Modified biotin-binding proteins, fusion proteins thereof, and uses

Abstract

To provide: modified biotin-binding proteins which can be expressed in a soluble form in a high yield in bacteria; and fusion proteins comprising the modified biotin-binding protein and an antigen.SOLUTION: A soluble biotin-binding protein comprises a specific amino acid sequence and / or any functional derivatives thereof. The invention also provides non-hemolytic variants of α-hemolysin from Staphylococcus aureus and a fusion protein comprising non-hemolytic variant of α-hemolysin and a biotin-binding domains. Immunogenic compositions comprising the proteins are used for inducing an immune response or for vaccinating a subject.SELECTED DRAWING: Figure 3

Description

Technical Field

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 61 / 484,934, filed May 11, 2011; U.S. Provisional Application No. 61 / 608,168, filed Mar. 8, 2012; and U.S. Provisional Application No. 61 / 609,974, filed Mar. 13, 2012, under 35 U.S.C. § 119(e), the contents of each of which are hereby incorporated by reference in their entirety.

[0002] Technical Field The present disclosure relates to biotin-binding proteins and fusion proteins / compositions containing such biotin-binding proteins. Also described herein are methods for expressing biotin-binding proteins and / or their fusion proteins in bacteria at high yields and in soluble form.

Background Art

[0003] Biotin-binding proteins and their derivatives can be widely used in various applications. However, the production or purification of recombinant biotin-binding proteins can be very difficult. When expressed in Escherichia coli (E. coli), most biotin-binding proteins tend to accumulate in inclusion bodies, and denaturation, refolding, and cumbersome downstream processing are required in the preparation of active proteins. Due to the low cost of production and the potential for further engineering technologies, expression in E. coli is preferred, and thus there is a great need for biotin-binding proteins that can be efficiently produced in E. coli. Furthermore, the expressing E. coli also offers the possibility of generating recombinant fusion proteins containing biotin-binding proteins for various applications.

[0004] Accordingly, there is a need in the art for biotin-binding proteins and fusion proteins containing biotin-binding proteins that can be expressed in E. coli in high yields in soluble form.

Summary of the Invention

[0005] One object of the present disclosure is to provide a recombinant biotin-binding protein that can be expressed in a soluble form at a high yield in Escherichia coli. Accordingly, the present disclosure provides a biotin-binding protein and a composition containing the same protein. In some embodiments, the recombinant biotin-binding protein is amino acids 45 to 179 of wild-type streptavidin (rhavi) contains an Escherichia coli signal sequence fused to the N-terminus of the amino acid sequence containing TIFF0007679431000001.tif17164. In some embodiments, the Escherichia coli signal sequence is TIFF0007679431000002.tif4128. The signal sequence can be fused to the sequence containing amino acids 45 to 179 of wild-type rhavi by a flexible peptide linker

[0006] This specification also provides a method for expressing a biotin-binding protein in a soluble form at a high yield in Escherichia coli. In some embodiments, the method includes the step of expressing a biotin-binding protein in Escherichia coli, and the native signal sequence of the biotin-binding protein is replaced with an Escherichia coli signal sequence. In some embodiments, the signal sequence is TIFF0007679431000003.tif4128

[0007] In yet another aspect, the present invention provides a biotin-binding fusion protein comprising a biotin-binding domain and a protein or peptide

[0008] In another aspect, lipidated biotin-binding proteins are provided herein. As used herein, the term "lipidated biotin-binding protein" refers to a biotin-binding protein that is covalently linked to a lipid. The lipidated biotin-binding protein is a ligand or agonist of Toll-like receptor 2. Accordingly, a method for inducing an immune response in a subject is also provided herein. The method includes administering to the subject a composition comprising a lipidated biotin-binding protein.

[0009] A method for expressing a lipidated biotin-binding protein in E. coli is also provided herein. In some embodiments, the method includes expressing a lipidated biotin-binding protein in E. coli, wherein the native signal sequence of the biotin-binding protein is replaced with an E. coli signal sequence containing a lipidation motif. In some embodiments, the signal sequence is TIFF0007679431000004.tif4128.

[0010] In yet another aspect, non-hemolytic derivatives of Staphylococcal aureus α-hemolysin (Hla) are provided herein. The Hla derivatives described herein can be in the form of fusion proteins, and the fusion proteins include both an Hla derivative domain and a biotin-binding domain. In some embodiments of this aspect, the biotin-binding domain is a biotin-binding protein described herein.

[0011] Like the lipidated biotin-binding proteins, Hla variants or their fusion proteins with the biotin-binding proteins described herein are also ligands or agonists of Toll-like receptors or other pattern recognition receptors (PRRs). Accordingly, a method for inducing an immune response in a subject is also provided herein. In some embodiments, the method includes administering to the subject a composition comprising a non-hemolytic Hla variant or its fusion protein with a biotin-binding protein described herein.

[0012] In yet another aspect, provided herein are immunogenic or vaccine compositions comprising a biotin-binding protein, a lipidated biotin-binding protein, and a biotin-binding fusion protein comprising a biotin-binding domain and an antigenic protein or peptide. In some embodiments of this aspect, the antigenic protein is a non-hemolytic derivative of Hla as described herein.

[0013] Also provided herein is a method of vaccinating a subject, such as a mammal, such as a human, with an immunogenic composition disclosed herein, the method comprising administering to the subject a vaccine composition disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0014]

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Modes for Carrying Out the Invention

[0015] Detailed Description of Exemplary Embodiments It should be understood that the present invention is not limited to the specific compositions, methodologies, protocols, reagents, etc. described herein and can accordingly vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined only by the appended claims.

[0016] While not wishing to be bound by theory, the low expression of biotin-binding proteins in the art may be due to misfolding caused by disulfide bonds in each monomer of the biotin-binding protein, which occurs only at very low levels or not at all in the cytoplasm of E. coli. Currently, the inventors have discovered that correct folding can be achieved by delivering into the periplasmic space of E. coli. Thus, the correct folding of the recombinant biotin-binding protein can be improved by replacing the complete native signal sequence of the biotin-binding protein with an E. coli secretion signal sequence. While not wishing to be bound by theory, this facilitates the translocation of the recombinant protein into the periplasmic space of E. coli cells. Thus, the translocation of the recombinant protein into the periplasmic space of E. coli can provide disulfide bonds that are functionally important in biotin-binding proteins (e.g., streptavidin), allowing the protein to be correctly folded in a soluble form and in high yield.

[0017] In one aspect, provided herein is a biotin-binding protein that can be expressed in E. coli in a soluble form and in high yield. As used herein, the term "biotin-binding protein" refers to a protein that non-covalently binds to biotin or an analog or derivative thereof. High yield means that the protein can be expressed in a soluble form in E. coli in an amount of about 10 mg / L, 11 mg / L, 12 mg / L, 13 mg / L, 14 mg / L, 15 mg / L, 20 mg / L, 25 mg / L, 30 mg / L, 35 mg / L, 30 mg / L, 35 mg / L, 40 mg / L, 45 mg / L, 50 mg / L or more.

[0018] In some embodiments, the biotin-binding protein can be a recombinant protein. The coding sequence of the biotin-binding protein can be optimized using E. coli expression codons to avoid any difficulties in expression in E. coli due to rare codons present in the original gene.

[0019] Generally, biotin-binding proteins contain a biotin-binding domain. As used herein, the term "biotin-binding domain" refers to a polypeptide sequence that binds to biotin. While a full biotin-binding protein can be used as the biotin-binding domain, only the biotin-binding portion of the protein can be used. In some embodiments, the biotin-binding domain is derived from streptavidin.

[0020] In some embodiments, the biotin-binding domain consists of, or consists essentially of, an amino acid sequence corresponding to amino acids 45-179 of wild-type streptavidin. The amino acid sequence of wild-type streptavidin is TIFF0007679431000005.tif17162.

[0021] In other words, the biotin-binding domain does not include (i.e., is lacking) amino acids 1-44 of wild-type streptavidin TIFF0007679431000006.tif4135. In some embodiments, the biotin-binding domain includes the amino acid sequence TIFF0007679431000007.tif17164.

[0022] In some embodiments, the biotin-binding domain includes an amino acid sequence having at least 50% identity, at least 55% identity, at least 60% identity, at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, preferably at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity, and more preferably at least 99.3% identity to SEQ ID NO:1.

[0023] Helppolainen et al. (Biochem J., 2007, 405:397-405) described removing only the first 24 residues of full-length streptavidin, but the inventors have found that the first 44 residues of full-length streptavidin are unnecessary for the core structure and function of streptavidin. Further, unexpectedly, substitution of the first 44 residues of full-length streptavidin with an E. coli signal peptide resulted in increased solubility and secretion in E. coli of the biotin protein described herein, thus amino acids 25-44 of full-length streptavidin TIFF0007679431000008.tif4128 reduces the solubility and secretion of streptavidin expressed in E. coli.

[0024] In the biotin-binding protein described herein, the biotin-binding domain can be extended at the N- or C-terminus by one or more amino acids, provided that the N-terminus of the biotin-binding domain does not contain an amino acid sequence corresponding to amino acid residues 1-44 of wild-type streptavidin. The inventors have discovered that by cleaving the first 44 amino acids at the N-terminus of wild-type streptavidin, the expression of the biotin-binding protein in soluble form in E. coli can be dramatically increased. Accordingly, the biotin-binding protein described herein has the sequence X 1 -X 2 -X 3 wherein X 2 is a peptide having an amino acid sequence corresponding to amino acids 45-179 of wild-type streptavidin, and X 1 and X 3 are independently either absent or are peptides of from 1 to about 100 amino acids, provided that the N-terminus of X 1 does not contain an amino acid sequence corresponding to the N-terminus of amino acids 1-44 of wild-type streptavidin.

[0025] In some embodiments, the biotin-binding protein can include a signal peptide conjugated to the N-terminus of the biotin-binding protein, i.e., X 1can include a signal peptide. The signal peptide can also be referred to as a leader peptide at the N-terminus, which may or may not be cleaved after translocation through the membrane. The secretion / signal peptide will be described in more detail below. In some embodiments, the signal sequence is TIFF0007679431000009.tif11152, or a derivative or functional portion thereof.

[0026] The signal peptide can be linked directly (e.g., via a bond) or indirectly (e.g., by a linker) to the N-terminus of the biotin-binding domain. In some embodiments, the signal peptide can be linked to the N-terminus of the biotin-binding domain by a peptide linker. The peptide linker sequence can be of any length. For example, the peptide linker sequence can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 amino acids in length or more. In some embodiments, the peptide linker is 4 amino acids in length.

[0027] The peptide linker sequence can include any amino acid sequence. For example, the peptide linker can include an amino acid sequence that can be cleaved by signal peptidase. In some embodiments, the peptide linker includes the amino acid sequence AQDP (SEQ ID NO: 8) or VSDP (SEQ ID NO: 9).

[0028] In the biotin-binding protein, the biotin-binding domain can be conjugated to a peptide of 1 to 100 amino acids at its C-terminus. Such a peptide at the C-terminus can be used as a purification tag, a linker to other domains, and the like.

[0029] In some embodiments, the biotin-binding protein contains one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) purification tags at its N or C terminus. Examples of purification tags include, but are not limited to, histidine tags, c-my tags, Halo tags, Flag tags, and the like. In some embodiments, the biotin-binding protein contains a histidine tag, e.g., (His) 6 (SEQ ID NO: 10) at its C terminus.

[0030] The purification tag can be conjugated to the biotin-binding protein by a peptide linker such that the tag is more likely to be exposed on the outside. The length of the linker can be at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) amino acid. The linker peptide can contain any amino acid sequence, non-limitingly. In some embodiments, the linker peptide contains the amino acid sequence TIFF0007679431000010.tif5130.

[0031] In some embodiments, the biotin-binding protein contains the amino acid sequence TIFF0007679431000011.tif4161 at its C terminus.

[0032] In some embodiments, the biotin-binding protein contains the amino acid sequence: TIFF0007679431000012.tif17165.

[0033] Compared to known biotin-binding proteins that form tetramers, the biotin-binding proteins described herein form dimers. Without wishing to be bound by theory, forming dimers can further improve the expression of the biotin-binding proteins described herein as soluble proteins in E. coli.

[0034] Biotin-binding proteins are known in the art, but the biotin-binding proteins described herein include significant differences from avidin and avidin-like proteins currently known in the art. First, currently known avidin is extremely difficult to express as a soluble protein in E. coli. However, as demonstrated by the inventors, the biotin-binding proteins described herein can be expressed as soluble proteins in E. coli with high yields.

[0035] The biotin-binding proteins described herein can be obtained in soluble form by expression in E. coli at high yields, for example, at least 30 mg per liter of culture broth. Therefore, the biotin-binding proteins described herein are more soluble than those described in the art, indicating a fundamental difference. Without wishing to be bound by theory, the difference in solubility may be due to fundamental physical and / or chemical and / or structural differences between the biotin-binding proteins described herein and other biotin-binding proteins known in the art.

[0036] Second, the biotin-binding proteins described herein include a biotin-binding domain consisting of amino acids 45-179 of wild-type streptavidin. Wild-type streptavidin and its partially truncated portions are known in the art, but there is no disclosure or suggestion in the art regarding biotin-binding proteins that contain the amino acid sequence of amino acids 45-179 of wild-type streptavidin and have an E. coli signal sequence that can result in a soluble protein that can be obtained in high yields in E. coli. According to Helppolainen et al. (Biochem J., 2007, 405: 397-405), amino acids 25-44 of wild-type streptavidin contain a putative signal sequence. However, as described herein, the inventors have discovered and demonstrated that substitution of such a putative signal sequence with an E. coli signal sequence leads to an increase in the expression of soluble biotin-binding proteins in E. coli.

[0037] Thirdly, the biotin-binding protein described herein has the amino acid sequence TIFF0007679431000013.tif4128 contains the peptide. This peptide at the C-terminus provides a histidine tag for purification and a site for insertion of other domains, such as an antigen domain, into the biotin protein. Furthermore, although Helppolainen et al. (Biochem J., 2007, 405:397-405) describe the expression of streptavidin in E. coli, Helppolainen et al. do not disclose or suggest conjugating an additional peptide to the C-terminus of the biotin-binding domain of streptavidin.

[0038] Fourthly, streptavidin has lower sequence homology to avidin (22.4% sequence identity and 35.0% similarity) compared to other avidin-like proteins. Thus, the biotin-binding protein described herein is different from avidin and other avidin-like proteins.

[0039] Fifth, the biotin-binding proteins described herein have a low isoelectric point (pI) compared to avidin and other avidin-like molecules. The isoelectric point of wild-type streptavidin is 4.0. (Helppolainen et al., Biochem J., 2007, 405:397-405). The isoelectric points of other known biotin-binding proteins are generally 6.1 or higher (see Helppolainen et al., Biochem J., 2007, 405:397-405). In comparison, the pI of the biotin-binding proteins described herein is 5.4. The acidic pI of the binding proteins described herein results in a reduction of non-specific binding. A problem in the use of currently known avidin and other avidin-like peptides is their non-specific binding. Currently known avidin and other avidin-like peptides can non-specifically bind not only to cells but also to biological substances such as DNA, proteins, and membranes. For example, in the detection of substances using avidin-biotin binding, avidin non-specifically binds to substances other than the target substance to be detected, increasing the background. One reason for the high non-specific binding of avidin is its high isoelectric point. Avidin is a strongly basic protein with an isoelectric point significantly higher than 10 and has an overall positive charge. Therefore, avidin is thought to easily bind to biological substances that often carry a negative charge. Thus, the low pI of the biotin-binding proteins described herein is advantageous over currently known avidin and other avidin-like peptides.

[0040] Sixth, the size of the biotin-binding protein described herein is relatively small compared to currently known avidin and other avidin-like proteins. The biotin-binding protein is smaller than 28 kDa (dimer size). However, most of the currently known avidin and other avidin-like proteins all have a size larger than 60 kDa (tetramer size). Wild-type streptavidin is said to have a size of about 29 kDa (dimer size). Using a biotin-binding protein of small size, the loading amount of the binding conjugate between the target molecules can be increased. For example, the biotin-binding protein can be used to conjugate a first target molecule with a second target molecule. One of the target molecules can be bound to one or more biotins or biotin-like molecules, and the second molecule can be bound, conjugated, or fused to the biotin-binding protein. Considering the small size of the biotin-protein described herein, the biotin or biotin-like molecules can be arranged closer together so as to enable more binding than when the second molecule is a larger currently known avidin and other avidin-like molecules.

[0041] Seventh, the biotin-binding protein described herein is a dimer. By forming a dimer, the expression of the biotin-binding protein described herein as a soluble protein in E. coli can be further improved. In addition, since the biotin-binding protein forms a dimer rather than a tetramer like all other known avidin-like proteins, 1) the structural complexity of the fusion antigen is reduced, 2) the difficulty of expressing the recombinant biotin-binding protein fusion protein is similarly reduced, 3) the steric hindrance of the operation of the biotin-binding protein fusion is minimized, which is advantageous for further operations using, for example, but not limited to, biotin, biotin mimics, or biotin derivatives, and 4) the solubility of the biotin-binding protein fusion is significantly increased. Thus, the fundamental difference between the biotin-binding protein described herein and those known in the art is demonstrated.

[0042] Eighth, the biotin-binding protein described herein reduces the risk of immunogenic compositions containing the same protein that induce allergic reactions related to eggs in a subject. Also, antibodies against the biotin-binding domain described herein do not have an obvious cross-reaction with egg avidin (and vice versa).

[0043] Furthermore, the biotin-binding protein described herein can have improved properties such as reduction of non-specific binding or further improvement of biotin binding while retaining the properties of wild-type streptavidin. The use of the biotin-binding protein described herein for measuring an analyte using avidin-biotin binding, for example, for detection in immunoassays or nucleic acid hybridization assays, can reduce the background, increase the sensitivity, and maintain the binding properties with biotin under stringent conditions.

[0044] This study clearly demonstrates the advantages and differences of the biotin-binding protein described herein compared to avidin and other avidin-like proteins. Thus, the biotin-binding protein described herein has the potential as a powerful and versatile tool in a wide range of applications utilizing avidin-biotin technology.

[0045] Without limitation, the biotin-binding protein can be used in any methodology, composition, or system that requires the use of an avidin-biotin system. As is well recognized by those skilled in the art, the avidin-biotin system can be used in numerous laboratory methods such as bioconjugates, target molecule detection, isolation of target molecules from samples, purification, or concentration, protein detection, nucleic acid detection, protein isolation, purification, or concentration, nucleic acid isolation, purification, or concentration, ELISA, flow cytometry, and the like.

[0046] Accordingly, exemplary uses of the recombinant biotin-binding proteins described herein include, but are not limited to, bioconjugates, target molecule detection, target molecule isolation, purification, or concentration from a sample, protein detection, nucleic acid detection, protein isolation, purification, or concentration, nucleic acid isolation, purification, or concentration, ELISA, flow cytometry, and the like.

[0047] In some embodiments, the biotin-binding proteins described herein can be used as part of an affinity pair in a multiple antigen presentation system (MAPS) as described in U.S. Provisional Application No. 61 / 48,934, filed May 11, 2012, U.S. Provisional Application No. 61 / 608,168, filed Mar. 8, 2012, U.S. Provisional Application No. 61 / 609,974, filed Mar. 13, 2012, and PCT Application PCT / US12 / 37412, filed May 11, 2012, the entire contents of each of which are incorporated herein by reference in their entirety. MAPS is also described in more detail below. Without wishing to be bound by theory, the use of the biotin-binding proteins described herein reduces the risk of MAPS inducing an allergic reaction to eggs in a subject. Also, antibodies to recombinant modified avidin do not have a significant cross-reactivity to egg avidin (and vice versa).

[0048] Lipid-added biotin-binding protein In another aspect, lipid-added biotin-binding proteins are provided herein. As used herein, the term "lipid-added biotin-binding protein" refers to a biotin-binding protein that is covalently conjugated to a lipid. The lipid moiety can be a diacyl lipid or a triacyl lipid.

[0049] The lipid-added biotin-binding protein can be produced using a lipid-added sequence. As described herein, the term "lipid-added sequence" refers to an amino acid sequence that promotes lipid addition of a polypeptide carrying the lipid-added sequence in bacteria, such as Escherichia coli. The lipid-added sequence can be present at the N-terminus or C-terminus of the protein. The lipid-added sequence can be linked to a recombinant biotin-binding protein to form a fusion protein that becomes lipid-added when expressed in Escherichia coli by conventional recombinant techniques. In some embodiments, the lipid-added sequence is located at the N-terminus of the biotin-binding protein.

[0050] Any lipid-added sequence known to those skilled in the art can be used. In some embodiments, the lipid-added sequence is TIFF0007679431000014.tif4128, or a derivative or functional portion thereof. Other exemplary lipid-added sequences include, but are not limited to, TIFF0007679431000015.tif11165, and derivatives or functional portions thereof.

[0051] In some embodiments, the lipid-added sequence can be fused to the biotin-binding protein via a peptide linker, where the peptide linker attaches the lipid-added sequence to the biotin-binding protein.

[0052] In some embodiments, the peptide linker comprises the amino acid sequence VSDP (SEQ ID NO: 9).

[0053] In one embodiment, the biotin-binding lipid protein comprises the amino acid sequence TIFF0007679431000016.tif17165.

[0054] The lipid-added biotin-binding protein is a ligand for Toll-like receptor (TLR). The lipid-added biotin-binding protein described herein can thus be used as a TLR ligand. For example, the lipid-added biotin-binding protein can be used in a composition for inducing TLR2 stimulation. This can be useful for inducing immunogenicity to other antigens / pathogens. Thus, the biotin-binding lipoprotein can be used in an immunogenic composition as a co-stimulatory factor or adjuvant for an antigen.

[0055] As used herein, the term "Toll-like receptor" is generally intended to refer to any Toll-like receptor of any organism of any species. The TLR can be derived from any mammalian species. TLRs have been identified in various mammalian species including, but not limited to, for example, humans, guinea pigs, and mice. A particular TLR can be identified by additionally referring to the species of origin (e.g., human, mouse, etc.), the specific receptor (e.g., TLR2, TLR3, TLR9, etc.), or both. In some embodiments, the lipid-added biotin-binding protein is a ligand for TLR2.

[0056] Toll-like receptors (TLRs) are a group of germline-encoded transmembrane proteins that promote pathogen recognition and activation of the innate immune system. Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) and are expressed by cells of the innate immune system, including macrophages, dendritic cells, and NK cells. Examples of known ligands for TLRs include gram-positive bacteria (TLR-2), bacterial endotoxin (TLR-4), flagellin protein (TLR-5), bacterial DNA (TLR-9), double-stranded RNA and poly I:C (TLR-3), and yeast (TLR-2). Other ligands that bind to intracellular pattern recognition receptors, scavenger receptors, or mannose-binding receptors can also be considered by the present invention. TLRs engage conserved pathogen-derived ligands and subsequently activate the TLR / IL-1R signaling pathway to induce various effector genes. Toll-like receptors (TLRs) represent an important group of PRRs that can sense pathogens or microbe-associated molecular patterns. They are widely expressed in blood, spleen, lung, muscle, and intestine by many types of cells, particularly dendritic cells (DCs), but also by macrophages, epithelial cells, and lymphocytes.

[0057] Some TLRs located on the cell surface are specific for microbial lipids and proteins, while other TLRs that bind to endosomal compartments inside the cell are specific for nucleic acids. Ligation of TLRs by their specific ligands results in conformational changes of the receptor and leads to downstream signaling mainly involving MyD88- and TRIF-dependent pathways. All other TLRs except TLR3 can signal through the MyD88 pathway to induce inflammatory cytokines, which involves activation of an intracellular protein kinase cascade including IκB kinase (IKK)-NF-κB, and extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK). The TRIF pathway, which is independent of MyD88, is utilized by both TLR3 and TLR4 and mediates the induction of type I interferons.

[0058] The recombinant biotin-binding lipoprotein described herein has enhanced immunogenicity. Without wishing to be bound by theory, the lipid moiety at the N-terminus of the lipoprotein or lipopeptide contributes to adjuvant activity. Accordingly, additional embodiments provide an immunogenic composition or vaccine composition for inducing an immune response, comprising an isolated biotin-binding lipoprotein or a suitable vector for its in vivo expression or both, and a suitable carrier, as well as a method for inducing an immune response or a protective response, comprising administering to a host an isolated recombinant biotin-binding lipoprotein, a vector expressing the recombinant biotin-binding lipoprotein, or a composition containing the recombinant lipoprotein or vector, in an amount sufficient to elicit a response.

[0059] An immune composition or immunogenic composition comprising a biotin-binding lipoprotein induces a local or systemic immune response. The response may be, but need not be, a protective response. It should be noted that, as used herein, the terms "immune composition" and "immunogenic composition" include "vaccine composition" (since such terms may be protective compositions). The lipidated biotin-binding proteins described herein can be used, without limitation, as antigens, adjuvants, or co-stimulatory factors in immune compositions, immunogenic compositions, or vaccine compositions. Further, since the lipidated biotin-binding protein contains a biotin-binding domain, the lipidated protein can be incorporated into the polymeric backbone of MAPS. Accordingly, a method for inducing an immune response in a host mammal is also provided herein. The method comprises administering to the host an immunogenic composition, an immune composition, or a vaccine composition comprising the lipidated biotin-binding protein described herein and a pharmaceutically acceptable carrier or diluent.

[0060] In some embodiments, the lipidated biotin-binding protein is a fusion protein comprising the lipidated biotin-binding protein and a protein or peptide.

[0061] Non-hemolytic Hla Hemolysin is an exotoxin produced by bacteria that causes lysis of red blood cells. Although highly immunogenic, their use in vaccines is limited because they cause lysis of red blood cells. Thus, in another aspect, variants of Staphylococcus aureus alpha-hemolysin (Hla), their fusion constructs with biotin-binding proteins, and their use are provided herein. These variants, designated herein as "mHla," are substantially non-hemolytic, i.e., have substantially low hemolytic activity. As used herein, the phrase "substantially non-hemolytic" means that it is unable to lyse red blood cells with wild-type Hla of equal titer. The term "wild-type Hla" has its ordinary definition associated with such phrase, i.e., Hla naturally secreted by a competent bacterial source. By definition, "wild-type Hla" does not include, for example, Hla fusion products derived via recombinant DNA techniques. In some embodiments, the hemolytic activity of mHla is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% lower than wild-type Hla of equal titer, and the amino acid sequence of SEQ ID NO: 59 corresponds to amino acids 27-319 of the wild-type Hla mature polypeptide. In some embodiments, mHla has no detectable hemolytic activity. The inventors have also found that the hemolytic activity of mHla can be further reduced by linking mHla to a biotin-binding protein. Thus, the present disclosure also describes fusion proteins comprising an mHla protein and a biotin-binding protein.

[0062] As provided herein, non-hemolytic Hla can be produced in which the residue W205 or W213 in wild-type Hla is substituted with alanine (A) or the tripeptide DRD209-211 is substituted with a tri-alanine peptide (AAA). The mutant Hla protein can be expressed and purified in an E. coli expression system. The mutant can be produced by site-directed mutagenesis using rapid cycling mutagenesis. For example, the nucleotide sequence of the nucleic acid encoding wild-type Hla can be altered such that a given amino acid in wild-type Hla is substituted with another amino acid.

[0063] In some embodiments, mHla is a fusion protein comprising mHla and a biotin-binding protein. In some embodiments, the biotin-binding mHla fusion protein has the amino acid sequence TIFF0007679431000017.tif50165.

[0064] The Hla variants described herein are ligands of Toll-like receptors (TLRs). The Hla variants described herein can thus be used as TLR ligands. For example, the Hla variant can be used in a composition for inducing TLR2 stimulation. This can be useful for inducing immunogenicity to other antigens / pathogens. Thus, the Hla variants described herein can be used in an immunogenic composition as a costimulatory factor or adjuvant for an antigen. Further, when mHla is fused with a biotin-binding protein, the fusion protein can conjugate with the polymer backbone of MAPS.

[0065] In some embodiments, mHla can be used as a costimulatory factor in an immunogenic composition or a vaccine composition.

[0066] Furthermore, since mHla induces an immune response in a subject, mHla can be used in an immunogenic composition or a vaccine composition for vaccinating the subject against Staphylococcus aureus.

[0067] The mHla described in this specification has enhanced immunogenicity. Accordingly, additional embodiments provide an immunogenic composition or vaccine composition for inducing an immune response, comprising mHla, or a suitable vector for its in vivo expression, or both, and a suitable carrier, as well as a method for inducing an immune response or a defensive response, comprising administering to a host an isolated mHla, a vector expressing mHla, or a composition containing mHla or the vector, in an amount sufficient to elicit a response.

[0068] An immune composition or immunogenic composition containing mHla can induce a local or systemic immune response. The response can be a defensive response, but this is not necessary. Accordingly, the non-hemolytic mutant of Hla described herein can be present as an antigen, adjuvant, or co-stimulatory factor in an immune composition, immunogenic composition, or vaccine composition.

[0069] Furthermore, provided herein is also a method for inducing an immune response in a host mammal. The method comprises administering to the host an immunogenic composition, immune composition, or vaccine composition comprising the non-hemolytic mutant of Hla described herein and a pharmaceutically acceptable carrier or diluent.

[0070] In another aspect, provided herein are fusion proteins comprising a biotin-binding protein described herein linked to an antigenic protein or peptide. These fusion proteins are also referred to herein as biotin-binding fusion proteins and antigen fusion proteins. The biotin-binding protein and the antigenic protein or peptide can be linked in any configuration, for example, the biotin-binding protein can be at the N-terminus of the fusion protein and the antigen peptide at the C-terminus, or vice versa.

[0071] In some embodiments, the biotin-binding protein and the antigenic protein or peptide are linked to each other by a peptide linker. The peptide linker can non-limitingly include any amino acid sequence and can be of any length. For example, the peptide linker sequence can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 amino acids or longer. In some embodiments, the peptide linker that links the antigen domain to the biotin-binding domain is 8 amino acids in length.

[0072] In some embodiments, the peptide linker that links the antigen domain to the biotin-binding domain has the amino acid sequence GGGGSSS (SEQ ID NO: 22).

[0073] In some embodiments, the antigenic protein is the non-hemolytic Hla described herein.

[0074] In some embodiments, the non-hemolytic Hla protein is a fusion protein comprising a biotin-binding protein and the non-hemolytic Hla described herein.

[0075] In some embodiments, the non-hemolytic Hla protein is a fusion protein comprising a lipid-added biotin-binding protein and the non-hemolytic Hla described herein.

[0076] Immunogenic composition In another aspect, immunogenic compositions are provided herein that include an antigen fusion protein, lipidated biotin binding, or a non-hemolytic variant of Hla as described herein. Additionally, provided herein are immunogenic compositions and vaccine compositions that include an immunogenic complex comprising at least one antigen fusion protein or multiple antigen fusion proteins attached to a polymer backbone, for use in inducing an immune response against each of the antigens attached to the polymer and optionally against the polymer itself when administered to a subject. Without wishing to be bound by theory, the immunogenic compositions described herein stimulate humoral and cellular immune responses; this can generate antibody responses and Th1 / Th17 responses against multiple protein antigens using a single MAPS immunogenic construct. The combination of B and T cell immunity against a living organism represents an optimal vaccine strategy against many diseases, including invasive infections and nasopharyngeal carriage associated with pneumococcal disease. In some embodiments, the immunogenic composition is a vaccine or is included in a vaccine.

[0077] Accordingly, embodiments herein provide immunogenic compositions and methods useful for enhancing an immune response in a subject, which can be used alone or in combination with or in admixture with essentially any existing vaccination approach.

