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WO2015169773A2 - Formes mutantes de spy0269 - Google Patents

Formes mutantes de spy0269 Download PDF

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Publication number
WO2015169773A2
WO2015169773A2 PCT/EP2015/059772 EP2015059772W WO2015169773A2 WO 2015169773 A2 WO2015169773 A2 WO 2015169773A2 EP 2015059772 W EP2015059772 W EP 2015059772W WO 2015169773 A2 WO2015169773 A2 WO 2015169773A2
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WIPO (PCT)
Prior art keywords
gas40
polypeptide
seq
amino acid
coiled
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PCT/EP2015/059772
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WO2015169773A3 (fr
Inventor
Romina D'AURIZIO
Marilena GALLOTTA
Guido Grandi
Immaculada Margarit Y Ros
Mariarosa Mora
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GlaxoSmithKline Biologicals SA
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GlaxoSmithKline Biologicals SA
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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • 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

Definitions

  • the invention relates to the fields of immunology and vaccinology.
  • it relates to mutated antigens derived from Streptococcus pyogenes spy0269 and their use in immunogenic compositions and vaccine compositions.
  • GAS Group A streptococcus
  • S. pyogenes S. pyogenes
  • GAS Group A streptococcus
  • Acute Streptococcus pyogenes infections may take the form of pharyngitis, scarlet fever (rash), impetigo, cellulitis, or erysipelas.
  • Invasive infections can result in necrotizing fasciitis, myositis and streptococcal toxic shock syndrome. Patients may also develop immune-mediated sequelae such as acute rheumatic fever and acute glomerulonephritis (Patterson 1996). Although S. pyogenes may be treated using antibiotics, a prophylactic vaccine to prevent the onset of disease is desired. Efforts to develop such a vaccine have been ongoing for many decades. While various GAS vaccine approaches have been suggested and some approaches are currently in clinical trials, to date, there are no GAS vaccines available to the public.
  • GAS40 also known as "Spy0269” (Ml), “SpyM3_0197” (M3), “SpyM18_0256” (M18) and “prgA” (Uniprot Accession No. Q9A1H3), is a putative surface exclusion protein that possesses a 130-amino-acid region showing similarity with the EzrA protein, which interacts with the cell division protein FtsZ.
  • GAS40 consistently provides protection in the animal model of systemic immunization and challenge and induction of bactericidal antibodies. Hence, GAS40 is a potential candidate for the production of a vaccine against GAS.
  • GAS vaccines based on the M protein contain antigenic determinants that have been determined to cross-rea with heart tissue, potentially resulting in a reaction important in rheumatic heart disease.
  • Molecular mimicry is defined as the theoretical possibility that sequence similarities between foreign and self-peptides are sufficient to result in the cross-activation of autoreactive T or B cells by pathogen-derived peptides. Upon the activation of B or T cells, it is believed that these "peptide mimic" specific T or B cells can cross-react with self-epitopes, thus leading to tissue pathology (autoimmunity).
  • Molecular mimicry is a phenomenon that has been recently discovered as one of several ways in which autoimmunity can be evoked. Therefore, vaccine candidates must be selected that will not elicit the disease it is intended to prevent.
  • GAS40 protein antigens such as GAS40 comprise antigenic determinants that might be problematic
  • historical fears concerning molecular mimicry and GAS vaccines per se may hinder the uptake and use of GAS vaccines.
  • GAS40 might produce autoimmune sequelae of GAS infection by molecular mimicry
  • the inventors have produced GAS40 polypeptides which are less similar to human proteins than wild type GAS40 proteins.
  • the Inventors have developed mutants of GAS40 polypeptides that elicit antibodies that are cross-reactive with wild-type GAS40 but which have reduced sequence identity to amino acid sequences present within human proteins.
  • the human proteins comprise coiled-coil domain containing 81 protein, UPF0492 protein C20orf94, janus kinase and microtubule-interacting protein 3, myosin or tropomyosin.
  • a purified recombinant streptococcal GAS40 antigen which comprises an epitope that elicits opsonic antibodies to Group A Streptococcus
  • the recombinant streptococcal GAS40 antigen comprising at least two substitutions in any of amino acids 235 to 243, and/or at least two substitutions in any of amino acids 684 to 692 and/or at least two substitutions in any of amino acids 699 to 706 and/or a deletion of Lysine at position 208 and/or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 amino acid substitutions at positions selected from the group consisting of amino acid positions 38, 52, 66, 148, 158, 179, 182, 186, 196, 203, 218, 225, 544, 558, 562, 576, 586, 614, 618, 621, 665, 672, 683 and 693, wherein the
  • the purified recombinant streptococcal GAS40 antigen comprises substitutions at amino acid positions T237, A241, A688, S692, A700 and A703.
  • the purified recombinant streptococcal GAS40 antigen comprises the substitutions T237S, A241V, A688S, S692T, A700S and A703S.
  • the recombinant streptococcal GAS40 polypeptide may comprise a deletion of Lysine at position 208.
  • the recombinant streptococcal GAS40 polypeptide may comprise amino acid substitutions at amino acid positions A38, Q52, T66, A148, S158, A179, A182, T186, A196, Q203, Q218, K225, A544, T558, S562, K576, A586, T614, A618, A621, T665, A672, Q683 and T693.
  • substitutions at amino acid positions may be A38L, Q52L, T66L, A148L, S158L, A179L, A182L, T186L, A196L, Q203L, Q218L, K225L, A544L, T558L, S562L, K576L, A586L, T614L, A618L, A621L, T665L, A672L, Q683L and T693L.
  • the recombinant streptococcal GAS40 polypeptide may comprise a combination of these mutations, for example, the substitutions T237S, A241V, A688S, S692T, A700S, A703S, A38L, Q52L, T66L, A148L, S158L, A179L, A182L, T186L, A196L, Q203L, Q218L, K225L, A544L, T558L, S562L, K576L, A586L, T614L, A618L, A621L, T665L, A672L, Q683L, T693L and a deletion of L208.
  • An aim of the present invention is to reduce any theoretical risk of serological cross-reactivity of
  • GAS40 antigens with mammalian tissue antigens are particularly, the mammal is a human.
  • the recombinant streptococcal GAS40 antigen does not contain 7-mers, 8-mers or 9-mers that are present in human proteins.
