HK1238144B - Group a streptococcus vaccine - Google Patents

Description

Group A Streptococcus vaccine

Technical Field

The present invention relates to the prevention and treatment of infectious diseases. More particularly, the present invention relates to vaccines for the treatment or prevention of diseases and conditions associated with Group A Streptococcus.

Background of the Invention

Vaccines against Streptococcus pyogenes (Lancefield Group A Streptococcus; GAS) have long been sought due to the debilitating diseases caused by the bacterium, particularly rheumatic fever and rheumatic heart disease. Today, rheumatic fever is a rare disease in most developed countries, but in developing countries it remains the leading cause of acquired heart disease in children, adolescents, and young adults. In addition, invasive GAS disease is a common cause of sepsis in children and adults and has a high mortality rate. Further adding to the burden of GAS disease is post-streptococcal glomerulonephritis, which may lead to high rates of late-stage renal failure in many GAS-endemic regions. GAS pharyngitis and impetigo are the GAS infections with the largest absolute number each year. Each year, GAS pharyngitis affects approximately 8%–15% of school-age children, and GAS impetigo is a very common infection in children, with a prevalence of 10–50%. Severe GAS-related diseases are a problem not only in developing countries but even in developed countries. In particular, virulent strains of GAS have emerged that are resistant to standard antibiotic therapy and cause debilitating diseases such as severe necrotizing fasciitis.

An important virulence factor of GAS is the M protein, which is strongly antiphagocytic and binds to serum factor H, disrupting the C3-convertase and preventing opsonization by C3b. Vaccines containing immunogenic peptides from the conserved C-repeat portion of the M protein have been developed, such as vaccines containing T-cell and B-cell epitopes from the C-repeat region, or the J8 and J14 peptide vaccines containing a single minimal B-cell epitope from the C-repeat region.

SUMMARY OF THE INVENTION

Surprisingly, the present inventors have found that an important factor in the immunity to group A streptococci induced by the J8-peptide is the activity of neutrophils. Although the opsonization analysis in vitro uses whole blood as the source of neutrophils and complement, it is unknown that neutrophils are needed for protection in vivo. Moreover, the opsonization analysis shows a relative moderate reduction in CFU (usually less than 10 times), while the protection induced by J8 in vivo can result in a reduction of several logarithmic levels of bacterial bioload. More specifically, at present, the inventors have found that neutrophil inhibitors (such as SpyCEP, which inactivates the neutrophil chemotactic substance interleukin 8) work for J8 in inducing immunity to group A streptococci.

In its broadest form, the present invention is thus directed to restoring or enhancing neutrophil activity to aid in M protein-induced immunity to group A Streptococcus.

One aspect of the present invention provides a method for eliciting an immune response to Group A Streptococcus bacteria in a mammal, the method comprising the steps of administering to the mammal a fragment, variant or derivative of an M protein; and a substance that promotes the restoration or enhancement of neutrophil activity; thereby eliciting an immune response to Group A Streptococcus bacteria in the mammal.

Another aspect of the present invention provides a method for immunizing a mammal against Group A Streptococcus bacteria, the method comprising administering to the mammal an M protein, a fragment, variant or derivative thereof; and a substance that promotes the restoration or enhancement of neutrophil activity, thereby immunizing the mammal against Group A Streptococcus bacteria.

Yet another aspect of the present invention provides a method for treating or preventing group A Streptococcus bacterial infection in a mammal, the method comprising administering to the mammal a fragment, variant or derivative thereof, or an antibody or antibody fragment thereof; and a substance that promotes the restoration or enhancement of neutrophil activity; thereby treating or preventing group A Streptococcus bacterial infection in the mammal.

Yet another aspect of the present invention provides a composition suitable for administration to a mammal, comprising: a fragment of the M protein, a variant or a derivative thereof, or an antibody or an antibody fragment thereof; and a substance that promotes the restoration or enhancement of neutrophil activity.

Related aspects of the invention provide for the administration of one or more isolated nucleic acids encoding fragments, variants, or derivatives thereof of the M protein and a substance that promotes restoration or enhancement of neutrophil activity, or administration of a composition comprising the one or more isolated nucleic acids.

In a specific embodiment, the fragment of the M protein is a conserved region of the M protein or comprises a conserved region of the M protein. In one embodiment, the fragment is an immunogenic fragment comprising the p145 peptide or an immunogenic fragment contained within the p145 peptide. In a specific embodiment, the immunogenic fragment is within the J8 peptide or a variant thereof or comprises the J8 peptide or a variant thereof. In certain embodiments, the variant comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of the following amino acid sequence or a fragment or variant thereof: SREAKKQSREAKKQVEKALKQVEKALC (SEQ ID NO: 59); SREAKKQSREAKKQVEKALKQSREAKC (SEQ ID NO: 60); SREAKKQVEKALKQSREAKKQVEKALC (SEQ ID NO: 61); and SREAKKQVEKALDASREAKKQVEKALC (SEQ ID NO: 62).

In a broad embodiment, the substance that promotes the restoration or enhancement of neutrophil activity is a protein or fragment thereof that generally directly or indirectly inhibits or suppresses neutrophils or neutrophil activity. Suitably, administration of the protein or fragment thereof elicits an immune response to the protein and/or to Group A Streptococcus.

In another broad embodiment, the substance that promotes restoration or enhancement of neutrophil activity is an antibody or antibody fragment that binds to a protein or fragment thereof that normally directly or indirectly inhibits or suppresses neutrophils or neutrophil activity.

In specific embodiments, the protein is SpyCEP or a fragment thereof.

In a preferred embodiment, the fragment comprises the amino acid sequence NSDNIKENQFEDFDEDWENF (SEQ ID NO: 18).

Yet another aspect of the invention provides an isolated peptide comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of NSDNIKENQFEDFDEDWENF (SEQ ID NO: 18); SREAKKQSREAKKQVEKALKQVEKALC (SEQ ID NO: 59); SREAKKQSREAKKQVEKALKQSREAKC (SEQ ID NO: 60); SREAKKQVEKALKQSREAKKQVEKALC (SEQ ID NO: 61); and SREAKKQVEKALDASREAKKQVEKALC (SEQ ID NO: 62).

Related aspects provide an isolated nucleic acid encoding the isolated peptide of the above aspects, a genetic construct comprising the isolated nucleic acid and/or a host cell comprising the genetic construct.

Yet another related aspect provides an antibody or antibody fragment that binds to or is elicited by the isolated peptide of the above aspects.

Yet another aspect of the invention provides a composition comprising the isolated peptide, isolated nucleic acid, genetic construct, host cell and/or antibody or antibody fragment of the preceding aspects.

As used herein, the indefinite articles ‘a’ and ‘an’ are used herein to refer to or encompass a singular or plural element or feature and should not be taken as meaning or limiting the “one” or “single” element or feature.

Unless the context requires otherwise, the terms "comprise," "comprises," and "comprising" or similar terms are intended to mean a non-exclusive inclusion, such that the stated list of elements or features includes not only those specified or listed elements, but may include additional elements or features that are not listed or specified.

In the context of amino acid sequences, by "consisting essentially of" is meant the recited amino acid sequence and one additional, two or three amino acids at the N-terminus or C-terminus.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Protective efficacy of J8-DT/Alum vaccination.

Figure 2: Neutrophils in J8-DT-mediated immunity. E: epidermis; D: dermis; SC: subcutaneous layer

Figure 3: Neutrophil depletion and efficacy of J8-DT vaccination.

Figure 4: Overview of genes with elevated expression in the M1T1 isolate of CovR/S mutant GAS.

Figure 5: Efficacy of J8-DT in protecting against 5448AP.

Figure 6: Degradation of IL-8 by different GAS strains.

Figure 7: Degradation of MIP-2 by different GAS strains.

Figure 8: Degradation of KC by different GAS strains.

Figure 9: Effects of different GAS isolates on the chemotactic ability of chemokines.

Figure 10: Protection against GAS5448AP strain by immunization with J8-DT-rSpyCEP/Alum.

Figure 11: Immunogenicity of J8-DT-SpyCEP as measured by the production of antigen-specific IgG.

FIG. 12 : Inhibition of IL-8 degradation activity of SpyCEP by rSpyCEP antiserum.

Figure 13: Epitope mapping of overlapping 20-mer peptides (10 amino acid overlap) spanning residues 35-587 of SpyCEP (SEQ ID NOs: 1-55 in descending order). Peptides in bold are those selected by epitope mapping.

Figure 14: Reactivity of antisera raised against recombinant SpyCEP with various peptide epitopes. B-cell epitope mapping of SpyCEP (553 amino acids) was performed using a peptide array. 20-mer peptides with 10 amino acid overlaps as shown in Figure 13 were probed with rSpyCEP antisera.

Figure 15: Identification of six (6) epitopes showing strong reactivity with antiserum raised against recombinant SpyCEP. S1 = SEQ ID NO: 15; S2 = SEQ ID NO: 18; S3 = SEQ ID NO: 19; S4 = SEQ ID NO: 24; S5 = SEQ ID NO: 30; and S6 = SEQ ID NO: 54.

Figure 16: Immunogenicity and recognition of the parent peptide by antisera raised against the SpyCEP epitope.

FIG17 : Inhibition of IL-8 degradation by antiserum against the SpyCEP epitope.

Figure 18: Titers and cross-recognition ELISA of murine α-p145-DT and α-J8-DT against self-peptides.

Figure 19: Titers of murine α-J8i variant-DT against self-peptide.

Figure 20: Cross-recognition ELISA.

