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    • 专利摘要:
    u L_ •S1 u_ The invention provides mutated antigens derived from Streptococcus pyogenes spy0269 and their use in immunogenic compositions and vaccine compositions. u L_ • S1 u_
    公开号:BE1022553B1
    申请号:E2015/5285
    申请日:2015-05-05
    公开日:2016-05-31
    发明作者:Romina D'AURIZIO；Marilena Gallotta；Guido Grandi；Y Ros Immaculada Margarit；Mariarosa Mora
    申请人:Glaxosmithkline Biologicals Sa；
    IPC主号:
    专利说明:

    MUTANTS OF SPY0269
    TECHNICAL FIELD The invention relates to the fields of immunology and vaccinology. In particular, it relates to mutated antigens derived from Streptococcus pyogenes Spy0269 and their use in immunogenic compositions and vaccine compositions.
    State of the art context
    Group A Streptococcus ("GAS", S. pyogenes) is a shell-shaped bacterium that does not form Gram-positive spores and generally appears in chains or pairs of cells. GAS is one of the most common pathogens of humans. It is estimated that between 5 and 15% of normal individuals harbor the bacteria, usually in the respiratory system, without showing any signs of the disease. When the defenses of the host are compromised, or when the body is able to exert its virulence, or when it is introduced into tissues or vulnerable hosts, however, an acute infection appears. Acute Streptococcus pyogenes can take the form of pharyngitis, scarlet fever (rash), impetigo, cellulite, or erysipelas. Invasive infections can produce necrotizing fasciitis, myositis, and streptococcal toxic shock syndrome. Patients may also develop immune-mediated sequelae such as rheumatic fever and acute glomerulonephritis (Patterson 1996). Although S. pyogenes can be treated using antibiotics, a prophylactic vaccine to prevent the onset of the disease is desired. Efforts to develop such a vaccine have continued for several decades. While various approaches to GAS vaccine have been suggested and some approaches are currently in clinical trials, to date, there is no publicly available anti-GAS vaccine. GAS40, also known as "Spy0269" (M1), "SpyM3_0197" (M3), "SpyM18_0256" (M18) and "prgA" (Uniprot Accession No. Q9A1H3), is a surface exclusion protein putative that has a 130 amino acid region with similarity to the EzrA protein, which interacts with the FtsZ cell division protein. GAS40 routinely provides protection in the animal model of systemic immunization and stimulation ("challenge") and induction of bactericidal antibodies. Therefore, GAS40 is a potential candidate for the production of a vaccine against a GAS. However, there have been some concerns in the past that M-protein-based GAS vaccines contain antigenic determinants that have been determined to be cross-reactive with cardiac tissue, potentially resulting in a significant reaction in the body. rheumatic heart disease.
    Molecular mimicry is defined as the theoretical possibility that sequence similarities between foreign peptides and auto-peptides are sufficient to cause cross-activation of autoreactive T or B cells by peptides derived from pathogens. Upon activation of B or T lymphocytes, it is believed that these T or B cells specific for "peptide mimetics" may exhibit cross-reactivity with auto-epitopes, thus leading to tissue pathology (autoimmunity). Molecular mimicry is a phenomenon that has recently been discovered to be one of several ways in which autoimmunity can be elicited. Therefore, candidate vaccines should be chosen that do not trigger the disease they are supposed to prevent.
    While there is no evidence that protein antigens like GAS40 include antigenic determinants that could be problematic, the past S®2 fears about molecular mimicry and anti-GAS vaccines per se may hinder the adoption and use of anti-GAS vaccines.
    In order to reduce these fears and the possibility that GAS40 could produce autoimmune sequelae of GAS infection by molecular mimicry, the inventors have produced GAS40 polypeptides that are less similar to human proteins than wild-type GAS40 proteins. Summary of the invention
    The inventors have developed mutants of GAS40 polypeptides that elicit antibodies that exhibit cross-reactivity with wild-type GAS40 but have reduced sequence identity with the amino acid sequences present in human proteins. In particular, human proteins include protein 81 containing supercoiled domains, UPF0492 protein C20orf94, Janus kinase and protein 3 interacting with microtubules, myosin or tropomyosin.
    Thus, in a first aspect, there is provided a purified recombinant streptococcal GAS40 antigen which comprises an epitope that elicits opsonic antibodies against Streptococcus Group A, the recombinant streptococcal GAS40 antigen comprising at least two substitutions in any of the 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 the level of 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 IBP amino acid positions are numbered according to SEQ ID NO: 2.
    In some embodiments, the purified recombinant streptococcal GAS40 antigen comprises substitutions at amino acid positions T237, A241, A688, S692, A700 and A703. For example, the purified recombinant streptococcal GAS40 antigen comprises the substitutions T237S, A241V, A688S, S692T, A700S and A703S. The recombinant streptococcal GAS40 polypeptide may comprise a Lysin deletion 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. For example, 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 object of the present invention is to reduce any theoretical risk of serological cross-reactivity of GAS40 antigens with mammalian tissue antigens. In particular, the mammal is a human being. In particular, the recombinant streptococcal GAS40 antigen does not contain 7-mer, 8-mer or 9-mer which are present in human proteins. Even more particularly, 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). Even more particularly, the polypeptide
    Streptococcal GAS40 recombinant does not include 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). Even more particularly, the amino acid sequences of 7-, 8- or 9-mer of the recombinant streptococcal GAS40 polypeptide positions 235 to 243 and / or positions 684 to 692 and / or 699 to 707, numbered according to SEQ ID NO : 2, have a 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).
    In particular, the recombinant streptococcal GAS40 polypeptide may share at least 92% identity with any one of SEQ ID NO: 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 NO: 4, 6, 7, 16, 17, 18, 24, 25 and 26.
    In some embodiments, there is provided a fragment of a recombinant GAS40 streptococcal polypeptide according to the first aspect. For example, a fragment that shares at least 97% identity with any one of SEQ ID NO: 20, 21, 22, 28, 29, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 44, 45, 46, 48, 49, 50, 52, 53, 54, 56, 57, 58, 60, 61, 62, 64, 65, 66, 67, 68, 69, 70, 72, 73, 74, 76, 77, 78, 80, 81, 82, 84, 85, 86, 88, 89, 90, 92, 93, 94, 96, 97, 98, 100, 101, 102, 104, 105,
    106, 108, 109, 110, 112, 113, 114, 116, 117 and 118. Further examples of fragments include polypeptide sequences comprising or consisting of any 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, 116, 7, 18 , 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114 and 118 The term "fragment" as used herein refers to a polypeptide which has an amino-terminal and / or carboxy-terminal deletion as compared to the full-length protein, but where the remaining amino acid sequence is identical to the corresponding positions of the full-length protein. The fragments generally have a length greater than 200 amino acids, in particular a length which is not greater than 850, 849, 848, 847, 846, 845 amino acids, more particularly a length which is not greater than 830. , 827, 826, 825, 824, 823, 822, 822, 821, 820 amino acids, still more particularly a length of not more than 460, 459, 458, 457, 456, 455, 454, 453, 452 , 451, 450 amino acids, including ranges between these values. For example, a length of 200 to 847 amino acids, a length of 200 to 823 amino acids, a length of 200 to 452 amino acids, a length of 450 to 850 amino acids, a length of 450 to 825 amino acids. For the avoidance of doubt, the fragments of the present invention (numbered according to SEQ ID NO: 2) will include substitutions corresponding to those described above where these amino acid positions are present in the fragment.
