 17 immune response assays following subcutaneous immunization with combinations of lipidated and non-lipidated proteins.
  • FIG. 3A shows systemic IL- 17A values from animals immunized with 10 ⁇ g lipidated SP0148, 10 ⁇ g non-lipidated pneumolysoid, or both.
  • FIG. 3B shows results from animals immunized with 1 ⁇ g lipidated SP2108, 10 ⁇ g non-lipidated SP1912, or both.
  • FIG. 3C shows results from animals immunized with 10 ⁇ g lipidated SP0148, 10 ⁇ g non-lipidated SP1912, or both.
  • 3C represents the response from a single animal, and lines represent the median value for the group.
  • Data in FIG. 3B are represented as the mean ⁇ SEM IL-17A concentration from four mice per group. In each case, immunization with a combination of lipidated and non-lipidated proteins resulted in a median 15 to 40-fold increase in IL-17A secretion, following stimulation with the non-lipidated protein.
  • FIG. 4A-B depicts exemplary results of T H 17 immune response assays following subcutaneous immunization with combinations of SP 1912, the synthetic free lipopeptide
  • FIG. 4A shows IL-17A values in blood.
  • FIG. 4B shows IL- 17A values in splenocytes. Each symbol represents the IL-17A response of an individual mouse, and lines represent the median value for the group. Tables below each graph indicated the ⁇ g dose of Pam3CSK4. Immunization with the combination of SP1912 and SP0148 or SP2108 proteins on alum primed SP1912-specific T H 17 responses as expected. Combining SP1912 with various doses of Pam3CSK4 on alum did not reproduce this effect. Therefore, the in trans effect on T H 17 immune response requires that a non-lipidated protein such as SP 1912 be combined with lipidated protein.
  • FIG. 5 depicts exemplary results of a TLR2 activation assay with IL-8 read-out in
  • HEK-TLR2 cells stimulated with lipidated and non-lipidated SP2108 and SP0148 proteins. Lines represent median values from at least four independent experiments. The lipidated proteins elicited robust IL-8 response in HEK-TLR2 cells, while non-lipidated mutants did not.
  • FIG. 6 depicts exemplary results of T H 17 immune response assays, following immunization of TLR2 _/" C57BL/6 mice (TLR2KO) or C57BL/6 (wild-type) with the indicated proteins on alum.
  • Systemic IL-17A values from immunized animals are shown as
  • FIG. 7 depicts exemplary results of ESI-MS analysis of SP0148.
  • Both peaks 1 and 2 matched nearly identically ( ⁇ 5 amu is well within the error of this technique) to the full-length non- lipidated form of SP0148 and the signal peptidase cleaved, lipidated (tri-palmitoylated) form, respectively. Peaks 3 and 4 had slightly greater masses, still consistent with tri-lipidation but possibly suggesting minor differences in the nature of the lipid adducts.
  • FIG. 8 depicts exemplary lipid adducts of SP0148 and SP2108.
  • FIG. 9 depicts exemplary results of T H 17 response assays following immunization with two doses of the indicated proteins absorbed to alum. Each bar represents the mean number of IL-17A spot forming units (SFU) per million splenocytes ⁇ standard deviation (SD) of the group of four mice. Immunization with either Lipo IsaA or SP2108_IsaA adsorbed to alum increased the frequency of IsaA-specific T H 17 cells 2-fold or 18-fold, respectively, compared to immunization with native non-lipidated IsaA.
  • SFU spot forming units
  • SD standard deviation
  • FIG. 10 depicts exemplary results of T H 17 response assays following
  • T H 17 cells and IL-17A expression have been described (WO2011/008548). Nevertheless, a general strategy to elicit a desirable T H 17 response is needed.
  • adjuvant refers to a substance that assists or modifies the
  • an "adjuvant” is a substance that enhances the immune response of a host organism to an antigen.
  • Adjuvants are added to antigens when the required immune response is quantitatively and/or qualitatively different to that induced by the antigen alone, as is often the case when the antigen is being delivered as a vaccine to an animal or person.
  • a substance is said to "enhance" an immune response of a host organism to an antigen (i.e.
  • an epitope refers to a molecule that contains one or more epitopes capable of stimulating a host's immune system to make a cellular antigen-specific immune response or a humoral antibody response when the antigen is presented in accordance with the present invention.
  • an epitope will include between about 3-15, generally about 5-15, amino acids.
  • Epitopes of a given protein can be identified using any number of epitope mapping techniques, well known in the art. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, N.J.
  • an "antigen” can be a subunit antigen, i.e., an antigen that is separate and discrete from a whole organism with which the antigen is associated in nature, as well as killed, attenuated or inactivated bacteria, viruses, parasites or other microbes.
  • Antibodies such as anti-idiotype antibodies, or fragments thereof, and synthetic peptide mimotopes, which can mimic an antigen or antigenic determinant, are also included as antigens.
  • antigens can be derived from any of several known viruses, bacteria, parasites and fungi, as well as any of the various tumor antigens.
  • excipient refers to substances that are commonly provided within finished dosage forms, and include vehicles (water, saline, buffer solutions, glycerol, polyethylene glycol, hyaluronic acid, ethanol, etc.), binders, disintegrants, fillers (diluents), lubricants, glidants (flow enhancers), compression aids, colors, sweeteners, preservatives, suspensing/dispersing agents, film formers/coatings, flavors and printing inks.
  • An "immunological response" to an antigen or a composition is the development in a subject of a humoral and/or a cellular immune response to molecules present in the antigen or composition of interest.
  • a “humoral immune response” refers to an immune response mediated by antibody molecules, while a “cellular immune response” is one mediated by T-lymphocytes and/or other white blood cells.
  • cytolytic T-cells CTLs
  • CTLs have specificity for peptide antigens that are presented in association with proteins encoded by the major histocompatibility complex (MHC) and expressed on the surfaces of cells. CTLs help induce and promote the intracellular destruction of intracellular microbes, or the lysis of cells infected with such microbes.
  • MHC major histocompatibility complex
  • Another aspect of cellular immunity involves an antigen- specific response by helper T-cells.
  • Helper T-cells act to help stimulate the function, and focus the activity, of nonspecific effector cells against cells displaying peptide antigens in association with MHC molecules on their surface.
  • a “cellular immune response” also refers to the production of cytokines, chemokines and other such molecules produced by activated T-cells and/or other white blood cells, including those derived from CD4+ and CD8+ T-cells.
  • a “T H 17 immune response” refers to a specialized cellular immune response mediated by T H 17 cells.
  • nucleic acid refers to DNA, RNA, as well as any of the known base analogs of DNA and RNA or chimeras formed therefrom.