Figure 21: Identification of immunodominant epitopes in recSpyCEP. To identify the immunodominant epitopes in recSpyCEP, peptide inhibition ELISA was performed. Groups of BALB/c mice (4-6 weeks) were immunized subcutaneously on days 0, 21 and 28 with SpyCEP epitope-DT conjugate or SpyCEP. One week after the last stimulation, the mice were bled via submandibular bleeding and the antisera were collected. The epitope antisera were incubated with autologous peptides at a concentration of 5 μg/ml or 0.5 μg/ml, and the inhibition of autologous peptide recognition was assessed (A). The peptide antisera were also incubated with SpyCEP at a concentration of 5 μg/ml or 0.5 μg/ml. The serum was then used in ELISA against immobilized peptides (B). Finally, SpyCEP antiserum was incubated with each peptide (S1-S6) at a concentration of 5 μg/ml or 0.5 μg/ml or with recSpyCEP, and its binding to each corresponding peptide or SpyCEP was assessed (C). Data for each column are mean ± SEM. Statistical analysis was performed using a two-way ANOVA with Bonferroni's multiple comparison test to determine significance between groups. *p < 0.05 and **p < 0.01, ***p < 0.001. ns is p < 0.05.

Figure 22: Binding efficacy of SpyCEP and antisera to the SpyCEP epitope to different GAS strains. To determine the specificity of recSpyCEP or SpyCEP epitope antisera to different GAS strains, flow cytometry was performed. The analysis measured the binding of epitope antisera (S1-S6) compared to SpyCEP antisera by FITC-conjugated IgG. The binding of 5448 and its animal-passaged derivatives (A), BSA10 and its animal-passaged derivatives (B), and throat isolates (pM1) and skin isolates (88/30) (C) are shown. The data for each column are mean ± SEM. Statistical analysis was performed using a two-tailed t-test to determine significance compared to the PBS control. *p<0.05, **p<0.01, and ***p<0.001.

Figure 23: Protective efficacy of J8-DT + S2-DT against GAS. Groups of BALB / c mice (4-6 weeks) were subcutaneously immunized with J8-DT, J8-DT + SpyCEP, J8-DT + S2-DT, SpyCEP, or PBS formulations on days 0, 21, and 28. Two weeks after the last challenge, mice were infected with GAS 5448AP via the skin infection route (A and B). On day 6 after infection, 5 mice/group were sacrificed and samples were collected to determine the GAS bioburden in the skin (A) and blood (B). Data represent two or more independent experiments, and results are shown as mean ± SD of 4-5 mice in each group. Two-way analysis of variance was used to determine the significance between the vaccinated and control groups. *p < 0.05, **p < 0.01, ***p < 0.001. ns is p < 0.05.

Figure 24: The protective efficacy of J8-DT+S2-DT against GAS. On day 0, day 21 and day 28, groups of BALB/c mice (4-6 weeks) were subcutaneously immunized with J8-DT, J8-DT+SpyCEP, J8-DT+S2-DT, SpyCEP or PBS formulations. Two weeks after the last stimulation, mice (A, B and C) were infected with GAS NS22.8 via the skin infection route. On day 3 and day 6 after infection, 5 mice/group were sacrificed and samples were collected to determine the GAS bioburden in the skin (A), blood (B) and spleen (C). The results are shown as the mean ± SD of 4-5 mice in each group. Two-way analysis of variance was used to determine the significance between the immunized and control groups. *p<0.05, **p<0.01, ***p<0.001. ns is p<0.05.

Detailed Description of the Invention

The present invention is based, at least in part, on the discovery that neutrophil activity is important for successful immunization against Group A Streptococcus with the J8 peptide. More specifically, it has been recognized that certain proteases of Group A Streptococcus, such as SpyCEP, exert a deleterious or inhibitory effect on neutrophils by proteolytically inactivating the neutrophil chemotactic substance interleukin-8. It has therefore been proposed that immunization with the J8 peptide or fragments of other M proteins, together with SpyCEP, would elicit an immune response to SpyCEP that would at least partially attenuate the ability of SpyCEP to inactivate interleukin-8 and thereby inhibit the response of neutrophils. This would thus synergistically enhance the immunological effect of the J8 immunization. In a related embodiment, anti-SpyCEP antibodies could be administered therapeutically to elicit an enhanced immune response to the J8 peptide. Furthermore, an immunodominant epitope of SpyCEP has been identified. In a specific form, the present invention may be suitable for treating or preventing infections caused by particularly virulent strains or isolates of Group A Streptococcus that are resistant to typical antibiotic treatments for Group A Streptococcus infections. These strains or isolates often cause serious infections of the skin (e.g., necrotizing fasciitis) and in some cases may contain the CovR/SCovR/S mutation.

Therefore, certain aspects of the present invention relate to administering to a mammal a fragment, variant, or derivative of the M protein and a substance that promotes restoration or enhancement of neutrophil activity, thereby eliciting an immune response in the mammal.

For purposes of this invention, "isolated" means material that has been removed from its natural state or otherwise manipulated by the hand of man. An isolated material may be substantially or essentially free from components normally associated with its natural state, or may have been manipulated so as to be in an artificial state along with components normally associated with its natural state. An isolated material may be in natural, chemically synthesized, or recombinant form.

As is well known in the art, "protein" means a polymer of amino acids. The amino acids can be natural or unnatural amino acids, D-amino acids or L-amino acids.

The term "protein" includes and encompasses "peptides," generally used to describe proteins having no more than fifty (50) amino acids, and "polypeptides," generally used to describe proteins having more than fifty (50) amino acids.

A "fragment" is a segment, domain, portion or region of a protein (e.g., M protein, p145, J14 or J8 or SpyCEP or a peptide or epitope of SpyCEP) that consists of less than 100% of the amino acid sequence of the protein. It will be understood that the fragment can be a single fragment or can be repeated alone or with other fragments.

Typically, a fragment may contain up to 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 100, 1050, 1100, 1250, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2250, 2300, 2400, 2500 150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550 or 1600 amino acids, consisting essentially of up to 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 100, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550 or 1600 amino acids or up to 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40, 50, 60, 750 or 1600 amino acids of the full-length protein. 0, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 100, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550 or 1600 amino acids.

Suitably, the fragment is an immunogenic fragment. In the context of the present invention, the term "immunogenicity" as used herein refers to the ability or potential to produce or elicit an immune response, such as to Group A Streptococcus or its molecular components (such as M protein), when an immunogenic fragment is administered to a mammal. Preferably, the immune response elicited by the immunogenic fragment is protective.

"Elicit an immune response" means causing or stimulating the production or activity of one or more elements of the immune system (including the cellular immune system, antibodies and/or the innate immune system). Suitably, the one or more elements of the immune system include B lymphocytes, antibodies and neutrophils.

As used generally herein, the terms "immunize," "vaccinate," and "vaccine" refer to methods and/or compositions that elicit a protective immune response against Group A Streptococcus, thereby at least partially preventing or minimizing subsequent infection by Group A Streptococcus.

As used herein, the terms "group A streptococcus," "Group A Streptococci," "Group A Streptococcal," "Group A Strep," and the abbreviation "GAS" refer to Streptococcus bacteria of Lancefield serotype A, which are Gram-positive, beta-hemolytic bacteria of the species Streptococcus pyogenes. An important virulence factor of GAS is the M protein, which is strongly antiphagocytic and binds to serum factor H, disrupting the C3-convertase and preventing opsonization by C3b. These also include toxic "mutants," such as those described in Graham et al., 2002, PNAS USA 99 13855, such as CovR/S or CovRS mutants, although not limited thereto.

Diseases and conditions caused by Group A Streptococcus include, but are not limited to, cellulitis, erysipelas, impetigo, scarlet fever, throat infections such as acute pharyngitis ("strep throat"), bacteremia, toxic shock syndrome, necrotizing fasciitis, acute rheumatic fever, and acute glomerulonephritis.

" Neutrophil " or neutrophil granulocyte used herein is a cell that forms a part of the polymorphonuclear cell family (PMN) together with basophils and eosinophils. Neutrophil granulocytes are relatively short-lived phagocytes formed by bone marrow stem cells and generally constitute 40% to 75% of white blood cells in mammals. As well as being phagocytes, neutrophils release soluble antimicrobial agents (such as granule proteins) and produce neutrophil extracellular bactericidal networks (neutrophil extracellular trap). Neutrophil responses to molecules such as interleukin-8 (IL-8), C5a, fMLP and leukotriene B4 promote the chemotaxis of neutrophils at sites of injury and/or acute inflammation.

As used herein, a "substance that promotes the restoration or enhancement of neutrophil activity" is a molecule that directly or indirectly at least partially increases, enhances or restores the production, migration and/or chemotaxis of neutrophils and/or one or more immune activities of neutrophils. In one embodiment, the substance triggers an immune response to a neutrophil inhibitor. In another embodiment, the substance binds to a neutrophil inhibitor and at least partially inactivates it. The neutrophil inhibitor can be a molecule derived from or derived from group A streptococcal bacteria. In one specific form, the neutrophil inhibitor is a serine protease or a fragment thereof that proteolytically cleaves interleukin 8. In a specific embodiment, the neutrophil inhibitor is SpyCEP or a fragment thereof. SpyCEP is a 170-kDa multi-domain serine protease expressed on the surface of the human pathogen Streptococcus pyogenes, which plays an important role in infection by catalyzing the cleavage and inactivation of the neutrophil chemoattractant interleukin-8. Non-limiting examples of SpyCEP amino acid sequences can be found under accession numbers YP597949.1 (Streptococcus pyogenes MGAS10270) and YP596076.1 (Streptococcus pyogenes MGAS9429). Thus, in one embodiment, the substance that promotes the restoration or enhancement of neutrophil activity is SpyCEP or an immunogenic fragment thereof. In another embodiment, the substance that promotes the restoration or enhancement of neutrophil activity is an antibody or antibody fragment that binds to SpyCEP. Specific embodiments of SpyCEP fragments are shown in Figure 13 (SEQ ID NO: 1-55). A preferred SpyCEP fragment is the amino acid sequence shown in SEQ ID NO: 18 (NSDNIKENQFEDFDEDWENF), or comprises the amino acid sequence shown in SEQ ID NO: 18 (NSDNIKENQFEDFDEDWENF). As will be described in more detail below, antisera against the SEQ ID NO: 18 peptide can block the degradation of IL8 as effectively as anti-rSpyCEP antibodies. Therefore, it is proposed that SEQ ID NO: 18 is a dominant epitope on SpyCEP that can induce functional antibodies, or contains a dominant epitope on SpyCEP that can induce functional antibodies.

