Staphylococcus protein variants and truncations

Abstract

The present 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 / involving SpA, Hla, Aur and LukE polypeptides. Also disclosed are methods of immunity induction using the polypeptides, polynucleotides, expression vectors, and immunogenic compositions.

Description

Technical Field

[0001] The present invention relates to the field of immunotechnology, including vaccine technology. Specifically, the present invention relates to novel variants of staphylococcal proteins and compositions comprising staphylococcal proteins. The present invention also relates to vectors, transformed cells and viruses and compositions comprising these. Background Art

[0002] Staphylococcus aureus is a Gram-positive opportunistic pathogen that is a major clinical challenge. In particular, multidrug-resistant methicillin-resistant Staphylococcus aureus (MRSA) strains cause serious hospital-acquired and community-acquired infections and are a huge global health problem. Antibiotic alternatives and vaccines for the treatment of S. aureus infections are urgently needed.

[0003] Despite years of research, no vaccine against S. aureus has yet to be tested in clinical trials. The traditional strategy for developing a vaccine has been to elicit opsonic antibodies against S. aureus surface proteins, leading to antibody-mediated clearance of the bacteria, but this strategy has not been successful. A different and more promising strategy is to develop vaccines that aim to neutralize one or more virulence factors of S. aureus. Virulence factors of S. aureus include several different proteins with toxic and immune evasion functions. The following is a brief description of specific S. aureus virulence factors and other S. aureus proteins currently disclosed.

[0004] IgG binding protein A

[0005] Immunoglobulin G binding protein A (SpA) is a protein containing 4 to 5 homologous immunoglobulin binding domains (E, D, A, B, C), each of which can bind to the constant region of IgG (Fcγ) and Ig fragments (Fab) involved in antigen binding. The SpA Fc binding site can also bind to von Willebrand factor (vWF). SpA induces immune evasion through multiple mechanisms. SpA binding to the Fcγ domain interferes with the antimicrobial functions of IgG, including complement fixation and opsonophagocytosis. Binding to Fab provides SpA with a powerful B cell superantigen function, where secreted SpA cross-links von Willebrand factor (vWF) containing Fab. H 3 B cell receptors and trigger all V H 3 secretion of antibodies, regardless of their antigenic specificity. Therefore, antibody-mediated protective immunity against S. aureus is severely impaired.

[0006] α-Hemolysin

[0007] α-Hemolysin (Hla), also known as α-toxin, is a member of the β-barrel toxin family and is the primary cytotoxic agent of Staphylococcus aureus. Hla is a monomer, and seven copies of Hla self-assemble to form a heptameric pore in the cell membrane that allows monovalent ion exchange, leading to DNA fragmentation and apoptosis. The heptameric pore consists of three structural regions: the cap, the rim, and the stem. Hla monomers may also oligomerize to form larger, Ca 2+ permissive pores, thereby inducing massive cell necrosis.

[0008] HtrA2

[0009] HtrA-like proteases are involved in the virulence of both Gram-positive and Gram-negative bacteria.

[0010] They are known to play roles in stress resistance and survival. In Streptococcus pyogenes, HtrA has been reported to interfere with the processing of extracellular virulence factors and play a role in controlling hemolytic activity. Staphylococcus aureus encodes two putative HtrA-like proteases, HtrA1 and HtrA2. HtrA2 of S. aureus is classified as a transmembrane protein that contains a domain predicted to be an active serine protease with an Asp / His / Ser catalytic triad.

[0011] Luke

[0012] The LukE protein is one of the two components of the lukED leukocidin. In addition to inducing leukocyte death, the pore-forming toxin LukED can also lyse erythrocytes. The 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 LukE recognizes the cell surface receptor, LukE alone does not form pores, only the heterodimers of LukE and LukD form pores.

[0013] Lysozyme

[0014] Aureolysin (Aur) is a zinc metalloproteinase. It is a Staphylococcus aureus virulence factor with multiple roles in immune evasion and toxicity. Aur activates glutamyl endopeptidase, a key virulence factor of S. aureus that cleaves certain human inflammatory mediators and immune components and inhibits 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. In addition, Aur activates prothrombin in human plasma and induces staphylococcal coagulation.

[0015] SAR2753

[0016] Lipase 1 / glyceride hydrolase 1 (SAR2753) is an enzyme that converts triglycerides + H 2 O is converted into diacylglycerol + fatty acids + H + SAR2753 may play a virulence role in S. aureus by degrading antimicrobial lipids produced by the host during infection.

[0017] SAR2723

[0018] N-acetylmuramoyl-L-alanine amidase (SAR2723) is a protein containing two amidase domains, LYZ2 / FlgJ and CHAP, which play a major role in peptidoglycan hydrolysis. Consistently, many proteins containing these domains are involved in cell wall metabolism and biogenesis.

[0019] Ea

[0020] The EsaA protein of Staphylococcus aureus is an essential 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 S. aureus T7SS is called T7b and 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, which contribute to the virulence of S. aureus. In addition, T7b-mediated secretion contributes to the production or suppression of specific cytokines during host infection, allowing S. aureus to manipulate the immune response.

[0021] Purpose of the Invention

[0022] It is an object of embodiments of the present invention to provide immunogens and immunogenic compositions that can be used for vaccination against SA infection. It is another object of the present invention to provide methods of immunizing / vaccinating against SA infection using these immunogens and immunogenic compositions. Summary of the invention

[0023] The present inventors have undertaken a comprehensive research program to identify new pharmaceutically acceptable immunogens that are derived from S. aureus antigens and are believed to be suitable for immunization / vaccination purposes. Part of the research has focused on reducing or eliminating the risks of administering immunogens, which may cause adverse effects similar to those exhibited by the wild-type antigens from which the immunogens were derived. In addition, the present inventors have identified optimized vaccine compositions that are believed to be able to induce useful immune responses against a variety of different S. aureus antigens, thereby effectively blocking various effector molecules of S. aureus.

[0024] Therefore, in a first aspect, the present invention relates to an immunogenic polypeptide consisting of or comprising:

[0025] i. A variant of the amino acid sequence of immunoglobulin G binding protein A (SpA), the variant comprising at least one of ae:

[0026] 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),

[0027] b. a sequence that is at least 85% identical to the amino acid sequence of IgBD D (SEQ ID NO: 16, residues 62-117),

[0028] c. a sequence that is at least 85% identical to the amino acid sequence of IgBD A (SEQ ID NO: 16, residues 120-175),

[0029] d. a sequence that is at least 85% identical to the amino acid sequence of IgBD B (SEQ ID NO: 16, residues 178-233),

[0030] e. a sequence that is at least 85% identical to the amino acid sequence of IgBD C (SEQ ID NO: 16, residues 236-291),

[0031] and

[0032] ii. one or more first mutations in each of at least one of items ae, wherein the one or more first mutations disrupt binding at the Fc binding site, and wherein the one or more first mutations are made at a position corresponding to an amino acid position selected from position 3, 8, 9, 64, 69, 70, 122, 127, 128, 180, 185, 186, 238, 243, 244 in SEQ ID NO: 16, and

[0033] iii. one or more second mutations in each of at least one of items ae, wherein the one or more second mutations disrupt binding at the Fab binding site, and wherein the one or more second mutations, where applicable, are made at a position corresponding to an amino acid position 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 in SEQ ID NO: 16,

[0034] iv. wherein, optionally, at least one of items ae, where applicable, does not comprise mutations that disrupt binding at the Fc binding site at positions corresponding to two amino acid positions 7+8, 68+69, 126+127, 184+185 and 242+243 of SEQ ID NO: 16, and

[0035] v. wherein at least one of items ae, where applicable, does not contain mutations that disrupt binding in the Fab binding site at the positions corresponding to the two amino acid positions 34+35 and 95+96 and 153+154 and 211+212 and 269+270 of SEQ ID NO: 16,

[0036] wherein the polypeptide is unable to bind to human IgG and human von Willebrand Factor, and wherein the substitution is preferably a non-conservative substitution.

[0037] In a second aspect, the present invention relates to an immunogenic polypeptide consisting of or comprising a variant of the amino acid sequence of α-hemolysin (Hla), said variant:

[0038] i. has at least 85% sequence identity with the sequence of SEQ ID NO: 2, and

[0039] 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,

[0040] wherein the polypeptide is incapable of participating in the formation of a heptameric structure with other HIa molecules, and wherein any substitution in ii) is preferably non-conservative.

[0041] In a third aspect, the present invention relates to an immunogenic polypeptide consisting of or comprising a variant of the amino acid sequence of LukE, said variant:

[0042] i. has at least 85% sequence identity with SEQ ID NO: 30, and

[0043] ii. does not contain a signal peptide corresponding to residues 1-28 of SEQ ID NO:29.

[0044] In a fourth aspect, the present invention relates to an immunogenic polypeptide consisting of or comprising a variant of the amino acid sequence of aurolysin (Aur), wherein:

[0045] i. having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 34, and

[0046] ii. comprising one or more amino acid substitutions in the HEXXH catalytic domain, wherein the HEXXH catalytic domain corresponds to amino acid positions 352-356 of SEQ ID NO: 34,

[0047] Wherein the polypeptide has reduced catalytic ability and wherein the substitution is preferably non-conservative.

[0048] In a fifth aspect, the present invention relates to an immunogenic polypeptide consisting of or comprising a variant of the amino acid sequence of N-acetylmuramoyl-L-alanine amidase (SAR 2723), wherein:

[0049] i. has at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 38, and

[0050] a. comprising one or more amino acid substitutions in the amidase active site TXEXX domain corresponding to amino acid residues 384-388 in SEQ ID NO: 37, and / or

[0051] b. comprising one or more substitutions in the amidase active site LXDYX domain corresponding to amino acid residues 409-413 in SEQ ID NO: 37, and / or

[0052] c. comprising a substitution corresponding to the conserved cysteine ​​at position 513 in SEQ ID NO: 37,

[0053] wherein the polypeptide has reduced catalytic ability, and wherein the substitution is preferably non-conservative.

[0054] In a sixth aspect, the present invention relates to a vaccine composition comprising selected polypeptides comprising at least 2 of items ad:

[0055] a) an HIa polypeptide or a variant thereof, wherein the variant preferably exhibits reduced or no hemolytic activity and / or preferably is capable of inducing antibodies that block the hemolytic activity of native HIa,

[0056] b) a LukE polypeptide or a variant thereof, wherein the variant preferably exhibits reduced or no leukocyte activity and / or preferably is capable of inducing antibodies that block the leukocyte activity of native LukE,

[0057] c) a 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 preferably is capable of inducing antibodies that block the interaction of native SpA with human IgG, and

[0058] d) an Aur polypeptide or a variant thereof, wherein the variant preferably exhibits reduced catalytic activity and is preferably capable of inducing antibodies that block the catalytic activity of native Aur,

[0059] The composition optionally further comprises a pharmaceutically acceptable carrier, vehicle or diluent, and optionally further comprises an immunogenic adjuvant, which is particularly selected from AlOH, SLA-SE and OMV (outer membrane vesicles).

[0060] In a seventh aspect, the present invention relates to a chimeric polypeptide comprising the amino acid sequence of a polypeptide selected according to the sixth aspect of the present invention, wherein the amino acid sequence is fused or linked via a linker.

[0061] In an eighth aspect, the present invention relates to a nucleic acid fragment encoding an immunogenic polypeptide according to any one of aspects 1 to 5 of the present invention or a chimeric polypeptide according to aspect 7 of the present invention, such as a DNA fragment or an RNA fragment.

[0062] In a ninth aspect, the present invention relates to a vector comprising a nucleic acid fragment according to the eighth aspect of the present invention.

[0063] In a tenth aspect, the present invention relates to a transformed cell or virus comprising and capable of expressing the nucleic acid fragment according to the eighth aspect of the present invention or the vector according to the ninth aspect of the present invention.

[0064] In the eleventh aspect, the present invention relates to an immunogenic composition comprising a nucleic acid fragment according to the eighth aspect of the present invention, a vector according to an embodiment of the ninth aspect of the present invention, or a transformed cell or virus according to the tenth aspect of the present invention and a pharmaceutically acceptable carrier, vehicle or diluent, and optionally an immunogenic adjuvant.

[0065] In the twelfth aspect, the present invention relates to an immunogenic composition comprising a nucleic acid fragment, a vector or a transformed cell or a virus capable of expressing a polypeptide selected according to the sixth aspect of the present invention, and a pharmaceutically acceptable carrier, vehicle or diluent, and an optional immunogenic adjuvant.

[0066] In the thirteenth aspect, the present invention relates to a method for inducing immunity in an animal, by administering at least once an immunogenic effective amount of an immunogenic polypeptide according to an embodiment of any one of the first to fifth aspects of the present invention, a vaccine composition according to the sixth aspect of the present invention, a chimeric polypeptide according to the seventh aspect of the present invention, a nucleic acid fragment according to the eighth aspect of the present invention, a vector according to the ninth aspect of the present invention, a transformed cell or virus according to the tenth aspect of the present invention, or an immunogenic composition according to the eleventh or twelfth aspect of the present invention, to induce adaptive immunity against Staphylococcus aureus in the animal.

[0067] In a fourteenth aspect, the present invention relates to an immunogenic polypeptide according to any one of the first to fifth aspects of the present invention for use as a medicament.

[0068] In a fifteenth aspect, the present invention relates to an immunogenic polypeptide according to any one of the first to fifth aspects of the present invention for use as a medicament for treating, preventing or ameliorating Staphylococcus aureus infection.

[0069] In a sixteenth aspect, the present invention relates to a chimeric polypeptide according to the seventh aspect of the invention for use as a medicament.

[0070] In a seventeenth aspect, the present invention relates to a chimeric polypeptide according to the seventh aspect of the present invention for use as a medicament for treating, preventing or ameliorating Staphylococcus aureus infection.

[0071] In an eighteenth aspect, the present invention relates to a nucleic acid fragment according to the eighth aspect of the invention or a vector according to the ninth aspect of the invention for use as a medicament.

[0072] In the nineteenth aspect, the present invention relates to the nucleic acid fragment according to the eighth aspect of the present invention or the vector according to the ninth aspect of the present invention, for use as a drug for treating, preventing or ameliorating Staphylococcus aureus infection.

[0073] In a twentieth aspect, the invention relates to a transformed cell or virus according to the tenth aspect of the invention for use as a medicament.

[0074] In a twenty-first aspect, the present invention relates to a transformed cell or virus according to the tenth aspect of the present invention, for use as a medicament for treating, preventing or ameliorating Staphylococcus aureus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 : Non-specific binding of different SpA variants to mouse IgG determined by direct ELISA (see Example 1.1).

[0076] Figure 2 : Non-specific binding of different SpA variants to human IgG determined by direct ELISA (see Example 1.1).

[0077] Figure 3 : Non-specific binding of different SpA variants to mouse serum IgG determined by direct ELISA (see Example 1.1).

[0078] Figure 4 : Non-specific binding of different SpA variants to mouse serum IgM determined by direct ELISA (see Example 1.1).

[0079] Figure 5: Non-specific binding of different SpA variants to rabbit serum IgG determined by direct ELISA (see Example 1.1).

[0080] Figure 6 : Binding of different SpA variants to vWF determined by direct ELISA. SpA_WT-49-339 binding was set to 100% and the relative SpA binding (percentage) was calculated based on this value (see Example 1.1).

[0081] Figure 7 : Determination of the non-specific binding of different SpA variants to human IgG by competitive ELISA (see Example 1.1).

[0082] Figure 8 :AB: The ability of immune sera from mice vaccinated with the different single and chimeric proteins to inhibit aurisin activity was determined by azocasein assay (see Example 1.2).

[0083] Fig. 9 : The ability of immune sera from mice vaccinated with LukE alone or with a chimeric protein containing LukE to inhibit LukED leukocyte activity was determined by XTT assay (see Example 1.3).

[0084] Fig.10 : The ability of immune sera from mice vaccinated with Hla alone or with a chimeric protein containing Hla to inhibit the hemolytic activity of Hla was determined by a red blood cell hemolysis assay (see Example 1.4).

[0085] Figure 11: Immunogenicity, half-maximal IgG data of mice immunized with single or chimeric proteins adjuvanted with SLA-SE (see Example 4).

[0086] Fig.12 : Immunogenicity, half-maximal IgG data of mice immunized with single or chimeric proteins adjuvanted with OMV+AIOH or AIOH (see Example 4). DETAILED DESCRIPTION OF THE INVENTION

[0088] definition

[0089] The term "polypeptide" herein means short peptides of 2 to 10 amino acid residues, oligopeptides of 11 to 100 amino acid residues, and polypeptides of more than 100 amino acid residues. In addition, the term is also intended to include proteins, i.e., functional biomolecules comprising at least one polypeptide; when comprising at least two polypeptides, these polypeptides may form a complex, be covalently linked or may be non-covalently linked. The polypeptides in the protein may be glycosylated and / or lipidated and / or contain prosthetic groups.

[0090] The term "subsequence" refers to any contiguous stretch of at least 3 amino acids or, where relevant, at least 3 nucleotides derived directly from a naturally occurring amino acid sequence or nucleic acid sequence, respectively.

[0091] The term "amino acid sequence" refers to the order in which amino acid residues connected by peptide bonds are arranged in the chain of peptides and proteins in the direction from N-terminus to C-terminus.

[0092] The term "adjuvant" has the usual meaning in the field of vaccine technology, i.e., a substance or composition of substances that 1) cannot induce a specific immune response to the immunogen of the vaccine by itself, but 2) can enhance the immune response to the immunogen. Or, in other words, vaccination with an adjuvant alone will not produce an immune response to the immunogen, vaccination with an immunogen may or may not produce an immune response to the immunogen, but vaccination with a combination of an immunogen and an adjuvant will induce an immune response to the immunogen that is stronger than the immune response to the immunogen induced by the immunogen alone.

