WO2013134656A1 - Induction of th17 immune response - Google Patents

Description

INDUCTION OF T_H17 IMMUNE RESPONSE

Cross Reference to Related Applications

[0001] The present application claims the benefit of U.S. provisional application Serial

No. 61/609,171, filed on March 9, 2012, the contents of which are herein incorporated by reference in their entirety.

Background

[0002] 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.

[0003] 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. Among the 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_H17 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. [0004] Numerous studies indicate that T_H17 cells play important roles in host defense, promoting pathogen clearance and limiting pathogen dissemination. Effects of T_H17-mediated immune response have 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 Defence Against Bacterial Mycobacterial and Fungal Pathogens, Immunology 126: pp. 177-185). Accordingly, compositions capable of inducing T_H17-mediated immune responses against Streptococcus pneumonia are desirable.

Summary

[0005] Surprisingly, it has been found that the ability of certain antigens to elicit a T_R17 response is dependent on lipidation. More surprisingly, it has been found that certain lipidated polypeptides serve as adjuvants for a T_H17 response. This elicited T_H17 response is believed to be useful in protecting subjects from colonization or otherwise increasing the immune response.

[0006] In certain aspects, the present disclosure provides a fusion protein comprising a first polypeptide having an amino acid sequence comprising SEQ ID NO: 7 and a second polypeptide from an organism susceptible to host T_H17 immune response, wherein the fusion protein elicits a host T_H17 immune response to said organism or pathogen. In some

embodiments, the fusion protein is lipidated. In some embodiments, the first polypeptide is fused to the N-terminus of the second polypeptide. In some embodiments, the first polypeptide comprises an exogenous signal sequence. In some embodiments, the first polypeptide is membrane-associated. In some embodiments, the second polypeptide does not include a lipobox motif.

[0007] In another aspect, 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_H17 immune response, wherein the fusion protein elicits a host T_R17 immune response to said organism or pathogen. [0008] In some embodiments, 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. In some embodiments, 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. In some embodiments, the composition further comprises a peptide or polypeptide from the same organism or pathogen as the second polypeptide.

[0009] In some embodiments, 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.

[0010] In another aspect, the present disclosure provides a method of eliciting or increasing a host T_H17 immune response, comprising administering to a subject in need thereof a fusion protein described herein, thereby eliciting or increasing a T_R17 immune response.

[0011] In another aspect, the present disclosure provides methods of making a fusion protein described herein. In some embodiments, the method comprises expressing in a host cell a nucleic acid encoding a fusion protein described herein. In some embodiments, the method further comprises isolating or purifying the fusion protein. In some embodiments, the method comprises producing the fusion protein synthetically (e.g., chemically). In some embodiments, 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.

[0012] In another aspect, the present disclosure provides fusion proteins produced by a method described herein. Brief Description of the Drawing

[0013] The present teachings described herein will be more fully understood from the following description of various illustrative embodiments, when read together with the accompanying drawings. It should be understood that the drawings described below are for illustration purposed only and are not intended to limit the scope of the present teachings in any way.

[0014] FIG. 1A-B depicts exemplary results of T_H17 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. 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.

[0015] FIG. 2 depicts exemplary representative results of a T_H17 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. At every dose, immunization with lipidated SP2108 resulted in 5 to 10-fold higher IL-17A secretion than immunization with non-lipidated SP2108.

[0016] FIG. 3A-C depicts exemplary results of T_H17 immune response assays following subcutaneous immunization with combinations of lipidated and non-lipidated proteins.

Protein(s) of immunization are indicated on the x axis. Protein used to stimulate blood cells are indicated by symbols or lines in the upper portion of each graph. FIG. 3A shows systemic IL- 17A values from animals immunized with 10 μg lipidated SP0148, 10 μg non-lipidated pneumolysoid, or both. FIG. 3B shows results from animals immunized with 1 μg lipidated SP2108, 10 μg non-lipidated SP1912, or both. FIG. 3C shows results from animals immunized with 10 μg lipidated SP0148, 10 μg non-lipidated SP1912, or both. Each symbol in FIG. 3A and FIG. 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.

[0017] FIG. 4A-B depicts exemplary results of T_H17 immune response assays following subcutaneous immunization with combinations of SP 1912, the synthetic free lipopeptide

Pam3CSK4, SP0148, or SP2108. 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 μg dose of Pam3CSK4. Immunization with the combination of SP1912 and SP0148 or SP2108 proteins on alum primed SP1912-specific T_H17 responses as expected. Combining SP1912 with various doses of Pam3CSK4 on alum did not reproduce this effect. Therefore, the in trans effect on T_H17 immune response requires that a non-lipidated protein such as SP 1912 be combined with lipidated protein.

[0018] 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.

[0019] FIG. 6 depicts exemplary results of T_H17 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/interquartile range. 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_H17 immune responses induced by the proteins of immunization are TLR2- dependent.

[0020] FIG. 7 depicts exemplary results of ESI-MS analysis of SP0148. The box at lower left lists observed masses for the individual peaks 1-4 of RP-HPLC fractionated SP0148 and compares them to the theoretical expected masses of various lipidated adducts of this protein (FL = full-length protein; DAG = diacylglycerol; FA = fatty acid). 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.

[0021] FIG. 8 depicts exemplary lipid adducts of SP0148 and SP2108.

[0022] FIG. 9 depicts exemplary results of T_H17 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_H17 cells 2-fold or 18-fold, respectively, compared to immunization with native non-lipidated IsaA.

[0023] FIG. 10 depicts exemplary results of T_H17 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_H17 cells 20-fold or 16-fold, respectively, compared to immunization with native non-lipidated IsaA.

Detailed Description

[0024] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein. [0025] A CD4+ T cell-dependent, antibody- and serotype-independent immune response has been shown to protect mice from colonization by Streptococcus pneumoniae. Further studies have elucidated the role of T_H17 cells and IL-17A expression in the response, and extended these observations to humans (Lu, Y.-J. et al., Interleukin-17A Mediates Acquired Immunity to Pneumococcal Colonization, PLoS Pathog 4(9): el 000159). A vaccine based on protein antigens that are well-conserved among pneumococcal serotypes may prevent immunologic escape through serotype replacement and significantly lower manufacturing cost. Further, a vaccine that elicits T cell immune responses, e.g., a T_H17 response, in addition to or in lieu of classic B cell responses, may have broader effectiveness, particularly if it targets both invasive disease and colonization. Streptococcus pneumoniae protein antigens and vaccine compositions that meet these criteria have been described (WO2011/008548). Nevertheless, a general strategy to elicit a desirable T_H17 response is needed.

A. Definitions

[0026] In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.

[0027] The term "adjuvant" refers to a substance that assists or modifies the

immunological action of a pharmaceutical composition, including but not limited to adjuvants that increase or diversify the immune response to an antigen. Typically, 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. is an adjuvant) if the immune response experienced by the host organism is greater when an antigen is applied to the host organism, in combination with the putative adjuvant, compared to the immune response experienced by the host organism when an antigen is applied without the putative adjuvant. Various immune cell assays can give a good indication of whether a substance is likely to be an effective adjuvant in a host organism (see for example, U.S. Pat. No. 6,406,705, which cites measuring the antibody forming capacity and number of lymphocyte subpopulations using a mixed leukocyte response assay and lymphocyte proliferation assay).

[0028] The term "antigen" as used herein 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. Normally, 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. Further, for purposes of the present invention, antigens can be derived from any of several known viruses, bacteria, parasites and fungi, as well as any of the various tumor antigens.

[0029] The terms "effective amount" and "pharmaceutically effective amount" of a composition, as provided herein, refer to a sufficient amount of the composition to have the desired effect, such as induce an immune response (e.g., systemic, mucosal, dermal) or inhibit colonization. As will be pointed out below, the exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular antigen of interest, the mode of administration, and the like. An appropriate "effective" amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

[0030] The term "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. [0031] 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. For purposes of the present invention, 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. One important aspect of cellular immunity involves an antigen-specific response by 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. 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_H17 immune response" refers to a specialized cellular immune response mediated by T_H17 cells.