[0078] In some embodiments, an immunogenic composition as disclosed herein comprises at least 2 antigens, or at least 3 antigens, or at least 5 antigens, or 2 - 10 antigens, or 10 - 15 antigens, or 15 - 20 antigens, or 20 - 50 antigens, or 50 - 100 antigens, or more than 100 antigens. In some embodiments, when an immunogenic composition as disclosed herein comprises at least 2 antigens, the antigens can be the same antigen or at least 2 different antigens. In some embodiments, the antigens can be from the same or different pathogens, or can be different epitopes or portions of the same antigenic protein, or can be the same antigen that is specific for different serotypes or seasonal variants of the same pathogen (e.g., influenza viruses A, B, and C).

[0079] In some embodiments, an immunogenic composition disclosed herein comprises an antigen from a pathogenic organism or abnormal tissue. In some embodiments, the antigen is a tumor antigen. In some embodiments, the antigen is at least 1 antigen selected from antigens of pathogens or parasites such as Streptococcus pneumoniae, Mycobacterium tuberculosis or M. tetanus, Bacillus anthracis, HIV, seasonal or pandemic influenza antigens (such as H1N1 or H5N1, etc.), Bordetella pertussis, Staphylococcus aureus, Neisseria meningitides, or Neisseria gonorrhoeae, HPV, Chlamydia trachomatis, HSV, or other herpes viruses, or antigens of Plasmodia. These antigens may comprise peptides, proteins, glycoproteins, or polysaccharides. In some embodiments, the antigen is a toxoid or a portion of a toxin.

[0080] In some embodiments, an immunogenic composition as disclosed herein comprises an antigenic polysaccharide such as, for example, Vi antigen (Salmonella typhi capsular polysaccharide), pneumococcal capsular polysaccharide, pneumococcal cell wall polysaccharide, Hib (Haemophilus influenzae type B) capsular polysaccharide, meningococcal capsular polysaccharide, and other bacterial capsules or cell wall polysaccharides, or any combination thereof. The polysaccharide can have a protein component, such as a glycoprotein, for example, one derived from a virus.

[0081] In some embodiments, the immunogenic composition disclosed herein further comprises at least one co-stimulatory factor that associates with a polymer or polysaccharide, and the co-stimulatory factor can associate directly or indirectly. For example, in some embodiments, the co-stimulatory factor can covalently bind to the polymer. For example, in some embodiments, the co-stimulatory factor can covalently bind to a first affinity molecule, which then cross-links to the polymer. For example, in some embodiments, the co-stimulatory factor can attach to a complementary affinity molecule, which associates with the first affinity molecule to link the co-stimulatory factor to the polymer. In some embodiments, the co-stimulatory factor is an adjuvant. In alternative embodiments, the co-stimulatory factor can be any known to those skilled in the art and can include any combination, for example, but not limited to, Toll-like receptor agonists (agonists for TLR2, 3, 4, 5, 7, 8, 9, etc.), NOD agonists, or agonists of the inflammasome.

[0082] In some embodiments, the co-stimulatory factor can be a lipidated biotin-binding protein, or a non-hemolytic variant of α-hemolysin, or a fusion protein of a biotin-binding protein described herein and mHla.

[0083] Another aspect of the invention relates to the use of an immunogenic composition as disclosed herein that is administered to a subject to induce an immune response in the subject. In some embodiments, the immune response is an antibody / B cell response, CD4 +T cell responses (including Th1, Th2, and Th17 cells), and / or CD8 + T cell responses. In some embodiments, at least one adjuvant is administered with the immunogenic composition.

[0084] Another aspect of the invention relates to a method for inducing an immune response against at least one antigen in a subject, comprising the step of administering to the subject an immunogenic composition as disclosed herein.

[0085] Another aspect of the invention relates to a method of vaccinating an animal, such as a bird, a mammal, or a human, against at least one antigen, comprising the step of administering a vaccine composition comprising an immunogenic composition as disclosed herein.

[0086] In all aspects as disclosed herein, the animal or subject can be a human. In some embodiments, the subject can be an agricultural or wild animal, or a domesticated animal. In some embodiments, a vaccine composition comprising an immunogenic composition as disclosed herein can be administered via subcutaneous, intranasal, oral, sublingual, intravaginal, rectal, intradermal, intraperitoneal, or intramuscular injection.

[0087] In all aspects as disclosed herein, the immune response is an antibody / B cell response, a CD4 + T cell response (including Th1, Th2, and Th17 responses), or a CD8+ T cell response against one or more protein / peptide antigens. In some embodiments, the immune response is an antibody / B cell response against a polymer, such as a pneumococcal polysaccharide. In some embodiments, at least one adjuvant is administered with the immunogenic composition.

[0088] Another aspect of the invention relates to the use of an immunogenic composition as disclosed herein for use in the diagnosis of exposure to a pathogen or an immunogenic substance.

[0089] Multiple antigen presentation system Also provided herein are immunogenic multivalent antigen presentation systems (MAPS) that are useful for the production of immunogenic compositions, such as those useful in vaccines. In particular, the invention relates to a composition comprising an immunogenic complex comprising at least one type of polymer, which may optionally be antigenic, such as a polysaccharide; at least one antigenic protein or peptide; and at least one pair of complementary affinity molecules comprising (i) a first affinity molecule that associates with the polymer and (ii) a complementary affinity molecule that associates with the protein or peptide, such that the first affinity molecule and the complementary affinity molecule act as an indirect linkage between the polymer and the antigenic protein or peptide. Thus, the polymer can attach at least one, or at least two, or a plurality of the same or different protein or peptide antigens. In some embodiments, the polymer is antigenic, for example, the polymer is a pneumococcal capsular polysaccharide. In some embodiments, the protein or peptide antigen is a recombinant protein or peptide antigen.

[0090] The immunogenic compositions as disclosed herein can simultaneously induce both a humoral response and a cellular response to one or more antigens. The immunogenic compositions provide a long-term memory response and potentially protect the subject from future infections. This enables a single immunogenic composition that enhances the high titers of functional anti-polysaccharide antibodies and is similar to or comparable to the antibody levels induced by conventional conjugate vaccines. Furthermore, various antigenic proteins can be used in the MAPS constructs to generate a robust anti-polysaccharide antibody response without being limited to a particular carrier protein.

[0091] In addition, the strong antibody response and Th17 / Th1 responses are specific to multiple protein antigens presented via the MAPS compositions. This presents a major advantage as a means to induce two forms of immunity with one construct. In addition to the more conventional immune response to antigenic polysaccharides conjugated to protein carriers, the present invention provides a T cell response to systemically injected proteins, more specifically Th17 and Th1 responses. Furthermore, the immunogenic composition can incorporate ligands onto the polymer backbone. This offers the possibility of enhancing specific B cell or T cell responses by modifying the protein / polymer ratio, complex size, or by incorporating specific co-stimulatory factors such as TLR2 / 4 ligands into the composition.

[0092] Compared to typical conjugation techniques that involve harsh treatment of proteins, this method avoids the risk of denaturation of other modifications of the peptide antigen. This provides the substantial advantage of preserving the antigenicity of the included proteins, increasing the likelihood that the proteins themselves function as antigens (rather than simply as carriers). Similarly, since there is no heavy chemical cross-linking, this method avoids unnecessary modification / damage of the polysaccharide backbone, allows biotinylation to be precisely controlled to react with specific functional groups of the polysaccharide, and allows the biotinylation level to be easily adjusted. This is advantageous in avoiding the typical processes of conjugation that can result in damage to important side chains or epitopes that can cause a decrease in immunogenicity and protection.

[0093] The affinity-based assemblies of the present application provide for an easy and highly flexible preparation of immunogenic compositions. These are highly specific and stable, can be kept in the cold for several months, and can retain their efficacy. The assembly process is simple enough to ensure high reproducibility; only a few steps are required, thereby reducing the risk of lot-to-lot variability and being highly advantageous industrially. MAPS assembly is highly efficient (≥95%) even with low concentrations of proteins and polysaccharides (such as 0.1 mg / ml); this is a major advantage as inefficiencies in conjugate preparation (typically, efficiencies are in the range of <50%) correspond to the main obstacle and the high cost of vaccines. With regard to formulation, it is easy to adjust the composition and physical properties of the final product. The protein:polymer ratio in the complex is adjustable; with moderate biotinylation of the polymer, the protein:polymer can be ≥10:1 (w / w); conversely, if it is of interest based on the immunological target, the ratio can be ≤1:10. In addition, the size of the immunogenic MAPS composition can be adjusted by the choice of polymer size. The present method of making MAPS provides ease in combining proteins and polymers with little modification. The potential multivalency of the final product by loading multiple protein antigens from the same or different pathogens (e.g., pneumococcus and tuberculosis) in a single immunogenic construct can be used to reduce the number of vaccines required to immunize subjects against two or more diseases, providing a composition. Furthermore, the MAPS composition is highly stable, dissociates only upon boiling, and maintains its immunogenicity even after several months at 4°C. The immunogenicity of the MAPS complex can be limited by the stability of the antigenic protein or peptide component, which can be extended by inclusion in the MAPS complex. The specific antigens used herein exhibited stability at room temperature and after at least one freeze-thaw cycle. This provides an important advantage over current vaccines that are hampered if the "cold chain" is not carefully maintained.

[0094] Accordingly, one aspect of the present invention relates to an immunogenic composition comprising a polymer, at least one protein or peptide antigen, and at least one pair of complementary affinity molecules, wherein the pair of complementary affinity molecules comprises a first affinity molecule that associates with the polymer and a complementary affinity molecule that associates with the protein or peptide antigen such that when the first affinity molecule associates with the complementary affinity molecule, the antigen is indirectly linked to the polymer.

[0095] In some embodiments, the first affinity molecule is cross-linked to the polymer using a cross-linking reagent, such as CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate), EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride), sodium cyanoborohydride, cyanogen bromide, or ammonium bicarbonate / iodoacetic acid. In some embodiments, the first affinity molecule is cross-linked to carboxyl, hydroxyl, amino, phenoxyl, hemiacetal, and mercapto functional groups of the polymer. In some embodiments, the first affinity molecule is covalently bound to the polymer.

[0096] In some embodiments, the first affinity molecule is biotin or a derivative thereof, or a molecule with a structure or physical properties similar to biotin, such as amine-PEG3-biotin ((+)-biotinylated-3-6,9-trioxaundecanediamine) or a derivative thereof.

[0097] In some embodiments, the protein or peptide antigen of the immunogenic composition is a fusion protein comprising an antigenic protein or peptide that fuses with a complementary affinity binding molecule. The fusion can be a gene construct, i.e., a recombinant fusion peptide or protein. In some embodiments, the antigen can be covalently bound to the complementary affinity molecule as a fusion protein. In alternative embodiments, the antigen non-covalently binds to the complementary affinity molecule.

[0098] In some embodiments, the complementary affinity molecule is a biotin-binding protein, or a derivative or functional portion thereof. In some embodiments, the complementary affinity molecule is an avidin-like protein, or a derivative or functional portion thereof, such as, but not limited to, streptavidin or a derivative thereof. In some embodiments, the complementary affinity molecule is avidin or streptavidin, or a derivative or functional portion thereof.

[0099] In some embodiments, the secretion signal peptide is located at the N-terminus of the avidin-like protein. Any signal sequence known to those of skill in the art can be used, and in some embodiments, the signal sequence is TIFF0007679431000018.tif4128, or a derivative or functional portion thereof. In some embodiments, the antigen can be fused to the complementary affinity molecule via a flexible linker peptide that attaches the antigen to the complementary affinity molecule.

[0100] In some embodiments, the polymeric component of the immunogen includes polymers derived from living organisms, such as polysaccharides. In some embodiments, the polymer can be purified and isolated from a natural source, or it can be synthesized in the same manner as a natural composition / structure, or it can be a synthetic (e.g., with an artificial composition / structure) polymer. In some embodiments, the polymer is derived from a living organism selected from the group consisting of eukaryotic cells such as bacteria, archaea, or fungi, insects, plants, or chimeras thereof. In some embodiments, the polymer is a polysaccharide derived from a pathogenic bacterium. In certain embodiments, the polysaccharide is pneumococcal capsular polysaccharide, pneumococcal cell wall polysaccharide, or typhoid Vi polysaccharide.

[0101] In some embodiments, the polymers of the immunogenic compositions disclosed herein can be branched-chain polymers, such as branched polysaccharides, or alternatively, linear polymers, such as single-chain polymers, such as polysaccharides. In some embodiments, the polymer is a polysaccharide, such as dextran or a derivative thereof. In some embodiments, the polymer, such as a dextran polysaccharide, can have an average molecular weight of from 425 kD to 500 kDa, or, in some embodiments, greater than 500 kDa. In some embodiments, the polymer, such as a dextran polysaccharide, can have an average molecular weight of from 60 kD to 90 kDa, or, in some embodiments, less than 70 kDa. Dextran polymers can be derived from bacteria such as Leuconostoc mesenteroides.

[0102] In some embodiments, an immunogenic composition as disclosed herein can include at least two antigens, or at least three antigens, or at least five antigens, or from 2 to 10 antigens, or from 10 to 15 antigens, or from 15 to 20 antigens, or from 20 to 50 antigens, or from 50 to 100 antigens, or more than 100 antigens. In some embodiments, when an immunogenic composition as disclosed herein includes at least two antigens, the antigens can be the same antigen or at least two different antigens. In some embodiments, the antigens can be derived from the same or different pathogens, or can be different epitopes or portions of the same antigenic protein, or can be the same antigen that is specific for different serotypes or seasonal variants of the same pathogen (e.g., influenza viruses A, B, and C).

[0103] In some embodiments, the immunogenic compositions disclosed herein comprise antigens from pathogenic organisms or abnormal tissues. In some embodiments, the antigen is a tumor antigen. In some embodiments, the antigen is at least one antigen selected from antigens of pathogens or parasites such as Streptococcus pneumoniae, Mycobacterium tuberculosis or Clostridium tetani, Bacillus anthracis, HIV, seasonal or pandemic influenza antigens (such as H1N1 or H5N1), Bordetella pertussis, Staphylococcus aureus, Neisseria meningitidis, or Neisseria gonorrhoeae, HPV, Chlamydia trachomatis, HSV, or other herpesviruses, or antigens of Plasmodium spp. These antigens may comprise peptides, proteins, glycoproteins, or polysaccharides. In some embodiments, the antigen is a toxoid or a portion of a toxin.

[0104] In some embodiments, the immunogenic compositions disclosed herein comprise antigenic polysaccharides such as, for example, Vi antigen (Salmonella typhi capsular polysaccharide), pneumococcal capsular polysaccharide, pneumococcal cell wall polysaccharide, Hib (Haemophilus influenzae type B) capsular polysaccharide, meningococcal capsular polysaccharide, polysaccharide of Bacillus anthracis (the causative agent of anthrax), and other bacterial capsules or cell wall polysaccharides, or any combination thereof. The polysaccharide may have protein components, such as those derived from viruses, glycoproteins.

[0105] In some embodiments, the immunogenic compositions disclosed herein further comprise at least one co-stimulatory factor that associates with a polymer or polysaccharide, and the co-stimulatory factor can associate directly or indirectly. For example, in some embodiments, the co-stimulatory factor can covalently bind to the polymer. For example, in some embodiments, the co-stimulatory factor can covalently bind to a first affinity molecule, which then cross-links to the polymer. For example, in some embodiments, the co-stimulatory factor can attach to a complementary affinity molecule, which associates with the first affinity molecule to link the co-stimulatory factor to the polymer. In some embodiments, the co-stimulatory factor is an adjuvant. In alternative embodiments, the co-stimulatory factor can be any known to those skilled in the art and can be in any combination, for example, but not limited to, Toll-like receptor agonists (agonists for TLR2, 3, 4, 5, 7, 8, 9, etc.), NOD agonists, or agonists of the inflammasome.

[0106] Another aspect of the invention relates to the use of an immunogenic composition as disclosed herein, which is administered to a subject to induce an immune response in the subject. In some embodiments, the immune response is an antibody / B cell response, CD4 + T cell response (including Th1, Th2, and Th17 cells), and / or CD8 + T cell response. In some embodiments, at least one adjuvant is administered with the immunogenic composition.

[0107] Another aspect of the invention relates to a method for inducing an immune response in a subject against at least one antigen, the method comprising administering to the subject an immunogenic composition as disclosed herein.

[0108] Another aspect of the invention relates to a method of vaccinating an animal, such as a bird, mammal, or human, against at least one antigen, the method comprising administering a vaccine composition comprising an immunogenic composition as disclosed herein.

[0109] In all aspects disclosed herein, the animal or subject can be a human. In some embodiments, the subject can be a farm animal or a wild animal, or a captive animal. In some embodiments, the vaccine composition comprising the immunogenic composition disclosed herein can be administered subcutaneously, intranasally, orally, sublingually, intravaginally, rectally, intradermally, intraperitoneally, intramuscularly, or via a skin patch for transdermal immunization.

[0110] In all aspects as disclosed herein, the immune response is an antibody / B cell response, a CD4 + T cell response (including Th1, Th2, and Th17 responses), or a CD8+ T cell response against one or more protein / peptide antigens. In some embodiments, the immune response is an antibody / B cell response against a polymer, such as a pneumococcal polysaccharide. In some embodiments, at least one adjuvant is administered with the immunogenic composition.

[0111] Another aspect of the invention relates to the use of the immunogenic composition disclosed herein for use in the diagnosis of exposure to a pathogen or an immunogenic substance.

[0112] Also provided herein is a method of vaccinating a subject, such as a mammal, such as a human, with the immunogenic composition disclosed herein, the method comprising administering the vaccine composition disclosed herein to the subject.

[0113] Generally, immunogenic compositions and compositions comprising immunogenic conjugates can contain at least one antigen or multiple antigens attached to a polymer backbone for use in inducing an immune response against each of the antigens attached to the polymer and optionally against the polymer itself when administered to a subject. This multiple antigen presentation system (MAPS) can stimulate humoral and cellular immune responses and generate anti-polysaccharide antibody responses and B cell / Th1 / Th17 responses against multiple protein antigens using a single MAPS immunogenic construct. The combination of B and T cell immunity against a living organism can represent an optimal vaccine strategy against many diseases, including invasive infections and nasopharyngeal carriage associated with pneumococcal disease. In some embodiments, the immunogenic composition is a vaccine or is included in a vaccine.

[0114] Accordingly, one aspect of the invention relates to an immunogenic composition (multiple antigen presentation system, or MAPS) comprising at least one polymer, such as one polysaccharide; at least one protein or peptide antigen; and at least one pair of complementary affinity molecules comprising (i) a first affinity molecule that associates with the polymer and (ii) a complementary affinity molecule that associates with the antigen and that serves to indirectly attach the antigen to the polymer (e.g., a first affinity molecule associates with a complementary affinity molecule to link the antigen to the polymer). Thus, the polymer can be used as a backbone for attaching at least one, or at least two, or more (e.g., multiple) of the same or different antigens. The immunogenic compositions disclosed herein can be used to simultaneously induce both humoral and cellular immunity against multiple antigens.

[0115] Accordingly, embodiments herein provide immunogenic compositions and methods useful for enhancing an immune response in a subject and can be used alone or in combination with or in admixture with essentially any existing vaccination approach.

[0116] MAPs are flexible and versatile compositions that can be designed and manufactured to elicit specific, broad, or diverse antigenic targets. Table 1 provides a simple illustrative guide for envisioning the flexibility of MAPs embodiments.

[0117] (Table 1) Versatility of the MAPS Platform TIFF0007679431000019.tif82166

[0118] Polymer One component of a MAP consists of a "backbone", typically made of a polymer. The polymer can be antigenic or non - antigenic. This can be made of a wide variety of substances as described herein, provided that the polymer serves as a means of presenting one or more associated antigens to the immune system in an immunogenic manner. In some embodiments, the polymer is a synthetic polymer. In some embodiments, the polymer is a natural polymer, such as a polysaccharide derived from or purified from bacterial cells. In some embodiments, the polysaccharide is derived from or purified from eukaryotic cells, such as fungal, insect, or plant cells. In still other embodiments, the polymer is derived from mammalian cells, such as virus - infected cells or cancer cells. Generally, such polymers are known in the art and are included for use in the methods and compositions disclosed herein.

[0119] In some embodiments, the polymer is a polysaccharide selected from any of the following: dextran, Vi polysaccharide of Salmonella typhi, pneumococcal capsular polysaccharide, pneumococcal cell wall polysaccharide (CWPS), meningococcal polysaccharide, Haemophilus influenzae type b polysaccharide, or any other polysaccharide of viral, prokaryotic, or eukaryotic origin.

[0120] In some embodiments, the polysaccharide consists of, or comprises, an antigenic sugar moiety. For example, in some embodiments, the polysaccharide for use in the methods and immunogenic compositions disclosed herein is the Vi polysaccharide of Salmonella typhi. The Vi capsular polysaccharide has been developed against bacterial enteric infections such as typhoid fever. Robbins et al., 150 J. Infect. Dis. 436 (1984); Levine et al., 7 Baillieres Clin. Gastroenterol. 501 (1993). Vi is a polymer of α-1→4-galacturonic acid having N-acetyl at the C-2 position and variable O-acetylation at the C-3 position. The pathogenicity of Salmonella typhi correlates with the expression of this molecule. Sharma et al., 101 PNAS 17492 (2004). The Vi polysaccharide vaccine of Salmonella typhi has several advantages. Side effects are infrequent and mild, and single-dose administration results in consistent immunogenicity and efficacy. The Vi polysaccharide can be reliably standardized by physicochemical methods validated for other polysaccharide vaccines, and Vi is stable at room temperature and can be co-administered with other vaccines without affecting immunogenicity and tolerance. Azze et al., 21 Vaccine 2758 (2003).

[0121] Thus, to attach at least one antigen to the Vi polysaccharide of Salmonella typhi, the polysaccharide may be cross-linked to a first affinity molecule as disclosed herein. In some embodiments, the antigen may be from the same or a different organism such that the resulting immunogenic composition confers at least some level of immunity against one pathogen, or two different pathogens. When the antigen confers protection against Streptococcus pneumoniae, an immunogenic composition having a polymer backbone of Vi polysaccharide can enhance the immunogenic response against both Salmonella typhi and Streptococcus pneumoniae. Other examples include combining sugars from encapsulated bacteria (such as Neisseria meningitidis, Staphylococcus aureus, Streptococcus pneumoniae, Hib, etc.) and tuberculosis antigens to provide an immunogenic composition that enhances the immune response against two different pathogens.

[0122] Other polysaccharide (PS) moieties that can be used in the present invention instead of dextran, bacterial cell wall polysaccharide (CWPS), etc. include cancer carbohydrate antigens.

[0123] Furthermore, with regard to pneumococcal polysaccharides, the polysaccharide can be derived from any of the more than 93 serotypes of pneumococci that have been identified to date, including, for example, but not limited to, serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F. Additional serotypes can be identified and included in the immunogenic compositions described herein. Two or more pneumococcal polysaccharides can be included as the polymer backbone of the immunogenic composition or in a vaccine comprising the present MAPS composition.

[0124] The polysaccharide can also be derived from the present invention, and the immunogenic composition comprises meningococcal capsular polysaccharides from at least one, two, three, or four of serogroups A, C, W, W135, or Y.

[0125] Further embodiments include any of the polysaccharides or oligosaccharides of Staphylococcus aureus of type 5, type 8, or both.

[0126] In some embodiments, the polymer is a chimeric polymer comprising two or more types of polymers. For example, the polymer of an immunogenic composition as disclosed herein can comprise a portion of polymer A and the remaining portion of polymer B. There is no limit to the amount of different types of polymers that can be used in a single MAPS backbone entity. In some embodiments, when the polymer is a branched polymer, the chain polymer can be polymer A and the branches can be at least one, or at least two, or at least three or more different polymers.

[0127] In some embodiments, the polymer is a branched polymer. In some embodiments, the polymer is a single-stranded polymer.

[0128] In some embodiments, the polymer is a polysaccharide comprising repeating units of at least 10 carbohydrate repeating units, or at least 20, or at least 50, or at least 75, or at least 100, or at least 150, or at least 200, or at least 250, or at least 300, or at least 350, or at least 400, or at least 450, or at least 500, or more than 500.

[0129] In one aspect of the invention, the polysaccharide (PS) can have a molecular weight of <500 kDa or >500 kDa. In another aspect of the invention, the PS has a molecular weight of <70 kDa.

[0130] In some embodiments, the polymer is a high molecular weight polymer, for example, the polymer comprises from about 425 to 500 kDa, for example, at least 300 kDa, or at least 350 kDa, or at least 400 kDa, or at least 425 kDa, or at least 450 kDa, or at least 500 kDa, or more than 500 kDa, but typically has an average molecular weight of less than 500 kDa.

[0131] In some embodiments, the polymer can be a low molecular weight polymer, for example, the polymer comprises from about 60 kDa to about 90 kDa, for example, at least 50 kDa, or at least 60 kDa, or at least 70 kDa, or at least 80 kDa, or at least 90 kDa, or at least 100 kDa, or more than 100 kDa, but generally has an average molecular weight of less than about 120 kDa.

[0132] In some embodiments, the polymer is harvested and purified from a natural source, and in other embodiments, the polymer is synthetic. Methods for producing synthetic polymers, including synthetic polysaccharides, are known to those skilled in the art and are encompassed by the compositions and methods disclosed herein.

[0133] Several of the polysaccharide polymers that can serve as a backbone for one or more antigens or antigen types are exemplified in Table 2.

[0134] (Table 2) Exemplary polysaccharide polymer MAPS backbones and related exemplary antigens TIFF0007679431000020.tif75166

[0135] Additional polymers that can be used in the immunogenic MAPS compositions described herein include polyethylene glycol-based polymers, poly(orthoester) polymers, polyacrylic carriers, PLGA, polyethyleneimine (PEI), polyamidoamine (PAMAM) dendrimers, β-amino ester polymers, polyphosphate esters (PPE), liposomes, polymersomes, nucleic acids, phosphorothioated oligonucleotides, chitosan, silk, polymeric micelles, protein polymers, virus particles, virus-like particles (VLP), or other microparticles. See, for example, El-Sayed et al., Smart Polymer Carriers for Enhanced Intracellular Delivery of Therapeutic Molecules, 5 Exp. Op. Biol. Therapy, 23 (2005). Biocompatible polymers developed for nucleic acid delivery can be adapted for use as a backbone herein. See, for example, BIOCOMPATIBLE POL. NUCL. ACID. DELIV. (Domb et al., eds., John Wiley & Sons, Inc. Hoboken, NJ, 2011).

[0136] For example, VLPs resemble viruses but are non-infectious as they do not contain any viral genetic material. Expression involving the recombinant expression of viral structural proteins, such as envelope or capsid components, can result in the self-assembly of VLPs. VLPs are generated from components of a wide variety of viral families, including parvoviruses (e.g., adeno-associated virus), retroviruses (e.g., HIV), and flaviviruses (e.g., hepatitis B or C virus). VLPs can be generated in a variety of cell culture systems, including mammalian cell lines, insect cell lines, yeast, and plant cells. Recombinant VLPs are particularly advantageous as viral components can be fused to the recombinant antigens described herein.

[0137] Antigen Fusion proteins and immunogenic compositions as disclosed herein can include any antigen that elicits an immune response in a subject. In some embodiments, at least one or more antigens are associated with the polymer of the composition. In some embodiments, at least 2, or at least 3, or at least 5, or at least 10, or at least 15, or at least 20, or at least 50, or at least 100, or more than 100 antigens may be associated with the polymers disclosed herein. In some embodiments, where the immunogenic composition includes more than one antigen, these antigens may be the same antigen, or these antigens may be a variety of different antigens associated with the polymer. In some embodiments, where the immunogenic composition includes more than one antigen, this antigen may be an antigen from the same pathogen or different pathogens, or alternatively, different antigens from the same pathogen, or similar antigens from different serotypes of pathogens.

[0138] The antigens for use in the fusion proteins, immunogenic compositions, and methods described herein can be any antigen, including but not limited to pathogenic peptides, toxins, toxoids, subunits thereof, or combinations thereof (e.g., cholera toxin, tetanus toxoid).

[0139] In some embodiments, the antigen that may be fused to a complementary affinity molecule can be any antigen associated with an infectious disease, or cancer or immune disease. In some embodiments, the antigen can be an antigen expressed by any of a variety of infectious agents, including viruses, bacteria, fungi, or parasites.

[0140] In some embodiments, the antigen is derived from (e.g., obtained from) a pathogenic organism. In some embodiments, the antigen is a cancer antigen or tumor antigen, e.g., an antigen derived from a tumor cell or cancer cell.

[0141] In some embodiments, the antigen derived from a pathogenic organism is an antigen associated with an infectious disease, which can be derived from any of a variety of infectious pathogens, including viruses, bacteria, fungi, or parasites.

[0142] In some embodiments, the target antigen is any antigen associated with a pathological condition, e.g., an infectious disease or pathogen, or an immune disease such as cancer or autoimmune disease. In some embodiments, the antigen can be expressed by any of a variety of infectious pathogens, including viruses, bacteria, fungi, or parasites. The target antigens for use in the methods and compositions disclosed herein can also include, for example, pathogenic peptides, toxins, toxoids, subunits thereof, or combinations thereof (e.g., cholera toxin, tetanus toxoid).

[0143] Non-limiting examples of infectious viruses include the Retroviridae; Picornaviridae (e.g., poliovirus, hepatitis A virus; enterovirus, human coxsackievirus, rhinovirus, echovirus); Calciviridae (such as strains causing gastroenteritis); Togaviridae (e.g., equine encephalitis virus, rubella virus); Flaviridae (e.g., dengue virus, encephalitis virus, yellow fever virus); Coronaviridae (e.g., coronavirus); Rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus); Filoviridae (e.g., Ebola virus); Paramyxoviridae (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza virus); Bunyaviridae (e.g., hantavirus, bunyavirus, phlebovirus, and nairovirus); Arenaviridae (hemorrhagic fever virus); Reoviridae (e.g., reovirus, orbivirus, and rotavirus); Birnaviridae; Hepadnaviridae (hepatitis B virus); Parvoviridae (parvovirus); Papovaviridae (papillomavirus, polyomavirus); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and HSV-2, varicella-zoster virus, cytomegalovirus (CMV), Marek's disease virus, herpesvirus); Poxviridae (smallpox virus, vaccinia virus, poxvirus); and Iridoviridae (African swine fever virus, etc.);and unclassified viruses (e.g., the causative agents of spongiform encephalopathies, the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), the agents of non-A, non-B hepatitis (class 1 = enterically transmitted; class 2 = parenterally transmitted (i.e., hepatitis C); Norwalk virus and related viruses, and astroviruses). The compositions and methods described herein are contemplated for use in the treatment of infections caused by these viral agents.;

[0144] Examples of fungal infections that can be addressed by including an antigen in this embodiment include aspergillosis, thrush (caused by Candida albicans), cryptococcosis (caused by Cryptococcus), and histoplasmosis. Thus, examples of infectious fungi include, but are not limited to, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans. Components of these life forms can be included as antigens in the MAPS described herein.

[0145] In one aspect of the present invention, the antigen is an infectious microorganism, such as Bordetella pertussis, Brucella, Enterococci, Neisseria meningitidis, Neisseria gonorrhoeae, Moraxella, Haemophilus (classifiable or non-classifiable), Pseudomonas, Salmonella, Shigella, Enterobacter, Citrobacter, Klebsiella, Escherichia coli, Helicobacter pylori, Clostridia, Bacteroides, Chlamydiaceae, Vibrio cholera, Mycoplasma, Treponemes, Borrelia burgdorferi (Lyme disease), Legionella pneumophilia, Mycobacteria (Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansaii, Mycobacterium gordonae, Mycobacterium leprae)such as Mycobacterium leprae), Staphylococcus aureus, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (Viridans group), Streptococcus faecalis, Streptococcus bovis, anaerobic Streptococcus genus, Streptococcus pneumoniae, pathogenic Campylobacter genus, Enterococcus genus, Haemophilus influenzae, Bacillus anthracis, Corynebacterium diphtheriae, Corynebacterium genus, Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Leptospira genus, Pasturella multocida, Bacteroides genus, Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, and Actinomyces israelii. The compositions and methods described herein are contemplated for use in the treatment or prevention of infectious diseases against these bacterial agents.