  • the GAS40 antigen does not contain a sequence selected from the group consisting of QLTEELAAQ (SEQ ID NO: 8), KQDLAKTTS (SEQ ID NO: 9) or EALAALQA (SEQ ID NO: 10).
  • the recombinant streptococcal GAS40 antigen does not contain 7-mers, 8-mers or 9-mers that are present in human proteins.
  • the GAS40 antigen does not contain a sequence selected from the group consisting of QLTEELAAQ (SEQ ID NO: 8), KQDLAKTTS (SEQ ID NO: 9) or EALAALQA (SEQ ID NO: 10).
  • the recombinant streptococcal does not contain 7-mers, 8-mers or
  • GAS40 polypeptide does not comprise an amino acid sequence having 100% sequence identity with a sequence selected from the group consisting of QLTEELAAQ (SEQ ID NO: 8), KQDLAKTTS (SEQ ID NO: 9) or EALAALQA (SEQ ID NO: 10).
  • the 7-, 8- or 9-mer amino acid sequences of the recombinant streptococcal GAS40 polypeptide from positions 235 to 243 and/or positions 684 to 692 and/or 699 to 707, numbered according to SEQ ID No: 2, have sequence identity of less than 100%, less than 95%, less than 90%, less than 85%), less than 80%>, less than 79%, less than 78%, less than 77%, less than 76%, less than 75%, less than 74%, less than 73%, less than 72%, less than 71 ), less than 70%), less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40% or less than 35% with a sequence selected from the group consisting of QLTEELAAQ (SEQ ID NO: 8), KQDLAKTTS (SEQ ID NO: 9) or EALAALQA (SEQ ID NO: 10).
  • QLTEELAAQ SEQ ID NO: 8
  • KQDLAKTTS SEQ ID NO:
  • the recombinant streptococcal GAS40 polypeptide may share at least 92% identity with any one of SEQ ID NOs: 4, 6, 7, 16, 17, 18, 24, 25 or 26. More particularly, the recombinant streptococcal GAS40 polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs: 4, 6, 7, 16, 17, 18, 24, 25 and 26.
  • a fragment of a recombinant streptococcal GAS40 polypeptide according to the first aspect.
  • fragments include polypeptide sequences comprising or consisting of any one of SEQ ID NOs: 4, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 1 12, 1 16, 7, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 1 10, 1 14 and 118.
  • fragment refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion as compared to the full length protein, but wherein the remaining amino acid sequence is identical to the corresponding positions of the full length protein. Fragments typically are greater than 200 amino acids long, particularly no more than 850, 849, 848, 847, 846, 845 amino acids long, yet more particularly no more than 830, 827, 826, 825, 824, 823, 822, 822, 821, 820 amino acids long, still yet more particularly no more than 460, 459, 458, 457, 456, 455, 454, 453, 452, 451, 450 amino acids long, including ranges between these values.
  • fragments of the present invention will comprise substitutions corresponding to those described above where those amino acid positions are present in the fragment.
  • an immunogenic composition comprising one or more GAS40 polypeptides of the first aspect.
  • such immunogenic compositions will comprise one or more additional GAS polypeptides such as, by way of non- limiting example, GAS57, GAS25 or mutants thereof.
  • Particularly immunogenic compositions of the invention will comprise an immunologically effective amount of the GAS40 polypeptide(s).
  • immunogenic compositions of the invention are vaccines.
  • Such compositions may comprise an adjuvant such as alum or MF59.
  • compositions comprise an immunologically effective amount of (i) a GAS40 polypeptide of the invention, (ii) a GAS57 double mutant of SEQ ID NO: 120, (iii) a GAS25 double mutant of SEQ ID NO: 121 and optionally (iv) a conjugate comprising a carrier protein selected from the group consisting of tetanus toxoid, diphtheria toxoid and CRM1 9 7 conjugated to at least one Group A Carbohydrate.
  • a carrier protein selected from the group consisting of tetanus toxoid, diphtheria toxoid and CRM1 9 7 conjugated to at least one Group A Carbohydrate.
  • a nucleic acid encoding the GAS40 polypeptide of the first aspect.
  • a cell comprising the nucleic acid of the third aspect.
  • a method of reducing the amino acid sequence similarity of a wild type GAS40 polypeptide to one or more human proteins comprises the steps of (a) identifying similarities between the amino acid sequence of said wild type GAS40 and human proteins; and (b) introducing one or more mutations in the sequence of said wild type GAS40.
  • said mutations are introduced in short amino-acid stretches that are common to wild type GAS40 and human proteins.
  • the human proteins are one or more of coiled-coil domain containing 81, UPF0492 protein C20orf94, janus kinase and microtubule-interacting protein 3, myosin or tropomyosin.
  • the mutations are introduced in the coiled-coil regions of GAS40 wherein said mutations idealize the coiled-coil regions.
  • FIG. 1 shows a diagrammatic representation of the structure of wild type GAS40.
  • FIG. 2 shows a prediction of the secondary structure of SEQ ID NO: 2 (the wild-type GAS40 polypeptide). The prediction indicates that the integrity of the coil-coiled propensity profile and leucine zipper motif would remain unaltered.
  • FIG. 3 shows a prediction of the secondary structure of SEQ ID NO: 4 (the 8-9mer mutant GAS40 polypeptide). The prediction indicates that the integrity of the coil-coiled propensity profile and leucine zipper motif would remain unaltered.
  • FIG. 4 shows the identification of the coiled-coil regions in GAS40. These regions were highlighted with a color scale from red to blue where red represents hydrophobic residues.
  • FIG. 5 shows the identification of the coiled-coil regions in GAS40. These regions were highlighted with a color scale from red to blue where red represents hydrophobic residues.
  • FIG. 6 shows the identification of the coiled-coil regions in GAS40. These regions were highlighted with a color scale from red to blue where red represents hydrophobic residues.
  • the invention provides a method of reducing the amino acid sequence similarity of a GAS40 polypeptide to one or more human proteins, which may be achieved by the introduction of mutations in the sequence of a wild type GAS40.
  • the purpose of this reduction in the amino acid sequence similarity of GAS40 polypeptide to one or more human proteins is to reduce the theoretical risk of eliciting of antibodies that might be cross-reactive with human proteins.
