WO2019145399A1 - Vaccines for prophylaxis of s. aureus infections - Google Patents

Abstract

Disclosed are proteins and nucleic acids as well as vaccine compositions where the immunogens are derived from/related to a Staphylococcus aureus having SEQ ID NO: 1 or the encoding sequences SEQ ID NOs: 2 and 3. Also disclosed are nucleic acids, vectors, transformed host organisms, as well as prophylactic and therapeutic uses and methods. Finally, also methods of preparation are part of the invention.

Description

VACCINES FOR PROPHYLAXIS OF S. AUREUS INFECTIONS

FIELD OF THE INVENTION

The present invention relates to the field of antimicrobial prophylaxis and therapy. In particular the present invention relates to novel vaccine compositions where the immunogens are derived from Staphylococcus aureus. The invention further relates to vectors,

transformed host organisms, as well as prophylactic and therapeutic uses and methods. Finally, also methods of preparation are part of the invention.

BACKGROUND OF THE INVENTION

Bacterial infections are in most instances successfully treated by administration of antibiotics to patients in need thereof. However, due to careless or thoughtless use of powerful antibiotics, many pathological germs become resistant against antibiotics over time. One threatening example is Staphyloccocus aureus. In particular in hospitals this bacterium is of relevance. So-called Methicillin Resistant S. Aureus (MRSA) strains jeopardize patient's survival in hospitals, in particular after surgery. Vaccination is considered to be a very effective method of preventing infectious diseases in human and veterinary health care. Vaccination is the administration of effective amounts of antigenic material (the vaccine) to produce immunity to a disease/disease-causing pathogenic agent. Vaccines have contributed to the eradication of smallpox, the near eradication of polio, and the control of a variety of diseases, including rubella, measles, mumps, chickenpox, typhoid fever.

Before "the genomic era", vaccines were based on killed or live attenuated, microorganisms, or parts purified from them. Subunit vaccines are considered as a modern upgrade of these types of vaccine, as the subunit vaccines contain one or more protective antigens, which are more or less the weak spot of the pathogen. Hence, in order to develop subunit vaccines, it is critical to identify the proteins, which are important for inducing protection and to eliminate others.

An antigen is said to be protective if it is able to induce protection from subsequent challenge by a disease-causing infectious agent in an appropriate animal model following immunization . The empirical approach to subunit vaccine development, which includes several steps, begins with pathogen cultivation, followed by purification into components, and then testing of antigens for protection. Apart from being time and labour consuming, this approach has several limitations that can lead to failure. It is not possible to develop vaccines using this approach for microorganisms, which cannot easily be cultured and only allows for the identification of the antigens, which can be obtained in sufficient quantities. The empirical approach has a tendency to focus on the most abundant proteins, which in some cases are not immuno-protective. In other cases, the antigen expressed during in vivo infection is not expressed during in vitro cultivation. Furthermore, antigen discovery by use of the empirical approach demands an extreme amount of proteins in order to discover the protective antigens, which are like finding needles in the haystack. This renders it a very expensive approach, and it limits the vaccine development around diseases, which is caused by pathogens with a large genome or disease areas, which perform badly in a cost-effective perspective.

The present applicant has previously filed patent applications relating to induction of immunity against Staphylococcus aureus. In international patent application publications WO 2012/136653, WO 2015/082536, WO 2017/144523, and in PCT application no.

PCT/EP2017/068694 are disclosed a number of polypeptides, nucleic acids, vectors, and compositions that are useful as vaccine agents.

OBJECT OF THE INVENTION

It is an object of embodiments of the invention to provide further polypeptides, nucleic acids, vectors, and compositions that are useful as vaccine agents that are able to induce protective immunity against infections with S. aureus. It is also an object of embodiments of the invention to provide useful tools for the recombinant production of such vaccine agents.

SUMMARY OF THE INVENTION

The present invention provides antigenic material in the form of polypeptides, nucleic acids and vaccine vectors (including cells and virus) that can induce protective immunity against S. aureus infection. This antigenic material is useful as (vaccine) immunogens per se but also in combination with any one of the immunogens disclosed in international patent application publications WO 2012/136653, WO 2015/082536, WO 2017/144523, and in PCT application no. PCT/EP2017/068694. Hence, in a first aspect the present invention relates to a polypeptide comprising or consisting of

a) the amino acid sequence SEQ ID NO: 1, or

b) an amino acid sequence consisting of at least or exactly 5 contiguous amino acid residues from SEQ ID NO: 1, or

c) an amino acid sequence having a sequence identity of at least 60% with the amino acid sequence of a),

d) an amino acid sequence having a sequence identity of at least 60% with the amino acid sequence of b), or

e) an assembly of amino acids derived from SEQ ID NO: 1, which has essentially the same 3D conformation as in the protein from which said assembly is derived so as to constitute a B-cell epitope,

said polypeptide being antigenic in a mammal.

A 2^nd aspect of the invention relates to an isolated nucleic acid fragment, which comprises or consists of a nucleotide sequence encoding a polypeptide according to the first of the invention as well as any embodiments of these aspects..

A 3^rd aspect of the invention relates to a vector comprising the nucleic acid of the 2^nd aspect of the invention or of any embodiments of the 3^rd aspect, such as a cloning vector or an expression vector.

A 4^th aspect of the invention relates to a cell which is transformed so as to carry the vector of 1) the 3^rd aspect of the present invention or 2) any embodiments of the 3^rd aspect. Also part of this aspect is a cell line derived from such a transformed cell of the present invention.

A 5^th aspect of the invention relates to a pharmaceutical composition comprising a

polypeptide of the 1^st aspect of the invention as well as any embodiments of this aspect, a nucleic acid fragment of the 2^nd aspect of the invention or the embodiments of the 2^nd aspect, a vector of the 3^rd aspect of the invention or of any embodiments thereof, or a cell of the 4^th aspect of the invention and any embodiments of the 4^th aspect, and a pharmaceutically acceptable carrier, vehicle or diluent

A 6^th aspects of the invention relates to a method for inducing immunity in an animal by administering at least once an immunogenically effective amount of a polypeptide of the first or second aspect of the invention as well as of embodiments this aspect, a nucleic acid fragment of the 2^nd aspect of the invention as well as any embodiment of the 2^nd aspect, a vector of the 3^rd aspect of the invention as well as any embodiment of the 3^rd aspect, a cell of the 4^th aspect of the invention as well as any embodiment thereof, or a pharmaceutical composition of the 5^th aspect of the invention as well as any embodiment thereof, so as to induce adaptive immunity against S. aureus in the animal .

Finally, in separate aspect relating to the 5^th aspect, the present invention also relates to the polypeptides of the invention, the nucleic acid or vector of the invention, the cells of the invention, or the pharmaceutical compositions of the invention for use as a pharmaceutical, in particular for use in the treatment, prophylaxis or amelioration of infection with S. aureus.

LEGENDS TO THE FIGURE

Fig . 1 shows survival plots after challenge infection in mice immunized with immunogens of the invention in a peritonitis model . Dotted lines indicates control, full lines indicates immunogen.

DETAILED DISCLOSURE OF THE INVENTION

Definitions

The term "polypeptide" is in the present context intended to mean both short peptides of from 2 to 10 amino acid residues, oligopeptides of from 11 to 100 amino acid residues, and polypeptides of more than 100 amino acid residues. Furthermore, the term is also intended to include proteins, i .e. functional biomolecules comprising at least one polypeptide; when comprising at least two polypeptides, these may form complexes, be covalently linked, or may be non-covalently linked . The polypeptide (s) in a protein can be glycosylated and/or lipidated and/or comprise prosthetic groups.

The term "subsequence" means any consecutive stretch of at least 3 amino acids or, when relevant, of at least 3 nucleotides, derived directly from a reference amino acid sequence or nucleic acid sequence, respectively

The term "amino acid sequence" is the order in which amino acid residues, connected by peptide bonds, lie in the chain in peptides and proteins.

The term "adjuvant" or "immunological adjuvant" has its usual meaning in the art of vaccine technology, i .e. a substance or a composition of matter which is 1) not in itself capable of mounting a specific immune response against the immunogen of the vaccine, but which is 2) nevertheless capable of enhancing the immune response against the immunogen. Or, in other words, vaccination with the adjuvant alone does not provide an immune response against the immunogen, vaccination with the immunogen may or may not give rise to an immune response against the immunogen, but the combined vaccination with immunogen and adjuvant induces an immune response against the immunogen which is stronger than that induced by the immunogen alone.

"Sequence identity" is in the context of the present invention determined by comparing 2 aligned sequences of equal length (e.g. DNA, RNA or amino acid) according to the following formula: (N_ref - N_dif) - 100/N_ref, wherein N_ref is the number of residues in one of the 2 sequences and N_drf is the number of residues which are non-identical in the two sequences when they are aligned over their entire lengths and in the same direction. So, two sequences 5'-ATTCGGAAC-3' and 5'- ATACGGGAC-3' will provide the sequence identity 77.78% (N_ref=9 and N_dif=2) . It will be understood that such a sequence identity determination requires that the two aligned sequences are aligned so that there are no overhangs between the two sequences: each amino acid in each sequence will have to be matched with a counterpart in the other sequence.

An "assembly of amino acids" means two or more amino acids bound together by physical or chemical means.

The "3D conformation" is the 3 dimensional structure of a biomolecule such as a protein. In monomeric polypeptides/proteins, the 3D conformation is also termed "the tertiary structure" and denotes the relative locations in 3 dimensional space of the amino acid residues forming the polypeptide.

"An immunogenic carrier" is a molecule or moiety to which an immunogen or a hapten can be coupled in order to enhance or enable the elicitation of an immune response against the immunogen/hapten. Immunogenic carriers are in classical cases relatively large molecules (such as tetanus toxoid, KLH, diphtheria toxoid etc.) which can be fused or conjugated to an immunogen/hapten, which is not sufficiently immunogenic in its own right - typically, the immunogenic carrier is capable of eliciting a strong cellular immune response against the combined substance constituted by the immunogen and the immunogenic carrier, and this in turn provides for improved responses against the immunogen antibody producing cells and cytotoxic cells. More recently, the large carrier molecules have to a certain extent been substituted by so-called promiscuous epitopes, i.e. shorter peptides that are recognized by a large fraction of MHC-haplotypes in a population, and which elicit antigen specific cellular immune responses. An "immunogen" is a substance of matter which is capable of inducing an adaptive immune response in a host, whose immune system is exposed to the immunogen. As such, immunogens are a subset of the larger genus "antigens", which are substances that can be recognized specifically by the immune system but which are not necessarily capable of inducing immunity - an antigen is, however, always capable of eliciting immunity, meaning that a host that has an established memory immunity against the antigen will mount a specific immune response against the antigen.