[0146] Additional parasitic pathogens from which the antigen can be derived include, for example, Entamoeba histolytica, Plasmodium falciparum, the genus Leishmania, Toxoplasma gondii, Rickettsia, and Helminths.

[0147] In another aspect of the invention, the antigen is a cleaved pneumococcal PsaA protein, pneumolysin toxoid pneumococcal serine / threonine protein kinase (StkP), pneumococcal serine / threonine protein kinase repeat unit (StkPR), pneumococcal PcsB protein, staphylococcal α-hemolysin, mycobacterial mtb protein ESAT-6, mycobacterial cell wall core antigen, Chlamydia CT144, CT242, or CT812 polypeptide or fragments thereof, Chlamydia DNA gyrase subunit B, Chlamydia sulfite synthase / phosphate dehydrogenase, Chlamydia cell division protein FtsY, Chlamydia methionyl-tRNA synthetase, Chlamydia DNA helicase (uvrD), Chlamydia ATP synthase subunit I (atpI), or Chlamydia metal-dependent hydrolase.

[0148] One embodiment of the invention provides an immunogenic composition that targets the pathogen Mycobacterium tuberculosis (TB), an intracellular bacterial parasite. An example of a TB antigen is TbH9 (also known as Mtb 39A). Other TB antigens include, but are not limited to, DPV (also known as Mtb8.4), 381, Mtb4l, Mtb40, Mtb32A, Mtb64, Mtb83, Mtb9.9A, Mtb9.8, Mtb16, Mtb72f, Mtb59f, Mtb88f, Mtb7lf, Mtb46f, and Mtb31f (the "f" indicates that it is a fusion or two or more proteins).

[0149] As described above, the antigen for use in the immunogenic composition of the present invention may be derived from the genus Chlamydia. The family Chlamydiaceae (comprising Chlamydiae and Chlamydophila) are obligate intracellular Gram-negative bacteria. Chlamydia trachomatis infection is one of the most common bacterial sexually transmitted infections, with perhaps 89 million new genital Chlamydia infections occurring each year. The Chlamydia of the present invention includes, for example, Chlamydia trachomatis, Chlamydophila pneumoniae, C. muridarum, C. suis, Chlamydophila abortus, Chlamydophila psittaci, Chlamydophila caviae, Chlamydophila felis, Chlamydophila pecorum, and Chlamydophila pneumoniae. Animal models of Chlamydia infection have demonstrated that T cells play an important role in both the elimination of primary infection and the prevention of reinfection of susceptible hosts. Therefore, specific benefits can be achieved by inducing a cellular immune response against Chlamydia infection using the immunogenic composition disclosed herein.

[0150] More specifically, the Chlamydia antigens useful in the present invention include DNA gyrase subunit B, sulfite synthase / bisphosphate phosphatase, cell division protein FtsY, methionyl-tRNA synthetase, DNA helicase (uvrD); ATP synthase subunit I (atpI), or a metal-dependent hydrolase (U.S. Patent Application Publication No. 20090028891). Additional Chlamydia trachomatis antigens include the CT144 polypeptide, a peptide having amino acid residues 67-86 of CT144, a peptide having amino acid residues 77-96 of CT144, the CT242 protein, a peptide having amino acids 109-117 of CT242, a peptide having amino acids 112-120 of the CT242 polypeptide, the CT812 protein (derived from the pmpD gene), a peptide having amino acid residues 103-111 of the CT812 protein; and several other antigenic peptides derived from Chlamydia trachomatis: including TIFF0007679431000021.tif24153. See International Publication No. 2009 / 020553. In addition, Chlamydia pneumoniae antigens including homologs of the aforementioned polypeptides (see U.S. Patent No. 6,919,187) can be used as antigens in immunogenic compositions and methods as disclosed herein.

[0151] Fungal antigens may be derived from Candida species and other yeasts, or other fungi (Aspergillus, other environmental fungi). With regard to other parasites, malaria, as well as worms and amoebas, can provide antigenic antigens for use in the immunogenic compositions and methods disclosed herein.

[0152] In some embodiments, when the antigen generates an anti-influenza immunogen, the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) are generally the optimal antigens. Both the nucleoprotein (NP) polypeptide and matrix (M) are internal viral proteins and are thus not normally considered in vaccine design for antibody-based immunity. Influenza vaccines are routinely used in humans and include vaccines derived from inactivated whole influenza virus, live attenuated influenza virus, or purified and inactivated materials from viral strains. For example, conventional influenza vaccines can be produced using three potentially threatening influenza virus strains. These strains are typically grown in fertilized chicken eggs, which requires a wide range of processes including inoculation and incubation of the eggs, egg collection, purification and inactivation of the virus, processing and storage until the final vaccine formulation of the virus or viral components, and aseptic filling into appropriate containers. Typically, this egg-based production cycle exceeds 70 weeks. In the event of a large-scale influenza pandemic, the availability of a potent and safe vaccine is a major concern. Additionally, there is a risk of impurities in the eggs such as antibiotics and contaminants, which can negatively affect the sterility of the vaccine. Furthermore, egg-derived influenza vaccines are contraindicated in those with severe allergies to egg proteins and those with a history of Guillain-Barré syndrome. The present invention provides an alternative to egg-based influenza vaccines that not only avoids selequae related to eggs but also provides a platform for using multiple influenza antigens on a highly controlled platform.

[0153] In some embodiments, the antigens for use in the immunogenic compositions disclosed herein may also include those used in biological warfare such as ricin, which can elicit a CMI response.

[0154] Furthermore, the present invention also provides an immunogenic composition comprising an antigen that elicits an immune response against cancer. In these conjugates, the antigen is an antigen expressed by a cancer or tumor, or an antigen derived from a tumor. In some embodiments, such an antigen is referred to herein as a "cancer antigen" and is typically a protein expressed primarily in cancer cells, and the conjugate thereof elicits both a strong humoral immunity and a strong cellular immunity against this protein. A number of cancer-related antigens have been identified, some of which are currently used to prepare experimental cancer therapeutic vaccines and are thus suitable for use in this embodiment. Antigens related to more than one type of cancer include carcinoembryonic antigen (CEA), cancer / testis antigens such as NY-ESO-1, Mucin-1 (MUC1) such as sialyl Tn (STn), gangliosides such as GM3 and GD2, p53 protein, and HER2 / neu protein (also known as ERBB2). Antigens specific to certain types of cancer include the mutant form of epidermal growth factor receptor called EGFRvIII, tyrosinase, MART1, gp100, melanocyte / melanoma differentiation antigens such as melanoma antigen gene (MAGE) and tyrosinase-related antigen, prostate-specific antigen, the fusion protein BCR-ABL, leukemia-associated antigens (LAA) such as Wilms tumor protein and proteinase 3, and idiotypic (Id) antibodies. See, for example, Mitchell, 3 Curr. Opin. Investig. Drugs 150 (2002), Dao & Scheinberg, 21 Best Pract. Res. Clin. Haematol. 391 (2008).

[0155] Another approach for generating an immune response against cancer is to use antigens derived from microorganisms that cause or are involved in the development of cancer. These vaccines have been used against cancers including hepatocellular carcinoma (hepatitis B virus, hepatitis C virus, Opisthorchis viverrin), lymphoma and nasopharyngeal carcinoma (Epstein–Barr virus), colorectal cancer, gastric cancer (Helicobacter pylori), bladder cancer (Schistosoma hematobium), T-cell leukemia (human T-cell leukemia virus), cervical cancer (human papillomavirus), etc. Clinical trials have been conducted to date for vaccines targeting bladder cancer, brain tumors, breast cancer, cervical cancer, kidney cancer, melanoma, multiple myeloma, leukemia, lung cancer, pancreatic cancer, prostate cancer, and solid tumors. See Pardoll et al., ABELOFF FS CLIN.ONCOL. (4th ed., Churchill Livingstone, Philadelphia 2008), Sioud, 360 Methods Mol.Bio. 277 (2007), Pazdur et al., 30 J.Infusion Nursing 30(3):173 (2007), Parmiani et al., 178 J.Immunol. 1975 (2007), Lollini et al., 24 Trends Immunol. 62 (2003), Schlom et al., 13 Clin.Cancer Res. 3776 (2007), Banchereau et al., 392 Nature 245 (1998), Finn, 358 New Engl.J.Med. 2704 (2008), Curigliano et al., 7 Exp.Rev.Anticancer Ther. 1225 (2007). Marek's disease virus, a herpesvirus that causes tumors in poultry, has long been managed by vaccines. Thus, this embodiment encompasses both preventive or prophylactic anti-cancer immunogenic compositions and therapeutic / curative cancer vaccines.

[0156] The intended proliferative diseases and cancers include AIDS-related cancers, acoustic neuromas, acute lymphoblastic leukemia, acute myeloid leukemia, adenocarcinomas, adrenocortical carcinomas, idiopathic myelofibrosis, alopecia, alveolar soft part sarcoma, anal cancer, angiosarcoma, astrocytoma, ataxia telangiectasia, basal cell carcinoma (skin), bladder cancer, bone cancer, bowel cancer, brain and CNS tumors, breast cancer, carcinoid tumors, cervical cancer, childhood brain tumors, childhood cancers, childhood leukemia, childhood soft tissue sarcoma, chondrosarcoma, choriocarcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colorectal cancer, cutaneous T-cell lymphoma, dermatofibrosarcoma protuberans, desmoplastic small round cell tumor, ductal carcinoma, endocrine cancer, endometrial cancer, epithelioma, esophageal cancer, Ewing sarcoma, extrahepatic bile duct cancer, eye cancers including, for example, uveal melanoma and retinoblastoma, fallopian tube cancer, Fanconi anemia, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal cancer, gastrointestinal carcinoid tumor, genitourinary cancer, germ cell tumor, gestational trophoblastic disease, glioma, gynecological cancer, hematological malignancies, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, hereditary breast cancer, Hodgkin disease, human papillomavirus-related cervical cancer, hydatidiform mole, hypopharyngeal cancer, islet cell cancer, Kaposi sarcoma, kidney cancer, laryngeal cancer, leiomyosarcoma, leukemia, Li-Fraumeni syndrome, lip cancer, liposarcoma, lung cancer, lymphedema, lymphoma, non-Hodgkin lymphoma, male breast cancer, malignant rhabdoid tumor of the kidney, medulloblastoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic cancer, mouth cancer, multiple endocrine neoplasia, mycosis fungoides, myelodysplastic syndrome, multiple myeloma, myeloproliferative diseases, nasal cancer, nasopharyngeal cancer, nephroblastoma, neuroblastoma, neurofibromatosis, Nijmegen breakage syndrome, non-melanoma skin cancer, non-small cell lung cancer (NSCLC), oral cancer, oropharyngeal cancer, osteosarcoma, ostomy ovarian cancer, pancreatic cancer, paranasal sinus cancer, parathyroid cancer, parotid gland cancer, penile cancer, peripheral neuroectodermal tumor, pituitary cancer, polycythemia vera, prostate cancer, renal cell cancer, retinoblastoma, rhabdomyosarcoma, Rothmund-Thomson syndrome, salivary gland cancer, sarcoma, schwannoma, Sézary syndrome, skin cancer, small cell lung cancer (SCLC), small intestine cancer, soft tissue sarcoma, spinal cord tumor, squamous cell carcinoma (skin), stomach cancer, synovial sarcoma, testicular cancer, thymic cancer, thyroid cancer, transitional cell carcinoma (bladder), transitional cell carcinoma (renal pelvis / ureter), trophoblastic carcinoma, urethral cancer, urological cancer, uterine sarcoma, uterine cancer, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and Wilms tumor.

[0157] In some embodiments, the antigens for use in the immunogenic compositions disclosed herein may include antigens of autoimmune diseases, and for example, these may be "self-antigens". Autoimmune diseases contemplated for diagnosis by the assays described herein include alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, Addison's disease, aplastic anemia, multiple sclerosis, autoimmune adrenal diseases, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue dermatitis, chronic fatigue syndrome, chronic inflammatory demyelinating syndrome (CFIDS), chronic inflammatory polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin syndrome, Crohn's disease, dermatitis herpetiformis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia, glomerulonephritis, Graves' disease, Guillain-Barré, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, insulin-dependent diabetes (type I), lichen planus, lupus, Meniere's disease, mixed connective tissue disease, myasthenia gravis, myocarditis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man syndrome, Takayasu arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, Wegener's syndrome, vasculitis, and vitiligo, but are not limited thereto. Assessing potential or actual CMI responsiveness in a subject having, suspected of having, or prone to having an autoimmune disease is generally important.

[0158] In some embodiments, the antigen for use in the immunogenic compositions disclosed herein may be an antigen associated with an inflammatory disease or condition. Examples of inflammatory disease states in which the antigen may be useful include, among others, acne, angina, arthritis, aspiration pneumonia, empyema, gastroenteritis, necrotizing enteritis, pelvic inflammatory disease, pharyngitis, pleurisy, chronic inflammatory demyelinating polyneuropathy, chronic inflammatory demyelinating polyradiculoneuropathy, and chronic inflammatory demyelinating polyneuropathy, but are not limited thereto.

[0159] In some embodiments, the antigen may be a native (i.e., full or whole) antigen or a functional portion of an antigen that contains more than one epitope. In some embodiments, the antigen is a peptide functional portion of the antigen. In this regard, "native" means that the antigen is the full-length antigen as it naturally occurs with its antigen polypeptide. This is in stark contrast to the delivery of only small portions or peptides of the antigen. Delivering a native antigen to cells enables or induces an immune response against all of the epitopes of the native antigen, not just a single or selected few peptide epitopes. Thus, the methods and immunogenic compositions described herein include native antigens associated with a polymer for a more sensitive and higher specificity immune response compared to the use of antigens based on single epitope peptides.

[0160] Alternatively, in some embodiments, the intact antigen can be divided into multiple portions depending on the size of the first antigen. Typically, when the whole antigen is a multimeric polypeptide, the whole protein can be divided into subunits and / or domains, where each individual subunit or domain of the antigen can be associated with a polymer by the methods disclosed herein. Alternatively, in some embodiments, the intact antigen can be inclusively divided into functional fragments or portions of the whole antigen, e.g., at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10, or at least 11, or at least 12, or at least 13, or at least 15, or at least 20, or at least 25, or more than 25 portions (e.g., fragments or segments), and each individual functional fragment of the antigen can be associated with a polymer according to a method as disclosed herein.

[0161] Fragmentation or splitting of the full-length antigen polypeptide may be an equal division of the full-length antigen polypeptide, or alternatively, in some embodiments, the fragmentation is asymmetric or unequal. As a non-limiting example, when splitting an antigen into two overlapping fragments, the antigen can be split into fragments of approximately the same (equal) size, or alternatively, one fragment can be approximately 45% of the full antigen and the other fragment can be 65%. As a further non-limiting example, the full antigen can be split into a combination of fragments of different sizes. For example, when splitting an antigen into two fragments, the fragments can be approximately 40% (including 40%) to approximately 70% (including 70%), or approximately 45% (including 45%) to approximately 65% (including 65%), or approximately 35% (including 35%) to approximately 75% (including 75%), or approximately 25% (including 25%) to approximately 85% (including 85%) of the full antigen. Any combination of overlapping fragments of the full-length full antigen is included for use in generating a panel of overlapping polypeptides of the antigen. By way of illustration only, when splitting an antigen into five parts, they can be split equally (i.e., each overlapping fragment is approximately 21% - 25% of the entire length of the antigen), or unevenly (i.e., the antigen can be split into the following five overlapping fragments: fragment 1 is approximately 25% of the size of the full-length antigen, fragment 2 is approximately 5%, fragment 3 is approximately 35%, fragment 4 is approximately 10%, and fragment 5 is approximately 25%, provided that each fragment overlaps with at least one other fragment).

[0162] Typically, the panel of antigenic portions may substantially encompass the entire length of the full (i.e., intact) antigen polypeptide. Thus, in some embodiments, the immunogenic composition comprises a polymer having many different and / or overlapping fragments of the same intact antigen. Duplicate protein fragments of the antigen can be produced much more rapidly and inexpensively and with improved stability compared to the use of peptide antigens alone. Further, in some embodiments, three-dimensional structure is important for epitope recognition and larger antigenic peptides or fragments may provide an advantage over peptide fragments, and thus antigens that are polypeptides larger than simple peptides are preferred.

[0163] One of ordinary skill in the art can split the full antigen into duplicate proteins of the antigen to create a panel of polypeptide antigens. By way of example only, the TB-specific antigen TB1 (CFP, also known as culture filtrate - 10 or CFP-10) can be split into at least 17 portions, for example, to generate a panel of 17 different polypeptides, each of which contains a different but overlapping TB-specific antigen TB1 (CFP) fragment. The culture filtrate protein (CFP-10) (Genbank AAC83445) is a 100 amino acid residue protein fragment of 10 kDa from Mycobacterium tuberculosis. This is also known as the L45 antigen homolog protein (LHP).

[0164] Target antigens for use in the methods and compositions described herein can be expressed by recombinant means and optionally can include affinity or epitope tags to facilitate purification, and the methods are well known in the art. To obtain the antigens of the present invention, chemical synthesis of any oligopeptide, free or conjugated to a carrier protein, can be used. Oligopeptides are considered a type of polypeptide. Antigens can be expressed as fusions with complementary affinity molecules such as, but not limited to, streptavidin or its derivatives or functional fragments. Alternatively, it is also possible to conjugate the target antigen to a complementary affinity molecule such as, but not limited to, streptavidin or its derivatives or functional fragments after the target antigen has been prepared.

[0165] Polypeptides can also be synthesized as branched structures such as those disclosed in U.S. Pat. Nos. 5,229,490 and 5,390,111. Antigenic polypeptides include, for example, synthetic or recombinant B-cell and T-cell epitopes, universal T-cell epitopes, and mixtures of T-cell epitopes from one organism or disease and B-cell epitopes from another.

[0166] Antigens can be obtained by recombinant means or chemical polypeptide synthesis and can also be purified using the physical and chemical properties of the antigen, such as by fractionation or chromatography of antigens obtained from natural sources or extracts. These techniques are known in the art.

[0167] In some embodiments, the antigen can be solubilized in water, a solvent such as methanol, or a buffer. Suitable buffers are Ca 2+ / Mg 2+It includes, but is not limited to, phosphate buffered saline (PBS) without, normal saline (150 mM NaCl in water), and Tris buffer. Antigens that are not soluble in neutral buffer can be solubilized with 10 mM acetic acid and then diluted to the desired dose with a neutral buffer such as PBS. In the case of antigens soluble only at acidic pH, after solubilization with dilute acetic acid, acetic acid-PBS may be used as a diluent at acidic pH. Glycerol can be a suitable non-aqueous solvent for use in the compositions, methods, and kits described herein.

[0168] Typically, when designing a protein vaccine against a pathogen, extracellular proteins or those exposed to the viral environment are often ideal candidates as antigenic components in the vaccine. Antibodies raised against such extracellular proteins are the first line of defense against the pathogen during infection. Antibodies bind to the pathogen's proteins, promote opsonization of the antibody, and mark the pathogen for ingestion and destruction by phagocytic cells such as macrophages. Opsonization by antibodies can also kill the pathogen by antibody-dependent cell-mediated cytotoxicity. Antibodies promote the release of lytic products from cells such as monocytes, neutrophils, eosinophils, and natural killer cells.

[0169] In one embodiment of the invention described herein, all wild-type proteins, which are antigens for use in the compositions disclosed herein, have sequences found in naturally occurring viruses as found in amino acid residues and have not been altered by selective growth conditions or molecular biological methods.

[0170] In one embodiment, the immunogenic composition described herein may comprise an antigen that is a glycosylated protein. In other words, each of the antigens of interest may be a glycosylated protein. In one embodiment of the immunogenic composition described herein, the antigen, or antigen fusion polypeptide, is O-linked glycosylated. In another embodiment of the immunogenic composition described herein, the antigen, or antigen fusion polypeptide, is N-linked glycosylated. In yet another embodiment of the immunogenic composition described herein, the antigen, or antigen fusion, is O-linked glycosylated and N-linked glycosylated. In other embodiments, other types of glycosylation, such as C-mannosylation, are possible. Glycosylation of proteins occurs primarily in eukaryotic cells. N-glycosylation is important for the folding of some eukaryotic proteins and results in co- and post-translational modification mechanisms that regulate the structure and function of membrane and secreted proteins. Glycosylation is an enzymatic process that links saccharides to form glycans and attaches them to proteins and lipids. In N-glycosylation, the glycan attaches to the amide nitrogen of the asparagine side chain during protein translation. The three main saccharides that form the glycan are glucose, mannose, and N-acetylglucosamine molecules. The N-glycosylation consensus is Asn-Xaa-Ser / Thr, where Xaa can be any of the known amino acids. O-linked glycosylation occurs in the latter stages of protein processing, probably within the Golgi apparatus. In O-linked glycosylation, N-acetyl-galactosamine, O-fucose, O-glucose, and / or N-acetylglucosamine are added to serine or threonine residues. One of ordinary skill in the art can use bioinformatics software, such as NetNGlyc 1.0 and NetOGlyc Prediction software from Technical University (Denmark), to find N- and O-glycosylation sites in the polypeptides of the present invention.The NetNglyc server predicts N-glycosylation sites in proteins using an artificial neural network that examines the sequence context around Asn-Xaa-Ser / Thr sequences. NetNGlyc 1.0 and NetOGlyc 3.1 Prediction software are accessible on the EXPASY website. In one embodiment, N-glycosylation occurs in the target antigen polypeptide of the fusion polypeptides described herein.

[0171] Pair of affinity molecules As disclosed herein, in some embodiments, the antigen is connected to the polymer via complementary affinity pairs. This connection between the antigen and the polymer is mediated by the polymer, which is connected to a first affinity molecule, which associates with a second (e.g., complementary) affinity molecule, which is attached to the antigen. Examples of complementary affinity pairs are biotin / biotin-binding proteins.

[0172] Examples of affinity complementary affinity pairs include, but are not limited to, biotin-binding proteins or avidin-like proteins that bind to biotin. For example, if the first affinity-binding molecule is biotin (which associates with the polymer), the complementary affinity molecule can be a biotin-binding protein or avidin-like protein, or derivatives thereof, such as, but not limited to, avidin, streptavidin, or streptavidin, or variants, derivatives, or functional moieties thereof.

[0173] In some embodiments, the first affinity-binding molecule is biotin, a biotin derivative, or a biotin mimetic, such as, but not limited to, amine-PEG3-biotin (((+)-biotinylated-3-6,9-trithiaundecanediamine), or derivatives or functional fragments thereof. Certain biotin mimetics have the formula where X a is R or L, X b is S or T, X c is Y or W, DX a AX b PXc (SEQ ID NO: 39) or CDX a AX b PX c Contains the sequence of CG (SEQ ID NO: 40) and has specific peptide motifs. These motifs can bind to avidin and neutravidin, but not to streptavidin. See, for example, Gaj et al., 56 Prot. Express. Purif. 54 (2006).

[0174] The binding of the first affinity molecule to the polymer and the complementary affinity molecule to the antigen may be non-covalent or by a chemical mechanism such as covalent bonding, affinity bonding, intercalation, coordination bonding, and complex formation. Covalent bonding results in a very stable bond and is particularly suitable for this embodiment. Covalent bonding can be achieved either by direct condensation of existing side chains or by incorporation of external binding molecules.

[0175] For example, in some embodiments, the antigen may be non-covalently bound to one of the pair in the complementary attachment pair. In alternative embodiments, the antigen may be covalently bonded or fused to one of the pair in the complementary attachment pair. Methods for creating fusion proteins are known in the art and are also discussed herein.

[0176] In other embodiments, the first affinity binding molecule is linked to the polymer by non-covalent or covalent bonding. In some embodiments, a cross-linking reagent is used to covalently bond the first affinity binding molecule to the polymers disclosed herein.

[0177] In some embodiments, the first affinity binding molecule associates with the complementary affinity molecule by non-covalent binding associations known in the art, including but not limited to electrostatic interactions, hydrogen bonding, hydrophobic interactions (i.e., van der Waals forces), hydrophilic interactions, and other non-covalent interactions. Other higher-order interactions with the intervening moiety are also contemplated.

[0178] In some embodiments, the complementary affinity molecule is an avidin-related polypeptide. In a specific embodiment, the complementary affinity molecule is a streptavidin such as recombinant streptavidin. In particular, the recombinant streptavidin is a modified streptavidin that can be expressed at high yield in E. coli. A typical yield is >30 mg per liter of E. coli culture. Streptavidin has lower sequence homology to egg avidin (22.4% sequence identity and 35.0% sequence similarity) compared to other avidin-like proteins. By using modified streptavidin, the risk that MAPS induces an allergic reaction by eggs in the subject is reduced. Furthermore, antibodies against recombinant modified streptavidin do not have an obvious cross-reaction with egg avidin (and vice versa).

[0179] More specifically, some embodiments include a modified streptavidin designed for recombinant expression in E. coli. The coding sequence of the streptavidin gene was optimized using E. coli expression codons to avoid any difficulties during expression in E. coli due to rare codons present in the original gene. After bioinformatics and structure-based analysis, the first 44 residues of the full-length streptavidin were found to be unnecessary for the core structure and function, so these were removed to simplify the construct. The correct folding of the recombinant protein was improved by adding an E. coli secretion signal sequence to the N-terminus of truncated streptavidin (45-179) to facilitate the translocation of the recombinant protein into the periplasmic space of E. coli cells where the functionally important disulfide bonds in streptavidin are correctly formed. Compared to known avidin-like proteins that form tetramers, the modified recombinant streptavidin forms dimers and further improves the expression of the recombinant streptavidin-antigen fusion as a soluble protein in E. coli.

[0180] Furthermore, as will be discussed in more detail elsewhere in this specification, a flexible linker region was added between the streptavidin and the antigen protein to improve the expression and solubility of the fusion antigen in E. coli. In addition, based on bioinformatics and structural analysis, different antigen constructs (either the full-length antigen or important functional domains) were cloned and expressed, or chimeric proteins were generated that contained two different antigens.

[0181] Additional affinity pairs that may be useful in the methods and compositions described herein include antigen-antibody, metal / ion-metal / ion-binding protein, lipid / lipid-binding protein, saccharide / saccharide-binding protein, amino acid / peptide / amino acid or peptide-binding protein, enzyme-substrate or enzyme-inhibitor, ligand-agonist / receptor, or biotin mimetic. When using alternative affinity pairs, alternative means of attaching each polymer and antigen, such as in vitro enzymatic reactions rather than gene fusions, may be utilized. More specifically, the antigen-antibody affinity pair results in a very strong and specific interaction. The antigen can be any epitope, including proteins, peptides, nucleic acids, lipids, polysaccharides / oligosaccharides, ions, etc. The antibody can be any type of immunoglobulin or the Ag-binding portion of an immunoglobulin such as a Fab fragment. For metal / ion-metal / ion-binding proteins, examples include Ni NTA vs. histidine-tagged proteins, or Zn vs. Zn-binding proteins. For lipid / lipid-binding proteins, an example is cholesterol vs. cholesterol-binding protein. Examples of saccharide / saccharide-binding proteins include maltose vs. maltose-binding protein, mannose / glucose / oligosaccharide vs. lectin. Enzyme-substrate / inhibitors include substrates derived from a wide range of substances, including proteins, peptides, amino acids, lipids, sugars, or ions. The inhibitor may be an analog of the actual substrate that can generally bind more tightly and even irreversibly to the enzyme. For example, trypsin vs. soybean trypsin inhibitor. The inhibitor can be a natural or synthetic molecule. For other ligand / agonist-receptors, the ligand may be derived from a wide range of substances, including proteins, peptides, amino acids, lipids, sugars, ions, agonists, and may be an analog of the actual ligand. An example is the LPS vs. TLR4 interaction.

[0182] Crosslinking reagent: When coupling protein molecules to other molecules, many divalent or polyvalent linking agents are useful. For example, representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzenes, and hexamethylenediamine. This list is not intended to be an exhaustive list of the various types of coupling agents known in the art, but rather is an exemplary list of more common coupling agents. See Killen & Lindstrom, 133 J. Immunol. 1335 (1984), Jansen et al., 62 Imm. Rev. 185 (1982), Vitetta et al.

[0183] In some embodiments, cross-linking reagents described in the literature are included for use in the methods, immunogenic compositions, and kits disclosed herein. See, for example, Ramakrishnan, et al., 44 Cancer Res. 201(1984) (describing the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester)), Umemoto et al., U.S. Patent No. 5,030,719 (describing the use of a halogenated acetylhydrazide derivative coupled to an antibody by an oligopeptide linker). Specific linkers include (a) EDC (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride, (b) SMPT (4-succinimidyl oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)-toluene (Pierce Chem. Co., Cat. (21558G)), (c) SPDP (succinimidyl-6[3-(2-pyridyldithio) propionamide] hexanoate (Pierce Chem. Co., Cat#21651G), (d) sulfo-LC-SPDP (sulfosuccinimidyl 6[3-(2-pyridyldithio)-propionamide(propianamide)] hexanoate (Pierce Chem. Co. Cat.#2165-G), and (f) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem. Co., Cat.#24510) conjugated with EDC.

[0184] The above-mentioned linkers or linking agents contain components with different properties, thus resulting in conjugates with different physicochemical characteristics. For example, the sulfo-NHS ester of alkyl carboxylate is more stable than the sulfo-NHS ester of aromatic carboxylate. The NHS-ester containing a linker is less soluble than the sulfo-NHS ester. Furthermore, the linker SMPT contains a sterically hindered disulfide bond and can form a conjugate with improved stability. The disulfide linkage may be cleaved in vitro, leading to a reduction in the available conjugate, and is generally less stable than other linkages. Sulfo-NHS can, in particular, enhance the stability of carbodiimide coupling. Carbodiimide coupling (such as EDC) forms an ester with higher hydrolysis resistance when used in combination with sulfo-NHS than when used alone in the carbodiimide coupling reaction.

[0185] Exemplary crosslinking molecules for use in methods and immunogenic compositions as disclosed herein include, but are not limited to, those described in Tables 3 and 4.

[0186] (Table 3) Exemplary homobifunctional crosslinkers * TIFF0007679431000022.tif84167 * A crosslinking reagent having the same type of reactive group at either end. The reagents are classified by which chemical groups are crosslinked (left column) and their chemical compositions (central column). The products are listed in order from the shortest in each cell.

[0187] (Table 4) Exemplary heterobifunctional crosslinkers * TIFF0007679431000023.tif137169 * A crosslinking reagent having different reactive groups at either end. The reagents are classified by which chemical groups are crosslinked (left column) and their chemical compositions (central column). The products are listed in order from the shortest in each cell.

[0188] Co-stimulatory factor In some embodiments, the immunogenic compositions disclosed herein comprise at least one costimulatory molecule. In some embodiments, the costimulatory factor is cross-linked to a polymer. In some embodiments, the costimulatory factor is associated with the polymer by complementary affinity pairs in the same manner as the antigen is associated with the polymer. In some embodiments, the complementary affinity pair that links the costimulatory factor to the polymer is the same or a different complementary affinity pair than the complementary affinity pair that links the antigen to the polymer.

[0189] In some embodiments, at least 1, or at least 2, or at least 3, or at least 5, or at least 10, or at least 15, or at least 20, or at least 50, or at least 100, or more than about 100 (including 100) costimulatory factors may be associated with a polymer as disclosed herein. In some embodiments, the costimulatory factors may be the same costimulatory factor, or they may be a diverse different set of costimulatory factors associated with the polymer.

[0190] In some embodiments, the costimulatory factor is a ligand / agonist of a Toll-like receptor, such as, but not limited to, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, etc. In some embodiments, the costimulatory factor is a NOD ligand / agonist, or an activator / agonist of the inflammasome. Without wishing to be bound by theory, the inflammasome is a multi-protein oligomer consisting of caspase 1, PYCARD, NALP, and optionally caspase 5 or caspase 11, and promotes the maturation of the inflammatory cytokines interleukin 1-β and interleukin 18.

