Modified streptococcal immunogens and their use

Abstract

This disclosure relates to immunogenic compositions comprising attenuated and / or dead streptococcal bacterial strains, including modifications that reduce, decrease, or prevent the functional expression of LytC (or its homologues). Such compositions may be used in vaccine compositions. Immunogenic compositions and / or vaccine compositions may be used in methods for inducing an immune response to at least one target streptococcal species and / or its serotype, and / or for preventing or mitigating infections and / or diseases or conditions in a subject caused by at least one target streptococcal species and / or its serotype.

Description

Technical Field

[0001] Related Applications This application claims priority to Australian Provisional Application No. 2023901596, filed on May 22, 2023, the entire content of which is incorporated herein by reference.

[0002] The present disclosure relates to immunogenic compositions. In one form, the present disclosure relates to attenuated or killed Streptococcus bacterial strains.

Background Art

[0003] No discussion of prior art throughout this specification should be taken as an admission that such prior art is widely known or forms part of the common general knowledge in the field.

[0004] Vaccines currently used against Streptococcus infections are generally polysaccharide vaccines (e.g., the PCV23 vaccine containing polysaccharides from 23 serotypes) containing purified capsular polysaccharides from multiple serotypes, or conjugate vaccines containing capsular polysaccharides conjugated to diphtheria toxoid or other protein antigens of non-Streptococcus or Streptococcus origin. These polysaccharide-targeted vaccines induce strictly serotype-dependent protection. Multivalent conjugate vaccines induce immunity only against some of the more than 100 recognized serotypes of Streptococcus pneumoniae, and since most of these serotypes have been shown to cause disease, serotype replacement has become an important issue associated with polysaccharide vaccines. Thus, the immunity induced by commercially available Streptococcus vaccines is insufficient to establish broad immunity against most Streptococcus species and / or Streptococcus serotypes within a particular pathogenic species. Therefore, an improved Streptococcus vaccine is needed.

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

[0006] This disclosure relates to the unexpected finding that the use of attenuated or dead whole-cell streptococcal vaccines, in which the lytC gene (or its homolog) is modified to attenuate, reduce, and / or prevent the functional expression of LytC (or its homolog), results in improved vaccine immunogenicity. [Overview of the Initiative]

[0007] This disclosure relates to attenuated or dead streptococcal bacterial strains, including modifications that reduce, diminish, or prevent the functional expression of LytC (or its homologues). Surprisingly, compared to parental vaccine strains lacking modification to lytC, mice and rabbits immunized with compositions containing streptococcal bacterial strains with lytC deletion produced IgG antibodies with enhanced binding to the surface of the pneumococcal strain. Furthermore, antibodies produced by immunization with strains with lytC deletion exhibit a higher functional opsonization phagocytic response, a recognized surrogate endpoint for pneumococcal vaccine efficacy. These findings suggest a novel, previously unrecognized mechanism for improving the immunogenicity of whole-cell vaccines by modifying or deleting the expression of the lytC gene, which could not have been predicted based on previous studies.

[0008] The present invention relates to at least the following embodiments.

[0009] Embodiment 1. An immunogenic composition comprising an attenuated and / or dead streptococcal bacterial strain, which includes modifications that reduce, decrease, or prevent the functional expression of LytC or its homologues. In some examples, the immunogenic composition may consist of, or be essentially derived from, an attenuated and / or dead streptococcal bacterial strain, which includes modifications that reduce, decrease, or prevent the functional expression of LytC or its homologues.

[0010] Embodiment 2. The immunogenic composition of Embodiment 1, wherein the modification comprises a deletion of the lytC gene or its homolog. In some examples, the modification may consist of, or be essentially, a deletion of the lytC gene or its homolog.

[0011] Embodiment 3. An immunogenic composition of Embodiment 1 or 2, which is a cross-protective immunogenic composition.

[0012] Embodiment 4. An immunogenic composition according to any one of Embodiments 1 to 3, wherein the attenuated and / or dead streptococcal bacterial strain is derived from an unmodified parent strain, and the attenuated and / or dead streptococcal bacterial strain improves the effectiveness of the immune response compared to the parent strain.

[0013] Embodiment 5. An immunogenic composition according to any one of Embodiments 1 to 4, wherein the attenuated and / or dead streptococcal bacterial strain is derived from an unmodified parent strain, and the attenuated and / or dead streptococcal bacterial strain has improved stability compared to the parent strain.

[0014] Embodiment 6. An immunogenic composition according to any one of Embodiments 1 to 5, wherein the attenuated and / or dead streptococcal bacterial strain further comprises a gene modification or deletion selected from the group consisting of lytA or its homologue, ply or its homologue, and psaA or its homologue.

[0015] Embodiment 7. An immunogenic composition according to any one of Embodiments 1 to 6, further comprising a weakened and / or dead streptococcal bacterial strain with a modified or deleted lytA gene, or a homolog thereof.

[0016] Embodiment 8. An immunogenic composition according to any one of Embodiments 1 to 7, further comprising a modified or deleted ply gene or homolog thereof in an attenuated and / or dead streptococcal bacterial strain.

[0017] Embodiment 9. An immunogenic composition according to any one of Embodiments 1 to 8, further comprising a toxoid-type Ply called PdT or its homologue, wherein the attenuated and / or dead streptococcal bacterial strain further comprises PdT.

[0018] Embodiment 10. An immunogenic composition according to any one of Embodiments 1 to 9, further comprising a modified or deleted psaA gene or homolog thereof in an attenuated and / or dead streptococcal bacterial strain.

[0019] Embodiment 11. An immunogenic composition of any one of Embodiments 1 to 10, wherein the attenuated and / or dead streptococcal bacterial strain further comprises a modification or deletion of the lytA gene or its homolog, a modification or deletion of the ply gene or its homolog, a modification or deletion of the psaA gene or its homolog, and further comprises a toxoid type Ply or its homolog called PdT.

[0020] Embodiment 12. An immunogenic composition according to any one of Embodiments 1 to 11, wherein an attenuated and / or dead streptococcal bacterial strain expresses a capsular polysaccharide.

[0021] Embodiment 13. An immunogenic composition according to any one of Embodiments 1 to 12, wherein the attenuated and / or dead streptococcal bacterial strain expresses reduced levels of capsular polysaccharides or does not express capsular polysaccharides.

[0022] Embodiment 14. An immunogenic composition according to any one of Embodiments 1 to 13, wherein the composition comprises a single attenuated and / or dead streptococcal bacterial strain, the composition comprising a modification that attenuates, reduces, or prevents the functional expression of LytC or its homologue.

[0023] Embodiment 15. An immunogenic composition according to any one of Embodiments 1 to 13, wherein the composition comprises a plurality of attenuated and / or dead streptococcal bacterial strains, each of which is modified to reduce, decrease, or prevent the functional expression of LytC or its homologue.

[0024] Embodiment 16. Attenuated and / or dead streptococcal bacterial strains include Streptococcus agalactiae, Streptococcus bovis, Streptococcus canis, Streptococcus dysgalactiae, Streptococcus equi, Streptococcus equinus, Streptococcus equisimilis, Enterococcus faecalis, Enterococcus faecium, Streptococcus iniae, and Streptococcus milleri. An immunogenic composition according to any one of Embodiments 1 to 15, wherein the species is selected from the group consisting of Streptococcus milleri, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus salivarius, Streptococcus sanguinis, Streptococcus suis, and Streptococcus uberis.

[0025] Embodiment 17. An immunogenic composition according to any one of Embodiments 1 to 16, wherein the attenuated and / or dead streptococcal bacterial strain comprises a strain of Streptococcus pneumoniae.

[0026] Embodiment 18. The immunogenic composition of Embodiment 17, wherein the Streptococcus pneumoniae strain is derived from Rx1.

[0027] Embodiment 19. An immunogenic composition according to any one of Embodiments 1 to 18, wherein an attenuated and / or dead streptococcal bacterial strain induces an immune response against at least one target streptococcal species and / or its serotype.

[0028] Embodiment 20. An immunogenic composition according to any one of Embodiments 1 to 19, wherein an attenuated and / or dead streptococcal bacterial strain induces an immune response against multiple target streptococcal species and / or their serotypes.

[0029] Embodiment 21. An immunogenic composition according to any one of Embodiments 1 to 20, wherein an attenuated and / or dead streptococcal bacterial strain induces a cross-protective immune response against at least one target streptococcal species and / or its serotype.

[0030] Embodiment 22. An immunogenic composition according to any one of Embodiments 1 to 21, wherein an attenuated and / or dead streptococcal bacterial strain induces a cross-protective immune response against multiple target streptococcal species and / or their serotypes.

[0031] Embodiment 23. An immunogenic composition according to any one of Embodiments 1 to 20, wherein the attenuated and / or dead streptococcal bacterial strain is derived from the same strain, serotype, or species as at least one target streptococcal species and / or its serotype.

[0032] Embodiment 24. An immunogenic composition according to any one of Embodiments 1 to 22, wherein the attenuated and / or dead streptococcal bacterial strain is derived from a strain, serotype, or species different from at least one target streptococcal species and / or its serotype.

[0033] Embodiment 25. At least one target streptococcal bacterial species and / or its serotype is Streptococcus agalactiae, Streptococcus bovis, Streptococcus canis, Streptococcus dysgalactiae, Streptococcus equi, Streptococcus equinus, Streptococcus equisimilis, Enterococcus faecalis, Enterococcus faecium, Streptococcus iniae, Streptococcus milleri An immunogenic composition according to any one of Embodiments 1 to 24, selected from the group consisting of Streptococcus milleri, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus salivarius, Streptococcus sanguinis, Streptococcus suis, and Streptococcus uberis.

[0034] Embodiment 26. An immunogenic composition from any one of Embodiments 1 to 25, which is a dead whole cell immunogenic composition.

[0035] Embodiment 27. An immunogenic composition from any one of Embodiments 1 to 26, wherein the whole-cell immunogenic composition is selected from the group consisting of chemically treated whole-cell immunogenic compositions, heat-treated whole-cell immunogenic compositions, irradiation-treated whole-cell immunogenic compositions, high hydrostatic pressure-treated whole-cell immunogenic compositions, pulsed electric field-treated whole-cell immunogenic compositions, ultrashort pulse laser-treated whole-cell immunogenic compositions, pressure-induced ultrasonic-treated whole-cell immunogenic compositions, UV irradiation, and microbial inactivation-treated whole-cell immunogenic compositions.

[0036] Embodiment 28. An immunogenic composition according to any one of Embodiments 1 to 27, wherein the whole-cell immunogenic composition is a photon-irradiated whole-cell immunogenic composition.

[0037] Embodiment 29. An immunogenic composition according to any one of Embodiments 1 to 28, wherein the whole-cell immunogenic composition is a gamma-ray irradiated whole-cell immunogenic composition.

[0038] Embodiment 30. An immunogenic composition according to any one of Embodiments 1 to 28, wherein the whole-cell immunogenic composition is an X-ray irradiated whole-cell immunogenic composition.

[0039] Embodiment 31. An immunogenic composition according to any one of Embodiments 1 to 31, wherein the whole-cell immunogenic composition is a whole-cell immunogenic composition irradiated with gamma rays and X-rays.

[0040] Embodiment 32. An immunogenic composition from any one of Embodiments 1 to 25, which is an attenuated whole-cell immunogenic composition.

[0041] Embodiment 33. An immunogenic composition according to any one of Embodiments 1 to 32, further comprising at least one of an adjuvant, a pharmaceutically acceptable excipient, and a pharmaceutically acceptable carrier.

[0042] Embodiment 34. An immunogenic composition according to any one of Embodiments 1 to 33, wherein the composition does not contain an adjuvant.

[0043] Embodiment 35. A vaccine composition comprising any one immunogenic composition from Embodiments 1 to 34.

[0044] Embodiment 36. Use of any one immunogenic composition from Embodiments 1 to 34 or the vaccine composition of Embodiment 35 as an immunogen.

[0045] Embodiment 37. Use of any one immunogenic composition from Embodiments 1 to 34 or the vaccine composition of Embodiment 35 as a vaccine.

[0046] Embodiment 38. A method for inducing an immune response to at least one target streptococcal species and / or serotype thereof in a subject, comprising administering a therapeutically effective dose to the subject one immunogenic composition of any one of Embodiments 1 to 34 or the vaccine composition of Embodiment 35.

[0047] Embodiment 39. A method for inducing a cross-protective immune response against at least one target streptococcal species and / or serotype thereof in a subject, comprising administering a therapeutically effective dose to one immunogenic composition of any one of Embodiments 1 to 34 or the vaccine composition of Embodiment 35 in the subject.

[0048] Embodiment 40. A method for preventing or mitigating an infection in a subject caused by at least one target streptococcal species and / or its serotype, comprising administering to the subject a therapeutically effective amount of any one immunogenic composition of Embodiments 1 to 34 or the vaccine composition of Embodiment 35.

[0049] Embodiment 41. A method for preventing or mitigating a disease or condition in a subject caused by at least one target streptococcal species and / or its serotype, comprising administering to the subject a therapeutically effective amount of any one immunogenic composition of Embodiments 1 to 34 or the vaccine composition of Embodiment 35.

[0050] Embodiment 42. Any one of Embodiments 38 to 42, wherein the attenuated and / or dead streptococcal bacterial strain is derived from the same strain, serotype, or species as at least one target streptococcal species and / or its serotype.

[0051] Embodiment 43. Any one of Embodiments 38 to 42, wherein the attenuated and / or dead streptococcal bacterial strain is derived from a strain, serotype, or species different from at least one target streptococcal species and / or its serotype.

[0052] Embodiment 44. Any one of Embodiments 38 to 42, wherein at least one target streptococcal species and / or its serotype comprises a plurality of target streptococcal species and / or its serotypes.

[0053] Embodiment 45. The target streptococcal species and / or its serotype is Streptococcus agalactiae, Streptococcus bovis, Streptococcus canis, Streptococcus dysgalactiae, Streptococcus equi, Streptococcus equinus, Streptococcus equisimilis, Enterococcus faecalis, Enterococcus faecium, Streptococcus iniae, Streptococcus milleri A method according to any one of embodiments 38 to 42, comprising a streptococcal bacterial species and / or its serotype selected from the group consisting of Streptococcus milleri, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus salivarius, Streptococcus sanguinis, Streptococcus suis, and Streptococcus uberis.

[0054] Embodiment 46. One of Embodiments 38 to 42, wherein an immunogenic composition or vaccine composition is administered to a target by a route of administration selected from the group consisting of intranasal, intravenous, intramuscular, subcutaneous, oral, intraocular, transmucosal, and transdermal administration.

[0055] Embodiment 47. The method of Embodiment 41, wherein the disease or condition is selected from the group consisting of respiratory tract infections, pneumonia, ear infections, ear pain, middle ear infections, otitis media, sinusitis, meningitis, conjunctivitis, bacteremia, sepsis, joint infections, bone infections, suppurative arthritis, osteomyelitis, soft tissue infections, cellulitis, myositis, periorbital cellulitis, abscess, peritonitis, cardiac infections, endocarditis, and pericarditis.

[0056] Embodiment 48. A method for preparing any one immunogenic composition from Embodiments 1 to 34 or the vaccine composition from Embodiment 35, (i) To produce photon-irradiated streptococcal bacteria by irradiating a preparation of a streptococcal bacterial strain containing modifications that attenuate, reduce, or prevent the functional expression of LytC or its homologue with photons, thereby killing the bacteria or physically weakening them. (ii) A method comprising combining photon-irradiated streptococcal bacteria with pharmaceutically acceptable excipients, diluents and / or carriers, and optionally an adjuvant.

[0057] Embodiment 49. The method of Embodiment 48, wherein photon irradiation includes exposing a streptococcal bacterial strain to gamma radiation.

[0058] Embodiment 50. The method of Embodiment 49, wherein photon irradiation includes exposing a streptococcal bacterial strain to X-ray radiation.

[0059] Embodiment 51. Any one of Embodiments 48 to 50, wherein photon irradiation includes exposing a streptococcal bacterial strain to gamma-ray and X-ray radiation.

[0060] Embodiment 52. Any one of Embodiments 48 to 51, wherein photon irradiation includes exposing a streptococcal bacterial strain to photon radiation with an energy of at least 0.01 MeV.

[0061] Embodiment 53. Use of a therapeutically effective amount of any one immunogenic composition of Embodiments 1 to 34 or the vaccine composition of Embodiment 35 in the preparation of a pharmaceutical product for inducing an immune response against at least one target streptococcal species and / or its serotype in a subject.

[0062] Embodiment 54. Use of a therapeutically effective amount of any one immunogenic composition of Embodiments 1 to 34 or the vaccine composition of Embodiment 35 in the preparation of a pharmaceutical product for inducing a cross-protective immune response against at least one target streptococcal species and / or its serotype in a subject.

[0063] Embodiment 55. Use of a therapeutically effective amount of any one immunogenic composition of Embodiments 1 to 34 or the vaccine composition of Embodiment 35 in the preparation of a pharmaceutical for preventing or mitigating an infection in a subject caused by at least one target streptococcal species and / or its serotype.

[0064] Embodiment 56. Use of a therapeutically effective amount of any one immunogenic composition of Embodiments 1 to 34 or the vaccine composition of Embodiment 35 in the preparation of a pharmaceutical for preventing or mitigating a disease or condition in a subject caused by at least one target streptococcal species and / or its serotype.

[0065] Embodiment 57. Use of any one of Embodiments 53 to 56, wherein the attenuated and / or dead streptococcal bacterial strain is derived from the same strain, serotype, or species as at least one target streptococcal species and / or its serotype.

[0066] Embodiment 58. Use of any one of Embodiments 53 to 56, wherein the attenuated and / or dead streptococcal bacterial strain is derived from a strain, serotype, or species different from at least one target streptococcal species and / or its serotype.

[0067] Embodiment 59. Use of any one of Embodiments 53 to 56, wherein at least one target streptococcal species and / or its serotype comprises multiple target streptococcal species and / or its serotypes.

[0068] Embodiment 60. The target streptococcal species and / or serotype is Streptococcus agalactiae, Streptococcus bovis, Streptococcus canis, Streptococcus dysgalactiae, Streptococcus equi, Streptococcus equinus, Streptococcus equisimilis, Enterococcus faecalis, Enterococcus faecium, Streptococcus iniae, Streptococcus milleri The use of any one of embodiments 53 to 56, comprising a streptococcal bacterial species and / or serotype thereof selected from the group consisting of Streptococcus milleri, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus salivarius, Streptococcus sanguinis, Streptococcus suis, and Streptococcus uberis.

[0069] Embodiment 61. Any one of Embodiments 53 to 56, wherein the immunogenic composition or vaccine composition is administered to a subject by a route of administration selected from the group consisting of intranasal, intravenous, intramuscular, subcutaneous, oral, intraocular, transmucosal, and transdermal administration.

[0070] Embodiment 62. Use of Embodiment 56, wherein the disease or condition is selected from the group consisting of respiratory tract infections, pneumonia, ear infections, ear pain, middle ear infections, otitis media, sinusitis, meningitis, conjunctivitis, bacteremia, sepsis, joint infections, bone infections, suppurative arthritis, osteomyelitis, soft tissue infections, cellulitis, myositis, periorbital cellulitis, abscess, peritonitis, cardiac infections, endocarditis, and pericarditis.

[0071] Embodiment 63. A therapeutically effective amount of any one immunogenic composition of Embodiments 1 to 34 or the vaccine composition of Embodiment 35 for use in inducing an immune response against at least one target streptococcal species and / or its serotype in a subject.

[0072] Embodiment 64. A therapeutically effective amount of any one immunogenic composition of Embodiments 1 to 34 or the vaccine composition of Embodiment 35 for use in inducing a cross-protective immune response against at least one target streptococcal species and / or its serotype in a subject.

[0073] Embodiment 65. A therapeutically effective amount of any one immunogenic composition of Embodiments 1 to 34 or the vaccine composition of Embodiment 35 for use in the prevention or mitigation of infections caused by at least one target streptococcal species and / or serotype thereof in a subject.

[0074] Embodiment 66. A method comprising administering to a subject a therapeutically effective amount of one immunogenic composition from any of Embodiments 1 to 34 or the vaccine composition from Embodiment 35 for use in the prevention or mitigation of a disease or condition in a subject caused by at least one target streptococcal species and / or its serotype.

[0075] Embodiment 67. Any one of the compositions from Embodiments 63 to 66, wherein the attenuated and / or dead streptococcal bacterial strain is derived from the same strain, serotype, or species as at least one target streptococcal species and / or its serotype.

[0076] Embodiment 68. Any one of Embodiments 63 to 66, wherein the attenuated and / or dead streptococcal bacterial strain is derived from a strain, serotype, or species different from at least one target streptococcal species and / or its serotype.

[0077] Embodiment 69. Any one of the compositions from Embodiments 63 to 66, wherein at least one target streptococcal species and / or its serotype comprises a plurality of target streptococcal species and / or its serotypes.

[0078] Embodiment 70. The target streptococcal species and / or serotype is Streptococcus agalactiae, Streptococcus bovis, Streptococcus canis, Streptococcus dysgalactiae, Streptococcus equi, Streptococcus equinus, Streptococcus equisimilis, Enterococcus faecalis, Enterococcus faecium, Streptococcus iniae, Streptococcus milleri A composition comprising any one of embodiments 63 to 66, comprising a streptococcal bacterial species and / or its serotype selected from the group consisting of Streptococcus milleri, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus salivarius, Streptococcus sanguinis, Streptococcus suis, and Streptococcus uberis.

[0079] Embodiment 71. Any one of the compositions from Embodiments 63 to 66, wherein the immunogenic composition or vaccine composition is administered to a target by a route of administration selected from the group consisting of intranasal, intravenous, intramuscular, subcutaneous, oral, intraocular, transmucosal, and transdermal administration.

