A live bacterial strain of the staphylococcus genus

Abstract

This invention relates to the field of biomedicine. In particular, this invention relates to bacteria derived from the genus *Staphylococcus* (…). Staphylococcus sp. Such as Staphylococcus aureus ( Staphylococcus aureus The present invention relates to live bacterial strains of the genus Staphylococcus, such as live bacterial strains of Staphylococcus aureus, having reduced saePQRS activity, reduced adsA activity and / or reduced capsule production; to the use of said live bacterial strains; to vaccines against bacterial infections comprising said live bacterial strains; and to methods for preventing and / or treating bacterial infections in subjects by administering said live bacterial strains.

Description

Technical Field

[0001] This invention relates to the field of biomedicine. In particular, this invention relates to bacteria derived from the genus *Staphylococcus* (…). Staphylococcus sp. Such as Staphylococcus aureus ( Staphylococcus aureus The present invention relates to live bacterial strains of the genus Staphylococcus, such as live bacterial strains of Staphylococcus aureus, having reduced saePQRS activity, reduced adsA activity and / or reduced capsule production; to the use of said live bacterial strains; to vaccines against bacterial infections comprising said live bacterial strains; and to methods for preventing and / or treating bacterial infections in subjects by administering said live bacterial strains. Background Technology

[0002] Staphylococcus aureus is a highly adaptable pathogen capable of infecting a wide range of tissues. Common manifestations of Staphylococcus aureus infections range from mild skin and soft tissue infections to invasive diseases. Furthermore, this pathogen has developed resistance to many antimicrobial agents, leaving healthcare providers with few options for treating infections. Therefore, developing new approaches to the treatment and prevention of Staphylococcus aureus infections is an urgent clinical need.

[0003] Live attenuated vaccines are weakened forms of pathogens designed to stimulate an immune response without causing disease. Virulence attenuation is achieved through natural selection via multiple passages of the pathogen in growth media, random mutagenesis, or genetic modification of target genes to induce attenuation. Genetic modification capable of achieving higher levels of virulence attenuation has become one of the most suitable methods for developing live attenuated vaccines. Target selection is crucial for the successful development of live attenuated vaccines.

[0004] Staphylococcus aureus has evolved multiple mechanisms to evade the killing effects of these potent innate immune cells. During pathogenicity, the production of secreted virulence factors is primarily regulated by a two-component system (TCS) capable of sensing the host environment and responding accordingly. Among these, the Sae TCS has been shown to be essential for evading human neutrophil killing. This system consists of the histidine kinase sensor SaeS, the response regulator SaeR, and two accessory proteins, SaeP and SaeQ. The sensor protein SaeS detects environmental signals (such as changes in pH or oxygen levels) and then phosphorylates the response regulator SaeR. The phosphorylated SaeR then binds to the promoter region of target genes, activating or inhibiting their transcription. Studies have demonstrated that the SaeR response regulator and its homologous sensor kinase SaeS play a crucial role in initiating infection. The Public University of Navarre selected a mutant strain lacking all TCS genes (including SaeRS) as a candidate for a live attenuated strain for vaccination purposes (EP3342854A1). To date, no one has reported that the saePQ gene (one of the TCS) has a virulence-attenuating function. On the contrary, the study by Collins, Madison M. et al. showed that the saeP and saeQ double mutant (USA300ΔsaePQ) enhanced the virulence of USA300 during mouse bacteremia[1].

[0005] Given the failure of past vaccines against Staphylococcus aureus, different approaches are needed to produce an effective vaccine. Summary of the Invention

[0006] This invention provides at least the following embodiments: Implementation Scheme 1. A live bacterial strain from the genus Staphylococcus, wherein, for example, compared with a corresponding control strain, i) The live bacterial strain has reduced activity of the sae two-component system, preferably lacking the activity of the sae two-component system; ii) Reduced expression of one or more genes of the SAE two-component system in the live bacterial strain; and / or iii) The live bacterial strain said to contain one or more mutations in one or more genes of the sae two-component system.

[0007] Implementation Scheme 2. The live bacterial strain according to Implementation Scheme 1, wherein, compared with the corresponding control strain, the expression of one or more genes of the sae two-component system 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.

[0008] Implementation Scheme 3. A live bacterial strain according to Implementation Scheme 1 or 2, wherein the expression of one or more genes selected from SaeS, SaeR, SaeP and SaeQ in the live bacterial strain is reduced, preferably, the expression of all SaeS, SaeR, SaeP and SaeQ in the live bacterial strain is reduced, more preferably, SaeS, SaeR, SaeP and SaeQ are not expressed in the live bacterial strain.

[0009] Implementation Scheme 4. The live bacterial strain according to Implementation Scheme 1, wherein the live bacterial strain contains one or more mutations in one or more genes selected from SaeS, SaeR, SaeP and SaeQ, preferably, all SaeS, SaeR, SaeP and SaeQ in the live bacterial strain are mutated.

[0010] Implementation Scheme 5. A live bacterial strain according to any one of Implementation Schemes 1 to 4, wherein the mutation in one or more genes of the sae two-component system causes a reduction or absence of expression of the one or more genes of the sae two-component system, or causes a reduction or absence of expression of one or more proteins of the sae two-component system.

[0011] Implementation Scheme 6. A live bacterial strain according to any one of Implementation Schemes 1 to 5, wherein the mutation includes the deletion of one or more genes of the sae two-component system, preferably, all genes of the sae two-component system in the live bacterial strain are completely or partially deleted, more preferably, all genes of the sae two-component system in the live bacterial strain are completely deleted.

[0012] Implementation Scheme 7. The live bacterial strain according to Implementation Scheme 6, wherein one or more genes selected from SaeS, SaeR, SaeP and SaeQ are deleted in the live bacterial strain, preferably, all SaeS, SaeR, SaeP and SaeQ are completely or partially deleted in the live bacterial strain, more preferably, all SaeS, SaeR, SaeP and SaeQ are completely deleted in the live bacterial strain.

[0013] Implementation Scheme 8. A live bacterial strain according to any one of Implementation Schemes 1 to 7, wherein, for example, compared with the corresponding control strain, the live bacterial strain also has reduced adenosine synthase A (adsA) activity, preferably, lacks adenosine synthase A (adsA) activity.

[0014] Implementation Scheme 9. The live bacterial strain according to Implementation Scheme 8, wherein the live bacterial strain contains a mutation in the adsA gene.

[0015] Implementation Scheme 10. The live bacterial strain according to Implementation Scheme 9, wherein the mutation causes a reduction or absence of expression of the adsA gene, or causes reduced or inactive expression of the adsA protein.

[0016] Implementation Scheme 11. A live bacterial strain according to Implementation Scheme 9 or 10, wherein the mutation includes the deletion of the adsA gene in the live bacterial strain, for example, the adsA gene is partially or completely deleted.

[0017] Implementation Scheme 12. A live bacterial strain according to any one of Implementation Schemes 1 to 11, for example, compared with a corresponding control strain, The live bacterial strains also exhibit reduced capsule production, preferably a lack of capsule production, and / or The expression of one or more genes related to capsular polysaccharide synthesis is reduced in the live bacterial strains, and / or The live bacterial strains contain one or more mutations in one or more genes related to the synthesis of capsular polysaccharides.

[0018] Implementation Scheme 13. The live bacterial strain according to Implementation Scheme 12, wherein, compared with the corresponding control strain, the 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, preferably, the live bacterial strain does not produce a capsule.

[0019] Implementation Scheme 14. The live bacterial strain according to Implementation Scheme 12 or 13, wherein, compared with the corresponding control strain, the production of capsular polysaccharide 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, preferably, the live bacterial strain does not produce capsular polysaccharide.

[0020] Implementation Scheme 15. A live bacterial strain according to any one of Implementation Schemes 12 to 14, wherein, compared with the corresponding control strain, the expression of one or more capsular polysaccharide synthesis-related 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.

[0021] Implementation Scheme 16. A live bacterial strain according to any one of Implementation Schemes 12 to 15, wherein the expression of all capsular polysaccharide synthesis-related genes in the live bacterial strain is reduced, preferably, the live bacterial strain does not express capsular polysaccharide synthesis-related genes.

[0022] Implementation Scheme 17. A live bacterial strain according to any one of Implementation Schemes 12 to 16, wherein all capsular polysaccharide synthesis-related genes in the live bacterial strain are mutated.

[0023] Implementation Scheme 18. A live bacterial strain according to any one of Implementation Schemes 12 to 17, wherein the mutation in the capsular polysaccharide synthesis-related gene causes a decrease or absence of expression of the capsular polysaccharide synthesis-related gene, or causes a decrease or absence of expression of capsular polysaccharide synthesis-related proteins.

[0024] Implementation Scheme 19. A live bacterial strain according to any one of Implementation Schemes 12 to 18, wherein the mutation includes the deletion of one or more capsular polysaccharide synthesis-related genes, preferably, all capsular polysaccharide synthesis-related genes in the live bacterial strain are partially or completely deleted, more preferably, all capsular polysaccharide synthesis-related genes in the live bacterial strain are completely deleted.

[0025] Implementation Scheme 20. A live bacterial strain according to any one of Implementation Schemes 12 to 19, wherein the one or more capsular polysaccharide synthesis-related 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.

[0026] Implementation Scheme 21. The live bacterial strain according to Implementation Scheme 20, wherein all of the polysaccharide synthesis genes of capA, capB, capC, capD, capE, capF, capG, capH, capI, capJ, capK, capL, capM, capN, capO and capP are deleted, preferably completely deleted.

[0027] Implementation Scheme 22. A live bacterial strain according to any one of Implementation Schemes 1 to 21, wherein the mutation is achieved by homologous recombination or by targeted mutagenesis, such as via CRISPR, TALEN or ZFN technology.

[0028] Implementation Scheme 23. A live bacterial strain according to any one of Implementation Schemes 1 to 22, wherein the live bacterial strain has reduced virulence compared with 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.

[0029] Implementation Scheme 24. A live bacterial strain according to any one of Implementation Schemes 1 to 23, wherein the live bacterial strain has increased immunogenicity compared with the 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.

[0030] Implementation Scheme 25. A live bacterial strain according to any one of Implementation Schemes 1 to 24, wherein the species from the genus Staphylococcus is selected from the group consisting of: Staphylococcus aureus, Staphylococcus capitella (…). Staphylococcus capitis ), Staphylococcus aureus ( Staphylococcus caprae Staphylococcus aureus ( Staphylococcus carnosus Staphylococcus epidermidis ( Staphylococcus epidermidis Staphylococcus intermedia ( Staphylococcus intermedius Staphylococcus aureus ( ), Staphylococcus aureus ( Staphylococcus lentus Staphylococcus ludensii ( Staphylococcus lugdunensis Staphylococcus piraceae ( ) Staphylococcus pettenkoferi ), mimicking Staphylococcus ( Staphylococcus simulans ), Staphylococcus aureus ( Staphylococcus vitulinus Staphylococcus xylose ( Staphylococcus xylosus ) and anaerobic staphylococcus subsp. Staphylococcus subspecies anaerobius ),

[0031] Preferably, the species from the genus Staphylococcus is Staphylococcus aureus.

[0032] Implementation Scheme 26. A live bacterial strain according to any one of Implementation Schemes 1 to 25, wherein the live bacterial strain is derived from a parent strain used as a clinical isolate.

[0033] Implementation Scheme 27. A live bacterial strain according to any one of Implementation Schemes 1 to 25, wherein the live bacterial strain is derived from a parent strain that has already acquired low virulence.

[0034] Implementation Scheme 28. A live bacterial strain according to any one of Implementation Schemes 1 to 27, wherein the live bacterial strain is derived from Staphylococcus aureus serotype 5 or serotype 8, more preferably, the live bacterial strain is derived from Staphylococcus aureus serotype 5.

[0035] Implementation Scheme 29. The live bacterial strain according to Implementation Scheme 28, wherein the live bacterial strain is derived from Staphylococcus aureus Newman strain, FPR3735, JE2 or ATCC29213.

[0036] Implementation Scheme 30. A live bacterial strain according to any one of Implementation Schemes 1 to 29, wherein the live bacterial strain is used as a live expression vector for expressing the target protein.

[0037] Implementation Scheme 31. A live bacterial strain according to any one of Implementation Schemes 1 to 30, wherein the live bacterial strain further comprises a coding sequence for the target protein and is thereby capable of expressing the target protein.

