Live bacterial strain with reduced capsule

Abstract

The present invention relates to the field of biomedicine. Specifically, the present invention relates to live bacterial strains with reduced capsule and their uses. More specifically, the present invention relates to a live bacterial strain with reduced capsule production (e.g., a live strain of Staphylococcus aureus), a vaccine against bacterial infection comprising the live strain, and a method for preventing and / or treating bacterial infection in a subject by administering the live strain.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine. Specifically, the present invention relates to live bacterial strains with reduced capsule production and uses thereof. More specifically, the present invention relates to a live bacterial strain with reduced capsule production (e.g., a live strain of Staphylococcus aureus), a vaccine against bacterial infection comprising the live strain, and a method for preventing and / or treating bacterial infection in a subject by administering the live strain. Background Art

[0002] The bacterial capsule is a ubiquitous structure on the cell surface of many bacteria. The capsule is composed of polysaccharides and can be homopolymers or heteropolymers of repeating monosaccharides linked by various glycosidic bonds, resulting in a diverse range of molecular compositions. Therefore, capsular polysaccharides have been used as a means of serotyping and identifying bacterial strains [1].

[0003] A large number of different capsular serotypes have been found in human pathogens. Bacterial capsules have multiple functions, including resistance to drying, adhesion, protection against nonspecific host immunity, protection against specific host immunity, and mediating the diffusion of molecules to the cell surface. Due to these interactions, the polysaccharide capsule is considered an important virulence factor for many bacterial pathogens [1].

[0004] Capsule gene clusters have been found in many Gram-positive bacteria, including Streptococcus pneumoniae, Staphylococcus aureus, and group B streptococci. Among them, Staphylococcus aureus (S. aureus) is one of the most common causes of community-acquired and healthcare-associated bacterial infections. S. aureus infections lead to a variety of clinical manifestations, ranging from skin and soft tissue infections to invasive diseases, including bloodstream infections, endocarditis, and sepsis. In addition, the emergence of methicillin-resistant S. aureus (MRSA) further highlights the serious threat of S. aureus infection as a global public health problem. Among encapsulated S. aureus clinical isolates, serotypes 5 and 8 are the most common. Capsular polysaccharides 5 (CP5) and 8 (CP8) have similar trisaccharide repeat units, but the linkage between sugars and the O-acetylation sites of the monosaccharide residues are different. However, previous reports have shown that 20% to 25% of human isolates of S. aureus are unable to produce CP5 or CP8 [2].

[0005] Several highly successful vaccines against other bacterial pathogens target CP, including vaccines against Haemophilus influenzae type b, Streptococcus pneumoniae (S. pneumoniae), and Neisseria meningitidis (N. meningitidis), as well as serogroups A, C, W, and Y. Given these successes, CP-based vaccine candidates for Staphylococcus aureus have been clinically tested. Examples include the use of the capsular proteins CP5 / CP8 in patients undergoing hemodialysis (StaphVAX, Nabi), the use of polyclonal immunoglobulins (Ig) with anti-CP5 and anti-CP8 antibodies in patients with complicated S. aureus bacteremia (Altastaph, Nabi), and the use of a combination of the capsular proteins CP5 / CP8, clumping factor A (ClfA), and manganese transporter (MntC) in patients undergoing orthopedic surgery (SA4Ag, Pfizer). In each case, despite high levels of antibodies elicited in vaccine recipients, vaccination failed to protect against infection [3]. In addition, GSK’s previous Phase I trial, which investigated the safety and immunogenicity of a vaccine containing four components (CP5, CP8, alpha-toxin (Hla), and ClfA), found that the vaccine had no effect on S. aureus carriage rates over a two-year period. Another GSK vaccine, PentaStaph, containing CP5, CP8, Hla, Panton-Valentine leukocidin (PVL), and wall teichoic acid, was also discontinued in Phase II [4].

[0006] As mentioned above, it has been shown that a large number of pathogenic S. aureus strains do not express capsular polysaccharides, which may be a significant factor in the inconsistent protection seen in past failed clinical trials. Importantly, one of the world's most critical clones, S. aureus USA300, has been found to be unable to form a detectable capsule due to a point mutation in the cap-5 promoter region, which is essential for CP5 biosynthesis [5]. Another study also demonstrated that the CP-negative phenotype persists in USA300 isolates and is a common characteristic trait of this highly successful MRSA lineage [6]. Therefore, vaccines composed of CP antigens have not been able to demonstrate high efficacy in preventing S. aureus infection.

[0007] Given the past failures of vaccines against S. aureus, a different approach is needed to produce an effective vaccine. Summary of the Invention

[0008] With the rapid development of genome modification technologies, the use of synthetic bacterial vectors in vaccines has become a promising strategy. The goal of this study was to identify general methods for constructing bacterial vaccine vectors that can be regulated with efficient antigen display and elicit immunogenicity in various infection models against multiple pathogens. A hypovirulent strain of Staphylococcus aureus was engineered by knocking out the capsule gene. This attenuated strain further demonstrated enhanced immunogenicity in mouse models compared to its wild-type strain expressing a functional capsule. Because capsule expression masks the bacterial surface and surface-associated proteins, removal of the capsule is expected to expose more cell surface proteins and enable higher phagocytosis rates, further enhancing antigen recognition by the host immune system. Furthermore, a diverse set of epitopes located on the capsule have been shown to be ineffective in protecting against S. aureus infection. By removing these immunodominant antigens from bacterial vectors, more protective epitopes become available for immune cell recognition, potentially eliciting more protective memory T cell and antibody responses.

[0009] In this regard, the present invention provides at least the following embodiments:

[0010] Embodiment 1. A live bacterial strain that does not produce a capsule or has reduced capsule production and / or has reduced expression of one or more capsular polysaccharide synthesis genes compared to a corresponding control strain.

[0011] Embodiment 2. The live bacterial strain according to embodiment 1, wherein capsule production in the live bacterial strain is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90% or more compared to a corresponding control strain, preferably, the live bacterial strain does not produce a capsule.

[0012] Embodiment 3. The live bacterial strain according to embodiment 1 or 2, wherein the production of capsular polysaccharides in the live bacterial strain is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90% or more compared to a corresponding control strain. Preferably, the live bacterial strain does not produce capsular polysaccharides.

[0013] Embodiment 4. The live bacterial strain according to any one of embodiments 1 to 3, wherein the expression of one or more capsular polysaccharide synthesis genes is reduced in the live bacterial strain.

[0014] Embodiment 5. The live bacterial strain of embodiment 4, wherein the expression of one or more capsular polysaccharide synthesis genes in the live bacterial strain is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more compared to a corresponding control strain.

[0015] Embodiment 6. The live bacterial strain according to embodiment 4, wherein the expression of all capsular polysaccharide synthesis genes in the live bacterial strain is reduced.

[0016] Embodiment 7. The live bacterial strain of embodiment 6, wherein the expression of all capsular polysaccharide synthesis genes in the live bacterial strain is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more compared to a corresponding control strain.

[0017] Embodiment 8. The live bacterial strain according to any one of embodiments 1 to 3, wherein no capsular polysaccharide synthesis genes are expressed in the live bacterial strain.

[0018] Embodiment 9. The live bacterial strain according to any one of embodiments 1 to 8, wherein the live strain comprises one or more mutations within the one or more capsular polysaccharide synthesis genes, preferably all capsular polysaccharide synthesis genes in the live bacterial strain are mutated.

[0019] Embodiment 10. The viable bacterial strain according to embodiment 9, wherein the mutation in the capsular polysaccharide synthesis gene results in reduced expression or no expression of the capsular polysaccharide synthesis protein, or results in the expression of a capsular polysaccharide synthesis protein with reduced activity or no activity.

[0020] Embodiment 11. The live bacterial strain according to any one of embodiments 1 to 10, wherein the mutation comprises a deletion of the one or more capsular polysaccharide synthesis genes, preferably a complete deletion or a partial deletion of all capsular polysaccharide synthesis genes in the live bacterial strain, more preferably a complete deletion of all capsular polysaccharide synthesis genes in the live bacterial strain.

[0021] Embodiment 12. The live bacterial strain according to any one of embodiments 9 to 11, wherein the mutation is achieved by homologous recombination or by targeted mutagenesis, such as via CRISPR, TALEN or ZFN technology.

[0022] Embodiment 13. The live bacterial strain of any one of embodiments 1 to 12, wherein the live bacterial strain has reduced virulence compared to a corresponding control strain, for example, the virulence of the live bacterial strain is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more.

[0023] Embodiment 14. The live bacterial strain according to any one of embodiments 1 to 13, wherein the live bacterial strain has increased immunogenicity compared to a corresponding control strain, for example, the immunogenicity of the live bacterial strain is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300% or more.

[0024] Embodiment 15. The live bacterial strain according to any one of embodiments 1 to 14, wherein the live bacterial strain is derived from a parent strain that is a clinical isolate.

[0025] Embodiment 16. The live bacterial strain according to any one of embodiments 1 to 14, wherein the live bacterial strain is derived from a parent strain that already has reduced virulence.

[0026] Embodiment 17. The live bacterial strain of any one of embodiments 1 to 16, wherein the live bacterial strain is derived from Staphylococcus aureus, Streptococcus pneumoniae, group B streptococci, Haemophilus influenzae, N. meningitidis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Bordetella pertussis, Salmonella typhi, or Acinetobacter baumannii.

