Staphylococcal protein variants and truncates

Abstract

This application discloses novel polypeptides, polynucleotides, expression vectors and novel immunogenic compositions derived from Staphylococcus aureus, in particular novel polypeptides, polynucleotides, expression vectors and compositions derived from / related to SpA, Hla, Aur and LukE polypeptides. Also disclosed is a method of immunization using polypeptides, polynucleotides, expression vectors, and immunogenic compositions.

Description

Technical Field

[0001] The present invention relates to the field of immunization techniques including vaccine technology. In particular, the present invention relates to novel variants of staphylococcal proteins, as well as compositions containing staphylococcal proteins. The present invention also relates to vectors and transformed cells and viruses, and compositions containing them.

Background Art

[0002] Staphylococcus aureus is a Gram-positive opportunistic pathogenic bacterium that is a major clinical problem. In particular, multi-drug resistant methicillin-resistant Staphylococcus aureus (MRSA) strains that cause severe nosocomial and community infections are a major global health problem. There is an urgent need for alternatives to antibiotics and vaccines for the treatment of Staphylococcus aureus infections.

[0003] Despite years of research, vaccines against Staphylococcus aureus have not yet passed the tests in clinical trials. Conventional strategies for developing vaccines that induce opsonizing antibodies against Staphylococcus aureus surface proteins and result in antibody-mediated clearance of the bacteria have not been successful. Another more promising strategy is to develop vaccines aimed at neutralizing one or more of the pathogenic factors of Staphylococcus aureus. The pathogenic factors of Staphylococcus aureus include several different proteins with toxic and immune evasion functions. A brief description of specific Staphylococcus aureus pathogenic factors and other Staphylococcus aureus proteins of the present disclosure is shown below.

[0004] Immunoglobulin G-binding protein A Staphylococcal protein A (SpA) is a protein containing 4 - 5 homologous immunoglobulin - binding domains (E, D, A, B, C), each of which binds to the constant region of IgG (Fcγ) and the Ig fragment (Fab) involved in antigen binding. The SpA Fc - binding site can also bind to von Willebrand factor (vWF). SpA induces immune evasion by several mechanisms. Binding of SpA to the Fcγ domain interferes with the antibacterial functions of IgG, including complement binding and opsonophagocytosis. Binding to Fab provides SpA with a potent B - cell superantigen function, and secreted SpA cross - links V H 3 - containing B - cell receptors, causing the secretion of all V H 3 antibodies regardless of their antigen specificity. As a result, antibody - mediated defensive immunity against Staphylococcus aureus is substantially impaired.

[0005] Alpha - hemolysin Alpha - hemolysin (Hla), also known as alpha - toxin, is a member of the beta - barrel toxin family and is a major cytotoxic agent of Staphylococcus aureus. Hla is a monomer, and 7 copies of Hla self - assemble to form a heptameric pore in the cell membrane, which allows the exchange of monovalent ions and leads to DNA fragmentation and apoptosis. The heptameric pore is composed of 3 structural regions: the cap, the rim, and the stem. Hla monomers can also oligomerize to form larger Ca 2+ permissive pores, which induce a large amount of necrosis.

[0006] HtrA2 HtrA-like proteases are involved in the pathogenicity of both Gram-positive and Gram-negative bacteria. They are known to play roles in stress tolerance and survival. In Streptococcus pyogenes, HtrA has been reported to intervene in the processing of extracellular pathogenic factors and play a role in the regulation of hemolytic activity. Two putative HtrA-like proteases, HtrA1 and HtrA2, are encoded by Staphylococcus aureus. Staphylococcus aureus HtrA2 is classified as a transmembrane protein and contains domains predicted to be an active serine protease with Asp / His / Ser catalytic triad residues.

[0007] LukE The LukE protein is one of two components of the lukED leucocidin. In addition to inducing cell death in leukocytes, the pore-forming toxin LukED can lyse erythrocytes. Target cells of lukED (in humans and mice) include neutrophils, monocytes, macrophages, dendritic cells, T cells, erythrocytes, and NK cells. LukE recognizes the receptors CCR5, CXCR1, and CXCR2, as well as the erythrocyte receptor DARC. After recognition of cell surface receptors by LukE, pores are not formed by LukE alone, but only by the heterodimer of LukE and LukD.

[0008] Aureolysin Aureolysin (Aur) is a zinc metalloprotease. It is a Staphylococcus aureus pathogenicity factor with multiple roles in both immune evasion and toxicity. Aur activates glutamyl endopeptidase, an important pathogenicity factor of Staphylococcus aureus that cleaves specific human inflammatory regulators and immune components and inhibits the activation of complement system components. Aur directly cleaves complement C3 and inhibits the deposition of C3b on the bacterial surface and the release of the chemoattractant C5a. Furthermore, Aur activates prothrombin in human plasma and induces staphylocoagulation.

[0009] SAR2753 Lipase 1 / glycerol ester hydrolase 1 (SAR2753) converts triacylglycerol + H 2 O to diacylglycerol + fatty acid + H + O. SAR2753 can play a role in toxicity in Staphylococcus aureus by degrading antibacterial lipids produced by the host during infection.

[0010] SAR2723 N-acetylmuramoyl-L-alanine amidase (SAR2723) is a protein containing two amidase domains, LYZ2 / FlgJ and CHAP, both of which have a major function in peptidoglycan hydrolysis. Consistently, many proteins containing these domains are involved in cell wall metabolism and biosynthesis.

[0011] EsaA The EsaA protein of Staphylococcus aureus is a basic protein of a specialized type VII protein secretion system (T7SS) present in many Gram-positive bacteria. It is a transmembrane protein with six transmembrane domains. The Staphylococcus aureus T7SS, called T7b, consists of six essential proteins, EsxA, EssC, EsaA, EssA, Essb, and EsaB. T7b mediates the secretion of different proteins, including the toxins EsaC and EsaD that contribute to the pathogenicity of Staphylococcus aureus. Furthermore, T7b-mediated secretion contributes to the production or suppression of specific cytokines during host infection, thereby enabling Staphylococcus aureus to manipulate the immune response.

Summary of the Invention

Problems to be Solved by the Invention

[0012] An object of embodiments of the present invention is to provide an immunogen and an immunogenic composition useful for vaccination against SA infection. A further object of the present invention is to provide a method of immunization / vaccination against SA infection using these immunogens and immunogenic compositions.

Means for Solving the Problem

[0013] The inventors have conducted an intensive research program to identify novel pharmaceutically acceptable immunogens derived from Staphylococcus aureus antigens and considered suitable for immunization / vaccination purposes. Part of the research has focused on reducing or eliminating the risk of administering immunogens that may cause harmful effects similar to those shown by the wild-type antigens from which the immunogens are derived. The inventors have also identified optimized vaccine compositions that are thought to induce useful immune responses against various distinct Staphylococcus aureus antigens in order to effectively block various effector molecules of Staphylococcus aureus.

[0014] Accordingly, in a first aspect, the present invention provides i. a-e: a. a sequence that is at least 85% identical to the amino acid sequence of immunoglobulin binding domain (IgBD) E (SEQ ID NO: 16, residues 1-56), b. a sequence that is at least 85% identical to the amino acid sequence of IgBD D (SEQ ID NO: 16, residues 62-117), c. a sequence that is at least 85% identical to the amino acid sequence of IgBD A (SEQ ID NO: 16, residues 120-175), d. a sequence that is at least 85% identical to the amino acid sequence of IgBD B (SEQ ID NO: 16, residues 178-233), e. a sequence that is at least 85% identical to the amino acid sequence of IgBD C (SEQ ID NO: 16, residues 236-291) A variant of the amino acid sequence of immunoglobulin G-binding protein A (SpA) comprising at least one of the above, and ii. one or more first mutations in each of at least one of a-e, which disrupt binding at the Fc binding site and occur at positions corresponding to the amino acid positions in SEQ ID NO: 16 selected from positions 3, 8, 9, 64, 69, 70, 122, 127, 128, 180, 185, 186, 238, 243, 244, and One or more second mutations in each of at least one of iii.a - e, which disrupt binding at the Fab binding site and, if applicable, occur at positions corresponding to the amino acid positions in SEQ ID NO: 16 selected from positions 34, 35, 37, 38, 41, 95, 96, 98, 99, 102, 153, 154, 156, 157, 160, 211, 212, 214, 215, 218, 269, 270, 272, 273, and 276, one or more second mutations An immunogenic polypeptide consisting of or comprising these, iv. Optionally, none of at least one of a - e, if applicable, contains a mutation that disrupts binding at the Fc binding site at positions corresponding to both of amino acid positions 7 + 8, 68 + 69, 126 + 127, 184 + 185, and 242 + 243 of SEQ ID NO: 16, v. None of at least one of a - e, if applicable, contains a mutation that disrupts binding at the Fab binding site at positions corresponding to both of amino acid positions 34 + 35 and 95 + 96 and 153 + 154 and 211 + 212, and 269 + 270 of SEQ ID NO: 16, Relates to an immunogenic polypeptide that the polypeptide cannot bind to human IgG and human von Willebrand factor, and the substitution is preferably a non - conservative substitution.

[0015] In a second aspect, the present invention relates to i. Having at least 85% sequence identity with the sequence of SEQ ID NO: 2, ii. An immunogenic polypeptide consisting of or comprising one or more amino acid deletions and / or substitutions in the first 12 consecutive N - terminal amino acid residues of the amino acid sequence of mature Hla, wherein the first 12 consecutive N - terminal amino acid residues correspond to positions 1 - 12 of SEQ ID NO: 2, a variant of the amino acid sequence of alpha - hemolysin (Hla), Relates to an immunogenic polypeptide that the polypeptide cannot participate in the formation of a heptameric structure with other Hla molecules, and any substitution in ii) is preferably non - conservative.

[0016] In a third aspect, the present invention relates to i. having at least 85% sequence identity with SEQ ID NO: 30, ii. an immunogenic polypeptide consisting of or comprising a variant of the amino acid sequence of LukE that does not contain the signal peptide corresponding to residues 1 to 28 of SEQ ID NO: 29.

[0017] In a fourth aspect, the present invention relates to i. having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 34, ii. an immunogenic polypeptide consisting of or comprising a variant of the amino acid sequence of Aureo lysin (Aur) that contains one or more amino acid substitutions in the HEXXH catalytic domain corresponding to amino acid positions 352 to 356 of SEQ ID NO: 34, wherein the polypeptide has reduced catalytic ability and the substitution is preferably non-conservative.

[0018] In a fifth aspect, the present invention relates to i. having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 38, a. containing one or more amino acid substitutions in the amidase active site TXEXX domain corresponding to amino acid residues 384 to 388 of SEQ ID NO: 37, and / or b. containing one or more substitutions in the amidase active site LXDYX domain corresponding to amino acid residues 409 to 413 of SEQ ID NO: 37, and / or c. containing a substitution of the conserved cysteine corresponding to position 513 of SEQ ID NO: 37, an immunogenic polypeptide consisting of or comprising a variant of the amino acid sequence of N-acetylmuramoyl-L-alanine amidase (SAR 2723), wherein the polypeptide has reduced catalytic ability and the substitution is preferably non-conservative.

[0019] In a sixth aspect, the present invention relates to a - d: a) An Hla polypeptide or a variant thereof, wherein the variant preferably exhibits a decrease in hemolytic activity or does not exhibit hemolytic activity, and / or can preferably induce an antibody that blocks the hemolytic activity of native Hla, an Hla polypeptide or a variant thereof, b) A LukE polypeptide or a variant thereof, wherein the variant preferably exhibits a decrease in leukocyte activity or does not exhibit leukocyte activity, and / or can preferably induce an antibody that blocks the leukocyte activity of native LukE, a LukE polypeptide or a variant thereof, c) An SpA polypeptide or a variant thereof, wherein the variant preferably exhibits a decrease in the ability to bind to human IgG and human von Willebrand factor, and / or can preferably induce an antibody that blocks the native SpA interaction with human IgG, an SpA polypeptide or a variant thereof, and d) A vaccine composition comprising a selection of polypeptides comprising at least two of an Aur polypeptide or a variant thereof, wherein the variant preferably exhibits a decrease in catalytic activity and can preferably induce an antibody that blocks the catalytic activity of native Aur, the composition may further comprise a pharmaceutically acceptable carrier, medium or diluent, and in particular may further comprise an immunogenic adjuvant selected from AlOH, SLA-SE and OMV (outer membrane vesicles).

[0020] In a seventh aspect, the present invention relates to a chimeric polypeptide comprising the amino acid sequence of a selection of the polypeptides according to the sixth aspect of the present invention, the amino acid sequences being fused or linked via a linker.

[0021] In an eighth aspect, the present invention relates to a nucleic acid fragment, such as a DNA fragment or an RNA fragment, encoding the immunogenic polypeptide according to any one of the first to fifth aspects of the present invention or the chimeric polypeptide according to the seventh aspect of the present invention.

[0022] In a ninth aspect, the present invention relates to a vector comprising the nucleic acid fragment described in the eighth aspect of the present invention.

[0023] In a tenth aspect, the present invention relates to a transformed cell or virus that contains the nucleic acid fragment described in the eighth aspect of the present invention or the vector described in the ninth aspect of the present invention and is capable of expressing it.

[0024] In an eleventh aspect, the present invention relates to an immunogenic composition comprising the nucleic acid fragment described in the eighth aspect of the present invention, the vector described in the embodiment of the ninth aspect of the present invention, or the transformed cell or virus described in the tenth aspect of the present invention, and a pharmaceutically acceptable carrier, medium or diluent, and optionally an immunogenic adjuvant.

[0025] In a twelfth aspect, the present invention relates to an immunogenic composition comprising a nucleic acid fragment, vector or transformed cell or virus capable of expressing a selection of the polypeptides described in the sixth aspect of the present invention, and a pharmaceutically acceptable carrier, medium or diluent, and optionally an immunogenic adjuvant.

[0026] In a thirteenth aspect, the present invention relates to a method for inducing immunity in an animal by administering at least once an immunogenically effective amount of the immunogenic polypeptide described in any one of the first to fifth aspects of the present invention, the vaccine composition described in the sixth aspect of the present invention, the chimeric polypeptide described in the seventh aspect of the present invention, the nucleic acid fragment described in the eighth aspect of the present invention, the vector described in the ninth aspect of the present invention, the transformed cell or virus described in the tenth aspect of the present invention, or the immunogenic composition described in the eleventh or twelfth aspect of the present invention, in order to induce adaptive immunity against Staphylococcus aureus in the animal.

[0027] In a fourteenth aspect, the present invention relates to an immunogenic polypeptide described in any one of the first to fifth aspects of the present invention for use as a medicament.

[0028] In a 15th aspect, the present invention relates to an immunogenic polypeptide according to any one of the 1st to 5th aspects of the present invention for use as a medicament in the treatment, prevention or amelioration of an infection by Staphylococcus aureus.

[0029] In a 16th aspect, the present invention relates to a chimeric polypeptide according to the 7th aspect of the present invention for use as a medicament.

[0030] In a 17th aspect, the present invention relates to a chimeric polypeptide according to the 7th aspect of the present invention for use as a medicament in the treatment, prevention or amelioration of an infection by Staphylococcus aureus.

[0031] In an 18th aspect, the present invention relates to a nucleic acid fragment according to the 8th aspect of the present invention or a vector according to the 9th aspect of the present invention for use as a medicament.

[0032] In a 19th aspect, the present invention relates to a nucleic acid fragment according to the 8th aspect of the present invention or a vector according to the 9th aspect of the present invention for use as a medicament in the treatment, prevention or amelioration of an infection by Staphylococcus aureus.

[0033] In a 20th aspect, the present invention relates to a transformed cell or virus according to the 10th aspect of the present invention for use as a medicament.

[0034] In a 21st aspect, the present invention relates to a transformed cell or virus according to the 10th aspect of the present invention for use as a medicament in the treatment, prevention or amelioration of an infection by Staphylococcus aureus.

Brief Description of the Drawings

[0035]

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[0036] Definitions As used herein, the term "polypeptide" is intended to mean any of a short peptide of 2-10 amino acid residues, an oligopeptide of 11-100 amino acid residues, and a polypeptide of more than 100 amino acid residues. Further, the term is also intended to include proteins, i.e., functional biomolecules containing at least one polypeptide, and when containing at least two polypeptides, these may form a complex, may be covalently linked, or may be non-covalently linked. The polypeptides in a protein may be glycosylated and / or lipidated and / or may contain prosthetic groups.

[0037] The term "subsequence" means any continuous stretch of at least 3 amino acids, or in the case of nucleic acid sequences, at least 3 nucleotides, each directly derived from a naturally occurring amino acid sequence or nucleic acid sequence, respectively.

[0038] The term "amino acid sequence" refers to the order in which amino acid residues linked by peptide bonds are present in a peptide or protein chain when listed in the N-terminal to C-terminal direction.

[0039] The term "adjuvant" has its ordinary meaning in the field of vaccine technology, namely, 1) a substance or composition that by itself is not capable of eliciting a specific immune response against the immunogen of a vaccine, but 2) is nevertheless capable of enhancing the immune response against the immunogen. Alternatively, in other words, vaccination with an adjuvant alone does not provide an immune response against the immunogen, and vaccination with an immunogen may or may not result in an immune response against the immunogen, but vaccination with a combination of an immunogen and an adjuvant elicits an immune response against the immunogen that is stronger than the immune response induced by the immunogen alone.

[0040] "Sequence identity", in the context of the present invention, is determined by comparing two optimally aligned sequences of equal length (e.g., DNA, RNA, or amino acids) according to the following formula: (N ref -N dif )·100 / N ref (wherein N ref is the number of residues in one of the two sequences and N dif is the number of residues that are not identical in the two sequences when they are aligned in the same direction over their full length). Thus, the two sequences 5'-ATTCGGAAC-3' and 5'-ATACGGGAC-3' provide 77.8% sequence identity (N ref =9 and N dif =2).

[0041] "3D structure" is the three-dimensional structure of a biomolecule such as a protein. In a monomeric polypeptide / protein, the 3D structure is also called the "tertiary structure" and indicates the relative positions of the amino acid residues that form the polypeptide in three-dimensional space.

[0042] An "immunogenic carrier" is a molecule or moiety to which an immunogen or hapten can bind in order to enhance or enable the induction of an immune response against the immunogen / hapten. Classically, an immunogenic carrier is a relatively large molecule (such as tetanus toxoid, KLH, diphtheria toxoid, etc.) that can be fused or conjugated to an immunogen / hapten that is not sufficiently immunogenic by itself, and typically, the immunogenic carrier can induce a strong T helper lymphocyte response against the combined substance composed of the immunogen and the immunogenic carrier, thereby improving the response of B lymphocytes and cytotoxic lymphocytes to the immunogen. More recently, large carrier molecules have been to some extent replaced by so-called promiscuous T helper epitopes, i.e., shorter peptides that are recognized by the majority of HLA haplotypes in the population and that induce a T helper lymphocyte response.

[0043] A "T helper lymphocyte response" is an immune response induced on the basis of a peptide that can bind to MHC class II molecules (e.g., HLA class II molecules) in antigen-presenting cells and that stimulates T helper lymphocytes in an animal species as a result of T cell receptor recognition of the complex of the peptide and the MHC class II molecule precursor.

[0044] An "immunogen" is a substance that can induce an adaptive immune response in a host in which the immune system is confronted with the immunogen. Thus, an immunogen is a subset of the larger genus of "antigens" that can be specifically recognized by the immune system (e.g., when bound by an antibody or when a fragment of an antigen bound to an MHC molecule is recognized by a T cell receptor), but that is not necessarily capable of inducing immunity, whereas an antigen is always capable of inducing immunity, meaning that a host with established memory immunity to an antigen initiates a specific immune response against the antigen.

[0045] "Adaptive immune response" is an immune response that responds to confrontation with an antigen or immunogen, the immune response being specific to the antigenic determinants of the antigen / immunogen, and examples of adaptive immune responses are the induction of antigen-specific antibody production or the antigen-specific induction / activation of T helper lymphocytes or cytotoxic lymphocytes.

[0046] "Defensive adaptive immune response" is an antigen-specific immune response induced in a subject as a reaction to immunization (artificial or natural) with an antigen, the immune response being able to protect the subject against subsequent challenge with the antigen or a pathology-related substance containing the antigen. Typically, prophylactic vaccination aims to establish a defensive adaptive immune response against one or several pathogens.

[0047] "Stimulation of the immune system" means that a substance or composition exhibits a general non-specific immune-stimulating effect. Some adjuvants and putative adjuvants (e.g., certain cytokines) share the ability to stimulate the immune system. The use of an immune stimulant results in an increase in the "attention" of the immune system, meaning that co- or subsequent immunization with an immunogen induces a significantly more effective immune response compared to the use of the immunogen alone.

[0048] The term "animal" is intended in this context generally to mean animal species (preferably mammals) such as Homo sapiens, Canis domesticus, etc., and is not intended to mean only one animal. However, since it is important that substantially all individuals immunized according to the method of the invention elicit an immune response against the immunogen of the invention, the term also denotes a population of such animal species.

[0049] As used herein, the term "antibody" refers to a polypeptide or group of polypeptides composed of at least one antibody binding site. An "antibody binding site" is a three-dimensional binding space having an internal surface shape and charge distribution complementary to the characteristics of an epitope of an antigen, which enables the binding of the antibody to the antigen. "Antibody" includes, for example, vertebrate antibodies, hybrid antibodies, chimeric antibodies, humanized antibodies, modified antibodies, monovalent antibodies, Fab proteins, and single domain antibodies.

[0050] "Specific binding" means the binding between two substances, which is more than the binding of either substance to a randomly selected substance, and also more than the simple association between substances that tend to aggregate because they share the same overall hydrophobicity or hydrophilicity. Thus, specific binding usually involves a combination of electrostatic and other interactions between two sterically complementary regions on two substances, which means that the substances can "recognize" each other in a complex mixture.

[0051] The term "vector" is used to refer to a carrier nucleic acid molecule that can be inserted to introduce a heterologous nucleic acid sequence into a cell capable of replicating and expressing it. This term further refers to certain biological agents useful for the same purpose, such as viral vectors and phages, both of which can introduce a heterologous nucleic acid sequence into a host cell.

[0052] The term "expression vector" refers to a vector containing a nucleic acid sequence encoding at least a part of a gene product that can be transcribed. In some cases, when the transcript is an mRNA molecule, it is then translated into a protein, polypeptide, or peptide.