A "hapten" is a (typically) small molecule, which can neither induce nor elicit an immune response, but if conjugated to an immunogenic carrier, a specific adaptive immune response can be induced against a hapten upon exposure of the immune system with the hapten carrier conjugate.

An "adaptive immune response" is an immune response in response to exposure to an antigen or immunogen, where the immune response is specific for antigenic determinants of the antigen/immunogen - examples of adaptive immune responses are induction of antigen specific antibody production or antigen specific induction/activation of cellular immune responses.

A "protective, adaptive immune response" is an antigen-specific immune response induced in a subject as a reaction to immunization (artificial or natural) with an antigen, where the immune response is capable of protecting the subject against subsequent challenges with the antigen or a pathology-related agent that includes the antigen. Typically, prophylactic vaccination aims at establishing a protective adaptive immune response against one or several pathogens.

"Stimulation of the immune system" means that a substance or composition of matter exhibits a general, non-specific immunostimulatory effect. A number of adjuvants and putative adjuvants (such as certain cytokines) share the ability to stimulate the immune system. The result of using an immunostimulating agent is an increased "alertness" of the immune system meaning that simultaneous or subsequent immunization with an immunogen induces a significantly more effective immune response compared to isolated use of the immunogen.

A "T-helper lymphocyte response" is an immune response elicited on the basis of a peptide, which is able to bind to an MHC class II molecule (e.g. an HLA class II molecule) in an antigen-presenting cell and which stimulates T-helper lymphocytes in an animal species as a consequence of T-cell receptor recognition of the complex between the peptide and the MHC Class II molecule presenting the peptide. Hybridization under "stringent conditions" is herein defined as hybridization performed under conditions by which a probe will hybridize to its target sequence, to a detectably greater degree than to other sequences. Stringent conditions are target-sequence-dependent and will differ depending on the structure of the polynucleotide. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified which are 100% complementary to a probe (homologous probing) . Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing) . Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution . Generally, stringent wash temperature conditions are selected to be about 5°C to about 2°C lower than the melting point (Tm) for the specific sequence at a defined ionic strength and pH. The melting point, or denaturation, of DNA occurs over a narrow

temperature range and represents the disruption of the double helix into its complementary single strands. The process is described by the temperature of the midpoint of transition, Tm, which is also called the melting temperature. Formulas are available in the art for the determination of melting temperatures.

As used herein, the term "antibody" refers to a polypeptide or group of polypeptides composed of at least one antibody combining site. An "antibody combining site" is the three- dimensional binding space with an internal surface shape and charge distribution

complementary to the features of an epitope of an antigen, which allows a binding of the antibody with the antigen. "Antibody" includes, for example, vertebrate antibodies, hybrid antibodies, chimeric antibodies, humanised antibodies, altered antibodies, univalent antibodies, Fab proteins, and single domain antibodies.

"Specific binding" denotes binding between two substances which goes beyond binding of either substance to randomly chosen substances and also goes beyond simple association between substances that tend to aggregate because they share the same overall

hydrophobicity or hydrophilicity. As such, specific binding usually involves a combination of electrostatic and other interactions between two conformationally complementary areas on the two substances, meaning that the substances can "recognize" each other in a complex mixture.

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 can be replicated and expressed . The term further denotes certain biological vehicles useful for the same purpose, e.g. viral and bacterial vectors - both these infectious agents are capable of introducing a heterologous nucleic acid sequence into a host and effect subsequence expression of a nucleic acid in the host. The term "expression vector" refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, when the transcription product is an mRNA molecule, this is in turn translated into a protein, polypeptide, or peptide. A "chimeric polypeptide" is a polypeptide, which is constituted by amino acid stretches derived from at least two different proteins, where these at least two stretches are fused to each other, optionally via a linker. By nature, a chimeric polypeptide does not occur in nature.

Typical linkers are flexible, meaning that they allow the joint polypeptides in a fusion construct to have a high degree of movement. Such flexible linkers are often rich in small, non-polar amino acid residues (such as glycine residues) but will often incorporate small polar amino acid residues such as serine or threonine residues, too. Such linkers are known as GS linkers.

A "linker" is an amino acid sequence, which is introduced between two other amino acid sequences in order to separate them spatially. Linkers are widely used in recombinant biotechnology and are reviewed in Chen X et al. (2013), Advanced drug delivery reviews 65(10) : 1357-1369. doi : 10.1016/j.addr.2012.09.039. A linker may be "rigid", meaning that it does substantially not allow the two amino acid sequences that it connects to move freely relative to each other. Likewise, a "flexible" linker allows the two sequences connected via the linker to move substantially freely relative to each other. In the fusion proteins, which are part of the present invention, both types of linkers are useful. Linkers of interest are listed in the following table:

Linker Type Amino Acid Sequence

Flexible GSGGGA (SEQ ID NO: 4)

Flexible GSGGGAGSGGGA (SEQ ID NO: 5)

Flexible GSGGGAGSGGGAGSGGGA (SEQ ID NO: 6)

Flexible GSGGGAGSGGGAGSGGGAGSGGGA (SEQ ID NO: 7)

Flexible GENLYFQSGG (SEQ ID NO: 8)

Rigid KPEPKPAPAPKP (SEQ ID NO: 9)

Rigid AEAAAKEAAAKA (SEQ ID NO: 10)

Rigid_ SACYCELS (SEQ ID NO: 11) Specific embodiments of the invention

The of the invention

In some embodiments the at least or exactly 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention constitute at least or exactly or at most 6, such as at least or exactly or at most 7, at least or exactly or at most 8, at least or exactly or at most 9, at least or exactly or at most 10, at least or exactly or at most 11, at least or exactly or at most 12, at least or exactly or at most 13, at least or exactly or at most 14, at least or exactly or at most 15, at least or exactly or at most 16, at least or exactly or at most 17, at least or exactly or at most 18, at least or exactly or at most 19, at least or exactly or at most 20, at least or exactly or at most 21, at least or exactly or at most 22, at least or exactly or at most 23, at least or exactly or at most 24, at least or exactly or at most 25, at least or exactly or at most 26, at least or exactly or at most 27 at least or exactly or at most 28, at least or exactly or at most 29, at least or exactly or at most 30, at least or exactly or at most 31, at least or exactly or at most 32, at least or exactly or at most 33, at least or exactly or at most 34, at least or exactly or at most 35, at least or exactly or at most 36, at least or exactly or at most 37, at least or exactly or at most 38, at least or exactly or at most 39, at least or exactly or at most 40, at least or exactly or at most 41, at least or exactly or at most 42, at least or exactly or at most 43, at least or exactly or at most 44, at least or exactly or at most 45, at least or exactly or at most 46, at least or exactly or at most 47, at least or exactly or at most 48, at least or exactly or at most 49, at least or exactly or at most 50, at least or exactly or at most 51, at least or exactly or at most 52, at least or exactly or at most 53, at least or exactly or at most 54, at least or exactly or at most 55, at least or exactly or at most 56, at least or exactly or at most 57, at least or 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most 142, at least or exactly or at most 143, at least or exactly or at most 144, at least or exactly or at most 145, at least or exactly or at most 146, at least or exactly or at most 147, at least or exactly or at most 148, at least or exactly or at most 149, at least or exactly or at most 150, at least or exactly or at most 151, at least or exactly or at most 152, at least or exactly or at most 153, at least or exactly or at most 154, at least or exactly or at most 155, at least or exactly or at most 156, at least or exactly or at most 157, at least or exactly or at most 158, at least or exactly or at most 159, at least or exactly or at most 160, at least or exactly or at most 161, at least or exactly or at most 162, at least or exactly or at most 163, at least or exactly or at most 164, at least or exactly or at most 165, at least or exactly or at most 166, at least or exactly or at most 167, at least or exactly or at most 168, at least or exactly or at most 169, at least or 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225, at least or exactly or at most 226, at least or exactly or at most 227, at least or exactly or at most 228, at least or exactly or at most 229, at least or exactly or at most 230, at least or exactly or at most 231, at least or exactly or at most 232, at least or exactly or at most 233, at least or exactly or at most 234, at least or exactly or at most 235, at least or exactly or at most 236, at least or exactly or at most 237, at least or exactly or at most 238, at least or exactly or at most 239, at least or exactly or at most 240, at least or exactly or at most 241, at least or exactly or at most 242, at least or exactly or at most 243, at least or exactly or at most 244, at least or exactly or at most 245, at least or exactly or at most 246, at least or exactly or at most 247, at least or exactly or at most 248, at least or exactly or at most 249, at least or exactly or at most 250, at least or exactly or at most 251, at least or exactly or at most 252, at least or exactly or 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exactly or at most 281, at least or exactly or at most 282, at least or exactly or at most 283, at least or exactly or at most 284, at least or exactly or at most 285, at least or exactly or at most 286, at least or exactly or at most 287, at least or exactly or at most 288, at least or exactly or at most 289, at least or exactly or at most 290, at least or exactly or at most 291, at least or exactly or at most 292, at least or exactly or at most 293, at least or exactly or at most 294, at least or exactly or at most 295, at least or exactly or at most 296, at least or exactly or at most 297, at least or exactly or at most 298, at least or exactly or at most 299, at least or exactly or at most 300, at least or exactly or at most 301, at least or exactly or at most 302, at least or exactly or at most 303, at least or exactly or at most 304, at least or exactly or at most 305, at least or exactly or at most 306, at least or exactly or at most 307, at least or exactly or at most 308, at 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336, at least or exactly or at most 337, at least or exactly or at most 338, at least or exactly or at most 339, at least or exactly or at most 340, at least or exactly or at most 341, at least or exactly or at most 342, at least or exactly or at most 343, at least or exactly or at most 344, at least or exactly or at most 345, at least or exactly or at most 346, at least or exactly or at most 347, at least or exactly or at most 348, at least or exactly or at most 349, at least or exactly or at most 350, at least or exactly or at most 351, at least or exactly or at most 352, at least or exactly or at most 353, at least or exactly or at most 354, at least or exactly or at most 355, at least or exactly or at most 356, at least or exactly or at most 357, at least or exactly or at most 358, at least or exactly or at most 359, at least or exactly or at most 360, at least or exactly or at most 361, at least or exactly or at most 362, at least or exactly or at most 363, or exactly or at most 364 contiguous amino acid residues.

Another way to phrase this is that the number of the contiguous amino acid residues from SEQ ID NO: 1 is at least or exactly or at most 365-n, where n is any integer between 1 and 360; that is, the at least 5 contiguous amino acids can be at least any number between 5 and the length of the reference sequence minus one, in increments of one.

