vaccine

Abstract

The present invention relates to a composition comprising at least one Group A Streptococcus antigen, an aluminum salt, and (a) a TLR7 agonist, or (b) a benzonaphthyridine compound. The present invention further relates to a method for producing an immunogenic composition comprising at least one antigen, an aluminum salt, and (a) a TLR7 agonist, and / or (b) a benzonaphthyridine compound. The present invention also relates to a vaccine comprising the composition, and methods and applications of the composition.

Description

[Technical Field]

[0001] The present invention relates to compositions comprising an antigen, an aluminum salt, and a TLR7 agonist and / or a benzonaphthyridine compound. The present invention also relates to methods for making such compositions, vaccines comprising the compositions, and methods or uses of the compositions. [Background technology]

[0002] Group A streptococci (GAS) cause a variety of diseases, ranging from superficial infections (pharyngitis, skin infections) to severe invasive diseases (cellulitis, birth bed sepsis, necrotizing fasciitis, streptococcal toxic shock syndrome), and are frequently associated with severe autoimmune sequelae such as acute rheumatic fever (ARF), rheumatic heart disease (RHD), and glomerulonephritis in low- and middle-income countries (LMICs) (Ralph et al. Curr. Top. Microbiol. Immunol. 2013; 368: 1-27).

[0003] Pharyngitis is the most frequent symptomatic GAS infection in children worldwide, with an estimated annual incidence of over 600 million cases (Carapetis et al.; The Lancet 2005; 5: 685-94), and is a significant driver of antibiotic use (Dooling et al.; Jama Pediatr. 2014; 168: 1073-4), which may ultimately lead to increased antimicrobial resistance, a growing public health crisis (Jansen et al.; Nature Medicine 2018; 24: 10-19). Pharyngitis can lead to RHD, a chronic inflammatory valvular heart disease that represents the major global burden of GAS. In 2015, there were an estimated 33 million cases of RHD and 319,000 deaths due to RHD, along with 10 million disability-adjusted life years (DALYs) lost (Watkins et al.; N. Engl. J. Med. 2017; 377: 713-722). Vaccination is the most feasible strategy for reducing the global GAS-related disease burden in the long term. However, there is still no commercially available vaccine against this pathogen (Vekemans et al.; Clin. Infect. Dis. 2019; 69: 877-883).

[0004] Group A carbohydrates (GACs) are surface polysaccharides containing a polyrhamnose backbone with alternating N-acetylglucosamine (GlcNAc) side chains. They are highly conserved and expressed across GAS strains, making them attractive vaccine candidates. Indeed, one of the major obstacles to vaccine strategy development represents the diversity of GAS serotypes relative to other non-glycosylated antigens (Walker et al.; Clin. Microbiol. Rev. 2014; 27: 264-301).

[0005] Human anti-GAC serum successfully stimulates phagocytosis of several GAS strains (Salvadori et al.; J. Infect. Dis. 1995; 171: 593-600), whereas tetanus toxoid (TT) or CRM 197It has been reported that mice immunized with GAC conjugated to a carrier protein were protected from GAS challenge (Kabanova et al.; Vaccine 2010; 29: 104-114 and Sabharwal et al.; J Infect. Dis. 2006; 193: 129-135). Furthermore, an inverse correlation was demonstrated between high anti-GAC antibody titers and the presence of GAS in the pharynx of Mexican children (Sabhawral et al., supra).

[0006] Conserved protein antigens are also being developed for vaccines against GAS. In particular, streptolysin O (SLO), SpyAD, and SpyCEP have been identified as promising vaccine candidates using a reverse vaccinology approach (Bensi et al.; Mol. Cell Proteomics 2012; 11, M111 015693). SLO has been shown to be an important virulence factor for GAS by preventing bacterial internalization into lysosomes where they can be destroyed (Cunningham; Clin. Microbiol. Rev. 2000; 13: 470-511). Furthermore, SLO promotes resistance of GAS to phagocytic clearance by neutrophils, facilitating GAS escape from innate immune killing. Inactivated SLO was demonstrated to be protective against GAS challenge in a mouse model (Vchiyama et al.; Front Immunol. 2015; 6: 581). SpyAD is a surface-exposed adhesin that mediates the interaction of GAS with host cells. Furthermore, deletion of the SpyAD gene in GAS strains results in impaired proper cell division in knockout mutants, suggesting its role in bacterial cell division (Gallotta et al.; Infect. Immun. 2014; 82: 2890-2901). Finally, SpyCEP is a multidomain protease with a catalytic domain responsible for cleaving interleukin (IL)-8 and other chemokines. IL-8 cleavage represents an immune evasion mechanism, preventing endothelial migration via the C-terminus of IL-8 and subsequent neutrophil recruitment (Edwards et al.; J. Infect. Dis. 2005; 192: 783-790 and Jobichen et al.; J. Infect. Dis. 2005; 192: 783-790).

[0007] These three protein antigens are highly conserved and widely present in clinical samples, and together with GAC, they can cover virtually all GAS clinical isolates (Davies et al.; Nat. Genet. 2019; 51: 1035-1043). Therefore, recombinant SLO, SpyAD, SpyCEP, and GAC-CRM 197 The formulation of multicomponent vaccines consisting of conjugates has been proposed (see Vekemans et al., supra).

[0008] Recombinant SLO, SpyAD, SpyCEP and GAC-CRM 197 A multivalent vaccine containing recombinant SLO, SpyAD, SpyCEP and GAC-CRM has the potential to be a superior vaccine. 197 It is an object of the present invention to further improve the immune response generated against a vaccine containing an antigen. Summary of the Invention

[0009] The present inventors have surprisingly found that compositions comprising at least one Group A Streptococcus antigen, an aluminum salt (such as aluminum hydroxide), and a TLR7 agonist and / or a benzonaphthyridine compound (such as LHD153R) are not only significantly more immunogenic than corresponding compositions lacking the TLR7 agonist and / or benzonaphthyridine compound, but also, when administered to mice, generate significantly greater numbers of functional antibodies against the at least one Group A Streptococcus antigen compared to corresponding compositions lacking the TLR7 agonist and / or benzonaphthyridine compound.

[0010] Furthermore, the inventors have surprisingly found that such compositions: (i) at least one antigen pre-adsorbed onto an aluminum salt and a TLR7 agonist and / or a benzonaphthyridine compound; (ii) a TLR7 agonist and / or a benzonaphthyridine compound pre-adsorbed onto at least one antigen and an aluminum salt; or (iii) at least one antigen pre-absorbed onto an aluminum salt and a TLR7 agonist and / or a benzonaphthyridine compound pre-adsorbed onto an aluminum salt; It has been found that the compound can be advantageously prepared using a method which comprises preparing a mixture of:

[0011] Thus, in a first aspect of the present invention there is provided an immunogenic composition comprising at least one Group A Streptococcus antigen, an aluminium salt and a TLR7 agonist.

[0012] In a second aspect of the present invention, there is provided an immunogenic composition comprising at least one Group A Streptococcus antigen, an aluminum salt and a benzonaphthyridine compound.

[0013] In a third aspect of the present invention, there is provided a method for producing an immunogenic composition containing at least one antigen, an aluminum salt, and (a) a TLR7 agonist and / or (b) a benzonaphthyridine compound, the method comprising the steps of: (i) at least one antigen pre-adsorbed onto an aluminum salt and a TLR7 agonist and / or a benzonaphthyridine compound; (ii) a TLR7 agonist and / or a benzonaphthyridine compound pre-adsorbed onto at least one antigen and an aluminum salt; or (iii) at least one antigen pre-adsorbed onto an aluminum salt, and a TLR7 agonist and / or a benzonaphthyridine compound pre-adsorbed onto an aluminum salt; and preparing a mixture of:

[0014] In a fourth aspect, there is provided an immunogenic composition obtained or obtainable from the method of the present invention.

[0015] In a fifth aspect of the invention, there is provided a vaccine comprising the immunogenic composition of the invention.

[0016] In a sixth aspect of the invention there is provided an immunogenic composition or vaccine of the invention for use in a method for preventing infection with Group A Streptococcus and / or preventing autoimmune disease following infection with Group A Streptococcus.

[0017] In a seventh aspect of the present invention, there is provided a method for preventing infection with Group A Streptococcus and / or autoimmune disease following infection with Group A Streptococcus, comprising administering an effective amount of the immunogenic composition or vaccine of the present invention.

[0018] In an eighth aspect of the present invention there is provided the use of an immunogenic composition or vaccine of the present invention for the preparation of a medicament for use in a method for the prevention of infection with group A streptococcus and / or autoimmune disease following infection with group A streptococcus. [Brief explanation of the drawings]

[0019] [Figure 1-1]Figure 1 is a graph showing data on the IgG responses observed in mice after vaccination with a vaccine containing a group A streptococcal antigen, aluminum hydroxide, and LHD153R, or a vaccine containing a group A streptococcal antigen and aluminum hydroxide but not LHD153R. Group A streptococcal antigen was administered at three different doses (low, medium, and high). The low dose corresponds to 78 ng of SpyAD polypeptide, 78 ng of SLO polypeptide, 78 ng of SpyCEP polypeptide, and 39 ng of GAC-CRM197. The medium dose corresponds to 625 ng of SpyAD polypeptide, 625 ng of SLO polypeptide, 625 ng of SpyCEP polypeptide, and 313 ng of GAC-CRM197. The high dose corresponds to 5000 ng of SpyAD polypeptide, 5000 ng of SLO polypeptide, 5000 ng of SpyCEP polypeptide, and 2500 ng of GAC-CRM197. Panel A shows the antibody response to SpyCEP (measured in RLU / ml) at 27 days post-vaccination, and panel B shows the antibody response to SpyCEP (measured in RLU / ml) at 42 days post-vaccination. Panel C shows the antibody response to SpyAD (measured in RLU / ml) at 27 days post-vaccination, and panel D shows the antibody response to SpyAD (measured in RLU / ml) at 42 days post-vaccination. Panel E shows the antibody response to SLO (measured in RLU / ml) at 27 days post-vaccination, and panel F shows the antibody response to SLO (measured in RLU / ml) at 42 days post-vaccination. Panel G shows the antibody response to GAC (measured in RLU / ml) at 27 days post-vaccination, and panel H shows the antibody response to GAC (measured in RLU / ml) at 42 days post-vaccination. [Figure 1-2] Continued from Figure 1-1. [Figure 1-3] Continued from Figure 1-2. [Figure 1-4] Continued from Figure 1-3. [Figure 2]Figure 2 is a graph showing the amount of functional antibodies generated in mice after vaccination with a vaccine containing a group A streptococcal antigen, aluminum hydroxide, and LHD153R, or a vaccine containing a group A streptococcal antigen and aluminum hydroxide without LHD153R. Group A streptococcal antigen was administered at two different doses (medium and high). The medium dose corresponds to 625 ng of SpyAD polypeptide, 625 ng of SLO polypeptide, 625 ng of SpyCEP polypeptide, and 313 ng of GAC-CRM197. The high dose corresponds to 5000 ng of SpyAD polypeptide, 5000 ng of SLO polypeptide, 5000 ng of SpyCEP polypeptide, and 2500 ng of GAC-CRM197. Panel A shows the amount of functional antibodies generated against SpyCEP, as determined using an IL-8 cleavage assay. Panel B shows the amount of functional antibodies generated against SLO, as determined using a hemolytic assay. [Figure 3-1]Figure 3 is a graph showing the IgG2 / IgG1 and IgG3 / IgG1 ratios observed in mice after vaccination with a vaccine containing group A streptococcal antigen, aluminum hydroxide, and LHD153R, or a vaccine containing group A streptococcal antigen and aluminum hydroxide but not LHD153R. The doses of group A streptococcal antigen used were 5,000 ng of SpyAD polypeptide, 5,000 ng of SLO polypeptide, 5,000 ng of SpyCEP polypeptide, and 2,500 ng of GAC-CRM197. For each panel, the left column corresponds to the vaccine containing only group A streptococcal antigen and aluminum hydroxide; the middle column corresponds to the vaccine containing group A streptococcal antigen, aluminum hydroxide, and LHD153R; and the right column corresponds to the vaccine containing group A streptococcal antigen but no adjuvant. Panel A shows the IgG2 / IgG1 ratio of antibodies raised against GAC. Panel B shows the IgG2 / IgG1 ratio of antibodies raised against SLO. Panel C shows the IgG2 / IgG1 ratio of antibodies raised against SpyAD. Panel D shows the IgG2 / IgG1 ratio of antibodies raised against SpyCEP. Panel E shows the IgG3 / IgG1 ratio of antibodies raised against GAC. Panel F shows the IgG3 / IgG1 ratio of antibodies raised against SLO. Panel G shows the IgG3 / IgG1 ratio of antibodies raised against SpyAD. Panel H shows the IgG3 / IgG1 ratio of antibodies raised against SpyCEP. [Figure 3-2] Continued from Figure 3-1. [Figure 3-3] Continued from Figure 3-2. [Figure 3-4] Continued from Figure 3-3. [Figure 4-1]Figure 4 is a graph showing data on IgG responses observed in mice after vaccination with a vaccine containing a group A streptococcal antigen, aluminum hydroxide, and LHD153R, or a vaccine containing a group A streptococcal antigen but no adjuvant. Group A streptococcal antigen was administered at three different doses (low, medium, and high). The low dose corresponds to 78 ng of SpyAD polypeptide, 78 ng of SLO polypeptide, 78 ng of SpyCEP polypeptide, and 39 ng of GAC-CRM197. The medium dose corresponds to 625 ng of SpyAD polypeptide, 625 ng of SLO polypeptide, 625 ng of SpyCEP polypeptide, and 313 ng of GAC-CRM197. The high dose corresponds to 5000 ng of SpyAD polypeptide, 5000 ng of SLO polypeptide, 5000 ng of SpyCEP polypeptide, and 2500 ng of GAC-CRM197. Panel A shows the antibody response to SpyCEP (measured in RLU / ml) at 27 days post-vaccination, and panel B shows the antibody response to SpyCEP (measured in RLU / ml) at 42 days post-vaccination. Panel C shows the antibody response to SpyAD (measured in RLU / ml) at 27 days post-vaccination, and panel D shows the antibody response to SpyAD (measured in RLU / ml) at 42 days post-vaccination. Panel E shows the antibody response to SLO (measured in RLU / ml) at 27 days post-vaccination, and panel F shows the antibody response to SLO (measured in RLU / ml) at 42 days post-vaccination. Panel G shows the antibody response to GAC (measured in RLU / ml) at 27 days post-vaccination, and panel H shows the antibody response to GAC (measured in RLU / ml) at 42 days post-vaccination. [Figure 4-2] Continued from Figure 4-1. [Figure 4-3] Continued from Figure 4-2. [Figure 4-4] Continued from Figure 4-3. [Figure 5]Figure 5 is a graph showing data on the amount of functional antibodies generated in mice after vaccination with a vaccine containing a group A streptococcal antigen, aluminum hydroxide, and LHD153R, or a vaccine containing a group A streptococcal antigen but no adjuvant. Group A streptococcal antigen was administered at two doses (medium and high). The medium dose corresponds to 625 ng of SpyAD polypeptide, 625 ng of SLO polypeptide, 625 ng of SpyCEP polypeptide, and 313 ng of GAC-CRM197. The high dose corresponds to 5000 ng of SpyAD polypeptide, 5000 ng of SLO polypeptide, 5000 ng of SpyCEP polypeptide, and 2500 ng of GAC-CRM197. Panel A shows the amount of functional antibody generated against SpyCEP, as determined using an IL-8 cleavage assay. Panel B shows the amount of functional antibody generated against SLO, as determined using a hemolytic assay. [Figure 6-1] FIG. 6 is a sequence listing. [Figure 6-2] Continued from Figure 6-1. [Figure 6-3] Continued from Figure 6-2. DETAILED DESCRIPTION OF THE INVENTION

[0020] general definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0021] In general, the term "comprising" is intended to mean including, but not limited to. For example, the phrase "an immunogenic composition comprising at least one Group A Streptococcus antigen, an aluminum salt, and a TLR7 agonist" should be interpreted to mean that the composition includes at least one Group A Streptococcus antigen, an aluminum salt, and a TLR7 agonist, but may include additional components.