As used herein, a "fragment of the M protein" is any fragment of the GAS M protein that is immunogenic and/or capable of binding to an antibody or antibody fragment. Typically, the fragment is the amino acid sequence of the C-repeat region of the GAS M protein or a fragment thereof, comprises the amino acid sequence of the C-repeat region of the GAS M protein or a fragment thereof, or is contained within the amino acid sequence of the C-repeat region of the GAS M protein or a fragment thereof. Non-limiting examples include p145, which is a 20-mer having the amino acid sequence LRRDLDASREAKKQVEKALE (SEQ ID NO: 56). In this regard, a fragment of the p145 amino acid sequence may be present in the J14 or J8 peptide.

As used herein, "J14 peptide" may comprise the amino acid sequence KQAEDKVKASREAKKQVEKALEQLEDRVK (SEQ ID NO: 57) or a fragment or variant thereof, a peptide having minimal B-cell and T-cell epitopes within p145, identified as a peptide of the C-region of the GAS M protein that does not contain potentially harmful T-cell self-epitopes but does contain opsonic B-cell epitopes. J14 is a chimeric peptide containing 14 amino acids from the C-region of the M protein (shown in bold) and flanked by yeast-derived GCN4 sequences that are essential for maintaining the correct helical folding and conformational structure of the peptide.

As used herein, a "J8 peptide" is a peptide comprising an amino acid sequence derived at least in part from or corresponding to a C-region peptide of the GAS M protein. The J8 peptide suitably comprises a B-cell conformational epitope and lacks potentially harmful T-cell self-epitopes. A preferred J8 peptide amino acid sequence is QAEDKVKQKQLEDKVQ (SEQ ID NO: 58) or a fragment or variant thereof, wherein the bold residues correspond to residues 344 to 355 of the GAS M protein. In this embodiment, J8 is a chimeric peptide that further comprises flanking GCN4 DNA-binding protein sequences that assist in maintaining the correct helical folding and conformational structure of the J8 peptide.

As used herein, a protein "variant" shares a definable nucleotide or amino acid sequence relationship with a reference amino acid sequence. The reference amino acid sequence can be the amino acid sequence of the M protein, SpyCEP or a fragment thereof as described above. A "variant" protein can be one or more amino acid deletions or replacements with different amino acids in the reference amino acid sequence. It is well known in the art that some amino acids can be replaced or deleted without changing the activity of the immunogenic fragment and/or protein (conservative substitutions). Preferably, the protein variant shares at least 70% or 75%, preferably at least 80% or 85%, or more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the reference amino acid sequence.

In a specific embodiment, variant protein or peptide can comprise one or more lysine residues at its N-terminus and/or C-terminus.Described multiple lysine residues (such as polylysine) can be a linear sequence of lysine residues, or can be a branched sequence of lysine residues.These other lysine residues can promote the solubility of increased peptide.

Non-limiting examples of J8 peptide variants include:

S R E A K K Q S R E A K K Q V E K A L K Q V E K A L C (SEQ ID NO: 59)

S R E A K K Q S R E A K K Q V E K A L K Q S R E A K C (SEQ ID NO: 60)

S R E A K K Q V E K A L K Q S R E A K K Q V E K A L C (SEQ ID NO: 61)

S R E A K K Q V E K A L D A S R E A K K Q V E K A L C (SEQ ID NO: 62)

Other variants may be based on heptads as described by Cooper et al., 1997.

For example, if an epitope is known to be located within the structural conformation of an α-helical protein, a model peptide can be synthesized to fold into that conformation. Based on the structure of the GCN4 leucine zipper (O'Shea et al., 1991), we designed a model α-helical coiled coil peptide. The first seven residues contain the sequence MKQLEDK (SEQ ID NO: 63), which contains several features found in the coiled coil seven-residue repeating motif (a-b-c-d-e-f-g)n that stabilizes the coil (Cohen & Parry, 1990). These features include large non-polar residues at positions a and d, acid/base pairs (Glu/Lys) at positions e and g (which generally facilitate ionic interactions between chains), and polar groups at positions b, c, and f (consistent with the predictions of Lupas et al. (1991)). The GCN4 peptide also contains a consensus valine at position a. It is also noted that when positions a and d are occupied by V and L, a coiled coil dimer is favored (Harbury et al., 1994). The model seven-residue repeat is derived from these common features of the GCN4 leucine zipper peptide (VKQLEDK; SEQ ID NO: 64), which has the potential to form an α-helical coiled coil. This peptide becomes the framework peptide. Overlapping fragments of the conformational epitope under study are embedded in the coiled coil peptide of the model to generate a chimeric peptide. Whenever the same residue is found in both the helical model peptide and the epitope sequence, an amino acid substitution (Cohen & Parry, 1990) designed to ensure the correct helical coiled coil conformation is incorporated into the chimeric peptide. The following substitutions are typically used: position a, V is substituted with I; position b, K is substituted with R; position c, Q is substituted with N; position d, L is substituted with A; position e, E is substituted with Q; position f: D is substituted with E; position g, K is substituted with R. All of these substituted residues are commonly found at their respective sites in the coiled coil protein (Lupas et al., 1991).

Terms commonly used herein to describe the sequence relationships between proteins and nucleic acids include "comparison window", "sequence identity", "percentage of sequence identity" and "substantial identity". Since each nucleic acid/protein can each (1) contain only one or more parts of the complete nucleic acid/protein sequence shared by the nucleic acid/protein, and (2) contain one or more parts that are different between nucleic acids/proteins, sequence comparison is generally performed by comparing sequences over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of typically 6, 9 or 12 consecutive residues that is compared to a reference sequence. For optimal alignment of each sequence, the comparison window can include additions or deletions (i.e., gaps) of about 20% or less compared to the reference sequence. The best sequence alignment of the comparison window can be carried out by computerized implementation of algorithm (Geneworks program of Intelligenetics; GAP, BESTFIT, FASTA and TFASTA in Wisconsin genetics software package 7.0, Genetics Computer Group, 575Science Drive Madison, WI, USA, incorporated herein by reference) or by inspection and by the best comparison (that is, producing the highest homology percentage on the comparison window) produced by any one of the various methods selected. It can also be referred to as such as Altschul etc., 1997, the BLAST program family disclosed in Nucl.Acids Res.25 3389, which is incorporated herein by reference. The detailed discussion of sequence analysis is found in Unit 19.3 of " Current Protocols in Molecular Biology Compilation " (John Wiley＆Sons Inc NY, 1995-1999) written by the people such as Ausubel.

The term "sequence identity" is used herein with its broadest implication, to include accurate Nucleotide or amino acid matching number, and it relates to the suitable alignment using standard algorithms, relates to the degree of identity of the sequence in the comparison window. Therefore, by following calculation " sequence identity percentage ratio ": at the sequence of two best alignments in the comparison window, determine that the position number of the identical nucleic acid base (as, A, T, C, G, I) all present in two sequences is to obtain the position number of coupling, the position number of coupling is divided by the total position number (that is, window size) in the comparison window, and the result is multiplied by 100 to obtain the sequence identity percentage ratio. For example, " sequence identity " can be understood as meaning by DNASIS computer program (for windows, version 2.5; Can be available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) " matching percentage ratio " calculated.

As understood in the art, "derivatives" as used herein are molecules such as proteins, fragments or variants thereof that have been altered, for example, by binding or complexing with other chemical moieties, post-translational modifications (e.g., phosphorylation, acetylation, etc.), glycosylation modifications (e.g., addition, removal or alteration of glycosylation) and lipidation modifications and/or the inclusion of additional amino acid sequences. In a specific embodiment, the additional amino acid sequence may comprise one or more lysine residues at its N-terminus and/or C-terminus. The plurality of lysine residues (e.g., polylysine) may be a linear sequence of lysine residues, or may be a branched sequence of lysine residues. These additional lysine residues may promote the solubility of the increased peptide.

A specific J8 peptide derivative is a "lipid core peptide" as described in Olive et al., 2002, Infect & Immun. 70 2734. In one embodiment, the lipid core peptide may comprise a plurality of J8 peptides (e.g., four J8 peptides) synthesized directly on the two amino groups of each lysine of a branched polylysine core coupled to a lipophilic anchor.

Other amino acid sequences can include the amino acid sequence of a fusion partner that generates a fusion protein. For example, the amino acid sequence of a fusion partner can help detect and/or purify the fusion protein. Non-limiting examples include metal binding fusion partners (e.g., polyhistidine), maltose binding protein (MBP), protein A, glutathione S-transferase (GST), fluorescent protein sequences (e.g., GFP), epitope tags (e.g., myc, FLAG, and hemagglutinin tags).

Other derivatives contemplated by the present invention include, but are not limited to, modifications to side chains, incorporation of unnatural amino acids and/or their derivatives during peptide or protein synthesis, and the use of cross-linking agents and other methods of imposing conformational constraints on the immunogenic proteins, fragments and variants of the invention.

In this regard, for broader methodology involving the chemical modification of proteins, one is referred to Chapter 15 of Coligan et al., ed., CURRENT PROTOCOLS IN PROTEIN SCIENCE (John Wiley & Sons NY 1995-2008).

Isolated immunogenic proteins, fragments and/or derivatives of the present invention can be produced by any method known in the art including, but not limited to, chemical synthesis, recombinant DNA technology, and proteolytic cleavage to produce peptide fragments.