[0093] In the context of the present invention, "sequence identity" is determined by comparing two optimally aligned sequences (eg, DNA, RNA or amino acids) of equal length according to the following formula: ref –N dif )·100 / N ref , where N ref is the number of residues in one of the two sequences, N dif is the number of residues that are not identical when the two sequences are aligned in the same direction over their entire length. Thus, the two sequences 5'-ATTCGGAAC-3' and 5'-ATACGGGAC-3' would provide 77.8% sequence identity (N ref =9 and N dif =2).

[0094] "3D conformation" is the three-dimensional structure of a biomolecule such as a protein. In a monomeric polypeptide / protein, the 3D conformation is also called "tertiary structure" and represents the relative positions of the amino acid residues forming the polypeptide in three-dimensional space.

[0095] An "immunogenic carrier" is a molecule or moiety that can be coupled to an immunogen or hapten to enhance or enable the induction of an immune response to the immunogen / hapten. Classically, the immunogenic carrier is a relatively large molecule (e.g., tetanus toxoid, KLH, diphtheria toxoid, etc.) to which an immunogen / hapten is fused or conjugated that is not sufficiently immunogenic on its own - typically, the immunogenic carrier is capable of inducing a strong T helper lymphocyte response to the combination of immunogen and immunogenic carrier, which in turn enhances the response of B lymphocytes and cytotoxic lymphocytes to the immunogen. Recently, large carrier molecules have been replaced to some extent by so-called promiscuous T helper epitopes, i.e., shorter peptides that are recognized by a large fraction of the HLA haplotypes in a population and are capable of inducing a T helper lymphocyte response.

[0096] A "T helper lymphocyte response" is an immune response based on a peptide that is able to bind to a class II MHC molecule (e.g., a class II HLA molecule) in an antigen presenting cell and stimulates T helper lymphocytes in an animal species due to recognition of a complex between the peptide and the class II MHC molecule present in the antigen presenting cell by the T cell receptor.

[0097] An "immunogen" is a substance that is capable of inducing an adaptive immune response in a host when the host's immune system is confronted with it. Immunogens are therefore a subset of the larger class of "antigens," which are substances that are specifically recognized by the immune system (e.g., when bound by antibodies, or when antigen fragments bound to MHC molecules are recognized by T-cell receptors), but are not necessarily capable of inducing immunity - however, antigens are always capable of eliciting immunity, meaning that a host that has established memory immunity to an antigen will develop a specific immune response to the antigen.

[0098] An "adaptive immune response" is an immune response generated in response to an antigen or immunogen that is specific for an antigenic determinant of the antigen / immunogen - 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.

[0099] A "protective, adaptive immune response" is an antigen-specific immune response induced in a subject in response to immunization (artificial or natural) with an antigen, wherein the immune response is capable of protecting the subject from subsequent challenge with the antigen or a pathology-related agent including the antigen. Typically, prophylactic vaccination is intended to establish a protective adaptive immune response against one or more pathogens.

[0100] "Stimulation of the immune system" refers to a substance or combination of substances that exhibits a general, non-specific immunostimulatory effect. Many adjuvants and putative adjuvants (e.g., certain cytokines) have the ability to stimulate the immune system. The result of using an immunostimulant is an increase in the "alertness" of the immune system, which means that immunization with the immunogen simultaneously or subsequently induces a more effective immune response than when the immunogen is used alone.

[0101] In this context, the term "animal" is generally intended to refer to an animal species (preferably a mammal), such as Homo sapiens, Canis familiaris, etc., rather than just a single animal. However, the term also refers to a population of such animal species, because it is important that individuals immunized according to the method of the present invention will essentially produce an immune response against the immunogen of the present invention.

[0102] The term "antibody" as used herein refers to a polypeptide or polypeptide group consisting of at least one antibody combining site. An "antibody combining site" is a three-dimensional binding space having an internal surface shape and charge distribution complementary to the characteristics of an antigenic epitope, which allows the antibody to bind to the antigen. An "antibody" includes, for example, vertebrate antibodies, hybrid antibodies, chimeric antibodies, humanized antibodies, altered antibodies, monovalent antibodies, Fab proteins, and single domain antibodies.

[0103] "Specific binding" refers to a binding between two substances that goes beyond the binding of either substance to a randomly selected substance or beyond a simple association between substances that tend to aggregate because of having the same overall hydrophobicity or hydrophilicity. Thus, specific binding typically involves a combination of electrostatic and other interactions between two conformationally complementary regions on the two substances, which means that the two substances can "recognize" each other in a complex mixture.

[0104] The term "vector" is used to refer to a carrier nucleic acid molecule into which a heterologous nucleic acid sequence can be inserted for introduction into a cell where it is replicated and expressed. The term also refers to certain biological vectors used for the same purpose, such as viral vectors and bacteriophages—both infectious agents capable of introducing heterologous nucleic acid sequences into host cells.

[0105] The term "expression vector" refers to a vector containing a nucleic acid sequence encoding at least part of a gene product that can be transcribed. In some cases, when the transcription product is an mRNA molecule, it can in turn be translated into a protein, polypeptide or peptide.

[0106] When referring to "amino acids", the 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, the 22 naturally occurring amino acids are preferred amino acids to appear in the polypeptides disclosed herein, since such polypeptides can be recombinantly produced.

[0107] The amino acid substitutions mentioned in the table below are considered "conservative". Therefore, substitutions not mentioned in the table are considered "non-conservative".

[0108]

[0109]

[0110] When variants of Staphylococcus aureus derived polypeptides are mutated as described herein, one embodiment requires that the amino acids are non-conservative, but the most important substitutions are those that do not result in significant changes in the secondary and tertiary structures of the substituted protein compared to the native protein. For example, proline introduces a fixed turn or bend in the secondary structure of the protein, so substitution with proline is usually avoided to avoid producing polypeptides that are unlikely to induce antibodies targeting the unsubstituted protein. In general, preferred mutations herein are those that make the mutant molecule less able to exert its pathological function (e.g., IgG and von Willebrand binding activity of SpA) but on the other hand retain the secondary and tertiary structure of the unmutated protein or protein fragment so that the mutant variant can induce non-mutated protein binding antibodies. For example, replacing a non-polar amino acid residue with a polar amino acid residue or vice versa will generally effectively interfere with the binding ability of the mutant variant, even if the 3D shape of the molecule remains almost unchanged.

[0111] When referring to an amino acid position or amino acid residue in a variant or homologous sequence, if the position or residue is said to "correspond to" an amino acid position in a reference amino acid sequence, it is intended herein to optimally align the reference sequence with the homologous / variant sequence; when doing so, the "corresponding" residue / position is the residue / position that is aligned with the indicated residue in the reference sequence. Optimal alignment of the two sequences must be achieved by preparing a global alignment of the reference sequence and the variant / homologous sequence using the EMBOSS Needle alignment, the online tool for which can be found at www.ebi.ac.uk / tools / psa (based on the Needleman-Wunsch algorithm), with the following settings for protein sequence alignment:

[0112] Matrix: Blosum62;

[0113] Gap open: 10;

[0114] Gap extend: 0.5;

[0115] End Gap Penalty: False;

[0116] End Gap Open: 10; and

[0117] End Gap Extend: 0.5.

[0118] When referring to "sequence identity" between amino acid sequences, the same alignment is used for the calculation. Therefore, optimal alignment of the two sequences must be achieved by preparing a global alignment of the reference sequence and variant / homologous sequence using the EMBOSS Needle alignment according to the parameters described above to obtain the percent sequence identity.

[0119] Specific embodiments of the present invention

[0120] Embodiments of the first aspect of the invention

[0121] The first aspect of the present invention relates to an immunogenic polypeptide consisting of or comprising:

[0122] i. A variant of the amino acid sequence of immunoglobulin G binding protein A (SpA), the variant comprising at least one of items ae:

[0123] a. a sequence that is at least 85% identical to the amino acid sequence of the immunoglobulin binding domain (IgBD) E (SEQ ID NO: 16, residues 1-56),

[0124] b. a sequence that is at least 85% identical to the amino acid sequence of IgBD D (SEQ ID NO: 16, residues 62-117),

[0125] c. a sequence that is at least 85% identical to the amino acid sequence of IgBD A (SEQ ID NO: 16, residues 120-175),

[0126] d. a sequence that is at least 85% identical to the amino acid sequence of IgBD B (SEQ ID NO: 16, residues 178-233),

[0127] e. a sequence that is at least 85% identical to the amino acid sequence of IgBD C (SEQ ID NO: 16, residues 236-291),

[0128] and

[0129] ii. the one or more first mutations in each of at least one of items ae, wherein the one or more first mutations disrupt binding at the Fc binding site, and wherein the one or more first mutations are made at a position corresponding to an amino acid position selected from position 3, 8, 9, 64, 69, 70, 122, 127, 128, 180, 185, 186, 238, 243, 244 in SEQ ID NO: 16, and

[0130] iii. the one or more second mutations in each of at least one of items ae, wherein the one or more second mutations disrupt binding at the Fab binding site, and wherein the one or more second mutations, where applicable, are made at a position corresponding to an amino acid position 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 in SEQ ID NO: 16,

[0131] iv. wherein, optionally, at least one of items ae, where applicable, does not comprise mutations that disrupt binding at the Fc binding site at positions corresponding to two amino acid positions 7+8, 68+69, 126+127, 184+185 and 242+243 of SEQ ID NO: 16, and

[0132] v. wherein at least one of items ae, where applicable, does not contain mutations that disrupt binding in the Fab binding site at positions corresponding to two amino acid positions 34+35 and 95+96 and 153+154 and 211+212 and 269+270 of SEQ ID NO: 16,

[0133] wherein the polypeptide is unable to bind to human IgG and human von Willebrand Factor, and wherein the substitution is preferably a non-conservative substitution.

[0134] The inventors have demonstrated that the specific mutational patterns in these novel SpA variants achieve the combined goals of being immunogenic and able to induce antibodies that bind to the wild-type protein, while exhibiting no effects on IgG and von Willebrand factor, which are characteristic of wild-type SpA.

[0135] In a first embodiment of the first aspect of the present invention, the one or more second mutations are made at least at the following positions or only at the following positions:

[0136] 1 or 2 or 3 or 4 or 5 of positions 34 and 96 and 154 and 212 and 270,

[0137] 1 or 2 or 3 or 4 or 5 of positions 35 and 95 and 154 and 212 and 270,

[0138] 1 or 2 or 3 or 4 or 5 of positions 35 and 96 and 153 and 212 and 270,

[0139] 1 or 2 or 3 or 4 or 5 of positions 35 and 96 and 154 and 211 and 270, 1 or 2 or 3 or 4 or 5 of positions 35 and 96 and 154 and 212 and 269, 1 or 2 or 3 or 4 or 5 of positions 34 and 95 and 154 and 212 and 270, 1 or 2 or 3 or 4 or 5 of positions 34 and 96 and 153 and 212 and 270, 1 or 2 or 3 or 4 or 5 of positions 34 and 96 and 154 and 211 and 270, 1 or 2 or 3 or 4 or 5 of positions 34 and 96 and 154 and 212 and 269, 1 or 2 or 3 or 4 or 5 of positions 35 and 95 and 153 and 212 and 270, 1 or 2 or 3 or 4 or 5 of positions 35 and 95 and 154 and 211 and 270, 1 or 2 or 3 or 4 or 5 of positions 35 and 95 and 154 and 212 and 269, 1 or 2 or 3 or 4 or 5 of positions 35 and 96 and 153 and 211 and 270, 1 or 2 or 3 or 4 or 5 of positions 35 and 96 and 153 and 212 and 269, 1 or 2 or 3 or 4 or 5 of positions 35 and 96 and 154 and 211 and 269, 1 or 2 or 3 or 4 or 5 of positions 34 and 95 and 153 and 212 and 270, 1 or 2 or 3 or 4 or 5 of positions 34 and 95 and 154 and 211 and 270, 1 or 2 or 3 or 4 or 5 of positions 34 and 95 and 154 and 212 and 269, 1 or 2 or 3 or 4 or 5 of positions 34 and 96 and 153 and 211 and 270, 1 or 2 or 3 or 4 or 5 of positions 34 and 96 and 153 and 212 and 269, 1 or 2 or 3 or 4 or 5 of positions 34 and 96 and 154 and 211 and 269, 1 or 2 or 3 or 4 or 5 of positions 35 and 95 and 153 and 211 and 270, 1 or 2 or 3 or 4 or 5 of positions 35 and 95 and 153 and 212 and 269, 1 or 2 or 3 or 4 or 5 of positions 35 and 96 and 153 and 211 and 269, 1 or 2 or 3 or 4 or 5 of positions 34 and 95 and 153 and 211 and 270, 1 or 2 or 3 or 4 or 5 of positions 34 and 95 and 153 and 212 and 269, 1 or 2 or 3 or 4 or 5 of positions 34 and 95 and 154 and 211 and 269,

[0140] 1 or 2 or 3 or 4 or 5 of positions 34 and 96 and 153 and 211 and 269,

[0141] 1 or 2 or 3 or 4 or 5 of positions 35 and 95 and 153 and 211 and 269,

[0142] 1 or 2 or 3 or 4 or 5 of positions 35 and 95 and 154 and 211 and 269,

[0143] 1 or 2 or 3 or 4 or 5 of positions 34 and 95 and 153 and 211 and 269, or 1 or 2 or 3 or 4 or 5 of positions 35 and 96 and 154 and 212 and 270.

[0144] In a second embodiment of the first aspect of the invention, the one or more second mutations are made at least or only at positions corresponding to positions selected from the group consisting of 34, 35, 95, 96, 153, 154, 211, 212, 269 and 270 in SEQ ID NO:16.

[0145] In a third embodiment of the first aspect of the invention, the one or more second mutations are made at positions corresponding to positions selected from the group consisting of 34, 35, 95, 96, 153, 154, 211, 212, 269 and 270 in SEQ ID NO:16.

[0146] In a fourth embodiment of the first aspect of the invention, the one or more second mutations are made at least or only at the positions corresponding to the positions in SEQ ID NO: 16 selected from the group consisting of: 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. 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, 15 3 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.

[0147] In a fifth embodiment of the first aspect of the invention, the one or more second mutations are made at least or only at the positions corresponding to the positions in SEQ ID NO: 16 selected from the group consisting of: 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. 12, 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 21 1, 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, 9 6 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, 153 and 2 11 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 and 211 and 270, and 154 and 212 and 270.

[0148] In a sixth embodiment of the first aspect of the invention, the one or more second mutations are made at least or only at the positions corresponding to the positions in SEQ ID NO: 16 selected from the group consisting of: 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, 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 27 0, 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 27 0, 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 270, 34 and 96 and 211 and 269, 34 and 96 and 211 and 270, 34 and 96 and 2 12 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 1 54 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, 3 5 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. ,

[0149] In a seventh embodiment of the first aspect of the invention, the one or more second mutations are made at least or only at positions corresponding to positions in SEQ ID NO: 16 selected from the group consisting of: 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. 70, 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, 3 5 and 96 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 15 3 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 21 1 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 26 9, 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.

[0150] In an eighth embodiment of the first aspect of the invention, the one or more first mutations are made at least or only at positions corresponding to positions selected from 8, 69, 127, 185 and 243 in SEQ ID NO:16.

[0151] In a ninth embodiment of the first aspect of the invention, the one or more first mutations are made at positions corresponding to positions in SEQ ID NO: 16 selected from the group consisting of: 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; 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 18 5 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 24 3; 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 1 85; 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 6 9 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 6 9 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 9 and 69 and 127 and 185; 3 and 9 and 69 and 127 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. ;

[0152] In the tenth embodiment of the first aspect of the present invention, the first mutation and / or the second mutation are non-conservative substitutions. However, as described above, this non-conservative substitution does not interfere with secondary structure and tertiary structure. In other words, the mutation should preferably retain the structure (to be able to induce effective antibodies that bind to the unmutated protein), but on the other hand, the mutation should preferably reduce or eliminate the undesirable function exhibited by the unmutated protein. This also results in a result, that is, if a mutation traditionally referred to as "conservative" has the effect of making the biological activity of the immunogen lower than that of the wild-type protein, then this mutation is useful.

[0153] In an eleventh embodiment of the first aspect of the invention, at least one of the one or more first mutations is as described in the eighth embodiment of the first aspect of the invention, and at least one of the one or more first mutations as described in the eighth embodiment of the first aspect of the invention is a mutation to lysine (K).

[0154] In the twelfth embodiment of the first aspect of the present invention, at least one of the one or more first mutations is as described in the eighth embodiment of the first aspect of the present invention, and all of the one or more first mutations as described in the eighth embodiment of the first aspect of the present invention are mutations to lysine (K).

[0155] In the thirteenth embodiment of the first aspect of the invention, at least one of the one or more second mutations is as described in the third embodiment of the first aspect of the invention, and at least one of the one or more second mutations as described in the third embodiment of the first aspect of the invention is a mutation to alanine (A) or arginine (R).

[0156] In the fourteenth embodiment of the first aspect of the invention, at least one of the one or more second mutations is as described in the third embodiment of the first aspect of the invention, and all of the one or more second mutations as described in the third embodiment of the first aspect of the invention are mutations to alanine (A) or arginine (R).

[0157] In the fifteenth embodiment of the first aspect of the invention, the first mutation is a mutation in the eleventh embodiment of the first aspect of the invention and the second mutation is a mutation in the thirteenth embodiment of the first aspect of the invention, or the first mutation is a mutation in the twelfth embodiment of the first aspect of the invention and the second mutation is a mutation in the thirteenth embodiment of the first aspect of the invention, or the first mutation is a mutation in the eleventh embodiment of the first aspect of the invention and the second mutation is a mutation in the fourteenth embodiment of the first aspect of the invention, or the first mutation is a mutation in the twelfth embodiment of the first aspect of the invention and the second mutation is a mutation in the fourteenth embodiment of the first aspect of the invention.

[0158] In a sixteenth embodiment of the first aspect of the invention, the variant has at least 85% sequence identity to the sequence of SEQ ID NO:16.

[0159] In a seventeenth embodiment of the first aspect of the invention the polypeptide is capable of inducing antibodies that block the ability of wild-type SpA to bind to IgG and von Willebrand Factor.

