Modified streptococcus immunogens and uses thereof

Abstract

The present disclosure relates to an immunogenic composition comprising an attenuated and / or killed Streptococcus bacterial strain comprising a modification that attenuates, reduces or prevents the functional expression of LytC (or homolog thereof). Such compositions are useful in vaccine compositions. The immunogenic composition and / or the vaccine composition may be used in a method of inducing an immune response against at least one target streptococcus species and / or serotype thereof and / or preventing or reducing an infection and / or disease or condition caused by at least one target streptococcus species and / or serotype thereof in a subject.

Description

Related applications

[0001] This application claims priority to Australian Provisional Application No. 2023901596, filed on 22 May 2023, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0002] This disclosure relates to an immunogenic composition. In one form, this disclosure relates to an attenuated or inactivated streptococcal bacterial strain. Background Technology

[0003] Any discussion of the prior art throughout this specification should in no way be construed as an admission that such prior art is well-known or constitutes part of common general knowledge in the field.

[0004] Currently used vaccines against streptococcal infection are typically polysaccharide vaccines containing purified capsular polysaccharides from multiple serotypes (e.g., the PCV23 vaccine containing polysaccharides from 23 serotypes), or conjugated vaccines containing capsular polysaccharides conjugated to diphtheria toxoid or other protein antigens (including those not of streptococcal or streptococcal origin). These polysaccharide-targeted vaccines induce strict serotype-dependent protection. Serotype substitution is a significant issue associated with polysaccharide vaccines because multivalent conjugated vaccines induce protection against only the more than 100 types of Streptococcus pneumoniae identified to date. Streptococcus pneumoniae Immunity is limited to a subset of serotypes, most of which have been shown to cause disease. Therefore, the immunity induced by commercially available streptococcal vaccines is insufficient to establish broad immunity against most streptococcal species and / or certain pathogenic serotypes. Therefore, an improved streptococcal vaccine is needed.

[0005] Streptococcal strains, such as Streptococcus pneumoniae, may possess autolysins such as CbpD, LytA, LytB, and LytC. LytC is a cell wall hydrolase that has been proposed to contribute to fratricide with LytA by promoting the lysis of non-competent Streptococcus pneumoniae cells (Eldholm et al., 2009).

[0006] This disclosure relates to an unexpected discovery, namely, the use of LytC Attenuated or inactivated whole-cell streptococcal vaccines with genes (or homologs) modified to attenuate, reduce, and / or prevent the expression of functional LytC (or homologs) provide improved vaccine immunogenicity. Summary of the Invention

[0007] This disclosure relates to an attenuated or bactericidal streptococcal bacterial strain comprising modifications that weaken, reduce, or prevent the functional expression of LytC (or its homologs). Surprisingly, this is in contrast to the lack of... lytCCompared to the modified parental vaccine strain, using a strain containing [a specific type of vaccine] lytC Mice and rabbits immunized with a composition of missing Streptococcus bacterial strains produced IgG antibodies with enhanced binding to the surface of Pneumococcal strains. Furthermore, by using a combination of strains carrying… lytC Antibodies produced by immunization with the missing strains exhibited a higher functional opsonization phagocytic response, a recognized surrogate endpoint for pneumococcal vaccine efficacy. These findings illustrate a previously unrecognized mechanism of action through modification. lytC Gene expression or deletion lytC A novel mechanism by which genes improve the immunogenicity of whole-cell vaccines. This is based on a mechanism that was not anticipated in previous studies.

[0008] This invention relates to at least the following embodiments: Implementation Scheme 1. An immunogenic composition comprising an attenuated and / or killable streptococcal bacterial strain, said strain comprising modifications that attenuate, reduce, or prevent the functional expression of LytC or its homologs. In some examples, the immunogenic composition may consist of or substantially comprise an attenuated and / or killable streptococcal bacterial strain comprising modifications that attenuate, reduce, or prevent the functional expression of LytC or its homologs.

[0009] Implementation Scheme 2. The immunogenic composition according to Implementation Scheme 1, wherein the modification includes lytC Deletion of a gene or its homolog. In some examples, the modification may be made by or substantially by the gene. lytC Deletion of a gene or its homolog.

[0010] Implementation Scheme 3. The immunogenic composition according to Implementation Scheme 1 or 2, wherein the immunogenic composition is a cross-protective immunogenic composition.

[0011] Implementation Scheme 4. The immunogenic composition according to any one of Implementation Schemes 1 to 3, wherein the attenuated and / or killable streptococcal bacterial strain is derived from a parent strain lacking the modification, and the attenuated and / or killable streptococcal bacterial strain improves the effectiveness of the immune response compared to the parent strain.

[0012] Implementation Scheme 5. The immunogenic composition according to any one of Implementation Schemes 1 to 4, wherein the attenuated and / or killable streptococcal bacterial strain is derived from a parent strain lacking the modification, and the attenuated and / or killable streptococcal bacterial strain has improved stability compared to the parent strain.

[0013] Implementation Scheme 6. The immunogenic composition according to any one of Implementation Schemes 1 to 5, wherein the attenuated and / or killable streptococcal bacterial strain further comprises a modification or deletion of a gene selected from the group consisting of: lytA or its homologs ply or its homologs and psaA Or its homologs.

[0014] Implementation Scheme 7. The immunogenic composition according to any one of Implementation Schemes 1 to 6, wherein the attenuated and / or killable streptococcal bacterial strain further comprises lytA Modification or deletion of a gene or its homolog.

[0015] Implementation Scheme 8. The immunogenic composition according to any one of Implementation Schemes 1 to 7, wherein the attenuated and / or killable streptococcal bacterial strain further comprises ply Modification or deletion of a gene or its homolog.

[0016] Implementation Scheme 9. The immunogenic composition according to any one of Implementation Schemes 1 to 8, wherein the attenuated and / or killable streptococcal bacterial strain further comprises a toxoid form of Ply named PdT or a homolog thereof.

[0017] Implementation Scheme 10. The immunogenic composition according to any one of Implementation Schemes 1 to 9, wherein the attenuated and / or killable streptococcal bacterial strain further comprises psaA Modification or deletion of a gene or its homolog.

[0018] Implementation Scheme 11. The immunogenic composition according to any one of Implementation Schemes 1 to 10, wherein the attenuated and / or killable streptococcal bacterial strain further comprises lytA Modification or deletion of genes or their homologs ply Modification or deletion of genes or their homologs and psaA The gene or its homolog is modified or deleted, and also contains a toxoid form of Ply named PdT or its homolog.

[0019] Implementation Scheme 12. An immunogenic composition according to any one of Implementation Schemes 1 to 11, wherein the attenuated and / or killed streptococcal bacterial strain expresses capsular polysaccharides.

[0020] Implementation Scheme 13. The immunogenic composition according to any one of Implementation Schemes 1 to 12, wherein the attenuated and / or killed streptococcal bacterial strain expresses reduced levels of capsular polysaccharides or does not express capsular polysaccharides.

[0021] Implementation Scheme 14. An immunogenic composition according to any one of Implementation Schemes 1 to 13, wherein the composition comprises a single attenuated and / or killable streptococcal bacterial strain, said strain comprising modifications that attenuate, reduce or prevent the functional expression of LytC or its homologs.

[0022] Implementation Scheme 15. An immunogenic composition according to any one of Implementation Schemes 1 to 13, wherein the composition comprises a plurality of attenuated and / or killable streptococcal bacterial strains, each strain comprising a modification that weakens, reduces, or prevents the functional expression of LytC or its homologs.

[0023] Implementation Scheme 16. The immunogenic composition according to any one of Implementation Schemes 1 to 15, wherein the attenuated and / or killable streptococcal bacterial strain belongs to a species selected from the group consisting of: agalactococci (Streptococcus agalactiae) Streptococcus agalactiae Streptococcus bovis () Streptococcus bovis Streptococcus canis ( Streptococcus canis Streptococcus pyogenes ( ), Streptococcus dysgalactiae Streptococcus equi ( Streptococcus equi Streptococcus equi ( Streptococcus equinus Streptococcus equi ( Streptococcus equisimilis ), Enterococcus faecalis ( Enterococcus faecalis ), Enterococcus faecalis ( Enterococcus faecium ), Dolphin Streptococcus ( Streptococcus iniae ), Streptococcus Miller ( Streptococcus milleri Streptococcus mutans ( ) Streptococcus mutans Streptococcus pneumoniae, Streptococcus pyogenes (Streptococcus pneumoniae) Streptococcus pyogenes ), Streptococcus salivarius ( Streptococcus salivarius ), Streptococcus sanguinis ( Streptococcus sanguinis Streptococcus suis ( Streptococcus suis ) and Streptococcus lactis ( Streptococcus uberis ).

[0024] Implementation Scheme 17. The immunogenic composition according to any one of Implementation Schemes 1 to 16, wherein the attenuated and / or killed streptococcal bacterial strains include Streptococcus pneumoniae strains.

[0025] Implementation Scheme 18. The immunogenic composition according to Implementation Scheme 17, wherein the Streptococcus pneumoniae strain is derived from Rx1.

[0026] Implementation Scheme 19. An immunogenic composition according to any one of Implementation Schemes 1 to 18, wherein the attenuated and / or killable streptococcal bacterial strain induces an immune response against at least one target streptococcal species and / or its serotype.

[0027] Implementation Scheme 20. An immunogenic composition according to any one of Implementation Schemes 1 to 19, wherein the attenuated and / or killable streptococcal bacterial strain induces an immune response against a variety of target streptococcal species and / or their serotypes.

[0028] Implementation Scheme 21. An immunogenic composition according to any one of Implementation Schemes 1 to 20, wherein the attenuated and / or killed streptococcal bacterial strain induces a cross-protective immune response against at least one target streptococcal species and / or its serotype.

[0029] Implementation Scheme 22. An immunogenic composition according to any one of Implementation Schemes 1 to 21, wherein the attenuated and / or killed streptococcal bacterial strain induces a cross-protective immune response against multiple target streptococcal species and / or their serotypes.

[0030] Implementation Scheme 23. The immunogenic composition according to any one of Implementation Schemes 1 to 20, wherein the attenuated and / or killed streptococcal bacterial strain is derived from a strain, serotype or species that is the same as the at least one target streptococcal species and / or its serotype.

[0031] Implementation Scheme 24. The immunogenic composition according to any one of Implementation Schemes 1 to 22, wherein the attenuated and / or killed streptococcal bacterial strain is derived from a strain, serotype or species that is different from the at least one target streptococcal species and / or its serotype.

[0032] Implementation Scheme 25. An immunogenic composition according to any one of Implementation Schemes 1 to 24, wherein the at least one target streptococcal bacterial species and / or its serotype is selected from the group consisting of: agalactiae, bovine streptococci, canine streptococci, dysgalactiae, equine streptococci, equine enterococci, equine-like streptococci, enterococci faecalis, enterococci faecium, dolphin streptococci, Miller streptococci, variant streptococci, pneumococci, pyogenic streptococci, saliva streptococci, blood streptococci, suis streptococci, and mammary streptococci.

[0033] Implementation Scheme 26. An immunogenic composition according to any one of Implementation Schemes 1 to 25, wherein the immunogenic composition is a killing whole-cell immunogenic composition.

[0034] Implementation Scheme 27. The immunogenic composition according to any one of Implementation Schemes 1 to 26, wherein the whole-cell-killing immunogenic composition is selected from the group consisting of: chemically treated whole-cell-killing immunogenic compositions, heat-treated whole-cell-killing immunogenic compositions, irradiated whole-cell-killing immunogenic compositions, high hydrostatic pressure-killing whole-cell-killing immunogenic compositions, pulsed electric field-killing whole-cell-killing immunogenic compositions, ultrashort pulse laser-killing whole-cell-killing immunogenic compositions, pressure-ultrasonic-killing whole-cell-killing immunogenic compositions, and UV irradiation and microbial inactivation-killing whole-cell-killing immunogenic compositions.

[0035] Implementation Scheme 28. The immunogenic composition according to any one of Implementation Schemes 1 to 27, wherein the whole-cell killing immunogenic composition is a whole-cell killing immunogenic composition by photon irradiation.

[0036] Implementation Scheme 29. The immunogenic composition according to any one of Implementation Schemes 1 to 28, wherein the whole-cell killing immunogenic composition is a whole-cell killing immunogenic composition by gamma irradiation.

[0037] Implementation Scheme 30. The immunogenic composition according to any one of Implementation Schemes 1 to 28, wherein the whole-cell killing immunogenic composition is a whole-cell killing immunogenic composition by X-ray irradiation.

[0038] Implementation Scheme 31. The immunogenic composition according to any one of Implementation Schemes 1 to 31, wherein the whole-cell killing immunogenic composition is a whole-cell killing immunogenic composition by gamma irradiation and X-ray irradiation.

[0039] Implementation Scheme 32. An immunogenic composition according to any one of Implementation Schemes 1 to 25, wherein the immunogenic composition is an attenuated whole-cell immunogenic composition.

[0040] Implementation Scheme 33. The immunogenic composition according to any one of Implementation Schemes 1 to 32 further comprises at least one of an adjuvant, a pharmaceutically acceptable excipient, and a pharmaceutically acceptable carrier.

[0041] Implementation Scheme 34. An immunogenic composition according to any one of Implementation Schemes 1 to 33, wherein the composition does not contain an adjuvant.

[0042] Implementation Scheme 35. A vaccine composition comprising an immunogenic composition according to any one of Implementation Schemes 1 to 34.

[0043] Implementation Scheme 36. Use of the immunogenic composition according to any one of Implementation Schemes 1 to 34 or the vaccine composition according to Implementation Scheme 35 as an immunogen.

[0044] Implementation Scheme 37. Use of the immunogenic composition according to any one of Implementation Schemes 1 to 34 or the vaccine composition according to Implementation Scheme 35 as a vaccine.

[0045] Implementation Scheme 38. A method for inducing an immune response in a subject against at least one target streptococcal species and / or its serotype, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition according to any one of Implementation Schemes 1 to 34 or a vaccine composition according to Implementation Scheme 35.

[0046] Implementation Scheme 39. A method for inducing a cross-protective immune response against at least one target streptococcal species and / or its serotype in a subject, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition according to any one of Implementation Schemes 1 to 34 or a vaccine composition according to Implementation Scheme 35.

[0047] Implementation Scheme 40. A method for preventing or reducing infection in a subject caused by at least one target streptococcal species and / or its serotype, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition according to any one of Implementation Schemes 1 to 34 or a vaccine composition according to Implementation Scheme 35.

[0048] Implementation Scheme 41. A method for preventing or reducing disease or condition in a subject caused by at least one target streptococcal species and / or its serotype, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition according to any one of Implementation Schemes 1 to 34 or a vaccine composition according to Implementation Scheme 35.

[0049] Implementation Scheme 42. The method according to any one of Implementation Schemes 38 to 42, wherein the attenuated and / or killed streptococcal bacterial strain is derived from a strain, serotype or species that is the same as the at least one target streptococcal species and / or its serotype.

[0050] Implementation Scheme 43. The method according to any one of Implementation Schemes 38 to 42, wherein the attenuated and / or killed streptococcal bacterial strain is derived from a strain, serotype or species that is different from the at least one target streptococcal species and / or its serotype.

[0051] Implementation Scheme 44. The method according to any one of Implementation Schemes 38 to 42, wherein the at least one target streptococcal species and / or its serotype includes multiple target streptococcal species and / or their serotypes.

[0052] Implementation Scheme 45. The method according to any one of Implementation Schemes 38 to 42, wherein the target streptococcal species and / or serotypes comprise streptococcal bacterial species and / or serotypes selected from the group consisting of: *Streptococcus agalactiae*, *Streptococcus bovis*, *Streptococcus canis*, *Streptococcus dysgalactiae*, *Streptococcus equi*, *Streptococcus equineus*, *Streptococcus faecalis*, *Streptococcus dolphinus*, *Streptococcus Miller*, *Streptococcus mutans*, *Streptococcus pneumoniae*, *Streptococcus pyogenes*, *Streptococcus salivarius*, *Streptococcus salivarius*, *Streptococcus sanguinis*, *Streptococcus suis*, and *Streptococcus lactis*.

[0053] Implementation Scheme 46. The method according to any one of Implementation Schemes 38 to 42, wherein the immunogenic composition or the vaccine composition is administered to the subject via an administration route selected from the group consisting of: intranasal, intravenous, intramuscular, subcutaneous, oral, intraocular, transmucosal, and transdermal administration.

[0054] Implementation Scheme 47. The method according to Implementation Scheme 41, wherein the disease or condition is selected from the group consisting of: respiratory tract infection, pneumonia, ear infection, earache, middle ear infection, otitis media, sinusitis, meningitis, conjunctivitis, bacteremia, sepsis, joint infection, bone infection, septic arthritis, osteomyelitis, soft tissue infection, cellulitis, myositis, periorbital cellulitis, abscess, peritonitis, cardiac infection, endocarditis, and pericarditis.

[0055] Implementation Scheme 48. A method for preparing an immunogenic composition according to any one of Implementation Schemes 1 to 34 or a vaccine composition according to Implementation Scheme 35, the method comprising: (i) A preparation of photon-irradiated streptococcal bacterial strains comprising modified strains that attenuate, reduce, or prevent the functional expression of LytC or its homologs, thereby killing or physically attenuating said bacteria to produce photon-irradiated streptococcal bacteria; and (ii) Combining the photon-irradiated streptococcal bacteria with pharmaceutically acceptable excipients, diluents and / or carriers, and optionally adjuvants.

[0056] Implementation Scheme 49. The method according to Implementation Scheme 48, wherein the photon irradiation comprises exposing the streptococcal bacterial strain to gamma radiation.

[0057] Implementation Scheme 50. The method according to Implementation Scheme 49, wherein the photon irradiation comprises exposing the streptococcal bacterial strain to X-ray radiation.

[0058] Implementation Scheme 51. The method according to any one of Implementation Schemes 48 to 50, wherein the photon irradiation comprises exposing the streptococcal bacterial strain to gamma radiation and X-ray radiation.

[0059] Implementation Scheme 52. The method according to any one of Implementation Schemes 48 to 51, wherein the photon irradiation comprises exposing the streptococcal bacterial strain to photon radiation with an energy of at least 0.01 MeV.

[0060] Implementation Scheme 53. Use of a therapeutically effective amount of the immunogenic composition according to any one of Implementation Schemes 1 to 34 or the vaccine composition according to Implementation Scheme 35 in the preparation of a medicament for inducing an immune response in a subject against at least one target streptococcal species and / or its serotype.

[0061] Implementation Scheme 54. Use of a therapeutically effective amount of the immunogenic composition according to any one of Implementation Schemes 1 to 34 or the vaccine composition according to Implementation Scheme 35 in the preparation of a medicament for inducing a cross-protective immune response against at least one target streptococcal species and / or its serotype in a subject.

[0062] Implementation Scheme 55. Use of a therapeutically effective amount of the immunogenic composition according to any one of Implementation Schemes 1 to 34 or the vaccine composition according to Implementation Scheme 35 in the preparation of a medicament for preventing or reducing infection in a subject caused by at least one target streptococcal species and / or its serotype.

[0063] Implementation Scheme 56. Use of a therapeutically effective amount of the immunogenic composition according to any one of Implementation Schemes 1 to 34 or the vaccine composition according to Implementation Scheme 35 in the preparation of a medicament for the prevention or reduction of a disease or condition in a subject caused by at least one target streptococcal species and / or its serotype.

[0064] Implementation Scheme 57. The use according to any one of Implementation Schemes 53 to 56, wherein the attenuated and / or killed streptococcal bacterial strain is derived from a strain, serotype or species that is the same as the at least one target streptococcal species and / or its serotype.

[0065] Implementation Scheme 58. The use according to any one of Implementation Schemes 53 to 56, wherein the attenuated and / or killed streptococcal bacterial strain is derived from a strain, serotype or species that is different from the at least one target streptococcal species and / or its serotype.

[0066] Implementation Scheme 59. The use according to any one of Implementation Schemes 53 to 56, wherein the at least one target streptococcal species and / or its serotype includes multiple target streptococcal species and / or their serotypes.

[0067] Implementation Scheme 60. The use according to any one of Implementation Schemes 53 to 56, wherein the target streptococcal species and / or serotypes comprise streptococcal bacterial species and / or serotypes selected from the group consisting of: agalactiae, bovine streptococci, canine streptococci, dysgalactiae, equine streptococci, equine enterococci, equine-like streptococci, enterococci faecalis, enterococci faecium, dolphin streptococci, Miller streptococci, variant streptococci, pneumococci, pyogenic streptococci, saliva streptococci, blood streptococci, suis streptococci, and mammary streptococci.

[0068] Implementation Scheme 61. The use according to any one of Implementation Schemes 53 to 56, wherein the immunogenic composition or the vaccine composition is administered to the subject via an administration route selected from the group consisting of: intranasal, intravenous, intramuscular, subcutaneous, oral, intraocular, transmucosal, and transdermal administration.

[0069] Implementation Scheme 62. According to the use described in Implementation Scheme 56, the disease or condition is selected from the group consisting of: respiratory tract infection, pneumonia, ear infection, earache, middle ear infection, otitis media, sinusitis, meningitis, conjunctivitis, bacteremia, sepsis, joint infection, bone infection, septic arthritis, osteomyelitis, soft tissue infection, cellulitis, myositis, periorbital cellulitis, abscess, peritonitis, cardiac infection, endocarditis, and pericarditis.

[0070] Implementation Scheme 63. A therapeutically effective amount of the immunogenic composition according to any one of Implementation Schemes 1 to 34 or the vaccine composition according to Implementation Scheme 35, for inducing an immune response in a subject against at least one target streptococcal species and / or its serotype.

[0071] Implementation Scheme 64. A therapeutically effective amount of the immunogenic composition according to any one of Implementation Schemes 1 to 34 or the vaccine composition according to Implementation Scheme 35, for inducing a cross-protective immune response in a subject against at least one target streptococcal species and / or its serotype.

[0072] Implementation Scheme 65. A therapeutically effective amount of the immunogenic composition according to any one of Implementation Schemes 1 to 34 or the vaccine composition according to Implementation Scheme 35, for preventing or reducing infection in a subject caused by at least one target streptococcal species and / or its serotype.

[0073] Implementation Scheme 66. A therapeutically effective amount of an immunogenic composition according to any one of Implementation Schemes 1 to 34 or a vaccine composition according to Implementation Scheme 35, for preventing or reducing disease or condition in a subject caused by at least one target streptococcal species and / or its serotype, the method comprising administering to the subject a therapeutically effective amount of the immunogenic composition according to any one of Implementation Schemes 1 to 34 or the vaccine composition according to Implementation Scheme 35.

[0074] Implementation Scheme 67. The composition according to any one of Implementation Schemes 63 to 66, wherein the attenuated and / or killed streptococcal bacterial strain is derived from a strain, serotype, or species that is the same as the at least one target streptococcal species and / or its serotype.

[0075] Implementation Scheme 68. The composition according to any one of Implementation Schemes 63 to 66, wherein the attenuated and / or killed streptococcal bacterial strain is derived from a strain, serotype or species that is different from the at least one target streptococcal species and / or its serotype.

[0076] Implementation Scheme 69. The composition according to any one of Implementation Schemes 63 to 66, wherein the at least one target streptococcal species and / or its serotype comprises multiple target streptococcal species and / or their serotypes.

[0077] Implementation Scheme 70. The composition according to any one of Implementation Schemes 63 to 66, wherein the target streptococcal species and / or serotypes comprise streptococcal bacterial species and / or serotypes selected from the group consisting of: agalactiae, bovine streptococci, canine streptococci, dysgalactiae, equine streptococci, equine enterococci, equine-like streptococci, enterococci faecalis, enterococci faecium, dolphin streptococci, Miller streptococci, variant streptococci, pneumococci, pyogenic streptococci, saliva streptococci, blood streptococci, suis streptococci, and mammary streptococci.

[0078] Implementation Scheme 71. The composition according to any one of Implementation Schemes 63 to 66, wherein the immunogenic composition or the vaccine composition is administered to the subject via an administration route selected from the group consisting of: intranasal, intravenous, intramuscular, subcutaneous, oral, intraocular, transmucosal, and transdermal administration.

[0079] Implementation Scheme 72. The composition according to Implementation Scheme 66, wherein the disease or condition is selected from the group consisting of: respiratory tract infection, pneumonia, ear infection, earache, middle ear infection, otitis media, sinusitis, meningitis, conjunctivitis, bacteremia, sepsis, joint infection, bone infection, septic arthritis, osteomyelitis, soft tissue infection, cellulitis, myositis, periorbital cellulitis, abscess, peritonitis, cardiac infection, endocarditis, and pericarditis. Attached Figure Description

[0080] Figure 1 A schematic diagram is provided illustrating the generation of GPN-002, GPN-002ΔlytC::KanR, GPN-003, and GPN-001ΔlytC::KanR from the GPN-001 derivative of Streptococcus pneumoniae Rx1.