Chemical synthesis includes solid phase synthesis and liquid phase synthesis. Such methods are well known in the art, although reference is made to the examples of chemical synthesis techniques provided in Chapter 15 of " Synthetic Vaccines " (SYNTHETIC VACCINES) (Blackwell Scientific Publications) edited by Nicholson and " Current Protein Science Experiment Guide " (CURRENT PROTOCOLS IN PROTEIN SCIENCE) (John Wiley & Sons, Inc.NY USA 1995-2008) edited by Coligan et al. In this regard, reference is also made to International Publication WO 99/02550 and International Publication WO 97/45444.

Recombinant proteins can be readily prepared by one skilled in the art using standard protocols as described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, 1989), in particular Sections 16 and 17; in Ausubel et al., eds., Current Protocols in Molecular Biology (John Wiley & Sons, Inc. NY USA 1995-2008), in particular Chapters 10 and 16; and in Coligan et al., eds., Current Protocols in Protein Science (John Wiley & Sons, Inc. NY USA 1995-2008), in particular Chapters 1, 5, and 6. Typically, production of a recombinant protein involves the expression of a nucleic acid encoding the protein in a suitable host cell.

Certain aspects and embodiments relate to administering one or more nucleic acids encoding fragments, variants, or derivatives thereof and substances that promote restoration or enhancement of neutrophil activity, or compositions comprising the one or more nucleic acids, to elicit an immune response to GAS and/or generate immunity against GAS infection.

As used herein, the term "nucleic acid" refers to single-stranded or double-stranded DNA and RNA. DNA includes genomic DNA and cDNA. RNA includes mRNA, RNA, RNAi, siRNA, cRNA and autocatalytic RNA. Nucleic acid can also be a DNA-RNA hybrid. Nucleic acid comprises a nucleotide sequence that typically comprises nucleotides comprising A, G, C, T or U bases. However, the nucleotide sequence can comprise other bases, such as modified purines (e.g., inosine, methylinosine and methyladenosine) and modified pyrimidines (e.g., thiouridine and methylcytosine).

In a preferred form, one or more isolated nucleic acids encoding fragments of the M protein, variants or derivatives thereof and substances that promote restoration or enhancement of neutrophil activity are in the form of a genetic construct suitable for administration to a mammal (e.g., a human). In a preferred form, the genetic construct is suitable for DNA vaccination of a mammal (e.g., a human).

Suitably, as is well known in the art, the genetic construct is in the form of a plasmid, phage, clay, yeast or bacterial artificial chromosome, or comprises a genetic component of a plasmid, phage, clay, yeast or bacterial artificial chromosome. The genetic construct can also be suitable for maintaining and propagating the isolated nucleic acid in bacteria or other host cells, suitable for manipulation by recombinant DNA techniques.

For the purpose of protein expression, genetic construct is an expression construct. Suitably, the expression construct comprises one or more nucleic acids operably connected to one or more other sequences in an expression vector. An "expression vector" can be a self-replicating extrachromosomal vector (such as a plasmid), or a vector that is integrated into the host genome.

"Operably linked" means that the additional nucleotide sequence is positioned relative to the nucleic acid of the present invention so as to preferably initiate, regulate or otherwise control transcription.

Regulatory nucleotide sequences are generally suitable for the host cells or tissues in which expression is desired. For various host cells, numerous types of suitable expression vectors and suitable regulatory sequences are known in the art.

Typically, the one or more regulatory nucleotide sequences may include, but are not limited to, a promoter sequence, a leader sequence or signal sequence, a ribosome binding site, a transcription start and stop sequence, a translation start and stop sequence, and an enhancer or activator sequence. The present invention contemplates constitutive or inducible promoters known in the art. As described above, the expression construct may also include additional nucleotide sequences encoding a fusion partner (usually provided by an expression vector) to express the recombinant protein of the present invention as a fusion protein.

It will be understood that the isolated nucleic acids encoding the M protein, fragment, variant or derivative thereof and the substance that promotes restoration or enhancement of neutrophil activity (e.g., SpyCEP or an immunogenic fragment thereof) can be administered as separate expression constructs or can be present in the same expression construct (e.g., a multicistronic expression construct).

Suitably, DNA vaccination is in the form of one or more plasmid DNA expression constructs. Plasmids typically contain viral promoters (such as SV40, RSV or CMV promoters). Intron A can be included to improve the stability of mRNA, thereby increasing protein expression. Plasmids can also contain multiple cloning sites, strong polyadenylation signal/transcription termination signal (such as bovine growth hormone or rabbit beta-globulin polyadenylation sequence). Plasmids can also contain Mason-Fisher monkey virus cis-acting transcription element (MPV-CTE), which has or does not have the envelope expression that HIV rev increases. Other modifications that can enhance expression include the insertion of enhancer sequences, synthetic intron sequences, adenovirus tripartite leader (TPL) sequences and/or the modification of polyadenylation and/or transcription termination sequences. The limiting examples of DNA vaccine plasmids is pVAC, which is commercially available from Invivogen.

A useful reference describing DNA vaccinology is DNA Vaccines, Methods and Protocols, Second Edition (Volume 127 of Methods in Molecular Medicine series, Humana Press, 2006).

As described above, the present invention provides compositions and/or methods for preventing or treating Group A Streptococcus-associated diseases, disorders, or conditions in mammals.

As used herein, "treating," "treat," or "treatment" refers to therapeutic intervention that at least partially ameliorates, eliminates, or alleviates the symptoms or pathological signs of a Group A Streptococcus-associated disease, disorder, or condition after it has begun to develop. Treatment need not be absolutely beneficial to the subject. Beneficial effects can be determined using any method or criteria known to those of ordinary skill in the art.

As used herein, "preventing," "prevent," or "prevention" refers to a process of action initiated prior to infection or exposure to Group A Streptococcus and/or prior to the onset of symptoms or pathological signs of a Group A Streptococcus-associated disease, disorder, or condition, to prevent infection and/or alleviate symptoms or pathological signs. It should be understood that such prevention need not be absolutely beneficial to the subject. "Prophylactic" treatment refers to treatment administered to a subject who does not exhibit signs of a Group A Streptococcus-associated disease, disorder, or condition, or to a subject who exhibits only early signs, for the purpose of reducing the risk of developing symptoms or pathological signs of a Group A Streptococcus-associated disease, disorder, or condition.

In the context of the present invention, "Group A Streptococcus-associated disease, disorder or condition" means any clinical pathology resulting from infection with Group A Streptococcus and includes, although not limited to, cellulitis, erysipelas, impetigo, scarlet fever, throat infections such as acute pharyngitis ("strep throat"), bacteremia, toxic shock syndrome, necrotizing fasciitis, acute rheumatic fever, and acute glomerulonephritis.

As described above, the treatment and/or immunization methods disclosed herein comprise administering to a mammal, alone or in combination, a fragment, variant, or derivative of an M protein and a substance that promotes the restoration or enhancement of neutrophil activity, thereby eliciting an immune response in the mammal. Alternatively, as described above, the treatment and/or immunization methods comprise administering, alone or in combination, one or more isolated nucleic acids encoding a fragment, variant, or derivative of an M protein and a substance that promotes the restoration or enhancement of neutrophil activity, thereby eliciting an immune response in the mammal.

As disclosed herein, other specific aspects and embodiments of the invention relate to the use of antibodies or antibody fragments to therapeutically treat GAS infection, such as by targeting SpyCEP to the site of infection (e.g., the skin). This can be combined with immunization with fragments of the M protein and/or administration of antibodies or antibody fragments against fragments of the M protein.

Antibodies and antibody fragments can be polyclonal or monoclonal, natural or recombinant. Antibody fragments include Fc, Fab or F (ab) ₂ fragments and/or can include single-chain Fv antibodies (scFv). Such scFv can be prepared, for example, according to the methods described in the article of U.S. Patent No. 5,091,513, European Patent No. 239,400 or Winter & Milstein, 1991, Nature 349:293. Antibodies can also include multivalent recombinant antibody fragments containing multiple scFv and dimerization-activated demibody (such as WO/2007/062466), such as double-chain antibodies (diabody), three-chain antibodies (triabody) and/or four-chain antibodies (tetrabody). For example, such antibodies can be prepared according to the methods described in Holliger et al., 1993 Proc Natl Acad Sci USA 90 6444; or Kipriyanov, 2009 Methods Mol Biol 562 177. Well-known protocols suitable for the production, purification and use of antibodies can be found, for example, in Chapter 2 of Coligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY (John Wiley & Sons NY, 1991-1994) and Harlow, E. & Lane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor, Cold Spring Harbor Laboratory, 1988.

Methods for producing polyclonal antibodies are well known to those skilled in the art. Exemplary protocols that can be used are described, for example, in Coligan et al., Current Protocols in Immunology, supra, and Harlow & Lane, 1988, supra. In specific embodiments, anti-SpyCEP polyclonal antibodies can be obtained or purified from human serum from individuals exposed to or infected with Group A Streptococcus. Alternatively, polyclonal antibodies can be elicited in a producing species, such as horses, from purified or recombinant SpyCEP or an immunogenic fragment thereof, and subsequently purified prior to administration.

Can use such as for example & Milstein, 1975, Nature 256, the standard method originally described in the article of 495, or by such as above Coligan et al. " the latest immunology experimental guide " (CURRENTPROTOCOLS IN IMMUNOLOGY) described in the most recent improvement to the standard method, by making the splenocyte or other antibody producing cell immortalized from the production species to produce monoclonal antibodies, the production species has been inoculated with one or more of the isolated protein, fragment, variant or derivative of the present invention. Therefore, for the purposes of the present invention, monoclonal antibodies can be induced by fragments, variants or derivatives of M protein and/or promote recovery or enhance neutrophil activity (such as SpyCEP). In certain embodiments, monoclonal antibodies or their fragments can be recombinant forms. If monoclonal antibodies are initially produced by the splenocytes of non-human mammals, recombinant forms may be particularly advantageous for making the monoclonal antibodies or fragments "humanized".

For embodiments involving therapeutic antibodies, a preferred fragment of the M protein may be the p145 peptide.

Preferred fragments of SpyCEP may comprise or consist of the amino acid sequence NSDNIKENQFEDFDEDWENF (SEQ ID NO: 18).

