WO2024083873A1 - Vaccin - Google Patents

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Publication number
WO2024083873A1
WO2024083873A1 PCT/EP2023/078896 EP2023078896W WO2024083873A1 WO 2024083873 A1 WO2024083873 A1 WO 2024083873A1 EP 2023078896 W EP2023078896 W EP 2023078896W WO 2024083873 A1 WO2024083873 A1 WO 2024083873A1
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WO
WIPO (PCT)
Prior art keywords
immunogenic composition
polypeptide
compound
benzonapthyridine
spycep
Prior art date
Application number
PCT/EP2023/078896
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English (en)
Inventor
Danilo Gomes Moriel
Margherita COCCIA
Emilia CAPPELLETTI
Anna Maria Rosaria COLUCCI
Original Assignee
Glaxosmithkline Biologicals Sa
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Publication of WO2024083873A1 publication Critical patent/WO2024083873A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A61K39/092—Streptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]

Definitions

  • the present invention relates to compositions comprising antigens, aluminium salts and TLR7 agonists and/or benzonapthyridine compounds.
  • the present invention also relates to methods of making such compositions, vaccines comprising the compositions, and methods of using or uses of the compositions.
  • Group A Streptococcus causes a diverse spectrum of diseases, from superficial infections (pharyngitis, skin infections) to severe invasive diseases (cellulitis, puerperal sepsis, necrotizing fasciitis, streptococcal toxic shock syndrome), with a high frequency of serious autoimmune sequelae in low- and middle-income countries (LMICs), such as acute rheumatic fever, (ARF); rheumatic heart disease, (RHD), and glomerulonephritis (Ralph et al.,' Curr. Top. Microbiol. Immunol. 2013; 368:1-27).
  • LMICs low- and middle-income countries
  • Pharyngitis is the most frequent symptomatic GAS infection in children across the world, with more than 600 million cases estimated annually (Carapetis et al , The Lancet 2005; 5: 685-94) and is an important driver of antibiotic use (Dooling et al:, Jama Pediatr. 2014; 168: 1073-4) that can ultimately result in increased antimicrobial resistance, a growing public health crisis (Jansen et al:, Nature Medicine 2018; 24: 10-19). Pharyngitis could lead to RHD, which is a chronic inflammatory heart valve condition representing the main global burden of GAS.
  • GAC Group A Carbohydrate
  • GAC is a surface polysaccharide comprising of a polyrhamnose backbone with alternating A-acetylglucosamine (GlcNAc) at the side chain. It represents an attractive vaccine candidate as it is highly conserved and expressed across GAS strains. Indeed, one of the major obstacles for vaccine strategy development is represented by GAS serotype diversity related to other non-carbohydrate antigens (Walker et al.,' Clin. Microbiol. Rev. 2014; 27: 264-301).
  • SLO Streptolysin O
  • SpyAD SpyAD
  • SpyCEP SpyCEP
  • SLO has been shown to be a key virulence factor of GAS by preventing internalization of the bacteria into lysosomes where they can be destroyed (Cunningham; Clin. Microbiol. Rev. 2000; 13: 470-511).
  • SLO promotes GAS resistance to phagocytic clearance by neutrophils, facilitating GAS escape from innate immune killing, and an inactivated SLO was demonstrated to be protective in a murine model against GAS challenge (Vchiyama et al.,' Front Immunol. 2015; 6: 581).
  • SpyAD is a surface-exposed adhesin that mediates GAS interaction with host cells.
  • deletion of the SpyAD gene in a GAS strain led to an impaired capacity of the knockout mutant to properly divide, suggesting also an important role in bacterial division (Gallotta etal.,' Infect. Immun. 2014; 82: 2890-2901).
  • SpyCEP is a multi-domain proteinase, with a catalytic domain responsible for the interleukin (IL)-8 and other chemokines cleavage.
  • Cleavage of IL-8 represents a mechanism of immune evasion, preventing IL-8 C-terminus-mediated endothelial translocation and subsequent recruitment of neutrophils (Edwards et al.,' J. Infect. Dis. 2005; 192: 783-790 and Jobichen et al.,' J. Infect. Dis. 2005; 192: 783-790).
  • Multivalent vaccines comprising recombinant SLO, SpyAD, SpyCEP and GAC-CRM197 have the potential to be excellent vaccines. However, it is an aim of the present invention to yet further improve the immune responses generated to vaccines comprising recombinant SLO, SpyAD, SpyCEP and GAC-CRM197 antigens.
  • a composition comprising at least one Group A Streptococcus antigen, an aluminium salt (such as aluminium hydroxide) and a TLR7 agonist and/or benzonapthyridine compound (such as LHD153R) is not only significantly more immunogenic than a corresponding composition lacking the TLR7 agonist and/or benzonapthyridine compound but when administered to mice elicits significantly more functional antibodies against the at least one Group A Streptococcus antigen compared to a corresponding composition lacking the TLR7 agonist and/or benzonapthyridine compound.
  • composition may be advantageously prepared using a method comprising preparing a mixture of:
  • an immunogenic composition comprising at least one Group A Streptococcus antigen, an aluminium salt and a TLR7 agonist.
  • an immunogenic composition comprising at least one Group A Streptococcus antigen, an aluminium salt and a benzonapthyridine compound.
  • an immunogenic composition comprising at least one antigen, an aluminium salt and (a) a TLR7 agonist, and/or (b) a benzonapthyridine compound comprising preparing a mixture of:
  • an immunogenic composition that is obtained or obtainable from the methods of the invention.
  • a vaccine comprising the immunogenic composition of the invention.
  • the immunogenic composition or vaccine or the invention for use in a method of preventing infection by and/or preventing autoimmune disease following infection by Group A Streptococcus.
  • a seventh aspect of the invention there is provided a method of preventing infection by and/or preventing autoimmune disease following infection by Group A Streptococcus comprising administering an effective amount of the immunogenic composition or vaccine of the invention.
  • an eighth aspect of the invention there is provided a use of the immunogenic composition or vaccine of the invention for preparing a medicament for use in a method of preventing infection by and/or preventing autoimmune disease following infection by Group A Streptococcus.
  • Figure 1 provides graphs presenting data on the IgG response seen in mice after vaccination with vaccines comprising Streptococcus A antigens, aluminium hydroxide and LHD153R or vaccines comprising Streptococcus A antigens and aluminium hydroxide without LHD153R.
  • the Streptococcus A antigens were 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 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.
  • Panel A depicts the antibody response (measured in RLU/ml) to SpyCEP at 27 days post-vaccination
  • Panel B depicts the antibody response (measured in RLU/ml) to SpyCEP at 42 days postvaccination
  • Panel C depicts the antibody response (measured in RLU/ml) to SpyAD at 27 days post-vaccination
  • Panel D depicts the antibody response (measured in RLU/ml) to SpyAD at 42 days post-vaccination
  • Panel E depicts the antibody response (measured in RLU/ml) to SLO at 27 days post-vaccination
  • Panel F depicts the antibody response (measured in RLU/ml) to SLO at 42 days post-vaccination
  • Panel G depicts the antibody response (measured in RLU/ml) to GAC at 27 days post-vaccination
  • Panel H depicts the antibody response (measured in RLU/ml) to GAC at 42 days post-vaccination.
  • Figure 2 provides graphs presenting data on the level of functional antibodies seen in mice seen in mice after vaccination with vaccines comprising Streptococcus A antigens, aluminium hydroxide and LHD153R or vaccines comprising Streptococcus A antigens and aluminium hydroxide without LHD153R.
  • the Streptococcus A antigens were 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 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.
  • Panel A depicts the level of functional antibodies raised against SpyCEP as determined using an IL-8 cleavage assay.
  • Panel B depicts the level of functional antibodies raised against SLO as determined using a hemolysis assay.
  • Figure 3 provides graphs presenting data on the ratio of IgG2/IgGl and IgG3/IgGl seen in mice after vaccination with vaccines comprising Streptococcus A antigens, aluminium hydroxide and LHD153R or vaccines comprising Streptococcus A antigens and aluminium hydroxide without LHD153R.
  • the dose of Streptococcus A antigens used was 5 000 ng of SpyAD polypeptide, 5 000 ng of SLO polypeptide, 5 000 ng of SpyCEP polypeptide and 2500 ng of GAC-CRM197.
  • the left hand column corresponds to a vaccine comprising Streptococcus A antigens and aluminium hydroxide only
  • the central column corresponds to a vaccine comprising Streptococcus A antigens, aluminium hydroxide and LHD153R
  • the right hand column corresponds to a vaccine comprising Streptococcus A antigens and no adjuvant.
  • Panel A describes the ratio of IgG2/IgGl ratio of antibodies raised against GAC.
  • Panel B describes the ratio of IgG2/IgGl ratio of antibodies raised against SLO.
  • Panel C describes the ratio of IgG2/IgGl ratio of antibodies raised against SpyAD.
  • Panel D describes the ratio of IgG2/IgGl ratio of antibodies raised against SpyCEP.
  • Panel E describes the ratio of IgG3/IgGl ratio of antibodies raised against GAC.
  • Panel F describes the ratio of IgG3/IgGl ratio of antibodies raised against SLO.
  • Panel G describes the ratio of IgG3/IgGl ratio of antibodies raised against SpyAD.