[0081] Figure 2 Photographs of the electrophoresis gels were provided, showing confirmation that the kanamycin resistance cassette was incorporated into GPN-002. lytC PCR products from the region.

[0082] Figure 3The optical density (OD) of washed GPN-002 and GPN-002∆lytC::kanR bacteria suspended in PBS containing 20% ​​glycerol at 25°C over time is provided. 600 The illustration is shown.

[0083] Figure 4 The optical density (OD) of washed GPN-002 and GPN-002∆lytC::kanR bacteria suspended in PBS containing 20% ​​glycerol at 25°C over time is provided. 600 The illustration is shown.

[0084] Figure 5 Photographs of the electrophoresis gel were provided, showing that GPN-003 was confirmed to be absent. lytA , ply / PdT replaces or does not exist. psaA and does not exist lytC PCR products.

[0085] Figure 6 Provided (top chart) Rx1 ply DNA sequence and protein translation of the region; and (see lower diagram) GPN-003. pdt DNA sequence and protein translation in the region.

[0086] Figure 7 The optical density (OD) of washed GPN-002 and GPN-003 bacteria suspended in PBS containing 20% ​​glycerol at 25°C over time is provided. 600 The illustration is shown.

[0087] Figure 8 Illustrations of antibody responses in mice immunized by ELISA with PBS (mock control), γ-PN2, and γ-PN3 using Rx1 as the capture antigen are provided, showing (A) total pneumococcal-specific IgG response at absorbance (450 / 620 nm); (B) total IgG response in titers; (C) IgG1; (D) IgG2a; (E) IgG2b; (F) IgG3. Data are presented as mean absorbance (450 / 620 nm) ± SEM from individual or pooled serum samples (n = 5 mice per group).

[0088] Figure 9The graphs show the IgG produced after simulated vaccination or vaccination with γ-PN2 or γ-PN3, where (A) is the percentage of IgG bound to Streptococcus pneumoniae serotype 2 strain D39, (B) is the geometric mean (GMFI) of IgG binding to Streptococcus pneumoniae serotype 2 strain D39, (C) is the percentage of IgG bound to Streptococcus pneumoniae serotype 6A strain P9, and (D) is the GMFI of IgG binding to Streptococcus pneumoniae serotype 6A strain P9. Data were analyzed by one-way ANOVA (** p < 0.01, **** p < 0.0001).

[0089] Figure 10 (A) IgG titers of individual rabbits before and after the third immunization, and (B) mean IgG titers (± SEM) within each vaccine group are provided graphically. Data were analyzed by one-way ANOVA, nd = not detected.

[0090] Figure 11 Flow cytometry analysis of rabbit IgG binding to Streptococcus pneumoniae serotype 7C cells after incubation with rabbit serum following vaccination with γ-PN2 or γPN3 is provided, where (A) is relative fluorescence, (B) is the percentage of serotype 7C cells bound by rabbit IgG antibodies, and (C) is the geometric mean fluorescence intensity of IgG antibodies bound to serotype 7C cells, indicating the magnitude of IgG binding. Data were analyzed by one-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns not significant).

[0091] Figure 12 Provides the relative values ​​of GPN-002 and GPN-003 as determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). gyrA Average transcription level prsA A diagram illustrating transcription levels.

[0092] Figure 13 Provided information showing 10 vaccinations 4 One CFU / mouse of Streptococcus pneumoniae serotype 2 strain D39; or 10,000 times higher doses (10 8 (A) Survival rate and (B) weight in grams of GPN-002 or GPN-003 mice (CFU / mouse).

[0093] Figure 14A schematic diagram is provided illustrating the generation of GPN-002, GPN-002ΔlytC::KanR, GPN-003, and GPN-001ΔlytC::KanR and GPN 001ΔlytC from the GPN-001 derivative of Streptococcus pneumoniae Rx1.

[0094] definition In the context of this specification, the term "a / an" is used herein to refer to one or more (i.e., at least one) grammatical object of the article. As an example, "element" means one element or more elements.

[0095] As used herein, when applied to the value of interest, the terms “about,” “approximately,” and “substantially” refer to a value similar to the stated value. In some embodiments, unless otherwise stated or apparent from the context, it is understood to refer to a range of + / - 10%, preferably + / - 9%, + / - 8%, + / - 7%, + / - 6%, + / - 5%, + / - 4%, + / - 3%, + / - 2%, or + / - 1%; or + / - 0.05% or + / - 0.1%.

[0096] As used herein, the term "attenuated" will be understood in the context of bacteria to mean that the bacteria do not cause significant pathological damage to the host during infection. For example, attenuated bacteria may only be able to cause nonpathogenic infection in the host to which they are administered, or may otherwise fail to establish infection in the host, or may establish infection in the host causing mild or acceptable symptoms. It should be understood that attenuated immunogenic compositions remain in the host for a period sufficient to induce an immune response in the host. However, the bacteria cannot establish a prolonged infection or a pathogenic infection harmful to non-immunely compromised hosts to which attenuated bacteria are administered.

[0097] It should be understood that the term "between" as used in this document when referring to numerical ranges encompasses the values ​​at each endpoint of the range. For example, a polypeptide with a length between 10 and 20 residues includes polypeptides with a length of 10 residues and polypeptides with a length of 20 residues.

[0098] The terms “comprise”, “comprised”, or “comprising” are used in this specification and claims in an inclusive sense, that is, specifying the presence of the stated feature but not excluding the presence of additional or further features.

[0099] As used herein, the term "complementary" is used to describe the relationship between the first and second nucleotide sequences according to base pairing rules, where an adenine (A) base pairs with a uracil (U) base in an RNA molecule or with a thymine (T) base in a DNA molecule; and a cytosine (C) base pairs with a guanine (G) base in both RNA and DNA molecules. For example, for a DNA polynucleotide molecule, the sequence "5′-AGTC-3′" is perfectly complementary to the sequence "3′-TCAG-5′"; note that in RNA sequences, uracil (U) is typically used instead of thymine (T).

[0100] The degree of complementarity between nucleic acid strands has a significant impact on the efficiency and strength of hybridization between nucleic acid strands. This is particularly important for amplification or detection methods that rely on binding between nucleic acids. For hybridization to occur, the nucleic acid sequence does not need to be “complete” (100%) complementary to its target. Complementarity can be “partial,” where only some nucleic acid bases match according to base pairing rules. It should be understood that two molecules are “specifically complementary” when they can hybridize under appropriate conditions; that is, when a first polynucleotide molecule containing the first nucleotide sequence and a second polynucleotide molecule containing the second nucleotide sequence can hybridize and form a double-stranded structure under conditions suitable for the reaction being performed (e.g., ligation, PCR, sequencing, etc.), the two molecules are specifically complementary. The term “specifically complementary” can be used interchangeably with “substantially complementary.” It should also be understood that two nucleotide molecules do not need to be complementary along their entire length. For example, a portion of the first polynucleotide molecule may be specifically complementary to and hybridize with a portion of the second polynucleotide molecule. In this example, the two molecules may not hybridize at non-specifically complementary portions. These terms can also be used to refer to individual nucleotides, especially in the context of oligonucleotides. For example, a particular nucleotide within an oligonucleotide may be noticed for its complementarity or lack thereof with a nucleotide in another nucleic acid chain, in contrast to or comparison with the complementarity between the rest of the oligonucleotide and the nucleic acid chain.

[0101] The transitional phrase "consisting of..." excludes any unspecified elements, steps, or components. If in a claim, this phrase would close the claim by excluding material other than the listed elements, unless it is an impurity typically associated with them. When the phrase "consisting of..." appears in a clause of the body of a claim, rather than immediately following the preamble, the phrase limits only the elements listed in that clause; other elements are not necessarily excluded from the claim as a whole.

[0102] The transitional phrase “consistent with essentially” can be used to describe a composition, process, or method that includes materials, steps, features, components, or elements other than those literally disclosed, provided that such additional materials, steps, features, components, or elements do not materially affect one or more essential and novel features of the claimed invention.

[0103] When used with reference to the compositions of this disclosure, the term "cross-protection" refers to a composition capable of inducing a cross-protective immune response.

[0104] As used herein, the term "cross-protective immune response" refers to an immune response induced by a specific antigen (i.e., a streptococcal bacterial strain) that provides protection against different target bacterial strains or species compared to the strain from which the antigen is derived. For example, if an immune response induced by a Streptococcus pneumoniae serotype 2 strain protects a subject from infection with or reduces illness caused by different Streptococcus pneumoniae serotypes (such as serotype 6), then that immune response is cross-protective. Similarly, if an immune response induced by a Streptococcus pneumoniae serotype 2 strain protects a subject from infection with or reduces illness caused by different streptococcal species (such as Streptococcus pyogenes), then that immune response is cross-protective.

[0105] As used herein, the terms “derivative” or “derived from” for bacterial strains refer to progeny bacterial strains that have at least one modification compared to a parent strain. The modification may be natural or intentionally introduced. It should be understood that a parent strain may be a direct parent (e.g., after the introduction of a single modification). Alternatively, a parent strain may be a more distant parent after the introduction of a series of modifications. For example, GPN-001, GPN-002, and GPN-003 described herein are all considered derivatives of Streptococcus pneumoniae Rx1; GPN-002 and GPN-003 are considered derivatives of GPN-001; and GPN-003 is considered a derivative of both GPN-001 and GPN-002.

[0106] As used in this article, the term “effectively” when used with reference to a particular parameter or result is intended to mean that the percentage of that parameter or result is sufficient to achieve the desired result.

[0107] As used herein, in the context of nucleic acid sequences or proteins, "expression" or "expresses" refers to the translation of mRNA into a polypeptide, the assembly of multiple polypeptides into a complete protein (e.g., an enzyme), and / or the post-translational modification of a polypeptide or a fully assembled protein (e.g., an enzyme). In this application, the terms "expression" and "production" and their grammatical equivalents are used interchangeably.

[0108] As used in this article, “expression” in the context of a particular bacterial characteristic or phenotype refers to the presence of that characteristic or phenotype.

[0109] As used herein, a “homology” of a protein refers to a group of proteins that perform the same biological function and are expressed by a homologous gene. A homologous gene is a gene that encodes a protein that has the same or similar biological function as a protein encoded by a second gene. Homologous genes and nucleic acid sequences can exist in the same or different organisms. Homologous genes include orthologs (i.e., genes expressed in different species that evolved from a common ancestral gene through speciation and encode proteins that retain the same function), but exclude paralogs (i.e., genes that are related through replication but have evolved to encode proteins with different functions). Homologous genes include naturally occurring alleles and artificially produced variants. The degeneracy of the genetic code provides the possibility of replacing at least one nucleotide of a gene or the protein-coding sequence with a different base without altering the amino acid sequence of the polypeptide produced by that gene. When optimally aligned, the homologous protein and nucleotide sequences (e.g., genes) of the present invention can have, for example, at least about 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over the full length of the reference gene or protein. In one embodiment, the homologous nucleotide sequence (e.g., gene) or homologous protein sequence of the present invention has at least about 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the reference common nucleotide or protein sequence over the full length of the reference sequence. The homologous nucleic acid sequences, genes, or proteins mentioned herein may be, for example, homologous nucleic acid sequences, genes, or proteins derived from other bacteria, other Firmicutes, other bacilli, other lactobacilli, other streptococci, and / or other streptococci.

[0110] Homologous gene / nucleic acid sequences and proteins, serving as reference sequences, can be identified by comparing homologous amino acid or nucleotide sequences (e.g., manually or using computer-based tools employing known homology-based search algorithms such as FASTA, BLAST, and Smith-Waterman). Local sequence alignment procedures (e.g., BLAST) can be used to find similar sequences by searching sequence databases, and the aggregated expectation value (E-value) can be used to measure sequence base similarity. Since a given sequence hit with the optimal E-value for a particular organism may not necessarily be an ortholog (i.e., having the same function / encoding a protein with the same function), or the only ortholog, a reciprocal query can be used to filter hit sequences with significant E-values ​​for ortholog identification. A reciprocal query may require searching a database of sequences from a basal organism that are sequence-similar to the query sequence for significant hits. A hit can be identified as an ortholog when the optimal hit of the reciprocal query is the query sequence itself or a protein encoded by a replicating gene after speciation.

[0111] As used herein, “sequence identity %” refers to the degree to which two best-aligned DNA or protein fragments remain unchanged within a component (e.g., nucleotide or amino acid sequence) alignment window. The “identity score” of the aligned segments of the test and reference sequences is calculated by dividing the number of common components shared by the sequences of the two aligned segments by the total number of sequence components in the reference segment within the alignment window, which is the smaller of the full test or full reference sequence. “Sequence identity %” is the identity score multiplied by 100. Such best alignments are understood as local alignments of the DNA sequence. For protein alignments, local alignments of the protein sequence allow for the introduction of gaps to achieve best alignment. The sequence identity percentage is calculated over the alignment length excluding gaps introduced by the alignment itself.

[0112] As used herein, in the context of immunity or immune response, the terms “induce,” “inducing,” “enhance,” and “enhancing” refer to an increase in immunity or immune response to levels that may not exist or are measurable.

[0113] As used herein, in the context of immunity or immune response induced by the compositions disclosed herein, the term "improved effectiveness" refers to a shift in the immune response against infection or disease compared to a baseline, such as that induced by a parental strain. This shift in immunity may, for example, refer to an increase in specific immunoglobulin subtypes (e.g., IgG1, IgG2a, IgG2b, IgG3, IgG4, etc., IgA, IgE, IgM, etc.), or a shift in Th1 / Th2 bias, etc.

[0114] As used herein, the term "immunogenic composition" refers to a composition or formulation that generates an immune response when administered to a subject.

[0115] As used herein, the term "vaccine composition" refers to a composition or formulation that, when administered to a subject, generates an immune response that protects the subject from infection by a pathogen or, alternatively, reduces disease caused by a pathogen.

[0116] The terms “immune response” and “immunity” are used interchangeably herein and refer to a subject’s response to a foreign antigen. An immune response may include inducing humoral (i.e., B cell) and / or cellular (i.e., T cell) responses. Suitablely, humoral immune responses can be assessed by measuring antigen-specific antibodies present in the serum of an immunized animal in response to the introduction of an antigen into the host. Immune responses can be assessed by various means, such as enzyme-linked immunosorbent assay (ELISA) of immunized mammalian serum, FACS analysis of antibodies bound to cell surface antigens, opsonization assays, and micro-neutralization assays of immunized animal serum. Optionally or additionally, cellular responses can be measured by FACS analysis, cytotoxic T cell (CTL) measurements of T cell responses from lymphocytes isolated from the spleen or other organs of an immunized animal, etc.

[0117] As used herein, the term "isolated" means material that is substantially or essentially free of the components that normally accompany it in its native state. For example, "isolated polynucleotide" as used herein refers to a polynucleotide that has been purified from a sequence flanking it in its native state, such as a DNA fragment that has been removed from a sequence normally adjacent to that fragment. Alternatively, "isolated peptide" or "isolated polypeptide" as used herein refers to a peptide or polypeptide molecule that has been isolated and / or purified in vitro from its native cellular environment and from its association with other components of the cell, i.e., it does not associate with substances in vivo.

[0118] As used herein, the term “tag” is used to describe the binding, connection, or inclusion of a biotag with a biomolecule, wherein the biotag can be used to detect the presence of a biomolecule or to quantify the absolute or relative amount of the biomolecule.

[0119] As used herein, the term "nucleotide" in its broadest sense refers to any compound and / or substance incorporated into or potentially incorporated into a polynucleotide chain. In one embodiment, a nucleotide is a compound and / or substance incorporated into or potentially incorporated into a polynucleotide chain via a phosphodiester bond. In one embodiment, "nucleotide" refers to a single nucleic acid residue (e.g., a nucleotide and / or a nucleoside). The term "nucleotide" may be used interchangeably with "nucleic acid". In one embodiment, "nucleotide" encompasses RNA as well as single-stranded and / or double-stranded DNA and / or cDNA.

[0120] As used herein, the term "messenger RNA" or "mRNA" refers to an RNA polynucleotide molecule that encodes at least one polypeptide. As used herein, mRNA encompasses both modified and unmodified RNA. mRNA may contain one or more coding and non-coding regions.

[0121] As used in this article, the phrase "weakening, reducing, or preventing modifications of functional LytC expression" refers to... lytC Mutations or deletions in the gene reduce or eliminate the expression of functional LytC protein, meaning that LytC has a function of reduction or elimination.

[0122] As used herein, the term "operably linked" or "operably coupled" refers to a functional relationship between two or more nucleic acid segments (such as genes and regulatory elements (including, but not limited to, promoters)) that subsequently regulate gene expression.

[0123] As used herein, the term "pharmaceuticalally acceptable" means a substance that, when administered to a subject, will not elicit a substantial adverse allergic or immune response. "Pharmaceuticalally acceptable carriers" include, but are not limited to, solvents, coatings, dispersants, wetting agents, isotonic agents, absorption delay agents, and disintegrants.

[0124] As used herein, the term "photon radiation" will be understood to encompass both gamma radiation (i.e., gamma rays) and X-ray radiation (i.e., X-rays). Therefore, a "photon-irradiated" material can be a material that has been exposed to gamma radiation and thus becomes "gamma-irradiated," a material that has been exposed to X-ray radiation and thus becomes "X-irradiated," or both. By way of non-limiting example only, in order to become photon-irradiated, a material can be subjected to photon radiation with energies of at least 0.01 MeV, at least 0.1 MeV, at least 0.5 MeV, 0.01 MeV to 0.5 MeV, 0.01 MeV to 1 MeV, 0.01 MeV to 10 MeV, 0.5 MeV to 20 MeV, 0.5 MeV to 15 MeV, 0.5 MeV to 10 MeV, 0.5 MeV to 5 MeV, 0.5 MeV to 2 MeV, or 1 MeV to 2 MeV (e.g., 1.25 MeV).

[0125] As used herein, the term "multiple" means more than one. In certain aspects or implementations, multiple may mean 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 or more, and any integer derived therefrom, and any range derived therefrom.

[0126] As used herein, the term "polynucleotide molecule" refers to a DNA or RNA nucleic acid molecule containing a chain of nucleotides, and may include oligonucleotide molecules or nucleic acids of interest. A polynucleotide molecule is encoded by a "polynucleotide sequence," which may also be referred to herein as a nucleic acid sequence. Those skilled in the art will understand that a nucleotide typically consists of three distinct chemical subunits: a pentose sugar molecule (pentose-glycan ring, deoxyribose in DNA or ribose in RNA), a nucleotide base (i.e., adenine (A), cytosine (C), guanine (G), thymine (T), or uracil (U)), and a phosphate group. Chemical convention names the carbon atoms in sugar molecules from 1' to 5', and this convention also specifies that polynucleotide molecules have a 5' end and a 3' end. In a polynucleotide molecule, the 3' carbon of the first nucleotide is linked to the 5' carbon of the next nucleotide. Those skilled in the art will understand that, unless otherwise specifically stated, polynucleotide sequences are read in the 5' to 3' direction.

[0127] The term "polynucleotide variant" refers to a polynucleotide that exhibits basic sequence identity with a reference polynucleotide sequence or hybridizes with a reference sequence under stringent conditions. This term also covers polynucleotides distinguished from a reference polynucleotide by the addition, deletion, or substitution of at least one nucleotide. Thus, the term "polynucleotide variant" includes polynucleotides in which one or more nucleotides have been added or deleted, or substituted with different nucleotides. In this regard, it is well understood in the art that certain changes, including mutations, additions, deletions, and substitutions, can be made to a reference polynucleotide, thereby altering the polynucleotide while preserving the biological function or activity of the reference polynucleotide. The term "polynucleotide variant" also includes naturally occurring allelic variants. The terms "peptide variant" and "polypeptide variant," etc., refer to peptides and polypeptides distinguished from a reference peptide or polypeptide by the addition, deletion, or substitution of at least one amino acid residue. In some examples, peptide or polypeptide variants are distinguished from reference peptides or polypeptides by one or more substitutions, which can be conserved or non-conserved. In some examples, peptide or polypeptide variants contain conserved substitutions, and in this regard, it is well understood in the art that some amino acids can be changed to other amino acids with broadly similar properties without altering the activity of the peptide or polypeptide. Peptide and polypeptide variants also include peptides and polypeptides in which one or more amino acids have been added or missing, or replaced by different amino acids.

[0128] As used herein, an "oligonucleotide" or "oligonucleotide molecule" is a single-stranded polynucleotide molecule that can be synthesized to have a user-specified sequence of interest. A "primer" is an example of an oligonucleotide molecule. Typically, at least a portion of an oligonucleotide molecule is specifically or completely complementary to the polynucleotide sequence of interest and hybridizes to a specifically complementary single-stranded polynucleotide molecule. Oligonucleotide molecules are generally considered to be short polynucleotide molecules; however, their length can vary. Their length may be suitable for at least one of a range of applications, including polymerase chain reaction (PCR) based applications, sequencing applications, molecular cloning, and molecular probes.

[0129] As used herein, in the context of a given infection and / or disease or condition caused by the infection, the terms “prevent,” “prevention,” and “preventing” will be understood to mean a reduced predisposition of a subject to develop an infection and / or disease or condition after exposure to a pathogenic organism that causes the infection, disease, or condition. A reduced predisposition to develop an infection and / or disease or condition will be understood to include both a weakened predisposition and a lack of any predisposition.

[0130] As used herein, the term "bacterial strain" is used to refer to a bacterial population (e.g., a bacterial culture) in which the vast majority, or virtually all, of the bacteria are descendants of a single bacterial organism. Therefore, the vast majority, or virtually all, of the bacteria within the population are identical or highly similar to each other and share a particular phenotype.

[0131] As used in this article, the term "streptococcal bacterial strain" refers to bacterial strains belonging to the family Streptococcus.

[0132] As used herein, the term "subject" includes any animal of economic, social, or research importance, including species of cattle, horses, sheep, primates, birds, and rodents. Thus, "subject" can be a mammal, such as, for example, a human, or a non-human mammal (e.g., a pig, cat, dog, cattle, horse, or sheep). The scope of the term also includes laboratory animals (e.g., rodents, rabbits, etc.), birds (e.g., poultry), fish, and crustaceans.

[0133] When applied to polynucleotide molecules, the term "synthesis" is intended to mean that the polynucleotide molecule is produced in vitro, including using bacterial amplification methods.

[0134] A "therapeutic effective dose" is at least the minimum concentration or amount required to produce a measurable improvement in a particular disease or condition. The therapeutic effective dose as defined herein can vary depending on factors such as the patient's disease state, age, sex, and weight. A therapeutic effective dose is also the amount at which the beneficial therapeutic effect outweighs any toxic or harmful effects.

[0135] As used herein, in the context of a given infection and / or disease or condition caused by the infection, the terms “treat” and “treating” will be understood to encompass reducing the number of pathogenic organisms in an infected subject and / or reducing any symptoms of the infection and / or symptoms of the disease or condition caused by the infection.

[0136] As used herein, the “wild-type” form of a given nucleic acid, protein, or microorganism (e.g., bacteria) will be understood to encompass the naturally occurring forms of the nucleic acid, protein, or microorganism and the biological functions they can possess. Detailed Implementation

[0137] The following detailed description conveys exemplary embodiments of the present disclosure with sufficient detail to enable those skilled in the art to practice the disclosure. The features or limitations of the various embodiments described are not necessarily limiting of other embodiments of the present disclosure or the disclosure as a whole. Therefore, the following detailed description does not limit the scope of the disclosure, which is defined only by the claims.

[0138] Therefore, the immunity induced by commercially available streptococcal vaccines is insufficient to establish broad immunity against most streptococcal species and / or certain serotypes within pathogenic species.

[0139] Vaccination with purified LytC has been shown to produce high levels of immunoglobulin G (IgG) antibodies and enhanced protection against pneumococcal infection in mice (Wisemann et al., 2001; Corsini et al., 2021). High titers of LytC-specific IgG antibodies have also been observed in serum samples from healthy volunteers in multiple studies, indicating that antibody responses are induced by natural exposure to Streptococcus pneumoniae.

[0140] This disclosure relates to an unexpected discovery, namely, the use of LytC Attenuated or inactivated whole-cell streptococcal vaccines, whose genes have been modified to attenuate, reduce, and / or prevent functional LytC expression, provide improved vaccine immunogenicity. This disclosure provides an immunogenic composition comprising an attenuated and / or inactivated streptococcal bacterial strain containing modifications that attenuate, reduce, or prevent functional LytC expression.