[0038] Implementation Scheme 32. The live bacterial strain according to Implementation Scheme 31, wherein the coding sequence of the target protein is introduced into the live bacterial strain, for example, through a nucleic acid expression construct.

[0039] Implementation Scheme 33. The live bacterial strain according to Implementation Scheme 32, wherein the introduced coding sequence of the target protein is integrated into the genome of the live bacterial strain.

[0040] Implementation Scheme 34. A live bacterial strain according to any one of Implementation Schemes 30 to 33, wherein the target protein is expressed and displayed on the cell surface of the live bacterial strain; or expressed and secreted by the cells of the live bacterial strain.

[0041] Implementation Scheme 35. A live bacterial strain according to any one of Implementation Schemes 30 to 34, wherein the target protein is an antigen.

[0042] Implementation Scheme 36. A live bacterial strain according to any one of Implementation Schemes 30 to 35, wherein the target protein is selected from EsxA, EsxB, mLukS-PV, mLukF-PV, mHla, mSpA, mTSST1, mSEB, mLukA, mLukB or any combination thereof.

[0043] Implementation Scheme 37. The live bacterial strain according to Implementation Scheme 36, wherein the target protein is

[0044] i) mHla; ii) EsxA and EsxB; iii) mHla, mLukS-PV and mLukF-PV; iv) mHla, EsxA, EsxB, mLukS-PV and mLukF-PV; or v) EsxA, EsxB, mLukS-PV, mLukF-PV, mHla, mSpA, mTSST1, mSEB, mLukA and mLukB.

[0045] Implementation Scheme 38. A live bacterial strain according to any one of Implementation Schemes 1 to 37, said live bacterial strain being used to prevent and / or treat bacterial infections in a subject.

[0046] Implementation Scheme 39. The live bacterial strain according to Implementation Scheme 38, wherein the bacterial infection is caused by a species derived from the live bacterial strain, such as a species from the genus Staphylococcus.

[0047] Implementation Scheme 40. The live bacterial strain according to Implementation Scheme 39, wherein the bacterial infection is caused by Staphylococcus aureus, Staphylococcus capitis, Staphylococcus capitis, Staphylococcus carinatum, Staphylococcus epidermidis, Staphylococcus intermedia, Staphylococcus slow-moving, Staphylococcus ludens, Staphylococcus pisciniformis, Staphylococcus mimicus, Staphylococcus calf, Staphylococcus xylose, or subspecies of anaerobic Staphylococcus or any combination thereof. ， The infection is preferably caused by Staphylococcus aureus.

[0048] Implementation Scheme 41. Use of any one of Implementation Schemes 1 to 40 in the preparation of compositions such as vaccines for the prevention or treatment of bacterial infections in subjects.

[0049] Implementation Scheme 42. A composition, such as a vaccine, for the prevention or treatment of bacterial infection in a subject, said composition comprising a live bacterial strain according to any one of Implementation Schemes 1 to 40.

[0050] Implementation Scheme 43. The composition according to Implementation Scheme 42, wherein the composition further comprises an adjuvant and / or a pharmaceutically acceptable carrier.

[0051] Implementation Scheme 44. A method for preventing and / or treating 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 Implementation Schemes 1 to 40, or a composition according to Implementation Scheme 42 or 43.

[0052] Implementation Scheme 45. The use according to Implementation Scheme 41, the composition according to any one of Implementation Schemes 42 to 43, or the method according to Implementation Scheme 44, wherein the bacterial infection is caused by a species derived from the live bacterial strain, such as a species from the genus Staphylococcus.

[0053] Implementation Scheme 46. The use, composition, or method according to Implementation Scheme 45, wherein the bacterial infection is caused by Staphylococcus aureus, Staphylococcus capitis, Staphylococcus capitis, Staphylococcus carinatum, Staphylococcus epidermidis, Staphylococcus intermedius, Staphylococcus slow-moving, Staphylococcus ludens, Staphylococcus pisciniformis, Staphylococcus mimicus, Staphylococcus calfii, Staphylococcus xylose, or anaerobic staphylococcal subsp., or any combination thereof. 。

[0054] Implementation Scheme 47. The use, composition, or method according to Implementation Scheme 46, wherein the bacterial infection is a Staphylococcus aureus infection.

[0055] Implementation Scheme 48. The use, composition, or method according to Implementation Scheme 47, wherein the Staphylococcus aureus infection is a skin infection, soft tissue infection, or invasive disease.

[0056] Implementation Scheme 49. The use, composition, or method according to Implementation Scheme 48, wherein the invasive disease is a bloodstream infection, endocarditis, Morell's disease, or sepsis.

[0057] Implementation Scheme 50. The use, composition, or method according to any one of Implementation Schemes 47 to 49, wherein the Staphylococcus aureus infection is methicillin-resistant Staphylococcus aureus. S. aureus MRSA infection or methicillin-sensitive Staphylococcus aureus S. aureus MSSA infection, preferably, said infection is recurrent Staphylococcus aureus infection.

[0058] Implementation Scheme 51. The use, composition, or method according to any one of Implementation Schemes 45 to 50, wherein the subject is a mammal, such as a human, mouse, rat, monkey, dog, pig, sheep, goat, cow, horse, donkey, domestic cattle, cat; or poultry, such as chicken, duck, goose. Attached Figure Description

[0059] Figure 1 The deletion regions of KO's target genes (adsA, CPs, saeRS, saePQRS).

[0060] Figure 2 Insertion regions of KI's target genes (mHla, EsxAB, mLukS-PV, EsxAB-mLukS-PV, mHla-mSpA, EsxAB-mLukSF-PV, mTSST1-mSEB, mLukAB).

[0061] Figure 3 IgG antibody titers in the enrolled animals.

[0062] Figure 4 The JE2ΔsaePQRS strain exhibited low virulence in animal systems models.

[0063] Figure 5 The NewmanΔsaePQRS strain exhibited low virulence in animal systems models.

[0064] Figure 6 The triple knockout ΔadsA ΔCP ΔsaePQRS strain showed lower virulence in a skin infection model than the double knockout ΔadsA ΔCP strain.

[0065] Figure 7 The three knockout ΔadsA ΔCP ΔsaePQRS strains showed a lower IgG response.

[0066] Figure 8 The three knockout strains ΔadsA ΔCP ΔsaePQRS provide protective efficacy when used as a vaccine in a mouse systemic model.

[0067] Figure 9 Three knockouts of ΔadsA, ΔCP, and ΔsaePQRS strains showed enhanced protective efficacy in a skin infection model.

[0068] Figure 10 Bacterial load of 10-antigen strains.

[0069] Figure 11 Virulence study of 10-antigen strains.

[0070] Figure 12 Study on the protective efficacy of 10-antigen strains.

[0071] Figure 13 Study on the protective efficacy of Sau0137 strain against HA-MRSA.

[0072] Figure 14 Study on the protective efficacy of Sau0140 strain against HA-MRSA and MU3.

[0073] Figure 15 Study on the protective efficacy of Sau0145 strain against HA-MRSA, FPR3735 and Sau-HK3117.

[0074] Figure 16 Study on the protective efficacy of Sau0144 strain against Sau-HK3117. Detailed Implementation

[0075] Before describing various aspects of the invention, it must be noted that, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “described” used herein and in the appended claims include plural references. The term “and / or” is intended to include any combination of items connected by the term, equivalent to listing all combinations individually. For example, “A, B, and / or C” covers “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 pertains.

[0076] "Sequence identity" has a recognized meaning in the art, and the percentage of sequence identity between two nucleotide sequences or amino acid sequences can be calculated using the disclosed techniques. Sequence identity can be measured along the entire length of a specified nucleotide or amino acid sequence or along a region of the sequence. In some embodiments, sequence identity is measured along the entire length of a specified nucleotide or amino acid sequence. Many methods for measuring sequence identity are available in the art and can be applied in this invention.

[0077] In one aspect, the present invention provides a live bacterial strain from a species of the genus Staphylococcus, wherein, for example, compared with a corresponding control strain, i) The live bacterial strain has reduced activity of the SAE two-component system, preferably lacking activity of the SAE two-component system; ii) Reduced expression of one or more genes in the SAE two-component system in live bacterial strains; and / or iii) The live bacterial strain contains one or more mutations in one or more genes of the sae two-component system.

[0078] The Sae two-component system was first discovered in Staphylococcus aureus, and the corresponding components can also be found in Staphylococcus aureus. Staphylococcus Found in other species of the genus Sae. The Sae two-component system has been shown to be essential for evading human neutrophil killing. This system consists of the histidine kinase sensor SaeS, the response regulator SaeR, and two accessory proteins, SaeP and SaeQ. The sensor protein SaeS detects environmental signals (such as changes in pH or oxygen levels) and then phosphorylates the response regulator SaeR. The phosphorylated SaeR then binds to the promoter region of a target gene, activating or inhibiting its transcription. Studies have demonstrated that the SaeR response regulator and its homologous sensor kinase SaeS play a crucial role in initiating infection.

[0079] "Activities of the Sae two-component system" include: SaeS's detection of environmental signals, SaeR's phosphorylation, and / or activation or inhibition of target gene transcription.

[0080] In some implementations, compared with the corresponding control strain, the expression of one or more genes of the Sae two-component system 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.

[0081] In some embodiments, the expression of SaeS and SaeR in the live bacterial strain is reduced. In some preferred embodiments, SaeS and SaeR are not expressed in the live bacterial strain.

[0082] In some embodiments, the expression of all SaeS, SaeR, SaeP, and SaeQ is reduced in the live bacterial strain. In some preferred embodiments, SaeS, SaeR, SaeP, and SaeQ are not expressed in the live bacterial strain.

[0083] The term "gene" in this article can refer to a protein-coding sequence, but it can also encompass expression regulatory elements / sequences, such as promoters and enhancers.

[0084] As used in this article, a "mutation" can be the addition, substitution, or deletion of one or more nucleotides.

[0085] Mutations in genes can cause reduced expression of encoded proteins or reduced expression of proteins with reduced activity. In some embodiments, mutations in genes cause the encoded protein to be not expressed or to be inactive. In some embodiments, the mutation is a frameshift mutation, which causes mistranslation of one or more genes.

[0086] The term "mutation" can include gene deletions, such as complete or partial deletions. In some embodiments, the live bacterial strains of the present invention contain the deletion of one or more genes. One or more genes in the strain may be completely deleted, such that one or more proteins encoded by the live bacterial strains of the present invention are absent. One or more genes may also be partially deleted, such that only one or more inactive truncated proteins are present in the live bacterial strains of the present invention.

[0087] Mutations in one or more genes can be achieved using various methods known in the art. In some embodiments, mutations are introduced into live bacterial strains via genetic engineering. In some embodiments, the mutations are not naturally occurring. For example, mutations such as deletions can be achieved via homologous recombination. In some embodiments, mutations are induced by targeted mutagenesis, such as via CRISPR, TALEN, or ZFN technologies.

[0088] In some embodiments, the live bacterial strain contains one or more mutations in one or more genes selected from SaeS, SaeR, SaeP, and SaeQ. Preferably, all SaeS, SaeR, SaeP, and SaeQ in the live bacterial strain are mutated.

[0089] In some implementations, mutations in one or more genes of the Sae two-component system can cause reduced or absent expression of one or more genes of the Sae two-component system, or cause reduced or inactive expression of one or more proteins of the Sae two-component system.

[0090] In some embodiments, the mutation includes the deletion of one or more genes of the sae two-component system, preferably, all genes of the sae two-component system are completely or partially deleted in the live bacterial strain, more preferably, all genes of the sae two-component system are completely deleted in the live bacterial strain.

[0091] In some implementations, one or more genes selected from SaeS, SaeR, SaeP, and SaeQ are deleted from the live bacterial strain.

[0092] In some implementations, SaeS and SaeR are completely or partially absent in the live bacterial strain.

[0093] In some preferred embodiments, all SaeS, SaeR, SaeP, and SaeQ in the live bacterial strain are completely or partially absent; more preferably, all SaeS, SaeR, SaeP, and SaeQ in the live bacterial strain are completely absent.

[0094] Exemplary protein sequences of SaeP, SaeQ, SaeS, and SaeR from *Staphylococcus aureus* (from the Newman strain) are shown in SEQ ID NO: 34-37, respectively. Exemplary coding sequences of SaeP, SaeQ, SaeS, and SaeR from *Staphylococcus aureus* (from the Newman strain) are shown in SEQ ID NO: 38-41, respectively. An exemplary protein sequence of SaeS from *Staphylococcus aureus* (from the JE2 strain) is shown in SEQ ID NO: 42. An exemplary coding sequence of SaeS from *Staphylococcus aureus* (from the JE2 strain) is shown in SEQ ID NO: 43.