In some embodiments, the polypeptide of the invention also has a sequence identity with the amino acid sequence of a) defined above of at least 61%, such as at least 63%, at least 63% at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81,%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%. Similarly, the polypeptide of the invention in some embodiments also has a sequence identity with the amino acid sequence of b) defined above of at least 61%, such as at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81,%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%.

In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least or exactly or at most 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,

25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,

49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,

73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,

97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,

115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,

133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,

151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,

169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186,

187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204,

205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222,

223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240,

241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258,

259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276,

277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294,

295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312,

313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330,

331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342„ 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, and 361 in SEQ ID NO: 1, if the number of the at least or exactly or at most 5 amino acid residues so permit - if the number of the at least or exactly or at most 5 amino acids is higher than 5, the N-terminal first residue will not be higher numbered than 366-L, where L is the number of amino acids defined for option b. For instance, if the number of the contiguous amino acid residues is exactly 60, the N-terminal first residue cannot be higher numbered than 366-60 = 306, meaning that the 60 amino acid residues in that particular case will be constituted by amino acid residues 306-365 of SEQ ID NO: 1. The polypeptide as disclosed in any of the embodiments above may be fused to an amino acid or amino acid sequence. Non-limiting examples of such fusion partners are

1) a methionine residue,

2) a heterologous amino acid sequence that comprises or constitutes a purification tag,

3) a heterologous amino acid sequence that comprises or constitutes an immunogenic carrier molecule or one or more separate T-helper epitopes,

4) a heterologous amino acid sequence that exerts adjuvant activity; and

5) any combination of 1-5.

If the polypeptide disclosed above is fused to a heterologous amino acid sequence, this fusion may be direct or via a suitable peptide linker, cf. the discussion of linkers above.

The polypeptide of the invention is in certain embodiments also covalently linked (i.e. fused or conjugated) to an immunogenic carrier molecule; or, phrased otherwise, the polypeptide of the invention also includes such an immunogenic carrier molecule in addition to the polypeptides of the present invention. The immunogenic carrier molecule is a typically polypeptide that induces T-helper lymphocyte responses in a majority of humans, such as immunogenic carrier proteins selected from the group consisting of keyhole limpet hemocyanin or a fragment thereof, tetanus toxoid or a fragment thereof, diphtheria toxoid or a fragment thereof. Other suitable carrier molecules are discussed infra. One further fusion partner, which is preferably incorporated is a "His tag", i.e. a stretch of amino acids, which is rich or only consists of histidinyl residues so as to facilitate protein purification.

In preferred embodiments, the polypeptide of the invention detailed above is capable of inducing an adaptive immune response against the polypeptide in a mammal, in particular in a human being. Preferably, the adaptive immune response is a protective adaptive immune response against infection with S. aureus, in particular multi-resistant S. aureus. The polypeptide may in these cases induce a humeral and/or a cellular immune response.

SEQ ID NO: 1 includes antigenic determinants (epitopes) that are as such recognized by antibodies and/or when bound to MHC molecules by T-cell receptors. For the purposes of the present invention, B-cell epitopes (i.e. antibody binding epitopes) are of particular relevance.

It is relatively uncomplicated to identify linear B-cell epitopes - one very simple approach entails that antibodies raised against S. aureus or S. aureus derived proteins disclosed herein are tested for binding to overlapping oligomeric peptides derived from SEQ ID NO: 1. Thereby, the regions of the S. aureus polypeptide which are responsible for or contribute to binding to the antibodies can be identified.

Alternatively, or additionally, one can produce mutated versions of the polypeptides of the invention, e.g. version where each single non-alanine residue in SEQ ID NO: 1 are point mutated to alanine - this method also assists in identifying complex assembled B-cell epitopes; this is the case when binding of the same antibody is modified by exchanging amino acids in different areas of the full-length polypeptide.

Also, in silico methods for B-cell epitope prediction can be employed : useful state-of-the-art systems for b-turn prediction is provided in Petersen B et al. (November 2010), Plos One 5(11) : el5079; prediction of linear B-cell epitopes, cf. : Larsen J E P et al. (April 2006), Immunome Research, 2:2; prediction of solvent exposed amino acids: Petersen B et al (July 2009), BMC Structural Biology, 9: 51.

Nucleic acid fragments of the invention; 2^nd aspect

The nucleic acid fragment of the invention referred to above preferably is a DNA fragment or an RNA fragment. Exemplary DNA fragments are provided as SEQ ID NO: 2 (DNA encoding SEQ ID NOs: 1, i.e. an exemplary polypeptide of the first aspect of the invention) and as SEQ ID NO: 3 (RNA encoding SEQ ID NO: 1). Also the complimentary sequences are embraced by the present invention.

Since the presently disclosed polypeptides can be encoded by a plethora of nucleic acid sequences due to the degeneracy of the genetic code, the skilled person will understand that none single nucleic acid sequence is particularly preferred as long as it encodes a polypeptide of the present invention. Rather, the skilled person will design suitable coding sequences that are codon optimised with respect to e.g. the expression system wherein recombinant production of the polypeptide is to take place. However, as a suitable starting point, the native DNA and RNA sequences are provided as SEQ ID NOs: 2 and 3.

It will be understood that nucleic acids that encode the polypeptide of the invention can have a sequence identity with the nucleotide sequence SEQ ID NO: 1 or 2 of at least 60%, such as at least 65%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%. 3^rd aspect - vectors of the invention

It will be understood that the nucleic acid fragments of the invention may be used for both production, carrier and vaccine purposes - the latter will require that the sequences are included in expression vectors that may lead to production of immunogenic proteins in the mammal receiving the vector. Or put differently, the nucleic acid is comprised in a vector capable of expressing the nucleic acid in man upon administration.

Such a vector of the invention often comprises in operable linkage and in the 5'-3' direction, an expression control region comprising an enhancer/promoter for driving expression of the nucleic acid, an optional signal peptide coding sequence, a nucleotide sequence of the invention, and optionally a terminator. Hence, such a vector constitutes an expression vector useful for effecting production in cells of the polypeptide of the invention. Since the polypeptides of the invention are protozoan of origin, recombinant production has to be effected in host cells that can express the coding nucleic acid. Bacterial host cells may be used. However, if the vector is to drive expression in eukaryotic cell (as would be the case for a DNA or RNA vaccine vector), the expression control region should be adapted to this particular use.

For production purposes it is therefore often convenient that the expression control region drives expression in a prokaryotic cell such as a bacterium, e.g. in E. coti, or in a eukaryotic cell such as a plant cell, an insect cell, or a mammalian cell. For vaccine purposes, the expression control region has to be able to drive expression in a mammalian, preferably human, cell.

Also, for production purposes, it is practical that the vector is capable of integrating the nucleic acid into the genome of a host cell - this is particularly useful if the vector is use in the production of stably transformed cells, where the progeny will also include the genetic information introduced via the vector. Alternatively, vectors incapable of being integrated into the genome of a piscine host cell are useful in e.g. nucleic acid vaccination.

An interesting production system is the use of plants. For instance, proteins can be produced at low cost in plants using an Agrobacterium transfection system to genetically modify plants to express genes that encode the protein of interest. One commercially available platform are those provided by i Bio CMO LLC (8800 HSC Pkwy, Bryan, TX 77807, USA) and i Bio, Inc (9 Innovatoin Way, Suite 100, Newark, DE 19711, USA) and disclosed in e.g. EP 2 853 599, EP 1 769 068, and EP 2 192 172. Hence, in such systems the vector is an Agrobacterium vector or other vector suitable for transfection of plants. The vector is typically selected from the group consisting of a virus, such as a virus which is non-pathogenic in mammals and in particular in humans, a bacterium such as a bacterium which is non-pathogenic in mammals such as humans, a plasmid, a minichromosome, and a cosmid.

Interesting vectors are viral vectors (in particular those useful as vaccine agents in humans). These may be selected from the group consisting of a retrovirus vector, such as a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, and a pox virus vector.

Certain pox virus vectors are preferred, in particular vaccinia virus vectors. A particularly preferred vaccinia virus vector is a modified vaccinia Ankara (MVA) vector.

Polypeptides of the invention may as indicated be encoded by a nucleic acid molecule comprised in a vector. A nucleic acid sequence can be "heterologous," which means that it is in a context foreign to the cell in which the vector is being introduced, which includes a sequence homologous to a sequence in the cell but in a position within the host cell where it is ordinarily not found.

Vectors include naked DNAs, RNAs, plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). One of skill in the art would be well equipped to construct a vector through standard recombinant techniques. In addition to encoding the polypeptides of this invention, a vector of the present invention may encode polypeptide sequences such as a "tag" or immunogenicity enhancing peptide (e.g. an immunogenic carrier or a fusion partner that stimulates the immune system, such as a cytokine or active fragment thereof). Useful vectors encoding such fusion proteins include pIN vectors, vectors encoding a stretch of histidines, and pGEX vectors, for use in generating glutathione S-transferase (GST) soluble fusion proteins for later purification and separation or cleavage.

Vectors of the invention may be used in a host cell to produce a polypeptide of the invention that may subsequently be purified for administration or the vector may be purified for direct administration for expression of the protein (as is the case when administering a nucleic acid vaccine).

Expression vectors can contain a variety of "control sequences," which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described infra. 1. Promoters and Enhancers

A "promoter" is a control sequence. The promoter is typically a region of a nucleic acid sequence at which initiation and rate of transcription are controlled . It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors. The phrases "operatively positioned," "operatively linked," "under control," and "under transcriptional control" mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and expression of that sequence. A promoter may or may not be used in conjunction with an "enhancer," which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

A promoter may be one naturally associated with a gene or sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment or exon. Such a promoter can be referred to as "endogenous." Similarly, an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally 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 cell, and promoters or enhancers not "naturally occurring," i .e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including polymerase chain reaction in connection with the compositions disclosed herein.

It may be important to employ a promoter and/or enhancer that effectively direct(s) the expression of the DNA segment in the cell type or organism chosen for expression. Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression. The promoters employed may be constitutive, tissue-specific, or inducible and in certain embodiments may direct high level expression of the introduced DNA segment under specified conditions, such as large-scale production of recombinant proteins or peptides.

