Immunization against group a streptococcus (GAS)

Abstract

A composition comprising isolated Streptococcus pyogenes extracellular vesicles (EVs) suspended in a vehicle, for use in eliciting an adaptive immune response against group A streptococcus (GAS) in a subject. The EVs may comprise antigens MtsA, Spy_1228, PrsA1, PrsA2, SPy_0319 and / or GlnP. A composition comprising isolated S. pyogenes protein(s) MtsA, SPy_1228, PrsA1, PrsA2, GlnP, SPy_0319 and / or MalX. The compositions may be used for inducing protective immunity in a subject against a GAS infection, such as streptococcal pharyngitis, scarlet fever, impetigo, streptococcal meningitis, streptococcal sinusitis, necrotizing fasciitis, cellulitis, streptococcal toxic shock syndrome, rheumatic fever and post-streptococcal glomerulonephritis.

Description

[0001] IMMUNIZATION AGAINST GROUP A STREPTOCOCCUS (GAS)

[0002] TECHNICAL FIELD

[0003] The present invention relates to means and methods for inducing adaptive immune response against group A streptococcus.

[0004] BACKGROUND TO THE INVENTION

[0005] Group A Streptococcus (GAS, also known as Streptococcus pyogenes), is a Gram-positive bacterium that is responsible for a wide range of both non-invasive and invasive diseases. Non-invasive diseases include conditions such as pharyngitis (strep throat) and impetigo, while invasive diseases include necrotizing fasciitis (flesh-eating disease) and streptococcal toxic shock syndrome.

[0006] GAS infections pose a significant global health burden. According to the World Health Organization, it is estimated that GAS diseases cause over half a million deaths annually, primarily in low-income and middle-income countries. Non-invasive diseases such as pharyngitis and impetigo are common worldwide, with millions of cases reported each year. Invasive diseases, although less common, are more severe and can be fatal.

[0007] Rheumatic heart disease (RHD), a complication of rheumatic fever which itself is a complication of untreated strep throat, is a significant contributor to the global disease burden of GAS. It is estimated that there are over 30 million cases of RHD worldwide, resulting in about 300,000 deaths annually.

[0008] The treatment of GAS infections depends on the severity and type of infection. For non- invasive diseases like strep throat and impetigo, oral antibiotics such as penicillin or amoxicillin are typically prescribed. Invasive GAS diseases require more aggressive treatment, often involving intravenous antibiotics and sometimes surgery. In the case of necrotizing fasciitis, surgical debridement of the infected tissue is usually necessary. For individuals with a history of rheumatic fever or RHD, long-term antibiotic prophylaxis is recommended to prevent recurrent GAS infections and further damage to the heart valves.

[0009] GAS vaccines

[0010] Vaccine development for GAS has been a challenging due to several factors. Firstly, GAS is characterized by a high degree of strain diversity. There are over 200 known serotypes of GAS, each with different M proteins, the major virulence factor of the bacteria. This diversity makes it difficult to develop a single vaccine that can provide broad protection against all strains. Secondly, there is a risk of autoimmune reactions. Some of the proteins in GAS are similar to human proteins, and there is a concern that a vaccine could trigger an immune response not only against the bacteria but also against the body's own tissues. This is particularly relevant given the link between GAS infections and autoimmune diseases such as rheumatic heart disease. Thirdly, there are technical challenges associated with vaccine production. For example, producing a multivalent vaccine that includes multiple M proteins is technically complex and costly. Lastly, there are regulatory and commercial challenges. The path to regulatory approval for a new vaccine is long and expensive, and there are somewhat limited commercial incentives for vaccine development given that GAS diseases predominantly affect low-income countries.

[0011] Nonetheless, there are currently at least eight candidates on a product development track, including four M protein-based candidates and four candidates designed around non-M protein antigens (reviewed by Walkinshaw et al. npj Vaccines (2023) 8:16; doi.org / 10.1038 / s41541-023-00609-x).

[0012] Extracellular vesicles

[0013] Group A Streptococcus (GAS) is known to produce extracellular vesicles (EVs) containing macromolecules such as RNAs, proteins and lipids (Resch et al,. 2016 mBio Vol 7 Issue 6 e00207-16). Bacterial EVs have been proposed as vaccine antigens (WO2018 / 123959) and therapeutics (W02019 / 051380)

[0014] Thus, an object of the present invention is the provision of alternative or improved means and methods for inducing adaptive immune response against GAS.

[0015] DEFINITIONS

[0016] The term extracellular vesicles (EVs) refer to membrane-derived vesicles released by bacteria, in particular Streptococcus pyogenes, such as strain 5448AP. The EVs can carry many different types of macromolecules, virulence factors and proteins, both soluble and membrane associated.

[0017] The term antigen refers to structures to which antibodies generated by the adaptive immune system of vertebrates specifically bind, or to which specific T-cell responses are elicited. Antigens are often proteins, peptides or polysaccharides (sometimes containing a lipid moiety). In the context of vaccines, an antigen is an ingredient in a vaccine against which a specific antibody or T-cell response is intended to be elicited in the host to which the vaccine is administered. Some vaccines contain several antigens. In the vaccine context, a vaccine antigen generally bears a structural resemblance to a pathogen antigen present in a pathogen against which the vaccine is intended to be used. A vaccine antigen may for example be a fragment or a modified version of the pathogen antigen, or a complete pathogen antigen presented in a non-pathogenic form e.g., as an inactivated pathogen or a recombinant protein produced in a suitable host.

[0018] The term adjuvant refers to a component added to vaccines and other immunogenic compositions that increases the effectiveness of the immune response to the antigen present in the vaccines or other immunogenic composition.

[0019] The term Group A Streptococcus (GAS) refers to the bacterium Streptococcus pyogenes.

[0020] MtsA refers to Streptococcus pyogenes iron ABC transporter substrate-binding lipoprotein which is part of the ATP-binding cassette (ABC) transport system MtsABC involved in iron import (Uniprot entry: P0A4G4). A reference sequence from Streptococcus pyogenes is presented in SEQ ID NO 1. In the context of the present invention, the MtsA protein may comprise a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, yet more preferably at least 95%, even more preferably at least 98%, most preferably complete sequence identity to SEQ ID NO: 1, or an immunologically effective fragment thereof.

[0021] SPy_1228 refers to a Streptococcus pyogenes plasma membrane lipoprotein of unknown function (Uniprot entry: Q99ZH4). A reference sequence from Streptococcus pyogenes is presented in SEQ ID NO 2. The term BMP is used interchangeably herein. In the context of the present invention, the SPy_1228 protein may comprise a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, yet more preferably at least 95%, even more preferably at least 98%, most preferably complete sequence identity to SEQ ID NO: 2, or an immunologically effective fragment thereof.