[0080] Embodiment 72. The composition of Embodiment 66, wherein the disease or condition is selected from the group consisting of respiratory tract infections, pneumonia, ear infections, ear pain, middle ear infections, otitis media, sinusitis, meningitis, conjunctivitis, bacteremia, sepsis, joint infections, bone infections, suppurative arthritis, osteomyelitis, soft tissue infections, cellulitis, myositis, periorbital cellulitis, abscess, peritonitis, cardiac infections, endocarditis, and pericarditis. [Brief explanation of the drawing]

[0081] [Figure 1] This document provides schematic diagrams showing the preparation of GPN-002, GPN-002ΔlytC::KanR, GPN-003, and GPN-001ΔlytC::KanR from GPN-001 derivative strains of Streptococcus pneumoniae (S. pneumoniae) Rx1. [Figure 2] This provides a photographic image of an electrophoretic gel showing a PCR product that confirms the integration of a kanamycin-resistant cassette in the lytC region of GPN-002. [Figure 3] This provides a graphical representation of the optical density (OD600) over time at 25°C for GPN-002 and GPN-002ΔlytC::kanR bacteria that have been washed and suspended in PBS containing 20% ​​glycerol. [Figure 4] This provides a graphical representation of the optical density (OD600) over time at 25°C for GPN-002 and GPN-002ΔlytC::kanR bacteria that have been washed and suspended in PBS containing 20% ​​glycerol. [Figure 5] This provides photographic images of an electrophoresis gel showing the PCR product that confirms the absence of lytA, ply / PdT substitution, psaA, and lytC in GPN-003. [Figure 6](Upper panel) Provides the DNA sequence and protein translation of the ply region of Rx1, and (Lower panel) provides the DNA sequence and protein translation of the pdt region of GPN-003. [Figure 7] This provides a graphical representation of the optical density (OD600) over time at 25°C for GPN-002 and GPN-003 bacteria that have been washed and suspended in PBS containing 20% ​​glycerol. [Figure 8] This document provides a graphical representation of antibody responses in mice immunized with PBS (false control), gamma-PN2, and gamma-PN3 using ELISA with Rx1 as the capture antigen, showing (A) total pneumococcal-specific IgG response as absorbance (450 / 620 nm), (B) total IgG response as titer, (C) IgG1, (D) IgG2a, (E) IgG2b, and (F) IgG3. Data are presented as mean absorbance (450 / 620 nm) ± SEM obtained from individual or pooled serum samples (n=5 mice per group). [Figure 9] This graph displays IgG levels after sham vaccination or vaccination with gamma-PN2 or gamma-PN3, where (A) is the percentage of IgG bound to Streptococcus pneumoniae serotype 2 D39, (B) is the geometric mean (GMFI) of fluorescence intensity of IgG bound to Streptococcus pneumoniae serotype 2 D39, (C) is the percentage of IgG bound to Streptococcus pneumoniae serotype 6A P9, and (D) is the GMFI of IgG bound to Streptococcus pneumoniae serotype 6A P9. Data were analyzed by one-way ANOVA (**p<0.01, ****p<0.0001). [Figure 10] This report provides graphs of (A) the IgG titers of individual rabbits before and after the third immunization within each vaccine group, and (B) the mean IgG titer (±SEM). Data were analyzed by one-way ANOVA (nd = not detected). [Figure 11]This graph provides a flow cytometry analysis of rabbit IgG binding to Streptococcus pneumoniae serotype 7C bacterial cells after incubation with rabbit serum following vaccination with either gamma-PN2 or gamma-PN3, where (A) is relative fluorescence, (B) is the percentage of serotype 7C cells bound to rabbit IgG antibody, and (C) is the geometric mean fluorescence intensity of IgG antibody bound to serotype 7C cells, indicating the degree of IgG binding. Data were analyzed by one-way ANOVA (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns indicates non-significant). [Figure 12] This provides a graphical representation of the average prsA transcription level relative to the gyrA transcription level determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in GPN-002 and GPN-003. [Figure 13] This provides a graph showing (A) survival rate and (B) body weight in grams for mice inoculated with either 104 CFU / mouse of Streptococcus pneumoniae serotype 2 D39 strain, or 10,000 times higher doses (108 CFU / mouse) of GPN-002 or GPN-003. [Figure 14] This document provides schematic diagrams illustrating the preparation of GPN-002, GPN-002ΔlytC::KanR, GPN-003, and GPN-001ΔlytC::KanR and GPN 001ΔlytC, derived from the GPN-001 derivative strain of Streptococcus pneumoniae (S. pneumoniae) Rx1.

[0082] definition In the context of this specification, the terms “a” and “an” are used herein to refer to one or more (i.e., at least one) grammatical objects of articles. For example, “an element” means one or more elements.

[0083] As used herein, the terms “about,” “approximately,” and “substantially,” when applied to the value in question, refer to a value that is substantially the same as the stated value. In certain embodiments, unless otherwise stated or made clear from the context, “about,” “approximately,” and “substantially,” refer to a range of + / -10%, preferably + / -9%, + / -8%, + / -7%, + / -6%, + / -5%, + / -4%, + / -3%, + / -2%, or + / -1%, or + / -0.05% or + / -0.1% of the stated value.

[0084] As used herein, the term “attenuated” in the context of bacteria is understood 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 a nonpathogenic infection in the host to which they are administered, or otherwise be unable to establish an infection in the host, or may establish an infection that causes mild or tolerable symptoms in the host. It should be understood that attenuated immunogenic compositions remain in the host for a period sufficient to induce an immune response. However, the bacteria cannot establish a long-term infection, nor can they establish a pathogenic infection that is harmful to a non-immune host to which the attenuated bacteria are administered.

[0085] When using the term "between" to refer to a range of values, it should be understood that the values ​​at each endpoint of the range are included. For example, polypeptides between 10 and 20 residues in length include polypeptides of length 10 and polypeptides of length 20.

[0086] The terms “comprise,” “comprises,” “comprised,” “comprising,” “including,” and “having” in this specification and in the claims are used in a comprehensive sense, that is, to identify the presence of the features described, but do not preclude the presence of additional or further features.

[0087] As used herein, the term “complementary” is used to describe the relationship between a first nucleotide sequence and a second nucleotide sequence according to the base pairing rule, in which adenine (A) bases pair with uracil (U) bases in RNA molecules or thymine (T) bases in DNA molecules, and cytosine (C) bases pair with guanine (G) bases in both RNA and DNA molecules. For example, in DNA polynucleotide molecules, the sequence “5'-AGTC-3'” is perfectly complementary to the sequence “3'-TCAG-5',” but it should be noted that in RNA sequences, uracil (U) is typically used instead of thymine (T).

[0088] The degree of complementarity between nucleic acid strands significantly affects the efficiency and strength of hybridization between them. This is particularly important in amplification or detection methods that rely on binding between nucleic acids. Nucleic acid sequences do not need to be "perfectly" (100%) complementary to their target sequence in order to hybridize. Complementarity can be "partial," where only some of the nucleic acid bases match according to base pairing rules. When two molecules can hybridize under appropriate conditions, it is understood that the two sequences are "specifically complementary"—that is, when the two molecules can hybridize to form a double-stranded structure under conditions appropriate for the reaction being performed (e.g., ligation, PCR, sequencing, etc.), then the first polynucleotide molecule containing the first nucleotide sequence is specifically complementary to the second polynucleotide molecule containing the second nucleotide sequence. The term "specifically complementary" can be used interchangeably with "substantially complementary." It should also be understood that the two nucleotide molecules do not need to be complementary throughout their entire length. For example, a portion of a first polynucleotide molecule may be specifically complementary to and hybridize with a portion of a second polynucleotide molecule. In this example, the two molecules may not hybridize over portions that are not specifically complementary. These terms can also be used with respect to individual nucleotides, particularly in the context of oligonucleotides. For example, a particular nucleotide within an oligonucleotide may be noted for its complementarity, or lack thereof, to a nucleotide in another nucleic acid chain, in contrast to, or in comparison to, the complementarity between the rest of the oligonucleotide and the nucleic acid chain.

[0089] The transitional phrase "consisting of" excludes any elements, processes, or components not explicitly stated. In the context of claims, such a transitional phrase excludes materials other than those enumerated in the claims, with the exception of impurities normally associated with the materials. If the phrase "consisting of" appears in a clause of the body of the claim rather than immediately following the preamble, it limits the elements described in that clause only, and other elements are not necessarily excluded from the claim as a whole.

[0090] The transitional phrase “essentially from” may be used to describe compositions, processes, or methods that include materials, processes, features, components, or elements in addition to those literally disclosed, provided that these additional materials, processes, features, components, or elements do not substantially affect the basic and novel features(s) of the claimed invention.

[0091] The term "cross-protective" as used in reference to the compositions of this disclosure refers to compositions that can induce a cross-protective immune response.

[0092] 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 is protective against a different target bacterial strain or species compared to the strain from which the antigen originates. For example, an immune response induced by serotype 2 of Streptococcus pneumoniae is cross-protective if it protects the subject from infection or mitigates disease caused by a different serotype of Streptococcus pneumoniae, such as serotype 6. Similarly, an immune response induced by serotype 2 of Streptococcus pneumoniae is cross-protective if it protects the subject from infection or mitigates disease caused by a different streptococcal species, such as Streptococcus pyogenes.

[0093] The terms “derived strain” or “derived from” as used herein in relation to bacterial strains are used to refer to progeny bacterial strains that have at least one modification compared to the parent strain. Modifications may be natural or intentionally introduced. It should be understood that the parent strain may be a direct parent (for example, after the introduction of a single modification). Alternatively, the 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 to be derived strains of Streptococcus pneumoniae (S. pneumoniae) Rx1, GPN-002 and GPN-003 are considered to be derived strains of GPN-001, and GPN-003 is considered to be a derived strain of GPN-001 and GPN-002.

[0094] As used herein, the term “effectively” is intended to refer to a sufficient proportion of the parameter or result to achieve the desired result when used in relation to a particular parameter or result.

[0095] As used herein, “expression” or “to express” in the context of nucleic acid sequences or proteins refers to the translation of mRNA into polypeptides, the assembly of multiple polypeptides into an intact protein (e.g., an enzyme), and / or post-translational modification of polypeptides or fully assembled proteins (e.g., enzymes). In this application, the terms “expression” and “production,” as well as their grammatical equivalents, are used interchangeably.

[0096] As used herein, "express" in the context of a particular bacterial characteristic or phenotype means the presence of that characteristic or phenotype.

[0097] As used herein, the “homologous” of a protein refers to a group of proteins that perform the same biological function and are expressed by homologous genes. A homologous gene is a gene that codes for a protein that has the same or similar biological function as the protein coded by a second gene. Homologous genes and nucleic acid sequences may exist in the same organism or in different organisms. Homologous genes include orthologs (i.e., genes that evolved from a common ancestral gene through speciation, code for a protein, and are expressed in different species but retain the same function), but do not include paralogs (i.e., genes that are related by duplication but have evolved to code for proteins with different functions). Homologous genes include naturally occurring alleles and artificially created variants. Genetic code degeneracy provides the possibility of substituting at least one nucleotide in the protein-coding sequence or coding sequence of a gene with a different base without altering the amino acid sequence of the polypeptide produced from the gene. When optimally aligned, the homologous protein and nucleotide sequences (e.g., genes) of the present invention may have, for example, at least about 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity across the entire 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 consensus nucleotide or protein sequence across the entire length of the reference sequence. The homologous nucleic acid sequences, genes, or proteins described herein may, for example, be homologous nucleic acid sequences, genes, or proteins from other bacteria, other Firmicutes, other Bacilli, other Lactobacillales, other Streptococci, and / or other Streptococci.

[0098] Gene / nucleic acid sequences and proteins that are homologs of a reference sequence can be identified by comparing homologous amino acid or nucleotide sequences (e.g., manually or using computer-based tools that employ known homology-based search algorithms such as FASTA, BLAST, and Smith-Waterman). Similar sequences can be found by searching a database of sequences using a local sequence alignment program (e.g., BLAST), and the similarity of sequence bases can be measured using the summary expectation (E value). A given sequence that hits with the best E value for a particular organism may not necessarily be an orthologue (i.e., it may have the same function / code a protein with the same function), or it may be the only orthologue. In such cases, cross-queries are used to filter hit sequences with significant E values ​​for orthologue identification. Cross-queries may involve searching a database of significant hits from sequences from the underlying organism that are similar to the query sequence. If the best hit of a cross-query is the query sequence itself, or a protein encoded by a duplicated gene after speciation, the hit can be identified as an orthologue.

[0099] As used herein, “sequence identity %” means the degree to which two optimally aligned DNA or protein segments are invariant across the entire alignment window of their components, e.g., nucleotide or amino acid sequences. The “percentage of identity” of aligned segments of a test sequence and a reference sequence is the number of identical components shared by the sequences of the two aligned segments divided by the total number of sequence components in the reference segment over the alignment window, which is the smaller of the complete test sequence or the complete reference sequence. “Sequence identity %” is the percentage of identity multiplied by 100. Such optimal alignment is understood to be considered a local alignment of the DNA sequence. In the case of protein alignment, local alignment of the protein sequence requires the introduction of gaps to achieve optimal alignment. The sequence identity percentage is calculated over the aligned length, excluding any gaps introduced by the alignment itself.

[0100] As used herein, the terms “inducing,” “inducing,” “enhancing,” and “enhancing” in relation to immunity or an immune response refer to an increase in immunity or an immune response beyond existing levels, whether nonexistent or measurable.

[0101] As used herein, the term “improved efficacy” in the context of immunity or immune response induced by the compositions of this disclosure refers to a shift in the immune response that increases protection against infection or disease compared to a baseline, such as the immune response induced by the parent strain. An immune shift may refer to, for example, an increase in a particular subtype of immunoglobulin (e.g., IgG1, IgG2a, IgG2b, IgG3, IgG4, etc., IgA, IgE, IgM, etc.), or a shift in Th1 / Th2 bias.

[0102] As used herein, the term “immunogenic composition” refers to a composition or formulation that elicits an immune response when administered to a subject.

[0103] As used herein, the term “vaccine composition” means a composition or formulation that, when administered to a subject, produces an immune response capable of protecting the subject from infection caused by a pathogenic substance or mitigating a disease caused by a pathogenic substance.

[0104] The terms “immune response” and “immunity” are used interchangeably herein and refer to the subject’s response to an exogenous antigen. An immune response may include the induction of humoral (i.e., B cell) responses and / or cellular (i.e., T cell) responses. Appropriately, 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 a variety of means, for example, by enzyme-linked immunosorbent assay (ELISA) of serum from immunized mammals, FACS analysis of antibodies that bind to cell surface antigens, opsonization assays, and microneutralization assays of serum from immunized animals to assess humoral responses. Alternatively, or in addition to these, cellular responses can be measured by FACS analysis, cytotoxic T cells (CTLs) for measuring T cell responses derived from lymphocytes isolated from the spleen or other organs of immunized animals, etc.

[0105] As used herein, the term “isolated” refers to a material that substantially or essentially does not contain the components normally associated with it in its natural state. For example, as used herein, “isolated polynucleotide” refers to a polynucleotide purified from a sequence adjacent to it in its naturally occurring state, e.g., a DNA fragment isolated from a sequence normally adjacent to the fragment. Alternatively, “isolated peptide” or “isolated polypeptide,” as used herein, refers to the in vitro isolation and / or purification of a peptide or polypeptide molecule from its natural cellular environment and from the association with other components of the cell, i.e., it is not associated with the in vivo material.

[0106] As used herein, the term “labeling” is used to describe the binding, ligation, or encapsulation of a biological tag to a biomolecule, where the biological tag may be used to detect the presence of a biomolecule or to quantify the absolute or relative amount of a biological molecule.

[0107] As used herein, the term “nucleotide” in its broadest sense refers to any compound and / or substance that is incorporated into or can be incorporated into a polynucleotide chain. In one embodiment, a nucleotide is a compound and / or substance that is incorporated into or can be incorporated into a polynucleotide chain via a phosphodiester bond. In one embodiment, “nucleotide” refers to an individual nucleic acid residue (e.g., a nucleotide and / or nucleoside). The term “nucleotide” may be used interchangeably with “nucleic acid.” In one embodiment, “nucleotide” includes RNA, as well as single-stranded and / or double-stranded DNA, and / or cDNA.

[0108] As used herein, the terms “messenger RNA” or “mRNA” refer 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.

[0109] As used herein, the phrase "modifications that attenuate, reduce, or prevent functional LytC expression" refers to mutations or deletions in the lytC gene that reduce or eliminate the expression of the functional LytC protein, meaning that the function of LytC is reduced or eliminated.

[0110] As used herein, the terms “operably connected” or “operably linked” refer 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.

[0111] As used herein, the term “pharmaceutically acceptable” means a substance that, when administered to a subject, does not cause a substantially harmful allergic or immunological reaction. “pharmaceutically acceptable carriers” include, but are not limited to, solvents, coatings, dispersants, wetting agents, isotonic agents and absorption retarders, and disintegrants.

[0112] As used herein, the term “photon-beam emission” will be understood to encompass both gamma-ray emission (i.e., gamma rays) and X-ray emission (i.e., X-rays). Therefore, a “photon-beam-irradiated” material may be a material exposed to gamma-ray emission and thus becoming “gamma-ray-irradiated,” a material exposed to X-ray emission and thus becoming “X-ray-irradiated,” or both. As a purely non-limiting example, to irradiate a material with photons, it may be subjected to photon emission of energy at least 0.01 MeV, at least 0.1 MeV, at least 0.5 MeV, between 0.01 MeV and 0.5 MeV, between 0.01 MeV and 1 MeV, between 0.01 MeV and 10 MeV, between 0.5 MeV and 20 MeV, between 0.5 MeV and 15 MeV, between 0.5 MeV and 10 MeV, between 0.5 MeV and 5 MeV, between 0.5 MeV and 2 MeV, or between 1 MeV and 2 MeV (e.g., 1.25 MeV).

[0113] As used herein, the term “plural” means two or more. In certain specific aspects or embodiments, plural 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, as well as any range derived therefrom.

[0114] As used herein, the term “polynucleotide molecule” refers to a DNA or RNA nucleic acid molecule containing a chain of nucleotides, and may include the nucleotide molecule or nucleic acid of interest. A polynucleotide molecule is encoded by a “polynucleotide sequence,” which may also be called a nucleic acid sequence herein. It will be understood by those skilled in the art that a nucleotide typically consists of three distinct chemical subunits: a five-carbon sugar molecule (pentose-sugar ring in DNA, deoxyribose, or ribose in RNA), a nucleic acid base (i.e., adenine (A), cytosine (C), guanine (G), thymine (T), or uracil (U)), and a phosphate group. By chemical convention, carbon atoms in sugar molecules are named 1' through 5', and this convention also stipulates that a polynucleotide molecule has a 5' end and a 3' end. In a polynucleotide molecule, the 3' carbon of the first nucleotide is ligated to the 5' carbon of the next nucleotide. Unless otherwise specified, it will be understood by those skilled in the art that a polynucleotide sequence is read in the 5' through 3' direction.

[0115] The term “polynucleotide variant” refers to a polynucleotide that exhibits substantial sequence identity with a reference polynucleotide sequence, or a polynucleotide that hybridizes with a reference sequence under stringent conditions. This term also encompasses polynucleotides distinguished from a reference polynucleotide by the addition, deletion, or substitution of at least one nucleotide. Therefore, 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 modifications, including mutations, additions, deletions, and substitutions, can be made to a reference polynucleotide, thereby allowing the modified polynucleotide to retain the biological function or activity of the reference polynucleotide. The term “polynucleotide variant” also includes naturally occurring allele variants. Terms such as “peptide variant” and “polypeptide variant” 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 certain examples, a peptide or polypeptide variant is distinguished from a reference peptide or polypeptide by one or more substitutions, which may be conserved or non-conserved. In certain cases, peptide or polypeptide variants include conservative substitutions, and in this regard, it is well understood in the art that some amino acids may be replaced with other amino acids having substantially 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 are added or deleted, or substituted with different amino acids.

[0116] As used herein, “oligonucleotide” or “oligonucleotide molecule” refers to a single-stranded polynucleotide molecule that can be synthesized to have a sequence of interest specified by the user. “Primer” is an example of an oligonucleotide molecule. Typically, at least a portion of an oligonucleotide molecule is specifically or completely complementary to the desired polynucleotide sequence and hybridizes to a specifically complementary single-stranded polynucleotide molecule. Oligonucleotide molecules are typically considered to be short polynucleotide molecules, but their lengths can vary. Their lengths may be suitable for use in at least one of a variety of applications, including polymerase chain reaction (PCR) based applications, sequencing applications, molecular cloning, and molecular probes.

[0117] As used herein, the terms “prevent,” “prevent,” and “prevent” in relation to a given infectious disease and / or disease or condition resulting from an infectious disease will be understood to mean that, even if the subject is exposed to the pathogenic organism causing the infectious disease, disease, or condition, the likelihood of developing that infectious disease and / or disease or condition is low. A low likelihood of developing an infectious disease and / or disease or condition will be understood to encompass both a reduced likelihood and an absence of likelihood.

[0118] As used herein, the term “bacterial strain” is used to refer to a population of bacteria (e.g., a bacterial culture) in which the majority or substantially all bacteria in the population originate from a single bacterial organism. Thus, the majority or substantially all bacteria in the population are identical or very similar to one another and share a particular phenotype.

[0119] As used herein, the term “Streptococcal bacterial strain” refers to a bacterial strain belonging to the family Streptococcus.

[0120] 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” may be mammals, e.g., humans or non-human mammals (e.g., pigs, cats, dogs, cattle, horses, or sheep). The scope of this term also includes laboratory animals (e.g., rodents, rabbits, etc.), birds (e.g., poultry), fish, and crustaceans.

[0121] The term "synthesis" applied to polynucleotide molecules is intended to mean that the polynucleotide molecules are produced in vitro, including by using bacterial amplification methods.

[0122] "Therapeutic dose" refers to the minimum concentration or amount necessary to produce at least a measurable improvement in a particular disease or condition. The therapeutic dose as used herein may vary depending on factors such as the patient's disease state, age, sex, and weight. The therapeutic dose is also defined as the amount in which the beneficial effect therapeutically outweighs any toxic or adverse effects.

[0123] As used herein, the terms “to treat” and “to treat” in relation to a given infectious disease and / or a disease or condition resulting from the infectious disease will be understood to include reducing the number of pathogenic organisms infecting the subject and / or alleviating any symptoms of the infectious disease and / or the symptoms of a disease or condition resulting from the infectious disease.