[0053] When referring to "amino acids", the present disclosure generally refers to proteinogenic amino acids, i.e., amino acids encoded by nucleic acids and appearing in the expression products of such nucleic acids, as well as non-proteinogenic amino acids. However, for the polypeptides disclosed herein, 22 naturally occurring amino acids are preferred, and such polypeptides can be produced recombinantly.

[0054] The amino acid substitutions described in the following table are considered to be "conservative". Substitutions not described in the table are consequently considered to be "non-conservative".

[0055] TIFF2025516318000001.tif141155

[0056] When variants of a polypeptide from Staphylococcus aureus are mutated as discussed herein, one embodiment requires that the amino acids be non-conservative, but the most important substitutions are those that do not cause dramatic changes in the secondary and tertiary structures of the substituted protein compared to the native protein. For example, proline is avoided because it introduces a fixed turn or bend into the secondary structure of a protein, and substitutions with proline generally do not provide polypeptides that are less likely to induce antibodies that target the unsubstituted protein. Generally, preferred mutations herein are those in which the mutant molecule has a reduced ability to perform its pathological functions (such as the IgG and von Willebrand binding activities of SpA), but on the other hand, preserve the secondary and tertiary structures of the non-mutated protein or protein fragment, thereby allowing the induction of non-mutated protein-binding antibodies by the mutated variant. For example, the exchange of a non-polar amino acid residue for a polar amino acid residue or vice versa is generally effective in interfering with the binding ability of the mutated variant while leaving the 3D shape of the molecule substantially unchanged.

[0057] When referring to an amino acid position or amino acid residue in a variant or homologous sequence, if that position or residue is said to "correspond" to an amino acid position in a reference amino acid sequence, it is intended herein that the reference sequence be optimally aligned with that of the homologous / variant sequence, and when this is done, the "corresponding" residue / position is that which aligns with the residue specified in the reference sequence. The optimal alignment of two sequences must be done by preparing a global alignment of the reference sequence and the variant / homologous sequence using the EMBOSS Needle alignment, and the online tool for this is available at www.ebi.ac.uk / tools / psa (based on the Needleman-Wunsch algorithm) for protein sequence alignment with the following settings: Matrix: Blosum62; Gap open: 10; Gap extend: 0.5; End gap penalty: none; End gap open: 10; and End gap extend: 0.5.

[0058] When referring to "sequence identity between amino acid sequences", it is calculated using the same alignment. Thus, the optimal alignment of two sequences must be done by preparing a global alignment of the reference sequence and the variant / homologous sequence using the EMBOSS Needle alignment with the parameters above to obtain the percentage of sequence identity.

[0059] Particular embodiments of the invention Embodiment of the first aspect of the present invention A first aspect of the invention is i.a - e: a. A sequence that is at least 85% identical to the amino acid sequence of immunoglobulin binding domain (IgBD) E (SEQ ID NO: 16, residues 1 - 56), b. A sequence that is at least 85% identical to the amino acid sequence of IgBD D (SEQ ID NO: 16, residues 62 - 117), a variant of the amino acid sequence of immunoglobulin G-binding protein A (SpA) comprising at least one of: a sequence that is at least 85% identical to the amino acid sequence of IgBD A (SEQ ID NO: 16, residues 120-175), a sequence that is at least 85% identical to the amino acid sequence of IgBD B (SEQ ID NO: 16, residues 178-233), a sequence that is at least 85% identical to the amino acid sequence of IgBD C (SEQ ID NO: 16, residues 236-291); and ii. one or more first mutations in at least one of a-e, which disrupt binding at the Fc binding site and occur at positions corresponding to the amino acid positions in SEQ ID NO: 16 selected from 3, 8, 9, 64, 69, 70, 122, 127, 128, 180, 185, 186, 238, 243, 244; and iii. one or more second mutations in at least one of a-e, which disrupt binding at the Fab binding site and occur at positions corresponding to the amino acid positions in SEQ ID NO: 16 selected from 34, 35, 37, 38, 41, 95, 96, 98, 99, 102, 153, 154, 156, 157, 160, 211, 212, 214, 215, 218, 269, 270, 272, 273, and 276, or an immunogenic polypeptide comprising or consisting of these; iv. optionally, none of at least one of a-e contains a mutation that disrupts binding at the Fc binding site at positions corresponding to both of amino acid positions 7+8, 68+69, 126+127, 184+185, and 242+243 of SEQ ID NO: 16, where applicable; v. optionally, none of at least one of a-e contains a mutation that disrupts binding at the Fab binding site at positions corresponding to both of amino acid positions 34+35, 95+96, 153+154, 211+212, and 269+270 of SEQ ID NO: 16, where applicable; Regarding an immunogenic polypeptide, the polypeptide cannot bind to human IgG and human von Willebrand factor, and the substitution is preferably a non-conservative substitution.

[0060] The specific mutation patterns in these novel variants of SpA are immunogenic, capable of inducing antibodies that bind to the wild-type protein, and at the same time do not show the effects on IgG and von Willebrand factor that are characteristics of the wild-type SpA, which has been demonstrated by the inventors to achieve a complex goal.

[0061] In the first embodiment of the first aspect of the present invention, the one or more second mutations are at least one or two or three or four or five of positions 34, 96, 154, 212, and 270, one or two or three or four or five of positions 35, 95, 154, 212, and 270, one or two or three or four or five of positions 35, 96, 153, 212, and 270, one or two or three or four or five of positions 35, 96, 154, 211, and 270, one or two or three or four or five of positions 35, 96, 154, 212, and 269, one or two or three or four or five of positions 34, 95, 154, 212, and 270, one or two or three or four or five of positions 34, 96, 153, 212, and 270, one or two or three or four or five of positions 34, 96, 154, 211, and 270, one or two or three or four or five of positions 34, 96, 154, 212, and 269, One, two, three, four, or five of the 35th, 95th, 153rd, 212th, and 270th positions One, two, three, four, or five of the 35th, 95th, 154th, 211th, and 270th positions One, two, three, four, or five of the 35th, 95th, 154th, 212th, and 269th positions One, two, three, four, or five of the 35th, 96th, 153rd, 211th, and 270th positions One, two, three, four, or five of the 35th, 96th, 153rd, 212th, and 269th positions One, two, three, four, or five of the 35th, 96th, 154th, 211th, and 269th positions One, two, three, four, or five of the 34th, 95th, 153rd, 212th, and 270th positions One, two, three, four, or five of the 34th, 95th, 154th, 211th, and 270th positions One, two, three, four, or five of the 34th, 95th, 154th, 212th, and 269th positions One, two, three, four, or five of the 34th, 96th, 153rd, 211th, and 270th positions One, two, three, four, or five of the 34th, 96th, 153rd, 212th, and 269th positions One, two, three, four, or five of the 34th, 96th, 154th, 211th, and 269th positions One, two, three, four, or five of the 35th, 95th, 153rd, 211th, and 270th positions One, two, three, four, or five of the 35th, 95th, 153rd, 212th, and 269th positions One, two, three, four, or five of the 35th, 96th, 153rd, 211th, and 269th positions One, two, three, four, or five of the 34th, 95th, 153rd, 211th, and 270th positions One, two, three, four, or five of the 34th, 95th, 153rd, 212th, and 269th positions One, two, three, four, or five of the 34th, 95th, 154th, 211th, and 269th positions One, two, three, four, or five of the 34th, 96th, 153rd, 211th, and 269th positions One, two, three, four, or five of the 35th, 95th, 153rd, 211th, and 269th positions One, two, three, four, or five of the 35th, 95th, 154th, 211th, and 269th positions One, two, three, four, or five of the 34th, 95th, 153rd, 211th, and 269th positions, or One, two, three, four, or five of the 35th, 96th, 154th, 212th, and 270th positions occur only at or at these positions.

[0062] In the second embodiment of the first aspect of the present invention, one or more second mutations occur only at or at positions corresponding to the positions in SEQ ID NO: 16 selected from the group consisting of at least 34, 35, 95, 96, 153, 154, 211, 212, 269, and 270.

[0063] In the third embodiment of the first aspect of the present invention, one or more second mutations occur at positions corresponding to the positions in SEQ ID NO: 16 selected from the group consisting of 34, 35, 95, 96, 153, 154, 211, 212, 269, and 270.

[0064] In a fourth embodiment of the first aspect of the present invention, one or more second mutations occur at or only at positions corresponding to the positions in SEQ ID NO: 16 selected from the group consisting of at least 34 and 95, 34 and 96, 34 and 153, 34 and 154, 34 and 211, 34 and 212, 34 and 269, 34 and 270, 35 and 95, 35 and 96, 35 and 153, 35 and 154, 35 and 211, 35 and 212, 35 and 269, 35 and 270, 95 and 153, 95 and 154, 95 and 211, 95 and 212, 95 and 269, 95 and 270, 96 and 153, 96 and 154, 96 and 211, 96 and 212, 96 and 269, 96 and 270, 153 and 211, 153 and 212, 153 and 269, 153 and 270, 154 and 211, 154 and 212, 154 and 269, 154 and 270, 211 and 269, 211 and 270, 212 and 269, and 212 and 270.

[0065] In the fifth embodiment of the first aspect of the present invention, one or more second mutations are at least 34 and 95 and 153, 34 and 95 and 154, 34 and 95 and 211, 34 and 95 and 212, 34 and 95 and 269, 34 and 95 and 270, 35 and 95 and 153, 35 and 95 and 154, 35 and 95 and 211, 35 and 95 and 212, 35 and 95 and 269, 35 and 95 and 270, 34 and 96 and 153, 34 and 96 and 154, 34 and 96 and 211, 34 and 96 and 212, 34 and 96 and 269, 34 and 96 and 270, 35 and 96 and 153, 35 and 96 and 154, 35 and 96 and 211, 35 and 96 and 212, 35 and 96 and 269, 35 and 96 and 270, 34 and 153 and 211, 34 and 153 and 212, 34 and 154 and 269, 34 and 154 and 270, 35 and 153 and 211, 35 and 153 and 212, 35 and 154 and 269, 35 and 154 and 270, 34 and 211 and 269, 34 and 212 and 270, 35 and 211 and 269, 35 and 212 and 270, 95 and 153 and 211, 95 and 153 and 212, 95 and 153 and 269, 95 and 153 and 270, 95 and 154 and 211, 95 and 154 and 212, 95 and 154 and 269, 95 and 154 and 270, 96 and 153 and 211, 96 and 153 and 212, 96 and 153 and 269, 96 and 153 and 270, 96 and 154 and 211, 96 and 154 and 212, 96 and 154 and 269, 96 and 154 and 270, 95 and 211 and 269, 95 and 211 and 270, 95 and 212 and 269, 95 and 212 and 270, 96 and 212 and 269, 96 and 212 and 270, 96 and 212 and 269, 96 and 212 and 270, 153 and 211 and 269, 153 and 212 and 269, 153 and 211 and 270, 153 and 212 and 270, 154 and 211 and 269,Occurs at a position corresponding to the position in SEQ ID NO: 16 selected from the group consisting of 154 and 212 and 269, 154 and 211 and 270, and 154 and 212 and 270, or only at these positions.

[0066] In the sixth embodiment of the first aspect of the present invention, one or more second mutations are at least 95 and 153 and 211 and 269, 95 and 153 and 211 and 270, 95 and 153 and 212 and 269, 95 and 153 and 212 and 270, 95 and 154 and 211 and 269, 95 and 154 and 211 and 270, 95 and 154 and 212 and 269, 95 and 154 and 212 and 270, 96 and 153 and 211 and 269, 96 and 153 and 211 and 270, 96 and 153 and 212 and 269, 96 and 153 and 212 and 270, 96 and 154 and 211 and 269, 96 and 154 and 211 and 270, 96 and 154 and 212 and 269, 96 and 154 and 212 and 270, 34 and 153 and 211 and 269, 34 and 153 and 211 and 270, 34 and 153 and 212 and 269, 34 and 153 and 212 and 270, 34 and 154 and 211 and 269, 34 and 154 and 211 and 270, 34 and 154 and 212 and 269, 34 and 154 and 212 and 270, 35 and 153 and 211 and 269, 35 and 153 and 211 and 270, 35 and 153 and 212 and 269, 35 and 153 and 212 and 270, 35 and 154 and 211 and 269, 35 and 154 and 211 and 270, 35 and 154 and 212 and 269, 35 and 154 and 212 and 270, 34 and 95 and 211 and 269, 34 and 95 and 211 and 270, 34 and 95 and 212 and 269, 34 and 95 and 212 and 270, 34 and 96 and 211 and 269, 34 and 96 and 211 and 270, 34 and 96 and 212 and 269, 34 and 96 and 212 and 270, 35 and 95 and 211 and 269, 35 and 95 and 211 and 270, 35 and 95 and 212 and 269, 35 and 95 and 212 and 270, 35 and 96 and 211 and 269, 35 and 96 and 211 and 270,Occurs at or only at positions corresponding to the positions in SEQ ID NO: 16 selected from the group consisting of 35 and 96 and 212 and 269, 35 and 96 and 212 and 270, 34 and 95 and 153 and 269, 34 and 95 and 153 and 270, 34 and 95 and 154 and 269, 34 and 95 and 154 and 270, 34 and 96 and 153 and 269, 34 and 96 and 153 and 270, 34 and 96 and 154 and 269, 34 and 96 and 154 and 270, 35 and 95 and 153 and 269, 35 and 95 and 153 and 270, 35 and 95 and 154 and 269, 35 and 95 and 154 and 270, 35 and 96 and 153 and 269, 35 and 96 and 153 and 270, 35 and 96 and 154 and 269, 35 and 96 and 154 and 270, 34 and 95 and 153 and 211, 34 and 95 and 153 and 212, 34 and 95 and 154 and 211, 34 and 95 and 154 and 212, 34 and 96 and 153 and 211, 34 and 96 and 153 and 212, 34 and 96 and 154 and 211, 34 and 96 and 154 and 212, 35 and 95 and 153 and 211, 35 and 95 and 153 and 212, 35 and 95 and 154 and 211, 35 and 95 and 154 and 212, 35 and 96 and 153 and 211, 35 and 96 and 153 and 212, 35 and 96 and 154 and 211, and 35 and 96 and 154 and 212.,

[0067] In the seventh embodiment of the first aspect of the present invention, one or more second mutations occur at positions corresponding to the positions in SEQ ID NO: 16 selected from the group consisting of at least 34 and 96 and 154 and 212 and 270, 35 and 95 and 154 and 212 and 270, 35 and 96 and 153 and 212 and 270, 35 and 96 and 154 and 211 and 270, 35 and 96 and 154 and 212 and 269, 34 and 95 and 154 and 212 and 270, 34 and 96 and 153 and 212 and 270, 34 and 96 and 154 and 211 and 270, 34 and 96 and 154 and 212 and 269, 35 and 95 and 153 and 212 and 270, 35 and 95 and 154 and 211 and 270, 35 and 95 and 154 and 212 and 269, 35 and 96 and 153 and 211 and 270, 35 and 96 and 153 and 212 and 269, 35 and 96 and 154 and 211 and 269, 34 and 95 and 153 and 212 and 270, 34 and 95 and 154 and 211 and 270, 34 and 95 and 154 and 212 and 269, 34 and 96 and 153 and 211 and 270, 34 and 96 and 153 and 212 and 269, 34 and 96 and 154 and 211 and 269, 35 and 95 and 153 and 211 and 270, 35 and 95 and 153 and 212 and 269, 35 and 96 and 153 and 211 and 269, 34 and 95 and 153 and 211 and 270, 34 and 95 and 153 and 212 and 269, 34 and 95 and 154 and 211 and 269, 34 and 96 and 153 and 211 and 269, 35 and 95 and 153 and 211 and 269, 35 and 95 and 154 and 211 and 269, 34 and 95 and 153 and 211 and 269, and 35 and 96 and 154 and 212 and 270, or only at these positions.

[0068] In the eighth embodiment of the first aspect of the present invention, one or more first mutations occur at positions corresponding to the positions in SEQ ID NO: 16 selected from at least 8, 69, 127, 185, and 243, or only at these positions.

[0069] In the ninth embodiment of the first aspect of the present invention, one or more first mutations are 3 and 8, 3 and 9, 3 and 69, 3 and 127; 3 and 185; 3 and 243; 8 and 9; 8 and 69; 8 and 127; 8 and 185; 8 and 243; 9 and 69; 9 and 127; 9 and 185; 9 and 243; 69 and 127; 69 and 185; 69 and 243; 127 and 185; 127 and 243; 185 and 243; 3 and 8 and 9; 3 and 8 and 69; 3 and 8 and 127; 3 and 8 and 185; 3 and 8 and 243; 3 and 9 and 69; 3 and 9 and 127; 3 and 9 and 185; 3 and 9 and 243; 3 and 69 and 127; 3 and 69 and 185; 3 and 69 and 243; 3 and 127 and 185; 3 and 127 and 243; 3 and 185 and 243; 8 and 9 and 69; 8 and 9 and 127; 8 and 9 and 185; 8 and 9 and 243; 8 and 69 and 127; 8 and 69 and 185; 8 and 69 and 243; 8 and 127 and 185; 8 and 127 and 243; 8 and 185 and 243; 9 and 69 and 127; 9 and 69 and 185; 9 and 69 and 243; 9 and 127 and 185; 9 and 127 and 243; 9 and 185 and 243; 69 and 127 and 185; 69 and 127 and 243; 69 and 185 and 243; 127 and 185 and 243; 3 and 8 and 9 and 69; 3 and 8 and 9 and 127; 3 and 8 and 9 and 185; 3 and 8 and 9 and 243; 3 and 8 and 69 and 127; 3 and 8 and 69 and 185; 3 and 8 and 69 and 243; 3 and 8 and 127 and 185; 3 and 8 and 127 and 243; 3 and 8 and 185 and 243; 3 and 9 and 69 and 127; 3 and 9 and 69 and 185; 3 and 9 and 69 and 243; 3 and 9 and 127 and 185; 3 and 9 and 127 and 243; 3 and 9 and 185 and 243; 3 and 69 and 127 and 185; 3 and 69 and 127 and 243; 3 and 69 and 185 and 243;3 and 127 and 185 and 243; 8 and 9 and 69 and 127; 8 and 9 and 69 and 185; 8 and 9 and 69 and 243; 8 and 9 and 127 and 185; 8 and 9 and 127 and 243; 8 and 9 and 185 and 243; 8 and 69 and 127 and 185; 8 and 69 and 127 and 243; 8 and 69 and 185 and 243; 8 and 127 and 185 and 243; 9 and 69 and 127 and 185; 9 and 69 and 127 and 243; 9 and 69 and 185 and 243; 9 and 127 and 185 and 243; 69 and 127 and 185 and 243; 3 and 8 and 9 and 69 and 127; 3 and 8 and 9 and 69 and 185; 3 and 8 and 9 and 69 and 243; 3 and 8 and 9 and 127 and 185; 3 and 8 and 9 and 127 and 243; 3 and 8 and 9 and 185 and 243; 3 and 8 and 69 and 127 and 185; 3 and 8 and 69 and 127 and 243; 3 and 8 and 69 and 185 and 243; 3 and 8 and 127 and 185 and 243; 3 and 9 and 69 and 127 and 185; 3 and 9 and 69 and 127 and 243; 3 and 9 and 69 and 185 and 243; 3 and 9 and 127 and 185 and 243; 3 and 69 and 127 and 185 and 243; 8 and 9 and 69 and 127 and 185; 8 and 9 and 69 and 127 and 243; 8 and 9 and 69 and 185 and 243; 8 and 9 and 127 and 185 and 243; 8 and 69 and 127 and 185 and 243; 9 and 69 and 127 and 185 and 243; 3 and 8 and 9 and 69 and 127 and 185; 3 and 8 and 9 and 69 and 127 and 243; 3 and 8 and 9 and 69 and 185 and 243; 3 and 8 and 9 and 127 and 185 and 243; 3 and 8 and 69 and 127 and 185 and 243;It occurs at positions corresponding to the positions in SEQ ID NO: 16 selected from 3 and 9 and 69 and 127 and 185 and 243, 8 and 9 and 69 and 127 and 185 and 243, and 3 and 8 and 9 and 69 and 127 and 185 and 243.;

[0070] In a tenth embodiment of the first aspect of the present invention, the first and / or second mutation is a non-conservative substitution. However, as pointed out above, such non-conservative substitutions do not interfere with the secondary and tertiary structures. In other words, the mutation should preferably (in order to allow the induction of an effective antibody that binds to the non-mutated protein) preserve the structure, while on the other hand, the mutation should preferably reduce or eliminate the undesirable functionality exhibited by the non-mutated protein. This also has the result that it is useful when a mutation that was conventionally called "conservative" has the effect of making the immunogen less biologically active than the wild-type protein.

[0071] In an eleventh embodiment of the first aspect of the present invention, at least one of the one or more first mutations is the same as in the eighth embodiment of the first aspect of the present invention, and at least one of the one or more first mutations that is the same as in the eighth embodiment of the first aspect of the present invention is a mutation to lysine (K).

[0072] In a twelfth embodiment of the first aspect of the present invention, at least one of the one or more second mutations is the same as in the eighth embodiment of the first aspect of the present invention, and all of the one or more first mutations that are the same as in the eighth embodiment of the first aspect of the present invention are mutations to lysine (K).

[0073] In the 13th embodiment of the first aspect of the present invention, at least one of the one or more second mutations is the same as in the 3rd embodiment of the first aspect of the present invention, and at least one of the one or more second mutations that is the same as in the 3rd embodiment of the first aspect of the present invention is a mutation to alanine (A) or arginine (R).

[0074] In the 14th embodiment of the first aspect of the present invention, at least one of the one or more second mutations is the same as in the 3rd embodiment of the first aspect of the present invention, and all of the one or more second mutations that are the same as in the 3rd embodiment of the first aspect of the present invention are mutations to alanine (A) or arginine (R).

[0075] In the 15th embodiment of the first aspect of the present invention, the first mutation is as described in the 11th embodiment of the first aspect of the present invention, and the second mutation is as described in the 13th embodiment of the first aspect of the present invention, or the first mutation is as described in the 12th embodiment of the first aspect of the present invention, and the second mutation is as described in the 13th embodiment of the first aspect of the present invention, or the first mutation is as described in the 11th embodiment of the first aspect of the present invention, and the second mutation is as described in the 14th embodiment of the first aspect of the present invention, or the first mutation is as described in the 12th embodiment of the first aspect of the present invention, and the second mutation is as described in the 14th embodiment of the first aspect of the present invention.