[0095] In some embodiments, SaeP has an amino acid sequence that is 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 with SEQ ID NO: 34.

[0096] In some embodiments, SaeQ has an amino acid sequence that is 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 with SEQ ID NO: 35.

[0097] In some embodiments, Saes has an amino acid sequence that is 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 with SEQ ID NO: 36 or SEQ ID NO: 42.

[0098] In some embodiments, SaeR has an amino acid sequence that is 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 with SEQ ID NO: 37.

[0099] In some embodiments, when comparing the activity or expression of the Sae two-component system, the "control strain" may refer to a strain of the same species whose Sae two-component system activity or Sae two-component system gene is unchanged. In some embodiments, when comparing the activity or expression of the Sae two-component system, the "control strain" may also refer to a strain of the same species that does not contain the mutations described above in the Sae two-component system gene.

[0100] In some embodiments, for example, the live bacterial strain also has reduced adenosine synthase A (adsA) activity compared to the corresponding control strain, and preferably, lacks adenosine synthase A (adsA) activity.

[0101] In some implementations, the expression of the adsA gene is reduced in live bacterial strains.

[0102] In some embodiments, the live bacterial strain contains a mutation in the adsA gene. In some embodiments, the mutation causes reduced or absent expression of the adsA gene, or causes reduced or inactive expression of the adsA protein.

[0103] Adenosine synthase A (adsA) is an important virulence factor in staphylococcal species, such as Staphylococcus aureus. An exemplary adsA of Staphylococcus aureus contains the amino acid sequence of SEQ ID NO: 1. However, it is well known to those skilled in the art that, due to polymorphism between species and strains, adsA may differ slightly from SEQ ID NO: 1 while maintaining the same or similar function.

[0104] In some embodiments, adsA has an amino acid sequence that is 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 with SEQ ID NO: 1.

[0105] In some embodiments, compared with a corresponding control strain, the adsA activity of the live bacterial strain 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. In some preferred embodiments, the live bacterial strain of the present invention has no adsA activity. The reduction or absence of adsA activity may result in attenuation of the virulence of the live bacterial strain of the present invention.

[0106] In some embodiments, the live bacterial strains of the present invention contain a mutation in the adsA gene encoding adsA. Such mutations may be the addition, substitution, or deletion of one or more nucleotides.

[0107] In some embodiments, mutations in the adsA gene result in reduced expression of the adsA protein, or in the expression of a mutant adsA protein with reduced activity. In some embodiments, mutations in the adsA gene result in the absence of adsA protein expression, or in the expression of an inactive mutant adsA protein.

[0108] In some embodiments, the mutation includes the deletion of the adsA gene, such as a complete or partial deletion of the adsA gene. The adsA gene in the strain may be completely deleted, resulting in the absence of the adsA gene in the live strain of the present invention. The adsA gene may also be partially deleted, resulting in the presence of only reduced or inactive adsA protein in the live strain of the present invention. In some embodiments, the mutation is a frameshift mutation, which causes mistranslation of the adsA protein. In some embodiments, the mutation in the adsA gene causes the deletion of a portion of adsA responsible for the production of adenosine.

[0109] In some embodiments, when comparing adsA activity, the "control strain" may refer to a strain of the same species with unchanged adsA activity or adsA gene. In some embodiments, when comparing adsA activity, the "control strain" may also refer to a strain of the same species that does not contain the adsA gene mutation as described above.

[0110] Mutations in the adsA gene can be achieved using various methods known in the art. In some embodiments, the mutation is introduced into a live bacterial strain via genetic engineering. In some embodiments, the mutation is not naturally occurring. For example, mutations such as deletions can be achieved via homologous recombination. In some embodiments, the mutation is performed via targeted mutagenesis, such as via CRISPR, TALEN, or ZFN technologies. In some other embodiments, the live bacterial strain of the present invention is derived from an attenuated parental live bacterial strain. For example, the parental live bacterial strain may have reduced adenosine synthase A (adsA) activity or no adenosine synthase A activity compared to a corresponding control strain. For example, the live bacterial strain of the present invention can be obtained by introducing mutations in one or more genes of the Sae two-component system into a parental live bacterial strain that already has reduced adenosine synthase A (adsA) activity or no adenosine synthase A activity.

[0111] In some embodiments, for example, the live bacterial strains of the present invention also have reduced capsule production, preferably, a lack of capsule production, compared to the corresponding control strains.

[0112] In some embodiments, for example, compared with corresponding control strains, the expression of one or more genes related to capsular polysaccharide synthesis is reduced in the live bacterial strains of the present invention.

[0113] In some implementations, the live bacterial strain contains one or more mutations in one or more genes related to the synthesis of capsular polysaccharides.

[0114] In some embodiments, when comparing capsular production or expression of genes related to capsular polysaccharide synthesis, the "control strain" may be a parent strain derived from a live bacterial strain of the present invention. In some embodiments, the "control strain" may refer to a strain of the same species whose capsular / capsular polysaccharide production or capsular polysaccharide synthesis-related genes are unchanged. In some embodiments, the "control strain" may also refer to a strain of the same species that does not contain mutations in one or more of the capsular polysaccharide synthesis-related genes as described above.

[0115] In some embodiments, compared with a corresponding control strain, capsule formation in the live bacterial strain 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. In some preferred embodiments, the live bacterial strain of the present invention does not produce capsules.

[0116] In some embodiments, compared with a corresponding control strain, the production of capsular polysaccharides in the live bacterial 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. In some preferred embodiments, the live bacterial strains of the present invention do not produce capsular polysaccharides.

[0117] In some implementations, compared with the corresponding control strain, the expression of one or more capsular polysaccharide synthesis-related 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.

[0118] In some implementations, the expression of all genes related to capsular polysaccharide synthesis is reduced in live bacterial strains.

[0119] In some embodiments, compared with corresponding control strains, the expression of all capsular polysaccharide synthesis-related 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. In some preferred embodiments, capsular polysaccharide synthesis-related genes are not expressed in the live bacterial strain.

[0120] As used herein, the term "capsular polysaccharide synthesis-related genes" refers to any gene involved in the production of capsular polysaccharides in bacteria, including regulatory genes, glycosyltransferase genes, synthase genes, etc. Capsular polysaccharide synthesis-related genes encode proteins involved in capsular polysaccharide synthesis. These genes can refer to protein-coding sequences, but can also encompass expression regulatory elements / sequences, such as promoters and enhancers.

[0121] In some embodiments, the live strain of the present invention contains one or more mutations in one or more genes related to capsular polysaccharide synthesis. The mutation may be the addition, substitution, or deletion of one or more nucleotides.

[0122] In some preferred embodiments, all capsular polysaccharide synthesis-related genes in the live bacterial strain are mutated. In some embodiments, mutations in capsular polysaccharide synthesis-related genes result in reduced expression of capsular polysaccharide synthesis-related proteins, or inactive expression of capsular polysaccharide synthesis-related proteins. In some embodiments, mutations in capsular polysaccharide synthesis-related genes result in the absence of expression of capsular polysaccharide synthesis-related proteins, or inactive expression of capsular polysaccharide synthesis-related proteins. In some embodiments, the mutation is a frameshift mutation, which causes mistranslation of one or more capsular polysaccharide synthesis-related genes.

[0123] In some embodiments, the mutation includes the deletion of genes related to capsular polysaccharide synthesis, for example, a complete or partial deletion of such genes. In some embodiments, the live bacterial strains of the present invention contain the deletion of one or more genes related to capsular polysaccharide synthesis. One or more genes related to capsular polysaccharide synthesis in the strain may be completely deleted, resulting in the absence of one or more capsular polysaccharide synthesis-related proteins in the live bacterial strains of the present invention. One or more genes related to capsular polysaccharide synthesis may also be partially deleted, resulting in the presence of only one or more inactive truncated capsular polysaccharide synthesis-related proteins in the live bacterial strains of the present invention. In some preferred embodiments, all genes related to capsular polysaccharide synthesis in the live bacterial strains of the present invention are completely or partially deleted. In some preferred embodiments, all genes related to capsular polysaccharide synthesis in the live bacterial strains of the present invention are completely deleted.

[0124] Mutations in one or more genes related to capsular polysaccharide synthesis can be achieved by various means known in the art. In some embodiments, the mutation is introduced into a live bacterial strain via genetic engineering. In some embodiments, the mutation is not naturally occurring. For example, mutations such as deletions can be achieved via homologous recombination. In some embodiments, the mutation is induced by targeted mutagenesis, such as via CRISPR, TALEN, or ZFN technologies.

[0125] Staphylococcus aureus from serotype 5 or serotype 8 typically contains a cluster of 16 capsular polysaccharide synthesis-related genes in its genome: capA, capB, capC, capD, capE, capF, capG, capH, capI, capJ, capK, capL, capM, capN, capO, and capP. Exemplary protein sequences of capA through capP in serotype 5 Staphylococcus aureus are shown in SEQ ID NO: 2 to 17, respectively. Exemplary coding sequences of capA through capP in serotype 5 Staphylococcus aureus are shown in SEQ ID NO: 18 to 33, respectively. Therefore, those skilled in the art will be able to identify capsular polysaccharide synthesis-related genes / gene clusters in other serotypes or isolates and thus perform the aforementioned mutations.

[0126] In some embodiments, one or more capsular polysaccharide synthesis-related genes encode one or more capsular polysaccharide synthesis-related 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 with any of SEQ ID NO: 2 to 17.

[0127] In some embodiments, for example, compared to corresponding control strains, the expression of one or more capsular polysaccharide synthesis-related genes selected from capA, capB, capC, capD, capE, capF, capG, capH, capI, capJ, capK, capL, capM, capN, capO, and capP is reduced in the live bacterial strains of the present invention. In some embodiments, one or more capsular polysaccharide synthesis-related genes selected from capA, capB, capC, capD, capE, capF, capG, capH, capI, capJ, capK, capL, capM, capN, capO, and capP are not expressed in the live bacterial strains of the present invention.

[0128] In some embodiments, the live bacterial strain contains one or more mutations in one or more capsular polysaccharide synthesis-related genes selected from capA, capB, capC, capD, capE, capF, capG, capH, capI, capJ, capK, capL, capM, capN, capO, and capP.

[0129] In some embodiments, the live strain of the present invention is missing, preferably completely missing. It has been reported that the deletion of the capF or capG gene can eliminate capsule formation in Staphylococcus aureus [3]. 。

[0130] In some embodiments, all capA, capB, capC, capD, capE, capF, capG, capH, capI, capJ, capK, capL, capM, capN, capO, and capP in the live strain of the present invention are missing, preferably completely missing.

[0131] In some embodiments, the live bacterial strains of the present invention cause a reduction in virulence.

[0132] For example, compared with the corresponding control strain, the virulence of the live bacterial strain of the present 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.

[0133] In some embodiments, the live bacterial strains of the present invention increase immunogenicity. Immunogenicity can refer to the ability to elicit an immune response (e.g., an antibody-mediated immune response) in a host.

[0134] For example, compared with the corresponding control strain, the immunogenicity of the live bacterial strain 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.

[0135] In some embodiments, the "control strain" may be a parent strain derived from the live bacterial strain of the present invention. In some embodiments, the "control strain" may refer to a strain of the same species whose Sae two-component system, adsA activity, and / or capsular / capsular polysaccharide production are unchanged. In some embodiments, the "control strain" may also refer to a strain of the same species that does not contain mutations in one or more of the Sae two-component system genes, adsA genes, and / or capsular polysaccharide synthesis-related genes as described above.

[0136] The live bacterial strains of the present invention can be derived from parental strains, which are wild-type strains 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 Sae two-component system genes, adsA genes, and / or capsular polysaccharide synthesis-related genes have not been genetically engineered. In some embodiments, the wild-type strain can be a clinically isolated strain.

[0137] The live bacterial strains of the present invention may be derived from parental strains that already possess low virulence. For example, the parental strain may be an attenuated strain. The live bacterial strains of the present invention may contain other modifications that can cause attenuation.