  • Panel H describes the ratio of IgG3/IgGl ratio of antibodies raised against SpyCEP.
  • Figure 4 provides graphs presenting data on the IgG response seen in mice after vaccination with vaccines comprising Streptococcus A antigens, aluminium hydroxide and LHD153R or Streptococcus A antigens with no adjuvant.
  • the Streptococcus A antigens were 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 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.
  • Panel A depicts the antibody response (measured in RLU/ml) to SpyCEP at 27 days post-vaccination
  • Panel B depicts the antibody response (measured in RLU/ml) to SpyCEP at 42 days post-vaccination
  • Panel C depicts the antibody response (measured in RLU/ml) to SpyAD at 27 days post-vaccination
  • Panel D depicts the antibody response (measured in RLU/ml) to SpyAD at 42 days post-vaccination
  • Panel E depicts the antibody response (measured in RLU/ml) to SLO at 27 days post-vaccination
  • Panel F depicts the antibody response (measured in RLU/ml) to SLO at 42 days postvaccination
  • Panel G depicts the antibody response (measured in RLU/ml) to GAC at 27 days post-vaccination
  • Panel H depicts the antibody response (measured in RLU/ml) to GAC at 42 days post-vaccination.
  • Figure 5 provides graphs presenting data on the level of functional antibodies seen in mice seen in mice after vaccination with vaccines comprising Streptococcus A antigens, aluminium hydroxide, and LHD153R or Streptococcus A antigens with no adjuvant.
  • the Streptococcus A antigens were administered at two different doses (medium and high).
  • the medium ng 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 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.
  • Panel A depicts the level of functional antibodies raised against SpyCEP as determined using an IL-8 cleavage assay.
  • Panel B depicts the level of functional antibodies raised against SLO as determined using a hemolysis assay.
  • the word “comprising” is replaced with the phrase “consisting of .
  • the term “consisting of” is intended to be limiting.
  • the phrase “An immunogenic composition consisting of at least one Group A Streptococcus antigen, an aluminium salt and a TLR7 agonist” should be understood to mean that the composition contains at least one Group A Streptococcus antigen, an aluminium salt and a TLR7 agonist and no further components.
  • the word “comprising” is replaced with the phrase “consisting essentially of”.
  • the term “consisting essentially of” means that specific further components can be present, namely those not materially affecting the essential characteristics of the subject matter.
  • the phrase “An immunogenic composition consisting essentially of at least one Group A Streptococcus antigen, an aluminium salt and a TLR7 agonist” may comprise components like buffers or other pharmaceutically acceptable excipients.
  • Amino acids “ corresponding to" specified positions of a specified SEQ ID NO may be amino acids at the specified positions of the particular SEQ ID NO recited.
  • the amino acid “corresponding to position 272 of SEQ ID NO: /” may be the amino acid at position 272 of SEQ ID NO: 1.
  • amino acids “corresponding to" positions of a specified SEQ ID NO may be amino acids from an alternative amino acid sequence which correspond to the specified positions of the specified SEQ ID NO.
  • the amino acid “corresponding to position 272 of SEQ ID NO: 1" may be the amino acid from an alternative amino acid sequence which corresponds to position 272 of SEQ ID NO: 1.
  • the sequences are aligned for optimal comparison purposes (e.g. , gaps can be introduced in a first sequence for optimal alignment with a second sequence).
  • the nucleotides or amino acid residues at each position are then compared.
  • a position in the first sequence is occupied by the same nucleotide or amino acid at the corresponding position in the second sequence, then the nucleotides or amino acids are identical at that position.
  • sequence comparison is carried out over the length of the reference sequence. For example, if the user wished 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), the skilled person would carry out an alignment over the length of SEQ ID NO: 1, and identify how many positions in the test sequence were identical to those of 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, the gaps or missing positions should be considered to be non-identical positions.
  • an alignment between two sequences can be accomplished using a mathematical algorithm.
  • the two amino acid or nucleic acid sequences are aligned using the Needleman and Wunsch (1970) algorithm which has been incorporated into the GAP program in the Accelrys GCG software package using either a Blosum 62 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.
  • At least one antigen is selected from:
  • the present invention provides an immunogenic composition comprising at least one Group A Streptococcus antigen. Similarly, the present invention provides methods of making an immunogenic composition comprising at least one antigen.
  • the at least one antigen may comprise an antigen from a bacterium 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.
  • Actinomyces e.g., A. israelii
  • Bacillus e.g., B. anthracis or B. cereus
  • Bartonella e.g., B. henselae, or B. quintana
  • Haemophilus e.g., FI. influenzae
  • Helicobacter e.g., II. pylori
  • Klebsiella e.g., K. pneumoniae and K. oxytoed
  • Legionella e.g., L. pneumophila
  • Leptospira e.g., L. interrogans, L. santarosai, L. wellii, L. noguchii
  • Listeria e.g., L. monocytogenes
  • Mycobacterium e.g., M. leprae, M. tuberculosis, or M. ulcerans
  • Mycoplasma e.g., M., M.
  • Neisseria e.g., N. gonorrhoeae or N. meningitidis
  • Pseudomonas e.g., P. aeruginosa
  • Rickettsia e.g., R. rickettsii
  • Salmonella e.g., S. Typhi, 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. epidermis, or S.
  • Streptococcus e.g., S. agalactiae, S. pneumoniae, or S. pyogenes
  • Treponema e.g., T. pallidum
  • Ureaplasma e.g., U. urealyticum
  • Vibrio e.g., V. cholerae
  • Yersinia e.g., Y. pestis, Y. enterocolitica, or Y. pseudotuberculosis.
  • the at least one antigen may comprise at least one Group A Streptococcus antigen.
  • Group A Streptococcus is intended to refer to and is synonymous with the term “Streptococcus pyogenes ” .
  • any instance of the term “Group A Streptococcus” may be replaced with the term “Streptococcus pyogenes”.
  • Streptococcus pyogenes is a species of pathogenic Gram-positive bacteria in the genus Streptococcus. Methods of determining whether a bacterium is Streptococcus pyogenes are well known to the skilled person. Specifically, all Streptococcus pyogenes strains express GAC, and the skilled person is aware of methods for detecting GAC.
  • Antigens from Streptococcus pyogenes include the antigens disclosed in W02005032582, W02006042027, W02009034473, W02009081274, and W02010076618.
  • Suitable antigens include a polysaccharide such as GAC, optionally as part of a polysaccharide- protein conjugate, a SpyCEP polypeptide, a SpyAD polypeptide and/or a SLO polypeptide.
  • the at least one Group A Streptococcus antigen comprises at least one recombinant polypeptide.
  • the SpyCEP polypeptide, the SpyAD polypeptide and/or a SLO polypeptide is/are a recombinant polypeptide(s).
  • the at least one Group A Streptococcus antigen comprises a polysaccharide - protein conjugate.
  • one of the polysaccharide and/or the protein is a Group A Streptococcus antigen.
  • the protein may be a Streptococcus pyogenes antigen like a SpyCEP polypeptide, a SpyAD polypeptide and/or a SLO polypeptide.
  • the polysaccharide may be a Streptococcus pyogenes antigen such as GAC.
  • polysaccharide refers to any linear or branched polymer consisting of monosaccharide residues, usually linked by glycosidic linkages, and thus includes oligosaccharides.
  • protein we mean or include any linear or branched molecule consisting of amino acid residues.
  • polysaccharide-protein conjugate refers to a molecule formed by a covalent linkage between a protein and a polysaccharide.
  • GAC (the Lancefield group A carbohydrate) is a polysaccharide comprising a polyrhamnose backbone with N-acetylglucosamine (GlcNAc) side chains, and may comprise the repeating units [ ⁇ 3)a-Rha(l ⁇ -2)[[i-GlcNAc(1 ⁇ 3)]a-Rha(l ⁇ ] (referred to herein as a “GAC subunit”).
  • GAC is the main component of the GAS cell wall.
  • GAC refers to a GAC polysaccharide derived from a strain of Streptococcus pyogenes.
  • GAC may refer to a fragment or subunit (repeating unit) of the GAC polysaccharide such as one or more GAC subunits.
  • GAC comprises 18 GAC subunits.
  • a fragment of a polysaccharide we mean polysaccharides that are truncated compared to the wild-type polysaccharide (e.g., have an average [mean] number of monosaccharide units that is smaller compared 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 polysaccharide with fewer monosaccharide units than wild-type, or genetic modification of polysaccharide producing strains.
  • polysaccharide we mean or include that one or more chemical group of the polysaccharide backbone and/or side chain(s) is modified compared to wild-type polysaccharide.
  • Polysaccharide modification can be achieved by any suitable means known in the art such as chemical reaction or genetic modification of polysaccharide producing strains.
  • Polysaccharide antigens such as GAC may be excellent antigens for stimulating B cell mediated immunity. However, it may be advantageous to conjugate GAC to a protein such as a carrier protein. Suitable proteins may include tetanus toxoid, diphtheria toxoid, CRM197 or a protein from Streptococcus pyogenes such as a SpyAD polypeptide, a SpyCEP polypeptide and/or a SLO polypeptide.