[0076] In the 16th embodiment of the first aspect of the present invention, the variant has at least 85% sequence identity with the sequence of SEQ ID NO: 16.

[0077] In the 17th embodiment of the first aspect of the present invention, the polypeptide can induce an antibody that blocks the ability of wild-type SpA to bind to IgG and von Willebrand factor.

[0078] Embodiment of the second aspect of the present invention The second aspect of the present invention is i. having at least 85% sequence identity with the sequence of SEQ ID NO: 2, ii. comprising one or more amino acid deletions and / or substitutions in the first 12 consecutive N-terminal amino acid residues of the amino acid sequence of mature Hla, wherein said first 12 consecutive N-terminal amino acid residues correspond to positions 1 to 12 of SEQ ID NO: 2, and being an immunogenic polypeptide consisting of or comprising a variant of the amino acid sequence of alpha-hemolysin (Hla), wherein the polypeptide is unable to participate in the formation of a heptameric structure with other Hla molecules, and any substitution in ii) is preferably non-conservative, relating to an immunogenic polypeptide.

[0079] Similar to the polypeptide of the first aspect of the present invention, the polypeptide of the second aspect has the ability to induce antibodies that bind to Hla due to precisely selected changes in its primary structure compared to the wild-type protein (Hla), while at the same time having a reduced biological reactivity.

[0080] In a first embodiment of the second aspect of the present invention, the variant comprises at least 2 amino acid alterations selected from deletions and substitutions in said first 12 consecutive N-terminal amino acid residues, for example, at least or exactly 2, at least or exactly 3, at least or exactly 4, at least or exactly 5, at least or exactly 6, at least or exactly 7, at least or exactly 8, at least or exactly 9, at least or exactly 10, at least or exactly 11, or exactly 12 amino acid alterations.

[0081] In the second embodiment of the second aspect of the present invention, the variant comprises at least two amino acid substitutions in the first 12 N-terminal amino acids below, for example, at least or exactly 2, at least or exactly 3, at least or exactly 4, at least or exactly 5, at least or exactly 6, at least or exactly 7, at least or exactly 8, at least or exactly 9, at least or exactly 10, at least or exactly 11, or exactly 12 amino acid substitutions.

[0082] In the third embodiment of the second aspect of the present invention, the variant comprises at least two amino acid deletions in the first 12 consecutive N-terminal amino acid residues, for example, at least or exactly 2, at least or exactly 3, at least or exactly 4, at least or exactly 5, at least or exactly 6, at least or exactly 7, at least or exactly 8, at least or exactly 9, at least or exactly 10, at least or exactly 11, or exactly 12 amino acid deletions.

[0083] In the fourth embodiment of the second aspect of the present invention, all of amino acid residues 1 to 12 are deleted.

[0084] In the fifth embodiment of the second aspect of the present invention, all of amino acid residues 1 to 12 are substituted.

[0085] In the sixth embodiment of the second aspect of the present invention, amino acid residues 1 to 12 are substituted with the sequence 5'-SETEVSVRSASS-3' (residues 1 to 12 of SEQ ID NO: 4).

[0086] In the seventh embodiment of the second aspect of the present invention, the variant comprises SEQ ID NO: 3.

[0087] In the eighth embodiment of the second aspect of the present invention, the immunogenic polypeptide comprises a substitution of histidine (H) to leucine (L) at the position corresponding to position 35 of SEQ ID NO: 2.

[0088] In the ninth embodiment of the second aspect of the present invention, the variant contains SEQ ID NO: 6.

[0089] In the tenth embodiment of the second aspect of the present invention, the variant lacks hemolytic activity or has a significantly reduced hemolytic activity.

[0090] In the eleventh embodiment of the second aspect of the present invention, the polypeptide can induce an antibody that blocks the hemolytic activity of wild-type Hla.

[0091] Embodiment of the third aspect of the present invention The third aspect of the present invention is i. having at least 85% sequence identity with SEQ ID NO: 30, ii. related to an immunogenic polypeptide consisting of or containing a variant of the amino acid sequence of LukE that does not contain the signal peptide corresponding to residues 1 to 28 of SEQ ID NO: 29.

[0092] In the first embodiment of the third aspect of the present invention, the polypeptide consists of SEQ ID NO: 30.

[0093] In the second embodiment of the third aspect of the present invention, the polypeptide can induce an antibody that blocks the leukotoxicity of functionally mature wild-type LukE.

[0094] Embodiment of the fourth aspect of the present invention The fourth aspect of the present invention relates to an immunogenic polypeptide consisting of or containing a variant of the amino acid sequence of aureolysin (Aur), wherein the variant i. has at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 34, ii. is an immunogenic polypeptide consisting of or containing a variant of the amino acid sequence of aureolysin (Aur) that contains one or more amino acid substitutions in the HEXXH catalytic domain corresponding to amino acid positions 352 to 356 of SEQ ID NO: 34, The present invention relates to an immunogenic polypeptide in which the catalytic ability of the polypeptide is reduced and the substitution is preferably non-conservative.

[0095] These variants of aureolysin have been demonstrated to induce antibodies that effectively target wild-type aureolysin, but are safe and pharmaceutically acceptable due to their low biological activity.

[0096] In the first embodiment of the fourth aspect of the present invention, in the variant, the conserved cysteine (C) corresponding to amino acid position 479 of SEQ ID NO: 34 is substituted.

[0097] In the second embodiment of the fourth aspect of the present invention, the conserved cysteine defined in the first embodiment of the fourth aspect of the present invention is substituted with serine (S).

[0098] In the third embodiment of the fourth aspect of the present invention, the glutamic acid residue (E) in the HEXXH catalytic domain corresponding to amino acid position 353 of SEQ ID NO: 34 is preferably substituted non-conservatively.

[0099] In the fourth embodiment of the fourth aspect of the present invention, the glutamic acid in the HEXXH catalytic domain defined in the third embodiment of the fourth aspect of the present invention is substituted with alanine (A).

[0100] In the fifth embodiment of the fourth aspect of the present invention, the variant consists of or comprises SEQ ID NO: 35.

[0101] In the sixth embodiment of the fourth aspect of the present invention, the variant comprises a sequence having at least 85% sequence identity with the amino acid sequence 210-509 of SEQ ID NO: 34.

[0102] In the seventh embodiment of the fourth aspect of the present invention, the variant shows a reduced ability to participate in the formation of antigen complexes compared to wild-type Aur.

[0103] In the eighth embodiment of the fourth aspect of the present invention, the polypeptide can induce an antibody that blocks the catalytic activity of wild-type Aur.

[0104] Embodiment of the fifth aspect of the present invention The fifth aspect of the present invention is i. having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 38, a. comprising one or more amino acid substitutions in the amidase active site TXEXX domain corresponding to amino acid residues 384-388 of SEQ ID NO: 37, and / or b. comprising one or more substitutions in the amidase active site LXDYX domain corresponding to amino acid residues 409-413 of SEQ ID NO: 37, and / or c. an immunogenic polypeptide consisting of or comprising a variant of the amino acid sequence of N-acetylmuramoyl-L-alanine amidase (SAR 2723) comprising a substitution of a conserved cysteine corresponding to position 513 of SEQ ID NO: 37, wherein the polypeptide has reduced catalytic ability and the substitutions are preferably non-conservative, relating to an immunogenic polypeptide.

[0105] These variants of SAR2723 have been demonstrated to induce antibodies that effectively target wild-type SAR2723, but are safe and pharmaceutically acceptable, like other polypeptides of the present invention disclosed above, due to their low biological activity.

[0106] In the first embodiment of the fifth aspect of the present invention, the immunogenic polypeptide comprises only feature i: a, only b, only c, only a and b, only a and c, only b and c, or a, b and c.

[0107] In the second embodiment of the fifth aspect of the present invention, the conserved cysteine is substituted with serine (S).

[0108] In the third embodiment of the fifth aspect of the present invention, the glutamic acid residue (E) in the active site TXEXX domain corresponding to amino acid position 386 of SEQ ID NO: 37 is preferably non-conservatively substituted, for example, with glutamine (Q).

[0109] In the fourth embodiment of the fifth aspect of the present invention, the aspartic acid (D) in the active site LXDYS domain corresponding to amino acid position 411 of SEQ ID NO: 37 is preferably non-conservatively substituted, for example, with asparagine (N).

[0110] In the fifth embodiment of the fifth aspect of the present invention, the variant consists of or comprises SEQ ID NO: 39.

[0111] In the sixth embodiment of the fifth aspect of the present invention, the polypeptide can block the catalytic activity of wild-type SAR2723 or induce an antibody that interferes with the development of the bacterial cell wall.

[0112] Embodiment of the sixth aspect of the present invention The sixth aspect of the present invention is a to d: a) An Hla polypeptide or a variant thereof, wherein the variant preferably shows a decrease in hemolytic activity or does not show hemolytic activity, and / or preferably can induce an antibody that blocks the hemolytic activity of native Hla, an Hla polypeptide or a variant thereof, b) A LukE polypeptide or a variant thereof, wherein the variant preferably shows a decrease in leukocyte activity or does not show leukocyte activity, and / or preferably can induce an antibody that blocks the leukocyte activity of native LukE, a LukE polypeptide or a variant thereof, c) An SpA polypeptide or a variant thereof, wherein the variant preferably shows a decrease in the ability to bind to human IgG and human von Willebrand factor, and / or preferably can induce an antibody that blocks the native SpA interaction with human IgG, an SpA polypeptide or a variant thereof, and d) A selection of polypeptides comprising at least two of an Aur polypeptide or a variant thereof, wherein the variant preferably exhibits a decrease in catalytic activity and can preferably induce an antibody that blocks the catalytic activity of native Aur, the composition optionally further comprising a pharmaceutically acceptable carrier, vehicle or diluent, and particularly may further comprise an immunogenic adjuvant selected from AlOH, SLA-SE and OMV (outer membrane vesicles), relates to a vaccine composition.

[0113] Such compositions can be composed of the above-mentioned variants of SpA, Hla, LukE, and Aur, but it is understood that polypeptides known in the art that exhibit comparable functional properties can also be used in this composition. However, in a first embodiment of the sixth aspect of the present invention, the Hla polypeptide variant is as described in the second aspect of the present invention, and / or the LukE polypeptide variant is as described in the third aspect of the present invention, and / or the SpA polypeptide variant is as described in the first aspect of the present invention, and / or the Aur polypeptide variant is preferably as described in the fourth aspect of the present invention.

[0114] In a second embodiment of the sixth aspect of the present invention, the Hla polypeptide variant has the amino acid sequence of SEQ ID NO: 5 or any other previously identified Hla polypeptide that shares the characteristic of being unable to form a heptamer (pore-forming) structure. Similarly, this embodiment also utilizes any existing SpA polypeptide variant and / or Aur polypeptide variant that shares the functionality discussed above for the SpA and Aur polypeptide variants of the present invention. Thus, this embodiment utilizes at least one immunogen having the same or comparable properties as those exhibited by the polypeptides of the first to fourth aspects of the present invention.

[0115] In a third embodiment of the sixth aspect of the present invention, the vaccine composition comprises at least three of a to d.

[0116] In a fourth embodiment of the sixth aspect of the present invention, the vaccine composition comprises - a and b, - a and c, - a and d, - b and c, - b and d, - c and d, - a, b, and c, - a, b, and d, - a, c, and d, - b, c, and d, or - a, b, c, and d and comprises.

[0117] In particular, a vaccine composition in which the Staphylococcus aureus immunogen comprises or consists of a and b is preferred, that is, the Staphylococcus aureus immunogen is a) an Hla polypeptide or a variant thereof, said variant preferably showing a reduced hemolytic activity or no hemolytic activity and / or preferably being able to induce an antibody that blocks the hemolytic activity of native Hla, an Hla polypeptide or a variant thereof, and b) a LukE polypeptide or a variant thereof, said variant preferably showing a reduced leukocyte activity or no leukocyte activity and / or preferably being able to induce an antibody that blocks the leukocyte activity of native LukE, a vaccine composition comprising or consisting of a LukE polypeptide or a variant thereof is preferred, and each of a and b is as disclosed herein.

[0118] In a fifth embodiment of the sixth aspect of the present invention, the vaccine composition comprises e) a SAR2723 polypeptide or a variant thereof, said variant preferably showing a reduced catalytic activity or no catalytic activity, preferably being able to induce an antibody that blocks the catalytic activity of native SAR2723, a SAR2723 polypeptide or a variant thereof, f) A SAR2753 polypeptide or variant thereof, wherein the variant preferably exhibits a decrease in lipase activity or does not exhibit lipase activity, and preferably can induce an antibody that blocks the lipase activity of native SAR2753, a SAR2753 polypeptide or variant thereof, g) A SAR0992 (HtrA2) polypeptide or variant thereof, and h) A SAR0280 (EsaA) polypeptide or variant thereof, wherein the variant preferably can induce an antibody that prevents EsxA / B secretion, a SAR0280 (EsaA) polypeptide or variant thereof further comprises at least one polypeptide selected from the group consisting of:

[0119] In the sixth embodiment of the sixth aspect of the present invention, - The SAR0992 polypeptide variant is a fragment or sequence variant of SEQ ID NO: 15 disclosed in WO2012 / 136653, particularly a fragment consisting of amino acid residues 1 to 409 of SEQ ID NO: 15 in WO2012 / 136653, or a variant of SAR0992 that can induce an antibody that blocks the catalytic activity of native SAR0992, for example, a truncation of SAR0992 at the C-terminus with respect to the transmembrane helix of SAR0992, or a mutant version of SAR0992 that includes a substitution of a serine residue corresponding to the serine residue at position 619 of SEQ ID NO: 15 disclosed in WO2012 / 136653, and / or the transmembrane helix is exchanged with a flexible linker, or the transmembrane helix is exchanged with a linker and the N-terminal and C-terminal portions of SAR0992 adjacent to the linker are exchanged with each other, and / or - The SAR0280 polypeptide variant is a fragment or sequence variant of SEQ ID NO: 13 disclosed in WO2012 / 136653, or a fusion of the most N-terminal extracellular fragment and the most C-terminal extracellular fragment of SAR0280, and / or - The SAR2723 polypeptide variant is as described in the fifth aspect of the present invention or is a fragment or sequence variant of SEQ ID NO: 13 disclosed in WO2015 / 082536, and / or - The SAR2753 polypeptide variant is a fragment or sequence variant of SEQ ID NO: 14 disclosed in WO2015 / 082536 or a polypeptide composed of amino acid SEQ ID NO: 42.

[0120] In the seventh embodiment of the sixth aspect of the present invention, the vaccine composition comprises at least two of e to h.

[0121] In the eighth embodiment of the sixth aspect of the present invention, the vaccine composition comprises at least three of e to h.

[0122] In the ninth embodiment of the sixth aspect of the present invention, the vaccine composition - e and f, - e and g, - e and h, - f and g, - f and h, - g and h, - e, f, and g, - e, f, and h, - e, g, and h, - f, g, and h, or - e, f, g, and h comprises.

[0123] Further details regarding the vaccine composition are found under the headings "Method of Immunization" and "Compositions of the Invention; Vaccines" below.

[0124] Also, a part of the sixth aspect of the present invention is a vaccine composition comprising at least one chimeric polypeptide of the seventh aspect of the present invention, which is detailed below under the consideration of the embodiments of the seventh aspect of the present invention. The vaccine composition further comprises a pharmaceutically acceptable carrier, vehicle or diluent and may further comprise an immunological adjuvant.

[0125] At least one chimeric polypeptide in this embodiment is preferably selected from CHIM_LukE_mHla_H35L_FS (SEQ ID NO: 56), CHIM_mc2716_mSpA_7_12_FS (SEQ ID NO: 57), and CHIM_2753_mc27723_FS (SEQ ID NO: 59), particularly from SEQ ID NOs: 56 and 57.

[0126] In an important embodiment, this vaccine composition comprises at least or exactly two chimeric polypeptides of the seventh aspect of the invention and its embodiments.

[0127] In some embodiments, a vaccine composition comprising at least one chimeric polypeptide of the seventh aspect also comprises at least or exactly one additional polypeptide selected from the polypeptides discussed above as e, g, f, and h in the embodiments of the sixth aspect of the invention, i.e., such a composition comprises at least one chimeric polypeptide and at least one of the polypeptides defined as e, g, f, or h above.

[0128] The additional polypeptide is preferably selected from the group consisting of SAR0992-1-409 (residues 1 to 409 of SEQ ID NO: 44) and SAR0280-28-820 (SEQ ID NO: 48).

[0129] Particularly important vaccine compositions include the chimeric polypeptides CHIM_LukE_mHla_H35L_FS (SEQ ID NO: 56) and CHIM_mc2716_mSpA_7_12_FS (SEQ ID NO: 57), and optionally a polypeptide consisting of amino acid residues 1 to 409 of SAR0992 (residues 1 to 409 of SEQ ID NO: 44). Particularly important compositions are mixed with a pharmaceutically acceptable carrier, vehicle or diluent, and further optionally an immunological adjuvant, to include the chimeric polypeptides CHIM_LukE_mHla_H35L_FS (SEQ ID NO: 56) and CHIM_mc2716_mSpA_7_12_FS (SEQ ID NO: 57), and optionally a polypeptide consisting of amino acid residues 1 to 409 of SAR0992 (residues 1 to 409 of SEQ ID NO: 44).

[0130] The adjuvant is, in a preferred embodiment, selected from the group consisting of AlOH, SLA-SE and OMV (outer membrane vesicles).

[0131] Embodiment of the seventh aspect of the present invention The seventh aspect of the present invention relates to a chimeric polypeptide comprising the amino acid sequence of a selection of the polypeptides described in the sixth aspect of the present invention, the amino acid sequences being fused or linked via a linker. In other words, instead of including some or all of the polypeptides considered in the sixth aspect of the present invention in a vaccine "cocktail", the immunogenic amino acid sequences are instead part of a fusion protein, and this approach may facilitate the production of the vaccine or eliminate any possible need to include an immunogenic carrier molecule fused to the polypeptide.

[0132] Particularly preferred chimeric polypeptides are composed of or include polypeptides a and b as considered above under the fourth embodiment of the sixth aspect above. In particular, the chimeric polypeptide is a Staphylococcus aureus immunogen a) an Hla polypeptide or a variant thereof, said variant preferably showing a reduced or no hemolytic activity and / or preferably being able to induce an antibody that blocks the hemolytic activity of native Hla, an Hla polypeptide or a variant thereof, and b) a LukE polypeptide or a variant thereof, wherein the variant preferably shows a decrease or no decrease in leukocyte activity and / or can preferably induce an antibody that blocks the leukocyte activity of native LukE, composed of or comprising a LukE polypeptide or a variant thereof, and each of a and b is as disclosed herein.

[0133] In a first embodiment of the seventh aspect of the present invention, the linker is flexible or rigid.

[0134] In a second embodiment of the seventh aspect of the present invention, the flexible linker as shown in the first embodiment of the seventh aspect of the present invention is GSGGGA (SEQ ID NO: 50) or GSGGGAGSGGGA (SEQ ID NO: 51), or the rigid linker as shown in the first embodiment of the seventh aspect of the present invention is KPEPKPAPAPKP (SEQ ID NO: 52).

[0135] Particularly preferred chimeric polypeptides are composed of or comprise polypeptides a and b as discussed above under the fourth embodiment of the sixth aspect above. In particular, the chimeric polypeptide is a Staphylococcus aureus immunogen a) an Hla polypeptide or a variant thereof as disclosed herein, wherein the variant preferably shows a decrease or no decrease in hemolytic activity and / or can preferably induce an antibody that blocks the hemolytic activity of native Hla, and b) a LukE polypeptide or a variant thereof as disclosed herein, wherein the variant preferably shows a decrease or no decrease in leukocyte activity and / or can preferably induce an antibody that blocks the leukocyte activity of native LukE, composed of or comprising a LukE polypeptide or a variant thereof, and each of a and b is as disclosed herein.

[0136] In the third embodiment of the seventh aspect of the present invention, the chimeric polypeptide thus has an amino acid sequence selected from the group consisting of CHIM_LukE_mHla_H35L_FS (SEQ ID NO: 56), CHIM_mc2716_mSpA_7_12_FS (SEQ ID NO: 57), CHIM_0992_mSpA_7_12_FS (SEQ ID NO: 58), CHIM_2753_mc2723_FS (SEQ ID NO: 59), CHIM_0280_2753_FS (SEQ ID NO: 60), CHIM_0992_2753_FS (SEQ ID NO: 61), CHIM_mc2723_Hla_H35L_t_FS (SEQ ID NO: 62), CHIM_mc2716_LukE_FS (SEQ ID NO: 63), CHIM_LukE_Hla_H35L_t_FS (SEQ ID NO: 64), CHIM_mc2716_mSpA_10_12_FS (SEQ ID NO: 65), CHIM_LukE_0280_FS (SEQ ID NO: 66), CHIM_m0992_0992_FL (SEQ ID NO: 67), CHIM_0992_Hla_H35L_t_FS (SEQ ID NO: 68), CHIM_LukE_mSpA_10_12_FS (SEQ ID NO: 82), CHIM_mc2723_mSpA_10_12_FS (SEQ ID NO: 83), CHIM_0992_mSpA_10_12_FS (SEQ ID NO: 84), and CHIM_LukE_mSpA_7_12_FS (SEQ ID NO: 85).

[0137] Embodiments of the eighth to twelfth aspects of the present invention The eighth aspect of the present invention relates to a nucleic acid or nucleic acid fragment encoding the polypeptide of the present invention, and the ninth aspect of the present invention relates to a vector containing the nucleic acid fragment described in the eighth aspect of the present invention. Similarly, the tenth to twelfth aspects relate to cells and compositions using the vectors and nucleic acids disclosed herein, and a more detailed discussion of these embodiments is found below.

[0138] Embodiment of the thirteenth aspect of the present invention A 13th aspect of the present invention relates to a method for inducing immunity in an animal by administering at least once an immunogenically effective amount of the immunogenic polypeptide according to any one of the 1st to 5th aspects of the present invention, the vaccine composition according to the 6th aspect of the present invention, the chimeric polypeptide according to the 7th aspect of the present invention, the nucleic acid fragment according to the 8th aspect of the present invention, the vector according to the 9th aspect of the present invention, the transformed cell or virus according to the 10th aspect of the present invention, or the immunogenic composition according to the 11th or 12th aspect of the present invention, in order to induce adaptive immunity against Staphylococcus aureus in the animal.