[0032] As used herein, the phrase "nucleic acid" refers to DNA, RNA, as well as any of the known base analogs of DNA and RNA or chimeras formed therefrom.

[0033] The term "peptide" refers to a polymer of amino acid residues and is not limited to a minimum or maximum length of the product. Thus, 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.

[0034] The term "pharmaceutical" refers to biologically active compounds such as antibiotics, antiviral agents, growth factors, hormones, and the like, discussed in more detail below. [0035] 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. Furthermore, 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.

[0036] A "signal sequence" is a sequence of amino acid residues at the N-terminus of a nascent protein, which targets the protein to a destination organelle. In the case of bacteria, the signal sequence targets the protein to the outer membrane or the cytoplasmic membrane. A signal sequence may include additional motifs that direct modification of the protein, e.g. a lipobox motif that directs lipidation. A signal sequence may be entirely or partially cleaved from the mature protein.

[0037] 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.

B. Specific first polypeptides for use in lipidated fusion protein adjuvants and immunogenic compositions

[0038] The disclosure includes fusion proteins, which are useful as adjuvants or self- adjuvanting antigens, and related compositions. A first group of fusion proteins of the disclosure includes fusion proteins comprising: (a) a first polypeptide having an amino acid sequence comprising SEQ ID NO: 7 and (b) a second polypeptide from an organism susceptible to host T_H17 immune response, wherein the fusion protein elicits a host T_H17 immune response to said organism or pathogen. 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_H17 immune response, wherein the fusion protein elicits a host T_R17 immune response to said organism or pathogen. In some embodiments, 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. In some embodiments, 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). The DNA and protein sequence of each gene and polypeptide may be found by searching for the Locus Tag in the publicly available database, Entrez Gene (on the NCBI NIH web site on the World Wide Web, at www.ncbi.nlm.nih.gov/sites/entrez?db=gene); the indicated sequences are also included in this application.

Table 1. Immunogenic polypeptides for lipidated fusion protein adjuvants

Locus tag name and description Protein DNA DNA GenBank

SEQ ID SEQ ID Accession No. No. No. (from March 30, 2010)

SP0148 signal sequence 1 -

SP0148 including signal sequence 2 6 NC_003028.3|: 145,513- 146,343*

SP2108 including signal sequence 3 NC_003028.3|:2020750- 2022021

SP0148 consensus including signal 4 - - sequence

SP2108 consensus including signal 5 - - sequence

SP2108 signal sequence 8

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.

[0039] Certain polypeptides of Table 1 and variants thereof, along with additional peptides, are described in greater detail below.

1. SP0148 (SEQ ID NO: 2) and variants thereof

[0040] The protein SP0148 is named "ABC transporter, substrate-binding protein".

Proteins of this class are 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. In addition, 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. In some embodiments, 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.

[0041] In some embodiments, a fusion protein comprises a first polypeptide and a second polypeptide wherein the first polypeptide comprises an S. pneumoniae polypeptide. In some embodiments, 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. In certain embodiments, a first polypeptide includes no more than 250, 275, 200, 175, 150, 125, or 100 consecutive amino acids of SP0148. In certain embodiments, the amino acids N-terminal to the cysteine that is part of the lipobox domain are not present (e.g., are cleaved).

[0042] In some embodiments, a first polypeptide comprises a signal sequence. In some embodiments, a first polypeptide described herein comprises a signal sequence from SP0148 comprising SEQ ID NO: 1. [0043] Endogenous SP0148 comprises a signal sequence that directs its secretion and potential lipidation. In some embodiments, the signal sequence of the polypeptide of SEQ ID NO: 2 is partially or fully processed by an expression host, e.g. E. coli. In some embodiments, a fusion protein described herein includes an SP0148 variant that comprise an exogenous lipidation sequence. In some embodiments, a signal sequence directs lipidation. Thus, 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). In some embodiments, a signal sequence is cleaved, leaving the cysteine that is part of the lipobox motif as the N-terminus of the first polypeptide.

[0044] 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.

[0045] Consensus sequences illustrating combinations of SP0148 sequences from different S. pneumoniae serotypes are provided as SEQ ID NO: 4. Accordingly, in certain embodiments, 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

polypeptide having an amino acid sequence comprising SEQ ID NO: 2). In particular embodiments, residues 1-23 are not present, leaving cysteine as the N-terminal residue.

2. SP2108 (SEQ ID NO: 3) and variants thereof

[0046] 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. In addition, SP2108 contains a signal sequence that directs its secretion and potential lipidation. In some embodiments, the signal sequence of the polypeptide of SEQ ID NO: 3 is partially or fully processed by an expression host, e.g. E. coli. In some embodiments, a fusion protein described herein includes an SP2108 variant that comprise an exogenous lipidation sequence. In some embodiments, a signal sequence directs lipidation. In some embodiments, 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. [0047] In some embodiments, 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. In certain embodiments, a first polypeptide includes no more than 250, 275, 200, 175, 150, 125, or 100 consecutive amino acids of SP2108. In certain embodiments, the amino acids N-terminal to the cysteine that is part of the lipobox domain are not present (e.g., are cleaved).

[0048] In some embodiments, a first polypeptide described herein comprises a signal sequence from SP2108 comprising SEQ ID NO: 8. In some embodiments, a SP2108 signal sequence comprises one or more intrinsic cysteine lipidation sites.

[0049] Consensus sequences illustrating combinations of SP2108 sequences from different serotypes are provided as SEQ ID NO: 5. Thus, in certain embodiments, 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). In particular embodiments, a fusion protein described herein does not include residues 1-24, and cysteine is the N-terminal residue.

3. Additional first polypeptides for use in fusion proteins

[0050] Additional polypeptides can be used as a first polypeptide in a fusion protein described herein. In some embodiments, a first polypeptide includes a cysteine. For a fusion protein included in the second group of fusion proteins described herein, the N-terminus of a first polypeptide is a cysteine. In any fusion protein described herein, the length of a first polypeptide can range from 1-500 or more residues. For the first group, 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. For the second group, examples of 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. [0051] In some embodiments, a first polypeptide of a fusion protein is membrane- associated. In this context, membrane-associated means that the polypeptide can be found in a biological membrane in its native environment.

[0052] In some embodiments, a first polypeptide described herein comprises RlpB signal sequence from E. coli comprising SEQ ID NO: 9. In other embodiments, residues 1-18 are not present, leaving cysteine as the N-terminal residue.

[0053] In some embodiments, a first polypeptide alone does not elicit a host T_H17 immune response.

[0054] In some embodiments, a first polypeptide is lipidated. Typically, a first polypeptide is lipidated at the cysteine of the lipobox motif (SEQ ID NO: 7). Typically, lipid moieties are covalently attached. Lipid moieties are generally fatty acids. Lipidation sites contain a cysteine residue which is the site of lipid attachment. In some embodiments, 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. These lipidated proteins are structurally divided into two groups, diacylated or triacylated lipoproteins, by the absence or presence of an amide-linked fatty acid. In another example, S-palmitoylation is the covalent attachment of palmitic acid to the cysteine residue through a thioester bond.

[0055] A first polypeptide can be lipidated by one or more lipids, such as two lipid moieties or three lipid moieties. When a first polypeptide has two 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. When a first polypeptide has three lipid moieties, 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,

methylpentadecanoate, 3-methyl-hexadecanoate, methyl-isomyristate, 5-methyl-hexadecanoate, octadecenoic acid, oleic acid and margaric acid methyl ester, particularly palmitate, stearate, and myristate. C. Second polypeptides for use in lipidated fusion protein adjuvants and immunogenic compositions

[0056] In some embodiments, a second polypeptide of a fusion protein described herein comprises a polypeptide from an organism susceptible to a host T_H17 immune response. In certain embodiments, the organism is a pathogen. In other embodiments, the organism is a host, such that the second polypeptide can be associated with a tumor cell or an autoimmune disorder.