[0027] Embodiment 18. The live bacterial strain according to any one of embodiments 1 to 17, wherein the live bacterial strain is a live strain of Staphylococcus aureus, preferably a live strain of Staphylococcus aureus derived from Staphylococcus aureus serotype 5 or serotype 8, more preferably a live strain of Staphylococcus aureus derived from Staphylococcus aureus serotype 5.

[0028] Embodiment 19. The live bacterial strain of embodiment 18, wherein the live Staphylococcus aureus strain is derived from the Staphylococcus aureus Newman strain.

[0029] Embodiment 20. The live bacterial strain according to embodiment 18 or 19, wherein the live Staphylococcus aureus strain further lacks or has reduced adenosine synthase A (AdsA) activity.

[0030] Embodiment 21. The live bacterial strain of embodiment 20, wherein the live S. aureus strain comprises a mutation in the AdsA gene encoding AdsA.

[0031] Embodiment 22. The live bacterial strain of any one of embodiments 18 to 21, wherein the one or more capsular polysaccharide synthesis genes are selected from the group consisting of: capA, capB, capC, capD, capE, capF, capG, capH, capI, capJ, capK, capL, capM, capN, capO, and capP.

[0032] Embodiment 23. The live bacterial strain of embodiment 22, wherein all of the polysaccharide synthesis genes capA, capB, capC, capD, capE, capF, capG, capH, capI, capJ, capK, capL, capM, capN, capO and capP in the live Staphylococcus aureus strain are deleted.

[0033] Embodiment 24. The live bacterial strain according to any one of embodiments 1 to 23, which is used as a live expression vector for expressing a target protein.

[0034] Embodiment 25. The live bacterial strain according to any one of embodiments 1 to 24, wherein the live bacterial strain further comprises a coding sequence for a protein of interest and is thereby capable of expressing the protein of interest.

[0035] Embodiment 26. The live bacterial strain according to embodiment 25, wherein the coding sequence for the protein of interest is introduced into the live bacterial strain, for example via a nucleic acid expression construct.

[0036] Embodiment 27. The live bacterial strain according to embodiment 26, wherein the introduced coding sequence of the target protein is integrated into the genome of the live bacterial strain.

[0037] Embodiment 28. The live bacterial strain according to any one of embodiments 24 to 27, wherein the target protein is expressed and displayed on the cell surface of the live bacterial strain; or the target protein is expressed and secreted out of the cells of the live bacterial strain.

[0038] Embodiment 29. The live bacterial strain according to any one of embodiments 24 to 28, wherein the target protein is selected from an antibody or an antigen, preferably, the target protein is an antigen,

[0039] For example, the antigen is selected from EsxA, EsxB, PmtA, PmtC, pdhC, mutated Hla, mutated Spa, etc.

[0040] Embodiment 30. The live bacterial strain according to any one of embodiments 1 to 29 for use in preventing and / or treating bacterial infections.

[0041] Embodiment 31. Use of a live bacterial strain according to any one of embodiments 1 to 30 for the preparation of a composition, such as a vaccine, for the prevention or treatment of a bacterial infection.

[0042] Embodiment 32. The use according to embodiment 31, wherein the bacterial infection is an infection caused by, for example, the species from which the live bacterial strain is derived.

[0043] Embodiment 33. A composition for preventing or treating a bacterial infection, such as a vaccine, comprising a live bacterial strain according to any one of embodiments 1 to 30.

[0044] Embodiment 34. The composition of embodiment 33, further comprising an adjuvant and / or a pharmaceutically acceptable carrier.

[0045] Embodiment 35. A method for preventing and / or treating a bacterial infection in a subject, the method comprising administering to the subject an effective amount of a live bacterial strain according to any one of embodiments 1 to 30, or a composition according to embodiment 33 or 34.

[0046] Embodiment 36. The use of any one of embodiments 31 to 32, the composition of any one of embodiments 33 to 34, or the method of embodiment 35, wherein the bacterial infection is a Staphylococcus aureus infection.

[0047] Embodiment 37. The use, composition or method of embodiment 36, wherein the S. aureus infection is a skin infection, a soft tissue infection or an invasive disease.

[0048] Embodiment 38. The use, composition or method of embodiment 37, wherein the invasive disease is a bloodstream infection, endocarditis or sepsis.

[0049] Embodiment 39. The use, composition or method of any one of embodiments 36 to 38, wherein the S. aureus infection is a methicillin-resistant S. aureus (MRSA) infection or a methicillin-sensitive S. aureus (MSSA) infection, preferably, the infection is a recurrent S. aureus infection.

[0050] Embodiment 40. A method for producing a live bacterial strain with reduced virulence and / or increased immunogenicity, the method comprising reducing capsule production in the live bacterial strain and / or reducing the expression of one or more capsular polysaccharide synthesis genes in the live bacterial strain.

[0051] Embodiment 41. The method of embodiment 40, wherein said method comprises reducing said production of capsular polysaccharides in said live bacterial strain.

[0052] Embodiment 42. The method of embodiment 40 or 41, wherein the method comprises reducing the expression of one or more capsular polysaccharide synthesis genes in the living bacterial strain.

[0053] Embodiment 43. The method of any one of Embodiments 40 to 42, wherein the method comprises introducing one or more mutations into the one or more capsular polysaccharide synthesis genes in the living bacterial strain.

[0054] Embodiment 44. The method of embodiment 43, wherein all capsular polysaccharide synthesis genes in the live bacterial strain are mutated.

[0055] Embodiment 45. The method according to embodiment 43, wherein one or more capsular polysaccharide synthesis genes are deleted, preferably all capsular polysaccharide synthesis genes are deleted in the live bacterial strain of the invention.

[0056] Embodiment 46. The method of any one of embodiments 40 to 45, wherein the live bacterial strain is derived from Staphylococcus aureus, for example, the live bacterial strain is derived from Staphylococcus aureus serotype 5 or serotype 8.

[0057] Embodiment 47. The method of embodiment 46, wherein the one or more capsular polysaccharide synthesis genes are selected from the group consisting of: capA, capB, capC, capD, capE, capF, capG, capH, capI, capJ, capK, capL, capM, capN, capO, and capP.

[0058] Embodiment 48. A method according to any one of embodiments 43 to 47, wherein the mutation, such as a deletion, is achieved by homologous recombination, such as double homologous recombination, or by targeted mutagenesis, such as via CRISPR, TALEN or ZFN technology.

[0059] Embodiment 49. The method according to any one of embodiments 40 to 48, wherein the method further comprises introducing a coding sequence for a target protein into the live bacterial strain, thereby enabling the live bacterial strain to express the target protein.

[0060] Embodiment 50. The method according to embodiment 49, wherein the coding sequence of the target protein is introduced into the living bacterial strain via a nucleic acid expression construct.

[0061] Embodiment 51. The method according to embodiment 49 or 50, wherein the introduced coding sequence of the target protein is integrated into the genome of the living bacterial strain.

[0062] Embodiment 52. The method according to any one of embodiments 49 to 51, wherein the target protein is selected from an antibody or an antigen, preferably an antigen,

[0063] For example, the antigen is selected from EsxA, EsxB, PmtA, PmtC, pdhC, mutated Hla, mutated Spa, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 .The deleted region of the capsular polysaccharide synthesis gene.

[0065] Figure 2 The ΔCP strain showed lower virulence in the skin abscess model.

[0066] Figure 3 A single subcutaneous immunization with the ΔCP strain increased the antibody titer.

[0067] Figure 4 Survival studies showed improved immunogenicity compared to the ΔadsA strain. DETAILED DESCRIPTION

[0068] Before describing various aspects of the present invention, it must be noted that, unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" used herein and in the appended claims include plural references. The term "and / or" is intended to include any combination of the items connected by the term, which is equivalent to listing all combinations individually. For example, "A, B, and / or C" encompasses "A", "B", "C", "A and B", "A and C", "B and C", and "A and B and C". Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0069] In one aspect, the present invention provides a live bacterial strain that produces no or reduced capsule and / or has reduced expression of one or more capsular polysaccharide synthesis genes compared to a corresponding control strain.

[0070] In some embodiments, capsule production in the live bacterial strains of the invention is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more compared to a corresponding control strain. In some preferred embodiments, the live bacterial strains of the invention do not produce a capsule.

[0071] In some embodiments, the production of capsular polysaccharides in the live bacterial strains of the invention is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more compared to a corresponding control strain. In some preferred embodiments, the live bacterial strains of the invention do not produce capsular polysaccharides.

[0072] In some embodiments, the expression of one or more capsular polysaccharide synthesis genes in the live bacterial strain is reduced. In some embodiments, the expression of all capsular polysaccharide synthesis genes in the live bacterial strain is reduced. In some embodiments, the expression of one or more capsular polysaccharide synthesis genes in the live bacterial strain is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more compared to the corresponding control strain. In some embodiments, the expression of all capsular polysaccharide synthesis genes in the live bacterial strain is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more compared to the corresponding control strain. In some preferred embodiments, no capsular polysaccharide synthesis genes are expressed in the live bacterial strain.

[0073] As used herein, the term "capsular polysaccharide synthesis gene" refers to any gene involved in the production of capsular polysaccharides in bacteria. A capsular polysaccharide synthesis gene can encode a capsular polysaccharide synthesis protein. A capsular polysaccharide synthesis gene can refer to a protein coding sequence, but can also encompass expression regulatory elements / sequences, such as promoters, enhancers, and the like.