  • peptide refers to a polymer of amino acid residues and is not limited to a minimum or maximum length of the product.
  • proteins, polypeptides, oligopeptides, dimers, multimers, and the like are included within the definition. Both full-length proteins and fragments thereof are encompassed by the definition.
  • the terms also include modifications, such as deletions, additions and substitutions (generally conservative in nature), to a native sequence, preferably such that the protein maintains the ability to elicit an immunological response within a subject to which the protein is administered.
  • a polypeptide is a continuous and unbranched peptide comprising twenty (20) or fifty (50) or more amino acids.
  • a "polynucleotide” is a nucleic acid polymer, which typically encodes a biologically active (e.g., immunogenic) protein or polypeptide. Depending on the nature of the polypeptide encoded by the polynucleotide, a polynucleotide can include as little as 10 nucleotides, e.g., where the polynucleotide encodes an antigen.
  • a "polynucleotide” can include both double- and single-stranded sequences and refers to, but is not limited to, cDNA from viral, prokaryotic or eukaryotic mR A, genomic R A and DNA sequences from viral (e.g. R A and DNA viruses and retroviruses) or prokaryotic DNA, and especially synthetic DNA sequences.
  • the term also captures sequences that include any of the known base analogs of DNA and RNA.
  • the term further includes modifications, such as deletions, additions and substitutions (generally conservative in nature), to a native sequence, preferably such that the nucleic acid molecule encodes, for example, an antigenic protein. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the antigens.
  • a "subject” refers to any organism to which a composition of this invention may be administered, e.g., for experimental, diagnostic, and/or therapeutic purposes. Typical subjects include mammals such as mice, rats, rabbits, non-human primates, and humans.
  • a second group of fusion proteins of the disclosure includes fusion proteins comprising (a) first polypeptide that includes a lipidated N-terminal cysteine and (b) a second polypeptide from an organism susceptible to host T H 17 immune response, wherein the fusion protein elicits a host T R 17 immune response to said organism or pathogen.
  • a fusion protein of the disclosure comprises or includes a polypeptide listed in Table 1 (or a portion of such polypeptide) or is encoded by a nucleotide sequence listed in Table 1.
  • fusion proteins of the second group do not include the N- terminal amino acids up to the cysteine that is part of the canonical lipobox domain (SEQ ID NO: 7).
  • SP0148 including signal sequence 2 6 NC_003028.3
  • SP2108 including signal sequence 3 NC_003028.3
  • E. coli RlpB signal sequence 9 - - *NB The database sequence incorrectly lists TTG (encoding Leu) at nucleotide positions 541-543.
  • the correct sequence as shown in SEQ ID NO: 6, has TTC at that codon and encodes Phe.
  • the database sequence further does not include a C-terminal Glu found in certain isolates.
  • SP0148 SEQ ID NO: 2 and variants thereof
  • the protein SP0148 is named "ABC transporter, substrate-binding protein”.
  • SP0148 is typically extracellular proteins that interact transiently with a transmembrane protein complex. Such complexes use energy generated by ATP hydrolysis to translocate specific substrates across a cell membrane.
  • SP0148 is a 276 or 277 (depending on the isolate) amino acid protein that contains a conserved PBPb (periplasmic binding protein) domain, spanning amino acids 40-246, which is typical of membrane-bound transport complexes.
  • PBPb peripheral binding protein domain
  • SP0148 has a bacterial extracellular solute -binding proteins family 3 domain which is largely co-extensive with the PBPb domain and extends from amino acid 40 to 244.
  • a fusion protein comprises a truncation mutant of SP0148 comprising or lacking one or more of said domains and motifs as a first polypeptide, albeit preserving at least one lipidatable cysteine.
  • a fusion protein comprises a first polypeptide and a second polypeptide wherein the first polypeptide comprises an S. pneumoniae polypeptide.
  • the S. pneumoniae polypeptide comprises at least 20 consecutive amino acid residues of SP0148.
  • a first polypeptide may also be a variant of the at least 20 amino acid residue fragment.
  • a first polypeptide includes no more than 250, 275, 200, 175, 150, 125, or 100 consecutive amino acids of SP0148.
  • the amino acids N-terminal to the cysteine that is part of the lipobox domain are not present (e.g., are cleaved).
  • a first polypeptide comprises a signal sequence.
  • a first polypeptide described herein comprises a signal sequence from SP0148 comprising SEQ ID NO: 1.
  • Endogenous SP0148 comprises a signal sequence that directs its secretion and potential lipidation.
  • the signal sequence of the polypeptide of SEQ ID NO: 2 is partially or fully processed by an expression host, e.g. E. coli.
  • a fusion protein described herein includes an SP0148 variant that comprise an exogenous lipidation sequence.
  • a signal sequence directs lipidation.
  • the lipidation signal may be, e.g., the signal sequence of SP2108 (SEQ ID NO: 8) or SP0148, or an E. coli signal sequence (SEQ ID NO: 9).
  • a signal sequence is cleaved, leaving the cysteine that is part of the lipobox motif as the N-terminus of the first polypeptide.
  • Variants of the amino acid sequence and nucleotide sequence of SP0148 may be found in U.S. Patent Application Publication No. 2005/0020813, U.S. Patent Nos. 7,378,514 and 7,504,110, and European Patent Application Nos. EP1572868 and EP1855717.
  • a fusion protein includes a first polypeptide having an amino acid sequence comprising, or consisting of, SEQ ID NO: 4, or a fragment thereof (e.g., in place of a
  • residues 1-23 are not present, leaving cysteine as the N-terminal residue.
  • SP2108 SEQ ID NO: 3
  • the polypeptide SP2108 is 423 amino acids in length and is alternatively known as MalX, maltose/maltodextrin ABC transporter, or maltose/maltodextrin-binding protein. Much of the protein (amino acids 3-423) is classified as a MalE (Maltose-binding periplasmic) domain.
  • SP2108 contains a signal sequence that directs its secretion and potential lipidation.
  • the signal sequence of the polypeptide of SEQ ID NO: 3 is partially or fully processed by an expression host, e.g. E. coli.
  • a fusion protein described herein includes an SP2108 variant that comprise an exogenous lipidation sequence.
  • a signal sequence directs lipidation.
  • a first polypeptide of a fusion protein is a truncation mutant of SP2108 comprising one or more of said domains and motifs as a first polypeptide, albeit preserving at least one lipidatable cysteine.
  • a fusion protein e.g., a lipidated fusion protein described herein comprises a first polypeptide from S. pneumoniae comprising at least 20 consecutive amino acid residues of SP2108.
  • a first polypeptide may also be a variant of the at least 20 amino acid residue fragment.