[0139] In a first embodiment of the 13th aspect of the present invention, when the immunogenic polypeptide according to any one of the 1st to 5th aspects of the present invention, the chimeric polypeptide according to the 7th aspect of the present invention, or a composition containing the immunogenic polypeptide or the chimeric polypeptide is administered, the animal receives 0.5 to 5,000 μg of the immunogenic polypeptide or chimeric polypeptide according to any one of the 1st to 5th aspects and the 7th aspect of the present invention per administration.

[0140] In a second embodiment of the 13th aspect of the present invention, the animal receives a first priming dose containing the immunogenic polypeptide or the chimeric polypeptide and one or more booster doses containing the immunogenic polypeptide or the chimeric polypeptide.

[0141] In a third embodiment of the 13th aspect of the present invention, the animal is a human.

[0142] In a fourth embodiment of the 13th aspect of the present invention, the administration is intended to induce protective immunity against Staphylococcus aureus.

[0143] In a fifth embodiment of the 13th aspect of the present invention, the protective immunity is effective in reducing the risk of attracting infection by Staphylococcus aureus or is effective in treating or improving infection by Staphylococcus aureus.

[0144] In the sixth embodiment of the thirteenth aspect of the present invention, administration is aimed at inducing an antibody specific to Staphylococcus aureus, and the antibody or B lymphocytes producing the antibody are then recovered from the animal.

[0145] In the seventh embodiment of the thirteenth aspect of the present invention, administration is aimed at inducing an antibody specific to Staphylococcus aureus, and B lymphocytes producing the antibody are then recovered from the animal and used for the preparation of monoclonal antibodies.

[0146] Further details regarding this aspect can be found in the following section which details immunization methods and vaccines.

[0147] Vector It is understood that the nucleic acid fragments of the present invention and nucleic acid fragments encoding the polypeptides of the compositions of the present invention can be used for any purpose of production, carrier, and vaccine, and the latter requires that the sequence be included in an expression vector that can result in the production of an immunogenic protein in a mammalian animal receiving the vector. In other words, the nucleic acid is included in a vector that can express the nucleic acid in humans when administered.

[0148] Such vectors often contain, operably linked in the 5'-3' direction, an expression control region including an enhancer / promoter for driving the expression of the nucleic acid, any signal peptide coding sequence, the nucleotide sequence to be expressed, and optionally a terminator. Thus, such vectors constitute expression vectors useful for bringing about the production of the polypeptide of the present invention or a polypeptide that is part of the composition of the present invention in cells. Since the polypeptide is of bacterial origin, recombinant production must be carried out in a host cell capable of expressing the coding nucleic acid. Bacterial host cells can preferably be used. However, when the vector drives expression in eukaryotic cells (as in the case of nucleic acid vaccine vectors), the expression control region should be adapted for this particular use.

[0149] Therefore, for production purposes, it is often convenient for the expression control region to drive expression in prokaryotic cells such as bacteria, for example Escherichia coli (E. coli), or eukaryotic cells such as plant cells, insect cells, or mammalian cells. For vaccine purposes, the expression control region must be able to drive expression in mammalian, preferably human cells.

[0150] Also, for production purposes, it is practical for the vector to be able to integrate nucleic acid into the genome of the host cell, which is particularly useful when the vector is used for the production of stably transformed cells whose progeny also contain the genetic information introduced via the vector. Alternatively, a vector that cannot integrate into the genome of a fish host cell is useful, for example, in nucleic acid vaccination.

[0151] An interesting production system is the use of plants. For example, an Agrobacterium transfection system can be used to genetically modify plants to express a gene encoding a target protein, allowing for low-cost production of the protein in plants. One commercially available platform is provided by iBio CMO LLC (8800 HSC Pkwy, Bryan, TX 77807, USA) and iBio, Inc (9 Innovation Way, Suite 100, Newark, DE 19711, USA) and is disclosed, for example, in European Patent No. 2853599, European Patent No. 1769068, and European Patent No. 2192172. Thus, in such a system, the vector is an Agrobacterium vector or other vector suitable for transfection of plants.

[0152] Vectors are typically selected from the group consisting of viruses such as viruses that are non-pathogenic in mammals, particularly humans, bacteria such as bacteria that are non-pathogenic in mammals such as humans, plasmids, minichromosomes, and cosmids.

[0153] Interesting vectors are viral vectors (especially those useful as vaccines in humans). These can be selected from the group consisting of retroviral vectors such as lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, and poxviral vectors. Certain poxviral vectors, particularly vaccinia virus vectors, are preferred. A particularly preferred vaccinia virus vector is the modified vaccinia Ankara (MVA) vector.

[0154] As shown, the polypeptide of the present invention or a polypeptide that is part of the composition of the present invention can be encoded by a nucleic acid molecule contained in a vector. The nucleic acid sequence may be "heterologous", which means that it is in a context foreign to the cell into which the vector is introduced and includes a sequence that is homologous to a sequence in the cell but is located at a position in the host cell where it is not normally found.

[0155] Vectors include naked DNA, RNA, plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YAC). Those skilled in the art have sufficient ability to construct vectors via standard recombinant techniques. In addition to encoding the polypeptide of the present invention that is part of the composition of the present invention, the vector can encode polypeptide sequences such as "tags" or immunogenic enhancing peptides (e.g., immunogenic carriers or fusion partners that stimulate the immune system, such as cytokines or active fragments thereof). Useful vectors for encoding such fusion proteins include pIN vectors, vectors encoding stretches of histidine residues, and pGEX vectors for use in producing glutathione S-transferase (GST) soluble fusion proteins for later purification and separation or cleavage.

[0156] The vector can be used to produce, in a host cell, the polypeptide of the invention or a polypeptide that is part of the composition of the invention, which can then be purified for administration, or the vector can be purified for direct administration for protein expression (as in the case of administering a nucleic acid vaccine).

[0157] Expression vectors can contain various "control sequences" that refer to nucleic acid sequences necessary for the transcription and optionally translation of a coding sequence operably linked in a particular host organism. In addition to the control sequences that govern transcription and translation, vectors and expression vectors can also perform other functions and can contain the nucleic acid sequences described below.

[0158] 1. Promoters and enhancers A "promoter" is a control sequence. A promoter is typically a region of a nucleic acid sequence where the initiation and rate of transcription are controlled. This can include genetic elements to which regulatory proteins and molecules can bind, such as RNA polymerase and other transcription factors. The phrases "operatively positioned", "operatively linked", "under control", and "under transcriptional control" mean that the promoter is in the correct functional position and / or orientation with respect to a nucleic acid sequence to control the initiation and expression of that sequence. A promoter may or may not be used in conjunction with an "enhancer", which refers to a cis-acting regulatory sequence involved in activating the transcription of a nucleic acid sequence.

[0159] A promoter may be one that is naturally associated with a gene or sequence, such as can be obtained by isolating a 5' non-coding sequence located upstream of a coding segment or exon. Such a promoter may be referred to as "endogenous". Similarly, an enhancer may be one that is naturally associated with a nucleic acid sequence and is located either downstream or upstream of that sequence. Alternatively, certain advantages can be obtained by placing a coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that does not normally associate with a nucleic acid sequence in its natural environment. A recombinant enhancer or heterologous enhancer also refers to an enhancer that does not normally associate with a nucleic acid sequence in its natural state. Such promoters or enhancers can include promoters or enhancers from other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, as well as promoters or enhancers that are not "naturally occurring", i.e., contain different elements of different transcriptional regulatory regions and / or mutations that alter expression. In addition to synthesizing the nucleic acid sequences of promoters and enhancers, the sequences can be produced using recombinant cloning and / or nucleic acid amplification techniques, including polymerase chain reaction, related to the compositions disclosed herein.

[0160] It may be important to use a promoter and / or enhancer that effectively induces the expression of a DNA segment in a cell type or organism selected for expression. Those of ordinary skill in the art of molecular biology generally know about the use of combinations of promoters, enhancers, and cell types for protein expression. The promoter used may be constitutive, tissue-specific, or inducible, and in certain embodiments, may induce high-level expression of the introduced DNA segment under specific conditions, such as the large-scale production of a recombinant protein or peptide.

[0161] Examples of inducible elements, which are regions of nucleic acid sequences that can be activated in response to specific stimuli, include immunoglobulin heavy chain, immunoglobulin light chain, T cell receptor, HLA DQα and / or DQβ, β-interferon, interleukin-2, interleukin-2 receptor, MHC class II 5, MHC class II HLA-DRα, β-actin, muscle creatine kinase (MCK), prealbumin (transthyretin), elastase I, metallothionein (MTII), collagenase, albumin, α-fetoprotein, γ-globulin, β-globulin, c-fos, c-HA-ras, insulin, neural cell adhesion molecule (NCAM), α1-antitrypsin, H2B (TH2B) histone, mouse and / or type I collagen, glucose-regulated proteins (GRP94 and GRP78), rat growth hormone, human serum amyloid A (SAA), troponin I (TN I), platelet-derived growth factor (PDGF), Duchenne muscular dystrophy, SV40, polyoma, retrovirus, papillomavirus, hepatitis B virus, human immunodeficiency virus, cytomegalovirus (CMV) IE, and simian sarcoma virus, but are not limited thereto.

[0162] Examples of inducible elements include MT II-phorbol ester (TFA) / heavy metals; MMTV (mouse mammary tumor virus)-glucocorticoid; β-interferon-poly(rI)x / poly(rC); adenovirus 5 E2-ElA; collagenase-phorbol ester (TPA); stromelysin-phorbol ester (TPA); SV40-phorbol ester (TPA); mouse MX gene-interferon, Newcastle disease virus; GRP78 gene-A23187; α-2-macroglobulin-IL-6; vimentin-serum; MHC class I gene H-2κb-interferon; HSP70-E1A / SV40 large T antigen; proliferin-phorbol ester / TPA; tumor necrosis factor-PMA; and thyroid stimulating hormone α gene-thyroid hormone.

[0163] Also considered useful in the present invention are the dectin-1 and dectin-2 promoters. Furthermore, any combination of promoters / enhancers (according to the eukaryotic promoter database EPDB) can also be used to drive the expression of a structural gene encoding an oligosaccharide processing enzyme, a protein folding assisting protein, a selectable marker protein or a heterologous protein of interest.

[0164] The specific promoter used to control the expression of a polynucleotide encoding a peptide or protein is not considered important as long as the polynucleotide can be expressed in the target cell. When fish cells are targeted (as in the case of nucleic acid vaccination), it is preferred to place the polynucleotide coding region adjacent to and under the control of a promoter that can be expressed in fish cells. Generally speaking, such a promoter can include any of a bacterial, fish or viral promoter as long as the promoter is effective in fish cells.

[0165] In various embodiments, particularly those where the recombinant production of polypeptides is desired, the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, and the Rous sarcoma virus long terminal repeat can be used to obtain high-level expression of the relevant polynucleotide. The use of other viral promoters or mammalian cell promoters or bacteriophage promoters well known in the art to achieve the expression of polynucleotides is also contemplated.

[0166] In embodiments where the vector is administered to a human for protein expression, desirable promoters for use with the vector are those that are not downregulated by cytokines, or, even if downregulated, are strong enough to produce an effective amount of the protein / polypeptide of the present invention (or useful in the compositions of the present invention) in humans to elicit an immune response. Non-limiting examples of these are CMV IE and RSV LTR. In other embodiments, promoters that are upregulated in the presence of cytokines are used. The MHC I promoter increases expression in the presence of IFN-γ.

[0167] In particular, tissue-specific promoters can be used when expression is desired in cells such as dendritic cells and macrophages where antigen expression is desired. Mammalian MHC I and MHC II promoters are examples of such tissue-specific promoters in humans, and it is contemplated that the corresponding fish promoters will be effective.

[0168] 2. Initiation signals and internal ribosome entry sites (IRES) Certain initiation signals may also be required for efficient translation of the coding sequence. These signals include the ATG start codon or adjacent sequences. It may be necessary to provide an exogenous translation control signal containing the ATG start codon. Those skilled in the art can readily determine this and provide the necessary signals. It is well known that the start codon must be "in-frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. The exogenous translation control signal and start codon may be either natural or synthetic and may be operable in bacterial or mammalian cells. The efficiency of expression can be enhanced by including appropriate transcriptional enhancer elements.

[0169] In certain embodiments of the invention, internal ribosome entry site (IRES) elements are used to create multi-gene messages or polycistronic messages. IRES elements can bypass the ribosome scanning model of 5'-methylated Cap-dependent translation and initiate translation at internal sites. IRES elements from two members of the picornavirus family (poliovirus and encephalomyocarditis), as well as IRESs from mammalian messages, have been described. IRES elements can be ligated to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES to create a polycistronic message. Thanks to the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter / enhancer to transcribe a single message (see U.S. Patent Nos. 5,925,565 and 5,935,819, which are incorporated herein by reference).

[0170] 3. Multiple Cloning Site The vector can include a multiple cloning site (MCS), a nucleic acid region containing multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant techniques to digest the vector. Frequently, the vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to allow an exogenous sequence to be ligated to the vector. Techniques involving restriction enzymes and ligation reactions are well known to those of ordinary skill in the art of recombinant techniques.

[0171] 4. Splicing Sites Most transcribed eukaryotic RNA molecules undergo RNA splicing to remove introns from the primary transcript. When relevant, vectors containing genomic eukaryotic sequences may require donor and / or acceptor splicing sites to ensure proper processing of the transcript for protein expression.

[0172] 5. Termination Signal Vectors or constructs disclosed herein generally include at least one termination signal. A "termination signal" or "terminator" consists of a DNA sequence involved in the specific termination of an RNA transcript by RNA polymerase. Thus, in certain embodiments, a termination signal that ends the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve the desired message level.

[0173] In eukaryotes, the terminator region may also include specific DNA sequences that allow for site-specific cleavage of the new transcript so as to expose the polyadenylation site. This signals a specialized endogenous polymerase to add a stretch of approximately 200 A residues (polyA) to the 3' end of the transcript. RNA molecules modified with this polyA tail are thought to be more stable and to be translated more efficiently. Thus, in other embodiments, including eukaryotes, it is preferred that the terminator include a signal for cleavage of the RNA, and more preferably that the terminator signal promote polyadenylation of the message.

[0174] Terminators contemplated for use in the present invention include any known transcriptional terminator described herein or known to those of skill in the art, including, but not limited to, viral termination sequences such as the bovine growth hormone terminator or the SV40 terminator. In certain embodiments, the termination signal may be the absence of a transcribable or translatable sequence, such as by truncation of the sequence.

[0175] 6. Polyadenylation Signal In expression, particularly in eukaryotic expression (related to nucleic acid vaccination), it typically contains a polyadenylation signal to effect proper polyadenylation of the transcript. The nature of the polyadenylation signal is not considered important for the successful practice of the present invention and / or any such sequence may be used. Preferred embodiments include the SV40 polyadenylation signal and / or the bovine growth hormone polyadenylation signal, which are convenient and / or known to function well in various target cells. Polyadenylation can increase the stability of the transcript or facilitate cytoplasmic transport.

[0176] 7. Origin of replication It may contain one or more origin of replication sites (often called "ori"), which are specific nucleic acid sequences at which replication is initiated, to propagate the vector in the host cell. Alternatively, when the host cell is yeast, an autonomously replicating sequence (ARS) can be used.

[0177] 8. Selectable markers and screenable markers In certain embodiments of the present invention, cells containing a nucleic acid construct can be identified in vitro or in vivo by encoding a screenable marker or a selectable marker in the expression vector. When transcribed and translated, the marker causes a distinguishable change in the cell, allowing easy identification of cells containing the expression vector. Generally, a selectable marker confers a property that enables selection. A positive selectable marker is one whose presence enables its selection, while a negative selectable marker is one whose presence prevents its selection. An example of a positive selectable marker is a drug resistance marker.

[0178] Typically, including a drug selection marker aids in the cloning and identification of transformants. For example, markers that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, or histidinol are useful selectable markers. In addition to markers that confer a phenotype allowing for the discrimination of transformants based on the implementation of conditions, other types of markers include screenable markers such as GFP for colorimetric analysis. Alternatively, screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) can be utilized. Those skilled in the art will also know how to use immunological markers that can be used in combination with FACS analysis. The marker used is not considered important as long as it can be expressed simultaneously with the nucleic acid encoding the protein of the present invention. Further examples of selectable and screenable markers are well known to those skilled in the art.

[0179] Transformed cell The transformed cells are useful as organisms for producing the polypeptides of the present invention and the compositions of the present invention, but are also useful as simple "containers" for the nucleic acids and vectors of the present invention.

[0180] Certain transformed cells containing the cells of the present invention can replicate a nucleic acid fragment containing the nucleic acid fragment of the present invention. Preferred transformed cells can express the nucleic acid fragment.

[0181] For recombinant production, since the proteins disclosed herein are of bacterial origin, it is convenient for the transformed cells to be prokaryotes such as bacteria. Generally, however, both prokaryotic and eukaryotic cells can be used.

[0182] Suitable prokaryotic cells are bacterial cells (preferably non-pathogenic) selected from the group consisting of Escherichia (e.g., E. coli), Bacillus (e.g., Bacillus subtilis), Salmonella, and Mycobacterium (e.g., Mycobacterium bovis BCG).

[0183] Eukaryotic cells may be in the form of yeast (e.g., Saccharomyces cerevisiae) and protozoa. Alternatively, the transformed eukaryotic cells are derived from multicellular organisms such as filamentous fungi, insect cells, plant cells, or mammalian cells.

[0184] For the purpose of production, it is advantageous for the transformed cells to be stably transformed by having the nucleic acid stably integrated into their genome. In certain embodiments, it is also preferred that the transformed cells secrete or carry on their surface the polypeptide of the present invention, as this facilitates the recovery of the produced polypeptide. A particular version of this embodiment is where the transformed cells are bacteria and the secretion of the polypeptide of the present invention is into the periplasmic space.

[0185] As described above, stably transformed cells are preferred, as they enable, inter alia, the establishment of cell lines consisting of the transformed cells as defined herein, and such cell lines are particularly preferred.

[0186] Further details regarding the cells and cell lines are provided below.

[0187] Cells suitable for recombinant nucleic acid expression of nucleic acid fragments such as those of the present invention are prokaryotes and eukaryotes. Examples of prokaryotic cells include Escherichia coli; members of the genus Staphylococcus such as Staphylococcus epidermidis; members of the genus Lactobacillus such as Lactobacillus plantarum; members of the genus Lactococcus such as Lactococcus lactis; members of the genus Bacillus such as Bacillus subtilis; members of the genus Corynebacterium such as Corynebacterium glutamicum; and members of the genus Pseudomonas such as Pseudomonas fluorescens. Examples of eukaryotic cells include mammalian cells; insect cells; members of the genus Saccharomyces (e.g., Saccharomyces cerevisiae), members of the genus Pichia (e.g., Pichia pastoris), members of the genus Hansenula (e.g., Hansenula polymorpha), members of the genus Kluyveromyces (e.g., Kluyveromyces lactis or Kluyveromyces fragilis), and members of the genus Schizosaccharomyces (e.g., Schizosaccharomyces pombe), such as yeast cells.

[0188] Techniques for the production, introduction into cells, and expression of recombinant genes are well known in the art. Examples of such techniques are provided in references such as Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987 - 2002, and Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989.

[0189] As used herein, the terms "cell", "cell line", and "cell culture" may be used interchangeably. All of these terms also include their progeny, which includes any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or accidental mutations. In the context of expressing a heterologous nucleic acid sequence, a "host cell" refers to a prokaryotic or eukaryotic cell and includes any transformable organism capable of replicating a vector or expressing a heterologous gene encoded by the vector. A host cell can be used and has been used as a recipient for a vector or virus. A host cell may be "transfected" or "transformed", which refers to the process by which an exogenous nucleic acid, such as a recombinant protein coding sequence, is introduced or transferred into the host cell. Transformed cells include primary subject cells and their progeny.

[0190] Host cells can be derived from prokaryotes or eukaryotes, including bacteria, yeast cells, insect cells, and mammalian cells, for the replication of vectors or the expression of some or all of the nucleic acid sequences. Many cell lines and cultures are available for use as host cells, and they can be obtained through the American Type Culture Collection (ATCC) (www.atcc.org), which serves as a living culture and an archive of genetic material, or from other depository institutions such as the Deutsche Sammlung vor Micrroorganismen und Zellkulturen (DSM). Appropriate hosts can be determined by those skilled in the art based on the vector backbone and the desired results. For example, plasmids or cosmids can be introduced into prokaryotic host cells for the replication of many vectors or the expression of the encoded proteins. Bacterial cells used as host cells for vector replication and / or expression include Staphylococcus strains, DH5α, JMI09, and KC8, as well as numerous commercially available bacterial hosts such as SURE® competent cells and SOLOP ACK™ gold cells (STRATAGENE®, La Jolla, CA). Alternatively, bacterial cells such as Escherichia coli LE392 can be used as host cells for phage viruses. Appropriate yeast cells include Saccharomyces cerevisiae, Saccharomyces pombe, and Pichia pastoris.

[0191] Examples of eukaryotic host cells for vector replication and / or expression include HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12. Many host cells from various cell types and organisms are available and would be known to those skilled in the art. Similarly, viral vectors can be used in combination with either eukaryotic host cells or prokaryotic host cells, particularly host cells that permit the replication or expression of the vector.

[0192] Some vectors may use control sequences that enable replication and / or expression in both prokaryotic and eukaryotic cells. Those skilled in the art will further understand the conditions for incubating and maintaining all of the above host cells to enable vector replication. Also understood and known are the techniques and conditions for large-scale production of vectors, as well as for the production of nucleic acids encoded by the vectors and their cognate polypeptides, proteins, or peptides.

[0193] Expression system There are numerous expression systems that include at least some or all of the compositions discussed above. Prokaryotic and / or eukaryotic-based systems can be used in the present invention to produce nucleic acid sequences, or their cognate polypeptides, proteins, and peptides. Many such systems are widely commercially available.

[0194] The insect cell / baculovirus system can result in high-level protein expression of heterologous nucleic acid segments, as described in U.S. Patent Nos. 5,871,986 and 4,879,236, both of which are incorporated herein by reference, and can be purchased, for example, from INVITROGEN® under the name MAXBAC® 2.0 and from CLONTECH® under the name BACPACKTM Baculovirus Expression System.