[0074] In some embodiments, the live strains of the present invention comprise one or more mutations in one or more capsular polysaccharide synthesis genes. The mutations may be additions, substitutions, or deletions of one or more nucleotides.

[0075] In some preferred embodiments, all capsular polysaccharide synthesis genes in the viable bacterial strain are mutated. In some embodiments, the mutation in the capsular polysaccharide synthesis gene results in reduced expression of the capsular polysaccharide synthesis protein, or results in expression of a capsular polysaccharide synthesis protein with reduced activity. In some embodiments, the mutation in the capsular polysaccharide synthesis gene results in no expression of the capsular polysaccharide synthesis protein, or results in expression of an inactive capsular polysaccharide synthesis protein. In some embodiments, the mutation is a frameshift mutation, which results in mistranslation of one or more capsular polysaccharide synthesis genes.

[0076] In some embodiments, the mutation comprises a deletion of a capsular polysaccharide synthesis gene, for example, a complete deletion or a partial deletion of a capsular polysaccharide synthesis gene. In some embodiments, the live bacterial strains of the present invention comprise a deletion of one or more capsular polysaccharide synthesis genes. One or more capsular polysaccharide synthesis genes may be completely deleted from the strain such that one or more capsular polysaccharide synthesis proteins are absent from the live bacterial strains of the present invention. One or more capsular polysaccharide synthesis genes may also be partially deleted such that only one or more inactive, truncated capsular polysaccharide synthesis proteins are present in the live bacterial strains of the present invention. In some preferred embodiments, all capsular polysaccharide synthesis genes in the live bacterial strains of the present invention are completely deleted or partially deleted. In some preferred embodiments, all capsular polysaccharide synthesis genes in the live bacterial strains of the present invention are completely deleted.

[0077] The mutation of one or more capsular polysaccharide synthesis genes can be achieved by various means known in the art. In some embodiments, the mutation is introduced into a living bacterial strain by genetic engineering. In some embodiments, the mutation is not a naturally occurring mutation. For example, a mutation such as a deletion can be achieved by homologous recombination, such as double homologous recombination. In some embodiments, the mutation is carried out by targeted mutagenesis, such as via CRISPR, TALEN or ZFN technology.

[0078] In some embodiments, the live bacterial strains of the invention have reduced or absent capsule / capsular polysaccharide production and / or mutations in one or more capsular polysaccharide synthesis genes leading to reduced virulence.

[0079] For example, the virulence of the live bacterial strains of the invention can be reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more compared to a corresponding control strain.

[0080] In some embodiments, the live bacterial strains of the present invention have reduced or no capsule / capsular polysaccharide production and / or mutations in one or more capsular polysaccharide synthesis genes leading to increased immunogenicity. Immunogenicity can refer to the ability to elicit an immune response (e.g., an antibody-mediated immune response) in a host.

[0081] For example, the immunogenicity of the live bacterial strains of the present invention can be increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, or more, compared to a corresponding control strain.

[0082] In some embodiments, a "control strain" can be a parent strain derived from a live bacterial strain of the present invention. In some embodiments, a "control strain" can refer to a strain of the same species whose capsule / capsular polysaccharide production or capsular polysaccharide synthesis genes are not altered. In some embodiments, a "control strain" can also refer to a strain of the same species that does not contain a mutation in one or more capsular polysaccharide synthesis genes as described above.

[0083] The live bacterial strains of the present invention can be derived from a parent strain that is a wild-type strain of the same species. In some embodiments, the wild-type strain can be a strain that has not been genetically engineered. In some embodiments, the wild-type strain can be a strain whose capsule / capsular polysaccharide production or capsular polysaccharide synthesis genes have not been genetically engineered. In some embodiments, the wild-type strain can be a clinical isolate.

[0084] The live bacterial strains of the present invention may be derived from a parent strain that already has reduced virulence. For example, the parent strain is an attenuated strain. The live bacterial strains of the present invention may contain other modifications (not within the capsular polysaccharide synthesis genes) that may result in attenuation.

[0085] The live bacterial strains of the present invention may be derived from any bacterial species that produces capsules / capsular polysaccharides. The live bacterial strains of the present invention may be derived from gram-positive bacteria or gram-negative bacteria. In some embodiments, the live bacterial strains of the present invention may be derived from pathogenic bacterial species.

[0086] In some embodiments, the bacteria can be selected from Staphylococcus aureus, Streptococcus pneumoniae, Group B Streptococcus, Haemophilus influenzae, Neisseria meningitidis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Bordetella pertussis, Salmonella typhi, Acinetobacter baumannii, and the like.

[0087] In some embodiments, the live bacterial strains of the present invention can be derived from any isolate of a particular bacterial species.

[0088] In some embodiments, the live bacterial strains of the present invention can be derived from bacterial strains of various serotypes. Typically, the serotype of a bacterial strain is determined by the capsular polysaccharide produced by the bacterial strain.

[0089] In some embodiments, the live bacterial strains of the present invention are derived from Staphylococcus aureus, and thus are also referred to as live Staphylococcus aureus strains of the present invention. In some embodiments, the live Staphylococcus aureus strains of the present invention are derived from different serotypes of Staphylococcus aureus, including but not limited to serotype 5, serotype 8, etc. In some specific embodiments, the live Staphylococcus aureus strains of the present invention are derived from serotype 5 Staphylococcus aureus. In some specific embodiments, the live Staphylococcus aureus strains of the present invention are derived from serotype 8 Staphylococcus aureus.

[0090] In some embodiments, the live S. aureus strains of the present invention are derived from the S. aureus Newman strain.

[0091] In some embodiments, the live Staphylococcus aureus strains of the present invention also lack or have reduced adenosine synthase A (AdsA) activity.

[0092] Adenosine synthase A (AdsA) is an important virulence factor of Staphylococcus aureus. An exemplary AdsA of Staphylococcus aureus comprises the amino acid sequence of SEQ ID NO: 1. However, those skilled in the art are aware that due to polymorphism between strains, the AdsA of Staphylococcus aureus may have minor differences from SEQ ID NO: 1 while maintaining the same or similar functions.

[0093] In some embodiments, the AdsA activity of the live Staphylococcus aureus strains of the present invention is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more compared to a corresponding control strain. In some preferred embodiments, the live Staphylococcus aureus strains of the present invention have no AdsA activity. A reduction or absence of AdsA activity may result in attenuation of the live Staphylococcus aureus strains of the present invention.

[0094] In some embodiments, the live Staphylococcus aureus strain of the present invention comprises a mutation in the AdsA gene encoding AdsA. Such a mutation may be an addition, substitution, or deletion of one or more nucleotides.

[0095] In some embodiments, the mutation in the AdsA gene results in reduced expression of the AdsA protein, or results in expression of a mutant AdsA protein with reduced activity. In some embodiments, the mutation in the AdsA gene results in no expression of the AdsA protein, or results in expression of an inactive mutant AdsA protein.

[0096] In some embodiments, the mutation comprises a deletion of the AdsA gene, such as a complete or partial deletion of the AdsA gene. The AdsA gene can be completely deleted from the strain, such that no AdsA gene is present in the viable strains of the invention. The AdsA gene can also be partially deleted, such that only AdsA protein with reduced or no activity is present in the viable strains of the invention. In some embodiments, the mutation is a frameshift mutation, which results in mistranslation of the AdsA protein. In some embodiments, the mutation in the AdsA gene results in the deletion of a portion of AdsA responsible for producing adenosine.

[0097] Mutation of the AdsA gene can be achieved by various means known in the art. In some embodiments, the mutation is introduced into a living bacterial strain by genetic engineering. In some embodiments, the mutation is not a naturally occurring mutation. For example, mutations such as deletions can be achieved by homologous recombination, such as double homologous recombination. In some embodiments, the mutation is performed by targeted mutagenesis, such as via CRISPR, TALEN or ZFN technology.

[0098] In some embodiments, when comparing AdsA activity, a "control strain" may refer to a strain of the same species in which the AdsA activity or AdsA gene is not altered. In some embodiments, when comparing AdsA activity, a "control strain" may also refer to a strain of the same species that does not contain a mutation in the AdsA gene as described above.

[0099] In some other embodiments, the live Staphylococcus aureus strains of the present invention are derived from a parental live Staphylococcus aureus strain that has been attenuated. For example, the parental live Staphylococcus aureus strain may have reduced or no adenosine synthase A (AdsA) activity compared to a corresponding control strain. For example, the live Staphylococcus aureus strains of the present invention can be obtained by introducing mutations in one or more capsular polysaccharide synthesis genes into a parental live Staphylococcus aureus strain that already has reduced or no adenosine synthase A (AdsA) activity.

[0100] In some embodiments, Staphylococcus aureus from serotype 5 or serotype 8 contains 16 capsular polysaccharide synthesis genes clustered in the genome: capA, capB, capC, capD, capE, capF, capG, capH, capI, capJ, capK, capL, capM, capN, capO, and capP. Exemplary protein sequences of capA to capP from serotype 5 Staphylococcus aureus are set forth in SEQ ID NOs: 2 to 17, respectively. Exemplary coding sequences of capA to capP from serotype 5 Staphylococcus aureus are set forth in SEQ ID NOs: 18 to 33, respectively. Thus, one skilled in the art will be able to identify capsular polysaccharide synthesis genes / gene clusters from other serotypes or isolates and make the above-described mutations accordingly.