    In a second aspect of the invention, there is provided an immunogenic composition comprising one or more GAS40 polypeptides according to the first aspect. Generally, such immunogenic compositions will comprise one or more additional GAS polypeptides such as, by way of non-limiting example, GAS57, GAS25 or mutants thereof. In particular, the immunogenic compositions of the invention will comprise an immunologically effective amount of the GAS40 polypeptide (s). Even more particularly, the immunogenic compositions of the invention are vaccines. Such compositions may include an adjuvant such as alum or MF59. Particular immunogenic 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 CRMi97 conjugated to at least one group A carbohydrate.
    In a third aspect of the invention, there is provided a nucleic acid encoding the GAS40 polypeptide according to the first aspect.
    In a fourth aspect of the invention there is provided a cell comprising the nucleic acid according to the third aspect.
    In a fifth aspect, there is provided a method of reducing the amino acid sequence similarity of a wild type GAS40 polypeptide with one or more human proteins, wherein said method 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 into the sequence of said wild-type GAS40. In particular, said mutations are introduced into short amino acid sequences which are common to wild-type GAS40 and human proteins. In particular, human proteins are one or more of the protein 81 containing supercoiled domains, UPF0492 protein C20orf94, Janus kinase and protein 3 interacting with microtubules, myosin or tropomyosin. Even more particularly, mutations are introduced in the supercoiled regions of GAS40 where said mutations "idealize" the supercoiled regions.
    Brief description of the drawings
    Figure 1 illustrates a diagrammatic representation of the wild-type GAS40 structure.
    Figure 2 illustrates a prediction of the secondary structure of SEQ ID NO: 2 (the wild-type GAS40 polypeptide). The prediction indicates that the integrity of the overcoiling propensity profile and the leucine zipper pattern will remain unchanged.
    Figure 3 illustrates a prediction of the secondary structure of SEQ ID NO: 4 (the mutant GAS40 polypeptide of 8 to 9-mer). The prediction indicates that the integrity of the overcoiling propensity profile and the leucine zipper pattern will remain unchanged.
    Figure 4 illustrates the identification of supercoiled regions in GAS40. These regions have been highlighted with a color scale from red to blue where red represents hydrophobic residues.
    Figure 5 illustrates the identification of supercoiled regions in GAS40. These regions have been highlighted with a color scale from red to blue where red represents hydrophobic residues.
    Figure 6 illustrates the identification of supercoiled regions in GAS40. These regions have been highlighted with a color scale from red to blue where red represents hydrophobic residues.
    Description of the invention
    Polypeptides of the Invention The invention provides a method of reducing the amino acid sequence similarity of a GAS40 polypeptide with one or more human proteins, which can be achieved by introducing mutations into the sequence of a wild-type GAS40. The purpose of this reduction of amino acid sequence similarity of the GAS40 polypeptide with a (Pii2 several human proteins is to reduce the theoretical risk of triggering antibodies that could be cross-reactive with human proteins. to reduce or eliminate the risk of autoimmune sequelae of GAS infection, said wild-type GAS40 polypeptide may comprise or comprise the sequence represented by 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 it may have sequence identity with 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% Generally, such proteins avoid the induction of antibodies that could cross-reactivity with human proteins which include protein 81 containing supercoiled domains (GI: 47271449), UPF0492 C20orf94 protein (GI: 61102723), Janus kinase and microtubule-interacting protein 3 (GI: 157502225), myosin or tropomyosin.
    In some embodiments, said mutations include single amino acid insertions, single amino acid deletions, and / or single amino acid substitutions. In some embodiments, 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.
    In a preferred embodiment of the invention, said mutations can be introduced in short sequences of amino acids, for example, 7-mer, 8-mer or 9-mer, which are common to GAS40 and human proteins. Examples of
    such short sequences of amino acids include QLTEELAAQ2 (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 proteins human, protein 81 containing supercoiled domains, UPF0492 protein C20orf94, and Janus kinase and protein 3 interacting with microtubules, respectively.
    In some embodiments of the invention, the mutations that are introduced have no significant effect on the secondary structure of GAS40. In other words, the secondary structure of the GAS40 polypeptides of the invention is preferably substantially the same as that of the wild-type GAS40 polypeptides. The secondary structure of polypeptides, such as GAS40, can be estimated by computer methods of ab initio prediction or circular dichroism, analytical ultracentrifugation, sedimentation equilibrium analyzes, X-ray crystallography, NMR spectroscopy, and the like. As illustrated in Figure 1, the wild-type GAS40 comprises a leader peptide at its N-terminus, two supercoiled 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 supercoiled region and between amino acid residues 673 and 701 in SEQ ID NO: 2) and a transmembrane domain (located between residues of amino acids 849 and 866 in SEQ ID NO: 2). The locations of these domains and regions in any GAS40 polypeptide can be predicted based on a pairwise alignment of a given sequence with SEQ ID NO: 2, for example by aligning the amino acid sequence of a GAS40 polypeptide. with SEQ ID NO: 2 and identifying the sequences that align with the respective domains / regions of SEQ ID NO: 2.
    Preferably, all the mutations that are introduced have no significant effect on the two supercoiled regions of alpha helix and leucine zipper. For example, deiiS2 substitutions may be introduced into each of SEQ ID NO: 8, SEQ ID NO: 9 and / or SEQ ID NO: 10. Preferably, said mutations include T237S, A241V, A688S, S692T, A700S and / or A703S with respect to the amino acid sequence of SEQ ID NO: 2. The locations of these substitutions in a GAS40 polypeptide of length and / or sequence different from SEQ ID NO: 2 are identified based on a pairwise alignment of a given sequence with SEQ ID NO: 2, for example by aligning the amino acid sequence of a GAS40 polypeptide of interest with SEQ ID NO: 2 and identifying the amino acids that correspond to T237 , A241, A688, S692, A700 and A703. SEQ ID NO: 4 is an example of a GAS40 polypeptide that includes all of these six substitutions (T237S, A241V, A688S, S692T, A700S and A703S). The invention also provides GAS40 polypeptides which have been produced by a method of the invention. In some embodiments, the invention provides a GAS40 polypeptide that contains a reduced number of 9-mer, 8-mer and / or 7-mer that are present in all human polypeptides. For example, the invention also provides a GAS40 polypeptide lacking one or more of the sequences: SEQ ID NO: 8, SEQ ID NO: 9 and / or SEQ ID NO: 10. In some embodiments, the GAS40 polypeptide has a secondary structure that is substantially the same as the wild type GAS40. Preferably, the secondary structure of the two supercoiled regions of alpha helix and leucine zipper that is localized within the C-terminal supercoiled region is substantially the same as wild-type GAS40. Examples of GAS40 polypeptide lacking one or more of the sequences: SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10 include 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 and 116. In some embodiments, the invention provides a mutant protein that 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 of the SEQ's 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.
    Surcoiling irregularities have recently been demonstrated in the M1 protein (Science 319 (5868): 1405-8). Similar structural irregularities occur in myosin and tropomyosin, providing a possible explanation for cross-reactive M protein-associated patterns in autoimmune sequelae of GAS infection. When substitutions are made within the supercoiled region of M1 resulting in optimal central residues for the formation and stability of dimer parallel supercoils ("idealization of the sequence"), fibrinogen binding, proinflammatory effects, and cross-reactivity of the antibodies were decreased. A similar "idealization" of the super-coils has been developed for the GAS40.