In certain aspects and embodiments, fragments, variants, or derivatives of the M protein and substances that promote restoration or enhancement of neutrophil activity, including antibodies or antibody fragments disclosed herein, may be administered to a mammal alone or in combination as a composition.

In preferred forms, the composition comprises an acceptable carrier, diluent or excipient.

"Acceptable carrier, diluent or excipient" means a solid or liquid filler, diluent or encapsulating material that can be safely used for systemic administration. Depending on the specific route of administration, a variety of carriers, diluents and excipients well known in the art can be used. These can be selected from the group consisting of sugars, starches, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solution, emulsifiers, isotonic saline and salts such as inorganic acid salts (including hydrochlorides, bromates and sulfates) and organic acid salts (such as acetates, propionates and malonates), water and pyrogen-free water.

An available reference describing acceptable carriers, diluents, and excipients is Remington's Pharmaceutical Sciences (Mack Publishing Co. N.J. USA, 1991), which is incorporated herein by reference.

Preferably, for the purpose of eliciting an immune response, certain immunological substances may be used in combination with the J8 peptide, fragment, variant or derivative and substances that promote restoration or enhancement of neutrophil activity, or with one or more genetic constructs encoding these.

As is well known in the art, the term "immunological substance" includes within its scope carriers, delivery materials, immunostimulants and/or adjuvants. As understood in the art, immunostimulants and adjuvants refer to or include one or more substances that enhance the immunogenicity and/or efficacy of a composition. Non-limiting examples of suitable immunostimulants and adjuvants include squalane and squalene (or other oils of plant or animal origin); block copolymers; detergents (such as -80); A, mineral oils (such as Drakeol or Marcol), vegetable oils (such as peanut oil); adjuvants derived from coryneform bacteria (such as Corynebacterium parvum); adjuvants derived from propionibacteria (such as Propionibacterium acne); Mycobacterium bovis (BCG or BCG); Bordetella pertussis pertussis antigens; tetanus toxoid; diphtheria toxoid; surfactants such as hexadecylamine, octadecylamine, octadecyl amino acid esters, lysolecithin, dimethyldioctadecyl ammonium bromide, N,N-dicoctadecyl-N′,N′-bis(2-hydroxyethyl-propylenediamine), methoxyhexadecylglycerol, and complex polyols; polyamines such as pyranose, dextran sulfate, and poly IC carbopol; peptides such as muramyl dipeptide and derivatives, dimethylglycine, and tuftsin; oily emulsions; and mineral gels such as aluminum phosphate, aluminum hydroxide, or alum; interleukins such as interleukin-2 and interleukin-12; monokines such as interleukin-1; tumor necrosis factor; interferons such as interferon-gamma; immunostimulatory DNA (such as CpG DNA), immunostimulatory combinations (such as saponin-aluminum hydroxide or Quil-A aluminum hydroxide); liposomes; and adjuvants; mycobacterial cell wall extracts; synthetic glycopeptides, such as muramyl dipeptide or other derivatives; Avridine; lipid A derivatives; dextran sulfate; DEAE-dextran alone or with aluminum phosphate; carboxypolymethylene, such as Carbopol'EMA; acrylic acid copolymer emulsions, such as Neocryl A640 (e.g., U.S. Patent No. 5,047,238); water-in-oil emulsifiers, such as Montanide ISA 720; poliovirus proteins, vaccinia proteins, or animal poxvirus proteins; or mixtures thereof.

Immunological substances can include carriers such as thyroglobulin; albumins such as human serum albumin; toxins, toxoids, or any mutant cross-reactive materials (CRMs) from toxins of tetanus, diphtheria, pertussis, Pseudomonas, Escherichia coli, Staphylococcus, and Streptococcus; polyamino acids such as poly(lysine:glutamic acid); influenza virus; rotavirus VP6, parvovirus VP1 and VP2; hepatitis B virus core protein; hepatitis B virus recombinant vaccine, etc. Alternatively, a fragment or epitope of a carrier protein or other immunogenic protein can be used. For example, a T cell epitope of a bacterial toxin, toxoid, or CRM can be used. In this regard, reference can be made to U.S. Patent No. 5,785,973, which is incorporated herein by reference.

Any suitable procedure for producing vaccine compositions is contemplated. Exemplary procedures include those described in, for example, New Generation Vaccines (1997, Levine et al., Marcel Dekker, Inc. New York, Basel, Hong Kong), which is incorporated herein by reference.

In some embodiments, compositions and vaccine can be administered to mammals in the form of attenuation or inactivated bacteria, and the attenuation or inactivated bacteria can be genetically modified to express J8 peptide, fragment, variant or derivative and/or promote recovery or enhance the material of neutrophil activity. The limiting examples of attenuated bacteria include Salmonella (Salmonella) species, such as Salmonella enterica var.Typhimurium or Salmonella typhi (Salmonella typhi). Alternatively, other enteropathogens such as Shigella (Shigella) species or Escherichia coli can be used in the form of attenuation. Attenuated Salmonella strains are constructed by inactivating genes in the aromatic amino acid biosynthetic pathway (Alderton et al., Avian Diseases 35435), introducing mutations into two genes in the aromatic amino acid biosynthetic pathway (as described in U.S. Pat. No. 5,770,214), or into other genes such as htrA (as described in U.S. Pat. No. 5,980,907), or into genes encoding outer membrane proteins such as ompR (as described in U.S. Pat. No. 5,851,519).

Any safe route of administration may be employed, including oral administration, rectal administration, parenteral administration, sublingual administration, buccal administration, intravenous administration, intraarticular administration, intramuscular administration, intradermal administration, subcutaneous administration, inhalation administration, intraocular administration, intraperitoneal administration, intracerebroventricular administration, topical administration, mucosal administration, and transdermal administration, although not limited thereto.

Dosage form comprises tablet, dispersant, suspension, injection, solution, syrup, lozenge, capsule, nasal spray, suppository, aerosol, transdermal patch etc.These dosage forms can also comprise specially designed injection or implantable controlled release device for this purpose, or be improved with other implant forms that act in addition in this way.Controlled release may be subject to the influence of hydrophobic polymer coating, and described hydrophobic polymer comprises acrylic resin, wax, higher fatty alcohol, polylactic acid and polyglycolic acid and some cellulose derivative (such as hydroxypropyl methylcellulose).In addition, controlled release may be subject to the influence of using other polymer matrix, liposome and/or microsphere.

The composition may be in the form of discrete units such as capsules, sachets, functional foods/feeds or tablets, each containing a predetermined amount of one or more therapeutic agents of the present invention; powders or granules; or solutions or suspensions in aqueous liquids, non-aqueous liquids, oil-in-water emulsions or oil-in-water liquid emulsions. Such compositions may be prepared by any pharmaceutical method, but all methods include the step of combining one or more substances as described above with a carrier constituting one or more necessary ingredients. Typically, the composition is prepared by uniformly or intimately mixing the substance of the present invention with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired presentation.

The above compositions can be administered in a manner compatible with the dosage form and in an effective amount. In the context of the present invention, the dosage administered to the patient should be sufficient to produce a beneficial response in the patient over an appropriate period of time. The amount of the substance to be administered can depend on the subject to be treated, including age, sex, weight, and overall health status thereof (a factor that depends on the judgment of the professional).

As used generally herein, the terms "patient," "individual," and "subject" are used in the context of any mammalian recipient of the treatments or compositions disclosed herein. Thus, the methods and compositions disclosed herein may have medical and/or veterinary applications. In a preferred form, the mammal is a human.

In order that the present invention may be more fully understood and put into practical application, reference is made to the following non-limiting examples.

Example

Materials and methods

Animals: BALB/c mice (female, 4-6 weeks old) were obtained from the Animal Resource Centre (ARC, Perth, Western Australia). All protocols were approved by the Animal Ethics Committee of Griffith University in accordance with the guidelines of the Australian National Health and Medical Research Council (NHMRC).

Bacterial strains and culture media: A number of GAS isolates obtained from various sources were used in this study. Streptococcus pyogenes M1 (emm1), 88/30 (emm97), BSA10 (emm124), and NS27 (emm91), NS1 (emm100), and 90/31 (emm57) were obtained from the Menzies School of Health Research, Darwin, Australia. Strain 5448AP (emm1) was obtained from the laboratory of Professor Mark Walker (University of Queensland, Brisbane, Australia). All strains were passaged in mice and made resistant to streptomycin (200 μg/ml) by serially placing them in increasing concentrations of streptomycin. To prepare the challenge inoculum, the GAS strain was grown in liquid medium containing Todd-Hewitt broth (THB; Oxoid, Australia) supplemented with 1% neopeptone (Difco) at 37° C. To determine the bacterial bioburden after infection, samples were plated on blood agar plates consisting of the liquid medium described above with 2% agar, 200 μg/ml streptomycin, and 2% horse blood.

Peptide Synthesis and Vaccine Formulation: Peptide J8 was synthesized and conjugated to DT from Auspep Pty Ltd (Australia) as described elsewhere (Batzloff et al., 2003). Recombinant SpyCEP was expressed and purified by GenScript USA Inc. All peptides were stored lyophilized or in solution at -80°C. Mice were vaccinated with a 30 μg/mouse dose of J8-DT or recombinant SpyCEP. For vaccination with the combined protein-peptide conjugate vaccine, 30 μg of the peptide conjugate J8-DT and 30 μg of recombinant SpyCEP were mixed per mouse and adsorbed onto aluminum hydroxide (Alhydrogel, alum) in a 1:1 (v/v) ratio.