[0160] Embodiments of the second aspect of the invention

[0161] A second aspect of the present invention relates to an immunogenic polypeptide consisting of or comprising a variant of the amino acid sequence of α-hemolysin (Hla), wherein:

[0162] i. has at least 85% sequence identity with the sequence of SEQ ID NO: 2, and

[0163] 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,

[0164] wherein the polypeptide is incapable of participating in the formation of a heptameric structure with other HIa molecules, and wherein any substitution in ii) is preferably non-conservative.

[0165] As with the polypeptides of the first aspect of the invention, the polypeptides of the second aspect, due to carefully selected changes in their primary structure relative to the wild-type protein (Hla), have the ability to induce antibodies that bind to Hla while exhibiting reduced biological reactivity.

[0166] In a first embodiment of the second aspect of the invention, the variant comprises at least two amino acid changes selected from deletion and substitution in the first 12 consecutive N-terminal amino acid residues, such as 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 changes.

[0167] In a second embodiment of the second aspect of the invention, the variant comprises at least two amino acid substitutions in the first 12 N-terminal amino acids under item 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.

[0168] In a third embodiment of the second aspect of the 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.

[0169] In a fourth embodiment of the second aspect of the invention, amino acid residues 1-12 are all deleted.

[0170] In a fifth embodiment of the second aspect of the invention, amino acid residues 1-12 are all substituted.

[0171] In a sixth embodiment of the second aspect of the invention, amino acid residues 1-12 are substituted with the sequence 5'-SETEVSVRSASS-3' (residues 1-12 in SEQ ID NO: 4).

[0172] In a seventh embodiment of the second aspect of the invention, the variant comprises SEQ ID NO:3.

[0173] In an eighth embodiment of the second aspect of the invention, the immunogenic polypeptide comprises a substitution from histidine (H) to leucine (L) at the position corresponding to position 35 of SEQ ID NO:2.

[0174] In a ninth embodiment of the second aspect of the invention, the variant comprises SEQ ID NO:6.

[0175] In a tenth embodiment of the second aspect of the invention, the variant lacks hemolytic activity or has significantly reduced hemolytic activity.

[0176] In an eleventh embodiment of the second aspect of the invention, the polypeptide is capable of inducing antibodies that block the hemolytic activity of wild-type Hla.

[0177] Embodiments of the third aspect of the present invention

[0178] A third aspect of the present invention relates to an immunogenic polypeptide consisting of or comprising a variant of the amino acid sequence of LukE, wherein:

[0179] i. has at least 85% sequence identity with SEQ ID NO: 30, and

[0180] ii. does not contain a signal peptide corresponding to residues 1-28 of SEQ ID NO:29.

[0181] In a first embodiment of the third aspect of the present invention, the polypeptide consists of SEQ ID NO:30.

[0182] In a second embodiment of the third aspect of the invention, the polypeptide is capable of inducing antibodies that block the leukotoxicity of functionally mature wild-type LukE.

[0183] Embodiments of the fourth aspect of the present invention

[0184] A fourth aspect of the present invention relates to an immunogenic polypeptide consisting of or comprising a variant of the amino acid sequence of aurolysin (Aur), wherein:

[0185] i. having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 34, and

[0186] ii. comprising one or more amino acid substitutions in the HEXXH catalytic domain, wherein the HEXXH catalytic domain corresponds to amino acid positions 352-356 of SEQ ID NO: 34,

[0187] Wherein the polypeptide has reduced catalytic ability and wherein the substitution is preferably non-conservative.

[0188] These variants of aurisin have been shown to be able to induce antibodies that effectively target wild-type aurisin, but are safe and pharmacologically acceptable due to their reduced biological activity.

[0189] In a first embodiment of the fourth aspect of the invention, in the variant, the conserved cysteine ​​(C) corresponding to amino acid position 479 of SEQ ID NO: 34 is substituted.

[0190] In a second embodiment of the fourth aspect of the invention, the conserved cysteine ​​defined in the first embodiment of the fourth aspect of the invention is substituted by serine (S).

[0191] In a third embodiment of the fourth aspect of the invention the glutamic acid residue (E) in the HEXXH catalytic domain corresponding to amino acid position 353 of SEQ ID NO: 34 is substituted, preferably non-conservatively substituted.

[0192] In a fourth embodiment of the fourth aspect of the invention, the glutamic acid in the HEXXH catalytic domain as defined in the third embodiment of the fourth aspect of the invention is substituted by alanine (A).

[0193] In a fifth embodiment of the fourth aspect of the invention, the variant consists of or comprises SEQ ID NO:35.

[0194] In a sixth embodiment of the fourth aspect of the invention, the variant comprises a sequence having at least 85% sequence identity to amino acid sequence 210-509 of SEQ ID NO:34.

[0195] In a seventh embodiment of the fourth aspect of the invention, the variant exhibits a reduced ability to participate in the formation of an antigen complex compared to wild-type Aur.

[0196] In an eighth embodiment of the fourth aspect of the invention, the polypeptide is capable of inducing antibodies that block the catalytic activity of wild-type Aur.

[0197] Embodiments of the fifth aspect of the present invention

[0198] A fifth aspect of the present invention relates to an immunogenic polypeptide consisting of or comprising a variant of the amino acid sequence of N-acetylmuramoyl-L-alanine amidase (SAR2723), wherein:

[0199] i. has at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 38, and

[0200] a. comprising one or more amino acid substitutions in the amidase active site TXEXX domain corresponding to amino acid residues 384-388 in SEQ ID NO: 37, and / or

[0201] b. comprising one or more substitutions in the amidase active site LXDYX domain corresponding to amino acid residues 409-413 in SEQ ID NO: 37, and / or

[0202] c. comprising a substitution corresponding to the conserved cysteine ​​at position 513 in SEQ ID NO: 37, wherein the polypeptide has reduced catalytic ability, and wherein the substitution is preferably non-conservative.

[0203] These variants of SAR2723 have been demonstrated to induce antibodies that effectively target wild-type SAR2723, but due to their reduced biological activity, the variants are safe and pharmacologically acceptable, like the other polypeptides of the invention disclosed above.

[0204] In a first embodiment of the fifth aspect of the invention, the immunogenic polypeptide comprises feature i of: only item a, only item b, only item c, only items a and b, only items a and c, only items b and c, or items a, b and c.

[0205] In a second embodiment of the fifth aspect of the invention, the conserved cysteine ​​is substituted by serine (S).

[0206] In a third embodiment of the fifth aspect of the invention, the glutamic acid residue (E) in the active site TXEXX domain corresponding to amino acid position 386 of SEQ ID NO: 37 is substituted, preferably non-conservatively, such as by glutamine (Q).

[0207] In a fourth embodiment of the fifth aspect of the invention, the aspartic acid residue (D) in the active site LXDYS domain corresponding to amino acid position 411 of SEQ ID NO: 37 is substituted, preferably non-conservatively, such as by asparagine (N).

[0208] In a fifth embodiment of the fifth aspect of the invention, the variant consists of or comprises SEQ ID NO:39.

[0209] In a sixth embodiment of the fifth aspect of the present invention, the polypeptide is capable of inducing antibodies that block the catalytic activity of wild-type SAR2723 or interfere with bacterial cell wall development.

[0210] Embodiments of the sixth aspect of the present invention

[0211] The sixth aspect of the present invention relates to a vaccine composition comprising selected polypeptides, which include at least two of items ad:

[0212] a) an HIa polypeptide or a variant thereof, wherein the variant preferably exhibits reduced or no hemolytic activity and / or preferably is capable of inducing antibodies that block the hemolytic activity of native HIa,

[0213] b) a LukE polypeptide or a variant thereof, wherein the variant preferably exhibits reduced or no leukocyte activity and / or preferably is capable of inducing antibodies that block the leukocyte activity of native LukE,

[0214] c) a 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 preferably is capable of inducing antibodies that block the interaction of native SpA with human IgG, and

[0215] d) an Aur polypeptide or a variant thereof, wherein the variant preferably exhibits reduced catalytic activity and is preferably capable of inducing antibodies that block the catalytic activity of native Aur,

[0216] The composition optionally further comprises a pharmaceutically acceptable carrier, vehicle or diluent, and optionally further comprises an immunogenic adjuvant, which is particularly selected from AlOH, SLA-SE and OMV (outer membrane vesicles).

[0217] It should be understood that such a composition may consist of variants of SpA, Hla, LukE and Aur as described above, but polypeptides known in the art that exhibit comparable functional properties may also be used in the composition. However, in a first embodiment of the sixth aspect of the invention, it is preferred that the Hla polypeptide variant is a variant according to the second aspect of the invention, and / or wherein the LukE polypeptide variant is a variant according to the third aspect of the invention, and / or wherein the SpA polypeptide variant is a variant according to the first aspect of the invention, and / or wherein the Aur polypeptide variant is a variant according to the fourth aspect of the invention.

[0218] In a second embodiment of the sixth aspect of the invention, the Hla polypeptide variant has the amino acid sequence of SEQ ID NO: 5 or any other previously identified Hla polypeptide, the common feature of which is that it cannot form a heptamer (pore-forming) structure. Similarly, this embodiment also utilizes any existing SpA polypeptide variant and / or Aur polypeptide variant, which have the functionality of the SpA and Aur polypeptide variants of the invention discussed above. Therefore, this embodiment utilizes at least one immunogen having the same or comparable properties as the polypeptides of the first to fourth aspects of the invention.

[0219] In a third embodiment of the sixth aspect of the present invention, the vaccine composition comprises at least 3 of items ad.

[0220] In a fourth embodiment of the sixth aspect of the present invention, the vaccine composition comprises the following:

[0221] -a and b;

[0222] -a and c;

[0223] -a and d;

[0224] -b and c;

[0225] -b and d;

[0226] -c and d;

[0227] -a, b and c;

[0228] -a, b and d;

[0229] -a, c and d;

[0230] - b, c and d; or

[0231] -a, b, c and d.

[0232] In particular, preferred are vaccine compositions wherein the Staphylococcus aureus immunogen comprises or consists of items a and b), i.e., vaccine compositions wherein the Staphylococcus aureus immunogen comprises or consists of the following a) and b): a) an Hla polypeptide or a variant thereof, wherein the variant preferably exhibits reduced or no hemolytic activity and / or is preferably capable of inducing antibodies that block the natural Hla hemolytic activity, and b) a LukE polypeptide or a variant thereof, wherein the variant preferably exhibits reduced or no leukocyte activity and / or is preferably capable of inducing antibodies that block the natural LukE leukocyte activity, wherein each of items a and b is as disclosed herein.

[0233] In a fifth embodiment of the sixth aspect of the present invention, the vaccine composition further comprises at least one polypeptide selected from the group consisting of:

[0234] e) a SAR2723 polypeptide or a variant thereof, wherein the variant preferably exhibits reduced or no catalytic activity and is preferably capable of inducing antibodies that block the catalytic activity of native SAR2723,

[0235] f) a SAR2753 polypeptide or variant, wherein the variant preferably exhibits reduced or no lipase activity and is preferably capable of inducing antibodies that block the lipase activity of native SAR2753,

[0236] g) SAR0992 (HtrA2) polypeptide or a variant thereof, and

[0237] h) SAR0280 (EsaA) polypeptide or a variant thereof, wherein said variant is preferably capable of inducing antibodies that prevent the secretion of EsxA / B.

[0238] In a sixth embodiment of the sixth aspect of the present invention,

[0239] - the SAR0992 polypeptide variant is a fragment or sequence variant of SEQ ID NO: 15 disclosed in WO2012 / 136653, in particular a fragment consisting of amino acid residues 1-409 of SEQ ID NO: 15 disclosed in WO2012 / 136653, or a variant of SAR0992, which is capable of inducing antibodies that block the catalytic activity of native SAR0992, such as a truncated form of the C-terminus of SAR0992 relative to the SAR0992 transmembrane helix, or a mutant version of SAR0992 comprising a substitution of the serine residue corresponding to the serine residue at position 619 in SEQ ID NO: 15 disclosed in WO2012 / 136653, and / or a mutant version of SAR0992, wherein the transmembrane helix is ​​exchanged with a flexible linker or wherein the transmembrane helix is ​​exchanged with a linker and the N-terminal and C-terminal parts of SAR0992 on both sides of the linker are exchanged with each other, and / or

[0240] - wherein 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 and most C-terminal extracellular fragments of SAR0280, and / or wherein

[0241] - the SAR2723 polypeptide variant is a fragment or sequence variant of SEQ ID NO: 13 according to the fifth aspect of the present invention or disclosed in WO2015 / 082536, and / or wherein

[0242] - The SAR2753 polypeptide variant is a fragment or sequence variant of SEQ ID NO: 14 disclosed in WO2015 / 082536 or a polypeptide comprising the amino acid sequence SEQ ID NO: 42.

[0243] In a seventh embodiment of the sixth aspect of the present invention, the vaccine composition comprises at least 2 of items eh.

[0244] In an eighth embodiment of the sixth aspect of the present invention, the vaccine composition comprises at least 3 of items eh.

[0245] In a ninth embodiment of the sixth aspect of the present invention, the vaccine composition comprises the following:

[0246] -e and f;

[0247] -e and g;

[0248] -e and h;

[0249] -f and g;

[0250] -f and h;

[0251] -g and h;

[0252] -e, f and g;

[0253] -e, f and h;

[0254] -e, g and h;

[0255] -f, g, and h; or

[0256] -e, f, g and h.

[0257] Further details on vaccine compositions can be found below under the headings "Methods of Immunization" and "Compositions of the Invention; Vaccines".

[0258] Part of the sixth aspect of the invention also relates to a vaccine composition, which comprises at least one chimeric polypeptide of the seventh aspect of the invention and is described in detail in the discussion of the embodiments of the seventh aspect of the invention below, and which further comprises a pharmaceutically acceptable carrier, vehicle or diluent, and optionally further comprises an immune adjuvant.

[0259] The 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 preferably selected from SEQ ID NOs: 56 and 57.

[0260] In an important embodiment, the vaccine composition comprises at least or exactly 2 chimeric polypeptides of the seventh aspect of the invention and embodiments thereof.

[0261] In some embodiments, the vaccine composition comprising at least one chimeric polypeptide of the seventh aspect further comprises at least one or exactly one additional polypeptide selected from the polypeptides discussed above in items e, g, f and h of the embodiments of the sixth aspect of the invention; that is, such a composition comprises at least one chimeric polypeptide and at least one polypeptide defined above in items e, g, f or h.

[0262] The additional polypeptide is preferably selected from SAR0992-1-409 (residues 1-409 of SEQ ID NO: 44) and SAR0280-28-820 (SEQ ID NO: 48).

[0263] A particularly important vaccine composition comprises the chimeric polypeptide 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 1-409 of SAR0992 (residues 1-409 of SEQ ID NO: 44), and a particularly important composition consists of the chimeric polypeptide 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 1-409 of SAR0992 (residues 1-409 of SEQ ID NO: 44), and is mixed with a pharmaceutically acceptable carrier, vehicle or diluent, and further optionally an immune adjuvant.

[0264] In a preferred embodiment, the adjuvant is selected from the group consisting of AlOH, SLA-SE and OMV (outer membrane vesicles).

[0265] Embodiment of the seventh aspect of the present invention

[0266] The seventh aspect of the invention relates to a chimeric polypeptide comprising the amino acid sequence of a polypeptide selected according to the sixth aspect of the invention, wherein the amino acid sequence is fused or linked via a linker. In other words, rather than including some or all of the polypeptides discussed in the sixth aspect of the invention in a vaccine "mixture", the immunogenic amino acid sequence is included as part of a fusion protein; this approach may facilitate vaccine production or avoid any need to include an immunogenic carrier molecule fused to the polypeptide.

[0267] Particularly preferred chimeric polypeptides consist of the polypeptides of items a and b discussed in the fourth embodiment of the sixth aspect above, or include the polypeptides of items a and b above. In particular, the chimeric polypeptide consists of or includes the following Staphylococcus aureus immunogens of items a) and b): a) Hla polypeptide or a variant thereof, wherein the variant preferably exhibits reduced hemolytic activity or no hemolytic activity and / or preferably can induce antibodies that block the hemolytic activity of natural Hla, and b) LukE polypeptide or a variant thereof, wherein the variant preferably exhibits reduced leukocyte activity or no leukocyte activity and / or preferably can induce antibodies that block the leukocyte activity of natural LukE, wherein each of items a and b is as disclosed herein.

[0268] In a first embodiment of the seventh aspect of the invention, the connector is flexible or rigid.

[0269] In a second embodiment of the seventh aspect of the invention, the flexible linker as shown in the first embodiment of the seventh aspect of the 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 invention is KPEPKPAPAPKP (SEQ ID NO: 52).

[0270] Particularly preferred chimeric polypeptides consist of the polypeptides of items a and b discussed in the fourth embodiment of the sixth aspect above, or include the polypeptides of items a and b discussed above. In particular, the chimeric polypeptide consists of or includes the following Staphylococcus aureus immunogens of items a) and b): a) the Hla polypeptide disclosed herein or a variant thereof, wherein the variant preferably exhibits reduced hemolytic activity or no hemolytic activity and / or preferably can induce antibodies that block the hemolytic activity of natural Hla, and b) the LukE polypeptide or a variant thereof described in detail herein, wherein the variant preferably exhibits reduced leukocyte activity or no leukocyte activity and / or preferably can induce antibodies that block the leukocyte activity of natural LukE, wherein each of items a and b is as disclosed herein.

[0271] In a third embodiment of the seventh aspect of the 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_0992_2753_FS (SEQ ID NO:65), 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).

[0272] Embodiments of the eighth to twelfth aspects of the present invention

[0273] The eighth aspect of the present invention relates to a nucleic acid or nucleic acid fragment encoding a polypeptide of the present invention, and the ninth aspect of the present invention relates to a vector comprising a nucleic acid fragment according to the eighth aspect of the present invention. Similarly, the tenth to twelfth aspects relate to cells and compositions utilizing the vectors and nucleic acids disclosed herein; a more detailed discussion of these embodiments is found below.