    A supercoiling is a structural motif in proteins, in which 2 to 7 alpha helices are wound together as the strands of a string (dimers and trimers are the most common types). The supercoils usually contain a repeating unit, hxxhcxc (SEQ ID NO: 14), hydrophobic (h) and charged (c) amino acid residues, which are 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 occupied by isoleucine, leucine or valine. However, supercoilings of tropomyosin and myosin deviate from the canonical structure of supercoils by subtle means, including insertions within heptads and charged residues, and alanine residues at heptatic positions. and D. Such sequences destabilizing the super-windings modify BE2 local conformation and energetics without interrupting the supercoiling. It is believed that the cross-reactivity of GAS M proteins attributable to molecular mimicry may result from the structural similarities between the supercoils of these proteins and those of myosin and tropomyosin. It has been found that GAS40 contains destabilizing sequences of similar supercoils. The invention provides a GAS40 polypeptide that contains mutations in the supercoiled regions of GAS40, preferably to reduce structural irregularities in the supercoiled regions, thereby "idealizing" said supercoiled regions and rendering the mutant protein potentially less structurally similar to myosin or tropomyosin. Preferably, hydrophilic residues at positions 1 and 4 of the supercoiling heptads are substituted with a hydrophobic residue such as leucine, and / or lysine 208 is deleted to correct a heptad frame shift. SEQ ID NO: 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 that include these features. In some embodiments, the invention provides a mutant protein that 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 of the SEQ IDs NO: 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 or 117. The invention also provides a GAS40 polypeptide with 9 or more copies of SEQ ID NO: 14, wherein said polypeptide optionally comprises less than 32 imperfect heptad repeats. Preferably, 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. In one embodiment, the invention provides a GAS40 polypeptide. wherein said polypeptide has n copies or more of SEQ ID NO: 14 and shares at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO: 2.
    In some embodiments, the GAS40 polypeptide contains any combination of the mutations presented above. For example, the invention provides a GAS40 polypeptide containing both: • a reduced number of 9-mer, 8-mer and / or 7-mer that are present in all human polypeptides (for example, lacking one or more several of the sequences: SEQ ID NO: 8, SEQ ID NO: 9 and / or SEQ ID NO: 10); and one or more "idealized" supercoiled regions in which the hydrophilic residues in positions 1 and 4 of the heptads forming the supercoils are substituted with a hydrophobic residue such as leucine, and / or the lysine 208 is deleted in order to correct an offset of heptad frame.
    Examples of such GAS40 polypeptides (i.e., those containing both: a reduced number of 9-mer, 8-mer and / or 7-mer that are present in all human polypeptides and one or more "idealized" supercoiled regions) include: SEQ ID NO: 7, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118. In one embodiment, the invention provides a polypeptide 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% identity with any of SEQ ID NO: 7, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66 , 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118.
    Preferably, the GAS40 polypeptides of the inventive2 retain the immunogenicity of the wild-type GAS40 polypeptides, for example, the GAS40 polypeptides of the invention will preferably elicit antibodies that are capable of binding wild-type GAS40 polypeptides. The GAS40 polypeptides of the invention preferably retain substantially the same level of immunogenicity as the wild-type GAS40 polypeptides. For example, the geometric means of the antibody titers resulting from the GAS40 polypeptides of the invention are not significantly different from those resulting from the wild-type GAS40 polypeptides when estimated by two t-tests for samples (where the differences are considered as significant if the P-value obtained by a one-sided analysis is <0.05, <0.01, or <0.001) when other variables (such as dose, route of administration, GAS40 strain, population group , etc.) are controlled. Methods for 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 have a length of at least: 177, 203, 250, 300, 400, 450, 460, 465, 470, 471, 500, 550, 600, 650, 700, 750 or 800 amino acids. Examples of such fragments include those cited in SEQ ID NO: 107, 111 and 115. The invention provides a method of reducing the risk of autoimmunogenicity of a GAS40 polypeptide, wherein said method comprises the following steps: the identification of short amino acid sequences (7-mer, 8-mer or 9-mer) that are common to human proteins and GAS40; and b) introducing mutations into said short amino acid sequences that are common to human proteins and GAS40. The invention also provides a method of reducing the risk of autoimmunogenicity of a GAS40 polypeptide, wherein said method comprises the following steps: a) identification of 2 supercoiled regions in GAS40; and b) the introduction of mutations to "idealize" the supercoiled structure. Step b) may involve the introduction of one or more of the following mutations: the substitution of one or more of the hydrophilic residues in positions 1 and 4 of the heptads supercoiled with a hydrophobic residue, preferably leucine; and deleting an amino acid residue, preferably lysine 208, to correct a heptad frame shift. The invention also provides a method for reducing the risk of autoimmunogenicity of a GAS40 polypeptide, wherein said method comprises the steps of: a) identifying short amino acid sequences that are common to human proteins and GAS40; b) introducing mutations into said short amino acid sequences that are common to human proteins and GAS40; c) the identification of overstretched regions in the GAS40; and d) the introduction of mutations to "idealize" the supercoiled structure. Step d) may involve the introduction of one or more of the following mutations: the substitution of one or more of the hydrophilic residues in positions 1 and 4 of the heptads supercoiled with a hydrophobic residue, preferably leucine; and deleting an amino acid residue, preferably lysine 208, to correct a heptad frame shift.
    Preferably, the 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 that can elicit antibodies that exhibit cross-reactivity with wild-type GAS40 polypeptides, but that do not elicit antibodies that cross-react with human polypeptides. The invention also provides a GAS40 polypeptide that can elicit antibodies that exhibit cross-reactivity with wild-type GAS40 polypeptides, but do not contain 7-merBE2 8-mer or 9-mer that are present in human proteins. The invention also provides a GAS40 polypeptide that can elicit antibodies that cross-react with wild-type GAS40 polypeptides and contains 9, 10, 15, 20, 25, 30, 31, 32 or more copies of SEQ ID NO: 14.
    Compositions The invention provides compositions comprising the GAS40 polypeptides of the invention. Such compositions may include 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). The compositions of the invention are useful for preventing S. pyogenes infection, reducing the risk of S. pyogenes infection, and / or treating a disease caused as a result of infection. S. pyogenes, such as bacteremia, meningitis, puerperal fever, scarlet fever, erysipelas, pharyngitis, impetigo, necrotizing fasciitis, myositis or toxic shock syndrome.
    The compositions containing GAS antigens are preferably immunogenic compositions, and more preferably are vaccine compositions. The pH of such compositions is preferably between 6 and 8, preferably about 7. The pH can be maintained by the use of a buffer. The composition may be sterile and / or pyrogen-free. The composition can be isotonic with respect to humans.
    The vaccines according to the invention can be used either prophylactically or therapeutically, but they will generally be prophylactic. Accordingly, the invention comprises a method of therapeutic or prophylactic treatment of Streptococcus pyogenic infection. The animal is preferably a mammal, most preferably a human being. 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 and / or preventing S. pyogenes infection.