[0057] In some embodiments, a second polypeptide lacks an intrinsic lipidation site.

[0058] In some embodiments, 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.

[0059] In some embodiments, a second polypeptide is not expressed by or derived from

Streptococcus pneumoniae. In some embodiments, 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.

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.

[0060] In some embodiments, 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.

Table 2. Representative second polypeptides and heterologous fusion proteins

D. Pharmaceutical compositions

[0062] The present disclosure also provides pharmaceutical compositions containing one or more fusion proteins described herein.

[0063] In some embodiments, all or a proportion of fusion proteins can be lipidated in a composition. In some embodiments, at least 0.1% of fusion proteins have a lipid moiety covalently attached. In some embodiments, at least 0.2%, 0.3%, 0.4%, 0.5%, 1%, 5% or 10% of fusion proteins contain a lipid moiety. In some embodiments, a pharmaceutical composition contains 0.1 %- 10%> or 0.1%>-25%> lipidated fusion proteins. 1. Additional Adjuvants

[0064] Some adjuvants, for example, pertussis toxin, stimulate mucosal immune responses, which are particularly important in defense against organisms entering through the digestive or respiratory tracts. Often, adjuvants are derived from bacterial components. For example, Freund's complete adjuvant, an adjuvant that is widely used in animal experiments, comprises killed Mycobacterium tuberculosis suspended in oil. Unfortunately, Freund's complete adjuvant has severe associated side effects and has therefore not been approved for use in humans. Similarly, 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. Successful adjuvants to date that have been used in human vaccines include mineral containing compositions, such as aluminium salts, and ADP- ribosylating toxins and detoxified derivatives thereof, saponin formulations, virosomes and virus like particles, non-toxic derivatives of enterobacterial lipopolysaccharide (LPS).

[0065] In some embodiments, 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

formulations, 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. In contrast, immunostimulatory adjuvants are sometimes derived from pathogens and can represent pathogen associated molecular patterns (PAMP), e.g., lipopolysaccharides (LPS), monophosphoryl lipid (MPL), or CpG-containing DNA, which activate cells of the innate immune system.

[0066] Alternatively, adjuvants may be classified as organic and inorganic. Inorganic adjuvants include alum salts such as aluminum phosphate, amorphous aluminum

hydroxyphosphate sulfate, and aluminum hydroxide, which are commonly used in human vaccines. Organic adjuvants comprise organic molecules including macromolecules. An example of an organic adjuvant is cholera toxin.

[0067] Adjuvants may also be classified by the response they induce. In some embodiments, the adjuvant induces the activation of T_RI cells or T_R2 cells. In other

embodiments, 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.

[0068] In certain embodiments, the adjuvant induces the activation of T_R17 cells. It may promote the CD4⁺ or CD8⁺ T cells to secrete IL-17, IL-22, or other cytokines. In some embodiments, an adjuvant that induces the activation of T_H17 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_H17 activation, and (2) cells from animals treated with the adjuvant and an irrelevant (control) polypeptide. An adjuvant that induces the activation of T_R17 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. Certain toxins, such as cholera toxin and labile toxin (produced by enterotoxigenic E. coli, or ETEC), activate a T_H17 response. Thus, in some embodiments, 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. 1644-1648, February 1995) and LTK63 (Williams 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. 1999 Sep;67(9):4400-6), and 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).

[0069] In some embodiments, the adjuvant comprises a VLP (virus-like particle). One such adjuvant platform, Alphavirus replicons, induces the activation of T_H17 cells using alphavirus and is produced by Alphavax. In certain embodiments of the Alphavirus replicon system, 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. In some embodiments, a vaccine formulation is administered to a patient in combination with a nucleic acid encoding a cytokine.

[0070] Certain classes of adjuvants activate toll-like receptors (TLRs), which are associated with potentiating a T_R17 response. 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 (for instance, as a fusion protein) 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).

Traditionally, MPL has been produced as a detoxified lipopolysaccharide (LPS) endotoxin obtained from gram negative bacteria, such as S. minnesota. In particular, 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. A number of synthetic TLR agonists (in particular, 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. Like MPL adjuvants, these synthetic compounds activate the innate immune system via TLR. Another type of TLR agonist is a synthetic phospholipid dimer, for example E6020 (Ishizaka ST et al. "E6020: a synthetic Toll-like receptor 4 agonist as a vaccine adjuvant." Expert Rev. Vaccines. 2007 Oct; 6(5):773-84.). Various TLR agonists (including 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. Another exemplary adjuvant that activates TLRs comprises a mixture of MPL, Trehalose Dicoynomycolate (TDM), and dioctadecyldimethylammonium bromide (DDA). Another TLR- activating adjuvant is R848 (resiquimod).

[0071] In some embodiments, the adjuvant is or comprises a saponin. Typically, 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

chromatography) to isolate the portions of the extract with the best adjuvant activity and with acceptable toxicity. Typical fractions of extract from Quillaja saponaria tree used as adjuvants are known as fractions A and C. A particular form of saponins that may be used in vaccine formulations described herein is immunostimulating complexes (ISCOMs). ISCOMs are an art- recognized class of adjuvants that generally comprise Quillaja saponin fractions and lipids (e.g., cholesterol and phospholipids such as phosphatidyl choline). In certain embodiments, an ISCOM is assembled together with a polypeptide or nucleic acid of interest. However, different saponin fractions may be used in different ratios. In addition, the different saponin fractions may either exist together in the same particles or have substantially only one fraction per particle (such that the indicated ratio of fractions A and C are generated by mixing together particles with the different fractions). In this context, "substantially" refers to less than 20%, 15%, 10%>, 5%, 4%, 3%, 2% or even 1%.

[0072] In certain embodiments, combinations of adjuvants are used. Three exemplary combinations of adjuvants are MPL and alum, E6020 and alum, and MPL and an ISCOM.

2. Additional components of a composition

[0073] In some embodiments, in addition to fusion proteins and/or adjuvants described above, a composition described herein (e.g., a pharmaceutical composition) comprising a lipidated fusion protein includes one or more additional components.

[0074] In some embodiments, an immunogenic lipidated fusion protein composition includes one or more polypeptides from the same organism as a second polypeptide, particularly an antigenic polypeptide. In some embodiments, 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. 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.

[0075] In certain embodiments, 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.

[0076] In certain embodiments, an immunogenic composition includes one or more buffers such as a mixture of sodium bicarbonate and ascorbic acid. In some embodiments, the immunogenic composition may be administered in saline, such as phosphate buffered saline (PBS), or distilled water.

[0077] In certain embodiments, 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.

[0078] In certain embodiments, an immunogenic composition includes one or more salts such as sodium chloride, ammonium chloride, calcium chloride, or potassium chloride.

[0079] In certain embodiments, a preservative is included in an immunogenic composition. In other embodiments, no preservative is used. A preservative is most often used in multi-dose vaccine vials, and is less often needed in single-dose vaccine vials. In certain embodiments, the preservative is 2-phenoxyethanol, methyl and propyl parabens, benzyl alcohol, and/or sorbic acid.

[0080] In certain embodiments, an immunogenic composition is a controlled release formulation.

E. Use of Fusion Proteins

[0081] Fusion proteins of the present disclosure are designed to elicit a host T_H17 immune response against an organism susceptible to host T_H17 immune response, especially when lipidated or when a subset (even as few as 0.1% or less) are lipidated. In some

embodiments, 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. In some embodiments, in addition to or separately from a T_H17 response, a composition stimulates a T_H1 -biased CD4⁺ T cell response, and/or a CD8 T cell response. In some embodiments, the composition stimulates an antibody response. In some embodiments, the composition stimulates a T_RI -biased CD4⁺ T cell response, T_H 17-biased CD4⁺ T cell response and/or a CD8 T cell response, and an antibody response.