[0141] Surprisingly, compared to the parental strain with functional LytC expression, the strain carrying... lytC Mice immunized with the missing streptococcal bacterial strain produced IgG antibodies that bind more strongly to the surface of encapsulated pneumococcal strains. Furthermore, mice carrying… lytC The antibodies induced by the missing strains exhibited a higher functional opsonization phagocytic response, a recognized surrogate endpoint for pneumococcal vaccine efficacy. These findings unexpectedly demonstrate that the missing strains… lytC Genes can improve the immunogenicity of whole-cell vaccines. This is something that previous studies could not have predicted.

[0142] This disclosure provides compositions capable of inducing cross-protective (i.e., heterotypic) immunity against different streptococcal species and / or different streptococcal serotypes. In one embodiment, the composition of this disclosure contains inactivated streptococcal bacteria. In one embodiment, the composition is an attenuated vaccine. The composition may also be capable of inducing immunity against a broad range of streptococcal species and / or serotypes, thereby reducing the potential impact of serotype replacement.

[0143] This document also provides methods for manufacturing the compositions disclosed herein, as well as pharmaceuticals and pharmaceutical compositions comprising immunogenic or vaccine compositions.

[0144] This disclosure also relates to a method for preventing or treating streptococcal infection in a subject. The method involves administering a composition of this disclosure to the subject. The composition can be administered for preventative or therapeutic purposes. The method can induce cross-protective immunity in the subject against multiple different streptococcal species and / or serotypes.

[0145] Streptococcus bacteria Streptococci are cocci belonging to the genus *Streptococcus* in the family Streptococciaceae. There are many different species of streptococci, some of which cause disease in humans and / or animals. Others are important in the manufacture of various fermented products.

[0146] Individual streptococcal species are classified into two key groups based on their hemolytic properties (α- and β-hemolytic). α-Hemolytic streptococci include *Streptococcus pneumoniae* and *Streptococcus viride*. The β-hemolytic group consists of group A and group B streptococci. Group B streptococci typically reside in the digestive system and vagina of women without adverse effects. Most people quickly develop natural immunity to group B streptococci, although they can cause more severe types of infection in newborns. Group A streptococci typically reside in the throat and on the skin surface and are a common cause of infection in adults and children. While most group A infections do not usually pose a serious threat to health (e.g., throat infections, cellulitis, impetigo, sinusitis, middle ear infections), group A streptococci can establish more serious invasive infections (e.g., pneumonia, sepsis, meningitis, necrotizing fasciitis) by penetrating deeper into the body's tissues and organs, and can cause serious sequelae, including acute post-streptococcal glomerulonephritis and acute rheumatic fever.

[0147] In addition, Enterococcus (fecal) streptococcus species are abundant in the intestines and may cause endocarditis and urinary tract infections.

[0148] Streptococcus pneumoniae (also known as pneumococcus) is a major human pathogen causing significant morbidity and mortality. It causes serious conditions including pneumonia, meningitis, bacteremia, sinusitis, and otitis media. Globally, an estimated 1.6 million people die annually from invasive pneumococcal disease, approximately one million of whom are children. There are many different Streptococcus pneumoniae serotypes (>100), which can be distinguished based on capsular chemical structure and immunogenicity. Capsular polysaccharides are considered essential virulence factors for Streptococcus pneumoniae because almost no non-capsulated strains are found among the Streptococcus pneumoniae that cause invasive pneumococcal disease, and antibodies against capsular polysaccharides can prevent infection with homologous Streptococcus pneumoniae serotypes. Therefore, capsular polysaccharides are used as vaccine antigens in current pneumococcal vaccines.

[0149] Streptococcal bacterial strains in the composition The immunogenic compositions disclosed herein are based on attenuated or completely eradicated streptococcal bacterial strains containing modifications that reduce, decrease, or prevent the functional expression of LytC (or its homologs). These modifications can be introduced into any suitable streptococcal bacterial strain.

[0150] Streptococcal bacterial strains can be, for example, α-, β-, or γ-hemolytic streptococci, as classified according to well-characterized hemolytic properties or, in the case of γ-hemolytic streptococci, the lack of hemolytic properties.

[0151] Non-limiting examples of suitable α-hemolytic streptococci include Streptococcus pneumoniae and viridans streptococci (e.g., Streptococcus mutans, Streptococcus sanguinis). 、 Streptococcus suis ( S. mitis ), oral streptococci ( S. oralis ), distant streptococci ( S. sobrinus ) 、 (Streptococcus Miller). Individual serotypes of these streptococcal species are also within the scope of this disclosure.

[0152] Non-limiting examples of suitable β-hemolytic streptococci include those classified under the Lancefield grouping (AH, L, N, and R / S groups) based on their carbohydrate composition according to cell wall bacterial antigens (polysaccharides). For example, β-hemolytic bacteria may include any one or more of the following: *Streptococcus pyogenes* (group A), *Streptococcus agalactiae* (group B), *Streptococcus equi* (group C), *Streptococcus equi* (group C), *Streptococcus vesicans ... S. zooepidemicus (Group C), Streptococcus pyogenes (Group C), Enterococcus faecalis (Group D), Streptococcus bovis (Group D), Streptococcus Miller (Group E), Streptococcus mutans (Group E), Streptococcus pharyngitis (Group C), Streptococcus pharyngitis (Group D), Streptococcus lactis (Group D), Streptococcus pharyngitis ... S. anginosus (Group F), Streptococcus canis (Group G), Streptococcus pyogenes (Group G), Streptococcus sanguinis (Group F ... S. sanguis (Group H), Streptococcus lactis (Group L), Lactococcus lactis (Group H), Lact ... Lactococcus lactis (N group) and Streptococcus suis (R / S group). Individual serotypes of these streptococcal species are also within the scope of this disclosure.

[0153] In one embodiment, the attenuated and / or killable streptococcal bacterial strains of this disclosure belong to species selected from the group consisting of: *Streptococcus agalactiae*, *Streptococcus bovis*, *Streptococcus canis*, *Streptococcus dysgalactiae*, *Streptococcus equi*, *Streptococcus equineus*, *Streptococcus equineus*, *Enterococcus faecalis*, *Enterococcus faecium*, *Streptococcus dolphinus*, *Streptococcus Miller*, *Streptococcus mutans*, *Streptococcus pneumoniae*, *Streptococcus pyogenes*, *Streptococcus salivarius*, *Streptococcus sanguinis*, *Streptococcus suis*, and *Streptococcus lactis*.

[0154] In one embodiment, the attenuated and / or inactivated streptococcal bacterial strain of this disclosure comprises one or more serotypes of Streptococcus pneumoniae. Therefore, the composition may comprise Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10B, 10C, 10F, 11A, 11B, 11C, 11D, 11F, 12A, 12B, 12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F, 19A, 19B, 19C, Any one or more of 19F, 20, 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24C, 24F, 25A, 25F, 27, 28A, 28F, 29, 31, 32A, 32F, 33A, 33B, 33C, 33D, 33F, 34, 35A, 35B, 35C, 35F, 36A, 36B, 37, 38, 39, 40, 41A, 41F, 42, 43, 44, 45, 46, 47A, 47F and / or 48.

[0155] However, those skilled in the art will understand that new bacterial strains and serotypes may be discovered. It should be understood that the attenuated and / or inactivated streptococcal bacterial strains of this disclosure may include streptococcal bacteria or serotypes not specifically listed herein. In one embodiment, the composition of this disclosure comprises any one or more of the following Streptococcus pneumoniae serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F.

[0156] Streptococcal bacterial derivatives In one implementation, the streptococcal bacterial strain is a streptococcal derivative.

[0157] Streptococcal derivatives can be recombinant forms of Streptococcus bacteria produced through artificial genetic manipulation, or naturally occurring mutant forms of Streptococcus bacteria. There are no particular restrictions; streptococcal derivatives can contain one or more genetic modifications that reduce pathogenicity.

[0158] By way of non-limiting example only, streptococcal derivatives may contain gene loci that disrupt or remove the capsular locus. cps Genetic alterations. For example, it can modify Streptococcus pneumoniae. cpsA, cpsB, cpsC, cpsD and / or cpsEAny one or more of the genes, or homologous genes from other streptococcal species, to prevent, disrupt, or modify capsule formation (e.g., through recombination). Optionally, the streptococcal derivative may have spontaneous mutations in these or other genes, resulting in naturally occurring non-capsulated streptococcal bacteria. The streptococcal derivative may lack all or at least some of the capsule loci.

[0159] In one embodiment, the streptococcal derivative lacking a capsule is Streptococcus pneumoniae strain Rx1 or an Rx1 derivative.

[0160] Alternatively or additionally, the streptococcal derivative may contain genetic alterations that reduce or prevent the production or activity of other target proteins. By way of non-limiting example only, the genetic alterations may be present in genes encoding one or more genes encoding choline-binding proteins; genes encoding other than... lytC In addition to one or more genes of other autolysins (e.g., Streptococcus pneumoniae) lytA or lytB (or homologous genes in other streptococcal bacteria); one or more genes that confer nutrients / cofactors (e.g., metal ions) required for growth (e.g., Streptococcus pneumoniae). psaA (or homologous genes in other streptococcal bacteria); one or more genes encoding protective antigens (e.g., Streptococcus pneumoniae). pspA (or homologous genes in other streptococcal bacteria); and / or one or more genes encoding virulence determinants or regulators (e.g., Streptococcus pneumoniae). codY, comC, comD, cps2A csp4A, glpO, mgrA, nanA, nanB, pavA, pcpA, phtA, phtB, phtD, phtE, piuA, piaA, ply, prtA, psaA, psrP, rrgA, rrgB, spxB (and homologs of these genes in other streptococcal bacteria).

[0161] Alternatively or additionally, the streptococcal derivative may contain genetic alterations that result in auxotrophic traits leading to pathogenicity and / or reduced in vivo growth. By way of non-limiting example only, the genetic alteration may be present in one or more genes encoding thymidine synthase.

[0162] Alternatively or additionally, the streptococcal derivative may comprise one or more (external) genes derived from streptococcal bacteria of the same species but different serotypes; streptococcal bacteria of different species; non-streptococcal bacteria; or human or non-human mammals (e.g., pigs, cats, dogs, cattle, horses, or sheep); laboratory animals (e.g., rodents or rabbits); birds; and / or a subject to be administered the recombinant streptococcal bacteria. In one embodiment, one or more external genes disrupt or otherwise inactivate one or more endogenous genes (e.g., any one or more genes listed immediately preceding the paragraph). In other embodiments, one or more external genes do not disrupt or inactivate any endogenous genes. By way of non-limiting example only, one or more external genes encode proteins that induce or enhance an immune response in a subject administered the streptococcal derivative. The immune response may be innate, adaptive, or both. In one embodiment, one or more external genes encode immunomodulators (e.g., cytokines, chemokines, antibodies, fusion proteins, peptides, proteins, and / or hormones). In other embodiments, one or more exogenous genes may contain antigens from another different bacterial family (e.g., Mycoplasma pneumoniae). Mycoplasma pneumoniae Antigen, Haemophilus influenzae ( Haemophilus influenzae ) antigen, Chlamydia pneumoniae ( Chlamydophila pneumoniae Antigen, Moraxella catarrhalis ( Moraxella catarrhalis Antigen, Staphylococcus aureus ( Staphylococcus aureus Antigens, viral antigens (e.g., adenovirus antigen, coronavirus antigen, influenza virus antigen, parainfluenza virus antigen, metapneumovirus antigen, rhinovirus antigen, respiratory syncytial virus antigen, HIV antigen, hepatitis virus antigen or herpes virus antigen, measles virus antigen, mumps virus antigen, papillomavirus virus antigen, rubella virus antigen, varicella-zoster virus antigen), fungal / yeast antigens, worm antigens and / or protozoan antigens.

[0163] Alternatively or additionally, the streptococcal derivative may contain genetic alterations that result in bacterial overexpression of one or more target genes. In this context, "overexpression" will be understood as an increased expression level under the same biological conditions compared to the expression of the same gene without genetic modification in the corresponding streptococcal bacteria. Overexpression of a given target gene can, for example, induce or enhance an immune response in a subject against a streptococcal strain that is a parent of the administered streptococcal derivative and / or against the streptococcal derivative itself. By way of non-limiting example only, the genetic alteration may increase one or more genes in the streptococcal derivative encoding proteins capable of activating the complement system (e.g., Streptococcus pneumoniae). cbpA, pspA, ply The production of homologs of these genes in other streptococcal bacteria.

[0164] Alternatively or additionally, the streptococcal derivative may contain genetic alterations that result in defects in DNA repair capacity. In one embodiment, it may be advantageous to use compositions of this disclosure containing a streptococcal derivative that reduces the ability to repair DNA damage caused by photon irradiation (e.g., gamma irradiation and / or X-ray irradiation) because the photon irradiation dose required for attenuation or inactivation can be reduced, while conversely increasing vaccine efficacy and safety. In one embodiment, the streptococcal derivative contains one or more genes that disrupt or inactivate proteins encoding proteins in a mismatch repair system (e.g., Streptococcus pneumoniae). hex Genetic alterations in the expression of a locus or homologs of that locus in other streptococcal bacteria. In other embodiments, the streptococcal derivative comprises disrupting or inactivating one or more genes encoding DNA alkylation repair proteins (e.g., Streptococcus pneumoniae DNA polymerase 4, ...). hexA , hexB , mutS , radC , recA , recF , recN , recO , uvrA , uvrB , uvrC , uvrD Genetic alterations in the expression of homologs of these genes in other streptococcal bacteria.

[0165] Alternatively or additionally, the streptococcal derivative may contain genetic alterations that promote the production of double-stranded RNA (dsRNA). The dsRNA may be mRNA or tRNA. There are no restrictions on the length of the dsRNA; it may be longer than 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 base pairs. Alternatively or concurrently, the length of the dsRNA may be: about 10 to about 70 base pairs (bp); about 10 to about 50 base pairs (bp); about 10 to about 30 base pairs (bp); about 20 to about 70 base pairs (bp); about 20 to about 60 base pairs (bp); about 20 to about 50 base pairs (bp); about 20 to about 40 base pairs (bp); about 20 to about 30 base pairs (bp); about 30 to about 70 base pairs (bp); about 40 to about 70 base pairs (bp); about 50 to about 70 base pairs (bp); about 60 to about 70 base pairs (bp); about 30 to about 60 base pairs (bp); about 30 to about 50 base pairs (bp); or about 30 to about 40 base pairs (bp). In one embodiment, the dsRNA is a component of an additional single-stranded, larger RNA molecule. Larger RNA molecules may contain multiple dsRNA components. dsRNA can be an internal or terminal component of the larger RNA molecule. In one embodiment, the dsRNA may contain a terminating stem-loop sequence. The dsRNA may originate from a self-complementary region within the larger RNA molecule. One or more coding regions / exons within a given gene of a streptococcal derivative can be engineered to include one or more self-complementary regions, thereby generating a dsRNA portion during transcription.

[0166] The dsRNA can be recognized by a Toll-like receptor (TLR) protein expressed in cells of a subject administered a streptococcal derivative. The TLR protein can be located in the endoplasmic reticulum and / or endosome compartments of the cell. The TLR protein can be Toll-like receptor 3 (TLR3) protein. There are no limitations; the cells can be any one or more of B lymphocytes, T lymphocytes, natural killer cells, and / or dendritic cells. Recognition of the dsRNA by the TLR3 protein can induce an immune response in the subject. The immune response can be an innate immune response. The immune response can be an interferon type 1 response and / or the release of inflammatory cytokines.

[0167] Typically, the streptococcal derivatives used in the compositions of this disclosure will have a significant degree of genetic similarity to the parental strains from which they are derived. As a non-limiting example, the "streptococcal derivative" as referred to herein may have sequence homology exceeding 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% to the parental streptococcal strain from which it is derived. As a further non-limiting example, the "streptococcal derivative" as referred to herein may contain one, two, three, four, five, or more genetic alterations in the regulatory sequences required to express the corresponding genes of its parental strain. These genetic alterations may increase, decrease, or prevent the expression of one or more of the discussed genes.

[0168] Techniques for genetic manipulation of bacteria are well known to those skilled in the art (see, for example, Vennison). Laboratory Manual for Genetic Engineering ", PHI Learning Pvt. Ltd., 2010; Zyskind and Bernstein," Recombinant DNA Laboratory Manual (Recombinant DNA Laboratory Manual) "Elsevier, 2014; Bose, "Methods in Molecular Biology" Genetic manipulation of Staphylococcus of Staphylococci) “Springer Protocols, Vol. 1106, pp. 101-111, 2014; Hakenbeck and Chhatwal, “ Molecular Biology of Streptococci ", Horizon Scientific Press, 2007; Morona et al., " Among the conserved capsular polysaccharide biosynthesis genes cpsA, cpsB, and cpsD The effect of mutations in the conserved bacteria on the virulence of Streptococcus pneumoniae capsular polysaccharide biosynthesis genes cpsA, cpsB and cpsD have on virulence of Streptococcus pneumoniae) ", J. Infect. Dis. 189: 1905-1913, 2004; Morona et al., " CpsD carboxyl terminus [YGX] 4 Mutation analysis of repeating domains revealed that CpsD is a biosynthetic component of the pneumococcal capsule. The required autophosphorylated tyrosine kinase (YGX) was synthesized. 4 repeat domain of CpsD, an autophosphorylating tyrosine kinase required for capsule biosynthesis in Streptococcus pneumonia) ", J. Bacteriol. 185: 3009-3019, 2003; McAllister et al., " Molecular analysis of the pSA permease complex of Streptococcus pneumoniae analysis of the psa permease complex of Streptococcus pneumoniae) ", Mol. Microbiol. 53:889-901, 2004; Mahdi et al.," A novel pneumococcus preferentially expressed during meningitis in mice Identification of a novel pneumococcal vaccine antigen preferentially expressed during meningitis in mice) ”, J. Clin. Invest. 122:2208-2220, 2012.

[0169] In one non-limiting embodiment, the attenuated and / or inactivated streptococcal bacterial strain comprises a Streptococcus pneumoniae strain derived from Rx1. Rx1 is a capsular-negative mutant derived from Streptococcus pneumoniae serotype 2 strain D39.

[0170] In one implementation, the attenuated and / or killable streptococcal bacterial strain further comprises selected free radicals. lytA , ply and psaA The modification or deletion of genes that make up the group.

[0171] In one implementation, the attenuated and / or killable streptococcal bacterial strain also includes lytA Gene modification or deletion. The autolysin gene can be deleted in Rx1-derived strains. lytA Genes or render them nonfunctional.

[0172] Alternatively or alternatively, the pneumococcal hemolysin gene may be deleted in Rx1-derived strains. ply ) or render it nonfunctional. For example, it could be made functional using another gene (such as ply (toxoid form) replacement ply Genes. In one implementation, the attenuated and / or killable streptococcal bacterial strain also contains ply Gene modification or deletion. In one implementation, the attenuated and / or killable streptococcal bacterial strain also contains a gene named PdT. ply The toxoid form.

[0173] In one implementation, modifications that weaken, reduce, or prevent functional LytC expression can be introduced into Rx1 (ΔLytA, PdT). GPN-001, as described herein, is an example of an Rx1 (ΔLytA, PdT) strain. A method for deriving strains with ΔLytA, PdT modifications is described in PCT Publication No. WO2016 / 149771, which is incorporated herein by reference in its entirety.

[0174] Alternatively or alternatively, the pneumococcal surface antigen A gene may be deleted. psaA Or otherwise render it nonfunctional. Streptococcus pneumoniae psaA Genes encoding Mn 2+ Transport and resistance to oxidative stress of pneumococcal surface antigen A (PsaA). As described herein. psaA Deletion mutants may be present in low Mn 2+ The growth in the environment is defective and / or there are defects in the competent cells of pneumococcus.

[0175] In one implementation, modifications that weaken, reduce, or prevent functional LytC expression can be introduced into Rx1 (ΔLytA, PdT, ΔPsaA). GPN-002, as described herein, is an example of an Rx1 (ΔLytA, PdT, ΔPsaA) strain. A method for deriving strains with ΔPsaA modifications is described in U.S. Publication No. 2021-0361757, which is incorporated herein by reference in its entirety. Such strains may exhibit reduced virulence, reduced competence, and / or low Mn content. 2+ Reduced growth in the environment. Furthermore, in Mn 2+ Under stress conditions, fermenter growth of strains modified with ΔLytA, PdT, and ΔpsaA may induce changes in gene expression that increase the production level of protective antigens. Therefore, improved protection can be provided by using immunogenic compositions with higher levels of protective antigen expression.

[0176] In one implementation, the attenuated and / or killable streptococcal bacterial strain also includes lytA Gene modification or deletion, ply Gene modification or deletion and psaA Gene modifications or deletions, and also include a gene named PdT. ply The toxoid form.

[0177] LytC This disclosure relates to an immunogenic composition comprising an attenuated and / or inactivated streptococcal bacterial strain containing modifications that weaken, reduce, or prevent the functional expression of LytC (or its homologs). In one embodiment, the modifications weaken, reduce, or prevent the functional expression of LytC.

[0178] LytC is a cell wall hydrolase that has been proposed to facilitate cannibalism with LytA by promoting the lysis of non-competent pneumococcal cells (Eldhom et al., 2009). LytC also plays a role in biofilm formation and adhesion to the nasopharyngeal epithelium. The activity of the LytC enzyme is highest at 30°C, suggesting that the physiological role of this enzyme may be to promote the colonization of pneumococci in the upper respiratory tract (Tomasz et al., 1975). Given the surface localization of LytC and the importance of this enzyme in key virulence characteristics, LytC has been studied in animal studies as a candidate for streptococcal vaccines. Vaccination with purified LytC has validated the protein's potential as a protective immunogen, with immunized mice exhibiting high levels of immunoglobulin G (IgG) antibodies and enhanced protection against pneumococcal infection (Wisemann et al., 2001; Corsini et al., 2021). In multiple studies, high titers of LytC-specific IgG antibodies have also been observed in serum samples from healthy volunteers, indicating that antibody responses were induced by natural exposure to Streptococcus pneumoniae (Holmlund et al., 2009; Croucher et al., 2017).

[0179] Modifications to LytC can be any suitable modifications, as long as they attenuate, reduce, or prevent the functional expression of LytC (or its homologs). For example, any suitable method known to those skilled in the art can be used for modification. lytC Genes. For example, the gene can be modified as described herein; however, it should be understood that modifications can be used... lytC Other suitable methods for generating the compositions disclosed herein. Techniques for genetic manipulation of bacteria are well known to those skilled in the art, as described elsewhere herein.

[0180] In one embodiment, the modification can prevent the functional expression of LytC (or its homologs). For example, the gene can be modified to prevent the expression of LytC (or its homologs), for example, by introducing a nonsense mutation to terminate the translation of LytC, thereby truncating and defunctionalizing any resulting protein expressed from the modified gene. Alternatively, the gene can be deleted, for example, using the methods described herein. In one embodiment, the gene can be modified such that the translated protein is a variant with reduced or weakened function. Alternatively, as those skilled in the art will understand, the expression level of LytC can be reduced or weakened, for example, by modifying a control sequence that promotes the expression level of LytC (or its homologs).

[0181] Modifications that weaken, reduce, or prevent the functional expression of LytC (or its homologs) can affect any product containing LytC. lytC The gene or its homolog is used on streptococcal bacterial strains. In one embodiment, the gene is used on streptococcal bacterial strains. lytCModifications of the species or their homologs may be performed on any suitable serotype of a species selected from the group consisting of: Streptococcus agalactiae, Streptococcus bovis, Streptococcus canis, Streptococcus dysgalactiae, Streptococcus equi, Streptococcus equineus, Streptococcus faecalis, Enterococcus faecium, Streptococcus dolphinus, Streptococcus Miller, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus salivarius, Streptococcus sanguinis, Streptococcus suis, and Streptococcus lactis.

[0182] In one embodiment, the attenuated and / or killing streptococcal bacterial strain containing modifications that weaken, reduce, or prevent functional LytC expression is an Rx1 (ΔlytA, PdT, ΔpsaA, ΔlytC) strain. GPN-003, as described herein, is an example of an Rx1 (ΔlytA, PdT, ΔpsaA, ΔlytC) strain. Methods for deriving strains possessing ΔlytC are described herein.

[0183] Target streptococcal species and / or their serotypes In one embodiment, the compositions of the present invention induce an immune response against at least one target streptococcal species and / or its serotype. The target streptococcal species and / or its serotype can be pathogenic bacteria capable of establishing harmful infections in a host organism.