[0022] PrsAl and PrsA2 refer to Streptococcus pyogenes parvulin-type peptidyl-prolyl isomerase 1 and 2, respectively (Uniprot entries P60811 and P60812). These foldase proteins play a major role in protein secretion by helping the post-translocational extracellular folding of several secreted proteins. These proteins have been implicated as virulence factors and play a role in biofilm formation, host adherence, and infection-induced cytotoxicity. Reference sequences from Streptococcus pyogenes are presented in SEQ ID NO: 3 (PrsAl) and SEQ ID NO: 4 (PrsA2). In the context of the present invention, the PrsAl protein may comprise a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, yet more preferably at least 95%, even more preferably at least 98%, most preferably complete sequence identity to SEQ ID NO: 3, or an immunologically effective fragment thereof. Similarly, in the context of the present invention, the PrsA2 protein may comprise a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, yet more preferably at least 95%, even more preferably at least 98%, most preferably complete sequence identity to SEQ ID NO: 4, or an immunologically effective fragment thereof.

[0023] GlnP refers to Streptococcus pyogenes ABC transmembrane type-1 domain-containing protein (Uniprot entry: Q99ZA4). A reference sequence from Streptococcus pyogenes is presented in SEQ ID NO 5. In the context of the present invention, the GlnP protein may comprise a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, yet more preferably at least 95%, even more preferably at least 98%, most preferably complete sequence identity to SEQ ID NO: 5, or an immunologically effective fragment thereof.

[0024] MalX is a maltose / maltodextrin-binding protein required for the uptake of maltose (a disaccharide of glucose) and maltodextrin (glucose oligosaccharides) from the host environment. Streptococcal MalX homologues are present in most streptococcal species including Streptococcus pyogenes (Uniprot Q99ZB1), Streptococcus pneumoniae, Streptococcus suis, Streptococcus e ui, Streptococcus mitis, and Streptococcus pseudopneumoniae. MalX is a lipoprotein and as such attached to a lipid (diacylglycerol) in the outer leaflet of the plasma membrane through covalent thioester bond linkage via its N- terminal cysteine residue of the mature protein. A reference sequence from Streptococcus pyogenes is presented in SEQ ID NO 6. In the context of the present invention, the MalX protein may comprise a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, yet more preferably at least 95%, even more preferably at least 98%, most preferably complete sequence identity to SEQ ID NO: 6, or an immunologically effective fragment thereof.

[0025] SPy_0319 refers to a poorly characterized protein classified as part of ABC-type transport system and periplasmic component / surface antigen (Uniprot entry Q9A1E4). It shares sequence similarity with lipoproteins involved in the transport of methionine. A reference sequence from Streptococcus pyogenes is presented in SEQ ID NO 7. The terms NIpA and MetQ are used synonymously herein to refer to the same protein. In the context of the present invention, the SPy_0319 protein may comprise a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, yet more preferably at least 95%, even more preferably at least 98%, most preferably complete sequence identity to SEQ ID NO: 7, or an immunologically effective fragment thereof.

[0026] The term protective immunity in the present context refers to immunization measures resulting in any degree of reduction in the likelihood of developing the condition for which the protective immunity is relevant, including a minor, substantial or major reduction in likelihood of developing the condition as well as total prevention. Preferably, the degree of likelihood reduction is at least a minor reduction.

[0027] The term sequence identity expressed in percentage is defined as the value determined by comparing two optimally aligned sequences over a comparison window, wherein a portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Unless indicated otherwise, the comparison window is the entire length of the sequence being referred to. In this context, optimal alignment is the alignment produced by the BLASTP algorithm as implemented online by the US National Center for Biotechnology Information (see The NCBI Handbook, 2ndedition [Available online at www.ncbi.nlm.nih.gov / books / NBK143764 / ]), with the following input parameters: Word length=3, Matrix=BLOSUIVI62, Gap cost=ll, Gap extension cost=l. The term isolated, when used herein in reference to a protein, refers to a protein that has been substantially separated, purified, or enriched from its natural environment, such as a whole cell, whole cell extract, or other complex biological matrix in which it is naturally found. An "isolated" protein exists in a form that is significantly more concentrated or pure than in its native state, having undergone one or more processes such as enrichment, purification, or recombinant production. For example, an isolated protein may be obtained through techniques including, but not limited to, chromatography, centrifugation, precipitation, or recombinant expression in a host cell followed by purification, resulting in a preparation where the protein may be substantially free of other cellular components, contaminants, or biomolecules present in the original cellular or extract context. The term "isolated" does not encompass a protein as it exists in an intact whole cell or in a crude whole cell extract where it remains in its naturally occurring concentration and environment without deliberate separation, concentration or enrichment.

[0028] The term isolated, when used in reference to extracellular vesicles (EVs) produced by bacterial cells, refers to EVs that have been substantially separated, purified, or enriched from their natural environment, such as the bacterial cells that produce them, the surrounding culture medium, or crude bacterial cell extracts. An "isolated" extracellular vesicle exists in a form that is significantly more concentrated or purified than in its native state, having undergone one or more processes such as enrichment, purification, or controlled production to separate the EVs from other cellular components, debris, or extracellular matrix materials. Such processes may include, but are not limited to, centrifugation, ultrafiltration, size-exclusion chromatography, or affinity-based separation techniques. The term "isolated" does not encompass extracellular vesicles as they exist in their naturally occurring state within a bacterial culture, associated with the surface of bacterial cells, or in an unprocessed bacterial cell supernatant where they remain in their native concentration and environment without deliberate separation, concentration or enrichment.

[0029] The term "comprising" is to be interpreted as including, but not being limited to.

[0030] Numerical values in the specification and claims of this application should be understood to include numerical values which are the same when reduced to the same number of significant figures and numerical values which differ from the stated value by less than the experimental error of conventional measurement technique of the type described in the present application to determine the value.

[0031] All ranges disclosed herein are inclusive of the recited endpoint and independently combinable (for example, the range of "from 2 to 10" is inclusive of the endpoints, 2 and 10, and all the intermediate values).

[0032] The term "about" can be used to include any numerical value that can vary without changing the basic function of that value. When used with a range, "about" also discloses the range defined by the absolute values of the two endpoints, e.g. "about 2 to about 4" also discloses the range "from 2 to 4." The term "about" may refer to plus or minus 10% of the indicated number.

[0033] BRIEF DESCRIPTION OF THE FIGURES

[0034] Figure 1. Characterization of GAS EVs. a) Cryo-EM image of GAS EVs isolated from liquid media, b) GAS EV size distribution determined by dynamic light scattering.

[0035] Figure 2. GAS-specific IgG and IgA response in sera and nasal wash, a) Immunization scheme. 5-week-old male C57BL / 6 mice were immunized intranasally with 50 pl of GAS EVs (10 pg) formulated with aluminium hydroxide (alum, lOmg / ml). The control group was immunized with alum in PBS. b) GAS-specific IgG and IgA levels from serum (left) and nasal wash (right), respectively, were analyzed by whole bacteria ELISA. Bars represent mean + standard error of the mean (SEM) of three independent experiments. Each symbol represents data from an individual mouse. Significance was determined by two-tailed unpaired t-test (IgG's) or Mann-Whitney test (IgA's). **, P < 0.01; ***, P <10.001.