    Some 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 that encodes the two different antigens. See, for example, Robinson &amp; Torres (1997) Seminars in Immunology 9: 2712-83; Donnelly et al. (1997) Ann. Rev Immunol 15: 617-648;
    Scott-Taylor &amp; Dalgleish (2000) Expert Opin Investig Drugs 9: 471-480; Apostolopoulos &amp; Plebanski (2000) Curr Opin Mol Ther 2: 441-447; Ilan (1999) Curr Opin Mol Ther 1: 116-120; Dubensky et al. (2000) Mol Med 6: 723-732; Robinson &amp; Pertmer (2000) Adv Virus Res 55: 1-74; Donnelly et al. (2000) Am J Respir Crit Care Med 162 (4 Pt 2): S190-193; Davis (1999) Mt. Sinai J. Med. 66: 84-90. Generally, the nucleic acid molecule is a DNA molecule, for example, in the form of a plasmid. GAS57 is also called 'Spy0416 (M1)', 'SpyM3_0298' (M3), 'SpyM18_0464' (M18), and 'prtS'. Spy0416 has been identified as a proteinase of the putative cell envelope. See WO 02/34771 and US 2006/0258849. Mutants of GAS57 useful in the invention include those with at least one amino acid modification (i.e., substitution, deletion, or insertion) at one or more of the amino acids D151, H279, particularly an amino acid sequence of SEQ ID NO: 120. GAS25 (Spy0167, streptolysin, SLO) is a potent pore-forming toxin that induces lysis of host cells and is described, inter alia, in WO 02/34771. LBE2 mutant forms of GAS25 exhibit 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%) compared to wild-type Spy0167 as determined by hemolysis assay, but they are immunogenic and preferably confer protection against lethal challenge by a GAS in a model. mouse. Mutants of Spy0167 for use in compositions of the invention include those with amino acid modification (i.e., substitution, deletion, or insertion) at one or more amino acids P427 and W535 as exemplified in SEQ ID NO: 121.
    The GAS polysaccharide (PS) is a cell wall polysaccharide present in all GAS strains. Antibody titers against PS are inversely correlated with disease and colonization in children. In some embodiments, the compositions of the invention comprise a polysaccharide antigen of GAS. The carbohydrate of GAS S. pyogenes generally displays a branched structure with an L-rhamnopyranose (Rhap) backbone consisting of alternating alpha- (1 ^ 2) and alpha- (1 ^ 3) bonds and residues of DN-acetylglucosamine ( GlcpNAc) linked in beta- (1 ^ 3) to alternating rhamnose cycles (Kreis et al., Int J Biol Macromol 17, 117-30, 1995). The polysaccharide antigens of GAS useful in the compositions of the invention have the formula:
    [(2) -α-Rhap- (1-3) -L-Rhap- (lh-R) 1-β-D-GlcpNAc wherein R is L-Rhamnose or D-GlcpMS2 terminal reductant and n is a number from about 3 to about 30. The polysaccharide antigen of GAS used according to the invention can be a substantially full length GAS carbohydrate, as it is found in nature, or it can be shorter than the natural length. full length can be depolymerized to give shorter fragments for use in which the invention is made, for example, by hydrolysis in mild acid, by heating, by exclusion chromatography, etc. However, it is preferable to use In particular, it is preferable to use saccharides with a molecular weight of about 10 kDa. The molecular weights can be measured by gel filtration against dextran standards. The saccharide can be chemically modified with respect to the GAS carbohydrate as found in nature. For example, the saccharide can be de-N-acetylated (partially or totally), N-propionated (partially or totally), etc. The effect of deacetylation etc., for example on immunogenicity, can be estimated by routine tests. In some embodiments, the GAS polysaccharide antigen may be conjugated to a carrier, such as CRM197 mutated diphtheria toxin.
    In one embodiment, the invention provides compositions comprising GAS57 and one or more GAS40 polypeptides of the invention. The sequences of the examples of GAS57 polypeptides are provided by SEQ ID NO: 119 and 120. Such compositions may also include other GAS antigens, particularly GAS25 having the sequence SEQ ID NO: 121.
    In some embodiments, the GAS40 polypeptides of the invention (and optionally GAS57) may be incorporated into an immunogenic composition, including a vaccine composition. Such compositions may be used to elicit antibodies in a mammal (eg, a human). In these compositions, the GAS40 polypeptides of the invention (and optionally GAS57, if present) can act as immunogens and / or as antigens. The invention provides pharmaceutical compositions comprising a GAS40 polypeptide of the invention. Such pharmaceutical compositions may also include additional polypeptides such as GAS57. The invention also provides methods of making a pharmaceutical composition involving the combination of a GAS40 polypeptide of the invention with a pharmaceutically acceptable carrier. Such methods may also include the step of adding additional polypeptides, such as GAS57.
    In some embodiments, the vaccine composition will comprise one or more pharmaceutically acceptable carriers, diluents and / or adjuvants. Adjuvants that can be used in compositions of the invention include, but are not limited to: • inorganic salts, such as aluminum salts and calcium salts, including hydroxides (e.g. oxyhydroxides), phosphates (eg, hydroxyphosphates, orthophosphates) and sulfates, etc. ; Oil-in-water emulsions, such as squalene emulsions in water, including MF59 (5% squalene, 0.5% Tween 80, and 0.5% Span 85, formulated as submicron particles) using a microfluidizer), Freund's Complete Adjuvant (CFA) and Freund's Incomplete Adjuvant (IFA); • saponin formulations such as QS21 and ISCOMs; • virosomes and pseudoviral particles (PPV); Bacterial or microbial derivatives, such as non-toxic derivatives of enterobacterial lipopolysaccharide (LPS), lipid A derivatives, immunostimulatory oligonucleotide 2, such as IC 31 ™; Human immunomodulators, including cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, interferons (by interferon γ), macrophage colony stimulating factor, and tumor necrosis factor, • bioadhesives and mucoadhesives, such as chitosan and its derivatives, microspheres of esterified hyaluronic acid or mucoadhesives, such as crosslinked derivatives of poly (acrylic acid), polyvinyl alcohol, polyvinylpyrrolidone, polysaccharides and carboxymethylcellulose • microparticles (that is, a particle with a diameter of ~ 100 nm to ~ 150 μm, more preferably a diameter of ~ 200 nm to ~ 30 μm, and most preferably a diameter of ~ 500 nm to ~ 10 μm) formed from materials that are biodegradable and non-toxic (e.g. , a poly (α-hydroxy acid), a polyhydroxybutyri that, a polyorthoester, a polyanhydride, a polycaprolactone, etc.); • liposomes; Polyoxyethylene ethers and polyoxyethylene esters; • PCPP formulations; 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- (1'-2'-dipalmitoyl-sn-glycero-3-hydroxy-phosphoryloxy) ethylamine MTP-PE); and imidazoquinolone compounds, including Imiquamod and its counterparts (eg, Resiquimod 3M). The invention may also include combinations of H 2 or more of the adjuvants identified above. For example, the following adjuvant compositions may be used in the invention: (1) a saponin and an oil-in-water emulsion; (2) a saponin (eg, QS21) + a nontoxic derivative of LPS (e.g., 3dMPL); (3) a saponin (eg, QS21) + a nontoxic derivative of LPS (e.g., 3dMPL) + cholesterol; (4) a saponin (eg, 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 80 ™, 5% Pluronic L121 block polymer, and thr-MDP, either microfluidized to a submicron emulsion or vortexed to generate an emulsion with a size larger particle; (7) RibiTM adjuvant system (RAS), (Ribi Immunochem) containing 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components of the monophosphoryl lipid A (MPL) group trehalose dimycolate (TDM) and cell wall skeleton (CWS), preferably MPL + CWS (Detox ™); and (8) one or more inorganic salts (such as an aluminum salt) + a nontoxic derivative of LPS (such as 3dMPL). The use of an adjuvant of aluminum hydroxide and / or aluminum phosphate is useful, particularly in children, and the antigens are generally adsorbed on these salts. Squalene emulsions in water are also preferred, particularly in the elderly. Useful adjuvant combinations include combinations of Th1 and Th2 adjuvants such as CpG and alum or resiquimod and alum. A combination of aluminum phosphate and 3dMPL can be used.