[0124] 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 that nucleic acid, protein, or microorganism, and the biological functions they may have. [Modes for carrying out the invention]

[0125] The following detailed description conveys exemplary embodiments of the Disclosure in sufficient detail to enable a person skilled in the art to implement the Disclosure. The features and limitations of the various embodiments described herein are not necessarily limited to other embodiments of the Disclosure or the Disclosure as a whole. Therefore, the following detailed description is not intended to limit the scope of the Disclosure as defined by the claims.

[0126] Therefore, immunity induced by commercially available streptococcal vaccines is insufficient to establish broad immunity against most streptococcal species and / or streptococcal serotypes within a particular pathogenic species.

[0127] Vaccination with purified LytC has been shown to result in elevated immunoglobulin G (IgG) antibodies and enhanced protection against pneumococcal infections in mice (Wisemann et al., 2001; Corsini et al., 2021). Furthermore, high titers of LytC-specific IgG antibodies have been observed in serum samples from healthy volunteers in multiple studies, indicating that the antibody response is induced as a result of natural exposure to Streptococcus pneumoniae (S. pneumoniae).

[0128] This disclosure relates to the unexpected finding that the use of attenuated or dead whole-cell streptococcal vaccines, in which the lytC gene is modified to attenuate, reduce, and / or prevent functional LytC expression, results in improved vaccine immunogenicity. This disclosure provides an immunogenic composition comprising attenuated and / or dead streptococcal bacterial strains, including modifications that attenuate, reduce, or prevent functional LytC expression.

[0129] Surprisingly, compared to the parental strain with functional LytC expression, mice immunized with a Streptococcus bacterial strain lacking lytC produced IgG antibodies with enhanced binding to the surface of the encapsulated Streptococcus pneumoniae strain. Furthermore, antibodies induced by the lytC-deficient strain showed a higher functional opsonization phagocytic response, a recognized surrogate endpoint for pneumococcal vaccine efficacy. These findings unexpectedly suggest that the immunogenicity of whole-cell vaccines may be improved by deleting the lytC gene, something that could not have been predicted based on previous studies.

[0130] This disclosure provides compositions that can induce cross-protective (i.e., heteromorphic) immunity against different streptococcal species and / or different streptococcal serotypes. In one embodiment, the composition of this disclosure contains dead streptococcal bacteria. In one embodiment, the composition is an attenuated vaccine. The composition can also induce immunity against a wide range of streptococcal species and / or serotypes and thus can reduce the potential effects of serotype substitution.

[0131] This specification also provides the compositions of the present disclosure, as well as pharmaceuticals and methods for producing pharmaceutical compositions, including immunogenic compositions or vaccine compositions.

[0132] This disclosure also relates to a method for preventing or treating streptococcal infections in subjects. The method comprises administering the composition of this disclosure to a subject. The composition may be administered for prophylactic or therapeutic purposes. The method may induce cross-protective immunity against multiple different streptococcal species and / or serotypes in a subject.

[0133] streptococcus bacteria Streptococci are a genus of cocci belonging to the family Streptococcus. There are numerous species of streptococci, some of which cause disease in humans and / or animals. Others are important in the production of various fermented products.

[0134] Individual streptococcal species are divided into two main groups based on their hemolytic properties (alpha-hemolytic and beta-hemolytic). Alpha-hemolytic streptococci include Streptococcus pneumoniae and Streptococcus viridans. The beta-hemolytic group consists of Group A and Group B streptococci. Group B streptococci usually inhabit the digestive system and the vagina in women and do not cause adverse effects. Although they can cause serious infections in newborns, most people rapidly develop innate immunity to Group B streptococci. Group A streptococci usually inhabit the throat and skin surface and are one of the common causes of infections in adults and children. Group A infections usually do not pose a significant threat to health (e.g., pharyngeal infections, cellulitis, impetigo, sinusitis, and middle ear infections), but Group A Streptococcus can invade deeper tissues and organs of the body, potentially establishing more severe invasive infections (e.g., pneumonia, sepsis, meningitis, and necrotizing fasciitis), as well as inducing serious complications including post-streptococcal glomerulonephritis and acute rheumatic fever.

[0135] Furthermore, the intestines contain numerous species of enterococcal (fecal) streptococci, which can cause endocarditis and urinary tract infections.

[0136] Streptococcus pneumoniae (also known as pneumococcus) is a significant human pathogen responsible for considerable morbidity and mortality. It causes serious conditions including pneumonia, meningitis, sinusitis, bacteremia, and otitis media. It is estimated that 1.6 million people worldwide die each year from invasive pneumococcal disease, approximately 1 million of whom are children. There are numerous serotypes (over 100) of Streptococcus pneumoniae, which are distinguished based on the chemical structure of their capsules and their immunogenicity. Since there are virtually no non-capsule strains of Streptococcus pneumoniae, the cause of invasive pneumococcal disease, and antibodies against capsular polysaccharides are protective against infection by homologous serotypes of Streptococcus pneumoniae, capsular polysaccharides are considered to be an essential pathogenic factor for Streptococcus pneumoniae. Therefore, capsular polysaccharides are used as vaccine antigens in pneumococcal vaccines currently in use.

[0137] Streptococcal bacterial strains of the composition The immunogenic compositions of this disclosure are based on attenuated or completely dead streptococcal bacterial strains, including modifications that reduce, decrease, or prevent the functional expression of LytC (or its homologues). The modifications can be introduced into any suitable streptococcal bacterial strain.

[0138] Streptococcal bacterial strains may be alpha-hemolytic, beta-hemolytic, or gamma-hemolytic streptococci, classified according to, for example, well-defined hemolytic properties, or, in the case of gamma-hemolytic streptococci, the absence of such properties.

[0139] Non-limiting examples of suitable alpha-hemolytic streptococcal bacteria include Streptococcus pneumoniae and green streptococci (e.g., S. mutans, S. sanguinis, S. mitis, S. oralis, S. sobrinus, S. milleri). Also included within the scope of this disclosure are individual serotypes of the above streptococcal species.

[0140] Non-limiting examples of suitable β-hemolytic streptococci include those classified by the Lancefield classification system based on the carbohydrate composition of bacterial antigens (polysaccharides) in the cell wall (groups A-H, L, N, and R / S). For example, β-hemolytic bacteria include Streptococcus pyogenes (group A), Streptococcus agalactie (group B), Streptococcus equisimilis (group C), Streptococcus equi (group C), Streptococcus zooepidemicus (group C), Streptococcus dysgalactiae (group C), and Enterococcus faecalis. Examples include one or more of the following: Streptococcus faecalis (Group D), Streptococcus bovis (Group D), Streptococcus milleri (Group E), Streptococcus mutans (Group E), Streptococcus anginosus (Group F), Streptococcus canis (Group G), Streptococcus dysgalactiae (Group G), Streptococcus sanguis (Group H), Streptococcus dysgalactiae (Group L), Lactococcus lactis (Group N), and Streptococcus suis (Group R / S). Also included within the scope of this disclosure are the individual serotypes of the above streptococcal species.

[0141] In one embodiment, the attenuated and / or dead streptococcal bacterial strains of the Disclosure include Streptococcus agalactiae, Streptococcus bovis, Streptococcus canis, Streptococcus dysgalactiae, Streptococcus equi, Streptococcus equinus, Streptococcus equisimilis, Enterococcus faecalis, Enterococcus faecium, Streptococcus iniae, and Streptococcus milleri. These are species selected from the group consisting of Streptococcus milleri, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus salivarius, Streptococcus sanguinis, Streptococcus suis, and Streptococcus uberis.

[0142] In one embodiment, the attenuated and / or dead streptococcal bacterial strain of the present disclosure comprises one or more serotypes of Streptococcus pneumoniae. Therefore, the composition comprises 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 , may include 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.

[0143] 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 dead streptococcal bacterial strains of this disclosure may include streptococcal bacteria or serotypes not specifically enumerated herein. In one embodiment, the composition of this disclosure comprises one or more of the serotypes of Streptococcus pneumoniae (S. pneumoniae) 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.

[0144] Streptococcus bacterial derivatives In one embodiment, the streptococcal bacterial strain is a streptococcal derivative strain.

[0145] Streptococcal strains may be recombinant streptococcal bacteria resulting from artificial genetic manipulation, or naturally occurring mutant streptococcal bacteria. While not limited to these, streptococcal strains may include one or more genetic modifications that reduce pathogenicity.

[0146] As a merely non-limiting example, streptococcal derivatives may include genetic modifications that disrupt or remove capsular loci (cps). For example, one or more of the cpsA, cpsB, cpsC, cpsD, and / or cpsE genes of Streptococcus pneumoniae, or homologous genes of other streptococcal species, may be modified (e.g., by recombination) to prevent, disrupt, or alter capsule production. Alternatively, streptococcal derivatives may have spontaneous mutations in these genes or other genes that result in naturally occurring non-capsular streptococcal bacteria. Streptococcal derivatives may lack all or at least some of the capsular loci.

[0147] In one embodiment, the capsule-less streptococcal derivative is the Streptococcus pneumoniae Rx1 strain or an Rx1 derivative.

[0148] In addition to or instead of the above, streptococcal derivatives may include genetic modifications that reduce or prevent the production or activity of other target proteins. These include, but are not limited to, one or more genes encoding choline-binding proteins, one or more genes encoding additional autolytic enzymes in addition to lytC (e.g., homologous genes of Streptococcus pneumoniae lytA or lytB, or other streptococcal bacteria), one or more genes conferring nutritional / cofactor (e.g., metal ions) requirements for growth (e.g., homologous genes of Streptococcus pneumoniae psaA or other streptococcal bacteria), or protective antigens (e.g., Streptococcus pneumoniae Genetic alterations may exist in ae)pspA or homologous genes of other streptococcal bacteria, and / or one or more genes encoding toxicity determinants or regulators (e.g., codY, comC, comD, cps2A, csp4A, glpO, mgrA, nanA, nanB, pavA, pcpA, phtA, phtB, phtD, phtE, piuA, piaA, ply, prtA, psaA, psrP, rrgA, rrgB, spxB of Streptococcus pneumoniae, and homologs of these genes of other streptococcal bacteria).

[0149] In addition, or instead, streptococcal derivatives may include genetic modifications that result in nutrient requirements with reduced in vivo pathogenicity and / or growth. As a merely non-limiting example, genetic modifications may be present in one or more genes encoding thymidylate synthase.

[0150] In addition, or instead, a streptococcal strain may contain one or more (external) genes derived from streptococcal bacteria of the same species but different serotypes, streptococcal bacteria from different species, non-streptococcal bacteria, or from humans or non-human mammals (e.g., pigs, cats, dogs, cattle, horses, or sheep), experimental animals (e.g., rodents or rabbits), birds, and / or subjects to whom recombinant streptococcal bacteria are administered. In one embodiment, one or more external genes disrupt or otherwise inactivate one or more endogenous genes (e.g., any one or more genes described in the preceding paragraph). In other embodiments, 1 One or more external genes do not disrupt or inactivate endogenous genes. As a merely non-limiting example, one or more external genes encode proteins that induce or enhance the immune response of a target to which a streptococcal strain is administered. The immune response may be an innate immune response, an adaptive immune response, 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 external genes encode antigens of bacteria from another different family (e.g., Mycoplasma pneumoniae). It may include pneumoniae antigens, Haemophilus influenzae antigens, Chlamydophila pneumoniae antigens, Moraxella catarrhalis antigens, Staphylococcus aureus antigens, viral antigens (e.g., adenovirus antigens, coronavirus antigens, influenza virus antigens, parainfluenza virus antigens, metapneumovirus antigens, rhinovirus antigens, respiratory syncytial virus antigens, HIV antigens, hepatitis virus antigens or herpesvirus antigens, measles virus antigens, mumps virus antigens, papillomavirus antigens, rubella virus antigens, varicella-zoster virus antigens), fungal / yeast antigens, helminthic antigens, and / or protozoan antigens.

[0151] In addition, or instead, streptococcal derivatives may include genetic modifications that cause the bacterium to overexpress one or more target genes. In this context, “overexpression” is understood to mean that, in the corresponding streptococcal bacterium, the expression level is increased compared to the expression of the same gene without genetic modification under the same biological conditions. Overexpression of a given target gene may induce or enhance an immune response in a target, for example, against the parent streptococcal strain of the administered streptococcal derivative and / or against the streptococcal derivative itself. As a merely non-limiting example, genetic modification may increase the production of one or more genes (e.g., cbpA, pspA, ply in Streptococcus pneumoniae, or homologs of these genes in other streptococcal bacteria) in a streptococcal derivative that encodes proteins capable of activating the complement system.

[0152] In addition, or instead, the streptococcal strain may include genetic modifications that result in defective DNA repair capabilities. In one embodiment, the use of compositions of the Disclosure, including a photon-irradiated (e.g., gamma-ray and / or X-ray) streptococcal strain with reduced ability to repair DNA damage resulting from photon irradiation, may be advantageous insofar as it can reduce the dose of photon irradiation required for attenuation or inactivation, while simultaneously increasing the efficacy and safety of the vaccine. In one embodiment, the streptococcal strain includes genetic modifications that interfere with or inactivate the expression of one or more genes encoding proteins in the mismatch repair system (e.g., the hex locus of Streptococcus pneumoniae or homologs of this locus in other streptococcal bacteria). In other embodiments, the streptococcal derivative strain includes genetic modification that interferes with or inactivates the expression of one or more genes encoding DNA alkylation repair proteins (e.g., DNA polymerase 4, hexA, hexB, mutS, radC, recA, recF, recN, recO, uvrA, uvrB, uvrC, uvrD of Streptococcus pneumoniae, or homologs of these genes of other streptococcal bacteria).

[0153] In addition, or instead, streptococcal strains may include genetic modifications that promote the production of double-stranded RNA (dsRNA). dsRNA may be mRNA or tRNA. The length of dsRNA may be, but is not limited to, greater than 10 base pairs, greater than 15 base pairs, greater than 20 base pairs, greater than 25 base pairs, greater than 30 base pairs, greater than 35 base pairs, greater than 40 base pairs, greater than 45 base pairs, greater than 50 base pairs, greater than 55 base pairs, greater than 60 base pairs, greater than 65 base pairs, or greater than 70 base pairs. In addition, or instead, the length of dsRNA can be approximately 10 to approximately 70 base pairs (bp), approximately 10 to approximately 50 base pairs (bp), approximately 10 to approximately 30 base pairs (bp), approximately 20 to approximately 70 base pairs (bp), approximately 20 to approximately 60 base pairs (bp), approximately 20 to approximately 50 base pairs (bp), approximately 20 to approximately 40 base pairs (bp), approximately 20 to approximately 30 base pairs (bp), approximately 30 to approximately 70 base pairs (bp), approximately 40 to approximately 70 base pairs (bp), approximately 50 to approximately 70 base pairs (bp), approximately 60 to approximately 70 base pairs (bp), approximately 30 to approximately 60 base pairs (bp), approximately 30 to approximately 50 base pairs (bp), or approximately 30 to approximately 40 base pairs (bp). In one embodiment, dsRNA is a component of another single-stranded, larger RNA molecule. Larger RNA molecules may contain multiple dsRNAs as components. dsRNAs can be internal or terminal components of larger RNA molecules. In one embodiment, a dsRNA may include a stop stem-loop sequence. dsRNAs may arise from self-complementary regions within larger RNA molecules. A coding region / exon(s) in a given gene of a streptococcal strain can be manipulated to include one or more self-complementary regions, thereby generating a dsRNA portion during transcription.

[0154] dsRNA may be recognizable by a Toll-like receptor (TLR) protein expressed by the target cells to which the streptococcal strain is administered. The TLR protein may be located in the endoplasmic reticulum and / or endosomal compartment of the cell. The TLR protein may be Toll-like receptor 3 (TLR3) protein. The cells may be one or more of the following, but are not limited to B lymphocytes, T lymphocytes, natural killer cells, and / or dendritic cells. Recognition of dsRNA by the TLR3 protein may induce an immune response in the target. The immune response may be an innate immune response. The immune response may be a type 1 interferon response and / or include the release of inflammatory cytokines.

[0155] In general, streptococcal derivatives used in the compositions of this disclosure have a significant degree of genetic similarity to the parental strains from which they are derived. As a non-limiting example, the “streptococcal derivatives” referred to herein may have sequence homology of more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 92%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% to the parental streptococcal strain from which they are derived. As a further non-limiting example, the “streptococcal derivatives” referred to herein may include genetic modifications in one, two, three, four, five, or more than five genes, or in the regulatory sequences necessary for the expression of those genes, when compared to their corresponding parental strains. Genetic modifications may increase, decrease, or prevent the expression of one or more of the genes in question.

[0156] Techniques for genetic manipulation of bacteria are well-known to those skilled in the art (for example, Vennison "Laboratory Manual for Genetic Engineering", PHI Learning Pvt. Ltd., 2010; Zyskind and Bernstein, "Recombinant DNA Laboratory Manual", Elsevier, 2014; Bose, "Genetic Manipulation of Staphylococci" in "Methods in Molecular Biology", Springer Protocols, volume 1106, pages 101 - 111, 2014; Hakenbeck and Chhatwal, "Molecular Biology of Streptococci", Horizon Scientific Press, 2007; Morona et al., "The effect that mutations in the conserved capsular polysaccharide biosynthesis genes cpsA, cpsB and cpsD have on virulence of Streptococcus pneumoniae", J. Infect. Dis. 189:1905 - 1913, 2004; Morona et al., "Mutational analysis of the carboxy-terminal [YGX]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", Mol. Microbiol. 53:889 - 901, 2004; mahdi et al.See "Identification of a novel pneumococcal vaccine antigen preferentially expressed during meningitis in mice," J. Clin. Invest. 122:2208-2220, 2012.

[0157] In one non-limiting embodiment, the attenuated and / or dead streptococcal bacterial strain includes Streptococcus pneumoniae derived from Rx1. Rx1 is a capsule-negative mutant derived from Streptococcus pneumoniae serotype 2 D39 strain.

[0158] In one embodiment, the attenuated and / or dead streptococcal bacterial strain further comprises a modification or deletion of a gene selected from the group consisting of lytA, ply, and psaA.

[0159] In one embodiment, the attenuated and / or dead streptococcal bacterial strain further comprises modification or deletion of the lytA gene. In the Rx1 derivative strain, the autolytic enzyme gene (lytA) gene may be deleted or rendered non-functional.

[0160] In addition, or instead, the pneumorylsin gene (ply) may be deleted or rendered non-functional in the Rx1 derivative. For example, the ply gene may be replaced with another gene, such as a gene encoding a toxoid type of ply. In one embodiment, the attenuated and / or dead streptococcal bacterial strain further comprises modification or deletion of the ply gene. In one embodiment, the attenuated and / or dead streptococcal bacterial strain further comprises a toxoid type of ply called PdT.

[0161] In one embodiment, modifications that attenuate, reduce, or prevent functional LytC expression can be introduced into Rx1(ΔLytA,PdT). GPN-001 described herein is an example of an Rx1(ΔLytA,PdT) strain. PCT International Publication 2016 / 149771 describes a method for inducing a strain with ΔLytA,PdT modifications, and this document is incorporated herein by reference in its entirety.

[0162] In addition, or instead, the pneumococcal surface antigen A gene (psaA) gene may be deleted or otherwise rendered non-functional. The psaA gene of Streptococcus pneumoniae is Mn 2+ It encodes pneumococcal surface antigen A (PsaA), which is involved in transport and resistance to oxidative stress. The psaA-deletion mutants described herein have low Mn 2+ It may have defects in growth in the environment and / or may be deficient in pneumococcal competence.

[0163] In one embodiment, modifications that attenuate, reduce, or prevent functional LytC expression can be introduced into Rx1(ΔLytA,PdT). GPN-002 described herein is an example of an Rx1(ΔLytA,PdT,ΔPsaA) strain. U.S. Patent Application Publication 2021-0361757 describes a method for inducing a strain having a ΔPsaA modification, and the above document is incorporated herein by reference in its entirety. Such strains have low Mn 2+ In the environment, it may exhibit reduced toxicity, reduced competence, and / or reduced growth. Furthermore, strains with ΔLytA, PdT, and ΔpsaA modifications are Mn 2+ Growing organisms in fermenters under stress conditions can induce changes in gene expression that increase the production level of protective antigens. Therefore, using immunogenic compositions with higher levels of protective antigen expression may result in improved levels of protection.

[0164] In one embodiment, the attenuated and / or dead streptococcal bacterial strain further comprises a modification or deletion of the lytA gene, a modification or deletion of the ply gene, and a modification or deletion of the psaA gene, and further comprises a toxoid type ply called PdT.

[0165] LytC This disclosure relates to immunogenic compositions comprising attenuated and / or dead streptococcal bacterial strains, including modifications that reduce, decrease, or prevent the functional expression of LytC (or its homologues). In one embodiment, the modifications reduce, decrease, or prevent the functional expression of LytC.

[0166] LytC is a cell wall hydrolase that, along with LytA, is proposed to be involved in fratricide (fratricide) by promoting the lysis of non-competent Streptococcus pneumoniae cells (Eldhom et al., 2009). LytC also plays some role in biofilm formation and adhesion to the nasopharyngeal epithelium. LytC enzyme activity is highest at 30°C, suggesting that its physiological role may be to promote colonization of Streptococcus pneumoniae in the upper respiratory tract (Tomasz et al., 1975). Given its surface localization and importance in key toxic traits, LytC has been investigated as a streptococcal vaccine candidate in animal studies. Vaccination with purified LytC has confirmed the protein's potential as a protective immunogen, resulting in high immunoglobulin (IgG) antibodies in immunized mice and enhanced protection against pneumococcal infections in mice (Wisemann et al., 2001; Corsini et al., 2021). Furthermore, high titers of LytC-specific IgG antibodies have been observed in serum samples from healthy volunteers in multiple studies, indicating that an antibody response is induced as a result of natural exposure to Streptococcus pneumoniae (holmlund et al. 2009; Croucher et al., 2017).

[0167] Modifications to LytC can be any suitable modification, as long as they attenuate, reduce, or prevent the functional expression of LytC (or its homologs). For example, the lytC gene can be modified using any suitable method known to those skilled in the art. For example, the gene may be modified as described herein, but it should be understood that the compositions of this disclosure may also be prepared using other suitable methods for modifying the lytC gene. Techniques for genetically modifying bacteria are well known to those skilled in the art, as described elsewhere in this specification.