[0101] In some embodiments, the one or more capsular polysaccharide synthesis genes encode one or more capsular polysaccharide synthesis proteins having an amino acid sequence having 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%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% sequence identity to any one of SEQ ID NOs: 2 to 17.

[0102] In some embodiments, all 16 polysaccharide synthesis genes are deleted in the live S. aureus strains of the invention.

[0103] In some embodiments, the live bacterial strains of the present invention are also used as live expression vectors for expressing target proteins. The target proteins can impart certain properties to the live bacterial strains of the present invention.

[0104] In some embodiments, the live bacterial strains of the present invention may comprise a coding sequence for a protein of interest and thereby be capable of expressing the protein of interest.

[0105] In some embodiments of various aspects, the target protein can be an endogenous protein, i.e., a protein of the bacterial species from which the living bacterial strain is derived. In some embodiments, the target protein can be an exogenous protein, i.e., a protein of a species different from the bacterial species from which the living bacterial strain is derived.

[0106] In some embodiments of various aspects, the coding sequence of the target protein is introduced into the living bacterial strain of the present invention, for example, via a nucleic acid expression construct. In some embodiments, the introduced coding sequence of the target protein is integrated into the genome of the living bacterial strain of the present invention.

[0107] As used herein, an "expression construct" refers to a vector, such as a recombinant vector, suitable for expressing a nucleotide sequence of interest in a host cell. "Expression" refers to the production of a functional product. For example, expression of a nucleotide sequence can refer to the transcription of the nucleotide sequence and / or the translation of RNA into a precursor or mature protein. An "expression construct" of the present invention can be a linear nucleic acid fragment, a circular plasmid, a viral vector, or, in some embodiments, can be RNA (such as mRNA) that is capable of translation.

[0108] In some embodiments of various aspects, the protein of interest can be expressed and displayed on the cell surface of the live bacterial strain of the present invention. In some embodiments, the protein of interest can be expressed and secreted from cells of the live bacterial strain of the present invention.

[0109] Target proteins include but are not limited to antibodies, antigens, etc.

[0110] In some preferred embodiments of various aspects, the target protein is an antigenic protein.Expression or display of the antigenic protein can further increase the immunogenicity of the live bacterial strain of the present invention.

[0111] In some embodiments of various aspects, the protein of interest is an antigenic protein of a species different from the bacterial species from which the live bacterial strain is derived.Expression or display of antigenic proteins of other species can confer immunogenicity to the live bacterial strain against the other species.

[0112] Exemplary antigenic proteins include, but are not limited to, EsxA, EsxB, PmtA, PmtC, pdhC, mutated Hla, mutated Spa, and the like.

[0113] In some embodiments, the live bacterial strains of the present invention are used to prevent and / or treat bacterial infections.

[0114] In one aspect, the present invention provides use of a live bacterial strain of the present invention in the preparation of a composition for preventing or treating a bacterial infection. In some embodiments, the composition is a vaccine.

[0115] In one aspect, the present invention provides a composition for preventing or treating a bacterial infection, comprising a live bacterial strain of the present invention. In some embodiments, the composition comprises an effective amount of a live bacterial strain of the present invention. In some embodiments, the composition is a vaccine.

[0116] In another aspect, the present invention provides a method for preventing and / or treating a bacterial infection in a subject, the method comprising administering to the subject an effective amount of a live bacterial strain of the present invention or a composition of the present invention.

[0117] As used herein, preventing and / or treating a bacterial infection also encompasses preventing and / or treating a disease or clinical signs or symptoms caused by a bacterial infection.

[0118] In some embodiments of various aspects, the bacterial infection is caused by a bacterial species derived from the live bacterial strains of the present invention.

[0119] In some embodiments of various aspects, the composition may further comprise an adjuvant. As used herein, "adjuvant" refers to an additional component in a vaccine that enhances the immune response, or an auxiliary molecule added to a vaccine, or an auxiliary molecule produced by the body after being induced by such additional components, such as, but not limited to, interferons, interleukins, or growth factors. "Adjuvants" as used herein may include aluminum hydroxide and aluminum phosphate, saponins, water-in-oil emulsions, oil-in-water emulsions, and water-in-oil-in-water emulsions.

[0120] In some embodiments of various aspects, the composition may further comprise a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antifungal agents, isotonic agents, and absorption delaying agents that are physiologically compatible. Non-limiting examples of pharmaceutically acceptable carriers include water, NaCl, normal saline, lactated Ringer's solution, standard sucrose, standard glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, saline solutions (such as Ringer's solution), alcohols, oils, gelatin, carbohydrates (such as lactose), amylose or starch, fatty acid esters, hydroxymethyl cellulose, polyvinyl pyrrolidone, and colorants.

[0121] In some embodiments of various aspects, the composition is formulated for intramuscular administration, intraperitoneal administration, subcutaneous administration, oral administration, or intranasal administration. In one embodiment, the composition is not for intravenous administration. In some embodiments, the composition is in lyophilized form, which can be reconstituted before use.

[0122] As used herein, "effective amount" refers to an amount of a substance, compound, material, or composition containing a compound (such as a live bacterial strain of the present invention or a composition of the present invention) that is at least sufficient to produce a prophylactic or therapeutic effect after administration to a subject. Thus, an effective amount is the amount necessary to prevent, cure, ameliorate, delay, or partially delay the symptoms of a disease or condition, such as a bacterial infection.

[0123] The actual dosage of the live strain or composition of the present invention to be administered to a subject can be determined according to the following physical and physiological factors: body weight, sex, severity of symptoms, type of disease to be treated, previous or current therapeutic interventions, the patient's disease of unknown etiology, time of administration, route of administration, etc. In any case, the amount of the live strain in the composition and the appropriate dosage for an individual subject will be determined by the medical personnel responsible for the administration.

[0124] In some embodiments of various aspects, the bacterial infection is caused by a bacterial strain of a serotype that is different from the serotype of the live bacterial strain of the invention.

[0125] In some embodiments of various aspects, the bacterial infection is a Staphylococcus aureus infection.

[0126] In some embodiments of various aspects, the S. aureus infection is a skin infection, a soft tissue infection, or an invasive disease. In some embodiments, the invasive disease is a bloodstream infection, endocarditis, or sepsis.

[0127] In some embodiments of various aspects, the S. aureus infection is a methicillin-resistant S. aureus (MRSA) infection or a methicillin-sensitive S. aureus (MSSA) infection. In some preferred embodiments, the infection is a recurrent S. aureus infection.

[0128] In one aspect, the present invention provides a method for reducing and / or increasing the immunogenicity of a live bacterial strain, or a method for producing a live bacterial strain with reduced virulence and / or increased immunogenicity, the method comprising reducing capsule production in the live bacterial strain and / or reducing the expression of one or more capsular polysaccharide synthesis genes in the live bacterial strain.

[0129] In some embodiments, the method comprises reducing capsular polysaccharide production in a live bacterial strain.

[0130] In some embodiments, the method comprises reducing expression of one or more capsular polysaccharide synthesis genes in the living bacterial strain.

[0131] In some embodiments, the method comprises introducing one or more mutations within one or more capsular polysaccharide synthesis genes into a living bacterial strain. Such mutations can be additions, substitutions or deletions of one or more nucleotides.

[0132] In some preferred embodiments, all capsular polysaccharide synthesis genes in the viable bacterial strain are mutated. In some embodiments, the mutation in the capsular polysaccharide synthesis gene results in reduced expression of the capsular polysaccharide synthesis protein, or results in expression of a capsular polysaccharide synthesis protein with reduced activity. In some embodiments, the mutation in the capsular polysaccharide synthesis gene results in no expression of the capsular polysaccharide synthesis protein, or results in expression of an inactive capsular polysaccharide synthesis protein. In some embodiments, the mutation is a frameshift mutation, which results in mistranslation of one or more capsular polysaccharide synthesis genes.

[0133] In some embodiments, the mutation comprises a deletion of a capsular polysaccharide synthesis gene, for example, a complete deletion or a partial deletion of a capsular polysaccharide synthesis gene. In some embodiments, one or more capsular polysaccharide synthesis genes are deleted. One or more capsular polysaccharide synthesis genes can be completely deleted from the strain so that one or more capsular polysaccharide synthesis proteins are not present in the viable bacterial strain of the present invention. One or more capsular polysaccharide synthesis genes can also be partially deleted so that only one or more inactive truncated capsular polysaccharide synthesis proteins are present in the viable bacterial strain of the present invention. In some preferred embodiments, all capsular polysaccharide synthesis genes in the viable bacterial strain of the present invention are completely deleted or partially deleted. In some preferred embodiments, all capsular polysaccharide synthesis genes in the viable bacterial strain of the present invention are completely deleted.

[0134] In some embodiments, the live bacterial strain is derived from Staphylococcus aureus. In some embodiments, the live bacterial strain is derived from Staphylococcus aureus serotype 5 or serotype 8.

[0135] In some embodiments, the one or more capsular polysaccharide synthesis genes encode one or more capsular polysaccharide synthesis proteins having an amino acid sequence having 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%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to any one of SEQ ID NOs: 2 to 17. In some embodiments, the one or more capsular polysaccharide synthesis proteins have the amino acid sequences set forth in SEQ ID NOs: 2 to 17, respectively.