Establishment of superficial skin infection model: In order to develop a superficial skin infection model for GAS, female BALB/c of inbred strain and Swiss mice of outbred strain (4-6 weeks old) were used. Mice were anesthetized by intraperitoneal (IP) injection (100 μl/10g mouse) of ketamine (100 mg/ml stock solution)/Xylazil-20 (20 mg/ml stock solution)/water in a ratio of 1:1:10. The fur of the back haunch of the mouse was removed using a trimming tool and a razor. The skin was cleaned with an ethanol swab and then mechanically scratched with the aid of a metal file. After the skin was scratched, the mice were infected with GAS. An inoculum (20 μl) of GAS containing a known CFU count was topically applied to the scratched skin. Once the inoculum was completely absorbed by the skin, the injured area was temporarily covered and the mice were raised in separate cages. In addition to the group of superficial infection, a group of mice infected with alveolar infection was used as a positive control. The mice were infected according to the method of Raeder and Boyle (1993). The mice were monitored daily for infection lesions and signs of disease according to an approved scoring table. The wound site was closely monitored to evaluate the status of the infection.

Histological sections: On the 3rd day after infection, scratch mice with or without GAS skin infection were sacrificed (n=3 per group). Tissue skin samples from the infection site were collected, fixed with formalin buffer, and embedded in paraffin for hematoxylin & eosin (H&E) staining. They were then cut into 5 μm thick tissue sections and stained with H&E and Giemsa and Gram stains to visualize Gram-positive organisms. The sections were scanned and read at high magnification using ImageScope software. For immunohistochemistry, the samples were frozen in OCT. Histological examinations were performed at different time points after neutrophil and macrophage depletion. Positive cells were counted in five areas of the scanned slides using ImageJ (National Institutes of Health, Bethesda, MD, USA) and expressed as the average number of positive cells per 10,000 ^μm .

Mouse immunization and challenge protocol: On day 0, BALB/c mice were immunized subcutaneously at the base of the tail with 30 μg J8-DT, 30 μg rSpyCEP, or 60 μg of a formulation containing 30 μg J8-DT and 30 μg rSpyCEP. All antigen preparations in PBS were formulated in aluminum gel as an adjuvant. Mice were challenged on days 21 and 28. Control mice received adjuvant only. Two weeks after the last immunization, mice were challenged with GAS using the skin infection route. After infection, mice were closely monitored and any mice showing signs of disease were sacrificed (based on a scoring table approved by the GU Animal Ethics Committee).

Sample collection and CFU determination: At defined time points after infection (days 3, 6, and 9), 5 mice were selected from each group. Blood samples were collected via cardiac puncture, and skin tissue was excised from the infected lesions located on the buttocks of infected mice. The skin samples were weighed and homogenized in saline. Appropriate dilutions were then inoculated on streptomycin blood agar plates in duplicate to determine the bacterial load at the infected lesions. The blood samples were diluted in PBS, and the appropriate dilutions were repeatedly inoculated on streptomycin blood agar plates to determine the bacterial load in the blood.

Cell depletion studies: Macrophages were depleted via IP administration of carrageenan (CGN) as previously described (Goldmann et al., 2004). To deplete skin macrophages, CGN was injected subcutaneously every 72 hours, resulting in >95% depletion of skin-resident macrophages. Flow cytometric analysis of splenocytes was used to optimize the dose and time course of CGN injections after labeling with FITC-conjugated anti-mouse Mac-1 and APC-conjugated F4/80 (BD Biosciences, New Jersey, USA). To deplete neutrophils, an anti-Ly6G mAb (clone 1A8) was used as previously described (Eyles et al., 2008). Neutrophil depletion was confirmed by flow cytometric analysis of blood, bone marrow, and spleen cells using CD11b-perCp-cy5.5 and Gr-1-APC mAbs.

Analysis of in vitro chemokine degradation: As previously described (Hidalgo-Grass et al., 2004), IL-8, MIP-2 and KC degradation were performed and quantified using a Quantikine kit (R&D systems, Minneapolis, MN, USA) by ELISA. The method was used to measure the amount of undegraded chemokines (IL-8, MIP-2 and KC) after incubation with GAS culture supernatant (S/N). In brief, in order to collect culture S/N, various GAS strains were grown to mid-logarithmic phase (OD ₆₀₀ 0.5), inoculated again into fresh THB and grown overnight at 37°C. The cell-free GAS culture S/N from each strain was then incubated at 37°C with recombinant chemokines (IL-8, MIP-2 and KC) of known concentrations. Samples were collected at 2 hours, 4 hours, 8 hours or 24 hours, and as described above, the amount of undegraded chemokines was determined by ELISA (R&D Systems).

Isolation and transwell migration analysis of neutrophils: Neutrophils were isolated from mouse bone marrow using a neutrophil isolation kit (Miltenyi Biotech, Germany). Neutrophils (2.5×10 ⁵ in 100 μl culture medium) were added to the upper chamber of a transwell system (Costar 24-well transwell, Corning NY) and then placed in the lower chamber containing a separate culture medium or complete or degraded chemokines. As a positive control, wells containing known concentrations of each recombinant chemokine were also used. After incubation at 37°C for 2 hours, cells were collected from the upper and lower chambers, and the number of live neutrophils transferred was determined using trypan blue exclusion. The percentage of migrated neutrophils was calculated by dividing the number of migrated neutrophils by the total number of neutrophils present.

SpyCEP Neutralization Assay: To evaluate the SpyCEP neutralization capacity of rSpyCEP antiserum, hyperimmune sera against rSpyCEP were generated in BALB/C mice. A panel of GAS strains, including 90/31, BSA10, and 5448AP, were grown to stationary phase. Cell-free GAS culture supernatants were incubated with recombinant chemokines and 50% normal serum or anti-SpyCEP serum at 37°C for 16 hours. Uncleaved IL-8 was measured using a Quantikine ELISA kit (R&D System).

SpyCEP epitope mapping: A peptide array containing 553 amino acids from the N-terminal region of SpyCEP was synthesized at GenScript (Genscript USA Inc). Of the 55 total peptides (SEQ ID NOs: 1-55), 20-mers were blotted onto a membrane in overlapping 10s. Following the ELISA protocol, the membrane was probed with antisera from mice immunized three times with 30 μg/dose of the rSpyCEP/Alum preparation. The six peptides most strongly recognized by the SpyCEP antiserum were selected for further investigation (SEQ ID NOs: 15, 18, 19, 24, 30, and 54).

Peptide Synthesis and Vaccine Formulation: At GenScript, six peptides (each 20-mer) identified by epitope mapping (as discussed above) were synthesized as free peptides or with an additional cysteine residue at the C-terminus. The C-terminal peptides were then conjugated to DT and used in mice for in vivo immunogenicity studies. All peptides were stored lyophilized or in solution at -20°C. Mice were vaccinated three times with rSpyCEP or SpyCEP peptide (S1-S6)-DT conjugates at a dose of 30 μg/mouse.

Determination of immunogenicity of each peptide: Serum samples were collected before and after each stimulation to determine the antibody titer against the immunizing peptide and against the parent protein (rSpyCEP). To determine the titer of peptide-specific IgG, plates were coated with 5 μg/ml of each of the six peptides (S1-S6). ELISA was performed using 2-fold serial dilutions of the antisera induced by each peptide. To determine the recognition of SpyCEP peptides by rSpyCEP, 2-fold serial dilutions of the rSpyCEP antisera were used. Finally, to determine the recognition of the parent peptide by the peptide antisera, plates were coated with rSpyCEP and the peptide antisera were used to assess their binding/recognition to rSpyCEP.

In vitro IL-8 protection assay: To evaluate the ability of peptide antisera to inhibit IL-8 degradation, an in vitro IL-8 protection assay was performed. GAS culture supernatants (S/N) were incubated with known concentrations of recombinant IL-8 and 1:2 dilutions of peptide antisera (S1-S6). The amount of undegraded IL-8 in the reaction mixture was quantified by ELISA using a Quantikine kit (R&D systems, Minneapolis, MN, USA) as previously described (Hidalgo-Grass et al., 2004). Briefly, to collect culture S/N, each GAS strain was grown to mid-logarithmic phase (OD ₆₀₀ 0.5), re-inoculated into fresh THB, and grown overnight at 37°C. Cell-free GAS culture S/N from each strain was then incubated with known concentrations of recombinant chemokine (IL-8) at 37°C in the presence or absence of antisera from each peptide. Samples were collected after 16 hours of incubation, and the amount of undegraded chemokine was determined by ELISA (R&D Systems) as described above.

Peptide inhibition ELISA: For peptide inhibition ELISA analysis, microtiter plates were coated overnight at 4°C with 100 μl of peptides S1 to S6 or recSpyCEP (in 75 mM sodium carbonate buffer (pH 9.6) at a final concentration of 5 μg/ml. The plates were washed with buffer (PBS containing 0.05% Tween 20, pH 7.2) and blocked at 37°C for 90 minutes with a buffer supplemented with 5% skim milk (blocking buffer). Anti-peptide or recSpyCEP serum was pre-incubated at 37°C for 30 minutes with 5 μg/ml or 2.5 μg/ml of each peptide or recSpyCEP. Thereafter, the test antiserum was added to the blocking plate and incubated at 37°C for 90 minutes. The plate was washed four times, and HRP-conjugated goat anti-mouse IgG (Biorad, Australia) was added and incubated at 37°C for another 90 minutes. After additional washes, plates were plated and OD450 was measured as described above.

Flow cytometry analysis: The binding of recSpyCEP or SpyCEP epitope antibodies to the GAS cell surface was analyzed by flow cytometry. Bacteria were grown overnight in THB containing 1% neopeptone and washed in PBS. Thereafter, bacteria (1×10 ⁷ colony forming units; cfu) were pre-incubated with 100 μl Fc blocker at room temperature for 15 minutes to block nonspecific binding sites. Peptide antiserum was then added at a dilution of 1:20. After incubation at room temperature for 1 hour, the bacteria were washed twice in PBS and then incubated with FITC-conjugated anti-mouse IgG (diluted 1/50 in PBS containing 2% BSA). Finally, the bacteria were washed and incubated in 1% formaldehyde (in PBS) at room temperature for 15 minutes. Samples were analyzed on a CyAn ADP analyzer (Beckman Coulter, Inc.). FITC-conjugated anti-mouse IgG alone was added as a negative control for each strain analyzed, and in addition, nonspecific mouse IgG served as a control.