[0168] In one embodiment, the modification may prevent the functional expression of LytC (or its homolog). For example, the gene may be modified to prevent the expression of LytC (or its homolog) by introducing a nonsense mutation such that translation of LytC is terminated, and as a result any protein expressed from the modified gene is shortened and nonfunctional. Alternatively, the gene may be deleted, for example, using the method described herein. In one embodiment, the gene may be modified so that the translated protein is a variant with reduced or attenuated function. Alternatively, for example, the expression level of LytC may be reduced or attenuated by modifying a regulatory sequence that promotes the expression level of LytC (or its homolog), as will be understood by those skilled in the art.

[0169] Any streptococcal bacterial strain possessing the lytC gene or its homolog can be modified to attenuate, reduce, or prevent the functional expression of LytC (or its homolog). In one embodiment, the modification of lytC or its homologue is performed on Streptococcus agalactiae, Streptococcus bovis, Streptococcus canis, Streptococcus dysgalactiae, Streptococcus equi, Streptococcus equinus, Streptococcus equisimilis, Enterococcus faecalis, Enterococcus faecium, Streptococcus iniae, and Streptococcus milleri. This can be performed on any suitable serotype of a species selected from the group consisting of Streptococcus milleri, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus salivarius, Streptococcus sanguinis, Streptococcus suis, and Streptococcus uberis.

[0170] In one embodiment, a weakened and / or dead streptococcal bacterial strain, which includes modifications to reduce, decrease, or prevent functional LytC expression, is the Rx1(ΔlytA, PdT, ΔpsaA, ΔlytC) strain. GPN-003 described herein is an example of the Rx1(ΔlytA, PdT, ΔpsaA, ΔlytC) strain. A method for inducing a strain having ΔlytC is described herein.

[0171] Target streptococcal species and / or their serotypes In one embodiment, the composition of the present invention induces an immune response against at least one target streptococcal species and / or its serotype. The target streptococcal species and / or its serotype may be pathogenic bacteria capable of establishing a harmful infection in a host organism.

[0172] In one embodiment, at least one target streptococcal bacterial species and / or its serotype is Streptococcus agalactiae, Streptococcus bovis, Streptococcus canis, Streptococcus dysgalactiae, Streptococcus equi, Streptococcus equinus, Streptococcus equisimilis, Enterococcus faecalis, Enterococcus faecium, Streptococcus iniae, Streptococcus milleri The group is selected from the following: Streptococcus milleri, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus salivarius, Streptococcus sanguinis, Streptococcus suis, and Streptococcus uberis.

[0173] In one embodiment, at least one target streptococcal bacterial species and / or its serotype of the present disclosure comprises one or more serotypes of Streptococcus pneumoniae. Thus, the composition comprises 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 , may include 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.

[0174] 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 dead streptococcal bacterial strains of this disclosure may include streptococcal bacteria or serotypes not specifically enumerated herein.

[0175] 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 its 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.

[0176] Culture of Streptococcal bacterial strains The attenuated and / or dead streptococcal bacterial strains of this disclosure can be cultured using any suitable standard technique known to those skilled in the art. For example, the attenuated and / or dead streptococcal bacterial strains can be cultured under standard conditions in a variety of suitable media, such as Soytone broth or Todd-Hewitt Yeast (THY) broth, for a suitable period of time until a desired cell density or optical density is obtained.

[0177] Death of streptococcal bacterial strains The streptococcal bacterial strains of this disclosure may be killed using any suitable method known in the art, provided that the killed bacteria are suitable for use in the compositions of this disclosure. For example, the streptococcal bacterial strains may be killed by chemical treatment, heat treatment, irradiation, high hydrostatic pressure, pulsed electric field, ultrashort pulse laser, ultrasound under pressure, UV irradiation, or a suitable protocol for microbial inactivation. In one embodiment, the streptococcal bacterial strains of this disclosure may be killed by photon irradiation.

[0178] Photon beam emission The Streptococcus bacteria in the compositions of the present disclosure may be exposed to photon beam radiation. As noted above, the term "photon beam radiation" is understood to encompass both gamma ray radiation (i.e., gamma rays) and x-ray radiation (i.e., x-rays). Thus, a "photon beam irradiated" Streptococcus bacterial strain of the present disclosure can be in a "gamma ray irradiated" state by exposure to gamma ray radiation (i.e., gamma rays), an "x-ray irradiated" state by exposure to x-ray radiation (i.e., x-rays), or both. As known to those skilled in the art, x-rays are identical to gamma rays except that x-rays are emitted when electrons pass through the electric field of the nucleus rather than the nucleus itself during radioactive decay. By way of merely non-limiting examples, to put a material in a photon beam irradiated state, the material can be subjected to photon beam radiation having an energy of at least 0.01 MeV, at least 0.1 MeV, at least 0.5 MeV, between 0.01 MeV and 0.5 MeV, between 0.01 MeV and 1 MeV, between 0.01 MeV and 10 MeV, between 0.5 MeV and 20 MeV, between 0.5 MeV and 15 MeV, between 0.5 MeV and 10 MeV, between 0.5 MeV and 5 MeV, between 0.5 MeV and 2 MeV, or between 1 MeV and 2 MeV (e.g., 1.25 MeV).

[0179] The Streptococcus bacteria in the compositions of the present disclosure can be gamma ray irradiated. Any suitable gamma ray radiation source can be used. Suitable gamma emitters include, but are not particularly limited to, Ba 137 Co 60 Cs 137 Ir 192 U 235 Se 75 and Yb 169 are included.

[0180] The gamma irradiation of streptococcal bacteria described herein can be carried out using commercially available equipment, such as Gammacell irradiation devices from Atomic Energy of Canada Ltd., Canada (e.g., Gammacell 40, Gammacell 220, Gammacell 1000, Gammacell 3000), gamma irradiation devices from JL Shepherd and Associates (San Fernando, California, USA), or Nordion Gamma Cell-1000 irradiation devices from Nordion Inc. (Kanata, Ontario, Canada). Other suitable equipment is described, for example, in U.S. Patent Nos. 3,557,370 and 3,567,938.

[0181] In addition, or alternatively, the Streptococcus bacteria in the vaccine of the present invention may be irradiated with X-rays. Any suitable X-ray source can be used. Suitable X-ray sources include, but are not limited to, the eXelis® germicidal X-ray equipment manufactured by IBA Industrial (Louvain-la-Neuve, Belgium). Other suitable equipment includes, for example, the RS2400® and RS3400® manufactured by Rad Source Technologies Inc. (Suwanee, Georgia, USA).

[0182] Generally, the streptococcal bacteria of the present disclosure are exposed to a dose of photon radiation (e.g., gamma-ray radiation and / or X-ray radiation) sufficient to attenuate or inactivate the streptococcal bacterial strains of the present disclosure. Preferably, the dose of photon radiation is sufficient to attenuate or inactivate the bacteria without substantially destroying the structure of the antigen (e.g., surface antigen). Thus, the immunogenicity of the antigenic determinant can be retained by the photon-irradiated bacteria and their derivatives. Preferably, the dose of photon radiation is administered to the bacteria for a sufficient period and at a sufficient level to ensure that all streptococcal bacterial strains of the present disclosure being treated are exposed without adversely affecting the structural integrity of the antigenic determinant.

[0183] As is known to those skilled in the art, the unit of measurement for absorbed radiation dose is the gray (Gy), which is defined as the energy of one joule stored in one kilogram of mass. The older unit of measurement for this is the rad, which represents "absorbed radiation dose," with 1 Gy = 100 rad.

[0184] Approximately 1 × 10⁻⁶ streptococcal bacteria for use in accordance with this disclosure 3 rad and approximately 2×10 9 rad (or approximately 10 Gy ~ approximately 2 × 10) 4 They may be exposed to photon radiation (e.g., gamma-ray radiation and / or X-ray radiation) with a total dose in the range of kGy. In certain embodiments of this disclosure, streptococcal bacteria or derivative strains may be exposed to about 1 × 10⁻⁶ 3 rad ~ approximately 2 × 10 9 rad, approximately 1 x 10 3 rad ~ approximately 1 × 10 9 rad, approximately 1 x 10 3 rad ~ approximately 1 × 10 8 rad, approximately 1X × 10 3 rad ~ approximately 1 × 10 7 rad, approximately 1 x 10 3 rad ~ approximately 1 × 10 6 rad, approximately 1 x 10 3 rad ~ approximately 1 × 10 5 rad, approximately 1 x 10 3 rad ~ approximately 1 × 10 4 rad, approximately 1 x 10 3 rad ~ approximately 2 × 10 9 rad, approximately 1 x 10 4 rad ~ approximately 2 × 10 9 rad, approximately 1 x 10 5 rad ~ approximately 2 × 10 9 rad, approximately 1 x 10 6 rad ~ approximately 2 × 10 9 rad, approximately 1 x 10 7 rad ~ approximately 2 × 10 9 rad, approximately 1 x 10 8 rad ~ approximately 2 × 10 9 rad, or approximately 1 × 10 9 rad ~ approximately 2 × 10 9Exposure to a total dose of X-rays and / or gamma rays in rads.

[0185] In one embodiment of the present disclosure, streptococcal bacteria are measured in approximately 6.5 × 10⁻⁶ units. 4 rad ~ approximately 2 × 10 7 Exposure to a total dose of photon radiation (e.g., X-ray radiation and / or gamma-ray radiation) in rad (approximately 0.65 kGy to approximately 200 kGy). In other embodiments of the present disclosure, the streptococcal bacterial strains of the present disclosure are subjected to approximately 10kGy to approximately 12kGy, approximately 12kGy to approximately 14kGy, approximately 14kGy to approximately 16kGy, approximately 10kGy to approximately 20kGy, approximately 14kGy to approximately 20kGy, approximately 20kGy to approximately 30kGy, approximately 20kGy to approximately 25kGy, approximately 25kGy to approximately 30kGy, approximately 30 to 35kGy, approximately 10kGy, approximately 11kGy, approximately 12kGy, approximately 13kGy, approximately 14kGy, approximately 15kGy, approximately 16kGy, approximately 17kGy, approximately 18kGy, approximately 19kGy, approximately 20kGy, Approximately 21kGy, approximately 22kGy, approximately 23kGy, approximately 24kGy, approximately 25kGy, approximately 26kGy, approximately 27kGy, approximately 28kGy, approximately 29kGy, approximately 30kGy, approximately 31kGy, approximately 32kGy, approximately 33kGy, approximately 34kGy, approximately 35kGy, approximately 20kGy, approximately 20kGy, approximately 12kGy to over approximately 14kGy, over 12kGy, over 14kGy, over 16kGy, over 18kGy, over 20kGy, over 22kGy, over 24kGy, over 26kGy, over 28kGy, over 30kGy, 35kGy, over 40kGy, 1.26 × 10 6 The total photon beam emission is rad (12.6 kGy), approximately 1 × 10⁻¹⁶. 6 The total photon radiation dose of a photon beam of rad (approximately 10 kGy), or approximately 1 × 10⁻¹⁶ 5 The subject is exposed to a total photon beam emission of rad (1 kGy).

[0186] The optimal dose of photon radiation (e.g., gamma-ray radiation and / or X-ray radiation) may be influenced by factors such as the culture medium in which the streptococcal bacteria of this disclosure are present, the number of bacteria to be treated, the temperature of the bacteria to be treated (e.g., frozen on dry ice or at room temperature), the availability of water, the availability of oxygen, and / or the subtype or strain being treated. Therefore, the effectiveness of the treatment can be enhanced by optimizing the total dose of photon radiation applied over the exposure period, the exposure time, and / or the level of photon radiation.

[0187] The total dose of photon radiation (e.g., X-ray radiation and / or gamma-ray radiation) may be administered to the streptococcal bacterial strain of the Disclosure cumulatively over a period of time. For example, photon radiation may be administered to the streptococcal bacterial strain of the Disclosure at a level lower than the total dose for a period of time sufficient to achieve the required total dose of photon radiation.

[0188] In one embodiment, the streptococcal bacterial strain of the present disclosure is cultured in a suitable bacterial medium that supports the growth of streptococcal strains, such as THY broth or soytone broth, to a suitable cell density, for example, 10 4 ~10 8 A cell density of colony-forming units (CFU) / mL can be obtained. In one embodiment, the cell density is 10 7 ~10 8 This is expressed as colony-forming units (CFU) / mL. In one embodiment, the cell density is approximately 10 8The concentration is expressed as colony-forming units (CFU) / mL. Bacteria can be concentrated using techniques known to those skilled in the art, such as centrifugation and filtration. In one embodiment, bacteria are washed with a suitable solution such as PBS, but those skilled in the art will understand that various buffer solutions are suitable. Bacteria can be washed using techniques such as tangential flow filtration and diafiltration. In one embodiment, bacterial cells are recentrifugated 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 to 1 × 10⁶ 4 ~1 × 10 12 CFU / mL, e.g., 1 × 10 4 CFU / mL, 1 x 10 5 CFU / mL, 1 x 10 6 CFU / mL, 1 x 10 7 CFU / mL, 1 x 10 8 CFU / mL, 1 x 10 9 CFU / mL, 1 x 10 11 CFU / mL, or 1 × 10 12 The preparation is resuspended at a concentration of CFU / mL. In one embodiment, the concentration of the preparation is 1 × 10⁻⁶. 10 The concentration is 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. Alternatively, the washed bacterial cells may be freeze-dried using standard techniques known to those skilled in the art prior to photon irradiation.

[0189] In one embodiment, the streptococcal bacterial strain preparation of the Disclosure is maintained in a frozen and / or lyophilized state while being exposed to photon radiation (e.g., gamma-ray radiation and / or X-ray radiation). This promotes the preservation of the biological integrity of the antigen, avoids unnecessary damage to the antigen, and thereby can enhance the immunogenicity of the photon-irradiated bacterial preparation, particularly its ability to induce cross-reactivity / cross-protective immunity against, for example, heterologous subtypes. Generally, a photon radiation dose of 10–30 kGy (as described herein) may be effective for processing the frozen and / or lyophilized streptococcal bacterial strain preparation of the Disclosure. Alternatively, photon emission levels of 20–40 kGy (e.g., greater than 20 kGy, greater than 22 kGy, greater than 24 kGy, greater than 26 kGy, greater than 28 kGy, greater than 30 kGy, greater than 32 kGy, greater than 34 kGy, greater than 36 kGy, or greater than 38 kGy, etc.) may be effective for processing the frozen and / or lyophilized streptococcal bacterial strain preparations of this disclosure.

[0190] As described above, treatment by photon emission is preferably sufficient to inactivate the streptococcal bacterial strains of this disclosure without substantially destroying the structure of the bacterial antigen. The attenuation and / or inactivation of the streptococcal bacterial strains of this disclosure can be evaluated using methods generally known in the art.

[0191] For example, bacterial attenuation and / or inactivation can be evaluated by determining the number of viable bacteria (i.e., colony-forming units) that form colonies on an agar plate after treatment with photon beam radiation (e.g., gamma-ray radiation and / or X-ray radiation).

[0192] The integrity of antigenic determinants can be assessed, for example, by the reactivity of purified native antigenic components with a panel of monospecific antisera produced using Western blotting of surface components, FACs analysis, or enzyme assays.

[0193] Attenuated streptococcal strains with modified lytC In one embodiment, a bacterial strain comprising modifications that attenuate, reduce, or prevent the functional expression of LytC (or its homolog) is attenuated. That is, this strain is nonpathogenic and therefore does not cause disease when inoculated into mice as a live strain. In one embodiment, the composition of the present disclosure comprises an attenuated whole-cell immunogenic composition.

[0194] In one embodiment, the attenuated whole-cell immunogenic composition of this disclosure may be used as a live attenuated immunogenic composition or vaccine composition. In one embodiment, the attenuated immunogenic composition or vaccine composition induces an immune response against at least one target streptococcal species and / or its serotype. In one embodiment, the attenuated immunogenic composition or vaccine composition improves the efficacy of the immune response compared to an unmodified parent strain. In one embodiment, the attenuated streptococcal bacterial strain is derived from an unmodified parent strain, and the attenuated streptococcal bacterial strain has improved stability compared to the parent strain.

[0195] Immune response The compositions of this disclosure may induce an immune response when administered to a subject. In one embodiment, the composition may induce a heterogeneous immune response. In one embodiment, the composition may induce a cross-protective immune response.

[0196] In one embodiment, attenuated and / or dead streptococcal bacterial strains containing lytC modification may induce an immune response with improved efficacy compared to the parent strain. For example, the composition may induce increased protection from infection and / or disease compared to a composition containing the parent strain. In one embodiment, the composition of the Disclosure induces a higher total level of antibody. In one embodiment, the composition of the Disclosure induces a higher total level of IgG antibody. In one embodiment, the composition induces an increase or decrease in a specific subtype of immunoglobulin (e.g., IgG1, IgG2a, IgG2b, IgG3, IgG4, etc., selected from IgA, IgE, IgM, etc.). In one embodiment, the composition induces a change in the ratio of one immunoglobulin subtype compared to another immunoglobulin subtype, e.g., a change in the ratio selected from IgG1:IgG2a, IgG1:IgG2b, IgG1:IgG3, etc. The change may be an increase or a decrease. In one embodiment, the composition enhances the antibody response to opsonize and kill a target streptococcal species and / or its serotype. In one embodiment, the composition enhances the antibody response to opsonize and kill Streptococcus pneumoniae. In one embodiment, the composition of the present disclosure induces enhancement of cellular responses, such as a shift in Th1 / Th2 bias.

[0197] Preventive and therapeutic methods The present invention provides a preventive method for preventing streptococcal infections in subjects. The present invention also provides a therapeutic method for treating streptococcal infections in subjects. The method involves administering to a subject, for example, in the form of a vaccine of the present disclosure, an immunogenic composition or vaccine composition comprising an attenuated and / or dead streptococcal bacterial strain, which includes modifications that reduce, decrease, or prevent the functional expression of LytC (or its homologues).

[0198] These methods induce or enhance an immune response against a target streptococcal bacterium. The immune response may be cross-protective / heterogeneous, insofar as it can induce or enhance an immune response against multiple serotypes of the streptococcal bacterium. The methods may also include administering multiple different attenuated and / or dead streptococcal bacterial strains, including lytC modifications, thereby inducing immunity against multiple species of streptococcal bacteria and their various serotypes.

[0199] This method targets the following streptococcal species and / or their serotypes: Streptococcus agalactiae, Streptococcus bovis, Streptococcus canis, Streptococcus dysgalactiae, Streptococcus equi, Streptococcus equinus, Streptococcus equisimilis, Enterococcus faecalis, Enterococcus faecium, Streptococcus iniae, and Streptococcus milleri. It may induce or enhance an immune response against one or more of the following: Streptococcus milleri, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus salivarius, Streptococcus sanguinis, Streptococcus suis, and Streptococcus uberis.

[0200] In one embodiment, the method includes preventing or treating streptococcal infections caused by a target streptococcal species and / or its serotype by administering a composition containing a weakened and / or dead streptococcal bacterial strain, which includes a lytC modification of the same streptococcal species, to a target.

[0201] In one embodiment, the streptococcal species or serotype causing the infection may differ from attenuated and / or dead streptococcal bacterial strains, which include modifications that reduce, decrease, or prevent the functional expression of LytC (or its homologues).