  • the carrier protein i.e. the protein in the polysaccharide-protein conjugate
  • CRM197 is a non-toxic mutant of diphtheria toxin which has a single mutation at position 52 substituting glutamic acid for glycine. The mutation at position 52 causes loss of the ADP-ribosyltransferase activity of native diphtheria toxin.
  • the polypeptide sequence of CRM197 is provided in SEQ ID NO: 7.
  • the polypeptide may be conjugated to the protein using any suitable conjugation method.
  • Attachment of the polysaccharide to the protein is preferably via a -NH2 group, e.g., through the side chain(s) of a lysine residue(s) or arginine residue(s) in the carrier polypeptide.
  • a -NH2 group e.g., through the side chain(s) of a lysine residue(s) or arginine residue(s) in the carrier polypeptide.
  • this group can react with an amine in the protein to form a conjugate by reductive amination.
  • Attachment to the carrier may also be via a -SH group, e.g., through the side chain(s) of a cysteine residue(s) in the carrier polypeptide.
  • the polysaccharide may be attached to the carrier protein via a linker molecule.
  • the polysaccharide will typically be activated or functionalised prior to conjugation. Activation may involve, for example, cyanylating reagents such as CDAP (l-cyano-4- dimethylamino pyridinium tetrafluoro borate). Other suitable techniques use carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU (see, e.g., the introduction to WO 98/42721). Direct linkages to the protein may comprise oxidation of the polysaccharide followed by reductive amination with the protein, as described in, for example, U.S. Pat No.
  • Linkages via a linker group may be made using any known procedure, for example, the procedures described in U.S. Pat No. 4,882,317 and U.S. Pat No. 4,695,624.
  • the linker is attached via an anomeric carbon of the polysaccharide.
  • a preferred type of linkage is an adipic acid linker, which may be formed by coupling a free -NH2 group (e.g., introduced to a polysaccharide by amination) with adipic acid (using, for example, diimide activation), and then coupling a protein to the resulting saccharide-adipic acid intermediate (see, e.g., 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 may be formed by coupling a free -NH group with glutaric acid in the same way.
  • Adipic and glutaric acid linkers may also be formed by direct coupling to the polysaccharide, i.e., without prior introduction of a free group, e.g., a free -NH group, to the polysaccharide, followed by coupling a protein to the resulting saccharide- adipic/glutaric acid intermediate.
  • a carbonyl linker which may be formed by reaction of a free hydroxyl group of a modified polysaccharide with CDI (Bethell G.S. et al. (1979) J. Biol. Chem. 254, 2572-4 and Heam M.T.W. (1981) J. Chromatogr.
  • linkers include P-propionamido (WO00/10599), nitrophenyl-ethylamine (Gever Qt al. (1979) Med. Microbiol. Immunol. 165, 171-288), haloacyl halides (U.S. Pat. No. 4,057,685), glycosidic linkages (U.S. Pat. Nos. 4,673,574; 4,761,283; and 4,808,700), 6-aminocaproic acid (U.S. Pat. No.
  • a bifunctional linker may be used to provide a first group for coupling to an amine group in the polysaccharide (e.g., introduced to the polysaccharide by amination) and a second group for coupling to the carrier (typically for coupling to an amine in the carrier).
  • the first group is capable of direct coupling to the polysaccharide, i.e., without prior introduction of a group, e.g., an amine group, to the polysaccharide.
  • the polysaccharide may be conjugated to the protein by a method in which the polysaccharide is exposed to random oxidation followed by reacting the oxidised polysaccharide with sodium cyanoborohydride, as disclosed in WO2022/101434 or in the Examples herein.
  • the at least one Group A Streptococcus antigen may comprise a SpyCEP polypeptide.
  • the protein in the polysaccharide-protein conjugate may comprise a SpyCEP polypeptide.
  • SpyCEP polypeptide refers to SpyCEP or a fragment or variant thereof. Suitable SpyCEP polypeptides comprise or consist 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.
  • the SpyCEP polypeptide may not comprise aspartate at a position corresponding to position 151 and/or serine at a position corresponding to position 617.
  • References to amino acids at a position corresponding to “position 151 and “position 617” refer to amino acids at a position corresponding to the 151 st or 617 th amino acid in SEQ ID NO: 1 or SEQ ID NO: 2.
  • the SpyCEP polypeptide comprises alanine at a position corresponding to position 151 and/or 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.
  • the SpyCEP polypeptide has reduced proteolytic activity against interleukin 8 (IL-8) relative to a wild type SpyCEP polypeptide.
  • the SpyCEP polypeptide has 50% or less proteolytic activity against interleukin 8 (IL-8) relative to a wild type SpyCEP polypeptide.
  • a suitable assay to determine proteolytic activity against interleukin 8 is set out in the section entitled “Assay to determine proteolytic activity against interleukin 8”.
  • the SpyCEP polypeptide may comprise: (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
  • the SpyCEP polypeptide is preferably immunogenic.
  • administration of the SpyCEP polypeptide into mice may raise antibodies.
  • the SpyCEP polypeptide may raise at least 50% (for example at least 80% or at least 95%) of the number of antibodies when administered to mice compared to a SpyCEP polypeptide of SEQ ID NO: 1 or SEQ ID NO: 2.
  • Suitable assays for determining the number of antibodies raised are set out in the section entitled “ Immunogenicity assays”.
  • 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.
  • the at least one Group A Streptococcus antigen may comprise a SpyAD polypeptide.
  • the protein in the polysaccharide-protein conjugate may comprise a SpyAD polypeptide.
  • SpyAD polypeptide refers to SpyAD or a fragment or variant thereof. Suitable SpyAD polypeptides comprise or consist 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.
  • the SpyAD polypeptide may comprise: (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; or
  • the SpyAD polypeptide is preferably immunogenic.
  • administration of the SpyAD polypeptide into mice may raise antibodies.
  • the SpyAD polypeptide may raise at least 50% (for example at least 80% or at least 95%) of the number of antibodies when administered to mice compared to a SpyAD polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4.
  • Suitable assays for determining the number of antibodies raised are set out in the section entitled “ Immunogenicity assays”.
  • the 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.
  • the 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.
  • the at least one Group A Streptococcus antigen may comprise a SLO (streptolysin O) polypeptide.
  • the protein in the polysaccharide-protein conjugate may comprise a SLO polypeptide.
  • SLO polypeptide or “streptolysin O polypeptide ” refers to streptolysin O or a fragment or variant thereof.
  • Suitable SLO polypeptides comprise or consist of SEQ ID NO: 5 or 6 or a fragment or variant thereof.
  • Another suitable SLO polypeptide is the SLO polypeptide ofNCBI reference sequence WP_010921831.1.
  • the SLO polypeptide may not comprise proline at a position corresponding to position 427 and/or tryptophan at a position corresponding to position 535.
  • References to amino acids at a position corresponding to “position 427 and “position 535” refer to amino acids at a position corresponding to the 427 th or 535 th amino acid in SEQ ID NO: 1 or SEQ ID NO: 2.
  • the SLO polypeptide comprises leucine at a position corresponding to position 427 and/or 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.
  • the SLO polypeptide has 50% or less hemolytic activity relative to a wild type SpyCEP polypeptide.
  • a suitable assay to determine proteolytic activity against interleukin 8 is set out in the section entitled “Assay to determine hemolytic activity”.
  • the SLO polypeptide may comprise:
  • 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
  • the SLO polypeptide is preferably immunogenic.
  • administration of the SLO polypeptide into mice may raise antibodies.
  • the SLO polypeptide may raise at least 50% (for example at least 80% or at least 95%) of the number of antibodies when administered to mice compared to a SLO polypeptide of SEQ ID NO: 5 or SEQ ID NO: 6.
  • Suitable assays for determining the number of antibodies raised are set out in the section entitled “Immunogenicity assays”.
  • 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.
  • the immunogenic composition of the invention comprises a TLR7 agonist and/or a benzonapthyridine compound.
  • Methods of the invention are methods of making immunogenic compositions that comprise a TLR7 agonist and/or a benzonapthyridine compound.
  • a TLR7 agonist is a compound that is capable of activating TLR7.
  • TLR7 agonists are small molecules.
  • the skilled person is aware of a number of different TLR7 agonists. Examples include 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.
  • the skilled person is also able to determine whether a given molecule is acting as a TLR agonist.
  • a suitable assay for determining whether a compound is a TLR7 agonist is provided in Russo et al.,- Blood; 117: 5683-5691.
  • the TLR7 agonist activity of a compound may be assessed by culturing pDCs with IL-3, stimulating the pDCs with the compound and measuring cytokine concentrations in the supernatant. Detection of cytokines and IFN-a in the supernatant is a marker of TLR7 agonist activity.
  • a TLR7 agonist may also be an agonist of another TLR such as TLR8. However, in some embodiments the TLR7 agonists preferentially activate TLR7 and do not activate other TLRs.
  • the TLR7 agonist may comprise or consist of a benzonapthyridine 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-(2- phosphonocthoxy)cthoxy)cthoxy)phcncthyl)bcnzo[f] [ 1 ,7]naphthyridin-8-yl)propanoic acid, or a pharmaceutically acceptable salt thereof.
  • the benzonapthyridine compound is a compound which comprises a benzonapthyridine moiety, as shown below.