[0022] In some embodiments of the invention, the word "comprising" is replaced with the phrase "consisting of." The term "consisting of" is intended to be limiting. For example, the phrase "an immunogenic composition consisting of at least one Group A Streptococcus antigen, an aluminum salt, and a TLR7 agonist" should be understood to mean that the composition contains at least one Group A Streptococcus antigen, an aluminum salt, and a TLR7 agonist, and does not contain additional components.

[0023] In some embodiments of the present invention, the word "comprising" is replaced with the phrase "consisting essentially of." The term "consisting essentially of" means that certain additional components may be present, i.e., components that do not significantly affect the essential characteristics of the contents. For example, the phrase "an immunogenic composition consisting essentially of at least one Group A Streptococcus antigen, an aluminum salt, and a TLR7 agonist" may include components such as buffers or other pharmaceutically acceptable excipients.

[0024] When referring to a numerical value, the term "about" or "around" refers to that value, but within a reasonable scientific error. Alternatively, if the value is within 10%, within 5%, or within 1% of x, then it is "about x" or "around x."

[0025] The singular forms "a," "an," and "the" include plural references unless the content clearly dictates otherwise.

[0026] An amino acid "corresponding to" a specified position in a particular SEQ ID NO can be the amino acid at a particular position in the particular SEQ ID NO described. For example, an amino acid "corresponding to position 272 in SEQ ID NO: 1" can be the amino acid at position 272 in SEQ ID NO: 1. Alternatively, an amino acid "corresponding to" a position in a particular SEQ ID NO can be an amino acid from an alternative amino acid sequence that corresponds to a particular amino acid in a particular SEQ ID NO. For example, an amino acid "corresponding to position 272 in SEQ ID NO: 1" can be the amino acid from an alternative amino acid sequence that corresponds to position 272 in SEQ ID NO: 1. Determining which amino acids in an alternative amino acid sequence "correspond" to a particular position in a particular SEQ ID NO is within the ability of one of ordinary skill in the art. For example, one of ordinary skill in the art need only perform a sequence alignment of the alternative amino acid sequence with a particular SEQ ID NO using an appropriate alignment algorithm, such as Needleman and Wunsch, described herein, to determine which regions of the alternative amino acid sequence align to a particular position in a particular SEQ ID NO. For example, one of ordinary skill in the art can align the alternative amino acid sequence with SEQ ID NO: 1 and determine which amino acids align and therefore correspond to, for example, position 272 in SEQ ID NO: 1.

[0027] For purposes of the present invention, to determine the percent identity of two sequences (such as two polynucleotide sequences or two polypeptide sequences), the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into the first sequence for optimal alignment with the second sequence). The nucleotide or amino acid residue at each position is then compared. If a position in the first sequence is occupied by the same nucleotide or amino acid as the corresponding position in the second sequence, then the nucleotide or amino acid is identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions in the reference sequence × 100).

[0028] Typically, sequence comparison is performed over the entire length of the reference sequence. For example, if a user wants to determine whether a given ("test") sequence is 95% identical to SEQ ID NO: 1, SEQ ID NO: 1 would be the reference sequence. To assess whether a sequence is at least 95% identical to SEQ ID NO: 1 (an example of a reference sequence), one skilled in the art would align the entire length of SEQ ID NO: 1 and identify how many positions in the test sequence are identical to those in SEQ ID NO: 1. If at least 95% of the positions are identical, the test sequence is at least 95% identical to SEQ ID NO: 1. If the test sequence is shorter than SEQ ID NO: 1, gaps or missing positions are considered non-identical positions.

[0029] Those skilled in the art are familiar with various computer programs available for aligning two sequences. For example, alignment between two sequences can be achieved using a mathematical algorithm. In one embodiment, two amino acid or nucleic acid sequences are aligned using the Needleman and Wunsch (1970) algorithm implemented in the GAP program of the Accelrys GCG software package, using either a Blosum62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6.

[0030] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0031] At least one antigen The present invention provides immunogenic compositions comprising at least one Group A Streptococcus antigen. Similarly, the present invention provides methods for producing immunogenic compositions comprising at least one antigen.

[0032] The at least one antigen may be selected from the group consisting of Actinomyces (e.g., A. israelii), Bacillus (e.g., B. anthracis or B. cereus), Bartonella (e.g., B. henselae or B. quintana), Bordetella (e.g., B. pertussis), Borrelia (e.g., B. burgdorferi, B. Borrelia garinii, B. afzelii, B. recurrentis), Brucella (e.g., B. abortus, B. canis, B. melitensis, or B. suis), Campylobacter (e.g., C. jejuni), Chlamydia (e.g., C. pneumoniae or C. trachomatis), Chlamydophila (e.g., C. psittaci), Clostridium (e.g., Clostridium botulinum, C. difficile, Clostridium perfringens, Clostridium tetani), Corynebacterium (e.g., Corynebacterium diphtheriae), Enterococcus (e.g., E. faecalis or E. faecium), Escherichia (e.g., Escherichia coli), Francisella (e.g., Francisella tularensis), Haemophilus (e.g., Haemophilus influenzae), Helicobacter (e.g., Helicobacter pylori), Klebsiella (e.g., Klebsiella pneumoniae and K. oxytoca), Legionella (e.g., L. pneumophila), Leptospira (e.g., L. interrogans, L. santarosai, L. weilii, L. noguchii), Listeria (e.g., L. monocytogenes), Mycobacterium (e.g., Mycobacterium leprae, Mycobacterium tuberculosis, or M. ulcerans), Mycoplasma (e.g., M. pneumoniae), Neisseria (e.g., Neisseria gonorrhoeae or Neisseria meningitidis), Pseudomonas (e.g., Pseudomonas aeruginosa), Rickettsia (e.g., R. rickettsii), Salmonella (e.g., S. Enteritidis, S. Paratyphi, S. Typhimurium, or S. Choleraesuis), Shigella (e.g., S. boydii, S. flexneri, S. sonnei, or S. dysenteriae), Staphylococcus (e.g., S. aureus, S. epidermidis, or S. saprophyticus), Streptococcus (e.g., S. agalactiae, S.The antigen may comprise an antigen derived from a bacterium selected from the group consisting of S. pneumoniae, S. pyogenes, Treponema (e.g., Treponema pallidum), Ureaplasma (e.g., U. urealyticum), Vibrio (e.g., V. cholerae), or Yersinia (e.g., Y. pestis, Y. enterocolitica, or Y. pseudotuberculosis).

[0033] The at least one antigen may comprise at least one Group A Streptococcus antigen. As used herein, the term "Group A Streptococcus" refers to and is intended to be synonymous with "Streptococcus pyogenes." Thus, any use of the term "Group A Streptococcus" can be replaced with the term "Streptococcus pyogenes." Streptococcus pyogenes is a pathogenic Gram-positive bacterium of the genus Streptococcus. Methods for determining whether a bacterium is Streptococcus pyogenes are well known to those skilled in the art. In particular, all strains of Streptococcus pyogenes express GAC, and those skilled in the art are familiar with methods for detecting GAC.

[0034] Antigens from Streptococcus pyogenes include those disclosed in WO2005032582, WO2006042027, WO2009034473, WO2009081274, and WO2010076618. Suitable antigens include polysaccharides such as GAC, optionally part of a polysaccharide-protein conjugate, SpyCEP polypeptides, SpyAD polypeptides, and / or SLO polypeptides.

[0035] Optionally, the at least one Group A Streptococcus antigen comprises at least one recombinant polypeptide. Optionally, the SpyCEP polypeptide, SpyAD polypeptide and / or SLO polypeptide is a recombinant polypeptide.

[0036] Optionally, at least one Group A Streptococcus antigen comprises a polysaccharide-protein conjugate. Optionally, one of the polysaccharides and / or proteins is a Group A Streptococcus antigen. For example, the protein can be a Streptococcus pyogenes antigen such as a SpyCEP polypeptide, a SpyAD polypeptide, and / or an SLO polypeptide. Alternatively or additionally, the polysaccharide can be a Streptococcus pyogenes antigen such as GAC.

[0037] Polysaccharide-protein conjugates The term "polysaccharide" refers to a linear or branched polymer of monosaccharide residues usually linked by glycosidic bonds, and thus includes oligosaccharides. "Protein" refers to or includes a linear or branched molecule of amino acid residues.

[0038] The term "polysaccharide-protein conjugate" refers to a molecule formed by the covalent bonding of a protein and a polysaccharide.

[0039] GAC (Lancefield group A carbohydrate) is a polysaccharide containing a polyrhamnose backbone with N-acetylglucosamine (GlcNAc) side chains and may contain repeating units of [→3)α-Rha(1→2)[β-GlcNAc(1→3)]α-Rha(1→] (referred to herein as "GAC subunit"). GAC is the major component of the GAS cell wall.

[0040] The term "GAC" refers to the GAC polysaccharide from a Streptococcus pyogenes strain. Alternatively, the term "GAC" can refer to a fragment or subunit (repeating unit) of the GAC polysaccharide, such as one or more GAC subunits. Optionally, the GAC comprises 18 GAC subunits.

[0041] A polysaccharide fragment refers to a polysaccharide that has been truncated relative to the wild-type polysaccharide (e.g., has a smaller average (statistical mean) number of monosaccharide units relative to the wild-type polysaccharide). Polysaccharide truncation can be achieved by any suitable means known in the art, such as chemical digestion, in vitro polysaccharide synthesis of a polysaccharide having fewer monosaccharide units than the wild-type, or genetic modification of a polysaccharide-producing strain.

[0042] A variant of a polysaccharide means or includes modifications of one or more chemical groups of the polysaccharide backbone and / or side chains compared to the wild-type polysaccharide. Modification of the polysaccharide can be achieved by any suitable means known in the art, such as chemical reaction or genetic modification of the polysaccharide-producing strain.

[0043] Polysaccharide antigens such as GAC can be excellent antigens for stimulating B cell-mediated immunity. However, it can be advantageous to conjugate GAC to a protein, such as a carrier protein. Suitable proteins include tetanus toxoid, diphtheria toxoid, and CRM. 197 or proteins derived from Streptococcus pyogenes such as a SpyAD polypeptide, a SpyCEP polypeptide and / or an SLO polypeptide.

[0044] Optionally, the carrier protein (i.e., the protein in the polysaccharide-protein conjugate) is a CRM 197 CRM 197 is a non-toxic mutant of diphtheria toxin with a single mutation at position 52, where glutamic acid is replaced by glycine. The mutation at position 52 abolishes the ADP-ribosyltransferase activity of naturally occurring diphtheria toxin. 197 The polypeptide sequence is set forth in SEQ ID NO:7.

[0045] The polypeptide can be conjugated to the protein using any suitable conjugation method.

[0046] The polysaccharide is preferably conjugated to the protein via an -NH group, for example, via the side chain of a lysine or arginine residue in the carrier polypeptide. If the polysaccharide has a free aldehyde group, this group can react with an amine in the protein to form a conjugate by reductive amination. Conjugation to the carrier can also be via an -SH group, for example, via the side chain of a cysteine ​​residue in the carrier polypeptide. Alternatively, the polysaccharide can be conjugated to the carrier protein via a linker molecule.

[0047] Polysaccharides are usually activated or functionalized before conjugation. Activation can be achieved using cyanylating reagents such as CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate). Other suitable methods use carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, and TSTU (see, for example, the introduction to WO 98 / 42721).

[0048] Direct linkage to a protein can involve oxidation of the polysaccharide followed by reductive amination with the protein, as described, for example, in U.S. Pat. Nos. 4,761,283 and 4,356,170. Linkage via a linker group can be carried out using known procedures, such as those described in U.S. Pat. Nos. 4,882,317 and 4,695,624. Typically, the linker is attached through the anomeric carbon of the polysaccharide. A preferred type of linkage is an adipic acid linker, which can be formed by coupling a free -NH group (e.g., introduced into the polysaccharide by amination) with adipic acid (e.g., using diimide activation), followed by coupling the resulting sugar-adipic acid intermediate with the protein (see, for example, EP-B-0477508, Mol. Immunol. (1985) 22, 907-919, and EP-A-0208375). A similar preferred type of linkage is a glutaric acid linker, which can be formed by similarly coupling a free -NH group with glutaric acid. Adipic acid and glutaric acid linkers can also be formed by direct attachment to the polysaccharide, i.e., by attaching a protein to the resulting sugar-adipic acid / glutaric acid intermediate without first introducing free groups, such as free -NH groups, into the polysaccharide. Another preferred type of linkage is a carbonyl linker, which can be formed by reacting free hydroxyl groups of the modified polysaccharide with CDI (Bethell GS et al. (1979) J. Biol. Chem. 254, 2572-4 and Hearn MTW (1981) J. Chromatogr. 218, 509-18); followed by reaction with the protein to form a carbamate bond.Other linkers include β-propionamide (WO 00 / 10599), nitrophenyl-ethylamine (Gevere et al. (1979) Med. Microbiol. Immunol. 165, 171-288), haloacyl halides (U.S. Pat. No. 4,057,685), glycosidic bonds (U.S. Pat. Nos. 4,673,574; 4,761,283; and 4,808,700), 6-aminocaproic acid (U.S. Pat. No. 4,459,286), N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP) (U.S. Pat. No. 5,204,098), adipic acid dihydrazide (ADH) (U.S. Pat. No. 4,965,338), C4-C12 moieties (U.S. Pat. No. 4,663,160), and the like. Carbodiimide condensation may also be used (WO2007 / 000343).

[0049] Bifunctional linkers can be used to provide a first group for attachment to an amine group in the polysaccharide (e.g., introduced into the polysaccharide by amination) and a second group for attachment to the carrier (typically attached to an amine in the carrier). Alternatively, the first group can be attached directly to the polysaccharide, i.e., without prior introduction of a group, e.g., an amine group, into the polysaccharide.

[0050] Alternatively, polysaccharides can be conjugated to proteins by subjecting the polysaccharide to random oxidation and then reacting the oxidized polysaccharide with sodium cyanoborohydride, as disclosed in WO2022 / 101434 or in the Examples herein.

[0051] SpyCEP Polypeptides At least one Group A Streptococcus antigen may comprise a SpyCEP polypeptide. Similarly, the protein in the polysaccharide-protein conjugate may comprise a SpyCEP polypeptide.

[0052] The term "SpyCEP polypeptide" refers to SpyCEP or a fragment or variant thereof. A suitable SpyCEP polypeptide comprises or consists of SEQ ID NO: 1 or 2, or a fragment or variant thereof. Another suitable SpyCEP polypeptide is the SpyCEP polypeptide of NCBI reference sequence WP_010921938.1.