[0195] In addition to the disclosed expression systems, other examples of expression systems include the COMPLETE CONTROL™ inducible mammalian expression system from STRATAGENE® that includes a synthetic ecdysone receptor, or its pET expression system, E. coli expression system. Another example of an inducible expression system is available from INVITROGEN®, which has the T-REX™ (tetracycline-regulated expression) system, an inducible mammalian expression system using a full-length CMV promoter. INVITROGEN® also offers a yeast expression system called the Pichia methanolica expression system, which is designed for high-level production of recombinant proteins in the methylotrophic yeast Pichia methanolica. Those skilled in the art will know how to express vectors such as expression constructs to produce nucleic acid sequences or their cognate polypeptides, proteins, or peptides.

[0196] Method of gene introduction Suitable methods for nucleic acid delivery to effect expression of the compositions or nucleic acid fragments of the invention include substantially any method by which a nucleic acid (e.g., DNA including viral and non-viral vectors) can be introduced into a cell, tissue or organism, as described herein or as known to those of skill in the art. Such methods include injection, including microinjection (U.S. Patent No. 5,789,215) (U.S. Patent Nos. 5,994,624; 5,981,274; 5,945,100; 5,780,448; 5,736,524; 5,702,932; 5,656,610; 5,589,466; 5,580,859); electroporation (U.S. Patent No. 5,384,253); calcium phosphate precipitation; use of DEAE dextran and subsequent polyethylene glycol; direct sonoporation; liposome-mediated transfection; particle bombardment (PCT Application Nos. WO94 / 09699 and 95 / 06128; U.S. Patent Nos. 5,610,042; 5,322,783; 5,563,055; 5,550,318; 5,538,877; 5,538,880); agitation with silicon carbide fibers (U.S. Patent Nos. 5,302,523 and 5,464,765); Agrobacterium-mediated transformation (U.S. Patent Nos. 5,591,616 and 5,563,055); or PEG-mediated transformation of protoplasts (U.S. Patent Nos. 4,684,611 and 4,952,500); direct delivery of DNA such as by dry / inhibition-mediated DNA uptake, but are not limited to these. Through the application of such techniques, an organelle, cell, tissue or organism can be stably or transiently transformed.

[0197] Composition of the present invention; Vaccine Compositions according to the invention, particularly vaccines, are prophylactic but can also be used therapeutically.

[0198] Such vaccines typically include an immunizing antigen, immunogen, polypeptide, protein, or nucleic acid in combination with a "pharmaceutically acceptable carrier" that usually includes any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition.

[0199] In some embodiments of the invention, a pharmaceutical composition such as a vaccine includes only a single antigen, immunogen, polypeptide, protein, nucleic acid, or vector of the invention, while in other embodiments, the pharmaceutical composition includes a "cocktail" of antigens or immunogens or polypeptides or proteins or nucleic acids or vectors.

[0200] In an interesting embodiment, the pharmaceutical composition is a vector as described herein that encodes at least two nucleic acid fragments of the invention and can effect their expression.

[0201] Another interesting embodiment of the pharmaceutical composition includes RNA as an active ingredient, i.e., at least one mRNA encoding a polypeptide of the invention.

[0202] Embodiments of the pharmaceutical composition of the invention include at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) distinct polypeptides as described above.

[0203] Another embodiment of the pharmaceutical composition of the invention includes at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) different nucleic acid molecules (e.g., DNA and RNA) each encoding one of the above polypeptides.

[0204] Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactivated virus particles.

[0205] Such carriers are well known to those skilled in the art. Furthermore, these carriers can function as immunostimulants (''adjuvants''). Further, the antigen or immunogen can be conjugated to a bacterial toxoid such as a toxoid derived from diphtheria, tetanus, cholera, H. pylori, etc.

[0206] Thus, the pharmaceutical composition of the present invention typically contains an immunological adjuvant which is generally an aluminum-based adjuvant or one of the other adjuvants described below.

[0207] Preferred adjuvants for enhancing the effectiveness of the composition include: (1) aluminum salts (alum) such as aluminum hydroxide (AlOH), aluminum phosphate, and aluminum sulfate; (2) oil-in-water emulsion formulations (with or without other specific immunostimulants such as muramyl peptides (see below) or bacterial cell wall components), for example (a) MF59 (WO90 / 14837; Chapter 10 in Vaccine design: the subunit and adjuvant approach, eds. Powell & Newman, Plenum Press 1995) formulated into submicron particles using a microfluidizer such as a Model 110Y Microfluidizer (Microfluidics, Newton, MA), containing 5% squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE (see below)); (b) SAF containing 10% squalene, 0.4% Tween 80, 5% pluronic-block polymer L121, and thr-MDP, which is either microfluidized into a submicron emulsion or vortexed to produce an emulsion with a larger particle size; and (c) 2% squalene, 0.Ribi adjuvant system (RAS) (Ribi Immunochem, Hamilton, MT) containing 2% Tween 80 and one or more bacterial cell components from the group consisting of monophosphoryl lipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL + CWS (DetoxTM); (3) saponin adjuvants such as Stimulon™ (Cambridge Bioscience, Worcester, MA) or particles generated therefrom such as ISCOMs (immunostimulating complexes) can be used; (4) complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA); (5) cytokines such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., gamma interferon), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; and (6) other substances that act as immunostimulants to enhance the effectiveness of the composition, but are not limited thereto. Alum and MF59™ adjuvant are preferably used in combination with CFA and IFA.

[0208] As described above, muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2''-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.

[0209] Other interesting adjuvants are disclosed in Didierlaurent AM et al., Expert Rev. Vaccines 2017; 16: 55-63; Gonzales-Lopez A. et al., Clin. Immunol. 2019; 209, 108275. Additionally, TLR4 ligand adjuvants such as SLA-SE are also preferred adjuvant systems (see Reed S.G. et al., Current Opinion in Immunology 2016, 41: 85-90; Liang H. et al., npj Vaccines 2020, 4:19, doi.org / 10.1038 / s41541-019-0116-6; van Hoeven N. et al., PLOS One, 2016, DOI:10.1371 / journal.pone.0149610; and Reed S. G. et al., Seminars in Immunology 2018, 39: 22-29).

[0210] Another preferred adjuvant system is disclosed in Agger EM et al., PLoS One. 2008; 3(9): e3116; van Dissel JD et al., Vaccine. 2014 Dec 12;32(52):7098-107. doi: 10.1016 / j.vaccine.2014.10.036. Epub 2014 Oct 30; and Dietrich J et al., PLoS One. 2014 Jun 23;9(6):e100879. doi: 10.1371 / journal.pone.0100879. eCollection 2014.

[0211] Another particularly preferred adjuvant system is outer membrane vesicles (OMVs); see Kaparakis-Liaskos, M. & Ferrero, R.L., 2015. Immune modulation by bacterial outer membrane vesicles. Nat Rev Immunol. 15:375-387; Russo, A.J. et al., 2018. Emerging insights into noncanonical inflammasome recognition of microbes. J Mol Biol. 430:207-216; and Tan K et al., Front Microbiol. 2018; 9: 783; doi: 10.3389 / fmicb.2018.00783. In one interesting embodiment, the OMVs are used in combination with aluminum hydroxide.

[0212] Another possibility for polypeptide vaccine formulations is to include the vaccine polypeptide within virus-like particles, i.e., non-infectious self-assembling structures composed of envelope or capsid proteins into which the protein is incorporated. The effect is the multiple presentation of the polypeptide of the invention on the surface of the VLP, which in turn provides improved immune recognition of the polypeptide. Thus, VLP also exerts an immunological adjuvant effect.

[0213] Immunogenic compositions (e.g., immune antigens or immunogens or polypeptides or proteins or nucleic acids, pharmaceutically acceptable carriers, and adjuvants) typically contain diluents such as water, saline, glycerol, ethanol, etc. In addition, auxiliary substances such as wetting or emulsifying agents, pH buffering substances may be present in such media.

[0214] Typically, an immunogenic composition is prepared as either a liquid solution or suspension for injectability and may also be prepared in solid form suitable for making a solution or suspension in a liquid vehicle prior to injection. The preparation may also be emulsified or encapsulated in liposomes for enhanced adjuvant effect, as described above, under a pharmaceutically acceptable carrier.

[0215] An immunogenic polypeptide composition for use as a vaccine comprises an immunologically effective amount of an antigenic polypeptide or immunogenic polypeptide, and optionally any other of the above components. "Immunologically effective amount" means that administration of that amount to an individual, either as a single dose or as part of a series of doses, is effective for treatment or prevention. This amount will vary depending on the health and physical condition of the individual being treated, the taxonomic group of the individual being treated (e.g., non-human primate, primate, etc.), the ability of the individual's immune system to synthesize antibodies or generally initiate an immune response, the degree of protection desired, the formulation of the vaccine, the assessment of the treating physician of the medical situation, and other relevant factors. The amount of immunogen is expected to fall within a relatively wide range that can be determined through routine testing. However, for the purpose of protein vaccination, the dosage per immunization typically ranges from 0.5 μg to 500 mg (although it is often not higher than 5,000 μg). Thus, the amount of the polypeptide of the present invention may be 1 to 400 μg, 2 to 350 μg, 4 to 300 μg, 5 to 250 μg, and 10 to 200 μg. Thus, the composition typically contains 0.1 to 500 μg of the protein of the present invention per gram of vaccine composition.

[0216] Immunogenic compositions have conventionally been administered parenterally, for example, by injection either subcutaneously, intramuscularly, or transdermally / transcutaneous (see, e.g., WO98 / 20734). Further formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications. In the case of nucleic acid vaccination, intravenous or intraarterial routes may also be applicable.

[0217] Drug treatment may be on a single-dose schedule or a multiple-dose schedule. The vaccine may be administered together with other immunomodulators.

[0218] As an alternative to protein-based vaccines, DNA vaccination (also referred to as nucleic acid vaccination or gene vaccination) can be used (see, for example, Robinson & Torres (1997) Seminars in Immunol 9: 271-283; Donnelly et al. (1997) Annu Rev Immunol 15: 617-648).

[0219] A further aspect of the invention, as described above, is the recognition that a mixed vaccine can be provided that enhances the immune response by vaccinated individuals by combining two or more polypeptide antigens disclosed herein to optimize the initial immune response and the duration of immunity. For the purposes of this aspect of the invention, multiple antigenic fragments derived from the same or longer protein can also be used, for example, the use of combinations of polypeptide sequence fragments of different lengths from one protein.

[0220] Accordingly, embodiments of the invention relate to a composition (or its use as a vaccine) comprising two different (i.e., non-identical) proteinaceous immunogens disclosed herein.

[0221] Immunization method The methods of this aspect of the invention generally relate to the induction of immunity and thus require prophylactic as well as therapeutic methods.

[0222] When an immunization method requires administration of a polypeptide of the invention or a polypeptide composition of the invention, an animal (e.g., a human) typically receives from 0.5 to 5,000 μg of polypeptide per administration; see the above instructions regarding dosage.

[0223] In a preferred embodiment, the immunization scheme includes an initial administration of the chimeric polypeptide, nucleic acid / vector, or composition of the invention, although one or more booster immunizations may be required.

[0224] A preferred embodiment includes that the administration is for the purpose of inducing protective immunity against Staphylococcus aureus. In this embodiment, it is particularly preferred that the protective immunity is effective in reducing the risk of attracting an infection by Staphylococcus aureus.

[0225] Some vaccine compositions of the invention induce humoral immunity, and thus the administration is preferably for the purpose of inducing antibodies specific for Staphylococcus aureus. However, as also mentioned, the immunization method may also be useful in antibody production, and thus in other embodiments, the administration is for the purpose of inducing antibodies specific for Staphylococcus aureus, and the B lymphocytes producing said antibodies are then recovered from the animal and used for the preparation of monoclonal antibodies.

[0226] The composition for immunization can contain a polypeptide, nucleic acid, vector, virus or cell as described above. The pharmaceutical composition contains a therapeutically effective amount thereof.

[0227] As used herein, the terms "therapeutically effective amount" or "prophylactically effective amount" refer to the amount of a therapeutic agent for treating, ameliorating, or preventing a desired disease or condition, or for showing a detectable prophylactic effect in a group of mammals such as humans. This effect can be detected, for example, by chemical markers or antigen levels. See above for the dosage range of an immunologically effective amount of a polypeptide. However, the effective amount for a given situation can be determined by routine experimentation and is within the scope of a clinician's judgment.

[0228] For the purposes of the present invention, the effective dose of the nucleic acid vaccine (vector) is a DNA or RNA construct of about 0.01 mg / kg to 50 mg / kg or 0.05 mg / kg to about 10 mg / kg in the animal to which it is administered.

[0229] The pharmaceutical composition can also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administering therapeutic agents such as antibodies or polypeptides, genes, and other therapeutic agents. This term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual to whom the composition is administered and can be administered without undue toxicity. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, and inactivated virus particles. Such carriers are known to those skilled in the art.

[0230] Pharmaceutically acceptable salts, such as mineral salts such as hydrochloride, hydrobromide, phosphate, sulfate; and organic acid salts such as acetate, propionate, malonate, benzoate, etc. can be used therein. A thorough discussion of pharmaceutically acceptable excipients is available in Remington’s Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

[0231] The pharmaceutically acceptable carrier in the therapeutic composition may include liquids such as water, physiological saline, glycerol and ethanol. Further, auxiliary substances such as wetting agents or emulsifiers, pH buffering substances may be present in such media. Typically, the therapeutic composition is prepared as either a liquid solution or suspension for injection, and solid forms suitable for making a solution or suspension in a liquid medium prior to injection can also be prepared. Liposomes are included within the definition of pharmaceutically acceptable carriers.

[0232] Specific amino acid sequences disclosed herein In this application, the following amino acid sequences are referred to by both the sequence numbers and the separate names (provided in parentheses) used herein. TIFF2025516318000002.tif130160 TIFF2025516318000003.tif252160 TIFF2025516318000004.tif252160 TIFF2025516318000005.tif252160 TIFF2025516318000006.tif249156 TIFF2025516318000007.tif251160 TIFF2025516318000008.tif237160 TIFF2025516318000009.tif253160 TIFF2025516318000010.tif235160 TIFF2025516318000011.tif251160 TIFF2025516318000012.tif63160

[0233] Table 1 shows an overview of all of the above amino acid sequences by reference to their nomenclature and any alternative nomenclature.

[0234] The following examples are provided for the purpose of illustrating various embodiments of the invention and are not meant to limit the invention in any way. Together with the methods described herein, these examples presently represent preferred embodiments, are exemplary, and are not intended to limit the scope of the invention. Those changes and other uses that are encompassed within the appended claims will be understood by those skilled in the art.

[0235] [Table 1]

[0236] [Example 1] Functional evaluation of staphylococcal protein variants 1.1 SpA variants Various SpA variants were tested for their ability to bind to Ig and von Willebrand factor (vWF).

[0237] Using ELISA, it was found that the following SpA variants did not bind to any of whole serum, purified human and mouse IgG, and mouse IgG, rabbit IgG, and mouse IgM from vWF in ELISA.

[0238] [Table 2]

[0239] More detailed data on the Ig-binding ability of SpA variants are shown below.

[0240] IgG affinity chromatography Based on disruptive mutations in SpA, an affinity chromatography assay was set up to evaluate the ability of different SpA constructs not to bind to human IgG. The principle of the assay was to immobilize a His-tagged SpA protein (neutralizing the His tag) on a chromatography column and then add purified human IgG (neutralizing the His tag of the SpA variant construct prior to testing for human IgG-SpA binding). The bound material was then eluted and run on an SDS-PAGE gel to visualize IgG protein binding.

[0241] The protocol was adopted from Kim et al., 2010; 2015 and slightly modified. Purified His-tagged SpA protein (100 μg (0.5 μg / μL) in 200 μL) was immobilized on Ni-NTA agarose (100 μL, Qiagen #30230, lot 151029515) at room temperature (RT) for 20 minutes, washed with binding buffer (50 mM Tris, 150 mM NaCl, pH 7.5) over 6 CV or 10 CV, and incubated with human IgG (100 μg (0.5 μg / μL) in 200 μL, Sigma #I2511-10MG, lot 048M4868V) at room temperature for 20 minutes. After washing with binding buffer supplemented with 30 mM imidazole over 20 CV or 10 CV, the bound protein was eluted with binding buffer supplemented with 500 mM imidazole over 5 CV and analyzed by SDS-PAGE (10 μL / fraction, 2.5 μg of starting material) (Any kDa Criterion TGX Stain Free Protein Gel and Pro Blue Safe Stain, Giotto Biotech).

[0242] Results from human IgG affinity chromatography showed that SpA_WT-49-339 (wild-type (WT) full-length SpA) (SEQ ID NO: 16) binds to the total amount of IgG loaded, while immobilized SpA KKAA -49-339 mutants and several SpA mutant variants, e.g., SpA_mut_7_12 (SEQ ID NO: 18) and SpA_mut_10_12 (SEQ ID NO: 17), did not bind to human IgG (Table 3).

[0243]

Table 3

[0244] Direct ELISA To evaluate the ability of different SpA mutant constructs to bind a wide range of immunoglobulins, direct ELISA was applied to assess the binding ability to human IgG, human vWF, mouse IgG and IgM, and rabbit IgG. The protocol described in Kim et al., 2015 was followed with slight adjustments according to the Ig evaluated in the assay.

[0245] To evaluate the inhibition of mouse IgG binding to SpA mutant variants, the protocol described in Kim et al., 2015 was used with the following adjustments. Briefly, wells were coated overnight at 4 °C with 300 ng of protein / well diluted in PBS (pH 7.6). Either a 5-fold serial dilution of purified mouse IgG (Sigma-Aldrich, #I8765-10MG, lot SLBW5857) starting from 40 ng / well or a 5-fold serial dilution of non-specific mouse serum starting from 1:1,000 was added to the plate. Dilutions of 1:5,000 or 1:2,000 of goat anti-mouse IgG antibody conjugated to alkaline phosphatase (Sigma-Aldrich, A3562, lot SLBK6489V) were added to the plate. As substrate, 3 mg / mL p-nitrophenyl phosphate (p-NPP; Sigma-Aldrich, P5869-25CAP, lot SLBV2576) in diethanolamine (DEA, pH 9.2; Sigma-Aldrich, D8885-500g, lot MKCB9120) was used. Absorbance at 405 nm was read using a Tecan Infinite M200Pro device, either 30 or 55 min after the reaction, respectively.

[0246] To evaluate the inhibition of purified human IgG binding to the SpA mutant variant, the protocol described by Kim et al., 2015 was used with the following modifications. Briefly, wells were coated overnight at 4 °C with 300 ng of SpA mutant protein / well diluted in PBS (pH 7.6). Starting from 40 ng / well, serially five-fold diluted human IgG (Sigma-Aldrich, #I2511-10MG, 048M4868V) was added to the plate, 1:10,000 alkaline phosphatase-conjugated goat anti-human IgG (Sigma-Aldrich, #A1543-1ML, lot 029M4838V) was used as the secondary antibody, and absorbance at 405 nm was read 37 min after the reaction using a Tecan Infinite M200Pro device.

[0247] To evaluate the inhibition of non-specific rabbit IgG binding to the SpA mutant variant, the protocol described by Kim et al., 2015 was used with the following modifications. Briefly, wells were coated overnight at 4 °C with 300 ng of SpA mutant protein / well diluted in PBS (pH 7.6). Starting from 1:1,000 dilution, serially three-fold diluted non-specific rabbit serum was added to the plate, 1:10,000 goat anti-rabbit IgG conjugated alkaline phosphatase (Sigma-Aldrich, A3687) was used as the secondary antibody, and absorbance at 405 nm was read 55 min after the reaction using a Tecan Infinite M200Pro device.

[0248] To evaluate the inhibition of non-specific mouse IgM binding to the SpA mutant variant, the protocol described by Kim et al., 2015 was used with the following adjustments. Briefly, wells were coated overnight at 4°C with 300 ng of SpA mutant protein / well diluted in PBS (pH 7.6). Non-specific mouse sera diluted 3-fold starting from a 1:1,000 dilution were added to the plates, and goat anti-mouse IgM conjugated alkaline phosphatase (Southern Biotech N1021-04, lot C7107RL27Z) at 1:2,000 was used as the secondary antibody. Absorbance at 405 nm was read after 108 minutes of the reaction using a Tecan Infinite M200Pro device.

[0249] To evaluate the inhibition of non-specific human von Willebrand factor (vWF) binding to the SpA mutant variant, the protocol described by Kim et al., 2015 was used with the following adjustments. Wells of a 96-well plate were coated overnight at 4°C with 300 ng of SpA mutant variant protein per well diluted in PBS (pH 7.6). To each well, 300 ng of human vWF (Invitrogen #RP-43132, lot UE2767197) in 1% bovine serum albumin (BSA) was added and incubated for 1 hour. Rabbit anti-human vWF antibody (Bio-Rad, #AHP062, lot 148125) was diluted 1:5,000 and incubated for 45 minutes. The secondary antibody, donkey anti-rabbit IgG conjugated to peroxidase (Sigma-Aldrich A3414-1mL, lot SLBV6847), was diluted 1:30,000 and incubated for 45 minutes. As the substrate, TMB (3,3’,5,5’-tetramethylbenzidine, Biolegend, #421101, lot B304351) was used according to the manufacturer's protocol. 1M H3PO4 (Sigma-Aldrich), the stop solution, was used. Absorbance at 405 nm was read after 45 minutes of the reaction using a Tecan Infinite M200Pro device.

[0250] The results summarized in Table 4 and Figures 1 - 6 indicate that the SpA_WT-49-339 (SEQ ID NO: 16) protein was able to bind to both human IgG, mouse IgG and IgM, rabbit IgG and human vWF. SpA KKAA -49-339 mutant did not bind to mouse IgM, mouse and rabbit-derived IgG, or vWF, but did bind to human IgG. The SpA mutant variants SpA_mut_7_12 (SEQ ID NO: 18) and SpA_mut_10_12 (SEQ ID NO: 17) were unable to bind to human IgG, mouse IgG, mouse IgM, rabbit IgM, or vWF. The SpA mutant variants SpA_mut_3_12 (SEQ ID NO: 20), SpA_mut_4_12 (SEQ ID NO: 19) and SpA_mut_10_15 (SEQ ID NO: 21) bound to human IgG but were unable to bind to mouse IgG, mouse IgM, rabbit IgG, or vWF.