[0138] The live bacterial strains of the present invention may be derived from species of the genus Staphylococcus. For example, the live bacterial strains of the present invention may be derived from Staphylococcus aureus, Staphylococcus capitis, Staphylococcus capitis, Staphylococcus carminans, Staphylococcus epidermidis, Staphylococcus intermedius, Staphylococcus slow-moving, Staphylococcus ludensundus, Staphylococcus pistilli, Staphylococcus mimicus, Staphylococcus calfii, subspecies of Staphylococcus anaerobicus, or Staphylococcus xylose.

[0139] In some embodiments, the live bacterial strains of the present invention may be derived from any isolate of a particular bacterial species.

[0140] In some embodiments, the live bacterial strains of the present invention may 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.

[0141] In some embodiments, the live bacterial strains of the present invention are derived from *Staphylococcus aureus*, and therefore also refer to the 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 and serotype 8. 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*.

[0142] In some embodiments, the live Staphylococcus aureus strain of the present invention is derived from Staphylococcus aureus Newman, FPR3735, JE2, or ATCC29213 strains. In some preferred embodiments, the live Staphylococcus aureus strain of the present invention is derived from Staphylococcus aureus Newman strain. The Staphylococcus aureus Newman strain is a serotype 5 strain and is commercially available from the National Type Culture Collection (NCTC) in the UK under accession number NCTC 8178.

[0143] In one aspect, the present invention provides a live bacterial strain from a species of the genus Staphylococcus, wherein, for example, compared with a corresponding control strain, a) The live bacterial strain has reduced activity of the SAE two-component system, preferably lacking SAE two-component system activity; the expression of one or more genes of the SAE two-component system is reduced in the live bacterial strain; and / or the live bacterial strain contains one or more mutations in one or more genes of the SAE two-component system; and b) The live bacterial strain has reduced adenosine synthase A (adsA) activity, preferably lacking adenosine synthase A (adsA) activity; reduced expression of the adsA gene in the live bacterial strain; and / or the live bacterial strain contains a mutation in the adsA gene.

[0144] These terms have the definitions described above.

[0145] In one aspect, the present invention provides a live bacterial strain from a species of the genus Staphylococcus, wherein, for example, compared with a corresponding control strain, a) The live bacterial strain has reduced activity of the SAE two-component system, preferably lacking SAE two-component system activity; the expression of one or more genes of the SAE two-component system is reduced in the live bacterial strain; and / or the live bacterial strain contains one or more mutations in one or more genes of the SAE two-component system; and b) The live bacterial strains of the present invention have reduced capsule production, preferably lacking capsule production; the expression of one or more capsular polysaccharide synthesis-related genes is reduced in the live bacterial strains of the present invention; and / or the live bacterial strains contain one or more mutations in one or more capsular polysaccharide synthesis-related genes.

[0146] These terms have the definitions described above.

[0147] In one aspect, the present invention provides a live bacterial strain from a species of the genus Staphylococcus, wherein, for example, compared with a corresponding control strain, a) The live bacterial strain has reduced activity of the SAE two-component system, preferably lacking SAE two-component system activity; the expression of one or more genes of the SAE two-component system is reduced in the live bacterial strain; and / or the live bacterial strain contains one or more mutations in one or more genes of the SAE two-component system; and b) The live bacterial strain has reduced adenosine synthase A (adsA) activity, preferably lacking adenosine synthase A (adsA) activity; the expression of the adsA gene is reduced in the live bacterial strain; and / or the live bacterial strain contains a mutation in the adsA gene; and c) The live bacterial strains of the present invention have reduced capsule production, preferably lacking capsule production; the expression of one or more capsular polysaccharide synthesis-related genes is reduced in the live bacterial strains of the present invention; and / or the live bacterial strains contain one or more mutations in one or more capsular polysaccharide synthesis-related genes.

[0148] These terms have the definitions described above.

[0149] In some embodiments of various aspects, the live bacterial strains of the present invention are also used as live expression vectors for expressing target proteins. The target proteins may confer certain characteristics upon the live bacterial strains of the present invention.

[0150] In some embodiments of various aspects, the live bacterial strains of the present invention may contain the coding sequence of the target protein and thereby be able to express the target protein.

[0151] In some embodiments, the target protein can be an endogenous protein, i.e., a protein derived from a bacterial species of a live bacterial strain. In some embodiments, the target protein can be a heterogeneous protein, i.e., a protein of a different species than the bacterial species derived from the live bacterial strain.

[0152] In some embodiments, the coding sequence of the target protein is introduced into the live bacterial strain of the present invention, for example, through a nucleic acid expression construct. In some embodiments, the introduced coding sequence of the target protein is integrated into the genome of the live bacterial strain of the present invention.

[0153] As used herein, “expression construct” refers to a vector, such as a recombinant vector suitable for expressing a target nucleotide sequence in a host cell. “Expression” means the production of a functional product. For example, the expression of a nucleotide sequence can refer to the transcription of a nucleotide sequence and / or the translation of RNA into a precursor or mature protein. The “expression construct” of this invention can be a linear nucleic acid fragment, a circular plasmid, a viral vector, or, in some embodiments, a translatable RNA (such as mRNA).

[0154] In some embodiments, the target protein may be expressed and displayed on the cell surface of the live bacterial strain of the present invention. In some embodiments, the target protein may be expressed and secreted by the cells of the live bacterial strain of the present invention.

[0155] Target proteins include, but are not limited to, antibodies and antigens.

[0156] In some preferred embodiments, the target protein is an antigenic protein. Expression or display of the antigenic protein can further increase the immunogenicity of the live bacterial strains of the present invention.

[0157] In some implementations, the target protein is an antigenic protein of a different species than the bacterial species derived from the live bacterial strain. Expressing or displaying other types of antigenic proteins can confer immunogenicity to said other species to the live bacterial strain.

[0158] Exemplary antigen proteins include, but are not limited to, EsxA, EsxB, mLukS-PV, mLukF-PV, mHla, mSpA, mTSST1, mSEB, mLukA, mLukB, etc., or any combination thereof.

[0159] EsxA may contain an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 99%, or even 100% sequence identity with SEQ ID NO: 44. EsxB may contain an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 99%, or even 100% sequence identity with SEQ ID NO: 46. mLukS-PV may contain an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 99%, or even 100% sequence identity with SEQ ID NO: 48. mLukF-PV may contain an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 99%, or even 100% sequence identity with SEQ ID NO: 50. mHla may contain an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 99%, or even 100% sequence identity with SEQ ID NO: 52. mSpA may contain an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 99%, or even 100% sequence identity with SEQ ID NO: 54. mTSST1 may contain an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 99%, or even 100% sequence identity with SEQ ID NO: 56. mSEB may contain an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 99%, or even 100% sequence identity with SEQ ID NO: 58. mLukA may contain an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 99%, or even 100% sequence identity with SEQ ID NO: 60. mLukB may contain an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 99%, or even 100% sequence identity with SEQ ID NO: 62.

[0160] In some embodiments, the live bacterial strains of the present invention express (e.g., overexpress) one or more antigens selected from EsxA, EsxB, mLukS-PV, mLukF-PV, mHla, mSpA, mTSST1, mSEB, mLukA, and mLukB. In some embodiments, the live bacterial strains of the present invention express (e.g., overexpress) antigen mHla. In some embodiments, the live bacterial strains of the present invention express (e.g., overexpress) antigens EsxA and EsxB. In some embodiments, the live bacterial strains of the present invention express (e.g., overexpress) antigens mHla and mLukS-PV. In some embodiments, the live bacterial strains of the present invention express (e.g., overexpress) antigens mHla, EsxA, EsxB, and mLukS-PV. In some embodiments, the live bacterial strains of the present invention express (e.g., overexpress) antigens EsxA, EsxB, mLukS-PV, mLukF-PV, mHla, mSpA, mTSST1, mSEB, mLukA, and mLukB.

[0161] In some embodiments, the live bacterial strains of the present invention are used to prevent and / or treat bacterial infections in subjects.

[0162] In one aspect, the present invention provides the use of the live bacterial strains of the invention in the preparation of compositions for the prevention or treatment of bacterial infections in subjects. In some embodiments, the composition is a vaccine.

[0163] In one aspect, the present invention provides a composition for preventing or treating bacterial infections in a subject, the composition comprising live bacterial strains of the present invention. In some embodiments, the composition comprises an effective amount of live bacterial strains of the present invention. In some embodiments, the composition is a vaccine.

[0164] In another aspect, the present invention provides a method for preventing and / or treating bacterial infections 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.

[0165] As used herein, “subject” may include, but is not limited to, mammals such as humans, mice, rats, monkeys, dogs, pigs, sheep, goats, cattle, horses, donkeys, domestic cattle, and cats; and poultry such as chickens, ducks, and geese.

[0166] As used in this article, prevention and / or treatment of bacterial infections also covers the prevention and / or treatment of diseases or clinical signs or symptoms caused by bacterial infections.

[0167] In some embodiments, the composition may also include an adjuvant. As used herein, "adjuvant" means 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, interferon, interleukin, or growth factors. "Adjuvant" 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.

[0168] In some embodiments across various aspects, the composition may also comprise a pharmaceutically acceptable carrier. As used herein, a “pharmaceutically acceptable carrier” includes any and all physiologically compatible solvents, dispersion media, coatings, antifungal agents, isotonic agents, and absorption delay agents. Non-limiting examples of pharmaceutically acceptable carriers include water, NaCl, physiological saline, lactated Ringer's solution, standard sucrose, standard glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, salt solutions (such as Ringer's solution), alcohols, oils, gelatin, carbohydrates (such as lactose), amylose or starch, fatty acid esters, hydroxymethyl cellulose, polyvinylpyrrolidone, and colorants.

[0169] In some embodiments, the composition is formulated for intramuscular, intraperitoneal, subcutaneous, oral, or intranasal administration. In one embodiment, the composition is not intended for intravenous administration. In some embodiments, the composition is in a lyophilized form that can be reconstituted prior to use.

[0170] As used herein, "effective amount" means an amount of a substance, compound, material, or composition containing a compound (such as the live bacterial strains of the present invention or the compositions of the present invention) sufficient to produce a preventive or therapeutic effect when administered to a subject. Therefore, an effective amount is the amount necessary to prevent, cure, improve, delay, or partially delay the symptoms of a disease or condition such as a bacterial infection.

[0171] The actual dose of the live strain or composition of the present invention to be administered to the subject may be determined based on the following physical and physiological factors: weight, sex, severity of symptoms, type of disease to be treated, previous or current treatment interventions, the patient's disease of unknown etiology, time of administration, route of administration, etc. In any case, the amount of live strain in the composition and the appropriate dose for the individual subject will be determined by the medical personnel responsible for administration.

[0172] In some embodiments, the bacterial infection is caused by a bacterial species derived from live bacterial strains of the present invention.

[0173] In some embodiments, the bacterial infection is caused by a bacterial strain with a serotype different from that of the live bacterial strain of the present invention.

[0174] In some implementations across various aspects, bacterial infections are caused by species from the genus Staphylococcus.

[0175] In some implementations across various aspects, the bacterial infection is caused by Staphylococcus aureus, Staphylococcus capitis, Staphylococcus capitis, Staphylococcus carinatum, Staphylococcus epidermidis, Staphylococcus intermedia, Staphylococcus slowi, Staphylococcus ludens, Staphylococcus piscini, Staphylococcus mimicus, Staphylococcus calfii, Staphylococcus anaerobicus subsp. spp., or Staphylococcus xylose, or any combination thereof.

[0176] In some implementation schemes, the bacterial infection is Staphylococcus aureus infection.

[0177] In some implementations, Staphylococcus aureus infection is a skin infection, soft tissue infection, or invasive disease. In some implementations, the invasive disease is a bloodstream infection, endocarditis, Morell's disease, or sepsis.

[0178] In various implementation schemes, Staphylococcus aureus infection is classified as methicillin-resistant Staphylococcus aureus. S. aureus MRSA infection or methicillin-sensitive Staphylococcus aureus S. aureus MSSA infection. In some preferred embodiments, the infection is recurrent Staphylococcus aureus infection.

[0179] In some embodiments, bacterial infections that can be prevented and / or treated by the present invention may depend on the target protein expressed by the live bacterial strains of the present invention.

[0180] In one aspect, the present invention provides a method for attenuating and / or increasing the immunogenicity of live bacterial strains, or a method for generating live bacterial strains with reduced virulence and / or increased immunogenicity, the method comprising:

[0181] a) Reduce capsule production in live bacterial strains and / or reduce the expression of one or more genes related to the synthesis of capsular polysaccharides in live bacterial strains; b) Reduce the activity of the Sae two-component system and / or reduce the expression of one or more genes in the Sae two-component system; and / or c) Reduce adenosine synthase A (adsA) activity and / or reduce AdsA gene expression.