  • a first polypeptide includes no more than 250, 275, 200, 175, 150, 125, or 100 consecutive amino acids of SP2108.
  • the amino acids N-terminal to the cysteine that is part of the lipobox domain are not present (e.g., are cleaved).
  • a first polypeptide described herein comprises a signal sequence from SP2108 comprising SEQ ID NO: 8.
  • a SP2108 signal sequence comprises one or more intrinsic cysteine lipidation sites.
  • a first polypeptide has an amino acid sequence comprising, or consisting of, SEQ ID NO: 5, or an immunogenic fragment thereof (e.g., in place of a first polypeptide having an amino acid sequence comprising SEQ ID NO: 3).
  • a fusion protein described herein does not include residues 1-24, and cysteine is the N-terminal residue.
  • a first polypeptide includes a cysteine.
  • the N-terminus of a first polypeptide is a cysteine.
  • the length of a first polypeptide can range from 1-500 or more residues.
  • examples of a first polypeptide are from 10 to 500 residues, such as 15 to 100 or 150 or 200 or 250 residues, or more specifically from 20 to 30 residues.
  • a first polypeptide are from 1 to 100 residues, such as 1 to 20 or 25 or 30 or 40 or 50, or more specifically from 1 to 10 residues.
  • a first polypeptide of a fusion protein is membrane-associated.
  • membrane-associated means that the polypeptide can be found in a biological membrane in its native environment.
  • a first polypeptide described herein comprises RlpB signal sequence from E. coli comprising SEQ ID NO: 9.
  • residues 1-18 are not present, leaving cysteine as the N-terminal residue.
  • a first polypeptide alone does not elicit a host T H 17 immune response.
  • a first polypeptide is lipidated.
  • a first polypeptide is lipidated at the cysteine of the lipobox motif (SEQ ID NO: 7).
  • lipid moieties are covalently attached.
  • Lipid moieties are generally fatty acids. Lipidation sites contain a cysteine residue which is the site of lipid attachment.
  • the cysteine is modified with a diacyl glycerol attached by a thioether linkage to the sulfur of the cysteine side chain and an amide-linked fatty acid to the new amino terminus.
  • lipidated proteins are structurally divided into two groups, diacylated or triacylated lipoproteins, by the absence or presence of an amide-linked fatty acid.
  • S-palmitoylation is the covalent attachment of palmitic acid to the cysteine residue through a thioester bond.
  • a first polypeptide can be lipidated by one or more lipids, such as two lipid moieties or three lipid moieties.
  • the lipids are typically attached to the polypeptide via a glycerol group and the glycerol forms a thioether with a cysteine of a first polypeptide.
  • the third lipid is attached to the N-terminal amine of a first polypeptide, where the N-terminal residue is typically cysteine.
  • Suitable lipid moieties include, palmitate, stearate, myristate,
  • a second polypeptide of a fusion protein described herein comprises a polypeptide from an organism susceptible to a host T H 17 immune response.
  • the organism is a pathogen.
  • the organism is a host, such that the second polypeptide can be associated with a tumor cell or an autoimmune disorder.
  • a second polypeptide lacks an intrinsic lipidation site.
  • the length of a second polypeptide can range from 1-500 or more residues.
  • Examples of a second polypeptide are from 10 to 500 residues, such as 15 to 100 or 150 or 200 or 250 residues, or more specifically from 20 to 30 residues.
  • a second polypeptide is not expressed by or derived from
  • a second polypeptide described herein is from Staphylococcus aureus, Klebsiella pneumoniae, Cibrobacter rodentium, any species of Candida, (e.g. C. albicans, C. glabrata, C. rugosa, C. parapsilosis, C. dubliniensis, or C.
  • Enterococcus faecium Chlamydia trachomatis, Chlamydia pneumoniae, Neisseria gonorrhoeae, or Pneumocystis carinii.
  • a second polypeptide induces a host mucosal immune response. In some embodiments, a second polypeptide induces a host dermal immune response. In some embodiments, a second polypeptide induces a systemic mucosal immune response. In some embodiments, a second polypeptide induces any combination of mucosal, dermal, or systemic immune response. In some embodiments, a second polypeptide inhibits colonization. In some embodiments, a second polypeptide induces IL-17A. In some embodiments, a second polypeptide induces IL-22. [0061] Representative second polypeptides and heterologous fusion proteins comprising a first polypeptide from SP2108 are listed in Table 2.
  • the Staphylococcus aureus protein IsaA (SEQ ID NO: 10) is a 20 kDa cell wall/secreted protein involved in transglycosylation.
  • the Staphylococcus aureus protein SceD (SEQ ID NO: 13) is a 25 kDa cell secreted protein also involved in transglycosylation.
  • the Chlamydia trachomatis protein CT144 is a predicted 25 kDa transmembrane protein.
  • compositions containing one or more fusion proteins described herein also provides pharmaceutical compositions containing one or more fusion proteins described herein.
  • all or a proportion of fusion proteins can be lipidated in a composition.
  • at least 0.1% of fusion proteins have a lipid moiety covalently attached.
  • at least 0.2%, 0.3%, 0.4%, 0.5%, 1%, 5% or 10% of fusion proteins contain a lipid moiety.
  • a pharmaceutical composition contains 0.1 %- 10%> or 0.1%>-25%> lipidated fusion proteins. 1. Additional Adjuvants
  • adjuvants for example, pertussis toxin, stimulate mucosal immune responses, which are particularly important in defense against organisms entering through the digestive or respiratory tracts.
  • adjuvants are derived from bacterial components.
  • Freund's complete adjuvant an adjuvant that is widely used in animal experiments, comprises killed Mycobacterium tuberculosis suspended in oil.
  • Freund's complete adjuvant has severe associated side effects and has therefore not been approved for use in humans.
  • other bacterial adjuvants such as killed Bordetella pertussis, bacterial polysaccharides, bacterial heat-shock proteins, and bacterial DNA have been found unsuitable for use in vaccines for humans.
  • lipidated fusion proteins and immunogenic compositions described herein include an adjuvant.
  • Adjuvants can be broadly separated into two classes, based on their principal mechanisms of action: vaccine delivery systems and immunostimulatory adjuvants (see, e.g., Singh et ah, Curr. HIV Res. 1 :309-20, 2003). In most vaccine
  • the adjuvant provides a signal to the immune system so that it generates a response to the antigen, and the antigen is required for driving the specificity of the response to the pathogen.
  • Vaccine delivery systems are often particulate formulations, e.g., emulsions, microparticles, immune-stimulating complexes (ISCOMs), nanoparticles, which may be, for example, particles and/or matrices, and liposomes.