[0184] In one embodiment, at least one target streptococcal bacterial species and / or its serotype is selected from the group consisting of: agalactiae, bovine streptococci, canine streptococci, dysgalactiae, equine streptococci, equine enterococci, equine-like streptococci, enterococci faecalis, enterococci faecium, dolphin streptococci, Miller streptococci, variant streptococci, pneumococci, pyogenic streptococci, salivary streptococci, blood streptococci, suis streptococci, and mammary streptococci.

[0185] In one embodiment, at least one target streptococcal bacterial species and / or serotype of the present disclosure comprises one or more serotypes of Streptococcus pneumoniae. Therefore, the composition may comprise Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10B, 10C, 10F, 11A, 11B, 11C, 11D, 11F, 12A, 12B, 12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F, 19A, 19B, 19C, Any one or more of 19F, 20, 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24C, 24F, 25A, 25F, 27, 28A, 28F, 29, 31, 32A, 32F, 33A, 33B, 33C, 33D, 33F, 34, 35A, 35B, 35C, 35F, 36A, 36B, 37, 38, 39, 40, 41A, 41F, 42, 43, 44, 45, 46, 47A, 47F and / or 48.

[0186] However, those skilled in the art will understand that new bacterial strains and serotypes may be discovered. It should be understood that the attenuated and / or inactivated streptococcal bacterial strains disclosed herein may include streptococcal bacteria or serotypes not specifically listed herein.

[0187] In one embodiment, the composition of the present invention induces an immune response against multiple target streptococcal species and / or their serotypes. In one embodiment, the composition of the present invention induces a cross-protective immune response against at least one target streptococcal species and / or their serotype. In one embodiment, the composition of the present invention induces a cross-protective immune response against multiple target streptococcal species and / or their serotypes.

[0188] Culture of Streptococcal bacterial strains The attenuated and / or killed streptococcal bacterial strains of this disclosure can be cultured using any suitable standard techniques known to those skilled in the art. For example, the attenuated and / or killed streptococcal bacterial strains can be cultured under standard conditions in a series of suitable culture media (e.g., Soytone broth, Todd-Hewitt yeast (THY) broth, etc.) for a suitable period of time until the desired cell density or optical density is obtained.

[0189] Killing of Streptococcal bacterial strains The Streptococcal bacterial strains disclosed herein can be killed using any suitable method known in the art, provided that the killed bacteria are suitable for use in the compositions disclosed herein. For example, Streptococcal bacterial strains can be killed using suitable methods for chemical treatment, heat treatment, irradiation treatment, high hydrostatic pressure, pulsed electric field, ultrashort pulse laser, pressure ultrasound, UV irradiation, or microbial inactivation. In one embodiment, the Streptococcal bacterial strains disclosed herein can be killed by photon irradiation.

[0190] Photon radiation The streptococcal bacteria in the compositions of this disclosure can be exposed to photon radiation. As stated above, the term "photon radiation" will be understood to encompass both gamma radiation (i.e., gamma rays) and X-ray radiation (i.e., X-rays). Therefore, the "photon-irradiated" streptococcal bacterial strains of this disclosure can be "gamma-irradiated" by exposure to gamma radiation (i.e., gamma rays), "X-irradiated" by exposure to X-ray radiation (i.e., X-rays), or both. As is known to those skilled in the art, X-rays are the same as gamma rays, except that they are emitted by electrons passing through the electric field of the atomic nucleus rather than by the atomic nucleus itself after radioactive decay. By way of non-limiting example only, in order to become photon irradiated, the material may be subjected to photon radiation with energies of at least 0.01 MeV, at least 0.1 MeV, at least 0.5 MeV, 0.01 MeV to 0.5 MeV, 0.01 MeV to 1 MeV, 0.01 MeV to 10 MeV, 0.5 MeV to 20 MeV, 0.5 MeV to 15 MeV, 0.5 MeV to 10 MeV, 0.5 MeV to 5 MeV, 0.5 MeV to 2 MeV, or 1 MeV to 2 MeV (e.g., 1.25 MeV).

[0191] The streptococcal bacteria in the compositions disclosed herein can be irradiated with gamma radiation. Any suitable gamma radiation source can be used. Suitable gamma emitters include, but are not limited to, Ba. 137 Co 60 Cs 137 Ir 192 U 235 Se 75 and Yb 169 .

[0192] The gamma irradiation of Streptococcus bacteria disclosed herein can be performed using commercially available devices, such as gammacell irradiators (e.g., gammacell 40 irradiator, gammacell 220 irradiator, gammacell 1000 irradiator, gammacell 3000 irradiator) manufactured by Atomic Energy of Canada Ltd., Canada, gamma irradiators manufactured by JL Shepherd and Associates (San Fernando, California, USA), or Nordion gamma cell-1000 irradiators manufactured by Nordion Inc. (Kanata, Ontario, Canada). Other suitable devices are described, for example, in U.S. Patent Nos. 3,557,370 and 3,567,938.

[0193] Alternatively or additionally, the streptococcal bacteria disclosed herein may be X-ray irradiated. Any suitable X-ray source may be used. Suitable X-ray sources include, but are not limited to, the eXelis® sterilization X-ray machine manufactured by IBA Industrial (Louvain-la-Neuve, Belgium). Other suitable devices include, for example, the RS2400® and RS3400® manufactured by Rad Source Technologies Inc. (Suwanee, Georgia, USA).

[0194] Typically, the Streptococcal bacteria of this disclosure are exposed to a dose of photon radiation (e.g., gamma radiation and / or X-ray radiation) sufficient to attenuate or inactivate the Streptococcal bacterial strains of this disclosure. Preferably, the dose of photon radiation is sufficient to attenuate or inactivate the bacteria without substantially destroying the structure of antigens (e.g., surface antigens). Thus, the immunogenicity of the antigenic determinants can be preserved by photon-irradiated bacteria and their derivatives. Preferably, the dose of photon radiation is applied to the bacteria over a period of time and at a level sufficient to ensure that all Streptococcal bacterial strains of this disclosure treated are exposed without adversely affecting the structural integrity of the antigenic determinants.

[0195] As is known to those skilled in the art, the measure of absorbed radiation dose is the gray (Gy), which is defined as 1 joule of energy deposited in 1 kilogram of mass. Its older unit of measurement is the rad, which stands for "absorbed radiation dose," where 1 Gy = 100 rads.

[0196] Streptococcal bacteria used according to this disclosure can be exposed to approximately 1 × 10⁻⁶. 3 Rad to approximately 2 × 10 9Rad (or about 10 Gy to about 2 × 10) 4 The total dose of photon radiation (e.g., gamma radiation and / or X-ray radiation) is in the range of kGy. In some embodiments of this disclosure, streptococcal bacteria or derivatives are exposed to about 1 × 10⁻⁶ kGy. 3 Rad to approximately 2 × 10 9 Rad, approximately 1 × 10 3 Rad to approximately 1 × 10 9 Rad, approximately 1 × 10 3 Radd to approximately 1 × 10 8 Rad, approximately 1 × 10 3 Radd to approximately 1 × 10 7 Rad, approximately 1 × 10 3 Rad to approximately 1 × 10 6 Rad, approximately 1 × 10 3 Radd to approximately 1 × 10 5 Rad, approximately 1 × 10 3 Radd to approximately 1 × 10 4 Rad, approximately 1 × 10 3 Rad to approximately 2 × 10 9 Rad, approximately 1 × 10 4 Rad to approximately 2 × 10 9 Rad, approximately 1 × 10 5 Rad to approximately 2 × 10 9 Rad, approximately 1 × 10 6 Rad to approximately 2 × 10 9 Rad, approximately 1 × 10 7 Rad to approximately 2 × 10 9 Rad, approximately 1 × 10 8 Rad to approximately 2 × 10 9 Rad or approximately 1 × 10 9 Rad to approximately 2 × 10 9 Rad's total dose of X-ray and / or gamma radiation.

[0197] In one embodiment of this disclosure, the Streptococcus bacteria of this disclosure are exposed to approximately 6.5 × 10⁻⁶. 4 Rad to approximately 2 × 10 7Total dose of photon radiation (e.g., X-ray and / or gamma radiation) of rad (approximately 0.65 kGy to approximately 200 kGy). In other embodiments of this disclosure, the Streptococcus bacterial strains of this disclosure are exposed to approximately 10 kGy to approximately 12 kGy, approximately 12 kGy to approximately 14 kGy, approximately 14 kGy to approximately 16 kGy, approximately 10 kGy to approximately 20 kGy, approximately 14 kGy to approximately 20 kGy, approximately 20 kGy to approximately 30 kGy, approximately 20 kGy to approximately 25 kGy, approximately 25 kGy to approximately 30 kGy, approximately 30 to 35 kGy, approximately 10 kGy, approximately 11 kGy, approximately 12 kGy, approximately 13 kGy, approximately 14 kGy, approximately 15 kGy, approximately 16 kGy, approximately 17 kGy, approximately 18 kGy, approximately 19 kGy, approximately 20 kGy, approximately 21 kGy, approximately 22 kGy, approximately 23 kGy, approximately 24 kGy, approximately 25 kGy, and approximately 26 kGy. kGy, approximately 27 kGy, approximately 28 kGy, approximately 29 kGy, approximately 30 kGy, approximately 31 kGy, approximately 32 kGy, approximately 33 kGy, approximately 34 kGy, approximately 35 kGy, approximately 20 kGy, approximately 20 kGy, over 10, approximately 12 kGy to approximately 14 kGy, over 12 kGy, over 14 kGy, over 16 kGy, over 18 kGy, over 20 kGy, over 22 kGy, over 24 kGy, over 26 kGy, over 28 kGy, over 30 kGy, over 35 kGy, over 40 kGy, 1.26 × 10 6 The total photon radiation dose of rad (12.6 kGy) is approximately 1 × 10⁻⁶. 6 The total photon radiation dose of rad (approximately 10 kGy) photons, or approximately 1 × 10 5 Total photon radiation dose of rad (1 kGy).

[0198] The optimal dose of photon radiation (e.g., gamma radiation and / or X-ray radiation) may be influenced by factors such as: the culture medium containing the Streptococcus bacteria of this disclosure, the number of present bacteria to be treated, the temperature of the present bacteria to be treated (e.g., frozen on dry ice or at room temperature), water availability, oxygen availability, and / or the subtype or strain being treated. Therefore, the total photon radiation dose, exposure time, and / or the level of photon radiation applied during exposure can be optimized to enhance the effectiveness of the treatment.

[0199] The total dose of photon radiation (e.g., X-ray radiation and / or gamma radiation) can be cumulatively applied to the Streptococcus bacterial strains of this disclosure over a period of time. For example, photon radiation can be applied to the Streptococcus bacterial strains of this disclosure at a level below the total dose for a period of time sufficient to achieve the desired total dose of photon radiation.

[0200] In one embodiment, the Streptococcus bacterial strain of this disclosure can be cultured in a suitable bacterial culture medium (such as THY broth, soy peptone broth, etc.) to support the growth of the Streptococcus strain, in order to obtain a suitable cell density, for example, 102. 4 Up to 10 8 A cell density of colony-forming units (CFU) / ml. In one embodiment, the cell density is 10-1. 7 Up to 10 8 Colony forming units (CFU) / ml. In one embodiment, the cell density is approximately 102. 8 Colony forming units (CFU) / ml. Bacteria can be concentrated, for example, using techniques known to those skilled in the art (such as centrifugation, filtration, etc.). In one embodiment, bacteria are washed with a suitable solution (such as PBS); however, those skilled in the art will understand that various buffer solutions are suitable. Techniques such as tangential flow filtration, percolation, etc., can be used to wash bacteria. In one embodiment, the bacterial cells are re-centrifuged and resuspended in PBS containing 5% to 20% glycerol. In one embodiment, the glycerol concentration is selected from 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. In one embodiment, bacterial cells are concentrated at 1 x 10⁻⁶ CFU / ml. 4 Up to 1x10 12 CFU / ml (e.g., 1x10) 4 1x10 5 1x10 6 1x10 7 1x10 8 1x10 9 1x10 11 Or 1x10 12 The solution was prepared at a concentration of 1 x 10⁻⁶ CFU / ml and then resuspended. In one embodiment, the prepared concentration was 1 x 10⁻⁶ CFU / ml. 10 CFU / ml. Streptococcal bacterial strain preparations can be photon irradiated on ice or dry ice (DI) at various irradiation doses (0.5–30 kGy) and temperature conditions as described herein. Optionally, washed bacterial cells can be lyophilized prior to photon irradiation using standard techniques known to those skilled in the art.

[0201] In one embodiment, the Streptococcal bacterial strain preparations of this disclosure are maintained in a frozen and / or lyophilized state while being exposed to photon radiation (e.g., gamma radiation and / or X-ray radiation). This may help maintain biological integrity and avoid unnecessary damage to antigens, thereby enhancing the immunogenicity of the photon-irradiated bacterial preparations, particularly their ability to induce cross-reactive / cross-protective immunity against, for example, heterologous subtypes. Typically, a photon radiation dose of 10–30 kGy (as described herein) may be effective for treating the frozen and / or lyophilized Streptococcal bacterial strain preparations of this disclosure. Alternatively, a photon radiation dose of 20–40 kGy (e.g., more than 20, more than 22, more than 24, more than 26, more than 28, more than 30, more than 32, more than 34, more than 36, or more than 38 kGy, etc.) may be effective for treating the frozen and / or lyophilized Streptococcal bacterial strain preparations of this disclosure.

[0202] As described above, preferably, treatment with photon radiation is sufficient to inactivate the Streptococcal bacterial strains of this disclosure without substantially destroying the structure of the bacterial antigens. The attenuation and / or inactivation of the Streptococcal bacterial strains of this disclosure can be evaluated using methods generally known in the art.

[0203] For example, bacterial attenuation and / or inactivation can be assessed by determining the number of viable bacteria (i.e., colony-forming units) that form colonies on agar plates after treatment with photon radiation (e.g., gamma radiation and / or X-ray radiation).

[0204] The integrity of antigenic determinants can be assessed by using, for example, Western blotting, FAC analysis, or enzymatic assays of surface components, through reactivity with monospecific antiserum generated against purified natural antigenic components.

[0205] With modification lytC Attenuated Streptococcus strains In one embodiment, the bacterial strain containing modifications that attenuate, reduce, or prevent the functional expression of LytC (or its homologs) is attenuated. That is, the strain is non-pathogenic and therefore does not cause disease when inoculated into mice as a live strain. In one embodiment, the compositions of this disclosure comprise attenuated whole-cell immunogenic compositions.

[0206] In one embodiment, the attenuated whole-cell immunogenic composition of this disclosure can be used as an attenuated live immunogenic or vaccine composition. In one embodiment, the attenuated immunogenic or vaccine composition induces an immune response against at least one target streptococcal species and / or its serotype. In one embodiment, the attenuated immunogenic or vaccine composition improves the effectiveness of the immune response compared to a parent strain lacking the modification. In one embodiment, the attenuated streptococcal bacterial strain is derived from a parent strain lacking the modification, and the attenuated streptococcal bacterial strain has improved stability compared to the parent strain.

[0207] Immune response The compositions disclosed herein can induce an immune response when administered to a subject. In one embodiment, the composition can induce a heterologous immune response. In one embodiment, the composition induces a cross-protective immune response.

[0208] In one implementation, compared to the parental strain, it contains... lytC Modified attenuated and / or killable streptococcal bacterial strains can induce immune responses with improved efficacy. For example, the composition can induce increased protection against infection and / or disease compared to a composition containing a parental strain. In one embodiment, the compositions of this disclosure induce higher total levels of antibodies. In one embodiment, the compositions of this disclosure induce higher total levels of IgG antibodies. In one embodiment, the compositions induce an increase or decrease in a specific immunoglobulin subtype (e.g., selected from IgG1, IgG2a, IgG2b, IgG3, IgG4, etc., IgA, IgE, IgM, etc.). In one embodiment, the compositions induce a change in the ratio of one immunoglobulin subtype to another, for example, a change in the ratio selected from IgG1:IgG2a, IgG1:IgG2b, IgG1:IgG3, etc. The change can be an increase or a decrease. In one embodiment, the compositions enhance antibody responses to opsonize and kill target streptococcal species and / or their serotypes. In one embodiment, the compositions enhance antibody responses to opsonize and kill capsular pneumococci. In one embodiment, the compositions of this disclosure induce enhanced cellular responses, such as a Th1 / Th2 bias shift.

[0209] Prevention and treatment methods This disclosure provides a method for preventing streptococcal infection in a subject. It also provides a treatment method for treating streptococcal infection in a subject. The method includes administering to the subject an immunogenic or vaccine composition comprising an attenuated and / or inactivated streptococcal bacterial strain containing modifications that weaken, reduce, or prevent the functional expression of LytC (or its homologs), for example, in the form of a vaccine of this disclosure.

[0210] The method induces or enhances an immune response against target streptococcal bacteria in a subject. The immune response can be cross-protective / heterologous, as it can induce or enhance an immune response against multiple serotypes of streptococci. The method may also include administering an immune response containing... lytC Modified streptococcal bacterial strains of various different attenuated and / or killable types, thereby generating immunity against multiple streptococcal species and their various serotypes.

[0211] The method can induce or enhance an immune response against any one or more of the following target streptococcal species and / or serotypes: agalactiae, bovine streptococci, canine streptococci, dysgalactiae, equine streptococci, equine enterococci, equine-like streptococci, enterococci faecalis, enterococci faecium, dolphin streptococci, Miller streptococci, variant streptococci, pneumococci, pyogenic streptococci, salivary streptococci, blood streptococci, suis streptococci, and mammary streptococci.

[0212] In one embodiment, the method includes preventing or treating streptococcal infection caused by a target streptococcal species and / or its serotype by administering to a subject a composition comprising an attenuated and / or inactivated streptococcal bacterial strain, wherein the attenuated and / or inactivated streptococcal bacterial strain comprises the same streptococcal species. lytC Modification.

[0213] In one implementation, the streptococcal species or serotype causing the infection may be different from attenuated and / or killable streptococcal bacterial strains containing modifications that weaken, reduce, or prevent the functional expression of LytC (or its homologs).

[0214] By way of non-limiting example only, the method can be used to: (i) To prevent or treat an infection, disease, or condition caused by any one or more serotypes of Streptococcus agalactiae by administering an immunogenic composition comprising one or more modified attenuated and / or killant Streptococcus agalactiae strains containing attenuated, reduced, or prevented functional expression of LytC (or its homologs), wherein said attenuated and / or killant Streptococcus agalactiae strains comprise one or more serotypes of Streptococcus agalactiae. The attenuated and / or killant Streptococcus agalactiae serotypes administered may differ from the one or more serotypes causing the infection, disease, or condition; (ii) To prevent or treat an infection, disease, or condition caused by any one or more serotypes of Streptococcus bovis by administering an immunogenic composition comprising one or more modified attenuated and / or killant Streptococcus bovis serotypes containing attenuated, reduced, or prevented functional expression of LytC (or its homologs), wherein the attenuated and / or killant Streptococcus bovis serotypes comprise one or more serotypes of Streptococcus bovis. The attenuated and / or killant Streptococcus bovis serotypes administered may differ from the one or more serotypes causing the infection, disease, or condition; (iii) To prevent or treat an infection, disease, or condition caused by any one or more serotypes of Streptococcus canis by administering an immunogenic composition comprising one or more modified attenuated and / or killant streptococcal bacterial strains containing attenuated, reduced, or prevented functional expression of LytC (or its homologs), wherein said attenuated and / or killant streptococcal bacterial strains comprise one or more serotypes of Streptococcus canis. The one or more attenuated and / or killant Streptococcus canis serotypes administered may be different from the one or more serotypes causing the infection, disease, or condition; (iv) To prevent or treat an infection, disease, or condition caused by any one or more serotypes of *Streptococcus lactis* by administering an immunogenic composition comprising one or more modified attenuated and / or killant *Streptococcus lactis* strains containing attenuated, reduced, or prevented functional expression of LytC (or its homologs), wherein said attenuated and / or killant *Streptococcus lactis* strains comprise one or more serotypes of *Streptococcus lactis*. The attenuated and / or killant *Streptococcus lactis* serotypes administered may differ from the one or more serotypes causing the infection, disease, or condition; (v) To prevent or treat an infection, disease, or condition caused by any one or more serotypes of *Streptococcus equi* by administering an immunogenic composition comprising one or more modified attenuated and / or killant *Streptococcus equi* bacterial strains containing attenuated, reduced, or prevented functional expression of LytC (or its homologs), wherein said attenuated and / or killant *Streptococcus equi* bacterial strains comprise one or more serotypes of *Streptococcus equi*. The one or more attenuated and / or killant *Streptococcus equi* serotypes administered may be different from the one or more serotypes causing the infection, disease, or condition; (vi) To prevent or treat an infection, disease, or condition caused by any one or more serotypes of *Streptococcus equi* by administering an immunogenic composition comprising one or more modified attenuated and / or killant *Streptococcus equi* bacterial strains containing attenuated, reduced, or prevented functional expression of LytC (or its homologs), wherein said attenuated and / or killant *Streptococcus equi* bacterial strains comprise one or more serotypes of *Streptococcus equi*. The one or more attenuated and / or killant *Streptococcus equi* serotypes administered may be different from the one or more serotypes causing the infection, disease, or condition; (vii) To prevent or treat an infection, disease, or condition caused by any one or more serotypes of Streptococcus agalactiae by administering an immunogenic composition comprising one or more modified attenuated and / or killant Streptococcus agalactiae strains containing attenuated, reduced, or prevented functional expression of LytC (or its homologs), wherein said attenuated and / or killant Streptococcus agalactiae strains comprise one or more serotypes of Streptococcus agalactiae. The attenuated and / or killant Streptococcus agalactiae serotypes administered may differ from the one or more serotypes causing the infection, disease, or condition; (viii) To prevent or treat an infection, disease, or condition caused by any one or more serotypes of Streptococcus equina by administering an immunogenic composition comprising one or more modified attenuated and / or killant streptococcal bacterial strains containing attenuated, reduced, or prevented functional expression of LytC (or its homologs), wherein said attenuated and / or killant streptococcal bacterial strains comprise one or more serotypes of Streptococcus equina. The one or more attenuated and / or killant Streptococcus equina serotypes administered may be different from the one or more serotypes causing the infection, disease, or condition; (ix) Prevention or treatment of infection, disease, or condition caused by any one or more serotypes of Enterococcus faecalis by administering an immunogenic composition comprising one or more modified, attenuated and / or killant streptococcal bacterial strains containing attenuated, reduced, or prevented functional expression of LytC (or its homologs), wherein said attenuated and / or killant streptococcal bacterial strains comprise one or more serotypes of Enterococcus faecalis. The one or more attenuated and / or killant Enterococcal serotypes administered may be different from the one or more serotypes causing the infection, disease, or condition; (x) To prevent or treat an infection, disease, or condition caused by any one or more serotypes of Enterococcus faecalis by administering an immunogenic composition comprising one or more modified, attenuated and / or killant streptococcal bacterial strains containing attenuated, reduced, or prevented functional expression of LytC (or its homologs), wherein said attenuated and / or killant streptococcal bacterial strains comprise one or more serotypes of Enterococcus faecalis. The one or more attenuated and / or killant Enterococcus faecalis serotypes administered may be different from the one or more serotypes causing the infection, disease, or condition; (xi) To prevent or treat an infection, disease, or condition caused by any one or more serotypes of *Streptococcus dolphinus* by administering an immunogenic composition comprising one or more modified attenuated and / or killant *Streptococcus dolphinus* bacterial strains containing attenuated, reduced, or prevented functional expression of LytC (or its homologs), wherein said attenuated and / or killant *Streptococcus dolphinus* bacterial strains comprise one or more serotypes of *Streptococcus dolphinus*. The one or more attenuated and / or killant *Streptococcus dolphinus* serotypes administered may be different from the one or more serotypes causing the infection, disease, or condition; (xii) To prevent or treat an infection, disease, or condition caused by any one or more serotypes of Streptococcus Müller by administering an immunogenic composition comprising one or more modified attenuated and / or killant streptococcal bacterial strains containing attenuated, reduced, or prevented functional expression of LytC (or its homologs), wherein said attenuated and / or killant streptococcal bacterial strains comprise one or more serotypes of Streptococcus Müller. The one or more attenuated and / or killant Streptococcus Müller serotypes administered may be different from the one or more serotypes causing the infection, disease, or condition; (xiii) Prevention or treatment of infection, disease, or condition caused by any one or more serotypes of Streptococcus mutans by administering an immunogenic composition comprising one or more modified attenuated and / or killant Streptococcus mutans strains containing attenuated, reduced, or prevented functional expression of LytC (or its homologs), wherein said attenuated and / or killant Streptococcus mutans strains comprise one or more serotypes of Streptococcus mutans. The one or more attenuated and / or killant Streptococcus mutans serotypes administered may be different from the one or more serotypes causing the infection, disease, or condition; (xiv) Prevention or treatment of infection, disease, or condition caused by any one or more serotypes of Streptococcus pneumoniae by administering an immunogenic composition comprising one or more modified attenuated and / or killant streptococcal bacterial strains containing attenuated, reduced, or prevented functional expression of LytC (or its homologs), wherein said attenuated and / or killant streptococcal bacterial strains comprise one or more serotypes of Streptococcus pneumoniae. The one or more attenuated and / or killant Streptococcus pneumoniae serotypes administered may be different from the one or more serotypes causing the infection, disease, or condition; (xv) To prevent or treat an infection, disease, or condition caused by any one or more serotypes of Streptococcus pyogenes by administering an immunogenic composition comprising one or more modified attenuated and / or killant streptococcal bacterial strains containing attenuated, reduced, or prevented functional expression of LytC (or its homologs), wherein said attenuated and / or killant streptococcal bacterial strains comprise one or more serotypes of Streptococcus pyogenes. The one or more attenuated and / or killant streptococcal serotypes administered may be different from the one or more serotypes causing the infection, disease, or condition; (xvi) To prevent or treat an infection, disease, or condition caused by any one or more serotypes of Streptococcus salivarius by administering an immunogenic composition comprising one or more modified attenuated and / or killant Streptococcus salivarius strains containing attenuated, reduced, or prevented functional expression of LytC (or its homologs), wherein said attenuated and / or killant Streptococcus salivarius strains comprise one or more serotypes of Streptococcus salivarius. The one or more attenuated and / or killant Streptococcus salivarius serotypes administered may be different from the one or more serotypes causing the infection, disease, or condition; (xvii) Prevention or treatment of infection, disease, or condition caused by any one or more serotypes of Streptococcus sanguinis by administration of an immunogenic composition comprising one or more modified attenuated and / or killant streptococcal bacterial strains containing attenuated, reduced, or prevented functional expression of LytC (or its homologs), wherein said attenuated and / or killant streptococcal bacterial strains comprise one or more serotypes of Streptococcus sanguinis. The one or more attenuated and / or killant streptococcal serotypes administered may be different from the one or more serotypes causing the infection, disease, or condition; (xviii) Prevention or treatment of infection, disease, or condition caused by any one or more serotypes of *Streptococcus suis* by administration of an immunogenic composition comprising one or more modified attenuated and / or killant *Streptococcus suis* bacterial strains containing attenuated, reduced, or prevented functional expression of LytC (or its homologs), wherein said attenuated and / or killant *Streptococcus suis* bacterial strains are one or more serotypes of *Streptococcus suis*. The one or more attenuated and / or killant *Streptococcus suis* serotypes administered may be different from the one or more serotypes causing the infection, disease, or condition; and / or (xix) Prevention or treatment of infection, disease, or condition caused by any one or more serotypes of Streptococcus mammologicus by administering an immunogenic composition comprising one or more modified attenuated and / or killant streptococcal bacterial strains that attenuate, reduce, or prevent the functional expression of LytC (or its homologs), wherein said attenuated and / or killant streptococcal bacterial strains are one or more serotypes of Streptococcus mammologicus. The one or more attenuated and / or killant Streptococcus mammologicus serotypes administered may be different from the one or more serotypes causing the infection, disease, or condition.