[0053] The SpyCEP polypeptide may not contain an aspartic acid at a position corresponding to position 151 and / or a serine at a position corresponding to position 617. References to amino acids at positions corresponding to "position 151" and "position 617" refer to the amino acids at positions corresponding to amino acids 151 and 617 of SEQ ID NO:1 or SEQ ID NO:2. Optionally, the SpyCEP polypeptide contains an alanine at a position corresponding to position 151 and / or an alanine at a position corresponding to position 617. Mutations at positions corresponding to positions 151 and 617 may reduce the proteolytic activity of the SpyCEP polypeptide, making it safer to administer as part of a vaccine. Optionally, the SpyCEP polypeptide may have reduced proteolytic activity against interleukin-8 (IL-8) compared to wild-type SpyCEP polypeptide. Optionally, the SpyCEP polypeptide has 50% or less proteolytic activity against interleukin-8 (IL-8) compared to wild-type SpyCEP polypeptide. A suitable assay for determining proteolytic activity towards interleukin-8 is described in the section entitled "Assay for measuring proteolytic activity towards interleukin-8."

[0054] SpyCEP polypeptides are (i) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a contiguous fragment of at least 1000, at least 1200, at least 1400, at least 1500, or at least 1550 amino acids of SEQ ID NO: 1 or 2; or (ii) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1 or 2; or (iii) the amino acid sequence of SEQ ID NO: 1 or 2 may include:

[0055] SpyCEP polypeptides are preferably immunogenic. For example, when administered to mice, the SpyCEP polypeptide can produce antibodies. When administered to mice, the SpyCEP polypeptide can produce at least 50% (e.g., at least 80% or at least 95%) of the number of antibodies produced compared to the SpyCEP polypeptide of SEQ ID NO: 1 or SEQ ID NO: 2. Suitable assays for determining the number of antibodies produced are described in the section entitled "Immunogenicity Assays."

[0056] The SpyCEP polypeptide may comprise an amino acid sequence that is at least 98%, at least 99%, or 100% identical to a contiguous fragment of at least 1400, at least 1500, or at least 1550 amino acids of SEQ ID NO: 1 or 2. The SpyCEP polypeptide may comprise an amino acid sequence that is at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1 or 2.

[0057] SpyAD polypeptide At least one Group A Streptococcus antigen may comprise a SpyAD polypeptide. Similarly, a protein in a polysaccharide-protein conjugate may comprise a SpyAD polypeptide.

[0058] The term "SpyAD polypeptide" refers to SpyAD or a fragment or variant thereof. A suitable SpyAD polypeptide comprises or consists of SEQ ID NO: 3 or 4, or a fragment or variant thereof. Another suitable SpyAD polypeptide is the SpyAD polypeptide of NCBI reference sequence WP_010921884.1.

[0059] The SpyAD polypeptide is (i) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a contiguous fragment of at least 600, at least 650, at least 700, at least 750, at least 770, or at least 800 amino acids of SEQ ID NO: 3 or 4; (ii) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3 or 4; or (iii) the amino acid sequence of SEQ ID NO: 3 or 4 may include:

[0060] SpyAD polypeptides are preferably immunogenic. For example, administration of a SpyAD polypeptide to mice can result in the generation of antibodies. A SpyAD polypeptide can generate at least 50% (e.g., at least 80% or at least 95%) of the number of antibodies generated when administered to mice compared to the SpyAD polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4. Suitable assays for determining the number of antibodies generated are described in the section entitled "Immunogenicity Assays."

[0061] A SpyAD polypeptide may comprise an amino acid sequence that is at least 98%, at least 99%, or 100% identical to a contiguous fragment of at least 750, at least 770, or at least 800 amino acids of SEQ ID NO: 3 or 4. A SpyCEP polypeptide may comprise an amino acid sequence that is at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3 or 4.

[0062] SLO polypeptide The at least one Group A Streptococcus antigen may comprise an SLO (streptolysin O) polypeptide. Similarly, the protein in the polysaccharide-protein conjugate may comprise an SLO polypeptide.

[0063] The term "SLO polypeptide" or "streptolysin O polypeptide" refers to streptolysin O or a fragment or variant thereof. A suitable SLO polypeptide comprises or consists of SEQ ID NO: 5 or 6, or a fragment or variant thereof. Another suitable SLO polypeptide is the SLO polypeptide of NCBI reference sequence WP_010921831.1.

[0064] The SLO polypeptide may not contain a proline at a position corresponding to position 427 and / or a tryptophan at a position corresponding to position 535. References to amino acids at positions corresponding to "position 427" and "position 535" refer to the amino acids at positions corresponding to amino acids 427 and 535 of SEQ ID NO:1 or SEQ ID NO:2. Optionally, the SLO polypeptide contains a leucine at a position corresponding to position 427 and / or a phenylalanine at a position corresponding to position 535. Mutations at positions corresponding to positions 427 and 535 may reduce the hemolytic activity of the SLO polypeptide, making it safer to administer as part of a vaccine. Optionally, the SLO polypeptide has 50% or less hemolytic activity compared to the wild-type SpyCEP polypeptide. Suitable assays for measuring proteolytic activity against interleukin-8 are described in the "Assay for Measuring Hemolytic Activity" section.

[0065] The SLO polypeptide is (i) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a contiguous fragment of at least 350, at least 400, at least 450, at least 500, or at least 530 amino acids of SEQ ID NO: 5 or 6; or (ii) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 5 or 6; or (iii) the amino acid sequence of SEQ ID NO: 5 or 6 may include:

[0066] The SLO polypeptide is preferably immunogenic. For example, administration of the SLO polypeptide to mice may result in the generation of antibodies. The SLO polypeptide may result in the generation of at least 50% (e.g., at least 80% or at least 95%) more antibodies when administered to mice compared to the SLO polypeptide of SEQ ID NO: 5 or SEQ ID NO: 6. Suitable assays for determining the number of antibodies generated are described in the section entitled "Immunogenicity Assays."

[0067] The SLO polypeptide may comprise an amino acid sequence that is at least 98%, at least 99%, or 100% identical to a contiguous fragment of at least 1400, at least 1500, or at least 1550 amino acids of SEQ ID NO: 5 or 6. The SLO polypeptide may comprise an amino acid sequence that is at least 98%, at least 99%, or 100% identical to SEQ ID NO: 5 or 6.

[0068] TLR7 agonists and / or benzonaphthyridine compounds The immunogenic composition of the present invention comprises a TLR7 agonist and / or a benzonaphthyridine compound.The method of the present invention is a method for producing an immunogenic composition comprising a TLR7 agonist and / or a benzonaphthyridine compound.

[0069] TLR7 agonists are compounds that can activate TLR7. Usually, TLR7 agonists are small molecules. Those skilled in the art know many different TLR7 agonists. For example, LHD153R, imiquimod, resiquimod, NKTR-262, RG-7854, DSP-0509, BDB-001, BDC-1001, LJC-165, SHR-2150, JNJ-4964, vesatolimod, RO-7119929, BNT-411, APR-003. Those skilled in the art can also determine whether a given molecule acts as a TLR agonist. A suitable assay for determining whether a compound is a TLR7 agonist is described in Russo et al.; Blood; 117:5683-5691. For example, the TLR7 agonist activity of a compound can be assessed by culturing pDCs with IL-3, stimulating the pDCs with a compound, and measuring cytokine concentrations in the supernatant. Detection of cytokines and IFN-α in the supernatant is a marker of TLR7 agonist activity.

[0070] A TLR7 agonist can also be an agonist of other TLRs, such as TLR8. However, in some embodiments, the TLR7 agonist preferentially activates TLR7 and not other TLRs.

[0071] The TLR7 agonist may comprise or consist of a benzonaphthyridine compound or a pharmaceutically acceptable salt thereof. The TLR7 agonist may be LHD153 and / or LHD153R. The TLR7 agonist may be 3-(5-amino-2-(2-methyl-4-(2-(2-(2-phosphonoethoxy)ethoxy)ethoxy)phenethyl)benzo[f][1,7]naphthyridin-8-yl)propanoic acid, or a pharmaceutically acceptable salt thereof.

[0072] Optionally, the benzonaphthyridine compound is a compound containing a benzonaphthyridine moiety as shown below: [ka]

[0073] Typically, the benzonaphthyridine compound is a compound of formula (I): Methods for preparing suitable benzonaphthyridine compounds of formula (I) are disclosed in WO2011 / 027222 and WO2013 / 131985.

[0074] Optionally, the benzonaphthyridine compound is a compound of formula (I) [ka] Formula (I) [In the formula, R 1 is H, C1-C6 alkyl, -C(R 5 )2OH, -L 1 R 5 , -L 1 R 6 , -L 2 R 5 , -L 2 R 6 , -OL 2 R 5 , or -OL 2 R 6 and; L 1 is —C(O)— or —O—; L 2 L 2 wherein the C1-C6 alkylene and C2-C6 alkenylene are optionally substituted with 1 to 4 fluoro groups, C1-C6 alkylene, C2-C6 alkenylene, arylene, heteroarylene, or -((CR 4 R 4 ) p O) q (CH2) p - and; Each L 3 is independently, L 3 C1-C6 alkylene in which the C1-C6 alkylene is optionally substituted with 1 to 4 fluoro groups and -((CR 4 R 4 ) p O) q (CH2) p -Selected from; L 4is arylene or heteroarylene; R 2 is H or C1-C6 alkyl R 3 is C1-C4 alkyl, -L 3 R 5 , -L 1 R 5 , -L 3 R 7 , -L 3 L 4 L 3 R 7 , -L 3 L 4 R 5 , -L 3 L 4 L 3 R 5 , -OL 3 R 5 , -OL 3 R 7 , -OL 3 L 4 R 7 , -OL 3 L 4 L 3 R 7 , -OR 8 , -OL 3 L 4 R 5 , -OL 3 L 4 L 3 R 5 , and -C(R 5 )2OH; Each R 4 is independently selected from H and fluoro; R 5 is -P(O)(OR 9 )2, R 6 is -CF2P(O)(OR 9 )2 or -C(O)OR 10 and; R 7 is -CF2P(O)(OR 9 )2 or -C(O)OR 10 and; R 8 is H or C1-C4 alkyl Each R9 is independently selected from H and C1-C6 alkyl; R 10 is H or C1-C4 alkyl; each p is independently selected from 1, 2, 3, 4, 5, and 6; and q is 1, 2, 3 or 4. or a pharmaceutically acceptable salt thereof.

[0075] Suitable benzonaphthyridine compounds include the following: 4-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)-1,1-difluorobutylphosphonic acid; 3-(5-amino-2-(4-(4,4-difluoro-4-phosphonobutoxy)-2-methylphenethyl)benzo[f][1,7]naphthyridin-8-yl)propanoic acid; 3-(5-amino-2-(4-(2-(3,3-difluoro-3-phosphonopropoxy)ethoxy)-2-methylphenethyl)benzo[f][1,7]naphthyridin-8-yl)propanoic acid; 3-(5-amino-2-(2-methyl-4-(2-(2-(2-phosphonoethoxy)ethoxy)ethoxy)phenethyl)benzo[f][1,7]naphthyridin-8-yl)propanoic acid; 4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenyl phosphate dihydrogen salt; (4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)methylphosphonic acid; 5-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)-1,1-difluoropentylphosphonic acid; 4-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)-1,1-difluorobutylphosphonic acid; 3-(2-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)ethoxy)-1,1-difluoropropylphosphonic acid; 2-(4-((4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)methyl)phenyl)-1,1-difluoroethylphosphonic acid; 2-(5-amino-2-(4-methoxy-2-methylphenethyl)benzo[f][1,7]naphthyridin-8-yl)-1,1-difluoro-2-oxoethylphosphonic acid; (E)-2-(5-amino-2-(4-methoxy-2-methylphenethyl)benzo[f][1,7]naphthyridin-8-yl)vinylphosphonic acid; 2-(5-amino-2-(4-methoxy-2-methyl(rnethyl)phenethyl)benzo[f][1,7]naphthyridin-8-yl)ethylphosphonic acid; (E)-2-(5-amino-2-(4-methoxy-2-methylphenethyl)benzo[f][1,7]naphthyridin-8-yl)-1-fluorovinylphosphonic acid; 3-((4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)methyl)phenylphosphonic acid; 5-Amino-2-(4-methoxy-2-methylphenethyl)benzo[f][1,7]naphthyridine-8-carbonylphosphonic acid; 3-(5-amino-2-(2-methyl-4-(3-phosphonopropoxy)phenethyl)benzo[f][1,7]naphthyridin-8-yl)propanoic acid; (4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenyl)(hydroxy)methylenediphosphonic acid; 3-(5-amino-2-(4-(2-(2-(3,3-difluoro-3-phosphonopropoxy)ethoxy)ethoxy)-2-methylphenethyl)benzo[f][1,7]naphthyridin-8-yl)propanoic acid; (5-amino-2-(4-methoxy-2-methylphenethyl)benzo[f][1,7]naphthyridin-8-yl)(hydroxy)methylenediphosphonic acid; 3-(5-amino-2-(2-methyl-4-(2-(2-phosphonoethoxy)ethoxy)phenethyl)benzo[f][1,7]naphthyridin-8-yl)propanoic acid; 2-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)ethylphosphonic acid; 6-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)hexylphosphonic acid; 6-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)-1,1-difluorohexylphosphonic acid; 4-((4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)methyl)benzylphosphonic acid; 2-(2-(2-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)ethoxy)ethoxy)ethylphosphonic acid; 3-[5-amino-2-(2-[4-[2-(3,3-difluoro-3-phosphonopropoxy)ethoxy]-2-methylphenyl]ethyl)benzo[f]1,7 naphthyridin-8-yl)propanoic acid; [5-[4-(2-{5-amino-8-methylbenzo[f]1,7-naphthyridin-2-yl)ethyl)-3-methylphenoxy]pentyl)phosphonic acid; (4-[4-(2-(5-amino-8-methylbenzo[f]1,7-naphthyridin-2-yl}ethyl)-3-methylphenoxy]butyl}phosphonic acid and pharmaceutically acceptable salts thereof.

[0076] Optionally, the benzonaphthyridine compound is 3-(5-amino-2-(2-methyl-4-(2-(2-(2-phosphonoethoxy)ethoxy)ethoxy)phenethyl)benzo[f][1,7]naphthyridin-8-yl)propanoic acid, or a pharmaceutically acceptable salt thereof.

[0077] Optionally, the benzonaphthyridine compound comprises or consists of an arginine salt of the compound of formula (I), (e.g., a D-arginine salt or an L-arginine salt, preferably an L-arginine salt). Optionally, the benzonaphthyridine compound is the arginine salt of 3-(5-amino-2-(2-methyl-4-(2-(2-(2-phosphonoethoxy)ethoxy)ethoxy)phenethyl)benzo[f][1,7]naphthyridin-8-yl)propanoic acid. Optionally, the benzonaphthyridine compound is the L-arginine salt of 3-(5-amino-2-(2-methyl-4-(2-(2-(2-phosphonoethoxy)ethoxy)ethoxy)phenethyl)benzo[f][1,7]naphthyridin-8-yl)propanoic acid.