  • adjuvants may be classified as organic and inorganic.
  • Inorganic adjuvants include alum salts such as aluminum phosphate, amorphous aluminum
  • Organic adjuvants comprise organic molecules including macromolecules.
  • An example of an organic adjuvant is cholera toxin.
  • Adjuvants may also be classified by the response they induce.
  • the adjuvant induces the activation of T R I cells or T R 2 cells. In other words, the adjuvant induces the activation of T R I cells or T R 2 cells.
  • the adjuvant induces the activation of B cells. In yet other embodiments, the adjuvant induces the activation of antigen-presenting cells. These categories are not mutually exclusive; in some cases, an adjuvant activates more than one type of cell.
  • the adjuvant induces the activation of T R 17 cells. It may promote the CD4 + or CD8 + T cells to secrete IL-17, IL-22, or other cytokines.
  • an adjuvant that induces the activation of T H 17 cells is one that produces at least a 2-fold, and in some cases a 10-fold, experimental sample to control ratio in the following assay. In the assay, an experimenter compares the IL-17, IL-22, or other cytokine levels secreted by two populations of cells: (1) cells from animals immunized with the adjuvant and a polypeptide known to induce T H 17 activation, and (2) cells from animals treated with the adjuvant and an irrelevant (control) polypeptide.
  • An adjuvant that induces the activation of T R 17 cells may cause the cells of population (1) to produce more than 2-fold, or more than 10-fold more IL-17, IL-22, or other cytokine than the cells of population (2).
  • Cytokines may be measured, for example, by ELISA or ELISPOT.
  • the adjuvant is a toxin. Cholera toxin was successfully used in the mouse model to induce protective immunity in conjunction with certain polypeptides from Table 1. One form of labile toxin is produced by Intercell.
  • Mutant derivates of labile toxin that are active as adjuvants but significantly less toxic can be used as well.
  • exemplary detoxified mutant derivatives of labile toxin include mutants lacking ADP-ribosyltransferase activity.
  • Particular detoxified mutant derivatives of labile toxin include LTK7 (Douce et al, "Mutants of Escherichia coli heat-labile toxin lacking ADP-ribosyltransferase activity act as nontoxic, mucosal adjuvants" PNAS Vol. 92, pp.
  • LTK63 Bacilliams et al, "Innate Imprinting by the Modified Heat-Labile Toxin of Escherichia coli (LTK63) Provides Generic Protection against Lung Infectious Disease” The Journal of Immunology, 2004, 173: 7435-7443), LT-G192 (Douce et al. "Genetically detoxified mutants of heat-labile toxin from Escherichia coli are able to act as oral adjuvants" Infect Immun.
  • LTR72 Mucosal adjuvanticity and immunogenicity of LTR72, a novel mutant of Escherichia coli heat-labile enterotoxin with partial knockout of ADP-ribosyltransferase activity. J Exp Med. 1998 Apr 6; 187(7): 1123-32).
  • the adjuvant comprises a VLP (virus-like particle).
  • VLP virus-like particle
  • Alphavirus replicons induces the activation of T H 17 cells using alphavirus and is produced by Alphavax.
  • alphavirus may be engineered to express an antigen of interest, a cytokine of interest (for example, IL-17 or a cytokine that stimulates IL-17 production), or both, and may be produced in a helper cell line. More detailed information may be found in U.S. Patent Nos. 5,643,576 and 6,783,939.
  • a vaccine formulation is administered to a patient in combination with a nucleic acid encoding a cytokine.
  • TLRs toll-like receptors
  • TLRs are well known proteins that may be found on leukocyte membranes, and recognize foreign antigens (including microbial antigens).
  • Administering a known TLR ligand together with an antigen of interest can promote the development of an immune response specific to the antigen of interest.
  • One exemplary adjuvant that activates TLRs comprises Monophosphoryl Lipid A (MPL).
  • MPL has been produced as a detoxified lipopolysaccharide (LPS) endotoxin obtained from gram negative bacteria, such as S. minnesota.
  • LPS lipopolysaccharide
  • sequential acid and base hydrolysis of LPS produces an immunoactive lipid A fraction (which is MPL), and lacks the saccharide groups and all but one of the phosphates present in LPS.
  • TLR-4 agonists are disclosed in Evans JT et al. "Enhancement of antigen-specific immunity via the TLR-4 ligands MPL adjuvant and Ribi.529.” Expert Rev Vaccines 2003 Apr;2(2):219-29.
  • TLR agonists include TLR-4 agonists.
  • TLR-4 agonists have been produced and/or sold by, for example, the Infectious Disease Research Institute (IRDI), Corixa, Esai, Avanti Polar Lipids, Inc., and Sigma Aldrich.
  • TLR-activating adjuvant comprises a mixture of MPL, Trehalose Dicoynomycolate (TDM), and dioctadecyldimethylammonium bromide (DDA).
  • TDM Trehalose Dicoynomycolate
  • DDA dioctadecyldimethylammonium bromide
  • R848 resiquimod
  • the adjuvant is or comprises a saponin.
  • the saponin is a triterpene glycoside, such as those isolated from the bark of the Quillaja saponaria tree.
  • a saponin extract from a biological source can be further fractionated (e.g., by
  • combinations of adjuvants are used.
  • Three exemplary combinations of adjuvants are MPL and alum, E6020 and alum, and MPL and an ISCOM.
  • a composition described herein e.g., a pharmaceutical composition
  • a composition described herein comprising a lipidated fusion protein includes one or more additional components.
  • an immunogenic lipidated fusion protein composition includes one or more polypeptides from the same organism as a second polypeptide, particularly an antigenic polypeptide.
  • a composition includes one or more polypeptides from Staphylococcus aureus, Klebsiella pneumoniae, Cibrobacter rodentium, any species of Candida, e.g. C. albicans, C. glabrata, C. rugosa, C. parapsilosis, C. dubliniensis, or C.
  • an immunogenic composition includes one or more stabilizers such as sugars (such as sucrose, glucose, or fructose), phosphate (such as sodium phosphate dibasic, potassium phosphate monobasic, dibasic potassium phosphate, or
  • monosodium phosphate glutamate (such as monosodium L-glutamate), gelatin (such as processed gelatin, hydrolyzed gelatin, or porcine gelatin), amino acids (such as arginine, asparagine, histidine, L-histidine, alanine, valine, leucine, isoleucine, serine, threonine, lysine, phenylalanine, tyrosine, and the alkyl esters thereof), inosine, or sodium borate.
  • amino acids such as arginine, asparagine, histidine, L-histidine, alanine, valine, leucine, isoleucine, serine, threonine, lysine, phenylalanine, tyrosine, and the alkyl esters thereof
  • inosine or sodium borate.