[0202] As merely an unrestricted example, this method can be used to prevent or treat the following: (i) By administering an immunogenic composition comprising one or more attenuated and / or dead streptococcal bacterial strains, including modifications that reduce, decrease, or prevent the functional expression of LytC (or its homologues), infections, diseases, or conditions caused by any one or more serotypes of Streptococcus agalactiae can be prevented or treated, where the attenuated and / or dead streptococcal bacterial strains comprise one or more serotypes of Streptococcus agalactiae. The administered attenuated and / or dead Streptococcus agalactiae serotype(s) may differ from the serotype(s) causing the infection, disease, or condition; (ii) By administering an immunogenic composition comprising one or more attenuated and / or dead streptococcal bacterial strains, including modifications that reduce, decrease, or prevent the functional expression of LytC (or its homologues), infections, diseases, or conditions caused by any one or more serotypes of Streptococcus bovis can be prevented or treated, wherein the attenuated and / or dead streptococcal bacterial strains comprise one or more serotypes of Streptococcus bovis. The administered attenuated and / or dead Streptococcus bovis serotype(s) may differ from the serotype(s) causing the infection, disease, or condition; (iii) Infections, diseases, or conditions caused by any one or more serotypes of Streptococcus canis can be prevented or treated by administering an immunogenic composition comprising one or more attenuated and / or dead streptococcal bacterial strains, which include modifications that reduce, decrease, or prevent the functional expression of LytC (or its homologues), wherein the attenuated and / or dead streptococcal bacterial strains comprise one or more serotypes of Streptococcus canis. The administered attenuated and / or dead Streptococcus canis serotype(s) may differ from the serotype(s) causing the infection, disease, or condition; (iv) By administering an immunogenic composition comprising one or more attenuated and / or dead streptococcal bacterial strains, including modifications that reduce, decrease, or prevent the functional expression of LytC (or its homologues), infections, diseases, or conditions caused by any one or more serotypes of Streptococcus dysgalactiae can be prevented or treated, wherein the attenuated and / or dead streptococcal bacterial strains comprise one or more serotypes of Streptococcus dysgalactiae. The administered attenuated and / or dead Streptococcus dysgalactiae serotype(s) may differ from the serotype(s) causing the infection, disease, or condition; (v) By administering an immunogenic composition comprising one or more attenuated and / or dead streptococcal bacterial strains, including modifications that reduce, decrease, or prevent the functional expression of LytC (or its homologues), infections, diseases, or conditions caused by any one or more serotypes of Streptococcus equi can be prevented or treated, where the attenuated and / or dead streptococcal bacterial strains comprise one or more serotypes of Streptococcus equi. The administered attenuated and / or dead Streptococcus equi serotype(s) may differ from the serotype(s) causing the infection, disease, or condition; (vi) Infections, diseases, or conditions caused by any one or more serotypes of Streptococcus equinus can be prevented or treated by administering an immunogenic composition comprising one or more attenuated and / or dead streptococcal bacterial strains, which include modifications that reduce, decrease, or prevent the functional expression of LytC (or its homologues), wherein the attenuated and / or dead streptococcal bacterial strain comprises one or more serotypes of Streptococcus equinus. The administered attenuated and / or dead Streptococcus equinus serotype(s) may differ from the serotype(s) causing the infection, disease, or condition; (vii) Infections, diseases, or conditions caused by any one or more serotypes of Streptococcus agalactiae can be prevented or treated by administering an immunogenic composition comprising one or more attenuated and / or dead streptococcal bacterial strains, which include modifications that reduce, decrease, or prevent the functional expression of LytC (or its homologues), wherein the attenuated and / or dead streptococcal bacterial strain comprises one or more serotypes of Streptococcus agalactiae. The administered attenuated and / or dead Streptococcus agalactiae serotype(s) may differ from the serotype(s) causing the infection, disease, or condition; (viii) By administering an immunogenic composition comprising one or more attenuated and / or dead streptococcal bacterial strains, including modifications that reduce, decrease, or prevent the functional expression of LytC (or its homologues), infections, diseases, or conditions caused by any one or more serotypes of Streptococcus equisimilis can be prevented or treated, wherein the attenuated and / or dead streptococcal bacterial strains comprise one or more serotypes of Streptococcus equisimilis. The administered attenuated and / or dead Streptococcus equisimilis serotype(s) may differ from the serotype(s) causing the infection, disease, or condition; (ix) By administering an immunogenic composition comprising one or more attenuated and / or dead streptococcal bacterial strains, including modifications that reduce, decrease, or prevent the functional expression of LytC (or its homologues), infections, diseases, or conditions caused by any one or more serotypes of Enterococcus faecalis can be prevented or treated, where the attenuated and / or dead streptococcal bacterial strain comprises one or more serotypes of Enterococcus faecalis. The administered attenuated and / or dead Enterococcus faecalis serotype(s) may differ from the serotype(s) causing the infection, disease, or condition; (x) By administering an immunogenic composition comprising one or more attenuated and / or dead streptococcal bacterial strains, including modifications that reduce, decrease, or prevent the functional expression of LytC (or its homologues), infections, diseases, or conditions caused by any one or more serotypes of Enterococcus faecium can be prevented or treated, where the attenuated and / or dead streptococcal bacterial strains comprise one or more serotypes of Enterococcus faecium. The administered attenuated and / or dead serotype(s) of Enterococcus faecium may differ from the serotype(s) causing the infection, disease, or condition; (xi) By administering an immunogenic composition comprising one or more attenuated and / or dead streptococcal bacterial strains, including modifications that reduce, decrease, or prevent the functional expression of (xi)LytC (or its homologues), infections, diseases, or conditions caused by any one or more serotypes of Streptococcus iniae can be prevented or treated, where the attenuated and / or dead streptococcal bacterial strains comprise one or more serotypes of Streptococcus iniae. The administered attenuated and / or dead Streptococcus iniae serotype(s) may differ from the serotype(s) causing the infection, disease, or condition; (xii) By administering an immunogenic composition comprising one or more attenuated and / or dead streptococcal bacterial strains, including modifications that reduce, decrease, or prevent the functional expression of LytC (or its homologues), infections, diseases, or conditions caused by any one or more serotypes of Streptococcus milleri can be prevented or treated, where the attenuated and / or dead streptococcal bacterial strains comprise one or more serotypes of Streptococcus milleri. The administered attenuated and / or dead Streptococcus milleri serotype(s) may differ from the serotype(s) causing the infection, disease, or condition; (xiii) By administering an immunogenic composition comprising one or more attenuated and / or dead streptococcal bacterial strains, including modifications that reduce, decrease, or prevent the functional expression of LytC (or its homologues), infections, diseases, or conditions caused by any one or more serotypes of Streptococcus mutans can be prevented or treated, where the attenuated and / or dead streptococcal bacterial strains comprise one or more serotypes of Streptococcus mutans. The administered attenuated and / or dead Streptococcus mutans serotype(s) may differ from the serotype(s) causing the infection, disease, or condition; An immunogenic composition comprising one or more attenuated and / or dead streptococcal bacterial strains, including modifications that reduce, decrease, or prevent the functional expression of (xiv)LytC (or its homologues), can be administered to prevent or treat infections, diseases, or conditions caused by any one or more serotypes of Streptococcus pneumoniae, where the attenuated and / or dead streptococcal bacterial strains comprise one or more serotypes of Streptococcus pneumoniae. The administered attenuated and / or dead Streptococcus pneumoniae serotype(s) may differ from the serotype(s) causing the infection, disease, or condition; By administering an immunogenic composition comprising one or more attenuated and / or dead streptococcal bacterial strains, including modifications that reduce, decrease, or prevent the functional expression of (xv)LytC (or its homologues), infections, diseases, or conditions caused by any one or more serotypes of Streptococcus pyogenes can be prevented or treated, where the attenuated and / or dead streptococcal bacterial strains comprise one or more serotypes of Streptococcus pyogenes. The administered attenuated and / or dead Streptococcus pyogenes serotype(s) may differ from the serotype(s) causing the infection, disease, or condition; (xvi) By administering an immunogenic composition comprising one or more attenuated and / or dead streptococcal bacterial strains, including modifications that reduce, decrease, or prevent the functional expression of (xvi)LytC (or its homologues), infections, diseases, or conditions caused by any one or more serotypes of Streptococcus salivarius can be prevented or treated, where the attenuated and / or dead streptococcal bacterial strains comprise one or more serotypes of Streptococcus salivarius. The administered attenuated and / or dead Streptococcus salivarius serotype(s) may differ from the serotype(s) causing the infection, disease, or condition; Infections, diseases, or conditions caused by any one or more serotypes of Streptococcus sanguinis can be prevented or treated by administering an immunogenic composition comprising one or more attenuated and / or dead streptococcal bacterial strains, which include modifications that reduce, decrease, or prevent the functional expression of (xvii)LytC (or its homologues), wherein the attenuated and / or dead streptococcal bacterial strains comprise one or more serotypes of Streptococcus sanguinis. The administered attenuated and / or dead Streptococcus sanguinis serotype(s) may differ from the serotype(s) causing the infection, disease, or condition; (xviii) By administering an immunogenic composition comprising one or more attenuated and / or dead streptococcal bacterial strains, including modifications that reduce, decrease, or prevent the functional expression of (xviii)LytC (or its homologs), infections, diseases, or conditions caused by any one or more serotypes of Streptococcus suis can be prevented or treated, where the attenuated and / or dead streptococcal bacterial strains can prevent or treat infections, diseases, or conditions that are one or more serotypes of Streptococcus suis. The administered attenuated and / or dead Streptococcus suis serotype(s) may differ from the serotype(s) causing the infection, disease, or condition; and / or An immunogenic composition comprising one or more attenuated and / or dead streptococcal bacterial strains, including a modification that reduces, diminishes, or prevents the functional expression of (xix)LytC (or its homologue), can prevent or treat infections, diseases, or conditions caused by any one or more serotypes of Streptococcus uberis, where the attenuated and / or dead streptococcal bacterial strain can prevent or treat infections, diseases, or conditions that are one or more serotypes of Streptococcus uberis. The administered attenuated and / or dead Streptococcus uberis serotype(s) may differ from the serotype(s) causing the infection, disease, or condition.

[0203] 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 involve inducing an immune response to multiple different Streptococcus pneumoniae serotypes in a subject by administering an immunogenic composition comprising one or more attenuated and / or dead streptococcal bacterial strains, including modifications that reduce, decrease, or prevent functional LytC expression. In one embodiment, the method involves administering a single attenuated and / or dead (e.g., gamma-irradiated and / or X-ray-irradiated) serotype of Streptococcus pneumoniae.

[0204] 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 at least one serotype of attenuated and / or dead Streptococcus pneumoniae to a subject, wherein the subjects are 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, 1 It may induce an immune response to one or more of the following: 8B, 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 may induce an immune response in a subject to one or more serotypes of Streptococcus pneumoniae (S. pneumoniae) 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 the Disclosure may induce an immune response to strains of streptococcal bacteria not specifically enumerated herein. For example, the compositions of the Disclosure may induce an immune response to emerging strains of Streptococcus pneumoniae (S. pneumoniae) to which a serotype number has not yet been assigned.

[0205] The attenuated and / or dead Streptococcus pneumoniae (S. pneumoniae) serotype(s) administered may differ from the serotype(s) causing the infection, disease, or pathological condition. In one embodiment, the attenuated and / or dead Streptococcus bacterial strain is derived from a strain, serotype, or species different from at least one target Streptococcus species and / or its serotype. Thus, in one embodiment, the induced immune response is cross-protective.

[0206] The disease or condition may be any disease or condition caused by infection with a specific species or serotype of Streptococcus bacteria. To give a merely non-limiting example, the disease or condition may be one or more of the following: pneumonia, ear infection, ear pain, middle ear infection, otitis media, sinusitis, meningitis, conjunctivitis, bacteremia, sepsis, joint infection, bone infection, suppurative arthritis, osteomyelitis, soft tissue infection, cellulitis, myositis, periorbital cellulitis, abscess, peritonitis, heart infection, endocarditis, and pericarditis.

[0207] The subjects may be any animals of economic, social, or research importance, including species of cattle, horses, sheep, primates, birds, and rodents. Therefore, the subjects may be mammals, such as humans or non-human mammals (e.g., pigs, cats, dogs, cattle, horses, or sheep). The subjects may be laboratory animals (e.g., rodents such as mice, rats, or guinea pigs, rabbits, etc.), birds (e.g., poultry), fish, or crustaceans.

[0208] Attenuated and / or dead streptococcal bacteria and / or their attenuated and / or dead derivative strains may be administered to a subject by any preferred route, including, for example, parenteral (e.g., intradermal, intravenous, intrathecal, intraperitoneal, subcutaneous, or intramuscular), oral, topical, or mucosal routes (e.g., intranasal). In some embodiments, administration is by a mucosal route. For example, administration may be intranasal.

[0209] Although not limited to a specific mechanism of action (multiple mechanisms are possible), this method applies to the subject, (i) Production of antibodies that specifically bind to antigens(s) of streptococcal bacteria that cause infectious diseases, illnesses, or pathological conditions. (ii) CD4 specific to the antigen(s) of a streptococcal bacterium causing an infectious disease, illness, or pathological condition. + T lymphocyte response, and / or (iii) CD8 specific to the antigen(s) of a streptococcal bacterium causing an infectious disease, illness, or pathological condition. + It can induce an immune response, including one or more T lymphocyte responses.

[0210] In one embodiment, the method may induce an immune response in a subject that is interleukin-17A (IL-17A) dependent, IL-17A independent, and / or includes 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 the antibody response to the antigen of interest.

[0211] As merely an unrestricted example, the immune response induced or enhanced in a subject by this 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 2 times, at least about 5 times, at least about 10 times, at least about 20 times, at least about 50 times, or at least about 100 times compared to a suitable control. A suitable control may be, for example, the same measurement of the immune response obtained before performing this method under otherwise identical, substantially identical, or identical conditions.

[0212] 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), liquid-phase assays (e.g., electrochemiluminescence assays), amplified luminescence proximity homogeneous assays, flow cytometry, intracellular cytokine staining, functional T cell assays, functional B cell assays, functional monocyte-macrophage assays, dendritic cell and reticular endothelial cell assays, measurement of natural killer cell response, oxidative burst assays, cytotoxicity-specific cell lysis assays, pentamer binding assays, and evaluation of phagocytosis and apoptosis.

[0213] Immunogenic compositions and vaccine composition preparations Attenuated and / or dead streptococcal bacterial strains, including modifications that reduce, diminish, or prevent the functional expression of LytC (or its homologs) as described herein, may be incorporated into a pharmaceutical composition. This composition can stimulate an immune response to a pathogenic organism capable of establishing an infection in a host that can lead to disease or pathological conditions. Therefore, this composition may be an immunogenic composition or vaccine composition, including prophylactic vaccines (i.e., vaccines administered for the prevention of infections and / or diseases / pathological conditions) and therapeutic vaccines (i.e., vaccines administered for the treatment of infections and / or diseases / pathological conditions). Accordingly, the vaccines of this disclosure may be administered to a recipient for the purpose of prevention, improvement, mitigation, or treatment. All such compositions will be understood to be collectively encompassed by the terms "compositions the disclosure" or "composition of the disclosure" as used herein. It will also be understood that vaccine compositions fall within the scope of immunogenic compositions of this disclosure.

[0214] Non-limiting examples of streptococcal bacteria suitable for incorporation into the compositions of this disclosure are described above in the subsections titled “Streptococcal Bacterial Strains” and “Streptococcal Bacterial Derivatives.”

[0215] In one embodiment, photon emission (e.g., gamma-ray emission and / or X-ray emission) is used to attenuate or inactivate the streptococcal bacteria and their derivatives in the composition. The streptococcal bacterial strains of the present disclosure may be subjected to photon emission before, during, or after combining them with other reagents to provide an immunogenic composition for a vaccine composition formulation.

[0216] formulation The compositions of this disclosure can be prepared using methods known to those skilled in the art. Non-limiting examples of preferred methods are described in Gennaro et al. (Eds), (1990), "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pennsylvania, USA, and methods for preparing vaccines are described in Voller et al., (1978), "New Trends and Developments in Vaccines," University Park Press, Baltimore, Maryland, USA.

[0217] The composition may include pharmaceutically acceptable carriers, excipients, diluents, and / or adjuvants. “pharmaceutically acceptable” carriers, excipients, diluents, and / or adjuvants as used herein are substances that do not cause adverse reactions (may be multiple) when administered to a specific recipient, such as a human or non-human animal. pharmaceutically acceptable carriers, excipients, excipients, and adjuvants are generally also compatible with other components of the vaccine. Non-limiting examples of suitable excipients, diluents, and carriers are listed in “Handbook of Pharmaceutical Excipients,” 4th Edition, (2003), Rowe et al. (Eds), The Pharmaceutical Press, London, American Pharmaceutical Association, Washington.

[0218] Non-limiting examples of pharmaceutically acceptable carriers, excipients, or diluents include: demineralized or distilled water; physiological saline; vegetable oils such as peanut oil, safflower oil, olive oil, cottonseed oil, corn oil, sesame oil, groundnut oil, or coconut oil; silicone oils containing polysiloxanes such as methylpolysiloxane, phenylpolysiloxane, and methylphenylpolysorboxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin, or squalane; methylcellulose, ethylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, hydroxypropylcellulose Examples include cellulose derivatives such as pyrumethylcellulose; lower alkanols, e.g., ethanol or isopropanol; lower alkanols; lower polyalkylene glycols or lower alkylene glycols, e.g., polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3-butylene glycol, or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate, or ethyl oleate; polyvinylpyrrolidone; agar; carrageenan; tragacanth gum or acacia gum; and petrolatum. Typically, one or more carriers will account for 10% to 99.9% by weight of the composition.

[0219] The compositions of this disclosure may be in a form suitable for administration by injection, in a formulation form suitable for oral ingestion (e.g., capsules, tablets, caplets, elixirs, etc.), in the form of an ointment, cream or lotion suitable for topical administration, in a form suitable for delivery as eye drops, in the form of an aerosol suitable for administration by inhalation such as intranasal inhalation or oral inhalation, or in a form suitable for parenteral administration, i.e., injection into the dermis, subcutaneously, intramuscularly or intravenously.

[0220] Solid compositions for oral administration may contain binders, sweeteners, disintegrants, diluents, flavorings, coatings, preservatives, lubricants, and / or time-delaying agents permitted under human and veterinary pharmaceutical regulations. Suitable binders include acacia gum, gelatin, corn starch, tragacanth gum, sodium alginate, carboxymethylcellulose, or polyethylene glycol. Suitable sweeteners include sucrose, lactose, glucose, aspartame, or saccharin. Suitable disintegrants include corn starch, methylcellulose, polyvinylpyrrolidone, guar gum, xanthan gum, bentonite, alginic acid, or agar. Suitable diluents include lactose, sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate, calcium silicate, or dicalcium phosphate. Suitable flavorings include peppermint oil, wintergreen oil, cherry flavoring, orange flavoring, or raspberry flavoring. 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, alpha-tocopherol, ascorbic acid, methylparaben, propylparaben, or sodium bisulfite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride, or talc. Suitable time retarders include glyceryl monostearate or glyceryl distearate.

[0221] A liquid composition for oral administration may include a liquid carrier in addition to the above-mentioned drug. Suitable liquid carriers include water, oils such as olive oil, peanut oil, sesame oil, sunflower oil, safflower oil, peanut oil, coconut oil, etc., liquid paraffin, ethylene glycol, propylene glycol, polyethylene glycol, ethanol, propanol, isopropanol, glycerol, fatty alcohols, triglycerides, or mixtures thereof.

[0222] A suspension containing a composition for oral administration may further contain a dispersant and / or a suspending agent. Suitable suspensions include sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone, sodium alginate, or acetyl alcohol. Suitable dispersants include polyoxyethylene esters of fatty acids such as lecithin and stearic acid, polyoxyethylene sorbitol monooleate or dioleate, polyoxyethylene sorbitol stearate, or polyoxyethylene sorbitol laurate, polyoxyethylene sorbitan monooleate or dioleate, stearate, or laurate.

[0223] To prepare the composition as an injectable solution or suspension, non-toxic, parenterally acceptable diluents or carriers, such as Ringer's solution, isotonic saline, phosphate-buffered saline, ethanol, and 1,2-propylene glycol, may be used.

[0224] Vaccine emulsions for oral administration may further contain one or more emulsifiers. Suitable emulsifiers include the dispersants exemplified above, or natural rubbers such as guar gum, acacia gum, or tragacanth gum.

[0225] A topical formulation of the composition comprises one active ingredient(s) (e.g., attenuated and / or dead Streptococcus bacteria and / or derivative strains thereof) together with one or more acceptable carriers and optionally any other therapeutic ingredients. Formulations suitable for topical administration include liquid or semi-liquid formulations suitable for penetration from the skin to the site of treatment, such as liniments, lotions, creams, ointments or pastes, as well as drops suitable for administration to the eyes, ears or nose.

[0226] When formulated as drops, the composition may include a sterile aqueous or oily liquid or suspension. These can be prepared by dissolving the active ingredient in an aqueous solution of a bactericide, fungicide, and / or any other suitable preservative, optionally containing a surfactant. The resulting solution is then clarified by filtration and transferred to a suitable container for sterilization. For example, after sterilization by filtration, the solution may be transferred to a container by aspiration. Examples of bactericides and fungicides suitable for inclusion in drops include phenylmercury nitrate or phenylmercury acetate (0.002%), benzalkonium chloride (0.01%), and chlorhexidine acetate (0.01%). Suitable solvents for preparing oily solutions include glycerol, diluting alcohol, and propylene glycol.

[0227] When formulated as a lotion, the composition may include elements suitable for application to the skin or eyes. An eye lotion may optionally include a sterile aqueous solution containing a bactericide and may be prepared by the same method as described above for the preparation of drops. A lotion or liniment for skin application may also contain substances that promote drying and provide a cooling sensation to the skin, such as alcohol or acetone, and / or humectants, such as glycerol or oils such as castor oil or peanut oil.

[0228] When formulated as a cream, ointment, or paste, the composition may be a semi-solid preparation of the active ingredient for external use. These can be prepared by mixing the active ingredient, either alone or in the form of an aqueous or non-aqueous solution or suspension, with an oily or non-oily base material. The base material may include hydrocarbons, such as hard, soft, or liquid paraffin, glycerol, beeswax, or metallic soap; mucus; oils of natural origin such as almond oil, corn oil, peanut oil, castor oil, or olive oil; lanolin or its derivatives; or fatty acids such as stearic acid or oleic acid together with alcohols such as propylene glycol or macrogol.

[0229] The composition may contain any suitable surfactant, such as anionic, cationic, or nonionic surfactants, such as sorbitan esters or their polyoxyethylene derivatives. It may also contain a suspending agent, such as natural rubber, cellulose derivatives, or inorganic substances, such as silicon silica, and other components, such as lanolin.

[0230] The composition may be administered in the form of liposomes. Liposomes are generally formed from monolayers or multilayers of hydrated liquid crystals, typically derived from phospholipids or other lipid substances, 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 include phospholipids and phosphatidylcholine (lecithin), both of which are natural and synthetic. Methods for forming liposomes are known in the art, and in this regard, see specifically Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, NY (1976), pp. 33 onwards.

[0231] Adjuvant The compositions of this disclosure may include adjuvants; however, the experimental data provided herein demonstrate that attenuated and / or dead streptococcal bacteria, and their derivatives, can induce immunity without the need for such adjuvants. Therefore, the compositions of this disclosure may or may not include adjuvants.

[0232] Generally, adjuvant activity in the context of vaccine compositions includes, but is not limited to, the ability to enhance (quantitatively or qualitatively) the immune response induced by immunogenic components in the vaccine (e.g., attenuated and / or dead Streptococcus bacteria and / or their derivatives). This can reduce the dose or level of immunogenic components required to elicit an immune response, and / or reduce the number or frequency of immunizations required to elicit a desired immune response.

[0233] Preferably, the adjuvant enhances the immune response induced and / or enhanced by the vaccine component(s), thereby improving the protective effect. Preferably, the adjuvant enables the induction of protective immunity using lower doses of other active components(s) (e.g., attenuated and / or dead Streptococcus bacteria and / or derivatives thereof).

[0234] Non-limiting examples of adjuvants suitable for inclusion in the compositions of this disclosure and methods for preparing them are described in "Vaccine Adjuvants: Preparation Methods and Research Protocols (Methods in Molecular Medicine)," (2000), Ohagan (Ed), Humana Press Inc. Any suitable adjuvant may be included in the vaccines of this disclosure.

[0235] Specific examples of such adjuvants include, but are not limited to, aluminum hydroxide; polypeptide adjuvants, including interferons, interleukins, and other cytokines; AMPHIGEN, oil-in-water and water-in-oil emulsions; and saponins such as QuilA.