[0036] Figure 3. Intranasal immunization with GAS EVs enhances recruitment of activated CD4+T cells to the lung. Mice were immunized as described in Figure 2a. Lung samples were collected 7 days after the last immunization. The percentage and total number of CD4+T cells (upper panels) and CD44+CD4+T cells (lower panels) was determined by flow cytometry. Bars represent mean + SEM of two independent experiments. Each symbol represents data from an individual mouse. Significance was determined by two-tailed unpaired t-test (% and # of CD44+CD4+T cells) or Mann-Whitney test (% and # of CD4+T cells). ***, P < 0.001; ****, P < 0.001. Figure 4. Intranasal immunization with GAS EVs elicits Thl7 and Thl responses in the lung.

[0037] Mice were immunized as described in Figure 2a. Lung samples were collected 7 days after the last immunization and stained for flow cytometry analysis (a,b,d and e) or cultured ex vivo to measure cytokines response to antigen stimulation (c and f). a) Percentage (%) and total number (#) of I L-17+CD44+CD4+T cells, b) % of RORyT+CD44+CD4+T cells, d) % and # of I FNy+CD44+CD4+T cells, e) % of T-bet+CD44+CD4+T cells. Lung cells were cultured ex vivo and stimulated with GAS EVs or media for 72h. Secretion of Thl7-specific IL17A (c) and Thl-specific I FNy (f) cytokines into the culture supernatant was measured by ELISA. Data are normalized to media stimulation controls. Bars represent mean + SEM of two (flow cytometry) or three (cytokines) independent experiments. Each symbol represents data from an individual mouse. Significance was determined by two-tailed unpaired t-test (% and

[0038] # of IL-17+T cells, % of I FNy+and RORyT+T cells) or Mann-Whitney test (# of IFNy+T cells, % of T-bet+T cells and IL-17A and IFNy secretion). **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

[0039] Figure 5. Intranasal immunization with GAS EVs induces a T-cell response in the spleen.

[0040] Mice were immunized as described in Figure 2a. Spleen samples were collected 7 days after the last immunization. The percentage (%) and total number (#) of I L-17+CD44+CD4+T cells (a) and I FNy+CD44+CD4+T cells (b) was determined by flow cytometry. Splenocytes were cultured ex vivo and stimulated with GAS EVs or media for 72h. Secretion of IL-17A (c) and IFNy (d) cytokines into the culture supernatant was measured by ELISA. Data are normalized to media stimulation controls. Bars represent mean + SEM of two (flow cytometry) or three (cytokines) independent experiments. Each symbol represents data from an individual mouse. Significance was determined by two-tailed unpaired t-test (# of I L-17+T cells, % and

[0041] # of I FNy+T cells and IFNy secretion) or Mann-Whitney test (% of I L-17+T cells and IL-17A secretion). *, P < 0.05; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

[0042] Figure 6. Identification of immunostimulatory antigens on GAS EVs. a) Western blot analysis of GAS EVs using sera from immunized mice detected at least six different proteins. The identity of these proteins was determined by co-immunoprecipitation coupled to mass- spectrometry (table to the right), b) antigen-specific IgG levels in sera vs recombinantly expressed MtsA, BMP, PrsAl, PrsA2, MetQ (SPy_0319) and GlnP were measured by ELISA. Bars represent mean + SEM. Statistical comparisons were made using Mann Whitney test. *,

[0043] P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.001; ns, not significant.

[0044] Figure 7. Identification of immunostimulatory antigens on GAS EVs. Isogenic mutants in each of the antigens were generated. The cell lysates of these mutants were probed by western blot using sera from immunized mice (left panel). GAPDH was used as a loading control. Right panel shows IgG binding to heat-inactivated GAS AP WT or an isogenic mutant lacking SPy_1228, MtsA and PrsA2 (GAS AP Aspy_1228 AmtsA, AprsA2), as tested by wholebacteria ELISA. The total IgG binding what slightly reduced for the mutant compared to the wild-type strain. Bars represent mean + SEM. Statistical comparisons were made using Mann Whitney test (*, P 0.05; **, P < 0.01; ***, P < 0.001, ****, P < 0.001).

[0045] Figure 8. IL-17A response to antigen stimulation. Lung cells (a) and splenocytes (b) from mice immunized with alum or GAS EVs (formulated with alum) were collected 1 week after the second immunization. Cells were cultured ex vivo and stimulated with 1 pg of recombinantly expressed proteins (MtsA, BMP (SPy_1228), PrsAl, PrsA2, MetQ (SPy_0319), GlnP or MalX) or media for 72h. The concentration of IL-17A in culture supernatants was measured by ELISA. Data are normalized to media stimulation controls and represented as mean + SEM of 2 independent experiments. Statistical comparisons were made using Mann Whitney test. **, P < 0.01; ***, P < 0.001; ****, P < 0.001.

[0046] Figure 9. IFNy response to antigen stimulation. Lung cells (a) and splenocytes (b) from mice immunized with alum or GAS EVs (formulated with alum) were collected 1 week after the second immunization. Cells were cultured ex vivo and stimulated with 1 pg of recombinantly expressed proteins (MtsA, BMP, PrsAl, PrsA2, MetQ (SPy_0319), GlnP or MalX) or media for 72h. The concentration of IFNy in culture supernatants was measured by ELISA. Data are normalized to media stimulation controls and represented as mean + SEM of 2 independent experiments. Statistical comparisons were made using Mann Whitney test. ***, P < 0.001.

[0047] Figure 10. Antigens identified in GAS EVs are conserved across GAS strains, a) Western blot analysis of GAS EVs and bacterial lysates of different invasive GAS isolates (M-type is indicated in brackets). Samples were probed with sera from GAS EVs-immunized mice (top panel) or anti-GAPDH as a loading control (bottom panel), b) IgG binding to different GAS strains was analyzed by whole-cell ELISA. Plates were coated with different invasive GAS strains, covering a range of M-types, and probed with sera from mice vaccinated with alum (light grey circles) or GAS EVs (blue circles). Bars represent mean + SEM of four independent experiments. Each symbol represents data from an individual mouse. Significance was determined by two-tailed unpaired t-test (S073, S221, S338 and S530) or Mann-Whitney test (AP, SOU, S137 and S150). ****, P < 0.0001.