[0078] Formula (I) [In the formula, R 1 is -(CH2)2C(O)OH R 2 is a C1 alkyl; and R 3 is -O(((CH2)2)O)2(CH2)2-P(O)(OH)2] The arginine salt of the compound may also be referred to as LHD153R.

[0079] aluminum salts The immunogenic composition of the present invention comprises an aluminum salt. The method of the present invention is a method for producing an immunogenic composition comprising an aluminum salt.

[0080] Aluminum salts can act as adjuvants and / or adsorbents. An "adjuvant" generally refers to a substance that enhances the immune response to an antigen. An "adsorbent" generally refers to a solid substrate to which other components of an immunogenic composition, such as at least one antigen, at least one group A streptococcal antigen, a TLR7 agonist, and / or a benzonaphthyridine compound, can bind, attach, or adsorb (e.g., via van der Waals interactions or hydrogen bonding). Specific aluminum salts include aluminum hydroxide (such as ALHYDROGEL®), aluminum phosphate, aluminum hydroxyphosphate, aluminum potassium sulfate, and alum.

[0081] Optionally, the aluminium salt comprises or consists of aluminium phosphate and / or aluminium hydroxide. Optionally, the aluminium salt comprises or consists of aluminium phosphate.

[0082] Mixture of components in immunogenic compositions The at least one Group A Streptococcus antigen may comprise a polysaccharide-protein conjugate. In such embodiments, the ratio of the concentration (w / v) of the polysaccharide in the polysaccharide-protein conjugate to the concentration (w / v) of the TLR7 agonist or benzonaphthyridine compound in the immunogenic composition is between 1:0.01 and 1:50, between 1:0.1 and 1:20, between 1:0.1 and 1:10, between 1:0.5 and 1:10, between 0.75:1 and 1:5, between 1:1 and 1:3, between 1:1.5 and 1:2.5, or around 1:2. Optionally, the ratio of the concentration (w / v) of the polysaccharide in the polysaccharide-protein conjugate to the concentration (w / v) of the TLR7 agonist or benzonaphthyridine compound in the immunogenic composition is between 1:1.5 and 1:2.5. Optionally, the ratio of the concentration (w / v) of polysaccharide in the polysaccharide-protein conjugate to the concentration (w / v) of aluminum in the immunogenic composition is between 0.25:1 and 1:250, between 1:25 and 1:200, between 1:5 and 1:100, between 0.75:10 and 1:50, between 1:10 and 1:30, between 1:15 and 1:25, or around 1:20. Optionally, the ratio of the concentration (w / v) of polysaccharide in the polysaccharide-protein conjugate to the concentration (w / v) of aluminum in the immunogenic composition is between 1:15 and 1:21.

[0083] The at least one Group A Streptococcus antigen may comprise a SpyCEP polypeptide. In such embodiments, the ratio of the concentration (w / v) of the SpyCEP polypeptide to the concentration (w / v) of the TLR7 agonist or benzonaphthyridine compound in the immunogenic composition is between 1:0.1 and 1:50, between 1:0.1 and 1:20, between 1:0.1 and 1:10, between 1:0.5 and 1:10, between 0.75:1 and 1:5, between 0.75:1 and 1:3, between 0.75:1 and 1:2.5, or around 1:1. Optionally, the ratio of the concentration (w / v) of the SpyCEP polypeptide to the concentration (w / v) of the TLR7 agonist or benzonaphthyridine compound in the immunogenic composition is between 0.75:1 and 1:2.5. Optionally, the ratio of the concentration (w / v) of SpyCEP polypeptide to the concentration (w / v) of aluminum in the immunogenic composition is between 0.25:1 and 1:250, between 1:25 and 1:200, between 1:5 and 1:100, between 0.75:10 and 1:50, between 0.75:10 and 1:30, between 1:5 and 1:15, or about 1:11. Optionally, the ratio of the concentration (w / v) of SpyCEP polypeptide to the concentration (w / v) of aluminum in the immunogenic composition is between 1:5 and 1:15.

[0084] The at least one Group A Streptococcus antigen may comprise a SpyAD polypeptide. In such embodiments, the ratio of the concentration (w / v) of the SpyAD polypeptide to the concentration (w / v) of the TLR7 agonist or benzonaphthyridine compound in the immunogenic composition is between 1:0.1 and 1:50, between 1:0.1 and 1:20, between 1:0.1 and 1:10, between 1:0.5 and 1:10, between 0.75:1 and 1:5, between 0.75:1 and 1:3, between 0.75:1 and 1:2.5, or around 1:1. Optionally, the ratio of the concentration (w / v) of the SpyAD polypeptide to the concentration (w / v) of the TLR7 agonist or benzonaphthyridine compound in the immunogenic composition is between 0.75:1 and 1:2.5. Optionally, the ratio of the concentration (w / v) of SpyAD polypeptide to the concentration (w / v) of aluminum in the immunogenic composition is between 0.25:1 and 1:250, between 1:25 and 1:200, between 1:5 and 1:100, between 0.75:10 and 1:50, between 0.75:10 and 1:30, between 1:5 and 1:15, or around 1:11. Optionally, the ratio of the concentration (w / v) of SpyAD polypeptide to the concentration (w / v) of aluminum in the immunogenic composition is between 1:5 and 1:15.

[0085] The at least one Group A Streptococcus antigen may comprise an SLO polypeptide. In such embodiments, the ratio of the concentration (w / v) of the SLO polypeptide to the concentration (w / v) of the TLR7 agonist or benzonaphthyridine compound in the immunogenic composition is between 1:0.1 and 1:50, between 1:0.1 and 1:20, between 1:0.1 and 1:10, between 1:0.5 and 1:10, between 0.75:1 and 1:5, between 0.75:1 and 1:3, between 0.75:1 and 1:2.5, or about 1:1. Optionally, the ratio of the concentration (w / v) of the SLO polypeptide to the concentration (w / v) of the TLR7 agonist or benzonaphthyridine compound in the immunogenic composition is between 0.75:1 and 1:2.5. Optionally, the ratio of the concentration (w / v) of SLO polypeptide to the concentration (w / v) of aluminum in the immunogenic composition is between 0.25:1 and 1:250, between 1:25 and 1:200, between 1:5 and 1:100, between 0.75:10 and 1:50, between 0.75:10 and 1:30, between 1:5 and 1:15, or about 1:11. Optionally, the ratio of the concentration (w / v) of SLO polypeptide to the concentration (w / v) of aluminum in the immunogenic composition is between 1:5 and 1:15.

[0086] Optionally, the immunogenic composition comprises (i) The polysaccharide is GAC and the protein is CRM 197 , SpyAD polypeptide, SpyCEP polypeptide, and SLO polypeptide; and (ii) the SpyAD polypeptide is at least 98% identical to SEQ ID NO:4, the SpyCEP polypeptide is at least 98% identical to SEQ ID NO:2, and the SLO polypeptide is at least 98% identical to SEQ ID NO:6.

[0087] Optionally, the immunogenic composition comprises (i) The polysaccharide is GAC and the protein is CRM 197 , SpyAD polypeptide, SpyCEP polypeptide, and SLO polypeptide; and (ii) the SpyAD polypeptide is at least 98% identical to SEQ ID NO:3, the SpyCEP polypeptide is at least 98% identical to SEQ ID NO:1, and the SLO polypeptide is at least 98% identical to SEQ ID NO:5.

[0088] In embodiments where the immunogenic composition comprises a polysaccharide-protein conjugate, a SpyAD polypeptide, a SpyCEP polypeptide, and an SLO polypeptide, the immunogenic composition may comprise these components in the following ratios: - the ratio of the concentration (w / v) of the polysaccharide in the polysaccharide-protein conjugate to the concentration (w / v) of the TLR7 agonist or benzonaphthyridine compound in the immunogenic composition is between 0.75:1 and 1:5; - the ratio of the concentration (w / v) of polysaccharide in the polysaccharide-protein conjugate to the concentration (w / v) of aluminum in the immunogenic composition is between 0.75:10 and 1:50; - the ratio of the concentration (w / v) of the SpyCEP polypeptide to the concentration (w / v) of the TLR7 agonist or benzonaphthyridine compound in the immunogenic composition is between 0.75:0.5 and 1:2.5; - the ratio of the concentration (w / v) of the SpyCEP polypeptide to the concentration (w / v) of aluminum in the immunogenic composition is between 1:5 and 1:25; - the ratio of the concentration (w / v) of the SpyAD polypeptide to the concentration (w / v) of the TLR7 agonist or benzonaphthyridine compound in the immunogenic composition is between 0.75:0.5 and 1:2.5; - the ratio of the concentration (w / v) of SpyAD polypeptide to the concentration (w / v) of aluminum in the immunogenic composition is between 1:3 and 1:25; - the ratio of the concentration (w / v) of the SLO polypeptide to the concentration (w / v) of the TLR7 agonist or benzonaphthyridine compound in the immunogenic composition is between 0.75:0.5 and 1:2.5; and - the ratio of the concentration (w / v) of the SLO polypeptide to the concentration (w / v) of aluminium in the immunogenic composition is between 1:3 and 1:25.

[0089] Optionally, the immunogenic composition comprises between 0.5 mg / ml and 10 mg / ml, between 0.5 mg / ml and 5 mg / ml, or around 1 mg / ml of an aluminum salt. Optionally, the immunogenic composition comprises between 0.01 mg / ml and 5 mg / ml, between 0.05 mg / ml and 2 mg / ml, or around 0.1 mg / ml of a TLR7 agonist and / or a benzonaphthyridine compound. Optionally, the at least one Group A Streptococcus antigen is GAC (optionally, CRM 197 and the immunogenic composition comprises between 5 μg and 75 μg, between 10 μg and 50 μg, or around 25 μg of the polysaccharide-protein conjugate. Optionally, at least one Group A Streptococcus antigen comprises a SpyAD polypeptide, and the immunogenic composition comprises between 10 μg and 150 μg, between 25 μg and 75 μg, or around 50 μg of the SpyCEP polypeptide. Optionally, at least one Group A Streptococcus antigen comprises a SpyCEP polypeptide, and the immunogenic composition comprises between 10 μg and 150 μg, between 25 μg and 75 μg, or around 50 μg of the SpyCEP polypeptide. Optionally, at least one Group A Streptococcus antigen comprises an SLO polypeptide, and the immunogenic composition comprises between 10 μg and 150 μg, between 25 μg and 75 μg, or around 50 μg of the SLO polypeptide.

[0090] Optionally, the immunogenic composition comprises: - aluminum salts between 0.5 mg / ml and 10 mg / ml; - Between 0.01 mg / ml and 5 mg / ml of a TLR7 agonist and / or benzonaphthyridine compound - GAC (optionally CRM 197 Between 5 μg and 75 μg of polysaccharide-protein conjugates containing GAC conjugated to - between 5 μg and 75 μg of SpyAD polypeptide; - between 5 μg and 75 μg of SpyCEP polypeptide; and - Between 5 μg and 75 μg of SLO polypeptide Includes:

[0091] Optionally, at least 50%, at least 75%, or at least 80% of the at least one antigen or at least one Group A Streptococcus antigen in the immunogenic composition is adsorbed to the aluminum salt. Optionally, at least 50%, at least 75%, or at least 80% of the TLR7 agonist and / or benzonaphthyridine compound is adsorbed to the aluminum salt. The amount of a test compound, such as a Group A Streptococcus antigen and / or a TLR agonist and / or a benzonaphthyridine compound, adsorbed to the aluminum salt can be determined by mixing the aluminum salt with the test compound, centrifuging the mixture at 14,000 × g for 2 to 5 minutes, and measuring the concentration of the test compound in the supernatant (e.g., calculating the concentration of the protein test compound using absorbance at 280 nm).

[0092] Immunogenicity of the components of the immunogenic composition The immunogenic composition may produce a greater number of antibodies when administered to mice than an equivalent immunogenic composition that does not contain a TLR7 agonist and / or a benzonaphthyridine compound. Optionally, the immunogenic composition produces a greater number of antibodies when administered to mice at a dose of 5000 ng than an equivalent immunogenic composition that does not contain a TLR7 agonist and / or a benzonaphthyridine compound. Optionally, the immunogenic composition produces a greater number of antibodies when administered to mice at a dose of 5000 ng than an equivalent immunogenic composition that does not contain a TLR7 agonist and / or a benzonaphthyridine compound, as measured using serum collected 27 days after administration.

[0093] The expression "an equivalent immunogenic composition that does not contain a TLR7 agonist and / or a benzonaphthyridine compound" refers to an immunogenic composition that is identical to the present immunogenic composition except that it does not contain a TLR7 agonist and / or a benzonaphthyridine compound. Administering "a dose of 5000 ng" to mice means that each / any protein antigen (such as a SpyCEP polypeptide, a SpyAD polypeptide, and / or a SLO polypeptide) in the immunogenic composition is administered at a dose of 5000 ng, and each / any polysaccharide conjugate antigen (such as a GAC-CRM 197The number of antibodies generated when administered to mice can be determined using the assays described under the heading "Immunogenicity Assays."

[0094] Optionally, the immunogenic composition, when administered to mice, exhibits a TLR7 agonist activity compared to an equivalent immunogenic composition that does not contain a TLR7 agonist and / or a benzonaphthyridine compound. (i) SpyCEP; (ii) SpyAD; (iii) SLO; and / or (iv) GAC The immunogenic composition may produce a greater number of antibodies against at least one of the antigens. For example, if at least one Group A Streptococcus antigen comprises a SpyCEP polypeptide, the immunogenic composition may produce a greater number of antibodies against SpyCEP when administered to mice, compared to an equivalent immunogenic composition that does not contain a TLR7 agonist and / or a benzonaphthyridine compound. Similarly, if at least one Group A Streptococcus antigen comprises a SpyAD polypeptide, the immunogenic composition may produce a greater number of antibodies against SpyAD when administered to mice, compared to an equivalent immunogenic composition that does not contain a TLR7 agonist and / or a benzonaphthyridine compound. Similarly, if at least one Group A Streptococcus antigen comprises an SLO polypeptide, the immunogenic composition may produce a greater number of antibodies against SLO when administered to mice, compared to an equivalent immunogenic composition that does not contain a TLR7 agonist and / or a benzonaphthyridine compound. Optionally, when the immunogenic composition comprises a SpyCEP polypeptide, the SpyCEP polypeptide administered to the mice is administered at a dose of 5000 ng. Optionally, when the immunogenic composition comprises a SpyAD polypeptide, the SpyAD polypeptide administered to the mice is administered at a dose of 5000 ng. Optionally, when the immunogenic composition comprises an SLO polypeptide, the SLO polypeptide administered to the mice is administered at a dose of 5000 ng.

[0095] Optionally, the immunogenic composition, when administered to mice at a dose of 5000 ng, exhibits a significant reduction in TLR7 agonist activity compared to an equivalent immunogenic composition that does not contain a TLR7 agonist and / or a benzonaphthyridine compound. (i) SpyCEP; (ii) SpyAD; (iii) SLO; and / or (iv) GAC This allows the patient to produce a greater number of antibodies against at least one of the antigens.

[0096] Optionally, the immunogenic composition generates a greater number of antibodies against all of the following antigens, SpyCEP, SpyAD, SLO, and GAC, when administered to mice compared to an equivalent immunogenic composition that does not contain a TLR7 agonist and / or a benzonaphthyridine compound. Optionally, the immunogenic composition generates a greater number of antibodies against all of the following antigens, SpyCEP, SpyAD, SLO, and GAC, when administered to mice at a dose of 5000 ng, compared to an equivalent immunogenic composition that does not contain a TLR7 agonist and / or a benzonaphthyridine compound.