[0236] For example, aluminum-based adjuvants can be used. Suitable aluminum-based adjuvants, though not particularly limited, include aluminum hydroxide, aluminum phosphate, and combinations thereof. Other specific examples of usable aluminum-based adjuvants are described in European Patent No. 1216053 and U.S. Patent No. 6,372,223.

[0237] Oil-in-water emulsions can be used as adjuvants in the compositions of this disclosure. Oil-in-water emulsions are well known in the art. Generally, oil-in-water emulsions contain metabolizable oils, such as fish oil, vegetable oil, or synthetic oil. Examples of suitable oil-in-water emulsions are described in European Patent No. 0399843, U.S. Patent No. 7,029,678, and PCT International Publication 2007 / 006939. Oil-in-water emulsions can be used in combination with other adjuvants and / or immunostimulants.

[0238] Other non-limiting examples of suitable adjuvants include immunostimulants such as granulocyte-macrophage colony-stimulating factor (GM-CSF), monophosphoryl lipid A (MPL), cholera toxin (CT) or its constituent subunits, thermolabile enterotoxin (LT) or its constituent subunits, Toll-like receptor ligand adjuvants (lipopolysaccharide (LPS) and its derivatives (e.g., monophosphoryl lipid A and 3-deacylated monophosphoryl lipid A), muramyl dipeptide (MDP), etc.), Toll-like receptor (TLR) agonists (e.g., TLR-2 agonists, TLR-3 agonists), and the F protein of respiratory encapsulation virus (RSV).

[0239] The adjuvants in the compositions of this disclosure may typically include emollients, emulsifiers, thickeners, preservatives, bactericides, and buffers. Conjugation of immunogenic peptides with lipids yields other types of “self-adjuvants,” such as the water-soluble lipopeptide Pam3Cys or its dipalmitoyl derivative Pam2Cys. Such adjuvants have the advantage of entering antigen-presenting cells (such as dendritic cells) along with their immunogenic components, thereby simultaneously enhancing antigen presentation and activating cells (see, for example, Brown and Jackson, (2005), “Lipid-based self-adjuvanting vaccines,” Current Drug Delivery, 23:83).

[0240] Suitable adjuvants are commercially available and include, for example, Freund's incomplete and complete adjuvants (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ); AS-2 (SmithKline Beecham, Philadelphia, Pa.); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron, or zinc; insoluble suspensions of acylated tyrosine; acylated sugars; cationic or anionic derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A. Cytokines such as GM-CSF, or interleukin-2, interleukin-7, or interleukin-12 can also be used as adjuvants.

[0241] In certain embodiments, adjuvants contained in the vaccines of this disclosure may induce a Th1-dominant immune response. Suitable adjuvants for inducing a Th1-dominant response include, for example, combinations of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL), and aluminum salts. For example, a composition or vaccine may be formulated with adjuvant AS04, which contains aluminum hydroxide (alum) and 3-O-deacylated monophosphoryl lipid A (MPL), as described in Thoelen et al. (2001), "A prophylactic hepatitis B vaccine with a novel adjuvant system," Vaccine, 19:2400-2403. Other known adjuvants that preferentially induce a Th1-dominant immune response include CpG-containing oligonucleotides, characterized in that the CpG dinucleotide is not methylated. Such oligonucleotides are known to those skilled in the art and are described, for example, in PCT International Publication No. 1996 / 02555. For example, immunostimulatory DNA sequences are also described in Sato et al., (1996), "Immunostimulatory DNA sequences necessary for effective intradermal gene immunization," Science, 273:352-354.

[0242] 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. Enhanced adjuvant systems may be used, including combinations of monophosphoryl lipid A with saponin derivatives, such as the combination of QS21 and 3D-MPL described in PCT International Publication 1994 / 00153, or a less reactive composition in which the reactivity of QS21 is suppressed with cholesterol, as described in PCT International Publication 1996 / 33739. Other alternative formulations include oil-in-water emulsions and tocopherols. An adjuvant formulation containing QS21, 3D-MPL, and tocopherol in an oil-in-water emulsion is described in PCT International Publication 1995 / 17210. The adjuvants contained in the compositions of this disclosure may include formulations containing QS21, 3D-MPL, and tocopherol in an oil-in-water emulsion, such as those described in PCT International Publication No. 1995 / 17210. In one embodiment, the compositions of this disclosure include the adjuvant Montanide ISA720 (M-ISA-720; Seppic, Fairfield, NJ), which is an adjuvant based on a naturally metabolizable oil.

[0243] Preferably, the adjuvant is a mucosal adjuvant effective to enhance mucosal immunity and / or systemic immunity against an immunogenic component administered via a mucosal route. Mucosal adjuvants are those that facilitate vaccine delivery and enhance the induction of protective immunity induced by other immunogenic components of the vaccine (e.g., liposomes, cochleates, attenuated live vectors, poly D,L-lactide-co-glycolide or PLGA, chitan, DNA vaccines, mucoadhesive substances), and those that play a role in stimulating immunity (e.g., innate immune-related toxin systems, cytokine systems, etc.). Without being limited to a particular mechanism, some of the beneficial effects of mucosal adjuvants are thought to be derived from the ability to help the immunogenic components of the vaccine pass through the mucosal barrier. When crossing the mucosal barrier, mucosal adjuvants are thought to enhance immunity, for example, by stimulating complement activation, cytokine induction, antibody production or antibody type switching, antigen-presenting cells, and / or by affecting the expression of HLA or MHC class I and / or class II.

[0244] Route of administration The compositions of the present disclosure can be administered to a recipient by standard routes including, but not limited to, parenteral (e.g., intradermal, intravenous, intrathecal, intraperitoneal, subcutaneous, or intramuscular), oral, topical, or mucosal routes (e.g., intranasal).

[0245] ]>For example, the composition can be administered by a mucosal route. Non-limiting examples of acceptable routes of mucosal vaccine administration include intranasal, intraocular, buccal, intra-genital (vaginal), rectal, intratracheal, skin, and the gastrointestinal tract.

[0246] In one embodiment, the composition of the present disclosure is administered by an intranasal route. Without being limited by theory or a particular mode of action(s), intranasal administration of the composition can be advantageous for enhancing immunity against certain streptococcal infections where bacteria infect the host from the mucosal surfaces of the upper and / or lower respiratory tract. Further, mucosal vaccination (e.g., intranasal vaccination) can induce mucosal immunity not only in the respiratory tract but also in distant mucosal sites including genital mucosa.

[0247] The intranasal compositions of the present disclosure can be formulated in liquid form, as a powder, as a cream, or as an emulsion, for example, as suitable for nasal drops, sprays or inhalation. It is also possible to utilize atomized or aerosolized intranasal vaccines. Administration of the composition to the mucosa of the upper and / or lower respiratory tract is also contemplated, such as by inhalation of a mist, powder or spray, or by intranasal administration of a nasal drop, applicator, powder, spray, mist, aerosol.

[0248] In one embodiment, the composition for intranasal administration is provided in the form of a lyophilized powder that can be reconstituted immediately before use. The powder vaccine formulation of the compositions of the present disclosure provides one means of overcoming the need for refrigerated storage and distribution associated with the stability and delivery of liquid-based vaccines. The dry powder formulation offers the advantages of being more stable and suppressing the growth of microorganisms.

[0249] The lyophilized composition can induce cross-protective immunity at a level comparable to that of the non-lyophilized composition. The composition can be lyophilized using any suitable technique known in the art. For example, a liquid preparation of attenuated and / or inactivated Streptococcus bacteria and / or its derivatives can be frozen in a dry ice-isopropanol slurry and lyophilized in a lyophilizer (e.g., Virti Model 10-324 Bench, Gardiner, NY) for a suitable time (e.g., 24 hours).

[0250] In one embodiment, the dried powder nasal vaccine of this disclosure is prepared by generating spray freeze-dried (SFD) particles (e.g., Costantino et al., (2002), "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. Effect of atomization conditions on particle size and stability", Pharm Res., 17:1374-1383; Maa et al., (1999), "Protein inhalation powders: spray drying vs spray freeze drying", Pharm Res, 16:249-254; Carrasquillo et al., (2001); "Non-aqueous encapsulation of excipient-stabilized spray-freeze dried BSA into poly(lactide-co-glycolide) microspheres results in release See "of native protein", J Control Release, 76:199-208; Carrasquillo et al., (2001), "Reduction of structural perturbations in bovine serum albumin by non-aqueous microencapsulation", J Pharm Pharmacol., 53:115-120; and U.S. Patent No. 6,569,458).

[0251] A preferred device for intranasal administration of the composition is a nasal spray device (e.g., devices commercially available from Pfeiffer GmbH, Valois and Becton Dickinson). Non-limiting examples of preferred devices are described, for example, Bommer, (1999), "Advances in Nasal drug delivery Technology," Pharmaceutical Technology Europe, pp. 26-33. Intranasal devices can produce droplets in the range of 1 to 500 μm. Preferably, a small percentage of droplets (e.g., less than 5%) are less than 10 μm to minimize the possibility of inhalation. Intranasal devices may be capable of two-dose delivery, i.e., delivering a single vaccine dose in two subdoses, one subdose to each nostril.

[0252] The compositions of this disclosure may be administered to a recipient alone or in combination with other additional therapeutic agents. In embodiments in which the vaccine is administered with therapeutic agents, the administration may be simultaneous or sequential (i.e., the agent may be administered after the vaccine, or vice versa). Therefore, when the vaccine of this disclosure is administered to a target in combination with another agent, both may be administered simultaneously in a single composition, simultaneously in separate compositions, or separately at different times.

[0253] Dosage Generally, the compositions of this disclosure are administered in a manner suitable to the route of administration and the recipient's physical characteristics (including health status) in order to induce a desired effect(s) (i.e., therapeutic effect, immunogenic effect, and / or protective effect).

[0254] For example, the appropriate dose of a given vaccine may depend on a variety of factors, including, but is not particularly limited, the physical characteristics of the subject (e.g., age, weight, sex), whether the compound is used as a single agent or an adjunct, the progression of a given streptococcal infection (i.e., pathological condition), and other factors that can be recognized by those skilled in the art. Various general considerations that may be taken into account when determining the appropriate dose of a given vaccine of this disclosure are, for example, described in Gennaro et al. (Eds), (1990), "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pennsylvania, USA; and Gilman et al., (Eds), (1990), "Goodman and Gilman's: The Pharmacological Bases of Therapeutics," Pergamon Press.

[0255] Generally, the compositions of this disclosure can be administered to a patient in an amount of about 5 micrograms to about 5 mg of the active ingredient(s) (i.e., attenuated and / or dead Streptococcus bacteria, and / or derivatives thereof). Dosages of about 50 micrograms to about 500 micrograms are particularly preferred.

[0256] Those skilled in the art will be able to determine, through routine experiments, the effective and non-toxic amount of attenuated and / or dead streptococcal bacteria, or derivatives thereof, to be included in the vaccines of the present disclosure for the desired therapeutic outcome.

[0257] Generally, the effective dose is expected to be in the range of approximately 0.0001 mg to 1000 mg of active ingredient(s) per kg of body weight per 24 hours (i.e., attenuated and / or dead streptococcal bacteria, or derivatives thereof); typically, approximately 0.001 mg to 750 mg per kg of body weight per 24 hours; approximately 0.01 mg to 500 mg per kg of body weight per 24 hours; approximately 0.1 mg to 500 mg per kg of body weight per 24 hours; approximately 0.1 mg to 250 mg per kg of body weight per 24 hours; or approximately 1.0 mg to 250 mg of active ingredient(s) per kg of body weight per 24 hours (i.e., attenuated and / or dead streptococcal bacteria, or derivatives thereof). More typically, the effective dose range is expected to be approximately 1.0 mg to 200 mg per kg of body weight per 24 hours; approximately 1.0 mg to 100 mg per kg of body weight per 24 hours; approximately 1.0 mg to 50 mg per kg of body weight per 24 hours; approximately 1.0 mg to 25 mg per kg of body weight per 24 hours; approximately 5.0 mg to 50 mg per kg of body weight per 24 hours; approximately 5.0 mg to 20 mg per kg of body weight per 24 hours; and approximately 5.0 mg to 15 mg per kg of body weight per 24 hours.

[0258] Alternatively, the effective dose is approximately 500 mg / m². 2 The active ingredient(s) may be (i.e., attenuated and / or dead Streptococcus bacteria, or derivative strains thereof). Generally, the effective dose is approximately 25 to 500 mg / m². 2 Preferably about 25 to 350 mg / m² 2 More comfortably, approximately 25 to 300 mg / m² 2 More preferably, about 25 to about 250 mg / m² 2 More preferably, about 50 to about 250 mg / m² 2 More preferably, about 75 to about 150 mg / m² 2 It is expected to be within the range of [a certain range].

[0259] Typically, in therapeutic applications, treatment extends over the duration of the infection, pathological condition, or disease. Furthermore, it will be apparent to those skilled in the art that the optimal dosage and interval of each administration are determined by the nature and severity of the infection, pathological condition, or disease being treated, the form, route, and site of administration, and the characteristics of the specific individual receiving treatment. Such optimal conditions can also be determined by prior art.

[0260] In many cases, it will be desirable to administer the vaccines of this disclosure several or more times. For example, the compositions of this disclosure may be administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, or more. The administrations may be at intervals of about 1 to about 12 weeks, 6 months, 12 months, etc., and in certain embodiments, at intervals of about 1 to about 4 weeks. In cases of repeated exposure to the specific pathogen targeted by the vaccines of this disclosure, periodic re-administration may be desirable.

[0261] Furthermore, it will be clear to those skilled in the art that it is possible to confirm the optimal treatment process using conventional examination methods for determining the treatment process.

[0262] The methods described herein may include administering a preliminary dose of the vaccine of this disclosure. A booster dose may be administered after the preliminary dose. The booster dose may be intended for re-vaccination. In various embodiments, the vaccine is administered at least once, twice, three times, or more times. The composition of this disclosure may be administered to an unsensitized recipient who is seronegative to a specific target strain(s) of streptococcal bacteria. Alternatively, the composition may be administered to a first-stimulated recipient who is seropositive to a specific target strain(s) of streptococcal bacteria.

[0263] Those skilled in the art will understand that numerous variations and / or modifications can be made to the Disclosure, as disclosed in the specific embodiments, without departing from the spirit or scope of the Disclosure as broadly described. Accordingly, these embodiments should be considered in all respects to be illustrative and not restrictive. [Examples]

[0264] Nucleic acid and amino acid sequences The nucleic acid sequences related to this example are listed in Table 1.

[0265]

Table 1-1

[0266]

Table 1-2

[0267]

Table 1-3

[0268] Example 1: Construction of GPN-001ΔlytC::kanR and GPN-002ΔlytC::kanR Streptococcus vaccine strains As shown in Figure 1, the lytC gene deletion was manipulated in both GPN-001 and GPN-002, which are unencapsulated vaccine strains derived from Streptococcus pneumoniae (S. pneumoniae) Rx1. GPN-001 has a deletion of the autolysin gene lytA, and the pneumolysin gene (ply) is replaced with a mutant ply gene encoding a toxoid type of Ply called PdT. GPN-002 is a derivative strain of the GPN-001 strain. GPN-002 further has a deletion of the gene (psaA) encoding the solute-binding component of the manganese uptake system.

[0269] [[ID=​​​​​In short, the lytC gene was deleted by substitution with a kanamycin-resistant cassette. Oligonucleotide primers were designed, and polymerase chain reaction (PCR) was performed to amplify the approximately 2 kilobase (kb) region immediately upstream of the lytC gene using primers lytC_F1 (SEQ ID NO: 2) and lytC_R1 (SEQ ID NO: 3), and the region downstream of the lytC gene using primers lytC_F2 (SEQ ID NO: 4) and lytC_R2 5'- (SEQ ID NO: 5). These primers further incorporated regions complementary to the kanamycin-resistant cassette.

[0271] Kanamycin-resistant cassettes were amplified using primers kanamycin R_F (SEQ ID NO: 6) and kanamycin R_R (SEQ ID NO: 7). All PCRs were performed using Phusion® High-Fidelity PCR Master Mix (New England BioLabs) containing HF buffer, with standard parameters. Oligonucleotide primers were 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. Linear lytC: The kanR fragments were transformed into GPN-001 and GPN-002 strains using a standard method. The success of lytC gene substitution by the kanamycin-resistant cassette was determined by evaluating the growth capacity on blood agar plates supplemented with manganese and kanamycin, and by size analysis by agarose gel electrophoresis after region amplification by PCR.

[0272] Transformants were identified by PCR amplification using a combination of oligonucleotide primers designed to test for the presence and orientation of the kanamycin-resistant cassette. For product A, it was expected that primers lytC_F1 5 (SEQ ID NO: 2) and lytC_R2 (SEQ ID NO: 5) would produce approximately 5.6 kb of product in parental strains GPN-001 and GPN-002, and approximately 4.6 kb of product in GPN-001ΔlytC and GPN-002ΔlytC. Products B and C were amplified to confirm the presence and orientation of the kanamycin-resistant 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 for products B and C did not produce any product in the parent strains GPN-001 and GPN-002, but it was expected that the GPN-001ΔlytC::kanR and GPN-002ΔlytC::kanR strains would produce a product of approximately 2.5 kb.

[0273] Results and Discussion The successful integration of the kanamycin-resistant cassette in the lytC region of GPN002 was confirmed by comparing the PCR product of the GPN-002 parent strain with the PCR products of three clones of the GPN-002ΔlytC::kanR strain. The sizes of products A, B, and C were determined by gel electrophoresis, and a reduction in the size of product A (approximately 4.6 kb) expected in the GPN-002ΔlytC::kanR strain, as well as the expected sizes of products B and C (approximately 2.5 kb) were observed in all clones (Figure 2).

[0274] Using the primers and methods employed in this example, we were able to induce the ΔlytC::kanR mutation in other Streptococcus pneumoniae strains, including both encapsulated and non-encapsulated forms.

[0275] After confirming the successful integration of the kanamycin-resistant cassette into the strain, further testing was conducted with one GPN-002ΔlytC::kanR clone.

[0276] Example 2: Deletion of lytC reduced bacterial lysis during vaccine production. Experiments were conducted to investigate whether the deletion of lytC in the vaccine strain improved vaccine stability during manufacturing by preventing bacterial lysis in buffer.

[0277] The stability of strains lacking lytC was evaluated by measuring cell density over time at 25°C. GPN-002 and GPN-002ΔlytC::kanR strains were measured in a standard animal-free soybean-based growth medium supplemented with 5 μM MnSO4, with optical density (approximately OD) measured at 600 nm. 600 1.2) The bacteria were grown until they were equivalent. The bacteria were washed and resuspended in phosphate-buffered saline (PBS) containing 20% ​​(w / v) glycerol. Only 100 μL of each sample or buffer was transferred to the wells of a 96-well flat-bottom plate. Spectrophotometer (BMG Labtech) was used to measure the OD (Optical Spectrophotometer). 600 The values ​​were measured at 25°C at 15-minute intervals.

[0278] Results and Discussion The data is shown over time as the average optical density ± average standard error at 600 nm at each time point (Figure 3), and also at representative time points of 0, 2, 4, 8, and 16 hours (Figure 4). GPN-002ΔlytC::kanR maintained a higher optical density compared to the parent GPN-002 strain during incubation in PBS glycerol buffer at 25°C.

[0279] These results indicate that the deletion of lytC in GPN-002 resulted in enhanced stability and reduced lysis in Streptococcus pneumoniae.

[0280] The same method can be used to test the stability of other streptococcal bacteria. Based on the results of this example, streptococcal bacterial derivatives with lytC deletion are expected to have improved stability compared to the parent strain.

[0281] Example 3: Construction and testing of GPN-003 with label-free deletion of the lytC gene The antibiotic resistance cassette was removed from GPN-002ΔlytC::kanR to create GPN-003, a vaccine strain more suitable for human use.

[0282] Deletion of kanamycin-resistant cassette from GPN-002ΔlytC::kanR The kanamycin-resistant cassette introduced into GPN-002 in the GPN-002ΔlytC::kanR strain was removed to induce a label-free deletion of the lytC coding region. Using PCR, approximately 2kb regions immediately upstream of the lytC gene were amplified using primers lytC_F1 (SEQ ID NO: 2) and lytC_MD_R (SEQ ID NO: 8), and approximately 20-30 base pairs (bp) of complementary homology were incorporated into the regions immediately upstream and downstream of the lytC gene. Linear fragments of these two products were generated by overlap extension PCR using primers lytC_F1 (SEQ ID NO: 2) and lytC_R2 (SEQ ID NO: 5).

[0283] Essentially as described above, linear label-free deletion fragments (lytC-) were transformed into GPN-002ΔlytC::kanR resistant strains, however, an enrichment step was performed after the addition of linear DNA to facilitate the selection of kanamycin-sensitive clones. Briefly, *Streptococcus pneumoniae* GPN-002ΔlytC::kanR competent cells were incubated with 100 ng / mL competence-stimulating peptide 1 (CSP-1) for 15 minutes. The linear lytC- fragments were added to the transformation reaction, cultured in the presence of kanamycin, and subsequently treated with ampicillin at standard concentrations to achieve enrichment of the deletion strains. Successful uptake was determined by evaluating the antibiotic resistance profile by patch testing on manganese-supplemented blood agar plates, kanamycin-supplemented blood agar plates, and unsupplemented blood agar plates, as well as by sequencing and size analysis by agarose gel electrophoresis. The resulting strain, *Streptococcus pneumoniae* GPN-002ΔlytC, which lacked label-free lytC, was named GPN-003.

[0284] Results and Discussion Genetic modifications to Rx1 present in GPN-003 were identified based on size using the following oligonucleotide primers (Figure 5): psaA: psaA_seq_F (sequence number 10) and psaA_seq_R (sequence number 11); lytA:lytA_seq_F(sequence number 12) and lytA_seq_R(sequence number 13); lytC:lytC_seq_F(sequence number 14) and lytC_seq_R(sequence number 15); and pdt: PdT_seq_F (sequence number 16) and PdT_seq_R (sequence number 17).