  • Such mutations may reduce or eliminate the risk of autoimmune sequelae of GAS infection.
  • Said wild type GAS40 polypeptide may consist or comprise the sequence shown in SEQ ID NO: 2, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91, 95, 99 or 103, or may have a sequence identity to SEQ ID NO: 2, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91, 95, 99 or 103 which is greater than 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
  • proteins avoid the elicitation of antibodies that might be cross-reactive with human proteins that comprise coiled-coil domain containing 81 (GI:47271449), UPF0492 protein C20orf94 (GI:61102723), janus kinase and microtubule-interacting protein 3 (GI: 157502225), myosin or tropomyosin.
  • said mutations include single amino acid insertions, single amino acid deletions and/or single amino acid substitutions.
  • a number of single amino acid mutations may be introduced into GAS40, for example: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , 50 or more amino acid mutations.
  • said mutations may be introduced into short amino-acid stretches, e.g. 7-mers, 8-mers or 9-mers, which are common to GAS40 and human proteins.
  • Examples of such short amino-acid stretches include QLTEELAAQ (SEQ ID NO: 8), QLTEELAA (SEQ ID NO: 128), KQDLAKTTS (SEQ ID NO: 9) and EALAALQA (SEQ ID NO: 10), which are found in the human proteins coiled-coil domain containing 81, UPF0492 protein C20orf94, and j anus kinase and microtubule-interacting protein 3, respectively.
  • the mutations that are introduced have no significant effect on the secondary structure of GAS40.
  • the secondary structure of GAS40 polypeptides of the invention is preferably substantially the same as that of wild-type GAS40 polypeptides.
  • the secondary structure of polypeptides, such as GAS40 can be assessed by ab initio computer prediction methods or by circular dichroism, analytical ultracentrifugation, sedimentation equilibrium analyses, X-ray crystallography, NMR spectroscopy, etc.
  • wild- type GAS40 comprises a leader peptide at its N-terminus, two coiled-coil regions (between amino acid residues 58 and 261 and between amino acid residues 556 and 733 in SEQ ID NO: 2), a leucine zipper (located within the C-terminal coiled-coil region and between amino acid residues 673 and 701 in SEQ ID NO: 2) and a transmembrane domain (located between amino acid residues 849 and 866 in SEQ ID NO: 2).
  • any GAS40 polypeptide can be predicted based on pairwise alignment of a given sequence to SEQ ID NO: 2, for example by aligning the amino acid sequence of a GAS40 polypeptide of interest to SEQ ID NO: 2 and identifying the sequences that align to the respective domains/regions of SEQ ID NO:2.
  • any mutations that are introduced have no significant effect on the two alpha- helical coiled-coil regions and the leucine zipper.
  • two substitutions may be introduced into each of the SEQ ID NO: 8, SEQ ID NO: 9 and/or SEQ ID NO: 10 sequences.
  • said mutations include T237S, A241V, A688S, S692T, A700S and/or A703S with respect to the amino acid sequence of SEQ ID NO: 2.
  • SEQ ID NO: 4 is an example of a GAS40 polypeptide which includes all six of these substitutions (T237S, A241V, A688S, S692T, A700S and A703S).
  • the invention also provides GAS40 polypeptides which have been produced by a method of the invention.
  • the invention provides a GAS40 polypeptide which contains a reduced number of 9-mers, 8-mers and/or 7-mers that are present in any human polypeptides.
  • the invention also provides a GAS40 polypeptide which lacks one or more of the sequences: SEQ ID NO: 8, SEQ ID NO: 9 and/or SEQ ID NO: 10.
  • the GAS40 polypeptide has a secondary structure that is substantially the same as wild-type GAS40.
  • the secondary structure of the two alpha-helical coiled-coil regions and the leucine zipper that is located within the C-terminal coiled-coil region is substantially the same as wild-type GAS40.
  • GAS40 polypeptide which lack one or more of the sequences: SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10 include SEQ ID NOs: 4, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112 and 116.
  • the invention provides a mutant protein which shares at least 95%, 96%, 96.5%, 97%, 97.2%, 97.4%, 97.6%, 97.8%, 98%, 98.2%, 98.4%, 98.6%, 98.8%, 99%, 99.2%, 99.4%, 99.6%, 99.8% or 100% identity with any one of SEQ ID NO: 4, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112 or 116.
  • a coiled coil is a structural motif in proteins, in which 2-7 alpha-helices are coiled together like the strands of a rope (dimers and trimers are the most common types).
  • Coiled coils usually contain a repeated pattern, hxxhcxc (SEQ ID NO: 14), of hydrophobic (h) and charged (c) amino- acid residues, referred to as a heptad repeat.
  • the positions in the heptad repeat are usually labeled abcdefg, where a and d are the hydrophobic positions, often being occupied by isoleucine, leucine or valine.
  • the coiled coils of tropomyosin and myosin deviate from the canonical coiled coil structure in subtle ways, including insertions within heptads and charged residues and alanine residues at a and d heptad positions.
  • Such coiled-coil destabilizing sequences alter the local conformation or energetics without interrupting the coiled coil. It is thought that cross-reactivity of GAS M proteins attributable to molecular mimicry may result from the structural similarities between the coiled coils of those proteins and those of myosin and tropomyosin. GAS40 has been found to contain similar coiled-coil destabilizing sequences.
  • the invention provides a GAS40 polypeptide which contains mutations in the coiled-coil regions of GAS40, preferably in order to reduce the structural irregularities in the coiled coil regions, thereby 'idealizing' said coiled-coil regions and making the mutant protein potentially less structurally similar to myosin or tropomyosin.
  • the hydrophilic residues in position 1 and 4 of the heptads forming the coiled coils are substituted with a hydrophobic residue such as leucine, and/or lysine 208 is deleted in order to correct a heptad frame-shift.
  • SEQ ID NOs: 6, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117 are examples of GAS40 polypeptides which include these features.
  • the invention provides a mutant protein which shares at least 90%, 91%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5% or 100% identity with any one of SEQ ID NOs: 6, 17, 21 , 25, 29, 33, 37, 41 , 45, 49, 53, 57, 61 , 65, 69, 73, 77, 81 , 85, 89, 93, 97, 101 , 105, 109, 1 13 or 1 17.