  • an immunogenic composition includes one or more surfactants such as polysorbate 80 (Tween 80), Triton X-100, Polyethylene glycol tert- octylphenyl ether t-Octylphenoxypolyethoxyethanol 4-(l,l,3,3-Tetramethylbutyl)phenyl- polyethylene glycol (TRITON X-100), Polyoxyethylenesorbitan monolaurate Polyethylene glycol sorbitan monolaurate (TWEEN 20), n-Tetradecyl-N,N-dimethyl-3-ammordo-l-propanesulfonate (Zwittergent3-14), and 4-(l,l,3,3-Tetramethylbutyl)phenol polymer with formaldehyde and oxirane (TYLOXAPOL).
  • a surfactant can be ionic or nonionic.
  • an immunogenic composition includes one or more salts such as sodium chloride, ammonium chloride, calcium chloride, or potassium chloride.
  • an immunogenic composition is a controlled release formulation.
  • fusion proteins described herein serve as adjuvants or as self-adjuvanting antigens. Fusion proteins described herein may stimulate an antibody response or a cell-mediated immune response, or both, in the mammal to which it is administered.
  • a composition stimulates a T H 1 -biased CD4 + T cell response, and/or a CD8 T cell response.
  • the composition stimulates an antibody response.
  • the composition stimulates a T R I -biased CD4 + T cell response, T H 17-biased CD4 + T cell response and/or a CD8 T cell response, and an antibody response.
  • a fusion protein that elicits a T H 17 immune response is one that activates T R 17 cells at least a 2-fold, 5 fold, 10 fold or 100 fold or more. In some embodiments, T R 17 cell activation is determined relative to untreated cells. In some
  • T R 17 cell activation is determined relative cells treated with a control polypeptide or fusion protein.
  • T H 17 cell activation comprises an increase in cytokine levels.
  • T R 17 cell activation comprises an increase in IL-17.
  • T R 17 cell activation comprises an increase in IL-22.
  • an experimenter compares the IL-17, IL-22, or other cytokine levels secreted by two populations of cells: (1) cells from animals immunized with a fusion protein described herein, and (2) cells from animals not treated with the fusion protein.
  • a fusion protein that induces the activation of T H 17 cells may cause the cells of population (1) to produce more than 2-fold, or more than 10-fold more IL-17, IL-22, or other cytokine than the cells of population (2).
  • Cytokines may be measured, for example, by ELISA or ELISPOT.
  • a T H 17 cell response is desirable in mounting an immune response to a composition (e.g., a pharmaceutical composition) disclosed herein.
  • an active T R 17 response is beneficial in clearing a pneumococcal infection.
  • an active T H 17 response is beneficial in clearing an infection in any one of the following organisms: Staphylococcus aureus, Klebsiella pneumoniae, Cibrobacter rodentium, any species of Candida, (e.g. C. albicans, C. glabrata, C. rugosa, C. parapsilosis, C. dubliniensis, or C.
  • Moraxella catarrhalis non-typable Haemophilus influenzae, Listeria monocytogenes, Salmonella enterica, Mycobacterium tuberculosis, Bordetella pertussis, Escherichia coli, Mycoplasma pneumoniae, Porphyromonas gingivalis, Mycobacterium bovis, Cryptococcus neoformans, Group A, B, or C Streptococcus, Enterococcus faecalis, Enterococcus faecium, Chlamydia trachomatis, Chlamydia pneumoniae, Neisseria gonorrhoeae, or Pneumocystis carinii.
  • a composition described herein is administered to a subject who has not been previously exposed to the organism which elicits an active T H 17 response. In certain embodiments, the subject has been previously exposed to the organism which elicits an active T H 17 response.
  • an active T H 17 response is beneficial in inducing a dermal, mucosal and/or systemic immune response in a subject.
  • an active T R 17 response is beneficial for inhibiting colonization in a subject. Colonization by any of the following organisms can be inhibited: Staphylococcus aureus, Klebsiella pneumoniae,
  • Cibrobacter rodentium any species of Candida, (e.g. C. albicans, C. glabrata, C. rugosa, C. parapsilosis, C. dubliniensis, or C. tropicalis), Moraxella catarrhalis, non-typable Haemophilus influenzae, Listeria monocytogenes, Salmonella enterica, Mycobacterium tuberculosis,
  • Bordetella pertussis Escherichia coli, Mycoplasma pneumoniae, Porphyromonas gingivalis, Mycobacterium bovis, Cryptococcus neoformans, Group A, B, or C Streptococcus, Enterococcus faecalis, Enterococcus faecium, Chlamydia trachomatis, Chlamydia pneumoniae, Neisseria gonorrhoeae, or Pneumocystis carinii. [0086] In addition, herein is provided a method of increasing IL-17 production by administering the compositions described herein to a subject.
  • this application provides a method of activating T H 17 cells by administering said compositions to a subject.
  • increased IL-17A levels result in increased killing of a pathogen, a tumor cell or a cell associated with an autoimmune condition by neutrophils or neutrophil-like cells, for instance by inducing recruitment and activation of neutrophils of neutrophil-like cells.
  • this pneumococcal killing is independent of antibodies and complement.
  • the amount of a fusion protein in a composition to be administered to a subject is typically selected as an effective amount, which induces a prophylactic or therapeutic response, as described above, in either a single dose or over multiple doses.
  • the dose is without significant adverse side effects.
  • a dose will comprise 1-1000 ⁇ g of each protein, in some instances 2-100 ⁇ g, for instance 4-40 ⁇ g.
  • the vaccine formulation comprises 1-1000 ⁇ g of the polypeptide and 1-250 ⁇ g of the adjuvant.
  • the appropriate amount of antigen to be delivered will depend on the age, weight, and health (e.g. immunocompromised status) of a subject. When present, typically an additional adjuvant will be present in amounts from 1 ⁇ g - 250 ⁇ g per dose, for example 50-150 ⁇ g, 75-125 ⁇ g or 100 ⁇ g.
  • compositions are administered in combination with antibiotics. This co-administration is particularly appropriate when the pharmaceutical composition is administered to a patient who has recently been exposed (or is suspected of having been recently exposed) to a pathogen.
  • antibiotics can be used to treat infections, including penicillin, amoxicillin, amoxicillin/clavulanate, cefuroxime, cefotaxime, ceftriaxone, and vancomycin.
  • the appropriate antibiotic may be selected based on the type and severity of the infection, as well as any known antibiotic resistance of the infection.