[0048] Fig 11. Antigen specific IgG. Mice were immunized with adjuvant (AS02-IS ke), GAS EVs (30 ug formulated with adjuvant) or recombinant proteins (5 ug of MalX, PrsA2, PrsAl, SPy_1228, GlnP, MtsA or NIpA (SPy_0319), also formulated with AS02-like adjuvant - denoted "rProt"). Two doses were delivered subcutaneously 2 weeks apart. Two weeks after the second immunization, sera were collected and measured for antigen specific IgGs. Antibody levels for MtsA, SPy_1228, PrsAl, PrsA2, NIpA (SPy_0319), GlnP and MalX were measured by ELISA. Bars represent mean + SEM. Statistical comparisons were made using a two-tailed unpaired t-test. ***, P < 0.001; ****, p < 0.001. Immunization with recombinant proteins resulted in high IgG levels (slightly higher than immunization with EVs) for PrsA2, PrsAl, Spy_1228 and GlnP. For MtsA and NIpA, immunization with EVs resulted in higher titers than recombinant proteins. For MalX, immunization with protein generated a robust IgG response, whereas the immunization with EVs resulted in poor immune response.

[0049] SUMMARY OF THE INVENTION

[0050] The present invention relates to the following items. The subject matter disclosed in the items below should be regarded disclosed in the same manner as if the subject matter were disclosed in patent claims.

[0051] 1. A composition comprising isolated Streptococcus pyogenes extracellular vesicles (EVs) and a pharmaceutically acceptable vehicle, for use in eliciting an adaptive immune response against group A streptococcus (GAS) in a subject.

[0052] 2. A composition comprising isolated 5. pyogenes protein(s) MtsA, SPy_1228, PrsAl, PrsA2, GlnP, SPy_0319 and / or MalX for use in eliciting an adaptive immune response against group A streptococcus (GAS) in a subject.

[0053] 3. The composition for use according to item 1 or 2, wherein the immune response is a T-cell response, preferably a Thl7-response. The composition for use according to any of the preceding items, wherein the immune response is an IgG response, IgA response, or a combined IgG and IgA response. The composition for use according to any of the preceding items, wherein the composition further comprises an adjuvant. The composition for use according to any of the preceding items, wherein the composition further comprises an adjuvant selected from the group consisting of (i) aluminium hydroxide; (ii) aluminium phosphate; (iii) a lipid-based substance such as squalene or quillaja saponin; and (iv) a protein or other immune stimulating component from a microbe such as diphtheria (preferably CRM), cholera (preferably choleratoxoid CTB or mmCT) or Escherichia coli (preferably non-toxic heat-labile toxin such as LTK63 or dmLT). The composition for use according to any of the preceding items, wherein the composition comprises immunogenic amount(s) of the 5. pyogenes protein(s) MtsA, SPy_1228, PrsAl, PrsA2, GlnP, SPy_0319 and / or MalX as antigen(s). The composition for use according to any of the preceding items, wherein the composition comprises EVs in an amount of at least 1 pg / ml, preferably at least 5 pg / ml, more preferably at least 10 pg / ml, even more preferably 25 pg / ml, still more preferably 50 pg / ml, yet more preferably 75 pg / ml, most preferably at least 100 pg / ml. The composition for use according to item 1 or 8, or any item dependent thereon, wherein the EVs comprise immunogenic amount(s) of the 5. pyogenes protein(s) MtsA, SPy_1228, PrsAl, PrsA2, GlnP and / or SPy_0319. The composition for use according to item 1 or 8, or any item dependent thereon, wherein the EVs comprise one or more of the 5. pyogenes protein(s) MtsA, SPy_1228, PrsAl, PrsA2, GlnP and / or SPy_0319 at the level of >0.001 pg / mg, preferably >0.01 pg / mg, even more preferably >0.02 pg / mg, yet more preferably >0.05 pg / mg, most preferably >0.1 pg / mg of the total protein in the EVs. The composition for use according to item 1 or 8, or any item dependent thereon, wherein the EVs comprise an immunogenic amount of the 5. pyogenes protein MtsA, preferably at the level of >0.001 pg / mg, more preferably >0.01 pg / mg, even more preferably >0.02 pg / mg, yet more preferably >0.05 pg / mg, most preferably >0.1 pg / mg of the total protein in the EVs. The composition for use according to item 1 or 8, or any item dependent thereon, wherein the EVs comprise an immunogenic amount of the 5. pyogenes protein SPy_1228, preferably at the level of >0.001 pg / mg, more preferably >0.01 pg / mg, even more preferably >0.02 pg / mg, yet more preferably >0.05 pg / mg, most preferably >0.1 pg / mg of the total protein in the EVs. The composition for use according to item 1 or 8, or any item dependent thereon, wherein the EVs comprise an immunogenic amount of the 5. pyogenes protein PrsAl, preferably at the level of >0.001 pg / mg, more preferably >0.01 pg / mg, even more preferably >0.02 pg / mg, yet more preferably >0.05 pg / mg, most preferably >0.1 pg / mg of the total protein in the EVs. The composition for use according to item 1 or 8, or any item dependent thereon, wherein the EVs comprise an immunogenic amount of the 5. pyogenes protein PrsA2, preferably at the level of >0.001 pg / mg, more preferably >0.01 pg / mg, even more preferably >0.02 pg / mg, yet more preferably >0.05 pg / mg, most preferably >0.1 pg / mg of the total protein in the EVs. The composition for use according to item 1 or 8, or any item dependent thereon, wherein the EVs comprise an immunogenic amount of the 5. pyogenes protein GlnP, preferably at the level of >0.001 pg / mg, more preferably >0.01 pg / mg, even more preferably >0.02 pg / mg, yet more preferably >0.05 pg / mg, most preferably >0.1 pg / mg of the total protein in the EVs. The composition for use according to item 1 or 8, or any item dependent thereon, wherein the EVs comprise an immunogenic amount of the 5. pyogenes protein SPy_0319, preferably at the level of >0.001 pg / mg, more preferably >0.01 pg / mg, even more preferably >0.02 pg / mg, yet more preferably >0.05 pg / mg, most preferably >0.1 pg / mg of the total protein in the EVs. The composition for use according to item 1 or 8, or any item dependent thereon, wherein the EVs comprise the 5. pyogenes protein MalX, preferably at the level of >0.001 pg / mg, more preferably >0.01 pg / mg, even more preferably >0.02 pg / mg, yet more preferably >0.05 pg / mg, most preferably >0.1 pg / mg of the total protein in the EVs. 18. The composition for use according to any of the preceding items, wherein the composition comprises the 5. pyogenes protein(s) MtsA, SPy_1228, PrsAl, PrsA2, GlnP and / or SPy_0319 associated with EVs at a concentration of at least 0.0001 pg / ml, or in an increasing order of preference: at least 0.005 pg / ml, at least 0.001 pg / ml, at least 0.01 pg / ml, at least 0.03 pg / ml, at least 0.05 pg / ml, at least 0.08 pg / ml, at least 0.1 pg / ml, at least 0.3 pg / ml, at least 0.5 pg / ml, at least 0.8 pg / ml or at least 1 pg / ml.

[0054] 19. The composition for use according to any of the preceding items, wherein the composition comprises the 5. pyogenes protein MtsA at a concentration of at least 0.0001 pg / ml, or in an increasing order of preference: at least 0.005 pg / ml, at least 0.001 pg / ml, at least 0.01 pg / ml, at least 0.03 pg / ml, at least 0.05 pg / ml, at least 0.08 pg / ml, at least 0.1 pg / ml, at least 0.3 pg / ml, at least 0.5 pg / ml, at least 0.8 pg / ml or at least 1 pg / ml.