[0274] Embodiments of the thirteenth aspect of the present invention

[0275] The thirteenth aspect of the present invention relates to a method for inducing immunity in an animal, by administering at least once an immunologically effective amount of an immunogenic polypeptide according to any one of the first to fifth aspects of the present invention, a vaccine composition according to the sixth aspect of the present invention, a chimeric polypeptide according to the seventh aspect of the present invention, a nucleic acid fragment according to the eighth aspect of the present invention, a vector according to the ninth aspect of the present invention, a transformed cell or virus according to the tenth aspect of the present invention, or an immunogenic composition according to the eleventh or twelfth aspect of the present invention, to induce adaptive immunity against Staphylococcus aureus in the animal.

[0276] In a first embodiment of the thirteenth aspect of the present invention, when administering an immunogenic polypeptide according to any one of the first to fifth aspects of the present invention, a chimeric polypeptide according to the seventh aspect of the present invention, or a composition comprising the immunogenic polypeptide or the chimeric polypeptide, the animal receives 0.5 to 5,000 μg of the immunogenic polypeptide or chimeric polypeptide according to any one of the first to fifth aspects and the seventh aspect of the present invention per administration.

[0277] In a second embodiment of the thirteenth aspect of the invention, the animal receives a prime administration comprising the immunogenic polypeptide or the chimeric polypeptide and one or more booster administrations comprising the immunogenic polypeptide or the chimeric polypeptide.

[0278] In a third embodiment of the thirteenth aspect of the invention, the animal is a human.

[0279] In a fourth embodiment of the thirteenth aspect of the present invention, the purpose of said administration is to induce protective immunity against S. aureus.

[0280] In a fifth embodiment of the thirteenth aspect of the present invention, the protective immunity is effective in reducing the risk of infection caused by Staphylococcus aureus, or is effective in treating or ameliorating Staphylococcus aureus infection.

[0281] In a sixth embodiment of the thirteenth aspect of the invention, the purpose of said administration is to induce antibodies specific for S. aureus, and wherein said antibodies or B lymphocytes producing said antibodies are subsequently recovered from the animal.

[0282] In a seventh embodiment of the thirteenth aspect of the present invention, the purpose of said administration is to induce antibodies specific for Staphylococcus aureus, and wherein B lymphocytes producing said antibodies are subsequently recovered from the animal and used to prepare monoclonal antibodies.

[0283] Further details on this can be found in the sections below detailing immunization methods and vaccines.

[0284] Carrier

[0285] It should be understood that the nucleic acid segments of the invention and nucleic acid segments encoding polypeptides of the compositions of the invention can be used for production, vector and vaccine purposes - the latter requiring that the sequence be contained in an expression vector, which can result in the production of an immunogenic protein in a mammal receiving the vector. Or in other words, the nucleic acid is contained in a vector that is capable of expressing the nucleic acid in humans after administration.

[0286] Such vectors usually comprise an expression control region in an operable connection in the 5'-3' direction, the expression control region comprising an enhancer / promoter for driving nucleic acid expression, an optional signal peptide coding sequence, a nucleotide sequence to be expressed, and an optional terminator. Therefore, such vectors constitute expression vectors that can be used to produce polypeptides of the present invention or polypeptides as a part of the composition of the present invention in cells. Since the polypeptides are derived from bacteria, recombinant production must be carried out in host cells capable of expressing the encoding nucleic acid. Bacterial host cells are preferably used. However, if the vector is to drive expression in eukaryotic cells (such as in the case of nucleic acid vaccine vectors), the expression control region should be adapted to this specific use.

[0287] Thus, for production purposes, it is often convenient for the expression control region to drive expression in prokaryotic cells (e.g., bacteria, such as E. coli) or eukaryotic cells (e.g., 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.

[0288] In addition, for production purposes, it is practical for the vector to be able to integrate the nucleic acid into the genome of the host cell - this is particularly useful if the vector is used to generate stably transformed cells, since the progeny will also include the genetic information introduced by the vector. Alternatively, vectors that are unable to integrate into the genome of fish host cells can be used, for example, for nucleic acid vaccination.

[0289] An interesting production system is to use plants. For example, plants can be genetically modified using an Agrobacterium transfection system to express a gene encoding a target protein, thereby producing protein in plants at low cost. iBio CMOLLC (8800HSC Pkwy, Bryan, TX 77807, USA) and iBio, Inc (9 Innovation Way, Suite 100, Newark, DE 19711, USA) provide a commercial platform, and are disclosed in, for example, EP2853599, EP1769068, and EP2192172. Therefore, in such a system, the vector is an Agrobacterium vector or other vectors suitable for transfecting plants.

[0290] The vector is usually selected from the group consisting of viruses (eg, viruses that are non-pathogenic to mammals, especially humans), bacteria (eg, bacteria that are non-pathogenic to mammals such as humans), plasmids, minichromosomes, and cosmids.

[0291] Vectors of interest are viral vectors (particularly those useful as human vaccine agents). These can be selected from retroviral vectors (e.g., lentiviral vectors), adenoviral vectors, adeno-associated viral vectors, and poxviral vectors. Certain poxviral vectors are preferred, particularly vaccinia virus vectors. A particularly preferred vaccinia virus vector is the modified vaccinia Ankara (MVA) vector.

[0292] As indicated, a polypeptide of the invention or a polypeptide as part of a composition of the invention can be encoded by a nucleic acid molecule contained in a vector. The nucleic acid sequence can be "heterologous," meaning that it is in an environment that is foreign to the cell into which the vector is introduced, including sequences that are homologous to sequences in the cell, but the sequence is not normally found in a location in the host cell.

[0293] Vectors include naked DNA, RNA, plasmids, cosmids, viruses (bacteriophages, animal viruses and plant viruses) and artificial chromosomes (e.g., YACs). One skilled in the art can construct vectors by standard recombinant techniques. In addition to encoding a polypeptide of the invention as part of the composition of the invention, the vector may also encode a polypeptide sequence, such as a "tag" or an immunogenicity enhancing peptide (e.g., an immunogenic carrier or a fusion partner that stimulates the immune system, such as a cytokine or an active fragment thereof). Useful vectors encoding such fusion proteins include pIN vectors, vectors encoding a stretch of histidine residues, and pGEX vectors for generating glutathione S-transferase (GST) soluble fusion proteins for subsequent purification and separation or cleavage.

[0294] The vectors may be used in host cells to produce a polypeptide of the invention or as part of a composition of the invention, which may then be purified for administration, or the vectors may be purified for direct administration to express the protein (such as when administering a nucleic acid vaccine).

[0295] Expression vectors may contain a variety of "control sequences," which refer to nucleic acid sequences necessary for the transcription and possible translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that control transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that can perform other functions, as described in detail below.

[0296] 1. Promoters and enhancers

[0297] A "promoter" is a control sequence. A promoter is generally a region of a nucleic acid sequence that controls the initiation and rate of transcription. It may contain regulatory proteins and molecules such as genetic elements to which RNA polymerase and other transcription factors can bind. The phrases "operably positioned," "operably linked," "under control," and "under transcriptional control" refer to a promoter being in the correct functional position and / or orientation relative to a nucleic acid sequence to control the initiation and expression of transcription of the sequence. A promoter may or may not be used in conjunction with an "enhancer," which refers to a cis-acting regulatory sequence that participates in the transcriptional activation of a nucleic acid sequence.

[0298] The promoter may be a promoter naturally associated with a gene or sequence, such as one that can be obtained by isolating a 5' non-coding sequence upstream of a coding segment or exon. Such a promoter may be referred to as "endogenous". Similarly, an enhancer may be an enhancer naturally associated with a nucleic acid sequence, located downstream or upstream of the sequence. Alternatively, placing the coding nucleic acid segment under the control of a recombinant or heterologous promoter will achieve certain advantages, and the recombinant or heterologous promoter refers to a promoter that is not usually associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer that is not associated with a nucleic acid sequence in its natural state. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral or eukaryotic cells, as well as promoters or enhancers that are not "naturally present", i.e., promoters or enhancers containing different elements of different transcriptional regulatory regions and / or mutations that alter expression. In addition to synthesizing nucleic acid sequences that produce promoters and enhancers, nucleic acid sequences of promoters and enhancers may also be produced using recombinant cloning and / or nucleic acid amplification techniques, including polymerase chain reaction, in combination with the components disclosed herein.

[0299] It may be important to use promoters and / or enhancers to effectively direct the expression of a DNA fragment in a selected cell type or organism. The use of promoters, enhancers, and cell type combinations for protein expression is generally known to those skilled in the art of molecular biology. The promoter employed may be constitutive, tissue-specific, or inducible, and in certain embodiments, may direct high-level expression of an introduced DNA fragment under specific conditions, such as large-scale production of a recombinant protein or peptide.

[0300] Inducible elements are regions of nucleic acid sequences that are activated in response to a specific stimulus, examples of which include, but are not limited to, immunoglobulin heavy chains, immunoglobulin light chains, T cell receptors, HLA DQα and / or DQβ, beta-interferon, interleukin 2, interleukin 2 receptor, class II MHC 5, class II MHC HLA-DRα, beta-actin, muscle creatine kinase (MCK), prealbumin (transthyretin), elastase I, metallothionein (MTII), collagenase, albumin, alpha-fetoprotein, gamma-globin, beta-globin, c-fos, c-HA-ras, insulin, neural cell adhesion molecule (NCAM), alphal-Antitrypain, 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, polyomavirus, retrovirus, papillomavirus, hepatitis B virus, human immunodeficiency virus, cytomegalovirus (CMV) IE, and gibbon ape leukemia virus.

[0301] Inducible elements include MT II-phorbol ester (TFA) / heavy metals; MMTV (mouse mammary tumor virus)-glucocorticoids; beta-interferon-poly (rl) x / poly (rc); adenovirus 5E2-EIA; collagenase-phorbol ester (TPA); stromelysin-phorbol ester (TPA); SV40-phorbol ester (TPA); mouse MX gene-interferon, Newcastle disease virus; GRP78 gene-A23187; alpha-2-macroglobulin-IL-6; vimentin-serum; class I MHC gene H-2Kb-interferon; HSP70-E1A / SV40 large T antigen; proliferation protein-phorbol ester / TPA; tumor necrosis factor-PMA; and thyroid stimulating hormone gene-thyroid hormone.

[0302] Also contemplated to be useful in the present invention are the dectin-1 and dectin-2 promoters. In addition, any promoter / enhancer combination (according to the eukaryotic promoter database EPDB) can also be used to drive the expression of structural genes encoding oligosaccharide processing enzymes, protein folding auxiliary proteins, selection marker proteins, or heterologous proteins of interest.

[0303] The specific promoter used to control the expression of a polynucleotide encoding a peptide or protein is not considered critical, as long as it is capable of expressing the polynucleotide 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 near and under the control of a promoter capable of expression in fish cells. In general, such promoters may include bacterial, fish or viral promoters, as long as the promoter is effective in fish cells.

[0304] In various embodiments, particularly those for the purpose of recombinant production of polypeptides, 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 levels of expression of the polynucleotides of interest. Other viral or mammalian cell or bacterial phage promoters known in the art are also contemplated for use to achieve expression of the polynucleotides.

[0305] In embodiments where a vector is administered to humans to express a protein, it is envisioned that the ideal promoter used with the vector is a promoter that is not down-regulated by cytokines, or a sufficiently strong promoter that, even if down-regulated, will produce an effective amount of the protein / polypeptide of the invention (or a protein, polypeptide that can be used in the composition of the invention) in humans to elicit an immune response. Non-limiting examples of these are CMV IE and RSV LTR. In other embodiments, a promoter that is up-regulated in the presence of cytokines is employed. In the presence of IFN-γ, the MHC I promoter increases expression.

[0306] Tissue-specific promoters may be used, particularly if expression is to be performed in cells in which the antigen is to be expressed, such as dendritic cells and macrophages. The mammalian MHC I and MHC II promoters are examples of such tissue-specific promoters in humans, and corresponding fish promoters are expected to be effective. 2. Promoter Signals and Internal Ribosome Binding Sites (IRES)

[0307] Efficient translation of the coding sequence may also require specific start signals. These signals include the ATG start codon or adjacent sequences. It may be necessary to provide exogenous translation control signals, including the ATG start codon. One of ordinary skill in the art can easily determine this and provide the necessary signals. As is well known, the start codon must be "in frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. Exogenous translation control signals and start codons can be natural or synthetic, and can also be operated in bacteria or mammalian cells. Expression efficiency can be enhanced by adding appropriate transcription enhancer elements.

[0308] In certain embodiments of the present invention, internal ribosome entry site (IRES) elements are used to create multi-gene or multi-cistronic information. IRES elements can bypass the ribosome scanning model of 5' methylated cap-dependent translation and start translation from internal sites. IRES elements from two members of the picornavirus family (poliomyelitis and encephalomyocarditis), as well as IRES from mammalian messengers, have been described. IRES elements can be connected to heterologous open reading frames. Multiple open reading frames can be transcribed together, and each frame is separated by IRES, thereby creating multi-cistronic information. With the help of IRES elements, each open reading frame can be accessed by ribosomes to perform efficient translation. Using a single promoter / enhancer to transcribe a single messenger can efficiently express multiple genes (see U.S. Patents 5,925,565 and 5,935,819, incorporated herein by reference).

[0309] 3. Multiple cloning site

[0310] The vector may include a multiple cloning site (MCS), which is a region of nucleic acid containing multiple restriction enzyme sites, any of which may be used in conjunction with standard recombinant techniques to digest the vector. Typically, the vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to enable ligation of exogenous sequences to the vector. Techniques involving restriction enzymes and ligation reactions are well known to those skilled in the art of recombinant technology.

[0311] 4. Splice Sites

[0312] Most transcribed eukaryotic RNA molecules undergo RNA splicing to remove introns from the primary transcript. If relevant, vectors containing genomic eukaryotic sequences may require donor and / or acceptor splice sites to ensure correct processing of the transcript for protein expression.

[0313] 5. Termination Signal

[0314] Vectors or constructs disclosed herein typically include at least one termination signal. A "termination signal" or "terminator" includes a DNA sequence that participates in the specific termination of RNA transcription by RNA polymerase. Therefore, in certain embodiments, it is contemplated that a termination signal for the production of RNA transcripts is used. A terminator may be required in vivo to achieve desired messenger levels.

[0315] In eukaryotic systems, the terminator region may also contain specific DNA sequences that allow site-specific cleavage of new transcripts, thereby exposing polyadenylation sites. This signals a specialized endogenous polymerase to add a stretch of about 200 A residues (poly A) to the 3' end of the transcript. RNA molecules modified with this poly A tail appear to be more stable and more efficiently translated. Therefore, in other embodiments involving eukaryotic organisms, it is preferred that the terminator contains a signal for cleaving the RNA, and more preferably, the terminator signal promotes polyadenylation of the messenger.

[0316] The terminator contemplated for use herein includes any transcription terminator described herein or known to those of ordinary skill in the art, including but not limited to, for example, the bovine growth hormone terminator or a viral terminator sequence, such as the SV40 terminator. In certain embodiments, for example, due to sequence truncation, the termination signal may be the absence of a transcribable or translatable sequence.

[0317] 6. Polyadenylation signal

[0318] In expression, particularly eukaryotic expression (such as that associated with nucleic acid vaccination), a polyadenylation signal will typically be included to achieve correct polyadenylation of the transcript. The nature of the polyadenylation signal is not believed to be important for the successful practice of the present invention, and / or any such sequence may be employed. 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 may increase the stability of the transcript or may promote cytoplasmic transport.

[0319] 7. Copy starting point

[0320] To propagate the vector in a host cell, the vector may contain one or more origins of replication (often referred to as "on"), i.e., specific nucleic acid sequences that initiate replication. Alternatively, if the host cell is yeast, an autonomously replicating sequence (ARS) may be used.

[0321] 8. Selectable and filterable markers

[0322] In certain embodiments of the invention, cells containing nucleic acid constructs can be identified in vitro or in vivo by encoding a screenable or selectable marker in an expression vector. When transcribed and translated, the marker imparts a recognizable change to the cell, thereby allowing easy identification of cells containing the expression vector. In general, a selectable marker is a marker that imparts a characteristic that allows selection. A positive selectable marker is a marker whose presence allows selection, while a negative selectable marker is a marker whose presence prevents selection. An example of a positive selectable marker is a drug resistance marker.

[0323] Usually, the addition of drug selection markers helps the cloning and identification of transformants, for example, markers that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, bleomycin or histidinol are useful selectable markers. In addition to conferring phenotypic markers so as to distinguish transformants according to implementation conditions, other types of markers also 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 also be utilized. Those skilled in the art also know how to use immune markers that can be used in combination with FACS analysis. The marker used is not important, as long as it can be expressed simultaneously with the nucleic acid encoding the protein of the present invention. Other examples of selectable and screenable markers are well known to those skilled in the art.

[0324] Transformed cells

[0325] Transformed cells can be used as organisms for producing the polypeptides of the present invention and the polypeptides of the compositions of the present invention, and can be used as simple "containers" for the nucleic acids and vectors of the present invention.

[0326] Certain transformed cells, including cells of the present invention, are capable of replicating nucleic acid fragments, including nucleic acid fragments of the present invention. Preferred transformed cells are capable of expressing nucleic acid fragments.

[0327] For recombinant production, since the proteins disclosed herein are derived from bacteria, it is convenient to use prokaryotic cells (such as bacteria) for transforming cells, but in general, both prokaryotic cells and eukaryotic cells can be used.

[0328] 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).

[0329] The eukaryotic cell can be in the form of yeast (eg, Saccharomyces cerevisiae) and protozoa. Alternatively, the transformed eukaryotic cell is derived from a multicellular organism, such as a filamentous fungus, an insect cell, a plant cell, or a mammalian cell.

[0330] For production purposes, it is advantageous that the transformed cell is stably transformed by stably integrating the nucleic acid into its genome, and in certain embodiments, it is also preferred that the transformed cell secretes or carries the polypeptide of the invention on its surface, as this facilitates the recovery of the produced polypeptide. A specific version of this embodiment is that the transformed cell is a bacterium and the polypeptide of the invention is secreted into the periplasmic space.