[0136] In some embodiments, mutations such as deletions can be achieved by homologous recombination, for example double homologous recombination. In some embodiments, mutations are performed by targeted mutagenesis, such as via CRISPR, TALEN or ZFN technology.

[0137] In some embodiments, the method further comprises introducing a coding sequence of the target protein into the living bacterial strain, thereby enabling the living bacterial strain to express the target protein. In some embodiments, the target protein can be an endogenous protein or an exogenous protein.

[0138] In some embodiments, the coding sequence of the target protein is introduced into a living bacterial strain via a nucleic acid expression construct. In some embodiments, the introduced coding sequence of the target protein is integrated into the genome of the living bacterial strain.

[0139] Target proteins include but are not limited to antibodies, antigens, etc.

[0140] In some preferred embodiments of various aspects, the protein of interest is an antigenic protein. In some embodiments of various aspects, the protein of interest is an antigenic protein of a species different from the bacterial species from which the live bacterial strain is derived.

[0141] Exemplary antigenic proteins include, but are not limited to, EsxA, EsxB, PmtA, PmtC, pdhC, mutated Hla, mutated Spa, and the like.

[0142] Example

[0143] A further understanding of the present invention may be obtained by reference to the specific embodiments shown herein, which are intended to illustrate the present invention only and are not intended to limit the scope of the invention. It will be apparent that various modifications and variations may be made to the present invention without departing from the spirit of the present invention, and such modifications and variations are also within the scope of the present invention.

[0144] Methods and Materials

[0145] Construction of CP-knockout Staphylococcus aureus variants

[0146] Staphylococcus aureus Newman strain (NM) and its isogenic ΔCP variant were grown in brain heart infusion (BHI) at 37°C. Plasmid pIMAY was used to construct an unmarked, non-polar CP deletion. Briefly, primers cap-NM-Up-F (AGCTCCACCGCGGTGCTAGACATCGGTAAACGTACT), cap-NM-Up-R (TATCAATCTTAATATTTTCTTTATTTAAAAAAATGATT), cap-NM-Down-F-V2 (ATATTAAGATTGATATAAAAAACCCCTAATCATGAAGT) and cap-NM-Down-R-V2 (TCCACTAGTTCTAGACTTATTTTCGAAAACATATTGCAC) were used to PCR amplify the 5'- and 3'- flanking regions of the CP from the chromosomal DNA of the Staphylococcus aureus Newman strain. Each PCR product was then mixed to generate an in-frame deletion pattern of the CP. Overlapping amplicons containing in-frame deletion patterns were subcloned into pIMAY to generate pIMAY-star-cap-Newman-KO-v2.0. The recombinant plasmid pIMAY-star-cap-Newman-KO-v2.0 was introduced into DH5a and then electrotransformed into Staphylococcus aureus RN4220 and subsequently electrotransformed into Staphylococcus aureus Newman. Two-step selection for allelic replacement was performed as previously described [7]. Briefly, transformants were first cultured in BHI containing Cm10 at 28°C for 2 to 3 days. These clones were screened by PCR to select the first recombinant strain with primers cap-Chr-1st-up (GCCAAATCTTCAGAGAGATACAAAC) and cap-Chr-1st-down V2 (CTAGTCACCGCTGGTTGTCTA). The selected clones were then passaged 5 to 7 times in BHI without antibiotics at 28°C for 5 to 7 generations. Secondary recombination was induced by culturing cells at 37°C, and Cm-sensitive colonies were screened. CP deletion was further confirmed by PCR using primers specific for the deleted sequence (cap-NM-Up-F / cap-NM-Down-R-V2) and internal primers (cap-Chr-F (CCGAACCCATCAACTAATACAG) / cap-Chr-RV2 (CTCTGCAATATAATCTCGAACGT)) and DNA sequencing.

[0147] Animal infection experiments

[0148] A single dose of 200 μl of freeze-dried bacterial suspension (1×10 8BALB / c mice (7 weeks old, male and female) were immunized intraperitoneally or subcutaneously with 8 × 10 CFU of NM wt strain. Animals were monitored daily for skin abscesses or weight loss for 14 days, and serum was sampled on days 14 and 35. On day 35, mice were challenged intraperitoneally with 200 μl of lyophilized NM wt strain (8 × 10 CFU), and survival was monitored for 14 days. Serum samples were tested for antibody titration by whole-bacteria ELISA (NMΔspaΔsbi).

[0149] Example 1. Construction and characterization of ΔCP Staphylococcus aureus strain

[0150] Sequence analysis of NM wt revealed 16 consecutive ORFs that were transcribed in the same orientation and were designated cap5A to cap5P. These 16 cap5 genes (cap5A-p) were clustered in a 16.7 kb chromosomal region and co-transcribed from a major promoter upstream of cap5A ( Figure 1 )[8,9]. All of these genes were targeted for markerless in-frame deletion and successfully eliminated from the NMwt and NMΔadsA strains to generate the corresponding NMΔCP and NMΔadsAΔCP strains. PCR results confirmed the deletion of all targeted CP genes in the parental strains.

[0151] Example 2. Characterization of ΔCP Staphylococcus aureus strains

[0152] 1.ΔCP strains showed lower virulence

[0153] In the mouse model, BALB / c mice immunized with the ΔCP strain showed fewer skin abscesses ( Figure 2 ).

[0154] 2.ΔCP strain showed higher immunogenicity

[0155] To measure antibody-mediated immune responses, BALB / c mice were immunized with the ΔCP strain and the antibody titers were determined by enzyme-linked immunosorbent assay (ELISA). The NMΔadsAΔCP strain showed significantly higher antibody titers on day 35 compared to the other strains ( Figure 3 ).

[0156] 3.ΔCP strain showed higher protective effect

[0157] Survival studies also showed improved protection over NW wt. The double knockout strain NMΔadsAΔCP showed 100% protection, while its isogenic strain NMΔadsA had only 40% protection ( Figure 4 ).

[0158] 4. The ΔCP strain exhibits cross-reactivity

[0159] The ΔCP strain can produce reactive antibodies against different serotypes.

[0160] Sequence information

[0161] SEQ ID NO 1 AdsA amino acid sequence

[0162] MKALLLKTSVWLVLLFSAMGLWQVSSAAEQHTPMKAHAVTTIDKATTDRQLVLPTKEAAHQSGEEAATNVSASAQGTADDTNNKVTSNAPSNKPSTAVSTTVNETHDVDAQQASTQKPTQSATFKLSNAKTASLSPRMFAANAPQTTTHKILHTNDIHGRLAEEKGRVIGMAKLKTVKEQEKPDLILDAGDAFQGLPLSNQSKGEEMAKAMNAVGYDAMAVGNHEFDFGYDQLKKLEGMLDFPMLSTNVYKDGKRAFKPSTIVTKNGIRYGIIGVTTPETKTKTRPEGIKGVEFRDPLQSVTAEMMRIYKDVDTFVVISHLGIDPSTQETWRGDYLVKQLSQNPQLKKRITVIDGHSHTVLQNGQIYNNDALAQTGTALANIGKVTFNYRNGEVSNIKPSLINVKDVENVTPNKALAEQINQADQTFRAQTAEVIIPNNTIDFKGERDDVRTRETNLGNAITDAMEAYGVKNFSKKTDFAVTNGGGIRASIAKGKVTRYDLISVLPFGNTIAQIDVKGSDVWTAFEHSLGAPTTQKDGKTVLTANGGLLHISDSIRVYYDMNKPSGKRINAIQILNKETGKFENIDLKRVYHVTMNDFTASGGDGYSMFGGPREEGISLDQVLASYLKTANLAKYDTTEPQRMLLGKPAVSEQPAKGQQGSKGSESGKDTQPIGKDKVMNPAKQPATGKVVLLPTHRGTVSSGAEGSDCALEGTAVSSKSGKQLTKMSASKGSGHEKQLPKTGTNQSSSPAAIFVLVAGIGLIATVRRRKAS

[0163] SEQ ID NO 2 capA (NWMN_0095):

[0164] MESTLELTKIKEVLQKNLKILIILPLLFLILISAIVTFFVLSPKYQANTQILVNQTKGDNPQFMAQEVQSNIQLVNTYKEIVKSPRILDEVSKDLNDKYSPSKLSSMLTITN QENTQLINIQVKSGHKQDSEKIANSFAKVTSKQIPKIMSVDNVSILSKADGTAVKVAPKTVNLIGAFFLGLVVALIYIFFKVIFDKRIKDEEDVEKELGLPVLGSIQKFN*

[0165] SEQ ID NO 3 capB(NWMN_0096):

[0166] MLLMSKKENTTTTLFVYEKKSTISEKFRGIRSNIMFSKANGEVKRLVTSEKPGAGKSTVVSNVAITYAQAGYKTLVIDGDMRKPTQNYIFNEQNNNGLSSLIIGRTTMSEAITS TEIENLDLLTAGPVPPNPSELIGSERFKELVDLFNKRYDIIIVDTPPVNTVTDAQLYARAIKDSLLVIDSEKNDKNEWKKALMEKAGSNILGVILNKTKVDKSSSYYHYGDE*

[0167] SEQ ID NO 4 capC(NWMN_0097):