[0191] In some embodiments, the co-stimulatory factor is a cytokine. In some embodiments, the cytokine is selected from the group consisting of GM-CSF, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-23, IFN-α, IFN-β, IFN-β, IFN-γ, MIP-1α, MIP-1β, TGF-β, TNFα, and TNFβ. In some embodiments, the co-stimulatory factor is an adjuvant, which, as discussed above, may be associated with the polymer or added to the MAPS composition before or simultaneously with administration to the subject. Adjuvants are further described elsewhere in this specification.

[0192] Production of recombinant protein Recombinant proteins may be conveniently expressed and purified by one of ordinary skill in the art or by using commercially available kits, such as the PROBOND™ Purification System (Invitrogen Corp., Carlsbad, CA). In some embodiments, the recombinant antigen can be synthesized and purified by protein purification methods using bacterial expression systems, yeast expression systems, baculovirus / insect cell expression systems, mammalian cell expression systems, or transgenic plant or animal systems known to those of ordinary skill in the art.

[0193] All of the proteins, polypeptides, and fusion polypeptides described herein can be synthesized and purified by protein and molecular methods well known to those of ordinary skill in the art. Molecular biology methods and recombinant heterologous protein expression systems are used. For example, recombinant proteins can be expressed in bacteria, mammals, insects, yeast, or plant cells; or transgenic plant or animal hosts.

[0194] In one embodiment, an isolated polynucleotide encoding the fusion polypeptide or non-fusion polypeptide described herein is provided herein. Using conventional polymerase chain reaction (PCR) cloning techniques, a chimeric or fusion coding sequence encoding the fusion polypeptide described herein can be constructed. The coding sequence can be cloned into a general-purpose cloning vector such as pUC19, pBR322, pBLUESCRIPT® vector (Stratagene, Inc.), or pCR TOPO® (Invitrogen). The resulting recombinant vector carrying the nucleic acid encoding the polypeptide described herein can then be used for further molecular biology manipulations such as site-directed mutagenesis to create the variant fusion polypeptides described herein, or can be subcloned into a protein expression vector or viral vector for protein synthesis in a variety of protein expression systems using a host cell selected from the group consisting of mammalian cell lines, insect cell lines, yeast, bacteria, and plant cells.

[0195] Each PCR primer should have at least 15 nucleotides that overlap with its corresponding template in the region to be amplified. The polymerase used in PCR amplification should have high fidelity, such as PfuULTRA® polymerase (Stratagene), to reduce sequence errors during the PCR amplification process. For example, during the construction of a fusion polypeptide, to facilitate ligation of several separate PCR fragments together and subsequent insertion into a cloning vector, the PCR primers should also have distinct unique restriction digestion sites at their adjacent ends that do not anneal to the DNA template during PCR amplification. The selection of the restriction digestion sites for each pair of specific primers should be such that the DNA sequence encoding the fusion polypeptide is in-frame and encodes the fusion polypeptide from start to finish without including a stop codon. At the same time, the selected restriction digestion sites should not be found in the coding DNA sequence of the fusion polypeptide. The coding DNA sequence of the polypeptide of interest can be ligated into one of the cloning vectors pBR322 or its derivatives for amplification, verification of the fidelity and reliability of the chimeric coding sequence, substitution for specific amino acid mutations / or site-directed mutagenesis at specific sites, and substitution in the polypeptide.

[0196] Alternatively, the coding DNA sequence of the polypeptide can also be PCR cloned into a vector using a TOPO® cloning method (Invitrogen, Inc., Carlsbad, CA) that includes topoisomerase-assisted TA vectors such as pCR®-TOPO, pCR®-Blunt II-TOPO, pENTR / D-TOPO®, and pENTR / SD / D-TOPO®. Both pENTR / D-TOPO® and pENTR / SD / D-TOPO® are directional TOPO-introducing vectors that enable the cloning of DNA sequences in the 5' to 3' direction into GATEWAY® expression vectors. Directional cloning in the 5' to 3' direction facilitates the unidirectional insertion of a DNA sequence into a protein expression vector, such that the promoter is upstream of the 5' ATG start codon of the DNA sequence encoding the fusion polypeptide, enabling protein expression promoted by the promoter. The recombinant vector carrying the coding DNA sequence of the fusion polypeptide can be transfected into and propagated in common cloning Escherichia coli such as XL1 Blue, SURE® (STRATAGENE®), and TOP-10 cells (Invitrogen).

[0197] One skilled in the art can clone and ligate the coding region of an antigen of interest together with the coding region of a complementary affinity molecule using specially designed oligonucleotide probes known in the art and the method of polymerase chain reaction (PCR) to construct a chimeric coding sequence of a fusion polypeptide containing the antigen or a fragment thereof and its complementary affinity molecule. One skilled in the art can also clone and ligate the chimeric coding sequence of the fusion protein into a selected vector, such as a bacterial expression vector, an insect expression vector, or a baculovirus expression vector. The coding sequences of the antigen and the target antigen polypeptide or a fragment thereof should be ligated in-frame, and the chimeric coding sequence should be ligated downstream of the promoter and between the promoter and the transcription terminator. Subsequently, the recombinant vector is transfected into ordinary cloning Escherichia coli such as XL1Blue. Next, recombinant Escherichia coli containing the introduced vector DNA is selected by antibiotic resistance to remove any Escherichia coli containing non-recombinant plasmid DNA. The selected transformed Escherichia coli is grown, and subsequently, the recombinant vector DNA can be purified for transfection into Spodoptera frugiperda (S. frugiperda) cells.

[0198] In some embodiments, the antigens disclosed herein may include a signal peptide for translocation into the periplasmic space of bacteria. The signal peptide is also referred to as a leader peptide at the N-terminus, which may or may not be cleaved after translocation through the membrane. An example of a signal peptide is as disclosed herein TIFF0007679431000024.tif4128. Another signal sequence is TIFF0007679431000025.tif4128. Other examples of signal peptides can be found in the SPdb (Signal Peptide Database) available at the World Wide Web site "proline.bic.nus.edu.sg / spdb / ".

[0199] In some embodiments, when the antigen is fused to a biotin-binding protein, the signal sequence may be located at the N-terminus of the biotin-binding protein. In some embodiments, the signal sequence is cleaved from the biotin-binding protein after translocation into the periplasmic space of E. coli.

[0200] In some embodiments, when the antigen is fused to a complementary affinity protein, the signal sequence may be located at the N-terminus of the complementary affinity protein. For example, when the antigen is fused to an avidin-like protein, the signal sequence may be located at the N-terminus of the complementary affinity protein. In some embodiments, the signal sequence is cleaved from the complementary affinity protein before the complementary affinity protein associates with the first affinity molecule.

[0201] In some embodiments, the antigens and / or complementary affinity proteins described herein do not have a signal sequence.

[0202] The polypeptides described herein can be expressed in a variety of expression host cells, such as bacterial, yeast, mammalian, insect, plant, algal cells such as Chlamydomonas, or cell-free expression systems. In some embodiments, the nucleic acid can be subcloned from a cloning vector into a recombinant expression vector suitable for expression of the fusion polypeptide in bacterial, mammalian, insect, yeast, or plant cells, or a cell-free expression system, such as a rabbit reticulocyte expression system. Some vectors are designed to introduce the coding nucleic acid for expression in mammalian, insect, and yeast cells in a single recombination reaction. For example, a portion of the GATEWAY® (Invitrogen) destination vector is designed for the construction of baculovirus, adenovirus, adeno-associated virus (AAV), retrovirus, and lentivirus, which allows for heterologous expression of the fusion polypeptide in the appropriate host cell upon infection of each host cell. Introduction of the gene into the destination vector is accomplished in two steps according to the manufacturer's instructions. There are GATEWAY® expression vectors for protein expression in insect, mammalian, and yeast cells. After transformation and selection in E. coli, the expression vector can be used for expression in the appropriate host.

[0203] Examples of other expression vectors and host cells include the strong CMV promoter-based pcDNA3.1 (Invitrogen) and pCINEO vectors (Promega) for expression in mammalian cell lines such as CHO, COS, HEK-293, Jurkat, and MCF-7; the vector pADENO-X™, pAd5F35, pLP-ADENO™-X-CMV (CLONTECH®), pAd / CMV / V5-DEST, pAd-DEST vectors (Invitrogen), which are replication-deficient adenoviral vectors for adenovirus-mediated gene transfer and expression in mammalian cells; the pLNCX2, pLXSN, and pLAPSN retroviral vectors for use with the RETRO-X™ system (Clontech) for retrovirus-mediated gene transfer and expression in mammalian cells; the pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells; adeno-associated virus expression vectors such as pAAV-MCS, pAAV-IRES-hrGFP, and pAAV-RC vectors (Stratagene) for adeno-associated virus-mediated gene transfer and expression in mammalian cells; the BACpak6 baculovirus (Clontech) and pFASTBAC™ HT (Invitrogen) for expression in Spodoptera frugiperda 9 (Sf9), Sf11, Tn-368, and BTI-TN-5B4-1 insect cell lines; the pMT / BiP / V5-His (Invitrogen) for expression in Drosophila Schneider S2 cells; the Pichia expression vectors pPICZα, pPICZ, pFLDα, and pFLD (Invitrogen) for expression in Pichia pastoris, and the vectors pMETα and pMET for expression in methylotrophic yeast; the pYES2 / GS and pYD1 (Invitrogen) vectors for expression in the yeast Saccharomyces cerevisiae.

[0204] Recent progress in the large-scale expression of heterologous proteins in Chlamydomonas reinhardtii has been described. Griesbeck., 34 Mol. Biotechnol. 213 (2006), Fuhrmann, 94 Methods Mol Med. 191 (2006). Foreign heterologous coding sequences are inserted into the genomes of the nucleus, chloroplast, and mitochondrion by homologous recombination. Chloroplast expression vector p64, which carries aminoglycoside adenyltransferase (aadA), the most versatile chloroplast selectable marker that confers resistance to spectinomycin or streptomycin, can be used to express foreign proteins in chloroplasts. The vector can be introduced into algae using a particle gene gun. Once inside the chloroplast, the foreign DNA is released from the gene gun particles and binds within the chloroplast genome by homologous recombination.

[0205] Also included in the present invention are complementary affinity molecules fused to an antigen. In some embodiments, the fusion construct may also optionally include a purification tag and / or a secretion signal peptide. These fusion proteins may be produced by any standard method. For example, for the production of a stable cell line expressing an antigen-complementary affinity molecule fusion protein, the PCR-amplified antigen nucleic acid may be cloned into the restriction sites of a derivative of a mammalian expression vector. For example, KA (Invitrogen), a derivative of pcDNA3, contains a DNA fragment encoding the influenza virus hemagglutinin tag (HA). Alternatively, vector derivatives encoding other tags such as the c-myc tag or the polyhistidine tag may be used. The antigen-complementary affinity molecule fusion expression construct may be co-transfected into a suitable mammalian cell line (e.g., COS, HEK293T, or NIH 3T3 cells) together with a marker plasmid using, for example, LIPOFECTAMINE™ (Gibco-BRL, Gaithersburg, MD) according to the manufacturer's instructions or other suitable transfection techniques known in the art. Suitable transfection markers include, for example, β-galactosidase or green fluorescent protein (GFP) expression plasmids, or any plasmid that does not contain the same detectable marker as the antigen-complementary affinity molecule fusion protein. The fusion protein-expressing cells may be selected and further cultured, or the tagged antigen-complementary affinity molecule fusion protein may be purified. In some embodiments, the antigen-complementary affinity molecule fusion protein is amplified together with a signal peptide. In an alternative embodiment, the cDNA encoding the antigen-complementary affinity molecule fusion protein is amplified without using a signal peptide and subcloned into a vector (pSecTagHis) having a strong secretion signal peptide. In another example, the antigen-complementary affinity molecule fusion protein may have an alkaline phosphatase (AP) tag or a histidine (His) tag for purification. Any method known to those skilled in the art for protein purification of the antigen and / or the antigen-complementary affinity molecule fusion protein is included for use in the methods of the present invention.

[0206] In some embodiments, any of the polypeptides described herein are produced by expression from a recombinant baculovirus vector. In another embodiment, any of the polypeptides described herein are expressed by insect cells. In yet another embodiment, any of the polypeptides described herein are isolated from insect cells. Protein expression by baculovirus in insect cells has several advantages, including high expression levels, ease of scale-up, production of proteins with post-translational modifications, and simple cell growth. Insect cells do not require CO 2 for growth and can be easily adapted to high-density suspension culture for large-scale expression. Many of the post-translational modification pathways present in mammalian systems are also utilized in insect cells, enabling the production of recombinant proteins that are antigenically, immunologically, and functionally similar to native mammalian proteins.

[0207] Baculoviruses are DNA viruses of the family Baculoviridae. These viruses are known to have a narrow host range, mainly restricted to insects of the order Lepidoptera (butterflies and moths). The baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV), which was the prototype baculovirus, replicates efficiently in susceptible cultured insect cells. AcNPV has a double-stranded circular DNA genome of approximately 130,000 base pairs and is well-characterized with respect to its host range, molecular biology, and genetics. The baculovirus expression vector system (BEVS) is a safe and rapid method for the abundant production of recombinant proteins in insect cells and insects. The baculovirus expression system is a powerful and versatile system for high-level recombinant protein expression in insect cells. Using the baculovirus expression system, expression levels of up to 500 mg / l have been reported, making it an ideal system for high-level expression. Recombinant baculoviruses that express foreign genes are constructed by homologous recombination between baculovirus DNA and a chimeric plasmid containing the gene sequence of interest. Recombinant viruses can be detected by their distinct plaque morphology and can be plaque-purified until they are homogeneous.

[0208] The recombinant fusion proteins described herein can be produced in insect cells including, but not limited to, cells derived from the lepidopteran species Spodoptera frugiperda. Other insect cells susceptible to infection by baculovirus, such as those derived from the species Bombyx mori, Galleria mellanoma, Trichplusia ni, or Lamanthria dispar, can also be used as suitable substrates for producing the recombinant proteins described herein. Baculovirus expression of recombinant proteins is known in the art. See U.S. Pat. Nos. 4,745,051, 4,879,236, 5,179,007, 5,516,657, 5,571,709, 5,759,809. It will be understood by those skilled in the art that the expression system is not limited to the baculovirus expression system. What is important is that the expression system leads to N-glycosylation of the expressed recombinant protein. The recombinant proteins described herein can also be expressed in other expression systems such as entomopoxvirus (an insect poxvirus), cytoplasmic polyhedrosis virus (CPV), and transformants of insect cells by constitutive expression of one or more recombinant genes. A significant number of baculovirus transfer vectors and corresponding appropriately modified host cells are commercially available, such as pAcGP67, pAcSECG2TA, pVL1392, pVL1393, pAcGHLT, and pAcAB4 from BD Biosciences, pBAC-3, pBAC-6, pBACgus-6, and pBACsurf-1 from NOVAGEN®, and pPolh-FLAG and pPolh-MAT from SIGMA ALDRICH®.

[0209] The region between the promoter and the transcription terminator may have multiple restriction enzyme digestion sites to facilitate the cloning of foreign coding sequences, which in this case are the coding DNA sequences of the antigen polypeptide and the complementary affinity molecule. Additional sequences, such as signal peptides and / or tag coding sequences, such as His-tag, MAT-tag, FLAG tag, enterokinase recognition sequence, honeybee melittin secretion signal, β-galactosidase, secretion, specific, accurate insertion, positive selection of recombinant virus, and / or glutathione S-transferase (GST) tag upstream of the MCS to facilitate purification of the recombinant protein may also be included.

[0210] In some embodiments, the fusion protein may include the N-terminal signal sequence disclosed herein. In some embodiments, the signal sequence is attached to the N-terminus of the complementary affinity molecule disclosed herein.

[0211] In some embodiments, the fusion polypeptide as described herein may include a spacer peptide, e.g., a 14-residue spacer that separates the antigen from the complementary affinity molecule TIFF0007679431000026.tif4128. The coding sequence of such a short spacer can be constructed by annealing complementary pairs of primers. One of ordinary skill in the art can design and synthesize oligonucleotides encoding the selected spacer. The spacer peptide should generally have non-polar amino acid residues such as glycine and proline.

[0212] Using standard techniques known to those skilled in the art, amino acid substitutions can be made in the antigen polypeptide sequence of the fusion polypeptides described herein, for example, in the nucleotide sequence encoding the fusion polypeptides of the antigens described herein, by introducing mutations including, for example, site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, the variant fusion polypeptides have, inclusively, less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the fusion polypeptides described herein.

[0213] Alternatively, certain silent or neutral missense mutations that do not alter the encoded amino acid sequence or the ability to facilitate transmembrane delivery may be made in the DNA coding sequence. These types of mutations are useful for optimizing codon usage or improving the expression and production of recombinant proteins.

[0214] Specific amino acid mutations and substitutions can be created using site-directed mutagenesis of the coding sequence of the fusion polypeptide in a vector. Site-directed mutagenesis can be performed, for example, using the QUICKCHANGE® Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer's instructions.

[0215] In one embodiment, for example, using a host cell selected from bacteria, mammals, insects, yeast, or plant cells, an expression vector containing the coding DNA sequence of the polypeptide described herein for the expression and purification of a recombinant polypeptide produced from a protein expression system is described herein. The expression vector should have the necessary 5' upstream and 3' downstream regulatory elements such as a promoter sequence, ribosome recognition and TATA box, and 3'UTR AAUAAA transcription termination sequence for efficient gene transcription and translation in their respective host cells. The expression vector preferably has a transcription promoter selected from the group consisting of CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, β-actin promoter, SV40 (simian virus 40) promoter, and muscle creatine kinase promoter, and a transcription terminator selected from the group consisting of SV40 poly(A) and BGH terminator, and more preferably has the cytomegalovirus immediate early promoter / enhancer sequence and adenovirus tripartite leader / intron sequence, and is an expression vector containing the SV40 origin of replication and poly(A) sequence. The expression vector can have additional coding regions such as regions encoding 6X-histidine, V5, thioredoxin, glutathione-S-transferase, c-Myc, VSV-G, HSV, FLAG, maltose-binding peptide, metal-binding peptide, HA, and "secretion" signals (honeybee melittin, α-factor, PHO, Bip) that can be incorporated into the expressed fusion polypeptide. Further, an enzyme digestion site can be incorporated after these coding regions to facilitate enzymatic removal when they are not needed. These additional nucleic acids are useful for the detection of fusion polypeptide expression, protein purification by affinity chromatography, improvement of the solubility of the recombinant protein in the host cell cytoplasm, and / or secretion of the expressed fusion polypeptide into the culture medium or spheroplasts of yeast cells.Expression of the fusion polypeptide may be constitutive in the host cell or it may be induced by, among other things, copper sulfate, sugars such as galactose, methanol, methylamine, thiamine, tetracycline, infection with baculovirus, and (isopropyl-β-D-thiogalactopyranoside) IPTG, a stable synthetic analog of lactose.

[0216] In another embodiment, the expression vector containing the polynucleotide described herein is, among others, viral vectors such as adenovirus, adeno-associated virus (AAV), retrovirus, and lentiviral vectors. Recombinant viruses provide a versatile system for gene expression studies and therapeutic applications.

[0217] In some embodiments, the fusion polypeptide described herein is expressed by viral infection of mammalian cells. The viral vector may be, for example, adenovirus, adeno-associated virus (AAV), retrovirus, and lentivirus. A simplified system for generating recombinant adenoviruses is presented by He et al., 95 PNAS 2509 (1998). The gene of interest is first cloned into a shuttle vector, such as pAdTrack-CMV. The resulting plasmid is linearized by digestion with the restriction endonuclease PmeI and subsequently co-transformed into E. coli BJ5183 cells using an adenovirus backbone plasmid, such as pADEASY-1, a Stratagene ADEASY™ adenoviral vector system. The recombinant adenoviral vector is selected for kanamycin resistance and the recombination is confirmed by restriction endonuclease analysis. Finally, the linearized recombinant plasmid is transfected into an adenovirus packaging cell line, such as HEK 293 cells (E1-transformed human fetal kidney cells) or 911 (E1-transformed human fetal retinal cells). Fallaux, et al. 7 Human Gene Ther. 215 (1996). Recombinant adenoviruses are produced within HEK 293 cells.

[0218] Recombinant lentiviruses have the advantage of delivering and expressing fusion polypeptides in both dividing and non-dividing mammalian cells. Lentiviruses based on HIV-1 can effectively transduce a broader host range than retroviral systems based on Moloney murine leukemia virus (MoMLV). The preparation of recombinant lentiviruses can be achieved, for example, by using pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, or pLenti vectors together with the VIRAPOWER™ Lentivirus Expression System (Invitrogen, Inc.).

[0219] Recombinant adeno-associated virus (rAAV) vectors can be applied to a wide variety of host cells, including many different human and non-human cell lines or tissues. rAAV can transduce a wide variety of cell types, and transduction does not depend on active host cell division. 8 High titers exceeding 1010 viral particles / mL can be easily obtained in the supernatant, and upon further concentration, 10 11 ~10 12 13 viral particles / mL are obtained. The transgene is integrated into the host genome, and thus expression is long-term and stable.

[0220] Large-scale preparation of AAV vectors is performed by triple plasmid co-transfection of packaging cell lines, such as AAV vectors carrying the coding nucleic acid, AAV RC vectors containing the AAV rep and cap genes, and the adenovirus helper plasmid pDF6, into semi-confluent 293 cells in 50 × 150 mm plates. The cells are harvested 3 days after transfection, and the virus is released by three freeze-thaw cycles or sonication.

[0221] AAV vectors can be purified in two different ways depending on the serotype of the vector. AAV2 vectors are purified by a one-step gravity flow column purification method based on their affinity for heparin. Auricchio et.al., 12 Human Gene Ther. 71 (2001), Summerford & Samulski, 72 J. Virol. 1438 (1998), Summerford & Samulski, 5 Nat. Med. 587 (1999). AAV2 / 1 and AAV2 / 5 vectors are currently purified by a three consecutive CsCl gradient.

[0222] Without wishing to be bound by theory, when a protein is expressed by a cell including a bacterial cell, the protein either localizes to a specific part in the cell or is secreted from the cell. For this reason, protein localization or protein sorting is the mechanism by which a cell transports a protein to an appropriate position in or outside the cell. The target of sorting may be the internal space of an organelle, any of several inner membranes, the outer membrane of the cell, or outside thereof via secretion. This delivery process is carried out based on the information contained in the protein itself. Correct sorting is extremely important for the cell, and errors can lead to diseases.

[0223] Although there are some exceptions, bacteria do not have membrane-bound organelles as found in eukaryotes, but can assemble proteins on various types of inclusions such as gas vesicles and storage granules. Also, depending on the bacterial species, bacteria can have a single plasma membrane (Gram-positive bacteria) or both an inner (cytoplasmic) membrane and an outer cell wall membrane with a water-containing space called the periplasm between the two (Gram-negative bacteria). Proteins can be secreted into the environment depending on the presence or absence of the outer membrane. The basic mechanism in the plasma membrane is the same as that of eukaryotes. Furthermore, bacteria direct proteins into or across the outer membrane. Systems for secreting proteins across the outer membrane of bacteria can be extremely complex and can play an important role in the pathogenesis. These systems can be represented as type I secretion, type II secretion, etc.

[0224] In most Gram-positive bacteria, there are specific proteins for export across the plasma membrane and subsequent covalent attachment to the bacterial cell wall. The sortase, a specialized enzyme, cleaves target proteins at characteristic recognition sites near the C-terminus of the protein, such as the LPXTG motif (SEQ ID NO: 42) (where X can be any amino acid), and then transports that protein onto the cell wall. A system similar to sortase / LPXTG, called exosortase / PEP-CTERM, which has the motif PEP-CTERM (SEQ ID NO: 43), has been proposed to exist in a wide range of Gram-negative bacteria.

[0225] Proteins with an appropriate N-terminal targeting signal are synthesized in the cytoplasm and then directed to specific protein transport pathways. During or immediately after their translocation across the cell membrane, the proteins are processed and folded into their active form. The translocated proteins are then either retained on the periplasmic side of the cell or released into the environment. Since signal peptides that direct proteins to the membrane are important determinants of transport pathway specificity, these signal peptides are classified according to the transport pathway they direct the protein to. The classification of signal peptides is based on the type of signal peptidase (SPase) responsible for signal peptide removal. Most of the proteins to be excreted are generally excreted from the cytoplasm via the "secretion (Sec) pathway". The best-known virulence factors secreted by Gram-positive pathogens (e.g., staphylococcal exotoxins, Bacillus anthracis protective antigen, Listeria monocytogenes listeriolysin O) have typical N-terminal signal peptides that can direct them to the Sec pathway. Proteins secreted via this system are translocated across the cell membrane in an unfolded state. Subsequent processing and folding of these proteins occur in the cell wall environment on the other side of the membrane. In addition to the Sec system, some Gram-positive bacteria also contain the Tat system, which can translocate folded proteins to the other side of the membrane. Pathogenic bacteria may contain transport systems for specific uses that are specifically involved only in the transport of a very small number of proteins. For example, in Mycobacterium, several gene clusters encoding proteins important for the Mycobacterium pathogenesis mechanism that are secreted into the environment via a specific pathway (ESAT-6) have been identified. Specific ATP-binding cassette (ABC) transporters direct the transport and processing of antibacterial small peptides called bacteriocins. Genes for endolysins involved in the occurrence of lysis are often located in the vicinity of genes encoding holin-like proteins, suggesting that these holins are involved in the excretion of endolysins to the cell wall (Wooldridge, BACT. SECRETED PROTS: SECRETORY MECHS. & ROLE IN PATHOGEN. (Caister Academic Press, 2009)).

[0226] In some embodiments, the signal sequence useful in the present invention is the OmpA signal sequence, although any signal sequence generally known to those skilled in the art that enables the transport and secretion of antibacterial factors outside bacteriophage-infected cells is also encompassed for use in the present invention.

[0227] Signal sequences that direct the secretion of proteins from bacterial cells are well known in the art and are disclosed, for example, in International Application No. 2005 / 071088. For example, some of the non-limiting examples of signal peptides shown in Table 5 can be used and attached to the amino or carboxyl terminus of an antibacterial peptide (Amp) or antibacterial polypeptide such that it is expressed by an antibacterial factor-modified bacteriophage, such as an AMP-modified bacteriophage. Attachment may be via a fusion or chimeric composition with a selected antigen or antigen-complementary affinity molecule fusion protein that results in secretion from bacteria infected with an antibacterial factor-modified bacteriophage, such as an AMP-modified bacteriophage.

[0228] (Table 5) Examples of signal peptides that direct the secretion of proteins or peptide antigens or antigen-complementary affinity molecule fusion proteins of bacterial cells TIFF0007679431000027.tif114165

[0229] The polypeptides described herein, such as antigen or antigen-complementary affinity molecule fusion proteins, can be expressed and purified by various methods known to those skilled in the art. For example, the fusion polypeptides described herein can be purified from any suitable expression system. The fusion polypeptides can be purified to substantial purity by standard techniques including selective precipitation with substances such as ammonium sulfate, column chromatography, immunoaffinity purification methods, etc., known in the art. See, for example, Scopes, Protein PURIFICATION: PRINCIPLES & PRACTICE (1982), U.S. Patent No. 4,673,641.

[0230] When purifying recombinant proteins, many procedures can be utilized. For example, a protein having established molecular adhesion properties can be reversibly fused to an optimal protein. Using an appropriate ligand, the protein can be selectively adsorbed to a purification column and then released from the column in a relatively pure form. Subsequently, the fusion protein is removed by enzymatic activity. Finally, the optimal protein can be purified using an affinity or immunoaffinity column.

[0231] After a protein is expressed in a host cell, the host cell can be lysed to release the expressed protein for purification. Various methods for lysing host cells are described in "Sample Preparation - Tools for Protein Research" by EMD Bioscience and Current Protocols in Protein Sciences (CPPS). Examples of purification methods include affinity chromatography such as metal ion affinity chromatography using nickel, cobalt, or zinc affinity resins for histidine-tagged fusion polypeptides. Methods for purifying histidine-tagged recombinant proteins are described by Clontech by using its TALON (registered trademark) cobalt resin and by NOVAGEN (registered trademark) in the 10th edition of its pET system manual. Another preferred purification method is immunoaffinity chromatography. For example, an anti-myc antibody-conjugated resin can be used to affinity purify a myc-tagged fusion polypeptide. When an appropriate protease recognition sequence is present, the fusion polypeptide can be cleaved from the histidine or myc tag, and the fusion polypeptide is released from the affinity resin while leaving the histidine tag and myc tag attached to the affinity resin.

[0232] Standard protein separation techniques for purifying recombinant and naturally occurring proteins are known in the art and include, for example, fractionation by solubility, size exclusion gel filtration, and various column chromatographies.

[0233] Fractionation by solubility: Often, as a first step, especially when the protein mixture is complex, the first salt fractionation can separate many of the unwanted host cell proteins (or proteins derived from the cell culture medium) from the protein of interest. A preferred salt is ammonium sulfate. Ammonium sulfate precipitates proteins by effectively reducing the amount of water in the protein mixture. The proteins then precipitate based on their solubility. The more hydrophobic a protein is, the more likely that protein is to precipitate at a lower ammonium sulfate concentration. A typical protocol involves adding saturated ammonium sulfate to the protein solution such that the resulting ammonium sulfate concentration is 20 - 30%. This concentration will precipitate the most hydrophobic proteins. Next, the precipitate is discarded (unless the protein of interest is hydrophobic), and ammonium sulfate is added to the supernatant to the concentration known to precipitate the protein of interest. The precipitate is then solubilized in buffer and, if necessary, the excess salt is removed either by dialysis or diafiltration. Other methods that depend on protein solubility, such as cold ethanol precipitation, are known to those skilled in the art and can be used to fractionate complex protein mixtures.

[0234] Size exclusion filtration: Using the optimal protein molecular weight, ultrafiltration through membranes of different pore sizes (e.g., AMICON® membranes or MILLIPORE® membranes) can be used to isolate proteins of larger or smaller sizes from it. As a first step, the protein mixture is ultrafiltered through a membrane with a pore size having a fractional molecular weight lower than the molecular weight of the protein of interest. The residue of the ultrafiltration is then ultrafiltered against a membrane with a fractional molecular weight higher than the molecular weight of the protein of interest. The recombinant protein passes through the membrane and into the filtrate. Chromatography can then be performed on the filtrate as described below.

[0235] Column chromatography: Optimal proteins can also be separated from other proteins based on their size, net surface charge, hydrophobicity, and affinity for ligands. Additionally, antibodies produced against recombinant or naturally occurring proteins can be conjugated to a column matrix to immunopurify the protein. All of these methods are well known in the art. It will be apparent to those skilled in the art that chromatographic techniques can be performed at any scale using equipment from many different manufacturers (e.g., Pharmacia Biotech). For example, the antigen polypeptide can be purified using a PA63 heptamer affinity column (Singh et al., 269, J. Biol. Chem. 29039 (1994)).

[0236] In some embodiments, to purify the fusion polypeptides described herein, for example, a combination of purification steps can be used, including (a) ion exchange chromatography, (b) hydroxyapatite chromatography, (c) hydrophobic interaction chromatography, and (d) size exclusion chromatography.

[0237] Cell-free expression systems are also contemplated. Cell-free expression systems offer several advantages over conventional cell-based expression methods, including the ability to easily change reaction conditions for favorable protein folding, reduced sensitivity to product toxicity, and reduced reaction dosage and processing time, making them suitable for high-throughput strategies such as rapid expression screening or large-scale protein production. In cell-free expression systems, plasmid or linear DNA can be used. Additionally, improved translation efficiency has resulted in yields exceeding 1 milligram of protein per milliliter of reaction mixture. A commercially available cell-free expression system is the TNT coupled reticulocyte lysate Systems (Promega), which uses an in vitro system based on rabbit reticulocytes.