[0331] As mentioned above, stably transformed cells are preferred - these are cells such that cell lines can be established which comprise transformed cells as defined herein - such cell lines are particularly preferred.

[0332] More detailed information about the cells and cell lines is provided below:

[0333] Cells suitable for recombinant nucleic acid expression of nucleic acid fragments (e.g., nucleic acid fragments 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 C. glutamicum; and members of the genus Pseudomonas, such as Pseudomonas fluorescens. Examples of eukaryotic cells include mammalian cells; insect cells; yeast cells, such as members of the genus Saccharomyces (e.g., S. cerevisiae), members of the genus Pichia (e.g., Pichia pastoris), members of the genus Hansenula (e.g., H. polymorpha), members of the genus Kluyveromyces (e.g., K. lactis or K. fragilis), and members of the genus Schizosaccharomyces (e.g., S. pombe).

[0334] The generation of recombinant genes, the introduction of cells and the techniques of recombinant gene expression 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.

[0335] As used herein, the terms "cell," "cell line," and "cell culture" are used interchangeably. All of these terms also include their progeny, including any and all progeny. It is understood that all progeny may not be identical due to intentional or unintentional mutations. In the case of expressing heterologous nucleic acid sequences, "host cell" refers to a prokaryotic or eukaryotic cell, including any transformable organism capable of replicating a vector or expressing a heterologous gene encoded by a vector. Host cells can and have been used as receptors for vectors or viruses. Host cells can be "transfected" or "transformed," which refers to the process of transferring or introducing exogenous nucleic acids (e.g., recombinant protein coding sequences) into host cells. Transformed cells include primary subject cells and their progeny.

[0336] Host cells can be from prokaryotes or eukaryotes, including bacteria, yeast cells, insect cells and mammalian cells, for replication of vectors or expression of some or all of the nucleic acid sequences. Many cell lines and cell cultures can be used as host cells, which can be obtained through the American Type Culture Collection (ATCC), which serves as an archive of living cultures and genetic materials (www.atcc.org), or obtained from other collections, such as the German Collection of Microorganisms (Deutsche Sammlung vor Micrroorganismen und Zellkulturen, DSM). Those skilled in the art can determine a suitable host based on the vector skeleton and the desired result. For example, plasmids or cosmids can be introduced into prokaryotic host cells to replicate many vectors or express encoded proteins. Bacterial cells used as host cells for vector replication and / or expression include staphylococcal strains, DH5α, JMI 09 and KC8, and many commercially available bacterial hosts, such as SURE (R) competent cells and SOLOP ACK (TM) Gold cells ( La Jolla, CA). Alternatively, bacterial cells such as E. coli LE392 can be used as host cells for bacteriophage viruses. Suitable yeast cells include Saccharomyces cerevisiae, Saccharomyces pombe, and Pichia pastoris.

[0337] Examples of eukaryotic host cells for replication and / or expression of vectors include HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12. Many host cells from various cell types and organisms are available and known to those skilled in the art. Similarly, viral vectors can be used in conjunction with eukaryotic or prokaryotic host cells, particularly host cells that allow vector replication or expression.

[0338] Some vectors may employ control sequences that enable them to be replicated and / or expressed in prokaryotic and eukaryotic cells. Those skilled in the art will further appreciate the conditions for incubating all of the above host cells to maintain the host cells and allow for vector replication. It is also necessary to understand and know the techniques and conditions that allow for large-scale production of vectors and production of vector-encoded nucleic acids and their cognate polypeptides, proteins or peptides.

[0339] Expression system

[0340] There are a variety of expression systems that include a portion or all of the above compositions. Systems based on prokaryotes and / or eukaryotes can be used in the present invention to produce nucleic acid sequences or their homologous polypeptides, proteins and peptides. Many such systems are commercially available and widely available.

[0341] The insect cell / baculovirus system can produce high levels of protein expression of heterologous nucleic acid fragments, as described in U.S. Pat. Nos. 5,871,986 and 4,879,236, both of which are incorporated herein by reference, and can be used, for example, from by 2.0 The name is obtained from Get BACPACK TM Baculovirus expression system.

[0342] In addition to the disclosed expression systems, examples of other expression systems include COMPLETE CONTROL TM Inducible mammalian expression system involving synthetic ecdysone inducible receptors, or its pET expression system, an E. coli expression system. Another example of an inducible expression system is available from It comes with T-REX TM (tetracycline regulated expression) system, an inducible mammalian expression system that uses the full-length CMV promoter.

[0343] A yeast expression system, called Pichia methanolica expression system, is also provided, which is designed to produce high levels of recombinant proteins in the methylotrophic yeast Pichia methanolica. Those skilled in the art know how to express vectors, such as expression constructs, to produce a nucleic acid sequence or its homologous polypeptides, proteins or peptides.

[0344] Gene transfer methods

[0345] Suitable methods for delivering nucleic acids to achieve expression of the compositions or nucleic acid fragments of the invention are contemplated to include virtually any method by which nucleic acids (e.g., DNA, including viral and non-viral vectors) can be introduced into cells, tissues, or organisms, as described herein or known to those of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA, such as by injection (U.S. Pat. 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, and 5,580,859), including microinjection (U.S. Pat. No. 5,789,215); by electroporation (U.S. Pat. No. 5,384,253); by calcium phosphate precipitation; by using DEAE dextran followed by polyethylene glycol; by direct sonic loading; by liposome-mediated transfection; by microparticle bombardment (PCT Application Nos. WO94 / 09699 and 95 / 06128; U.S. Pat. Nos. 5,610,042; 5,322,7835,563,055; 5,550,318; 5,538,877 and 5,538,880); by agitation with silicon carbide fibers (U.S. Pat. Nos. 5,302,523 and 5,464,765); by Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,591,616 and 5,563,055); or by PEG-mediated transformation of protoplasts (U.S. Pat. Nos. 4,684,611 and 4,952,500); by desiccation / inhibition-mediated DNA uptake. By applying techniques such as these, organelles, cells, tissues or organisms can be stably or transiently transformed.

[0346] Composition of the present invention; Vaccine

[0347] The compositions according to the invention, in particular vaccines, are prophylactic, but can also be used therapeutically.

[0348] Such vaccines comprise an immunizing antigen, immunogen, polypeptide, protein or nucleic acid, typically in combination with a "pharmaceutically acceptable carrier," which includes any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition.

[0349] In some embodiments of the invention, the pharmaceutical composition, such as a vaccine, comprises only one antigen, immunogen, polypeptide, protein, nucleic acid or vector of the invention, but in other embodiments, the pharmaceutical composition comprises a "mixture" of antigens or immunogens or polypeptides or proteins or nucleic acids or vectors.

[0350] In an interesting embodiment, the pharmaceutical composition is a vector as mentioned herein, which encodes and enables the expression of at least two nucleic acid fragments of the invention.

[0351] Another interesting embodiment of the pharmaceutical composition comprises as active ingredient RNA, ie at least one mRNA encoding a polypeptide of the present invention.

[0352] An embodiment of the pharmaceutical composition of the invention comprises at least 2 (eg, 2, 3, 4, 5, 6, 7, 8, 9 or 10) different polypeptides as described above.

[0353] Another embodiment of the pharmaceutical composition of the invention comprises at least 2 (eg, 2, 3, 4, 5, 6, 7, 8, 9 or 10) different nucleic acid molecules (eg, DNA and RNA), each encoding a polypeptide as discussed above.

[0354] Suitable carriers are generally large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (eg, oil droplets or liposomes), and inactive viral particles.

[0355] Such carriers are well known to those of ordinary skill in the art. In addition, these carriers can act as immunostimulants ("adjuvants"). In addition, the antigen or immunogen can be conjugated with a bacterial toxoid, such as toxoids from diphtheria, tetanus, cholera, Helicobacter pylori, etc.

[0356] Therefore, the pharmaceutical composition of the present invention usually contains an immunoadjuvant, which is usually an aluminum-based adjuvant or one of the following other adjuvants:

[0357] Preferred adjuvants for enhancing the effectiveness of the composition include, but are not limited to: (1) aluminum salts (alum), such as aluminum hydroxide (AlOH), aluminum phosphate, aluminum sulfate, etc.; (2) oil-in-water emulsion formulations (with or without other specific immunostimulants, such as muramyl peptides (see below) or bacterial cell wall components), such as (a) MF59 (WO90 / 14837; Vaccine design: the subunit and adjuvant approach, Chapter 10, Powell & Newman, ed., Plenum Press, 1995), which contains 5% squalene, 0.5% Tween 80 and 0.5% Span 85 (optionally containing varying amounts of MTP-PE (see below), although this is not required), using a microfluidizer (e.g., Model 110Y microfluidizer (Microfluidics, Newton, MA)) formulated into submicron particles, (b) SAF containing 10% squalane, 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 larger particle size, and (c) Ribi Adjuvant System (RAS), (Ribi Immunochem, Hamilton, MT) containing 2% squalene, 0.2% Tween 80 and one or more bacterial cell wall components selected from monophosphoryl lipid A (MPL), dimycoate trehalose (TDM) and cell wall skeleton (CWS), preferably MPL + CWS (Detox TM ); (3) saponin adjuvants such as Stimulon can be used TM (Cambridge Bioscience, Worcester, MA) or particles produced therefrom such as ISCOM (immunostimulatory complex); (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. Alum and MF59 TM Adjuvants are preferably used with CFA and IFA.

[0358] 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-isoglutamine-L-alanine-2″-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), and the like.

[0359] Didierlaurent AM et al. (Expert Rev. Vaccines 2017; 16: 55-63) and Gonzales-Lopez A. et al. (Clin. Immunol. 2019; 209, 108275) disclose other adjuvants of interest. In addition, TLR4 ligand adjuvants, such as SLA-SE, are also preferred adjuvant systems (see, Reed SG 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 SG et al., Seminars in Immunology 2018, 39:22-29).

[0360] 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).

[0361] Another particularly preferred adjuvant system is OMV (outer membrane vesicles), see Kaparakis-Liaskos, M. & Ferrero, RL, 2015. Immune modulation by bacterial outer membrane vesicles. Nat Rev Immunol. 15: 375-387; Russo, AJ 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 an embodiment of interest, OMV is used in combination with aluminum hydroxide.

[0362] Another possibility for polypeptide vaccine formulations is to include the vaccine polypeptide in virus-like particles, i.e., non-infectious self-assembled structures composed of envelope or capsid proteins into which proteins are incorporated. The effect is that the polypeptide of the invention is presented multiple times on the surface of the VLP, which in turn provides improved immune recognition of the polypeptide. Thus, the VLP also acts as an immune adjuvant.

[0363] 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 agents or emulsifiers, pH buffer substances, etc., may also be present in such carriers.

[0364] Typically, the immunogenic composition is prepared as an injectable liquid solution or suspension; it can also be prepared in a solid form suitable for dissolution or suspension in a liquid carrier before injection. As discussed above in the pharmaceutically acceptable carrier, the preparation can also be emulsified or encapsulated in liposomes to enhance the adjuvant effect.

[0365] The immunogenic polypeptide composition used as a vaccine comprises an immunologically effective amount of an antigenic or immunogenic polypeptide, and any other components described above as needed. "Immunologically effective amount" means that the amount is effective for treatment or prevention when it is administered to an individual as a single dose or as part of a series of doses. The amount depends on the health and physical condition of the individual being treated, the classification group of the individual to be treated (e.g., non-human primates, primates, etc.), the ability of the individual's immune system to synthesize antibodies or generally produce an immune response, the degree of protection required, the formulation of the vaccine, the evaluation of the medical condition by the treating physician, and other relevant factors. It is expected that the amount of the immunogen will fall within a relatively wide range, which can be determined by routine experiments. However, for the purpose of protein vaccination, the amount of each immunization administration is generally between 0.5 μg and 500 mg (but generally not more than 5,000 μg). Therefore, the amount of the polypeptide of the present invention can be 1 to 400 μg, 2 to 350 μg, 4 to 300 μg, 5 to 250 μg, and 10 to 200 μg. Therefore, each gram of vaccine composition generally contains 0.1-500 μg of the protein of the present invention.

[0366] Immunogenic compositions are usually administered parenterally, for example by injection, subcutaneous injection, intramuscular injection or percutaneous / transdermal injection (see, for example WO98 / 20734). Other formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories and transdermal applications. For nucleic acid vaccination situations, intravenous or intra-arterial routes may also be applicable.

[0367] Dosage therapy may be a single dose regimen or a multiple dose regimen. The vaccine may be administered with other immunomodulators.

[0368] As an alternative to protein-based vaccines, DNA vaccination (also known as nucleic acid vaccination or genetic vaccination) can be used (see, e.g., Robinson & Torres (1997) Seminars in Immunol 9:271-283; Donnelly et al. (1997) Annu Rev Immunol 15:617-648).

[0369] As mentioned above, another aspect of the present invention is the recognition that a combination vaccine can be provided in which two or more polypeptide antigens disclosed herein are combined to enhance the immune response of the vaccinated individual, including optimizing the initial immune response and the duration of immunity. For the purpose of this aspect of the present invention, multiple antigen fragments from the same longer protein can also be used, for example, a combination of polypeptide sequence fragments of different lengths from one protein is used.

[0370] Thus, embodiments of the invention relate to compositions (or their use as vaccines) comprising two different (ie, non-identical) protein immunogens as disclosed herein.

[0371] Immunization methods

[0372] The methods of this aspect of the invention generally involve inducing immunity, and thus both prophylactic and therapeutic methods are also desired.

[0373] When the immunization method requires administration of a polypeptide of the invention or a polypeptide composition of the invention, the animal (eg, human) typically receives 0.5 to 5,000 μg of the polypeptide per administration, see above for instructions regarding dosage.

[0374] In a preferred embodiment, the immunization regimen includes a primary administration of a chimeric polypeptide, nucleic acid / vector or composition of the invention, but may require one or more subsequent booster administrations.

[0375] Preferred embodiments include administration with the purpose of inducing protective immunity against S. aureus. In this embodiment, it is particularly preferred that the protective immunity is effective in reducing the risk of infection with S. aureus.

[0376] Some vaccine compositions of the invention induce humoral immunity, and thus preferably are administered with the purpose of inducing antibodies specific for S. aureus. However, as mentioned, the immunization method can also be used for antibody production, and thus in other embodiments, the purpose of administration is to induce antibodies specific for S. aureus, wherein the B lymphocytes producing the antibodies are subsequently recovered from the animal and used to prepare monoclonal antibodies.

[0377] As described above, the composition for immunization may comprise a polypeptide, a nucleic acid, a vector, a virus or a cell. The pharmaceutical composition will comprise a therapeutically effective amount of a polypeptide, a nucleic acid, a vector, a virus or a cell.

[0378] The term "therapeutically effective amount" or "prophylactically effective amount" as used herein refers to the amount of a therapeutic agent that treats, ameliorates or prevents a desired disease or condition, or an amount that exhibits a detectable prophylactic effect in a mammalian population (e.g., humans), which effect can be detected by, for example, chemical markers or antigen levels. For dosage ranges of immunologically effective amounts of polypeptides, see above. However, the effective amount in a particular case can be determined by routine experiments and is within the judgment of the clinician.

[0379] For the purposes of the present invention, an effective dosage of a nucleic acid vaccine (vector) is about 0.01 mg / kg to 50 mg / kg or 0.05 mg / kg to about 10 mg / kg of DNA or RNA construct in the administered animal.

[0380] The pharmaceutical composition may 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. The term refers to any drug carrier that does not itself induce the production of antibodies that are harmful to the individual receiving the composition and does not produce excessive toxicity when administered. Suitable carriers can be large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive viral particles. Such carriers are well known to those of ordinary skill in the art.

[0381] Pharmaceutically acceptable salts can be used herein, for example, mineral acid salts, such as hydrochlorides, hydrobromides, phosphates, sulfates, etc.; and organic acid salts, such as acetates, propionates, malonates, benzoates, etc. A detailed discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0382] Pharmaceutically acceptable carriers in therapeutic compositions may include liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances such as wetting agents or emulsifiers, pH buffering substances, etc. may also be present in such carriers. Typically, the therapeutic composition is prepared as an injectable liquid solution or suspension; it can also be prepared in a solid form suitable for dissolution or suspension in a liquid carrier prior to injection. Liposomes are included in the definition of pharmaceutically acceptable carriers.