[0182] In some embodiments, the method includes introducing one or more mutations into one or more genes related to capsular polysaccharide synthesis, one or more genes of the Sae two-component system, and / or the adsA gene in a live bacterial strain. Such mutations may be the addition, substitution, or deletion of one or more nucleotides.

[0183] In some preferred embodiments, all capsular polysaccharide synthesis-related genes, all genes of the Sae two-component system, and / or the adsA gene in the live bacterial strain are mutated. In some embodiments, mutations in genes cause reduced expression or decreased activity of the encoded protein. In some embodiments, mutations in genes cause no expression or inactive expression of the encoded protein. In some embodiments, the mutations are frameshift mutations, which cause mistranslation of one or more genes.

[0184] In some embodiments, the mutation includes gene deletion, such as complete or partial deletion of a gene. In some embodiments, genes related to capsular polysaccharide synthesis, genes of the Sae two-component system, and / or the adsA gene are deleted. Genes related to capsular polysaccharide synthesis, genes of the Sae two-component system, and / or the adsA gene in the strain may be completely deleted. Genes related to capsular polysaccharide synthesis, genes of the Sae two-component system, and / or the adsA gene may also be partially deleted, such that only one or more inactive truncated proteins are present in the live bacterial strain of the present invention. In some preferred embodiments, all genes related to capsular polysaccharide synthesis, all genes of the Sae two-component system, and / or the adsA gene in the live bacterial strain of the present invention are completely or partially deleted. In some preferred embodiments, genes related to capsular polysaccharide synthesis, genes of the Sae two-component system, and / or the adsA gene in the live bacterial strain of the present invention are completely deleted.

[0185] In some embodiments, the live bacterial strains are derived from species of the genus *Staphylococcus*. In some embodiments, the live bacterial strains are derived from species selected from: *Staphylococcus aureus*, *Staphylococcus capitis*, *Staphylococcus carinatum*, *Staphylococcus epidermidis*, *Staphylococcus intermedius*, *Staphylococcus slow-moving*, *Staphylococcus ludens*, *Staphylococcus petrificus*, *Staphylococcus calf*, *Staphylococcus anaerobicus* subspecies, or *Staphylococcus xylosus*. In some preferred embodiments, the live bacterial strains are derived from *Staphylococcus aureus*. In some embodiments, the live bacterial strains are derived from *Staphylococcus aureus* serotype 5 or serotype 8.

[0186] In some embodiments, the method further includes introducing the coding sequence of the target protein into a live bacterial strain, thereby enabling the live bacterial strain to express the target protein. In some embodiments, the target protein may be an endogenous protein or an exogenous protein.

[0187] In some embodiments, the coding sequence of the target protein is introduced into a live 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 live bacterial strain.

[0188] Target proteins include, but are not limited to, antibodies and antigens.

[0189] In some preferred embodiments, the target protein is an antigenic protein. In some embodiments, the target protein is an antigenic protein of a different species than the bacterial species derived from the live bacterial strain.

[0190] Exemplary antigen proteins include, but are not limited to, EsxA, EsxB, mLukS-PV, mLukF-PV, mHla, mSpA, mTSST1, mSEB, mLukA, mLukB, etc., or any combination thereof.

[0191] Example

[0192] A further understanding of the invention can be obtained by referring to the specific embodiments shown herein, which are for illustrative purposes only and not for limiting the scope of the invention. It will be apparent that various modifications and variations can be made to the invention without departing from its spirit, and such modifications and variations are also within the scope of the invention.

[0193] Methods and Materials

[0194] wild type strain

[0195] In this invention, the wild-type (wt-type) strains involved are listed in Table 1.

[0196] Table 1. List of wild-type strains involved

[0197] Strain construction and DNA sequencing confirmation

[0198] Newman ΔadsA ΔCP ΔsaePQRS, Newman ΔsaeRS, Newman ΔadsA ΔsaeRS, and Newman ΔadsAΔCPΔsaeRS are built based on Newman; JE2 ΔadsA ΔsaeRS and JE2 ΔadsA ΔsaePQRS are built based on JE2.

[0199] Staphylococcus aureus Newman strain was cultured in brain heart infusion (BHI) at 37°C. The pIMAY plasmid was used. Label-free, nonpolar deletion knockout plasmids of the target KO genes (adsA, CPs, saeros, saePQRS) were constructed using vectors (Schuster, Christopher F et al., Microbiology (Reading, England) vol. 165,5(2019): 572-584. doi:10.1099 / mic.0.000791). Agarose gel electrophoresis results of KO gene (adsA, CPs, saeros, saePQRS, agrA) PCR were analyzed. Figure 1 The arrows indicate the expected bands for the KO mutant and wild-type strains, respectively. In short, the upstream and downstream flanking regions of the target KO gene were amplified from the chromosomal DNA of *Staphylococcus aureus* Newman strain using the corresponding primers (Table 3) via PCR. The PCR products were separated by 1% agarose gel electrophoresis (using a 5kbPlus DNA ladder as the molecular weight standard). The individual PCR products were then combined to construct the in-frame deletion pattern of the target knockout gene. The overlapping amplicons containing the in-frame deletion pattern were subcloned into pIMAY. In order to generate the corresponding recombinant plasmid, the recombinant plasmid was transformed into DH5α, and then transformed into Escherichia coli (E. coli). E. coliThe transformants were then transformed into parental strains (wt strains or engineered single / double KO strains) in DC10B. Allelic substitution two-step screening was performed as previously described [2]. In short, the transformants were first cultured in BHI containing chloramphenicol (10 μg / ml) at 37°C for 2 to 3 days. Clones were screened by PCR using specific primers (Table 3) to select the first recombinant strains. Next, the selected clones were passaged 5 to 7 times in antibiotic-free BHI at 28°C and screened on plates containing PCPA ((4-chloro-DL-phenylalanine)) for the second recombination and plasmid loss. Then, chloramphenicol-sensitive colonies were screened. The deletion of the target gene was first confirmed by PCR using specific primers for the deleted sequence (Table 3) and further confirmed by DNA sequencing. Finally, the adsA gene was successfully removed from Newman wt to generate Newman ΔadsA, the CPs gene was successfully removed from Newman ΔadsA to generate Newman ΔadsAΔCP, the saePQRS gene was successfully removed from Newman ΔadsAΔCP to generate Newman ΔadsAΔCPΔsaePQRS, and the saeRS gene was successfully removed from Newman wt, Newman ΔadsA, or Newman ΔadsAΔCP to generate Newman ΔsaeRS, Newman ΔadsA ΔsaeRS, and Newman ΔadsAΔCPΔsaeRS.

[0200] Due to the high similarity of the genomic sequences between Newman and JE2 strains, we were able to use the same primer set to knock out selected genes in the JE2 strain other than adsA. Therefore, only the JE2-adsA primer sequence is listed separately in Table 2. Following the same approach as with Newman, we successfully removed the adsA gene from JE2 wt to generate JE2 ΔadsA, and successfully removed the saeRS or saePQRS genes from JE2 ΔadsA to generate JE2 ΔadsA ΔsaeRS or JE2 ΔadsA ΔsaePQRS. The constructed strains are shown in Table 2.

[0201] Table 2 List of strains involved in the construction

[0202] Table 3. Primer List

[0203] Construction of strains expressing one or more antigens

[0204] Newman ΔadsA ΔCP ΔsaePQRS + mHla (Sau0137), Newman ΔadsA ΔCP ΔsaePQRS + EsxAB (Sau0144), Newman ΔadsA ΔCP ΔsaePQRS + mHla + mLukS-PV (Sau0140), Newman ΔadsA ΔCP ΔsaePQRS + mHla + EsxAB + mLukS-PV (Sau0145), Newman ΔadsA ΔCP ΔsaePQRS + mHla + mSpa + mLukSF-PV + EsxAB + mLukAB +mSEB + mTSST1 (Sau0105) were constructed based on Newman ΔadsAΔCPΔsaePQRS (Sau0126); and NewmanΔadsA ΔCP + mHla + mSpa + mLukSF-PV + EsxAB + mLukAB + mSEB + mTSST1 (Sau0102) and Newman ΔadsA ΔCP ΔagrA + mHla + mSpa + mLukSF-PV + EsxAB + mLukAB + mSEB + mTSST1 (Sau0103) are constructed based on Newman ΔadsA ΔCP (Sau0021).

[0205] Staphylococcus aureus Newman ΔadsA ΔCP ΔsaePQRS or Newman ΔadsA ΔCP was cultured in brain heart infusion (BHI) at 37°C. The plasmid pIMAY was used. The vector was used to construct plasmids encoding antigen genes (mHla, EsxAB, mLukS-PV, EsxAB-mLukS-PV, mHla-mSpA, EsxAB-mLukSF-PV, mTSST1-mSEB, mLukAB) (Schuster, Christopher F et al., Microbiology (Reading, England) vol. 165,5(2019): 572-584. doi:10.1099 / mic.0.000791). Gel electrophoresis results of PCR detection of the inserted genes were shown in... Figure 2In short, using the appropriate primers (Table 4), the upstream and downstream flanking regions of the target insert gene were amplified from the chromosomal DNA of *Staphylococcus aureus* strains Newman ΔadsA ΔCP ΔsaePQRS or Newman ΔadsA ΔCP by PCR, and the PCR products were separated by 1% agarose gel electrophoresis (using 5kb Plus DNA Ladder as the molecular weight standard). The arrows indicate the expected bands of the knock-in mutant and wild-type strains, respectively. Suitable insertion sites were selected, and antigen gene expression cassettes (mHla, EsxA+EsxB, mLukS-PV, EsxA+EsxB+LukS-PV, mHla+mSpA, EsxA+EsxB+mLukS-PV+LukF-PV, mTSST1+mSEB, mLukA+mLukB) were synthesized, with the upstream and downstream gene sequences of the insertion sites placed at opposite ends of the expression cassettes, respectively. Subsequently, the overlapping amplicon containing the antigen gene was subcloned into pIMAY. The recombinant plasmids were then transformed into DH5α, then into Escherichia coli DC10B, and subsequently into parental strains (Newman ΔadsAΔCPΔsaePQRS / Newman ΔadsAΔCP or engineered single / double / triple KI strains). Allelic substitution two-step screening was performed as previously described [2]. In short, the transformants were first cultured in BHI containing chloramphenicol (10 μg / ml) at 37°C for 2 to 3 days. Clones were screened by PCR using specific primers (Table 3) to select the first recombinant strains. Next, the selected clones were passaged 5 to 7 times in antibiotic-free BHI at 28°C and screened on plates containing PCPA (4-chloro-DL-phenylalanine) for the second recombination and plasmid loss. Then, chloramphenicol-sensitive colonies were screened. The insertion of the target antigen gene was first confirmed by PCR using the primers in Table 4 and further DNA sequencing. Finally, the mHla gene was successfully inserted into Newman ΔadsAΔCPΔsaePQRS to generate the Newman ΔadsA ΔCP ΔsaePQRS + mHla strain; the EsxAB gene was successfully inserted into Newman ΔadsAΔCPΔsaePQRS to generate the Newman ΔadsA ΔCP ΔsaePQRS + EsxAB strain; the antigen mLukS-PV gene was successfully inserted into Newman ΔadsAΔCPΔsaePQRS + mHla strain to generate the Newman ΔadsA ΔCP ΔsaePQRS + mHla + mLukS-PV strain; and the EsxAB-mLukS-PV gene was successfully inserted into Newman ΔadsAΔCPΔsaePQRS + mHla strain to generate the Newman ΔadsA ΔCP ΔsaePQRS + mHla + EsxAB + mLukS-PV strain; the genes mHla-mSpA, EsxAB-mLukSF-PV, mTSST1-SEB, and mLukAB were successfully inserted into Newman ΔadsAΔCPΔsaePQRS or Newman ΔadsA ΔCP to generate Newman ΔadsAΔCP ΔsaePQRS + mHla + mSpa + mLukSF-PV + EsxAB + mLukAB + mSEB + mTSST1 strain and Newman ΔadsA ΔCP + mHla + mSpa + mLukSF-PV + EsxAB + mLukAB + mSEB + mTSST1 strain.