    The vaccines of the invention may be prophylactic (i.e., to prevent disease) or therapeutic (i.e., to reduce or eliminate symptoms of disease).
    Preferably, when said compositions of the invention are administered to a large number of patients, the immunity against GAS is produced in some or all of the patients and a small percentage of patients develop autoimmune sequelae of the infection. by a GAS, such as rheumatic fever or acute glomerulonephritis, compared to the percentage of patients who develop said autoimmune sequelae when wild-type GAS40 is administered.
    Nucleic Acids The invention also provides nucleic acids encoding the GAS40 polypeptides of the invention. For example, the nucleic acid sequences represented by SEQ ID NO: 1, 3 and 5 encode the GAS40 polypeptides represented by SEQ ID NO: 2, 4 and 6. The invention also provides a nucleic acid comprising nucleotide sequences having sequence identity with such nucleotide sequences. The identity between the sequences is preferably determined by the Smith-Waterman homology search algorithm. Such nucleic acids include those using codon alternatives to encode the same amino acid. The invention comprises a nucleic acid comprising sequences complementary to these sequences (for example, for antisense or probing, or for use as primers).
    The 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 (by example, PCR, SDA, SSSR, LCR, TMA, NASBA, etc.) and other techniques with nucleic acids.
    A nucleic acid according to the invention can take various forms (for example, single-stranded, double-stranded, vectors, labeled, etc.). The nucleic acids of the invention may be circular or branched, but will generally be linear. Unless otherwise indicated or required, any embodiment of the invention that uses a nucleic acid may use both the double-stranded form and each of the two complementary single-stranded forms that constitute the double-stranded form.
    The nucleic acids of the invention are preferably provided in a purified or substantially purified form, i.e., substantially free of other nucleic acids (e.g., lacking naturally occurring nucleic acids), particularly other nucleic acids of E. coli or host cell, being generally at least about 50% (by weight), and usually at least about 90% pure.
    The nucleic acids of the invention can be prepared in many ways, for example by chemical synthesis (e.g., DNA synthesis by phosphoramidites) in whole or in part, by digestion of longer nucleic acids using nucleases ( for example, restriction enzymes), by joining shorter nucleic acids or nucleotides (e.g., using ligases or polymerases), from genomic or cDNA libraries, etc.
    A nucleic acid of the invention may be attached to a solid support (eg, a bead, a plate, a filter, a film, a slide, a microchip support, a resin, etc.). A nucleic acid of the invention may be labeled, for example with a radioactive or fluorescent label, or a biotin label. This is particularly useful when the nucleic acid is to be used in detection techniques, for example when the nucleic acid is a primer or as a probe.
    The term "nucleic acid" generally includes a polymeric form of nucleotides of any length, which contains deoxyribonucleotides, ribonucleotides, and / or their analogs. It includes DNA, RNA, DNA / RNA hybrids. It also includes DNA or RNA analogs, such as those containing modified backbones (e.g., peptide nucleic acids (PNAs) or phosphorothioates) or modified bases. Thus, the invention comprises mRNA, tRNA, rRNA, ribozymes, DNA, cDNA, recombinant nucleic acids, branched nucleic acids, plasmids, vectors, probes, primers, etc. When the nucleic acid of the invention takes the form of RNA, it may or may not include a 5 'cap.
    The nucleic acids of the invention may be part of a vector, i.e. they may be part of a nucleic acid construct designed for the transduction / transfection of one or more cell types. . The vectors may be, for example, "cloning vectors" that are designed for the isolation, propagation and replication of inserted nucleotides, "expression vectors" that are designed for the expression of a nucleotide sequence. in a host cell, "viral vectors" that are designed to cause the production of a recombinant virus or a pseudoviral particle, or "shuttle vectors", which include the attributes of more than one type of vector. The preferred vectors are plasmids, as mentioned above. A "host cell" comprises an individual cell or a cell culture that can or has been a recipient of an exogenous nucleic acid. Host cells comprise the progeny of a single host cell, and the offspring may not necessarily be completely identical (in morphology or complement to total DNA) to the original parent cell because of a mutation and / or a natural, accidental, or deliberate change. Host cells comprise cells transfected or infected in vivo or in vitro with a nucleic acid of the invention.
    When a nucleic acid is DNA, it will be understood that "U" in an RNA sequence will be replaced by "T" in the DNA. Similarly, when a nucleic acid is RNA, it will be understood that "T" in a DNA sequence will be replaced by "U" in the RNA.
    The term "complement" or "complementary" when used in conjunction with nucleic acids refers to the Watson-Crick base pairing. Thus, the complement of C is G, the complement of G is C, the complement of A is T (or U), and the complement of T (or U) is A. It is also possible to use bases such as I (inosine purine), for example to supplement pyrimidines (C or T).
    The nucleic acids of the invention can be used, for example: to produce polypeptides; as hybridization probes for the detection of a 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 oligonucleotides forming triple strands. The invention provides a method of producing a nucleic acid of the invention, wherein the nucleic acid is synthesized in part or in whole using a 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.
    For some embodiments of the invention, the nucleic acids preferably have a length of at least 1340 nucleotides (e.g., 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, or 2400 nucleotides or longer).
    Cells of the Invention The invention also provides a cell comprising a nucleic acid of the invention. Said cell can be stably transformed with said nucleic acid. The invention also provides a cell that expresses a GAS40 polypeptide of the invention.
    Production of GAS protein antigens
    The redundancy of the genetic code is well known. Thus, any nucleic acid molecule (polynucleotide) that encodes one of the GAS antigens described herein can be used to recombinantly produce this protein. The nucleic acid molecules encoding the wild-type GAS antigens can also be isolated from the appropriate S. pyogenes bacterium using standard nucleic acid purification techniques or they can be synthesized using an amplification technique, like the polymerase chain reaction (PCR), or using an automated synthesizer. See Caruthers et al., Nucl. Acids Res. Symp. Ser. 215, 223, 1980; Horn et al., Nucl. Acids Res. Symp. Ser. 225, 232, 1980; Hunkapiller et al., Nature 310, 105-11, 1984; Grantham et al., Nucleic Acids Res. 9, r43-r74, 1981. The cDNA molecules can be made using standard molecular biology techniques, using mRNA as a template. The cDNA molecules can then be replicated using well-known molecular biology techniques of the state of the art. An amplification technique, such as PCR, can be used to obtain additional copies of the polynucleotides of the invention, using either genomic DNA or cDNA as template.