  • Fusion protein formulations and pharmaceutical compositions described herein can be delivered by administration to an individual, such as by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, intradermal, subcutaneous, subdermal, transdermal, intracranial, intranasal, mucosal, anal, vaginal, oral, buccal route or they can be inhaled) or they can be administered by topical application.
  • systemic administration e.g., intravenous, intraperitoneal, intramuscular, intradermal, subcutaneous, subdermal, transdermal, intracranial, intranasal, mucosal, anal, vaginal, oral, buccal route or they can be inhaled
  • the route of administration is intramuscular.
  • the route of administration is subcutaneous.
  • the route of administration is mucosal.
  • the route of administration is transdermal or intradermal
  • Fusion protein compositions described herein may be suitable for administration to a human patient, and vaccine or immunogenic composition preparation may conform to USFDA guidelines.
  • the composition is suitable for administration to a non-human animal.
  • the vaccine or immunogenic composition is substantially free of either endotoxins or exotoxins.
  • Endotoxins may include pyrogens, such as lipopolysaccharide (LPS) molecules.
  • LPS lipopolysaccharide
  • the vaccine or immunogenic composition may also be substantially free of inactive protein fragments which may cause a fever or other side effects.
  • the composition contains less than 1%, less than 0.1%, less than 0.01%, less than 0.001%, or less than 0.0001% of endotoxins, exotoxins, and/or inactive protein fragments.
  • the vaccine or immunogenic composition has lower levels of pyrogens than industrial water, tap water, or distilled water.
  • Other composition components may be purified using methods known in the art, such as ion-exchange chromatography, ultrafiltration, or distillation.
  • the pyrogens may be inactivated or destroyed prior to administration to a patient.
  • Raw materials for vaccines such as water, buffers, salts and other chemicals may also be screened and depyrogenated. All materials in the vaccine may be sterile, and each lot of the vaccine may be tested for sterility.
  • the endotoxin levels in the vaccine fall below the levels set by the USFDA, for example 0.2 endotoxin (EU)/kg of product for an intrathecal injectable composition; 5 EU/kg of product for a non-intrathecal injectable composition, and 0.25-0.5 EU/mL for sterile water.
  • EU endotoxin
  • the preparation comprises less than 50%>, 20%>, 10%>, or
  • the desired molecule is present in the substantial absence of other biological macromolecules, such as other proteins (particularly other proteins which may substantially mask, diminish, confuse or alter the characteristics of the component proteins either as purified preparations or in their function in the subject reconstituted mixture).
  • other biological macromolecules such as other proteins (particularly other proteins which may substantially mask, diminish, confuse or alter the characteristics of the component proteins either as purified preparations or in their function in the subject reconstituted mixture).
  • the composition comprising purified subunit proteins contains less than 5%, 2%, 1%, 0.5%, 0.2%, 0.1% of protein from host cells in which the subunit proteins were expressed, relative to the amount of purified subunit.
  • the desired polypeptides are substantially free of nucleic acids and/or carbohydrates.
  • the composition contains less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.2%, or less than 0.1% host cell DNA and/or R A.
  • At least 80%), 90%), 95%o, 99%o, or 99.8%> (by dry weight) of biological macromolecules of the same type are present in the preparation (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 5000, can be present).
  • the composition has low or no toxicity, within a reasonable risk-benefit ratio.
  • the composition comprises ingredients at
  • a composition, and any component within it might have an LD 50 value in rats of greater than 100 g/kg, greater than 50g/kg, greater than 20 g/kg, greater than 10 g/kg, greater than 5 g/kg, greater than 2 g/kg, greater than 1 g/kg, greater than 500 mg/kg, greater than 200 mg/kg, greater than 100 mg/kg, greater than 50 mg/kg, greater than 20 mg/kg, or greater than 10 mg/kg.
  • a composition that comprises a toxin such as botulinum toxin (which can be used as an adjuvant) should contain significantly less than the LD 50 of botulinum toxin.
  • formulations suitable for introduction of a composition vary according to route of administration.
  • routes of administration such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, , transdermal, intravaginal, intrarectal, intraperitoneal, intranasal, transcutaneous, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • the cells can be administered intravenously or parenterally.
  • Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the polypeptides or packaged nucleic acids suspended in diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a
  • Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, tragacanth, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes,
  • Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.
  • the pharmaceutical compositions can be encapsulated, e.g., in liposomes, or in a formulation that provides for slow release of the active ingredient.
  • a fusion protein described herein, alone or in combination with other suitable components, can be made into aerosol formulations (e.g., they can be "nebulized") to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. Aerosol formulations can be delivered orally or nasally.
  • pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen, and the like. Aerosol formulations can be delivered orally or nasally.
  • Suitable formulations for vaginal or rectal administration include, for example, suppositories, which consist of the polypeptides or packaged nucleic acids with a suppository base.
  • Suitable suppository bases include natural or synthetic triglycerides or paraffin
  • gelatin rectal capsules which consist of a combination of the polypeptides or packaged nucleic acids with a base, including, for example, liquid triglycerides, polyethylene glycols, and paraffin hydrocarbons.
  • Fusion proteins and compositions described herein may be produced using a variety of techniques.
  • a polypeptide e.g., a first polypeptide and/or a second polypeptide described herein
  • a suitable host cell may be bacterial, yeast, mammalian, insect, or other type of cell.
  • the host cell may be genetically engineered to express an exogenous copy of a nucleic acid sequence encoding a fusion protein, using known techniques.
  • a nucleic acid sequence encoding a fusion protein is operably linked to appropriate regulatory sequences such as a strong promoter and a polyadenylation sequence.
  • the promoter is inducible or repressible.
  • Other regulatory sequences may provide for secretion or excretion of a fusion protein or retention of a fusion protein in the cytoplasm or in the membrane, depending on how one wishes to purify the fusion protein.
  • the nucleic acid encoding a fusion protein may be present on an extrachromosomal plasmid, or may be integrated into the host genome.
  • One of skill in the art will recognize that it is not necessary to use a nucleic acid 100% identical to the naturally-occurring sequence. Rather, some alterations to these sequences are tolerated and may be desirable. For instance, the nucleic acid may be altered to take advantage of the degeneracy of the genetic code such that the encoded polypeptide remains the same.
  • a nucleic acid sequence encoding a fusion protein is codon-optimized to improve expression in a particular host.
  • the nucleic acid may be produced, for example, by PCR or by chemical synthesis.
  • a fusion protein may be isolated from it.
  • the isolation may be accomplished, for example, by affinity purification techniques or by physical separation techniques (e.g., a size column).
  • a first polypeptide and a second polypeptide described herein are recombinantly produced in cells, purified, and are conjugated or joined, e.g., using chemical means known in the art.
  • a fusion protein is produced synthetically.