[0238] Formulation and method of use of immunocomposition Specific embodiments of the invention provide for the use of the immunogenic compositions disclosed herein for inducing an immune response in an animal. More specifically, the compositions induce both humoral and cellular immunity and, in many cases, mucosal immunity. Embodiments of the invention provide at least partial protection from, or partial amelioration after, infection, particularly by Streptococcus pneumoniae. Streptococcus pneumoniae causes a number of diseases such as meningitis, pneumonia, bacteremia, and otitis media. Approximately one million children worldwide die each year from pneumococcal disease. Streptococcus pneumoniae has been extensively studied and at least some of its genome has been sequenced. See, for example, U.S. Patent No. 7,141,418. Antibodies to the capsular polysaccharides that define the known serotypes confer serotype-specific protection, although other immune defense mechanisms have also been described. See Malley et al., 88 J. Mol. Med. 135 (2010). These other defense mechanisms include, but are not limited to, antibodies to non-capsular antigens and T cell responses to pneumococcal components. The application of PCV7, a protein-polysaccharide conjugate vaccine, has significantly reduced disease. See Black et al., 24 (S2) Vaccine 79 (2006), Hansen et al., 25 Pediatr. Infect. Dis. J. 779 (2006). However, recent studies have shown that the lack of other serotypes in PCV7 has led to the emergence of new alternative pneumococcal serotypes. See Pichichero & Casey, 26 (S10) Pediatr. Infect. Dis. J. S12 (2007).

[0239] Certain pneumococcal antigens common to all serotypes of this kind, such as surface proteins PspA, PspC, PsaA, and the cytotoxin pneumolysin or pneumolysin mutants, have been shown to have the potential for immune defense despite encapsulation (Basset et al., 75 Infect. Immun. 5460 (2007), Briles et al., 18 Vaccine 1707 (2000)), and the use of genomics and mutant libraries has identified dozens of additional species-common proteins (Hava & Camilli, 45 Mol. Microbiol. 1389 (2002), Wizemann et al., 60 Infect. Immun. 1593 (2001)). In animal models, immunity has been induced by individual antigens (Alexander et al., 62 Infect. Immun. 5683 (1994), Balachandran et al., 70 Infect. Immun. 2526 (2002), Chung et al., 170 J. Immunol. 1958 (2003), Glover et al., 76 Infect. Immun. 2767 (2008), Wu et al., 175 J. Infect. Dis. 839 (1997)), but vaccines based on common antigens have not been approved for use in humans to date.

[0240] In one embodiment, provided herein is a method of vaccinating a mammal comprising administering an immunogenic composition comprising at least one or more antigens attached to a polymer backbone, such as a polysaccharide or carbohydrate polymer, for use in inducing an immune response against the one or more antigens attached to the polymer when administered to a subject. In some embodiments, the immune response is a humoral and / or cellular immune response.

[0241] Accordingly, one aspect of the invention relates to a method for inducing an immune response in a subject, which comprises administering to the subject an immunogenic composition comprising at least one type of polymer, such as a polysaccharide, at least one antigen, (i) a first affinity molecule that associates with the polymer, such as the polysaccharide, and (ii) a complementary affinity molecule that associates with the antigen so as to attach the antigen to the polymer, such as the polysaccharide (e.g., the first affinity molecule associates with the complementary affinity molecule to link the antigen to the polymer, such as the polysaccharide), and at least a pair of complementary affinity molecules.

[0242] Accordingly, one aspect of the invention relates to a method for simultaneously inducing humoral and / or cellular immunity against multiple antigens, for example, when the immunogenic composition administered to the subject comprises a polymer that contains at least one, or at least two or more, for example, a plurality of the same or different antigens.

[0243] One aspect of the invention relates to a method of immunizing or vaccinating a subject, such as a bird or a mammal, such as a human, against a pathogen, the method comprising administering an immunological composition as disclosed herein that comprises at least one antigen derived from one or more pathogens. In some embodiments, the subject can be simultaneously immunized against at least one, or at least two, or at least two, or at least three, or at least five, or at least ten, or at least fifteen, or at least about twenty, or at least fifty, or at least about one hundred, or more than one hundred different pathogens, wherein the polymer of the immunogenic composition is attached with the corresponding different antigens.

[0244] In some embodiments, a subject can be administered several different immunogenic compositions disclosed herein. For example, a subject can be administered a composition comprising a polymer along with one or more antigens, such as antigens A, B, C, and D, etc., and can also be administered a composition comprising a polymer comprising a different single antigen or a different series of antigens, such as antigens W, X, Y, and Z, etc. Alternatively, a subject can be administered a composition comprising polymer A along with one or more antigens, such as antigens A, B, C, and D, etc., and can also be administered a composition comprising polymer B comprising the same, such as antigens A, B, C, and D, etc., or a different series of antigens. The present invention contemplates methods for immunizing a subject with as many antigens as desired, such as using various different immunogenic conjugates described herein, to enable immunization with over 100 antigens.

[0245] In one embodiment, the immunogenic composition described herein comprises a pharmaceutically acceptable carrier. In another embodiment, the immunogenic composition described herein is formulated for administration to birds, mammals, or humans as a vaccine or in a vaccine. Suitable formulations can be found, for example, in Remington's Pharmaceutical Sciences (2006), or Introduction to Pharmaceutical Dosage Forms (4th ed., Lea & Febiger, Philadelphia, 1985).

[0246] In one embodiment, the immunogenic composition described herein comprises a pharmaceutically acceptable carrier that is essentially non-toxic and non-therapeutic. Examples of such carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, and polyethylene glycol. For all administrations, conventional depot forms are preferably used. Such forms include, for example, microcapsules, nanocapsules, liposomes, plasters, inhalation forms, nasal sprays, sublingual tablets, and sustained release preparations. For examples of sustained release compositions, see U.S. Patent Nos. 3,773,919, 3,887,699, European Patent No. 58,481A, European Patent No. 158,277A, Canadian Patent No. 1176565, Sidman et al., 22 Biopolymers 547 (1983), Langer et al., 12 Chem. Tech. 98 (1982). Proteins can generally be formulated at a concentration of about 0.1 mg / ml to 100 mg / ml per use per patient.

[0247] In one embodiment, other materials can be added to the vaccine formulation, including antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues) polypeptides such as polyarginine or tripeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrin; chelating agents such as EDTA; and sugar alcohols such as mannitol or sorbitol.

[0248] In some embodiments, the present MAPS immunogen composition is administered with at least one adjuvant. An adjuvant is a heterogeneous group of substances that enhance the immune response to an antigen administered concurrently. In some cases, the adjuvant improves the immune response such that less of the vaccine is required. An adjuvant acts to bring an antigen (a substance that stimulates a specific protective immune response) into contact with the immune system and affects the type of immunity generated as well as the quality (magnitude or duration) of the immune response. An adjuvant can also reduce the toxicity of an antigen and provide solubility to some vaccine components. Almost all adjuvants currently used for enhancing the immune response to an antigen are particulate or form particles with the antigen. In the book VACCINE DESIGN-SUBUNIT & ADJUVANT APPROACH (Powell & Newman, Eds., Plenum Press, 1995), many known adjuvants are described with respect to both their immunological activity and their chemical properties. Non-particulate types of adjuvants consist of a group of substances that act as immunological signaling substances and are formed by the immune system under normal conditions as a result of immunological activation following administration of a particulate adjuvant system.

[0249] Adjuvants for immunogenic compositions and vaccines are well known in the art. By way of example, monoglycerides and fatty acids (e.g., a mixture of monoolein, oleic acid, and soybean oil), inorganic salts such as aluminum hydroxide gel and aluminum phosphate gel or calcium phosphate gel, oil emulsions and surfactant-based formulations such as MF59 (an oil-in-water emulsion stabilized with a microfluidized detergent), QS21 (purified saponin), AS02[SBAS2] (oil-in-water emulsion + MPL + QS-21), MPL-SE, Montanide ISA-51 and ISA-720 (stabilized water-in-oil emulsions), particulate adjuvants such as virosomes (a monolayer liposome vehicle incorporating influenza hemagglutinin), AS04([SBAS4]Al salt with MPL), ISCOMS (a structural complex of saponin and lipid), polylactide coglycolide (PLG), microbial derivatives (natural and synthetic) such as monophosphoryl lipid A (MPL), Detox (MPL + M. Phlei cell wall skeleton), AGP[RC-529] (synthetic acylated monosaccharides), Detox-PC, DC_Chol (a lipidic immunostimulatory factor capable of self-organizing within liposomes), OM-174 (a lipid A derivative), CpG motifs (synthetic oligonucleotides containing immunostimulatory CpG motifs), or other DNA structures, modified LT and CT (genetically modified bacterial toxins that provide a non-toxic adjuvant effect), endogenous human immunomodulatory factors such as hGM-CSF or hIL-12 (cytokines that can be administered either as a protein or as a coded plasmid), Immudaptin (C3d tandem array), MoGM-CSF, TiterMax-G, CRL-1005, GERBU, TERamide, PSC97B, Adjumer, PG-026, GSK-I, GcMAF, B-alethine, MPC-026, Adjuvax, CpG ODN, Betafectin, Alum, and MF59, as well as inert vehicles such as gold particles, but are not limited thereto. Additional adjuvants are known in the art, see, for example, U.S. Patent No. 6,890,540, U.S. Patent Publication No. 2005;0244420, PCT / SE97 / 01003.

[0250] In some embodiments, the adjuvant can be a particle and can have the characteristic of being slowly biodegradable. Care must be taken to ensure that the adjuvant does not form toxic metabolites. Preferably, in some embodiments, such adjuvants that can be used as a substrate are mainly substances of body origin. These include lactic acid polymers, polyamino acids (proteins), carbohydrates, lipids, and biocompatible polymers with low toxicity. Combinations of these groups of substances originating from the body, or combinations of substances resulting from the body and biocompatible polymers, can also be used. Lipids are preferred substances as they exhibit a biodegradable structure and the fact that they are important elements in all biological membranes.

[0251] In one embodiment, the immunogenic composition described herein for administration must be sterile for administration to a subject. Sterility is readily achieved by filtration through a sterile filtration membrane (e.g., a 0.2 micron membrane) or by gamma irradiation.

[0252] In some embodiments, the immunogenic compositions described herein further comprise pharmaceutical excipients including, but not limited to, biocompatible oils, saline, preservatives, carbohydrates, proteins, amino acids, osmotic agents, carrier gases, pH regulators, organic solvents, hydrophobic substances, enzyme inhibitors, water-absorbing polymers, surfactants, absorption promoters, and antioxidants. Representative examples of carbohydrates include soluble saccharides such as hydroxypropyl cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose, hyaluronic acid, chitosan, alginates, glucose, xylose, galactose, fructose, maltose, sucrose, dextran, chondroitin sulfate, and the like. Representative examples of proteins include albumin, gelatin, and the like. Representative examples of amino acids include glycine, alanine, glutamic acid, arginine, lysine, and their salts. Such pharmaceutical excipients are known in the art.

[0253] In some embodiments, the immunogenic MAPS compositions are administered in combination with other therapeutic materials including, for example, gamma-interferon, cytokines, chemotherapeutic agents, or anti-inflammatory or antiviral agents. In some embodiments, the immunogenic compositions disclosed herein can be administered together with one or more costimulatory molecules and / or adjuvants disclosed herein.

[0254] In some embodiments, the immunogenic composition is administered in pure or substantially pure form, but may also be administered as a pharmaceutical composition, formulation, or preparation. Such formulations include the MAPS described herein, together with one or more pharmaceutically acceptable carriers and optionally other therapeutic materials. Other therapeutic materials include compounds that enhance antigen presentation, such as gamma interferon, cytokines, chemotherapeutic agents, or anti-inflammatory agents. The formulations can be conveniently presented in unit dosage form and may be prepared by methods well known in the pharmaceutical art. For example, in Plotkin and Mortimer, VACCINES (2nd ed., W.B. Saunders Co., 1994), methods for vaccinating animals or humans to induce an immune response specific to a particular pathogen, as well as methods for preparing antigens, determining suitable dosages of antigens, and analyzing the induction of an immune response are described.

[0255] Formulations suitable for intravenous, intramuscular, intranasal, oral, sublingual, intravaginal, rectal, subcutaneous, or intraperitoneal administration preferably include a sterile aqueous solution of the active ingredient containing a solution that is preferably isotonic with the recipient's blood. Such formulation is conveniently prepared by dissolving the solid active ingredient in water containing a physiologically compatible substance such as sodium chloride (e.g., 0.1 M to 2.0 M), glycine, and equivalents, and having a buffered pH compatible with physiological conditions to produce an aqueous solution, and rendering the solution sterile. These may be present in single or multiple dose containers, for example, sealed ampoules or vials.

[0256] Liposome suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods well known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0257] Formulations for intranasal delivery are described in U.S. Patent Nos. 5,427,782, 5,843,451, and 6,398,774.

[0258] The formulations of the immunogenic compositions can incorporate stabilizers. Exemplary stabilizers are polyethylene glycol, proteins, saccharides, amino acids, inorganic acids, and organic acids, which can be used either alone or as mixtures. Two or more stabilizers may be used in an aqueous solution at appropriate concentrations and / or pH. The specific osmotic pressure in such an aqueous solution is generally in the range of 0.1 to 3.0 osmoles, preferably in the range of 0.80 to 1.2. The pH of the aqueous solution is adjusted to be in the range of pH 5.0 to 9.0, preferably in the range of pH 6 to 8.

[0259] When an oral preparation is desired, the immunogenic composition can be combined with typical carriers such as, among others, lactose, sucrose, starch, talc, magnesium stearate, crystalline cellulose, methylcellulose, carboxymethylcellulose, glycerin, sodium alginate, or gum arabic.

[0260] In some embodiments, the immunogenic compositions described herein can be administered intravenously, intranasally, intramuscularly, subcutaneously, intraperitoneally, sublingually, intravaginally, rectally, or orally. In some embodiments, the route of administration is oral, intranasal, subcutaneous, or intramuscular. In some embodiments, the route of administration is intranasal administration.

[0261] Vaccination can be carried out by conventional methods. For example, the immunogenic composition can be used in a suitable diluent, such as saline or water, or a complete or incomplete adjuvant. The immunogenic composition can be administered by any route appropriate for inducing an immune response. The immunogenic composition can be administered once or at periodic intervals until an immune response is induced. The immune response can be detected by a variety of methods known to those skilled in the art, including, but not limited to, antibody production, cytotoxicity assays, proliferation assays, and cytokine release assays. For example, a blood sample can be taken from an immunized mammal and analyzed by ELISA for the presence of antibodies against the antigen of the immunogenic composition (see de Boer et.al., 115 Arch Virol. 147 (1990)), and the titer of these antibodies can be determined by methods known in the art.

[0262] The exact dosage to be employed in the formulation will also depend on the route of administration and should be determined according to the judgment of the practitioner and the circumstances of each patient. For example, a total protein in the range of 25 μg to 900 μg can be administered monthly for 3 months.

[0263] Ultimately, the attending physician determines the amount of the immunogenic composition or vaccine composition to be administered to a particular individual. As with all immunogenic compositions or vaccines, the immunologically effective amount of the immunogen must be determined empirically. Factors to be considered include immunogenicity, whether the immunogen complexes or covalently binds to an adjuvant or carrier protein or other carrier, the route of administration, and the number of immunizing doses to be administered. Such factors are known in the art of vaccines, and making such determinations without undue experimentation is well within the skill of an immunologist.

[0264] In one embodiment, the immunogenic composition or vaccine composition described herein, when administered to a mouse, is 5LD of the immunogenic composition containing the antigen (against which the symptoms of the disease are prevented)50 In at least 20% of the animals challenged with an attack infection, an immune response can be elicited that prevents the symptoms of the disease. Methods of vaccinating and challenging animals are known to those skilled in the art. For example, for each mouse per vaccination, an aliquot of 10 μg of the immunogenic composition or vaccine composition as disclosed herein can be prepared in 100 μl of PBS and / or by the addition of incomplete Freund's adjuvant and intramuscularly injected. Alternatively, parenteral, intraperitoneal, and plantar injections can be used. The dose of plantar injection can be reduced to up to 50 μl. Mice can be immunized with the immunogenic composition or vaccine composition as disclosed herein on three separate occasions, with an interval of several days, for example 14 days, in between.

[0265] The effectiveness of vaccination can be tested by challenge infection with a pathogen. Seven days after the last administration of the immunogenic composition, the immunized mice are challenged intranasally with the pathogenic organism from which the antigen is derived. Ether-anesthetized mice (10 g - 12 g) can be infected intranasally with 50 μl of PBS-diluted allantoic cavity fluid containing 5 LD 50 of the pathogen. Protection can be measured by monitoring the survival and body weight of the animals, which is evaluated throughout a 21-day observation period. Severely affected mice are euthanized. 1 LD 50 of A / Mallard / Pennsylvania / 10218 / 84 is equal to 100 - 1000 of the 50% tissue culture infective dose (TCID50) assay.

[0266] In other embodiments, the immunized mice can be challenged with various different pathogenic organisms, such as different pathogenic organisms from which each of the antigens attached to the polymer is derived. For example, the immunogenic composition can include five different antigens attached to a polymer, such as a polysaccharide, where each antigen is derived from five different pathogenic organisms, and the immunized mice can be challenged with the five different pathogenic organisms either sequentially (in any order) or simultaneously. One of ordinary skill in the art would be able to determine the LD 50 of each pathogenic organism used to challenge the immunized mice by methods known in the art. See, for example, LaBarre & Lowy, 96 J. Virol. Meths. 107 (2001), Golub, 59 J. Immunol. 7 (1948).

[0267] Kit The present invention also provides a kit for producing an immunogenic composition as disclosed herein that is useful, for example, for researchers to prepare an immunogenic composition using a preferred antigen for research purposes of evaluating the effect of an antigen or combination of antigens on an immune response. Such a kit can be prepared from readily available materials and reagents. For example, such a kit can include any one or more of the following materials: a container containing a polymer, such as a polysaccharide, crosslinked with a plurality of first affinity molecules; and a container containing a complementary affinity molecule that associates with the first affinity molecule, where the complementary affinity molecule associates with the antigen.

[0268] In another embodiment, the kit can include a container containing a polymer, such as a polysaccharide, a container containing a plurality of first affinity molecules, and a container containing a crosslinking reagent for crosslinking the first affinity molecules to the polymer.

[0269] In some embodiments, the kit further comprises means for attaching the complementary affinity molecule to the antigen, which can be by means of a crosslinking reagent or by means of some intermediate fusion protein. In some embodiments, the kit can comprise at least one co-stimulatory factor that can be added to the polymer. In some embodiments, the kit comprises crosslinking reagents such as, but not limited to, CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate), EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride), sodium cyanoborohydride, cyanogen bromide, ammonium bicarbonate / iodoacetic acid for binding cofactors to the polymer.

[0270] Depending on the intended use of the kit, the specific target antigen, and the needs of the user, various kits and components can be prepared for use in the methods described herein.

[0271] The present invention can further be described in any of the following numbered paragraphs in any of the embodiments. 1. Amino acid sequence A soluble biotin-binding protein comprising TIFF0007679431000028.tif17152 and any functional derivative thereof. 2. The biotin-binding protein according to paragraph 1, produced in soluble form at a level of at least 10 mg per liter of culture medium in Escherichia coli (E. coli). 3. The biotin-binding protein according to claim 1 or 2, which is a dimer. 4. The biotin-binding protein according to any one of claims 1 to 3, comprising a bacterial signal sequence at the N-terminus. 5. The bacterial signal sequence is TIFF0007679431000029.tif4128, the biotin-binding protein according to paragraph 4. 6. The biotin-binding protein according to paragraph 4 or 5, wherein the signal sequence is linked to the biotin protein by a peptide linker. 7. The peptide linker has an amino acid sequence TIFF0007679431000030.tif4128 and is the biotin-binding protein according to paragraph 6. 8. The biotin-binding protein according to any one of claims 1 to 7, which contains a purification tag at the C-terminus. 9. The biotin-binding protein according to paragraph 8, wherein the purification tag is selected from the group consisting of a histidine tag, a c-my tag, a Halo tag, a Flag tag, and any combination thereof. 10. The amino acid sequence of the histidine tag is TIFF0007679431000031.tif4128 and is the biotin-binding protein according to paragraph 9. 11. The biotin-binding protein according to any one of claims 8 to 10, wherein the purification tag is linked to the biotin-binding protein via a peptide linker. 12. The peptide linker has an amino acid sequence TIFF0007679431000032.tif4129 and is the biotin-binding protein according to paragraph 11. 13. The amino acid sequence is TIFF0007679431000033.tif24152 and is the biotin-binding protein according to any one of paragraphs 1 to 12. 14. A composition comprising the biotin-binding protein according to any one of claims 1 to 13. 15. A fusion protein comprising a biotin-binding protein and a protein or peptide. 16. The fusion protein according to paragraph 15, wherein the protein or peptide is fused to the biotin-binding protein by a peptide linker. 17. The peptide linker has an amino acid sequence TIFF0007679431000034.tif4128 and is the fusion protein according to paragraph 15. 18. The fusion protein according to any one of claims 15 to 17, wherein the protein or peptide is an antigen selected from the group consisting of a pneumococcal antigen, a tuberculosis antigen, an anthrax antigen, an HIV antigen, a seasonal or pandemic influenza antigen, an influenza antigen, a pertussis antigen, a staphylococcus aureus antigen, a meningococcal antigen, a Haemophilus antigen, an HPV antigen, or a combination thereof. 19. The fusion protein according to any one of claims 15 to 18, wherein the antigen is a non-hemolytic variant of staphylococcus aureus alpha-hemolysin. 20. The fusion protein according to paragraph 19, wherein the non-hemolytic variant of staphylococcus aureus alpha-hemolysin contains a mutation at amino acid residues 205, 213, or 209-211 of the wild-type staphylococcus aureus alpha-hemolysin. 21. The fusion protein according to paragraph 19, wherein the non-hemolytic variant of staphylococcus aureus alpha-hemolysin contains one of the following mutations in the wild-type staphylococcus aureus alpha-hemolysin: (i) residue 205 W→A, (ii) residue 213 W→A, or (iii) residue 209-211 DRD→AAA. 22. The non-hemolytic variant of staphylococcus aureus alpha-hemolysin is TIFF0007679431000035.tif139146, and the fusion protein according to paragraph 19, which contains an amino acid sequence selected from the group consisting of functional variants, parts, and derivatives thereof. 23. The fusion protein according to any one of claims 15 to 22, which contains a bacterial signal sequence at the N-terminus. 24. The bacterial signal sequence is TIFF0007679431000036.tif4128, and the fusion protein according to paragraph 23. 25. The fusion protein according to paragraph 23 or 24, wherein the signal sequence is linked to the biotin protein by a peptide linker. 26. The peptide linker of the fusion protein according to paragraph 25 contains the amino acid sequence TIFF0007679431000037.tif4128. 27. The fusion protein according to any one of claims 15 to 26, comprising a purification tag at the C-terminus. 28. The fusion protein according to paragraph 27, wherein the purification tag is selected from the group consisting of a histidine tag, a c-my tag, a Halo tag, a Flag tag, and any combination thereof. 29. The fusion protein according to paragraph 27, wherein the histidine tag has the amino acid sequence TIFF0007679431000038.tif4128. 30. The fusion protein according to any one of claims 27 to 29, wherein the purification tag is linked to the biotin-binding protein via a peptide linker. 31. The fusion protein according to paragraph 30, wherein the peptide linker has the amino acid sequence TIFF0007679431000039.tif4129. 32. The fusion protein according to any one of claims 15 to 31, wherein the biotin-binding protein is the biotin-binding protein according to any one of claims 1 to 13. 33. TIFF0007679431000040.tif219145. The fusion protein according to any one of claims 15 to 32, comprising an amino acid sequence selected from the group consisting of. 34. A composition comprising the fusion protein according to any one of claims 15 to 33. 35. A mutant α-hemolysin (mHla) protein comprising a mutation at amino acid residues 205, 213, or 209 - 211 of wild-type Staphylococcus aureus α-hemolysin, wherein the mutant α-hemolysin has a lower hemolytic activity than wild-type α-hemolysin (Hla) of equal titer. 36. The mutant α-hemolysin according to paragraph 35, wherein the hemolytic activity of the mutant α-hemolysin is at least 25% lower than that of wild-type Hla of equal titer. 37. A mutant α-hemolysin according to paragraph 35 or 36, comprising one of the following mutations in wild-type Staphylococcus aureus α-hemolysin: (i) residue 205 W→A, (ii) residue 213 W→A, or (iii) residue 209-211 DRD→AAA. 38. A mutant α-hemolysin according to any of claims 13-15, comprising an amino acid sequence selected from the group consisting of TIFF0007679431000041.tif139145, and functional variants, portions, and derivatives thereof. 39. A composition comprising a mutant α-hemolysin according to any of claims 35-38. 40. A fusion protein comprising an α-hemolysin and a biotin-binding domain, having a hemolytic activity lower than that of wild-type α-hemolysin (Hla) with equal titers. 41. The fusion protein according to paragraph 18, wherein the α-hemolysin is a mutant hemolysin according to any of claims 35-38, or the α-hemolysin consists of the amino acid sequence of amino acids 27-319 of wild-type α-hemolysin of Staphylococcus aureus. 42. The fusion protein according to paragraph 19, wherein the biotin-binding domain consists of the amino acid sequence of SEQ ID NO: 1. 43. The fusion protein according to any of claims 40-42, wherein the biotin-binding domain and the mutant α-hemolysin are linked by a peptide linker. 44. The peptide linker of the fusion protein according to paragraph 43 comprises the amino acid sequence TIFF0007679431000042.tif4128. 45. The fusion protein according to any of claims 40-44, comprising a bacterial signal sequence at the N-terminus. 46. The bacterial signal sequence of the fusion protein according to paragraph 45 is TIFF0007679431000043.tif4128. 47. The fusion protein according to paragraph 45 or 46, wherein the signal sequence is linked to the biotin protein by a peptide linker. 48. The peptide linker is an amino acid sequence The fusion protein according to paragraph 47, comprising TIFF0007679431000044.tif4128. 49. The fusion protein according to any one of claims 40 to 48, comprising a purification tag at the C-terminus. 50. The fusion protein according to paragraph 49, wherein the purification tag is selected from the group consisting of a histidine tag, a c-my tag, a Halo tag, a Flag tag, and any combination thereof. 51. The histidine tag is an amino acid sequence The fusion protein according to paragraph 50, comprising TIFF0007679431000045.tif4128. 52. The fusion protein according to any one of claims 49 to 51, wherein the purification tag is linked to the biotin-binding protein via a peptide linker. 53. The peptide linker is an amino acid sequence The fusion protein according to paragraph 52, comprising TIFF0007679431000046.tif4129. 54. The fusion protein according to any one of claims 40 to 53, wherein the biotin-binding domain is the biotin-binding protein according to any one of claims 1 to 13. 55. The fusion protein according to any one of claims 40 to 54, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28. 56. The fusion protein according to any one of claims 40 to 55, wherein the hemolytic activity of the fusion protein is at least 25% lower than that of wild-type Hla with an equal titer. 57. A composition comprising the fusion protein according to any one of claims 40 to 56. 58. A method for inducing an immune response in a subject, comprising the step of administering the composition according to paragraph 14, 34, 39, or 57 to the subject. 59. A method for vaccinating a mammal against a pathogen having at least one antigen, comprising the step of administering the composition according to paragraph 14, 34, 39, or 57. 60. The method according to claim 58 or 59, wherein the subject is a human. 61. The method according to claim 58 or 59, wherein the subject is a farm animal or a wild animal. 62. The method according to claim 58 or 59, wherein the subject is a domestic animal. 63. The method according to claim 58 or 59, wherein the administration is via subcutaneous, intranasal, intradermal, or intramuscular injection. 64. The method according to paragraph 58, wherein the immune response is an antibody / B cell response. 65. The method according to paragraph 58, wherein the immune response is a CD4+ T cell response comprising a Th1, Th2, or Th17 response. 66. The method according to paragraph 58, wherein the immune response is a CD8+ T cell response. 67. The composition according to any one of claims 14, 34, 39, or 57 for use in the diagnosis of exposure to a pathogen or an immune threat. 68. The amino acid sequence of SEQ ID NO: 1 A lipidated biotin-binding protein comprising TIFF0007679431000047.tif17152 and any functional derivative thereof. 69. The lipidated biotin-binding protein according to paragraph 68, which is produced in soluble form at a level of at least 10 mg per liter of culture medium in Escherichia coli (E. coli). 70. The lipidated biotin-binding protein according to claim 68 or 69, which is a dimer. 71. The lipidated biotin-binding protein according to any one of claims 68 to 70, which comprises a lipidation sequence at the N-terminus. 72. The lipidation sequence is TIFF0007679431000048.tif4128, the lipidated biotin-binding protein according to paragraph 71. 73. The lipidated biotin-binding protein according to paragraph 71 or 72, wherein the lipidation sequence is linked to the biotin protein by a peptide linker. 74. The peptide linker has the amino acid sequence The lipid-added biotin-binding protein described in paragraph 73, including TIFF0007679431000049.tif4128. 75. The lipid-added biotin-binding protein according to any one of claims 68 to 74, comprising a purification tag at the C-terminus. 76. The lipid-added biotin-binding protein according to paragraph 75, wherein the purification tag is selected from the group consisting of a histidine tag, a c-my tag, a Halo tag, a Flag tag, and any combination thereof. 77. The histidine tag has the amino acid sequence The lipid-added biotin-binding protein according to paragraph 76, including TIFF0007679431000050.tif4128. 78. The lipid-added biotin-binding protein according to any one of claims 75 to 77, wherein the purification tag is linked to the biotin-binding protein via a peptide linker. 79. The peptide linker has the amino acid sequence The lipid-added biotin-binding protein according to paragraph 78, including TIFF0007679431000051.tif5130. 80. The amino acid sequence The lipid-added biotin-binding protein according to any one of paragraphs 68 to 79, including TIFF0007679431000052.tif17151. 81. A composition comprising the lipid-added biotin-binding protein according to any one of claims 68 to 80. 82. A fusion protein comprising a lipid-added biotin-binding protein and a protein or peptide. 83. The fusion protein according to paragraph 82, wherein the protein or peptide is fused to the lipid-added biotin-binding protein by a peptide linker. 84. The peptide linker has the amino acid sequence The fusion protein according to paragraph 83, including TIFF0007679431000053.tif4128. 85. The fusion protein according to any one of claims 82 to 84, wherein the protein or peptide is an antigen selected from the group consisting of a pneumococcal antigen, a tuberculosis antigen, an anthrax antigen, an HIV antigen, a seasonal or pandemic influenza antigen, an influenza antigen, a pertussis antigen, a staphylococcus aureus antigen, a meningococcal antigen, a Haemophilus antigen, an HPV antigen, or a combination thereof. 86. The fusion protein according to any one of paragraph 85, wherein the antigen is a non-hemolytic variant of staphylococcus aureus alpha-hemolysin. 87. The fusion protein according to paragraph 86, wherein the non-hemolytic variant of staphylococcus aureus alpha-hemolysin contains a mutation at amino acid residues 205, 213, or 209-211 of the wild-type staphylococcus aureus alpha-hemolysin. 88. The fusion protein according to paragraph 86, wherein the non-hemolytic variant of staphylococcus aureus alpha-hemolysin contains one of the following mutations in the wild-type staphylococcus aureus alpha-hemolysin: (i) residue 205 W→A, (ii) residue 213 W→A, or (iii) residue 209-211 DRD→AAA. 89. The non-hemolytic variant of staphylococcus aureus alpha-hemolysin is TIFF0007679431000054.tif139145, and the fusion protein according to paragraph 86, comprising an amino acid sequence selected from the group consisting of functional variants, portions, and derivatives thereof. 90. The fusion protein according to any one of claims 82 to 89, comprising a lipid addition sequence at the N-terminus. 91. The lipid addition sequence is TIFF0007679431000055.tif4128, the fusion protein according to paragraph 90. 92. The fusion protein according to paragraph 90 or 91, wherein the signal sequence is linked to the biotin protein by a peptide linker. 93. The peptide linker has an amino acid sequence TIFF0007679431000056.tif4128, the fusion protein according to paragraph 92. 94. A fusion protein according to any one of claims 82 to 93, comprising a purification tag at the C-terminus. 95. The fusion protein according to paragraph 94, wherein the purification tag is selected from the group consisting of a histidine tag, a c-my tag, a Halo tag, a Flag tag, and any combination thereof. 96. The histidine tag has the amino acid sequence The fusion protein according to paragraph 95, comprising TIFF0007679431000057.tif4128. 97. The fusion protein according to any one of claims 93 to 96, wherein the purification tag is linked to the biotin-binding protein via a peptide linker. 98. The peptide linker has the amino acid sequence The fusion protein according to paragraph 97, comprising TIFF0007679431000058.tif4129. 99. The fusion protein according to any one of claims 82 to 98, wherein the lipid-added biotin-binding protein is the biotin-binding protein according to any one of claims 68 to 80. 100. The fusion protein according to any one of claims 82 to 99, comprising an amino acid sequence selected from the group consisting of TIFF0007679431000059.tif199152. 101. A composition comprising the lipid-added biotin-binding protein according to any one of claims 82 to 100. 102. A method for inducing an immune response in a subject, comprising the step of administering the composition according to paragraph 81 or 101 to the subject. 103. A method for vaccinating a mammal against a pathogen having at least one antigen, comprising the step of administering the composition according to paragraph 81 or 101. 104. The method according to claim 102 or 103, wherein the subject is a human. 105. The method according to claim 102 or 103, wherein the subject is a farm animal or a wild animal. 106. The method according to claim 102 or 103, wherein the subject is a domestic animal. 107. The method according to claim 102 or 103, wherein the administration is via subcutaneous, intranasal, intradermal, or intramuscular injection. 108. The method according to paragraph 102, wherein the immune response is an antibody / B cell response. 109. The method according to paragraph 102, wherein the immune response is a CD4+ T cell response comprising a Th1, Th2, or Th17 response. 110. The method according to paragraph 102, wherein the immune response is a CD8+ T cell response. 111. The composition according to any one of claims 81 or 101, for use in the diagnosis of exposure to a pathogen or immune threat.