[0055] 20. The composition for use according to any of the preceding items, wherein the composition comprises the 5. pyogenes protein SPy_1228 at a concentration of at least 0.0001 pg / ml, or in an increasing order of preference: at least 0.005 pg / ml, at least 0.001 pg / ml, at least 0.01 pg / ml, at least 0.03 pg / ml, at least 0.05 pg / ml, at least 0.08 pg / ml, at least 0.1 pg / ml, at least 0.3 pg / ml, at least 0.5 pg / ml, at least 0.8 pg / ml or at least 1 pg / ml.

[0056] 21. The composition for use according to any of the preceding items, wherein the composition comprises the 5. pyogenes protein PrsAl at a concentration of at least 0.0001 pg / ml, or in an increasing order of preference: at least 0.005 pg / ml, at least 0.001 pg / ml, at least 0.01 pg / ml, at least 0.03 pg / ml, at least 0.05 pg / ml, at least 0.08 pg / ml, at least 0.1 pg / ml, at least 0.3 pg / ml, at least 0.5 pg / ml, at least 0.8 pg / ml or at least 1 pg / ml

[0057] 22. The composition for use according to any of the preceding items, wherein the composition comprises the 5. pyogenes protein PrsA2 at a concentration of at least 0.0001 pg / ml, or in an increasing order of preference: at least 0.005 pg / ml, at least 0.001 pg / ml, at least 0.01 pg / ml, at least 0.03 pg / ml, at least 0.05 pg / ml, at least 0.08 pg / ml, at least 0.1 pg / ml, at least 0.3 pg / ml, at least 0.5 pg / ml, at least 0.8 pg / ml or at least 1 pg / ml. The composition for use according to any of the preceding items, wherein the composition comprises the 5. pyogenes protein GlnP at a concentration of at least 0.0001 pg / ml, or in an increasing order of preference: at least 0.005 pg / ml, at least 0.001 pg / ml, at least 0.01 pg / ml, at least 0.03 pg / ml, at least 0.05 pg / ml, at least 0.08 pg / ml, at least 0.1 pg / ml, at least 0.3 pg / ml, at least 0.5 pg / ml, at least 0.8 pg / ml or at least 1 pg / ml. The composition for use according to any of the preceding items, wherein the composition comprises the 5. pyogenes protein SPy_0319 at a concentration of at least 0.0001 pg / ml, or in an increasing order of preference: at least 0.005 pg / ml, at least 0.001 pg / ml, at least 0.01 pg / ml, at least 0.03 pg / ml, at least 0.05 pg / ml, at least 0.08 pg / ml, at least 0.1 pg / ml, at least 0.3 pg / ml, at least 0.5 pg / ml, at least 0.8 pg / ml or at least 1 pg / ml. The composition for use according to any of the preceding items, wherein the composition comprises the 5. pyogenes protein MalX at a concentration of at least 0.0001 pg / ml, or in an increasing order of preference: at least 0.005 pg / ml, at least 0.001 pg / ml, at least 0.01 pg / ml, at least 0.03 pg / ml, at least 0.05 pg / ml, at least 0.08 pg / ml, at least 0.1 pg / ml, at least 0.3 pg / ml, at least 0.5 pg / ml, at least 0.8 pg / ml or at least 1 pg / ml. The composition for use according to any of the preceding items, wherein the composition comprises each of the 5. pyogenes protein(s) MtsA, SPy_1228, PrsAl, PrsA2, GlnP and SPy_0319 at a concentration of at least 0.0001 pg / ml, or in an increasing order of preference: at least 0.005 pg / ml, at least 0.001 pg / ml, at least 0.01 pg / ml, at least 0.03 pg / ml, at least 0.05 pg / ml, at least 0.08 pg / ml, at least 0.1 pg / ml, at least 0.3 pg / ml, at least 0.5 pg / ml, at least 0.8 pg / ml or at least 1 pg / ml. The composition for use according to any of the preceding items, wherein the composition is for use by injection (including intramuscular, intracutaneous, subcutaneous, intravenous), buccal administration, oral administration or intranasal administration. The composition for use according to any of the preceding items, wherein the composition further comprises one or more group A streptococcus antigen(s) in addition to MtsA, SPy_1228, PrsAl, PrsA2, GlnP, MalX and / or SPy_0319. 29. The composition for use according to any of the preceding items, wherein the composition is essentially free of Streptococcus pyogenes whole cells.

[0058] 30. The composition for use according to any of the preceding items, wherein the composition is essentially free of Streptococcus pyogenes whole cell lysate.

[0059] 31. The composition for use according to any of the preceding items, wherein the composition is an immunogenic composition.

[0060] 32. The composition for use according to any of the preceding items, wherein the composition is a vaccine.

[0061] 33. The composition for use according to any of the preceding items, for use in a method for inducing protective immunity in a subject against a GAS infection.

[0062] 34. The composition for use according to any of the preceding items, for use in a method for inducing protective immunity in a subject against colonization by GAS.

[0063] 35. The composition for use according to any of the preceding items, for use in a method for inducing protective immunity in a subject against a GAS infection selected from streptococcal pharyngitis, scarlet fever, impetigo, streptococcal meningitis, streptococcal sinusitis, necrotizing fasciitis, cellulitis, streptococcal toxic shock syndrome, rheumatic fever, and post-streptococcal glomerulonephritis.

[0064] The arrangement of the present disclosure into sections with headings and subheadings is merely to improve legibility and is not to be interpreted limiting in any way, in particular, the division does not in any way preclude or limit combining features under different headings and subheadings with each other. All references are hereby incorporated by reference.

[0065] DETAILED DESCRIPTION

[0066] The inventors have characterized Streptococcus pyogenes (GAS) extracellular vesicles (EVs) and found that following intranasal immunization with the EVs, both humoral and T-cell immune response was elicited in mice (Examples 1-4). Main immunostimulatory proteins were identified in Example 5. Thl7 immunological memory recall was also demonstrated (Example 6). The immunostimulatory antigens identified in GAS EVs were shown to be conserved across different M-types (Example 7). Using recombinant versions of the identified immunostimulatory GAS-proteins for immunization was demonstrated in Example 8. Compositions for use in eliciting an adaptive immune response against GAS

[0067] In a first aspect, the present invention provides a composition comprising isolated Streptococcus pyogenes extracellular vesicles (EVs) and a pharmaceutically acceptable vehicle, for use in eliciting an adaptive immune response against group A streptococcus (GAS) in a subject, preferably a human. The EVs may be suspended in the vehicle.