Examples of inducible elements, which are regions of a nucleic acid sequence that can be activated in response to a specific stimulus, include but are not limited to Immunoglobulin Heavy Chain, Immunoglobulin Light Chain, T Cell Receptor, HLA DQa and/or DQ , b- Interferon, Interleukin-2, Interleukin-2 Receptor, MHC Class II 5, MHC Class II HLA-DRa, b- Actin, Muscle Creatine Kinase (MCK), Prealbumin (Transthyretin), Elastase I, Metallothionein (MTII), Collagenase, Albumin, a-Fetoprotein, y-Globin, b-Globin, c-fos, c-HA-ras, Insulin, Neural Cell Adhesion Molecule (NCAM), al-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, Polyoma, Retroviruses, Papilloma Virus, Hepatitis B Virus, Human Immunodeficiency Virus, Cytomegalovirus (CMV) IE, and Gibbon Ape Leukemia Virus.

Inducible Elements include MT II - Phorbol Ester (TFA)/Heavy metals; MMTV (mouse mammary tumor virus) - Glucocorticoids; b-Interferon - poly(rl)x/poly(rc); Adenovirus 5 E2 - EIA; Collagenase - Phorbol Ester (TPA); Stromelysin - Phorbol Ester (TPA); SV40 - Phorbol Ester (TPA); Murine MX Gene - Interferon, Newcastle Disease Virus; GRP78 Gene - A23187; a-2-Macroglobulin - IL-6; Vimentin - Serum; MHC Class I Gene H-2k±> - Interferon; HSP70 - E1A/SV40 Large T Antigen; Proliferin - Phorbol Ester/TPA; Tumor Necrosis Factor - PMA; and Thyroid Stimulating Hormone a Gene - Thyroid Hormone.

Also contemplated as useful in the present invention are the dectin-1 and dectin-2 promoters. Additionally any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of structural genes encoding oligosaccharide processing enzymes, protein folding accessory proteins, selectable marker proteins or a heterologous protein of interest.

The particular promoter that is employed to control the expression of peptide or protein encoding polynucleotide of the invention is not believed to be critical, so long as it is capable of expressing the polynucleotide in a targeted cell. Where a piscine cell is targeted (as is the case in nucleic acid vaccination), it is preferable to position the polynucleotide coding region adjacent to and under the control of a promoter that is capable of being expressed in a piscine cell. Generally speaking, such a promoter might include either a bacterial, piscine or viral promoter as long as the promoter is effective in piscine cells.

In various embodiments - in particular those where recombinant production of the polypeptide of the invention is the aim - the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, and the Rous sarcoma virus long terminal repeat can be used to obtain high level expression of a related polynucleotide to this invention. The use of other viral or mammalian cellular or bacterial phage promoters, which are well known in the art, to achieve expression of polynucleotides is contemplated as well. In embodiments in which a vector is administered to humans for expression of the protein, it is contemplated that a desirable promoter for use with the vector is one that is not down- regulated by cytokines or one that is strong enough that even if down-regulated, it produces an effective amount of the protein/polypeptide of the current 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. The MHC I promoter increases expression in the presence of IFN-y.

Tissue specific promoters can be used, particularly if expression is in cells in which expression of an antigen is desirable, such as dendritic cells and macrophages. The mammalian MHC I and MHC II promoters are examples of such tissue-specific promoters in man and it is contemplated that corresponding piscine promoters will be effective.

2. Initiation Signals and Internal Ribosome Binding Sites (IRES)

A specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous

translational control signals, including the ATG initiation codon, may need to be provided.

One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be "in-frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. The exogenous translational control signals and initiation codons can be either natural or synthetic and may be operable in bacteria or mammalian cells. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements.

In certain embodiments of the invention, the use of internal ribosome entry sites (IRES) elements are used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap dependent translation and begin translation at internal sites. IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) have been described, as well an IRES from a mammalian message. IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating

polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message (see U.S. Patents 5,925,565 and 5,935,819, herein incorporated by reference). 3. Multiple Cloning Sites

Vectors can include a multiple cloning site (MCS), which is a nucleic acid region that contains multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant technology to digest the vector. Frequently, a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to enable exogenous sequences to be ligated to the vector. Techniques involving restriction enzymes and ligation reactions are well known to those of skill in the art of recombinant technology.

4. Splicing Sites

Most transcribed eukaryotic RNA molecules will undergo RNA splicing to remove introns from the primary transcripts. If relevant in the context of vectors of the present invention, vectors containing genomic eukaryotic sequences may require donor and/or acceptor splicing sites to ensure proper processing of the transcript for protein expression.

5. Termination Signals

The vectors or constructs of the present invention will generally comprise at least one termination signal. A "termination signal" or "terminator" is comprised of the DNA sequences involved in specific termination of an RNA transcript by an RNA polymerase. Thus, in certain embodiments a termination signal that ends the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve desirable message levels.

In eukaryotic systems, the terminator region may also comprise specific DNA sequences that permit site-specific cleavage of the new transcript so as to expose a polyadenylation site.

This signals a specialized endogenous polymerase to add a stretch of about 200 A residues (a "poly(A) tail") to the 3' end of the transcript. RNA molecules modified with this poly(A) tail appear to more stable and are translated more efficiently. Thus, in other embodiments involving eukaryotes, it is preferred that that terminator comprises a signal for the cleavage of the RNA, and it is more preferred that the terminator signal promotes polyadenylation of the RNA.

Terminators contemplated for use in the invention include any known terminator of transcription described herein or known to one of ordinary skill in the art, including but not limited to, for example, the bovine growth hormone terminator or viral termination sequences, such as the SV40 terminator. In certain embodiments, the termination signal may be a lack of transcribable or translatable sequence, such as due to a sequence truncation. 6. Polyadenylation Signals

In expression, particularly eukaryotic expression (as is relevant in nucleic acid vaccination), one will typically include a polyadenylation signal to effect proper polyadenylation of the transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and/or any such sequence may be employed. Preferred embodiments include the SV40 polyadenylation signal and/or the bovine growth hormone polyadenylation signal, convenient and/or known to function well in various target cells. Polyadenylation may increase the stability of the transcript or may facilitate cytoplasmic transport. Consequently, the corresponding encoded RNA fragment preferably comprises a poly(A) tail. In particular for nucleic acid vaccination purposes, it is preferred that the DNA fragments of the invention include a polyadenylation signal and that the RNA fragments include a poly(A) tail.

7. Origins of Replication

In order to propagate a vector in a host cell, it may contain one or more origins of replication sites (often termed "on"), which is a specific nucleic acid sequence at which replication is initiated. Alternatively an autonomously replicating sequence (ARS) can be employed if the host cell is yeast.

8. Selectable and Screenable Markers

In certain embodiments of the invention, cells containing a nucleic acid construct of the present invention may be identified in vitro or in vivo by encoding a screenable or selectable marker in the expression vector. When transcribed and translated, a marker confers an identifiable change to the cell permitting easy identification of cells containing the expression vector. Generally, a selectable marker is one that confers a property that allows for selection. A positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection. An example of a positive selectable marker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning and identification of transformants, for example, markers that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin or histidinol are useful selectable markers. In addition to markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions, other types of markers including screenable markers such as GFP for colorimetric analysis. Alternatively, screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized. One of skill in the art would also know how to employ immunologic markers that can be used in conjunction with FACS analysis. The marker used is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a protein of the invention. Further examples of selectable and screenable markers are well known to one of skill in the art.

The transformed cells of the invention - 4^th aspect

Transformed cells of the invention are useful as organisms for producing the polypeptide of the invention, but also as simple "containers" of nucleic acids and vectors of the invention. Also, the transformed cells find use as immunization and vaccine agents.

Certain transformed cells of the invention are capable of replicating the nucleic acid fragment of the 2^nd aspect of the invention. Preferred transformed cells of the invention are capable of expressing the nucleic acid fragment.

For recombinant production it is convenient, but not a prerequisite that the transformed cell according is prokaryotic, such as a bacterium, but generally both prokaryotic cells and eukaryotic cells may be used.

Suitable prokaryotic cells are bacterial cells selected from the group consisting of Escherichia (such as E. coli ), Bacillus (e.g. Bacillus subtiHs), Salmonella, and Mycobacterium (preferably non-pathogenic, e.g. M. bovis BCG). Generally, if prokaryotic cells are to be used as vaccine agents, they should be non-pathogenic and/or attenuated.

Eukaryotic cells can be in the form of yeasts (such as baker's yeast, Saccharomyces cerevisiae) and protozoans. Alternatively, the transformed eukaryotic cells are derived from a multicellular organism such as a fungus (typically filamentous), an insect cell, a plant cell, or a mammalian cell.

For production purposes, it is advantageous that the transformed cell of the invention is is stably transformed by having the nucleic acid defined above for option i) stably integrated into its genome, and in certain embodiments it is also preferred that the transformed cell secretes or carries on its surface the polypeptide of the invention, since this facilitates recovery of the polypeptides produced. A particular version of this embodiment is one where the transformed cell is a bacterium and secretion of the polypeptide of the invention is into the periplasmic space. As noted above, stably transformed cells are preferred - these i.a. allows that cell lines comprised of transformed cells as defined herein may be established - such cell lines are particularly preferred aspects of the invention.

Further details on cells and cell lines are presented in the following :

Suitable cells for recombinant nucleic acid expression of the nucleic acid fragments of the present invention are prokaryotes and eukaryotes. Examples of prokaryotic cells include E. coli ; members of the Staphylococcus genus, such as S. epidermidis ; members of the

Lactobacillus genus, such as L. plantarunrr, members of the Lactococcus genus, such as L. lactis ; members of the Bacillus genus, such as B. subtilis ; members of the Corynebacterium genus such as C. glutamicuirr, and members of the Pseudomonas genus such as Ps.

fluorescens. Examples of eukaryotic cells include mammalian cells; insect cells; yeast cells such as members of the Saccharomyces genus (e.g. S. cerevisiae) , members of the Pichia genus (e.g. P. pastohs), members of the Hansenula genus (e.g. H. polymorpha ), members of the Kluyveromyces genus (e.g. K. lactis or K. fragilis ) and members of the

Schizosaccharomyces genus (e.g. S. pombe).

Techniques for recombinant gene production, introduction into a cell, and recombinant gene expression are well known in the art. Examples of such techniques are provided in references such as Current Protocols in Molecular Biology, John Wiley, ISSN 1934-3647, and Green MR and Sambrook J et a/., Molecular Cloning, A Laboratory Manual, 4^th Edition, Cold Spring Harbor Laboratory Press, 2012, ISBN 978-1-936113-41-5 and ISBN 978-1-936113-42-2.