[0272] Some selected definitions For convenience, certain terms employed throughout this application (including the specification, examples, and appended claims) are summarized herein. 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 pertains.

[0273] As used in this specification and the appended claims, the singular forms include plural references and vice versa, unless the context clearly dictates otherwise. The term "or" is inclusive, unless modified by, for example, "either". Unless otherwise indicated by way of operation example or otherwise, all numbers representing amounts of materials or reaction conditions used herein are to be understood as being modified in all instances by the term "about".

[0274] As used herein, the term "immunogenic composition" is defined as a composition capable of inducing an immune response, such as an antibody or cellular immune response, when administered to a subject. The immunogenic compositions of the present invention may or may not be immunoprotective or therapeutic. When the immunogenic composition of the present invention prevents, ameliorates, alleviates, or eliminates a disease from a subject, the immunogenic composition may optionally be referred to as a vaccine. However, when used herein, the term "immunogenic composition" is not intended to be limited to vaccines.

[0275] As used herein, the term "antigen" refers to any substance that promotes an immune response directed against the substance. In some embodiments, the antigen is a peptide or polypeptide, and in other embodiments, it can be any chemical substance or moiety, such as a carbohydrate that elicits an immune response directed against the substance.

[0276] The term "associate", as used herein, refers to the linkage of two or more molecules by non-covalent or covalent bonds. In some embodiments, when the linkage of two or more molecules occurs by covalent bonds, the two or more molecules can be fused together or cross-linked together. In some embodiments, when the binding of two or more molecules occurs by non-covalent bonds, the two or more molecules can form a complex.

[0277] The term "complex", as used herein, refers to a population of two or more molecules that are spatially connected by means other than covalent interactions, e.g., they can be connected by electrostatic interactions, hydrogen bonds, or hydrophobic interactions (i.e., van der Waals forces).

[0278] As used herein, the term "fused" means that at least one protein or peptide is physically associated with a second protein or peptide. In some embodiments, the fusion is typically a covalent linkage, however, other types of linkages, including linkages via, for example, electrostatic interactions or hydrophobic interactions, are encompassed by the term "fused". Covalent linkages can include linkages as fusion proteins or chemical bonds formed via, for example, disulfide bonds formed between two cysteine residues.

[0279] As used herein, the terms "fusion polypeptide" or "fusion protein" mean a protein created by joining together two or more polypeptide sequences. The fusion polypeptides encompassed by the present invention include the translation products of chimeric gene constructs that join a DNA sequence encoding one or more antigens, or fragments or mutants thereof, with a DNA sequence encoding a second polypeptide so as to form a single open reading frame. In other words, a "fusion polypeptide" or "fusion protein" is a recombinant protein of two or more proteins joined by peptide bonds. In some embodiments, the second protein to which the antigen is fused is a complementary affinity molecule capable of interacting with a first affinity molecule of a complementary affinity pair.

[0280] The terms "polypeptide" and "protein" can be used synonymously to refer to a polymer of amino acid residues linked by peptide bonds and, for the purposes of the present invention as claimed, having a typical minimum length of at least 25 amino acids. The terms "polypeptide" and "protein" can include multimeric proteins, such as proteins containing two or more domains or subunits. The term "peptide", as used herein, refers to an array of peptide-bonded amino acids containing less than 25 amino acids, e.g., from about 4 to 25 amino acids in length. Proteins and peptides can consist of linearly arranged amino acids linked by peptide bonds, regardless of whether they are produced biologically, recombinantly, or synthetically and regardless of whether they consist of natural or non-natural amino acids, and are included within this definition. Both full-length proteins and fragments thereof of 25 amino acids or more are included by the definition of protein. The term also includes polypeptides having post-translational modifications such as co-translational modification of the polypeptide (e.g., signal peptide cleavage) and, e.g., disulfide bond formation, glycosylation, acetylation, phosphorylation, lipidation, proteolytic cleavage (e.g., cleavage by a metalloprotease), etc. Further, as used herein, "polypeptide" refers to a protein that includes modifications such as deletions, additions, and substitutions (which are generally conservative in nature as would be known to one of ordinary skill in the art) to the native sequence, so long as the protein maintains the desired activity. These modifications can be artificial through site-directed mutagenesis or can be accidental through mutations in the host producing the protein or errors in PCR amplification or other recombinant DNA methods.

[0281] By "signal sequence" is meant a nucleic acid sequence that, when operably linked to a nucleic acid molecule, facilitates the secretion of the product encoded by the nucleic acid molecule (e.g., a protein or peptide). In some embodiments, the signal sequence is preferably located 5' to the nucleic acid molecule.

[0282] As used herein, the term "N-glycosylated" or "N-glycosylation" refers to the covalent attachment of a sugar moiety to an asparagine residue in a polypeptide. The sugar moiety can include, but is not limited to, glucose, mannose, and N-acetylglucosamine. Glycan modifications, such as sialylation, are also included.

[0283] An "antigen-presenting cell" or "APC" is a cell that expresses major histocompatibility complex (MHC) molecules and can present foreign antigens that are complexed with MHC on its surface. Examples of antigen-presenting cells are dendritic cells, macrophages, B cells, fibroblasts (skin), thymic epithelial cells, thyroid epithelial cells, glial cells (brain), pancreatic beta cells, and vascular endothelial cells.

[0284] The term "functional portion" or "functional fragment" when used in the context of "functional portion of an antigen" refers to a portion of an antigen or antigen polypeptide that mediates the same effect as the full-length antigen portion, e.g., induces an immune response in a subject or mediates an association with other molecules, e.g., by including at least one epitope.

[0285] As used herein, a "portion" of a target antigen can be at least 3 amino acids in length and can be, for example, at least 6, at least 8, at least 10, at least 14, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 25 or more amino acids.

[0286] The term "cytotoxic T lymphocyte" or "CTL" refers to a lymphocyte that induces apoptosis in targeted cells. CTLs form antigen-specific conjugates with target cells through the interaction of processed antigen (Ag) and TCR on the surface of the target cell, resulting in apoptosis of the targeted cells. Apoptotic bodies are eliminated by macrophages. The term "CTL response" is used to refer to the primary immune response mediated by CTL cells.

[0287] The term "cell-mediated immunity" or "CMI", as used herein, refers to an immune response that does not involve antibodies or complement, but rather involves, for example, the activation of macrophages, natural killer (NK) cells, antigen-specific cytotoxic T lymphocytes (T cells), and the release of various cytokines in response to a target antigen. Put another way, CMI refers to immune cells (such as T cells and other lymphocytes) that bind to the surface of other cells (such as antigen-presenting cells (APCs)) presenting a target antigen and trigger a response. The response can involve either other lymphocytes and / or other white blood cells (leukocytes), as well as the release of cytokines. Cell-mediated immunity protects the body by (i) activating antigen-specific cytotoxic T lymphocytes (CTLs) that can destroy somatic cells presenting epitopes of foreign antigens on their surface, such as cells infected with viruses and intracellular bacteria, (2) activating macrophages and NK cells to enable the destruction of intracellular pathogens, and (3) stimulating cells to secrete various cytokines that affect the functions of other cells involved in adaptive and innate immune responses.

[0288] The term "immune cell", as used herein, refers to any cell that can release cytokines in response to direct or indirect antigenic stimulation. In this specification, the term "immune cell" includes lymphocytes, including natural killer (NK) cells, T cells (CD4+ and / or CD8+ cells), B cells, macrophages and monocytes, Th cells, Th1 cells, Th2 cells, white blood cells, dendritic cells, macrophages, mast cells and monocytes, and any other cell capable of producing cytokine molecules in response to direct or indirect antigenic stimulation. Typically, immune cells are lymphocytes, such as T cell lymphocytes.

[0289] As used herein, the term "cytokine" refers to a molecule released from immune cells in response to antigenic stimulation. Examples of such cytokines include, but are not limited to, GM-CSF, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-17A, IL-17F, or other members of the IL-17 family, IL-22, IL-23, IFN-α, IFN-β, IFN-γ, MIP-1α, MIP-1β, TGF-β, TNFα, or TNFβ. The term "cytokine" does not include antibodies.

[0290] As used herein, the term "subject" refers to any animal that is useful for inducing an immune response. The subject can be a wild, domestic, commercial, or companion animal such as a bird or a mammal. The subject can be a human. In one embodiment of the invention, it is contemplated that an immunogenic composition as disclosed herein may also be suitable for therapeutic or prophylactic treatment in humans, although it is also applicable to warm-blooded vertebrates such as mammals (particularly higher primates) such as non-human primates, sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, cows, as well as non-mammals such as chickens, ducks, or turkeys. In another embodiment, the subject is a wild animal such as a bird for diagnosing avian influenza, etc. In some embodiments, the subject is an experimental animal or animal substitute as a disease model. The subject may be a subject in need of veterinary treatment where induction of an immune response against an antigen is useful for preventing a disease and / or controlling the spread of a disease such as SIV, STL1, SFV, or in the case of livestock, hoof and mouth disease, or in the case of birds, Marek's disease or avian influenza, and other such diseases.

[0291] As used herein, the term "pathogen" refers to a living organism or molecule that causes disease or illness in a subject. For example, pathogens include, but are not limited to, viruses, fungi, bacteria, parasites, and other infectious organisms or molecules therefrom, as well as taxonomically related macroscopic organisms within categories such as algae, fungi, yeast, protozoa, etc.

[0292] "Cancer cell" refers to a cancerous, pre-cancerous, or transformed cell in vivo, in vitro, or in tissue culture that has an altered phenotype, either spontaneous or induced, which does not necessarily require the uptake of new genetic material. Transformation can result from infection with a transforming virus and the integration of new genomic nucleic acid or the uptake of exogenous nucleic acid, but can also occur spontaneously or after exposure to carcinogens, thereby mutating endogenous genes. Transformation / cancer is associated with, for example, morphological changes in a suitable animal host such as a nude mouse, immortalization of cells, abnormal growth control, lesion formation, anchorage independence, malignancy, loss of contact inhibition and density limitation of growth, growth factor or serum dependence, tumor-specific markers, invasion or metastasis, and tumor growth. See, for example, Freshney, CULTURE ANIMAL CELLS: MANUAL BASIC TECH. (3rd ed., 1994).

[0293] The term "wild-type" generally refers to a natural, normal polynucleotide sequence or a portion thereof that encodes a protein, or a protein sequence or a portion thereof, as it normally exists in vivo.

[0294] The term "mutant" refers to a living organism or cell having any change in its genetic material, in particular, a change with respect to a wild-type polynucleotide sequence (i.e., deletion, substitution, addition, or modification), or any change with respect to a wild-type protein sequence. The term "variant" may be used synonymously with "mutant". It is often assumed that a change in the genetic material results in a change in the function of the protein, but the terms "mutant" and "variant" refer to a change in the sequence of a wild-type protein, regardless of whether the change modifies (e.g., increases, decreases, confers a new function) the function of the protein or has no effect on the function of the protein (e.g., the mutation or variant is silent).

[0295] The term "pharmaceutically acceptable" refers to compounds and compositions that can be administered to mammals without undue toxicity. The term "pharmaceutically acceptable carrier" excludes tissue culture media. Exemplary pharmaceutically acceptable salts include, but are not limited to, mineral salts such as hydrochloride, hydrobromide, phosphate, sulfate, etc., and salts of organic acids such as acetate, propionate, malonate, benzoate, etc. Pharmaceutically acceptable carriers are known in the art.

[0296] Proteins or polypeptides often contain amino acids other than the 20 amino acids generally referred to as the 20 natural amino acids, and many amino acids, including the terminal amino acids, can be modified in a given polypeptide either by natural processes such as glycosylation and other post-translational modifications or by chemical modification techniques well known in the art. It will be understood that known modifications that may be present in the polypeptides of the present invention include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a polynucleotide or polynucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of a covalent cross-link, formation of cystine, formation of pyroglutamic acid, formulation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of an amino acid to a protein such as arginylation, and ubiquitination, but are not limited thereto.

[0297] As used herein, the terms "homologous" or "homologue" are used interchangeably and when used to describe a polynucleotide or polypeptide, two polynucleotides or polypeptides, or their designated sequences, when optimally aligned and compared using, for example, BLAST, version 2.2.14 with default parameters for alignment, are at least 70% nucleotides, usually at least about 75% to 99%, such as at least about 98 - 99% nucleotides, with appropriate nucleotide insertions or deletions, or amino acid insertions or deletions, indicating identity. For polypeptides, there should be at least 50% amino acid identity in the polypeptide. The terms "homolog" or "homologous" also, as used herein, refer to homology with respect to structure. The determination of a homolog of a gene or polypeptide can be readily ascertained by one of ordinary skill in the art. When in the context with a specified percentage, the specified percentage of homology means at least that percentage of amino acid similarity. For example, 85% homology refers to at least 85% amino acid similarity.

[0298] As used herein, the term "heterologous" in relation to a nucleic acid sequence, protein, or polypeptide means that these molecules are not native in that cell. For example, in the context of a protein expression vector, a nucleic acid sequence encoding a fusion antigen polypeptide described herein, such as inserted into a cell, is a heterologous nucleic acid sequence.

[0299] Regarding array comparison, typically one array serves as the reference array against which a test array is compared. When using an array comparison algorithm, the test and reference arrays are input into a computer, and if necessary, subsequence coordinates are specified and array algorithm program parameters are specified. The array comparison algorithm then calculates the percent identity of the array for one or more test arrays relative to the reference array based on the specified program parameters. If necessary or desired, the optimal alignment of the arrays for comparison can be done in any of a variety of ways, which are well known in the art.

[0300] As used herein, the term "variant" can refer to a polypeptide or nucleic acid that differs from a native polypeptide or nucleic acid by one or more amino acid or nucleic acid deletions, additions, substitutions, or side chain modifications, but still retains one or more specific functions or biological activities of the native molecule. Amino acid substitutions include modifications in which an amino acid is replaced with a different natural or non-conventional amino acid residue. Such substitutions can be classified as "conservative", in which case the amino acid residue contained in the polypeptide is replaced with a similar characteristic of another natural amino acid with respect to either polarity, side chain function, or size. Substitutions encompassed by the variants described herein can also be "non-conservative", where the amino acid residue present in the peptide is replaced with an amino acid having different properties (e.g., substitution of a charged or hydrophobic amino acid with alanine), or alternatively, a natural amino acid is replaced with a non-conventional amino acid. When used in relation to a polynucleotide or polypeptide, the term "variant" encompasses modifications in the primary, secondary, or tertiary structure, respectively, as compared to the respective reference polynucleotide or polypeptide (e.g., as compared to a wild-type polynucleotide or polypeptide).

[0301] When used with reference to a variant of an antigen or a functional derivative of an antigen, as compared to the original antigen, the term "substantially similar" means that a particular target sequence varies from the sequence of the antigen polypeptide by one or more substitutions, deletions, or additions, but retains the function of the antigen, such that it elicits an immune response in a subject, including at least 50% or more, for example at least 60%, 70%, 80%, 90% or more. When determining polynucleotide sequences, all target polynucleotide sequences capable of encoding substantially similar amino acid sequences are considered to be substantially similar to the reference polynucleotide sequence, regardless of differences in the codon sequence. A nucleotide sequence is "substantially similar" to a given antigen nucleic acid sequence if (a) the nucleotide sequence hybridizes to the coding region of the native antigen sequence, or (b) the nucleotide sequence is capable of hybridizing to the nucleotide sequence of the native antigen under moderately stringent conditions and has a biological activity similar to that of the native antigen protein, or (c) the nucleotide sequence is degenerate as a result of the genetic code with respect to the nucleotide sequence defined in (a) or (b). Substantially similar proteins can typically be at least about 80% similar to the corresponding sequence of the native protein.

[0302] Variants can, as described hereinafter, include conservative or non-conservative amino acid changes. Polynucleotide changes can result in amino acid substitutions, additions, deletions, fusions, and cleavages in the polypeptide encoded by the reference sequence. Variants can also include insertions and substitutions of amino acids and other molecules that do not normally occur in the peptide sequence on which the variant is based, for example, insertions of ornithine, which do not normally occur in human proteins, including, but not limited to, insertions, deletions, or substitutions of amino acids. "Conservative amino acid substitutions" result from replacing one amino acid with another having similar structural and / or chemical properties. Tables of conservative substitutions providing functionally similar amino acids are known in the art. For example, the following six groups each contain amino acids that are conservative substitutions for one another: (1) alanine (A), serine (S), threonine (T); (2) aspartic acid (D), glutamic acid (E); (3) asparagine (N), glutamine (Q); (4) arginine (R), lysine (K); (5) isoleucine (I), leucine (L), methionine (M), valine (V); and (6) phenylalanine (F), tyrosine (Y), tryptophan (W). See, for example, Creighton, PROTEINS (W.H. Freeman & Co., 1984).

[0303] The choice of conserved amino acids can be made based on the location of the amino acid in the peptide to be substituted, e.g., whether the amino acid is on the outside of the peptide and exposed to the solvent or on the inside and not exposed to the solvent. The selection of such conservative amino acid substitutions is within the skill of one of ordinary skill in the art. Thus, one can select conservative amino acid substitutions suitable for amino acids on the outside of a protein or peptide (i.e., amino acids exposed to the solvent). These substitutions include, but are not limited to: substitution of Y with F, substitution of T with S or K, substitution of P with A, substitution of E with D or Q, substitution of N with D or G, substitution of R with K, substitution of G with N or A, substitution of T with S or K, substitution of D with N or E, substitution of I with L or V, substitution of F with Y, substitution of S with T or A, substitution of R with K, substitution of G with N or A, substitution of K with R, substitution of A with S, K, or P.

[0304] Alternatively, one can select conservative amino acid substitutions suitable for amino acids on the inside of a protein or peptide (i.e., amino acids not exposed to the solvent). For example, the following conservative substitutions can be used: substitution of T with A or S when Y is substituted with F, substitution of I with L or V, substitution of W with Y, substitution of M with L, substitution of N with D, substitution of G with A, substitution of T with A or S, substitution of D with N, substitution of I with L or V, substitution of F with Y or L, substitution of S with A or T, and substitution of A with S, G, T, or V. In some embodiments, LF polypeptides that include non-conservative amino acid substitutions are also encompassed within the term "variant." As used herein, the term "non-conservative" substitution refers to the substitution of an amino acid residue with a different amino acid residue having different chemical properties. Non-limiting examples of non-conservative substitutions include aspartic acid (D) replaced with glycine (G); asparagine (N) replaced with lysine (K); and alanine (A) replaced with arginine (R).

[0305] As used herein, the term "derivative" refers to a chemically modified peptide, for example, by ubiquitination, labeling, pegylation (derivatization with polyethylene glycol), or addition of other molecules. A molecule is also a "derivative" of another molecule when it contains additional chemical moieties that are not normally part of the molecule. Such moieties can improve the solubility, absorption, biological half-life, etc. of the molecule. The moieties can alternatively reduce the toxicity of the molecule or eliminate or mitigate undesirable side effects of the molecule. Moieties capable of mediating such effects are disclosed in REMINGTON'S PHARMACEUTICAL SCIENCES (21st ed., Tory, ed., Lippincott Williams & Wilkins, Baltimore, MD, 2006).

[0306] When used with "derivative" or "variant", the term "functional" refers to a protein molecule having a biological activity that is substantially similar to the biological activity of an entity or a molecule that is a derivative or variant thereof. "Substantially similar" in this context means that the biological activity, for example, the antigenicity of a polypeptide, is at least 50% active, for example, at least 60% active, 70% active, 80% active, 90% active, 95% active, 100% active, or even higher activity (i.e., the variant or derivative has activity greater than the wild type), for example, 110% active, 120% active, or more, relative to a reference, for example, the corresponding wild-type polypeptide.

[0307] When used to describe a nucleic acid molecule, the term "recombinant" means a polynucleotide of genomic, cDNA, viral, semi-synthetic, and / or synthetic origin, in which all or a portion of the polynucleotide sequences that are associated in nature are not associated for their origin or manipulation. When the term "recombinant" is used with respect to a peptide, polypeptide, protein, or recombinant fusion protein, it means a polypeptide produced by expression from a recombinant polynucleotide. When the term "recombinant" is used with respect to a host cell, it means a host cell into which a recombinant polynucleotide has been incorporated. Recombinant is also used herein with respect to a material (e.g., a cell, nucleic acid, protein, or vector) to refer to the material being modified by the introduction of a heterologous material (e.g., a cell, nucleic acid, protein, or vector).

[0308] The term "vector" refers to a nucleic acid molecule that can transport a heterologous nucleic acid linked to a host cell or mediate its expression, and a plasmid is a species of the genus encompassed by the term "vector". The term "vector" typically refers to a nucleic acid sequence containing an origin of replication and other entities necessary for replication and / or maintenance in a host cell. A vector capable of directing the expression of a gene and / or nucleic acid sequence to which they are functionally linked is referred to herein as an "expression vector". Generally, useful expression vectors are often in the form of "plasmids", which do not bind to chromosomes in their vector form and typically refer to circular double-stranded DNA molecules containing entities or encoded DNA for stable or transient expression. Other expression vectors that can be used in the methods as disclosed herein include, but are not limited to, plasmids, episomes, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophages, or viral vectors, and such vectors can integrate into the host genome or replicate autonomously in certain cells. Vectors can be DNA or RNA vectors. Other forms of expression vectors known to those skilled in the art that perform equivalent functions, such as self-replicating extrachromosomal vectors or vectors that integrate into the host genome, can also be used. Preferred vectors are those capable of autonomous replication and / or expression of the nucleic acids to which they are linked.

[0309] The terms "reduced" or "reducing" or "decreasing", when used herein, generally mean a statistically significant decrease in amount relative to a reference. To avoid misunderstanding, "reduced", as the term is defined herein, means a statistically significant decrease of at least 10% compared to a reference level, such as at least 20%, at least 30%, at least 40%, at least 50%, or at least 60%, or at least 70%, or at least 80%, at least 90% or more, up to a 100% decrease (i.e., non-existent level compared to a reference sample), or any decrease from 10 to 100% compared to a reference level.

[0310] When used herein, the term "low" generally means lower by a statistically significant amount. To avoid misunderstanding, "low" means a statistically significant value that is at least 10% lower than the reference level, for example, at least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower than the reference level, and includes values that are up to 100% lower (i.e., non - existent level compared to the reference sample) than the reference level.

[0311] When used herein, the terms "increased" or "increasing" generally mean an increase by a statistically significant amount as the term is defined herein. For example, compared to the reference level, it includes an increase of at least 2 - fold, at least 3 - fold, at least 4 - fold, at least 5 - fold, at least 10 - fold or more, an increase of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more, i.e., a statistically significant increase of at least 10% compared to the reference level.

[0312] When used herein, the term "high" generally means higher by a statistically significant amount compared to the reference level. For example, at least 2 - fold higher, at least 3 - fold higher, at least 4 - fold higher, at least 5 - fold higher, at least 10 - fold or more higher than the reference level, i.e., at least 10% higher than the reference level, such as at least 20% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 60% higher, at least 70% higher, at least 80% higher, at least 90% higher, at least 100% higher, meaning a statistically significant value that is higher than the reference.

[0313] As used herein, the term "comprising" means that other elements may also be present in addition to the recited elements, and is meant to be inclusive rather than limiting.

[0314] The term "consisting of" refers to the compositions, methods, and respective components described herein, excluding any elements not recited in the description of the embodiment.

[0315] As used herein, the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristics of that embodiment of the invention.

[0316] It is further understood that all base sizes or amino acid sizes, and all molecular weights or molecular mass values given for nucleic acids or polypeptides are approximate and are provided for illustrative purposes. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described herein.

[0317] As used herein, the term "biotin" refers to the compound biotin itself, as well as its analogs, derivatives, and variants. Thus, the term "biotin" includes biotin (cis-hexahydro-2-oxo-1H-thieno[3,4]imidazole-4-pentanoic acid), as well as any derivatives and analogs thereof, including biotin-like compounds. Such compounds include, for example, biotin-e-N-lysine, biocytin hydrazide, 2-iminobiotin, and amino or sulfhydryl derivatives of biotinyl-E-aminocaproic acid-N-hydroxysuccinimide ester, sulfo-succinimidyl iminobiotin, biotin bromoacetyl hydrazide, p-diazobenzoyl biocytin, 3-(N-maleimidopropionyl) biocytin, desthiobiotin, and equivalents. The term "biotin" also includes biotin variants that can specifically bind to one or more of streptavidin, avidin, streptavidin, the tamavidin moiety, or other avidin-like peptides.

[0318] The foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, but it will be readily apparent to those skilled in the art that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims in light of the teachings of the present invention. The following are intended to be illustrative of the present invention. However, the practice of the present invention is not limited or restricted in any way by the examples.

Examples

[0319] Example 1: Expressing a high-yield and soluble recombinant biotin-binding protein and its fusion protein in Escherichia coli The recombinant rhesus abzyme (rRhavi) used in these studies is an N-terminal modified version that contains only residues 45-179 of the wild-type protein. To optimize the expression level of rRhavi in E. coli, the gene sequence (45-179) encoding the rhesus abzyme polypeptide was redesigned by using E. coli-preferred expression codons, and then synthesized and cloned into the PET21b vector. To facilitate correct folding and obtain a high yield of soluble recombinant protein, a DNA sequence encoding an E. coli periplasm localization signal sequence (19 amino acids, TIFF0007679431000060.tif4128) was introduced at the 5' end of the synthetic gene of rRhavi. This signal sequence is predicted to be automatically removed from the recombinant protein after targeting the periplasm of E. coli during the expression process.

[0320] A DNA sequence encoding a flexible linker region and His tag TIFF0007679431000061.tif4128 was directly inserted at the 3' end of the synthetic rRhavi gene. This helps in the purification of the recombinant biotin-binding protein. Further, an antigen can be inserted into a linker having flexible linkers on both sides, for example, the antigen can be inserted between amino acids S and V of the linker. The antigen is thus separated from the biotin-binding protein by the peptide linker TIFF0007679431000062.tif4128 and from the His tag by the peptide linker TIFF0007679431000063.tif4128, which can stabilize the fusion protein.

[0321] To construct a rhesus abzyme antigen fusion protein, a DNA sequence encoding a flexible linker region consisting of seven amino acids was used to help stabilize the fusion protein. TIFF0007679431000064.tif4128 can be directly inserted into the 3'-end of the synthetic rRhavi gene. The gene encoding the candidate antigen (full-length or desired fragment) was amplified from the genomic DNA of the pathogen of interest by normal PCR procedures and inserted into the rRhavi expression vector just beyond the linker region.

[0322] For protein expression, the plasmid containing the target construct was transformed into Escherichia coli strain BL21(DE3) using standard heat shock procedures. A single colony was freshly picked from the plate (or the glycerol stock was used later) and inoculated into 30 ml of Luria-Bertani (LB) medium containing ampicillin (Amp+) for overnight culture at 37°C. On the second day, a 5 ml starter culture was inoculated into 1 liter of LB medium / Amp+ and grown at 37°C until OD 600 reached 1. After cooling the medium to 16°C, IPTG at a final concentration of 0.2 mM was added to the culture for overnight induction.

[0323] The protein was purified from the periplasmic fraction using a modified osmotic shock protocol. Briefly, bacterial cells from 6 liters of culture were collected and resuspended in 120 ml of buffer containing 30 mM Tris (pH 8.0), 20% sucrose, and 1 mM EDTA. After stirring at room temperature for 20 minutes, the cells were reprecipitated by centrifugation at 10,000 rpm for 10 minutes. The supernatant was collected as fraction 1 and the cells were resuspended in 80 ml of ice-cold solution containing 5 mM MgCl2, protease inhibitor, and deoxyribonuclease. After stirring at 4°C for 20 minutes, the mixture was centrifuged at 13,000 rpm for 20 minutes and the supernatant was collected as fraction 2. The final concentration of 150 mM NaCl, 10 mM MgCl 2After adding [ID=] and 10 mM imidazole, the combined supernatant of fraction 1 and fraction 2 was applied onto a Ni-NTA column. The protein eluted from the Ni-NTA column was further purified by gel filtration using a superdex 200 column operating on an AKTA purification device. The peak fractions containing the target protein were stored and concentrated. The protein concentration was measured using a BCA protein assay kit from Bio-Rad. The purified protein was aliquoted, rapidly frozen in liquid nitrogen, and stored at -80 °C for future use.

[0324] The construct of the biotin-binding protein is schematically shown in Figure 1, and the SDS-PAGE of the purified biotin-binding protein is shown in Figure 2.

[0325] The construct of the fusion protein containing the biotin-binding protein is schematically shown in Figure 3, and an exemplary SDS-PAGE of the purified fusion protein is shown in Figure 4.

[0326] Example 2: Lipid-added derivative of biotin-binding protein Using a method similar to that described in Example 1, a lipid-added derivative of recombinant biotin-binding protein was produced. The lipid-added derivative used in this study is an N-terminal modified version of wild-type streptavidin containing only residues 45 - 179 of the wild-type protein. To optimize the expression level of rRhavi in E. coli, the gene sequence (45 - 179) encoding the streptavidin polypeptide was redesigned by using E. coli-preferred expression codons, and then synthesized and cloned into the PET21b vector. To promote lipid addition, proper folding, and obtain a high yield of soluble recombinant protein, a DNA sequence encoding a lipid-added sequence (19 amino acids, TIFF0007679431000065.tif4128) was introduced at the 5'-end of the synthetic gene of rRhavi. Lipid addition can be added onto the Cys residue of the lipid-added sequence by bacteria, such as E. coli, during the process of expression.