    If desired, the polynucleotides can be modified using methods generally known from the state of the art for modifying antigen coding sequences for various reasons, including but not limited to modifications for modify the cloning, processing and / or expression of the mRNA polypeptide or product. DNA rearrangement by random fragmentation and PCR reassembly of synthetic gene fragments and oligonucleotides can be used to modify the nucleotide sequences. For example, site-directed mutagenesis can be used to insert new restriction sites, to modify glycosylation patterns, to change codon preference, to produce splice variants, to introduce mutations, and so on.
    Sequence modifications, such as the addition of a marker sequence for purification or codon optimization, can be used to facilitate expression. For example, the N-terminal leader sequence may be replaced by a sequence encoding a marker protein such as polyhistidine ("HIS") or glutathione S-transferase ("GST"). Such marker proteins can be used to facilitate the purification, detection, and stability of the expressed protein. Codons preferred by a particular prokaryotic or eukaryotic host may be selected to increase the level of expression of the protein or to produce an RNA transcript having desirable properties, such as a half-life that is longer than that of a protein. transcript generated from the existing sequence in the natural state. These methods are well known in the state of the art and are further described in WO 05/032582.
    A nucleic acid molecule that encodes a GAS antigen for use in the invention may be inserted into an expression vector that contains the elements necessary for transcription and translation of the inserted coding sequence. The methods that are well known to those skilled in the art can be used to construct expression vectors containing appropriate coding sequences and transcriptional control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.
    Host cells for the production of 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, baculoviruses, mammalian cells, etc.
    A host cell strain may be selected for its ability to modulate expression of the inserted sequences or to process the expressed polypeptide in the desired manner. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing that cleaves a "prepro" form of the polypeptide can also be used to facilitate proper insertion, folding, and / or function. Different host cells that have specific cell machinery and characteristic mechanisms for post-translational activities are available from the American Type Culture Collection (ATCC;
    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 may be introduced into host cells using well established techniques which include, but are not limited to, transferrin-polycation conjugated DNA transfer, transfection with naked nucleic acids. or encapsulated, liposome mediated cell fusion, intracellular transport of DNA coated latex beads, protoplast fusion, viral infection, electroporation, "gene gun" methods, and transfection mediated by DEAE or calcium phosphate.
    Host cells transformed with expression vectors can be cultured under conditions suitable for expression and recovery of the protein from the cell culture. The protein produced by a transformed cell can be secreted or intracellularly contained according to the nucleotide sequence and / or the expression vector used. It will be understood by those skilled in the art that the expression vectors may be designed to contain signal sequences that direct the secretion of soluble antigens through a prokaryotic or eukaryotic cell membrane.
    The signal or export sequences can be included in a recombinantly produced GAS antigen so that the antigen can be purified from the cell culture medium using known methods. Alternatively, recombinantly produced GAS antigens can be isolated from the modified host cells and separated from other components in the cell, such as proteins, carbohydrates, or lipids, using methods well known in the art. 'art. Such methods include, but are not limited to, 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. The purity of the preparations can be estimated by any means known from the state of the art, such as SDS polyacrylamide gel electrophoresis or RP-HPLC analysis. Where appropriate, mutant Spy0167 proteins may be solubilized, for example, with urea.
    The antigens of GAS can be synthesized, for example ## EQU1 ## using solid phase techniques. See, for example, 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 an Applied Biosystems 431A Peptide Synthesizer instrument (Perkin Elmer). Optionally, fragments of the GAS antigens can be synthesized separately and combined using chemical methods to produce a full length molecule. Overview
    The term "comprising" includes "including" as well as "consisting of", for example, a composition "comprising" X may consist exclusively of X or it may comprise something else, for example, X + Y.
    The term "substantially" does not exclude "completely", for example, a composition that is "substantially devoid" of Y may be completely devoid of Y. When necessary, the term "substantially" may be omitted from the definition. of the invention. The term "essentially consisting of" means that the composition, process or structure may comprise additional components, steps and / or parts, but only if the additional components, steps and / or parts do not materially alter the basic and novel features of the claimed composition, process and / or structure. The term "consisting of" is generally understood to mean that the invention as claimed is limited to those items specifically mentioned in the claim (and this may include their equivalents, as long as the doctrine of equivalents is applicable).
    The term "about" as used herein2 when reference is made to a measurable value such as quantity, time duration, and the like is intended to encompass variations of ± 20% or ± 10%, more preferably ± 5%, even more preferably ± 1%, and still more preferably ± 0.1% from the specified value, as long as such variations are suitable for carrying out the disclosed methods.
    The term "substantially" does not exclude "completely", for example, a composition that is "substantially free" of Y may be completely devoid of Y. For example, "substantially free" of Y may be understood as a composition containing no not more than 5% of Y, not more than 4% of Y, not more than 3% of Y, not more than 2% of Y, not more than 1% of Y, or not more than 0.1% of Y Where necessary, the term "substantially" may be omitted from the definition of the invention.
    The term "mutant" refers to a gene or gene product that exhibits changes in its sequence and / or functional properties (i.e., modified characteristics) when compared to the gene. or the wild-type gene product. Thus, wild-type sequences and wild-type sequence fragments, which do not include substitutions of the present invention, are excluded. For example, 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 can be excluded.
    All GenBank accession numbers provided herein are incorporated by reference.
    Unless otherwise indicated, a process comprising a step of mixing two or more components requires no specific mixing order. Thus, the components can be mixed in any order. When BE2 has three components, then two components can be combined with each other, and then the combination can be combined with the third component, etc.
    When animal (and particularly bovine) materials are used in cell culture, they should be obtained from sources that are free of transmissible spongiform encephalopathy (TSE), and in particular free of bovine spongiform encephalopathy (BSE). Overall, it is best to grow cells in the complete absence of animal material.
    When a compound is administered to the body as part of a composition, then this compound may alternatively be replaced by a suitable precursor. Sequence identity between polypeptide sequences is preferably determined by a pair alignment algorithm using the Needleman-Wunsch global alignment algorithm (Needleman & Wunsch (1970) J. Mol Biol., 48, 443 -453), using the default parameters (for example, with a gap opening penalty = 10.0, and with a gap extension penalty = 0.5, using the score matrix EBLOSUM62). This algorithm is conveniently implemented in the needle tool in the EMBOSS package (Rice et al (2000) Trends Genet 16: 276-277). Sequence identity should be calculated over the entire length of the polypeptide sequence of the invention.
    Examples
    Example 1 - Bioinformatic analysis to identify short sequences of amino acids common to human proteins potentially responsible for a mimicry of linear epitopes, leading to autoimmune diseases
    In order to detect short sequences of amino acids that are common to both GAS40 antigen and one or more human proteins and potentially responsible for mimicry of linear epitopes, the amino acid sequences of GAS40 have been - divided into 7-seas, 8-seas, 9-seas and 10-seas overlapping, with a sliding window of 1 amino acid residue. Each of these fragments was compared to a collection of human proteins available at ftp://ftp.ncbi.nih.gov/genomes/H_sapiens/protein/. The same analysis was carried out on the tetanus toxin sequence (GI: 135624), the safety of which is widely accepted, and the M1 and M5 proteins of Streptococcus pyogenes (Streptococcus pyogenes M1 GAS, GI: 15675799, Streptococcus pyogenes serotype M5 , GI: 126669), which have also been reported to contain epitopes that exhibit cross-reactivity with human tissues (Baird et al., 1991). The results of this analysis are summarized in Table 1.