[0082] In some embodiments, a fusion protein that elicits a T_H17 immune response is one that activates T_R17 cells at least a 2-fold, 5 fold, 10 fold or 100 fold or more. In some embodiments, T_R17 cell activation is determined relative to untreated cells. In some

embodiments, T_R17 cell activation is determined relative cells treated with a control polypeptide or fusion protein. In some embodiments T_H17 cell activation comprises an increase in cytokine levels. In some embodiments T_R17 cell activation comprises an increase in IL-17. In some embodiments, T_R17 cell activation comprises an increase in IL-22. In one exemplary assay, 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 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_H17 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.

[0083] While not wishing to be bound by theory, in some embodiments a T_H17 cell response is desirable in mounting an immune response to a composition (e.g., a pharmaceutical composition) disclosed herein. In certain embodiments, an active T_R17 response is beneficial in clearing a pneumococcal infection. In some embodiments, an active T_H17 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. 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.

[0084] In certain embodiments, a composition described herein is administered to a subject who has not been previously exposed to the organism which elicits an active T_H17 response. In certain embodiments, the subject has been previously exposed to the organism which elicits an active T_H17 response.

[0085] In some embodiments, an active T_H17 response is beneficial in inducing a dermal, mucosal and/or systemic immune response in a subject. In some embodiments, an active T_R17 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. Furthermore, this application provides a method of activating T_H17 cells by administering said compositions to a subject. In certain embodiments, 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. In certain embodiments, this pneumococcal killing is independent of antibodies and complement.

However, specific antibody production and complement activation may be useful additional mechanisms that contribute to clearing of a pneumococcal infection.

F. Doses and Routes of Administration

1. Dosage forms, amounts, and timing

[0087] 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. Preferably, the dose is without significant adverse side effects. Such amount will vary depending upon which specific fusion protein is employed. Generally, it is expected that a dose will comprise 1-1000 μg of each protein, in some instances 2-100 μg, for instance 4-40 μg. In some aspects, the vaccine formulation comprises 1-1000 μg of the polypeptide and 1-250 μg of the adjuvant. In some embodiments, 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 μg - 250 μg per dose, for example 50-150 μg, 75-125 μg or 100 μg.

[0088] In some embodiments, only one dose of the composition is administered to achieve the results described above. In other embodiments, following an initial administration, subjects receive one or more boost administrations, for a total of two, three, four or five administrations. Advantageously, the number is three or fewer. It may be advantageous to administer split doses of compositions which may be administered by the same or different routes. [0089] In some embodiments, the 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. Many 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.

2. Routes of administration

[0090] 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. In some embodiments, the route of administration is intramuscular. In other embodiments, the route of administration is subcutaneous. In yet other embodiments, the route of administration is mucosal. In certain embodiments, the route of administration is transdermal or intradermal

[0091] Certain routes of administration are particularly appropriate for compositions comprising specified adjuvants. In particular, transdermal administration is one suitable route of administration for compositions comprising toxins (e.g. cholera toxin or labile toxin); in other embodiments, the administration is intranasal. Compositions formulated with Alphavirus replicons may be administered, for example, by the intramuscular or the subcutaneous route. Compositions comprising Monophosphory Lipid A (MPL), Trehalose Dicoynomycolate (TDM), and dioctadecyldimethylammonium bromide (DDA) are suitable {inter alia) for intramuscular and subcutaneous administration. A composition comprising resiquimod may be administered topically or subcutaneously, for example. 3. Formulations

[0092] 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. In some embodiments, the composition is suitable for administration to a non-human animal. In some embodiments, the vaccine or immunogenic composition is substantially free of either endotoxins or exotoxins. Endotoxins may include pyrogens, such as lipopolysaccharide (LPS) molecules. The vaccine or immunogenic composition may also be substantially free of inactive protein fragments which may cause a fever or other side effects. In some embodiments, 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. In some embodiments, 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. In other embodiments, 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. Thus, in certain embodiments 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.

[0093] In certain embodiments, the preparation comprises less than 50%>, 20%>, 10%>, or

5% (by dry weight) contaminating protein. In certain embodiments, 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). In certain embodiments, at least 80%>, 90%>, 95%, 99%, or 99.8% (by dry weight) of biological macromolecules of the same type present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 5000, can be present). In some embodiments, 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. In some embodiments, the desired polypeptides are substantially free of nucleic acids and/or carbohydrates. For instance, in some embodiments, 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. In certain embodiments, 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).

[0094] It is preferred that the composition has low or no toxicity, within a reasonable risk-benefit ratio. In certain embodiments, the composition comprises ingredients at

concentrations that are less than LD₅₀ measurements for the animal being vaccinated. LD₅₀ measurements may be obtained in mice or other experimental model systems, and extrapolated to humans and other animals. Methods for estimating the LD₅₀ of compounds in humans and other animals are well-known in the art. A composition, and any component within it, might have an LD₅₀ 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 LD₅₀ of botulinum toxin.

[0095] The formulations suitable for introduction of a composition vary according to route of administration. Formulations suitable for parenteral 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. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials. [0096] Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. In the case of adoptive transfer of therapeutic T cells, the cells can be administered intravenously or parenterally.

[0097] 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

predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions. 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,

disintegrating agents, and pharmaceutically compatible carriers. 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.

[0098] 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.

[0099] 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

hydrocarbons. In addition, it is also possible to use 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. G. Preparation and Storage of Fusion Protein Compositions

[0100] Fusion proteins and compositions described herein may be produced using a variety of techniques. For example, a polypeptide (e.g., a first polypeptide and/or a second polypeptide described herein) may be produced using recombinant DNA technology in a suitable host cell. 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. Typically, a nucleic acid sequence encoding a fusion protein is operably linked to appropriate regulatory sequences such as a strong promoter and a polyadenylation sequence. In some embodiments, 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. In some embodiments, 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.

[0101] Once a recombinant cell line has been produced, 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).

[0102] In some embodiments, 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. In some embodiments, a fusion protein is produced synthetically. [0103] In a further aspect of the present disclosure, there is provided a method of manufacture comprising mixing one or more fusion proteins with a carrier and/or an adjuvant.

[0104] In some embodiments, 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. In some embodiments, the isolated polypeptides are incorporated into a microparticle or a nanoparticle.

[0105] In some embodiments, fusion proteins for inclusion in the compositions are not purified. In some embodiments, 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.

[0106] Fusion proteins described herein can be packaged in packs, dispenser devices, and kits for administering nucleic acid compositions to a mammal. For example, packs or dispenser devices that contain one or more unit dosage forms are provided. Typically, 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.

[0107] The disclosure is further illustrated by the following examples. The example is provided for illustration purposes only. It is not to be construed as limiting the scope or content of the disclosure in any way.

Examples

Example 1. Effect of lipidation on T_H17 immune response and protection from

colonization

Generation of recombinant lipoproteins and non-lipidated mutants

[0108] Two potentially lipidated substrate binding proteins, SP2108 and SP0148, from

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

(WO2011/008548; Moffitt K. et al. (2011), TH17-based vaccine design for prevention of Streptococcus pneumoniae colonization, Cell Host Microbe 9(2):pp. 158-65).

[0109] Secretion and lipidation are directed by a signal sequence comprising a conserved

"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. To generate non-lipidated forms of SP2108 and SP0148, site-directed

mutagenesis was used to change the invariant cysteine to aspartate (for SP2108Alipid or SP2108CD: MSSKFMKSAAVLGTATLASLLLVAD (SEQ ID NO: 19), and for SP0148Alipid or SP0148CD: MKKI VKYS SLAAL ALVAAGVLAAD (SEQ ID NO: 20)).