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

Host cells may be derived from prokaryotes or eukaryotes, including bacteria, yeast cells, insect cells, and mammalian cells for replication of the vector or expression of part or all of the nucleic acid sequence(s). Numerous cell lines and cultures are available for use as a host cell, and they can be obtained through the American Type Culture Collection (ATCC), which is an organization that serves as an archive for living cultures and genetic materials

( www.atcc.org^') or from other depository institutions such as Deutsche Sammlung vor Micrroorganismen und Zellkulturen (DSM). An appropriate host can be determined by one of skill in the art based on the vector backbone and the desired result. A plasmid or cosmid, for example, can be introduced into a prokaryote host cell for replication of many vectors or expression of encoded proteins. Bacterial cells used as host cells for vector replication and/or expression include Staphylococcus strains, DH5a, JMI 09, and KC8, as well as a number of commercially available bacterial hosts such as SURE(R) Competent Cells and SOLOP ACK(TM) Gold Cells (STRATAGENE®, La Jolla, CA). Alternatively, bacterial cells such as E. coli LE392 could be used as host cells for phage viruses. Appropriate yeast cells include Saccharomyces cerevisiae, Saccharomyces pombe, and Pichia pastohs.

Examples of eukaryotic host cells for replication and/or expression of a vector include HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12. Many host cells from various cell types and organisms are available and would be known to one of skill in the art. Similarly, a viral vector may be used in conjunction with either a eukaryotic or prokaryotic host cell, particularly one that is permissive for replication or expression of the vector.

Some vectors may employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells. One of skill in the art would further understand the conditions under which to incubate all of the above described host cells to maintain them and to permit replication of a vector. Also understood and known are techniques and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides.

Expression Systems

Numerous expression systems exist that comprise at least a part or all of the compositions discussed above. Prokaryote- and/or eukaryote-based systems can be employed for use with the present invention to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Many such systems are commercially and widely available.

The insect cell/baculovirus system can produce a high level of protein expression of a heterologous nucleic acid segment, such as described in U.S. Patents 5,871,986, 4,879,236, both herein incorporated by reference, and which can be bought, for example, under the name MAXBAC® 2.0 from INVITROGEN® and BACPACK™ Baculovirus expression system from CLONTECH® In addition to the disclosed expression systems of the invention, other examples of expression systems include STRATAGENE®'s COMPLETE CONTROL™ Inducible Mammalian Expression System, which involves a synthetic ecdysone-inducible receptor, or its pET Expression System, an E. coli expression system. Another example of an inducible expression system is available from INVITROGEN® , which carries the T-REX™ (tetracycline-regulated expression) System, an inducible mammalian expression system that uses the full-length CMV promoter. INVITROGEN® also provides a yeast expression system called the Pichia methanolica Expression System, which is designed for high-level production of recombinant proteins in the methylotrophic yeast Pichia methanolica. One of skill in the art would know how to express a vector, such as an expression construct, to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide.

Methods of Gene Transfer

Suitable methods for nucleic acid delivery to effect expression of compositions of the present invention are believed to include virtually any method by which a nucleic acid (e.g., DNA, including viral and nonviral vectors) can be introduced into a cell, a tissue or an organism, as described herein or as would be known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA such as by injection (U.S. Patents 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. Patent 5,789,215); by electroporation (U.S. Patent 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 microprojectile bombardment (PCT Application Nos. WO 94/09699 and 95/06128; U.S.

Patents 5,610,042; 5,322,783 5,563,055, 5,550,318, 5,538,877 and 5,538,880); by agitation with silicon carbide fibers (U.S. Patents 5,302,523 and 5,464,765); by

Agrobacterium mediated transformation (U.S. Patents 5,591,616 and 5,563,055); or by PEG mediated transformation of protoplasts (U.S. Patents 4,684,611 and 4,952,500); by desiccation/inhibition mediated DNA uptake. Through the application of techniques such as these, organelle(s), cell(s), tissue(s) or organism(s) may be stably or transiently

transformed.

Recently, the development of RNA vaccines has shown great promise. Hence technology for RNA vaccine delivery and expression are within the ambit of the present application.

Generally the teachings provided in Deering R.P. et a/., Expert Opin Drug Deliv. 2014 Jun; ll(6) :885-99 can be followed in order to effect vaccination with RNA. Compositions of the invention : vaccines

Compositions, in particular vaccines, according to the invention are prophylactic but may also be used therapeutically.

Such vaccines comprise immunising antigen(s), immunogen(s), polypeptide(s), protein(s) or nucleic acid(s), usually in combination with "pharmaceutically acceptable carriers", which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition.

In some embodiments of the invention, the pharmaceutical compositions such as vaccines include merely one single antigen, immunogen, polypeptide, protein, nucleic acid or vector of the invention, but in other embodiments, the pharmaceutical compositions comprise

"cocktails" of the antigens or of the immunogens or of the polypeptides or of the protein or of the nucleic acids or of the vectors of the invention.

In particularly interesting embodiments, the pharmaceutical composition is a vector mentioned herein, which encodes and can effect expression of at least 2 nucleic acid fragments of the invention.

Another interesting embodiment of a pharmaceutical composition comprises RNA as the active principle, i .e. at least one mRNA encoding a polypeptide of the invention.

An embodiment of a pharmaceutical composition of the invention at least 2 (such as 2, 3, 4, 5,6, 7, 8, 9, or 10) distinct polypeptides of the invention described above.

Another embodiment of the pharmaceutical composition of the invention comprises at least 2 (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10) distinct nucleic acid molecules (such as DNA and RNA) each encoding a polypeptide of the invention.

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

Such carriers are well known to those of ordinary skill in the art. Additionally, these carriers may function as immunostimulating agents ("adjuvants") . Furthermore, the antigen or immunogen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H . pylori, etc. pathogen, cf. the description of immunogenic carriers supra. The pharmaceutical compositions of the invention thus typically contain an immunological adjuvant, which is commonly an aluminium based adjuvant or one of the other adjuvants described in the following :

Preferred adjuvants to enhance effectiveness of the composition include, but are not limited to: (1) aluminium salts (alum), such as aluminium hydroxide, aluminium phosphate, aluminium sulphate, etc. ; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59 (WO 90/14837; Chapter 10 in Vaccine design: the subunit and adjuvant approach, eds. Powell & Newman, Plenum Press 1995), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE (see below), although not required) formulated into submicron particles using a microfluidizer such as Model HOY microfluidizer (Microfluidics, Newton, MA), (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP (see below) either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, 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 from the group consisting of monophosphoryl lipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL + CWS (DetoxTM) ; (3) saponin adjuvants such as Stimulon™ (Cambridge Bioscience, Worcester, MA) may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes); (4)

Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (5) cytokines, such as interleukins (eg. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (eg.

gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc. ; and (6) other substances that act as immunostimulating agents to enhance the effectiveness of the composition. Alum and MF59™ adjuvants are preferred together with CFA and IFA.

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

Another possibility for a polypeptide vaccine formulation is to include the vaccine

polypeptide(s) of the present invention in a virus-like particle, i.e. a non-infectious self assembling structure composed of envelope or capsid proteins, where the protein(s) of the invention are incorporated. The effect is multiple presentations of the polypeptides of the invention on the surface of the VLP, which in turn provides for improved immune recognition of the polypeptides. Hence, VLPs exert immunological adjuvant effects, too. The immunogenic compositions (e.g. the immunising antigen or immunogen or polypeptide or protein or nucleic acid, pharmaceutically acceptable carrier, and adjuvant) typically will contain diluents, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.

Typically, the immunogenic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect, as discussed above under pharmaceutically acceptable carriers.

Immunogenic compositions used as vaccines comprise an immunologically effective amount of the antigenic or immunogenic polypeptides, as well as any other of the above-mentioned components, as needed. By "immunologically effective amount", it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated (eg . non-human primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies or generally mount an immune response, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount of immunogen will fall in a relatively broad range that can be determined through routine trials. However, for the purposes of protein vaccination, the amount administered per immunization is typically in the range between 0.5 pg and 500 mg (however, often not higher than 5,000 pg) . The amount of polypeptide of the invention can therefore be between 1 and 400 pg, between 2 and 350 pg, between 4 and 300 pg, between 5 and 250 pg, and between 10 and 200 pg . Hence, the composition will typically contain between 0.1-500 pg of protein of the invention per g of vaccine composition.

The immunogenic compositions are conventionally administered parenterally, eg, by injection, either subcutaneously, intramuscularly, or transdermally/transcutaneously (eg . W0 98/20734) . Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications. In the case of nucleic acid vaccination, also the intravenous or intraarterial routes may be applicable.

Dosage treatment may be a single dose schedule or a multiple dose schedule. The vaccine may be administered in conjunction with other immunoregulatory agents. As an alternative to protein-based vaccines, DNA vaccination (also termed nucleic acid vaccination or gene vaccination) may be used (eg. Robinson & Torres (1997) Seminars in Immunol 9: 271-283; Donnelly et al. (1997) Annu Rev Immunol 15 : 617-648).

A further aspect of the invention is as mentioned above the recognition that combination vaccines can be provided, wherein 2 or more polypeptide antigens disclosed herein and in the above-referenced filings by the present assignee are combined to enhance the immune response by the vaccinated individual, including to optimize initial immune response and duration of immunity. For the purposes of this aspect of the invention, multiple antigenic fragments derived from the same, longer protein can also be used, such as the use of a combination of different lengths of polypeptide sequence fragments from one protein.

Thus, embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising at least 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein.

In addition to this, the present invention also relates to immunogenic compositions that include a polypeptide of the present invention derived from SEQ ID NO: 1 (i.e. being one of the fragments or sequence variants disclosed herein) or being identical to SEQ ID NO: 1 in combination with any one of the polypeptides disclosed as SEQ ID NOs: 1-19 in WO

2012/136653 or with any one of the fragments and variants of these polypeptides disclosed on pages 9-18 in WO 2012/136653.

Also, the present invention also relates to immunogenic compositions that include a polypeptide of the present invention derived from SEQ ID NO: 1 (i.e. being one of the fragments or sequence variants disclosed herein) or being identical to SEQ ID NO: 1 in combination with any one of the polypeptides disclosed as SEQ ID NOs: 1-16 and 49 in WO 2015/082536 or with any of the fragments and variants of these polypeptides disclosed on pages 10-25 in WO 2015/082536.

Further, the present invention also relates to immunogenic compositions that include a polypeptide of the present invention derived from SEQ ID NO: 1 (i.e. being one of the fragments or sequence variants disclosed herein) or being identical to SEQ ID NO: 1 in combination with any one of the polypeptides disclosed as SEQ ID NOs: 1-2 in WO

2017/144523 or with any of the fragments and variants of these polypeptides disclosed on pages 10-19 in WO 2017/144523.