  • the invention also provides a GAS40 polypeptide with 9 or more copies of SEQ ID NO: 14, wherein optionally said polypeptide comprises less than 32 imperfect heptad repeats.
  • said imperfect heptad repeats contain one or two mutations in SEQ ID NO: 14 and/or share between 71 and 86%) identity with SEQ ID NO: 14.
  • the invention provides a GAS40 polypeptide, wherein said polypeptide has n or more copies of SEQ ID NO: 14 and shares at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO: 2.
  • the GAS40 polypeptide contains any combination of the mutations outlined above.
  • the invention provides a GAS40 polypeptide containing both:
  • GAS40 polypeptides examples include: SEQ ID NOs: 7, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 1 10, 1 14, 1 18.
  • the invention provides a GAS40 polypeptide, wherein said polypeptide shares at least 80%, 85%, 90%, 91%, 92%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5% or 100% with any one of SEQ ID NOs: 7, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 1 10, 1 14, 1 18.
  • the GAS40 polypeptides of the invention retain the immunogenicity of wild-type GAS40 polypeptides, for example the GAS40 polypeptides of the invention will preferably elicit antibodies which are able to bind wild-type GAS40 polypeptides.
  • the GAS40 polypeptides of the invention preferably retain substantially the same level of immunogenicity as wild-type GAS40 polypeptides.
  • the geometric means of the antibody titers resulting from the GAS40 polypeptides of the invention are preferably not significantly different from those resulting from wild-type GAS40 polypeptides when assessed by two sample t tests (wherein differences are considered significant if the P value obtained by one-tailed analysis is ⁇ 0.05, ⁇ 0.01 , or ⁇ 0.001) when other variables (such as dose, delivery route, GAS40 strain, population group, etc.) are controlled.
  • Methods of determining the immunogenicity of bacterial polypeptides are well known in the art.
  • the invention also provides fragments of the GAS40 polypeptides of the invention, wherein said fragments are at least: 177, 203, 250, 300, 400, 450, 460, 465, 470, 471 , 500, 550, 600, 650, 700, 750 or 800 amino acids in length.
  • fragments include those recited in SEQ ID NOs: 107, 1 1 1 and 1 15.
  • the invention provides a method of reducing the risk of auto-immunogenicity of a GAS40 polypeptide, wherein said method comprises the steps of: a) identifying short (7-mer, 8-mer or 9- mer) amino acid stretches that are common to human proteins and GAS40; and b) introducing mutations into said short amino acid stretches that are common to human proteins and GAS40.
  • the invention also provides a method of reducing the risk of auto-immunogenicity of a GAS40 polypeptide, wherein said method comprises the steps of: a) identifying coiled-coil regions in GAS40; and b) introducing mutations to idealize the coiled-coil structure.
  • Step b) may involve in the introduction of one or more of the following mutations: substitution of one or more of the hydrophilic residues in position 1 and 4 of the coiled coil heptads with a hydrophobic residue, preferably leucine; and deletion of an amino acid residue, preferably lysine 208, in order to correct a heptad frame-shift.
  • the invention also provides a method of reducing the risk of auto-immunogenicity of a GAS40 polypeptide, wherein said method comprises the steps of: a) identifying short amino acid stretches that are common to human proteins and GAS40; b) introducing mutations into said short amino acid stretches that are common to human proteins and GAS40; c) identifying coiled-coil regions in GAS40; and d) introducing mutations to idealize the coiled-coil structure.
  • Step d) may involve in the introduction of one or more of the following mutations: substitution of one or more of the hydrophilic residues in position 1 and 4 of the coiled coil heptads with a hydrophobic residue, preferably leucine; and deletion of an amino acid residue, preferably lysine 208, in order to correct a heptad frame-shift.
  • GAS40 polypeptides of the invention do not cause molecular mimicry and/or do not cause cross-activation of autoreactive T or B cells.
  • the invention provides a GAS40 polypeptide which can elicit antibodies that are cross- reactive with wild-type GAS40 polypeptides, but which does not elicit antibodies that are cross- reactive with human polypeptides.
  • the invention also provides a GAS40 polypeptide which can elicit antibodies that are cross-reactive with wild-type GAS40 polypeptides, but which does not contain 7-mers, 8-mers or 9-mers that are present in human proteins.
  • the invention also provides a GAS40 polypeptide which can elicit antibodies that are cross-reactive with wild-type GAS40 polypeptides and contains 9, 10, 15, 20, 25, 30, 31 , 32 or more copies of SEQ ID NO: 14.
  • compositions comprising the GAS40 polypeptides of the invention.
  • Such compositions may comprise additional polypeptides, such as other polypeptides from GAS (e.g. GAS57, GAS25 and/or GAS polysaccharide antigen).
  • These compositions may be for use as medicaments (e.g., as immunogenic compositions or vaccines).
  • Compositions of the invention are useful for preventing S. pyogenes infection, reducing the risk of S. pyogenes infection, and/or treating disease caused as a result of S.
  • pyogenes infection such as bacteremia, meningitis, puerperal fever, scarlet fever, erysipelas, pharyngitis, impetigo, necrotizing fasciitis, myositis or toxic shock syndrome.
  • compositions containing GAS antigens are preferably immunogenic compositions, and are more preferably vaccine compositions.
  • the pH of such compositions preferably is between 6 and 8, preferably about 7.
  • the pH can be maintained by the use of a buffer.
  • the composition can be sterile and/or pyrogen free.
  • the composition can be isotonic with respect to humans.
  • Vaccines according to the invention may be used either prophylactically or therapeutically, but will typically be prophylactic. Accordingly, the invention includes a method for the therapeutic or prophylactic treatment of a Streptococcus pyogenes infection.
  • the animal is preferably a mammal, most preferably a human.
  • the methods involve administering to the animal a therapeutic or prophylactic amount of the immunogenic compositions of the invention.
  • the invention also provides the immunogenic compositions of the invention for treating, reducing the risk or, and/or preventing a S. pyogenes infection.
  • compositions comprise GAS40 polypeptides of the invention and at least one or at least two different GAS antigens, as described below.
  • Other compositions of the invention comprise at least one nucleic acid molecule which encodes the two different antigens. See, e.g., Robinson & Torres (1997) Seminars in Immunology 9:271-283; Donnelly et al. (1997) Ann. Rev Immunol 15:617-648; Scott-Taylor & Dalgleish (2000) Expert Opin Investig Drugs 9:471-480; tendopoulos & Plebanski (2000) Curr Opin Mol Ther 2:441-447; Ilan (1999) Curr Opin Mol Ther 1 : 116-120; Dubensky et al.