Intranasal immunization, followed by assays for T_H17 immune response and colonization

[0110] The model of intranasal immunization with cholera toxin adjuvant (CT; List

Biologies), T_H17 immune response and colonization assays of C57BL/6 mice are previously described (WO2011/008548; Moffitt K. et al. (2011), T_H17-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 μg of the indicated lipidated (SP2108, SP0148) or non-lipidated (SP2108Alipid, SP0148Alipid) antigen and 1 μg of CT in 10 μΐ volumes instilled intranasally. Positive and negative controls were immunized, respectively, with killed, unencapsulated S. pneumoniae (WCB) and CT alone. 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 10⁷ 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.

Subcutaneous immunization, followed by assay for TH17 immune response

[0111] The model of subcutaneous immunization with alum, T_H17 immune response and colonization assays of C57BL/6 mice are previously described (WO2011/008548). Briefly, C57BL/6 mice were immunized subcutaneously three times, two weeks apart, with the indicated dose of lipidated (SP2108) or non-lipidated (SP2108CD) protein adsorbed on 250 μg alum, in 100 μΐ volumes. Negative controls were immunized with alum alone, and SP2108 or SP2108CD without adjuvant (no alum indicated on the x-axis). As a positive control, one group received 5μg SP2108 + ^g of CT intranasally administered in ΙΟμΙ volume. One week following their last immunization, animals were bled; aliquots of whole blood were stimulated with SP2108 antigen for six days, and then culture supernatants were evaluated for IL-17A by ELISA.

[0112] Figure 1 shows representative results of T_H17 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.

[0113] Figure 2 shows representative results of a T_H17 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. At every dose, immunization with lipidated SP2108 resulted in 5 to 10-fold higher IL-17A secretion than immunization with non-lipidated SP2108.

Example 2. in trans effect of lipidation on T_H17 immune response

[0114] The ability of a lipidated protein to induce or augment in trans a specific T_H17 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_R17 immune response), singly or in combination with a lipidated protein (SP2108, SP0148) adsorbed on 250 μg alum, in 100 μΐ 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.

[0115] Figure 3 shows representative results of T_H17 immune response assays following subcutaneous immunization with combinations of lipidated and non-lipidated proteins.

Protein(s) of immunization are indicated on the x axis. Protein used to stimulate blood cells are indicated by symbols or lines in the upper portion of each graph. FIG. 3A shows systemic IL- 17A values from animals immunized with 10 μg lipidated SP0148, 10 μg non-lipidated pneumolysoid, or both. FIG. 3B shows results from animals immunized with 1 μg lipidated SP2108, 10 μg non-lipidated SP1912, or both. FIG. 3C shows results from animals immunized with 10 μg lipidated SP0148, 10 μg non-lipidated SP1912, or both. Each symbol in FIG. 3A and FIG. 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. Example 3. Synthetic free lipopeptide combined with antigen does not elicit T_H1 immune response

[0116] To determine whether the in trans effect of lipidation described in Example 2 above could be achieved with free lipopeptide, studies were conducted with the non-lipidated protein SP1912 combined with Pam3CSK4, a synthetic free lipopeptide known to be a potent activator of TLR2 (InvivoGen), or the lipidated proteins SP2108 and SP0148. Briefly, C57BL/6 mice were immunized subcutaneously into the scruff of the neck three times, two weeks apart, with 10 μg SP1912, singly or in combination with 2-50 μg Pam3CSK4, 10 μg SP0148 or 1 μg SP2108, adsorbed on 250μg alum. Negative controls were immunized with alum alone. Three weeks following their last immunization, animals were euthanized and spleens and blood were collected. Heparinized whole blood was incubated with overlapping peptides (OLP) for six days. Culture supernatants were then collected and assayed for IL-17A levels by ELISA.

Similarly, 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.

[0117] Figure 4 shows representative results of T_H17 immune response assays following subcutaneous immunization with combinations of SP 1912, the synthetic free lipopeptide

Pam3CSK4, SP0148, or SP2108. 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 μg dose of Pam3CSK4. Immunization with the combination of SP1912 and SP0148 or SP2108 proteins on alum primed SP1912-specific T_H17 responses as expected. Combining SP1912 with various doses of Pam3CSK4 on alum did not reproduce this effect. Therefore, the in trans effect on T_H17 immune response requires that a non-lipidated protein such as SP 1912 be combined with lipidated protein.

Example 4. Signaling pathways upstream of T_H17 immune response

[0118] Induction of T_H17 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). To evaluate whether SP2108 and SP0148 induce T_H17 immune responses via TLR2, and whether the effect is dependent on lipidation, in vitro and in vivo studies were conducted.

[0119] The potential of SP2108 and SP0148 to activate TLR2 was first evaluated using a

Human Embryonic Kidney(HEK)-TLR2 reporter system in which TLR2 activation is marked by release of IL-8 (J Immunol. 2004 Feb 15;172(4):2431-8). Briefly, HEK cells transfected with TLR2 receptors (HEK-TLR2; InvivoGen, USA) were plated at 5xl0⁴ in 96-well format or 5xl0⁵ in 24-well format. Wells were stimulated for 18 hours with 1- 0.01 μg/ml of Pam3CSK4 (a potent TLR2 agonist; InvivoGen), lipidated SP2108, lipidated SP0148, or the non-lipidated mutants SP2108Alipid and SP0148Alipid. Supematants were harvested and IL-8 was measured by ELISA.

[0120] 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.

[0121] Dependence of the TH17 immune response induced by SP2108 and SP0148 on

TLR2 was then evaluated using TLR2 knock-out mice. Briefly, TLR2^_/" C57BL/6 mice and their wild-type C57BL/6 counterparts (Jackson Laboratories; n=10 animals/group) were immunized subcutaneously three times, two weeks apart, with 1-10 μg SP2108, SP0148, or SP1912 protein (shown previously to elicit weak or no TH17 immune response) adsorbed on 250 μg alum, in 200 μΐ volumes. Positive controls were immunized with killed, unencapsulated S. pneumoniae (WCB). One week following their last immunization, animals were bled; aliquots of whole blood were stimulated with protein for six days, and then culture supematants were evaluated for IL-17A by ELISA.

[0122] Figure 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_R17 immune responses induced by the proteins of immunization are TLR2-dependent.

[0123] Combining the results shown in Figures 5 and 6 with those shown in Figure 4

(Example 3 above) suggests that activation of TLR2 is necessary but likely not sufficient to elicit a T_H17 immune response.

Example 5. Lipid profiles; effect on ΤΉ17 immune response and protection from

colonization

[0124] Purified preparations of 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).

[0125] Figure 7 shows representative results of electrospray ionization mass

spectrometry (ESI-MS) analysis of SP0148. The box at lower left lists observed masses for the individual peaks 1-4 of reverse-phased high-performance liquid chromatography (RP-HPLC) fractionated SP0148 and compares them to the theoretical expected masses of various lipidated adducts of this protein. 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. Peaks A, B and C are unresolved mixtures of lipidated and non- lipidated protein.

[0126] To gain greater insight into the actual identities of the lipid chains present in each peak, material was subjected to gas chromatography mass spectroscopic analysis (GC-MS). For these analyses, 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. [0127] To determine the effect of different SP0148 and SP2108 lipid profiles on T_H17 immune response and protection from colonization, material from each of peaks 1-4 is used to immunize C57BL/6 mice. Briefly, C57BL/6 mice are immunized subcutaneously two to three times, two weeks apart, with protein of peaks 1-4 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 10⁶-10⁷ live type 6B pneumococci intranasally. One week following intranasal challenge, nasal washes are collected and pneumococcal CFU are counted after overnight culture on blood agar plates supplemented with gentamicin.