Moreover, the present invention also relates to immunogenic compositions that include a polypeptide of the present invention derived from SEQ ID NO: 1 (i.e. being one of the fragments or sequence variants disclosed herein) or being identical to SEQ ID NO: 1 in combination with any one of the chimeric polypeptides disclosed in PCT/EP2017/068694, in particular those chimeric polypeptides disclosed on pages 10-46 and defined in the claims in PCT/EP2017/068694.

The present invention also relates to immunogenic compositions that include a nucleic acid that encodes and is capable of effecting expression of a (poly)peptide of the present invention derived from SEQ ID NO: 1 (i.e. the (poly)peptide being one of the fragments or sequence variants disclosed herein) or being identical to SEQ ID NO: 1, in combination with a nucleic acid encoding and being capable of effecting expression of 1) any one of the polypeptides disclosed as SEQ ID NOs: 1-19 in WO 2012/136653 or 2) any one of the fragments and variants of these polypeptides disclosed on pages 9-18 in WO 2012/136653. Such a nucleic acid is typically an expression vector, either DNA-based or RNA-based. It is also within the ambit of the present invention that the 2 nucleic acids may be fused and part of one single nucleic acid that is capable of expressing two different immunogens or one single immunogen which constitutes a fusion protein. Thus the "combination of nucleic acids" can be in the form of separate nucleic acids that each encode a (poly)peptide or it can be in the form of a single nucleic acid that includes coding sequences from both of the

(poly)peptides.

The present invention also relates to immunogenic compositions that include a nucleic acid that encodes and is capable of effecting expression of a (poly)peptide of the present invention derived from SEQ ID NO: 1 (i.e. the (poly)peptide being one of the fragments or sequence variants disclosed herein) or being identical to SEQ ID NO: 1, in combination with a nucleic acid encoding and being capable of effecting expression of 1) any one of the polypeptides disclosed as SEQ ID NOs: 1-16 and 49 in WO 2015/082536 or 2) any one of the fragments and variants of these polypeptides disclosed on pages 10-25 in WO 2015/082536. Such a nucleic acid is typically an expression vector, either DNA-based or RNA-based. It is also within the ambit of the present invention that the 2 nucleic acids may be fused and part of one single nucleic acid that is capable of expressing two different immunogens or one single immunogen which constitutes a fusion protein. Thus the "combination of nucleic acids" can be in the form of separate nucleic acids that each encode a (poly)peptide or it can be in the form of a single nucleic acid that includes coding sequences from both of the

(poly)peptides.

The present invention also relates to immunogenic compositions that include a nucleic acid that encodes and is capable of effecting expression of a (poly)peptide of the present invention derived from SEQ ID NO: 1 (i.e. the (poly)peptide being one of the fragments or sequence variants disclosed herein) or being identical to SEQ ID NO: 1, in combination with a nucleic acid encoding and being capable of effecting expression of 1) any one of the polypeptides disclosed as SEQ ID NOs: 1-2 in WO 2017/144523 or 2) any one of the fragments and variants of these polypeptides disclosed on pages 10-19 in WO 2017/144523. Such a nucleic acid is typically an expression vector, either DNA-based or RNA-based. It is also within the ambit of the present invention that the 2 nucleic acids may be fused and part of one single nucleic acid that is capable of expressing two different immunogens or one single immunogen which constitutes a fusion protein. Thus the "combination of nucleic acids" can be in the form of separate nucleic acids that each encode a (poly)peptide or it can be in the form of a single nucleic acid that includes coding sequences from both of the

(poly)peptides.

The present invention also relates to immunogenic compositions that include a nucleic acid that encodes and is capable of effecting expression of a (poly)peptide of the present invention derived from SEQ ID NO: 1 (i.e. the (poly)peptide being one of the fragments or sequence variants disclosed herein) or being identical to SEQ ID NO: 1, in combination with a nucleic acid encoding and being capable of effecting expression of 1) any one of the chimeric polypeptides disclosed in the claims of PCT/EP2017/068694 as well as on pages 10-42 in PCT/EP2017/068694. Such a nucleic acid is typically an expression vector, either DNA-based or RNA-based. It is also within the ambit of the present invention that the 2 nucleic acids may be fused and part of one single nucleic acid that is capable of expressing two different immunogens or one single immunogen which constitutes a fusion protein. Thus the

"combination of nucleic acids" can be in the form of separate nucleic acids that each encode a (poly)peptide or it can be in the form of a single nucleic acid that includes coding sequences from both of the (poly)peptides.

The compositions may in the above cases comprise a cocktail of several proteins or nucleic acids disclosed in the patent applications discussed above.

The disclosures of WO 2012/136653, WO 2015/082536, WO 2017/144523, and

PCT/EP2017/068694 are for these reasons incorporated in their entirety by reference herein.

Immunization methods

The method of this aspect of the invention generally relates to induction of immunity and as such also entails methods that are prophylactic as well as therapeutic.

When immunization methods entail that a polypeptide of the invention or a composition comprising such a polypeptide is administered the animal (e.g. the human) typically receives between 0.5 and 5,000 pg of the polypeptide of the invention per administration, cf. the above indications concerning dosages.

In preferred embodiments, the immunization scheme includes that the a primary

administration of the polypeptide(s), the nucleic acids/vectors, or the composition(s) of the invention, but it may be necessary to follow up with one or more booster administrations.

Preferred embodiments comprise that the administration is for 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 attracting infection with S. aureus.

As mentioned herein, the some vaccines of the invention induce humoral immunity, so it is preferred that the administration is for the purpose of inducing antibodies specific for S aureus.

But, as also mentioned the immunization method may also be useful in antibody production, so in other embodiments the administration is for the purpose of inducing antibodies specific for S. aureus wherein B-lymphocytes producing said antibodies are subsequently recovered from the animal and used for preparation of monoclonal antibodies.

Compositions for immunization can as mentioned above comprise polypeptides, nucleic acids, or vectors of the invention. The pharmaceutical compositions will comprise a therapeutically effective amount thereof.

The term "therapeutically effective amount" or "prophylactically effective amount" as used herein refers to an amount of a therapeutic agent to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable preventative effect in a group of mammals such as humans The effect can be detected by, for example, chemical markers or antigen levels. Reference is made to the ranges for dosages of immunologically effective amounts of polypeptides, cf. above. However, the effective amount for a given situation can be determined by routine experimentation and is within the judgement of the clinician.

For purposes of the present invention, an effective dose will be from about 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the DNA constructs in the animal to which it is administered.

A pharmaceutical composition can also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be

administered without undue toxicity. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art.

Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's

Pharmaceutical Sciences (Mack Pub. Co., N. J. 1991) .

Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol . Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. Typically, the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared . Liposomes are included within the definition of a

pharmaceutically acceptable carrier.

EXAMPLE

Testing of immunogens of the present invention in a murine model of peritonitis

Female NMRI mice were immunized with recombinant polypeptide in combination with the adjuvant aluminium hydroxide (Alhydrogel®, Brenntag, Cat. no. 21645-51-2) and Freund's incomplete adjuvant. In experiment 1, 20 mice were administered the vaccine, in experiment 2, only 8 mice received the vaccinations. As control, the adjuvant alone was administered . Each mouse was immunized subcutaneously three times at approximately two week intervals. At each immunization the mice were immunized with a formulation of 100

Alhydrogel adjuvant mixed with 25 pg polypeptide followed by addition of an equal volume of Freund's incomplete adjuvant. Only aluminium hydroxide was used as adjuvant in the following immunizations (100 pi mixed with 25 pg polypeptide) .

Blood samples were collected from each animal approximately ten days after the last immu nization for analysis of antibody titre. Four days prior to challenge, temperature transponders (BMDS, cat. no. IPTT-300) were inserted into each mouse. The mice were briefly anaesthetized by inhalation of isoflurane, and a temperature transponder inserted underneath the skin on the lower back or side of the mouse. Using a compatible wireless scanner (BMDS Smart Probe; BMDS, cat. no. DAS- 7007s) body temperature could be registered when placing the scanner close to the transponders underneath the skin of the mouse.

Preparation of bacterial inoculum

The bacteria used in the animal model of peritonitis were prepared in advance and frozen at -80°C in aliquots; bacterial matter was streaked out on a blood agar plate and incubated at 37°C overnight. The following day, a single colony of S. aureus was used for the inoculation of 30 mL tryptic soy broth (TSB) media. The culture was incubated overnight at 37°C, with continuous shaking. The following day 1 L of TSB media was inoculated with 10 mL of the overnight culture and incubated at 37°C under continuous shaking for 6 hours. The bacterial suspension was centrifuged at 3000xG for 10 minutes and the pellet washed twice in 400 mL sterile PBS. After each wash the bacterial suspension was centrifuged at 3000xG for 10 minutes. The bacterial pellet was resuspended in 10-15 mL PBS and glycerol added to a final concentration of 16%. The suspension was thoroughly mixed, aliquoted in 1 mL aliquots and stored at -80°C. The number of colony forming units (CFU) per mL was determined for the frozen stock, as aliquots were thawed on ice and serially diluted in sterile saline. The dilutions were plated on TSB agar plates and incubated overnight at 37°C. The number of CFU per mL was established the following day. The procedure was repeated with an additional aliquot to confirm homogeny among the aliquots. Immediately prior to challenge, aliquots were thawed and diluted in sterile saline to the desired number of CFU.

The mice were housed at the at the animal facility of the Panum Institute, University of Copenhagen.

The animals were kept in an environment characterized by a 12-hours light-dark cycle and temperature and humidity control. The mice had access to food and water ad libitum. The experimental procedures were carried out in accordance with the guidelines of the Danish National Animal Ethics Committee.

The experiments were performed in class 2 certified facilities at the Biomedical Laboratory. Each mouse was challenged intraperitoneally with 6.5 x 10⁹ CFU S. aureus strain MRSA252. The seven days following the challenge, the mice were assessed daily to register symptoms and development of disease. To ensure a consistent evaluation of all animals, each animal was scored individually following the criteria for clinical symptoms set forth here: Score

Normal behaviour 0 = no; 1 = yes

Normal appearance 0 = no; 1 = yes

Eye condition 0 = normal ; 1 = mild irritation; 2 = one closed ; 3 = both closed Diarrhea 0 = no; 1 = yes

Changes in fur 0 = no; 1 = yes

Hunchback 0 = no; 1 = yes

Activity 0 = normal ; 1 = low activity; 2 = no activity

The mice were individually assessed on their physical appearance and behaviour, noting the presence or absence of the given characteristics.

Apart from the registration of clinical symptoms, body weight and temperature of each animal was registered daily following challenge. The weight loss was calculated as a percentage of the body weight registered prior to challenge. Animals were euthanized if either of the following humane endpoints were reached : a body temperature below 34°C or a weight loss above 20% of the initial body weight. Additionally, mice scored 3 over three successive days, without signs of improvements such as weight gain, or 4 once were euthanized .