[0206] Table 4. Primers

[0207] Mouse animal infection experiment

[0208] For safety and systemic infection models, each BALB / c mouse was administered a single dose of freshly cultured bacterial suspension (2 × 10⁻⁶) via intraperitoneal or intravenous injection. 8 CFU or 2×10 7 Immunization was administered with CFU. Animal survival was monitored daily for several days. On day 49, Newman strain (2 × 10⁻⁶) was administered intraperitoneally. 8 CFU was used to challenge the bacteria, and survival rates were monitored for several consecutive days. For the skin infection model, each BALB / c or C57BL / 6 mouse was subcutaneously injected with a single dose of freshly cultured bacterial suspension (1×10⁻⁶ CFU). 7 CFU or 5×10 7 Immunization was performed using CFU. Animal weight loss was monitored daily for 14 days, and blood samples were collected from the BALB / c group on days 21 and 28 for antibody titer determination using a whole-bacterial ELISA (Newman Δspa Δsbi). BALB / c group mice were subcutaneously injected with Newman (1×10⁻⁶ CFU) on day 42. 8 CFU or 2×10 8 CFU), HA-MRSA (4×10) 8 CFU), FPR3735 (8×10) 8 CFU), Sau-HK3117 (2×10) 8 CFU) and MU3 strain (8×10 8 The patient was challenged with CFU and monitored for skin abscesses and necrosis for several consecutive days.

[0209] The procedure for whole bacterial ELISA (Newman Δspa Δsbi) is as follows.

[0210] To detect specific antibodies against the Newman strain, a mutant strain (Δspa Δsbi) lacking both Protein A and Protein Sbi was used to avoid nonspecific binding to IgG or IgM antibodies. In summary, 96-well ELISA plates were coated with Newman Δspa Δsbi and incubated overnight at 4°C in 100 mM, pH 9.6 carbonate-bicarbonate buffer to fix the bacteria to the bottom of the wells. After coating, the plates were washed five times with PBS to remove any unfixed bacteria. 200 μL of blocking buffer (PBS containing 5% skim milk) was added to each well, and the plates were blocked for 2 hours at room temperature to block any remaining sites. The liquid in the plates was aspirated, and the plates were washed five times with washing buffer (PBS solution of 0.005% Tween 20) and incubated for 2 hours at room temperature with serially diluted mouse serum in dilution buffer (PBS solution of 2% skim milk). After incubation, wash the plate five times with washing buffer, add 100 μL of secondary antibody (horseradish peroxidase-labeled anti-mouse IgG) diluted 1:5000 with dilution buffer to each well, and incubate for 1 hour. Wash the plate five times with washing buffer, then add 100 μL of 3,3',5,5'-tetramethylbenzidine to each well. After 20 minutes, stop the reaction by adding 50 μL of 1M H2SO4 to each well, and read the absorbance value at 450 nm.

[0211] sheep animal infection experiment

[0212] In Northwest China, Morel disease (MD, caused by the anaerobic subspecies of Staphylococcus aureus, clinically manifested only as abscesses) broke out in breeding centers during the summer as temperatures rose. The incidence decreased as the weather cooled. Environmental infection was present, and most sheep were infected or exposed. Infection was more likely to occur if animals suffered trauma (e.g., injuries from shearing). When multisite sequencing and 16S identification were used to identify the isolated strains, the bacteria isolated from sheep abscesses were found to be highly homologous to the anaerobic subspecies of Staphylococcus aureus strain MVF-7 (ATCC 35844).

[0213] 1) Screening of animals for inclusion

[0214] First, healthy sheep under one year old with clear backgrounds and no obvious Staphylococcus aureus infection were selected as the experimental group. A total of 294 animals were recruited for this experiment. In addition, serum IgG responses against Staphylococcus aureus were screened from the total of 294 healthy sheep, and 72 sheep with low IgG responses (below 1.5) were recruited. All 72 selected animals were randomly assigned to other groups.

[0215] 2) Experimental design and animal grouping

[0216] This experiment was divided into three groups: two immunization groups (Sau0105 group and Sau0137 group) and one placebo group (physiological saline). The Sau0105 group consisted of 30 animals, the Sau0137 group of 12 animals, and the placebo group of 30 animals (as shown in Table 5). The median and distribution of antibody levels in the final grouped animals were basically consistent, indicating that the background levels of the three groups were similar (e.g., ...). Figure 3 (As shown).

[0217] Table 5. Experimental Design and Animal Grouping

[0218] 3) Monitoring of sheep after vaccination

[0219] Animals were immunized on day 0 according to the grouping plan and observed for 42 days.

[0220] Following immunization, monitor the animals' health and for any abnormalities at the injection site. Record any other adverse reactions to assess the safety of the vaccine in sheep.

[0221] Following immunization, the incidence of abscesses (MD) in animals was observed and recorded periodically, including the appearance, recovery, and disappearance of abscesses, in order to study the efficacy of the vaccine against sheep abscesses (MD).

[0222] Example 1. Characterization of ΔsaePQRS strain

[0223] 1.1 The virulence of ΔsaePQRS strain is lower than that of ΔsaeRS strain.

[0224] Knocking out saePQRS resulted in lower virulence than knocking out saeRS. In the BALB / c mouse model, mice immunized (intraperitoneally) with the ΔsaePQRS strain (JE2 ΔadsAΔsaePQRS) showed lower virulence than mice injected with the same dose (2 × 10⁻⁶). 8 Mice with JE2 ΔadsA ΔsaeRS (CFU) had a higher survival rate. Figure 4 Similar results were observed in Newman strains. Using ΔsaePQRS and ΔsaeRS strains with different genetic backgrounds (Newman ΔsaePQRS / Newman ΔsaeRS / Newman ΔadsA ΔCP ΔsaePQRS / Newman ΔadsA ΔCP ΔsaeRS) (2 × 10⁻⁶) 7 Mice were immunized with CFU (intravenous). On day 15, Newman ΔsaePQRS and Newman ΔadsA ΔCP ΔsaePQRS showed higher survival rates than Newman ΔsaeRS and Newman ΔadsA ΔCP ΔsaeRS, respectively. Figure 5 ).

[0225] 1.2 Double knockout of ΔadsA and ΔsaePQRS strains showed reduced virulence and increased protective efficacy.

[0226] In a skin infection model, the double-knockout ΔadsA ΔsaePQRS strain (JE2 ΔadsA ΔsaePQRS strain) showed lower virulence and higher protective efficacy than the single-knockout ΔadsA strain (JE2 ΔadsA). See also Figure 4 .

[0227] 1.3 The virulence of the triple knockout ΔadsA ΔCP ΔsaePQRS strain was lower than that of the double knockout ΔadsA ΔCP strain.

[0228] In a systemic infection model, the virulence of triple knockout ΔadsA ΔCP ΔsaePQRS strains (Newman ΔCP ΔadsA ΔsaePQRS and JE2 ΔadsA ΔsaePQRS - JE2 strain with non-functional CP) was lower than that of double knockout ΔadsA ΔCP strains (Newman ΔCP ΔadsA and JE2 ΔadsA), such as... Figure 4 and Figure 5 As shown.

[0229] The inventors further investigated Sau0126 (Newman ΔadsA ΔCP ΔsaePQRS) in a skin infection model to examine the attenuation effect of additional removal of saePQRS in the ΔadsA ΔCP genetic background. The results showed a significant reduction in virulence. At the same immunization dose (2 × 10⁻⁶ subcutaneously), [the virulence was significantly reduced]. 8 Under CFU, mice injected with Sau0126 showed no obvious abscesses at the injection site, while its parent strain Sau0021 (Newman ΔadsA ΔCP) caused visible skin abscesses in both BALB / c and C57BL / 6 mouse models. Figure 6 The results showed that removing saePQRS did indeed further reduce toxicity.

[0230] 1.4 The triple knockout of ΔadsA, ΔCP, and ΔsaePQRS strains showed lower IgG responses in systemic infection models. The answer is yes, but the efficacy is retained.

[0231] To measure antibody-mediated immune responses, BALB / c mice were immunized with Newman, Sau0021, Sau0126, and PBS (5 × 10⁶ mice subcutaneously). 7 (n=10), and antibody titers were determined by enzyme-linked immunosorbent assay (ELISA).

[0232] Compared with Newman wt and Sau0021, ΔsaePQRS strain Sau0126 showed significantly lower antibody titers on both day 21 and day 28. Figure 7However, during the challenge experiment conducted subsequently in the same set of safety evaluation experiments (immune route: intraperitoneal injection of 2 × 10⁻⁶), 8 Attack route: Intraperitoneal injection of 2×10 8 (n=5), the ΔsaePQRS strain retained its protective effect. Mice immunized with Sau0021 and Sau0126 achieved 100% survival after challenge, while the control group had only a 50% survival rate. Figure 8 The results showed that removing saePQRS did not reduce the efficacy of its parent strain.

[0233] 1.5 The triple knockout of ΔadsA, ΔCP, and ΔsaePQRS strains showed enhanced protective efficacy in a skin infection model.

[0234] The inventors further investigated the protective efficacy of the triple knockout ΔadsA ΔCP ΔsaePQRS strain Sau0126 in a skin infection model. After additional removal of saePQRS in the ΔadsA ΔCP genetic background, Sau0126 exhibited enhanced protection against the Newman wt strain. Compared to the parental strain Sau0021, active immunization with Sau0126 significantly reduced abscess size. Figure 9 Immunization route: subcutaneous injection of 5×10 7 Attack route: Subcutaneous injection of 2×10 8 (n=5).

[0235] Example 2. Characterization of the above-mentioned strains having one or more antigens.

[0236] To improve the protective efficacy and coverage of the strains as vaccines, the inventors additionally expressed one or more antigens derived from different Staphylococcus aureus substrains, namely EsxA (amino acid sequence: SEQ ID NO: 44, coding sequence: SEQ ID NO: 45), and / or EsxB (amino acid sequence: SEQ ID NO: 46, coding sequence: SEQ ID NO: 47), and / or mLukS-PV (amino acid sequence: SEQ ID NO: 48, coding sequence: SEQ ID NO: 49), and / or mLukF-PV (amino acid sequence: SEQ ID NO: 50, coding sequence: SEQ ID NO: 51), and / or mHla (amino acid sequence: SEQ ID NO: 52, coding sequence: SEQ ID NO: 53), and / or mSpA (amino acid sequence: SEQ ID NO: 54, coding sequence: SEQ ID NO: 55), and / or mTSST1 (amino acid sequence: SEQ ID NO: 56, coding sequence: SEQ ID NO: 57), namely EsxA (amino acid sequence: SEQ ID NO: 44, coding sequence: SEQ ID NO: 45), and / or mTSST1 (amino acid sequence: SEQ ID NO: 56, coding sequence: SEQ ID NO: 57), namely EsxA (amino acid sequence: SEQ ID NO: 45), and / or mTSST1 (amino acid sequence: SEQ ID NO: 57), namely mHla (amino acid sequence: SEQ ID NO: 58), and / or mTSST1 (amino acid sequence: SEQ ID NO: 58), namely mHla (amino acid sequence: SEQ ID NO: 59), and / or mTSST1 (amino acid sequence: SEQ ID NO: 59 ... namely mHla (amino 57), and / or mSEB (amino acid sequence: SEQ ID NO: 58, coding sequence: SEQ ID NO: 59), and / or mLukA (amino acid sequence: SEQ ID NO: 60, coding sequence: SEQ ID NO: 61), and / or mLukB (amino acid sequence: SEQ ID NO: 62, coding sequence: SEQ ID NO: 63). The strains involved, possessing one or more antigens, are shown in Table 2.

[0237] 2.1 Sau0105 (Newman ΔadsA ΔCP ΔsaePQRS + 10-antigen) strain in the kidneys Load reduction

[0238] The role of ΔsaePQRS in reducing virulence was further investigated using a 10-antigen strain in a bacterial load assay. Results showed that the Newman ΔadsA ΔCP ΔsaePQRS + 10-antigen strain had a significantly lower bacterial load than the control strains Newman ΔadsA ΔCP + 10-antigen and Newman ΔadsA ΔCP. Furthermore, to specifically investigate the role of saePQRS, another 10-antigen strain was constructed by removing the different TCS gene agrA. Compared to ΔadsA ΔCP and Newman ΔadsA ΔCP + 10-antigen, the bacterial load of the ΔagrA strain was not different, indicating that the main effect comes from the removal of the specific target saePQRS, rather than any other TCS gene. Figure 10 2×10 intravenous injection 7 (n=2).