Example 6. Exemplary Chlamydia trachomatis proteins lipidated via heterologous lipidation sequences; effect on TH17 immune response and protection from colonization

[0128] 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.

[0129] To determine the effect of lipidation via heterologous lipidation sequences on

TR17 immune response and protection from colonization, lipidated and non-lipidated

recombinant proteins described above are used to immunize C57BL/6 mice. Briefly, 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 10⁶-10⁷ live type 6B pneumococci intranasally. One week following intranasal challenge, nasal washes are collected and pneumococcal CFU are counted after overnight culture on gentamicin blood agar plates. Example 7. Exemplary Staphyloccocus aureus proteins lipidated via heterologous lipidation sequences: effect on TH17 immune response.

[0130] Lipidated recombinant proteins comprising the non-lipidated protein IsaA of

Staphlococcus aureus strain USA300 FPR3757 (SEQ ID NO: 10) 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.

[0131] To determine the effect of lipidation via heterologous lipidation sequences on antigen-specific T_H17 immune responses, 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 μg alum per dose. Negative controls were immunized with proteins alone or alum alone. One to two weeks following their last immunization, antigen-specific TR17 responses in spleen and blood 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. 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. After 40 h of co-culture with peptides, the ELISPOT plates were developed using biotinylated anti-IL-17A secondary antibody followed by peroxidase staining. Spot forming units were quantified using an automated ELISPOT reader. Antigen-specific TR17 cell responses in circulation were measured in whole blood stimulated with IsaA OLPs for six days, after which cell-free culture supernatants were evaluated for IL-17A levels by ELISA. [0132] Figure 9 shows representative results of T_H17 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_H17 cells 2-fold or 18-fold, respectively, compared to immunization with native non-lipidated IsaA.

[0133] Figure 10 shows representative results of T_H17 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_H17 cells 20-fold or 16-fold, respectively, compared to immunization with native non-lipidated IsaA.

Equivalents

[0134] It is to be understood that while the disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

SEQUENCES

SEQ ID NO: 1

SP0148 signal sequence

MKKIVKYSSLAALALVAAGVLAAC

SEQ ID NO: 2

SP0148 including signal sequence (277 amino acids with C-terminal E)

MKKIVKYSSLAALALVAAGVLAACSGGAKKEGEAASKKEI IVATNGSPKPFIYEENGELTGYEIEWRAIFKDSDKY

DVKFEKTEWSGVFAGLDADRYNMAVNNLSYTKERAEKYLYAAPIAQNPNVLWKKDDSSIKSLDDIGGKSTEWQAT

TSAKQLEAYNAEHTDNPTILNYTKADFQQIMVRLSDGQFDYKIFDKIGVETVIKNQGLDNLKVIELPSDQQPYVYPL

LAQGQDELKSFVDKRIKELYKDGTLEKLSKQFFGDTYLPAEADIKE

SEQ ID NO: 3

SP2108 including signal sequence

>gi I 14973620 I gb I AAK76167.1 I maltose/maltodextrin ABC transporter,

maltose/maltodextrin-binding protein (Streptococcus pneumoniae TIGR4)

MSSKFMKSAAVLGTATLASLLLVACGSKTADKPADSGSSEVKELTVYVDEGYKSYIEEVAKAYEKEAGVKVTLKTGD ALGGLDKLSLDNQSGNVPDVMMAPYDRVGSLGSDGQLSEVKLSDGAKTDDTTKSLVTAANGKVYGAPAVIESLVMYY NKDLVKDAPKTFADLENLAKDSKYAFAGEDGKTTAFLADWTNFYYTYGLLAGNGAYVFGQNGKDAKDIGLANDGSIV GINYAKSWYEKWPKGMQDTEGAGNLIQTQFQEGKTAAI IDGPWKAQAFKDAKVNYGVA IPTLPNGKEYAAFGGGKA WVIPQAVKNLEASQKFVDFLVATEQQKVLYDKTNEIPANTEARSYAEGKNDELTTAVIKQFKNTQPLPNISQMSAVW DPAKNMLFDAVSGQKDAKTAANDAVTLIKETIKQKFGE

SEQ ID NO: 4

SP0148 consensus including signal sequence

MKKIVKYSSLAALALVAAGVLAACSGGAKKEGEAASKKEI IVATNGSPKPFIYEENGELT G L Q S R N

GYEIEWRAIFKDSDKYDVKFEKTEWSGVFAGLDADRYNMAVNNLSYTKERAEKYLYAAP X I

IAQNPNVLWKKDDSSIKSLDDIGGKSTEWQATTSAKQLEAYNAEHTDNPTILNYTKAD E

LQQIMVRLSDGQFDYKIFDKIGVETVIKNQGLDNLKVIELPSDQQPYVYPLLAQGQDELK F Y S

SFVDKRIKELYKDGTLEKLSKQFFGDTYLPAEADIK (E)

SEQ ID NO: 5

SP2108 consensus including signal sequence

MSSKFMKSAAVLGTATLASLLLVACGSKTADKPADSGSSEVKELTVYVDEGYKSYIEEVA T T V A

KAYEKEAGVKVTLKTGDALGGLDKLSLDNQSGNVPDVMMAPYDRVGSLGSDGQLSEVKLS I I X

DGAKTDDTTKSLVTAANGKVYGAPAVIESLVMYYNKDLVKDAPKTFADLENLAKDSKYAF T

AGEDGKTTAFLADWTNFYYTYGLLAGNGAYVFGQNGKDAKDIGLANDGSIVGINYAKSWY

A G P A X

EKWPKGMQDTEGAGNLIQTQFQEGKTAAI IDGPWKAQAFKDAKVNYGVA IPTLPNGKEY

H

AAFGGGKAWVIPQAVKNLEASQKFVDFLVATEQQKVLYDKTNEIPANTEARSYAEGKNDE

A S A

LTTAVIKQFKNTQPLPNISQMSAVWDPAKNMLFDAVSGQKDAKTAANDAVTLIKETIKQK S

FGE

SEQ ID NO: 6

SP0148 including signal sequence (nucleotides)

ATGAAAAAAATCGTTAAATACTCATCTCTTGCAGCCCTTGCTCTTGTTGCTGCAGGTGTGCTTGCGGCTTGCTCAGG GGGTGCTAAGAAAGAAGGAGAAGCAGCTAGCAAGAAAGAAATCATCGTTGCAACCAATGGATCACCAAAGCCATTTA TCTATGAAGAAAATGGCGAATTGACTGGTTACGAGATTGAAGTCGTTCGCGCTATCTTTAAAGATTCTGACAAATAT GATGTCAAGTTTGAAAAGACAGAATGGTCAGGTGTCTTTGCTGGTCTTGACGCTGATCGTTACAATATGGCTGTCAA CAATCTTAGCTACACTAAAGAACGTGCGGAGAAATACCTCTATGCCGCACCAATTGCCCAAAATCCTAATGTCCTTG TCGTGAAGAAAGATGACTCTAGTATCAAGTCTCTCGATGATATCGGTGGAAAATCGACGGAAGTCGTTCAAGCCACT ACATCAGCTAAGCAGTTAGAAGCATACAATGCTGAACACACGGACAACCCAACTATCCTTAACTATACTAAGGCAGA CTTCCAACAAATCATGGTACGTTTGAGCGATGGACAATTTGACTATAAGATTTTTGATAAAATCGGTGTTGAAACAG TGATCAAGAACCAAGG GGACAACT GAAAG A CGAACTTCCAAGCGACCAACAACCGTACGTTTACCCACTT CTTGCTCAGGGTCAAGATGAGTTGAAATCGTTTGTAGACAAACGCATCAAAGAACTTTATAAAGATGGAACTCTTGA AAAATTGTCTAAACAATTCTTCGGAGACACTTATCTACCGGCAGAAGCTGATATTAAAGAGTAA