  • fusion proteins for inclusion in the compositions may be produced in cell culture.
  • One method comprises providing one or more expression vectors and cloning nucleotides encoding one or more polypeptides disclosed herein, then expressing and isolating the polypeptides.
  • the isolated polypeptides are incorporated into a microparticle or a nanoparticle.
  • fusion proteins for inclusion in the compositions are not purified.
  • an expression vector comprising nucleotides encoding one or more polypeptides disclosed herein (e.g., a fusion protein) is used as a delivery system and in some cases administered as plasmid DNA.
  • Fusion proteins described herein can be packaged in packs, dispenser devices, and kits for administering nucleic acid compositions to a mammal.
  • packs or dispenser devices that contain one or more unit dosage forms are provided.
  • instructions for administration of the compounds will be provided with the packaging, along with a suitable indication on the label that the compound is suitable for treatment of an indicated condition, such as those disclosed herein.
  • Streptococcus pneumoniae were identified as antigens from proteomic screens using murine and human CD4+ T cells.
  • the antigens were shown to elicit a TH17 immune response as evidenced by induction of IL-17A, and to be protective in colonization challenge experiments in mice.
  • the antigens were also confirmed to be lipidated. Methods of the mouse screens, IL-17A assay, colonization assay and expression of the lipidated proteins are previously described
  • lipobox motif immediately upstream of an invariant cysteine residue.
  • the invariant cysteine serves as the attachment site for di- or tri-glyceride chains that confer mature lipoprotein conformation.
  • T H 17 immune response and colonization assays of C57BL/6 mice are previously described (WO2011/008548; Moffitt K. et al. (2011), T H 17-based vaccine design for prevention of Streptococcus pneumoniae colonization, Cell Host Microbe 9(2):pp. 158-65). Briefly, C57BL/6 mice were immunized twice one week apart with 4 ⁇ g of the indicated lipidated (SP2108, SP0148) or non-lipidated (SP2108Alipid, SP0148Alipid) antigen and 1 ⁇ g of CT in 10 ⁇ volumes instilled intranasally. Positive and negative controls were immunized, respectively, with killed, unencapsulated S. pneumoniae (WCB) and CT alone.
  • mice Two weeks following their last immunization, animals were bled; aliquots of whole blood were stimulated with WCB, and then culture supernatants were evaluated for IL-17A by ELISA. Animals were then challenged intranasally with 10 7 live type 6B pneumococci. One week following intranasal challenge, nasal washes were collected and pneumococcal CFU were counted after overnight culture on blood agar plates supplemented with gentamicin.
  • FIG. 1 shows representative results of T H 17 response and colonization assays, following intranasal immunization with lipidated and non-lipidated forms of SP2108 or SP0148 in the presence of CT adjuvant.
  • Systemic WCB-specific IL-17A values from immunized animals are shown in FIG. 1 A. Each symbol represents the response from a single animal, and lines represent the median value for the group.
  • CFU of bacteria per nasal lavage are shown in FIG. IB. Lines represent median values and p- values were calculated using Mann- Whitney test compared to CT immunized controls. Immunization with lipidated antigens resulted in 1-2 log higher IL-17A secretion compared with non-lipidated mutants, correlating with more significant protection from colonization.
  • lipidated protein The ability of a lipidated protein to induce or augment in trans a specific T H 17 immune response to a second, non-lipidated protein was tested by immunizing mice with combinations of proteins. Briefly, C57BL/6 mice were immunized subcutaneously three times, two weeks apart, with a non-lipidated protein (pneumolysoid (ply), SP1912; both previously shown to elicit weak or no T R 17 immune response), singly or in combination with a lipidated protein (SP2108, SP0148) adsorbed on 250 ⁇ g alum, in 100 ⁇ volumes. Negative controls were immunized with alum alone. Three weeks following their last immunization, animals were bled; aliquots of whole blood were stimulated with protein for six days and then culture supernatants were evaluated for IL-17A by ELISA.
  • pneumolysoid ply
  • SP1912 both previously shown to elicit weak or no T R 17 immune response
  • FIG. 3 shows representative results of T H 17 immune response assays following subcutaneous immunization with combinations of lipidated and non-lipidated proteins.
  • FIG. 3A shows systemic IL- 17A values from animals immunized with 10 ⁇ g lipidated SP0148, 10 ⁇ g non-lipidated pneumolysoid, or both.
  • FIG. 3B shows results from animals immunized with 1 ⁇ g lipidated SP2108, 10 ⁇ g non-lipidated SP1912, or both.
  • FIG. 3C shows results from animals immunized with 10 ⁇ g lipidated SP0148, 10 ⁇ g non-lipidated SP1912, or both.
  • 3C represents the response from a single animal, and lines represent the median value for the group.
  • Data in FIG. 3B are represented as the mean ⁇ SEM IL-17A concentration from four mice per group.
  • immunization with a combination of lipidated and non-lipidated proteins resulted in a median 15 to 40-fold increase in IL-17A secretion, following stimulation with the non-lipidated protein.
  • Example 3 Synthetic free lipopeptide combined with antigen does not elicit T H 1 immune response
  • a single-cell suspension of splenocytes was seeded at 200,000 cells per well, stimulated with OLPs and supernatants tested for the presence of IL-17A after six days.
  • FIG. 4 shows representative results of T H 17 immune response assays following subcutaneous immunization with combinations of SP 1912, the synthetic free lipopeptide
  • FIG. 4A shows IL-17A values in blood.
  • FIG. 4B shows IL- 17A values in splenocytes. Each symbol represents the IL-17A response of an individual mouse, and lines represent the median value for the group. Tables below each graph indicated the ⁇ g dose of Pam3CSK4. Immunization with the combination of SP1912 and SP0148 or SP2108 proteins on alum primed SP1912-specific T H 17 responses as expected. Combining SP1912 with various doses of Pam3CSK4 on alum did not reproduce this effect. Therefore, the in trans effect on T H 17 immune response requires that a non-lipidated protein such as SP 1912 be combined with lipidated protein.
  • T H 17 immune response is believed to be enhanced by upstream activation of Toll-Like Receptor 2 (TLR2) and Toll-Like Receptor 4 (TLR4) (JLeukoc Biol. 2010 July; 88(1): 5-7; doi: 10.1189/ilb.0110057).