[0068] Using the EVs according to the first aspect as antigens has certain advantages. The EVs contain multiple proteins acting as antigens, ensuring a broad host exposure to relevant GAS antigens increasing the odds of protection against multiple strains. The EVs also contain bacterial components (both protein and non-protein) that may trigger host immune responses leading to more robust adaptive immune response (thus providing adjuvant-like effects).

[0069] In a second aspect, the present invention provides a composition comprising isolated 5. pyogenes protein(s) MtsA, SPy_1228, PrsAl, PrsA2, GlnP, SPy_0319 and / or MalX for use in eliciting an adaptive immune response against group A streptococcus (GAS) in a subject, preferably a human.

[0070] Isolated proteins may be advantageous in that they can be readily produced in large quantities in industrial processes (e.g. recombinantly) and may therefore be most cost- effective. When using several antigens, the relative amounts of the antigens are convenient to tailor when combining different isolated proteins in a composition.

[0071] The EVs according to the first aspect may be combined with one or more isolated proteins of the second aspect, as will be explained in more detail below. Such combination allows convenient adjustment of the relative amounts of the antigens.

[0072] Any combination of the 5. pyogenes protein(s) MtsA, SPy_1228, PrsAl, PrsA2, GlnP, SPy_0319 and / or MalX may be collectively referred to as "the antigen(s)" herein.

[0073] The immune response may be a T-cell response, preferably a Thl7-response. The immune response may alternatively or additionally be an IgG response, IgA response, or a combined IgG and IgA response.

[0074] The composition of the first or the second aspect may comprise EVs in an amount of at least 1 pg / ml, preferably at least 5 pg / ml, more preferably at least 10 pg / ml, even more preferably 25 pg / ml, still more preferably 50 pg / ml, yet more preferably 75 pg / ml, most preferably at least 100 pg / ml.

[0075] In certain embodiments, the composition of the first or the second aspect is free, essentially free or substantially devoid of Streptococcus pyogenes whole cells (live or killed). In certain embodiments, the composition contains less than 103Streptococcus pyogenes whole cells per ml, more preferably less than 102cells / ml, most preferably less than 10 cells / ml.

[0076] In certain embodiments, the composition is free, essentially free or substantially devoid of Streptococcus pyogenes whole cell lysate. In certain embodiments, the composition contains unfractionated whole cell lysate components corresponding to the constituents of less than 105Streptococcus pyogenes whole cells per ml, more preferably less than 103 / ml, most preferably less than 102 / ml. The composition of the first or the second aspect is preferably not an unfractionated Streptococcus pyogenes lysate.

[0077] The composition of the first or the second aspect may be formulated as an immunogenic composition or as a vaccine.

[0078] As a non-limiting example, the aforementioned EVs may be obtained as follows. EVs are produced culturing 5. pyogenes (e.g. strain 5448 AP) to late log phase in Todd Hewitt liquid media. Bacterial culture is centrifuged and filtered to obtain cell-free supernatant. Cell-free supernatant is concentrated by tangential flow filtration followed by ultracentrifugation. EVs are purified by density gradient and washed with PBS, using subsequent steps of ultracentrifugation. Finally, EVs are resuspended in a vehicle (see Example 1).

[0079] Adjuvants

[0080] The composition of the first or the second aspect may further comprise an adjuvant, many of which are known in the art. The adjuvant may be selected from aluminium hydroxide, aluminium phosphate, a lipid-based substance such as squalene or quillaja saponin, an immune-stimulating component or a protein from a microbe such as diphtheria (preferably CRM), cholera (preferably choleratoxoid CTB or mmCT) or Escherichia coli (preferably nontoxic heat-labile toxin such as LTK63 or dmLT). Immunogenic components

[0081] The composition of the first or the second aspect may comprise immunogenic amount(s) of the 5. pyogenes protein(s) MtsA, SPy_1228, PrsAl, PrsA2, GlnP, SPy_O319 and / or MalX as antigen(s). The aforementioned EVs may comprise immunogenic amount(s) of the 5. pyogenes protein(s) MtsA, SPy_1228, PrsAl, PrsA2, GlnP and / or SPy_O319 as antigens. The amount(s) may be according to an embodiment in Table 1.

[0082] TABLE 1: Minimum levels of designated protein antigens in various embodiments of the first aspect OR the second aspect of the invention. A blank cell indicates that the antigen is optional i.e. may be absent or present at any level. For the first aspect: the sign "+" denotes >0.001 pg / mg, preferably >0.01 pg / mg, even more preferably >0.02 pg / mg, yet more preferably >0.05 pg / mg, most preferably >0.1 pg / mg of the total protein in the EVs. For the second aspect: The sign "+" denotes a concentration of at least 0.0001 pg / ml, or in an increasing order of preference: at least 0.005 pg / ml, at least 0.001 pg / ml, at least 0.01 pg / ml, at least 0.03 pg / ml, at least 0.05 pg / ml, at least 0.08 pg / ml, at least 0.1 pg / ml, at least 0.3 pg / ml, at least 0.5 pg / ml, at least 0.8 pg / ml or at least 1 pg / ml.

[0083] The EVs may comprise one or more of the 5. pyogenes protein(s) MtsA, SPy_1228, PrsAl, PrsA2, GlnP and / or SPy_O319 at the level of >0.001 pg / mg, preferably >0.01 pg / mg, even more preferably >0.02 pg / mg, yet more preferably >0.05 pg / mg, most preferably >0.1 pg / mg of the total protein in the EVs.

[0084] The EVs may comprise an immunogenic amount of the 5. pyogenes protein MtsA, preferably at the level of >0.001 pg / mg, more preferably >0.01 pg / mg, even more preferably >0.02 pg / mg, yet more preferably >0.05 pg / mg, most preferably >0.1 pg / mg of the total protein in the EVs. The EVs may comprise an immunogenic amount of the 5. pyogenes protein SPy_1228, preferably at the level of >0.001 pg / mg, more preferably >0.01 pg / mg, even more preferably >0.02 pg / mg, yet more preferably >0.05 pg / mg, most preferably >0.1 pg / mg of the total protein in the EVs.

[0085] The EVs may comprise an immunogenic amount of the 5. pyogenes protein PrsAl, preferably at the level of >0.001 pg / mg, more preferably >0.01 pg / mg, even more preferably >0.02 pg / mg, yet more preferably >0.05 pg / mg, most preferably >0.1 pg / mg of the total protein in the EVs.

[0086] The EVs may comprise an immunogenic amount of the 5. pyogenes protein PrsA2, preferably at the level of >0.001 pg / mg, more preferably >0.01 pg / mg, even more preferably >0.02 pg / mg, yet more preferably >0.05 pg / mg, most preferably >0.1 pg / mg of the total protein in the EVs.

[0087] The EVs may comprise an immunogenic amount of the 5. pyogenes protein GlnP, preferably at the level of >0.001 pg / mg, more preferably >0.01 pg / mg, even more preferably >0.02 pg / mg, yet more preferably >0.05 pg / mg, most preferably >0.1 pg / mg of the total protein in the EVs.