  • the nucleic acid molecule is a DNA molecule, e.g., in the form of a plasmid.
  • GAS 57 is also referred to as 'Spy0416 (Ml)', 'SpyM3_0298' (M3), 'SpyM18_0464' (M18), and 'prtS'.
  • Spy0416 has been identified as a putative cell envelope proteinase. See WO02/34771 and US 2006/0258849.
  • GAS57 mutants useful in the invention include those with at an amino acid alteration (i.e., a substitution, deletion, or insertion) at one or more of amino acids D151, H279, particularly an amino acid sequence of SEQ ID NO: 120.
  • GAS25 (Spy0167, streptolysin, SLO) is a potent pore-forming toxin which induces host cell lysis and is described, inter alia, in WO 02/34771.
  • Mutant forms of GAS25 have at least 50% less hemolytic activity than wild-type Spy0167 (e.g., 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100%) relative to wild-type Spy0167 as determined by a hemolysis assay but are immunogenic and preferably confer protection against GAS lethal challenge in a mouse model.
  • Spy0167 mutants for use in compositions of the invention include those with an amino acid alteration (i.e., a substitution, deletion, or insertion) at one or more of amino acids P427 and W535 as exemplified in SEQ ID NO: 121.
  • GAS polysaccharide is a cell-wall polysaccharide present in all GAS strains. Antibody titers to PS correlate inversely with disease and colonization in children.
  • compositions of the invention comprise a GAS polysaccharide antigen.
  • S. pyogenes GAS carbohydrate typically features a branched structure with an L-rhamnopyranose (Rhap) backbone consisting of alternating alpha-(l ⁇ 2) and alpha-(l ⁇ 3) links and D-N-acetylglucosamine (GlcpNAc) residues beta-(l ⁇ 3)-connected to alternating rhamnose rings (Kreis et al., Int. J. Biol. Macromol. 17, 117-30, 1995).
  • GAS polysaccharide antigens useful in compositions of the invention have the formula:
  • the GAS polysaccharide antigen used according to the invention may be a substantially full-length GAS carbohydrate, as found in nature, or it may be shorter than the natural length. Full- length polysaccharides may be depolymerized to give shorter fragments for use with the invention, e.g., by hydrolysis in mild acid, by heating, by sizing chromatography, etc. However, it is preferred to use saccharides of substantially full-length. In particular, it is preferred to use saccharides with a molecular weight of about 10 kDa.
  • the saccharide may be chemically modified relative to the GAS carbohydrate as found in nature.
  • the saccharide may be de N acetylated (partially or fully), N propionated (partially or fully), etc.
  • the effect of de-acetylation etc., for example on immunogenicity can be assessed by routine assays.
  • the GAS polysaccharide antigen is conjugated to a carrier, such as the mutated diphtheria toxin CRM197.
  • the invention provides compositions comprising GAS57 and one or more GAS40 polypeptides of the invention. Sequences of exemplary GAS57 polypeptides are provided in SEQ ID NOs: 119 and 120. Such compositions may also comprise other GAS antigens, particularly GAS25 having SEQ ID NO: 121. In some embodiments, the GAS40 polypeptides of the invention (and optionally GAS57) can be incorporated into an immunogenic composition, including a vaccine composition. Such compositions can be used to raise antibodies in a mammal (e.g. a human). In these compositions, the GAS40 polypeptides of the invention (and optionally GAS57, if present) may act as immunogens and/or as antigens. The invention provides pharmaceutical compositions comprising a GAS40 polypeptide of the invention. Such pharmaceutical compositions may also comprise additional polypeptides such as GAS57.
  • the invention also provides processes for making a pharmaceutical composition involving combining a GAS40 polypeptide of the invention with a pharmaceutically acceptable carrier. Such processes may also comprise the step of adding additional polypeptides, such as GAS57.
  • the vaccine composition will comprise one or more pharmaceutically acceptable carriers, diluents and/or adjuvants.
  • Adjuvants which may be used in compositions of the invention include, but are not limited to:
  • mineral salts such as aluminium salts and calcium salts, including hydroxides (e.g. oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates) and sulphates, etc.;
  • oil-in-water emulsions such as squalene-water emulsions, including MF59 (5% Squalene, 0.5% Tween 80, and 0.5% Span 85, formulated into submicron particles using a microfluidizer), complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IF A);
  • squalene-water emulsions including MF59 (5% Squalene, 0.5% Tween 80, and 0.5% Span 85, formulated into submicron particles using a microfluidizer), complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IF A);
  • saponin formulations such as QS21 and ISCOMs
  • VLPs virus like particles
  • bacterial or microbial derivatives such as non toxic derivatives of enterobacterial lipopolysaccharide (LPS), Lipid A derivatives, immunostimulatory oligonucleotides, such as IC 31TM;
  • human immunomodulators including cytokines, such as interleukins (e.g. IL-1 , IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, interferons (e.g. interferon ⁇ ), macrophage colony stimulating factor, and tumor necrosis factor;
  • cytokines such as interleukins (e.g. IL-1 , IL-2, IL-4, IL-5, IL-6, IL-7, IL-12
  • interferons e.g. interferon ⁇
  • macrophage colony stimulating factor e.g. interferon ⁇
  • tumor necrosis factor tumor necrosis factor
  • bioadhesives and mucoadhesives such as chitosan and derivatives thereof, esterified hyaluronic acid microspheres or mucoadhesives, such as cross linked derivatives of poly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides and carboxymethylcellulos;
  • microparticles i.e. a particle of -lOOnm to ⁇ 150 ⁇ in diameter, more preferably ⁇ 200nm to
  • ⁇ 30 ⁇ in diameter, and most preferably ⁇ 500nm to - ⁇ in diameter) formed from materials that are biodegradable and non toxic e.g. a poly(a-hydroxy acid), a polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a polycaprolactone, etc.
  • materials that are biodegradable and non toxic e.g. a poly(a-hydroxy acid), a polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a polycaprolactone, etc.