[0168] MIDIHNHILPNIDDGPTNETEMMDLLKQATTQGVTEIIVTSHHLHPRYTTPIEKVKSCLNHIESLEEVQALNLKFYYGQEIRITDQILNDIDRKVINGINDSRYLLIEFPSNEVPHYTDQLFFELQS KGFVPIIAHPERNKAISQNLDILYDLINKGALSQVTTASLAGISGKKIRKLAIQMIENNLTHFIGSDAHNTEIRPFLMKDLFNDKKLRDYYEDMNGFISNAKLVVDDKKIPKRMPQQDYKQKRWFGL*

[0169] SEQ ID NO 5 capD(NWMN_0098):

[0170] *

[0171] SEQ ID NO 6 capE(NWMN_0099):

[0172] MFDDKILLITGGTGSFGNAVMKQFLDSNIKEIRIFSRDEKKQDDIRKKYNNSKLKFYIGDVRDSQSVETAMRDVDYVFAAALKQVPSCEFFPVEAVKTNIIGTENVLQSAIHQNVKKVICLSTDKAAYPINAMGISKAMMEKVFVAKSRNIRSEQTLICGTRYGNVMASR *

[0173] SEQ ID NO 7 capF(NWMN_0100):

[0174] MNIVITGAKGFVGKNLKADLTSTTDHHIFEVHRQTKEEELESALLKADFVVHLAGVNRPEHDKEFSLGNVSYLDHVLDILTRNTKKPAILLSSSIQATQDNPYGESKLQGEQLLREYAEEYGNTVYIYRWPNLFGKWCKPNYNSVIATFCYKIARNEEIQVNDRNVELTLNYVDDIVAEIKRAIE GTPTIENGVPTVPNVFKVTLGEIVDLLYKFKQSRLDRTLPKLDNLFEKDLYSTYLSYLPSTDFSYPLLMNVDDRGSFTEFIKTPDRGQVSVNISKPGITKGNWHHTKNEKFLVVSGKGVIRFRHVNDDEIIEYYVSGDKLEVVDIPVGYTHNIENLGDTDMVTIMWVNEMFDPNQPDTYFLEV*

[0175] SEQ ID NO 8 capG(NWMN_0101):

[0176] MEKLKLMTIVGTRPEIIRLSSTIKACDQYFNQILVHTGQNYDYTLNQIFFDDLELRQPDHYLEAVGSNLGETMGNIIAKTYDVLLREQPDALLILGDTNSCLAAVSAKRLKIPVFHMEAGNRCFDQNVPEEINRKIVDHVSDVNLPYTEHSRRYLLDEGFNKANIFVTGSPMTEVIEAHRDKINHSDVLNKLGLEPQQYILVSAHREENIDNEKNFKSLMNAINDIAKKYKMPVIYSTHPRSWKKIEESKFEFDPLVKQLKPFGFFDYNALQKDAFVVLSDSGTLSEESSILKFPGVLIRTSTERPEVLDKGTVIVGGITYNNLIQSVELAREMQNNNEPMIDAIDYKDTNVSTKVVKIIQSYKDIINRNTWRK*

[0177] SEQ ID NO 9 capH(NWMN_0102):

[0178] MRIAIEKIIGLLKNQSSKESNVKIHRLAYITNSKFDGNNYIDRWCKIRNSHIGEYSYIGFGSDFNNVEVGRYCSISSDVKIGLGKHPTHFFSSSPIFYSNNNPFNIKQKFIDFNDQPSRTTIKNDVWIGANVIIMDGLTINTGAVIAAGSVVTKNVGAYEVVGGVPAKVIKKRFDNKTIEKLLESKWWEKTPDKLKGFSVEYLNKKDT*

[0179] SEQ ID NO 10 capI(NWMN_0103):

[0180] MRILNIVSSNIVQDPRVLKQIETIKGVTDDYKIVGMNNSQATNKRLENLDCNYRLLGSKVDPKNILSKLIKRIRFATGVIREIKAYKPDVIHANDFDVLLMVYLSNYKKANIVYDAHEIYAKNAFINKVPLISKFVESIEKHIVKHRVNAFVTVSHAAKEYYQSKGYKKEANVITNAPILNDSREFKEIENFKEIVYQGQIVMDRGYEEFIIASSAFKQNAPSFIIRGFGPHEEVIKELISYNPENIRLDKPVEVKELVDKLAESNVGVVLTKPVSINFEYTVSNKIFECIHAGLPVILSPVKEHIYLNEKYKFGIVLKEVTPLEIEKAVRKLRDNHDLFNHLRQNAIKASKILNWQIESERLVELYKF*

[0181] SEQ ID NO 11 capJ(NWMN_0104):

[0182] MKFFVLCAIISMNIFIVISTFTKEVLGFPIEPVYYSTMVGIALITTVFAIYKIIVTQEIPRGLILLIAICLLYLAFYYFSPDKEEKLAKNNILFFLTWAVPAAISGIYIKYINKATVERFFKLVFFIFSVSFIFVILIPKLTGEIPSYINFGLMNYQNASYLSAFTAGLGIYFIMKGSVKHKWIYVLFTIIDIPIVFIPGGRGGAILLILYGLFAFILITFKRGIPIAVKSIMYIFALSISSVLIYFLFTKGSNTRTFSYLQGGTLNLEGTSGRGPIYEKGIYFIQQSSLLGYGPFNYYKLIGNIPHNIIIELILSFGLLGFFIIMICILLLVYKMIRNYDPNTIDLLVMFIAIYPITLLMFSSNYLVVSEFWFVLFYFITKGRRHHG*

[0183] SEQ ID NO 12 capK(NWMN_0105):

[0184] MAKKVFIMDSVKTIIGTLLIALGLQFLAYPIINQRVGNEAFGSILTIYTIITITSVVLGNTLNNIRLINMNLYKSNHYYWKFASILLISILIESIALIIVFLYFFNLNIIDIIFLILLNILMCLRIYLNVFFRMTLKYNQILYIALIQFLGLLIGLFLYYLTQNWIVCFITSELFATIYTLVKLRGLTIGEYQSEDNNVVKDYVMLLSTNSLNNLNLYLDRLILLPIIGGTAVTISFLSTFIGKMLATFLYPINNVVLSYISVNESDNIKKQYLKTNLIAIAALCLVMIICYPITIIIVSLLYNIDSSLYSKFIILGNIGVLFNAVSIMIQTLNTKHASITLQANYMTLHTITFIFITILMTIAFGLNGFFWTTLFSNIIKYVILNIIGLKSKFINKKDVD*

[0185] SEQ ID NO 13 capL(NWMN_0106):

[0186] MSEKKILILCQYFYPEYVSSATLPTQLAEDLIANHINVDVMCGWPYEYSNHKQVSKTEMHRGIRIRRLKYSRFNNKSKVGRIINFFSLFSKFVINIPKMLKYDQILVYSNPPILPLIPDVLHRLLKKKYSFVVYDIAPDNAIKTGATRPGSMIDKLMRYINRHVYKNAENVIVLGTEMKNYLLNHQISKNADNIHVIPNWYDMRQLQDNRIYNDTFKAYREQYDKILLYSGNMGQLQDMETLISFLKLNKDQSQTLTILCGHGKKFADVKTAIEDHRIENVKMFEFLTGTDYADVLKIADVCIASLIKEGVGLGVPSKNYGYLAAKKALVLIMDKQSDIVQHVEQYDAGIQIDNGDAHAIYNFINTHSSKELHEMGERAHQLFKDKYTREINTMKYYNLLK*

[0187] SEQ ID NO 14 capM(NWMN_0107):

[0188] MKRLFDVVSSIYGLVVLSPILLITALLIKMESPGPAIFKQKRPTINNELFNIYKFRSMKIDTPNVATDLMDSTSYITKTGKVIRKTSIDELPQLLNVLKGEMSIVGPRALYNQYELIEKRTKANVHTIRPGVTGLAQVMGRDDITDDQKVAYDHYYLTHQSMMLDMYIIYKTIKNIVTSEGVHH*

[0189] SEQ ID NO 15 capN(NWMN_0108):

[0190] MRKNILITGVHGYIGNALKDKLIEQGHQVDQINVRNQLWKSTFKDYDVLIHTAALVHNNSPQARLSDYMQVNMLLTKQLAQKAKAEDVKQFIMSTMAVYGKEHVGKSDQVDTQTPMNPTTNYGISKFAEQALQELISDSFKVAI VRPPMIYGAHCPGNFQRLMQLSKRLPIIPNINNQRSALYIKHLTAFIDQLISLEVTWGYHPQDSfyFDTSSVMYEIRRRQSHRKTVLINMPSMLNKYFNKLSVFRKLFGNLIYSNTLYENNNALEIIPGKMSLVIADMETTTKDKA*

[0191] SEQ ID NO 16 capO(NWMN_0109):

[0192] MKLTVVGLGYIGLPTSIMFAKHGVDVLGVDINQQTIDKLQSGQISIEEPGLQEVYEEVLSSGKLKVSTTPDASDVFIIAVPTPNNDDQYRSCDISLVMRALDSILSFLEKGNTIIVESTIAPKTMDDFVKPVIENLGFTIGEDIYLVHCPERVLPGKILEELVHNNRIIGGVTEACIEAGKRVYRTFVQGEMIETDARTAEMSKLMENTYRDVNIALANELTKICNNLNINVLDVIEMANKHPRVNIHQPGPGVGGHCLAVDPYFIIAKDPENAKLIQTGREINNSMPAYVVDTTKQIIKVLSGNKVTVFGLTYKGDVDDIRESPAFDIYELLNQEPDIEVCAYDPHVELDFVEHDMSHAVKDASLVLILSDHSEFKNLSDSHFDKMKHKVIFDTKNVVKSSFEDVSYYNYGNIFNFIDK*