  • the benzonapthyridine compound is a compound of formula (I).
  • Methods of manufacturing suitable benzonapthyridine compounds of formula (I) are disclosed in WO20 11/027222 and WO2013/131985.
  • the benzonapthyridine compound comprises or consists of a compound of Formula (I),
  • R 1 is H, Cl-C6alkyl, -C(R 5 ) 2 OH, -L' R 5 , - R 6 , -L 2 R 5 , -L 2 R 6 , -OL 2 R 5 , or -OL 2 R 6 ;
  • L 1 is -C(O)- or -O-;
  • L 2 is Cl-C6alkylene, C2-C6alkenylene, arylene, heteroarylene or - ((CR 4 R 4 ) p O)q(CH2)p- wherein the Cl-C6alkylene and C2-C6alkenylene of L 2 are optionally substituted with 1 to 4 fluoro groups; each L 3 is independently selected from Cl-C6alkylene and -((CR 4 R 4 ) p O) q (CH2) P - wherein the Cl-C6alkylene of L 3 is optionally substituted with 1 to 4 fluoro groups;
  • L 4 is arylene or heteroarylene
  • R 2 is H or Cl-C6alkyl
  • R 3 is selected from Cl-C4alkyl, -L 3 R 5 , -L’ 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 ) 2 OH; each R 4 is independently selected from H and fluoro;
  • R 5 is -P(O)(OR 9 ) 2 ,
  • R 6 is -CF 2 P(O)(OR 9 ) 2 or -C(O)OR 10 ;
  • R 7 is -CF 2 P(O)(OR 9 ) 2 or -C(O)OR 10 ;
  • R 8 is H or Cl-C4alkyl; each R 9 is independently selected from H and Cl-C6alkyl;
  • R 10 is H or Cl-C4alkyl; 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.
  • Suitable benzonapthyridine compounds include:
  • the benzonapthyridine compound is 3-(5-amino-2-(2-methyl-4-(2-(2-(2-(2- phosphonoethoxy)ethoxy)ethoxy)phenethyl)benzo[l [ 1 ,7]naphthyridin-8-yl)propanoic acid, or a pharmaceutically acceptable salt thereof.
  • the benzonapthyridine compound comprises or consists of an arginine salt, (for example, a D-arginine salt or an L-arginine salt, preferably an L-arginine salt), of the compound of formula (I).
  • the benzonapthyridine compound is an arginine salt of 3-(5-amino-2-(2-methyl-4-(2-(2-(2-(2- phosphonoethoxy)ethoxy)ethoxy)phenethyl)benzo[l [ 1 ,7]naphthyridin-8-yl)propanoic acid.
  • the benzonapthyridine compound is an L-arginine salt of 3-(5-amino-2- (2-methyl-4-(2-(2-(2-(2- phosphonoethoxy)ethoxy)ethoxy)phenethyl)benzo[l [ 1 ,7]naphthyridin-8-yl)propanoic acid.
  • R 1 is -(CH 2 ) 2 C(O)OH
  • R 2 is C 1 alkyl
  • R 3 is -O(((CH 2 ) 2 )O) 2 (CH 2 ) 2 -P(O)(OH) 2 may also be referred to as LHD153R.
  • the immunogenic composition of the invention comprises an aluminium salt.
  • Methods of the invention are methods of making immunogenic compositions that comprise an aluminium salt.
  • the aluminium salt may act as an adjuvant and/or an absorbent.
  • An '"adjuvant generally refers to a substance that increase the immune response to an antigen.
  • An “absorbent” generally refers to a solid substrate to which other components of the immunogenic composition, such as the at least one antigen, at least one Group A Streptococcus antigen, the TLR7 agonist and/or the benzonap thyridine compound may bind, attach or adsorb (for example, by Van der Waals interactions or hydrogen bonding).
  • Particular aluminium salts include aluminium hydroxide (such as ALHYDROGEL®), aluminium phosphate, aluminium hydroxyphosphate, potassium aluminium sulphate and alum.
  • the aluminium salt comprises or consists of aluminium phosphate and/or aluminium hydroxide.
  • the aluminium salt comprises or consists of aluminium phosphate.
  • the at least one Group A Streptococcus antigen may comprise a polysaccharide -protein conjugate.
  • 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 benzonapthyridine compound in the immunogenic composition may be 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.
  • 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 benzonapthyridine compound in the immunogenic composition is between 1:1.5 and 1:2.5.
  • the ratio of the concentration (w/v) of the polysaccharide in the polysaccharide-protein conjugate 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 1:10 and 1:30, between 1:15 and 1:25, or around 1:20.
  • the ratio of the concentration (w/v) of the polysaccharide in the polysaccharide-protein conjugate to the concentration (w/v) of aluminium in the immunogenic composition is between 1:15 and 1:21.
  • the at least one Group A Streptococcus antigen may comprise a SpyCEP polypeptide.
  • the ratio of the concentration (w/v) of the SpyCEP polypeptide to the concentration (w/v) of the TLR7 agonist or benzonapthyridine compound in the immunogenic composition may be 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.
  • the ratio of the concentration (w/v) of the SpyCEP polypeptide to the concentration (w/v) of the TLR7 agonist or benzonapthyridine compound in the immunogenic composition is between 0.75:1 and 1:2.5.
  • the ratio of the concentration (w/v) of the SpyCEP 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.
  • the ratio of the concentration (w/v) of the SpyCEP polypeptide to the concentration (w/v) of aluminium in the immunogenic composition is between 1:5 and 1:15.
  • the at least one Group A Streptococcus antigen may comprise a SpyAD polypeptide.
  • the ratio of the concentration (w/v) of the SpyAD polypeptide to the concentration (w/v) of the TLR7 agonist or benzonapthyridine compound in the immunogenic composition may be 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.
  • the ratio of the concentration (w/v) of the SpyAD polypeptide to the concentration (w/v) of the TLR7 agonist or benzonapthyridine compound in the immunogenic composition is between 0.75:1 and 1:2.5.
  • the ratio of the concentration (w/v) of the 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.
  • the ratio of the concentration (w/v) of the SpyAD polypeptide to the concentration (w/v) of aluminium in the immunogenic composition is between 1:5 and 1:15.
  • the at least one Group A Streptococcus antigen may comprise a SLO polypeptide.
  • the ratio of the concentration (w/v) of the SLO polypeptide to the concentration (w/v) of the TLR7 agonist or benzonapthyridine compound in the immunogenic composition may be 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.
  • the ratio of the concentration (w/v) of the SLO polypeptide to the concentration (w/v) of the TLR7 agonist or benzonapthyridine compound in the immunogenic composition is between 0.75:1 and 1:2.5.
  • the ratio of the concentration (w/v) of the SLO 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.
  • the ratio of the concentration (w/v) of the SLO polypeptide to the concentration (w/v) of aluminium in the immunogenic composition is between 1:5 and 1:15.
  • the immunogenic composition :
  • (i) comprises a polysaccharide -protein conjugate, wherein the polysaccharide is GAC and the protein is CRM197, a SpyAD polypeptide, a SpyCEP polypeptide and a SLO polypeptide; and
  • 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
  • the SLO polypeptide is at least 98% identical to SEQ ID NO: 6.
  • the immunogenic composition :
  • (i) comprises a polysaccharide -protein conjugate, wherein the polysaccharide is GAC and the protein is CRM197, a SpyAD polypeptide, a SpyCEP polypeptide and a SLO polypeptide; and
  • 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
  • the SLO polypeptide is at least 98% identical to SEQ ID NO: 5.
  • the immunogenic composition comprises a polysaccharide - protein conjugate, a SpyAD polypeptide, a SpyCEP polypeptide and a SLO polypeptide
  • the immunogenic composition may comprise these components at 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 benzonapthyridine compound in the immunogenic composition is between 0.75:1 and 1:5; the ratio of the concentration (w/v) of the polysaccharide in the polysaccharide - protein conjugate to the concentration (w/v) of aluminium 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 benzonapthyridine compound in the immunogenic composition is between 0.75:0.5 and 1:2.5; the ratio of the concentration of the concentration
  • the immunogenic composition comprises between 0.5 mg/ml and 10 mg/ml, between 0.5 mg/ml and 5mg/ml, or around 1 mg/ml of the aluminium salt.
  • 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 the TLR7 agonist and/or the benzonapthyridine compound.
  • the at least one Group A Streptococcus antigen comprises a polysaccharide-protein conjugate comprising GAC (optionally GAC conjugated to CRM197), and the immunogenic composition comprises between 5 pg and 75 pg, between lOpg and 50 pg, or around 25 pg of polysaccharide -protein conjugate
  • the at least one Group A Streptococcus antigen comprises a SpyAD polypeptide and the immunogenic composition comprises between lOpg and 150pg, between 25pg and 75pg, or around 50 pg of the SpyCEP polypeptide.
  • the at least one Group A Streptococcus antigen comprises a SpyCEP polypeptide and the immunogenic composition comprises between lOpg and 150pg, between 25pg and 75pg, or around 50 pg of the SpyCEP polypeptide.
  • the at least one Group A Streptococcus antigen comprises a SLO polypeptide and the immunogenic composition comprises between lOpg and 150pg, between 25pg and 75pg, or around 50 pg of the SLO polypeptide.