  • muramyl peptides including N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N- acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), and N acetylmuramyl-L-alanyl-D- isoglutaminyl-L-alanine-2-( -2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine MTP- PE); and
  • the invention may also comprise combinations of one or more of the adjuvants identified above.
  • the following adjuvant compositions may be used in the invention: (1) a saponin and an oil-in-water emulsion; (2) a saponin (e.g. QS21) + a non-toxic LPS derivative (e.g. 3dMPL); (3) a saponin (e.g. QS21) + a non-toxic LPS derivative (e.g. 3dMPL) + a cholesterol; (4) a saponin (e.g.
  • QS21 3dMPL + IL 12 (optionally + a sterol); (5) combinations of 3dMPL with, for example, QS21 and/or oil-in-water emulsions; (6) SAF, containing 10% squalane, 0.4% Tween 80TM, 5% pluronic-block polymer L121, and thr-MDP, either micro fluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion.
  • SAF containing 10% squalane, 0.4% Tween 80TM, 5% pluronic-block polymer L121, and thr-MDP, either micro fluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion.
  • RibiTM adjuvant system (RAS), (Ribi Immunochem) containing 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL + CWS (DetoxTM); and (8) one or more mineral salts (such as an aluminum salt) + a non-toxic derivative of LPS (such as 3dMPL).
  • MPL monophosphorylipid A
  • TDM trehalose dimycolate
  • CWS cell wall skeleton
  • LPS such as 3dMPL
  • an aluminium hydroxide and/or aluminium phosphate adjuvant is useful, particularly in children, and antigens are generally adsorbed to these salts. Squalene-in-water emulsions are also preferred, particularly in the elderly.
  • Useful adjuvant combinations include combinations of Thl and Th2 adjuvants such as CpG & alum or resiquimod & alum.
  • a combination of aluminium phosphate and 3dMPL may be used.
  • Vaccines of the invention may be prophylactic (i.e. to prevent disease) or therapeutic (i.e. to reduce or eliminate the symptoms of a disease).
  • compositions of the invention when said compositions of the invention are administered to a large number of patients, immunity against GAS is produced in some or all of the patients and a reduced percentage of patients develop autoimmune sequelae of GAS infection, such as rheumatic fever or acute glomerulonephritis, compared to the percentage of patients that develop said autoimmune sequelae when wild type GAS40 is administered.
  • autoimmune sequelae of GAS infection such as rheumatic fever or acute glomerulonephritis
  • the invention also provides nucleic acids encoding the GAS40 polypeptides of the invention.
  • nucleic acid sequences shown in SEQ ID NOs: 1 , 3 and 5 encode the GAS40 polypeptides depicted in SEQ ID NOs: 2, 4 and 6.
  • the invention also provides nucleic acid comprising nucleotide sequences having sequence identity to such nucleotide sequences. Identity between sequences is preferably determined by the Smith- Waterman homology search algorithm. Such nucleic acids include those using alternative codons to encode the same amino acid.
  • the invention includes nucleic acid comprising sequences complementary to these sequences (e.g. for antisense or probing, or for use as primers).
  • Nucleic acids of the invention can be used in hybridization reactions (e.g. Northern or Southern blots, or in nucleic acid microarrays or 'gene chips') and amplification reactions (e.g. PCR, SDA, SSSR, LCR, TMA, NASBA, etc.) and other nucleic acid techniques.
  • hybridization reactions e.g. Northern or Southern blots, or in nucleic acid microarrays or 'gene chips'
  • amplification reactions e.g. PCR, SDA, SSSR, LCR, TMA, NASBA, etc.
  • Nucleic acid according to the invention can take various forms (e.g. single-stranded, double-stranded, vectors, labeled etc.). Nucleic acids of the invention may be circular or branched, but will generally be linear. Unless otherwise specified or required, any embodiment of the invention that utilizes a nucleic acid may utilize both the double-stranded form and each of two complementary single-stranded forms which make up the double-stranded form.
  • Nucleic acids of the invention are preferably provided in purified or substantially purified form i.e. substantially free from other nucleic acids (e.g. free from naturally-occurring nucleic acids), particularly from other E. coli or host cell nucleic acids, generally being at least about 50% pure (by weight), and usually at least about 90%) pure.
  • Nucleic acids of the invention may be prepared in many ways e.g. by chemical synthesis (e.g. phosphoramidite synthesis of DNA) in whole or in part, by digesting longer nucleic acids using nucleases (e.g. restriction enzymes), by joining shorter nucleic acids or nucleotides (e.g. using ligases or polymerases), from genomic or cDNA libraries, etc.
  • nucleases e.g. restriction enzymes
  • ligases or polymerases e.g. using ligases or polymerases
  • Nucleic acids of the invention may be part of a vector i. e. part of a nucleic acid construct designed for transduction/transfection of one or more cell types.
  • Vectors may be, for example, "cloning vectors” which are designed for isolation, propagation and replication of inserted nucleotides, "expression vectors” which are designed for expression of a nucleotide sequence in a host cell, "viral vectors” which is designed to result in the production of a recombinant virus or viruslike particle, or “shuttle vectors", which comprise the attributes of more than one type of vector.
  • Preferred vectors are plasmids, as mentioned above.
  • a "host cell” includes an individual cell or cell culture which can be or has been a recipient of exogenous nucleic acid.
  • Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change.
  • Host cells include cells transfected or infected in vivo or in vitro with nucleic acid of the invention.
  • nucleic acid is DNA
  • U in a RNA sequence
  • T in the DNA
  • RNA RNA
  • T in a DNA sequence
  • complement or “complementary” when used in relation to nucleic acids refers to Watson-Crick base pairing.
  • the complement of C is G
  • the complement of G is C
  • the complement of A is T (or U)
  • the complement of T is A.
  • bases such as I (the purine inosine) e.g. to complement pyrimidines (C or T).
  • Nucleic acids of the invention can be used, for example: to produce polypeptides; as hybridization probes for the detection of nucleic acid in biological samples; to generate additional copies of the nucleic acids; to generate ribozymes or antisense oligonucleotides; as single-stranded DNA primers or probes; or as triple-strand forming oligonucleotides.
  • the invention provides a process for producing nucleic acid of the invention, wherein the nucleic acid is synthesized in part or in whole using chemical means.
  • the invention provides vectors comprising nucleotide sequences of the invention (e.g. cloning or expression vectors) and host cells transformed with such vectors.