[0251] [Table 4]

[0252] Competitive ELISA Competitive ELISA was applied to address the blockade of SpA:IgG interaction by SpA mutant polyclonal immune sera. To evaluate the inhibition of human IgG binding to SpA mutants by chicken IgY, a protocol described by Kim et al. 2010 was used with slight modification.

[0253] Briefly, wells were coated with 200 ng per well of immobilized SpA_WT-49-339 (SEQ ID NO: 15) diluted in PBS (pH 7.6). IgY from eggs of chickens immunized with different SpA variants (Davids Biotechnologie GmbH) adjuvanted with AddaVax™ (InvivoGen) was serially diluted (1:10 to 1:320) and added to the plates. The plates were incubated at room temperature for 1 hour. 50 nanograms (50 ng) of human IgG (Sigma-Aldrich l2511, lot number O48M4868V) per well was added and the plates were incubated at room temperature for 1 hour. A 1:10,000 dilution of goat anti-human IgG antibody conjugated to alkaline phosphatase (Sigma-Aldrich A1541, lot number 029M4838V) was used as the secondary antibody. As substrate, 3 mg / mL p-nitrophenyl phosphate (p-NPP) (Sigma-Aldrich, P5869-25CAP, lot number SLBV2576) in diethanolamine (DEA, pH 9.2) (Sigma-Aldrich, D8885-500g, lot number MKCB9120) was used. Absorbance at 495 nm was read 55 minutes after the reaction using a Tecan Infinite M200Pro instrument. The PBS sample was set as 100% (control without inhibition) and the percentage of human IgG binding was calculated using the following formula: (Average absorbance at 405 nm) ÷ (Absorbance of PBS at 405 nm) × 100%.

[0254] The results showed that non-specific binding of human IgG could compete with specific IgY antibodies against some of the SpA mutant variants tested in the competitive ELISA (Figure 7). Specific IgY against the chimeric protein CHIM_mc2716_mSpA_7_12_FS (SEQ ID NO: 57) effectively blocked SpA binding of human IgG in a dose-dependent manner (63% binding to human IgG at a 1:40 dilution, SpA_WT-49-339 had 82% human IgG binding). IgY specific for several SpA mutant variants; CHIM_mc2716_mSpA_7_12_FS (SEQ ID NO: 57), SpA_mut_7_12 (SEQ ID NO: 18), SpA_mut_10_12 (SEQ ID NO: 17), SpA_mut_4_12 (SEQ ID NO: 19), SpA_mut_3_12 (SEQ ID NO: 20), CHIM_0992_mSpA_7_12_FS (SEQ ID NO: 58), CHIM_mc2716_mSpA_10_12_FS (SEQ ID NO: 65), CHIM_mc2723_mSpA_10_12_FS (SEQ ID NO: 83), CHIM_0992_mSpA_10_12_FS (SEQ ID NO: 84) and CHIM_LukE_mSpA_7_12_FS (SEQ ID NO: 85) all resulted in a different degree of reduction in human IgG binding compared to SpA_WT-49-339 (SEQ ID NO: 15). The pre-immune serum (negative control) collected before the first immunization did not have the ability to block non-specific binding of human IgG to SpA_WT-49-339. SpA KKAA IgY specific for -49-339 (positive control) blocked SpA_WT-49-339 binding of human IgG.

[0255] 1.2 Aureolysin variants An azocasein hydrolysis assay was set up to analyze the possibility that vaccine candidate-specific antibodies inhibit aureolysin enzyme activity. Aureolysin activity was determined using azocasein, a chromogenic derivative of casein, as a substrate. Aureolysin degrades azocasein to yield TCA-soluble (trichloroacetic acid) azopeptides with high UV absorbance that can be quantified spectrophotometrically. Thus, aureolysin was incubated with azocasein and azocasein degradation was evaluated. Thermolysin, a heat-stable neutral metalloprotease enzyme produced by the Gram-positive bacterium Bacillus thermoproteolyticus (Sigma-Aldrich, P1512), was used as a positive control. First, commercially available aureolysin and USA300HOU_2637 were tested for casein hydrolysis activity. However, since none of these aureolysins showed enzyme activity in the assay (data not shown), a protocol for the purification of aureolysin from Staphylococcus aureus USA300 cultures was set up.

[0256] The protocol for the purification of aureolysin from Staphylococcus aureus USA300 cultures was as follows. 1 L of Staphylococcus aureus USA300 HOU cell cultures were grown in TSB (Tryptic Soy Broth) medium at 37 °C for 24 h with stirring at 170 r.p.m. The cultures were harvested by centrifugation (4,100 r.p.m, 4 °C for 30 min), and the supernatant was filtered using a 0.22 μm sterile filter. The supernatant was concentrated using tangential flow filtration (TFF) (General Electric Healthcare, AKTA Flux) with a 10 kDa hollow fiber (General Electric Healthcare, AKTA Flux, UFP-10-C-3MA, batch 17000576). The precipitation of the sterile supernatant was performed at 4 °C using 60% ammonium sulfate (according to Banbula et al., 1998). After adding 1 M Tris (pH 8.0) at a ratio of 1:10 (v / v) to stabilize the pH, precipitation was carried out. The suspension was centrifuged at 10,000×g at 4 °C for 30 min. The pellet was resuspended in 10 mM Tris-HCl, pH 7.6 and 5 mM CaCl 2 2. The dialysis was carried out with 10 mM Tris-HCl pH 7.6 and 5 mM CaCl 2It was carried out overnight at 4 °C (10 kDa membrane cut-off). Subsequently, two-step chromatography was performed. (1) Akta purification: anion exchange - Mono Q 5 / 50 GL (General Electric Healthcare, 17-5166-01, lot number 10244496) - elution with a gradient of 0 - 0.5 M NaCl, (2) size exclusion chromatography (SEC): SUPERDEX 75 pg 16 / 600 (General Electric Healthcare, 28-9893-33, lot number 10244501). The fractions were analyzed by SDS-PAGE (using TGX Stain-Free Protein Gel, Bio-Rad, and Pro Blue Safe Stain, Giotto Biotech) and Western blot (primary antibody produced in rabbit at 1 μg / mL; goat anti-rabbit IgG / HRP conjugate diluted 1:2,000 (Dako, P0448, lot number 20066477)).

[0257] Purified aureolysin from Staphylococcus aureus cultures showed a purity of over 90% (estimated by SDS), and this purified aureolysin also showed activity in the casein hydrolysis assay. The quality of the aureolysin preparation (good peptide coverage of the mature form) was confirmed by mass spectrometry (data not shown).

[0258] The protocol for the azocasein (activity) assay is as follows. For this assay, 1 μg of thermolysin (from Geobacillus stearothermophilus, Sigma-Aldrich, P1512) was used as a positive control. 1 microgram (1 μg) of thermolysin was used as a negative control with 10 mM EDTA (EDTA acts as an inhibitor of metalloproteases). Mcllavaine buffer (pH 7.4, 10 mM CaCl 2 (19.07 mL of 0.2 M Na 2 HPO 4 4, 1.73 mL of 0.1 M citric acid, 0.2 mL of 1 M CaCl 2) It was prepared using a 10 mg / mL solution of azocasein. A mixture was prepared from 200 μL of the azocasein solution, PBS supplemented with 2 μg of purified aureolysin, 20 μL or 50 μL of serum from mice immunized with different antigens, or adjuvant alone (negative control). The mixture was incubated at 37 °C for 1 hour with shaking (1,000 r.p.m) in a thermomixer at room temperature. The reaction was stopped by adding 400 μL of 5% TCA (trichloroacetic acid). The samples were centrifuged at 10,000×g for 3 minutes at 4 °C. Next, 300 μL of the supernatant was transferred to a 96-well plate, and the absorbance at 400 nm was read using a Tecan Infinite M200Pro device (serial number: 1206001022).

[0259] The activity of aureolysin purified from a Staphylococcus aureus culture (designated SA. aureolysin) could be inhibited using immune sera from mice immunized with an aureolysin chimeric protein construct (Figure 8). The immune sera from mice that received CHIM_mc2716_mSpA_7_12_FS (SEQ ID NO: 57) adjuvanted with SLA-SE showed a strong ability to inhibit enzymatic aureolysin activity in a dose-dependent manner, as observed by the decrease in absorbance at 400 nm when compared to sera from mice immunized with PBS or SLA-SE adjuvant (Figure 8A). This dose-dependent aureolysin inhibition was also observed for immune sera collected from mice immunized with CHIM_mc2716_mSpA_10_12_FS (SEQ ID NO: 65) and adjuvanted with SLA-SE (Figure 8B). The assay control confirmed the assay setup (Figure 8C). The assay was repeated multiple times to obtain comparable results.

[0260] 1.3 LukE variants The LukE protein is one of the two components of LukED leukocidin. The LukE and LukD subunits combine together to create a pore-forming toxin in the membranes of immune cells and red blood cells, resulting in lysis and death. The target immune cells of LukED (in humans and mice) include, among others, neutrophils, monocytes, and macrophages. Therefore, LukE-specific functional antibodies can inhibit the formation of LukED toxin and its cytotoxic activity against human neutrophil cells.

[0261] Mice immunized with LukE or a chimeric protein containing LukE and adjuvanted with SLA-SE were evaluated for the inhibition of leukocyte activity of antibodies in serum by a cytotoxity inhibition assay (XTT assay). The human promyelocytic leukemia (HL-60) cell line was cultured according to the reported culture conditions as described by the ATCC. HL-60 cells were differentiated into neutrophil-like cells by adding 0.78% dimethylformamide (DMF) to the culture medium and incubating at 37 °C for 5 days with 5% CO 2 2. For the assay, 3.5 - 4×10 5 cells per well were used. The assay was performed in 96-well plates by adding components to each well in the following order (applied in duplicate).

[0262] - Test serum (2-fold dilutions from 1:50 to 1:400 dilution) with a final volume of 50 μL diluted in culture medium

[0263] - Toxins; LukE only, LukD only, and a mixture of LukE and LukD, with a final volume of 40 μL diluted in culture medium to the required final concentration.

[0264] - 3.5 - 4×10 5 HL-60 differentiated cells in a final volume of 10 μL.

[0265] After plating, the plates were incubated with 5% CO 2It was incubated at 37 °C for 24 h. After incubation with Cell Proliferation Kit II (XTT assay reagent, Sigma-Aldrich) for 16 h, cell viability was measured by reading the absorbance at 470 / 690 nm according to the manufacturer's protocol. The colorimetric assay is based on the reduction of the yellow tetrazolium salt XTT (sodium 3’-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate) to the orange formazan dye by metabolically active cells. Finally, data were acquired using a SpectraMax Reader II Instrument.

[0266] Immunization of mice with LukE-29-311 adjuvanted with SLA-SE (SEQ ID NO: 30; positive control), or a chimeric protein containing LukE adjuvanted with SLA-SE, induced a large amount of functional antibodies that could neutralize the cytotoxic activity of LukED as compared to sera from mice immunized with SLA-SE adjuvant alone (Figure 9).

[0267] The leukotoxicity of functional wild-type LukE can be blocked / neutralized by antibodies induced by the CHIM_LukE_mHla_H35L_FS (SEQ ID NO: 56) chimeric protein. Sera from mice immunized with several other LukE chimeric proteins adjuvanted with SLA-SE induced high cell viabilities comparable to the levels observed for the positive control, native LukE-29-311 immune serum (Figure 9). This assay also showed that the toxin subunits LukE and LukD alone did not affect cell viability across all chimeric proteins tested (data not shown).

[0268] 1.4 Hla variants To evaluate the efficacy of different chimeric proteins including the design of the Hla subunit, a hemolysis inhibition assay was set up. Using different chimeric proteins adjuvanted with SLA-SE, mice were immunized subcutaneously (s.c.) twice in the flank using a two-week schedule. Immune sera were collected 14 days after the second immunization to evaluate the induction of specific antibodies. The immune sera were serially diluted two-fold (1:10 to 1:640) in PBS containing 0.5% bovine serum albumin (BSA) in 96-well plates. Each serum dilution was incubated with 20 ng of recombinant Hla (rHla) for 20 minutes at room temperature. Solutions of water only and PBS containing 0.5% BSA were used as positive and negative controls, respectively. A 2 percent (%) solution of rabbit red blood cells from rabbit defibrinated blood was added to the plates and the plates were incubated at 37 °C for 30 minutes. After centrifugation at 1,000 × g for 5 minutes at 4 °C, the supernatant was transferred to a new 96-well plate. 100 microliters (100 μL) of PBS was added to each well and the absorbance at 540 nm was read using a plate reader. The percentage of hemolysis was calculated relative to hemolysis induced by water (100% hemolysis).

[0269] Serum from mice immunized with adjuvant alone (SLA-SE) did not inhibit erythrocyte hemolysis (Figure 10). Positive control serum collected from mice that received Hla_H35L-27-319 (SEQ ID NO: 5) together with SLA-SE showed inhibition of hemolysis, which was lost at a dilution of 1:320. Both control sera together verified the setting of the hemolysis inhibition assay. Immune serum from mice immunized with the chimeric protein CHIM_LukE_mHla_H35L_FS (SEQ ID NO: 56) together with SLA-SE showed a strong ability to inhibit hemolysis even at the highest dilution tested (1:640). This chimeric protein immune serum inhibited hemolysis by 53% at a dilution of 1:640, while sera from mice that received placebo (SLA-SE) and positive control (Hla_H35L-27-319) both inhibited hemolysis by only about 1-3% at the same dilution. Different chimeric proteins containing all Hla subunits were able to induce hemolysis-inhibiting immune sera to varying degrees. However, the highest hemolysis inhibition effect was observed with immune serum from mice that received CHIM_mc2723_Hla_H35L_t_FS (SEQ ID NO: 62), which showed 77% hemolysis inhibition at a dilution of 1:640. The assay was repeated and comparable results were obtained.

[0270] [Example 2] Efficacy of prophylactic vaccines in an animal exposure model The effectiveness of the prophylactic vaccine protein was evaluated using a murine model of peritonitis induced by Staphylococcus aureus infection, using CD-1 mice (female, 6 - 8 weeks old, 6 - 12 mice per group, Charles River, Germany). Prior to exposure, on days 0 and 14 (two immunizations) or days 0, 14, and 28 (three immunizations), the mice were immunized two or three times either intramuscularly (i.m.) in the hind limb (divided) or subcutaneously (s.c.) in the flank with a prophylactic vaccine product adjuvanted with either outer membrane vesicles (OMV, InvivoGen, E. coli OMV InvivoFit™) containing aluminum hydroxide gel (AlOH, Alhydrogel, InvivoGen, catalog code: vac-alu-250, lot number 5531), aluminum hydroxide gel (AlOH, Alhydrogel, InvivoGen, catalog code: vac-alu-250, lot number 5531) alone, or SLA-SE (AAHI, the Access to Advanced Health Institute) as described in Table 5. The protein was formulated at a 1:1 [vol / vol] ratio with the above adjuvant diluted in 10 mM Tris + 144 mM NaCl, pH 7.2 buffer. The doses of the single protein or chimeric construct are shown in Table 5. As negative controls, mice were immunized with adjuvant alone or saline.

[0271] The OMV + AlOH adjuvant was prepared as follows. 500 μg of OMV from InvivoGen was resuspended in 1 mL of supplied sterile endotoxin-free water and aliquoted into 200 μL aliquots. For an 8-mouse group, 96 μL of the OMV suspension was mixed with 144 μL of sterile buffer. Then, 240 μL of redispersed aluminum hydroxide gel was added to the OMV / buffer to a final concentration of 5 μL OMV / 50 μL, mixed well, and stored on ice in the refrigerator for at least 1 hour prior to immunization.

[0272] Two or four weeks after the last immunization, mice were exposed by intraperitoneal (i.p.) injection with different exposure CFU doses as described in Table 5 using either Staphylococcus aureus USA300 LAC strain (NCBI: CP000730.1, Diep et al., 2006), Staphylococcus aureus MRSA252 strain (NCBI: BX571856.1), Staphylococcus aureus Newman strain (NCBI: AP009351.1) or Staphylococcus aureus USA400 (NCBI: NZ_CP019574.1).

[0273] The protocol for generating different Staphylococcus aureus strain exposure doses was as follows. An overnight culture was diluted in fresh tryptic soy broth (TSB) to an optical density (OD 600nm ) of 0.5 at 600 nm and grown at 37 °C for approximately 2.5 h until an OD 600nm of 1.2 was achieved. The bacteria were then harvested by centrifugation, washed twice and concentrated in 2% PBS + 16% glycerol. The cultures were aliquoted and immediately stored at -80 °C for later use. The final exposure dose administered to the animals was experimentally verified by plating the cultures on agar plates and counting the CFUs the next day.

[0274] Animals were monitored for 7 days after bacterial exposure, tested for predetermined clinical signs of infection, and the decision to euthanize the mice was made using humane endpoint criteria. The total survival time was recorded.

[0275] Survival of the exposed mice showed that immunization with EDEN Combo-1 consisting of CHIM_mc2716_mSpA_7_12_FS (SEQ ID NO: 57), CHIM_2753_mc2723_FS (SEQ ID NO: 59), SAR0280-28-820 (SEQ ID NO: 48) and SAR0992-1-409, either with or without the toxoid CHIM_LukE_mHla_H35L_FS (SEQ ID NO: 56), adjuvanted with OMV+AlOH and given as three intramuscular immunizations, resulted in highly significant protection (87.5% survival, p = <0.0001) as shown in Table 5 compared to SHAM saline controls.

[0276] When immunized intramuscularly three times with EDEN Combo-1 adjuvanted with OMV+AlOH without the toxoid chimeric protein, 50% survival was achieved against the USA300 LAC strain compared to the SHAM saline control (p = 0.0013, ** ). EDEN Combo-1 containing the toxoid chimeric protein adjuvanted with AlOH was also observed to induce high protection against the USA300 LAC strain (p = <0.0001, **** ). In this study, immunization with EDEN Combo-1 antigen with or without the toxoid chimeric protein resulted in strong protection regardless of the adjuvant. The log-rank Mantel-Cox test was used for statistical evaluation.

[0277] After exposure to USA300 LAC or MRSA252, the LukE-29-311 (SEQ ID NO: 30) SLA-SE adjuvanted vaccine provided significant protection. Furthermore, vaccination with any of USA300HOU_2637_E353A-28-509 (SEQ ID NO: 35), SAR2723-28-509 (SEQ ID NO: 39), or SAR0280-28-820 (SEQ ID NO: 48) adjuvanted with SLA-SE provided significant protection against exposure to USA300 LAC. Immunization with either SAR2753-291-680 (SEQ ID NO: 42) or a portion of the chimeric protein constructs in Table 5 adjuvanted with SLA-SE all provided protection against exposure to MRSA252. Immunization with SAR0992-1-409 adjuvanted with SLA-SE provided significant protection against exposure to the USA300 LAC, USA400, and Newman strains. The log-rank Mantel-Cox test was used for statistical evaluation.

[0278]

Table 5

[0279] [Example 3] Efficacy of preventive vaccine in skin model Using a skin abscess animal model of Staphylococcus aureus infection with BALB / c mice (6-week-old females, 12 mice per group), the efficacy of the preventive vaccine protein was evaluated. Before exposure, on days 0 and 14 (two immunizations) or days 0, 14, and 28 (three immunizations), the mice were immunized intramuscularly (i.m.) in the hind limb (divided) or subcutaneously (s.c.) in the flank with one of the preventive vaccine products adjuvanted with either aluminum hydroxide (AlOH, Alhydrogel, InvivoGen, catalog code: vac-alu-250, lot number 5531) or SLA-SE (AAHI, the Access to Advanced Health Institute) either two or three times. The protein was formulated at a 1:1 [vol / vol] ratio with the above adjuvant diluted in 10 mM Tris + 144 mM NaCl, pH 7.2 buffer. The doses of the single protein or chimeric construct are shown in Table 6. As negative controls, the mice were immunized with adjuvant alone or saline.

[0280] Two weeks after the last immunization, the mice were exposed by subcutaneous (s.c.) injection using either the Staphylococcus aureus USA300 LAC strain (Diep et al., 2006) or the Staphylococcus aureus Newman strain (Baba et al., 2008) at different exposure CFU doses as described in Table 6.

[0281] The protocol for generating different exposure doses of the Staphylococcus aureus strains was as follows. The overnight culture was diluted to an optical density (OD 600nm ) of 0.5 at 600 nm in fresh tryptic soy broth (TSB) and 1.2 OD 600nmIt was grown at 37°C for approximately 2.5 hours until achievement. Subsequently, the bacteria were harvested by centrifugation, washed twice, and concentrated in 2% PBS + 16% glycerol. The culture was aliquoted and immediately stored at -80°C for later use. The final exposure dose administered to the animals was experimentally verified by plating the culture on agar plates and counting the CFUs the next day.

[0282] Abscess formation (size and cutaneous necrotic lesions) was monitored at 24-hour intervals over 10 days post-infection. The size of the abscess and the size of the overlying cutaneous necrotic lesions were determined using the standard formula for the area of an ellipse: [A = π × lesion length / 2 × lesion width / 2]. For the statistical evaluation of the area under the curve (AUC), a two-way analysis of variance (group × time) was performed on the cutaneous lesion area parameter, followed by Dunnett's multiple comparison test (Kruskal–Wallis test). A difference between groups was considered statistically significant if the p-value < 0.05. Statistical analysis and AUC data were analyzed using GraphPad Prism version 9. A highly significant difference was considered if the p-value ≤ 0.0001 and was indicated as " **** ". "ns" (not significant) refers to a p-value > 0.05.

[0283] As a result of immunization with EDEN Combo-1, which consists of a combination of CHIM_mc2716_mSpA_7_12_FS (SEQ ID NO: 57), CHIM_2753_mc2723_FS (SEQ ID NO: 59), SAR0280-28-820 (SEQ ID NO: 48), and SAR0992-1-409 adjuvanted with AlOH, USA300 LAC abscess formation was significantly impaired, and thus, significant protection (p = 0.0114 * ) was conferred compared to AlOH adjuvant alone.