  • the immunogenic composition comprises: between 0.5 mg/ml and 10 mg/ml of the aluminium salt; between 0.01 mg/ml and 5 mg/ml of the TLR7 agonist and/or the benzonapthyridine compound;
  • At least 50%, at least 75%, or at least 80% of the at least one antigen or the at least one Group A streptococcus antigen in the immunogenic composition is adsorbed to the aluminium salt.
  • at least 50%, at least 75%, or at least 80% of the TLR7 agonist and/or benzonapthyridine compound is adsorbed to the aluminium salt.
  • the level of adsorption of a test compound such as a Group A streptococcal antigen and/or a TLR agonists and/or a benzonapthyridine compound to an aluminium salt may be measured by mixing the aluminium salt with the test compound, centrifuging the mixture at 14,000 x g for 2-5 minutes and then measuring the concentration of the test compound in the supernatant (for example using absorbance at 280 nm to calculate the concentration of a protein test compound).
  • a test compound such as a Group A streptococcal antigen and/or a TLR agonists and/or a benzonapthyridine compound
  • the immunogenic composition may raise a greater number of antibodies when administered to mice compared to an equivalent immunogenic composition that does not comprise the TLR7 agonist and/or benzonap thyridine compound.
  • the immunogenic composition raises a greater number of antibodies when administered to mice at a dose of 5000 ng compared to an equivalent immunogenic composition that does not comprise the TLR7 agonist and/or benzonapthyridine compound.
  • the immunogenic composition raises a greater number of antibodies when administered to mice at a dose of 5000 ng compared to an equivalent immunogenic composition that does not comprise the TLR7 agonist and/or benzonapthyridine compound when measured using serum collected 27 days after administration.
  • equivalent immunogenic composition that does not comprise the TLR7 agonist and/or benzonapthyridine compound we mean an immunogenic composition which is identical to the immunogenic composition except that it lacks the TLR7 agonist and/or the benzonapthyridine compound.
  • each/any protein antigen(s) such as a SpyCEP polypeptide, a SpyAD polypeptide and/or a SLO polypeptide
  • each/any polysaccharide conjugate antigen such as GAC-CRM197
  • the number of antibodies raised when administered to mice may be determined using an assay described under the heading ""immunogenicity assays”.
  • the immunogenic composition raises a greater number of antibodies against at least one of the following antigens:
  • the immunogenic composition when administered to mice compared to an equivalent immunogenic composition that does not comprise the TLR7 agonist and/or benzonapthyridine compound.
  • the immunogenic composition may raise a greater number of antibodies against SpyCEP when administered to mice compared to an equivalent immunogenic composition that does not comprise the TLR7 agonist and/or benzonapthyridine compound.
  • the immunogenic composition may raise a greater number of antibodies against SpyAD when administered to mice compared to an equivalent immunogenic composition that does not comprise the TLR7 agonist and/or benzonapthyridine compound.
  • the immunogenic composition may raise a greater number of antibodies against SLO when administered to mice compared to an equivalent immunogenic composition that does not comprise the TLR7 agonist and/or benzonapthyridine compound.
  • the immunogenic composition comprises a SpyCEP polypeptide
  • the SpyCEP polypeptide that is administered to mice is administered at a dose of 5000ng.
  • the immunogenic composition comprises a SpyAD polypeptide
  • the SpyAD polypeptide that is administered to mice is administered at a dose of 5000ng.
  • the immunogenic composition comprises a SLO polypeptide
  • the SLO polypeptide that is administered to mice is administered at a dose of 5000ng.
  • the immunogenic composition raises a greater number of antibodies against at least one of the following antigens:
  • the immunogenic composition raises 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 comprise the TLR7 agonist and/or benzonapthyridine compound.
  • the immunogenic composition raises a greater number of antibodies against all of the following antigens SpyCEP, SpyAD, SLO and GAC when administered to mice at a 5000 ng dose compared to an equivalent immunogenic composition that does not comprise the TLR7 agonist and/or benzonapthyridine compound.
  • the immunogenic composition raises a greater number of antibodies against SpyCEP when administered to mice at a 5000 ng dose (optionally when the number of antibodies is measured using serum collected 27 days after administration) compared to an equivalent immunogenic composition that does not comprise the TLR7 agonist and/or benzonapthyridine compound.
  • the immunogenic composition raises at least 2 times or at least 5 times the number of antibodies against SpyCEP when administered to mice at a 5000 ng dose (optionally when the number of antibodies is measured using serum collected 27 days after administration) compared to an equivalent immunogenic composition that does not comprise the TLR7 agonist and/or benzonapthyridine compound.
  • the immunogenic composition raises a greater number of antibodies against SpyAD when administered to mice at a 5000 ng dose (optionally when the number of antibodies is measured using serum collected 27 days after administration) compared to an equivalent immunogenic composition that does not comprise the TLR7 agonist and/or benzonapthyridine compound.
  • the immunogenic composition raises at least 2 times the number of antibodies against SpyAD when administered to mice at a 5000 ng dose (optionally when the number of antibodies is measured using serum collected 27 days after administration) compared to an equivalent immunogenic composition that does not comprise the TLR7 agonist and/or benzonapthyridine compound.
  • the immunogenic composition raises a greater number of antibodies against SLO when administered to mice at a 5000 ng dose (optionally when the number of antibodies is measured using serum collected 27 days after administration) compared to an equivalent immunogenic composition that does not comprise the TLR7 agonist and/or benzonapthyridine compound.
  • the immunogenic composition raises at least 2 times or at least 5 times the number of antibodies against SLO when administered to mice at a 5000 ng dose (optionally when the number of antibodies is measured using serum collected 27 days after administration) compared to an equivalent immunogenic composition that does not comprise the TLR7 agonist and/or benzonapthyridine compound.
  • the immunogenic composition raises a greater number of antibodies against GAC-CRM197 when administered to mice at a 5000 ng dose (which correlates to 2500 ng of GAC-CRM197 as explained in the definition of 5000 ng dose given above) (optionally when the number of antibodies is measured using serum collected 27 days after administration) compared to an equivalent immunogenic composition that does not comprise the TLR7 agonist and/or benzonap thyridine compound.
  • the immunogenic composition raises at least 2 times or at least 5 times the number of antibodies against GAC-CRM197 when administered to mice at a 5000 ng dose (optionally when the number of antibodies is measured using serum collected 27 days after administration) compared to an equivalent immunogenic composition that does not comprise the TLR7 agonist and/or benzonap thyridine compound.
  • the immunogenic composition when administered to mice at a 5000 ng dose, raises at least 2 times the number of antibodies against SpyAD, SpyCEP, SLO and GAC- CRM197 compared to an equivalent immunogenic composition that does not comprise the TLR7 agonist and/or benzonap thyridine compound when measured using serum collected 27 days after administration.
  • the immunogenic composition may raise a greater number of functional antibodies when administered to mice compared to an equivalent immunogenic composition that does not comprise the TLR7 agonist and/or benzonap thyridine compound.
  • the immunogenic composition raises a greater number of functional antibodies when administered to mice at a dose of 5000 ng compared to an equivalent immunogenic composition that does not comprise the TLR7 agonist and/or benzonap thyridine compound.
  • the immunogenic composition raises a greater number of functional antibodies when administered to mice at a dose of 5000 ng compared to an equivalent immunogenic composition that does not comprise the TLR7 agonist and/or benzonap thyridine compound when measured using serum collected 27 days after administration.
  • the number of functional antibodies raised may be determined using the assays described under the headings “Assay to determine proteolytic activity against interleukin 8” and “Assay to determine hemolytic activity”.
  • the immunogenic composition raises a greater number of functional antibodies against SpyCEP and SLO when administered to mice compared to an equivalent immunogenic composition that does not comprise the TLR7 agonist and/or benzonap thyridine compound.
  • the immunogenic composition may raise a greater number of functional antibodies against SpyCEP when administered to mice compared to an equivalent immunogenic composition that does not comprise the TLR7 agonist and/or benzonap thyridine compound.
  • the immunogenic composition may raise a greater number of functional antibodies against SLO when administered to mice compared to an equivalent immunogenic composition that does not comprise the TLR7 agonist and/or benzonap thyridine compound.
  • the immunogenic composition raises a greater number of functional antibodies against SpyCEP when administered to mice at a 5000 ng dose (optionally when the number of antibodies is measured using serum collected 42 days after administration wherein a booster dose of the immunogenic composition was administered at day 28) compared to an equivalent immunogenic composition that does not comprise the TLR7 agonist and/or benzonapthyridine compound.
  • the immunogenic composition raises at least 2 times, at least 5 times or at least 7.5 times the number of functional antibodies against SpyCEP when administered to mice at a 5000 ng dose (optionally when the number of antibodies is measured using serum collected 42 days after administration wherein a booster dose of the immunogenic composition was administered at day 28) compared to an equivalent immunogenic composition that does not comprise the TLR7 agonist and/or benzonapthyridine compound.
  • the immunogenic composition raises a greater number of functional antibodies against SLO when administered to mice at a 5000 ng dose (optionally when the number of antibodies is measured using serum collected 42 days after administration wherein a booster dose of the immunogenic composition was administered at day 28) compared to an equivalent immunogenic composition that does not comprise the TLR7 agonist and/or benzonapthyridine compound.