  • nucleotide sequences of the invention e.g. cloning or expression vectors
  • nucleic acids are preferably at least 1340 nucleotides in length ⁇ e.g. 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, or 2400 nucleotides or longer).
  • Cells of the invention are preferably at least 1340 nucleotides in length ⁇ e.g. 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, or 2400 nucleotides or longer).
  • the invention also provides a cell comprising a nucleic acid of the invention. Said cell may be stably transformed with said nucleic acid.
  • the invention also provides a cell that expresses a GAS40 polypeptide of the invention.
  • nucleic acid molecule which encodes one of the GAS antigens described herein can be used to produce that protein recombinantly.
  • Nucleic acid molecules encoding wild-type GAS antigens also can be isolated from the appropriate S. pyogenes bacterium using standard nucleic acid purification techniques or can be synthesized using an amplification technique, such as the polymerase chain reaction (PCR), or by using an automatic synthesizer. See Caruthers et al., Nucl. Acids Res. Symp. Ser. 215, 223, 1980; Horn et al., Nucl. Acids Res. Symp. Ser.
  • cDNA molecules can be made with standard molecular biology techniques, using mRNA as a template. cDNA molecules can thereafter be replicated using molecular biology techniques well known in the art. An amplification technique, such as PCR, can be used to obtain additional copies of polynucleotides of the invention, using either genomic DNA or cDNA as a template.
  • polynucleotides can be engineered using methods generally known in the art to alter antigen-encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the polypeptide or mRNA product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to engineer the nucleotide sequences.
  • site directed mutagenesis can be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.
  • Sequence modifications such as the addition of a purification tag sequence or codon optimization, can be used to facilitate expression.
  • the N-terminal leader sequence may be replaced with a sequence encoding for a tag protein such as polyhistidine ("HIS") or glutathione S-transferase ("GST").
  • tag proteins may be used to facilitate purification, detection, and stability of the expressed protein.
  • Codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half life which is longer than that of a transcript generated from the naturally occurring sequence.
  • a nucleic acid molecule which encodes a GAS antigen for use in the invention can be inserted into an expression vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • Methods which are well known to those skilled in the art can be used to construct expression vectors containing coding sequences and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.
  • Host cells for producing GAS antigens can be prokaryotic or eukaryotic.
  • E. coli is a preferred host cell, but other suitable hosts include Lactococcus lactis, Lactococcus cremoris, Bacillus subtilis, Vibrio cholerae, Salmonella typhi, Salmonella typhimurium, Neisseria lactamica, Neisseria cinerea, Mycobacteria (e.g., M. tuberculosis), yeasts, baculovirus, mammalian cells, etc.
  • a host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed polypeptide in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
  • Post translational processing which cleaves a "prepro" form of the polypeptide also can be used to facilitate correct insertion, folding and/or function.
  • Different host cells which have specific cellular machinery and characteristic mechanisms for post translational activities are available from the American Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, VA 20110-2209) and can be chosen to ensure the correct modification and processing of a foreign protein. See WO 01/98340.
  • Expression constructs can be introduced into host cells using well-established techniques which include, but are not limited to, transferrin-polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome-mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, "gene gun” methods, and DEAE- or calcium phosphate-mediated transfection.
  • Host cells transformed with expression vectors can be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
  • the protein produced by a transformed cell can be secreted or contained intracellularly depending on the nucleotide sequence and/or the expression vector used.
  • expression vectors can be designed to contain signal sequences which direct secretion of soluble antigens through a prokaryotic or eukaryotic cell membrane.
  • Signal export sequences can be included in a recombinantly produced GAS antigen so that the antigen can be purified from cell culture medium using known methods.
  • recombinantly produced GAS antigens can be isolated from engineered host cells and separated from other components in the cell, such as proteins, carbohydrates, or lipids, using methods well-known in the art. Such methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis.
  • a preparation of purified GAS antigens is at least 80% pure; preferably, the preparations are 90%, 95%, or 99% pure.
  • Purity of the preparations can be assessed by any means known in the art, such as SDS-polyacrylamide gel electrophoresis or RP-HPLC analysis. Where appropriate, mutant Spy0167 proteins can be solubilized, for example, with urea.
  • GAS antigens can be synthesized, for example, using solid phase techniques. See, e.g., Merrifield, J. Am. Chem. Soc. 85, 2149 54, 1963; Roberge et al., Science 269, 202 04, 1995. Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer). Optionally, fragments of GAS antigens can be separately synthesized and combined using chemical methods to produce a full-length molecule.
  • composition comprising
  • X may consist exclusively of X or may include something additional e.g. X + Y.
  • compositions which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention.
  • Consisting essentially of means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do no materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • the term “consisting of is generally taken to mean that the invention as claimed is limited to those elements specifically recited in the claim (and may include their equivalents, insofar as the doctrine of equivalents is applicable).
  • substantially does not exclude “completely” e.g. a composition which is “substantially free” from Y may be completely free from Y.
  • “substantially free” from Y can be understood as a composition containing not more than 5%> Y, not more than 4%> Y, not more than 3%> Y, not more than 2%> Y, not more than 1%> Y, or not more than 0.1%) Y.
  • the word “substantially” may be omitted from the definition of the invention.
  • mutant refers to a gene or gene product that displays modifications in sequence and/or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product.
  • modifications in sequence and/or functional properties i.e., altered characteristics
  • sequences not including at least two or more positions selected from the group consisting of T237, A241, A688, S692, A700, A703, A38, Q52, T66, A148, S158, A179, A182, T186, A196, Q203, Q218, K225, A544, T558, S562, K576, A586, T614, A618, A621, T665, A672, Q683, T693 may be excluded. All GenBank Accession numbers provided herein are incorporated by reference.
  • a process comprising a step of mixing two or more components does not require any specific order of mixing.
  • components can be mixed in any order. Where there are three components then two components can be combined with each other, and then the combination may be combined with the third component, etc.
  • animal (and particularly bovine) materials are used in the culture of cells, they should be obtained from sources that are free from transmissible spongiform encaphalopathies (TSEs), and in particular free from bovine spongiform encephalopathy (BSE). Overall, it is preferred to culture cells in the total absence of animal-derived materials.