Challenge model for evaluating vaccine efficacy: Inbred female BALB/c mice (4-6 weeks old) were anesthetized by intraperitoneal (IP) injection (100 μl/10 g mouse) of ketamine (100 mg/ml stock)/Xylazil-20 (20 mg/ml stock)/water in a ratio of 1:1:10. The hair on the neck of the mice was removed using a trimming tool and a razor. After a superficial scratch on the skin, a GAS inoculum (20 μl) containing 1×10 ⁶ CFU count was applied topically. Once the inoculum was completely absorbed by the skin, the injured area was temporarily covered and the mice were placed in individual cages for maintenance. Mice were fed a streptomycin (200 μg/ml) aqueous solution 24 hours before infection and maintained on a streptomycin (200 μg/ml) aqueous solution throughout the study. Mice were monitored daily for infection lesions and signs of disease according to a scoring table approved by the IBC of Griffith University. The injured area was closely monitored to evaluate the infection status.

Organ collection and CFU determination: At different time points after infection (days 3, 6, and 9), a limited number of mice from each group were sacrificed. Blood samples were collected by cardiac puncture, spleens were removed, and skin biopsies were obtained from the infected lesions located in the neck. Skin and spleen samples were homogenized and then appropriate dilutions were plated on streptomycin blood agar plates in duplicate. After infection, mice were closely monitored and any mice showing signs of disease (based on the scoring sheet) were sacrificed.

result

In Figure 1, the protective efficacy of J8-DT/Alum vaccination is shown by comparing different GAS strains, M1 (throat isolate), and skin isolates 88/30 and BSA10. Compared to M1, J8-DT/Alum vaccination was less effective against both skin isolates. As shown in Figure 2, the efficacy of J8 immunization correlated with the presence of neutrophils (PMNs) in the skin of GAS-infected mice.

Therefore, experiments were performed to measure the effect of neutrophil depletion on the response to J8-DT/Alum, the results of which are shown in Figure 3. Neutrophil depletion had a deleterious effect on the efficacy of J8-DT/Alum vaccination.

The data in Figures 1-3 show that neutrophils may play a role in determining the efficacy of J8 immunity against GAS infection. Figure 4 provides an overview of bacterial genes that are upregulated in GAS isolates with CovR/S mutations, such as 5448AP (M1T1 isolate). As shown in Figure 5, the protective efficacy of J8-DT/Alum against GAS isolate 5448AP is relatively weak. Given that neutrophils appear to promote the immune response to the J8 peptide, a candidate bacterial gene that is highly expressed in GAS isolate 5448AP is SpyCEP (70kD serine protease), which cuts and inactivates the neutrophil chemotactic substance interleukin 8 (see Figure 4). Figure 6 shows the results of the experiment, in which 5448AP exhibited particularly strong IL-8 degradation. The 5448AP isolate also exhibited strong degradation activity against the mouse functional homologs of IL-8: CXCL1/MIP-2 (Figure 7) and CXCL1/KC (Figure 8). Functionally, this degradation correlates with inhibition of neutrophil chemotaxis as shown in FIG9 .

The data in Figures 1-9 show that the serine protease SpyCEP is highly expressed in CovR/SCovR/S mutant GAS bacteria, which negatively regulates IL-8 by proteolytic degradation, thereby inhibiting or suppressing the chemotaxis of neutrophils. Therefore, experiments were conducted to determine whether targeting SpyCEP will improve, restore or increase the immune response to the J8 peptide. The results shown in Figure 10 show that the immunization performed with J8-DT-recombinant SpyCEP/Alum is far more effective than the immunization performed with J8-DT peptide-conjugate or recombinant SpyCEP alone, and the combination of J8 peptide and recombinant SpyCEP acts synergistically to protect against infection with 5448AP GAS bacteria. Further, Figure 11 shows that the immunization performed with J8-DT-rSpyCEP results in a stronger IgG antibody response than with separate J8-DT or SpyCEP. These data have created the possibility that antibodies against SpyCEP may be beneficial therapeutic agents that can be directly applied to the GAS infection site (skin) to treat infection. As shown in FIG12 , anti-SpyCEP antibodies (in the form of antisera from mice immunized with recombinant SpyCEP) inhibited the IL-8 degradation activity of SpyCEP.

We prepared a series of overlapping 20-mer peptides (peptide arrays on nitrocellulose membranes) (SEQ ID NO:1-55 in Figure 13) from the residue 35-587 of SpyCEP to determine which ones are recognized by the recSpyCEP antiserum. From the data in Figure 14, we identified 6 putative peptide epitopes (S1-6 with the amino acid sequence shown in Figure 15; SEQ ID NO:15,18,19,24,30 and 54). Then we prepared those individual synthetic peptides and conjugated each to DT. Mice were immunized, and we showed that antiserum can recognize recSpyCEP, although some antiserum is stronger than other antiserum (Figure 16). Then we carried out IL-8 protection analysis, and showed that anti-peptide antibodies can block the degradation of SpyCEP to IL-8 to varying degrees, and the antiserum of a peptide (S2; SEQ ID NO:18) can effectively block the degradation of IL8 (Figure 17) as well as anti-recSpyCEP. Therefore, we identified a dominant epitope on SpyCEP that can induce functional antibodies (SEQ ID NO: 18).

Data testing the immunogenicity of p145, J8, and J8 peptide variants designated as follows are shown in Figures 18-20:

●p145 LRRDLDASREAKKQVEKALE(SEQ ID NO:56)

●J8 QAEDKVKQSREAKKQVEKALKQLEDKVQ(SEQ ID NO:58)

●J8i V1 SREAKKQSREAKKQVEKALKQVEKALC(SEQ ID NO:59)

●J8i V2 SREAKKQSREAKKQVEKALKQSREAKC(SEQ ID NO:60)

●J8i V3 SREAKKQVEKALKQSREAKKQVEKALC(SEQ ID NO:61)

●J8i V4 SREAKKQVEKALDASREAKKQVEKALC (SEQ ID NO:62).

Hyperimmune serum was generated in female Balb/c mice aged 4-6 weeks using a 28-day immunization schedule. Mice were immunized subcutaneously with 30 mg of DT-conjugated peptide (J8, p145, J8iV1, J8iV2, J8iV3, or J8iV4) in PBS and Alum. Immunizations occurred on days 0, 21, and 28. Blood/serum was collected by submandibular bleeding on days 35, 42, and 49.

We have previously observed that antibodies induced by vaccination with J8-DT in mice and humans only weakly recognize the native sequence p145. Therefore, we designed four variant peptides based on the 12-mer J8 insert sequence. These variant peptides were designed to maintain a seven-residue periodicity of hydrophobic and hydrophilic amino acids, thereby maintaining the α-helical structure of the peptide. These are the J8iV1, J8iV2, J8iV3, and J8iV4 peptides listed above.

Figure 18 (right panel) shows that the titers of p145-specific antibodies induced by J8-DT vaccination were very low (average of approximately 2000, with titers below 200 in some mice). In contrast, the titers of J8-specific antibodies after J8-DT vaccination were approximately 600,000. The high titers against J8 must mean that most of the antibodies recognize non-streptococcal flanking sequences located in the amino-terminal and carboxyl-terminal segments of J8, although this has not yet been formally demonstrated.

Figure 19 shows that J8iV1, J8iV2, J8iV3 and J8iV4 all induce antibodies (about 100,000) of high titer for themselves.Importantly, all induce high titer (>10,000) for p145, wherein J8iV3-DT induces about 30,000 titer (Figure 20, right figure). Possibly, the titer of new peptide for itself is higher than the titer for p145, because p145 contains other amino acids that do not exist in the new peptide, and some of these may cover the epi-position that is identified by the antiserum for new variant. However, the high titer (about 100,000) of variant J8 peptide for itself is significant, because they exclusively derive from streptococcal sequence. Therefore, new variant induces 100,000 titer, wherein all antibodies all identify streptococcal sequence, yet J8 induces about 2,000 streptococcal titer.

The immunodominance of S2 was further assessed using peptide inhibition analysis. Antisera for recSpyCEP and each epitope were pre-incubated with each immune antigen, after which their binding to the antigen was analyzed. After pre-incubation with the self-peptide, the binding of the epitope antiserum to the immobilized self-peptide was greatly weakened (40-80% inhibition) (Figure 21 A). Pre-incubation of the epitope antiserum with recSpyCEP also resulted in inhibition of the recognition of the immobilized self-peptide, wherein when the antiserum for S2-DT was incubated with recSpyCEP, the highest inhibition (Figure 21 B) was observed, indicating that the epitope identified by the S2-DT immunity was presented on the surface of recSpyCEP. Moreover, when the antiserum for SpyCEP was incubated with each of the 6 peptides, we observed that peptide S2 (SEQ ID NO:18) inhibited binding to the greatest extent, and was comparable to the inhibition caused by recSpyCEP (Figure 21 C). These data indicate that the immune response to SpyCEP is primarily limited by antibody recognition of S2 (SEQ ID NO: 18) by ELISA, and that S2 (SEQ ID NO: 18) can elicit an immune response similar to that to the entire recSpyCEP. Therefore, S2 (SEQ ID NO: 18) is an immunodominant epitope of SpyCEP.