SEQ ID NO: 7

Canonical lipobox motif

[LIVMFESTAGPC] - [LVIAMFTG] - [IVMSTAGCP] - [AGS] -C

SEQ ID NO: 8

SP2108 signal sequence

MSSKFMKSAAVLGTATLASLLLVAC

SEQ ID NO: 9

E. coli RlpB signal sequence

MRYLATLLLSLAVLITAGCG

SEQ ID NO: 10

Staphylococcus aureus protein IsaA lacking native signal sequence

MAEVNVDQAH LVDLAHNHQD QLNAAPIKDG AYDIHFVKDG FQYNFTSNGT TSWSYEAANG WQTAGFSNVA GADYTTSYNQ GSNVQSVSYN AQSSNSNVEA VSAPTYHNYS TSTTSSSVRL SNGNTAGATG SSAAQIMAQR TGVSASTWAA I IARESNGQV NAYNPSGASG LFQTMPGWGP TNTVDQQINA AVKAYKAQGL GAWGF

SEQ ID NO: 11

SP2108 lipidation signal sequence-IsaA fusion protein

MSSKFMKSAA VLGTATLASL LLVACAEVNV DQAHLVDLAH NHQDQLNAAP IKDGAYDIHF VKDGFQYNFT SNGTTWSWSY EAANGQTAGF SNVAGADYTT SYNQGSNVQS VSYNAQSSNS NVEAVSAPTY HNYSTSTTSS SVRLSNGNTA GATGSSAAQI MAQRTGVSAS TWAAI IARES NGQVNAYNPS GASGLFQTMP GWGPTNTVDQ QINAAVKAYK AQGLGAWGF

SEQ ID NO: 12

SP2108-IsaA fusion protein

MSSKFMKSAA VLGTATLASL LLVACGSKTA DKPADSGSSE VKELTVYVDE GYKSYIEEVA KAYEKEAGVK

VTLKTGDALG GLDKLSLDNQ SGNVPDVMMA PYDRVGSLGS DGQLSEVKLS DGAKTDDTTK SLVTAANGKV

YGAPAVIESL VMYYNKDLVK DAPKTFADLE NLAKDSKYAF AGEDGKTTAF LADWTNFYYT YGLLAGNGAY

VFGQNGKDAK DIGLANDGSI VGINYAKSWY EKWPKGMQDT EGAGNLIQTQ FQEGKTAAI I DGPWKAQAFK

DAKVNYGVAT IPTLPNGKEY AAFGGGKAWV IPQAVKNLEA SQKFVDFLVA TEQQKVLYDK TNEIPANTEA

RSYAEGKNDE LTTAVIKQFK NQPLPNISQM SAVWDPAKNM LFDAVSGQKD AKTAANDAVT LIKETIKQKF GEAEVNVDQA HLVDLAHNHQ DQLNAAPIKD GAYDIHFVKD GFQYNFTSNG TTWSWSYEAA NGQTAGFSNV AGADYTTSYN QGSNVQSVSY NAQSSNSNVE AVSAPTYHNY STSTTSSSVR LSNGNTAGAT GSSAAQIMAQ RTGVSAS WA AIIARESNGQ VNAY PSGAS GLFQTMPGWG PTNTVDQQIN AAVKAYKAQG LGAWGF

SEQ ID NO: 13

Staphylococcus aureus protein SceD lacking native signal sequence

MSEADLNKAS LAQMAQSNDQ TLNQKPIEAG AYNYTFDYEG FTYHFESDGT HFAWNYHATG TNGADMSAQA PATNNVAPSA VQANQVQSQE VEAPQNAQTQ QPQASTSNNS QVTATPTESK SSEGSSVNVN AHLKQIAQRE SGGNIHAVNP TSGAAGKYQF LQSTWDSVAP AKYKGVSPAN APESVQDAAA VKLYNTGGAG HWVTA

SEQ ID NO: 14

SP2108 lipidation signal sequence-SceD fusion protein

MSSKFMKSAA VLGTATLASL LLVACSEADL NKASLAQMAQ SNDQTLNQKP IEAGAYNYTF DYEGFTYHFE SDGTHFAWNY HATGTNGADM SAQAPATNNV APSAVQANQV QSQEVEAPQN AQTQQPQAST SNNSQVTATP TESKSSEGSS VNVNAHLKQI AQRESGGNIH AVNPTSGAAG KYQFLQSTWD SVAPAKYKGV SPANAPESVQ DAAAVKLYNT GGAGHWVTA

SEQ ID NO: 15

SP2108-SceD fusion protein

MSSKFMKSAA VLGTATLASL LLVACGSKTA DKPADSGSSE VKELTVYVDE GYKSYIEEVA KAYEKEAGVK

VTLKTGDALG GLDKLSLDNQ SGNVPDVMMA PYDRVGSLGS DGQLSEVKLS DGAKTDDTTK SLVTAANGKV

YGAPAVIESL VMYYNKDLVK DAPKTFADLE NLAKDSKYAF AGEDGKTTAF LADWTNFYYT YGLLAGNGAY

VFGQNGKDAK DIGLANDGSI VGINYAKSWY EKWPKGMQDT EGAGNLIQTQ FQEGKTAAI I DGPWKAQAFK

DAKVNYGVAT IPTLPNGKEY AAFGGGKAWV IPQAVKNLEA SQKFVDFLVA TEQQKVLYDK TNEIPANTEA

RSYAEGKNDE LTTAVIKQFK NQPLPNISQM SAVWDPAKNM LFDAVSGQKD AKTAANDAVT LIKETIKQKF

GESEADLNKA SLAQMAQSND QTLNQKPIEA GAYNYTFDYE GFTYHFESDG THFAWNYHAT GTNGADMSAQ

APATNNVAPS AVQANQVQSQ EVEAPQNAQT QQPQASTSNN SQVTATPTES KSSEGSSVNV NAHLKQIAQR

ESGGNIHAVN PTSGAAGKYQ FLQSTWDSVA PAKYKGVSPA NAPESVQDAA AVKLYNTGGA GHWVTA

SEQ ID NO: 16

Chlamydia trachomatis protein CT144

MTTPDNNTID VSFPTFVRLN VATTDLADGN KSNAVTITET ATANYVNVTQ DLTSSTAKLE CTQDLIAQGK

LIVTNPKSDI SFGGRVNLAD NTVNYSNGGA EVSFTNINSR QGKQYVPYGL YKNGEPKISM RSALSGGHVG

SGDTGGWGAE VLWDAYTEQL KDMTDGAVTL NSSNRGKLSF TASPEAPVLF RLSVFMRKNG DWLDNGVGGR

VMLYVNTTDS AGKTVRRLLG IAVCLGSTWY TTVPMFWCAA TYYATSSGFF QLIVGERNFR VSSLSWSWR LPWP SEQ ID NO: 17

SP2108 lipidation signal sequence-CT144 fusion protein

MSSKFMKSAA VLGTATLASL LLVACGNKSN AVTITETATA NYVNVTQDLT SSTAKLECTQ DLIAQGKLIV TNPKSDISFG GRVNLADNTV NYSNGGAEVS FTNINSRQGK QYVPYGLYKN GEPKISMRSA LSGGHVGSGD TGGWGAEVLW DAYTEQLKDM TDGAVTLNSS NRGKLSFTAS PEAPVLFRLS VFMRKNGDWL DNGVGGRVML YVNTTDSAGK TVRRLLGIAV CLGSTWYTTV PMFWCAATYY ATSSGFFQLI VGERNFRVSS LSWSWRLPV VP