[0383] Specific amino acid sequences disclosed herein

[0384] In this application, the following amino acid sequences are referred to by the sequence ID NO and the individual designations used herein (provided in parentheses):

[0385] SEQ ID NO: 1 (Hla-wild type full length)

[0386]

[0387] SEQ ID NO: (Hla_wt-27-319)

[0388]

[0389] SEQ ID NO:3 (Hla_t-39-319)

[0390]

[0391]

[0392] SEQ ID NO:4 (mHla-27-319)

[0393]

[0394] SEQ ID NO:5 (Hla_H35L-27-319)

[0395]

[0396] SEQ ID NO:6(Hla_H35L_t-39-319)

[0397]

[0398] SEQ ID NO:7 (mHla_H35L-27-319)

[0399]

[0400] SEQ ID NO: 15 (SpA_wild type full length)

[0401]

[0402] SEQ ID NO: 16 (SpA_wt_t: sequence listed in the order of domains EDABC)

[0403]

[0404] SEQ ID NO:17 (SpA_10_12)

[0405]

[0406] SEQ ID NO:18 (SpA_7_12)

[0407]

[0408] SEQ ID NO:19 (SpA_4_12)

[0409]

[0410] SEQ ID NO:20 (SpA_3_12)

[0411]

[0412] SEQ ID NO:21 (SpA_10_15)

[0413]

[0414] SEQ ID NO:29(wt_LukE)

[0415]

[0416]

[0417] SEQ ID NO:30(LukE-29-311)

[0418]

[0419] SEQ ID NO:33(USA300HOU_2637)

[0420]

[0421] SEQ ID NO:34(USA300HOU_2637-28-509)

[0422]

[0423] SEQ ID NO:35(mc2716)

[0424]

[0425] SEQ ID NO:37(2723;SAR2723)

[0426]

[0427] SEQ ID NO:38(wt_2723-28-619)

[0428]

[0429] SEQ ID NO:39(mc2723)

[0430]

[0431] SEQ ID NO:42(2753-291-680)

[0432]

[0433] SEQ ID NO:44(SAR0992)

[0434]

[0435] SEQ ID NO:45(m0992;SAR0992_S 619A-428-769)

[0436]

[0437] SEQ ID NO:46(CHIM_m0992_0992_FL)

[0438]

[0439] SEQ ID NO:47(wt_SAR0280)

[0440]

[0441]

[0442] SEQ ID NO:48(0280-28-820;SAR0280-28-820)

[0443]

[0444] SEQ ID NO:49(s0280;SAR0280-949-978)

[0445]

[0446] SEQ ID NO:56(CHIM_LukE_mHla_H35L_FS)

[0447]

[0448] SEQ ID NO:57(CHIM_mc2716_mSpA_7_12_FS)

[0449]

[0450] SEQ ID NO:58(CHIM_0992_mSpA_7_12_FS)

[0451]

[0452] SEQ ID NO:59(CHIM_2753_mc2723_FS)

[0453]

[0454]

[0455] SEQ ID NO:60(CHIM_0280_2753_FS)

[0456]

[0457] SEQ ID NO:61(CHIM_0992_2753_FS)

[0458]

[0459] SEQ ID NO:62(CHIM_mc2723_Hla_H35L_t_FS)

[0460]

[0461]

[0462] SEQ ID NO:63(CHIM_mc2716_LukE_FS)

[0463]

[0464] SEQ ID NO:64(CHIM_LukE_Hla_H35L_t_FS)

[0465]

[0466] SEQ ID NO:65(CHIM_mc2716_mSpA_10_12_FS)

[0467]

[0468]

[0469] SEQ ID NO:66(CHIM_LukE_0280_FS)

[0470]

[0471] SEQ ID NO:67(CHIM_0992_m0992_FS)

[0472]

[0473] SEQ ID NO:68(CHIM_0992_Hla_H35L_t_FS)

[0474]

[0475] SEQ ID NO:82(CHIM_LukE_mSpA_10_12_FS)

[0476]

[0477] SEQ ID NO:83(CHIM_mc2723_mSpA_10_12_FS)

[0478]

[0479] SEQ ID NO:84(CHIM_0992_mSpA_10_12_FS)

[0480]

[0481]

[0482] SEQ ID NO:85(CHIM_LukE_mSpA_7_12_FS)

[0483]

[0484] Table 1 summarizes all of the above amino acid sequences by reference to their nomenclature and any alternative nomenclature.

[0485] The following examples are provided to illustrate various embodiments of the present invention and are not intended to limit the present invention in any way. The present examples and the methods described herein currently represent preferred embodiments, are exemplary, and are not intended to limit the scope of the present invention. Those skilled in the art will appreciate variations and other uses that are included within the scope of the appended claims.

[0486]

[0487] Table 1. Overview of protein constructs of the present invention

[0488] Example 1: Functional evaluation of Staphylococcal protein variants

[0489] 1.1 SpA variants

[0490] A range of different SpA variants were tested for their ability to bind to Ig and von Willebrand factor (vWF).

[0491] The following SpA variants were found not to bind to any mouse IgG, rabbit IgG and mouse IgM in whole serum, purified human and mouse IgG, and vWF in ELISA:

[0492]

[0493] Table 2. SpA variants were found to have abolished binding to Ig and vWF. There were two or three mutations in each Fab binding site and two mutations in each Fc binding site in the E, D, A, B and C domains. In the nomenclature SpA_X_Y, X is a number indicating a specific combination of mutations in the Fab binding site and Y is a number indicating a specific combination of mutations in the Fc binding site.

[0494] More detailed data concerning the Ig binding capacity of the SpA variants are given below.

[0495] IgG affinity chromatography

[0496] To evaluate the ability of different SpA constructs not to bind human IgG, an affinity chromatography assay was established based on disruptive mutations in SpA. The principle of this assay is to immobilize 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 constructs before testing human-IgG-SpA binding). The bound material is then eluted and run on an SDS-PAGE gel to visualize binding to the IgG protein.

[0497] This protocol was adopted from Kim et al., 2010, 2015 with minor modifications. Purified His-tagged SpA protein (100 μg (0.5 μg / μL) in 200 μL) was immobilized on Ni-NTA agarose (100 μL, Qiagen #30230, batch number 151029515) for 20 minutes at room temperature (RT), washed with 6CV or 10CV binding buffer (50 mM Tris, 150 mM NaCl, pH 7.5), and incubated with human IgG (100 μg (0.5 μg / μL) in 200 μL, Sigma #I2511-10MG, batch number 048M4868V) for 20 minutes at room temperature (RT). After washing with 20 CV or 10 CV of binding buffer supplemented with 30 mM imidazole, bound proteins were eluted with 5 CV of binding buffer supplemented with 500 mM imidazole and analyzed by SDS-PAGE (10 μL / fraction, 2.5 μg starting material) (any kDa Criterion TGX Stain Free Protein Gel and Pro Blue Safe Stain, Giotto Biotech).

[0498] The results of human IgG affinity chromatography showed that SpA_WT-49-339 (wild-type (WT) full-length SpA) (SEQ ID NO: 16) bound to the entire amount of loaded IgG, while the immobilized SpA KKAAThe -49-339 mutant and several SpA mutant variants, such as 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).

[0499] SpA variants Binding to IgG SpA-WT-49-339 yes <![CDATA[SpA KKAA -49-339]]> no SpA_mut_3_12 no SpA_mut_4_12 no SpA_mut_7_12 no SpA_mut_10_12 no SpA_mut_10_15 no

[0500] Table 3. Human IgG binding capacity of SpA mutant variants

[0501] Direct ELISA

[0502] To evaluate the binding capacity of different SpA mutant constructs to various immunoglobulins, direct ELISA was applied to evaluate the binding capacity to human IgG, human vWF, mouse IgG and IgM, and rabbit IgG. Minor adjustments were made to the protocol described in Kim et al., 2015, depending on the Ig evaluated in the assay.

[0503] To evaluate the inhibition of mouse IgG binding to SpA mutant variants, the protocol described by Kim et al., 2015 was used with the following modifications: Briefly, wells were coated with protein diluted in PBS (pH 7.6) (300 ng protein / well) overnight at 4°C. Purified mouse IgG (Sigma-Aldrich, #I8765-10MG, Lot SLBW5857) was diluted fivefold starting from 40 ng / well or nonspecific mouse serum was diluted fivefold starting from 1:1,000 and then added to the plates. Goat anti-mouse IgG antibody conjugated to alkaline phosphatase (Sigma-Aldrich, A3562, Lot SLBK6489V) was added to the plates at a dilution of 1:5,000 or 1:2,000, respectively. 3 mg / mL p-nitrophenyl phosphate (p-NPP; Sigma-Aldrich, P5869-25CAP, Lot No. SLBV2576) in diethanolamine (DEA, pH 9.2; Sigma-Aldrich, D8885-500g, Lot No. MKCB9120) was used as substrate. After 30 or 55 minutes of reaction, respectively, the absorbance at 405 nm was read using a Tecan Infinite M200 Pro instrument.

[0504] To evaluate the inhibition of purified human IgG binding to SpA mutant variants, the protocol described by Kim et al., 2015 was used with the following modifications: Briefly, wells were coated with SpA mutant proteins diluted in PBS (pH 7.6) (300 ng protein / well) overnight at 4°C. Human IgG (Sigma-Aldrich, #I2511-10MG, 048M4868V) was diluted 5-fold starting from 40 ng / well and added to the plate, and goat anti-human IgG conjugated to alkaline phosphatase (Sigma-Aldrich, #A1543-1ML, lot number 029M4838V) was used as a secondary antibody at 1:10,000, and absorbance was read at 405 nm using a Tecan Infinite M200Pro instrument after 37 minutes of reaction.

[0505] To assess the inhibition of nonspecific rabbit IgG binding to SpA mutant variants, the protocol described in Kim et al. 2015 was used with the following modifications: Briefly, wells were coated with SpA mutant proteins diluted in PBS (pH 7.6) (300 ng protein / well) overnight at 4°C. Nonspecific rabbit serum was diluted threefold starting from a 1:1,000 dilution and added to the plates, and goat anti-rabbit IgG conjugated to alkaline phosphatase (Sigma-Aldrich, A3687) was used as a secondary antibody at 1:10,000, and absorbance was read at 405 nm using a Tecan Infinite M200Pro instrument after 55 minutes of reaction.

[0506] To assess the inhibition of nonspecific mouse IgM binding to SpA mutant variants, the protocol described by Kim et al., 2015 was used with the following modifications: Briefly, wells were coated with SpA mutant proteins diluted in PBS (pH 7.6) (300 ng protein / well) overnight at 4°C. Nonspecific mouse sera were diluted threefold starting from a 1:1,000 dilution and added to the plates, and goat anti-mouse IgM conjugated to alkaline phosphatase (SouthernBiotech N1021-04, lot number C7107RL27Z) was used as a secondary antibody at 1:2,000, and absorbance was read at 405 nm using a Tecan InfiniteM200Pro instrument after 108 minutes of reaction.

[0507] To assess the inhibition of nonspecific human von Willebrand factor (vWF) binding to SpA mutant variants, the protocol described by Kim et al., 2015, was used with the following modifications: 96-well plates (300 ng protein / well) were coated with SpA mutant variant proteins diluted in PBS (pH 7.6) overnight at 4°C. 300 ng of human vWF (Invitrogen #RP-43132, Lot UE2767197) in 1% bovine serum albumin (BSA) was added to each well and incubated for 1 hour. Goat anti-human vWF antibody (Bio-Rad, #AHP062, Lot 148125) was diluted 1:5,000 and incubated for 45 minutes. A secondary antibody conjugated to peroxidase, donkey anti-goat IgG (Sigma-Aldrich A3414-1mL, Lot SLBV6847) was diluted 1:30,000 and incubated for 45 minutes. TMB (3,3',5,5'-tetramethylbenzidine, Biolegend, #421101, batch number B304351) was used as substrate according to the manufacturer's protocol. A stop solution of 1 M H 3 PO 4 (Sigma-Aldrich). After 45 minutes of reaction, the absorbance at 405 nm was read using a Tecan Infinite M200Pro instrument.

[0508] Table 4 and Figure 1-6 The results summarized in show that the SpA_WT-49-339 (SEQ ID NO: 16) protein is able to bind to human IgG, mouse IgG and IgM, rabbit IgG and human vWF. SpA KKAA The -49-339 mutant does not bind to mouse IgM, mouse and rabbit IgG, and vWF, but can bind to human IgG. SpA mutant variants SpA_mut_7_12 (SEQ ID NO: 18) and SpA_mut_10_12 (SEQ ID NO: 17) cannot bind to human IgG, mouse IgG, mouse IgM, rabbit IgM, or vWF. 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) do bind to human IgG, but not to mouse IgG, mouse IgM, rabbit IgG, or vWF.

[0509]

[0510] Table 4. Overview of SpA variants binding to IgG and IgM of various animal origins and human vWF

[0511] Competitive ELISA

[0512] Competitive ELISA was performed to address the blocking of SpA:IgG interaction by polyclonal immune sera against SpA variants. To evaluate the inhibitory effect of chicken IgY on human IgG binding to SpA mutants, the protocol described by Kim et al., 2010 was modified slightly.

[0513] Briefly, wells were coated with immobilized SpA_WT-49-339 (SEQ ID NO: 15) diluted in PBS (pH 7.6) (200 ng per well). IgY from egg yolks of chickens immunized with different SpA variants (Davids Biotechnologie GmbH) was mixed with AddaVax TM (InvivoGen) adjuvant was diluted twice (from 1:10 to 1:320) and added to the culture plate. The plate was incubated at room temperature for 1 hour. 50 nanograms (50ng) of human IgG (Sigma-Aldrich l2511, batch number O48M4868V) was added to each well and incubated at room temperature for 1 hour. Goat anti-human IgG antibody (Sigma-Aldrich A1541, batch number 029M4838V) conjugated with alkaline phosphatase diluted 1:10,000 was used as a secondary antibody. 3mg / mL p-nitrophenyl phosphate (p-NPP) (Sigma-Aldrich P5869-25CAP, batch number SLBV2576) in diethanolamine (DEA, pH9.2) (Sigma-Aldrich, D8885-500g, batch number MKCB9120) was used as a substrate. After 55 minutes of reaction, the absorbance at 495 nm was read using a Tecan Infinite M200 Pro instrument. The PBS sample was set to 100% (no inhibition control), and the human IgG binding percentage was calculated using the following formula:

[0514] (Average absorbance at 405 nm) ÷ (PBS absorbance at 405 nm) × 100%

[0515] The results showed that specific IgY antibodies against several SpA mutant variants tested in a competitive ELISA exceeded nonspecific binding to human IgG ( Figure 7). Specific IgY against the chimeric protein CHIM_mc2716_mSpA_7_12_FS (SEQ ID NO: 57) effectively blocked the binding of SpA to human IgG in a dose-dependent manner (the binding rate to human IgG was 63% at a dilution of 1:40, while the binding rate of SpA_WT-49-339 to human IgG was 82%). IgY specific for the following 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 reduction in the binding of human IgG to SpA_WT-49-339 (SEQ ID NO: 15) to varying degrees. The pre-immune serum collected before the first immunization (negative control) could not block the non-specific binding of human IgG to SpA_WT-49-339. KKAA -49-339-specific IgY (positive control) blocked the binding of SpA_WT-49-339 to human IgG.

[0516] 1.2 Lysozyme variants

[0517] Azocasein hydrolysis assay was established to analyze the potential of vaccine candidate-specific antibodies to inhibit the enzymatic activity of auriostigmin. Azocasein, a chromogenic derivative of casein, was used as a substrate to measure auriostigmin activity. Auriostigmin degrades azocasein to generate trichloroacetic acid (TCA)-soluble azopeptides with high UV absorbance that can be quantified spectrophotometrically. Therefore, auriostigmin was incubated with azocasein and the degradation of azocasein was assessed. Thermolysin, a heat-stable neutral metalloprotease produced by the Gram-positive bacterium Bacillus thermoproteolyticus (Sigma-Aldrich, P1512), was used as a positive control. Initially, commercial auriostigmin and USA300HOU_2637 were tested for caseinolytic activity.

[0518] However, since neither of these two aurisin exhibited enzymatic activity in the assay (data not shown), a protocol was established to purify aurisin from cultures of S. aureus USA300.

[0519] The protocol for purifying lysogenin from Staphylococcus aureus USA300 culture is as follows: 1L of Staphylococcus aureus USA300HOU cell culture was grown in tryptic soy broth (TSB) medium at 37°C and 170 rpm for 24 hours. The culture was harvested by centrifugation (4,100 rpm, 30 minutes at 4°C) 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) and a 10 kDa hollow fiber (General Electric Healthcare, AKTA Flux, UFP-10-C-3MA, batch 17000576). The sterile supernatant was precipitated with 60% ammonium sulfate at 4°C (according to Banbula et al., 1998). To stabilize the pH, 1M Tris (pH 8.0) was added at a ratio of 1:10 (v / v) before precipitation. The suspension was centrifuged at 10,000 × g for 30 min at 4 °C. The pellet was resuspended in 10 mM Tris-HCl (pH 7.6) and 5 mM CaCl 2 10 mM Tris-HCl (pH 7.6) and 5 mM CaCl 2Dialysis was performed overnight at 4°C (10 kDa membrane cutoff). This was followed by two chromatography steps; (1) Akta purification: anion exchange - Mono Q5 / 50GL (General Electric Healthcare, 17-5166-01, lot #10244496) - eluted with a 0-0.5 M NaCl gradient, (2) size exclusion chromatography (SEC): SUPERDEX 75pg 16 / 600 (General Electric Healthcare, 28-9893-33, lot #10244501). These fractions were analyzed by SDS-PAGE (using TGX stain-free protein gels, Bio-Rad and using Pro Blue Safe Stain, Giotto Biotech) and Western blotting (primary antibody raised in rabbit 1 μg / mL; goat anti-rabbit IgG / HRP-conjugate (Dako, P0448, lot #20066477) diluted 1:2,000).

[0520] Lysogenin purified from S. aureus culture had a purity of >90% (estimated by SDS) and also showed activity in the casein hydrolysis assay. The quality of the lysogenin preparation (good peptide coverage of the mature form) was confirmed by mass spectrometry analysis (data not shown).

[0521] The protocol for the azocasein (activity) assay was 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 with 10 mM EDTA as a negative control (EDTA acts as an inhibitor of metalloproteinases). Mcllavaine buffer (pH 7.4, 10 mM CaCl 2 (19.07mL 0.2M Na 2 HPO 4 , 1.73 mL 0.1 M citric acid, 0.2 mL 1 M CaCl 2) Prepare a 10 mg / mL azocasein solution. 200 μL of azocasein solution was mixed with PBS supplemented with 2 μg of purified aurocin and 20 μL or 50 μL of mouse serum immunized with different antigens or with adjuvant alone (negative control). At room temperature, the mixture was shaken (1,000 rpm) at 37°C in a thermomixer for 1 hour. 400 μL of 5% trichloroacetic acid (TCA) was added to terminate the reaction. The sample was centrifuged at 10,000 × g for 3 minutes at 4°C. Next, 300 μL of 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).

[0522] The immune sera of mice immunized with the aurisin chimeric protein construct could inhibit the activity of aurisin purified from Staphylococcus aureus culture (named SA. aurisin) ( Figure 8 ). The immune serum of mice that received CHIM_mc2716_mSpA_7_12_FS (SEQ ID NO: 57) adjuvanted with SLA-SE showed a strong ability to inhibit the activity of lysozyme in a dose-dependent manner ( Figure 8 A), the absorbance at 400 nm was reduced compared with sera from mice immunized with PBS or SLA-SE adjuvant. This dose-dependent inhibition of lysogenin was also observed in immune sera collected from mice immunized with CHIM_mc2716_mSpA_10_12_FS (SEQ ID NO: 65) adjuvanted with SLA-SE ( Figure 8 B). Assay controls confirm the assay setup ( Figure 8 C). The test has been repeated several times with comparable results.