[0215] In one embodiment, the method is used to prevent or treat infections, diseases, or conditions caused by one or more serotypes of Streptococcus pneumoniae. The method may include inducing an immune response against multiple different Streptococcus pneumoniae serotypes in a subject by administering an immunogenic composition to the subject, said immunogenic composition comprising one or more attenuated and / or killable streptococcal bacterial strains containing modifications that attenuate, reduce, or prevent functional LytC expression. In one embodiment, the method includes administering a single attenuated and / or killable (e.g., gamma-irradiated and / or X-ray-irradiated) serotype of Streptococcus pneumoniae.

[0216] In one embodiment, the method includes preventing or treating an infection, disease, or condition caused by one or more serotypes of Streptococcus pneumoniae. The method includes administering to a subject attenuated and / or inactivated Streptococcus pneumoniae serotypes at least one, and may induce an immune response in the subject against any one or more of the following Streptococcus pneumoniae serotypes: 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10B, 10C, 10F, 11A, 11B, 11C, 11D, 11F, 12A, 12B, 12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17 F, 18A, 18B, 18C, 18F, 19A, 19B, 19C, 19F, 20, 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24C, 24F, 25A, 25F, 27, 28A, 28F, 29, 31, 32A, 32F, 33A, 33B, 33C, 33D, 33F, 34, 35A, 35B, 35C, 35F, 36A, 36B, 37, 38, 39, 40, 41A, 41F, 42, 43, 44, 45, 46, 47A, 47F and / or 48. In one embodiment, the method can induce an immune response in a subject against any one or more of the following Streptococcus pneumoniae serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F. However, it should be understood that the compositions of this disclosure can induce an immune response against streptococcal bacterial strains not specifically listed herein. For example, the compositions of this disclosure can induce an immune response against newly emerging Streptococcus pneumoniae strains that have not yet been assigned serotype numbers.

[0217] The applied attenuated and / or inactivated Streptococcus pneumoniae serotypes may differ from one or more serotypes that cause infection, disease, or condition. In one embodiment, the attenuated and / or inactivated Streptococcus bacterial strain is derived from a strain, serotype, or species that is different from the at least one target Streptococcus species and / or its serotype. Therefore, in one embodiment, the induced immune response is cross-protective.

[0218] The disease or condition can be any disease or condition caused by infection with a specific species or serotype of streptococcus. By way of non-limiting example only, the disease or condition can be any one or more of the following: pneumonia, ear infection, earache, middle ear infection, otitis media, sinusitis, meningitis, conjunctivitis, bacteremia, sepsis, joint infection, bone infection, septic arthritis, osteomyelitis, soft tissue infection, cellulitis, myositis, periorbital cellulitis, abscess, peritonitis, heart infection, endocarditis, and pericarditis.

[0219] Subjects can be any animal of economic, social, or research importance, including cattle, horses, sheep, primates, birds, and rodent species. Therefore, subjects can be mammals, such as humans, or non-human mammals (e.g., pigs, cats, dogs, cattle, horses, or sheep). Subjects can be laboratory animals (e.g., rodents such as mice, rats, or guinea pigs; rabbits, etc.), birds (e.g., poultry), fish, or crustaceans.

[0220] Attenuated and / or inactivated streptococcal bacteria and / or their attenuated and / or inactivated derivatives may be administered to the subject via any suitable route, including, for example, parenteral (e.g., intradermal, intravenous, intraspinal, intraperitoneal, subcutaneous, or intramuscular), oral, local, or mucosal routes (e.g., intranasal). In one embodiment, administration is via a mucosal route. For example, administration may be intranasal.

[0221] Without being limited to one or more specific mechanisms of action, the method can induce an immune response in a subject that includes one or more of the following: (i) To produce antibodies that specifically bind to one or more antigens of streptococcal bacteria that cause infection, disease or condition; (ii) CD4 antigens specific to one or more antigens of streptococcal bacteria that cause infection, disease, or condition. + T lymphocyte response; and / or (iii) CD8 antigens specific to one or more antigens of streptococcal bacteria that cause infection, disease, or condition. + T lymphocyte response.

[0222] In one embodiment, the method can induce an immune response in a subject, which can be interleukin-17A (IL-17A) dependent, IL-17A independent, and / or involve activation of the innate immune system, including the production of cytokines (e.g., IFN-γ) and / or activation of Toll-like receptors (e.g., TLR-3). This may help lower the activation threshold of B cells and / or enhance the quality or quantity of antibody responses against the antigen of interest.

[0223] By way of non-limiting example only, the immune response induced or enhanced in subjects by the method may be increased by at least about 10%, at least about 20%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, at least about two times, at least about five times, at least about ten times, at least about twenty times, at least about fifty times, or at least about 100 times when compared with a suitable control. A suitable control may be, for example, a measurement of the same immune response prior to the method performed under otherwise similar, substantially the same, or identical conditions.

[0224] Methods for detecting and quantifying immune responses are well known to those skilled in the art and include, for example, solid-phase heterogeneous assays (e.g., enzyme-linked immunosorbent assays), solution-phase assays (e.g., electrochemiluminescence assays), amplified luminescence adjacent homogeneous assays, flow cytometry, intracellular cytokine staining, functional T cell assays, functional B cell assays, functional monocyte-macrophage assays, dendritic and reticuloendothelial cell assays, natural killer cell response measurements, oxidative burst assays, cytotoxicity-specific cell lysis assays, pentamer binding assays, and evaluation of phagocytosis and apoptosis.

[0225] Immunogenic compositions and vaccine composition formulations Attenuated and / or inactivating streptococcal bacterial strains described herein, comprising modifications that weaken, reduce, or prevent the functional expression of LytC (or its homologs), may be incorporated into pharmaceutical compositions. Such compositions can stimulate an immune response against pathogenic organisms capable of establishing an infection in a host, which may ultimately lead to disease or condition. Therefore, such compositions may be immunogenic compositions or vaccine compositions, including prophylactic vaccines (i.e., vaccines administered for the purpose of preventing infection and / or disease / condition) and therapeutic vaccines (i.e., vaccines administered for the purpose of treating infection and / or disease / condition). Thus, the vaccines of this disclosure may be administered to recipients for preventative, ameliorative, palliative, or therapeutic purposes. It should be understood that all such compositions are collectively covered herein by reference to “various compositions of this disclosure” or “compositions of this disclosure.” It should also be understood that vaccine compositions fall within the scope of the immunogenic compositions of this disclosure.

[0226] Non-limiting examples of suitable streptococcal bacteria suitable for inclusion in compositions of this disclosure are described above in the title “ Streptococcal bacterial strains "and" Streptococcal bacterial derivatives In the section "".

[0227] In one embodiment, the streptococcal bacteria and their derivatives of the composition are attenuated or inactivated by photon radiation (e.g., gamma radiation and / or X-ray radiation). Photon radiation may be applied to them before, during, or after combining the streptococcal bacterial strains of this disclosure with one or more other reagents to provide an immunogenic composition or vaccine composition formulation.

[0228] preparation The compositions disclosed herein can be prepared using methods known to those skilled in the art. Non-limiting examples of suitable methods are described in Gennaro et al. (ed.), (1990), “ Remington's Pharmaceutical Sciences Pharmaceutical Sciences) “, Mack Publishing Co., Easton, Pennsylvania, USA, and vaccine preparation methods are generally described in Voller et al., (1978), “ New Trends and Developments in Vaccines and Developments in Vaccines) ”, University Park Press, Baltimore, Maryland, USA.

[0229] The composition may comprise a pharmaceutically acceptable carrier, excipient, diluent, and / or adjuvant. A "pharmaceutically acceptable" carrier, excipient, diluent, and / or adjuvant, as contemplated herein, is a substance that does not produce one or more adverse reactions when administered to a particular recipient (such as a human or non-human animal). Pharmaceutically acceptable carriers, excipients, diluents, and adjuvants are generally also compatible with other components of the vaccine. Non-limiting examples of suitable excipients, diluents, and carriers can be found in […]. Handbook of Pharmaceutical Excipients 4th edition, (2003) Rowe et al. (eds.), The Pharmaceutical Press, London, American Pharmaceutical Association, Washington.

[0230] Non-limiting examples of pharmaceutically acceptable carriers, excipients, or diluents include demineralized or distilled water; saline solutions; and plant-based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, corn oil, sesame oil, and arachid oil. Oils such as coconut oil; silicone oils, including polysiloxanes such as methylpolysiloxane, phenylpolysiloxane, and polysolpoxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin, or squalane; cellulose derivatives such as methylcellulose, ethylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, or hydroxypropyl methylcellulose; lower alkanols (e.g., ethanol or isopropanol); lower aryl alkanols; lower polyalkylene glycols or lower alkylene glycols such as polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3-butanediol, or glycerol; fatty acid esters such as isopropyl palmitate, isopropyl myristate, or ethyl oleate; polyvinylpyrridone; agar; carrageenan; tragacanth or gum arabic; and mineral oil gels. Typically, one or more carriers will constitute 10% to 99.9% by weight of the composition.

[0231] The compositions disclosed herein may be in the form suitable for injection, in the form suitable for oral administration (e.g., such as capsules, tablets, pouches, elixirs), in the form suitable for topical application (ointment, cream or lotion), in the form suitable for delivery as eye drops, in the form suitable for inhalation (such as intranasal or oral inhalation) (aerosol form), or in the form suitable for parenteral administration (i.e., intradermal, subcutaneous, intramuscular or intravenous injection).

[0232] The solid form of the composition for oral administration may contain binders, sweeteners, disintegrants, diluents, flavoring agents, coating agents, preservatives, lubricants, and / or delay agents acceptable in human and veterinary pharmaceutical practice. Suitable binders include gum arabic, gelatin, corn starch, tragacanth gum, sodium alginate, carboxymethyl cellulose, or polyethylene glycol. Suitable sweeteners include sucrose, lactose, glucose, aspartame, or saccharin. Suitable disintegrants include corn starch, methylcellulose, polyvinylpyrrolidone, guar gum, xanthan gum, bentonite, alginate, or agar. Suitable diluents include lactose, sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate, calcium silicate, or dicalcium phosphate. Suitable flavoring agents include peppermint oil, wintergreen oil, cherry, orange, or raspberry flavorings. Suitable coating agents include polymers or copolymers of acrylic acid and / or methacrylic acid and / or their esters, waxes, fatty alcohols, zein, shellac, or gluten. Suitable preservatives include sodium benzoate, vitamin E, α-tocopherol, ascorbic acid, methylparaben, propylparaben, or sodium bisulfite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride, or talc. Suitable delay agents include glyceryl monostearate or glyceryl distearate.

[0233] In addition to the reagents mentioned above, the liquid form of the composition for oral administration may also contain a liquid carrier. Suitable liquid carriers include water, oils (such as olive oil, peanut oil, sesame oil, sunflower oil, safflower oil, arachis oil, coconut oil), liquid paraffin, ethylene glycol, propylene glycol, polyethylene glycol, ethanol, propanol, isopropanol, glycerol, fatty alcohols, triglycerides, or mixtures thereof.

[0234] Suspensions containing compositions for oral administration may also contain dispersants and / or suspending agents. Suitable suspending agents include sodium carboxymethyl cellulose, methylcellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone, sodium alginate, or acetyl alcohol. Suitable dispersants include lecithin, polyoxyethylene esters of fatty acids (such as stearic acid), polyoxyethylene sorbitan mono- or di-oleate, stearate, or laurate, polyoxyethylene dehydrated sorbitan mono- or di-oleate, stearate, or laurate, etc.

[0235] For the preparation of compositions as injectable solutions or suspensions, non-toxic, parenteral-acceptable diluents or carriers such as Ringer's solution, isotonic saline, phosphate-buffered saline, ethanol, and 1,2-propylene glycol can be used.

[0236] Vaccine emulsions intended for oral administration may also contain one or more emulsifiers. Suitable emulsifiers include dispersants or natural gums, such as guar gum, gum arabic, or tragacanth gum, as exemplified above.

[0237] Topical formulations of the composition comprise one or more active ingredients (e.g., attenuated and / or inactivated streptococcal bacteria and / or their derivatives), one or more acceptable carriers, and optionally any other therapeutic ingredient. Formulations suitable for topical application include liquid or semi-liquid formulations, such as liniments, lotions, creams, ointments, or pastes, suitable for penetration through the skin to the site of treatment, and drops suitable for application to the eyes, ears, or nose.

[0238] When formulated into drops, the composition may comprise a sterile aqueous or oily solution or suspension. These can be prepared by dissolving the active ingredient in an aqueous solution of a bactericide and / or fungicide and / or any other suitable preservative, optionally including a surfactant. The resulting solution can then be clarified by filtration, transferred to a suitable container, and sterilized. Sterilization can be achieved, for example, by filtration followed by aseptic transfer to the container. Examples of bactericides and fungicides suitable for inclusion in drops are phenylmercuric nitrate or phenylmercuric acetate (0.002%), benzalkonium chloride (0.01%), and chlorhexidine acetate (0.01%). Suitable solvents for preparing oily solutions include glycerol, diluted alcohols, and propylene glycol.

[0239] When formulated as a lotion, the composition includes those suitable for application to the skin or eyes. Eye washes may contain a sterile aqueous solution optionally containing a bactericidal agent and may be prepared by a method similar to that described above for preparing drops. Lotions or liniments for application to the skin may also include agents that accelerate drying and cooling of the skin, such as alcohols or acetone, and / or moisturizers (such as glycerin), or oils (such as castor oil or peanut oil).

[0240] When formulated as creams, ointments, or pastes, the compositions can be semi-solid formulations of active ingredients for external application. They can be prepared by mixing an active ingredient in fractionated or powdered form (alone or in a solution or suspension in an aqueous or non-aqueous fluid) with an oily or non-oily matrix. The matrix may include hydrocarbons such as hard paraffin, soft paraffin, or liquid paraffin, glycerin, beeswax, metallic soaps; gels; oils of natural origin such as almond oil, corn oil, peanut oil, castor oil, or olive oil; lanolin or derivatives thereof, or fatty acids such as stearic acid or oleic acid, and alcohols such as propylene glycol or polyethylene glycol.

[0241] The composition may contain any suitable surfactant, such as anionic, cationic, or nonionic surfactants, such as sorbitol esters or their polyoxyethylene derivatives. It may also contain suspending agents, such as natural gums, cellulose derivatives, or inorganic materials, such as silica, and other ingredients, such as lanolin.

[0242] The composition can be administered in the form of liposomes. Liposomes are typically derived from phospholipids or other lipid substances and are formed from monolayers or multilayers of hydrated liquid crystals dispersed in an aqueous medium. Any non-toxic, physiologically acceptable, and metabolizable lipid capable of forming liposomes can be used. The composition in liposome form may contain stabilizers, preservatives, excipients, etc. Preferred lipids are natural and synthetic phospholipids and phosphatidylcholine (lecithin). Methods for forming liposomes are known in the art, and for this purpose, please refer specifically to: Prescott (ed.), Methods in Cell Biology, Volume XIV, Academic Press, New York, NY (1976), page 33 and subsequent pages.

[0243] adjuvant One or more adjuvants may be included in the compositions disclosed herein, although experimental data provided herein confirm that attenuated and / or inactivated streptococcal bacteria and their derivatives can induce immunity without the need for such adjuvants. Therefore, the compositions disclosed herein may or may not contain adjuvants.

[0244] Typically, adjuvant activity in the context of a vaccine composition includes, but is not limited to, enhancing (quantitatively or qualitatively) the ability to induce an immune response by an immunogenic component in the vaccine (e.g., attenuated and / or inactivated streptococcal bacteria and / or derivatives). This can reduce the dose or level of the immunogenic component required to generate an immune response and / or reduce the number or frequency of immunizations required to generate the desired immune response.

[0245] Preferably, the adjuvant will enhance and / or strengthen the immune response induced by one or more components of the vaccine, thereby improving protective efficacy. Preferably, the adjuvant will enable the induction of protective immunity using lower doses of one or more other active components (e.g., attenuated and / or inactivated streptococcal bacteria and / or their derivatives).

[0246] Non-limiting examples of adjuvants suitable for inclusion in the compositions disclosed herein and methods for their preparation are described in " epidemic Vaccine Adjuvants: Preparation Methods and Research Protocol (Molecular Medicine Approach) Methods and Research Protocols (Methods in Molecular Medicine)) “, (2000), Ohagan (ed.), Humana Press Inc. Any suitable adjuvant may be included in the vaccine disclosed herein.

[0247] Specific examples of such adjuvants include, but are not limited to, aluminum hydroxide; peptide adjuvants, including interferon, interleukin and other cytokines; AMPHIGEN, oil-in-water and water-in-oil emulsions; and saponins, such as QuilA.

[0248] For example, aluminum-based adjuvants can be used. Suitable aluminum-based adjuvants include, but are not limited to, aluminum hydroxide, aluminum phosphate, and combinations thereof. Other specific examples of aluminum-based adjuvants that can be used are described in European Patent No. 1216053 and U.S. Patent No. 6,372,223.

[0249] Oil-in-water emulsions can be used as adjuvants in the compositions disclosed herein. Oil-in-water emulsions are well known in the art. Typically, oil-in-water emulsions will contain metabolizable oils, such as fish oil, vegetable oil, or synthetic oil. Examples of suitable oil-in-water emulsions include those described in European Patent No. 0399843, U.S. Patent No. 7,029,678, and PCT Publication No. WO 2007 / 006939. Oil-in-water emulsions can be used in combination with other adjuvants and / or immunostimulants.

[0250] Non-limiting examples of other suitable adjuvants include immunostimulants such as granulocyte-macrophage colony-stimulating factor (GM-CSF), monophospholipid A (MPL), cholera toxin (CT) or a constituent subunit thereof, heat-labile enterotoxin (LT) or a constituent subunit thereof, Toll-like receptor ligand adjuvants (such as lipopolysaccharide (LPS) and its derivatives (e.g., monophospholipid A and 3-deacylated monophospholipid A)), muramyl dipeptide (MDP), Toll-like receptor (TLR) agonists (e.g., TLR-2, TLR-3 agonists), and the F protein of respiratory syncytial virus (RSV).

[0251] The adjuvants in the compositions disclosed herein typically include emollients, emulsifiers, thickeners, preservatives, bactericides, and buffers. Another type of “self-adjuvant” is provided by conjugating an immunogenic peptide to a lipid (such as the water-soluble lipopeptide Pam3Cys or its dipalmitoyl derivative Pam2Cys). Such adjuvants have the advantage of accompanying the immunogenic component into antigen-presenting cells (such as dendritic cells) and thus simultaneously producing enhanced antigen presentation and cell activation (see, for example, Brown and Jackson, (2005), “…”). Lipid-based self-adjuvanted vaccines vaccines) ”, Current Drug Delivery, 23:83).

[0252] Suitable adjuvants are commercially available, such as Freund's incomplete adjuvant and complete adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ); AS-2 (SmithKlineBeecham, Philadelphia, Pa.); aluminum salts, such as aluminum hydroxide gel (alum) or aluminum phosphate; calcium, iron, or zinc salts; insoluble suspensions of acylated tyrosine; acylated sugars; cationic or anionic derived polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipids A and Quil A. Cytokines, such as GM-CSF or interleukin-2, -7, or -12, may also be used as adjuvants.

[0253] In some embodiments, the adjuvant included in the vaccine disclosed herein can induce a predominantly Th1-type immune response. Suitable adjuvants for inducing a predominantly Th1-type response include, for example, monophosphoryl lipid A, preferably a combination of 3-de-O-acylated monophosphoryl lipid A (3D-MPL) and an aluminum salt. For example, the composition or vaccine may be made with aluminum hydroxide (alum) and 3- O - Adjuvant AS04 formulation of deacylated monophosphorylated lipid A (MPL), such as Thoelen et al. (2001), " With novel adjuvants Systemic prophylactic hepatitis B vaccine (A prophylactic hepatitis B vaccine with a novel) adjuvant system) As described in Vaccine, 19:2400-2403. Other known adjuvants that preferentially induce Th1-type immune responses include oligonucleotides containing CpG. These oligonucleotides are characterized in that the CpG dinucleotides are unmethylated. Such oligonucleotides are known to those skilled in the art and are described, for example, in PCT Publication No. WO 1996 / 02555. Immunostimulatory DNA sequences have also been described, for example, in Sato et al. (1996). Immune stimulation required for effective intradermal gene therapy Immunostimulatory DNA sequences necessary for effective intradermal gene immunization) See Science, 273:352-354.

[0254] Another example of an adjuvant is a saponin, preferably QS21 (Aquila Biopharmaceuticals Inc., Framingham, Mass.), which can be used alone or in combination with other adjuvants. For example, enhanced adjuvant systems involving combinations of monophosphoryl lipid A and saponin derivatives can be utilized, such as the combination of QS21 and 3D-MPL as described in PCT Publication No. WO 1994 / 00153, or compositions with lower reactivity where QS21 is quenched by cholesterol, as described in PCT Publication No. WO 1996 / 33739. Other alternative formulations include oil-in-water emulsions and tocopherols. Adjuvant formulations involving QS21, 3D-MPL, and tocopherol in oil-in-water emulsions are described in PCT Publication No. WO 1995 / 17210. Adjuvants included in the compositions of this disclosure may include formulations relating to QS21, 3D-MPL, and tocopherol in oil-in-water emulsions, such as those described in PCT Publication No. WO 1995 / 17210. In one embodiment, the compositions of this disclosure comprise the adjuvant Montanide ISA720 (M-ISA-720; Seppic, Fairfield, NJ) (an adjuvant based on a natural metabolizable oil).