[0097] Optionally, the immunogenic composition, when administered to mice at a 5000 ng dose (optionally, when antibody counts are measured using serum collected 27 days after administration), generates a greater number of antibodies against SpyCEP than an equivalent immunogenic composition that does not contain the TLR7 agonist and / or the benzonaphthyridine compound. Optionally, the immunogenic composition, when administered to mice at a 5000 ng dose (optionally, when antibody counts are measured using serum collected 27 days after administration), generates at least two-fold or at least five-fold more antibodies against SpyCEP than an equivalent immunogenic composition that does not contain the TLR7 agonist and / or the benzonaphthyridine compound.

[0098] Optionally, the immunogenic composition, when administered to mice at a dose of 5000 ng (optionally, when antibody counts are measured using serum collected 27 days after administration), produces a greater number of antibodies against SpyAD than an equivalent immunogenic composition that does not contain the TLR7 agonist and / or the benzonaphthyridine compound. Optionally, the immunogenic composition, when administered to mice at a dose of 5000 ng (optionally, when antibody counts are measured using serum collected 27 days after administration), produces at least twice as many antibodies against SpyAD as an equivalent immunogenic composition that does not contain the TLR7 agonist and / or the benzonaphthyridine compound.

[0099] Optionally, the immunogenic composition, when administered to mice at a 5000 ng dose (optionally, when antibody counts are measured using serum collected 27 days after administration), generates a greater number of antibodies against SLO than an equivalent immunogenic composition that does not contain the TLR7 agonist and / or the benzonaphthyridine compound. Optionally, the immunogenic composition, when administered to mice at a 5000 ng dose (optionally, when antibody counts are measured using serum collected 27 days after administration), generates at least two-fold or at least five-fold more antibodies against SLO than an equivalent immunogenic composition that does not contain the TLR7 agonist and / or the benzonaphthyridine compound.

[0100] Optionally, the immunogenic composition comprises a 5000 ng dose (which is defined above as a 5000 ng dose, and 2500 ng of GAC-CRM 197 and corresponding to the TLR7 agonist and / or benzonaphthyridine compound), the GAC-CRM antibody produced significantly more antibodies than an equivalent immunogenic composition that did not contain a TLR7 agonist and / or benzonaphthyridine compound when administered to mice (optionally measuring antibody counts using serum collected 27 days after administration). 197 Optionally, the immunogenic composition generates a greater number of antibodies against GAC-CRM when administered to mice at a dose of 5000 ng (optionally measuring antibody numbers using serum collected 27 days after administration) compared to an equivalent immunogenic composition that does not contain a TLR7 agonist and / or a benzonaphthyridine compound. 197The number of antibodies against the virus is increased by at least two or at least five times.

[0101] Optionally, when administered to mice at a 5000 ng dose, the immunogenic composition exhibits a significantly greater immunogenicity than SpyAD, SpyCEP, SLO, and GAC-CRM compared to an equivalent immunogenic composition that does not contain a TLR7 agonist and / or a benzonaphthyridine compound, as measured using serum collected 27 days after administration. 197 Generate at least double the number of antibodies against

[0102] The immunogenic composition may produce a greater number of functional antibodies when administered to mice than an equivalent immunogenic composition that does not contain a TLR7 agonist and / or a benzonaphthyridine compound. Optionally, the immunogenic composition produces a greater number of functional antibodies when administered to mice at a dose of 5000 ng than an equivalent immunogenic composition that does not contain a TLR7 agonist and / or a benzonaphthyridine compound. Optionally, the immunogenic composition produces a greater number of functional antibodies when administered to mice at a dose of 5000 ng than an equivalent immunogenic composition that does not contain a TLR7 agonist and / or a benzonaphthyridine compound, as measured using serum collected 27 days after administration. The number of functional antibodies produced can be determined using the assays described under the headings "Assay for Measuring Proteolytic Activity Against Interleukin-8" and "Assay for Measuring Hemolytic Activity."

[0103] Optionally, the immunogenic composition, when administered to mice, produces a greater number of functional antibodies against SpyCEP and SLO than an equivalent immunogenic composition that does not contain a TLR7 agonist and / or a benzonaphthyridine compound. For example, if at least one Group A Streptococcus antigen comprises a SpyCEP polypeptide, the immunogenic composition may, when administered to mice, produce a greater number of functional antibodies against SpyCEP than an equivalent immunogenic composition that does not contain a TLR7 agonist and / or a benzonaphthyridine compound. Similarly, if at least one Group A Streptococcus antigen comprises an SLO polypeptide, the immunogenic composition may, when administered to mice, produce a greater number of functional antibodies against SLO than an equivalent immunogenic composition that does not contain a TLR7 agonist and / or a benzonaphthyridine compound.

[0104] Optionally, the immunogenic composition, when administered to mice at a dose of 5000 ng (optionally where a booster dose of the immunogenic composition is administered on day 28 and serum collected on day 42 is used to measure antibody counts), produces a greater number of functional antibodies to SpyCEP than an equivalent immunogenic composition that does not contain the TLR7 agonist and / or the benzonaphthyridine compound. Optionally, the immunogenic composition, when administered to mice at a dose of 5000 ng (optionally where a booster dose of the immunogenic composition is administered on day 28 and serum collected on day 42 is used to measure antibody counts), produces at least 2-fold, at least 5-fold, or at least 7.5-fold the number of functional antibodies to SpyCEP than an equivalent immunogenic composition that does not contain the TLR7 agonist and / or the benzonaphthyridine compound.

[0105] Optionally, the immunogenic composition, when administered to mice at a dose of 5000 ng, produces a greater number of functional antibodies to SLO than an equivalent immunogenic composition that does not contain the TLR7 agonist and / or the benzonaphthyridine compound (optionally, where antibody counts are measured using serum collected on day 42 after a booster dose of the immunogenic composition is administered on day 28). Optionally, the immunogenic composition, when administered to mice at a dose of 5000 ng, produces at least two-fold, or at least five-fold, the number of functional antibodies to SLO than an equivalent immunogenic composition that does not contain the TLR7 agonist and / or the benzonaphthyridine compound (optionally, where antibody counts are measured using serum collected on day 42 after a booster dose of the immunogenic composition is administered on day 28).

[0106] Optionally, the immunogenic composition, when administered to mice at a dose of 5000 ng, produces at least 5 times the number of functional antibodies against SpyCEP and SLO compared to an equivalent immunogenic composition that does not contain a TLR7 agonist and / or a benzonaphthyridine compound, as measured using serum collected on day 42 after a booster dose of the immunogenic composition is administered on day 28.

[0107] Immunogenicity assays The level of immunogenicity (the number of antibodies generated against a given antigen, such as SpyCEP, SpyAD, SLO, or GAC) can be determined in one of several ways known to those of skill in the art.

[0108] For example, one skilled in the art may use an ELISA-based method or a fluorescent immunoassay. When using a fluorescent immunoassay, the amount of antibody can be measured in units of RLU / ml. Fluorescent beads can also be used, with each bead coupled to a unique bead region. The beads are associated with a unique spot on the fluorescence spectrum. Different antigens can be coupled to beads in different regions. Suitable antigens include GAC, SpyCEP (e.g., proteins of SEQ ID NO: 1 or 2), SpyAD (e.g., proteins of SEQ ID NO: 3 or 4), and SLO (e.g., proteins of SEQ ID NO: 5 or 6). For example, beads in one region can be coupled to the SpyCEP antigen, and beads in a second region can be coupled to the SpyAD antigen. Serum from a mouse immunized with the immunogenic composition of the present invention can be applied to the beads and allowed to bind to the bead-bound antigen. The remaining serum can then be removed in a washing step, and a detection mixture containing a biotinylated detection antibody (which binds to the mouse antibody) and a streptavidin-phycoerythrin (PE) reporter can be applied. The beads can be excited one by one to determine the bead region and the corresponding antigen. A second laser then determines the magnitude of the signal from the PE, which is proportional to the amount of bound analyte, allowing the user to determine the number of antibodies bound to each antigen.

[0109] A suitable fluorescent immunoassay uses a Luminex® instrument. A suitable fluorescent (Luminex®) assay is described in Example 2.

[0110] To determine whether a test immunogenic composition generates a greater number of antibodies when administered to mice compared to a reference immunogenic composition (e.g., an equivalent immunogenic composition that does not contain a TLR7 agonist and / or a benzonaphthyridine compound), the amount of antibody generated by the test immunogenic composition can be measured using any one of the assays described above, the amount of antibody generated by the reference immunogenic composition can be measured using the same assay, and the two antibody amounts can be compared.

[0111] Assay for measuring proteolytic activity against interleukin-8 The number of functional antibodies against SpyCEP (anti-SpyCEP antibodies) can be measured using an IL-8 cleavage assay. Wild-type SpyCEP has the proteolytic activity to cleave IL-8, and the ability of antibodies generated against SpyCEP to block IL-8 cleavage by SpyCEP is a measure of the functionality of the antibody.

[0112] A suitable IL-8 cleavage assay involves preincubating wild-type SpyCEP with serum from mice immunized with serial dilutions of the immunogenic composition being tested or a buffer-only control. Human IL-8 is then added, and the reaction is incubated at 37°C for 2 hours. The reaction mixture can then be diluted 20-fold and incubated with a monoclonal antibody against IL-8 to measure the amount of IL-8 remaining (before cleavage). The amount of IL-8 remaining should be compared to the amount of IL-8 remaining in a control experiment (an identical experiment, except that the serum is from the mice prior to administration of the immunogenic composition being tested). The IC50 can be calculated by determining the serum concentration that achieves 50% inhibition of hemolysis (50% less reduction in IL-8 compared to the control experiment).

[0113] A suitable IL-8 cleavage assay is described in Example 2. To determine whether a test immunogenic composition produces a greater number of functional anti-SpyCEP antibodies when administered to mice compared to a reference immunogenic composition (e.g., an equivalent immunogenic composition that does not contain a TLR7 agonist and / or a benzonaphthyridine compound), the amount of functional anti-SpyCEP antibodies produced by the test immunogenic composition can be measured using one of the assays described above, and the amount of functional anti-SpyCEP antibodies produced by the reference immunogenic composition should be measured using the same assay, and the two amounts of functional anti-SpyCEP antibodies should be compared.

[0114] Hemolytic activity assay The number of functional antibodies (anti-SLO antibodies) generated against SLO can be measured using a hemolytic assay. Because wild-type SLO is hemolytic, the ability of antibodies generated against SLO to block the hemolytic activity of SLO is a measure of antibody functionality.

[0115] A suitable hemolysis assay involves preparing a suspension of red blood cells (e.g., rabbit red blood cells). In parallel, a mixture of serially diluted serum from mice immunized with the immunogenic composition to be tested can be prepared and preincubated with a fixed concentration of wild-type SLO toxin to form a test preincubation mixture. Rabbit blood cells can then be mixed with the preincubation mixture and incubated at 37°C for 30 minutes. The amount of hemolysis can then be measured by centrifuging the blood cells at 1000 x g for 5 minutes and observing the amount of hemoglobin released. The amount of released hemoglobin can be measured by absorbance at 540 nm. The amount of released hemoglobin should be compared to the amount of hemoglobin released in a control experiment (an identical experiment, except that the serum is from mice prior to administration of the immunogenic composition to be tested). The serum titer corresponding to the dilution factor providing 50% inhibition of cleavage (i.e., 50% less hemoglobin released compared to the control experiment) is determined.

[0116] A suitable hemolysis assay is described in Example 2. To determine whether a test immunogenic composition produces a greater number of functional anti-SLO antibodies when administered to mice compared to a reference immunogenic composition (e.g., an equivalent immunogenic composition that does not contain a TLR7 agonist and / or a benzonaphthyridine compound), the amount of functional anti-SLO antibodies produced by the test immunogenic composition can be measured using any of the assays described above, and the amount of functional anti-SLO antibodies produced by the reference immunogenic composition should be measured using the same assay, and the two amounts of functional anti-SLO antibodies should be compared.

[0117] pharmaceutically acceptable excipients The immunogenic composition may further comprise a pharmaceutically acceptable excipient.

[0118] Exemplary "pharmaceutically acceptable excipients" include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, sucrose, trehalose, lactose, and lipid aggregates (such as oil droplets or liposomes). Such carriers are well known to those skilled in the art. Pharmaceutically acceptable excipients can also include diluents, such as water, saline, or glycerol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, can be present. Sterile, pyrogen-free Tris-buffered saline is a preferred carrier, particularly when an aluminum adjuvant is used, because phosphate in phosphate-buffered saline can inhibit aluminum binding to outer membrane vesicles.

[0119] Typical pharmaceutical excipients may include one or more of Tris buffer, histidine, sodium chloride, and sodium phosphate. Optionally, the immunogenic composition includes Tris buffer, histidine, sodium chloride, and sodium phosphate. Optionally, the immunogenic composition includes Tris buffer at a concentration of between 0.5 mM and 25 mM, between 1 mM and 10 mM, or around 2 mM. Optionally, the immunogenic composition includes histidine at a concentration of between 1 mM and 50 mM, between 5 mM and 25 mM, or around 10 mM. Optionally, the immunogenic composition includes sodium chloride at a concentration of between 50 mM and 300 mM, between 75 mM and 250 mM, or around 129 mM. Optionally, the immunogenic composition includes sodium phosphate at a concentration of between 1 mM and 25 mM, between 2 mM and 10 mM, or around 4 mM.

[0120] Thus, the immunogenic compositions of the present invention may be useful as vaccines. Vaccines according to the present invention may be either prophylactic (i.e., to prevent infection) or therapeutic (i.e., to treat infection), but are typically prophylactic. Immunogenic compositions used as vaccines contain an immunologically effective amount of antigen and, optionally, other components. By "immunologically effective amount," it is meant that administration of that amount to an individual, either in a single dose or as part of a series of doses, is effective for treatment or prevention. This amount will vary depending on the health and physical condition of the individual being treated, their age, the taxonomic group of the individual being treated (e.g., non-human primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the vaccine formulation, the treating physician's evaluation of the medical situation, and other relevant factors. It is expected that the amount will fall within a relatively broad range that can be determined by routine testing. The immunogenic compositions of the present invention, particularly when packaged in a multi-dose format, may also contain an antibacterial agent.

[0121] Treatment method The present invention also provides the immunogenic composition or vaccine of the present invention for use in a method for preventing infection with Group A Streptococcus. The present invention also provides a method for preventing infection with Group A Streptococcus and / or autoimmune diseases following infection, comprising administering an effective amount of the immunogenic composition or vaccine of the present invention. The present invention also provides use of the immunogenic composition or vaccine of the present invention for preparing a medicament for use in a method for preventing infection with Group A Streptococcus and / or autoimmune diseases following infection.

[0122] The term "treating" includes both therapeutic and prophylactic or preventative treatment aimed at preventing or alleviating infection. For example, treatment can include, for example, directly affecting or curing an infection, suppressing, inhibiting, preventing, reducing the severity of, delaying the onset of, alleviating associated symptoms, or a combination thereof. "Preventing" can refer, inter alia, to delaying the onset of symptoms, preventing recurrence of disease, and the like. "Treatment" can also include "suppressing" or "inhibiting" an infection or disease, for example, reducing the severity, number, incidence, or latency of symptoms, ameliorating symptoms, reducing secondary symptoms, reducing secondary infections, prolonging patient survival, or a combination thereof.