  • the immunogenic composition raises at least 2 times, or at least 5 times the number of functional antibodies against SLO when administered to mice at a 5000 ng dose (optionally when the number of antibodies is measured using serum collected 42 days after administration wherein a booster dose of the immunogenic composition was administered at day 28) compared to an equivalent immunogenic composition that does not comprise the TLR7 agonist and/or benzonapthyridine compound.
  • the immunogenic composition when administered to mice at a 5000 ng dose, raises at least 5 times the number of functional antibodies against SpyCEP and SLO compared to an equivalent immunogenic composition that does not comprise the TLR7 agonist and/or benzonapthyridine compound when measured using serum collected 42 days after administration wherein a booster dose of the immunogenic composition was administered at day 28.
  • Immunogenicity levels (the number of antibodies raised against a given antigen such as SpyCEP, SpyAD, SLO or GAC) may be determined in one of several ways known to the skilled person.
  • the skilled person may use an ELISA based method or may use a fluorescence immunoassay.
  • a fluorescence immunoassay the level of antibodies can be measured in units of RLU/ml.
  • Fluorescence beads may be used, where each bead is coupled to a unique bead region. The beads are associated with unique spots on the fluorescence spectrum.
  • Different antigens may be coupled to the beads in the different regions. Suitable antigens include GAC, SpyCEP (for example a protein of SEQ ID NO: 1 or 2), SpyAD (for example a protein of SEQ ID NO: 3 or 4) and SLO (for example a protein of SEQ ID NO: 5 or 6).
  • beads from a first region may be coupled to SpyCEP antigen
  • beads from a second region may be coupled to SpyAD antigen.
  • Sera from mice immunised with the immunogenic composition of the invention may be applied to the beads and allowed to bind to the antigens coupled to the beads.
  • the remaining serum may then be removed with a washing step, and a detection mixture comprising biotinylated detection antibodies (that bind to mice antibodies) and a streptavidin-phycoerythrin (PE) reporter applied.
  • the beads can be excited one by one to determine the bead region and corresponding antigen.
  • a second laser determines that magnitude of the PE-derived signal, which is proportional to the amount of analyte bound. The user can, thereby, determining the number of bound antibodies associated with each antigen.
  • a suitable fluorescence immunoassay uses the Luminex ® machine.
  • a suitable fluorescence (Luminex ®) assay is described in Example 2.
  • test immunogenic composition raises greater numbers of antibodies when administrated to mice compared to a reference immunogenic composition (for example an equivalent immunogenic composition that does not comprise a TLR7 agonist and/or benzonapthyridine compound)
  • a reference immunogenic composition for example an equivalent immunogenic composition that does not comprise a TLR7 agonist and/or benzonapthyridine compound
  • the level of antibodies raised by the test immunogenic composition may be measured using one of the assays above, and the level of antibodies raised by the reference immunogenic composition should be measured using an identical assay, and the two antibody levels compared.
  • the number of functional antibodies against SpyCEP may be measured using an IL-8 cleavage assay.
  • Wild type SpyCEP has a proteolytic activity that allows it to cleave IL-8, and so the ability of antibodies raised against SpyCEP to prevent IL-8 cleavage by SpyCEP is a measure of how functional the antibodies are.
  • a suitable IL-8 cleavage assay may involve pre-incubating wild-type SpyCEP with serum from mice immunised with serial dilutions of the immunogenic composition to be tested or a buffer only control. Human IL-8 can then be added and the reaction incubated at 37°C for 2 hours. The reaction mixture may then be diluted 20-fold and incubated with monoclonal antibodies directed against IL-8 to determine the level of IL-8 remaining (uncleaved). The level of IL-8 remaining should be compared with the level of IL-8 remaining in a control experiment (an equivalent experiment except that the serum is from mice prior to administration of the immunogenic composition to be tested). The IC50 may be calculated by determining the serum concentration that achieved 50% inhibition of hemolysis (50% less reduction in IL-8 compared to the control experiment).
  • a suitable IL-8 cleavage assay is described in Example 2.
  • test immunogenic composition raises greater numbers of functional anti-SpyCEP antibodies when administered to mice compared to a reference immunogenic composition (for example an equivalent immunogenic composition that does not comprise a TLR7 agonist and/or benzonapthyridine compound)
  • a reference immunogenic composition for example an equivalent immunogenic composition that does not comprise a TLR7 agonist and/or benzonapthyridine compound
  • the level of functional anti-SpyCEP antibodies raised by the test immunogenic composition may be measured using one of the assays above, and the level of functional anti-SpyCEP antibodies raised by the reference immunogenic composition should be measured using an identical assay, and the two functional anti-SpyCEP antibody levels compared.
  • the number of functional antibodies raised against SLO may be measured using a hemolysis assay. Wild type SLO is haemolytic, and so the ability of antibodies raised against SLO to prevent the haemolytic activity by SLO is a measure of how functional the antibodies are.
  • a suitable hemolysis assay may involve preparing a red blood cell suspension (e.g. rabbit red blood cells).
  • a red blood cell suspension e.g. rabbit red blood cells
  • one may prepare mixtures of a serial dilution of serum from mice immunised with the immunogenic composition to be tested and pre-incubate them with a fixed concentration of wild type SLO toxin to form a test pre-incubated mixture.
  • the rabbit blood cells may then be mixed with the pre-incubated mixture and the incubated for 30 minutes at 37°C.
  • the blood cells may then be centrifuged for 5 minutes at 1000 x g, and the level of hemolysis can be measured by looking at how much haemoglobin is released.
  • the level of haemoglobin released may be measured by reading absorbance at 540 nm.
  • Th level of haemoglobin released should be compared to the level of haemoglobin released in a control experiment (an equivalent experiment except that the serum is from mice prior to administration of the immunogenic composition to be tested).
  • the serum titre corresponding to the dilution factor which provides 50% inhibition of cleavage i.e. 50% of the haemoglobin released compared to the control experiment, is determined.
  • a suitable hemolysis assay is described in Example 2.
  • a test immunogenic composition raises greater numbers of functional anti-SLO antibodies when administered to mice compared to a reference immunogenic composition (for example an equivalent immunogenic composition that does not comprise a TLR7 agonist and/or benzonapthyridine compound)
  • the level of functional anti-SLO antibodies raised by the test immunogenic composition may be measured using one of the assays above, and the level of functional anti-SLO antibodies raised by the reference immunogenic composition should be measured using an identical assay, and the two functional anti-SLO antibody levels compared.
  • the immunogenic composition may further comprise a pharmaceutically acceptable excipient.
  • Typical "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 metabolised 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).
  • Pharmaceutically acceptable excipients may also contain diluents, such as water, saline, glycerol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present.
  • Tris-buffered physiologic saline is a suitable carrier particularly when using aluminium adjuvants since the phosphate in phosphate buffered saline may interfere with outer membrane vesicle binding to aluminium.
  • Typical pharmaceutical excipients may include one of more of Tris buffer, histidine, sodium chloride and sodium phosphate.
  • the immunogenic composition comprises tris buffer, histidine, sodium chloride and sodium phosphate.
  • the immunogenic composition comprises tris buffer at a concentration between 0.5 mM and 25 mM, between 1 mM and 10 mM, or around 2 mM.
  • the immunogenic composition comprises histidine at a concentration between 1 mM and 50 mM, between 5 mM and 25mM, or around 10 mM.
  • the immunogenic composition comprises sodium chloride at a concentration between 50mM and 300mM, between 75mM and 250mM, or around 129 mM.
  • the immunogenic composition comprises sodium phosphate at a concentration between 1 mM and 25 mM, between 2 mM and 10 mM, or around 4mM.
  • immunogenic compositions of the invention may be useful as vaccines.
  • Vaccines according to the invention may either be prophylactic (i.e. to prevent infection) or therapeutic (i.e. to treat infection), but will typically be prophylactic.
  • Immunogenic compositions used as vaccines comprise an immunologically effective amount of antigen(s), as well as any other components, as needed.
  • immunologically effective amount it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g. non-human primate, primate, etc.), the capacity of the individual's immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
  • Immunogenic compositions of the invention may include an antimicrobial, particularly when packaged in multiple dose formats.
  • the present invention also provides the immunogenic composition or the vaccine of the invention for use in a method of preventing infection by Group A Streptococcus.
  • the present invention provides a method of preventing infection and/or autoimmune disease following infection by Group A Streptococcus comprising administering an effective amount of the immunogenic composition or vaccine of the invention.
  • the present invention also provides a use of the immunogenic composition or the vaccine of the invention for preparing a medicament for use in a method of preventing infection and/or autoimmune disease following infection by Group A Streptococcus.
  • the term “treating” includes both therapeutic treatment and prophylactic or preventative treatment, wherein the object is to prevent or lessen infection.
  • treating may include directly affecting or curing, suppressing, inhibiting, preventing, reducing the severity of, delaying the onset of, reducing symptoms associated with, for example, infection, or a combination thereof.
  • Preventing may refer, inter alia, to delaying the onset of symptoms, preventing relapse of a disease, and the like.