[0285] The sequence of the lytC deletion in GPN-003 was further confirmed using the Sanger sequencing method (Australian Genome Reference Facility, Australia). The DNA sequence after the deletion of the region originally containing lytC (including an adjacent region of approximately 200 bp) is shown in Sequence ID No. 18. The sequence of the 3' end of the pdt region of GPN-003 was confirmed using the Sanger sequencing method (AGRF) and is shown in Sequence ID No. 19, with protein translation shown in Figure 6 (lower panel) and Sequence ID No. 25. For reference, the sequence of the 3' end of the ply gene of Rx1 is shown in Sequence ID No. 26, with protein translation shown in Figure 6 (upper panel) and Sequence ID No. 24. Table 2 summarizes the size of the product and the properties of the modification compared to Rx1 confirmed in GPN-003.

[0286] [Table 2]

[0287] 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. GPN-002 and GPN-003 strains were grown in a standard animal-free soybean-based growth medium supplemented with 5 μM MnSO4 until their optical density at 600 nm was equivalent. The bacteria were washed and resuspended in phosphate-buffered saline (PBS) containing 20% ​​(w / v) glycerol. Only 100 μL of each sample or buffer was transferred to the wells of a 96-well flat-bottom plate. Spectrophotometric analysis (BMG Labtech) was performed. 600 The values ​​were measured at 25°C at 15-minute intervals.

[0288] Results and Discussion The percentage of cell density relative to the starting optical density at 600 nm was calculated. The data is shown over time as the mean percentage relative to the input at each time point + / - mean standard error (Figure 7).

[0289] Compared to the parent strain GPN-002, GPN-003, which has an unlabeled deletion of lytC, exhibits enhanced stability and reduced lysis. In other words, removing the lytC gene from the vaccine strain reduced bacterial lysis when the bacteria were stored at room temperature for extended periods. This indicates that the deletion of lytC in Streptococcus pneumoniae bacteria leads to enhanced stability.

[0290] The same method can be used to test the stability of other streptococcal bacteria. Based on the results of this example, it is expected that streptococcal bacteria with lytC deletion will also exhibit improved stability compared to the parent strain.

[0291] Example 5: Analysis of total IgG and subclass IgG responses from mice vaccinated with gamma-ray irradiated GPN-003 (gamma-PN3). Preparation of gamma-PN2 vaccine and gamma-PN3 vaccine To evaluate whether a vaccine based on the gamma-irradiated GPN-003 strain induced a similar antibody response against strains containing 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, 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 gamma-PN2 and gamma-PN3, respectively. The vaccines' ability to induce a pneumococcus-specific antibody response and their effects on IgG subclasses were examined.

[0292] Vaccination of mice with gamma-PN2 vaccine and gamma-PN3 vaccine. Uninbred Swiss mice were intramuscularly (IM) vaccinated with 50 μg of total protein each of gamma-PN2 and gamma-PN3 three times at two-week intervals. Control mice were sham-vaccinated with PBS containing 20% ​​(w / v) glycerol. Serum was collected from all mice two weeks after the last vaccination. Gamma-PN2 and gamma-PN3 were administered in the absence of adjuvants.

[0293] Detection of IgG response after vaccination Serum samples were tested for *Streptococcus pneumoniae*-specific IgG responses using a standard direct enzyme-linked immunosorbent assay (ELISA). Rx1, an unencapsulated derivative of D39 (serotype 2), was used as the coated antigen.

[0294] Results and Discussion Figure 8(A) shows the pneumococcal specific IgG response as absorbance (450 / 620 nm), and Figure 8(B) shows it as titer. The data are shown as the average absorbance (450 / 620 nm) ± SEM obtained from individual or pooled serum samples (n=5 mice per group). Figure 8(C) shows the IgG1 subclass response measured by ELISA using Rx1 as the coated antigen, Figure 8(D) shows the IgG2a response, Figure 8(E) shows the IgG2b response, and Figure 8(F) shows the IgG3 response. The ratios of these subclasses in each sample are shown in Table 3.

[0295] [Table 3]

[0296] Gamma-PN2 and gamma-PN3 induced a significant pneumococcal-specific IgG antibody response in mice after immunization compared to placebo-vaccinated mice. Notably, gamma-PN3-vaccinated mice had significantly higher titers of pneumococcal-specific IgG1, IgG2a, IgG2b, and IgG3 than gamma-PN2-vaccinated mice.

[0297] Surprisingly, these results showed that deletion of the lytC 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 pathogenic factor. For example, vaccination with purified LytC has shown that this protein is promising as a protective immunogen, resulting in high immunoglobulin (IgG) antibodies in immunized mice and enhanced protection against pneumococcal infections in mice (Wizemann et al., 2001; Corsini et al., 2021). In addition, high titers of LytC-specific IgG antibodies have been observed in serum samples from healthy volunteers in multiple studies, indicating that an anti-LytC antibody response is induced as a result of natural exposure to Streptococcus pneumoniae.

[0298] The same method can be used to test the antibody response of other streptococcal bacteria. Based on the results of this example, it is expected that streptococcal bacteria with lytC deletion or reduced LytC function will also have an improved antibody response compared to the parent strain.

[0299] Example 6: Gamma-PN3 vaccination enhances the ability to induce antibodies that bind to serotypes of Streptococcus pneumoniae (S. pneumoniae). To determine whether the gamma-PN3 antibody can bind to fully encapsulated Streptococcus pneumoniae isolates, IgG binding to all Streptococcus pneumoniae was evaluated by flow cytometry.

[0300] To produce antibodies for analysis, mice were immunized with gamma-PN2 and gamma-PN3 (50 μg of total protein in 50 μL of PBS containing 20% ​​(w / v) glycerol per mouse) as described herein. Control mice were sham-vaccinated with PBS containing only 20% (w / v) glycerol. Mice were immunized three times at two-week intervals. Serum was collected from all mice two weeks after the final immunization.

[0301] Detection of IgG binding to gamma-PN3 and gamma-PN2-induced Streptococcal bacteria by flow cytometry. Serum samples obtained from each group were pooled (n=5), and IgG binding to serotype 2 D39 and serotype 6A P9 of *Streptococcus pneumoniae* was tested by flow cytometry using a standard protocol. In short, *Streptococcus pneumoniae* D39 or 6A strains were cultured using a standard protocol. 1 × 10⁻⁶ 7 Live bacteria derived from colony-forming units (CFUs) of strain D39 or P9 were incubated with mouse serum pooled at a 1:50 dilution in PBS-BSA. Bacterial cells were centrifuged, washed twice, and primary IgG bound to the surface of Streptococcus pneumoniae was detected using an anti-mouse IgG secondary antibody (FITC-conjugated).

[0302] Results and Discussion The graph in Figure 9 shows the percentage of IgG-bound cells (D39 in Panel A and P9 in Panel C) and geometric mean fluorescence intensity (GMFI; D39 in Panel B and P9 in Panel D) detected by flow cytometry after incubation of pooled mouse serum from the vaccine and control groups with each serotype. Higher fluorescence intensity indicates a greater amount of IgG bound to the surface of pneumococcal cells. The data were analyzed by one-way ANOVA. ** p<0.01, **** p<0.0001).

[0303] The results indicate that gamma-PN3-inducing antibodies can bind to multiple serotypes of Streptococcus pneumoniae, including different serotypes of the capsular strain. Furthermore, the level of binding by gamma-PN3-inducing antibodies was significantly higher than that of gamma-PN2-inducing antibodies for both serotypes. This suggests that deletion of the lytC gene enhances the antibody response to antigens present on capsular bacteria, including bacteria of different serotypes, compared to the parent strain of GPN-003. These results indicate that vaccination with gamma-PN3 induces cross-serotype protection against Streptococcus pneumoniae.

[0304] The same method can be used to test the antibody response of other streptococcal bacteria. Based on the results of this example, streptococcal bacteria with lytC deletion or reduced lytC function are expected to have an improved antibody response compared to their parent strain, including an increased cross-protective antibody response against encapsulated bacteria.

[0305] Example 7: Induction of a gamma-PN3-specific serum antibody response to Streptococcus pneumoniae (S. pneumoniae) in rabbits intramuscularly vaccinated with gamma-PN3. To determine whether gamma-PN3 can induce a serum antibody response in rabbits, immunization experiments were conducted.

[0306] Rabbit vaccination Gamma-PN2 and gamma-PN3 were prepared as specified herein. Uninbred rabbits were intramuscularly (IM) vaccinated with gamma-PN2 or gamma-PN3 (500 μg of total protein in 0.5 mL of PBS per rabbit). The rabbits were immunized three times at 3-week intervals. Gamma-PN2 and gamma-PN3 were administered in the absence of adjuvants.

[0307] Detection of IgG response after vaccination Serum was collected from all rabbits prior to immunization (labeled "pre-bleeding"), and again 3 weeks after final immunization. Individual serum samples were tested for total IgG by direct ELISA using gamma-PN3 as the coated antigen, following a standard protocol. Data were analyzed by one-way ANOVA (nd = not detected) (Figure 10).

[0308] Results and Discussion Figure 10 shows the IgG titers of individual rabbits before immunization and after the third immunization (Panel A), and the mean IgG titers (±SEM) within each vaccine group (Panel B). Both vaccination with gamma-PN2 and gamma-PN3 resulted in high-titer antibody responses against non-encapsulated Streptococcus pneumoniae strains in rabbits after IM immunization.

[0309] The ability of other streptococcal bacteria to induce specific serum antibodies can be tested using the same method as detailed above. Based on the results of this example, it is expected that immunizing rabbits with other streptococcal bacteria that have lytC deletion or reduced LytC function will increase the antibody response to the vaccine strain.

[0310] Example 8: Binding of gamma-PN2 and gamma-PN3 inducing antibodies to Streptococcus pneumoniae (S. pneumoniae) To further investigate the binding of gamma-PN2 and gamma-PN3 inducing antibodies to fully encapsulated Streptococcus pneumoniae (S. pneumoniae), flow cytometry analysis was performed.

[0311] Rabbit vaccination with GPN-002 and GPN-003 To produce antibodies for this analysis, uninbred rabbits were intramuscularly (IM) vaccinated with gamma-PN2 or gamma-PN3 (500 μg of total protein in 0.5 mL of PBS per rabbit). The rabbits were immunized three times at 3-week intervals. Serum was collected from all rabbits before immunization (labeled "pre-bleeding"), and again 3 weeks after the final immunization.

[0312] Detection of IgG binding to gamma-PN3 and gamma-PN2-induced Streptococcal bacteria by flow cytometry. Flow cytometry was used to test IgG binding to serotype 7C of Streptococcus pneumoniae (S. pneumoniae) in individual serum samples. In short, 1 × 10⁻⁶ 6 CFUs of Streptococcus pneumoniae serotype 7C were incubated with immunized rabbit serum diluted 1:200 in PBS-BSA. Bacterial cells were pelleted and washed twice, after which primary IgG bound to the surface of the pneumococci was detected using an anti-rabbit IgG secondary antibody (FITC-conjugated).

[0313] Results and Discussion As shown in Figure 11, flow cytometry detected the binding of IgG induced by vaccination with either GPN-002 or GPN-003 to serotype 7C of Streptococcus pneumoniae. (A) shows relative fluorescence, (B) shows the percentage of serotype 7C cells bound to rabbit IgG antibody, and (C) shows the mean fluorescence intensity of IgG antibody bound to serotype 7C cells (indicating the degree of IgG binding) (Panel C). Data were analyzed by one-way ANOVA. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, ns indicates no statistical significance).

[0314] These results demonstrate that gamma-PN2 and gamma-PN3-inducing antibodies can bind with high affinity to different serotypes of Streptococcus pneumoniae against the parental vaccine strain. Furthermore, IgG binding levels against different serotypes of Streptococcus pneumoniae were higher in gamma-PN3-vaccinated animals than in gamma-PN2-vaccinated animals. This indicates that the gamma-PN3 vaccine induces more antibodies capable of binding to different serotypes of Streptococcus pneumoniae than gamma-PN2. Therefore, deletion of the lytC gene increased the antibody response. This indicates that GPN-003 induces cross-protective immunity, including against encapsulated strains.

[0315] The ability of other streptococcal bacteria to induce antibodies that bind to capsular bacteria can be tested using the same method as detailed above. Based on the results of this example, it is expected that immunizing rabbits with other streptococcal bacteria lacking lytC or with reduced lytC function will increase the cross-protective antibody response against capsular bacteria.

[0316] Example 9: Analysis of the function of gamma-PN2 and gamma-PN3 in mediating the opsonin phagocytic killing action of Streptococcus pneumoniae (S. pneumoniae).

[0317] Host defense against pneumococcal infection is thought to be related to opsonin-dependent phagocytosis. Therefore, we investigated the in vitro opsonin-phagocytic killing activity (OPA) of functionally antibody-active pneumococcal-specific antibodies after vaccination of rabbits with gamma-PN2 or gamma-PN3.

[0318] Rabbits were intramuscularly (IM) vaccinated with gamma-PN2 or gamma-PN3 (500 μg of total protein in 0.5 mL of PBS per rabbit). Three immunizations were performed on the rabbits at 3-week intervals. Serum was collected from all rabbits prior to immunization (labeled "pre-bleeding"), and again 3 weeks after the final immunization. Individual or pooled serum samples were tested for opsonin-mediated phagocytic activity against a panel of Streptococcus pneumoniae serotypes (serotypes 6A, 6B, 9N, 23A, 24F, and 33F) using the opsonin-mediated phagocytic assay protocol established by Nahm and Burton (2014). Titer was determined as the reciprocal of the maximum dilution of rabbit serum that killed more than 50% of the input CFU. If no cell death is detected in undiluted serum (or 1:4 dilution if mixed with other assay components), this is expressed as a titer of ≤4.

[0319] Results and Discussion Table 4 shows the opsonin phagocytic index (OI) titers.

[0320] [Table 4]

[0321] The results indicate that gamma-PN3 induced higher opsonizing phagocytic killing activity than gamma-PN2 against all tested Streptococcus pneumoniae serotypes. This suggests that deletion of the lytC gene enhances the antibody response, opsonizing and killing capsular Streptococcus pneumoniae. These results also indicate that gamma-PN3 induces cross-protection responses against different Streptococcus pneumoniae serotypes. The ability of gamma-PN3 to induce cross-protection responses against further Streptococcus pneumoniae serotypes or other streptococcal bacterial species can be tested using the same methods described herein.

[0322] Furthermore, the ability of other streptococcal bacteria to induce antibodies that opsonize various streptococcal bacteria can be tested using the methods detailed above. Based on the results of this example, it is expected that immunizing rabbits with other streptococcal bacteria that lack lytC or have reduced LytC function will increase the opsonization response to various streptococcal bacteria.

[0323] Example 10: Effect of lytC mutation on transcription of conserved antigenic lipoprotein prsA To further investigate the improved immunogenicity demonstrated by vaccine strains containing lytC deletion, antigen gene expression in the GPN-003 strain was evaluated. Quantitative reverse transcription PCR (qRT-PCR) was performed to assess whether the mutation in lytC induced changes in the expression of the conserved lipoprotein prsA. The prsA gene encodes a conserved lipoprotein associated with serotype-independent protection.

[0324] Quantitative reverse transcription PCR GPN-002 and GPN-003 strains were subjected to the same optimal density (OD) in soytone medium (supplemented with 2.14 μM MnSO4). 600The cells were grown to 0.6%). The cultured samples were immediately transferred to RNAProtect Bacteria Reagent (Qiagen) and incubated at room temperature for 5 minutes. The cell material was centrifuged, the supernatant was removed, and the bacterial pellet was stored at -80°C. The samples were enzymatically lysozyme and mutanoricin, and RNA was extracted using the RNeasy Mini Kit (Qiagen) along with on-column DNase I treatment (Qiagen). Total RNA was quantified using a Nanodrop spectrophotometer and diluted to 2 ng / μL. qRT-PCR was performed using the SYBR q-PCR master mix and the QuantStudio 7 Flex Real-Time PCR System (Thermofisher Scientific). Primers for prsA amplification were designed using UGENE extension primers 3 prsA_F (SEQ ID NO: 20) and prsA_R (SEQ ID NO: 21). The levels of gene transcription were normalized for constitutively expressed gyrase A gene (gyrA) using primers gyrA_F (SEQ ID NO: 22) and gyrA_R (SEQ ID NO: 23). The data are shown as the mean value of prsA transcription relative to gyrA expression.

[0325] Results and Discussion As shown in Figure 12, a comparison of prsA gene expression revealed that GPN-003 showed higher expression than GPN-002. These data suggest that mutations in lytC led to enhanced gene expression of important immunogens in vitro.

[0326] Furthermore, the level of prsA expression in streptococcal bacteria can be tested using the method detailed above. Based on the results of this example, it is expected that other streptococcal bacteria with lytC deletion or reduced LytC function will also show increased prsA expression.

[0327] Example 11: GPN-002 and GPN-003 are non-toxic in mice. To investigate the potential of GPN-003 as a weakened vaccine, the toxicity of GPN-002 and GPN-003 was determined in mice.

[0328] Female Swiss mice (5 mice per group) were given a lethal dose of Streptococcus pneumoniae serotype 2 (strain D39, 10%). 4 CFU / mouse), or a 10,000 times higher dose of GPN-002 or GPN-003 (10 8 CFU (concentrated fungicide / mouse) was administered intraperitoneally. The mice were monitored for overall survival, and their body weight was measured daily for up to 7 days.

[0329] Results and Discussion As shown in Figure 13(A), all mice inoculated with a lethal dose of D39 reached a near-fatal state within 24 hours. Conversely, mice administered much higher doses of GPN-002 or GPN-003 showed no clinical symptoms of infection and no weight loss (Figure 13(B)).

[0330] These data provide evidence that GPN-002 and GPN-003 are non-toxic and can be used as attenuated vaccines.

[0331] Predictive Example 1: Construction of GPN-001 strain with label-free deletion of lytC By removing the antibiotic resistance cassette from GPN001ΔlytC::kanR, GPN001ΔlytC can be produced. Removing the antibiotic resistance gene yields a vaccine strain more suitable for human use.

[0332] The kanamycin-resistant cassette introduced into GPN-001 in the GPN-001ΔlytC::kanR strain described earlier in this specification was removed as shown in Figure 14 to produce an unlabeled deletion of the lytC coding region. Oligonucleotide primers were designed, and approximately 2kb regions immediately upstream of the lytC gene were PCR amplified using primers lytC_F1 (SEQ ID NO: 2) and lytC_MD_R (SEQ ID NO: 8), and approximately 20-30 base pairs (bp) of complementary homology were incorporated into the regions immediately upstream and downstream of the lytC region using primers lytC_MD_F (SEQ ID NO: 9) and lytC_R2 (SEQ ID NO: 5). Linear fragments of these two products were generated by overlap extension PCR, both using primers lytC_F1 (SEQ ID NO: 2) and lytC_R2 (SEQ ID NO: 5).

[0333] Alternatively, the linear fragment can be directly amplified from the GPN-003 strain by PCR. Essentially, as described above, the linear, label-free deletion fragment (lytC-) is transformed into the GPN-001ΔlytC::kanR resistant strain, however, an enrichment step is performed after the addition of the linear DNA to facilitate the selection of kanamycin-sensitive clones. Briefly, Streptococcus pneumoniae (S. pneumoniae) GPN-001ΔlytC competent cells are incubated with 100 ng / mL of competence-stimulating peptide 1 (CSP-1) for 15 minutes. The linear lytC- fragment is added to the transformation reaction, cultured in the presence of kanamycin, and subsequently treated with ampicillin to achieve enrichment of the deletion strain. The success of uptake is determined by evaluating the antibiotic resistance profile by patch testing on kanamycin-supplemented and non-supplemented blood agar plates, sequencing, and size analysis by agarose gel electrophoresis. The resulting strain, Streptococcus pneumoniae (GPN-001ΔlytC-), possesses an unlabeled lytC deletion suitable for clinical use.

[0334] All gene modifications to Rx1 introduced into GPN-001ΔlytC are confirmed based on size using the following oligonucleotide primers: lytA:lytA_seq_F(sequence number 12) and lytA_seq_R(sequence number 13); lytC:lytC_seq_F(sequence number 14) and lytC_seq_R(sequence number 15); and pdt: PdT_seq_F (sequence number 16) and PdT_seq_R (sequence number 17).

[0335] The sequence of the lytC deletion in GPN-001ΔlytC will be further confirmed using the Sanger sequencing method. The sequence of the 3' end of the pdt region of GPN-001ΔlytC will also be confirmed using the Sanger sequencing method.

[0336] Predictive Example 2: Analysis of total IgG and subclass IgG responses from mice vaccinated with gamma-PN1 and gamma-PN1ΔlytC. Vaccination experiments in animal models will be conducted to evaluate whether a vaccine developed based on the GPN-001ΔlytC strain induced an altered antibody response compared to GPN-001. The GPN-001 and GPN-001ΔlytC strains will be inactivated by exposure to gamma radiation as described herein, and the resulting vaccines will be named gamma-PN1 and gamma-PN1ΔlytC, respectively. The vaccines' ability to induce a pneumococcal-specific antibody response and their effects on IgG subclasses will be examined.

[0337] Uninbred Swiss mice were intramuscularly (IM) vaccinated with 50 μg of total protein gamma-PN1 and gamma-PN1ΔlytC three times at two-week intervals. Control mice were sham-vaccinated with PBS containing 20% ​​(w / v) glycerol. Serum was collected from all mice two weeks after the last vaccination. Serum samples were tested for the total IgG response specific to Streptococcus pneumoniae (S. pneumoniae) by direct enzyme-linked immunosorbent assay (ELISA). Rx1, an unencapsulated derivative of D39 (serotype 2), was used as the coating antigen. The response to IgG subclasses IgG1, IgG2a, IgG2b, and IgG3 was tested by ELISA using Rx1, an unencapsulated derivative of D39 (serotype 2), as the coating antigen.

[0338] Predictive Example 3: The ability of antibodies induced by gamma-PN1 and gamma-PN1ΔlytC to bind to serotypes of Streptococcus pneumoniae (S. pneumoniae).