[0327] For protein expression, the plasmid containing the target construct was transformed into E. coli strain BL21(DE3) using standard heat shock procedures. A single colony was freshly picked from the plate (or the glycerol stock was used later) and inoculated into 30 ml of Luria-Bertani (LB) medium containing ampicillin (Amp+) for overnight culture at 37°C. On the second day, a 5 ml starter culture was inoculated into 1 liter of LB medium / Amp+ and grown at 37°C until OD 600 = 1 was reached. After cooling the medium to 16°C, IPTG at a final concentration of 0.2 mM was added to the culture for overnight induction.

[0328] Lipid-added streptavidin was purified from the E. coli membrane fraction. E. coli cells were collected and resuspended in lysis buffer (20 mM Tris, 500 mM NaCl, pH 8.0) containing protease inhibitors, deoxyribonuclease, 10 mM Mg 2+ and lysozyme. Cells were disrupted by one freeze-thaw cycle, and the supernatant was removed after centrifugation at 13,000 rpm for 45 minutes. The cell pellet was then resuspended in lysis buffer containing 0.5% SDOC and homogenized by a bead beater. The lysate was then applied for centrifugation at 13,000 rpm for 45 minutes, and the supernatant was collected for affinity purification. Lipid-added rhavi was eluted using lysis buffer containing 0.5% SDOC and 300 mM Im.

[0329] The protein eluted from the Ni-NTA column was further purified by gel filtration using a superdex 200 column operating on an AKTA purification device. The peak fractions containing the target protein were stored and concentrated. The protein concentration was measured using a BCA protein assay kit from Bio-Rad. The purified protein was aliquoted, rapidly frozen in liquid nitrogen, and stored at -80°C for future use.

[0330] The lipid-added biotin-binding protein produced is schematically shown in Figure 5, and the SDS-PAGE of the purified lipid-added biotin-binding protein is shown in Figure 6.

[0331] Example 3: TLR2 Activity of Lipid-Added Biotin-Binding Protein The TLR2 activity of lipid-added biotin-binding protein was tested in HEK TLR2 cells. HEK TLR2 cells were placed in a 24-well plate at 5 × 10 5 cells / well in a volume of 500 μl. Lipid-added biotin-binding protein was added at different concentrations for stimulation overnight at 37°C. The supernatant was collected on the second day for IL-8 measurement by ELISA. As a control, HEK 293 cells were used to stimulate under the same conditions.

[0332] The TLR2 activity of lipid-added biotin-binding protein was determined. The results show that lipid-added biotin-binding protein induced the production of IL-8 from HEK TLR2 but not from HEK 293 cells (Figure 7).

[0333] Example 4: Non-Hemolytic Mutants and Fusion Proteins of Hla The DNA sequence encoding the wild-type Hla mature polypeptide (amino acids 27 - 319) was cloned from the Staphylococcus aureus genome. All non-hemolytic mutants of Hla were generated by site-directed mutagenesis using quick change. To create the Hla-biotin-binding fusion protein, the DNA sequence encoding wild-type Hla or mutant Hla was inserted following the biotin-binding protein gene, across the linker region. All constructs were cloned into PET21b as described above and transformed into E. coli for expression.

[0334] Non-hemolytic mutants of Hla were produced. Exemplary non-hemolytic variants of Hla are schematically shown in Figure 8. SDS-PAGE of purified wild-type or non-hemolytic variants and fusion proteins of Hla are shown in Figures 9 and 10.

[0335] Example 5: Hemolytic Activity of Wild-Type Hla, Mutant Hla, and Fusion Proteins Using rabbit blood cells, the hemolytic activities of wild-type Hla, mutant Hla, and their fusion proteins with biotin-binding protein were analyzed. Erythrocytes from 250 μl of rabbit blood were precipitated, washed twice with PBS, and then resuspended in 10 ml of PBS. Wild-type Hla, mutant Hla, and the fusion proteins were diluted with PBS at the indicated concentrations and then added into a 96-well plate at 100 μl / well. The blood cells were added into the 96-well plate containing Hla or the fusion proteins at 25 μl / well and then incubated at 37 °C for 30 minutes. After centrifugation at 2000 rpm for 5 minutes, the supernatant was collected and analyzed by an ELISA reader at OD450.

[0336] The hemolytic activities of wild-type Hla, mutant Hla, and their fusion proteins were analyzed. The results demonstrate that the mutant Hla has a much lower hemolytic activity than wild-type Hla (Figure 11). Furthermore, the Hla fusion protein containing biotin-binding protein had an even lower hemolytic activity than the unfused mutant Hla protein (Figure 12).

[0337] Example 6: Stimulatory activity of mutant Hla fusion protein C57 WT macrophage cells were stimulated with a non-hemolytic Hla mutant fusion protein. The cells were seeded into a 24-well plate at 5×10 5 cells / well. The mutant Hla fusion protein was diluted in growth medium and added into the wells at the indicated concentrations for stimulation overnight at 37 °C. On the second day after centrifugation at 2000 rpm for 5 minutes, the supernatant was collected and then analyzed for cytokine secretion by ELISA. The stimulatory activity of the mutant Hla fusion protein was analyzed. The results showed that the mutant Hla fusion protein (rhavi-Hla209) induced the production of multiple inflammatory cytokines, including TNF-α, IL-6, Il-23, IL-1β, and IL-17 (Figure 13).

[0338] Example 7. Lipid-added biotin-binding protein and mutant Hla fusion protein promote immune response against other antigens MAPS-based vaccine constructs were prepared from any one of biotinylated serotype 1 pneumococcal capsular polysaccharide, streptavidin fusion TB antigen, and non-lipid-added streptavidin, lipid-added streptavidin, or rhavi-Hla209. Mice were immunized with different MAPS constructs, the T cell responses against the TB antigen in different immune groups were analyzed, and compared after three immunizations. Briefly, whole blood from different mouse groups was stimulated with purified TB protein in vitro at 37 °C for 6 days, and the cytokine concentrations in the supernatant were detected by ELISA.

[0339] The results showed that the mouse groups that received the MAPS complex containing lipid-added streptavidin or containing rhavi-Hla209 produced better Th17 (IL-17A) and Th1 cell (IFN-γ) responses against the TB antigen (Figure 14). This indicated that lipid-added streptavidin and rhavi-Hla209 could play the role of co-stimulatory factors / adjuvants in MAPS vaccine formation.

[0340] It is understood that the foregoing detailed description and examples are illustrative only and are not to be construed as limitations on the scope of the present invention. Various changes and modifications of the disclosed embodiments that would be apparent to those skilled in the art may be made without departing from the spirit and scope of the present invention. Further, all patents and other publications referred to are hereby expressly incorporated herein by reference for the purpose of, for example, explaining and disclosing the methodologies described in such publications that may be used in connection with the present invention. These publications are provided only with respect to their disclosure prior to the filing date of the present application. In this regard, nothing herein should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or the content of these documents are based on the information available to the applicant and do not constitute any admission as to the date or the accuracy of the content of these documents.

[0341] All patents and other publications referred to in this specification and the examples are hereby expressly incorporated by reference herein for all purposes. These publications are provided only as of their disclosure prior to the filing date of this application. In no event should anything be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or any other reason. All statements as to the date or representation as to the content of these documents are based on the information available to the applicant and do not constitute any admission as to the date or the accuracy of the content of these documents.

[0342] Preferred embodiments are described and illustrated in detail herein, but various modifications, additions, substitutions, and equivalents can be made without departing from the spirit of the invention, and thus it will be apparent to those skilled in the art that these are considered to be within the scope of the invention as defined in the appended claims.

[0343] Furthermore, it will be understood by those skilled in the art that any one of the various embodiments described and illustrated herein can be further modified to incorporate features shown in any of the other embodiments disclosed herein to the extent not already indicated.