[0255] Preferably, the adjuvant is a mucosal adjuvant that effectively enhances mucosal immunity and / or systemic immunity to an immunogenic component administered via the mucosal route. Mucosal adjuvants can be broadly classified into those that promote vaccine delivery to enhance protective immunity induced by other immunogenic components of the vaccine (e.g., liposomes, cochlear implants, attenuated live carriers, poly(D,L-lactide-co-glycolic acid) or PLGA, chitin, DNA vaccines, mucosal adhesives), and those that have immunostimulatory effects (e.g., toxin-based, cytokine-based, etc., associated with innate immunity). Without being limited to a specific mechanism, it is assumed that the beneficial effects of mucosal adjuvants derive in part from their ability to facilitate the crossing of the immunogenic component in the vaccine across the mucosal barrier. After crossing the mucosal barrier, mucosal adjuvants can enhance immunity, for example, through complement activation, induction of cytokines, stimulation of antibody production or antibody type switching, stimulation of antigen-presenting cells, and / or influence of HLA or MHC class I and / or II expression.

[0256] Application route The compositions disclosed herein can be administered to recipients via standard routes, including but not limited to parenteral (e.g., intradermal, intravenous, intraspinal, intraperitoneal, subcutaneous, or intramuscular), oral, topical, or mucosal routes (e.g., intranasal).

[0257] For example, the composition can be administered via a mucosal route. Non-limiting examples of acceptable routes of administration for mucosal vaccines include intranasal, ocular, buccal, genital (vaginal), rectal, intratracheal, skin, and gastrointestinal routes.

[0258] In one embodiment, the compositions of this disclosure are administered via an intranasal route. Without being limited to the theory or one or more specific modes of action, intranasal administration of the compositions may be advantageous for enhancing immunity against certain streptococcal infections, where bacteria infect the host via the mucosal surfaces of the upper and / or lower respiratory tracts. Furthermore, mucosal vaccination (e.g., intranasal vaccination) can induce mucosal immunity not only in the respiratory tract but also at distant mucosal sites, including the genital mucosa.

[0259] The intranasal compositions disclosed herein can be formulated, for example, in liquid form as nasal drops, sprays, or formulated as powders, creams, or emulsions suitable for inhalation. Intranasal vaccines via nebulization or aerosolization can also be utilized. Application of the compositions to the mucous membranes of the upper and / or lower respiratory tracts via inhalation of mists, powders, or sprays, or by intranasal administration of nasal drops, swabs, powders, sprays, mists, aerosols, etc., is also contemplated.

[0260] In one embodiment, the composition for intranasal administration is provided in the form of a freeze-dried powder that can be reconstituted immediately prior to use. The powdered vaccine formulations of the disclosed compositions provide a means of overcoming the refrigerated storage and dispensing requirements associated with the stability and delivery of liquid-based vaccines. The dry powder formulation offers the advantage of greater stability and does not support microbial growth.

[0261] Freeze-dried compositions can induce cross-protective immunity levels similar to those of non-freeze-dried compositions. Compositions can be freeze-dried using any suitable technique known in the art. For example, liquid formulations of attenuated and / or inactivated streptococcal bacteria and / or their derivatives can be frozen in a dry ice-isopropanol slurry and freeze-dried for a suitable period of time (e.g., 24 hours) in a freeze dryer (e.g., Virtis Model 10-324 Bench, Gardiner, NY).

[0262] In one embodiment, the dry powder nasal vaccine of this disclosure is produced by generating spray freeze-dried (SFD) particles (see, for example, Costantino et al., (2002), " Protein spray freeze-drying. 2. Formulation variables affecting particle size and stability. The effects of protein spray freeze drying. 2. Effect of formulation variables on (particle size and stability) ", J Pharm Sci., 91:388-395; Costantino, et al., (2000), " Protein spray-freeze drying. Effects of atomization conditions on particle size and stability. drying. Effect of atomization conditions on particle size and stability)",Pharm Res.,17:1374-1383; Maa et al., (1999), " Protein inhalation powder: spray drying vs. spray freeze drying (Protein inhalation powders: spray drying vs spray freeze drying) ", PharmRes, 16:249-254; Carrasquillo et al., (2001); " Excipient-stabilized spray freeze-dried BSA non-aqueous packaging Encapsulation in poly(lactide-co-glycolic acid) microspheres leads to the release of native proteins (non-aqueous encapsulation). of excipient-stabilized spray-freeze dried BSA into poly(lactide-co- glycolide) microspheres results in release of native protein) ", J ControlRelease,76:199-208; Carrasquillo et al., (2001), " Reducing bovine serum albumin levels through non-aqueous microencapsulation Structural perturbations in bovine serum albumin (Reduction of structural perturbations in bovine serum albumin by) non-aqueous microencapsulation) ", J Pharm Pharmacol., 53:115-120; and US Patent No. 6,569,458).

[0263] Preferred devices for intranasal application of the composition are nasal spray devices (e.g., devices commercially available from Pfeiffer GmBH, Valois, and Becton Dickinson). Non-limiting examples of suitable devices are described, for example, in Bommer, (1999), " Advances in Nasal Drug Delivery Technology) In Pharmaceutical Technology Europe, pp. 26-33, the intranasal device can produce droplets ranging from 1 to 500 μm. Preferably, only a small percentage of droplets (e.g., <5%) are below 10 μm to minimize the chance of inhalation. The intranasal device can be capable of dual-dose delivery, i.e., delivering two sub-dose of a single vaccine dose, one sub-dose to each nostril.

[0264] The compositions of this disclosure can be administered to a recipient alone or in combination with one or more other additional therapeutic agents. In embodiments where the vaccine is administered together with one or more therapeutic agents, the administration can be simultaneous or sequential (i.e., administration of the vaccine followed by administration of one or more agents, or vice versa). Thus, when the vaccine of this disclosure is administered to a subject in combination with another agent, both can be administered simultaneously in a single composition, simultaneously in separate compositions, or separately at different times.

[0265] dose Typically, the compositions disclosed herein are administered in a manner compatible with the route of administration and the recipient’s physical characteristics (including health status), and in a manner that induces one or more desired effects (i.e., therapeutic efficacy, immunogenicity, and / or protection).

[0266] For example, the appropriate dose of a given vaccine may depend on a variety of factors, including but not limited to the subject’s physical characteristics (e.g., age, weight, sex), whether the compound is used as a single agent or adjunctive therapy, the progression of the given streptococcal infection (i.e., pathological state), and other factors that a person skilled in the art may recognize. Various general considerations that may be taken into account when determining the appropriate dose of the vaccine given in this disclosure are described, for example, in Gennaro et al. (ed.), (1990), “ Remington Pharmaceutical Science ", Mack Publishing Co., Easton, Pennsylvania, USA; and Gilman et al., (eds.), (1990), " Goodman and Gilman: The Pharmacological Basis of Therapeutics Pharmacological Bases of Therapeutics) ”, Pergamon Press.

[0267] Typically, the compositions disclosed herein can be administered to a patient in amounts of about 5 micrograms to about 5 mg of one or more active components (i.e., attenuated and / or inactivated streptococcal bacteria and / or derivatives). Doses of about 50 micrograms to about 500 micrograms are particularly preferred.

[0268] Those skilled in the art will be able to determine, through routine experiments, the effective, non-toxic amount of attenuated and / or inactivated streptococcal bacteria or their derivatives contained in the vaccine disclosed herein to achieve the desired therapeutic outcome.

[0269] Generally, the expected effective dose is in the following ranges: about 0.0001 mg to about 1000 mg per kg body weight per 24 hours, for one or more active ingredients (i.e., attenuated and / or inactivated streptococcal bacteria or derivatives); generally, about 0.001 mg to about 750 mg per kg body weight per 24 hours; about 0.01 mg to about 500 mg per kg body weight per 24 hours; about 0.1 mg to about 500 mg per kg body weight per 24 hours; about 0.1 mg to about 250 mg per kg body weight per 24 hours; about 1.0 mg to about 250 mg per kg body weight per 24 hours. More generally, the expected effective dose range is from about 1.0 mg to about 200 mg per kg of body weight every 24 hours; from about 1.0 mg to about 100 mg per kg of body weight every 24 hours; from about 1.0 mg to about 50 mg per kg of body weight every 24 hours; from about 1.0 mg to about 25 mg per kg of body weight every 24 hours; from about 5.0 mg to about 50 mg per kg of body weight every 24 hours; from about 5.0 mg to about 20 mg per kg of body weight every 24 hours; and from about 5.0 mg to about 15 mg per kg of body weight every 24 hours.

[0270] Optionally, the effective dose can be up to about 500 mg / m². 2One or more active ingredients (i.e., attenuated and / or inactivated streptococcal bacteria or derivatives). Typically, the expected effective dose is from about 25 to about 500 mg / m². 2 (Preferred to be about 25 to about 350 mg / m³) 2 More preferably about 25 to about 300 mg / m³ 2 More preferably about 25 to about 250 mg / m³ 2 Even more preferred is about 50 to about 250 mg / m 2 And even more preferably about 75 to about 150 mg / m³ 2 Within the range of ).

[0271] Typically, in therapeutic applications, treatment will sustain the duration of an infection, disease state, or condition. Furthermore, it will be apparent to those skilled in the art that the optimal amount and interval of a single dose will be determined by the nature and extent of the infection, disease state, or condition being treated, the form, route, and site of administration, and the nature of the specific individual being treated. Likewise, such optimal conditions can be determined using conventional techniques.

[0272] In many cases, it will be desirable to administer the vaccine of this disclosure several or more times. For example, the composition of this disclosure can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. Administration can be at intervals of about one to about twelve weeks, six months, twelve months, etc., and in some embodiments, at intervals of about one to about four weeks. In cases of repeated exposure to the specific pathogen targeted by the vaccine of this disclosure, periodic re-administration may be desirable.

[0273] It will also be apparent to those skilled in the art that conventional treatment procedure determination tests can be used to determine the optimal treatment procedure.

[0274] The methods described herein may include administering an initial dose of the vaccine of this disclosure. A booster dose may follow the initial dose. The booster may be used for the purpose of re-vaccination. In various embodiments, the vaccine is administered at least once, twice, three times, or more. The compositions of this disclosure may be administered to initial recipients, i.e., individuals who are seronegative to one or more specific target streptococcal bacterial strains. Optionally, the compositions may be administered to sensitized recipients, i.e., individuals who are seropositive to one or more specific target streptococcal bacterial strains.

[0275] Those skilled in the art will understand that many changes and / or modifications may be made to this disclosure as disclosed in the specific embodiments without departing from the spirit or scope of the disclosure as broadly described. Therefore, this disclosure should be considered illustrative rather than restrictive in all respects.

[0276] Example Nucleic acid and amino acid sequence The nucleic acid sequences related to this embodiment are listed in Table 1.

[0277] Table 1: Polynucleotide and amino acid sequences

[0278]

[0279]

[0280]

[0281]

[0282]

[0283] Example 1: Construction of Streptococcal vaccine strains GPN-001∆lytC::kanR and GPN-002∆lytC::kanR Will lytC Gene deletion engineering was applied to GPN-001 and GPN-002, both of which are non-capsulated vaccine strains derived from Streptococcus pneumoniae Rx1, such as... Figure 1 As shown. GPN-001 possesses an autolysin gene. lytA The absence of the pneumococcal hemolysin gene ( ply The mutation has been replaced with a Ply toxin-like variant encoded as PdT. ply Genes. GPN-002 is a derivative of the GPN-001 strain. GPN-002 also possesses a gene encoding the solute-binding component of the manganese uptake system. psaA The absence of ).

[0284] lytC is missing from GPN-001 and GPN-002 to form GPN-001∆lytC::kanR and GPN-002∆ lytC::kanR strain Use such as Figure 1 The conventional methods presented in GPN-001 and GPN-0002 have been removed. lytC Genes. Before the deletion. lytC The DNA sequence of the gene (including a flanking region of approximately 200 bp) is shown in SEQ ID NO: 1.

[0285] Simply put, it is achieved by replacing the missing kanamycin (kanamycin) resistance kit. lytC Genes. Oligonucleotide primers were designed to amplify adjacent genes via polymerase chain reaction (PCR) using primers lytC_F1 (SEQ ID NO: 2) and lytC_R1 (SEQ ID NO: 3). lytCUpstream of the gene and amplification were performed using primers lytC_F2 (SEQ ID NO: 4) and lytC_R2 (SEQ ID NO: 5). lytC The primers are located approximately 2 kilobases (kb) downstream of the gene. These primers also incorporate a region complementary to the kanamycin resistance cassette.

[0286] The kanamycin resistance cassette was amplified using primers kanamycin R_F (SEQ ID NO: 6) and kanamycin R_R (SEQ ID NO: 7). All PCRs were performed using standard parameters with HF buffer (New England BioLabs) and oligonucleotide primers purchased from Sigma / Merck. Linear fragments of these three products were generated by overlap extension PCR using primers lytC_F1 (SEQ ID NO: 2) and lytC_R2 (SEQ ID NO: 5) with standard parameters. The linear lytC::kanR fragments were transformed into GPN-001 and GPN-002 strains using standard methods. Successful replacement of the kanamycin resistance cassette with the cassette was confirmed by evaluating growth ability on blood agar plates supplemented with manganese and kanamycin, and by size analysis by agarose gel electrophoresis after PCR amplification of the region. lytC Gene.

[0287] Transformants were confirmed by PCR amplification using a combination of oligonucleotide primers designed to test the presence or absence and orientation of the kanamycin resistance cassette. For product A, primers lytC_F1 5 (SEQ ID NO: 2) and lytC_R2 (SEQ ID NO: 5) were expected to produce approximately 5.6 kb of product in parental strains GPN-001 and GPN-002, and approximately 4.6 kb of product in GPN-001∆lytC and GPN-002∆lytC. Products B and C were amplified to confirm the presence and orientation of the kanamycin resistance cassette. Product B was amplified using primers lytC_F1 (SEQ ID NO: 2) and kanamycin R_R (SEQ ID NO: 7). Product C was amplified using primers kanamycin R_F (SEQ ID NO: 6) and lytC_R2 (SEQ ID NO: 5). PCR using primer sets B and C was expected to produce no product in parental strains GPN-001 and GPN-002, but to produce approximately 2.5 kb of product in strains GPN-001∆lytC::kanR and GPN-002∆lytC::kanR.

[0288] Results and discussion By comparing the PCR products of three clones of the parental strain GPN-002 and the GPN-002∆lytC::kanR strain, the kanamycin resistance cassette in GPN002 was confirmed. lytC Successful incorporation of the region. The sizes of products A, B, and C, as described above, were determined by gel electrophoresis. A decrease in the expected size of product A (approximately 4.6 kb) was observed in the GPN-002∆lytC::kanR strain, and a size of approximately 2.5 kb (expected) for products B and C was observed in all clones. Figure 2 ).

[0289] The primers and methods used in this embodiment can be used to generate the ∆lytC::kanR mutation in other Streptococcus pneumoniae strains (including both encapsulated and non-encapsulated strains).

[0290] After confirming that the kanamycin resistance cassette was successfully integrated into the strain, further research was conducted using GPN-002∆lytC::kanR clone 1.

[0291] Example 2: lytC The absence of [something] reduces bacterial lysis during vaccine production. Experiments were conducted to study the vaccine strains. lytC Does the absence of [a specific ingredient] improve vaccine stability during manufacturing by preventing bacterial lysis in the buffer solution?

[0292] Assessing the presence of carriers by measuring cell density over time at 25°C lytC Stability of the missing strains. GPN-002 and GPN-002∆lytC::kanR strains were grown in standard soybean-based animal-free growth medium supplemented with 5 µM MnSO4 until the equivalent optical density at 600 nm (approximately OD) was reached. 600 1.2). Wash and resuspend the bacteria in phosphate-buffered saline (PBS) containing 20% ​​(w / v) glycerol. Transfer 100 µL of each sample or buffer-only to the wells of a 96-well plate. Measure OD at 15-minute intervals at 25°C using a spectrophotometer (BMG Labtech). 600 .

[0293] Results and discussion The data is presented as the average optical density at 600 nm at each time point over time, plus or minus the standard error of the average. Figure 3 ), and presented as representative time points at times = 0, 2, 4, 8, and 16 hours ( Figure 4Compared to its parent strain GPN-002, GPN-002∆lytC::kanR maintained a higher optical density during incubation in PBS glycerol buffer at 25°C.

[0294] The results show that in GPN-002 lytC The absence of this component leads to increased stability and reduced lysis of Streptococcus pneumoniae.

[0295] The same method can be used to test the stability of other streptococcal bacteria. Based on the results of this embodiment, it is expected that... lytC The missing streptococcal bacterial derivatives exhibit improved stability compared to the parent strain.

[0296] Example 3: Carrying lytC Construction and testing of unlabeled deletion of GPN-003 gene Remove the antibiotic resistance cassette from GPN-002∆lytC::kanR to generate GPN-003 (a vaccine strain more suitable for human use).

[0297] The kanamycin resistance kit is missing from GPN-002∆lytC::kanR. Remove the kanamycin resistance cassette from GPN-002 introduced into the GPN-002∆lytC::kanR strain to generate lytC Unlabeled deletion of the coding region. Immediately adjacent regions were amplified using PCR primers lytC_F1 (SEQ ID NO: 2) and lytC_MD_R (SEQ ID NO: 8). lytC The upstream of the gene and the gene were amplified using primers lytC_MD_F (SEQ ID NO: 9) and lytC_R2 (SEQ ID NO: 5). lytC A region approximately 2 kb downstream of the gene is incorporated into and immediately adjacent to it. lytC Approximately 20-30 base pairs (bp) of complementary homologous regions upstream and downstream of the target region. Linear fragments of both products were generated by overlap extension PCR using primers lytC_F1 (SEQ ID NO: 2) and lytC_R2 (SEQ ID NO: 5).

[0298] Except for an enrichment step following the addition of linear DNA to facilitate the selection of kanamycin-sensitive clones, the linear unlabeled deletion fragment (lytC-) was essentially transformed into the GPN-002∆lytC::kanR resistant strain, as described above. In short, *Streptococcus pneumoniae* GPN-002∆lytC::kanR competent cells were incubated with 100 ng / mL competent stimulating peptide 1 (CSP-1) for 15 min. The linear lytC- fragment was added to the transformation reaction, and enrichment of the deletion strain was achieved by culturing in the presence of kanamycin followed by treatment with standard concentrations of ampicillin. Successful incorporation was confirmed by patch testing on blood agar plates containing manganese, kanamycin, and unreplenished blood to assess the antibiotic resistance profile, sequencing, and size analysis by agarose gel electrophoresis. The resulting strain was *Streptococcus pneumoniae* GPN-002∆lytC (which carried the unlabeled...). lytC The missing part was named GPN-003.

[0299] Results and discussion The following oligonucleotide primers are used based on size ( Figure 5 Confirmation of genetic modifications in GPN-003 relative to Rx1: psaA : psaA_seq_F (SEQ ID NO: 10) and psaA_seq_R (SEQ ID NO: 11); lytA : lytA_seq_F (SEQ ID NO: 12) and lytA_seq_R (SEQ ID NO: 13); lytC : lytC_seq_F (SEQ ID NO: 14) and lytC_seq_R (SEQ ID NO: 15); and pdt : PdT_seq_F (SEQ ID NO: 16) and PdT_seq_R (SEQ ID NO: 17).

[0300] GPN-003 lytC The deleted sequence was further confirmed using Sanger sequencing (Australian Genome Reference Facility, Australia). The deletion initially carried... lytC The DNA sequence following the region (including approximately 200 bp flanking region) is shown in SEQ ID NO: 18. GPN-003 pdtThe 3' end sequence of the region was confirmed by Sanger sequencing (AGRF) and is shown in SEQ ID NO: 19, and protein translation is shown in... Figure 6 (See the lower image) and SEQ ID NO: 25. Rx1 ply The sequence at the 3' end of the gene is shown in SEQ ID NO: 26, and the protein translation is shown in... Figure 6 (See the top image) and SEQ ID NO: 24 for reference. The confirmed size and modification properties of the product relative to Rx1 in GPN-003 are summarized in Table 2.

[0301] Table 2. Rx1 and GPN-003 strains lytA , ply、psaA and lytC Summary of the sizes of amplified PCR products

[0302] Example 4: Stability of GPN-002 and GPN-003 strains The stability of GPN-002 and GPN-003 strains was compared by measuring cell density over time at 25°C. Both strains were grown in standard soybean-based animal-free growth medium supplemented with 5 µM MnSO4 until the equivalent optical density at 600 nm was reached. The bacteria were washed and resuspended in phosphate-buffered saline (PBS) containing 20% ​​(w / v) glycerol. 100 µL of each sample or buffer-only solution was transferred to the wells of a 96-well plate. OD was measured at 25°C at 15-minute intervals using a spectrophotometer (BMGLabtech). 600 .

[0303] Results and discussion The percentage of cell density was calculated relative to the initial optical density at 600 nm. Data are expressed as the average percentage relative to the input at each time point, plus / minus the standard error over time. Figure 7 ).

[0304] Compared with the parent strain GPN-002, it has lytC The unlabeled deletion GPN-003 showed enhanced stability and reduced lysis compared to GPN-002. In other words, removing [the deletion] from the vaccine strain... lytC The gene leads to a reduction in bacterial lysis after prolonged storage at room temperature. This confirms the presence of [a specific gene] in Streptococcus pneumoniae. lytC The absence of [something] provides enhanced stability.

[0305] The same method can be used to test the stability of other streptococcal bacteria. Based on the results of this embodiment, it is expected that... lytC The missing streptococcal bacteria exhibited improved stability compared to the parent strain.

[0306] Example 5: Analysis of total IgG and IgG subclass responses from mice vaccinated with gamma-irradiated GPN-003 (γ-PN3). Preparation of γ-PN2 and γ-PN3 vaccines To assess whether a vaccine based on the gamma-irradiated GPN-003 strain induced an antibody response similar to that of strains containing the functional LytC protein, vaccination experiments were conducted in animal models. GPN-002 and GPN-003 strains were cultured as described above. The bacteria were washed and resuspended in phosphate-buffered saline (PBS) containing 20% ​​(w / v) glycerol and inactivated by exposure to 26 kGy of gamma radiation. The resulting vaccines were named γ-PN2 and γ-PN3, respectively. The ability of these vaccines to induce pneumococcal-specific antibody responses and their effects on IgG subclasses were investigated.

[0307] Mice were vaccinated with γ-PN2 and γ-PN3 vaccines. Outbred Swiss mice were vaccinated intramuscularly (IM) three times with 50 μg of total protein in γ-PN2 and γ-PN3, two weeks apart. Control mice were vaccinated simulantly with PBS containing 20% ​​(w / v) glycerol. Serum was collected from all mice two weeks after the final vaccination. γ-PN2 and γ-PN3 were administered in the absence of adjuvants.

[0308] Detection of IgG response after vaccination Using standard techniques, the pneumococcal-specific IgG response in serum samples was tested by direct enzyme-linked immunosorbent assay (ELISA). Rx1, an uncapsulated derivative of D39 (serotype 2), was used as the coating antigen.

[0309] Results and discussion Figure 8 (A) shows the pneumococcal-specific IgG response as absorbance (450 / 620 nm), and Figure 8 (B) shows the response as a titer. Data are presented as mean absorbance (450 / 620 nm) ± SEM from individual or pooled serum samples (n = 5 mice per group). The IgG1 subclass response, as determined by ELISA using Rx1 as the coating antigen, is shown in... Figure 8 In (C), the IgG2a response is... Figure 8 In (D), the IgG2b response is... Figure 8 In (E), the IgG3 response is shown to be... Figure 8(F) The proportions of these subclasses in each sample are presented in Table 3.

[0310] Table 3. Ratios of IgG subclasses induced in mice after vaccination with γ-PN2 and γ-PN3.

[0311] Compared with mice simulating vaccination, γ-PN2 and γ-PN3 induced a significant pneumococcal-specific IgG antibody response in mice after immunization. Notably, mice vaccinated with γ-PN3 had significantly higher titers of pneumococcal-specific IgG1, IgG2a, IgG2b, and IgG3 than mice vaccinated with γ-PN2.