[0123] The term "preventing or treating Group A Streptococcus infection" in the context of the use of an immunogenic composition in the manufacture of an immunogenic composition / medicament for the methods / uses of the invention includes generating an immune response in a subject. The immune response may be protective and may result in the generation of antibodies, such as IgG antibodies.

[0124] The subject of the present invention is a mammal, optionally a human. If the vaccine is for prophylactic use, the human may be an adult, i.e., the subject may be 18 years of age or older. If the vaccine is for prophylactic use, the human may be a child, i.e., the subject may be younger than 18 years of age. If the vaccine is for prophylactic use, the child may be 12 to 72 months of age, preferably 24 to 59 months of age, more preferably 6 to 12 months of age.

[0125] If the vaccine is for prophylactic use, the child may be around 9 months of age. If the vaccine is for therapeutic use, the human is preferably a child. Pediatric vaccines can also be administered to adults to assess safety, dosage, and immunogenicity, for example.

[0126] Method for producing immunogenic compositions The present invention further provides a method for producing an immunogenic composition containing at least one antigen, an aluminum salt, and (a) a TLR7 agonist, and / or (b) a benzonaphthyridine compound, comprising: (i) at least one antigen pre-adsorbed onto an aluminum salt and a TLR7 agonist and / or a benzonaphthyridine compound; (ii) at least one antigen and a TLR7 agonist and / or a benzonaphthyridine compound pre-adsorbed onto an aluminum salt; or (iii) at least one antigen pre-adsorbed onto an aluminum salt, and a TLR7 agonist and / or a benzonaphthyridine compound pre-adsorbed onto an aluminum salt. The present invention provides a method for producing an immunogenic composition, comprising preparing a mixture of:

[0127] The mixture can be prepared in any suitable manner. For example, the mixture can be prepared by adding two components (such as alum and at least one antigen) together and mixing them by vortexing or simply flicking. The mixture can be prepared in any suitable mixing vessel. For example, the mixture can be prepared in large quantities in a large tank for later use in syringe filling, or can be prepared directly in a ready-to-use syringe for injection into a patient.

[0128] The present invention further provides an immunogenic composition obtained or obtainable from the method for producing an immunogenic composition of the present invention. [Table 1] [Example]

[0129] Example 1 - Preparation of vaccine antigens The vaccine is formulated to contain four different antigens: the first antigen is a conjugate of GAC and CRM197, the second antigen is the SpyCEP polypeptide of SEQ ID NO: 1, the third antigen is the SpyAD polypeptide of SEQ ID NO: 3, and the fourth antigen is the SLO polypeptide of SEQ ID NO: 5.

[0130] SpyAD The upstream process starts from the thawing of one vial of MCB GMP inoculum (E. coli BL21(DE3) pET24 strain expressing SpyAD antigen), followed by the preparation of the inoculum solution (25 °C, 200 rpm, 0.7 < OD < 2.0), and then the fermentation process (the fermentation medium contains yeast extract, 30 °C, pH 7.2, pO2 set point 30%). The expression of the recombinant protein SpyAD is initiated by adding isopropyl-β-D-thiogalactoside (IPTG) when the OD value of the culture reaches 6.0 ± 1.0 (induction phase). This process ends 4 hours after induction. At the end of fermentation, the broth is sampled and centrifuged (12,227 g × 30 min). The obtained pellet is recovered and stored at -70 °C.

[0131] The recombinant protein SpyAD is extracted from the biomass using a homogenizer (Avestin, 10 Ksi, 2 cycles), and the lysate is clarified by centrifugation (12,227 g x 30 min) and tangential filtration (Sartorius, Sartobran 300). The subsequent purification consists of three chromatography steps: - Capto Q (Cytiva, buffer A: 30 mM Tris-HCl 90 mM NaCl, pH 9; buffer B: 30 mM Tris-HCl 150 mM NaCl, pH 9, step elution), - Butyl Sepharose 4 FF hs (Cytiva, buffer A: 450 mM (NH4)2SO4, 10 mM KPi, pH 7.2; buffer B: 200 mM (NH4)2SO4, 10 mM KPi, pH 7.2; step elution), - Ceramic hydroxyapatite (CHT) (type I - 40 μm, Biorad, buffer A: 10 mM NaPi pH 7.2, buffer B: 200 mM NaPi, pH 7.2, gradient elution).

[0132] The first TFF (Tangential Flow Filtration) step (30KDa cut-off, TMP 1.1 barg, DP 0.2 barg) between the Butyl step and the CHT step is necessary to exchange the buffer and add the intermediate of SpyAD to the CHT loading buffer. The second TFF step (30KDa cut-off, TMP 1.1 barg, DP 0.2 barg) after the CHT step is necessary to put the purified SpyAD antigen into the preparation buffer (10 mM KPi, pH 7.2) and reach the target drug substance (DS) concentration. Then, the pre-bulk is filtered through a 0.2 μm membrane (Sartorius, Sartobran 150) to become the final bulk.

[0133] SLO The upstream process starts from the thawing of one vial of MCB GMP inoculum (E. coli BL21(DE3) pET24 strain expressing the SLO antigen), followed by the preparation of the inoculum solution (25°C, 200 rpm, 3 < OD < 7) and the fermentation process (the fermentation medium contains yeast extract, 25°C, pH 7.2, pO2 set point 30%). The expression of the recombinant protein SLO is initiated by the addition of IPTG when the OD value of the culture reaches 5.0 ± 2.0 (induction phase). This process ends 9 hours after induction. At the end of fermentation, the broth is harvested and centrifuged (12,227 g x 30 min). The obtained pellet is recovered and stored at -70°C.

[0134] The recombinant protein SLO is extracted from the biomass by a homogenizer (Avestin, 10 Ksi, 2 cycles), and the lysate is clarified by centrifugation (12,227 g x 30 min) and orthogonal filtration (Sartorius, Sartobran 300). The subsequent purification consists of three chromatography steps: - Butyl Sepharose 4 FF hs (Cytiva, Buffer A: 2M NaCl, 20mM NaPi, pH 7; Buffer B: 1.5M NaCl, 20mM NaPi, pH 7; Buffer C: 20mM NaPi, pH 7, step elution), - Capto Q (Cytiva, buffer 20 mM NaPi pH 7, negative chromatography), - Ceramic hydroxyapatite (type I - 40 μm, Biorad, buffer A: 10 mM NaPi pH 6.8, buffer B: 130 mM NaPi pH 6.8, buffer C: 250 mM NaPi pH 6.8, step elution).

[0135] The purified SLO antigen is placed in preparation buffer (10 mM KPi, pH 7.2), and a final TFF step (30 KDa cut-off, TMP 1.0 barg, DP 1.0 barg) after the CHT step is required to reach the target DS concentration. Subsequently, the bulk is filtered through a 0.2 μm membrane (Sartorius, Sartobran 150) to obtain the final bulk.

[0136] SpyCEP The upstream process starts from the thawing of one vial of MCB GMP inoculum (E. coli BL21(DE3) pET24 strain expressing SpyCEP antigen), followed by the preparation of the inoculum solution (30 °C, 200 rpm, 1 < OD < 5) and the fermentation process (the fermentation medium contains yeast extract, 30 °C, pH 7.2, pO2 set point 30%). The expression of the recombinant protein SpyCEP is initiated by the addition of IPTG and a temperature decrease to 25 °C when the culture reaches an OD value ≥3.5 (induction phase). This process ends when the final OD of the culture reaches 30 ± 5. At the end of fermentation, the broth is harvested and centrifuged (12,227 g × 30 min). The obtained pellet is recovered and stored at -70 °C.

[0137] The recombinant protein SpyCEP is extracted from the biomass using a homogenizer (Avestin, 10 Ksi, 2 cycles), and the lysate is clarified by centrifugation (12,227 g x 30 min) and orthogonal filtration (Sartorius, Sartobran 300). The subsequent purification consists of three chromatography steps: - Capto Q (Cytiva, Buffer A: 10 mM NaPi pH 7.5; Buffer B: 10 mM NaPi, 0.2M NaCl pH 7.5, step elution), - Ceramic hydroxyapatite (Type I - 40 μm, Biorad, Buffer A: 10 mM NaPi pH 7.5, Buffer B: 80 mM NaPi, pH 7.5, step elution). - Phenyl Sepharose 6 FF hs (Cytiva, Buffer A: 2M NaCl, 10 mM NaPi, pH 7.5; Buffer B: 10 mM NaPi, pH 7.5; step elution). After the CHT step, a final TFF step (30 kDa cutoff, TMP 0.8 barg, DP 0.5 barg) is required to bring the purified SpyCEP antigen into formulation buffer (10 mM KPi, pH 7.2) and reach the target DS concentration. The prebulk is then filtered through a 0.2 μm membrane (Sartorius, Sartobran 150) to obtain the final bulk.

[0138] GAC-rCRM The upstream process begins with thawing two MCB GMP seed cultures (Streptococcus pyogenes emml knockout), followed by inoculum preparation and fermentation. The fermentation medium contains certified animal-free yeast extract. Fermentation is stopped when growth continues to slow. At this point, the OD measured is stable (or lower) compared to the previous sampling point. The final expected OD is 17 ± 5. At the end of the fermentation, an inactivation step follows. This consists of heating the culture in the bioreactor to 100°C for 2 hours. The culture is then cooled to 20°C, and the broth is harvested and centrifuged. The resulting pellet is collected and stored at -70°C.

[0139] GAC is extracted from biomass by generating HNO2 from NaNO2 and H3PO4 at pH 3.2 and room temperature for 24 hours. GAC is separated from the lysate by centrifugation and clarified by orthogonal filtration. Subsequent purification consists of the following steps: - 30K membrane TFF (TMP 0.7barg, DP 0.6barg) - Q Sepharose FF (Cytiva, buffer 10 mM NaPi, 0.3 M NaCl, pH 7.2, negative chromatography) - If the GAC concentration is lower than the target concentration, an additional TFF step (30K membrane, TMP 0.7 barg, DP 0.6 barg) may be required to further concentrate the purified GAC. The GAC intermediate is then filtered through a 0.2 μm membrane (hold point).

[0140] The GAC intermediate was then activated with 200 mM NaIO4 solution (final concentration 8 mM) and left in a constant temperature chamber at 25 °C for 30 min. The reaction was quenched with Na2SO3 solution (final concentration 16 mM) by stirring at room temperature for 15–30 min. The oxidized GAC was purified and exchanged into borate buffer, pH 8, using a TFF 10K (TMP 0.5 barg, DP 1.0 barg) and centrifugal concentrators (3–10 kDa); the target GACox concentration was 130–140 mg / mL.

[0141] rCRM (recombinant CRM) 197 ) is concentrated to 65-70 mg / mL and exchanged into 50 mM borate buffer, pH 8, prior to the conjugation step by TFF and / or centrifugal concentrator (10 KDa membrane cutoff).

[0142] The conjugation step was performed at 37°C for 24 hours (with gentle mixing) using a 1:1 (w / w) ratio of GACox and rCRM at 30 mg / mL. The reaction was initiated by the addition of a 200 mg / mL NaBH3CN solution (final concentration 5 mg / mL). The reaction was terminated by diluting 10-fold with PBX 1X and adding a 20 mg / mL NaBH4 solution at a ratio of 1:0.5 = mg GACox:mg NaBH4 for 2-4 hours at room temperature.

[0143] GAC-rCRM is purified and exchanged into PBS 1X by TFF 50K (TMP 0.3 barg, DP 0.5 barg) before filtering through a 0.2 μm membrane (Sartorius, Sartobran 150) to produce the final bulk.

[0144] mixture These four antigens were mixed with adjuvants at the concentrations shown in the table below. The adjuvants used were aluminum hydroxide alone or a combination of aluminum hydroxide and LHD153R. [Table 2]

[0145] Example 2 - Comparison of immunogenicity of formulations containing alum or alum and LHD153R in mice Mouse immunization protocol Groups of eight CD1 mice were immunized with one of the six formulations listed above. Immunizations were administered intraperitoneally with 200 μl of each formulation on days 0 and 28. Blood samples were collected on days 27 and 42, and antibodies produced were assayed using the following assays.

[0146] Evaluation of anti-SLO, anti-SpyAD, anti-SpyCEP and anti-GAC antibody levels Antibody levels against SLO, SpyAD, SpyCEP, and GAC in blood samples collected from mice immunized as described in the section entitled "Mouse Immunization Protocol" were measured using the Luminex assay described by Keeley et al., Methods Protoc. 2022, 5, 55, with the following modifications: using anti-mouse or anti-rat II antibody PE, multiple serum dilutions were tested (a minimum of 300 dilutions, with 3-fold intervals up to 7-fold dilutions), and antibody concentrations were calculated as the median of all results falling within the dynamic range of the standard curve. The results of this assay are shown in Figure 1.

[0147] IL-8 cleavage inhibition assay Individual sera isolated from blood samples collected from mice immunized as described in the section entitled "Mouse Immunization Protocol" after the second immunization were tested in an IL-8 cleavage ELISA assay to assess their ability to inhibit SpyCEP proteolytic activity. This assay was performed according to Bensi et al. (Multiple high-throughput approach for highly selective identification of vaccine candidates: The Group A Streptococcus case. Mol Cell Proteomics 2012, 11, (6), M111 015693.) with some modifications. Briefly, SpyCEP (5 ng / mL) was preincubated with four different dilutions (1:100, 1:300, 1:900, and 1:2700) of mouse polyclonal anti-SpyCEP serum in PBS containing 0.5 mg / mL BSA for 5 min at 4°C. SpyCEP preincubated with buffer alone and with preimmune serum served as negative controls. Human IL-8 (Gibco, 10 ng / ml) was then added, and the reactions were incubated at 37°C (reactions without enzyme served as controls). After 2 hours, each reaction mixture was diluted 20-fold and incubated in a 96-well plate coated with a blend of monoclonal antibodies (Life Technologies) against different epitopes of IL-8. The amount of IL-8 in each sample and control reaction (no enzyme) was determined using an IL-8 standard curve according to the manufacturer's protocol. Each serum dilution was tested in duplicate, and the average values ​​with error bars are plotted on the graph. The results are shown in Figure 2, panel A, and are expressed as the minimum serum dilution required to inhibit 50% of native SpyCEP activity (IC50) for each serum tested.

[0148] In vitro hemolysis assay Individual sera isolated from blood samples collected before and after the second immunization of mice immunized as described in the section entitled "Mouse Immunization Protocol" were tested in a hemolytic assay to assess their ability to block SLO hemolytic activity. This assay was performed as previously described by Bensi et al., with minor modifications. Briefly, red blood cell suspensions were prepared by washing rabbit red blood cells (Emozoo) four times with PBS and resuspending them in PBS (20% rabbit red blood cell suspension in PBS). Eight serial two-fold dilutions of anti-SLO serum or negative control pre-immunization serum diluted in PBS containing 0.5% BSA were prepared in a 96-well round-bottom plate and preincubated with 900 units / mL SLO toxin (Sigma, diluted in PBS containing 40 mM dithiothreitol (Invitrogen)) for 30 minutes at room temperature (final volume 150 μL). After adding rabbit blood cell suspension (50 μL), incubation continued for 30 minutes at 37°C. Finally, the plate was centrifuged at 1000 × g for 5 minutes, and the supernatant was carefully transferred to a 96-well flat-bottom plate. The absorbance of released hemoglobin was read at 540 nm. The results are shown in Figure 2, panel B, and are expressed as the minimum serum dilution required to inhibit 50% of hemolysis (IC50) for each serum tested.