[0088] The EVs may comprise an immunogenic amount of the 5. pyogenes protein SPy_0319, preferably at the level of >0.001 pg / mg, more preferably >0.01 pg / mg, even more preferably >0.02 pg / mg, yet more preferably >0.05 pg / mg, most preferably >0.1 pg / mg of the total protein in the EVs.

[0089] The EVs may comprise an immunogenic amount of the 5. pyogenes protein MalX, preferably at the level of >0.001 pg / mg, more preferably >0.01 pg / mg, even more preferably >0.02 pg / mg, yet more preferably >0.05 pg / mg, most preferably >0.1 pg / mg of the total protein in the EVs.

[0090] The composition of the first or the second aspect may comprise the 5. pyogenes protein(s) MtsA, SPy_1228, PrsAl, PrsA2, GlnP and / or SPy_0319 associated with the EVs at a concentration of at least 0.0001 pg / ml, or in an increasing order of preference: at least 0.005 pg / ml, at least 0.001 pg / ml, at least 0.01 pg / ml, at least 0.03 pg / ml, at least 0.05 pg / ml, at least 0.08 pg / ml, at least 0.1 pg / ml, at least 0.3 pg / ml, at least 0.5 pg / ml, at least 0.8 pg / ml or at least 1 pg / ml. Associated in this context may refer to covalent association, ionic association, lipid-anchoring or transmembrane configuration with the EVs.

[0091] Antigen concentrations

[0092] The 5. pyogenes protein(s) MtsA, SPy_1228, PrsAl, PrsA2, GlnP, SPy_0319 and / or MalX may additionally be present as non-EV associated form in the composition of the first aspect. For instance, purified recombinant or purified proteins can be added to the composition of the first aspect.

[0093] The concentrations specified below represent the total amount of the protein present in the composition, whether EV associated or not. The composition of the first or the second aspect may comprise of the 5. pyogenes protein MtsA at a concentration of at least 0.0001 pg / ml, or in an increasing order of preference: at least 0.005 pg / ml, at least 0.001 pg / ml, at least 0.01 pg / ml, at least 0.03 pg / ml, at least 0.05 pg / ml, at least 0.08 pg / ml, at least 0.1 pg / ml, at least 0.3 pg / ml, at least 0.5 pg / ml, at least 0.8 pg / ml or at least 1 pg / ml.

[0094] The composition of the first or the second aspect may comprise the 5. pyogenes protein SPy_1228 at a concentration of at least 0.0001 pg / ml, or in an increasing order of preference: at least 0.005 pg / ml, at least 0.001 pg / ml, at least 0.01 pg / ml, at least 0.03 pg / ml, at least 0.05 pg / ml, at least 0.08 pg / ml, at least 0.1 pg / ml, at least 0.3 pg / ml, at least 0.5 pg / ml, at least 0.8 pg / ml or at least 1 pg / ml.

[0095] The composition of the first or the second aspect may comprise the 5. pyogenes protein PrsAl at a concentration of at least 0.0001 pg / ml, or in an increasing order of preference: at least 0.005 pg / ml, at least 0.001 pg / ml, at least 0.01 pg / ml, at least 0.03 pg / ml, at least 0.05 pg / ml, at least 0.08 pg / ml, at least 0.1 pg / ml, at least 0.3 pg / ml, at least 0.5 pg / ml, at least 0.8 pg / ml or at least 1 pg / ml.

[0096] The composition of the first or the second aspect may comprise the 5. pyogenes protein PrsA2 at a concentration of at least 0.0001 pg / ml, or in an increasing order of preference: at least 0.005 pg / ml, at least 0.001 pg / ml, at least 0.01 pg / ml, at least 0.03 pg / ml, at least 0.05 pg / ml, at least 0.08 pg / ml, at least 0.1 pg / ml, at least 0.3 pg / ml, at least 0.5 pg / ml, at least 0.8 pg / ml or at least 1 pg / ml.

[0097] The composition of the first or the second aspect may comprise the 5. pyogenes protein GlnP at a concentration of at least 0.0001 pg / ml, or in an increasing order of preference: at least 0.005 pg / ml, at least 0.001 pg / ml, at least 0.01 pg / ml, at least 0.03 pg / ml, at least 0.05 pg / ml, at least 0.08 pg / ml, at least 0.1 pg / ml, at least 0.3 pg / ml, at least 0.5 pg / ml, at least 0.8 pg / ml or at least 1 pg / ml.

[0098] The composition of the first or the second aspect may comprise the 5. pyogenes protein SPy_0319 at a concentration of at least 0.0001 pg / ml, or in an increasing order of preference: at least 0.005 pg / ml, at least 0.001 pg / ml, at least 0.01 pg / ml, at least 0.03 pg / ml, at least 0.05 pg / ml, at least 0.08 pg / ml, at least 0.1 pg / ml, at least 0.3 pg / ml, at least 0.5 pg / ml, at least 0.8 pg / ml or at least 1 pg / ml. The composition of the first or the second aspect may comprise the 5. pyogenes protein MalX at a concentration of at least 0.0001 pg / ml, or in an increasing order of preference: at least 0.005 pg / ml, at least 0.001 pg / ml, at least 0.01 pg / ml, at least 0.03 pg / ml, at least 0.05 pg / ml, at least 0.08 pg / ml, at least 0.1 pg / ml, at least 0.3 pg / ml, at least 0.5 pg / ml, at least 0.8 pg / ml or at least 1 pg / ml.

[0099] The composition of the first or the second aspect may comprise each of the 5. pyogenes protein(s) MtsA, SPy_1228, PrsAl, PrsA2, GlnP and SPy_0319 at a concentration of at least 0.0001 pg / ml, or in an increasing order of preference: at least 0.005 pg / ml, at least 0.001 pg / ml, at least 0.01 pg / ml, at least 0.03 pg / ml, at least 0.05 pg / ml, at least 0.08 pg / ml, at least 0.1 pg / ml, at least 0.3 pg / ml, at least 0.5 pg / ml, at least 0.8 pg / ml or at least 1 pg / ml.

[0100] The composition of the first or the second aspect may further comprise one or more group A streptococcus antigen(s) in addition to MtsA, SPy_1228, PrsAl, PrsA2, GlnP, MalX and / or SPy_0319.

[0101] The EVs may be from EVs released by 5. pyogenes strain 5448 AP.

[0102] The EVs may have a diameter in the range of about 40 to about 400 nm as determined by dynamic light scattering or electron microscopy.

[0103] Unit dose

[0104] The composition of the first or the second aspect may be provided as a unit dose, which herein refers to a dose to be administered to an individual for eliciting an adaptive immune response against GAS. The amounts of components in a unit dose may vary depending on the subject to be immunized, for instance children may receive lower doses than adults. A unit dose may be provided in liquid form or in dried (e.g. lyophilized) form that is reconstituted prior to administration. For injectable vaccines, the volume administered to an individual is dictated practical constraints but would typically be in the order of 0.1-0.5 ml.