    Table 1
    Two 9-mer matches and an 8-mer match were detected for the GAS40. The first 9-mer has the sequence represented by QLTEELAAQ (SEQ ID NO: 8), and is located in position 235 to 243 of GAS40 which is in the first supercoiled domain. This 9-mer is also found in a 562 amino acid human protein known as "81 protein containing supercoiled domains" (GI: 47271449). The second 9-mer has the sequence shown as BE2 SEQ ID NO: 9, and 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 represented by SEQ ID NO: 10 and is located at position 699 to 706, which bridges the C-terminal boundary of the leucine zipper domain. This 8-mer is also found in a human protein of 844 amino acids known as "Janus kinase and microtubule-interacting protein 3" (GI: 157502225).
    In order to reduce the sequence identity of the above-mentioned 9-mer and 8-mer with the corresponding human protein sequences, multiple mutations have been introduced into these 9-mer and 8-mer. In order to avoid a reduction in the immunological efficacy of GAS40, six amino acid substitutions (two in each of the 8/9-mer correspondences) that were designed to have no effect on the structure were introduced. overall protein (alpha helical overwrap / leucine zipper). The two substitutions that were performed on the first 9-mer (QLTEELAAQ) were T237S and A241V, resulting in the mutated sequence of 9-mer: QLsEELvAQ (SEQ ID NO: 11). The two substitutions that were carried out on SEQ ID NO: 9
    were A688S and S692T, resulting in the mutated sequence of 9-mer: KQDLsKTTt (SEQ ID NO: 12). The two substitutions that were performed on SEQ ID NO: 10 were A700S and A703S, resulting in the mutated sequence of 8-mer: EsLAsLQA (SEQ ID NO: 13).
    Figure 1 shows a prediction of the secondary structure of the wild-type and mutant GAS40 protein. As shown, the prediction indicates that the integrity of the overcoiling propensity profile and the leucine zipper pattern will remain unchanged by the above six mutations. BE2
    Example 2 - Bioinformatic analysis to identify possible supercoiling irregularities similar to human myosin and tropomyosin in GAS40
    Surcoiling irregularities have recently been demonstrated in the M1 protein (McNamara et al., 2008). Similar structural irregularities occur in myosin and tropomyosin, providing a possible explanation for cross-reactive M protein-associated patterns in autoimmune sequelae of GAS infection. When substitutions were made within the super-rolled region of M1 producing optimal central residues for the formation and stability of dimer parallel supercoils ("sequence idealization"), fibrinogen binding, proinflammatory effects, and cross-reactivity antibodies were decreased. A similar "idealization" of the supercoiling approach was followed in the case of GAS40 to mitigate any potential cross-reactivity.
    Using the Jalview software package (http://www.jalview.org/), the supercoiled regions of the GAS40 have been identified and highlighted with a color scale from red to blue where red represents hydrophobic residues and blue represents residues. hydrophilic. Hydrophilic residues in positions 1 and 4 of the supercoiling heptads were substituted by the hydrophobic leucine residue to "idealize" the supercoiling structure, and lysine 208 was deleted to correct a heptad frame shift. A list of mutations that have been introduced into GAS40 is presented in Table 2.
    Table 2
    2
    Example 3 - Cloning and general DNA techniques 3.1 GAS40 without homologies of 8 and 9-seas
    Three rare AGA codons encoding arginine (Arg 359, Arg 360 and Arg 849) were mutated in a common codon for CGT arginine to optimize expression of GAS40. The nucleotide sequence of GAS40 containing the CGT AGA substitutions of the arginine codons, but excluding the sequences of the leader peptide and the transmembrane domain, is represented by SEQ ID NO: 1 and the corresponding amino acid sequence is represented by SEQ ID NO: 2.
    A pET21b + vector containing the GAS40 gene with optimized codons was used as a template to create the 8-9-mer mutant of GAS40. The introduction of the 6 mutations was carried out in 3 steps, each incorporating 2 mutations in one of the sequences homologous to the human, producing two intermediate constructs and one final construct incorporating the three mutations.
    In each step, the Polymerase Incomplete Primer Extension (PIPE) cloning method (Klock and Lesley 2009) was used to amplify the entire plasmid using two primers designed to create two mutations (substitutions) and to be complementary to each other. another, such that the linearized PCR product will be able to self-hybridize to recreate a viable mutant plasmid. The primers used in each of the three steps are shown below:
    Table 3_
    The nucleotide and amino acid sequences of the GAS40 8-9-mer mutant, with the deleted leader and transmembrane domain sequences, are represented by SEQ ID NO: 3 and 4, respectively. 3.2 GAS40 with "idealized" super-windings
    A "multi-mutant" GAS40 protein with optimized codons containing "idealized" supercoils has been produced by chemical synthesis. In order to optimize the expression, the GC content was adjusted to prolong the half-life of the mRNA. The use of codons was also adapted to the preference of Escherichia coli. The nucleotide and amino acid sequences of GAS40 with "idealized" (multi-mutant) supercoils, with deleted leader and transmembrane domain sequences, are represented by SEQ ID NO: 5 and 6.
    Example 4 Expression and Purification of Recombinant GAS40 and its Derivatives
    His-tagged mutant derivatives of recombinant GAS40 were expressed in E. coli cells. coli BL21 (DE3) and purified by affinity chromatography on affinity chromatography columns with immobilized metal ions.
    The cells were first incubated at 30 ° C with shaking at 180 rpm until they reached a culture density of OD600nm = 0.4 to 0.6, then the cells were induced with 1 mM IPTG, and incubated for another 3 hours at 25 ° C with shaking at 180 rpm. Cells were then recovered by centrifugation and frozen at -20 ° C.
    Frozen pellets of E. coli were suspended in lysis buffer (10 ml B-PER ™ (Pierce), 0.1 mM MgCl 2, 100 units DNase I (Sigma), and 1 mg / ml lysozyme (Sigma)) and mixed for 30 to 40 minutes at room temperature. The resulting lysates were centrifuged at 3000040000 xg for 20-25 minutes, and the supernatants were then loaded onto columns equilibrated with Wash Buffer A (50 mM NaH2PO4, 300 mM NaCl, pH 8.0) (Poly -Prep with 1 ml of Ni-Activated Chelating Sepharose Fast Flow resin). The loaded resin was washed three times with Wash Buffer A and three times with Wash Buffer B (70 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH 8.0). Proteins were eluted with elution buffer (250 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH 8.0) in Eppendorf tubes containing 1 mM DTT. Total eluted proteins were quantified with Bradford's reagent, intensively dialyzed with PBS and then analyzed by polyacrylamide gel electrophoresis with SDS (see Figure 7) using Criterion ™ premixed gels at 4-12% (Bio -Rad).
    Example 5 - Immunization and Infection
    To test whether GAS mutants are capable of inducing protection against lethal challenge in mice, five-week old mice were immunized intraperitoneally three times (day 0, day 21 and day 35). ) with either hßi2-labeled wild-type GAS40 or either the GAS40 8-9-mer mutant or the multi-mutant GAS40, each of which was combined with an alum adjuvant (20 μg protein in 2 mg / ml d aluminum hydroxide). Mice immunized with the adjuvant alone were used as negative controls. Two weeks after the third immunization, blood samples were taken. A few days later, the immunized mice were stimulated ("challenged") intranasally with 108 cfu (50 μ!) Of strains of GAS M1 3348. The survival of the mice was monitored for a period of 10 to 14 days.