[0312] The result surprisingly showed that the missing lytC The gene enhanced the antibody response. This was unexpected, as LytC is considered an immunogen due to its surface location on streptococcal bacteria and its function as a virulence factor. For example, vaccination with purified LytC has demonstrated the protein's potential as a protective immunogen, with immunized mice exhibiting high levels of immunoglobulin G (IgG) antibodies and enhanced protection against pneumococcal infection in mice (Wizemann et al., 2001; Corsini et al., 2021); and high titers of LytC-specific IgG antibodies have been observed in serum samples from healthy volunteers in multiple studies, indicating that natural exposure to Streptococcus pneumoniae induces an anti-LytC antibody response.

[0313] The same method can be used to test antibody responses to other streptococcal bacteria. Based on the results of this embodiment, it is expected that... lytC Streptococcal bacteria lacking or with reduced LytC function will have improved antibody responses compared to their parental strains.

[0314] Example 6: Enhanced ability of γ-PN3 vaccination to induce antibodies binding to Streptococcus pneumoniae serotypes To determine whether γ-PN3 antibodies could bind to fully encapsulated Streptococcus pneumoniae isolates, flow cytometry was used to assess the binding of IgG to whole Streptococcus pneumoniae.

[0315] To generate antibodies for analysis, mice were immunized with IM vaccine containing γ-PN2 and γ-PN3 (50 μg total protein per mouse in 50 μL of PBS containing 20% ​​(w / v) glycerol), as described herein. Control mice were immunized only with PBS containing 20% ​​(w / v) glycerol. Mice were immunized three times, two weeks apart. Serum was collected from all mice two weeks after the final immunization.

[0316] The binding of γ-PN3 and γ-PN2-induced IgG to capsular streptococcal bacteria was detected by flow cytometry. Serum from each group (n = 5) was pooled, and the binding of IgG to Streptococcus pneumoniae serotype 2 strain D39 and Streptococcus pneumoniae serotype 6A strain P9 was tested by flow cytometry using a standard protocol. Simply put, Streptococcus pneumoniae D39 or 6A strains were cultured using a standard protocol. 1 × 10⁻⁶ 7 Live bacteria from strain D39 or P9, comprising colony-forming units (CFU), were incubated with conjugated mouse serum diluted 1:50 in PBS-BSA. The bacterial cells were centrifuged and washed twice, and the primary IgG bound to the surface of the pneumococcal bacteria was detected by anti-mouse IgG secondary antibody (FITC conjugation).

[0317] Results and discussion Figure 9 The charts show the percentage of IgG-bound cells (D39 in subplot A and P9 in subplot C) and geometric mean fluorescence intensity (GMFI; D39 in subplot B and P9 in subplot D) detected by flow cytometry after each serotype was incubated with combined mouse serum from the vaccine and control groups. Greater fluorescence intensity indicates more IgG binding to the surface of pneumococcal cells. Data were analyzed by one-way ANOVA (** p < 0.01, **** p < 0.0001).

[0318] The results showed that γ-PN3-induced antibodies could bind to multiple serotypes of pneumococcus, including capsular strains of different serotypes. Furthermore, for both serotypes, the binding levels of γ-PN3-induced antibodies were significantly higher than those induced by γ-PN2. This indicates that compared to the parental strain GPN-003, lytC The gene deletion enhanced the antibody response to antigens present on capsular bacteria, including different serotypes. This result indicates that vaccination with γ-PN3 induces cross-serotype protection against capsular streptococci.

[0319] The same method can be used to test antibody responses to other streptococcal bacteria. Based on the results of this embodiment, it is expected that... lytC Streptococcal bacteria lacking or with reduced LytC function will have improved antibody responses compared to their parental strains, including increased cross-protective antibody responses against encapsulated bacteria.

[0320] Example 7: Induction of Streptococcus pneumoniae γ-PN3-specific serum antibody response in rabbits vaccinated intramuscularly with γ-PN3 An immunologic experiment was conducted to determine whether γ-PN3 could induce a serum antibody response in rabbits.

[0321] Rabbit vaccination γ-PN2 and γ-PN3 were prepared as described herein. Outcrossed rabbits were vaccinated intramuscularly (IM) with either γ-PN2 or γ-PN3 (500 µg total protein per rabbit in 0.5 mL PBS). Rabbits received three immunizations at 3-week intervals. γ-PN2 and γ-PN3 were administered in the absence of adjuvants.

[0322] Detection of IgG response after vaccination Serum was collected from all rabbits before immunization (denoted as 'pre-bleed') and 3 weeks after final immunization. Total IgG in individual serum samples was tested by direct ELISA using a standard protocol with γ-PN3 as the coating antigen. Data were analyzed by one-way ANOVA (nd = not detected). Figure 10 ).

[0323] Results and discussion Figure 10 The figures show the IgG titers for each individual rabbit before and after the third immunization (Figure A), and the mean IgG titer (± SEM) within each vaccine group (Figure B). Following IM immunization, vaccination with both γ-PN2 and γ-PN3 resulted in high-titer antibody responses against non-capsulated Streptococcus pneumoniae strains in rabbits.

[0324] The ability of other streptococcal bacteria to induce specific serum antibodies can be tested using the same method detailed above. Based on the results of this embodiment, it is anticipated that [the following method] will be used... lytC Rabbits immunized with other streptococcal bacteria that lack or have reduced LytC function will also show increased antibody responses against vaccine strains.

[0325] Example 8: Binding of γ-PN2 and γ-PN3 induced antibodies to Streptococcus pneumoniae To further investigate the binding of γ-PN2 and γ-PN3 induced antibodies to completely encapsulated Streptococcus pneumoniae, flow cytometry analysis was performed.

[0326] Rabbits were vaccinated with GPN-002 and GPN-003. To generate antibodies for this analysis, outcrossed rabbits were vaccinated intramuscularly (IM) with either γ-PN2 or γ-PN3 (500 µg total protein per rabbit in 0.5 mL PBS). Rabbits received three immunizations at 3-week intervals. Serum was obtained from all rabbits before immunization (referred to as 'pre-blood collection') and 3 weeks after the final immunization.

[0327] The binding of γ-PN3 and γ-PN2-induced IgG to capsular streptococcal bacteria was detected by flow cytometry. Flow cytometry was used to test the binding of IgG to Streptococcus pneumoniae serotype 7C in individual serum samples. Simply put, 1 × 10⁻⁶ IgG was used to test the binding of IgG to Streptococcus pneumoniae serotype 7C in individual serum samples. 6 One CFU of Streptococcus pneumoniae serotype 7C was incubated with immune rabbit serum diluted 1:200 in PBS-BSA. The bacterial cells were precipitated and washed twice, and any primary IgG bound to the surface of the Streptococcus pneumoniae was detected using anti-rabbit IgG secondary antibody (FITC conjugation).

[0328] Results and discussion Flow cytometry was used to detect the binding of IgG induced by vaccination with GPN-002 or GPN-003 to Streptococcus pneumoniae serotype 7C, such as... Figure 11 As shown: (A) shows relative fluorescence, (B) the percentage of serotype 7C cells bound by rabbit IgG antibodies, and (C) the mean fluorescence intensity of IgG antibodies bound to serotype 7C cells, indicating the order of IgG binding (subplot C). Data were analyzed by one-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns not significant).

[0329] These results show that antibodies induced by γ-PN2 and γ-PN3 can bind with high affinity to serotypes of Streptococcus pneumoniae that are different from the parental vaccine strains. Furthermore, the binding levels of IgG to different serotypes of Streptococcus pneumoniae in animals vaccinated with γ-PN3 were higher than those in animals vaccinated with γ-PN2. This indicates that the γ-PN3 vaccine induces more antibodies capable of binding to different serotypes of capsular bacteria than γ-PN2. Therefore, lytC The deletion of the gene increased the antibody response. This suggests that GPN-003 induces cross-protective immunity, including against encapsulated strains.

[0330] The ability of other streptococcal bacteria to induce binding to encapsulated bacteria can be tested using the same methods detailed above. Based on the results of this embodiment, it is anticipated that antibodies with [specific characteristics] can be used... lytC Rabbits immunized with other streptococcal bacteria that lack or have reduced LytC function will also show increased cross-protective antibody responses against encapsulated bacteria.

[0331] Example 9: Analysis of the function of γ-PN2 and γ-PN3 induced antibodies in mediating opsonization and killing of Streptococcus pneumoniae. Host protection against pneumococcal infection is thought to be associated with opsonin-dependent phagocytosis. Therefore, the in vitro opsonization and phagocytic activity (OPA) of pneumococcal-specific antibodies with functional antibody activity was investigated after rabbits were vaccinated with γ-PN2 or γ-PN3.

[0332] Rabbits were vaccinated intramuscularly (IM) with γ-PN2 or γ-PN3 (500 µg total protein in 0.5 mL PBS per rabbit). Rabbits received three immunizations at 3-week intervals. Serum was obtained from all rabbits before immunization (referred to as 'pre-collection') and 3 weeks after the final immunization. The opsonization phagocytosis killing assay protocol established by Nahm and Burton, 2014 was used to test the opsonization phagocytosis killing activity of individual or pooled serum samples against a group of Streptococcus pneumoniae serotypes (serotypes 6A, 6B, 9N, 23A, 24F, and 33F). Titer was determined as the reciprocal of the highest dilution of rabbit serum that produced 50% or more of the input CFU killing. If no killing was detected with pure serum (a 1:4 dilution when mixed with other assay components), this was expressed as a titer ≤ 4.

[0333] Results and discussion Table 4 presents the conditioning phagocytic index (OI) titers.

[0334] Table 4. Optonogenic phagocytic index (OI) titers of rabbit serum after vaccination with γ-PN2 or γ-PN3 compared to pre-collection serum against various Streptococcus pneumoniae serotypes.

[0335] The results showed that γ-PN3 induced higher opsonization and phagocytic activity than γ-PN2 against all tested Streptococcus pneumoniae serotypes. This indicates that... lytC The deletion of the gene enhanced the antibody response to opsonize and kill capsular pneumococci. This result also indicates that γ-PN3 induces cross-protective responses against different Streptococcus pneumoniae serotypes. This ability of γ-PN3 to induce cross-protective responses against other Streptococcus pneumoniae serotypes or other streptococcal species can be tested using the same methods described herein.

[0336] Furthermore, the ability of other streptococcal bacteria to induce and modulate antibodies against a range of streptococcal bacteria can be tested using the methods detailed above. Based on the results of this embodiment, it is anticipated that antibodies with... lytC Rabbits immunized against other streptococcal bacteria with missing or reduced LytC function will also show increased opsonization responses against a range of streptococcal bacteria.

[0337] Example 10: lytC Mutations on conserved antigen lipoproteins prsA The effect of transcription To further explore the contents lytC The improved immunogenicity exhibited by the missing vaccine strain was assessed by evaluating the expression of antigen genes in the GPN-003 strain. Quantitative reverse transcription PCR (qRT-PCR) was performed to evaluate... lytCDo mutations in lipoproteins induce conserved lipoproteins? prsA Changes in expression. prsA The gene encodes a conserved lipoprotein that is associated with serum type-independent protection.

[0338] Quantitative reverse transcription PCR Strains GPN-002 and GPN-003 were grown to the same optimal density (OD) in soybean peptone medium (supplemented with 2.14 μM MnSO4). 600 0.6). Immediately transfer the culture sample to the RNAProtect bacterial reagent (Qiagen) and incubate at room temperature for 5 minutes. Centrifuge the cell material, remove the supernatant, and store the bacterial pellet at -80°C. Lyse the sample with lysozyme and mutalysin, and extract RNA using the RNeasy Mini Kit (Qiagen) with on-column DNase I treatment (Qiagen). Quantify total RNA using a Nanodrop spectrophotometer and dilute to 2 ng / μL. Perform qRT-PCR using the SYBR q-PCR master mix and a QuantStudio 7 Flex real-time PCR system (Thermofisher Scientific). Designed for amplification using UGENE extension primers 3 prsA_F (SEQ ID NO: 20) and prsA_R (SEQ ID NO: 21). prsA Primers were used to normalize the gene transcription level to constitutively expressed gyrase A gene using primers gyrA_F (SEQ ID NO: 22) and gyrA_R (SEQ ID NO: 23). gyrA The data is presented in a relative manner. gyrA average expression prsA Transcription.

[0339] Results and discussion like Figure 12 As shown, prsA A comparison of gene expression showed higher expression in GPN-003 compared to GPN-002. These data indicate... lytC Mutations in the cells lead to enhanced in vitro gene expression of key immunogens.

[0340] In addition, the methods detailed above can be used to test for other streptococcal bacteria. prsA The level of expression. Based on the results of this embodiment, it is expected to have... lytC Other streptococcal bacteria with missing or reduced LytC function will also have increased [function / effect]. prsA Express.

[0341] Example 11: GPN-002 and GPN-003 are non-toxic in mice. To explore the potential of GPN-003 as an attenuated vaccine, the virulence of GPN-002 and GPN-003 was determined in mice.

[0342] Female Swiss mice (n=5 per group) were intraperitoneally inoculated with a lethal dose of Streptococcus pneumoniae serotype 2 (strain D39, 10). 4 (1 CFU / mouse) or 10,000 times higher doses of GPN-002 or GPN-003 (10 8 (1 CFU / mouse). The overall survival rate of mice was monitored and they were weighed daily for up to 7 days.

[0343] Results and discussion like Figure 13 As shown in (A), all mice vaccinated with the lethal dose of D39 reached a morbid state within 24 hours. In contrast, mice receiving higher doses of GPN-002 or GPN-003 did not exhibit clinical signs of infection and did not experience weight loss. Figure 13 (B)).

[0344] These data provide evidence that GPN-002 and GPN-003 are non-toxic and can be used as attenuated vaccines.

[0345] Predictive Example 1: Construction of an unlabeled GPN-001 strain carrying lytC deletion The antibiotic resistance box can be removed from GPN001∆lytC::kanR to generate GPN001∆ lytC Removing antibiotic resistance genes produces vaccine strains that are more suitable for human use.

[0346] like Figure 14 As shown, the kanamycin resistance cassette introduced into GPN-001 in the strain GPN-001∆lytC::kanR described earlier in this paper will be removed to generate lytC Unlabeled deletion of the coding region. Oligonucleotide primers were designed for PCR amplification of adjacent regions. lytC Upstream of the gene (using primers lytC_F1 (SEQ ID NO: 2) and lytC_MD_R (SEQ ID NO: 8)) and lytC A region approximately 2 kb downstream of the gene (using primers lytC_MD_F (SEQ ID NO: 9) and lytC_R2 (SEQ ID NO: 5)) was incorporated into the immediately following region. lytCThe complementary homology between the upstream and downstream regions is approximately 20-30 base pairs (bp). Linear fragments of both products will be generated by overlap extension PCR using primers lytC_F1 (SEQ ID NO: 2) and lytC_R2 (SEQ ID NO: 5).

[0347] Optionally, the linear fragment can be amplified directly from the GPN-003 strain by PCR. Except for an enrichment step following the addition of linear DNA to facilitate the selection of kanamycin-sensitive clones, the linear unlabeled deletion fragment (lytC-) is essentially transformed into the GPN-001∆lytC::kanR resistant strain, as described above. In short, *Streptococcus pneumoniae* GPN-001∆lytC competent cells are incubated with 100 ng / mL competent stimulating peptide 1 (CSP-1) for 15 min. The linear lytC- fragment is added to the transformation reaction, and enrichment of the deletion strain is achieved by culturing in the presence of kanamycin followed by ampicillin treatment. Successful incorporation is confirmed by patch testing on blood agar plates containing kanamycin and unreplenished blood to assess the antibiotic resistance profile, sequencing, and size analysis by agarose gel electrophoresis. The resulting *Streptococcus pneumoniae* GPN-001∆lytC- strain will carry an unlabeled fragment suitable for clinical use. lytC Missing.

[0348] The following oligonucleotide primers will be used to confirm, based on size, all genetic modifications introduced relative to Rx1 in GPN-001∆lytC: lytA : lytA_seq_F (SEQ ID NO: 12) and lytA_seq_R (SEQ ID NO: 13); lytC : lytC_seq_F (SEQ ID NO: 14) and lytC_seq_R (SEQ ID NO: 15); and pdt : PdT_seq_F (SEQ ID NO: 16) and PdT_seq_R (SEQ ID NO: 17).

[0349] GPN-001∆lytC lytC The missing sequence will be further confirmed by Sanger sequencing. GPN-001∆lytC pdt The sequence at the 3' end of the region will also be confirmed by Sanger sequencing.

[0350] Predictive Example 2: Analysis of total IgG and IgG subclass responses from mice vaccinated with γ-PN1 and γ-PN1∆lytC To assess whether vaccines developed based on the GPN-001∆lytC strain induce altered antibody responses compared to GPN-001, vaccination experiments in animal models will be conducted. As described herein, both GPN-001 and GPN-001∆lytC strains will be inactivated by exposure to gamma radiation, and the resulting vaccines will be named γ-PN1 and γ-PN1∆lytC, respectively. The ability of these vaccines to induce pneumococcal-specific antibody responses and their effects on IgG subclasses will be investigated.

[0351] Outcrossed Swiss mice were intramuscularly (IM) vaccinated three times with 50 μg of total protein in γ-PN1 and γ-PN1∆lytC, two weeks apart. Control mice were given a dummy vaccination with PBS containing 20% ​​(w / v) glycerol. Serum was collected from all mice two weeks after the final vaccination. The pneumococcal-specific total IgG response in serum samples was tested by direct enzyme-linked immunosorbent assay (ELISA). Rx1, an uncapsulated derivative of D39 (serotype 2), was used as the coating antigen. The IgG subclass (IgG1, IgG2a, IgG2b, and IgG3) response was tested by ELISA using Rx1, an uncapsulated derivative of D39 (serotype 2), as the coating antigen.

[0352] Predictive Example 3: Antibodies induced by γ-PN1 and γ-PN1∆lytC have the ability to bind to Streptococcus pneumoniae serotypes.

[0353] To generate antibodies for analysis, mice will be vaccinated with γ-PN1 and GPN-001∆lytC (50 μg total protein per mouse in 50 μL PBS / glycerol). Control mice will be vaccinated with PBS / glycerol alone. Mice will be immunized three times, two weeks apart. Serum will be collected from all mice two weeks after the final immunization. Serum from each group (n=5) will be pooled and flow cytometry will be used to test for binding of IgG to encapsulated serotype 2 (strain D39) and 6A (strain P9). Simply put, 1 × 10⁻⁶ antibodies will be pooled. 7 One colony-forming unit (CFU) of live bacteria from each serotype was incubated with co-conjugated mouse serum diluted 1:50 in PBS-BSA. Bacterial cells were centrifuged and washed twice, and the binding of primary IgG to the surface of *Streptococcus pneumoniae* was detected by anti-mouse IgG secondary antibody (FITC conjugation). These data will show the percentage of IgG-bound cells and geometric mean fluorescence intensity detected by flow cytometry after incubation with co-conjugated mouse serum from the vaccine and control groups for each serotype. Greater fluorescence intensity indicates more IgG binding to the surface of *Streptococcus pneumoniae* cells.

[0354] Predictive Example 4: Induction of pneumococcal-specific serum antibody response in rabbits after immunization with a vaccine carrying the lytC mutation Immunization experiments will be conducted to determine the efficacy of γ-PN1, γ-PN2, γ-PN3, and γ-PN1Δ compared to vaccines based on parental strains. lytC::KanR or γ-PN1Δ lytC Whether it induces a higher serum antibody response in rabbits.

[0355] γ-PN1, γ-PN1Δ will be used lytC ::kanR、γ-PN1Δ lytC Outcrossed rabbits will be vaccinated with IM vaccine containing γ-PN2 or γ-PN3 (500 μg total protein in 0.5 mL PBS per rabbit). Rabbits will receive three immunizations at 3-week intervals, with serum samples collected before the first immunization (referred to as 'pre-collection') and 3 weeks after the final immunization. Total IgG from individual serum samples will be tested by direct ELISA using whole-cell Rx1 as the coating antigen.

[0356] Predictive Example 5: Binding of antibodies induced by γ-PN1, γ-PN1∆lytC::kanR, γ-PN1∆lytC, γ-PN2, or γ-PN3 to Streptococcus pneumoniae. To evaluate γ-PN1, γ-PN1Δ lytC ::kanR、γ-PN1Δ lytC The binding of antibodies induced by γ-PN2 or γ-PN3 (targeting pneumococcal proteins) to Streptococcus pneumoniae will be analyzed by flow cytometry.

[0357] To generate antibodies for this analysis, γ-PN1 and γ-PN1Δ will be used. lytC ::kanR、γ-PN1Δ lytC Outcrossed rabbits will be vaccinated intramuscularly (IM) with γ-PN2 or γ-PN3 (500 µg total protein in 0.5 mL PBS per rabbit). Rabbits will receive three immunizations at 3-week intervals. Serum will be collected from all rabbits before immunization (referred to as 'pre-blood collection') and 3 weeks after the final immunization. Flow cytometry will be used to test the binding of IgG in individual serum samples to different serotypes of Streptococcus pneumoniae from the parental strain. Simply put, 1 x 10 6One CFU of capsulated viable bacteria was incubated with rabbit serum diluted 1:200 in PBS-BSA. The bacterial cells were centrifuged and washed twice, and any primary IgG bound to the surface of the pneumococcus was detected using anti-rabbit IgG secondary antibody (FITC conjugated). The percentage of pneumococcal cells bound by the rabbit IgG antibody and the average fluorescence intensity of the IgG antibody bound to the pneumococcal cells will be shown, indicating the magnitude of IgG binding.

[0358] The result will define γ-PN1, γ-PN1Δ lytC γ-PN1Δ lytC- Serum antibodies induced by γ-PN2 or γ-PN3 vaccination bind to Streptococcus pneumoniae.

[0359] Predictive Example 6: Analysis of the opsonization and phagocytic killing effect of antibodies induced by γ-PN1, γ-PN1ΔlytC::kanR, γ-PN1ΔlytC, γ-PN2, or γ-PN3 on Streptococcus pneumoniae. Host protection against pneumococcal infection is primarily mediated by opsonin-dependent phagocytosis. Therefore, the opsonization-phagocytic activity (OPA) of pneumococcal-specific antibodies is considered a useful in vitro measure of functional antibody activity. This will be further investigated using γ-PN1 and γ-PN1Δ... lytC ::kanR、γ-PN1Δ lytC OPA titers were determined after rabbits were vaccinated with γ-PN2 or γ-PN3.

[0360] γ-PN1, γ-PN1Δ will be used lytC ::kanR、γ-PN1Δ lytC Rabbits will be vaccinated intramuscularly (IM) with γ-PN2 or γ-PN3 (500 µg total protein in 0.5 mL PBS per rabbit). Rabbits will receive three immunizations at 3-week intervals. Serum will be collected from all rabbits before immunization (referred to as 'pre-blood collection') and 3 weeks after the final immunization. The opsonization phagocytosis assay protocol established by Nahm and Burton (2014) will be used to test the opsonization phagocytosis activity of individual or pooled serum samples against a group of Streptococcus pneumoniae serotypes.

[0361] The opsonization index (OI) titer will be determined as the reciprocal of the highest three-fold sequential dilution of rabbit serum that produced 50% or more of the input CFU kill. If no kill was detected with pure serum (at a 1:4 dilution when mixed with other assay components), the OI will be expressed as a titer ≤ 4.

[0362] The result will define γ-PN1, γ-PN1Δ lytC ::kanR、γ-PN1Δ lytCOPA response of γ-PN2 and / or γ-PN3 to different Streptococcus pneumoniae serotypes.

[0363] Predictive Example 7: The effect of lytC deficiency on the antibody profile induced by vaccination Experiments will be conducted to evaluate lytC Does the absence of [a specific substance] alter the binding profile of serum IgG with a group of known pneumococcal antigens?

[0364] Swiss mice will be administered γ-PN1 and γ-PN1∆ at intervals of two weeks. lytC ::kanR、γ-PN1Δ lytC Mice were vaccinated three times with 50 µg of total protein from pneumococcal antigens 1, 2, 3, and 4. Control mice were given a dummy vaccination with PBS / glycerol. Serum was harvested 14 days after the final vaccination to test the binding of IgG to a panel of 289 pneumococcal antigen proteins. Control mice were used to calculate pneumococcal-specific antibody titers.

[0365] Predictive Example 8: Gene Expression Analysis in Strains Carrying lytC Deficiency Compared to Parental Strains Experiments will be conducted to evaluate the results. lytC Any changes in gene transcription induced by deletion.