[0193] SEQ ID NO 17 capP(NWMN_0110):

[0194] MCLNFREDNVMKKIMVIFGTRPEAIKMAPLVKEIDHNGNFEANIVITAQHRDMLDSVLSIFDIQADHDLNIMQDQQTLAGLTANALAKLDSIINEEQPDMILVHGDTTTTFVGSLAAFYHQIPVGHVEAGLRTHQKYSPFPEELNRVMVSNIAELNFAPTVIAAKNLLFENKDKERIFITGNTVIDALSTTVQNDFVSTIINKHKGKKVVLLTAHRRENIGEPMHQIFKAVRDLADEYKDVVFIYPMHRNPKVRAIAEKYLSGRNRIELIEPLDAIEFHNFTNQSYLVLTDSGGIQEEAPTFGKPVLVLRNHTERPEGVEAGTSRVIGTDYDNIVRNVKQLIEDDEAYQRMSQANNPYGDGQASRRICEAIEYYFGLRTDKPDEFVPLRHK*

[0195] SEQ ID NO 18 capA(NWMN_0095):

[0196] ATGGAAAGTACATTAGAATTAACAAAAATTAAAGAAGTATTACAAAAAAACTTGAAGATTTTAATTATTTTACCGCTATTATTTTTAATTATTAGCGCTATTGTTACATTTTTCGTCTTATCACCTAAATATCAAGCTAATACTCAAATTTTAGTGAATCAAACTAGGGTGACAATCCTCAGTTTATGGCGCAAGAGGTTCAAAGTAATATTCAACTTGTAAATACGTATAAAGAAATTGTTAAAAGTCCTAGAATTTTAGATGAGGTGTCAAAGGACTTAAATGATAAGTATTCACCATCTAAATTGTCGAGTATGTTGACAATTACAAACC AAGAAAATACGCAACTTATCAACATCCAAGTTAAAAGTGGTCATAAACAAGATTCGGAAAAAATTGCGAATAGCTTCGCTAAAGTTACAAGTAAACAAATTCCGAAGATTATGAGTGTGGATAACGTATCAATTTTATCTAAAGCAGACGGTACAGCAGTTAAAGTCGCACCAAAAACTGTAGTGAATCTAATCGGTGCATTCTTTTTAGGATTAGTTGTCGCGCTTATATATATCTTCTTCAAAGTAATTTTCGATAAGCGAATTAAAGATGAAGAAGATGTAGAGAAAGAATTAGGATTGCCTGTATTGGGTTCAATTCAAAAATTTAATTAA

[0197] SEQ ID NO 19 capB(NWMN_0096):

[0198] TTGCTACTTATGTCAAAAAAGGAAAATACGACAACAACACTATTTGTATATGAAAAACCAAAATCAACAATTAGTGAAAAGTTTCGAGGTATACGTTCAAACATCATGTTTTCAAAAGCAAATGGTGAAGTAAAGCGCTTATTGGTTACTTCTGAAAAGCCTGGTGCAGGTAAAAGTACAGTTGTATCGAATGTAGCGATTACTTATGCACAAGCAGGCTATAAGACATTAGTTATTGATGGCGATATGCGTAAGCCAACACAAAACTATATTTTTAATGAGCAAAATAATAATGGACTATCAAGCTTAATCATTGGTCGAACGACTATGTCAGAAGCAATTACGTCG ACAGAAAATTGAAAATTTAGATTTGCTAACAGCTGGCCCTGTACCTCCAAATCCATCTGAGTTAATTGGGTCTGAAAGGTTCAAAGAATTAGTTGATCTGTTTAATAAACGTTACGACATTATTATTGTCGATACACCGCCAGTTAATACTGTGACTGATGCACAACTATATGCGCGTGCTATTAAAGATAGTCTGTTAGTAATTGATAGTGAAAAAAATGATAAAAATGAAGTTAAAAAAGCAAAAGCACTTATGGAAAAAGCAGGCAGTAACATTCTAGGTGTCATTTTGAACAAGACAAAGGTCGATAAATCTTCTAGTTATTATCACTATTATGGAGATGAATAA

[0199] SEQ ID NO 20 capC(NWMN_0097):

[0200] ATGATTGATATTCATAACCATATATTGCCTAATATCGATGACGGTCCGACAATGAAACAGAGATGATGGATCTTTTAAAACAAGCGACAACACAAGGTGTTACAGAAATCATTGTAACATCACATCACTTACATCCTCGATATACCACACCTATAGAAAAAGTGAAATCATGTTTAAACCATATTGAAAGCTTAGAGGAAGTACAAGCACTAAATCTAAAGTTTTATTATGGTCAGGAAATAAGAATTACCGATCAAATCCTTAATGATATTGATCGAAAGTTATTAACGGTATTAATGATTCACGCTATTTACTAATAGAATTTCCATCAAATGAAGTTCCACACTATACTGATCAATTATTTTTCGAATTACAGAGTA AAGGCTTTGTACCGATTATTGCACATCCAGAGCGGAATAAAGCAATAAGTCAAAACCTTGACATACTATACGATTTAATTAACAAAGGTGCTTTAAGTCAAGTGACAACGGCGTCATTAGCGGGTATTTCCGGTAAAAATTAGAAAATTAGCAATTCAAATGATTGAAAACAATCTGACACATTTCATC GGTCAGATGCGCATAACACAGAAATCAGACCGTTCTTAATGAAAGACTTATTTAATGATAAGAAATTACGTGATTATTATGAAGATATGAACGGATTTATTAGTAATGCGAAGTTAGTTGTTGATGATAAAAGATTCCTAAACGAATGCCACAACAAGATTATAAACAGAAAAGATGGTTTGGGTTATAA

[0201] SEQ ID NO 21 capD(NWMN_0098):

[0202]

[0203] SEQ ID NO 22 capE(NWMN_0099):

[0204]

[0205] SEQ ID NO 23 capF(NWMN_0100):

[0206]

[0207] SEQ ID NO 24 capG(NWMN_0101):

[0208]

[0209] SEQ ID NO 25 capH(NWMN_0102):

[0210] ATGAGGATAGCGATTGAAAAGATAATTGGTTTGCTGAAAAACCAGTCCTCTAAAGAATCGAATGTTAAGATTCATCGCTTGGCGTATATTACAAACTCAAAATTTGATGGCAATAACTATATAGATAGATGGTGTAAAATCAGGAATTCTCACATTGGTGAATACAGTTATATTGGATTTGGTAGTGATTTTAATAATGTAGAAGTAGGAAGATATTGTTCGATATCTTCGGATGTAAAAATTGGGTTAGGAAAACATCCTACACACTTTTTTAGCTCATCACCGATTTTTTATTCTAATAATAATCCATTTA ACATAAAGCAAAAGTTTATAGACTTTAATGACCAACCAAGCCGTACAACAATTAAAAATGATGTGTGGATTGGTGCAAATGTAATTATTATGGATGGTTTAACAATAAATACTGGTGCAGTCATAGCAGCCGGCTCAGTTGTTACTAAAAATGTAGGAGCATATGAGGTTGTTGGTGGTGTTCCTGCAAAAGTGATTAAGAAGCGATTTGACAATAAAACAATTGAAAAACTTTTGGAAAGCAAGTGGTGGGAGAAAACGCCTGACAAACTAAAAGGATTTTCGGTTGAATATTTAAATAAAAAGGATACTTAA

[0211] SEQ ID NO 26 capI(NWMN_0103):

[0212]

[0213] SEQ ID NO 27 capJ(NWMN_0104):

[0214]

[0215] SEQ ID NO 28 capK(NWMN_0105):

[0216]

[0217] SEQ ID NO 29 capL(NWMN_0106):

[0218]

[0219] SEQ ID NO 30 capM(NWMN_0107):

[0220] ATGAAGCGATTATTCGATGTAGTGAGTTCAATATATGGTTTAGTAGTTTTAAGTCCGATTCTGTTAATTACAGCATTACTAATTAAAATGGAATCACCTGGACCAGCCATTTTCAAACAAAAAAGACCGACGATTAATAATGAATTGTTTAATATTTATAAGTTTAGATCAATGAAAATAGACACACCTAATGTTGCAACTGATTTAATGGATTCAACATCGTATATAACAAAGACAGGGAAGGTCATTCGTAAGACCTCTATTGATGAATTGCCACAA TTATTGAATGTTTTAAAAGGAGAAATGTCAATTGTAGGTCCTAGACCAGCGCTTTATAATCAATACGAATTAATCGAAAAACGTACAAAAGCGAACGTGCATACGATTAGACCAGGGTGTGACAGGACTAGCTCAAGTGATGGGGAGAGATGATATCACTGATGATCAAAAAGTAGCGTATGATCATTATTACTTAACACATCAATCTATGATGCTTGATATGTATATCATATATAAAACAATTAAAAATATCGTTACTTCAGAAGGTGTGCATCACTAA