[0523] 1.3LukE variants

[0524] LukE protein is one of the two components of LukED leukocidin. LukE and LukD subunits combine to form pore-forming toxins on immune cell and erythrocyte membranes, leading to cell lysis and killing. LukED's target immune cells (in humans and mice) include neutrophils, monocytes, and macrophages, among others. Therefore, LukE-specific functional antibodies can inhibit the formation of LukED toxins and their cytotoxic activity against human neutrophils.

[0525] The inhibition of leukocyte activity by antibodies in the sera of mice immunized with chimeric proteins containing LukE adjuvanted with LukE or SLA-SE was evaluated by cytotoxic inhibition assay (XTT assay). The human promyelocytic leukemia (HL-60) cell line was cultured under the reported culture conditions as described by ATCC. The cells were cultured by adding 0.78% dimethylformamide (DMF) to the culture medium and incubating at 37°C with 5% CO. 2 HL-60 cells were differentiated into neutrophil-like cells by incubation for 5 days. In this assay, 3.5-4 × 10 5 The assay was performed in a 96-well plate and components were added to each well in the following order (replicates were applied):

[0526] - Test serum diluted in culture medium in a final volume of 50 μL (two-fold dilutions from 1:50 to 1:400)

[0527] -Toxins; LukE only, LukD only, and LukE together with LukD, diluted in culture medium to the desired final concentration in a final volume of 40 μL.

[0528] -3.5-4×10 5 HL-60 differentiated cells in a final volume of 10 μL.

[0529] After inoculation, the culture plates were incubated at 37°C and 5% CO 2 After incubation for 24 h under the conditions of 4% paraformaldehyde and 1% paraformaldehyde, the cells were incubated for 24 h. After incubation for 16 h with the Cell Proliferation Kit II (XTT Detection Reagent, Sigma-Aldrich), 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-tetrazolyl]-bis(4-methoxy-6-nitro)benzenesulfonate hydrate) to an orange formazan dye by metabolically active cells. Finally, the data were acquired using a SpectraMax Reader II instrument.

[0530] Compared with the sera of mice immunized with SLA-SE adjuvant alone, immunization of mice with LukE-29-311 (SEQ ID NO: 30; positive control) adjuvanted with SLA-SE or a chimeric protein comprising LukE adjuvanted with SLA-SE induced a large amount of functional antibodies capable of neutralizing the cytotoxic activity of LukED ( Fig. 9 ).

[0531] The leukocyte toxicity of functional wild-type LukE can be blocked / neutralized by antibodies induced by CHIM_LukE_mHla_H35L_FS (SEQ ID NO: 56) chimeric protein. The sera of mice immunized with several other SLA-SE adjuvanted LukE chimeric proteins can induce high cell viability, which is comparable to the level of positive control natural LukE-29-311 immune serum ( Fig. 9 The assay also showed that the toxin subunits LukE and LukD alone had no effect on the cell viability of all the chimeric proteins tested (data not shown).

[0532] 1.4Hla variants

[0533] To evaluate the efficacy of different chimeric proteins containing Hla subunit designs, a hemolysis inhibition test was established. Using a two-week schedule, mice were immunized subcutaneously (sc) twice in the flank with different SLA-SE adjuvanted chimeric proteins. Immune sera were collected 14 days after the second immunization to evaluate the induction of specific antibodies. The immune sera were diluted two-fold in a 96-well plate with PBS containing 0.5% bovine serum albumin (BSA) (from 1:10 to 1:640). Each serum dilution was incubated with 20ng recombinant Hla (rHIa) at room temperature for 20 minutes. Water alone and PBS solutions containing 0.5% BSA were used as positive and negative controls, respectively. A two percent (2%) rabbit erythrocyte solution derived from defibrinated rabbit blood was added to the culture plate, and the plate was 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. One hundred microliters (100 μL) of PBS was added to each well, and then the absorbance at 540 nm was read using a plate reader. The percentage of hemolysis was calculated based on the hemolysis caused by water (100% hemolysis).

[0534] Sera from mice immunized with adjuvant only (SLA-SE) were unable to inhibit hemolysis of erythrocytes ( Fig.10). The positive control serum collected from mice that received SLA-SE adjuvanted Hla_H35L-27-319 (SEQ ID NO: 5) showed hemolysis inhibition, which disappeared at a dilution of 1:320. The two control sera together verified the establishment of the hemolysis inhibition test. The immune serum of mice immunized with the chimeric protein CHIM_LukE_mHla_H35L_FS (SEQ ID NO: 56) together with SLA-SE showed strong hemolysis inhibition ability even at the highest tested dilution (1:640). This chimeric protein immune serum inhibited 53% hemolysis at a dilution of 1:640, while the serum of mice that received the placebo (SLA-SE) and the positive control (Hla_H35L-27-319) only inhibited about 1-3% hemolysis at the same dilution. Different chimeric proteins containing Hla subunits can induce hemolysis inhibition immune serum to varying degrees. However, the highest hemolysis inhibition effect was observed in immune sera from mice receiving CHIM_mc2723_Hla_H35L_t_FS (SEQ ID NO: 62), showing 77% hemolysis inhibition at a dilution of 1: 640. The assay was repeated with comparable results.

[0535] Example 2: Prophylactic Vaccine Efficacy in Animal Challenge Model

[0536] The efficacy of the prophylactic vaccine proteins was evaluated using an animal model of Staphylococcus aureus peritonitis using CD-1 mice (female, 6-8 weeks old, 6-12 per group, Charles River, Germany). Before challenge, mice were immunized twice or three times, either by hind leg muscle (im) injection on days 0 and 14 (two immunizations) or by flank subcutaneous (sc) injection on days 0, 14 and 28 (three immunizations) with the prophylactic vaccine products shown in Table 5, which were formulated with outer membrane vesicles (OMV, InvivoGen, E. coli OMV InvivoFit TM) and aluminum hydroxide gel (AIOH, Alhydrogel, InvivoGen, catalog code: vac-alu-250, batch number #5531), aluminum hydroxide gel (AIOH, Alhydrogel, InvivoGen, catalog code: vac-alu-250, batch number #5531) alone or SLA-SE (AAHI, the Access to Advanced Health Institute). The protein was formulated with the above adjuvants diluted in 10mM Tris + 144mM NaCl, pH 7.2 buffer at a ratio of 1:1 [vol / vol]. The dosage of the individual proteins or chimeric constructs is shown in Table 5. As negative controls, mice were immunized with adjuvant or saline alone.

[0537] OMV+AIOH adjuvant was prepared as follows: 500 μg of InvivoGen OMVs were resuspended in 1 mL of sterile, endotoxin-free water provided and then dispensed into 200 μL aliquots. For groups of 8 mice, 96 μL of OMV suspension was mixed with 144 μL of sterile buffer. Then, 240 μL of redispersed aluminum hydroxide gel was added to the OMV / buffer solution to a final concentration of 5 μL OMV / 50 μL, mixed rigorously, and stored on ice in a refrigerator for at least one hour before immunization.

[0538] Two or four weeks after the last immunization, mice were challenged by intraperitoneal (ip) injection of S. aureus USA300 LAC strain (NCBI: CP000730.1, Diep et al., 2006), S. aureus MRSA252 strain (NCBI: BX571856.1), S. aureus Newman strain (NCBI: AP009351.1) or S. aureus USA400 (NCBI: NZ_CP019574.1) at different challenge CFU doses shown in Table 5.

[0539] To generate challenge doses of different S. aureus strains, the protocol was as follows: overnight cultures were diluted to an optical density at 600 nm (OD 600nm ) was 0.5, placed in fresh tryptic soy broth (TSB), and grown at 37°C for about 2.5 hours until OD 600nm The cells 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 subsequent use. The final challenge dose administered to the animals was experimentally verified by plating on agar plates and counting CFU the next day.

[0540] Animals were monitored for 7 days after bacterial challenge for predefined clinical signs of infection and humane endpoint criteria were used to determine whether mice should be sacrificed. Overall survival was recorded.

[0541] The survival rate of challenged mice showed that compared with SHAM saline controls, as shown in Table 5, 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 adjuvanted with OMV+AIOH (with or without toxoid CHIM_LukE_mHla_H35L_FS (SEQ ID NO:56) achieved a very significant protective effect (survival rate of 87.5%, p=<0.0001) after three intramuscular immunizations.

[0542] Compared with the SHAM saline control, when three intramuscular injections of EDEN Combo-1 adjuvanted with OMV+AIOH without toxoid chimeric protein were used for immunization, the survival rate against the USA300 LAC strain reached 50% (p=0.0013, **). It was also observed that EDEN Combo-1 adjuvanted with AIOH containing toxoid chimeric protein induced high protection against the USA300 LAC strain (p=<0.0001, ****). In this study, regardless of the adjuvant used, immunization with EDEN Combo-1 antigen with or without toxoid chimeric protein can produce strong protection. For statistical evaluation, the Log-Rank Mantel-Cox test was used.

[0543] After USA300 LAC or MRSA252 attack, LukE-29-311 (SEQ ID NO:30) SLA-SE adjuvanted vaccine produced significant protective effect. In addition, the use of SLA-SE adjuvanted USA300HOU_2637_E353A-28-509 (SEQID NO:35), SAR2723-28-509 (SEQ ID NO:39) or SAR0280-28-820 (SEQ ID NO:48) vaccination has a significant protective effect to the attack of USA300 LAC. With SAR2753-291-680 (SEQ ID NO:42) or some chimeric protein constructs in Table 5, all have a protective effect to the attack of MRSA252. Immunization with SAR0992-1-409 adjuvanted with SLA-SE provided significant protection against challenge with USA300 LAC, USA400 and Newman strains.For statistical evaluation, the Log-Rank Mantel-Cox test was used.

[0544]

[0545] Table 5. Sepsis challenge model protection data. *For combinations, the antigen doses shown are the total doses given. In combinations with OMV+AIOH or AIOH adjuvant alone, the dose of each chimeric protein was 20 μg and the dose of each single antigen was 10 μg.

[0546] Example 3: Prophylactic Vaccine Efficacy in Skin Model

[0547] The efficacy of the preventive vaccine protein was evaluated using a skin abscess animal model infected with Staphylococcus aureus using BALB / c mice (6-week-old females, 12 per group). Before the attack, mice were immunized twice or three times, injected into the hind leg muscle (im) on day 0 and day 14 (twice immunization) or injected subcutaneously (sc) in the flank on day 0, day 14 and day 28 (three times immunization) with the preventive vaccine products shown in Table 6, which were adjuvanted with aluminum hydroxide (AIOH, Alhydrogel, InvivoGen, catalog code: vac-alu-250, batch number #5531) or SLA-SE (AAHI, the Access to Advanced Health Institute). The protein was prepared with the above adjuvant diluted in 10mM Tris+144mMNaCl, pH 7.2 buffer at a ratio of 1:1 [vol / vol]. The dosage of a single protein or chimeric construct is shown in Table 6. As a negative control, mice were immunized with adjuvant or saline alone.

[0548] Two weeks after the last immunization, S. aureus USA300 LAC strain (Diep et al., 2006) or S. aureus Newman strain (Baba et al., 2008) were injected subcutaneously (sc) at different challenge CFU doses shown in Table 6.

[0549] To generate challenge doses of different S. aureus strains, the protocol was as follows: overnight cultures were diluted to an optical density at 600 nm (OD 600nm ) was 0.5, placed in fresh tryptic soy broth (TSB), and grown at 37°C for about 2.5 hours until OD 600nm The cells 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 subsequent use. The final challenge dose administered to the animals was experimentally verified by plating on agar plates and counting CFU the next day.

[0550] Abscess formation (size and skin necrotic lesions) was monitored at 24-h intervals for 10 days after infection. The size of abscesses and associated overlying skin necrotic lesions was determined using the standard formula for the area of ​​an ellipse: [A = π × lesion length / 2 × lesion width]. For statistical evaluation of the area under the curve (AUC), a two-way analysis of variance (group × time) was performed for the skin lesion area parameter, followed by Dunnett's multiple comparison test (Kruskal-Wallis test). Differences between groups were considered statistically significant when p value < 0.05. Statistical analysis and AUC data were analyzed using GraphPad Prism version 9. Differences were considered highly significant when p value ≤ 0.0001 and are indicated by "****". "ns" (not significant) refers to p value > 0.05.

[0551] Immunization with AIOH-adjuvanted EDEN Combo-1, 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, significantly impaired USA300LAC abscess formation, thus producing a significant protective effect compared to the use of AIOH adjuvant alone (p=0.0114*).

[0552] Immunization with SLA-SE adjuvanted Hla_H35L-39-319 (SEQ ID NO: 6), LukE-29-311 (SEQ ID NO: 30), or SAR2753-291-680 (SEQ ID NO: 42) significantly impaired abscess formation after USA300 LAC and Newman challenge compared to immunization with SLA-SE adjuvant alone. Immunization with SLA-SE adjuvanted USA300HOU_2637_E353A-28-509 (SEQ ID NO: 35) significantly impaired (*) abscess formation after USA300 LAC challenge. Immunization with SLA-SE adjuvanted SAR2723-28-619 (SEQ ID NO: 39) or SAR0992-1-409 significantly impaired abscess formation after Newman challenge. Immunization with different chimeric constructs adjuvanted with SLA-SE also resulted in lower AUC and impaired abscess formation when challenged with S. aureus USA300 LAC or S. aureus Newman strain. All groups were tested only against SLA-SE adjuvant alone.

[0553]

[0554] Table 6. Staphylococcus aureus skin abscess data

[0555] *For combinations, the antigen doses shown are the total doses administered. In combinations with AIOH adjuvant, the dose of each chimeric protein was 20 μg and the dose of each single antigen was 10 μg. In combinations with SLA-SE adjuvant, the dose of each single antigen was 20 μg.

[0556] Example 4: Prophylactic Vaccine Immunogenicity IgG Data

[0557] Total immunoglobulin G (IgG) responses after immunization with prophylactic vaccine proteins were assessed using standard ELISA assays. Mice were immunized three times with 30 μg of each single protein or chimeric construct formulated with adjuvant SLA-SE as shown in FIG11a, or with 20 μg of each chimeric construct 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 the addition of toxoid chimeric construct CHIM_LukE_mHla_H35L_FS (SEQ ID NO: 56), and with OMV (InvivoGen, E. coli OMV InvivoFit TM ) + AIOH or AIOH (InvivoGen) alone as adjuvant. The adjuvant was diluted in 10mM Tris + 144mM NaCl, pH 7.2 buffer. Immunization was by intramuscular (im) injection into the hind leg or by subcutaneous (sc) injection into the flank on days 0, 14 and 28. Blood samples were drawn from the tail vein 12 days after immunization to assess antigen-specific total IgG titers.

[0558] Standard ELISA detection protocol: 96-well Nunc-Immuno TM MaxiSorp plates (Sigma-Aldrich) were coated with single or chimeric proteins, and the half-maximal titer of IgG in the sera of mice immunized with the corresponding proteins was measured. The threshold of detection sensitivity was set at 10 3 Each point in Figure 11a-c represents one immunized mouse. Each ELISA run included a plate control to verify the efficiency of the assay.

[0559] As shown in Figure 11a, compared with the use of SLA-SA adjuvant alone, 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: 19), and SpA_mut_8_20 (SEQ ID NO: 21). All tested immune sera from mice immunized with prophylactic vaccines of either USA300HOU_2637_E353A-28-509 (SEQ ID NO: 18) or USA300HOU_2637_E353A-28-509 (SEQ ID NO: 35) had high levels of antigen-specific total IgG antibodies ( FIG. 11c ).

[0560] As shown in Figure 11b, immune sera from mice immunized with EDEN Combo-1 adjuvanted with OMV+AIOH or AIOH alone induced high levels of antigen-specific total IgG antibodies against the individual and chimeric proteins contained in EDEN Combo-1, compared with OMV+AIOH and AIOH adjuvant alone (Figure 11c).

[0561] GraphPad Prism 9.0 version was used to analyze data by calculating the EC50 of each serum dilution curve using the Hill equation to establish a nonlinear fitting model, and the group mean of the IgG titer of each immunization was calculated using Row Statistics, which calculated the mean value of each data set column. For statistical evaluation, one-way ANOVA test and Tukey's multiple comparison test were used. When p value ≤ 0.05, it was considered that there was a significant difference. When p value ≤ 0.0001, it was considered that there was a highly significant difference and was represented by "****". "ns" (not significant) refers to p value> 0.05. For IgG subclasses, single sample t and Wilcoxon test were applied.

[0562] Examples Reference List

[0563] 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

[0564] 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.

[0565] Banbula A, Potempa J, Travis J, Fernandez - Catalán 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.

[0566] 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.

[0567] Kim HK et al., 2010 Nontoxigenic protein A vaccine for methicillin - resistant Staphylococcus aureus infections in mice. J Exp Med 207: 1863 - 1870.

[0568] Kim HK et al., 2015 Protein A suppresses immune responses during Staphylococcus aureus bloodstream infection in guinea pigs. mBio 6.

Claims

1. An immunogenic polypeptide consisting of or comprising a variant of the amino acid sequence of α-hemolysin (Hla), wherein: i. has at least 85% sequence identity with the sequence of SEQ ID NO: 2, and 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, wherein the polypeptide is incapable of participating in the formation of a heptameric structure with other HIa molecules, and wherein any substitution in ii) is preferably non-conservative.

2. An immunogenic polypeptide according to claim 1, wherein the variant comprises at least two amino acid changes selected from deletion and substitution in the first 12 consecutive N-terminal amino acid residues, such as 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 changes.

3. An 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 item ii), such as 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. An immunogenic polypeptide according to claim 1 or 2, wherein the variant comprises at least two amino acid deletions in the first 12 consecutive N-terminal amino acid residues, such as 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 of claim 1, wherein amino acid residues 1-12 are all deleted.