  • TLR2 Toll-Like Receptor 2
  • TLR4 Toll-Like Receptor 4
  • HEK-TLR2 Human Embryonic Kidney(HEK)-TLR2 reporter system in which TLR2 activation is marked by release of IL-8
  • HEK-TLR2 Human Embryonic Kidney(HEK)-TLR2 reporter system in which TLR2 activation is marked by release of IL-8
  • HEK-TLR2 Human Embryonic Kidney(HEK)-TLR2 reporter system in which TLR2 activation is marked by release of IL-8
  • Figure 5 shows representative results of a TLR2 activation assay with IL-8 readout in HEK-TLR2 cells stimulated with lipidated and non-lipidated SP2108 and SP0148 proteins. Lines represent median values from at least four independent experiments. The lipidated proteins elicited robust IL-8 response in HEK-TLR2 cells, while non-lipidated mutants did not.
  • FIG. 6 shows representative results of TH17 immune response assays, following immunization of TLR2 _/" C57BL/6 mice (TLR2KO) or C57BL/6 (wild-type) with the indicated proteins on alum.
  • Systemic IL-17A values from immunized animals are shown as medians/IQR. P-values were calculated using the Mann- Whitney test.
  • Knocking TLR2 out resulted in 3.5 to 5- fold reduction of IL17-A secretion compared to wild-type animals, demonstrating that T R 17 immune responses induced by the proteins of immunization are TLR2-dependent.
  • SP0148 and SP2108 protein were analyzed to determine their lipid profiles. Briefly, the proteins were fractionated by reverse-phase HPLC (RP-HPLC), then subjected to electrospray injection mass spectroscopy (ESI-MS).
  • RP-HPLC reverse-phase HPLC
  • ESI-MS electrospray injection mass spectroscopy
  • Figure 7 shows representative results of electrospray ionization mass
  • ESI-MS spectrometry
  • lipids were first removed from the lipoproteins via acid catalyzed transmethylation and separated via solvent extraction prior to GC-MS analysis. As shown in Figure 8, among the more than half-dozen lipid chains identified, the predominant ones were palmitic acid (more than 50 % abundance), followed by stearic acid and myristic acid. This distribution of lipid chains is well within that reported previously for other exogenously expressed lipoproteins produced by E. coli hosts.
  • Example 6 Exemplary Chlamydia trachomatis proteins lipidated via heterologous lipidation sequences; effect on TH17 immune response and protection from colonization
  • Lipidated or non-lipidated recombinant proteins comprising either the signal sequence of SP2108 (SEQ ID NO: 8), SP0148 (SEQ ID NO: 1) or E. coli RlpB (SEQ ID NO: 9) or the same signal sequences in which the invariant cysteine residue required for lipidation is mutated to aspartate, are fused to Chlamydia trachomatis non-lipidated proteins CT242, CT144, CT062, CT572, CT043, CT725, CT067, CT788-tmr, CT328 or CT476 and expressed by standard methods.
  • C57BL/6 mice are immunized subcutaneously two to three times, two weeks apart, with lipidated and non- lipidated recombinant proteins adsorbed on alum. Negative controls are immunized with alum alone. Three weeks following their last immunization, animals are bled; aliquots of whole blood are stimulated with protein for six days and then culture supematants are evaluated for IL-17A by ELISA. Animals are then challenged with 10 6 -10 7 live type 6B pneumococci intranasally.
  • Example 7 Exemplary Staphyloccocus aureus proteins lipidated via heterologous lipidation sequences: effect on TH17 immune response.
  • Staphlococcus aureus strain USA300 FPR3757 fused either to the lipidation signal sequence of the naturally lipidated antigen SP2108 (Lipo IsaA: SEQ ID NO: 11) or to the complete sequence of SP2108 (SP2108_IsaA: SEQ ID NO: 12) were constructed and expressed by standard methods.
  • the proteins were prepared by affinity chromatography and FPLC, and refolded in the presence of 10% sucrose. The percent of lipidated proteins was determined using reversed phase-high performance liquid chromatography (RP-HPLC). Representative preparations of Lipo IsaA and SP2108_IsaA were 0.95%> and 3.2% lipidated respectively.
  • lipidated recombinant proteins described above and non-lipidated IsaA were used to immunize C57BL/6 mice.
  • Groups of four C57BL/6 mice were immunized subcutaneously either two times or three times, two weeks apart, with 200 pmol Lipo IsaA (SEQ ID NO: 11), SP2108_IsaA (SEQ ID NO: 12) or non-lipidated IsaA (SEQ ID NO: 10) adsorbed on 250 ⁇ g alum per dose.
  • Negative controls were immunized with proteins alone or alum alone.
  • IL-17A ELISPOT and IL-17 whole blood assay were determined by IL-17A ELISPOT and IL-17 whole blood assay (WBA), respectively.
  • Splenocytes were harvested, depleted of contaminating RBC and pulsed with lug/mL of a pool of overlapping peptides (OLPs) that span the complete IsaA sequence, or control OLPs.
  • OLPs overlapping peptides
  • the IsaA peptide pool contained 49 peptides consisting of 15-mer peptides with 13- amino acid overlap, covering the complete protein sequence.
  • the splenocytes were plated at 600,000 per well on PVDF membranes pre-coated with mouse anti-IL-17A capture antibody.
  • FIG. 9 shows representative results of T H 17 response assays following immunization with two doses of the indicated proteins absorbed to alum. Each bar represents the mean number of IL-17A spot forming units (SFU) per million splenocytes ⁇ SD of the group of four mice.
  • Immunization with either Lipo IsaA or SP2108_IsaA adsorbed to alum increased the frequency of IsaA-specific T H 17 cells 2-fold or 18-fold, respectively, compared to immunization with native non-lipidated IsaA.
  • FIG. 10 shows representative results of T H 17 response assays following immunization with three doses of the indicated proteins absorbed to alum. Each bar represents the mean number of IL-17A spot forming units (SFU) per million splenocytes ⁇ SD of the group of four mice. Immunization with either Lipo IsaA or SP2108_IsaA adsorbed to alum increased the frequency of IsaA-specific T H 17 cells 20-fold or 16-fold, respectively, compared to immunization with native non-lipidated IsaA.
  • SFU spot forming units
  • SP0148 including signal sequence (277 amino acids with C-terminal E)
  • maltose/maltodextrin-binding protein (Streptococcus pneumoniae TIGR4)
  • SP0148 including signal sequence (nucleotides)

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Abstract

L'invention concerne des protéines de fusion et des compositions pharmaceutiques qui élicitent un hôte de TH17. Les protéines de fusion peuvent soumises à une lipidation. Les protéines de fusion sont utilisées en tant qu'adjuvants ou antigènes auto-adjuvants. L'invention concerne également des procédés pour éliciter ou augmenter une réponse d'hôte de TH17 au moyen de protéines de fusion.
PCT/US2013/029907 2012-03-09 2013-03-08 Induction de réponse immunitaire de th17 WO2013134656A1 (fr)

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