SEQ ID NO: 18

SP2108-CT144 fusion protein

MSSKFMKSAA VLGTATLASL LLVACGSKTA DKPADSGSSE VKELTVYVDE GYKSYIEEVA KAYEKEAGVK

VTLKTGDALG GLDKLSLDNQ SGNVPDVMMA PYDRVGSLGS DGQLSEVKLS DGAKTDDTTK SLVTAANGKV

YGAPAVIESL VMYYNKDLVK DAPKTFADLE NLAKDSKYAF AGEDGKTTAF LADWTNFYYT YGLLAGNGAY

VFGQNGKDAK DIGLANDGSI VGINYAKSWY EKWPKGMQDT EGAGNLIQTQ FQEGKTAAI I DGPWKAQAFK

DAKVNYGVAT IPTLPNGKEY AAFGGGKAWV IPQAVKNLEA SQKFVDFLVA TEQQKVLYDK TNEIPANTEA

RSYAEGKNDE LTTAVIKQFK NQPLPNISQM SAVWDPAKNM LFDAVSGQKD AKTAANDAVT LIKETIKQKF

GEGNKSNAVT ITETATANYV NVTQDLTSST AKLECTQDLI AQGKLIVTNP KSDISFGGRV NLADNTVNYS

NGGAEVSFTN INSRQGKQYV PYGLYKNGEP KISMRSALSG GHVGSGDTGG WGAEVLWDAY TEQLKDMTDG

AVTLNSSNRG KLSFTASPEA PVLFRLSVFM RKNGDWLDNG VGGRVMLYVN TTDSAGKTVR RLLGIAVCLG

STWYTTVPMF WCAATYYATS SGFFQLIVGE RNFRVSSLSW SWRLPWP

Claims

We claim:

1. A lipidated fusion protein comprising:

(a) a first polypeptide comprising a lipidation sequence having an amino acid sequence comprising SEQ ID NO: 7; and

(b) a second polypeptide from an organism susceptible to a T_H 17 immune response in a host,

wherein the lipidated fusion protein elicits the T_H17 immune response to said organism in the host upon administration to the host.

2. A lipidated fusion protein comprising:

(a) a first amino acid or polypeptide comprising a lipidated N-terminal cysteine; and

(b) a second polypeptide from an organism susceptible to a T_H17 immune response in a host,

wherein the lipidated fusion protein elicits the T_H17 immune response to said organism in the host upon administration to the host.

3. The fusion protein of claims 1 or 2, wherein the second polypeptide is not expressed by Streptococcus pneumoniae.

4. The fusion protein of any one of claims 1-3, wherein the organism comprises:

Staphylococcus aureus, Klebsiella pneumoniae, Cibrobacter rodentium, any species of Candida, 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.

The fusion protein of any one of claims 1-4, wherein the second polypeptide lacks an intrinsic lipidation site.

The fusion protein of any one of claims 1-5, wherein the first polypeptide is membrane- associated.

The fusion protein of any one of claims 1 and 3-6, wherein the first polypeptide comprises a signal sequence of at least 15 amino acids.

The fusion protein of claim 7, wherein the first polypeptide comprises an amino acid sequence at least 80% homologous to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 , SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 1.

The fusion protein of claim 8, wherein the first polypeptide comprises an amino acid sequence comprising SEQ ID NO: 8.

10. The fusion protein of claim 9, wherein the first polypeptide comprises an amino acid sequence comprising SEQ ID NO: 3.

11. The fusion protein of claim 8, wherein the first polypeptide comprises an amino acid sequence comprising SEQ ID NO: 5.

12. The fusion protein of claim 8, wherein the first polypeptide comprises an amino acid sequence comprising SEQ ID NO: 1.

13. The fusion protein of claim 12, wherein the first polypeptide comprises an amino acid sequence comprising SEQ ID NO: 2.

14. The fusion protein of claim 8, wherein the first polypeptide comprises an amino acid sequence comprising SEQ ID NO: 4.

15. The fusion protein of claim 8, wherein the first polypeptide comprises an amino acid sequence comprising SEQ ID NO: 9.

16. The fusion protein of claim 2, wherein the first polypeptide comprises an amino acid sequence at least 80% homologous to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and wherein the amino acid residues N-terminal to lipidated N-terminal cysteine are removed.

17. The fusion protein of any one of claims 1-16, wherein one or more lipid moieties are covalently attached to the fusion protein.

18. The fusion protein of claim 17, wherein two lipid moieties are attached to the fusion protein.

19. The fusion protein of claim 17, wherein three lipid moieties are attached to the fusion protein.

20. The fusion protein of claim 17, wherein the one or more lipid moieties are independently selected from palmitate, stearate, myristate, methylpentadecanoate, 3-methyl- hexadecanoate, methyl-isomyristate , 5-methyl-hexadecanoate, octadecenoic acid, oleic acid, and margaric acid methyl ester.

21. The fusion protein of claim 20, wherein the one or more lipid moieties are independently selected from palmitate, stearate and myristate.

22. A composition comprising the fusion protein of any one of claims 1-21.

23. The composition of claim 22, wherein at least 0.1% of the fusion proteins contain a lipid moiety.

24. The composition of claim 22, further comprising alum.

25. The composition of claim 22, further comprising cholera toxin or its nontoxic mutant derivatives.

26. The composition of claim 22, further comprising E. coli heat-labile toxin or its nontoxic mutant derivatives.

27. The composition of any one of claims 22-26, wherein the composition induces a mucosal immune response.

28. The composition of any one of claims 22-26, wherein the composition induces a dermal immune response.

29. The composition of any one of claims 22-26, wherein the composition induces a systemic immune response.

30. The composition of any one of claims 22-29, wherein the composition induces any

combination of mucosal, dermal, or systemic immune responses.

31. The composition of any one of claims 22-26, wherein the composition inhibits

colonization.

32. The composition of any one of claims 22-26, wherein the composition induces IL-17A. The composition of any one of claims 22-26, further comprising a third polypeptide from the same organism as the second polypeptide.

A method of producing a fusion protein comprising fusing a first polypeptide that includes a signal sequence comprising SEQ ID NO: 7 to a second polypeptide from said organism, thereby creating a fusion protein.

The method of claim 34, wherein the second polypeptide is not from S. pneumoniae.

The method of claim 34, wherein the fusion protein is lipidated.

The method of claim 34, wherein the first polypeptide is fused to the N-terminus of the second polypeptide.

The method of claim 34, wherein the first polypeptide comprises an amino acid sequence at least 80% homologous to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 1.

39. The method of claim 38, wherein the first polypeptide has an amino acid sequence

comprising SEQ ID NO: 8.

40. The method of claim 39, wherein the first polypeptide has an amino acid sequence comprising SEQ ID NO: 3.

41. The method of claim 38, wherein the first polypeptide has an amino acid sequence

comprising SEQ ID NO: 5.

42. The method of claim 38, wherein the first polypeptide has an amino acid sequence

comprising SEQ ID NO: 1.

43. The method of claim 42, wherein the first polypeptide has an amino acid sequence

comprising SEQ ID NO: 2.

44. The method of claim 38, wherein the first polypeptide has an amino acid sequence

comprising SEQ ID NO: 4.

45. The method of claim 38, wherein the first polypeptide has an amino acid sequence

comprising SEQ ID NO: 9.

46. A method of inducing a mucosal immune response comprising administering to a subject the composition of any one of claims 22-30.

47. A method of inducing a dermal immune response comprising administering to a subject the composition of any one of claims 22-30.

48. A method of inducing a systemic immune response comprising administering to a subject the composition of any one of claims 22-30.

49. A method of inducing any combination of mucosal, dermal, or systemic immune

responses comprising administering to a subject the composition of any one of claims 22- 30.

50. A method of inhibiting colonization comprising administering to a subject the

composition of any one of claims 1-30 and 33.

51. The method of claims 45 or 46, wherein the subject has not previously been exposed to the organism of the second polypeptide.

52. The method of claims 45 or 46, wherein the subject has previously been exposed to the organism of the second polypeptide.