[0149] statistics The Mann-Whitney two-tailed test was used to compare immune responses induced by two different antigens, and the Kruskal-Wallis test with Dunn's post hoc analysis was used for comparisons of more than two groups. A Wilcoxon matched-pairs signed-rank two-tailed test was performed to compare responses induced by the same antigen on days 27 and 42.

[0150] Antibody type assessment Individual sera isolated from blood samples collected from mice immunized as described in the "Mouse Immunization Protocol" section after the second immunization were tested to determine the antibody isotypes produced using a Luminex-based assay operating on the same principle as described in the "Evaluation of Anti-SLO, Anti-SpyAD, Anti-SpyCEP, and Anti-GAC Antibody Levels" section. This assay was repeated in parallel to measure the RLU / mL of each individual serum at the highest dose of vaccine tested using anti-mouse IgG1-PE, anti-mouse IgG2a-PE, anti-mouse IgG2b-PE, anti-mouse IgG2c-PE, and anti-mouse IgG3-PE as secondary antibodies in a standard assay. The results are shown in Figure 3 and are expressed as a ratio relative to the IgG1 of each individual serum.

[0151] Example 3 - Comparison of immunogenicity of LHD153R + formulations with or without alum in mice Mouse immunization protocol Groups of eight CD1 mice were immunized with one of six formulations, as shown in the table below. For immunization, 200 μl of each formulation was administered intraperitoneally on days 0 and 28. Blood samples were collected on days 27 and 42, and the antibodies produced were examined using the following assays. [Table 3]

[0152] Evaluation of generated antibodies The amounts of antibodies to SLO, SpyAD, SpyCEP, and GAC in blood samples taken from mice immunized as described in the section entitled "Mouse Immunization Protocol" were measured using the assay described under "Evaluation of Anti-SLO, Anti-SpyAD, Anti-SpyCEP, and Anti-GAC Antibody Levels." The results of this assay are shown in Figure 4.

[0153] Pre-immunization and post-second immunization individual sera isolated from blood samples collected from mice immunized as described in the section entitled "Mouse Immunization Protocol" were tested for functional antibodies in an IL-8 cleavage ELISA assay and a hemolytic assay, as described under the headings "IL-8 Cleavage Inhibition Assay" and "In Vitro Hemolytic Assay," respectively. The results are shown in Figure 5.

[0154] The invention described herein also relates to the following aspects: Embodiment 1. An immunogenic composition comprising at least one Group A Streptococcus antigen, an aluminum salt, and a TLR7 agonist. Embodiment 2. An immunogenic composition comprising at least one Group A Streptococcus antigen, an aluminum salt, and a benzonaphthyridine compound.

[0155] Embodiment 3. A method for producing an immunogenic composition containing at least one antigen, an aluminum salt, and (a) a TLR7 agonist and / or (b) a benzonaphthyridine compound, comprising: (i) at least one antigen pre-adsorbed onto an aluminum salt, and a TLR7 agonist and / or a benzonaphthyridine compound; (ii) at least one antigen and a TLR7 agonist and / or a benzonaphthyridine compound pre-adsorbed onto an aluminum salt; or (iii) at least one antigen pre-adsorbed onto an aluminum salt, and a TLR7 agonist and / or a benzonaphthyridine compound pre-adsorbed onto an aluminum salt. preparing a mixture of:

[0156] Embodiment 4. An immunogenic composition obtained or obtainable from the method of embodiment 3. Aspect 5. The at least one antigen is selected from the group consisting of Actinomyces (e.g., A. israelii), Bacillus (e.g., B. anthracis or B. cereus), Bartonella (e.g., B. henselae or B. quintana), Bordetella (e.g., Borrelia pertussis), Borrelia (e.g., B. burgdorferi, B. Borrelia garinii, B. afzelii, B. recurrentis), Brucella (e.g., B. abortus, B. canis, B. melitensis, or B. suis), Campylobacter (e.g., C. jejuni), Chlamydia (e.g., C. pneumoniae or C. trachomatis), Chlamydophila (e.g., C. psittaci), Clostridium (e.g., Clostridium botulinum, C. difficile, Clostridium perfringens, Clostridium tetani), Corynebacterium (e.g., Corynebacterium diphtheriae), Enterococcus (e.g., E. faecalis or E. faecium), Escherichia (e.g., Escherichia coli), Francisella (e.g., Francisella tularensis), Haemophilus (e.g., Haemophilus influenzae), Helicobacter (e.g., Helicobacter pylori), Klebsiella (e.g., Klebsiella pneumoniae and K. oxytoca), Legionella (e.g., L. pneumophila), Leptospira (e.g., L. interrogans, L. santarosai, L. weilii, L. noguchii), Listeria (e.g., L. monocytogenes), Mycobacterium (e.g., Mycobacterium leprae, Mycobacterium tuberculosis, or M. ulcerans), Mycoplasma (e.g., M. pneumoniae), Neisseria (e.g., Neisseria gonorrhoeae or Neisseria meningitidis), Pseudomonas (e.g., Pseudomonas aeruginosa), Rickettsia (e.g., R. rickettsii), Salmonella (e.g., S. Enteritidis, S. Paratyphi, S. Typhimurium, or S. Choleraesuis), Shigella (e.g., S. boydii, S. flexneri, S. sonnei, or S. dysenteriae), Staphylococcus (e.g., S. aureus, S. epidermidis, or S. saprophyticus), Streptococcus (e.g., S. agalactiae, S.5. The immunogenic composition or method of aspect 3 or 4, comprising an antigen derived from a bacterium selected from the group consisting of S. pneumoniae, S. pyogenes, Treponema (e.g., Treponema pallidum), Ureaplasma (e.g., U. urealyticum), Vibrio (e.g., V. cholerae), or Yersinia (e.g., Y. pestis, Y. enterocolitica, or Y. pseudotuberculosis).

[0157] Embodiment 6. The immunogenic composition or method of any one of embodiments 3 to 5, wherein the at least one antigen comprises an antigen from Streptococcus. Aspect 7. The immunogenic composition or method of any one of the preceding aspects, wherein the aluminum salt comprises or consists of aluminum phosphate or aluminum hydroxide.

[0158] Embodiment 8. The immunogenic composition or method of any one of the preceding embodiments, wherein the aluminum salt comprises or consists of aluminum hydroxide. Aspect 9. The immunogenic composition or method of any one of Aspects 1, or 3 to 8, wherein the TLR7 agonist comprises or consists of a benzonaphthyridine compound.

[0159] Aspect 10. The benzonaphthyridine compound is a compound of formula (I) [ka] Formula (I) [In the formula, R 1 is H, C1-C6 alkyl, -C(R 5 )2OH, -L 1 R 5 , -L 1 R 6 , -L 2 R 5 , -L 2 R 6 , -OL 2 R 5 , or -OL 2 R 6 and; L 1 is —C(O)— or —O—; L 2 represents C1-C6 alkylene, C2-C6 alkenylene, arylene, heteroarylene, or -((CR 4 R 4 ) p O) q (CH2) p - and; Each L 3 are independently selected from C1-C6 alkylene, in which the C1-C6 alkylene is optionally substituted with 1 to 4 fluoro groups, and -((CR 4 R 4 ) p O) q (CH2) p -Selected from; L 4 is arylene or heteroarylene; R 2 is H or C1-C6 alkyl; R 3 is C1-C4 alkyl, -L 3 R 5 , -L 1 R 5 , -L 3 R 7 , -L 3 L 4 L 3 R 7 , -L 3 L 4 R 5 , -L 3 L 4 L 3 R 5 , -OL 3 R 5 , -OL 3 R 7 , -OL 3 L 4 R 7 , -OL 3 L 4 L 3 R 7 , -OR 8 , -OL 3 L 4R 5 , -OL 3 L 4 L 3 R 5 , and -C(R 5 )2OH; Each R 4 is independently selected from H and fluoro; R 5 is -P(O)(OR 9 )2, R 6 is -CF2P(O)(OR 9 )2 or -C(O)OR 10 and; R 7 is -CF2P(O)(OR 9 )2 or -C(O)OR 10 and; R 8 is H or C1-C4 alkyl; Each R 9 is independently selected from H and C1-C6 alkyl; R 10 is H or C1-C4 alkyl; each p is independently selected from 1, 2, 3, 4, 5, and 6; and q is 1, 2, 3 or 4. 10. The immunogenic composition or method according to any one of aspects 2 to 9, comprising or consisting of:

[0160] Aspect 11. The benzonaphthyridine compound is a compound of formula (I) [In the formula, R 1 -L 2 R 6 and; R 2 is C1-C6 alkyl; R 3 -OL 3 R 5 or -OL 3 R 7 and; R 5 is -P(O)(OH)2; R6 is -C(O)OH; R 7 is -CF2P(O)(OH)2; L 2 is C1-C6 alkylene; L 3 is -((CR 4 R 4 ) p O) q (CH2) p - and; R 4 is H; q is 1 or 2; and p is 2.] 11. The immunogenic composition or method according to aspect 10, comprising or consisting of:

[0161] Aspect 12. The benzonaphthyridine compound is a compound of formula (I) [In the formula, R 1 is (CH2)2C(O)OH; R 2 is C1 alkyl; and R 3 is -O(((CH2)2)O)2(CH2)2-P(O)(OH)2. 12. The immunogenic composition or method of aspect 10 or 11, comprising or consisting of:

[0162] Aspect 13. The immunogenic composition or method of any one of Aspects 10 to 12, wherein the benzonaphthyridine compound is an arginine salt. Aspect 14. The immunogenic composition or method of any one of the preceding aspects, wherein the benzonaphthyridine compound and / or TLR7 agonist is LHD153R.

[0163] Embodiment 15. The immunogenic composition or method of any one of embodiments 3 to 14, wherein the at least one antigen comprises at least one Group A Streptococcus antigen. Embodiment 16. The immunogenic composition or method of any one of Embodiments 1, 2, or 7-15, wherein the at least one Group A Streptococcus antigen comprises at least one recombinant polypeptide.

[0164] Embodiment 17. The immunogenic composition or method of any one of Embodiments 1, 2, or 7-16, wherein the at least one Group A Streptococcus antigen comprises a polysaccharide-protein conjugate. Aspect 18. The immunogenic composition or method of Aspect 17, wherein the ratio of the concentration (w / v) of the polysaccharide in the polysaccharide-protein conjugate to the concentration (w / v) of the TLR7 agonist or benzonaphthyridine compound in the immunogenic composition is between 1:0.01 and 1:50, between 1:0.1 and 1:20, between 1:0.1 and 1:10, between 1:0.5 and 1:10, between 0.75:1 and 1:5, between 1:1 and 1:3, between 1:1.5 and 1:2.5, or around 1:2.

[0165] Aspect 19. The immunogenic composition or method of Aspect 18, wherein in the immunogenic composition, the ratio of the concentration (w / v) of the polysaccharide in the polysaccharide-protein conjugate to the concentration (w / v) of the TLR7 agonist or the benzonaphthyridine compound is between 1:1.5 and 1:2.5.

[0166] Aspect 20. The immunogenic composition or method of any one of Aspects 17 to 19, wherein in the immunogenic composition, the ratio of the concentration of polysaccharide (w / v) in the polysaccharide-protein conjugate to the concentration of aluminum (w / v) is between 0.25:1 and 1:250, between 1:25 and 1:200, between 1:5 and 1:100, between 0.75:10 and 1:50, between 1:10 and 1:30, between 1:15 and 1:25, or around 1:21.

[0167] Aspect 21. The immunogenic composition or method according to any one of Aspects 17 to 20, wherein in the immunogenic composition, the ratio of the concentration (w / v) of polysaccharide in the polysaccharide-protein conjugate to the concentration (w / v) of aluminum is between 1:15 and 1:25.

[0168] Embodiment 22. The immunogenic composition or method of any one of embodiments 17 to 21, wherein the polysaccharide of the polysaccharide-protein conjugate is GAC. Embodiment 23 The immunogenic composition or method according to any one of embodiments 17 to 22, wherein the protein of the polysaccharide-protein conjugate is CRM197, a SpyCEP polypeptide, an SLO polypeptide, or a SpyAD polypeptide.

[0169] Embodiment 24. The immunogenic composition or method according to any one of embodiments 17 to 23, wherein the protein of the polysaccharide-protein conjugate is CRM197. Embodiment 25 The immunogenic composition or method of any one of embodiments 1, 2, or 7-24, wherein the at least one Group A Streptococcus antigen comprises a SpyCEP polypeptide.

[0170] Aspect 26. The immunogenic composition or method of Aspect 25, wherein the ratio of the concentration (w / v) of the SpyCEP polypeptide to the concentration (w / v) of the TLR7 agonist or benzonaphthyridine compound in the immunogenic composition is between 1:0.1 and 1:50, between 1:0.1 and 1:20, between 1:0.1 and 1:10, between 1:0.5 and 1:10, between 0.75:1 and 1:5, between 0.75:1 and 1:3, between 0.75:1 and 1:2.5, or around 1:1.

[0171] Aspect 27. The immunogenic composition or method of Aspect 26, wherein the ratio of the concentration (w / v) of the SpyCEP polypeptide to the concentration (w / v) of the TLR7 agonist or the benzonaphthyridine compound in the immunogenic composition is between 0.75:1 and 1:2.5.

[0172] Aspect 28. The immunogenic composition or method according to any one of Aspects 25 to 27, wherein the ratio of the concentration (w / v) of the SpyCEP polypeptide to the concentration (w / v) of aluminum in the immunogenic composition is between 0.25:1 and 1:250, between 1:25 and 1:200, between 1:5 and 1:100, between 0.75:10 and 1:50, between 0.75:10 and 1:30, or between 1:5 and 1:15, or around 1:11.

[0173] Aspect 29. The immunogenic composition or method according to any one of Aspects 25 to 28, wherein in the immunogenic composition, the ratio of the concentration (w / v) of the SpyCEP polypeptide to the concentration (w / v) of aluminum is between 1:5 and 1:15.

[0174] Aspect 30. The immunogenic composition or method according to any one of Aspects 23 or 25 to 29, wherein the SpyCEP polypeptide does not contain an aspartic acid at a position corresponding to position 151 and / or a serine at a position corresponding to position 617.

[0175] Embodiment 31 The immunogenic composition or method of any one of embodiments 23 or 25 to 30, wherein the SpyCEP polypeptide comprises an alanine at a position corresponding to position 151 and / or an alanine at a position corresponding to position 617.

[0176] 32. The SpyCEP polypeptide is (i) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a contiguous fragment of at least 1000, at least 1200, at least 1400, at least 1500, or at least 1550 amino acids of SEQ ID NO: 1 or 2; or (ii) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1 or 2; or (iii) the amino acid sequence of SEQ ID NO: 1 or 2 32. The immunogenic composition or method according to any one of aspects 23 or 25 to 31, comprising:

[0177] Embodiment 33 The immunogenic composition or method of embodiment 32, wherein the SpyCEP polypeptide comprises an amino acid sequence that is at least 98%, at least 99%, or 100% identical to a contiguous fragment of at least 1400, at least 1500, or at least 1550 amino acids of SEQ ID NO: 1 or 2.