[0366] Pneumococcal vaccine strains GPN-001, GPN-001∆lytC, GPN-002, GPN-002∆lytC, and / or GPN-003 will be cultured in standard animal-free soybean-based medium until mid-log phase, and RNA will be isolated using conventional techniques (e.g., Qiagen RNeasy Mini Kit). Transcriptomic RNA sequencing analysis will be performed essentially as described above. Briefly, ribosomal RNA content will be depleted using established methods (e.g., Epicentre Bacterial Ribozero Kit, Illumina), and a coded library will be generated (e.g., Ultra-Directional RNA Kit, New England Biolabs). The prepared library will be sequenced (e.g., Illumina HiSeq2500), and reads will be aligned to the genome of Pneumococcus Rx1 (GenBank accession CP079923) (e.g., BOWTIE2). Various tools will be used to derive the counts and differential gene expression for each gene (e.g., SAMtools, BEDtools, R, DESeq library).

[0367] Predictive Example 9: Proteomic Analysis of Strains Carrying lytC Deficiency Compared to Parental Strains Experiments will be conducted to evaluate the results. lytC Any changes in protein abundance induced by deletion.

[0368] Streptococcal vaccine strains GPN-001, GPN-001∆lytC, GPN-002, GPN-002∆lytC, and / or GPN-003 will be cultured in standard animal-free soybean-based medium, harvested by centrifugation, and lysed using a combination of chemical and mechanical methods. Comparative proteomic separation and protein identification will be performed using standard techniques, such as 2D differential gel electrophoresis [2D-DIGE] and mass spectrometry.

[0369] Predictive Example 10: Constructing a strain carrying a capsular infection in an alternative non-capsulated background strain lytA、lytC、psaA and pdT Modified vaccine strains To date, experiments have been conducted based on derivatives of Streptococcus pneumoniae Rx1 strain (an unencapsulated derivative of serotype 2 strain D39). However, similar results are expected for any unencapsulated strain.

[0370] Experiments will be conducted to genetically modify the strain to remove the gene from the capsule biosynthetic locus. cps The capsule gene can be removed by means of culture that leads to spontaneous loss of the capsule gene. The capsule gene can be removed using conventional genetic methods or by standard in vitro sequential passage of any serotype, including but not limited to serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10B, 10C, 10F, 11A, 11B, 11C, 11D, 11F, 12A, 12B, 12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F, 19A, 19B, 19C, 19F, 20, 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 2 4C, 24F, 25A, 25F, 27, 28A, 28F, 29, 31, 32A, 32F, 33A, 33B, 33C, 33D, 33F, 34, 35A, 35B, 35C, 35F, 36A, 36B, 37, 38, 39, 40, 41A, 41F, 42, 43, 44, 45, 46, 47A, 47F and / or 48.

[0371] For example, genes at the cps locus (including but not limited to) can be analyzed using conventional methods. cpsA / wzg / capA、 cpsB / wzh / capB、cpsC / wzd / capC、cpsD / wze / capD、cpsE / wchA / capE、cpsT、cpsF、cpsG、cpsH / wzy、cpsI、cpsJ / wzx、cpsK、cpsP、cpsL、cpsM、cpsN、cpsO Genetic modifications will be performed to eliminate capsule production / expression. The absence or reduction of the capsule will be confirmed by measuring capsule content, serological typing, and microscopic examination. Experiments will be conducted to introduce further modifications into one or more of the following genes using standard genetic manipulation methods: lytA、ply / pdt、psaA and lytC .

[0372] As a non-limiting example, experiments will be conducted to remove from a fully characterized TIGR4 strain (serotype 4). cps4E Genes. Oligonucleotide primers will be designed to amplify... cps4E The 2 kb regions upstream and downstream of the gene, and the region complementary to the antibiotic resistance cassette, will be incorporated. A linear fragment covering the 2 kb flanking region of the antibiotic resistance cassette will be generated by overlap extension PCR and confirmed by plating on blood agar plates supplemented with antibiotics. Experiments will then be performed to introduce further modifications in one or more of the following genes using standard genetic manipulation methods: lytA、ply / pdt、psaA and lytC The resulting strain can be defined as TIGR4. ∆ cps4E∆lytA PdT∆psaA∆lytC, which is similar to the GPN-003 strain described in this paper.

[0373] The resulting vaccine strain will be tested in animal models at several time points via intranasal, intramuscular, subcutaneous, or intraperitoneal routes in immunized animals (e.g., mice or rabbits) as a live attenuated vaccine or an inactivated vaccine (e.g., through chemical or physical methods, such as exposure to high-energy photons). Serum will be collected two weeks after final immunization to assess the immune response, such as antibody titers (e.g., total IgG titers), antibody binding (e.g., IgG binding to encapsulated bacteria), antibody function (e.g., opsonization of phagocytic responses), and protective efficacy.

[0374] Data from these experiments will confirm the use of alternative non-capsulated strains (such as Streptococcus pneumoniae TIGR4) ∆ Immunogenicity of the vaccine generated by cps4E∆lytA PdT∆psaA∆lytC).

[0375] Predictive Example 11: Constructing a capsulated background strain engineered to express low levels of capsule on its cell surface. lytA、lytC、psaA and pdT Modified vaccine strains To date, experiments have been conducted based on Streptococcus pneumoniae Rx1 (a non-capsulated derivative of serotype 2 strain D39). However, similar results are expected for strains engineered to express lower levels of capsule (which also allows exposure of key surface antigens). Experiments will be conducted to engineer encapsulated bacterial strains to introduce genetic modifications that reduce, but do not eliminate, capsular polysaccharide expression or alter their cell surface localization or attachment. Strains from any serotype can be engineered to reduce capsular levels on the cell surface, including but not limited to serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10B, 10C, 10F, and 11. A, 11B, 11C, 11D, 11F, 12A, 12B, 12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F ,18A,18B,18C,18F,19A,19B,19C,19F,20,21,22A,22F,23A,23B,23F,24A,24B,2 4C, 24F, 25A, 25F, 27, 28A, 28F, 29, 31, 32A, 32F, 33A, 33B, 33C, 33D, 33F, 34, 35A, 35B, 35C, 35F, 36A, 36B, 37, 38, 39, 40, 41A, 41F, 42, 43, 44, 45, 46, 47A, 47F and / or 48.

[0376] One example of how this can be achieved is by modifying genes such as Cps2A, LytR, and Psr, which are associated with capsule retention at the cell wall. Optionally, cpsE Certain point mutations in the gene have been shown to reduce total capsular production in clinical isolates. Mutations in vaccine strains will be constructed individually or in combination using conventional methods to remove or alter these genes, with the expectation of resulting in a reduction in capsular quantity. The reduction in cell-associated capsular quantity will be confirmed by measurements of capsular abundance. The ability to further expose protein antigens on the surface and cell membrane will be assessed by using serum comparative antibodies generated in animals using a non-capsular vaccine strain (e.g., GPN-003) to bind to the modified strain and the parent strain, or by using specific proteins representing different levels of protein exposure on the surface (e.g., PspA, PcpA, PrtA, PhtE, PiuA, AdcA, etc.). Experiments will be conducted to generate capsular vaccine strains with these modifications by introducing further modifications into one or more of the following genes using conventional genetic manipulation methods: lytA、ply / pdt、psaA and lytC For example, vaccine strains can be prepared using the strain Streptococcus pneumoniae D39∆cps2K∆lytR∆lytAPdT∆psaA∆lytC.

[0377] The resulting vaccine will be tested in animal models at several time points via intranasal, intramuscular, subcutaneous, or intraperitoneal routes in immunized animals (e.g., mice or rabbits) as a live attenuated vaccine or an inactivated vaccine (e.g., through chemical or physical methods, such as exposure to high-energy photons). Serum will be collected two weeks after final immunization to assess immune response (e.g., antibody titer (total IgG titer), antibody binding (IgG binding to encapsulated bacteria), antibody function (opsonization of phagocytic response), and protective efficacy).

[0378] Data from these experiments will confirm the immunogenicity of vaccines generated from encapsulated strains engineered to alter surface capsule abundance.

[0379] Predictive Example 12: Constructing a strain carrying a capsular expression in an alternative background strain cultured in a manner that reduces capsular expression. lytA、lytC、psaA and pdT Modified vaccine strains To date, experiments have been conducted based on Streptococcus pneumoniae Rx1 (a non-capsulated derivative of serotype 2 strain D39). However, similar results are expected for encapsulated strains that have been cultured in a manner that allows exposure to key surface antigens, resulting in lower capsular polysaccharide expression. Strains from any serotype can be cultured to reduce overall capsular expression, including but not limited to serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10B, 10C, 10F, and 11. A, 11B, 11C, 11D, 11F, 12A, 12B, 12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F ,18A,18B,18C,18F,19A,19B,19C,19F,20,21,22A,22F,23A,23B,23F,24A,24B,2 4C, 24F, 25A, 25F, 27, 28A, 28F, 29, 31, 32A, 32F, 33A, 33B, 33C, 33D, 33F, 34, 35A, 35B, 35C, 35F, 36A, 36B, 37, 38, 39, 40, 41A, 41F, 42, 43, 44, 45, 46, 47A, 47F and / or 48.

[0380] Experiments will be conducted to generate capsulated vaccine strains with these modifications by introducing modifications into one or more of the following genes using conventional genetic manipulation methods: lytA、ply / pdt、psaA and lytC One method for regulating capsule expression is by overloading bacteria with zinc. Zinc overload affects the activity of phosphoglucomutase, an enzyme required for the production of key sugar precursors in capsular polysaccharide biosynthesis, resulting in fewer capsules. Several methods can be used to overload bacteria with zinc, including: culturing bacteria in a medium supplemented with a molar excess of zinc ions relative to manganese ions (e.g., by supplementing the medium with ZnSO4); and engineering strains to remove the zinc export protein gene. czcD Alternatively, through chemical treatment during growth, such as with an ion carrier (e.g., zinc pyrithione, PBT2, etc.), the chemical treatment increases the transport of zinc across the biomembrane.

[0381] The resulting vaccine will be tested in animal models at several time points via intranasal, intramuscular, subcutaneous, or intraperitoneal routes, either as a live attenuated vaccine or an inactivated vaccine (e.g., through chemical or physical methods, such as exposure to high-energy photons), by immunizing animals (e.g., mice or rabbits). Serum will be collected two weeks after final immunization to assess immune response (e.g., antibody titers [e.g., total IgG titers], antibody binding [e.g., IgG binding to encapsulated bacteria], and antibody function [opsonization of phagocytic response]) and protective efficacy.

[0382] References Corsini, B., Aguinagalde, L., Ruiz, S., Domenech, M., and Yuste, J. (2021). Vaccination with LytA, LytC, or Pce of Streptococcus Pneumoniae Protects Against Sepsis by Inducing IgGs That Activate the Complement System. Vaccines , 9 (2), 186. Croucher, NJ, Campo, JJ, Le, TQ, Liang, X., Bentley, SD, Hanage, WP, and Lipsitch, M. (2017). Diverse evolutionary patterns of pneumococcal antigens identified by pangenome-wide immunological screening. Proceedings of the National Academy of Sciences , 114 (3), E357-E366. Eldholm, Vegard et al. (2009) "Fratricide in Streptococcus pneumoniae: contributions and role of the cell wall hydrolases CbpD, LytA and LytC". Microbiology , 155.7,2223-2234. Holmlund, E., Quiambao, B., Ollgren, J., Jaakkola, T., Neyt, C., Poolman, J.,... and Käyhty, H. (2009). Antibodies to pneumococcal proteins PhtD, CbpA, and LytC in Filipino pregnant women and their infants in relation to pneumococcal carriage. Clinical and Vaccine Immunology , 16 (6), 916-923. Nahm, MH, and Burton, RL (2014) Protocol for multiplexed opsonophagocytic killing assay (UAB-MOPA) for antibodies against Streptococcus pneumoniae (Revision E.02, December 2014). The Bacterial Respiratory Pathogen Reference Laboratory of the US NIH, WHO Reference Laboratory for Pneumococcal Serology, Departments of Pathology and Microbiology, University of Alabama at Birmingham, Birmingham AL 35294-2170 USA. https: / / www.vaccine.uab.edu / UAB-MOPA.pdf Tomasz, A. and Waks, S. (1975). Enzyme replacement in a bacterium: phenotypic correction by the experimental introduction of the wild-type enzyme into a live enzyme defective mutant pneumococcus. Biochemical and Biophysical Research Communications , 65 (4), 1311-1319. Wizemann, TM, Heinrichs, JH, Adamou, JE, Erwin, AL, Kunsch, C., Choi, GH,... and Koenig, S. (2001). Use of a whole genome approach to identify vaccine molecules affording protection against Streptococcus pneumoniaeinfection. Infection and immunity , 69 (3), 1593-1598.

Claims

1. An immunogenic composition comprising an attenuated and / or killable streptococcal bacterial strain, said strain comprising modifications that reduce, decrease, or prevent the functional expression of LytC or its homologs.

2. The immunogenic composition according to claim 1, wherein the modification includes lytC Deletion of a gene or its homolog.

3. The immunogenic composition according to claim 1 or 2, wherein the immunogenic composition is a cross-protective immunogenic composition.

4. The immunogenic composition according to any one of claims 1 to 3, wherein the attenuated and / or killable streptococcal bacterial strain is derived from a parental strain lacking the modification, and the attenuated and / or killable streptococcal bacterial strain improves the effectiveness of the immune response compared to the parental strain.

5. The immunogenic composition according to any one of claims 1 to 4, wherein the attenuated and / or killable streptococcal bacterial strain is derived from a parent strain lacking the modification, and the attenuated and / or killable streptococcal bacterial strain has improved stability compared to the parent strain.

6. The immunogenic composition according to any one of claims 1 to 5, wherein the attenuated and / or killable streptococcal bacterial strain further comprises a gene modification or deletion selected from the group consisting of: lytA or its homologs ply or its homologs and psaA Or its homologs.

7. The immunogenic composition according to any one of claims 1 to 6, wherein the attenuated and / or killable streptococcal bacterial strain further comprises lytA Modification or deletion of a gene or its homolog.

8. The immunogenic composition according to any one of claims 1 to 7, wherein the attenuated and / or killable streptococcal bacterial strain further comprises ply Modification or deletion of a gene or its homolog.

9. The immunogenic composition according to any one of claims 1 to 8, wherein the attenuated and / or killable streptococcal bacterial strain further comprises a substance named PdT. Ply Toxoid forms or their homologs.

10. The immunogenic composition according to any one of claims 1 to 9, wherein the attenuated and / or killable streptococcal bacterial strain further comprises psaA Modification or deletion of a gene or its homolog.

11. The immunogenic composition according to any one of claims 1 to 10, wherein the attenuated and / or killable streptococcal bacterial strain further comprises lytA Modification or deletion of genes or their homologs ply Modification or deletion of genes or their homologs and psaA Modifications or deletions of genes or their homologs, and also including genes named PdT. ply Toxoid forms or their homologs.

12. The immunogenic composition according to any one of claims 1 to 11, wherein the attenuated and / or killed streptococcal bacterial strain expresses capsular polysaccharide.

13. The immunogenic composition according to any one of claims 1 to 12, wherein the attenuated and / or killed streptococcal bacterial strain expresses reduced levels of capsular polysaccharides or does not express capsular polysaccharides.

14. The immunogenic composition according to any one of claims 1 to 13, wherein the composition comprises a single attenuated and / or killable streptococcal bacterial strain, said strain comprising modifications that attenuate, reduce, or prevent the functional expression of LytC or its homologs.

15. The immunogenic composition according to any one of claims 1 to 13, wherein the composition comprises a plurality of attenuated and / or killable streptococcal bacterial strains, each strain comprising a modification that weakens, reduces, or prevents the functional expression of LytC or its homologs.

16. The immunogenic composition according to any one of claims 1 to 15, wherein the attenuated and / or killable streptococcal bacterial strain is a species selected from the group consisting of: *Streptococcus agalactiae* (…). Streptococcus agalactiae Streptococcus bovis () Streptococcus bovis Streptococcus canis ( Streptococcus canis Streptococcus pyogenes ( ), Streptococcus dysgalactiae Streptococcus equi ( Streptococcus equi Streptococcus equi ( Streptococcus equinus Streptococcus equi ( Streptococcus equisimilis ), Enterococcus faecalis ( Enterococcus faecalis ), Enterococcus faecalis ( Enterococcus faecium ), Dolphin Streptococcus ( Streptococcus iniae ), Streptococcus Miller ( Streptococcus milleri Streptococcus mutans ( ) Streptococcus mutans Streptococcus pneumoniae () Streptococcus pneumoniae ), Streptococcus pyogenes ( Streptococcus pyogenes ), Streptococcus salivarius ( Streptococcus salivarius ), Streptococcus sanguinis ( Streptococcus sanguinis Streptococcus suis ( Streptococcus suis ) and Streptococcus lactis ( Streptococcus uberis ).

17. The immunogenic composition according to any one of claims 1 to 16, wherein the attenuated and / or killed streptococcal bacterial strain includes Streptococcus pneumoniae strains.

18. The immunogenic composition of claim 17, wherein the Streptococcus pneumoniae strain is derived from Rx1.

19. The immunogenic composition according to any one of claims 1 to 18, wherein the attenuated and / or killed streptococcal bacterial strain induces an immune response against at least one target streptococcal species and / or its serotype.

20. The immunogenic composition according to any one of claims 1 to 19, wherein the attenuated and / or killed streptococcal bacterial strain induces an immune response against a variety of target streptococcal species and / or their serotypes.

21. The immunogenic composition according to any one of claims 1 to 20, wherein the attenuated and / or killed streptococcal bacterial strain induces a cross-protective immune response against at least one target streptococcal species and / or its serotype.

22. The immunogenic composition according to any one of claims 1 to 21, wherein the attenuated and / or killed streptococcal bacterial strain induces a cross-protective immune response against multiple target streptococcal species and / or their serotypes.

23. The immunogenic composition according to any one of claims 1 to 20, wherein the attenuated and / or killed streptococcal bacterial strain is derived from a strain, serotype, or species that is the same as the at least one target streptococcal species and / or its serotype.

24. The immunogenic composition according to any one of claims 1 to 22, wherein the attenuated and / or killed streptococcal bacterial strain is derived from a strain, serotype or species that is different from the at least one target streptococcal species and / or its serotype.

25. The immunogenic composition according to any one of claims 1 to 24, wherein the at least one target streptococcal bacterial species and / or its serotype is selected from the group consisting of: agalactiae, bovine streptococci, canine streptococci, dysgalactiae, equine streptococci, equine enterococci, equine-like streptococci, enterococci faecalis, enterococci faecium, dolphin streptococci, Miller streptococci, variant streptococci, pneumococci, pyogenic streptococci, salivary streptococci, blood streptococci, suis streptococci, and mammary streptococci.

26. The immunogenic composition according to any one of claims 1 to 25, wherein the immunogenic composition is a killing whole-cell immunogenic composition.

27. The immunogenic composition according to any one of claims 1 to 26, wherein the whole-cell-killing immunogenic composition is selected from the group consisting of: chemically treated whole-cell-killing immunogenic compositions, heat-treated whole-cell-killing immunogenic compositions, irradiated whole-cell-killing immunogenic compositions, high hydrostatic pressure-killing whole-cell-killing immunogenic compositions, pulsed electric field-killing whole-cell-killing immunogenic compositions, ultrashort pulse laser-killing whole-cell-killing immunogenic compositions, pressure-ultrasonic-killing whole-cell-killing immunogenic compositions, and UV irradiation and microbial inactivation-killing whole-cell-killing immunogenic compositions.

28. The immunogenic composition according to any one of claims 1 to 27, wherein the whole-cell-killing immunogenic composition is a whole-cell-killing immunogenic composition by photon irradiation.

29. The immunogenic composition according to any one of claims 1 to 28, wherein the whole-cell killing immunogenic composition is a whole-cell killing immunogenic composition by gamma radiation.

30. The immunogenic composition according to any one of claims 1 to 29, wherein the whole-cell-killing immunogenic composition is a whole-cell-killing immunogenic composition killed by X-ray radiation.

31. The immunogenic composition according to any one of claims 1 to 30, wherein the whole-cell-killing immunogenic composition is a whole-cell-killing immunogenic composition killed by gamma irradiation and X-ray irradiation.

32. The immunogenic composition according to any one of claims 1 to 25, wherein the immunogenic composition is an attenuated whole-cell immunogenic composition.

33. The immunogenic composition according to any one of claims 1 to 32, further comprising at least one of an adjuvant, a pharmaceutically acceptable excipient, and a pharmaceutically acceptable carrier.

34. The immunogenic composition according to any one of claims 1 to 33, wherein the composition does not contain an adjuvant.

35. A vaccine composition comprising the immunogenic composition according to any one of claims 1 to 34.

36. Use of the immunogenic composition according to any one of claims 1 to 34 or the vaccine composition according to claim 35 as an immunogen.

37. Use as a vaccine by any one of the immunogenic compositions according to claims 1 to 34 or the vaccine composition according to claim 35.

38. A method for inducing an immune response in a subject against at least one target streptococcal species and / or its serotype, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition according to any one of claims 1 to 34 or a vaccine composition according to claim 35.

39. A method for inducing a cross-protective immune response in a subject against at least one target streptococcal species and / or its serotype, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition according to any one of claims 1 to 34 or a vaccine composition according to claim 35.

40. A method for preventing or reducing infection in a subject caused by at least one target streptococcal species and / or its serotype, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition according to any one of claims 1 to 34 or a vaccine composition according to claim 35.

41. A method for preventing or reducing disease or condition in a subject caused by at least one target streptococcal species and / or its serotype, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition according to any one of claims 1 to 34 or a vaccine composition according to claim 35.

42. The method according to any one of claims 38 to 42, wherein the attenuated and / or killed streptococcal bacterial strain is derived from a strain, serotype, or species that is the same as the at least one target streptococcal species and / or its serotype.

43. The method according to any one of claims 38 to 42, wherein the attenuated and / or killed streptococcal bacterial strain is derived from a strain, serotype or species that is different from the at least one target streptococcal species and / or its serotype.

44. The method according to any one of claims 38 to 42, wherein the at least one target streptococcal species and / or its serotype comprises multiple target streptococcal species and / or their serotypes.

45. The method according to any one of claims 38 to 42, wherein the target streptococcal species and / or serotype comprises streptococcal bacterial species and / or serotypes selected from the group consisting of: *Streptococcus agalactiae*, *Streptococcus bovis*, *Streptococcus canis*, *Streptococcus dysgalactiae*, *Streptococcus equi*, *Streptococcus equineus*, *Streptococcus faecalis*, *Streptococcus dolphinus*, *Streptococcus Miller*, *Streptococcus mutans*, *Streptococcus pneumoniae*, *Streptococcus pyogenes*, *Streptococcus salivarius*, *Streptococcus salivarius*, *Streptococcus sanguinis*, *Streptococcus suis*, and *Streptococcus lactis*.

46. ​​The method according to any one of claims 38 to 42, wherein the immunogenic composition or the vaccine composition is administered to the subject via an administration route selected from the group consisting of: intranasal, intravenous, intramuscular, subcutaneous, oral, intraocular, transmucosal, and transdermal administration.

47. The method of claim 41, wherein the disease or condition is selected from the group consisting of: respiratory tract infection, pneumonia, ear infection, earache, middle ear infection, otitis media, sinusitis, meningitis, conjunctivitis, bacteremia, sepsis, joint infection, bone infection, septic arthritis, osteomyelitis, soft tissue infection, cellulitis, myositis, periorbital cellulitis, abscess, peritonitis, cardiac infection, endocarditis, and pericarditis.

48. A method for preparing an immunogenic composition according to any one of claims 1 to 34 or a vaccine composition according to claim 35, the method comprising: (i) A preparation of photon-irradiated streptococcal bacterial strains comprising modified strains that attenuate, reduce, or prevent the functional expression of LytC or its homologs, thereby killing or physically attenuating said bacteria to produce photon-irradiated streptococcal bacteria; and (ii) Combining the photon-irradiated streptococcal bacteria with pharmaceutically acceptable excipients, diluents and / or carriers, and optionally adjuvants.

49. The method of claim 48, wherein the photon irradiation comprises exposing the streptococcal bacterial strain to gamma radiation.

50. The method of claim 49, wherein the photon irradiation comprises exposing the streptococcal bacterial strain to X-ray radiation.

51. The method according to any one of claims 48 to 50, wherein the photon irradiation comprises exposing the streptococcal bacterial strain to gamma radiation and X-ray radiation.

52. The method according to any one of claims 48 to 51, wherein the photon irradiation comprises exposing the streptococcal bacterial strain to photon radiation with an energy of at least 0.01 MeV.

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