A formulation of USAHOU-1728-88-453 (SEQ ID NO: 1) was tested in duplicate trials (experiment 1 using 20 animals, experiment 2 using 8 animals)

Survival of animals is provided in the survival plots in Fig . 1A and IB and as is clear, both experiments indicates increased survival of challenge infected animals upon administration of the experimental vaccines: the results show that immunization with USAHOU-1728-88-452 protected mice against a lethal infection with S. aureus MRSA252 when compared to adjuvant alone. The experiment in Fig . 1A shows a significant protection over control (p=0.0375), whereas the degree of protection reported in Fig . IB was not significant (p=0.0902) due to the small group of animals vaccinated .

BIOLOGICAL SEQUENCES

The amino acid sequence of USAHOU-1728-88-452 referred to in the present application is the following :

SEQ ID NO: 1 (USAHOU-1728-88-452)

GFLNKSKNEQ AALKAQQAAI KEEASANNLS DTSQEAQEIQ EAKREAQAEA DKSVAVSNKE SKAVALKAQQ AAIKEEASAN NLSDTSQEAQ EIQEAKKEAQ AETDKSAAVS NEEPKAVALK AQQAAIKEEA SANNLSDISQ EAQEVQEAKK EAQAEKDSDT LTKDASAAKV EVSKPESQAE RLANAAKQKQ AKLTPGSKES QLTEALFAEK PVAKNDLKEI PQLVTKKNDV SETETVNIDN KDTVKQKEAK FENGVITRKA DEKTTNNTAV DKKSGKQSKK TTPSNKRNAS KASTNKTSGQ KKQHNKKSSQ GAKKQSSSSK STQKNNQTSN KNSKTTNAKS SNASKTPNAK VEKAKSKIEK

RTFND

Claims

1. A polypeptide comprising

a) the amino acid sequence SEQ ID NO: 1, or

b) an amino acid sequence consisting of at least or exactly 5 contiguous amino acid residues from SEQ ID NO: 1, or

c) an amino acid sequence having a sequence identity of at least 60% with the amino acid sequence of a),

d) an amino acid sequence having a sequence identity of at least 60% with the amino acid sequence of b), or

e) an assembly of amino acids derived from SEQ ID NO: 1, which has essentially the same 3D conformation as in the protein from which said assembly is derived so as to constitute a B-cell epitope,

said polypeptide being antigenic in a mammal.

2. The polypeptide according to claim 1, wherein the at least or exactly 5 contiguous amino acids are at least or exactly or at most 6, such as at least or exactly or at most 7, at least or exactly or at most 8, at least or exactly or at most 9, at least or exactly or at most 10, at least or exactly or at most 11, at least or exactly or at most 12, at least or exactly or at most 13, at least or exactly or at most 14, at least or exactly or at most 15, at least or exactly or at most 16, at least or exactly or at most 17, at least or exactly or at most 18, at least or exactly or at most 19, at least or exactly or at most 20, at least or exactly or at most 21, at least or exactly or at most 22, at least or exactly or at most 23, at least or exactly or at most 24, at least or exactly or at most 25, at least or exactly or at most 26, at least or exactly or at most 27 at least or exactly or at most 28, at least or exactly or at most 29, at least or exactly or at most 30, at least or exactly or at most 31, at least or exactly or at most 32, at least or exactly or at most 33, at least or exactly or at most 34, at least or exactly or at most 35, at least or exactly or at most 36, at least or exactly or at most 37, at least or exactly or at most 38, at least or exactly or at most 39, at least or exactly or at most 40, at least or exactly or at most 41, at least or exactly or at most 42, at least or exactly or at most 43, at least or exactly or at most 44, at least or exactly or at most 45, at least or exactly or at most 46, at least or exactly or at most 47, at least or exactly or at most 48, at least or exactly or at most 49, at least or exactly or at most 50, at least or exactly or at most 51, at least or exactly or at most 52, at least or exactly or at most 53, at least or exactly or at most 54, at least or exactly or at most 55, at least or exactly or at most 56, at least or exactly or at most 57, at least or exactly or at most 58, at least or exactly or at most 59, at least or exactly or at most 60, at least or exactly or at most 61, at least or exactly or at most 62, at least or exactly or at most 63, at least or exactly or at most 64, at least or exactly or at most 65, at least or exactly or at most 66, at least or exactly or at most 67, at least or exactly or at most 68, at least or exactly or at most 69, at least or exactly or at most 70, at least or exactly or at most 71, at least or exactly or at most 72, at least or exactly or at most 73, at least or exactly or at most 74, at least or exactly or at most 75, at least or exactly or at most 76, at least or exactly or at most 77, at least or exactly or at most 78, at least or exactly or at most 79, at least or exactly or at most 80, at least or exactly or at most 81, at least or exactly or at most 82, at least or exactly or at most 83, at least or exactly or at most 84, at least or exactly or at most 85, at least or exactly or at most 86, at least or exactly or at most 87, at least or exactly or at most 88, at least or exactly or at most 89, at least or exactly or at most 90, at least or exactly or at most 91, at least or exactly or at most 92, at least or exactly or at most 93, at least or exactly or at most 94, at least or exactly or at most 95, at least or exactly or at most 96, at least or exactly or at most 97, at least or exactly or at most 98, at least or exactly or at most 99, at least or exactly or at most 100, at least or exactly or at most 101, at least or exactly or at most 102, at least or exactly or at most 103, at least or exactly or at most 104, at least or exactly or at most 105, at least or exactly or at most 106, at least or exactly or at most 107, at least or exactly or at most 108, at least or exactly or at most 109, at least or exactly or at most 110, at least or exactly or at most 111, at least or exactly or at most 112, at least or exactly or at most 113, at least or exactly or at most 114, at least or exactly or at most 115, at least or exactly or at most 116, at least or exactly or at most 117, at least or exactly or at most 118, at least or exactly or at most 119, at least or exactly or at most 120, at least or exactly or at most 121, at least or exactly or at most 122, at least or exactly or at most 123, at least or exactly or at most 124, at least or exactly or at most 125, at least or exactly or at most 126, at least or exactly or at most 127, at least or exactly or at most 128, at least or exactly or at most 129, at least or exactly or at most 130, at least or exactly or at most 131, at least or exactly or at most 132, at least or exactly or at most 133, at least or exactly or at most 134, at least or exactly or at most 135, at least or exactly or at most 136, at least or exactly or at most 137, at least or exactly or at most 138, at least or exactly or at most 139, at least or exactly or at most 140, at least or exactly or at most 141, at least or exactly or at most 142, at least or exactly or at most 143, at least or exactly or at most 144, at least or exactly or at most 145, at least or exactly or at most 146, at least or exactly or at most 147, at least or exactly or at most 148, at least or exactly or at most 149, at least or exactly or at most 150, at least or exactly or at most 151, at least or exactly or at most 152, at least or exactly or at most 153, at least or exactly or at most 154, at least or exactly or at most 155, at least or exactly or at most 156, at least or exactly or at most 157, at least or exactly or at most 158, at least or exactly or at most 159, at least or exactly or at most 160, at least or exactly or at most 161, at least or exactly or at most 162, at least or exactly or at most 163, at least or exactly or at most 164, at least or exactly or at most 165, at least or exactly or at most 166, at least or exactly or at most 167, at least or exactly or at most 168, at least or exactly or at most 169, at least or exactly or at most 170, at least or exactly or at most 171, at least or exactly or at most 172, at least or exactly or at most 173, at least or exactly or at most 174, at least or exactly or at most 175, at least or exactly or at most 176, at least or exactly or at most 177, at least or exactly or at most 178, at least or exactly or at most 179, at least or exactly or at most 180, at least or exactly or at most 181, at least or exactly or at most 182, at least or exactly or at most 183, at least or exactly or at most 184, at least or exactly or at most 185, at least or exactly or at most 186, at least or exactly or at most 187, at least or exactly or at most 188, at least or exactly or at most 189, at least or exactly or at most 190, at least or exactly or at most 191, at least or exactly or at most 192, at least or exactly or at most 193, at least or exactly or at most 194, at least or exactly or at most 195, at least or exactly or at most 196, at least or exactly or at most 197, at least or exactly or at most 198, at least or exactly or at most 199, at least or exactly or at most 200, at least or exactly or at most 201, at least or exactly or at most 202, at least or exactly or at most 203, at least or exactly or at most 204, at least or exactly or at most 205, at least or exactly or at most 206, at least or exactly or at most 207, at least or exactly or at most 208, at least or exactly or at most 209, at least or exactly or at most 210, at least or exactly or at most 211, at least or exactly or at most 212, at least or exactly or at most 213, at least or exactly or at most 214, at least or exactly or at most 215, at least or exactly or at most 216, at least or exactly or at most 217, at least or exactly or at most 218, at least or exactly or at most 219, at least or exactly or at most 220, at least or exactly or at most 221, at least or exactly or at most 222, at least or exactly or at most 223, at least or exactly or at most 224, at least or exactly or at most 225, at least or exactly or at most 226, at least or exactly or at most 227, at least or exactly or at most 228, at least or exactly or at most 229, at least or exactly or at most 230, at least or exactly or at most 231, at least or exactly or at most 232, at least or exactly or at most 233, at least or exactly or at most 234, at least or exactly or at most 235, at least or exactly or at most 236, at least or exactly or at most 237, at least or exactly or at most 238, at least or exactly or at most 239, at least or exactly or at most 240, at least or exactly or at most 241, at least or exactly or at most 242, at least or exactly or at most 243, at least or exactly or at most 244, at least or exactly or at most 245, at least or exactly or at most 246, at least or exactly or at most 247, at least or exactly or at most 248, at least or exactly or at most 249, at least or exactly or at most 250, at least or exactly or at most 251, at least or exactly or at most 252, at least or exactly or at most 253, at least or exactly or at most 254, at least or exactly or at most 255, at least or exactly or at most 256, at least or exactly or at most 257, at least or exactly or at most 258, at least or exactly or at most 259, at least or exactly or at most 260, at least or exactly or at most 261, at least or exactly or at most 262, at least or exactly or at most 263, at least or exactly or at most 264, at least or exactly or at most 265, at least or exactly or at most 266, at least or exactly or at most 267, at least or exactly or at most 268, at least or exactly or at most 269, at least or exactly or at most 270, at least or exactly or at most 271, at least or exactly or at most 272, at least or exactly or at most 273, at least or exactly or at most 274, at least or exactly or at most 275, at least or exactly or at most 276, at least or exactly or at most 277, at least or exactly or at most 278, at least or exactly or at most 279, at least or exactly or at most 280, at least or exactly or at most 281, at least or exactly or at most 282, at least or exactly or at most 283, at least or exactly or at most 284, at least or exactly or at most 285, at least or exactly or at most 286, at least or exactly or at most 287, at least or exactly or at most 288, at least or exactly or at most 289, at least or exactly or at most 290, at least or exactly or at most 291, at least or exactly or at most 292, at least or exactly or at most 293, at least or exactly or at most 294, at least or exactly or at most 295, at least or exactly or at most 296, at least or exactly or at most 297, at least or exactly or at most 298, at least or exactly or at most 299, at least or exactly or at most 300, at least or exactly or at most 301, at least or exactly or at most 302, at least or exactly or at most 303, at least or exactly or at most 304, at least or exactly or at most 305, at least or exactly or at most 306, at least or exactly or at most 307, at least or exactly or at most 308, at least or exactly or at most 309, at least or exactly or at most 310, at least or exactly or at most 311, at least or exactly or at most 312, at least or exactly or at most 313, at least or exactly or at most 314, at least or exactly or at most 315, at least or exactly or at most 316, at least or exactly or at most 317, at least or exactly or at most 318, at least or exactly or at most 319, at least or exactly or at most 320, at least or exactly or at most 321, at least or exactly or at most 322, at least or exactly or at most 323, at least or exactly or at most 324, at least or exactly or at most 325, at least or exactly or at most 326, at least or exactly or at most 327, at least or exactly or at most 328, at least or exactly or at most 329, at least or exactly or at most 330, at least or exactly or at most 331, at least or exactly or at most 332, at least or exactly or at most 333, at least or exactly or at most 334, at least or exactly or at most 335, at least or exactly or at most 336, at least or exactly or at most 337, at least or exactly or at most 338, at least or exactly or at most 339, at least or exactly or at most 340, at least or exactly or at most 341, at least or exactly or at most 342, at least or exactly or at most 343, at least or exactly or at most 344, at least or exactly or at most 345, at least or exactly or at most 346, at least or exactly or at most 347, at least or exactly or at most 348, at least or exactly or at most 349, at least or exactly or at most 350, at least or exactly or at most 351, at least or exactly or at most 352, at least or exactly or at most 353, at least or exactly or at most 354, at least or exactly or at most 355, at least or exactly or at most 356, at least or exactly or at most 357, at least or exactly or at most 358, at least or exactly or at most 359, at least or exactly or at most 360, at least or exactly or at most 361, at least or exactly or at most 362, at least or exactly or at most 363, or at least or exactly or at most 364 contiguous amino acid residues.