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

7. An immunogenic polypeptide according to claim 6, wherein amino acid residues 1-12 are substituted by the sequence 5'-SETEVSVRSASS-3' (residues 1-12 in 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 from 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 is capable of inducing antibodies that block the hemolytic activity of wild-type Hla.

13. An immunogenic polypeptide consisting of or comprising: i. A variant of the amino acid sequence of immunoglobulin G binding protein A (SpA), the variant comprising at least one of the ae items: 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), and ii. the one or more first mutations in each of at least one of items ae, wherein the one or more first mutations disrupt binding at the Fc binding site, and wherein the one or more first mutations are made at a position corresponding to an amino acid position selected from position 3, 8, 9, 64, 69, 70, 122, 127, 128, 180, 185, 186, 238, 243, 244 in SEQ ID NO: 16, and iii. the one or more second mutations in each of at least one of items ae, wherein the one or more second mutations disrupt binding at the Fab binding site, and wherein the one or more second mutations, where applicable, are made at a position corresponding to an amino acid position 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 in SEQ ID NO: 16, iv. wherein, optionally, at least one of items ae, where applicable, does not comprise mutations that disrupt binding at the Fc binding site at positions corresponding to two amino acid positions 7+8, 68+69, 126+127, 184+185 and 242+243 of SEQ ID NO: 16, and v.Among them, At least one of items ae, where applicable, does not contain mutations that disrupt binding in the Fab binding site at the positions corresponding to two amino acid positions 34+35 and 95+96 and 153+154 and 211+212 and 269+270 of SEQ ID NO: 16, wherein the polypeptide is unable to bind to human IgG and human von Willebrand Factor, and wherein the substitution is preferably a non-conservative substitution.

14. The polypeptide according to claim 13, wherein the one or more second mutations are made at least at the following positions or only at the following positions: 1 or 2 or 3 or 4 or 5 of positions 34 and 96 and 154 and 212 and 270, 1 or 2 or 3 or 4 or 5 of positions 35 and 95 and 154 and 212 and 270, 1 or 2 or 3 or 4 or 5 of positions 35 and 96 and 153 and 212 and 270, 1 or 2 or 3 or 4 or 5 of positions 35 and 96 and 154 and 211 and 270, 1 or 2 or 3 or 4 or 5 of positions 35 and 96 and 154 and 212 and 269, 1 or 2 or 3 or 4 or 5 of positions 34 and 95 and 154 and 212 and 270, 1 or 2 or 3 or 4 or 5 of positions 34 and 96 and 153 and 212 and 270, 1 or 2 or 3 or 4 or 5 of positions 34 and 96 and 154 and 211 and 270, 1 or 2 or 3 or 4 or 5 of positions 34 and 96 and 154 and 212 and 269, 1 or 2 or 3 or 4 or 5 of positions 35 and 95 and 153 and 212 and 270, 1 or 2 or 3 or 4 or 5 of 154 and 211 and 270, 1 or 2 or 3 or 4 or 5 of 35 and 95 and 154 and 212 and 269, 1 or 2 or 3 or 4 or 5 of 35 and 96 and 153 and 211 and 270, 1 or 2 or 3 or 4 or 5 of 35 and 96 and 153 and 212 and 269, 1 or 2 or 3 or 4 or 5 of 35 and 96 and 154 and 211 and 269, 1 or 2 or 3 or 4 or 5 of 34 and 95 and 153 and 212 and 270, 1 or 2 or 3 or 4 or 5 of 34 and 95 and 154 and 211 and 270, 1 or 2 or 3 or 4 or 5 of 34 and 95 and 154 and 212 and 1 or 2 or 3 or 4 or 5 of positions 34 and 96 and 153 and 211 and 270, 1 or 2 or 3 or 4 or 5 of positions 34 and 96 and 153 and 212 and 269, 1 or 2 or 3 or 4 or 5 of positions 34 and 96 and 154 and 211 and 269, 1 or 2 or 3 or 4 or 5 of positions 35 and 95 and 153 and 211 and 270, 1 or 2 or 3 or 4 or 5 of positions 35 and 95 and 153 and 212 and 269, 1 or 2 or 3 or 4 or 5 of positions 35 and 96 and 153 and 211 and 269, 1 or 2 or 3 or 4 or 5 of positions 34 and 95 and 153 and 211 and 270 or 3 or 4 or 5, 1 or 2 or 3 or 4 or 5 of positions 34 and 95 and 153 and 212 and 269, 1 or 2 or 3 or 4 or 5 of positions 34 and 95 and 154 and 211 and 269, 1 or 2 or 3 or 4 or 5 of positions 34 and 96 and 153 and 211 and 269, 1 or 2 or 3 or 4 or 5 of positions 35 and 95 and 153 and 211 and 269, 1 or 2 or 3 or 4 or 5 of positions 35 and 95 and 154 and 211 and 269, 1 or 2 or 3 or 4 or 5 of positions 34 and 95 and 153 and 211 and 269, or 1 or 2 or 3 or 4 or 5 of positions 35 and 96 and 154 and 212 and 270.

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

16.

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

16.

17. A polypeptide according to claim 13 or 14, wherein the one or more second mutations are made at least or only at positions corresponding to positions in SEQ ID NO: 16 selected from the group consisting of: 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.

18. A polypeptide according to claim 13 or 14, wherein the one or more second mutations are made at least or only at positions corresponding to positions in SEQ ID NO: 16 selected from the group consisting of: 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 54 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 and 211 and 270, and 154 and 212 and 270.

19. A polypeptide according to claim 13 or 14, wherein the one or more second mutations are made at least or only at positions corresponding to positions in SEQ ID NO: 16 selected from the group consisting of: 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 2 69, 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 15 3 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.

20. A polypeptide according to claim 13 or 14, wherein the one or more second mutations are made at least or only at positions corresponding to positions in SEQ ID NO: 16 selected from the group consisting of: 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.

21. The polypeptide according to any one of claims 13 to 20, wherein the one or more first mutations are made at least or only at positions corresponding to positions selected from the group consisting of 3, 8, 9, 69, 127, 185 and 243 in SEQ ID NO:

16.

22. A polypeptide according to claim 21, wherein the one or more first mutations are made at least or only at positions corresponding to positions in SEQ ID NO: 16 selected from the group consisting of: 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.

23. The immunogenic polypeptide according to any one of claims 13 to 22, wherein the first mutation and / or the second mutation is a non-conservative substitution.

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 wherein at least one of the one or more first mutations as described in claim 21 is a mutation to lysine (K).

25. The immunogenic polypeptide according to claim 24, wherein all mutations in the one or more first mutations as claimed in claim 21 are mutations to lysine (K).

26. The immunogenic polypeptide according to any one of claims 13 to 25, wherein at least one of the one or more second mutations is as described in claim 16, and wherein at least one of the one or more second mutations as described in claim 16 is a mutation to alanine (A) or arginine (R).

27. The immunogenic polypeptide according to claim 26, wherein all mutations in the one or more second mutations as claimed in claim 16 are mutations to alanine (A) or arginine (R).

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

29. The immunogenic polypeptide according to any one of claims 1 to 28, wherein the variant has at least 85% sequence identity with the sequence of SEQ ID NO:

16.

30. An immunogenic polypeptide according to any one of claims 1 to 29, wherein the polypeptide is capable of inducing antibodies that block the ability of wild-type SpA to bind to IgG and von Willebrand Factor.

31. An immunogenic polypeptide consisting of or comprising a variant of the amino acid sequence of LukE, wherein: i. has at least 85% sequence identity with SEQ ID NO: 30, and ii. does not contain a signal peptide corresponding to residues 1-28 of SEQ ID NO:

29.

32. The immunogenic polypeptide according to claim 31, wherein the polypeptide consists of SEQ ID NO:

30.

33. An immunogenic polypeptide according to claim 31 or 32, wherein the polypeptide is capable of inducing antibodies that block the leukotoxicity of functionally mature wild-type LukE.

34. An immunogenic polypeptide consisting of or comprising a variant of the amino acid sequence of aurolysin (Aur), wherein: i. having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 34, and ii. comprising one or more amino acid substitutions in a HEXXH catalytic domain, wherein the HEXXH catalytic domain corresponds to amino acid positions 352-356 of SEQ ID NO: 34, Wherein the polypeptide has reduced catalytic ability and wherein the substitution is preferably non-conservative.

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.

36. The immunogenic polypeptide according to claim 35, wherein the conserved cysteine ​​is substituted by serine (S).

37. The immunogenic polypeptide according to any one of claims 34 to 36, wherein the glutamic acid residue (E) in the HEXXH catalytic domain corresponding to amino acid position 353 of SEQ ID NO: 34 is substituted, preferably non-conservatively substituted.

38. An immunogenic polypeptide according to claim 37, wherein the glutamic acid in the HEXXH catalytic domain is substituted by alanine (A).

39. The immunogenic polypeptide according to any one of claims 34 to 38, wherein the variant consists of or comprises SEQ ID NO:

35.

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 to amino acid sequence 210-509 of SEQ ID NO:

34.

41. An immunogenic polypeptide according to any one of claims 34 to 40, wherein the variant exhibits a reduced ability to participate in the formation of an antigenic complex compared to wild-type Aur.

42. An immunogenic polypeptide according to any one of claims 34 to 41, wherein the polypeptide is capable of inducing antibodies that block the catalytic activity of wild-type Aur.

43. An immunogenic polypeptide consisting of or comprising a variant of the amino acid sequence of N-acetylmuramoyl-L-alanine amidase (SAR 2723), said variant having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 38, and a. comprising one or more amino acid substitutions in the amidase active site TXEXX domain corresponding to amino acid residues 384-388 in 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 in SEQ ID NO: 37, and / or c. comprising a substitution corresponding to the conserved cysteine ​​at position 513 in SEQ ID NO: 37, wherein the polypeptide has reduced catalytic ability, and wherein the substitution is preferably non-conservative.

44. An immunogenic polypeptide according to claim 43, comprising feature i of: only item a, only item b, only item c, only items a and b, only items a and c, only items b and c, or items a, b and c.

45. An immunogenic polypeptide according to any one of claims 43 or 44, wherein the conserved cysteine ​​is substituted by serine (S).

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, such as a non-conservative substitution, and preferably substituted with glutamine (Q).

47. An immunogenic polypeptide according to any one of claims 43 to 46, wherein the aspartic acid residue (D) in the active site LXDYS domain corresponding to amino acid position 411 of SEQ ID NO: 37 is substituted, such as a non-conservative substitution, and preferably substituted with asparagine (N).

48. The immunogenic polypeptide according to any one of claims 43 to 47, wherein the variant consists of or comprises SEQ ID NO:

39.

49. An immunogenic polypeptide according to any one of claims 43 to 48, wherein the polypeptide is capable of inducing antibodies that block the catalytic activity of wild-type SAR2723 or interfere with bacterial cell wall development.

50. A vaccine composition comprising selected polypeptides comprising at least 2 of items ad: a) an HIa polypeptide or a variant thereof, wherein the variant preferably exhibits reduced or no hemolytic activity and / or preferably is capable of inducing antibodies that block the hemolytic activity of native HIa, b) a LukE polypeptide or a variant thereof, wherein the variant preferably exhibits reduced or no leukocyte activity and / or preferably is capable of inducing antibodies that block the leukocyte activity of native LukE, c) a 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 preferably is capable of inducing antibodies that block the interaction of native SpA with human IgG, and d) an Aur polypeptide or a variant thereof, wherein said variant preferably exhibits reduced catalytic activity and is preferably capable of inducing antibodies that block the catalytic activity of native Aur, The composition optionally further comprises a pharmaceutically acceptable carrier, vehicle or diluent, and optionally further comprises an immunogenic adjuvant, in particular selected from AlOH, SLA-SE and OMV (outer membrane vesicles).

51. The vaccine composition according to claim 50, wherein the Hla polypeptide variant is a variant according to any one of claims 1-12, and / or wherein the LukE polypeptide variant is a variant according to any one of claims 31-33 and / or wherein the SpA polypeptide variant is a variant according to any one of claims 13-30 and / or wherein the Aur polypeptide variant is a variant according to any one of claims 34-42.

52. The vaccine composition according to claim 50, wherein the Hla polypeptide variant has the amino acid sequence of SEQ ID NO:

5.

53. A vaccine composition according to any one of claims 50 to 52, comprising at least 3 of items ad.

54. A vaccine composition according to any one of claims 50 to 52, comprising: -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.

55. A vaccine composition according to any one of claims 50 to 54, further comprising at least one polypeptide selected from the group consisting of: e) a SAR2723 polypeptide or a variant thereof, wherein the variant preferably exhibits reduced or no catalytic activity and is preferably capable of inducing antibodies that block the catalytic activity of native SAR2723, f) a SAR2753 polypeptide or variant, wherein the variant preferably exhibits reduced or no lipase activity and is preferably capable of inducing antibodies that block the lipase activity of native SAR2753, g) SAR0992 (HtrA2) polypeptide or a variant thereof, and h) SAR0280 (EsaA) polypeptide or a variant thereof, wherein said variant is preferably capable of inducing antibodies that prevent the secretion of EsxA / B.

56. A vaccine composition according to claim 55, wherein - the SAR0992 polypeptide variant is a fragment or sequence variant of SEQ ID NO: 15 disclosed in WO2012 / 136653, in particular a fragment consisting of amino acid residues 1-409 of SEQ ID NO: 15 disclosed in WO2012 / 136653, or a variant of SAR0992, which is capable of inducing antibodies that block the catalytic activity of native SAR0992, such as a truncated form of the C-terminus of SAR0992 relative to the SAR0992 transmembrane helix, or a mutant version of SAR0992 comprising a substitution of the serine residue corresponding to the serine residue at position 619 in SEQ ID NO: 15 disclosed in WO2012 / 136653, and / or a mutant version of SAR0992, wherein the transmembrane helix is ​​exchanged with a flexible linker or wherein the transmembrane helix is ​​exchanged with a linker and the N-terminal and C-terminal parts of SAR0992 on both sides of the linker are exchanged with each other, and / or - wherein 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 and most C-terminal extracellular fragments of SAR0280, and / or wherein - the SAR2723 polypeptide variant is a fragment or sequence variant of SEQ ID NO: 13 according to any one of claims 43 to 49 or disclosed in WO2015 / 082536, and / or wherein - The SAR2753 polypeptide variant is a fragment or sequence variant of SEQ ID NO: 14 disclosed in WO2015 / 082536 or a polypeptide consisting of the amino acid sequence SEQ ID NO:

42.

57. A vaccine composition according to claim 55 or 56, comprising at least 2 of items eh.

58. A vaccine composition according to claim 55 or 56, comprising at least 3 of items eh.

59. A vaccine composition according to any one of claims 55 to 58, comprising: -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.

60. A chimeric polypeptide comprising the amino acid sequence of a selected polypeptide as defined in 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 GSGGGA (SEQ ID NO: 50) or GSGGGAGSGGGA (SEQ ID NO: 51), or wherein the rigid linker is KPEPKPAPAPKP (SEQ ID NO: 52).

63. The chimeric polypeptide according to any one of claims 60-62, 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), 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 immunoadjuvant.

65. The vaccine composition according to claim 64, wherein the 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 selected from SEQ ID NOs: 56 and 57.

66. A vaccine composition according to claim 64 or 65 comprising at least or exactly 2 chimeric polypeptides according to any one of claims 60-63.

67. A vaccine composition according to any one of claims 64 to 66, further comprising at least one or exactly one additional polypeptide selected from the group consisting of a polypeptide as defined in 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 SAR0992-1-409 (residues 1-409 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 1-409 of SAR0992 (residues 1-409 of SEQ ID NO: 44).

70. A vaccine composition according to claim 69, which consists of 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 1-409 of SAR0992 (residues 1-409 of SEQ ID NO: 44), and is mixed with a pharmaceutically acceptable carrier, vehicle or diluent, and further optionally an immune adjuvant.

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

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

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 and capable of expressing the nucleic acid fragment according to claim 64 or the vector according to claim 66.

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, vehicle or diluent, and optionally an immunogenic adjuvant.

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

78. A method of inducing immunity in an animal by administering at least once an immunologically effective amount of an immunogenic polypeptide according to any one of claims 1-49, a vaccine composition according to any one of claims 50-59, a chimeric polypeptide according to any one of claims 60-63, 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, to induce adaptive immunity against Staphylococcus aureus in the animal.

79. The method according to claim 70, wherein: When administering the immunogenic polypeptide of any one of claims 1-49, the chimeric polypeptide of any one of claims 60-63, or a composition comprising the immunogenic polypeptide or the chimeric polypeptide, the animal receives 0.5 to 5,000 μg of the immunogenic polypeptide or chimeric polypeptide of any one of claims 1-49 and 60-63 per administration.

80. The method according to claim 70 or 71, wherein the animal receives a prime administration comprising the immunogenic polypeptide or the chimeric polypeptide and one or more booster administrations comprising the immunogenic polypeptide or the chimeric polypeptide.

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

82. The method according to any one of claims 70 to 73, wherein the purpose of said administration is to induce protective immunity against Staphylococcus aureus (S. aureus).

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

84. A method according to any one of claims 70 to 75, wherein the purpose of said administration is to induce antibodies specific for Staphylococcus aureus, and wherein said antibodies or B lymphocytes producing said antibodies are subsequently recovered from the animal.

85. A method according to any one of claims 70 to 75, wherein the purpose of said administration is to induce antibodies specific for Staphylococcus aureus, and wherein B lymphocytes producing said antibodies are subsequently recovered from the animal and used to prepare monoclonal antibodies.

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 for treating, preventing or ameliorating Staphylococcus aureus infection.

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

89. A chimeric polypeptide according to any one of claims 60 to 63 for use as a medicament for treating, preventing or ameliorating Staphylococcus aureus infection.

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 for treating, preventing or ameliorating Staphylococcus aureus infection.

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

93. The transformed cell or virus according to claim 67, for use as a medicament for treating, preventing or ameliorating Staphylococcus aureus infection.

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