  • Treating may also include “suppressing” or “inhibiting” an infection or illness, for example reducing severity, number, incidence or latency of symptoms, ameliorating symptoms, reducing secondary symptoms, reducing secondary infections, prolonging patient survival, or combinations thereof.
  • the term “preventing or treating Group A Streptococcus infection” in the method/immunogenic composition for use/use of the immunogenic composition in the manufacture of a medicament of the invention comprises raising an immune response in a subject.
  • the immune response may be protective and may raises antibodies, such as IgG antibodies.
  • the subject of the invention is a mammal, optionally a human.
  • the human may be an adult i.e. subject is 18 years old or above 18 years old.
  • the human may be a child i.e. below 18 years old.
  • the child may be between 12 to 72 months, preferably between 24 to 59 months, more preferably between 6 to 12 months.
  • the child may be around 9 months.
  • the human is preferably a child.
  • a vaccine intended for children may also be administered to adults e.g. to assess safety, dosage, or immunogenicity.
  • the invention further provides a method of making an immunogenic composition comprising at least one antigen, an aluminium salt and (a) a TLR7 agonist, and/or (b) a benzonapthyridine compound comprising preparing a mixture of:
  • the mixtures may be prepared by any suitable method.
  • the mixtures may be prepared by adding two components together (like the alum and the at least one antigen) and vortexing them or simply flicking them to mix them together.
  • the mixtures may be prepared in any suitable mixing vessel.
  • the mixtures may be prepared in bulk in large vats for use in later syringe filling, or may be prepared directly in syringes ready to use to inject a patient.
  • the present invention further provides an immunogenic composition that is obtained or obtainable from the methods of making an immunogenic composition of the invention.
  • Vaccines are prepared containing four separate antigens.
  • the first antigen was a conjugate of GAC and CRM 197
  • the second antigen is an SpyCEP polypeptide of SEQ ID NO: 1
  • the third antigen is a SpyAD polypeptide of SEQ ID NO: 3
  • the fourth antigen is an SLO polypeptide of SEQ ID NO: 5.
  • the upstream process begins with thawing one MCB GMP seed (E. coli BL21 (DE3) pET24 strain expressing the SpyAD antigen), followed by inoculum preparation (25°C, 200 rpm, 0.7 ⁇ OD ⁇ 2.0) and then the fermentation process (fermentation medium contains yeast extract, 30°C, pH 7.2, pO2 set point 30%).
  • the recombinant protein SpyAD expression is triggered by addition of isopropyl-
  • IPTG isopropyl-
  • the process ends 4 hours after induction.
  • the broth is harvested and centrifuged (12,227 g x 30 min). The pellet obtained is collected and stored at -70°C.
  • the recombinant protein SpyAD is extracted from biomass with a homogenizer (Avestin, 10 Ksi, 2 cycles), then the lysate is clarified by centrifugation (12,227 g x 30 min) and orthogonal filtration (Sartorius, Sartobran 300).
  • the subsequent purification consists of three chromatographic steps:
  • CHT Ceramic Hydroxyapatite
  • a first TFF (tangential flow filtration) step (30KDa cut-off, TMP 1.1 barg, DP 0.2 barg) between Butyl and CHT steps is required to exchange buffer and put the SpyAD intermediate in the CHT loading buffer.
  • a second TFF step (30KDa cut-off, TMP 1.1 barg, DP 0.2 barg) after the CHT step is needed to put the purified SpyAD antigen in the formulation buffer (10 mM KPi, pH 7.2) and reach the target drug substance (DS) concentration.
  • the Pre-Bulk is then filtered through a 0.2 pm membrane (Sartorius, Sartobran 150) to generate the final Bulk.
  • the upstream process begins with thawing one MCB GMP seed (E. coli BL21 (DE3) pET24 strain expressing the SLO antigen), followed by inoculum preparation (25°C, 200 rpm, 3 ⁇ OD ⁇ 7) and then the fermentation process (fermentation medium contains yeast extract, 25°C, pH 7.2, pO2 set point 30%).
  • the recombinant protein SLO expression is triggered by addition of IPTG when the culture reaches an OD value of 5.0 ⁇ 2.0 (induction phase). The process ends 9 hours after induction.
  • the broth is harvested and centrifuged (12,227 g x 30 min). The pellet obtained is collected and stored at -70°C.
  • the recombinant protein SLO is extracted from biomass with a homogenizer (Avestin, 10 Ksi, 2 cycles), then the lysate is clarified by centrifugation (12,227 g x 30 min) and orthogonal filtration (Sartorius, Sartobran 300).
  • the subsequent purification consists of three chromatographic steps:
  • Ceramic Hydroxyapatite (Type 1 - 40 pm, 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).
  • a final TFF step (30KDa cut-off, TMP 1.0 barg, DP 1.0 barg) after the CHT step is needed to put the purified SLO antigen in the formulation buffer (10 mM KPi, pH 7.2) and reach the target DS concentration.
  • the Pre-Bulk is then filtered through a 0.2 pm membrane (Sartorius, Sartobran 150) to generate the final Bulk.
  • the upstream process begins with thawing one MCB GMP seed (E. coli BL21(DE3) pET24 strain expressing the SpyCEP antigen), followed by inoculum preparation (30°C, 200 rpm, 1 ⁇ OD ⁇ 5) and then the fermentation process (fermentation medium contains yeast extract, 30°C, pH 7.2, pCh set point 30%).
  • the recombinant protein SpyCEP expression is triggered by addition of IPTG and decreasing temperature at 25°C when the culture reaches an OD value >3.5 (induction phase). The process ends when the final culture's OD is 30 ⁇ 5.
  • the broth is harvested and centrifuged (12,227 g x 30 min). The pellet obtained is collected and stored at -70°C.
  • the recombinant protein SpyCEP is extracted from biomass with a homogenizer (Avestin, 10 Ksi, 2 cycles), then the lysate is clarified by centrifugation (12,227 g x 30 min) and orthogonal filtration (Sartorius, Sartobran 300).
  • the subsequent purification consists of three chromatographic steps:
  • Ceramic Hydroxyapatite (Type 1 - 40 pm, 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),
  • a final TFF step (30KDa cut-off, TMP 0.8 barg, DP 0.5 barg) after the CHT step is needed to put the purified SpyCEP antigen in the formulation buffer (10 mM KPi, pH 7.2) and reach the target DS concentration.
  • the Pre -Bulk is then filtered through a 0.2 pm membrane (Sartorius, Sartobran 150) to generate the final Bulk.
  • the upstream process begins with thawing two MCB GMP seeds (Streptococcus pyogenes emml knockout), followed by inoculum preparation and then the fermentation process.
  • the fermentation medium contains Yeast Extract certified animal free. The Fermentation is stopped when the growth slows down consistently. At this point the OD value measured remains stable (or even at a lower value) compared with the previous sampling point. The final OD expected is 17 ⁇ 5.
  • an inactivation phase will follow. This consists in heating the culture in the bioreactor at 100°C for 2 hours. Afterwards, the culture is cooled down reaching 20°C and the broth is harvested and centrifuged. The pellet obtained is collected and stored at -70°C.
  • the GAC is extracted from biomass generating HNO2 starting from NaNCL and H3PO4, at pH 3.2 at room temperature for 24 hours.
  • the GAC is separated from lysate by centrifugation and clarified by orthogonal filtration.
  • the subsequent purification consists of the following steps:
  • TFF step (30K membrane, TMP 0.7 barg, DP 0.6 barg) could be required to further concentrate the purified GAC, if the GAC concentration is lower than target concentration
  • the GAC intermediate is then filtered through a 0.2 pm membrane (hold point).
  • the GAC intermediate is activated with a solution of 200 mM NalCL (8mM final concentration) and left for 30 minutes in a climate chamber at 25°C. Quenching is performed using a Na2SOs solution (final concentration of 16 mM), mixing for 15-30 minutes at RT.
  • the oxidized GAC is purfied and exchanged in Borate buffer pH 8 with TFF 10K (TMP 0.5 barg, DP 1.0 barg) and centrifuge concentrators (3-10 KDa): the target GACox concentration is 130-140 mg/mL.
  • the rCRM (recombinant CRM197) is concentrated up to 65-70 mg/mL and exchanged in 50 mM Borate buffer pH 8 before conjugation step with TFF and/or centrifuge concentrators (lOKDa membrane cut-off).
  • the conjugation step is done with a concentration of GACox and rCRM of 30 mg/mL with a ratio of 1 : 1 (w/w) for 24 hours at 37°C (mild mixing).
  • the reaction starts with the addition of 200 mg/mL NaBHsCN solution (final concentration 5 mg/mL).
  • the GAC-rCRM is purified and exchanged in PBS IX by TFF 5 OK (TMP 0.3 barg, DP 0.5 barg), then filtered through a 0.2 pm membrane (Sartorius, Sartobran 150) to generate the final Bulk.
  • the adjuvant used was either aluminium hydroxide alone, or a combination of aluminium hydroxide and LHD153R.
  • Example 2 Comparing the Immunogenicity of the Formulations comprising Alum or Alum and LHD153R in Mice
  • Groups of 8 CD1 mice were immunized with one of the 6 formulations set out above.
  • the immunisations involved intraperitoneal immunization of 200 pl of each formulation at day 0 and day 28. Blood samples were collected at days 27 and 42, and the antibodies raised were studied using the following assays.