[0284] Immunization with any of Hla_H35L-39-319 (SEQ ID NO: 6), LukE-29-311 (SEQ ID NO: 30), or SAR2753-291-680 (SEQ ID NO: 42) adjuvanted with SLA-SE significantly impaired abscess formation after exposure to USA300 LAC and Newman, compared to immunization with SLA-SE adjuvant alone. Immunization with USA300HOU_2637_E353A-28-509 (SEQ ID NO: 35) adjuvanted with SLA-SE significantly impaired abscess formation after exposure to USA300 LAC ( * ). Immunization with either SAR2723-28-619 (SEQ ID NO: 39) or SAR0992-1-409 adjuvanted with SLA-SE significantly impaired abscess formation after exposure to Newman. Immunization with different chimeric constructs adjuvanted with SLA-SE also had a lower AUC and impaired abscess formation when exposed to either Staphylococcus aureus USA300 LAC or Staphylococcus aureus Newman strain. All groups were tested against SLA-SE alone as an adjuvant.

[0285] [Table 6]

[0286] [Example 4] Prophylactic vaccine immunogenic IgG data The total immunoglobulin G (IgG) response after immunization with the prophylactic vaccine protein was evaluated using a standard ELISA assay. Mice were immunized three times at 30 μg per single protein or chimeric construct for formulation with the adjuvant SLA-SE as described in Figure 11a, or immunized with 20 μg per chimeric construct formulated with either OMV (InvivoGen, Escherichia coli OMV InvivoFit™) + AlOH or AlOH (InvivoGen) alone as an adjuvant as described in Figure 11b, and 10 μg of a single protein in EDEN Combo-1 consisting of CHIM_mc2716_mSpA_7_12_FS (SEQ ID NO: 57), CHIM_2753_mc2723_FS (SEQ ID NO: 59), SAR0280-28-820 (SEQ ID NO: 48) and SAR0992-1-409, with or without addition of the toxoid chimeric construct CHIM_LukE_mHla_H35L_FS (SEQ ID NO: 56). The adjuvant was diluted in 10 mM Tris + 144 mM NaCl, pH 7.2 buffer. Immunization was performed by intramuscular (i.m.) administration to the hind limb or subcutaneous (s.c.) administration to the flank on days 0, 14 and 28. Blood samples were collected from the tail vein 12 days after immunization to evaluate the antigen-specific total IgG titer.

[0287] It was carried out according to the standard ELISA assay protocol. 96-well Nunc-Immuno™ MaxiSorp plates (Sigma-Aldrich) were coated with single or chimeric proteins, and the IgG half-maximal titers in sera from mice immunized with each protein were determined. The threshold of assay sensitivity was set at the maximum half-titer of 10 3 . Each dot in Figures 11a - c represents one immunized mouse. To verify the assay efficiency, plate controls were included in each ELISA run.

[0288] As shown in Figure 11a, all of the tested immune sera from mice immunized with a prophylactic vaccine containing CHIM_mc2716_LukE_FS (SEQ ID NO: 63), CHIM_LukE_Hla_H35L_t_FS (SEQ ID NO: 64), CHIM_LukE_0280_FS (SEQ ID NO: 66), CHIM_0992_2753_FS (SEQ ID NO: 61), SAR2723-28-619 (SEQ ID NO: 39), SAR2753-291-680 (SEQ ID NO: 42), SAR0280-28-820 (SEQ ID NO: 48), SAR0992-1-409, Hla_H35L-27-319 (SEQ ID NO: 5), SpA_mut_10_12 (SEQ ID NO: 17), SpA_mut_7_12 (SEQ ID NO: 18), or USA300HOU_2637_E353A-28-509 (SEQ ID NO: 35) adjuvanted with SLA-SE had high levels of antigen-specific total IgG antibodies compared to SLA-SA adjuvant alone (Figure 11c).

[0289] As shown in Figure 11b, immune sera from mice immunized with EDEN Combo-1 adjuvanted with either OMV+AlOH or AlOH alone induced high levels of antigen-specific total IgG antibodies against the single and chimeric proteins contained in EDEN Combo-1 compared to both OMV+AlOH and AlOH adjuvant alone (Figure 11c).

[0290] The data were analyzed by calculating the EC50 of each serum dilution curve using the Hill's equation for modeling non-linear fits using GraphPad Prism version 9.0, and the group mean of the IgG titers for each immunization was calculated using row statistics that calculated the mean for each column of each dataset. For statistical evaluation, a one-way analysis of variance test and Tukey's multiple comparison test were used. A significant difference was considered when the p-value ≤ 0.05. A highly significant difference was considered when the p-value ≤ 0.0001 and was indicated by " **** ". "ns" (non-significant) refers to a p-value > 0.05. For IgG subclasses, a one-sample t-test and Wilcoxon test were applied.

[0291] Reference list of examples Ashley M. Vaughan (ed.), Malaria Vaccines: Methods and Protocols, Methods in Molecular Biology, vol. 1325, DOI 10.1007 / 978-1-4939-2815-6_16, Springer Science+Business Media New York 2015 Baba T, Bae T, Schneewind O, Takeuchi F, Hiramatsu K. J Bacteriol. 2008 Jan;190(1):300-10. doi: 10.1128 / JB.01000-07. Epub 2007 Oct 19. PMID: 17951380; PMCID: PMC2223734. Banbula A, Potempa J, Travis J, Fernandez-Catalan C, Mann K, Huber R, Bode W, Medrano F. Structure. 1998 Sep 15;6(9):1185-93. doi: 10.1016 / s0969-2126(98)00118-x. PMID: 9753696. Diep BA, Gill SR, Chang RF, Phan TH, Chen JH, Davidson MG, Lin F, Lin J, Carleton HA, Mongodin EF, Sensabaugh GF, Perdreau-Remington F. Lancet. 2006 Mar 4;367(9512):731-9. doi: 10.1016 / S0140-6736(06)68231-7. PMID: 16517273. Kim HK et al., 2010 Nontoxigenic protein A vaccine for methicillin-resistant Staphylococcus aureus infections in mice. J Exp Med 207:1863-1870. Kim HK et al., 2015 Protein A suppresses immune responses during Staphylococcus aureus bloodstream infection in guinea pigs. mBio 6.

Claims

1. i. having at least 85% sequence identity with the sequence of SEQ ID NO: 2, ii. comprising one or more amino acid deletions and / or substitutions in the first 12 consecutive N-terminal amino acid residues of the amino acid sequence of mature Hla, wherein the first 12 consecutive N-terminal amino acid residues correspond to positions 1 to 12 of SEQ ID NO: 2, an immunogenic polypeptide consisting of or comprising a variant of the amino acid sequence of alpha-hemolysin (Hla), wherein the polypeptide is unable to participate in the formation of a heptameric structure with other Hla molecules, and any substitution in ii) is preferably non-conservative, an immunogenic polypeptide.

2. The immunogenic polypeptide according to claim 1, wherein the variant comprises at least two amino acid alterations selected from deletions and substitutions in the first 12 consecutive N-terminal amino acid residues, for example, at least or exactly 2, at least or exactly 3, at least or exactly 4, at least or exactly 5, at least or exactly 6, at least or exactly 7, at least or exactly 8, at least or exactly 9, at least or exactly 10, at least or exactly 11, or exactly 12 amino acid alterations.

3. The immunogenic polypeptide according to claim 1 or 2, wherein the variant comprises at least two amino acid substitutions in the first 12 N-terminal amino acids under ii), for example, at least or exactly 2, at least or exactly 3, at least or exactly 4, at least or exactly 5, at least or exactly 6, at least or exactly 7, at least or exactly 8, at least or exactly 9, at least or exactly 10, at least or exactly 11, or exactly 12 amino acid substitutions.

4. The immunogenic polypeptide according to claim 1 or 2, wherein the variant comprises at least two amino acid deletions in said first 12 consecutive N-terminal amino acid residues, for example, at least or exactly 2, at least or exactly 3, at least or exactly 4, at least or exactly 5, at least or exactly 6, at least or exactly 7, at least or exactly 8, at least or exactly 9, at least or exactly 10, at least or exactly 11, or exactly 12 amino acid deletions.

5. The immunogenic polypeptide according to claim 1, wherein all of amino acid residues 1 to 12 are deleted.

6. The immunogenic polypeptide according to claim 1, wherein all of amino acid residues 1 to 12 are substituted.

7. The immunogenic polypeptide according to claim 6, wherein amino acid residues 1 to 12 are substituted with the sequence 5'-SETESVRSASS-3' (residues 1 to 12 of SEQ ID NO: 4).

8. The immunogenic polypeptide according to any one of claims 1 to 7, wherein the variant comprises SEQ ID NO:

3.

9. The immunogenic polypeptide according to any one of claims 1 to 7, comprising a substitution of histidine (H) to leucine (L) at the position corresponding to position 35 of SEQ ID NO:

2.

10. The immunogenic polypeptide according to claim 9, wherein the variant comprises SEQ ID NO:

6.

11. The immunogenic polypeptide according to any one of claims 1 to 10, wherein the variant lacks hemolytic activity or has significantly reduced hemolytic activity.

12. The immunogenic polypeptide according to any one of claims 1 to 11, wherein the polypeptide can induce an antibody that blocks the hemolytic activity of wild-type Hla.

13. i. a to e: a. A sequence that is at least 85% identical to the amino acid sequence of immunoglobulin binding domain (IgBD) E (SEQ ID NO: 16, residues 1 to 56), b. A sequence that is at least 85% identical to the amino acid sequence of IgBD D (SEQ ID NO: 16, residues 62 to 117), c. A sequence that is at least 85% identical to the amino acid sequence of IgBD A (SEQ ID NO: 16, residues 120 to 175), d. A sequence that is at least 85% identical to the amino acid sequence of IgBD B (SEQ ID NO: 16, residues 178 to 233), e. A sequence that is at least 85% identical to the amino acid sequence of IgBD C (SEQ ID NO: 16, residues 236 to 291) A variant of the amino acid sequence of immunoglobulin G-binding protein A (SpA) that includes at least one of them, and ii. One or more first mutations in each of at least one of a to e, which disrupt binding at the Fc binding site and occur at positions corresponding to the amino acid positions in SEQ ID NO: 16 selected from positions 3, 8, 9, 64, 69, 70, 122, 127, 128, 180, 185, 186, 238, 243, 244, one or more first mutations, and iii. One or more second mutations in each of at least one of a to e, which disrupt binding at the Fab binding site and, where applicable, occur at positions corresponding to the amino acid positions in SEQ ID NO: 16 selected from positions 34, 35, 37, 38, 41, 95, 96, 98, 99, 102, 153, 154, 156, 157, 160, 211, 212, 214, 215, 218, 269, 270, 272, 273, and 276, one or more second mutations Comprising or containing these immunogenic polypeptides, iv. Optionally, none of at least one of a to e, where applicable, contains a mutation that disrupts binding at the Fc binding site at positions corresponding to both of amino acid positions 7 + 8, 68 + 69, 126 + 127, 184 + 185, and 242 + 243 of SEQ ID NO: 16, v. None of at least one of a to e, where applicable, contains a mutation that disrupts binding at the Fab binding site at positions corresponding to both of amino acid positions 34 + 35 and 95 + 96 and 153 + 154 and 211 + 212, and 269 + 270 of SEQ ID NO: 16, An immunogenic polypeptide in which the polypeptide is unable to bind to human IgG and human von Willebrand factor, and the substitution is preferably a non-conservative substitution.

14. One or more second mutations are at least One or two or three or four or five of positions 34 and 96 and 154 and 212 and 270, One or two or three or four or five of positions 35 and 95 and 154 and 212 and 270, One or two or three or four or five of positions 35 and 96 and 153 and 212 and 270, One or two or three or four or five of the 35th, 96th, 154th, 211th, and 270th positions, One or two or three or four or five of the 35th, 96th, 154th, 212th, and 269th positions, One or two or three or four or five of the 34th, 95th, 154th, 212th, and 270th positions, One or two or three or four or five of the 34th, 96th, 153rd, 212th, and 270th positions, One or two or three or four or five of the 34th, 96th, 154th, 211th, and 270th positions, One or two or three or four or five of the 34th, 96th, 154th, 212th, and 269th positions, One or two or three or four or five of the 35th, 95th, 153rd, 212th, and 270th positions, One or two or three or four or five of the 35th, 95th, 154th, 211th, and 270th positions, One or two or three or four or five of the 35th, 95th, 154th, 212th, and 269th positions, One or two or three or four or five of the 35th, 96th, 153rd, 211th, and 270th positions, One or two or three or four or five of the 35th, 96th, 153rd, 212th, and 269th positions, One or two or three or four or five of the 35th, 96th, 154th, 211th, and 269th positions, One or two or three or four or five of the 34th, 95th, 153rd, 212th, and 270th positions, One or two or three or four or five of the 34th, 95th, 154th, 211th, and 270th positions, One or two or three or four or five of the 34th, 95th, 154th, 212th, and 269th positions, One or two or three or four or five of the 34th, 96th, 153rd, 211th, and 270th positions, One or two or three or four or five of the 34th, 96th, 153rd, 212th, and 269th positions, One or two or three or four or five of positions 34, 96, 154, 211, and 269, One or two or three or four or five of positions 35, 95, 153, 211, and 270, One or two or three or four or five of positions 35, 95, 153, 212, and 269, One or two or three or four or five of positions 35, 96, 153, 211, and 269, One or two or three or four or five of positions 34, 95, 153, 211, and 270, One or two or three or four or five of positions 34, 95, 153, 212, and 269, One or two or three or four or five of positions 34, 95, 154, 211, and 269, One or two or three or four or five of positions 34, 96, 153, 211, and 269, One or two or three or four or five of positions 35, 95, 153, 211, and 269, One or two or three or four or five of positions 35, 95, 154, 211, and 269, One or two or three or four or five of positions 34, 95, 153, 211, and 269, or One or two or three or four or five of positions 35, 96, 154, 212, and 270 wherein the polypeptide according to claim 13 occurs only at or at these positions.

15. The polypeptide according to claim 13 or 14, wherein one or more second mutations occur only at or at positions corresponding to positions in SEQ ID NO: 16 selected from the group consisting of at least 34, 35, 95, 96, 153, 154, 211, 212, 269, and 270.

16. The polypeptide according to claim 15, wherein one or more second mutations occur at positions corresponding to the positions in SEQ ID NO: 16 selected from the group consisting of at least 34, 35, 95, 96, 153, 154, 211, 212, 269, and 270, or occur only at these positions.

17. One or more second mutations are at least 34 and 95, 34 and 96, 34 and 153, 34 and 154, 34 and 211, 34 and 212, 34 and 269, 34 and 270, 35 and 95, 35 and 96, 35 and 153, 35 and 154, 35 and 211, 35 and 212, 35 and 269, 35 and 270, 95 and 153, 95 and 154, 95 and 211, 95 and 212, 95 and 269, 95 and 270, 96 and 153, 96 and 154, 96 and 211, 96 and 212, 96 and 269, 96 and 270, 153 and 211, 153 and 212, 153 and 269, 153 and 270, 154 and 211, 154 and 212, 154 and 269, 154 and 270, 211 and 269, 211 and 270, 212 and 269, and 212 and 270 The polypeptide according to claim 13 or 14, wherein one or more second mutations occur at positions corresponding to the positions in SEQ ID NO: 16 selected from the group consisting of these, or occur only at these positions.

18. One or more second mutations are at least 34 and 95 and 153, 34 and 95 and 154, 34 and 95 and 211, 34 and 95 and 212, 34 and 95 and 269, 34 and 95 and 270, 35 and 95 and 153, 35 and 95 and 154, 35 and 95 and 211, 35 and 95 and 212, 35 and 95 and 269, 35 and 95 and 270, 34 and 96 and 153, 34 and 96 and 154, 34 and 96 and 211, 34 and 96 and 212, 34 and 96 and 269, 34 and 96 and 270, 35 and 96 and 153, 35 and 96 and 154, 35 and 96 and 211, 35 and 96 and 212, 35 and 96 and 269, 35 and 96 and 270, 34 and 153 and 211, 34 and 153 and 212, 34 and 154 and 269, 34 and 154 and 270, 35 and 153 and 211, 35 and 153 and 212, 35 and 154 and 269, 35 and 154 and 270, 34 and 211 and 269, 34 and 212 and 270, 35 and 211 and 269, 35 and 212 and 270, 95 and 153 and 211, 95 and 153 and 212, 95 and 153 and 269, 95 and 153 and 270, 95 and 154 and 211, 95 and 154 and 212, 95 and 154 and 269, 95 and 154 and 270, 96 and 153 and 211, 96 and 153 and 212, 96 and 153 and 269, 96 and 153 and 270, 96 and 154 and 211, 96 and 154 and 212, 96 and 154 and 269, 96 and 154 and 270, 95 and 211 and 269, 95 and 211 and 270, 95 and 212 and 269, 95 and 212 and 270, 96 and 212 and 269, 96 and 212 and 270, 96 and 212 and 269, 96 and 212 and 270, 153 and 211 and 269, 153 and 212 and 269, 153 and 211 and 270, 153 and 212 and 270, 154 and 211 and 269, 154 and 212 and 269, 154, 211, 270, and 154 and 212 and 270 The polypeptide according to claim 13 or 14, which occurs at a position corresponding to the position at SEQ ID NO: 16 selected from the group consisting of, or only at these positions.

19. One or more second mutations are at least 95 and 153 and 211 and 269, 95 and 153 and 211 and 270, 95 and 153 and 212 and 269, 95 and 153 and 212 and 270, 95 and 154 and 211 and 269, 95 and 154 and 211 and 270, 95 and 154 and 212 and 269, 95 and 154 and 212 and 270, 96 and 153 and 211 and 269, 96 and 153 and 211 and 270, 96 and 153 and 212 and 269, 96 and 153 and 212 and 270, 96 and 154 and 211 and 269, 96 and 154 and 211 and 270, 96 and 154 and 212 and 269, 96 and 154 and 212 and 270, 34 and 153 and 211 and 269, 34 and 153 and 211 and 270, 34 and 153 and 212 and 269, 34 and 153 and 212 and 270, 34 and 154 and 211 and 269, 34 and 154 and 211 and 270, 34 and 154 and 212 and 269, 34 and 154 and 212 and 270, 35 and 153 and 211 and 269, 35 and 153 and 211 and 270, 35 and 153 and 212 and 269, 35 and 153 and 212 and 270, 35 and 154 and 211 and 269, 35 and 154 and 211 and 270, 35 and 154 and 212 and 269, 35 and 154 and 212 and 270, 34 and 95 and 211 and 269, 34 and 95 and 211 and 270, 34 and 95 and 212 and 269, 34 and 95 and 212 and 270, 34 and 96 and 211 and 269, 34 and 96 and 211 and 270, 34 and 96 and 212 and 269, 34 and 96 and 212 and 270, 35 and 95 and 211 and 269, 35 and 95 and 211 and 270, 35 and 95 and 212 and 269, 35 and 95 and 212 and 270, 35 and 96 and 211 and 269, 35 and 96 and 211 and 270, 35 and 96 and 212 and 269, 35 and 96 and 212 and 270, 34 and 95 and 153 and 269, 34 and 95 and 153 and 270, 34 and 95 and 154 and 269, 34 and 95 and 154 and 270, 34 and 96 and 153 and 269, 34 and 96 and 153 and 270, 34 and 96 and 154 and 269, 34 and 96 and 154 and 270, 35 and 95 and 153 and 269, 35 and 95 and 153 and 270, 35 and 95 and 154 and 269, 35 and 95 and 154 and 270, 35 and 96 and 153 and 269, 35 and 96 and 153 and 270, 35 and 96 and 154 and 269, 35 and 96 and 154 and 270, 34 and 95 and 153 and 211, 34 and 95 and 153 and 212, 34 and 95 and 154 and 211, 34 and 95 and 154 and 212, 34 and 96 and 153 and 211, 34 and 96 and 153 and 212, 34 and 96 and 154 and 211, 34 and 96 and 154 and 212, 35 and 95 and 153 and 211, 35 and 95 and 153 and 212, 35 and 95 and 154 and 211, 35 and 95 and 154 and 212, 35 and 96 and 153 and 211, 35 and 96 and 153 and 212, 35 and 96 and 154 and 211, and 35 and 96 and 154 and 212 The polypeptide according to claim 13 or 14, which occurs at a position corresponding to the position in SEQ ID NO: 16 selected from the group consisting of, or only at these positions.

20. One or more second mutations are at least 34 and 96 and 154 and 212 and 270, 35 and 95 and 154 and 212 and 270, 35 and 96 and 153 and 212 and 270, 35 and 96 and 154 and 211 and 270, 35 and 96 and 154 and 212 and 269, 34 and 95 and 154 and 212 and 270, 34 and 96 and 153 and 212 and 270, 34 and 96 and 154 and 211 and 270, 34 and 96 and 154 and 212 and 269, 35 and 95 and 153 and 212 and 270, 35 and 95 and 154 and 211 and 270, 35 and 95 and 154 and 212 and 269, 35 and 96 and 153 and 211 and 270, 35 and 96 and 153 and 212 and 269, 35 and 96 and 154 and 211 and 269, 34 and 95 and 153 and 212 and 270, 34 and 95 and 154 and 211 and 270, 34 and 95 and 154 and 212 and 269, 34 and 96 and 153 and 211 and 270, 34 and 96 and 153 and 212 and 269, 34 and 96 and 154 and 211 and 269, 35 and 95 and 153 and 211 and 270, 35 and 95 and 153 and 212 and 269, 35 and 96 and 153 and 211 and 269, 34 and 95 and 153 and 211 and 270, 34 and 95 and 153 and 212 and 269, 34 and 95 and 154 and 211 and 269, 34 and 96 and 153 and 211 and 269, 35 and 95 and 153 and 211 and 269, 35 and 95 and 154 and 211 and 269, 34 and 95 and 153 and 211 and 269, and 35 and 96 and 154 and 212 and 270 The polypeptide according to claim 13 or 14, which occurs at a position corresponding to the position at SEQ ID NO: 16 selected from the group consisting of, or only at these positions.

21. One or more first mutations occur at a position corresponding to the position at SEQ ID NO: 16 selected from at least 3, 8, 9, 69, 127, 185, and 243, or only at these positions, the polypeptide according to any one of claims 13 to 20.