[0221] SEQ ID NO 31 capN(NWMN_0108):

[0222] ATGAGAAAAAATATTTTAATTACAGGCGTACATGGATATATCGGTAATGCTTTAAAAGATAAGCTTATTGAACAAGGACATCAAGTAGATCAAATTAATGTTAGGAATCAATTATGGAAGTCGACCTCGTTCAAAGATTATGATGTTTTAATTCATACAGCAGCTTTGGTTCACAACAATTCACCTCAAGCAAGGCTATCTGATTATATGCAAGTGAATATGTTGCTGACGAAACAATTGGCACAAAAGGCTAAAGCTGAAGACGTTAAACAATTTATTTTTATGAGTACTATGGCAGTTTATGGAAAAGAAGGTCATGTTGGTAAATCAGATCAAGTTGATACACAAACACCAATGAACCCTACGACCAACTATGGTATTTCCAAAAAGTTCGCTGAACAAGCATTACAAGAATTGATTAGTGATTCGTTTAAAGTAGCAATT GTGAGACCACCAATGATTTATGGTGCACATTGCCCAGGAAATTTCCAACGGTTAATGCAATTGTCAAAGCGATTGCCAATCATTCCCAAATATTAACAATCAGCGCAGTGCATTATATATTAAACATCTGACAGCATTTATTGATCAATTAATATCATTAGAAGTGACAGGTGTGTACCATCCTCAAGATAGTTTTTACTTTGATACATCGTCAGTAATGTATGAAATACGTCGCCAATCACATCGTAAAACGGTATTGATCAACATGCCTTCAATGCTAAATAAGTATTTTAATAAGTTGTCGGTCTTTAGAAAATTATTCGGCAATTTAATATACAGCAATACGTTATATGAAAATAATAATGCACTTGAAATTATTCCTGGAAAAATGTCACTTGTTATTGCGGACATCATGGATGAAACGACAACCAAAGATAAGGCATAA

[0223] SEQ ID NO 32 capO(NWMN_0109):

[0224]

[0225] SEQ ID NO 33 capP(NWMN_0110):

[0226]

[0227] References

[0228] 1. Roberts, I.S., THE BIOCHEMISTRY AND GENETICS OF CAPSULAR POLYSACCHARIDE PRODUCTION IN BACTERIA. Annual Review of Microbiology, 1996. 50(1): p. 285-315.

[0229] 2. Cocchiaro, J.L., et al., Molecular characterization of the capsule locus from non-typeable Staphylococcus aureus. Molecular microbiology, 2006. 59(3): p. 948-960.

[0230] 3. Teymournejad, O. and C.P. Montgomery, Evasion of Immunological Memory by S. aureus Infection: Implications for Vaccine Design. Frontiers in Immunology, 2021. 12: p. 430.

[0231] 4. Clegg, J., et al., Staphylococcus aureus Vaccine Research and Development: The Past, Present and Future, Including Novel Therapeutic Strategies. Frontiers in Immunology, 2021. 12(2693).

[0232] 5.Bagnoli,F.,S.Bertholet,and G.Grandi,Inferring reasons for thefailure of Staphylococcus aureus vaccines in clinical trials.Frontiers incellular and infection microbiology,2012.2:p.16-16.6.Boyle-Vavra,S.,et al.,USA300 and USA500 clonal lineages of Staphylococcus aureus do not produce acapsular polysaccharide due to conserved mutations in the cap5 locus.MBio,2015.6(2):p.e02585-14.

[0233] 7.Bae,T.and O.Schneewind,Allelic replacement in Staphylococcus aureuswith inducible counter-selection.Plasmid,2006.55(1):p.58-63.

[0234] 8.Wann,E.R.,et al.,Genetic analysis of the cap5 locus ofStaphylococcus aureus.FEMS microbiology letters,1999.170(1):p.97-103.

[0235] 9.Sau,S.,et al.,The Staphylococcus aureus allelic genetic loci forserotype 5and 8capsule expression contain the type-specific genes flanked bycommon genes.Microbiology,1997.143(7):p.2395-2405.

[0236] The foregoing description of specific embodiments will fully reveal the general nature of the present invention so that others can easily modify and / or adjust such specific embodiments for various applications without departing from the general concept of the present invention by applying the knowledge in the technology of the relevant fields (including the contents of the documents cited and incorporated herein by reference) without excessive experimentation. Therefore, based on the teachings and guidance presented herein, such adjustments and modifications are intended to be within the meaning and scope of the equivalents of the disclosed embodiments. It should be understood that the wording or terminology herein is for descriptive and not limiting purposes, so that the terms or wording of this specification are interpreted by those skilled in the art based on the teachings and guidance presented herein, in conjunction with the knowledge of those skilled in the relevant art.

[0237] Although various embodiments of the present disclosure have been described above, it should be understood that they are presented by way of example and not limitation. It will be apparent to those skilled in the relevant art that various changes in form and detail can be made without departing from the spirit and scope of the present disclosure. Therefore, the present disclosure should not be limited by any of the above exemplary embodiments, but should be limited only in accordance with the appended claims and their equivalents.

[0238] All references cited herein are incorporated by reference in their entirety and for all purposes to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

Claims

1. A live bacterial strain that, compared to a corresponding control strain, does not produce capsular polysaccharide; wherein the live bacterial strain has no adenosine synthase A (AdsA) activity; and wherein the live bacterial strain is derived from the Staphylococcus aureus Newman strain.

2. The live bacterial strain according to claim 1, wherein the live bacterial strain comprises one or more mutations within one or more of the capsular polysaccharide synthesis genes capA, capB, capC, capD, capE, capF, capG, capH, capI, capJ, capK, capL, capM, capN, capO, and capP, which result in non-expression of the capsular polysaccharide synthesis protein or expression of an inactive capsular polysaccharide synthesis protein.

3. The live bacterial strain according to claim 2, wherein the mutation comprises a complete or partial deletion of the capsular polysaccharide synthesis gene in the live bacterial strain.

4. The live bacterial strain according to claim 3, wherein the capsular polysaccharide synthesis gene in the live bacterial strain is completely deleted.

5. The live bacterial strain according to claim 2, wherein the mutation is achieved by homologous recombination or by targeted mutagenesis.

6. The live bacterial strain according to claim 5, wherein the mutation is achieved via CRISPR, TALEN, or ZFN technology.

7. The live bacterial strain according to claim 1, wherein the live bacterial strain has reduced virulence compared to a corresponding control strain.

8. The live bacterial strain according to claim 1, wherein the live bacterial strain has increased immunogenicity compared to a corresponding control strain.

9. The live bacterial strain according to claim 1, wherein the Staphylococcus aureus live strain comprises a mutation in the AdsA gene encoding AdsA.

10. The live bacterial strain according to claim 9, wherein the mutation in the AdsA gene results in non-expression of the AdsA protein or expression of an inactive mutant AdsA protein.

11. The live bacterial strain according to claim 10, wherein the mutation comprises a complete or partial deletion of the AdsA gene in the live bacterial strain.

12. The live bacterial strain according to claim 11, wherein the AdsA gene in the live bacterial strain is completely deleted.

13. The live bacterial strain according to claim 10, wherein the mutation is achieved by homologous recombination or by targeted mutagenesis.

14. The live bacterial strain according to claim 13, wherein the mutation is achieved via CRISPR, TALEN, or ZFN technology.

15. The live bacterial strain according to any one of claims 1 to 14, wherein the live bacterial strain is used as a live expression vector for expressing a target protein.

16. The live bacterial strain according to claim 15, wherein the live bacterial strain further comprises a coding sequence of the target protein and is thereby capable of expressing the target protein.

17. The live bacterial strain according to claim 16, wherein the coding sequence of the target protein is introduced into the live bacterial strain by a nucleic acid expression construct.

18. The live bacterial strain according to claim 17, wherein the introduced coding sequence of the target protein is integrated into the genome of the live bacterial strain.

19. The live bacterial strain according to claim 15, wherein the target protein is expressed and displayed on the cell surface of the live bacterial strain; or the target protein is expressed and secreted out of the cells of the live bacterial strain.

20. The live bacterial strain according to claim 15, wherein the target protein is selected from an antibody or an antigen.

21. The live bacterial strain according to claim 20, wherein the target protein is an antigen selected from EsxA, EsxB, PmtA, PmtC, pdhC, mutant Hla, mutant Spa.

22. Use of the live bacterial strain according to any one of claims 1 to 21 in the preparation of a composition for preventing or treating a bacterial infection, wherein the bacterial infection is a Staphylococcus aureus infection.

23. A composition for preventing or treating a bacterial infection, the composition comprising the live bacterial strain according to any one of claims 1 to 22, wherein the bacterial infection is a Staphylococcus aureus infection.

24. The composition according to claim 23, wherein the composition further comprises an adjuvant and / or a pharmaceutically acceptable carrier.

25. The use according to claim 22 or the composition according to claim 23 or 24, wherein the Staphylococcus aureus infection is a skin infection, a soft tissue infection or an invasive disease.

26. The use or composition according to claim 25, wherein the invasive disease is a bloodstream infection, endocarditis or sepsis.

27. The use according to claim 22 or the composition according to claim 23 or 24, wherein the Staphylococcus aureus infection is a methicillin-resistant Staphylococcus aureus (MRSA) infection or a methicillin-sensitive Staphylococcus aureus (MSSA) infection.

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