  • SpyCEP (5 ng/mL) was preincubated with mouse polyclonal anti-SpyCEP serum at four different dilutions (1:100, 1:300, 1:900, 1:2700) for 5 minutes at 4 °C in PBS 0.5 mg/ml BSA. Pre-incubation of SpyCEP with buffer only and with pre-immune serum were used as negative controls. Then, human IL-8 (Gibco, 10 ng/ml) was added and the reaction was incubated at 37°C (reaction without enzyme was used as control).
  • each reaction mix was diluted 20-fold and incubated in 96-well plates coated with a blend of monoclonal antibodies directed against distinct epitopes of IL-8 (Life Technologies).
  • the amount of IL-8 in each sample and in the control reaction (without the enzyme) was determined according to the manufacturer's protocols, using a standard curve of IL-8.
  • Each serum dilution was tested twice, and the mean value with error bar was reported in the graph. Results are shown in panel A of Figure 2 and expressed as the minimal sera dilution required to inhibit 50% of native SpyCEP activity (IC50) for each serum tested.
  • the assay was repeated in parallel to determine the RLU/mL on each individual sera at the highest vaccine dose tested using as secondary antibody antimouse-IgGl-PE, antimouse-IgG2a- PE, antimouse-IgG2b-PE, antimouse-IgG2c-PE and antimouse-IgG3-PE antibodies in standard assay. Results are shown in Figure 3 and expressed as a ratio compared to IgGl for each individual sera.
  • Groups of 8 CD1 mice were immunized with one of the six formulations described in the table below.
  • the immunisations involved intraperitoneal immunization of 200 pl of each formulation at day 0 and day 28. Blood samples were collected at days 27 and 42, and the antibodies raised were studied using the following assays.
  • An immunogenic composition comprising at least one Group A Streptococcus antigen, an aluminium salt and a TLR7 agonist.
  • An immunogenic composition comprising at least one Group A Streptococcus antigen, an aluminium salt and a benzonapthyridine compound.
  • a method of making an immunogenic composition comprising at least one antigen, an aluminium salt and (a) a TLR7 agonist, and/or (b) a benzonapthyridine compound comprising preparing a mixture of:
  • the at least one antigen comprises an antigen from a bacterium 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,
  • 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., C. botulinum, C. difficile, C. perfringens , C. tetani), Corynebac terium (e.g., C. diphtheriae), Enterococcus (e.g., E.faecalis, or E. faecium), Escherichia (e.g., E. coll) , Francisella (e.g., F. tularensis), Haemophilus (e.g., H. influenzae), Helicobacter (e.g., H.
  • Klebsiella e.g., K. pneumoniae and K. oxytoed
  • Legionella e.g., L. pneumophila
  • Leptospira e.g., L. interrogans, L. santarosai, L. wellii, L. noguchii
  • Listeria e.g., L. monocytogenes
  • Mycobacterium e.g., M. leprae, M. tuberculosis, or M. ulcerans
  • Mycoplasma e.g., M. pneumoniae , Neisseria (e.g., N. gonorrhoeae or N.
  • Pseudomonas e.g., P. aeruginosa
  • Rickettsia e.g., R. rickettsii
  • Salmonella e.g., S. Typhi, 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. epidermis, or
  • Streptococcus e.g., S. agalactiae, S. pneumoniae, or S. pyogenes
  • Treponema e.g., T. pallidum
  • Ureaplasma e.g., U. urealyticum
  • Vibrio e.g., V. cholerae
  • Yersinia e.g., Y. pestis, Y. enterocolitica, or Y. pseudotuberculosis.
  • aluminium salt comprises or consists of aluminium phosphate or aluminium hydroxide.
  • aluminium salt comprises or consists of aluminium hydroxide.
  • TLR7 agonist comprises or consists of a benzonapthyridine compound.
  • R 1 is H, Cl-C6alkyl, -C(R 5 ) 2 OH, -L’ R 5 , - R 6 , -L 2 R 5 , -L 2 R 6 , -OL 2 R 5 , or -OL 2 R 6 ;
  • L 1 is -C(O)- or -O-;
  • L 2 is Cl-C6alkylene, C2-C6alkenylene, arylene, heteroarylene or - ((CR 4 R 4 ) p O)q(CH2)p- wherein the Cl-C6alkylene and C2-C6alkenylene of L 2 are optionally substituted with 1 to 4 fluoro groups; each L 3 is independently selected from Cl-C6alkylene and -((CR 4 R 4 ) p O) q (CH2) P - wherein the Cl-C6alkylene of L 3 is optionally substituted with 1 to 4 fluoro groups;
  • L 4 is arylene or heteroarylene
  • R 2 is H or Cl-C6alkyl
  • R 3 is selected from Cl-C4alkyl, -L 3 R 5 , -L' 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 ) 2 OH; each R 4 is independently selected from H and fluoro;
  • R 5 is -P(O)(OR 9 ) 2 ,
  • R 6 is -CF 2 P(O)(OR 9 ) 2 or -C(O)OR 10 ;
  • R 7 is -CF 2 P(O)(OR 9 ) 2 or -C(O)OR 10 ;
  • R 8 is H or Cl-C4alkyl; each R 9 is independently selected from H and Cl-C6alkyl;
  • R 10 is H or Cl-C4alkyl; 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.
  • benzonapthyridine compound comprises or consists of the compound of formula (I), wherein:
  • R 1 is -L 2 R 6 ;
  • R 2 is Cl-C6alkyl
  • R 3 is -OL 3 R 5 or -OL 3 R 7 ;
  • R 5 is -P(O)(OH) 2 ;
  • R 6 is -C(O)OH
  • R 7 is -CF 2 P(O)(OH) 2 ;
  • L 2 is Cl-C6alkylene
  • L 3 is -((CR 4 R 4 )pO)q(CH 2 ) P -;
  • R 4 is H; q is 1 or 2; and p is 2; or a pharmaceutically acceptable salt thereof.
  • R 1 is -(CH 2 ) 2 C(O)OH
  • R 2 is C 1 alkyl
  • R 3 is -O(((CH 2 ) 2 )O) 2 (CH 2 ) 2 -P(O)(OH) 2 ; or a pharmaceutically acceptable salt thereof.
  • 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 benzonapthyridine 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.
  • the immunogenic composition or method of any one of aspects 17 to 19, wherein the ratio of the concentration (w/v) of the polysaccharide in the polysaccharide-protein conjugate 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 1:10 and 1:30, between 1:15 and 1:25, or around 1:21.
  • the ratio of the concentration (w/v) of the SpyCEP polypeptide to the concentration (w/v) of the TLR7 agonist or benzonap thyridine 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.
  • the ratio of the concentration (w/v) of the SpyCEP 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, or between 1:5 and 1:15 or around 1:11.
  • the SpyCEP 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 1000, at least 1200, at least 1400, at least 1500, or at least 1550 amino acids of SEQ ID NO: 1 or 2; or
  • the ratio of the concentration (w/v) of the SpyAD polypeptide to the concentration (w/v) of the TLR7 agonist or benzonap thyridine 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.
  • 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 benzonap thyridine compound in the immunogenic composition is between 0.75:1 and 1:2.5.
  • 39. The immunogenic composition or method of any one of aspects 36 to 38, wherein the ratio of the concentration (w/v) of the 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.
  • 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; or
  • 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.
  • the immunogenic composition or method of aspect 45 wherein the ratio of the concentration (w/v) of the SLO polypeptide to the concentration (w/v) of the TLR7 agonist or benzonap thyridine 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.
  • the immunogenic composition or method of aspect 46 wherein the ratio of the concentration (w/v) of the SLO polypeptide to the concentration (w/v) of the TLR7 agonist or benzonap thyridine compound in the immunogenic composition is between 0.75:1 and 1:2.5.
  • the ratio of the concentration (w/v) of the SLO 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.
  • 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
  • immunogenic composition or method of any one of the preceding aspects, wherein the immunogenic composition raises a greater number of antibodies when administered to mice compared to an equivalent immunogenic composition that does not comprise the TLR7 agonist and/or the benzonapthyridine compound.
  • the immunogenic composition or method of aspect 60 wherein the immunogenic composition raises 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 comprise the TLR7 agonist and/or the benzonapthyridine compound.
  • a vaccine comprising the immunogenic composition of any one of aspects 1, 2, or 4 to 66.
  • a method of preventing infection by and/or preventing autoimmune disease following infection by Group A Streptococcus comprising administering an effective amount of the immunogenic composition of aspects 1, 2, or 4 to 66 or the vaccine of aspect 67 to a subject.

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Abstract

La présente invention concerne des compositions comprenant au moins un antigène de streptocoque de groupe A, un sel d'aluminium et (a) un agoniste de TLR7, ou (b) un composé de benzonapthyridine. La présente invention concerne en outre des procédés de fabrication d'une composition immunogène comprenant au moins un antigène, un sel d'aluminium et (a) un agoniste de TLR7, et/ou (b) un composé de benzonapthyridine. La présente invention concerne également des vaccins comprenant les compositions, ainsi que des procédés d'utilisation et des utilisations des compositions.
PCT/EP2023/078896 2022-10-18 2023-10-17 Vaccin WO2024083873A1 (fr)

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