[0344] Array information SEQUENCE LISTING <110> THE CHILDREN'S MEDICAL CENTER CORPORATION <120> MODIFIED BIOTIN-BINDING PROTEIN, FUSION PROTEINS THEREOF AND APPLICATIONS <150> US 61 / 609,974 <151> 2012-03-13 <150> US 61 / 608,168 <151> 2012-03-08 <150> US 61 / 484,934 <151> May 11, 2011 <160> 59 <170> PatentIn version 3.5 <210> 1 <211> 135 <212> PRT <213> Rhizobium sp. <400> 1 Phe Asp Ala Ser Asn Phe Lys Asp Phe Ser Ser Ile Ala Ser Ala Ser 1 5 10 15 Ser Ser Trp Gln Asn Gln Ser Gly Ser Thr Met Ile Ile Gln Val Asp 20 25 30 Ser Phe Gly Asn Val Ser Gly Gln Tyr Val Asn Arg Ala Gln Gly Thr 35 40 45 Gly Cys Gln Asn Ser Pro Tyr Pro Leu Thr Gly Arg Val Asn Gly Thr 50 55 60 Phe Ile Ala Phe Ser Val Gly Trp Asn Asn Ser Thr Glu Asn Cys Asn 65 70 75 80 Ser Ala Thr Gly Trp Thr Gly Tyr Ala Gln Val Asn Gly Asn Asn Thr 85 90 95 Glu Ile Val Thr Ser Trp Asn Leu Ala Tyr Glu Gly Gly Ser Gly Pro 100 105 110 Ala Ile Glu Gln Gly Gln Asp Thr Phe Gln Tyr Val Pro Thr Thr Glu 115 120 125 Asn Lys Ser Leu Leu Lys Asp 130 135 <210> 2 <211> 19 <212> PRT <213> Escherichia coli <400> 2 Met Lys Lys Ile Trp Leu Ala Leu Ala Gly Leu Val Leu Ala Phe Ser 1 5 10 15 Ala Ser Ala <210> 3 <211> 17 <212> PRT <213> Escherichia coli <400> 3 Met Lys Lys Val Ala Ala Phe Val Ala Leu Ser Leu Leu Met Ala Gly 1 5 10 15 Cys <210> 4 <211> 179 <212> PRT <213> Rhizobium sp. <400> 4 Met Ile Ile Thr Ser Leu Tyr Ala Thr Phe Gly Thr Ile Ala Asp Gly 1 5 10 15 Arg Arg Thr Ser Gly Gly Lys Thr Met Ile Arg Thr Asn Ala Val Ala 20 25 30 Ala Leu Val Phe Ala Val Ala Thr Ser Ala Leu Ala Phe Asp Ala Ser 35 40 45 Asn Phe Lys Asp Phe Ser Ser Ile Ala Ser Ala Ser Ser Ser Trp Gln 50 55 60 Asn Gln Ser Gly Ser Thr Met Ile Ile Gln Val Asp Ser Phe Gly Asn 65 70 75 80 Val Ser Gly Gln Tyr Val Asn Arg Ala Gln Gly Thr Gly Cys Gln Asn 85 90 95 Ser Pro Tyr Pro Leu Thr Gly Arg Val Asn Gly Thr Phe Ile Ala Phe 100 105 110 Ser Val Gly Trp Asn Asn Ser Thr Glu Asn Cys Asn Ser Ala Thr Gly 115 120 125 Trp Thr Gly Tyr Ala Gln Val Asn Gly Asn Asn Thr Glu Ile Val Thr 130 135 140 Ser Trp Asn Leu Ala Tyr Glu Gly Gly Ser Gly Pro Ala Ile Glu Gln 145 150 155 160 Gly Gln Asp Thr Phe Gln Tyr Val Pro Thr Thr Glu Asn Lys Ser Leu 165 170 175 Leu Lys Asp <210> 5 <211> 44 <212> PRT <213> Rhizobium sp. <400> 5 Met Ile Ile Thr Ser Leu Tyr Ala Thr Phe Gly Thr Ile Ala Asp Gly 1 5 10 15 Arg Arg Thr Ser Gly Gly Lys Thr Met Ile Arg Thr Asn Ala Val Ala 20 25 30 Ala Leu Val Phe Ala Val Ala Thr Ser Ala Leu Ala 35 40 <210> 6 <211> 20 <212> PRT <213> Rhizobium sp. <400> 6 Met Ile Arg Thr Asn Ala Val Ala Ala Leu Val Phe Ala Val Ala Thr 1 5 10 15 Ser Ala Leu Ala 20 <210> 7 <211> 23 <212> PRT <213> Unknown <220> <223> Description of Unknown: Signal peptide <400> 7 Met Ala Pro Phe Glu Pro Leu Ala Ser Gly Ile Leu Leu Leu Leu Trp 1 5 10 15 Leu Ile Ala Pro Ser Arg Ala 20 <210> 8 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 8 Ala Gln Asp Pro 1 <210> 9 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 9 Val Ser Asp Pro 1 <210> 10 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic 6xHis tag <400> 10 His His His His His His 1 5 <210> 11 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 11 Val Asp Lys Leu Ala Ala Ala Leu Glu 1 5 <210> 12 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 12 Gly Gly Gly Gly Ser Ser Ser Val Asp Lys Leu Ala Ala Ala Leu Glu 1 5 10 15 <210> 13 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 13 Val Asp Lys Leu Ala Ala Ala Leu Glu His His His His His 1 5 10 <210> 14 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 14 Gly Gly Gly Gly Ser Ser Ser Val Asp Lys Leu Ala Ala Ala Leu Glu 1 5 10 15 His His His His His His 20 <210> 15 <211> 180 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 15 Met Lys Lys Ile Trp Leu Ala Leu Ala Gly Leu Val Leu Ala Phe Ser 1 5 10 15 Ala Ser Ala Ala Gln Asp Pro Phe Asp Ala Ser Asn Phe Lys Asp Phe 20 25 30 Ser Ser Ile Ala Ser Ala Ser Ser Ser Trp Gln Asn Gln Ser Gly Ser 35 40 45 Thr Met Ile Ile Gln Val Asp Ser Phe Gly Asn Val Ser Gly Gln Tyr 50 55 60 Val Asn Arg Ala Gln Gly Thr Gly Cys Gln Asn Ser Pro Tyr Pro Leu 65 70 75 80 Thr Gly Arg Val Asn Gly Thr Phe Ile Ala Phe Ser Val Gly Trp Asn 85 90 95 Asn Ser Thr Glu Asn Cys Asn Ser Ala Thr Gly Trp Thr Gly Tyr Ala 100 105 110 Gln Val Asn Gly Asn Asn Thr Glu Ile Val Thr Ser Trp Asn Leu Ala 115 120 125 Tyr Glu Gly Gly Ser Gly Pro Ala Ile Glu Gln Gly Gln Asp Thr Phe 130 135 140 Gln Tyr Val Pro Thr Thr Glu Asn Lys Ser Leu Leu Lys Asp Gly Gly 145 150 155 160 Gly Gly Ser Ser Ser Val Asp Lys Leu Ala Ala Ala Leu Glu His His 165 170 175 His His His His 180 <210> 16 <211> 21 <212> PRT <213> Escherichia coli <400> 16 Met Asn Ser Lys Lys Leu Cys Cys Ile Cys Val Leu Phe Ser Leu Leu 1 5 10 15 Ala Gly Cys Ala Ser 20 <210> 17 <211> 19 <212> PRT <213> Escherichia coli <400> 17 Met Arg Tyr Ser Lys Leu Thr Met Leu Ile Pro Cys Ala Leu Leu Leu 1 5 10 15 Ser Ala Cys <210> 18 <211> 24 <212> PRT <213> Escherichia coli <400> 18 Met Phe Val Thr Ser Lys Lys Met Thr Ala Ala Val Leu Ala Ile Thr 1 5 10 15 Leu Ala Met Ser Leu Ser Ala Cys 20 <210> 19 <211> 18 <212> PRT <213> Escherichia coli <400> 19 Met Ile Lys Arg Val Leu Val Val Ser Met Val Gly Leu Ser Leu Val 1 5 10 15 Gly Cys <210> 20 <211> 156 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 20 Met Lys Lys Val Ala Ala Phe Val Ala Leu Ser Leu Leu Met Ala Gly 1 5 10 15 Cys Val Ser Asp Pro Phe Asp Ala Ser Asn Phe Lys Asp Phe Ser Ser 20 25 30 Ile Ala Ser Ala Ser Ser Ser Trp Gln Asn Gln Ser Gly Ser Thr Met 35 40 45 Ile Ile Gln Val Asp Ser Phe Gly Asn Val Ser Gly Gln Tyr Val Asn 50 55 60 Arg Ala Gln Gly Thr Gly Cys Gln Asn Ser Pro Tyr Pro Leu Thr Gly 65 70 75 80 Arg Val Asn Gly Thr Phe Ile Ala Phe Ser Val Gly Trp Asn Asn Ser 85 90 95 Thr Glu Asn Cys Asn Ser Ala Thr Gly Trp Thr Gly Tyr Ala Gln Val 100 105 110 Asn Gly Asn Asn Thr Glu Ile Val Thr Ser Trp Asn Leu Ala Tyr Glu 115 120 125 Gly Gly Ser Gly Pro Ala Ile Glu Gln Gly Gln Asp Thr Phe Gln Tyr 130 135 140 Val Pro Thr Thr Glu Asn Lys Ser Leu Leu Lys Asp 145 150 155 <210> 21 <211> 458 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic polypeptide <400> 21 Met Lys Lys Ile Trp Leu Ala Leu Ala Gly Leu Val Leu Ala Phe Ser 1 5 10 15 Ala Ser Ala Ala Gln Asp Pro Phe Asp Ala Ser Asn Phe Lys Asp Phe 20 25 30 Ser Ser Ile Ala Ser Ala Ser Ser Ser Trp Gln Asn Gln Ser Gly Ser 35 40 45 Thr Met Ile Ile Gln Val Asp Ser Phe Gly Asn Val Ser Gly Gln Tyr 50 55 60 Val Asn Arg Ala Gln Gly Thr Gly Cys Gln Asn Ser Pro Tyr Pro Leu 65 70 75 80 Thr Gly Arg Val Asn Gly Thr Phe Ile Ala Phe Ser Val Gly Trp Asn 85 90 95 Asn Ser Thr Glu Asn Cys Asn Ser Ala Thr Gly Trp Thr Gly Tyr Ala 100 105 110 Gln Val Asn Gly Asn Asn Thr Glu Ile Val Thr Ser Trp Asn Leu Ala 115 120 125 Tyr Glu Gly Gly Ser Gly Pro Ala Ile Glu Gln Gly Gln Asp Thr Phe 130 135 140 Gln Tyr Val Pro Thr Thr Glu Asn Lys Ser Leu Leu Lys Asp Gly Gly 145 150 155 160 Gly Gly Ser Ser Ser Ala Asp Ser Asp Ile Asn Ile Lys Thr Gly Thr 165 170 175 Thr Asp Ile Gly Ser Asn Thr Thr Val Lys Thr Gly Asp Leu Val Thr 180 185 190 Tyr Asp Lys Glu Asn Gly Met His Lys Lys Val Phe Tyr Ser Phe Ile 195 200 205 Asp Asp Lys Asn His Asn Lys Lys Leu Leu Val Ile Arg Thr Lys Gly 210 215 220 Thr Ile Ala Gly Gln Tyr Arg Val Tyr Ser Glu Glu Gly Ala Asn Lys 225 230 235 240 Ser Gly Leu Ala Trp Pro Ser Ala Phe Lys Val Gln Leu Gln Leu Pro 245 250 255 Asp Asn Glu Val Ala Gln Ile Ser Asp Tyr Tyr Pro Arg Asn Ser Ile 260 265 270 Asp Thr Lys Glu Tyr Met Ser Thr Leu Thr Tyr Gly Phe Asn Gly Asn 275 280 285 Val Thr Gly Asp Asp Thr Gly Lys Ile Gly Gly Leu Ile Gly Ala Asn 290 295 300 Val Ser Ile Gly His Thr Leu Lys Tyr Val Gln Pro Asp Phe Lys Thr 305 310 315 320 Ile Leu Glu Ser Pro Thr Asp Lys Lys Val Gly Trp Lys Val Ile Phe 325 330 335 Asn Asn Met Val Asn Gln Asn Trp Gly Pro Tyr Ala Ala Ala Ser Trp 340 345 350 Asn Pro Val Tyr Gly Asn Gln Leu Phe Met Lys Thr Arg Asn Gly Ser 355 360 365 Met Lys Ala Ala Asp Asn Phe Leu Asp Pro Asn Lys Ala Ser Ser Leu 370 375 380 Leu Ser Ser Gly Phe Ser Pro Asp Phe Ala Thr Val Ile Thr Met Asp 385 390 395 400 Arg Lys Ala Ser Lys Gln Gln Thr Asn Ile Asp Val Ile Tyr Glu Arg 405 410 415 Val Arg Asp Asp Tyr Gln Leu His Trp Thr Ser Thr Asn Trp Lys Gly 420 425 430 Thr Asn Thr Lys Asp Lys Trp Ile Asp Arg Ser Ser Glu Arg Tyr Lys 435 440 445 Ile Asp Trp Glu Lys Glu Glu Met Thr Asn 450 455 <210> 22 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 22 Gly Gly Gly Gly Ser Ser Ser 1 5 <210> 23 <211> 293 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 23 Ala Asp Ser Asp Ile Asn Ile Lys Thr Gly Thr Thr Asp Ile Gly Ser 1 5 10 15 Asn Thr Thr Val Lys Thr Gly Asp Leu Val Thr Tyr Asp Lys Glu Asn 20 25 30 Gly Met His Lys Lys Val Phe Tyr Ser Phe Ile Asp Asp Lys Asn His 35 40 45 Asn Lys Lys Leu Leu Val Ile Arg Thr Lys Gly Thr Ile Ala Gly Gln 50 55 60 Tyr Arg Val Tyr Ser Glu Glu Gly Ala Asn Lys Ser Gly Leu Ala Trp 65 70 75 80 Pro Ser Ala Phe Lys Val Gln Leu Gln Leu Pro Asp Asn Glu Val Ala 85 90 95 Gln Ile Ser Asp Tyr Tyr Pro Arg Asn Ser Ile Asp Thr Lys Glu Tyr 100 105 110 Met Ser Thr Leu Thr Tyr Gly Phe Asn Gly Asn Val Thr Gly Asp Asp 115 120 125 Thr Gly Lys Ile Gly Gly Leu Ile Gly Ala Asn Val Ser Ile Gly His 130 135 140 Thr Leu Lys Tyr Val Gln Pro Asp Phe Lys Thr Ile Leu Glu Ser Pro 145 150 155 160 Thr Asp Lys Lys Val Gly Trp Lys Val Ile Phe Asn Asn Met Val Asn 165 170 175 Gln Asn Ala Gly Pro Tyr Asp Arg Asp Ser Trp Asn Pro Val Tyr Gly 180 185 190 Asn Gln Leu Phe Met Lys Thr Arg Asn Gly Ser Met Lys Ala Ala Asp 195 200 205 Asn Phe Leu Asp Pro Asn Lys Ala Ser Ser Leu Leu Ser Ser Gly Phe 210 215 220 Ser Pro Asp Phe Ala Thr Val Ile Thr Met Asp Arg Lys Ala Ser Lys 225 230 235 240 Gln Gln Thr Asn Ile Asp Val Ile Tyr Glu Arg Val Arg Asp Asp Tyr 245 250 255 Gln Leu His Trp Thr Ser Thr Asn Trp Lys Gly Thr Asn Thr Lys Asp 260 265 270 Lys Trp Ile Asp Arg Ser Ser Glu Arg Tyr Lys Ile Asp Trp Glu Lys 275 280 285 Glu Glu Met Thr Asn 290 <210> 24 <211> 293 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 24 Ala Asp Ser Asp Ile Asn Ile Lys Thr Gly Thr Thr Asp Ile Gly Ser 1 5 10 15 Asn Thr Thr Val Lys Thr Gly Asp Leu Val Thr Tyr Asp Lys Glu Asn 20 25 30 Gly Met His Lys Lys Val Phe Tyr Ser Phe Ile Asp Asp Lys Asn His 35 40 45 Asn Lys Lys Leu Leu Val Ile Arg Thr Lys Gly Thr Ile Ala Gly Gln 50 55 60 Tyr Arg Val Tyr Ser Glu Glu Gly Ala Asn Lys Ser Gly Leu Ala Trp 65 70 75 80 Pro Ser Ala Phe Lys Val Gln Leu Gln Leu Pro Asp Asn Glu Val Ala 85 90 95 Gln Ile Ser Asp Tyr Tyr Pro Arg Asn Ser Ile Asp Thr Lys Glu Tyr 100 105 110 Met Ser Thr Leu Thr Tyr Gly Phe Asn Gly Asn Val Thr Gly Asp Asp 115 120 125 Thr Gly Lys Ile Gly Gly Leu Ile Gly Ala Asn Val Ser Ile Gly His 130 135 140 Thr Leu Lys Tyr Val Gln Pro Asp Phe Lys Thr Ile Leu Glu Ser Pro 145 150 155 160 Thr Asp Lys Lys Val Gly Trp Lys Val Ile Phe Asn Asn Met Val Asn 165 170 175 Gln Asn Trp Gly Pro Tyr Asp Arg Asp Ser Ala Asn Pro Val Tyr Gly 180 185 190 Asn Gln Leu Phe Met Lys Thr Arg Asn Gly Ser Met Lys Ala Ala Asp 195 200 205 Asn Phe Leu Asp Pro Asn Lys Ala Ser Ser Leu Leu Ser Ser Gly Phe 210 215 220 Ser Pro Asp Phe Ala Thr Val Ile Thr Met Asp Arg Lys Ala Ser Lys 225 230 235 240 Gln Gln Thr Asn Ile Asp Val Ile Tyr Glu Arg Val Arg Asp Asp Tyr 245 250 255 Gln Leu His Trp Thr Ser Thr Asn Trp Lys Gly Thr Asn Thr Lys Asp 260 265 270 Lys Trp Ile Asp Arg Ser Ser Glu Arg Tyr Lys Ile Asp Trp Glu Lys 275 280 285 Glu Glu Met Thr Asn 290 <210> 25 <211> 293 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 25 Ala Asp Ser Asp Ile Asn Ile Lys Thr Gly Thr Thr Asp Ile Gly Ser 1 5 10 15 Asn Thr Thr Val Lys Thr Gly Asp Leu Val Thr Tyr Asp Lys Glu Asn 20 25 30 Gly Met His Lys Lys Val Phe Tyr Ser Phe Ile Asp Asp Lys Asn His 35 40 45 Asn Lys Lys Leu Leu Val Ile Arg Thr Lys Gly Thr Ile Ala Gly Gln 50 55 60 Tyr Arg Val Tyr Ser Glu Glu Gly Ala Asn Lys Ser Gly Leu Ala Trp 65 70 75 80 Pro Ser Ala Phe Lys Val Gln Leu Gln Leu Pro Asp Asn Glu Val Ala 85 90 95 Gln Ile Ser Asp Tyr Tyr Pro Arg Asn Ser Ile Asp Thr Lys Glu Tyr 100 105 110 Met Ser Thr Leu Thr Tyr Gly Phe Asn Gly Asn Val Thr Gly Asp Asp 115 120 125 Thr Gly Lys Ile Gly Gly Leu Ile Gly Ala Asn Val Ser Ile Gly His 130 135 140 Thr Leu Lys Tyr Val Gln Pro Asp Phe Lys Thr Ile Leu Glu Ser Pro 145 150 155 160 Thr Asp Lys Lys Val Gly Trp Lys Val Ile Phe Asn Asn Met Val Asn 165 170 175 Gln Asn Trp Gly Pro Tyr Ala Ala Ala Ser Trp Asn Pro Val Tyr Gly 180 185 190 Asn Gln Leu Phe Met Lys Thr Arg Asn Gly Ser Met Lys Ala Ala Asp 195 200 205 Asn Phe Leu Asp Pro Asn Lys Ala Ser Ser Leu Leu Ser Ser Gly Phe 210 215 220 Ser Pro Asp Phe Ala Thr Val Ile Thr Met Asp Arg Lys Ala Ser Lys 225 230 235 240 Gln Gln Thr Asn Ile Asp Val Ile Tyr Glu Arg Val Arg Asp Asp Tyr 245 250 255 Gln Leu His Trp Thr Ser Thr Asn Trp Lys Gly Thr Asn Thr Lys Asp 260 265 270 Lys Trp Ile Asp Arg Ser Ser Glu Arg Tyr Lys Ile Asp Trp Glu Lys 275 280 285 Glu Glu Met Thr Asn 290 <210> 26 <211> 473 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 26 Met Lys Lys Ile Trp Leu Ala Leu Ala Gly Leu Val Leu Ala Phe Ser 1 5 10 15 Ala Ser Ala Ala Gln Asp Pro Phe Asp Ala Ser Asn Phe Lys Asp Phe 20 25 30 Ser Ser Ile Ala Ser Ala Ser Ser Ser Trp Gln Asn Gln Ser Gly Ser 35 40 45 Thr Met Ile Ile Gln Val Asp Ser Phe Gly Asn Val Ser Gly Gln Tyr 50 55 60 Val Asn Arg Ala Gln Gly Thr Gly Cys Gln Asn Ser Pro Tyr Pro Leu 65 70 75 80 Thr Gly Arg Val Asn Gly Thr Phe Ile Ala Phe Ser Val Gly Trp Asn 85 90 95 Asn Ser Thr Glu Asn Cys Asn Ser Ala Thr Gly Trp Thr Gly Tyr Ala 100 105 110 Gln Val Asn Gly Asn Asn Thr Glu Ile Val Thr Ser Trp Asn Leu Ala 115 120 125 Tyr Glu Gly Gly Ser Gly Pro Ala Ile Glu Gln Gly Gln Asp Thr Phe 130 135 140 Gln Tyr Val Pro Thr Thr Glu Asn Lys Ser Leu Leu Lys Asp Gly Gly 145 150 155 160 Gly Gly Ser Ser Ser Ala Asp Ser Asp Ile Asn Ile Lys Thr Gly Thr 165 170 175 Thr Asp Ile Gly Ser Asn Thr Thr Val Lys Thr Gly Asp Leu Val Thr 180 185 190 Tyr Asp Lys Glu Asn Gly Met His Lys Lys Val Phe Tyr Ser Phe Ile 195 200 205 Asp Asp Lys Asn His Asn Lys Lys Leu Leu Val Ile Arg Thr Lys Gly 210 215 220 Thr Ile Ala Gly Gln Tyr Arg Val Tyr Ser Glu Glu Gly Ala Asn Lys 225 230 235 240 Ser Gly Leu Ala Trp Pro Ser Ala Phe Lys Val Gln Leu Gln Leu Pro 245 250 255 Asp Asn Glu Val Ala Gln Ile Ser Asp Tyr Tyr Pro Arg Asn Ser Ile 260 265 270 Asp Thr Lys Glu Tyr Met Ser Thr Leu Thr Tyr Gly Phe Asn Gly Asn 275 280 285 Val Thr Gly Asp Asp Thr Gly Lys Ile Gly Gly Leu Ile Gly Ala Asn 290 295 300 Val Ser Ile Gly His Thr Leu Lys Tyr Val Gln Pro Asp Phe Lys Thr 305 310 315 320 Ile Leu Glu Ser Pro Thr Asp Lys Lys Val Gly Trp Lys Val Ile Phe 325 330 335 Asn Asn Met Val Asn Gln Asn Ala Gly Pro Tyr Asp Arg Asp Ser Trp 340 345 350 Asn Pro Val Tyr Gly Asn Gln Leu Phe Met Lys Thr Arg Asn Gly Ser 355 360 365 Met Lys Ala Ala Asp Asn Phe Leu Asp Pro Asn Lys Ala Ser Ser Leu 370 375 380 Leu Ser Ser Gly Phe Ser Pro Asp Phe Ala Thr Val Ile Thr Met Asp 385 390 395 400 Arg Lys Ala Ser Lys Gln Gln Thr Asn Ile Asp Val Ile Tyr Glu Arg 405 410 415 Val Arg Asp Asp Tyr Gln Leu His Trp Thr Ser Thr Asn Trp Lys Gly 420 425 430 Thr Asn Thr Lys Asp Lys Trp Ile Asp Arg Ser Ser Glu Arg Tyr Lys 435 440 445 Ile Asp Trp Glu Lys Glu Glu Met Thr Asn Val Asp Lys Leu Ala Ala 450 455 460 Ala Leu Glu His His His His His His 465 470 <210> 27 <211> 473 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 27 Met Lys Lys Ile Trp Leu Ala Leu Ala Gly Leu Val Leu Ala Phe Ser 1 5 10 15 Ala Ser Ala Ala Gln Asp Pro Phe Asp Ala Ser Asn Phe Lys Asp Phe 20 25 30 Ser Ser Ile Ala Ser Ala Ser Ser Ser Trp Gln Asn Gln Ser Gly Ser 35 40 45 Thr Met Ile Ile Gln Val Asp Ser Phe Gly Asn Val Ser Gly Gln Tyr 50 55 60 Val Asn Arg Ala Gln Gly Thr Gly Cys Gln Asn Ser Pro Tyr Pro Leu 65 70 75 80 Thr Gly Arg Val Asn Gly Thr Phe Ile Ala Phe Ser Val Gly Trp Asn 85 90 95 Asn Ser Thr Glu Asn Cys Asn Ser Ala Thr Gly Trp Thr Gly Tyr Ala 100 105 110 Gln Val Asn Gly Asn Asn Thr Glu Ile Val Thr Ser Trp Asn Leu Ala 115 120 125 Tyr Glu Gly Gly Ser Gly Pro Ala Ile Glu Gln Gly Gln Asp Thr Phe 130 135 140 Gln Tyr Val Pro Thr Thr Glu Asn Lys Ser Leu Leu Lys Asp Gly Gly 145 150 155 160 Gly Gly Ser Ser Ser Ala Asp Ser Asp Ile Asn Ile Lys Thr Gly Thr 165 170 175 Thr Asp Ile Gly Ser Asn Thr Thr Val Lys Thr Gly Asp Leu Val Thr 180 185 190 Tyr Asp Lys Glu Asn Gly Met His Lys Lys Val Phe Tyr Ser Phe Ile 195 200 205 Asp Asp Lys Asn His Asn Lys Lys Leu Leu Val Ile Arg Thr Lys Gly 210 215 220 Thr Ile Ala Gly Gln Tyr Arg Val Tyr Ser Glu Glu Gly Ala Asn Lys 225 230 235 240 Ser Gly Leu Ala Trp Pro Ser Ala Phe Lys Val Gln Leu Gln Leu Pro 245 250 255 Asp Asn Glu Val Ala Gln Ile Ser Asp Tyr Tyr Pro Arg Asn Ser Ile 260 265 270 Asp Thr Lys Glu Tyr Met Ser Thr Leu Thr Tyr Gly Phe Asn Gly Asn 275 280 285 Val Thr Gly Asp Asp Thr Gly Lys Ile Gly Gly Leu Ile Gly Ala Asn 290 295 300 Val Ser Ile Gly His Thr Leu Lys Tyr Val Gln Pro Asp Phe Lys Thr 305 310 315 320 Ile Leu Glu Ser Pro Thr Asp Lys Lys Val Gly Trp Lys Val Ile Phe 325 330 335 Asn Asn Met Val Asn Gln Asn Trp Gly Pro Tyr Asp Arg Asp Ser Ala 340 345 350 Asn Pro Val Tyr Gly Asn Gln Leu Phe Met Lys Thr Arg Asn Gly Ser 355 360 365 Met Lys Ala Ala Asp Asn Phe Leu Asp Pro Asn Lys Ala Ser Ser Leu 370 375 380 Leu Ser Ser Gly Phe Ser Pro Asp Phe Ala Thr Val Ile Thr Met Asp 385 390 395 400 Arg Lys Ala Ser Lys Gln Gln Thr Asn Ile Asp Val Ile Tyr Glu Arg 405 410 415 Val Arg Asp Asp Tyr Gln Leu His Trp Thr Ser Thr Asn Trp Lys Gly 420 425 430 Thr Asn Thr Lys Asp Lys Trp Ile Asp Arg Ser Ser Glu Arg Tyr Lys 435 440 445 Ile Asp Trp Glu Lys Glu Glu Met Thr Asn Val Asp Lys Leu Ala Ala 450 455 460 Ala Leu Glu His His His His His His 465 470 <210> 28 <211> 473 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 28 Met Lys Lys Ile Trp Leu Ala Leu Ala Gly Leu Val Leu Ala Phe Ser 1 5 10 15 Ala Ser Ala Ala Gln Asp Pro Phe Asp Ala Ser Asn Phe Lys Asp Phe 20 25 30 Ser Ser Ile Ala Ser Ala Ser Ser Ser Trp Gln Asn Gln Ser Gly Ser 35 40 45 Thr Met Ile Ile Gln Val Asp Ser Phe Gly Asn Val Ser Gly Gln Tyr 50 55 60 Val Asn Arg Ala Gln Gly Thr Gly Cys Gln Asn Ser Pro Tyr Pro Leu 65 70 75 80 Thr Gly Arg Val Asn Gly Thr Phe Ile Ala Phe Ser Val Gly Trp Asn 85 90 95 Asn Ser Thr Glu Asn Cys Asn Ser Ala Thr Gly Trp Thr Gly Tyr Ala 100 105 110 Gln Val Asn Gly Asn Asn Thr Glu Ile Val Thr Ser Trp Asn Leu Ala 115 120 125 Tyr Glu Gly Gly Ser Gly Pro Ala Ile Glu Gln Gly Gln Asp Thr Phe 130 135 140 Gln Tyr Val Pro Thr Thr Glu Asn Lys Ser Leu Leu Lys Asp Gly Gly 145 150 155 160 Gly Gly Ser Ser Ser Ala Asp Ser Asp Ile Asn Ile Lys Thr Gly Thr 165 170 175 Thr Asp Ile Gly Ser Asn Thr Thr Val Lys Thr Gly Asp Leu Val Thr 180 185 190 Tyr Asp Lys Glu Asn Gly Met His Lys Lys Val Phe Tyr Ser Phe Ile 195 200 205 Asp Asp Lys Asn His Asn Lys Lys Leu Leu Val Ile Arg Thr Lys Gly 210 215 220 Thr Ile Ala Gly Gln Tyr Arg Val Tyr Ser Glu Glu Gly Ala Asn Lys 225 230 235 240 Ser Gly Leu Ala Trp Pro Ser Ala Phe Lys Val Gln Leu Gln Leu Pro 245 250 255 Asp Asn Glu Val Ala Gln Ile Ser Asp Tyr Tyr Pro Arg Asn Ser Ile 260 265 270 Asp Thr Lys Glu Tyr Met Ser Thr Leu Thr Tyr Gly Phe Asn Gly Asn 275 280 285 Val Thr Gly Asp Asp Thr Gly Lys Ile Gly Gly Leu Ile Gly Ala Asn 290 295 300 Val Ser Ile Gly His Thr Leu Lys Tyr Val Gln Pro Asp Phe Lys Thr 305 310 315 320 Ile Leu Glu Ser Pro Thr Asp Lys Lys Val Gly Trp Lys Val Ile Phe 325 330 335 Asn Asn Met Val Asn Gln Asn Trp Gly Pro Tyr Ala Ala Ala Ser Trp 340 345 350 Asn Pro Val Tyr Gly Asn Gln Leu Phe Met Lys Thr Arg Asn Gly Ser 355 360 365 Met Lys Ala Ala Asp Asn Phe Leu Asp Pro Asn Lys Ala Ser Ser Leu 370 375 380 Leu Ser Ser Gly Phe Ser Pro Asp Phe Ala Thr Val Ile Thr Met Asp 385 390 395 400 Arg Lys Ala Ser Lys Gln Gln Thr Asn Ile Asp Val Ile Tyr Glu Arg 405 410 415 Val Arg Asp Asp Tyr Gln Leu His Trp Thr Ser Thr Asn Trp Lys Gly 420 425 430 Thr Asn Thr Lys Asp Lys Trp Ile Asp Arg Ser Ser Glu Arg Tyr Lys 435 440 445 Ile Asp Trp Glu Lys Glu Glu Met Thr Asn Val Asp Lys Leu Ala Ala 450 455 460 Ala Leu Glu His His His His His His 465 470 <210> 29 <211> 20 <212> PRT <213> Chlamydia trachomatis <400> 29 Asn Val Thr Gln Asp Leu Thr Ser Ser Thr Ala Lys Leu Glu Cys Thr 1 5 10 15 Gln Asp Leu Ile 20 <210> 30 <211> 20 <212> PRT <213> Chlamydia trachomatis <400> 30 Ala Lys Leu Glu Cys Thr Gln Asp Leu Ile Ala Gln Gly Lys Leu Ile 1 5 10 15 Val Thr Asn Pro 20 <210> 31 <211> 9 <212> PRT <213> Chlamydia trachomatis <400> 31 Ser Asn Leu Lys Arg Met Gln Lys Ile 1 5 <210> 32 <211> 9 <212> PRT <213> Chlamydia trachomatis <400> 32 Ala Ala Leu Tyr Ser Thr Glu Asp Leu 1 5 <210> 33 <211> 9 <212> PRT <213> Chlamydia trachomatis <400> 33 Phe Gln Glu Lys Asp Ala Asp Thr Leu 1 5 <210> 34 <211> 9 <212> PRT <213> Chlamydia trachomatis <400> 34 Gln Ser Val Asn Glu Leu Val Tyr Val 1 5 <210> 35 <211> 9 <212> PRT <213> Chlamydia trachomatis <400> 35 Leu Glu Phe Ala Ser Cys Ser Ser Leu 1 5 <210> 36 <211> 9 <212> PRT <213> Chlamydia trachomatis <400> 36 Ser Gln Ala Glu Gly Gln Tyr Arg Leu 1 5 <210> 37 <211> 9 <212> PRT <213> Chlamydia trachomatis <400> 37 Gly Gln Ser Val Asn Glu Leu Val Tyr 1 5 <210> 38 <211> 9 <212> PRT <213> Chlamydia trachomatis <400> 38 Gln Ala Val Leu Leu Leu Asp Gln Ile 1 5 <210> 39 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (2)..(2) <223> Arg or Leu <220> <221> MOD_RES <222> (4)..(4) <223> Ser or Thr <220> <221> MOD_RES <222> (6)..(6) <223> Tyr or Trp <400> 39 Asp Xaa Ala Xaa Pro Xaa 1 5 <210> 40 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (3)..(3) <223> Arg or Leu <220> <221> MOD_RES <222> (5)..(5) <223> Ser or Thr <220> <221> MOD_RES <222> (7)..(7) <223> Tyr or Trp <400> 40 Cys Asp Xaa Ala Xaa Pro Xaa Cys Gly 1 5 <210> 41 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 41 Gly Ser Pro Gly Ile Ser Gly Gly Gly Gly Gly Ile Leu Glu 1 5 10 <210> 42 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (3)..(3) <223> Any amino acid <400> 42 Leu Pro Xaa Thr Gly 1 5 <210> 43 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 43 Pro Glu Pro 1 <210> 44 <211> 24 <212> PRT <213> Listeria monocytogenes <400> 44 Met Lys Lys Ile Met Leu Val Ile Thr Leu Ile Leu Val Ser Pro Ile 1 5 10 15 Ala Gln Gln Thr Glu Ala Lys Asp 20 <210> 45 <211> 29 <212> PRT <213> Lactococcus lactis <400> 45 Met Lys Lys Lys Ile Ile Ser Ala Ile Leu Met Ser Thr Val Ile Leu 1 5 10 15 Ser Ala Ala Ala Pro Leu Ser Gly Val Tyr Ala Asp Thr 20 25 <210> 46 <211> 31 <212> PRT <213> Bacillus anthracis <400> 46 Met Lys Lys Arg Lys Val Leu Ile Pro Leu Met Ala Leu Ser Thr Ile 1 5 10 15 Leu Val Ser Ser Thr Gly Asn Leu Glu Val Ile Gln Ala Glu Val 20 25 30 <210> 47 <211> 29 <212> PRT <213> Listeria monocytogenes <400> 47 Met Asn Met Lys Lys Ala Thr Ile Ala Ala Thr Ala Gly Ile Ala Val 1 5 10 15 Thr Ala Phe Ala Ala Pro Thr Ile Ala Ser Ala Ser Thr 20 25 <210> 48 <211> 54 <212> PRT <213> Listeria monocytogenes <400> 48 Met Gln Lys Thr Arg Lys Glu Arg Ile Leu Glu Ala Leu Gln Glu Glu 1 5 10 15 Lys Lys Asn Lys Lys Ser Lys Lys Phe Lys Thr Gly Ala Thr Ile Ala 20 25 30 Gly Val Thr Ala Ile Ala Thr Ser Ile Thr Val Pro Gly Ile Glu Val 35 40 45 Ile Val Ser Ala Asp Glu 50 <210> 49 <211> 28 <212> PRT <213> Bacillus anthracis <400> 49 Met Lys Lys Leu Lys Met Ala Ser Cys Ala Leu Val Ala Gly Leu Met 1 5 10 15 Phe Ser Gly Leu Thr Pro Asn Ala Phe Ala Glu Asp 20 25 <210> 50 <211> 31 <212> PRT <213> Staphylococcus aureus <400> 50 Met Ala Lys Lys Phe Asn Tyr Lys Leu Pro Ser Met Val Ala Leu Thr 1 5 10 15 Leu Val Gly Ser Ala Val Thr Ala His Gln Val Gln Ala Ala Glu 20 25 30 <210> 51 <211> 59 <212> PRT <213> Listeria monocytogenes <400> 51 Met Thr Asp Lys Lys Ser Glu Asn Gln Thr Glu Lys Thr Glu Thr Lys 1 5 10 15 Glu Asn Lys Gly Met Thr Arg Arg Glu Met Leu Lys Leu Ser Ala Val 20 25 30 Ala Gly Thr Gly Ile Ala Val Gly Ala Thr Gly Leu Gly Thr Ile Leu 35 40 45 Asn Val Val Asp Gln Val Asp Lys Ala Leu Thr 50 55 <210> 52 <211> 53 <212> PRT <213> Bacillus subtillis <400> 52 Met Ala Tyr Asp Ser Arg Phe Asp Glu Trp Val Gln Lys Leu Lys Glu 1 5 10 15 Glu Ser Phe Gln Asn Asn Thr Phe Asp Arg Arg Lys Phe Ile Gln Gly 20 25 30 Ala Gly Lys Ile Ala Gly Leu Gly Leu Gly Leu Thr Ile Ala Gln Ser 35 40 45 Val Gly Ala Phe Gly 50 <210> 53 <211> 471 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 53 Met Lys Lys Val Ala Ala Phe Val Ala Leu Ser Leu Leu Met Ala Gly 1 5 10 15 Cys Val Ser Pro Asp Phe Asp Ala Ser Asn Phe Lys Asp Phe Ser Ser 20 25 30 Ile Ala Ser Ala Ser Ser Ser Trp Gln Asn Gln Ser Gly Ser Thr Met 35 40 45 Ile Ile Gln Val Asp Ser Phe Gly Asn Val Ser Gly Gln Tyr Val Asn 50 55 60 Arg Ala Gln Gly Thr Gly Cys Gln Asn Ser Pro Tyr Pro Leu Thr Gly 65 70 75 80 Arg Val Asn Gly Thr Phe Ile Ala Phe Ser Val Gly Trp Asn Asn Ser 85 90 95 Thr Glu Asn Cys Asn Ser Ala Thr Gly Trp Thr Gly Tyr Ala Gln Val 100 105 110 Asn Gly Asn Asn Thr Glu Ile Val Thr Ser Trp Asn Leu Ala Tyr Glu 115 120 125 Gly Gly Ser Gly Pro Ala Ile Glu Gln Gly Gln Asp Thr Phe Gln Tyr 130 135 140 Val Pro Thr Thr Glu Asn Lys Ser Leu Leu Lys Asp Gly Gly Gly Gly 145 150 155 160 Ser Ser Ser Ala Asp Ser Asp Ile Asn Ile Lys Thr Gly Thr Thr Asp 165 170 175 Ile Gly Ser Asn Thr Thr Val Lys Thr Gly Asp Leu Val Thr Tyr Asp 180 185 190 Lys Glu Asn Gly Met His Lys Lys Val Phe Tyr Ser Phe Ile Asp Asp 195 200 205 Lys Asn His Asn Lys Lys Leu Leu Val Ile Arg Thr Lys Gly Thr Ile 210 215 220 Ala Gly Gln Tyr Arg Val Tyr Ser Glu Glu Gly Ala Asn Lys Ser Gly 225 230 235 240 Leu Ala Trp Pro Ser Ala Phe Lys Val Gln Leu Gln Leu Pro Asp Asn 245 250 255 Glu Val Ala Gln Ile Ser Asp Tyr Tyr Pro Arg Asn Ser Ile Asp Thr 260 265 270 Lys Glu Tyr Met Ser Thr Leu Thr Tyr Gly Phe Asn Gly Asn Val Thr 275 280 285 Gly Asp Asp Thr Gly Lys Ile Gly Gly Leu Ile Gly Ala Asn Val Ser 290 295 300 Ile Gly His Thr Leu Lys Tyr Val Gln Pro Asp Phe Lys Thr Ile Leu 305 310 315 320 Glu Ser Pro Thr Asp Lys Lys Val Gly Trp Lys Val Ile Phe Asn Asn 325 330 335 Met Val Asn Gln Asn Ala Gly Pro Tyr Asp Arg Asp Ser Trp Asn Pro 340 345 350 Val Tyr Gly Asn Gln Leu Phe Met Lys Thr Arg Asn Gly Ser Met Lys 355 360 365 Ala Ala Asp Asn Phe Leu Asp Pro Asn Lys Ala Ser Ser Leu Leu Ser 370 375 380 Ser Gly Phe Ser Pro Asp Phe Ala Thr Val Ile Thr Met Asp Arg Lys 385 390 395 400 Ala Ser Lys Gln Gln Thr Asn Ile Asp Val Ile Tyr Glu Arg Val Arg 405 410 415 Asp Asp Tyr Gln Leu His Trp Thr Ser Thr Asn Trp Lys Gly Thr Asn 420 425 430 Thr Lys Asp Lys Trp Ile Asp Arg Ser Ser Glu Arg Tyr Lys Ile Asp 435 440 445 Trp Glu Lys Glu Glu Met Thr Asn Val Asp Lys Leu Ala Ala Ala Leu 450 455 460 Glu His His His His His His 465 470 <210> 54 <211> 471 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 54 Met Lys Lys Val Ala Ala Phe Val Ala Leu Ser Leu Leu Met Ala Gly 1 5 10 15 Cys Val Ser Pro Asp Phe Asp Ala Ser Asn Phe Lys Asp Phe Ser Ser 20 25 30 Ile Ala Ser Ala Ser Ser Ser Trp Gln Asn Gln Ser Gly Ser Thr Met 35 40 45 Ile Ile Gln Val Asp Ser Phe Gly Asn Val Ser Gly Gln Tyr Val Asn 50 55 60 Arg Ala Gln Gly Thr Gly Cys Gln Asn Ser Pro Tyr Pro Leu Thr Gly 65 70 75 80 Arg Val Asn Gly Thr Phe Ile Ala Phe Ser Val Gly Trp Asn Asn Ser 85 90 95 Thr Glu Asn Cys Asn Ser Ala Thr Gly Trp Thr Gly Tyr Ala Gln Val 100 105 110 Asn Gly Asn Asn Thr Glu Ile Val Thr Ser Trp Asn Leu Ala Tyr Glu 115 120 125 Gly Gly Ser Gly Pro Ala Ile Glu Gln Gly Gln Asp Thr Phe Gln Tyr 130 135 140 Val Pro Thr Thr Glu Asn Lys Ser Leu Leu Lys Asp Gly Gly Gly Gly 145 150 155 160 Ser Ser Ser Ala Asp Ser Asp Ile Asn Ile Lys Thr Gly Thr Thr Asp 165 170 175 Ile Gly Ser Asn Thr Thr Val Lys Thr Gly Asp Leu Val Thr Tyr Asp 180 185 190 Lys Glu Asn Gly Met His Lys Lys Val Phe Tyr Ser Phe Ile Asp Asp 195 200 205 Lys Asn His Asn Lys Lys Leu Leu Val Ile Arg Thr Lys Gly Thr Ile 210 215 220 Ala Gly Gln Tyr Arg Val Tyr Ser Glu Glu Gly Ala Asn Lys Ser Gly 225 230 235 240 Leu Ala Trp Pro Ser Ala Phe Lys Val Gln Leu Gln Leu Pro Asp Asn 245 250 255 Glu Val Ala Gln Ile Ser Asp Tyr Tyr Pro Arg Asn Ser Ile Asp Thr 260 265 270 Lys Glu Tyr Met Ser Thr Leu Thr Tyr Gly Phe Asn Gly Asn Val Thr 275 280 285 Gly Asp Asp Thr Gly Lys Ile Gly Gly Leu Ile Gly Ala Asn Val Ser 290 295 300 Ile Gly His Thr Leu Lys Tyr Val Gln Pro Asp Phe Lys Thr Ile Leu 305 310 315 320 Glu Ser Pro Thr Asp Lys Lys Val Gly Trp Lys Val Ile Phe Asn Asn 325 330 335 Met Val Asn Gln Asn Trp Gly Pro Tyr Asp Arg Asp Ser Ala Asn Pro 340 345 350 Val Tyr Gly Asn Gln Leu Phe Met Lys Thr Arg Asn Gly Ser Met Lys 355 360 365 Ala Ala Asp Asn Phe Leu Asp Pro Asn Lys Ala Ser Ser Leu Leu Ser 370 375 380 Ser Gly Phe Ser Pro Asp Phe Ala Thr Val Ile Thr Met Asp Arg Lys 385 390 395 400 Ala Ser Lys Gln Gln Thr Asn Ile Asp Val Ile Tyr Glu Arg Val Arg 405 410 415 Asp Asp Tyr Gln Leu His Trp Thr Ser Thr Asn Trp Lys Gly Thr Asn 420 425 430 Thr Lys Asp Lys Trp Ile Asp Arg Ser Ser Glu Arg Tyr Lys Ile Asp 435 440 445 Trp Glu Lys Glu Glu Met Thr Asn Val Asp Lys Leu Ala Ala Ala Leu 450 455 460 Glu His His His His His His 465 470 <210> 55 <211> 471 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 55 Met Lys Lys Val Ala Ala Phe Val Ala Leu Ser Leu Leu Met Ala Gly 1 5 10 15 Cys Val Ser Pro Asp Phe Asp Ala Ser Asn Phe Lys Asp Phe Ser Ser 20 25 30 Ile Ala Ser Ala Ser Ser Ser Trp Gln Asn Gln Ser Gly Ser Thr Met 35 40 45 Ile Ile Gln Val Asp Ser Phe Gly Asn Val Ser Gly Gln Tyr Val Asn 50 55 60 Arg Ala Gln Gly Thr Gly Cys Gln Asn Ser Pro Tyr Pro Leu Thr Gly 65 70 75 80 Arg Val Asn Gly Thr Phe Ile Ala Phe Ser Val Gly Trp Asn Asn Ser 85 90 95 Thr Glu Asn Cys Asn Ser Ala Thr Gly Trp Thr Gly Tyr Ala Gln Val 100 105 110 Asn Gly Asn Asn Thr Glu Ile Val Thr Ser Trp Asn Leu Ala Tyr Glu 115 120 125 Gly Gly Ser Gly Pro Ala Ile Glu Gln Gly Gln Asp Thr Phe Gln Tyr 130 135 140 Val Pro Thr Thr Glu Asn Lys Ser Leu Leu Lys Asp Gly Gly Gly Gly 145 150 155 160 Ser Ser Ser Ala Asp Ser Asp Ile Asn Ile Lys Thr Gly Thr Thr Asp 165 170 175 Ile Gly Ser Asn Thr Thr Val Lys Thr Gly Asp Leu Val Thr Tyr Asp 180 185 190 Lys Glu Asn Gly Met His Lys Lys Val Phe Tyr Ser Phe Ile Asp Asp 195 200 205 Lys Asn His Asn Lys Lys Leu Leu Val Ile Arg Thr Lys Gly Thr Ile 210 215 220 Ala Gly Gln Tyr Arg Val Tyr Ser Glu Glu Gly Ala Asn Lys Ser Gly 225 230 235 240 Leu Ala Trp Pro Ser Ala Phe Lys Val Gln Leu Gln Leu Pro Asp Asn 245 250 255 Glu Val Ala Gln Ile Ser Asp Tyr Tyr Pro Arg Asn Ser Ile Asp Thr 260 265 270 Lys Glu Tyr Met Ser Thr Leu Thr Tyr Gly Phe Asn Gly Asn Val Thr 275 280 285 Gly Asp Asp Thr Gly Lys Ile Gly Gly Leu Ile Gly Ala Asn Val Ser 290 295 300 Ile Gly His Thr Leu Lys Tyr Val Gln Pro Asp Phe Lys Thr Ile Leu 305 310 315 320 Glu Ser Pro Thr Asp Lys Lys Val Gly Trp Lys Val Ile Phe Asn Asn 325 330 335 Met Val Asn Gln Asn Trp Gly Pro Tyr Ala Ala Ala Ser Trp Asn Pro 340 345 350 Val Tyr Gly Asn Gln Leu Phe Met Lys Thr Arg Asn Gly Ser Met Lys 355 360 365 Ala Ala Asp Asn Phe Leu Asp Pro Asn Lys Ala Ser Ser Leu Leu Ser 370 375 380 Ser Gly Phe Ser Pro Asp Phe Ala Thr Val Ile Thr Met Asp Arg Lys 385 390 395 400 Ala Ser Lys Gln Gln Thr Asn Ile Asp Val Ile Tyr Glu Arg Val Arg 405 410 415 Asp Asp Tyr Gln Leu His Trp Thr Ser Thr Asn Trp Lys Gly Thr Asn 420 425 430 Thr Lys Asp Lys Trp Ile Asp Arg Ser Ser Glu Arg Tyr Lys Ile Asp 435 440 445 Trp Glu Lys Glu Glu Met Thr Asn Val Asp Lys Leu Ala Ala Ala Leu 450 455 460 Glu His His His His His His 465 470 <210> 56 <211> 23 <212> PRT <213> Unknown <220> <223> Description of Unknown: Signal peptide <400> 56 Met Lys Lys Ile Trp Leu Ala Leu Ala Gly Leu Val Leu Ala Phe Ser 1 5 10 15 Ala Ser Ala Ala Gln Asp Pro 20 <210> 57 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 57 Leu Glu His His His His His His 1 5 <210> 58 <211> 21 <212> PRT <213> Unknown <220> <223> Description of Unknown: Signal peptide <400> 58 Met Lys Lys Val Ala Ala Phe Val Ala Leu Ser Leu Leu Met Ala Gly 1 5 10 15 Cys Val Ser Asp Pro 20 <210> 59 <211> 293 <212> PRT <213> Staphylococcus aureus <400> 59 Ala Asp Ser Asp Ile Asn Ile Lys Thr Gly Thr Thr Asp Ile Gly Ser 1 5 10 15 Asn Thr Thr Val Lys Thr Gly Asp Leu Val Thr Tyr Asp Lys Glu Asn 20 25 30 Gly Met His Lys Lys Val Phe Tyr Ser Phe Ile Asp Asp Lys Asn His 35 40 45 Asn Lys Lys Leu Leu Val Ile Arg Thr Lys Gly Thr Ile Ala Gly Gln 50 55 60 Tyr Arg Val Tyr Ser Glu Glu Gly Ala Asn Lys Ser Gly Leu Ala Trp 65 70 75 80 Pro Ser Ala Phe Lys Val Gln Leu Gln Leu Pro Asp Asn Glu Val Ala 85 90 95 Gln Ile Ser Asp Tyr Tyr Pro Arg Asn Ser Ile Asp Thr Lys Glu Tyr 100 105 110 Met Ser Thr Leu Thr Tyr Gly Phe Asn Gly Asn Val Thr Gly Asp Asp 115 120 125 Thr Gly Lys Ile Gly Gly Leu Ile Gly Ala Asn Val Ser Ile Gly His 130 135 140 Thr Leu Lys Tyr Val Gln Pro Asp Phe Lys Thr Ile Leu Glu Ser Pro 145 150 155 160 Thr Asp Lys Lys Val Gly Trp Lys Val Ile Phe Asn Asn Met Val Asn 165 170 175 Gln Asn Trp Gly Pro Tyr Asp Arg Asp Ser Trp Asn Pro Val Tyr Gly 180 185 190 Asn Gln Leu Phe Met Lys Thr Arg Asn Gly Ser Met Lys Ala Ala Asp 195 200 205 Asn Phe Leu Asp Pro Asn Lys Ala Ser Ser Leu Leu Ser Ser Gly Phe 210 215 220 Ser Pro Asp Phe Ala Thr Val Ile Thr Met Asp Arg Lys Ala Ser Lys 225 230 235 240 Gln Gln Thr Asn Ile Asp Val Ile Tyr Glu Arg Val Arg Asp Asp Tyr 245 250 255 Gln Leu His Trp Thr Ser Thr Asn Trp Lys Gly Thr Asn Thr Lys Asp 260 265 270 Lys Trp Ile Asp Arg Ser Ser Glu Arg Tyr Lys Ile Asp Trp Glu Lys 275 280 285 Glu Glu Met Thr Asn 290

Claims

1. 1. A lipidated biotin-binding protein comprising a biotin-binding protein comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1 and a lipidation sequence fused to said biotin-binding protein, wherein said biotin-binding protein does not comprise amino acids 1-44 of wild-type rhizavidin protein.

2. 2. The lipidated biotin-binding protein of claim 1, comprising a biotin-binding protein comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1 and a lipidation sequence fused to said biotin-binding protein.

3. The lipidated biotin-binding protein of any one of claims 1 to 2, wherein the lipidation sequence is fused to the biotin-binding protein by a peptide linker.

4. 4. The lipidated biotin-binding protein of claim 3, wherein the peptide linker comprises the amino acid sequence of SEQ ID NO:9 or a sequence having at least 90% sequence identity to SEQ ID NO:

9.

5. The lipidated biotin-binding protein of any one of claims 1 to 4, wherein the lipidation sequence has an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:

19.

6. 6. The lipidated biotin-binding protein according to any one of claims 1 to 5, comprising the amino acid sequence of SEQ ID NO:

20.

7. The lipidated biotin-binding protein of any one of claims 1 to 6, wherein the lipidation sequence is a ligand for a Toll-like receptor (TLR).

8. 8. The lipidated biotin-binding protein of claim 7, wherein the ligand for TLR is selected from the group consisting of TLR-2, TLR-4, TLR-5, TLR-9, and TLR-3.

9. 9. The lipidated biotin-binding protein of any one of claims 1 to 8, wherein the lipidation sequence is at the N-terminus of the biotin-binding protein comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

1.

10. A vaccine composition comprising the lipidated biotin-binding protein according to any one of claims 1 to 9.

11. The vaccine composition of claim 10, further comprising a pharma- ceutically acceptable carrier.

12. 10. Use of a lipidated biotin-binding protein according to any of claims 1 to 9 in the manufacture of a medicament for inducing an immune response in a subject, wherein the immune response is against an antigenic polysaccharide and / or an antigenic polypeptide or peptide.

13. The use according to claim 12, wherein the immune response is an antibody or B cell response.

14. The use according to claim 12, wherein the immune response is a CD4+ T cell response (including a Th1, Th2 or Th17 response) and / or a CD8+ T cell response.

15. 15. The use according to claim 14, wherein the CD4+ T cell response comprises Th1, Th2 and Th17 responses.

16. The immune response an antibody response; or B Cell and T Cell Responses The use according to claim 12,

17. A pharmaceutical composition comprising the lipidated biotin-binding protein of any one of claims 1 to 9 and a pharma- ceutically acceptable carrier.

18. A cell comprising the lipidated biotin-binding protein according to any one of claims 1 to 9.

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