  • TSEs transmissible spongiform encaphalopathies
  • BSE bovine spongiform encephalopathy
  • a compound is administered to the body as part of a composition then that compound may alternatively be replaced by a suitable prodrug.
  • the amino acid sequences of GAS40 was subdivided into overlapping 7-mers, 8-mers, 9-mers and 10-mers, with a sliding window of 1 amino acid residue. Each of these fragments was compared to a collection of human proteins available from ftp://ftp.ncbi.nih.gov/genomes/H_sapiens/protein/.
  • the first 9-mer has the sequence shown in QLTEELAAQ (SEQ ID NO: 8), and it is located at position 235-243 of GAS40 which is in the first coiled-coil domain. This 9-mer is also found in a 562-amino acid human protein known as "coiled-coil domain containing 81 " (GI: 47271449).
  • the second 9-mer has the sequence shown in SEQ ID NO: 9, and it is located at position 684-692 of GAS40, which is in the leucine zipper domain. This 9-mer is also found in a 408-amino acid human protein known as "UPF0492 protein C20orf94 " (GI:61102723).
  • the 8-mer has the sequence shown in SEQ ID NO: 10 and it is located at position 699-706, which bridges the C-terminal boundary of the leucine zipper domain. This 8-mer is also found in a 844-amino acid human protein known as "janus kinase and microtubule-interacting protein 3 " (GI: 157502225).
  • SEQ ID NO: 9 The two substitutions that were made to SEQ ID NO: 9 were A688S and S692T, resulting in the mutated 9-mer sequence: KQDLsKTTt (SEQ ID NO: 12).
  • SEQ ID NO: 10 The two substitutions that were made to SEQ ID NO: 10 were A700S and A703S, resulting in the mutated 8-mer sequence: EsLAsLQA (SEQ ID NO: 13).
  • Figure 1 shows a prediction of the secondary structure of GAS40 wild-type and mutant proteins. As shown, the prediction indicates that the integrity of the coil-coiled propensity profile and leucine zipper motif would remain unaltered by the above-mentioned six mutations.
  • GAS40 Three rare AGA codons encoding arginine (Arg 359, Arg 360 and Arg 849) were mutagenized to the common arginine CGT codon in order to optimize expression of GAS40.
  • a pET21b+ vector containing the codon-optimised GAS40 gene was used as template to create the GAS40 8-9-mers mutant.
  • nucleotide and amino acid sequences of the GAS40 8-9-mers mutant, with leader peptide and transmembrane domain sequences deleted, are shown in SEQ ID NOs: 3 and 4, respectively.
  • GAS40 'multi-mutant' protein containing idealized coiled-coils was produced by chemical synthesis. In order to optimise expression, GC-content was adjusted to prolong mRNA half- life. Codon usage was also adapted to the bias of Escherichia coli.
  • the nucleotide and amino acid sequences of GAS40 with idealized coiled-coils (multi-mutant), with the leader peptide and transmembrane domain sequences deleted, are shown in SEQ ID NOs: 5 and 6.
  • Recombinant GAS40 his-tagged mutant derivatives were expressed in BL21(DE3) E. coli cells and purified by affinity chromatography on immobilized metal ion affinity chromatography columns.
  • the frozen E. coli pellets were suspended in lysis buffer (10 ml B-PERTM (Pierce), 0.1 mM
  • the loaded resin was washed three times with wash buffer A and three times with wash buffer B (70 mM imidazole, 50 mM NaH 2 P0 4 , 300 mM NaCl, pH 8.0). Proteins were eluted with elution buffer (250 mM imidazole, 50 mM NaH 2 P0 4 , 300 mM NaCl, pH 8.0) in Eppendorf tubes containing 1 mM DTT. Total elution proteins were quantified with Bradford reagent, extensively dialyzed against PBS and then analysed by SDS-polyacrylamide gel electrophoresis (see Figure 7) using 4-12% CriterionTM pre-cast gels (Bio-Rad).
  • mice were immunized intraperitoneally three times (day 0, day 21 and day 35) with either his-tagged wild-type GAS40, GAS40 8-9-mers mutant or GAS40 multi-mutant, which were each combined with an alum adjuvant (20 ⁇ g protein in 2 mg/ml aluminium hydroxide). Mice immunized with adjuvant alone were used as negative controls. Two weeks after the third immunization, blood samples were taken. A few days afterwards, immunized mice were challenged intranasally with 10 8 cfu (50 ⁇ ) of the Ml 3348 GAS strains. Mice survival was monitored for a 10-14 day period.
  • the non-GLP human tissue cross-reactivity study was optimized and run at Charles River Laboratories/ PAI in Frederick, MD.
  • a panel of selected human tissues was screened because of their association with specific post-GAS infection pathology: cartilage, brain (cerebrum), heart, heart valve, kidney and skeletal muscle. Integrity of the tissue was confirmed by staining with beta 2 microglobulin. Pooled sera from mice with the highest titers were optimized for use at dilutions of 1: 10,000 and 1 :40,000. Spot control slides of the individual antigens were incubated with the pooled anti-sera from the respective group of animals in order to confirm antigen-specific responses.

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Abstract

L'invention concerne des antigènes mutants dérivés de Streptococcus pyogenes spy0269 et leur utilisation dans des compositions immunogènes et des compositions vaccinales.
PCT/EP2015/059772 2014-05-07 2015-05-05 Formes mutantes de spy0269 Ceased WO2015169773A2 (fr)

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JP2020117470A (ja) * 2019-01-25 2020-08-06 公益財団法人ヒューマンサイエンス振興財団 抗体並びに劇症型溶血性レンサ球菌感染症を予防及び／又は治療するための薬剤
WO2022256310A1 (fr) * 2021-06-01 2022-12-08 The Regents Of The University Of California Antigène vaccinal protecteur contre une infection streptococcique

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EP2897635A1 (fr) * 2012-09-18 2015-07-29 Novartis AG Vésicules de membrane externe

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JP2020117470A (ja) * 2019-01-25 2020-08-06 公益財団法人ヒューマンサイエンス振興財団 抗体並びに劇症型溶血性レンサ球菌感染症を予防及び／又は治療するための薬剤
WO2022256310A1 (fr) * 2021-06-01 2022-12-08 The Regents Of The University Of California Antigène vaccinal protecteur contre une infection streptococcique

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