[0178] Embodiment 34 The immunogenic composition or method of embodiment 32, wherein the SpyCEP polypeptide comprises an amino acid sequence that is at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1 or 2.

[0179] Embodiment 35 The immunogenic composition or method of embodiment 32, wherein the SpyCEP polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or 2. Embodiment 36 The immunogenic composition or method of any one of embodiments 1, 2, or 7 to 35, wherein the at least one Group A Streptococcus antigen comprises a SpyAD polypeptide.

[0180] Embodiment 37. The immunogenic composition or method of embodiment 36, wherein the ratio of the concentration (w / v) of the SpyAD polypeptide to the concentration (w / v) of the TLR7 agonist or benzonaphthyridine compound in the immunogenic composition is between 1:0.1 and 1:50, between 1:0.1 and 1:20, between 1:0.1 and 1:10, between 1:0.5 and 1:10, between 0.75:1 and 1:5, between 0.75:1 and 1:3, between 0.75:1 and 1:2.5, or around 1:1.

[0181] Aspect 38. The immunogenic composition or method of Aspect 37, wherein the ratio of the concentration (w / v) of the SpyAD polypeptide to the concentration (w / v) of the TLR7 agonist or benzonaphthyridine compound in the immunogenic composition is between 0.75:1 and 1:2.5.

[0182] Embodiment 39. An immunogenic composition or method according to any one of embodiments 36 to 38, wherein the ratio of the concentration (w / v) of SpyAD polypeptide to the concentration (w / v) of aluminium in the immunogenic composition is between 0.25:1 and 1:250, between 1:25 and 1:200, between 1:5 and 1:100, between 0.75:10 and 1:50, between 0.75:10 and 1:30, between 1:5 and 1:15, or around 1:11.

[0183] Embodiment 40. The immunogenic composition or method of any one of embodiments 36 to 39, wherein the ratio of the concentration (w / v) of SpyAD polypeptide to the concentration (w / v) of aluminum in the immunogenic composition is between 1:5 and 1:15.

[0184] Embodiment 41 The SpyAD polypeptide comprises: (i) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a contiguous fragment of at least 600, at least 650, at least 700, at least 750, at least 770, or at least 800 amino acids of SEQ ID NO: 3 or 4; (ii) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3 or 4; or (iii) the amino acid sequence of SEQ ID NO: 3 or 4 41. The immunogenic composition or method according to any one of aspects 23 or 36 to 40, comprising:

[0185] Embodiment 42. The immunogenic composition or method of embodiment 41, wherein the SpyAD polypeptide comprises an amino acid sequence that is at least 98%, at least 99%, or 100% identical to a contiguous fragment of at least 700, at least 750, or at least 800 amino acids of SEQ ID NO: 3 or 4.

[0186] Embodiment 43 The immunogenic composition or method of embodiment 41, wherein the SpyAD polypeptide comprises an amino acid sequence that is at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3 or 4.

[0187] Embodiment 44 The immunogenic composition or method of embodiment 41, wherein the SpyAD polypeptide comprises the amino acid sequence of SEQ ID NO: 3 or 4.

[0188] Embodiment 45. The immunogenic composition or method of any one of embodiments 1, 2, or 7-44, wherein the at least one Group A Streptococcus antigen comprises an SLO polypeptide.

[0189] Embodiment 46. The immunogenic composition or method of embodiment 45, wherein the ratio of the concentration (w / v) of the SLO polypeptide to the concentration (w / v) of the TLR7 agonist or benzonaphthyridine compound in the immunogenic composition is between 1:0.1 and 1:50, between 1:0.1 and 1:20, between 1:0.1 and 1:10, between 1:0.5 and 1:10, between 0.75:1 and 1:5, between 0.75:1 and 1:3, between 0.75:1 and 1:2.5, or around 1:1.

[0190] Embodiment 47. The immunogenic composition or method of embodiment 46, wherein the ratio of the concentration (w / v) of the SLO polypeptide to the concentration (w / v) of the TLR7 agonist or benzonaphthyridine compound in the immunogenic composition is between 0.75:1 and 1:2.5.

[0191] Embodiment 48. The immunogenic composition or method of any one of embodiments 45 to 47, wherein the ratio of the concentration (w / v) of the SLO polypeptide to the concentration (w / v) of aluminum in the immunogenic composition is between 0.25:1 and 1:250, between 1:25 and 1:200, between 1:5 and 1:100, between 0.75:10 and 1:50, between 0.75:10 and 1:30, between 1:5 and 1:15, or around 1:11.

[0192] Aspect 49. The immunogenic composition or method of any one of Aspects 45 to 48, wherein the ratio of the concentration (w / v) of the SLO polypeptide to the concentration (w / v) of aluminum in the immunogenic composition is between 1:5 and 1:15.

[0193] Embodiment 50. The immunogenic composition or method of any one of embodiments 23 or 45 to 49, wherein the SLO polypeptide does not contain a proline at the position corresponding to position 427 and / or a tryptophan at the position corresponding to position 535.

[0194] Embodiment 51 The immunogenic composition or method of any one of embodiments 23 or 45 to 50, wherein the SLO polypeptide comprises a leucine at a position corresponding to 427 and / or a phenylalanine at a position corresponding to 535.

[0195] 52. SLO polypeptide (i) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a contiguous fragment of at least 350, at least 400, at least 450, at least 500, or at least 530 amino acids of SEQ ID NO: 5 or 6; or (ii) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 5 or 6; or (iii) the amino acid sequence of SEQ ID NO: 5 or 6 52. The immunogenic composition or method of any one of aspects 23 or 45 to 51, comprising:

[0196] Embodiment 53 The immunogenic composition or method of embodiment 52, wherein the SLO polypeptide comprises an amino acid sequence that is at least 98%, at least 99%, or 100% identical to a contiguous fragment of at least 450, at least 500, or at least 530 amino acids of SEQ ID NO: 5 or 6.

[0197] Embodiment 54 The immunogenic composition or method of embodiment 52, wherein the SLO polypeptide comprises an amino acid sequence that is at least 98%, at least 99%, or 100% identical to SEQ ID NO: 5 or 6.

[0198] Embodiment 55 The immunogenic composition or method of embodiment 52, wherein the SLO polypeptide comprises the amino acid sequence of SEQ ID NO: 5 or 6.

[0199] Embodiment 56 The immunogenic composition or method of any one of the preceding embodiments, wherein at least 50%, at least 75%, or at least 80% of the at least one antigen or at least one Group A Streptococcus antigen in the immunogenic composition is adsorbed onto an aluminum salt.

[0200] Embodiment 57 The immunogenic composition or method of any one of the preceding embodiments, wherein at least 50%, at least 75%, or at least 80% of the TLR7 agonist and / or benzonaphthyridine compound is adsorbed onto the aluminum salt.

[0201] Aspect 58 The immunogenic composition or method of any one of the preceding aspects, wherein the immunogenic composition is more immunogenic than an equivalent immunogenic composition that does not contain a TLR7 agonist and / or a benzonaphthyridine compound.

[0202] Aspect 59 The immunogenic composition or method of any one of the preceding aspects, wherein the immunogenic composition, when administered to a mouse, generates a greater number of antibodies compared to an equivalent immunogenic composition that does not contain a TLR7 agonist and / or a benzonaphthyridine compound.

[0203] Aspect 60. An immunogenic composition, when administered to mice, inhibits the expression of the following antigens, compared to an equivalent immunogenic composition that does not contain a TLR7 agonist and / or a benzonaphthyridine compound: (i) SpyCEP; (ii) SpyAD; (iii) SLO; and / or (iv) GAC; 60. The immunogenic composition or method of embodiment 59, wherein the immunogenic composition or method generates a greater number of antibodies against at least one of the following:

[0204] Aspect 61 The immunogenic composition or method of Aspect 60, wherein the immunogenic composition, when administered to mice, produces a greater number of antibodies against all of the following antigens: SpyCEP, SpyAD, SLO, and GAC, compared to an equivalent immunogenic composition that does not contain a TLR7 agonist and / or a benzonaphthyridine compound.

[0205] Embodiment 62. The immunogenic composition or method of any one of embodiments 59 to 61, wherein the number of antibodies is measured using a fluorescent immunoassay in which the antigen is bound to beads and the beads containing the antigen-antibody complex are detected by fluorescence measurement.

[0206] Aspect 63 The immunogenic composition or method of any one of the preceding aspects, wherein the immunogenic composition, when administered to a mouse, produces a greater number of functional antibodies compared to an equivalent immunogenic composition that does not contain a TLR7 agonist and / or a benzonaphthyridine compound.

[0207] Aspect 64. An immunogenic composition, when administered to mice, exhibits a TLR7 agonist and / or a benzonaphthyridine compound-free immunogenic activity compared to an equivalent immunogenic composition that does not contain a TLR7 agonist and / or a benzonaphthyridine compound. (i) SpyCEP; and / or (ii) SLOs; 64. The immunogenic composition or method of embodiment 63, wherein the immunogenic composition or method generates a greater number of functional antibodies against the

[0208] Embodiment 65 The immunogenic composition or method of embodiment 64, wherein the immunogenic composition, when administered to mice, produces a greater number of functional antibodies against SpyCEP and SLO compared to an equivalent immunogenic composition that does not contain a TLR7 agonist and / or a benzonaphthyridine compound.

[0209] Embodiment 66 The immunogenic composition or method of any one of embodiments 1 to 65, wherein the immunogenic composition further comprises a pharmaceutically acceptable excipient. Aspect 67. A vaccine comprising the immunogenic composition of any one of Aspects 1, 2, or 4 to 66.

[0210] Embodiment 68. The immunogenic composition of any one of embodiments 1, 2, or 4 to 66, or the vaccine of embodiment 67, for use in a method for preventing infection with group A streptococcus and / or preventing an autoimmune disease following infection with group A streptococcus.

[0211] Aspect 69. A method for preventing infection with Group A Streptococcus and / or an autoimmune disease following infection with Group A Streptococcus, comprising administering to a subject an effective amount of the immunogenic composition of any one of aspects 1, 2, or 4 to 66, or the vaccine of aspect 67.

[0212] Aspect 70. Use of an immunogenic composition according to any one of aspects 1, 2 or 4 to 66, or a vaccine according to aspect 67, for the preparation of a medicament for use in a method for preventing infection with group A streptococcus and / or preventing an autoimmune disease following infection with group A streptococcus.

Claims

1. An immunogenic composition comprising at least one Group A Streptococcus antigen, an aluminum salt, and a TLR7 agonist.

2. An immunogenic composition comprising at least one Group A Streptococcus antigen, an aluminum salt, and a benzonapthyridine compound.

3. 1. A method for producing an immunogenic composition containing at least one antigen, an aluminum salt, and (a) a TLR7 agonist and / or (b) a benzonaphthyridine compound, the method comprising: (i) at least one antigen pre-adsorbed onto an aluminum salt, and a TLR7 agonist and / or a benzonaphthyridine compound; (ii) at least one antigen and a TLR7 agonist and / or a benzonaphthyridine compound pre-adsorbed onto an aluminum salt; or (iii) at least one antigen pre-adsorbed onto an aluminum salt, and a TLR7 agonist and / or a benzonaphthyridine compound pre-adsorbed onto an aluminum salt. The above method comprising preparing a mixture of:

4. An immunogenic composition obtained or obtainable from the method of claim 3.

5. The immunogenic composition or method of any one of claims 1 to 3, wherein the aluminium salt comprises or consists of aluminium phosphate or aluminium hydroxide.

6. The immunogenic composition or method of any one of claims 1 to 4, wherein the aluminium salt comprises or consists of aluminium hydroxide.

7. The immunogenic composition or method according to any one of claims 1 to 6, wherein the benzonaphthyridine compound and / or the TLR7 agonist is LHD153R.

8. 8. The immunogenic composition or method of any one of claims 1 to 7, wherein the at least one Group A Streptococcus antigen comprises a polysaccharide-protein conjugate, wherein the polysaccharide of the polysaccharide-protein conjugate is GAC and the protein of the polysaccharide-protein conjugate is CRM197.

9. The immunogenic composition or method of any one of claims 1 to 8, wherein the at least one Group A Streptococcus antigen or at least one antigen comprises a SpyCEP polypeptide.

10. The immunogenic composition or method of claim 9, wherein the SpyCEP polypeptide comprises an alanine at a position corresponding to position 151 and / or an alanine at a position corresponding to position 617.

11. The immunogenic composition or method of claim 9 or 10, wherein the SpyCEP polypeptide comprises an amino acid sequence that is at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1 or 2.

12. The immunogenic composition or method of any one of claims 1 to 11, wherein the at least one Group A Streptococcus antigen or at least one antigen comprises a SpyAD polypeptide.

13. The immunogenic composition or method of claim 12, wherein the SpyAD polypeptide comprises an amino acid sequence at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3 or 4.

14. The immunogenic composition or method of any one of claims 1 to 13, wherein the at least one Group A Streptococcus antigen or at least one antigen comprises an SLO polypeptide.

15. 15. The immunogenic composition or method of claim 14, wherein the SLO polypeptide comprises a leucine at a position corresponding to position 427 and / or a phenylalanine at a position corresponding to position 535.

16. 16. The immunogenic composition or method of claim 14 or 15, wherein the SLO polypeptide comprises an amino acid sequence at least 98%, at least 99%, or 100% identical to SEQ ID NO: 5 or 6.

17. 17. The immunogenic composition or method of any one of claims 1 to 16, wherein the immunogenic composition is more immunogenic than an equivalent immunogenic composition that does not contain a TLR7 agonist and / or a benzonaphthyridine compound.

18. 18. The immunogenic composition or method of any one of claims 1 to 17, wherein the immunogenic composition, when administered to mice, produces a greater number of antibodies compared to an equivalent immunogenic composition that does not contain a TLR7 agonist and / or a benzonaphthyridine compound.

19. 20. The immunogenic composition or method of claim 18, wherein the immunogenic composition, when administered to mice, produces a greater number of antibodies against all of the following antigens: SpyCEP, SpyAD, SLO, and GAC, compared to an equivalent immunogenic composition that does not contain a TLR7 agonist and / or a benzonaphthyridine compound.

20. the immunogenic composition, when administered to mice, exhibits a significant reduction in TLR7 agonist activity compared to an equivalent immunogenic composition that does not contain a TLR7 agonist and / or a benzonaphthyridine compound; (i) SpyCEP, and / or (ii) SLO 20. The immunogenic composition or method of any one of claims 1 to 19, which results in the generation of a greater number of functional antibodies against

21. The immunogenic composition or method of any one of claims 1 to 20, wherein the immunogenic composition further comprises a pharmaceutically acceptable excipient.

22. A vaccine comprising the immunogenic composition of any one of claims 1, 2, and 4 to 21.

23. An immunogenic composition according to any one of claims 1, 2 and 4 to 21, or a vaccine according to claim 22, for use in a method for preventing infection with group A streptococcus.

24. An immunogenic composition according to any one of claims 1, 2 and 4 to 21, or a vaccine according to claim 22, for use in a method for preventing autoimmune disease following infection with group A streptococcus.

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