[0239] 2.2 The Sau0105 strain (Newman ΔadsA ΔCP ΔsaePQRS + 10-antigen) exhibits low virulence.

[0240] Through a systemic infection model (intravenous injection of 2×10) 7 Survival studies (n=5) confirmed that the ΔsaePQRS +10- antigen strain had low virulence.

[0241] The survival rate of Sau0105 (Newman ΔadsA ΔCP ΔsaePQRS + 10-antigen) was higher than that of the control strains Newman ΔadsA ΔCP and Newman ΔadsA ΔCP + 10-antigen. Figure 11 ).

[0242] 2.3 In the skin infection model, Sau0105 (Newman ΔadsA ΔCP ΔsaePQRS + 10-antigen) It provides protective efficacy when used as a vaccine.

[0243] The inventors further investigated the ΔsaePQRS+ 10-antigen strain in a skin infection model to test protective efficacy (immunization route: subcutaneous injection of 1×10). 7 Attack route: Subcutaneous injection of 1×10 8 (n=10). Compared with the control group, immunization with Newman ΔadsA ΔCP ΔsaePQRS + 10- antigen showed a significant reduction in the area of ​​skin necrosis. Figure 12 ).

[0244] 2.4 In a mouse skin infection model, strain Sau0137 (Newman ΔadsA ΔCP ΔsaePQRS+) mHla provides protective efficacy when used as a vaccine.

[0245] To examine the protective efficacy of strains with fewer than 10 antigens, the inventors further investigated the protective efficacy of strain Sau0137 (Newman ΔadsA ΔCP ΔsaePQRS+ mHla) in a mouse skin infection model (immunization route: subcutaneous injection 1×10). 7 Attack route: Subcutaneous injection of 4×10 8 n=20 ）。 Sau0137 showed significantly higher protective activity against HA-MRSA strains than the PBS group. Figure 13 ).

[0246] 2.5 In a mouse skin infection model, strain Sau0140 (Newman ΔadsA ΔCP ΔsaePQRS+) mHla mLukS) provides protective efficacy when used as a vaccine.

[0247] To examine the protective efficacy of strains with fewer than 10 antigens, the inventors further investigated the protective efficacy of strain Sau0140 (Newman ΔadsA ΔCP ΔsaePQRS+ mHla mLukS) in a mouse skin infection model (immunization route: subcutaneous injection 1×10). 7Attack route: Subcutaneous injection of 4×10 8 HA-MRSA or 8×10 8 MU3, ​​n=20). Sau0140 showed significantly higher protective activity against HA-MRSA or MU3 strains than the PBS group. Figure 14 Notably, Sau0140 showed higher protective efficacy against MU3 compared to 4C-Sta Ag (intramuscular immunization, 10 μg / protein, with 1 / 10 alum adjuvant). 4C-Sta Ag is a four-component Staphylococcus aureus vaccine composed of recombinant proteins HlaH35L, EsxAB, FhuD2, and Csa1A, developed by GSK.

[0248] 2.6 In a mouse skin infection model, strain Sau0145 (Newman ΔadsA ΔCP ΔsaePQRS+) mHla mLukS EsxAB provides protective efficacy when used as a vaccine.

[0249] To examine the protective efficacy of strains with fewer than 10 antigens, the inventors further investigated the protective efficacy of strain Sau0145 (Newman ΔadsA ΔCP ΔsaePQRS+ mHla mLukS EsxAB) in a mouse skin infection model (immunization route: subcutaneous injection 1×10). 7 Attack route: Subcutaneous injection of 8×10 8 FPR3735 or 4×10 8 HA-MRSA or 2×10 8 Sau-HK3117, n=20 ）。 Sau0145 showed significantly higher protective activity against HA-MRSA, FPR3735, or Sau-HK3117 strains than the PBS group. Figure 15 ).

[0250] 2.7 In a mouse skin infection model, strain Sau0144 (Newman ΔadsA ΔCP ΔsaePQRS+) EsxAB provides protective efficacy when used as a vaccine.

[0251] To examine the protective efficacy of strains with fewer than 10 antigens, the inventors further investigated the protective efficacy of strain Sau0144 in a mouse skin infection model (Newman ΔadsA ΔCP ΔsaePQRS+ EsxAB) (immunization route: subcutaneous injection 1×10 7 Attack route: Subcutaneous injection of 2×10 8 Sau-HK3117, n=20 ） Sau0144 showed significantly higher protective activity against HA-MRSA strains than the PBS group. Figure 16 ).

[0252] 2.8 In the sheep systemic infection model, Sau0137 (Newman ΔadsA ΔCP ΔsaePQRS+ mHla) Sau0105 (Newman ΔadsA ΔCP ΔsaePQRS + 10-antigen) provides protective efficacy when used as a vaccine.

[0253] Safety study results: No obvious redness or swelling was observed at the injection site in the vaccine group after immunization. The animals generally showed good health, with no signs of fatigue or recumbency. These observations preliminarily demonstrate the safety of the vaccine in sheep.

[0254] Efficacy study results: Throughout the observation period, each animal was examined at all observation points, and its disease status was recorded. When an animal was first found to have an abscess infection, it was considered a primary case. If an animal recovered from its initial infection and subsequently developed an infection again, it was counted as a relapsed case. At the end of the observation period, the number of observed cases was tallied, and the proportion of cases within each group was calculated based on the number of animals that completed the experiment. This data was used to determine the overall efficacy of the vaccine.

[0255] Incidence rate within a group = Number of cases in the group / Base number in the group

[0256] Vaccine efficacy = 1 - (incidence rate in the vaccine group / incidence rate in the control group)

[0257] The experimental results showed that at the end of the experiment (day 42): Sau0105 was 60% effective in preventing the incidence of Staphylococcus aureus-induced abscess (MD), while Sau0137 was 100% effective. This indicates that both Sau0105 and Sau0137 immunizations can effectively prevent the occurrence of sheep abscess (MD), with Sau0137 showing better efficacy (as shown in Table 6).

[0258] Table 6 Results of systemic infection experiments in sheep

[0259] According to this disclosure, all live bacterial strains, compositions, uses, and / or methods disclosed and claimed herein can be prepared and performed without excessive experimentation. While the live bacterial strains, compositions, uses, and / or methods of the present invention have been described according to preferred embodiments, it will be apparent to those skilled in the art that changes may be made to the live bacterial strains, compositions, uses, and / or methods described herein, as well as the steps or order of steps of the methods, without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain chemically and physiologically relevant reagents can be substituted for the reagents described herein while obtaining the same or similar results. All such similar substitutions and modifications that are apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the invention as defined in the appended claims.

[0260] Sequence information (partial)

[0261] SEQ ID NO: 1 adsA amino acid sequence

[0262] SEQ ID NO: 2 capA (NWMN_0095) amino acid sequence

[0263] SEQ ID NO: 3 capB (NWMN_0096) amino acid sequence

[0264] SEQ ID NO: 4 capC (NWMN_0097) amino acid sequence

[0265] SEQ ID NO: 5 capD (NWMN_0098) amino acid sequence

[0266] SEQ ID NO: 6 capE (NWMN_0099) amino acid sequence

[0267] SEQ ID NO: 7 capF (NWMN_0100) amino acid sequence

[0268] SEQ ID NO: 8 capG (NWMN_0101) amino acid sequence

[0269] SEQ ID NO: 9 capH (NWMN_0102) amino acid sequence

[0270] SEQ ID NO: 10 capI (NWMN_0103) amino acid sequence

[0271] SEQ ID NO: 11 capJ (NWMN_0104) amino acid sequence

[0272] SEQ ID NO: 12 capK (NWMN_0105) amino acid sequence

[0273] SEQ ID NO: 13 capL (NWMN_0106) amino acid sequence

[0274] SEQ ID NO: 14 capM (NWMN_0107) amino acid sequence

[0275] SEQ ID NO: 15 capN (NWMN_0108) amino acid sequence

[0276] SEQ ID NO: 16 capO (NWMN_0109) amino acid sequence

[0277] SEQ ID NO: 17 capP (NWMN_0110) amino acid sequence

[0278] SEQ ID NO: 18 capA (NWMN_0095) nucleotide sequence

[0279] SEQ ID NO: 19 capB (NWMN_0096) nucleotide sequence

[0280] SEQ ID NO: 20 capC (NWMN_0097) nucleotide sequence

[0281] SEQ ID NO: 21 capD (NWMN_0098) nucleotide sequence

[0282] SEQ ID NO: 22 capE (NWMN_0099) nucleotide sequence

[0283] SEQ ID NO: 23 capF (NWMN_0100) nucleotide sequence

[0284] SEQ ID NO: 24 capG (NWMN_0101) nucleotide sequence

[0285] SEQ ID NO: 25 capH (NWMN_0102) nucleotide sequence

[0286] SEQ ID NO: 26 capI (NWMN_0103) nucleotide sequence

[0287] SEQ ID NO: 27 capJ (NWMN_0104) nucleotide sequence

[0288] SEQ ID NO: 28 capK (NWMN_0105) nucleotide sequence

[0289] SEQ ID NO: 29 capL (NWMN_0106) nucleotide sequence

[0290] SEQ ID NO: 30 capM (NWMN_0107) nucleotide sequence

[0291] SEQ ID NO: 31 capN (NWMN_0108) nucleotide sequence

[0292] SEQ ID NO: 32 capO (NWMN_0109) nucleotide sequence

[0293] SEQ ID NO: 33 capP (NWMN_0110) nucleotide sequence

[0294] SEQ ID NO: 34 saeP amino acid sequence (Newman)

[0295] SEQ ID NO: 35 saeQ amino acid sequence (Newman)

[0296] SEQ ID NO: 36 saeS amino acid sequence (Newman)

[0297] SEQ ID NO: 37 saeR amino acid sequence (Newman)

[0298] SEQ ID NO: 38 saeP nucleotide sequence (Newman)

[0299] SEQ ID NO: 39 saeQ nucleotide sequence (Newman)

[0300] SEQ ID NO: 40 saeS nucleotide sequence (Newman)

[0301] SEQ ID NO: 41 saeR nucleotide sequence (Newman)

[0302] SEQ ID NO: 42 saeS amino acid sequence (JE2)

[0303] SEQ ID NO: 43 saeS nucleotide sequence (JE2)

[0304] SEQ ID NO: 44 EsxA nucleotide sequence (codon optimization)

[0305] SEQ ID NO: 45 EsxA amino acid sequence from Newman

[0306] SEQ ID NO: 46 EsxB nucleotide sequence (codon optimization)

[0307] SEQ ID NO: 47 EsxB amino acid sequence from Newman

[0308] SEQ ID NO: 48 mLukS-PV nucleotide sequence

[0309] SEQ ID NO: 49 mLukS-PV amino acid sequence

[0310] SEQ ID NO: 50 mLukF-PV nucleotide sequence

[0311] SEQ ID NO: 51 mLukF-PV amino acid sequence

[0312] SEQ ID NO: 52 mHla nucleotide sequence

[0313] SEQ ID NO: 53 mHla amino acid sequence

[0314] SEQ ID NO: 54 mSpA nucleotide sequence

[0315] SEQ ID NO: 55 mSpA amino acid sequence

[0316] SEQ ID NO: 56 mTSST1 nucleotide sequence

[0317] SEQ ID NO: 57 mTSST1 amino acid sequence

[0318] SEQ ID NO: 58 mSEB nucleotide sequence

[0319] SEQ ID NO: 59 mSEB amino acid sequence

[0320] SEQ ID NO: 60 mLukA nucleotide sequence

[0321] SEQ ID NO: 61 mLukA amino acid sequence

[0322] SEQ ID NO: 62 mLukB nucleotide sequence

[0323] SEQ ID NO: 63 mLukB amino acid sequence

[0324] References

[0325] 1. Collins, Madison M., et al. “The accessory gene saeP of the SaeR / Stwo-component gene regulatory system impacts Staphylococcus aureus virulenceduring neutrophil interaction.” Frontiers in microbiology 11 (2020): 561.

[0326] 2. Bae, T. and O. Schneewind, “Allelic replacement in Staphylococcusaureus with inducible counter-selection”. Plasmid, 2006. 55(1): p. 58-63.

[0327] 3. Kneidinger, B., et al. 2003. “Three highly conserved proteins catalyze the conversion of UDP-Nacetyl-D-glucosamine to precursors for the biosynthesis of O antigen inPseudomonas aeruginosa O11 and capsule inStaphylococcus aureus type 5-implications for the UDP-N-acetyl-L-fucosaminebiosynthetic pathway.” J. Biol. Chem. 278:3615–3627.