We then tested the binding efficacy of SpyCEP and epitope antisera to various GAS strains. SpyCEP is expressed on the surface of GAS and shed. To assess recognition of the native antigen, FACS analysis was used to compare the binding of different epitope-specific antibodies to various GAS strains. GAS strain 5448 and its animal-passaged derivative 5448AP (a CovR/S mutant known to express high levels of SpyCEP) were used together. Similarly, wild-type BSA10 and its animal-passaged derivative pBSA10 (also a CovR/S mutant) were used in parallel. Reference strain 2031 (emm1) and skin isolate 88/30 from the Northern Territory of Australia were also included. Our data showed that in all cases, the binding of antibodies induced by recSpyCEP vaccination to GAS isolates was comparable to the binding of antibodies induced by S2-DT immunization to GAS isolates. Antibodies against other epitopes showed variable levels of binding to GAS (Figures 22A, B, and C). Furthermore, as measured by mean fluorescence intensity (MFI) data, surface expression of SpyCEP and S2 was found to be highest among all tested strains (data not shown). These data confirm the immunodominance of the S2 epitope on native SpyCEP. However, the data do not indicate that antibodies against the S2 epitope will functionally impair the CXC chemokine protease of SpyCEP.

Then, we asked whether S2-DT could enhance the efficacy of the J8-DT vaccine. We have previously shown that the combination of recSpyCEP and J8-DT results in significantly better protection against invasive infection of the CovR/S mutant strain of GAS than independent J8-DT or independent recSpyCEP (manuscript submitted). J8-DT and recSpyCEP or S2-DT were mixed in a weight ratio of 1:1 and tested in our newly developed skin challenge model (manuscript submitted). Control mice were vaccinated with each antigen or PBS vaccine. After vaccination, mice were challenged with 5448AP GAS. Vaccination with J8-DT+S2-DT resulted in a significant reduction in the bacterial bioburden in the skin and blood of mice infected with 5448AP (Figures 23A and B). It was also found that the protection level provided by the J8-DT+S2-DT vaccination was comparable to that provided by the J8-DT+SpyCEP vaccination. To confirm these findings, the challenge experiment (Figures 24A and B) was repeated with another CovR/S mutant NS88.2. Again, we found that vaccination with J8-DT+SpyCEP or J8-DT+S2-DT resulted in significantly enhanced protection compared to J8-DT alone. Vaccination with J8-DT or SpyCEP alone did not provide any protection compared to the PBS control.

in conclusion

Neutrophil activity appears to be a key factor in the efficacy of vaccination with the J8 peptide. SpyCEP is an IL-8 degrading protease that is highly expressed by virulent GAS bacteria, such as those with the CovR/S mutation. Immunization with a combination of the J8 peptide and rSpyCEP has a synergistic effect on GAS infection caused by the CovR/S GAS mutant 5448AP, compared to either the J8 peptide or SpyCEP alone. It is also clear that anti-SpyCEP antibodies can effectively neutralize the IL-8 degrading activity of SpyCEP, thereby providing a therapeutic intervention for existing GAS infections. Moreover, we have identified a dominant epitope on SpyCEP that can induce functional antibodies (SEQ ID NO: 18). We are currently planning active and passive vaccine studies using GAS stimulation. The advantage of this epitope is that it enables us to avoid the use of the entire recombinant SpyCEP protein in the vaccine, thereby improving the safety profile. We believe that the optimal vaccine is a mixture of J8 and this epitope. In confirmation of this prediction, vaccination with J8-DT+SpyCEP or J8-DT+S2-DT resulted in significantly enhanced protective efficacy compared to J8-DT alone. Furthermore, variant J8 peptides have been developed that induce high titers against both themselves and the Streptococcus peptide p145, presumably because they are exclusively derived from the amino acid sequence of Streptococcus p145.

Throughout the specification, the goal has been to describe preferred embodiments of the present invention without limiting the present invention to any one embodiment or specific collection of features. Various changes and modifications may be made to the embodiments described and illustrated herein without departing from the broad spirit and scope of the present invention.

All computer programs, algorithms, patents, and scientific literature mentioned herein are incorporated by reference in their entirety.

Claims (21)

1. The use of fragments of the M protein and substances that promote the restoration or enhancement of neutrophil activity in the preparation of drugs for inducing an immune response against group A streptococcal bacteria in mammals. The M protein fragment is a J8 variant peptide, which consists of an amino acid sequence selected from the following: SREAKKQSREAKKQVEKALKQVEKALC (SEQ ID NO: 59); SREAKKQSREAKKQVEKALKQSREAKC (SEQ ID NO: 60); SREAKKQVEKALKQSREAKKQVEKALC (SEQ ID NO: 61); and SREAKKQVEKALDASREAKKQVEKALC (SEQ ID NO: 62); The substance that promotes the recovery or enhancement of neutrophil activity is a fragment of SpyCEP protein or an antibody thereof, the fragment of SpyCEP protein consisting of the following amino acid sequence: NSDNIKENQFEDFDEDWENF (SEQ ID NO:18). 2. The use of fragments of the M protein and substances that promote the restoration or enhancement of neutrophil activity in the preparation of drugs for immunizing mammals against group A streptococcal bacteria. The M protein fragment is a J8 variant peptide, which consists of an amino acid sequence selected from the following: SREAKKQSREAKKQVEKALKQVEKALC (SEQ ID NO: 59); SREAKKQSREAKKQVEKALKQSREAKC (SEQ ID NO: 60); SREAKKQVEKALKQSREAKKQVEKALC (SEQ ID NO: 61); and SREAKKQVEKALDASREAKKQVEKALC (SEQ ID NO: 62); The substance that promotes the recovery or enhancement of neutrophil activity is a fragment of SpyCEP protein or an antibody thereof, the fragment of SpyCEP protein consisting of the following amino acid sequence: NSDNIKENQFEDFDEDWENF (SEQ ID NO:18). 3. Fragments of the M protein, or its antibodies or antibody fragments; and the use of substances that promote the restoration or enhancement of neutrophil activity in the preparation of medicaments for the treatment or prevention of group A streptococcal bacterial infections in mammals. The M protein fragment is a J8 variant peptide, which consists of an amino acid sequence selected from the following: SREAKKQSREAKKQVEKALKQVEKALC (SEQ ID NO: 59); SREAKKQSREAKKQVEKALKQSREAKC (SEQ ID NO: 60); SREAKKQVEKALKQSREAKKQVEKALC (SEQ ID NO: 61); and SREAKKQVEKALDASREAKKQVEKALC (SEQ ID NO: 62); The substance that promotes the recovery or enhancement of neutrophil activity is a fragment of SpyCEP protein or an antibody thereof, the fragment of SpyCEP protein consisting of the following amino acid sequence: NSDNIKENQFEDFDEDWENF (SEQ ID NO:18). 4. The use as described in any one of claims 1 to 3, comprising a fragment encoding an M protein and one or more isolated nucleic acids encoding a substance that promotes the restoration or enhancement of neutrophil activity, or a composition comprising said one or more isolated nucleic acids. 5. The use as described in any one of claims 1 to 3, wherein the substance that promotes the recovery or enhancement of neutrophil activity is a fragment of the SpyCEP protein, the fragment of the SpyCEP protein directly or indirectly inhibiting or suppressing neutrophils or neutrophil activity. 6. The use according to any one of claims 1 to 3, wherein the substance that promotes the recovery or enhancement of neutrophil activity is an antibody or antibody fragment of a SpyCEP protein, said antibody or antibody fragment binding to a protein or fragment thereof that directly or indirectly inhibits or blocks neutrophil activity. 7. The use as described in any one of claims 1 to 3, wherein the neutrophil activity is the chemotaxis of neutrophils. 8. The use as described in any one of claims 1 to 3, wherein the SpyCEP protein or a fragment thereof is a protease or a fragment thereof that hydrolyzes and inactivates interleukin-8 protein. 9. The use as claimed in any one of claims 1 to 3, wherein the mammal is a human. 10. A composition comprising a fragment of the M protein, or an antibody or antibody fragment thereof; and a substance that promotes the restoration or enhancement of neutrophil activity. The M protein fragment is a J8 variant peptide, which consists of an amino acid sequence selected from the following: SREAKKQSREAKKQVEKALKQVEKALC (SEQ ID NO: 59); SREAKKQSREAKKQVEKALKQSREAKC (SEQ ID NO: 60); SREAKKQVEKALKQSREAKKQVEKALC (SEQ ID NO: 61); and SREAKKQVEKALDASREAKKQVEKALC (SEQ ID NO: 62); The substance that promotes the recovery or enhancement of neutrophil activity is a fragment of SpyCEP protein or an antibody thereof, the fragment of SpyCEP protein consisting of the following amino acid sequence: NSDNIKENQFEDFDEDWENF (SEQ ID NO:18). 11. The composition of claim 10, comprising a fragment encoding the M protein and one or more isolated nucleic acids encoding the substance that promotes the restoration or enhancement of neutrophil activity, or a combination comprising the one or more isolated nucleic acids. 12. The composition of claim 10 or 11, wherein the substance that promotes the restoration or enhancement of neutrophil activity is a fragment of SpyCEP protein, the fragment of SpyCEP protein directly or indirectly inhibiting or suppressing neutrophil activity. 13. The composition of claim 10 or 11, wherein the substance that promotes the restoration or enhancement of neutrophil activity is an antibody or antibody fragment of a SpyCEP protein, said antibody or antibody fragment binding to a protein or fragment thereof that directly or indirectly inhibits or blocks neutrophil activity. 14. The composition of claim 10 or 11, wherein the neutrophil activity is the chemotaxis of neutrophils. 15. The composition of claim 10 or 11, wherein the SpyCEP protein or a fragment thereof is a protease or a fragment thereof that hydrolyzes and inactivates interleukin-8 protein. 16. An isolated peptide, said isolated peptide consisting of the following amino acid sequence: NSDNIKENQFEDFDEDWENF (SEQ ID NO: 18). 17. An antibody or antibody fragment that binds to, or is induced by, the isolated peptide of claim 16. 18. An isolated nucleic acid encoding the isolated peptide of claim 16. 19. A genetic construct comprising the isolated nucleic acid as described in claim 18. 20. A host cell comprising the genetic construct of claim 19. 21. A composition comprising the isolated peptide of claim 16; and/or one or more antibodies or antibody fragments of claim 17; and/or the isolated nucleic acid of claim 18; and/or the genetic construct of claim 19; and/or the host cell of claim 20.