3. The polypeptide according to clam 1 or 2, wherein the sequence identity with the amino acid sequence of a), which is defined in c), is at least 65%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% .

4. The polypeptide according to clam 1 or 2, wherein the sequence identity with the amino acid sequence of b), which is defined in d), is at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% .

5. The polypeptide according to any one of claims 1-4, wherein the at least 5 contiguous amino acid residues has an N-terminal amino acid residue corresponding to any one of amino acid residues 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,

24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,

48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,

72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,

96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,

115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342„ 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, and 361 in SEQ ID NO: 1, with the proviso that the selected amino acid i esidue satisfies the formula N < 365-n+ l, where N is the number of the selected residue, and n is the number of consecutive amino acid residues.

6. An isolated nucleic acid fragment, which comprises a nucleotide sequence encoding a polypeptide according to any one of the preceding claims.

7. The nucleic acid fragment according to claim 6, which is a DNA or an RNA fragment.

8. A vector comprising the nucleic acid according to claim 5 or 6, such as a cloning vector or an expression vector.

9. The vector according to claim 8, which comprises in operable linkage and in the 5'-3' direction, an expression control region comprising an enhancer/promoter for driving expression of the nucleic acid fragment defined in claim 6, optionally a signal peptide coding sequence, a nucleotide sequence defined in claim 6, and optionally a terminator.

10. The vector according to claim 8 or 9, wherein the expression control region drives expression in prokaryotic cell such as a bacterium, e.g. in E coli.

11. The vector according to claim any one of claims 8-10, which is capable of autonomous replication.

12. The vector according to any one of claims 8-11, which is capable of being integrated into the genome of a host cell.

13. The vector according to any one of claims 8-11, which is incapable of being integrated into the genome of a mammalian host cell.

14. The vector according to any one of claims 8-13, which is selected from the group consisting of a virus, such as an attenuated virus, a bacteriophage, a plasmid, a

minichromosome, and a cosmid.

15. A cell which is transformed so as to carry the vector according to any one of claims 8- 14.

16. The transformed cell according to claim 15, which is capable of replicating the nucleic acid fragment defined in claim 6.

17. The transformed cell according to claim 15 or 16, which is capable of expressing the nucleic acid fragment defined in claim 6.

18. The transformed cell according to any one of claims 15-17, which is selected from a prokaryotic cell and a eukaryotic cell.

19. The transformed cell according to any one of claims 15-18, which is a bacterial cell, which is preferably non-pathogenic, selected from the group consisting of Escherichia (such as E. coli .), Bacillus (e.g. Bacillus subtilis), Salmonella, and Mycobacterium, e.g. M. bovis BCG.

20. The transformed cell according to any one of claims 15-19, which is stably

transformed by having the nucleic acid defined in claim 6 stably integrated into its genome.

21. The transformed cell according to any one of claims 15-20, which secretes or carries on its surface the polypeptide according to any one of claims 1-5.

22. The transformed cell according to claim 21, wherein the cell is a bacterium and secretion is into the periplasmic space.

23. A cell line derived from a transformed cell according to any one of claims 15-22.

24. A pharmaceutical composition comprising the polypeptide according to any one of claims 1-5, a nucleic acid fragment according to any one of claims 6-7, a vector according to any one of claims 8-14, or a cell according to any one of claims 15-20, and a

pharmaceutically acceptable carrier, vehicle or diluent.

25. The pharmaceutical composition according to claim 24, which further comprises an immunological adjuvant.

26. The pharmaceutical composition according to claim 25, wherein the adjuvant is an aluminium based adjuvant.

27. The pharmaceutical composition according to any one of claims 24-26, which comprises the polypeptide according to any one of claims 1-5 and which further comprises:

- any one of the polypeptides disclosed as SEQ ID NOs: 1-19 in WO 2012/136653 or with any one of the fragments and variants of these polypeptides disclosed on pages 9-18 in WO 2012/136653, and/or

- any one of the polypeptides disclosed as SEQ ID NOs: 1-16 and 49 in WO 2015/082536 or with any of the fragments and variants of these polypeptides disclosed on pages 10-25 in WO 2015/082536, and/or

- any one of the polypeptides disclosed as SEQ ID NOs: 1-2 in WO 2017/144523 or with any of the fragments and variants of these polypeptides disclosed on pages 10-19 in WO

2017/144523, and/or

- any one of the chimeric polypeptides disclosed in PCT/EP2017/068694, in particular those chimeric polypeptides disclosed on pages 10-46 and defined in the claims in

PCT/EP2017/068694.

28. The pharmaceutical composition according to any one of claims 24-26, which comprises the nucleic acid fragment according to any one of claims 6-7, and which further comprises:

- a nucleic acid encoding and being capable of effecting expression of 1) any one of the polypeptides disclosed as SEQ ID NOs: 1-19 in WO 2012/136653 or 2) any one of the fragments and variants of these polypeptides disclosed on pages 9-18 in WO 2012/136653, and/or

- a nucleic acid encoding and being capable of effecting expression of 1) any one of the polypeptides disclosed as SEQ ID NOs: 1-16 and 49 in WO 2015/082536 or 2) any one of the fragments and variants of these polypeptides disclosed on pages 10-25 in WO 2015/082536, and/or

- a nucleic acid encoding and being capable of effecting expression of 1) any one of the polypeptides disclosed as SEQ ID NOs: 1-2 in WO 2017/144523 or 2) any one of the fragments and variants of these polypeptides disclosed on pages 10-19 in WO 2017/144523, and/or

- a nucleic acid encoding and being capable of effecting expression of 1) any one of the chimeric polypeptides disclosed in the claims of PCT/EP2017/068694 as well as on pages 10- 42 in PCT/EP2017/068694.

29. A method for inducing immunity in an animal by administering at least once an immunogenically effective amount of the polypeptide according to any one of claims 1-5, a nucleic acid fragment according to any one of claims 6-7, a vector according to any one of claims 8-14, a cell according to any one of claims 15-22, or a pharmaceutical composition according to any one of claims 24-28 so as to induce adaptive immunity against S. aureus in the animal.

30. The method according to claim 29, wherein, when the polypeptide according to any one of claims 1-5 or a composition comprising said polypeptide is administered, the animal receives between 0.5 and 5,000 pg of the polypeptide according to any one of claims 1-5 per administration.

31. The method according to claim 29 or 30, wherein the animal receives a first priming administration comprising said polypeptide and one or more booster administrations comprising said polypeptide.

32. The method according to any one of claims 29-31, wherein the animal is a human being.

33. The method according to any one of claims 29-32, wherein the administration is for the purpose of inducing protective immunity against S. aureus.

34. The method according to claim 33, wherein the protective immunity is effective in reducing the risk of attracting infection with S. aureus or is effective in treating or

ameliorating infection with S. aureus.

35. The method according to any one of claims 29-32, wherein the administration is for the purpose of inducing antibodies specific for S. aureus and wherein said antibodies or B- lymphocytes producing said antibodies are subsequently recovered from the animal.

36. The method according to any one of claims 29-32, wherein the administration is for the purpose of inducing antibodies specific for S. aureus and wherein B-lymphocytes producing said antibodies are subsequently recovered from the animal and used for preparation of monoclonal antibodies.

37. The polypeptide according to any one of claims 1-5 for use as a pharmaceutical.

38. The polypeptide according to any one of claims 1-5 for use as a pharmaceutical in the treatment, prophylaxis or amelioration of infection with S. aureus.

39. The nucleic acid fragment according to any one of claims 6-7 or the vector according to any one of claims 8-14 for use as a pharmaceutical.

40. The nucleic acid fragment according to any one of claims 6-7 or the vector according to any one of claims 8-14 for use as a pharmaceutical in the treatment, prophylaxis or amelioration of infection with S. aureus.

41. The transformed cell according to any one of claims 15-22 for use as a

pharmaceutical.

42. The transformed cell according to any one of claims 15-22 for use as a pharmaceutical in the treatment, prophylaxis or amelioration of infection with S. aureus.