22. One or more first mutations occur at least 3 and 8, 3 and 9, 3 and 69, 3 and 127, 3 and 185, 3 and 243, 8 and 9, 8 and 69, 8 and 127, 8 and 185, 8 and 243, 9 and 69, 9 and 127, 9 and 185, 9 and 243, 69 and 127, 69 and 185, 69 and 243, 127 and 185, 127 and 243, 185 and 243, 3 and 8 and 9, 3 and 8 and 69, 3 and 8 and 127, 3 and 8 and 185, 3 and 8 and 243, 3 and 9 and 69, 3 and 9 and 127, 3 and 9 and 185, 3 and 9 and 243, 3 and 69 and 127, 3 and 69 and 185, 3 and 69 and 243, 3 and 127 and 185, 3 and 127 and 243, 3 and 185 and 243, 8 and 9 and 69, 8 and 9 and 127, 8 and 9 and 185, 8 and 9 and 243, 8 and 69 and 127, 8 and 69 and 185, 8 and 69 and 243, 8 and 127 and 185, 8 and 127 and 243, 8 and 185 and 243, 9 and 69 and 127, 9 and 69 and 185, 9 and 69 and 243, 9 and 127 and 185, 9 and 127 and 243, 9 and 185 and 243, 69 and 127 and 185, 69 and 127 and 243, 69 and 185 and 243, 127 and 185 and 243, 3 and 8 and 9 and 69, 3 and 8 and 9 and 127, 3 and 8 and 9 and 185, 3 and 8 and 9 and 243, 3 and 8 and 69 and 127, 3 and 8 and 69 and 185, 3 and 8 and 69 and 243, 3 and 8 and 127 and 185, 3 and 8 and 127 and 243, 3 and 8 and 185 and 243, 3 and 9 and 69 and 127, 3 and 9 and 69 and 185, 3 and 9 and 69 and 243, 3 and 9 and 127 and 185, 3 and 9 and 127 and 243, 3 and 9 and 185 and 243 3 and 69 and 127 and 185, 3 and 69 and 127 and 243, 3 and 69 and 185 and 243, 3 and 127 and 185 and 243, 8 and 9 and 69 and 127, 8 and 9 and 69 and 185, 8 and 9 and 69 and 243, 8 and 9 and 127 and 185, 8 and 9 and 127 and 243, 8 and 9 and 185 and 243, 8 and 69 and 127 and 185, 8 and 69 and 127 and 243, 8 and 69 and 185 and 243, 8 and 127 and 185 and 243, 9 and 69 and 127 and 185, 9 and 69 and 127 and 243, 9 and 69 and 185 and 243, 9 and 127 and 185 and 243, 69 and 127 and 185 and 243, 3 and 8 and 9 and 69 and 127, 3 and 8 and 9 and 69 and 185 3 and 8 and 9 and 69 and 243, 3 and 8 and 9 and 127 and 185, 3 and 8 and 9 and 127 and 243, 3 and 8 and 9 and 185 and 243, 3 and 8 and 69 and 127 and 185, 3 and 8 and 69 and 127 and 243, 3 and 8 and 69 and 185 and 243, 3 and 8 and 127 and 185 and 243, 3 and 9 and 69 and 127 and 185, 3 and 9 and 69 and 127 and 243, 3 and 9 and 69 and 185 and 243, 3 and 9 and 127 and 185 and 243, 3 and 69 and 127 and 185 and 243, 8 and 9 and 69 and 127 and 185, 8 and 9 and 69 and 127 and 243, 8 and 9 and 69 and 185 and 243, 8 and 9 and 127 and 185 and 243, 8 and 69 and 127 and 185 and 243, 9 and 69 and 127 and 185 and 243, 3 and 8 and 9 and 69 and 127 and 185, 3 and 8 and 9 and 69 and 127 and 243, 3 and 8 and 9 and 69 and 185 and 243, 3 and 8 and 9 and 127 and 185 and 243, 3 and 8 and 69 and 127 and 185 and 243, 3 and 9 and 69 and 127 and 185 and 243, 8 and 9 and 69 and 127 and 185 and 243, and 3 and 8 and 9 and 69 and 127 and 185 and 243 at a position corresponding to the position in SEQ ID NO: 16 selected therefrom, or only at or at these positions, the polypeptide according to claim 21. **Claim 23** The immunogenic polypeptide according to any one of claims 13 to 22, wherein the first and / or second mutation is a non-conservative substitution. **Claim 24** The immunogenic polypeptide according to any one of claims 13 to 23, wherein at least one of the one or more first mutations is as described in claim 21, and at least one of the one or more first mutations as described in claim 21 is a mutation to lysine (K). **Claim 25** The immunogenic polypeptide according to claim 24, wherein all of the one or more first mutations as described in claim 21 are mutations to lysine (K). **Claim 26** At least one of the one or more second mutations is as described in claim 16, and at least one of the one or more second mutations that is as described in claim 16 is a mutation to alanine (A) or arginine (R). The immunogenic polypeptide according to any one of claims 13 to 25.

27. All of the one or more second mutations that are as described in claim 16 are mutations to alanine (A) or arginine (R). The immunogenic polypeptide according to claim 26.

28. The first mutation is as described in claim 24, and the second mutation is as described in claim 26, or the first mutation is as described in claim 25, and the second mutation is as described in claim 26, or the first mutation is as described in claim 24, and the second mutation is as described in claim 27, or the first mutation is as described in claim 25, and the second mutation is as described in claim 27. The immunogenic polypeptide according to any one of claims 13 to 27.

29. The variant has at least 85% sequence identity with the sequence of SEQ ID NO:

16. The immunogenic polypeptide according to any one of claims 1 to 28.

30. The polypeptide can induce an antibody that blocks the ability of wild-type SpA to bind to IgG and von Willebrand factor. The immunogenic polypeptide according to any one of claims 1 to 29.

31. i. It has at least 85% sequence identity with SEQ ID NO: 30, ii. It consists of or contains a variant of the amino acid sequence of LukE that does not contain the signal peptide corresponding to residues 1 to 28 of SEQ ID NO:

29. The immunogenic polypeptide.

32. The polypeptide consists of SEQ ID NO:

30. The immunogenic polypeptide according to claim 31.

33. The polypeptide can induce an antibody that blocks the leukotoxicity of functionally mature wild-type LukE. The immunogenic polypeptide according to claim 31 or 32.

34. i. It has at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 34, ii. An immunogenic polypeptide consisting of or comprising a variant of the amino acid sequence of aureolysin (Aur) that contains one or more amino acid substitutions in the HEX X H catalytic domain corresponding to amino acid positions 352 to 356 of SEQ ID NO: 34, wherein the polypeptide has reduced catalytic ability and the substitution is preferably non-conservative. **Claim 35** The immunogenic polypeptide according to claim 34, wherein in the variant, the conserved cysteine (C) corresponding to amino acid position 479 of SEQ ID NO: 34 is substituted. **Claim 36** The immunogenic polypeptide according to claim 35, wherein the conserved cysteine is substituted with serine (S). **Claim 37** The immunogenic polypeptide according to any one of claims 34 to 36, wherein the glutamic acid residue (E) in the HEX X H catalytic domain corresponding to amino acid position 353 of SEQ ID NO: 34 is preferably substituted non-conservatively. **Claim 38** The immunogenic polypeptide according to claim 37, wherein the glutamic acid in the HEX X H catalytic domain is substituted with alanine (A). **Claim 39** The immunogenic polypeptide according to any one of claims 34 to 38, wherein the variant consists of or comprises SEQ ID NO:

35. **Claim 40** The immunogenic polypeptide according to any one of claims 34 to 39, wherein the variant comprises a sequence having at least 85% sequence identity with the amino acid sequence 210 - 509 of SEQ ID NO:

34. **Claim 41** The immunogenic polypeptide according to any one of claims 34 to 40, wherein the variant exhibits a decrease in the ability to participate in the formation of antigen complexes as compared to wild-type Aur. **Claim 42** The immunogenic polypeptide according to any one of claims 34 to 41, wherein the polypeptide is capable of inducing an antibody that blocks the catalytic activity of wild-type Aur. **Claim 43** having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 38, a. containing one or more amino acid substitutions in the amidase active site TXEXX domain corresponding to amino acid residues 384 - 388 of SEQ ID NO: 37, and / or b. containing one or more substitutions in the amidase active site LXDYX domain corresponding to amino acid residues 409 - 413 of SEQ ID NO: 37, and / or c. An immunogenic polypeptide consisting of or comprising a variant of the amino acid sequence of N-acetylmuramoyl-L-alanine amidase (SAR 2723) that contains a substitution of a conserved cysteine corresponding to position 513 of SEQ ID NO: 37, wherein the polypeptide has a reduced catalytic ability and the substitution is preferably non-conservative, the immunogenic polypeptide. **Claim 44** The immunogenic polypeptide according to claim 43, comprising the following features i: a only, b only, c only, a and b only, a and c only, b and c only, or a, b, and c. **Claim 45** The immunogenic polypeptide according to any one of claims 43 or 44, wherein the conserved cysteine is substituted with serine (S). **Claim 46** The immunogenic polypeptide according to any one of claims 43 to 45, wherein the glutamic acid residue (E) in the active site TXEXx domain corresponding to amino acid position 386 of SEQ ID NO: 37 is substituted, for example non-conservatively, preferably with glutamine (Q). **Claim 47** The immunogenic polypeptide according to any one of claims 43 to 46, wherein the aspartic acid (D) in the active site LXDYS domain corresponding to amino acid position 411 of SEQ ID NO: 37 is substituted, for example non-conservatively, preferably with asparagine (N). **Claim 48** The immunogenic polypeptide according to any one of claims 43 to 47, wherein the variant consists of or comprises SEQ ID NO:

39. **Claim 49** The immunogenic polypeptide according to any one of claims 43 to 48, wherein the polypeptide can block the catalytic activity of wild-type SAR2723 or induce an antibody that interferes with the development of the bacterial cell wall. **Claim 50** a - d: a) An Hla polypeptide or a variant thereof, wherein the variant preferably exhibits a decrease in hemolytic activity or does not exhibit hemolytic activity and / or can preferably induce an antibody that blocks the hemolytic activity of native Hla, the Hla polypeptide or a variant thereof, b) A LukE polypeptide or a variant thereof, wherein the variant preferably exhibits a decrease in leukocyte activity or does not exhibit leukocyte activity and / or can preferably induce an antibody that blocks the leukocyte activity of native LukE, the LukE polypeptide or a variant thereof, c) An SpA polypeptide or a variant thereof, wherein the variant preferably exhibits a reduced ability to bind to human IgG and human von Willebrand factor, and / or can preferably induce an antibody that blocks the natural SpA interaction with human IgG, an SpA polypeptide or a variant thereof, and d) An Aur polypeptide or a variant thereof, wherein the variant preferably exhibits a reduced catalytic activity and can preferably induce an antibody that blocks the catalytic activity of natural Aur, a selection of polypeptides comprising at least two of the Aur polypeptides or variants thereof, a vaccine composition, wherein the composition may further comprise a pharmaceutically acceptable carrier, medium or diluent, and in particular may further comprise an immunogenic adjuvant selected from AlOH, SLA-SE and OMV (outer membrane vesicles). **Claim 51** The vaccine composition according to claim 50, wherein the Hla polypeptide variant is as described in any one of claims 1 to 12, and / or the LukE polypeptide variant is as described in any one of claims 31 to 33, and / or the SpA polypeptide variant is as described in any one of claims 13 to 30, and / or the Aur polypeptide variant is as described in any one of claims 34 to 42. **Claim 52** The vaccine composition according to claim 50, wherein the Hla polypeptide variant has the amino acid sequence of SEQ ID NO:

5. **Claim 53** The vaccine composition according to any one of claims 50 to 52, comprising at least three of a to d. **Claim 54** - a and b, - a and c, - a and d, - b and c, - b and d, - c and d, - a, b, and c, - a, b, and d, - a, c, and d, - b, c, and d, or - a, b, c, and d The vaccine composition according to any one of claims 50 to 52, comprising. **Claim 55** e) An SAR2723 polypeptide or a variant thereof, wherein the variant preferably exhibits a reduced catalytic activity or no catalytic activity and can preferably induce an antibody that blocks the catalytic activity of natural SAR2723, an SAR2723 polypeptide or a variant thereof, f) an SAR2753 polypeptide or variant thereof, wherein said variant preferably exhibits a decrease in lipase activity or does not exhibit lipase activity, and preferably can induce an antibody that blocks the lipase activity of native SAR2753, an SAR2753 polypeptide or variant thereof, g) an SAR0992 (HtrA2) polypeptide or variant thereof, and h) an SAR0280 (EsxA) polypeptide or variant thereof, wherein said variant preferably can induce an antibody that prevents EsxA / B secretion, an SAR0280 (EsxA) polypeptide or variant thereof The vaccine composition according to any one of claims 50 to 54, further comprising at least one polypeptide selected from **Claim 56** - The SAR0992 polypeptide variant is a fragment or sequence variant of SEQ ID NO: 15 disclosed in WO2012 / 136653, particularly a fragment consisting of amino acid residues 1 to 409 of SEQ ID NO: 15 of WO2012 / 136653, or a variant of SAR0992 that can induce an antibody that blocks the catalytic activity of native SAR0992, for example, a truncation of SAR0992 at the C-terminus relative to the transmembrane helix of SAR0992, or a mutant version of SAR0992 that includes a substitution of a serine residue corresponding to the serine residue at position 619 of SEQ ID NO: 15 disclosed in WO2012 / 136653, and / or the transmembrane helix is replaced with a flexible linker, or the transmembrane helix is replaced with a linker and the N-terminal and C-terminal portions of SAR0992 adjacent to the linker are exchanged with each other, and / or - The SAR0280 polypeptide variant is a fragment or sequence variant of SEQ ID NO: 13 disclosed in WO2012 / 136653, or a fusion of the most N-terminal extracellular fragment and the most C-terminal extracellular fragment of SAR0280, and / or - The SAR2723 polypeptide variant is as described in any one of claims 43 to 49, or a fragment or sequence variant of SEQ ID NO: 13 disclosed in WO2015 / 082536, and / or The vaccine composition according to claim 55, wherein the -SAR2753 polypeptide variant is a fragment or sequence variant of SEQ ID NO: 14 disclosed in WO2015 / 082536, or a polypeptide composed of amino acid SEQ ID NO:

42.

57. The vaccine composition according to claim 55 or 56, comprising at least two of e to h.

58. The vaccine composition according to claim 55 or 56, comprising at least three of e to h.

59. - e and f, - e and g, - e and h, - f and g, - f and h, - g and h, - e, f, and g, - e, f, and h, - e, g, and h, - f, g, and h, or - e, f, g, and h The vaccine composition according to any one of claims 55 to 58, comprising.

60. A chimeric polypeptide comprising the amino acid sequence of a selection of the polypeptides according to any one of claims 50 to 59, wherein the amino acid sequences are fused or linked via a linker.

61. The chimeric polypeptide according to claim 60, wherein the linker is flexible or rigid.

62. The chimeric polypeptide according to claim 60, wherein the flexible linker is GSGGGGA (SEQ ID NO: 50) or GSGGGGAGSGGGGA (SEQ ID NO: 51), or the rigid linker is KPEPKPAPAPKP (SEQ ID NO: 52).

63. A chimeric polypeptide according to any one of claims 60 to 62, having an amino acid sequence selected from the group consisting of CHIM_LukE_mHla_H35L_FS (SEQ ID NO: 56), CHIM_mc2716_mSpA_7_12_FS (SEQ ID NO: 57), CHIM_0992_mSpA_7_12_FS (SEQ ID NO: 58), CHIM_2753_mc2723_FS (SEQ ID NO: 59), CHIM_0280_2753_FS (SEQ ID NO: 60), CHIM_0992_2753_FS (SEQ ID NO: 61), CHIM_mc2723_Hla_H35L_t_FS (SEQ ID NO: 62), CHIM_mc2716_LukE_FS (SEQ ID NO: 63), CHIM_LukE_Hla_H35L_t_FS (SEQ ID NO: 64), CHIM_mc2716_mSpA_10_12_FS (SEQ ID NO: 65), CHIM_LukE_0280_FS (SEQ ID NO: 66), CHIM_m0992_0992_FL (SEQ ID NO: 67), CHIM_0992_Hla_H35L_t_FS (SEQ ID NO: 68), CHIM_LukE_mSpA_10_12_FS (SEQ ID NO: 82), CHIM_mc2723_mSpA_10_12_FS (SEQ ID NO: 83), CHIM_0992_mSpA_10_12_FS (SEQ ID NO: 84), and CHIM_LukE_mSpA_7_12_FS (SEQ ID NO: 85).

64. A vaccine composition comprising at least one chimeric polypeptide according to any one of claims 60 to 63, further comprising a pharmaceutically acceptable carrier, vehicle or diluent, and optionally further comprising an immunological adjuvant.

65. The vaccine composition according to claim 64, wherein at least one chimeric polypeptide is selected from CHIM_LukE_mHla_H35L_FS (SEQ ID NO: 56), CHIM_mc2716_mSpA_7_12_FS (SEQ ID NO: 57), and CHIM_2753_mc27723_FS (SEQ ID NO: 59), preferably from SEQ ID NOs: 56 and 57.

66. The vaccine composition according to claim 64 or 65, comprising at least or exactly two chimeric polypeptides according to any one of claims 60 to 63.

67. A vaccine composition according to any one of claims 64 to 66, further comprising at least or exactly one additional polypeptide selected from the polypeptides according to any one of claims 50 to 52, 55, and 56.

68. The vaccine composition according to claim 67, wherein the additional polypeptide is selected from the group consisting of SAR0992-1-409 (residues 1 to 409 of SEQ ID NO: 44) and SAR0280-28-820 (SEQ ID NO: 48).

69. A vaccine composition according to any one of claims 64 to 68, comprising the chimeric polypeptides CHIM_LukE_mHla_H35L_FS (SEQ ID NO: 56) and CHIM_mc2716_mSpA_7_12_FS (SEQ ID NO: 57), and optionally, a polypeptide consisting of amino acid residues 1 to 409 of SAR0992 (residues 1 to 409 of SEQ ID NO: 44).

70. A vaccine composition according to claim 69, comprising a pharmaceutically acceptable carrier, medium or diluent, and optionally, mixed with an immunological adjuvant, the chimeric polypeptides CHIM_LukE_mHla_H35L_FS (SEQ ID NO: 56) and CHIM_mc2716_mSpA_7_12_FS (SEQ ID NO: 57), and optionally, a polypeptide consisting of amino acid residues 1 to 409 of SAR0992 (residues 1 to 409 of SEQ ID NO: 44).

71. The vaccine composition according to claim 70, comprising an immunological adjuvant selected from the group consisting of AlOH, SLA-SE, and OMV (outer membrane vesicles).

72. A nucleic acid fragment, such as a DNA fragment or an RNA fragment, encoding an immunogenic polypeptide according to any one of claims 1 to 49 or a chimeric polypeptide according to any one of claims 60 to 63.

73. A vector comprising the nucleic acid fragment according to claim 64.

74. The vector according to claim 65, which is an expression vector.

75. A transformed cell or virus comprising the nucleic acid fragment according to claim 64 or the vector according to claim 66 and capable of expressing.

76. An immunogenic composition comprising the nucleic acid fragment according to claim 64, the vector according to claim 65 or 66, or the transformed cell or virus according to claim 67, and a pharmaceutically acceptable carrier, medium or diluent, and optionally, an immunogenic adjuvant.

77. A nucleic acid fragment, vector, transformed cell or virus capable of expressing a selected polypeptide according to any one of claims 50 to 59, and a pharmaceutically acceptable carrier, vehicle or diluent, and optionally an immunogenic adjuvant, an immunogenic composition.

78. A method for inducing immunity in an animal by administering at least once an immunogenically effective amount of an immunogenic polypeptide according to any one of claims 1 to 49, a vaccine composition according to any one of claims 50 to 59, a chimeric polypeptide according to any one of claims 60 to 63, a nucleic acid fragment according to claim 64, a vector according to claim 65 or 66, a transformed cell or virus according to claim 67, or an immunogenic composition according to claim 68 or 69, in order to induce adaptive immunity against Staphylococcus aureus in the animal.

79. When an immunogenic polypeptide according to any one of claims 1 to 49, a chimeric polypeptide according to any one of claims 60 to 63, or a composition comprising said immunogenic polypeptide or said chimeric polypeptide is administered, the animal receives 0.5 to 5,000 μg of an immunogenic polypeptide or chimeric polypeptide according to any one of claims 1 to 49 and 60 to 63 per administration, the method according to claim 70.

80. The method according to claim 70 or 71, wherein the animal receives a first priming dose comprising said immunogenic polypeptide or said chimeric polypeptide and one or more booster doses comprising said immunogenic polypeptide or said chimeric polypeptide.

81. The method according to any one of claims 70 to 72, wherein the animal is a human.

82. The method according to any one of claims 70 to 73, wherein the administration is for the purpose of inducing protective immunity against Staphylococcus aureus.

83. The method according to claim 74, wherein the protective immunity is effective to reduce the risk of attracting an infection by Staphylococcus aureus or is effective to treat or ameliorate an infection by Staphylococcus aureus.

84. The method according to any one of claims 70 to 75, wherein the administration is for the purpose of inducing an antibody specific for Staphylococcus aureus, and the antibody or B lymphocytes producing the antibody are subsequently recovered from the animal.

85. The method according to any one of claims 70 to 75, wherein administration is aimed at inducing an antibody specific for Staphylococcus aureus, and B lymphocytes producing the antibody are then recovered from an animal and used for the preparation of a monoclonal antibody.

86. An immunogenic polypeptide according to any one of claims 1 to 49 for use as a medicament.

87. An immunogenic polypeptide according to any one of claims 1 to 49 for use as a medicament in the treatment, prevention or amelioration of an infection by Staphylococcus aureus.

88. A chimeric polypeptide according to any one of claims 60 to 63 for use as a medicament.

89. A chimeric polypeptide according to any one of claims 60 to 63 for use as a medicament in the treatment, prevention or amelioration of an infection by Staphylococcus aureus.

90. A nucleic acid fragment according to claim 64 or a vector according to claim 65 or 66 for use as a medicament.

91. A nucleic acid fragment according to claim 64 or a vector according to claim 65 or 66 for use as a medicament in the treatment, prevention or amelioration of an infection by Staphylococcus aureus.

92. A transformed cell or virus according to claim 67 for use as a medicament.

93. A transformed cell or virus according to claim 67 for use as a medicament in the treatment, prevention or amelioration of an infection by Staphylococcus aureus.

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