[0339] To produce antibodies for analysis, mice are immunized with gamma-PN1 and GPN-001ΔlytC (50 μg of total protein in 50 μL of PBS / glycerol per mouse). Control mice are given a placebo vaccine with PBS / glycerol only. Mice are immunized three times at 2-week intervals. Serum is collected from all mice 2 weeks after the final immunization. Serum from each group is pooled (n=5), and the binding of IgG to capsular serotype 2 (D39 strain) and 6A (P9 strain) is tested using flow cytometry. In short, 1 × 10⁻⁶ 7Live bacteria from each serotype of colony-forming units (CFU) are incubated with mouse serum pooled at a 1:50 dilution in PBS-BSA. The bacterial cells are centrifuged, washed twice, and primary IgG bound to the surface of the Streptococcus pneumoniae is detected using an anti-mouse IgG secondary antibody (FITC-conjugated). These data represent the percentage of cells bound with IgG, as well as the geometric mean fluorescence intensity detected by flow cytometry after incubation with pooled mouse serum from the vaccine and control groups against each serotype. Higher fluorescence intensity indicates a greater amount of IgG bound to the surface of the Streptococcus pneumoniae cells.

[0340] Predictive Example 4: Induction of a Streptococcus pneumoniae (S. pneumoniae)-specific serum antibody response in immunized rabbits with a vaccine containing a mutation in lytC. Immunotherapy experiments will be conducted to determine whether gamma-PN1, gamma-PN2, gamma-PN3, gamma-PN1ΔlytC::KanR, or gamma-PN1ΔlytC induce a higher serum antibody response in rabbits compared to a vaccine based on the parental strain.

[0341] Uninbred rabbits are immunized with IM vaccine containing gamma-PN1, gamma-PN1ΔlytC::kanR, gamma-PN1ΔlytC, gamma-PN2, or gamma-PN3 (500 μg of total protein in 0.5 mL of PBS per rabbit). The rabbits are immunized three times at 3-week intervals, with serum collected before the first immunization (called "pre-bleeding") and 3 weeks after the last immunization. Individual serum samples are tested for total IgG directly by ELISA using whole cell Rx1 as the coated antigen.

[0342] Predictive Example 5: Binding of gamma-PN1, gamma-PN1ΔlytC::kanR, gamma-PN1ΔlytC, gamma-PN2, or gamma-PN3 inducing antibodies to Streptococcus pneumoniae. Flow cytometry analysis will be performed to evaluate the binding of gamma-PN1, gamma-PN1ΔlytC::kanR, gamma-PN1ΔlytC, gamma-PN2, or gamma-PN3 inducing antibodies (targeting pneumococcal proteins) to fully encapsulated Streptococcus pneumoniae.

[0343] To produce antibodies for this analysis, uninbred rabbits are intramuscularly (IM) vaccinated with gamma-PN1, gamma-PN1ΔlytC::kanR, gamma-PN1ΔlytC, gamma-PN2, or gamma-PN3 (500 μg of total protein in 0.5 mL of PBS per rabbit). The rabbits are immunized three times at 3-week intervals. Serum is collected from all rabbits before immunization (labeled "pre-bleeding"), and again 3 weeks after the final immunization. Individual serum samples are tested for IgG binding to serotypes of Streptococcus pneumoniae (S. pneumoniae) different from the parent strain using flow cytometry. In short, 1 × 10⁻⁶ 6 Live capsular bacteria (CFUs) were incubated with rabbit serum diluted 1:200 in PBS-BSA. The bacterial cells were centrifuged and washed twice. Primary IgG bound to the surface of the pneumococci was then detected using an anti-rabbit IgG secondary antibody (FITC-bound). The percentage of pneumococcal cells bound to rabbit IgG antibody and the average fluorescence intensity of the IgG antibody bound to the pneumococcal cells (indicating the degree of IgG binding) are shown.

[0344] The results clearly demonstrate the binding of serum antibodies induced by vaccination with gamma-PN1, gamma-PN1ΔlytC, gamma-PN1ΔlytC-, gamma-PN2, or gamma-PN3 to capsular Streptococcus pneumoniae.

[0345] Prophetic Example 6: Opsonin phagocytic sterilization of Streptococcus pneumoniae by gamma-PN1, gamma-PN1ΔlytC::kanR, gamma-PN1ΔlytC, gamma-PN2, or gamma-PN3 inducing antibodies. Host defense against pneumococcal infection is primarily mediated by opsonin-dependent phagocytosis. Therefore, the opsonin-phagocytic killing activity (OPA) of pneumococcal-specific antibodies is considered a useful in vitro measure of functional antibody activity. OPA titers are determined after vaccination of rabbits with gamma-PN1, gamma-PN1ΔlytC::kanR, gamma-PN1ΔlytC, gamma-PN2, or gamma-PN3.

[0346] Rabbits are intramuscularly (IM) vaccinated with gamma-PN1, gamma-PN1ΔlytC::kanR, gamma-PN1ΔlytC, gamma-PN2, or gamma-PN3 (500 μg of total protein in 0.5 mL of PBS per rabbit). The rabbits are immunized three times at 3-week intervals. Serum is collected from all rabbits before immunization (labeled "pre-bleeding"), and from all rabbits three weeks after the final immunization. Individual or pooled serum samples are tested for opsonin-phagocytic activity against a panel of Streptococcus pneumoniae serotypes using the opsonin-phagocytic assay protocol established by Nahm and Burton (2014).

[0347] The opsonin phagocytic index (OI) titer is determined as the reciprocal of the largest 3-fold serial dilution of rabbit serum that kills more than 50% of the input CFU. If no mortality is detected in undiluted serum (or 1:4 dilution if mixed with other assay components), the OI is expressed as a titer of ≤4.

[0348] The results reveal the OPAs for different serotypes of Streptococcus pneumoniae (S. pneumoniae): gamma-PN1, gamma-PN1ΔlytC::kanR, gamma-PN1ΔlytC, gamma-PN2, and / or gamma-PN3.

[0349] Predictive Example 7: Effect of lytC deletion on antibody profiles induced by vaccination Experiments will be conducted to evaluate whether the deletion of lytC alters the binding profile of serum IgG to a panel of known pneumococcal antigens.

[0350] Swiss mice were vaccinated with 50 μg of total protein each of gamma-PN1, gamma-PN1ΔlytC::kanR, gamma-PN1ΔlytC, gamma-PN2, and gamma-PN3 for three doses every other week. Control mice were given a placebo vaccine of PBS / glycerol. Fourteen days after the last vaccination, serum was collected and tested for IgG binding to a panel of 289 pneumococcal antigenic proteins. Pneumococcal-specific antibody titers were calculated using control mice.

[0351] Predictive Example 8: Analysis of gene expression in strains with lytC deletion relative to the parent strain. Experiments will be conducted to evaluate arbitrary changes in gene transcription induced by the deletion of lytC.

[0352] Pneumococcal (S. pneumoniae) vaccine strains GPN-001, GPN-001ΔlytC, GPN-002, GPN-002ΔlytC, and / or GPN-003 are cultured in a standard animal-free soybean-based medium until the mid-log phase, and RNA is isolated using conventional techniques (e.g., Qiagen RNeasy Mini Kit). Transcriptome RNA sequencing analysis is performed essentially as described above. Briefly, ribosomal RNA content is depleted using established methods (e.g., Epicentre Bacterial Ribozero Kit, Illumina) to create a barcoded library (e.g., Ultra-Directional RNA kit, New England Biolabs). The prepared library is sequenced (e.g., using Illumina HiSeq2500), and the reads are aligned to the *Streptococcus pneumoniae* (Rx1, GenBank accession number CP079923) genome (e.g., BOWTIE2). Gene counts and differential gene expression are derived using various tools (e.g., SAMtools, BEDtools, R, DESeq Library).

[0353] Predictive Example 9: Proteomic analysis of a strain with lytC deletion relative to the parent strain. Experiments will be conducted to evaluate arbitrary changes in protein abundance induced by lytC deletion.

[0354] Pneumococcus pneumoniae vaccine strains GPN-001, GPN-001ΔlytC, GPN-002, GPN-002ΔlytC, and / or GPN-003 are cultured in a standard animal-free soybean-based medium, collected by centrifugation, and lysed using a combination of chemical and mechanical methods. Comparative proteome separation and protein identification are performed using standard techniques (e.g., two-dimensional differential gel electrophoresis [2D-DIGE] and mass spectrometry).

[0355] Predictive Example 10: Construction of vaccine strains with lytA, lytC, psaA, and pdT modifications in alternative non-encapsulated background strains. The experiments conducted so far have been based on a derivative strain of Streptococcus pneumoniae (S. pneumoniae) strain Rx1, which is an unencapsulated derivative of serotype 2 D39. However, similar results are expected for unencapsulated strains.

[0356] Experiments are conducted to genetically modify strains by removing genes from the capsular biosynthesis locus (cps gene) or by culturing the strains in a way that induces spontaneous loss of the capsular gene. Using conventional genetic methods, or, without being particularly limited, serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10B, IOC, 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, 22 Capsular genes can be removed by standard in vitro serial passage of any serotype including F, 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.

[0357] For example, but not limited to, genes at the cps locus, including 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, and cpsO, can be genetically modified using conventional methods to suppress capsule production / expression. Capsule absence or reduction is confirmed by measurement of capsule content, serotyping assay, and microscopy, and experiments are carried out to introduce further modifications to one or more of the following genes, namely lytA, ply / pdt, psaA, and lytC, using conventional gene manipulation methods.

[0358] As one non-limiting example, experiments are performed to remove the cps4E gene from a well-characterized TIGR4 strain (serotype 4). The upstream and downstream 2kb regions of the cps4E gene are amplified, and oligonucleotide primers are designed to incorporate regions complementary to the antibiotic resistance cassette. Overlap extension PCR is used to generate a linear fragment of the 2kb adjacent region containing the antibiotic resistance cassette, which is confirmed by seeding onto an antibiotic-supplemented blood agar plate. Then, experiments are performed using conventional genetic engineering techniques to introduce further modifications to one or more of the following genes, namely lytA, ply / pdt, psaA, and lytC. The resulting strain can be defined as TIGR4Δcps4EΔlytA PdTΔpsaAΔlytC, which is similar to the GPN-003 strain described herein.

[0359] The obtained vaccine strains are tested in animal models as live or inactivated vaccines attenuated (e.g., by chemical or physical methods such as exposure to high-energy photons) via intranasal, intramuscular, subcutaneous, or intraperitoneal routes at several time points. Serum is collected two weeks after final immunization and evaluated for immunological responses, e.g., antibody titer (e.g., total IgG titer), antibody binding (e.g., IgG binding to capsular bacteria), antibody function (e.g., opsonin phagocytic response), and protective efficacy.

[0360] The data obtained from these experiments will confirm the immunogenicity of vaccines prepared based on alternative non-encapsulated strains of Streptococcus pneumoniae (S. pneumoniae) such as TIGR4Δcps4EΔlytA PdTΔpsaAΔlytC.

[0361] Predictive Example 11: Construction of vaccine strains with modifications of lytA, lytC, psaA, and pdT in a capsular background strain manipulated to express a smaller amount of capsule on the cell surface. The experiments conducted so far have been based on *Streptococcus pneumoniae* Rx1, a non-capsule derivative of serotype 2 D39. However, similar results are expected for strains manipulated to express lower levels of capsule, which also allows exposure to important surface antigens. Experiments will be conducted to manipulate capsule-type bacterial strains by introducing genetic modifications that reduce but do not eliminate the expression of capsular polysaccharides, or to alter their localization or adhesion to the cell surface. While not particularly limited, serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10B, IOC, 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, 2 Strains derived from any serotype, including 3B, 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, can be manipulated to reduce the level of capsule on the cell surface.

[0362] One method that can achieve this is through modification of genes such as Cps2A, LytR, and Psr, which are thought to be involved in capsule retention in the cell wall. Alternatively, specific point mutations in cpsE have been shown to reduce total capsule production in clinical isolates. Mutations in vaccine strains to remove or modify these genes are constructed using conventional methods alone or in combination and are expected to result in a decrease in capsule volume. The decrease in cell-associated capsules is confirmed by measuring the capsule abundance. The ability to further expose surface and cell membrane protein antigens is evaluated by comparing antibody binding between modified strains and parental strains using serum produced in animals with an uncapsule-type vaccine strain (e.g., GPN-003) or using specific proteins representing classes of proteins exposed to varying degrees on the surface (e.g., PspA, PcpA, PrtA, PhtE, PiuA, AdcA, etc.). To create capsular vaccine strains with these modifications, experiments are carried out by introducing further modifications to one or more of the following genes, namely lytA, ply / pdt, psaA, and lytC, using conventional gene manipulation techniques. For example, it is possible to create vaccine strains using the Streptococcus pneumoniae strain D39Δcps2KΔlytRΔlytAPdTΔpsaAΔlytC.

[0363] The obtained vaccine is tested in animal models as a live vaccine or an inactivated vaccine, attenuated (e.g., by chemical or physical methods such as exposure to high-energy photons) by immunizing animals (e.g., mice or rabbits) at several time points via intranasal, intramuscular, subcutaneous, or intraperitoneal routes. Serum is collected two weeks after final immunization and evaluated for immunological response (e.g., antibody titer (total IgG titer), antibody binding (IgG binding to capsular bacteria), antibody function (opsonization phagocytic response), and protective efficacy).

[0364] The data obtained from these experiments will be used to confirm the immunogenicity of vaccines created based on capsule-type strains that have been manipulated to alter the amount of surface capsule present.

[0365] Predictive Example 12: Construction of vaccine strains with modifications of lytA, lytC, psaA, and pdT in alternative capsular background strains cultured in a manner that reduces capsular expression. The experiments conducted so far have been based on *Streptococcus pneumoniae* Rx1, an uncapsulated derivative of serotype 2 D39. However, similar results are expected for capsulated strains cultured in a manner that results in lower expression of capsular polysaccharides, which enable exposure to important surface antigens. While not particularly limited, serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10B, IOC, 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, 23 The overall capsular expression can be reduced by culturing strains derived from any serotype including A, 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.

[0366] To produce capsular vaccine strains with these modifications, experiments are carried out by introducing modifications to one or more of the following genes, namely lytA, ply / pdt, psaA, and lytC, using conventional gene manipulation techniques. One approach to regulating capsular expression is to overload bacteria with zinc. Zinc excess affects the activity of the phosphoglucum-comutase enzyme required for the production of important sugar precursors for capsular polysaccharide biosynthesis, leading to a reduction in capsules. Bacteria can be overloaded with zinc using various methods, 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), manipulating the strain to remove the zinc efflux protein gene (czcD), or performing chemical treatments during growth, such as using ionophores that increase the transmembrane transfer of zinc (e.g., zinc pyrithione, PBT2, etc.).

[0367] The obtained vaccine is tested in animal models as a live vaccine or an inactivated vaccine, attenuated (e.g., by chemical or physical methods such as exposure to high-energy photons) by immunizing animals (e.g., mice or rabbits) at several time points via intranasal, intramuscular, subcutaneous, or intraperitoneal routes. Serum is collected two weeks after final immunization and evaluated for immunological response (e.g., antibody titer [total IgG titer], antibody binding [IgG binding to capsular bacteria], antibody function [opsonization response]) and protective efficacy.

[0368] References Corsini, B., Aguinagalde, L., Ruiz, S., Domenech, M., & 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.

[0369] Croucher,N.J.,Campo,J.J.,Le,T.Q.,Liang,X.,Bentley,S.D.,Hanage,W.P.,& 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。

[0370] 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。

[0371] Holmlund,E.,Quiambao,B.,Ollgren,J.,Jaakkola,T.,Neyt,C.,Poolman,J.,...& Kayhty,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。

[0372] Nahm,M.H.and Burton,R.L.(2014)Protocol for multiplexed opsonophagocytic killing assay(UAB-MOPA)for antibodies against Streptococcus pneumoniae(Version 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

[0373] Tomasz,A.,& 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。

[0374] Wizemann,T.M.,Heinrichs,J.H.,Adamou,J.E.,Erwin,A.L.,Kunsch,C.,Choi,G.H.,...& Koenig,S.(2001).Use of a whole genome approach to identify vaccine molecules affording protection against Streptococcus pneumoniae infection.Infection and immunity,69(3),1593-1598。

Claims

1. An immunogenic composition comprising a modified strain of Streptococcus bacteria that reduces, diminishes, or prevents the functional expression of LytC or its homologues, thereby weakening, reducing, or preventing its functional expression.

2. The immunogenic composition according to claim 1, wherein the modification comprises the deletion of the lytC gene or its homolog.

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

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

5. The immunogenic composition according to any one of claims 1 to 4, wherein the attenuated and / or dead streptococcal bacterial strain is derived from the parent strain lacking the modification, and the attenuated and / or dead 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 dead streptococcal bacterial strain further comprises a modification or deletion of a gene selected from the group consisting of lytA or its homologue, ply or its homologue, and psaA or its homologue.

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

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

9. The immunogenic composition according to any one of claims 1 to 8, wherein the attenuated and / or dead streptococcal bacterial strain further comprises a toxoid-type ply called PdT or its homologue.

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

11. The immunogenic composition according to any one of claims 1 to 10, wherein the attenuated and / or dead streptococcal bacterial strain further comprises a modification or deletion of the lytA gene or its homolog, a modification or deletion of the ply gene or its homolog, and a modification or deletion of the psaA gene or its homolog, and further comprises a toxoid type ply or its homolog called PdT.

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

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

14. The immunogenic composition according to any one of claims 1 to 13, wherein the composition comprises a single attenuated and / or dead streptococcal bacterial strain that includes modifications to reduce, decrease, or prevent the functional expression of LytC or its homologue.

15. The immunogenic composition according to any one of claims 1 to 13, wherein the composition comprises a plurality of attenuated and / or dead streptococcal bacterial strains, each of which is modified to reduce, decrease, or prevent the functional expression of LytC or its homologue.

16. The aforementioned weakened and / or dead streptococcal bacterial strains include Streptococcus agalactiae, Streptococcus bovis, Streptococcus canis, Streptococcus dysgalactiae, Streptococcus equi, Streptococcus equinus, Streptococcus equisimilis, Enterococcus faecalis, Enterococcus faecium, Streptococcus iniae, Streptococcus milleri, and Streptococcus mutans. The immunogenic composition according to any one of claims 1 to 15, wherein the species is selected from the group consisting of Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus salivarius, Streptococcus sanguinis, Streptococcus suis, and Streptococcus uberis.

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

18. The immunogenic composition according to 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 dead 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 dead streptococcal bacterial strain induces an immune response against a plurality 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 dead streptococcal bacterial strain induces a cross-protective immune response against the 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 dead streptococcal bacterial strain induces a cross-protective immune response against a plurality of 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 dead streptococcal bacterial strain is derived from the same strain, serotype, or species 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 dead streptococcal bacterial strain is derived from a strain, serotype, or species different from the at least one target streptococcal species and / or its serotype.

25. The aforementioned at least one target streptococcal bacterial species and / or its serotype is Streptococcus agalactiae, Streptococcus bovis, Streptococcus canis, Streptococcus dysgalactiae, Streptococcus equi, Streptococcus equinus, Streptococcus equisimilis, Enterococcus faecalis, Enterococcus faecium, Streptococcus iniae, Streptococcus milleri, and Streptococcus mutans. An immunogenic composition according to any one of claims 1 to 24, selected from the group consisting of Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus salivarius, Streptococcus sanguinis, Streptococcus suis, and Streptococcus uberis.

26. The immunogenic composition according to any one of claims 1 to 25, which is a dead whole cell immunogenic composition.

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

28. The immunogenic composition according to any one of claims 1 to 27, wherein the immunogenic composition that kills whole cells is a photon-irradiated immunogenic composition that kills whole cells.

29. The immunogenic composition according to any one of claims 1 to 28, wherein the immunogenic composition that kills whole cells is a immunogenic composition that kills whole cells by gamma radiation.

30. The immunogenic composition according to any one of claims 1 to 29, wherein the whole-cell immunogenic composition is an X-ray emission whole-cell immunogenic composition.

31. The immunogenic composition according to any one of claims 1 to 30, wherein the immunogenic composition that kills whole cells is an immunogenic composition that kills whole cells by gamma irradiation and X-ray irradiation.

32. The immunogenic composition according to any one of claims 1 to 25, which 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 described in 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 of the immunogenic composition according to any one of claims 1 to 34 or the vaccine composition according to claim 35.

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

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

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

42. The method according to any one of claims 38 to 42, wherein the attenuated and / or dead streptococcal bacterial strain is derived from the same strain, serotype, or species 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 dead streptococcal bacterial strain is derived from a strain, serotype, or species 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 a plurality of target streptococcal species and / or its serotypes.

45. The aforementioned target streptococcal species and / or serotypes include Streptococcus agalactiae, Streptococcus bovis, Streptococcus canis, Streptococcus dysgalactiae, Streptococcus equi, Streptococcus equinus, Streptococcus equisimilis, Enterococcus faecalis, Enterococcus faecium, Streptococcus iniae, Streptococcus milleri, and Streptococcus mutans. The method according to any one of claims 38 to 42, comprising a streptococcal bacterial species and / or serotype thereof selected from the group consisting of Streptococcus mutans), Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus salivarius, Streptococcus sanguinis, Streptococcus suis, and Streptococcus uberis.

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 by a route of administration selected from the group consisting of intranasal, intravenous, intramuscular, subcutaneous, oral, intraocular, transmucosal, and transdermal administration.

47. The method according to claim 41, wherein the disease or condition is selected from the group consisting of respiratory tract infections, pneumonia, ear infections, ear pain, middle ear infections, otitis media, sinusitis, meningitis, conjunctivitis, bacteremia, sepsis, joint infections, bone infections, suppurative arthritis, osteomyelitis, soft tissue infections, cellulitis, myositis, periorbital cellulitis, abscess, peritonitis, cardiac infections, 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, (i) Irradiating a preparation of the streptococcal bacterial strain, which includes modifications that reduce, decrease, or prevent the functional expression of LytC or its homologue, with photons to kill the bacteria or physically weaken them, thereby producing photon-irradiated streptococcal bacteria, (ii) A method comprising combining the photon-irradiated streptococcal bacteria with a pharmaceutically acceptable excipient, diluent and / or carrier, and optionally an adjuvant.

49. The method according to claim 48, wherein the photon beam irradiation includes exposing the streptococcal bacterial strain to gamma-ray radiation.

50. The method according to claim 49, wherein the photon irradiation includes 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 includes exposing the streptococcal bacterial strain to gamma-ray radiation and X-ray radiation.

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

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