[0328] 4. Paul D. Fey, et al. 2013. “A Genetic Resource for Rapid and Comprehensive Phenotype Screening of Nonessential Staphylococcus aureusGenes.” mBio 4(1): e00537-12. doi:10.1128 / mBio.00537-12.

[0329] All references cited herein are incorporated herein by reference in their entirety and for all purposes, to the extent that each individual publication, patent, or patent application is specifically and individually indicated as being incorporated herein by reference in its entirety for all purposes.

Claims

1. A species from the genus Staphylococcus ( Staphylococcus sp. ) types of live bacterial strains, among which, For example, compared with the corresponding control strain, i) The live bacterial strain has reduced activity of the sae two-component system, preferably lacking the activity of the sae two-component system; ii) Reduced expression of one or more genes of the SAE two-component system in the live bacterial strain; and / or iii) The live bacterial strain said to contain one or more mutations in one or more genes of the sae two-component system.

2. The live bacterial strain according to claim 1, wherein, compared with the corresponding control strain, the expression of one or more genes of the sae two-component system 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.

3. The live bacterial strain according to claim 1 or 2, wherein the expression of one or more genes selected from SaeS, SaeR, SaeP and SaeQ is reduced in the live bacterial strain; preferably, the expression of SaeS and SaeR or the expression of all SaeS, SaeR, SaeP and SaeQ is reduced in the live bacterial strain; more preferably, the live bacterial strain does not express SaeS and SaeR or does not express SaeS, SaeR, SaeP and SaeQ.

4. The live bacterial strain according to claim 1, wherein the live bacterial strain contains one or more mutations in one or more genes selected from SaeS, SaeR, SaeP and SaeQ, preferably, all SaeS, SaeR, SaeP and SaeQ in the live bacterial strain are mutated.

5. The live bacterial strain according to any one of claims 1 to 4, wherein the mutation in one or more genes of the sae two-component system causes a reduction or absence of expression of the one or more genes of the sae two-component system, or causes a reduction or absence of expression of one or more proteins of the sae two-component system.

6. The live bacterial strain according to any one of claims 1 to 5, wherein the mutation comprises the deletion of one or more genes of the sae two-component system, preferably, all genes of the sae two-component system in the live bacterial strain are completely or partially deleted, more preferably, all genes of the sae two-component system in the live bacterial strain are completely deleted.

7. The live bacterial strain according to claim 6, wherein one or more genes selected from SaeS, SaeR, SaeP and SaeQ are deleted in the live bacterial strain, preferably, SaeS and SaeR or all SaeS, SaeR, SaeP and SaeQ are completely or partially deleted in the live bacterial strain, more preferably, SaeS and SaeR or all SaeS, SaeR, SaeP and SaeQ are completely deleted in the live bacterial strain.

8. The live bacterial strain according to any one of claims 1 to 7, wherein, for example, compared with the corresponding control strain, the live bacterial strain also has reduced adenosine synthase A (adsA) activity, preferably, lacks adenosine synthase A (adsA) activity.

9. The live bacterial strain according to claim 8, wherein the live bacterial strain contains a mutation in the adsA gene.

10. The live bacterial strain according to claim 9, wherein the mutation causes a decrease or absence of expression of the adsA gene, or causes reduced or inactive expression of the adsA protein.

11. The live bacterial strain according to claim 9 or 10, wherein the mutation includes the deletion of the adsA gene in the live bacterial strain, for example, the adsA gene is partially or completely deleted.

12. The live bacterial strain according to any one of claims 1 to 11, for example, compared with the corresponding control strain, The live bacterial strains also exhibit reduced capsule production, preferably a lack of capsule production, and / or The expression of one or more genes related to capsular polysaccharide synthesis is reduced in the live bacterial strains, and / or The live bacterial strains contain one or more mutations in one or more genes related to the synthesis of capsular polysaccharides.

13. The live bacterial strain according to claim 12, wherein, compared with the corresponding control strain, the 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, preferably, the live bacterial strain does not produce a capsule.

14. The live bacterial strain according to claim 12 or 13, wherein, compared with the corresponding control strain, the production of capsular polysaccharide 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, preferably, the live bacterial strain does not produce capsular polysaccharide.

15. The live bacterial strain according to any one of claims 12 to 14, wherein, compared with the corresponding control strain, the expression of one or more capsular polysaccharide synthesis-related 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.

16. The live bacterial strain according to any one of claims 12 to 15, wherein the expression of all capsular polysaccharide synthesis-related genes is reduced in the live bacterial strain, preferably, the live bacterial strain does not express capsular polysaccharide synthesis-related genes.

17. The live bacterial strain according to any one of claims 12 to 16, wherein all capsular polysaccharide synthesis-related genes in the live bacterial strain are mutated.

18. The live bacterial strain according to any one of claims 12 to 17, wherein the mutation in the capsular polysaccharide synthesis-related gene causes a decrease or absence of expression of the capsular polysaccharide synthesis-related gene, or causes reduced or inactive expression of capsular polysaccharide synthesis-related proteins.

19. The live bacterial strain according to any one of claims 12 to 18, wherein the mutation comprises the deletion of one or more capsular polysaccharide synthesis-related genes, preferably, all capsular polysaccharide synthesis-related genes in the live bacterial strain are partially or completely deleted, more preferably, all capsular polysaccharide synthesis-related genes in the live bacterial strain are completely deleted.

20. The live bacterial strain according to any one of claims 12 to 19, wherein the one or more capsular polysaccharide synthesis-related 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.

21. The live bacterial strain according to claim 20, wherein all of the capsular polysaccharide synthesis-related genes of capA, capB, capC, capD, capE, capF, capG, capH, capI, capJ, capK, capL, capM, capN, capO and capP are deleted, preferably completely deleted.

22. The live bacterial strain according to any one of claims 1 to 21, wherein the mutation is achieved by homologous recombination or by targeted mutagenesis, such as via CRISPR, TALEN or ZFN technology.

23. The live bacterial strain according to any one of claims 1 to 22, wherein the live bacterial strain has reduced virulence compared with the 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.

24. The live bacterial strain according to any one of claims 1 to 23, wherein the live bacterial strain has increased immunogenicity compared with the 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.

25. The live bacterial strain according to any one of claims 1 to 24, wherein the species from the genus Staphylococcus is selected from the group consisting of: Staphylococcus aureus ( Staphylococcus aureus ), Staphylococcus aureus ( Staphylococcus capitis ), Staphylococcus aureus ( Staphylococcus caprae Staphylococcus aureus ( Staphylococcus carnosus Staphylococcus epidermidis ( Staphylococcus epidermidis Staphylococcus intermedia ( Staphylococcus intermedius Staphylococcus aureus ( ), Staphylococcus aureus ( Staphylococcus lentus Staphylococcus ludensii ( Staphylococcus lugdunensis Staphylococcus piraceae ( ) Staphylococcus pettenkoferi ), mimicking Staphylococcus ( Staphylococcus simulans ), Staphylococcus aureus ( Staphylococcus vitulinus ), Anaerobic Staphylococcus subsp. Staphylococcus subspecies anaerobius ) and Staphylococcus xylose ( Staphylococcus xylosus Preferably, the species from the genus Staphylococcus is Staphylococcus aureus.

26. The live bacterial strain according to any one of claims 1 to 25, wherein the live bacterial strain is derived from a parent strain used as a clinical isolate.

27. The live bacterial strain according to any one of claims 1 to 25, wherein the live bacterial strain is derived from a parent strain that has already acquired low virulence.

28. The live bacterial strain according to any one of claims 1 to 27, wherein the live bacterial strain is derived from Staphylococcus aureus serotype 5 or serotype 8, more preferably, the live bacterial strain is derived from Staphylococcus aureus serotype 5.

29. The live bacterial strain according to claim 28, wherein the live bacterial strain is derived from Staphylococcus aureus Newman strain, FPR3735, JE2 or ATCC29213.

30. The live bacterial strain according to any one of claims 1 to 29, wherein the live bacterial strain is used as a live expression vector for expressing the target protein.

31. The live bacterial strain according to any one of claims 1 to 30, wherein the live bacterial strain further comprises a coding sequence for the target protein and is thereby capable of expressing the target protein.

32. The live bacterial strain of claim 31, wherein the coding sequence of the target protein is introduced into the live bacterial strain, for example, through a nucleic acid expression construct.

33. The live bacterial strain of claim 32, wherein the introduced coding sequence of the target protein is integrated into the genome of the live bacterial strain.

34. The live bacterial strain according to any one of claims 30 to 33, wherein the target protein is expressed and displayed on the cell surface of the live bacterial strain; or expressed and secreted by the cells of the live bacterial strain.

35. The live bacterial strain according to any one of claims 30 to 34, wherein the target protein is an antigen.

36. The live bacterial strain according to any one of claims 30 to 35, wherein the target protein is selected from EsxA, EsxB, mLukS-PV, mLukF-PV, mHla, mSpA, mTSST1, mSEB, mLukA, mLukB or any combination thereof.

37. The live bacterial strain according to claim 36, wherein the target protein is i) mHla; ii) EsxA and EsxB; iii) mHla, mLukS-PV and mLukF-PV; iv) mHla, EsxA, EsxB, mLukS-PV and mLukF-PV; or v) EsxA, EsxB, mLukS-PV, mLukF-PV, mHla, mSpA, mTSST1, mSEB, mLukA and mLukB.

38. The live bacterial strain according to any one of claims 1 to 37, wherein the live bacterial strain is used for the prevention and / or treatment of bacterial infection in a subject.

39. The live bacterial strain of claim 38, wherein the bacterial infection is caused by a species derived from the live bacterial strain, such as a species from the genus Staphylococcus.

40. The live bacterial strain of claim 39, wherein the bacterial infection is caused by Staphylococcus aureus, Staphylococcus capitis, Staphylococcus capitis, Staphylococcus carinatum, Staphylococcus epidermidis, Staphylococcus intermedius, Staphylococcus slow-moving, Staphylococcus ludens, Staphylococcus pistilli, Staphylococcus mimicus, Staphylococcus calfii, Staphylococcus aureus subsp. anaerobicus, or Staphylococcus xylose, or any combination thereof. ， The infection is preferably caused by Staphylococcus aureus.

41. Use of any of the live bacterial strains according to any one of claims 1 to 40 in the preparation of compositions such as vaccines for the prevention or treatment of bacterial infections in subjects.

42. A composition, such as a vaccine, for the prevention or treatment of bacterial infection in a subject, said composition comprising a live bacterial strain according to any one of claims 1 to 40.

43. The composition of claim 42, wherein the composition further comprises an adjuvant and / or a pharmaceutically acceptable carrier.

44. 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 claims 1 to 40, or a composition according to claim 42 or 43.

45. The use according to claim 41, the composition according to any one of claims 42 to 43, or the method according to claim 44, wherein the bacterial infection is caused by a species derived from the live bacterial strain, such as a species from the genus Staphylococcus.

46. ​​The use, composition, or method according to claim 45, wherein the bacterial infection is caused by Staphylococcus aureus, Staphylococcus capitis, Staphylococcus capitis, Staphylococcus carinatum, Staphylococcus epidermidis, Staphylococcus intermedius, Staphylococcus stenoticus, Staphylococcus ludens, Staphylococcus pisciniformis, Staphylococcus mimicus, Staphylococcus calfii, Staphylococcus aerobicus subsp. anaerobicus, or Staphylococcus xylose, or any combination thereof.

47. The use, composition, or method according to claim 46, wherein the bacterial infection is a Staphylococcus aureus infection.

48. The use, composition, or method according to claim 47, wherein the Staphylococcus aureus infection is a skin infection, soft tissue infection, or invasive disease.

49. The use, composition, or method according to claim 48, wherein the invasive disease is a bloodstream infection, endocarditis, Morell's disease, or sepsis.

50. The use, composition, or method according to any one of claims 47 to 49, wherein the Staphylococcus aureus infection is methicillin-resistant Staphylococcus aureus. S. aureus MRSA infection or methicillin-sensitive Staphylococcus aureus S. aureus MSSA infection, preferably, said infection is recurrent Staphylococcus aureus infection.

51. The use, composition, or method according to any one of claims 45 to 50, wherein the subject is a mammal, such as a human, mouse, rat, monkey, dog, pig, sheep, goat, cow, horse, donkey, domestic cattle, or cat; or poultry, such as chicken, duck, or goose.

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