    As shown in the table below, both the multi-mutant and the 9-mer mutant increased the survival rate of immunized mice from 4.2 to 4.3 times the negative control survival rate. This indicates that the mutant proteins produce high levels of protection. Indeed, the mutant proteins were even more effective than the wild-type GAS40.
    Table 4
    Example 6 Cross Reactivity of Mouse Antisera Against Human Tissues
    The development of autoimmune diseases is associated with dEE2 persistent high titre levels of high affinity antibodies. In order to estimate the potential of GAS40 to elicit antibodies recognizing specific human tissue components, a pilot cross-reactivity study with mouse antisera directed against GAS40 was performed. Sera were obtained from groups of 8 inbred CD1 mice immunized as shown in Table 10.
    Table 10
    The cross-reactivity study of non-GLP human tissue was optimized and performed at Charles River Laboratories / PAI in Frederick, MD. A panel of selected human tissues was screened for their association with a specific GAS postinfection pathology: cartilage, brain, heart, heart valve, kidney and skeletal muscle. The 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 1: 10,000 and 1: 40,000 dilutions. Individual antigen deposition control slides were incubated with the pooled antisera from the pools. respective animals to confirm the specific responses of the antigens. No specific cell membrane staining of the test article was observed in any group suggesting that antibodies to GAS40 failed to recognize membrane elements that could biologically influence the processes. Specific staining was observed in the cytoplasm with antisera from mice treated with M6, M12 and GAS40 and was observed more frequently in the heart valve (M6 and M12) and the heart (M6, M12). and GAS40). Cytoplasmic staining has been considered to be of little toxicological relevance because it is believed that, in general, the cytoplasmic compartment is of limited access to antibodies in vivo. It is the nature of this test that allows the cytoplasmic compartment to be exposed and available for binding by elements in mouse antisera. The results of this preliminary screening showed that (1) the immunohistochemical test was reproducible between the series, (2) there were slight differences between tissue staining from different donors, (3) staining decreased with increasing the dilution of antisera, and (4) adding high stringency washes to reduce nonspecific binding of unpurified mouse antibodies decreased or eliminated staining. This latter observation has indirectly confirmed that high affinity antibodies directed against human tissue elements have not been elicited in mice. GAS40 mutants can be tested using similar protocols.
    While some embodiments of the present invention have been specifically described and exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made without departing from the scope and spirit of the present invention as set forth in the following claims. TABLE OF SEQUENCE IDENTIFICATION NUMBERS USED BE2
    References
    Baird, RW, MS, Kraus, Bronze W, HR Hill, Veasey LG, and JB Dale. "Epitopes of streptococcal group A protein shared with antigens of articular cartilage and synovium." The Journal of Immunology 146 (1991): 3132-3137.
    McNamara, C, AS Zinkernagel, Macheboeuf P, MW Cunningham, V Nizet, and P Ghosh. "Coiled-coil irregularities and instabilities in group A Streptococcus M1 are required for virulence." Science 319 (2008): 1405-1408. O'Hagan, Derek T., ed. Adjuvant Vaccine: Preparation Methods and Research Protocols (Volume 42 of the Molecular Medicine Methods). Humana Press, 2000.
    Patterson, MJ. "Streptococcus." In Medical Microbiology, edited by S Baron. Galveston (TX): University of Texas Medical
    Branch at Galveston, 1996.
    Powell, Michael F., and Mark J Newman,. Vaccine Design: The
    Subunit and Adjuvant Approach. Plenum Press, 1995.
    Remington, Joseph Price. Remington: The Science and Practice of Pharmacy. 20th. Edited by Alfonso R. Gennaro. Lippincott Williams and Wilkins, 2000.
    权利要求:
    Claims (16)
    [1]
    A recombinant streptococcal GAS40 polypeptide which comprises an epitope that elicits opsonic antibodies against Streptococcus Group A and further comprises: (i) at least two substitutions in any one of amino acids 235 to 243, at least two substitutions in any of amino acids 684 to 692 and at least two substitutions in any of amino acids 699 to 700, and / or (ii) a lysine deletion at position 208 and amino acid substitutions at positions of amino acids 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, where the amino acid positions are numbered according to SEQ ID NO: 2.
    [2]
    The recombinant streptococcal GAS40 polypeptide according to claim 1 which comprises substitutions at amino acid positions T237, A241, A688, S692, A700 and A703.
    [3]
    The recombinant streptococcal GAS40 polypeptide according to claim 2 which comprises the substitutions T237S, A241V, A688S, S692T, A700S and A703S.
    [4]
    A recombinant streptococcal GAS40 polypeptide according to any one of claims 1 to 3 which comprises a deletion of lysine at position 208 and amino acid substitutions 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.
    [5]
    The recombinant streptococcal GAS40 polypeptide according to claim 1, wherein said polypeptide shares at least 92% identity with any one of SEQ ID NO: 4, 6, 7, 16, 17.2, 18, 24, 25 or 26 .
    [6]
    The recombinant streptococcal GAS40 polypeptide according to claim 5, wherein said polypeptide comprises a sequence selected from the group consisting of SEQ ID NO: 4, 6, 7, 16, 17, 18, 24, 25 and 26.
    [7]
    The fragment of a recombinant GAS40 streptococcal polypeptide according to any one of the preceding claims, wherein the fragment shares at least 97% identity with any one of SEQ ID NOS: 20, 21, 22, 28, 29, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 44, 45, 46, 48, 49, 50, 52, 53, 54, 56, 57, 58, 60, 61, 62, 64, 65, 66, 67, 68, 69, 70, 72, 73, 74, 76, 77, 78, 80, 81, 82, 84, 85, 86, 88, 89, 90, 92, 93, 94, 96, 97, 98, 100, 101, 102, 104, 105, 106, 108, 109, 110, 112, 113, 114, 116, 117 and 118.
    [8]
    The fragment of claim 7 wherein the polypeptide comprises or consists of 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, 116, 7, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54 , 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114 and 118.
    [9]
    A polypeptide comprising the recombinant streptococcal GAS40 polypeptide of claims 1-6 or the fragment of a recombinant streptococcal GAS40 polypeptide according to claims 7-8.
    [10]
    An immunogenic composition comprising the recombinant streptococcal GAS40 polypeptide of claims 1-6 or the fragment of a recombinant streptococcal GAS40 polypeptide according to claims 7-8.
    [11]
    The immunogenic composition of claim 10, wherein said composition comprises one or more additional GAS polypeptides.
    [12]
    The immunogenic composition of claim 11, wherein said composition comprises a GAS57 polypeptide of SEQ ID NO: 120 and a GAS25 polypeptide of SEQ ID NO: 121 and optionally a GAS polysaccharide.
    [13]
    An immunogenic composition according to any one of claims 10 to 12, wherein said composition is a vaccine.
    [14]
    14. An immunogenic composition according to any one of claims 10 to 13, which comprises an adjuvant.
    [15]
    Nucleic acid encoding the recombinant streptococcal GAS40 polypeptide according to claims 1-6 or the fragment of a recombinant streptococcal GAS40 polypeptide according to claims 7-8.
    [16]
    A cell comprising the nucleic acid of claim 15.
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