[0105] Thus, herein is disclosed an immunogenic composition of the first aspect as a unit dose, containing aforementioned EVs and optionally additional antigens and components as disclosed for the composition of the first aspect. The amounts of EVs and optional additional antigens in the unit dose may correspond to 0.5 ml of a composition of the first aspect. For instance, a unit dose may contain the 5. pyogenes protein MtsA at a concentration of at least 0.00005 pg, or in an increasing order of preference: at least 0.0025 pg, at least 0.0005 pg, at least 0.005 pg, at least 0.015 pg, at least 0.025 pg, at least 0.04 pg, at least 0.05 pg, at least 0.15 pg, at least 0.25 pg, at least 0.4 pg or at least 0.5 pg. Same applies for SPy_1228, PrsAl, PrsA2, SPy_0319, MalX and / or GlnP, based on the disclosure of the first aspect, mutatis mutandis.

[0106] Similarly, a composition of the first aspect as a unit dose may contain the antigens as disclosed in Table 1, wherein "+" denotes >0.001 pg / mg, preferably >0.01 pg / mg, even more preferably >0.02 pg / mg, yet more preferably >0.05 pg / mg, most preferably >0.1 pg / mg of the total protein in the EVs.

[0107] The unit dose may comprise EVs in an amount of at least 0.5 pg, preferably at least 2.5 pg, more preferably at least 5 pg, even more preferably 12.5 pg, still more preferably 25 pg, yet more preferably 37.5 pg, most preferably at least 50 pg.

[0108] A unit dose of the first aspect may contain the antigens as disclosed in Table 1 wherein "+" denotes presence of the antigen an amount of at least 0.00005 pg, or in an increasing order of preference: at least 0.0025 pg, at least 0.0005 pg, at least 0.005 pg, at least 0.015 pg, at least 0.025 pg, at least 0.04 pg, at least 0.05 pg, at least 0.15 pg, at least 0.25 pg, at least 0.4 pg or at least 0.5 pg. The amounts refer to total amount present and includes both EVs- associated and non-associated protein.

[0109] Herein is further disclosed an immunogenic composition of the second aspect as a unit dose, containing components as disclosed for the composition of the second aspect.

[0110] The amounts of the antigen(s) in the unit dose may correspond to 0.5 ml of a composition of the second aspect. For instance, a unit dose may contain the 5. pyogenes protein MtsA at an amount of at least 0.00005 pg, or in an increasing order of preference: at least 0.0025 pg, at least 0.0005 pg, at least 0.005 pg, at least 0.015 pg, at least 0.025 pg, at least 0.04 pg, at least 0.05 pg, at least 0.15 pg, at least 0.25 pg, at least 0.4 pg or at least 0.5 pg. Same applies for SPy_1228, PrsAl, PrsA2, SPy_0319, MalX and / or GlnP, based on the disclosure of the second aspect, mutatis mutandis. Similarly, a unit dose of the second aspect may contain the antigens as disclosed in Table 1, wherein "+" denotes presence of the antigen an amount of at least 0.00005 pg, or in an increasing order of preference: at least 0.0025 pg, at least 0.0005 pg, at least 0.005 pg, at least 0.015 pg, at least 0.025 pg, at least 0.04 pg, at least 0.05 pg, at least 0.15 pg, at least 0.25 pg, at least 0.4 pg or at least 0.5 pg. The amounts refer to total amount present and includes both EVs-associated and non-associated protein.

[0111] Particular medical uses

[0112] The composition of the first or the second aspect may be for use by injection (including intramuscular, intracutaneous, subcutaneous, intravenous), buccal administration, oral administration or intranasal administration.

[0113] The composition of the first or the second aspect may be for use in a method for inducing protective immunity in a subject against a GAS infection.

[0114] The composition of the first or the second aspect may be for use in a method for inducing protective immunity in a subject against colonization by GAS.

[0115] The composition of the first or the second aspect may be for use in a method for inducing protective immunity in a subject against a GAS infection selected from streptococcal pharyngitis, scarlet fever, impetigo, streptococcal meningitis, streptococcal sinusitis, necrotizing fasciitis, cellulitis, streptococcal toxic shock syndrome, rheumatic fever and poststreptococcal glomerulonephritis.

[0116] The composition of the first or the second aspect may be for use in a method involving a single administration, two administrations, three administrations or more than three administrations to the subject. The administrations can be separated in time by at least 7 days, at least 14 days, at least 28 days, at least two months, at least 6 months, 7-14 days, 7- 28 days, 28 days to two months, 28 days to 6 months or any other combination of aforementioned times. Multiple administrations may be performed using the same mode of administration. Multiple administrations may be performed using the same composition. EXAMPLES

[0117] The following examples are not to be regarded as limiting. For further information on the experimental details, the skilled reader is directed to a separate section titled Materials and Methods.

[0118] Example 1: Characterization of S. pyogenes-derived extracellular vesicles.

[0119] Extracellular vesicles spontaneously released by the 5. pyogenes strain 5448 AP (GAS EVs) were purified from cell-free supernatant. 5. pyogenes strain 5448 AP was grown to an ODeoo 0.6 in Todd Hewitt, bacterial culture was centrifugated and filtrated to obtain cell-free supernatant. Cell-free supernatant was concentrated by tangential flow filtration followed by ultracentrifugation. GAS EVs were purified by density gradient and washed using ultracentrifugation. GAS EVS were visualized by cryo-electron microscopy (Cryo-EM). GAS EVs appear as single membrane vesicles of different sizes (Fig 1A), with no evidence for the presence of a cell-wall. As determined by dynamic light scattering (DLS), the size distribution of GAS EVS ranges from 40 to 400 nm in diameter, with a median size of 145 nm (Fig IB). GAS EVs are loaded with ~700 proteins, as determined by mass-spectrometry analysis (data not shown). The cargo of these vesicles includes surface antigens, virulence factors and other proteins.

[0120] Example 2: Intranasal immunization with GAS EVs generates GAS-specific antibodies.

[0121] To determine the immunogenicity of GAS EVs, male C57BL / 6 mice were immunized intranasally with 50uL of GAS EVs (10 ug) formulated with aluminum hydroxide (alum, 10 mg / ml). The control group was immunized with PBS and adjuvant alone (alum, lOmg / ml). Two immunizations, 14 days apart, were delivered intranasally (Figure 2A). Twenty-one days after the primary immunization, sera and nasal washes were collected and used to measure GAS-specific IgG and IgA levels, respectively. The levels of GAS-specific IgG in sera were significantly increased in the GAS EVs-immunized group as compared to the control group. Similarly, IgA levels in nasal washes were significantly increased following GAS EVs- immunization (Figure 2B). These results indicate that intranasal delivery of GAS EVs elicits the production of IgG's and IgA's that recognize antigens on the surface of GAS.

[0122] Example 3: Intranasal immunization with GAS EVs induces Thl7 and Thl responses in lung. Previous reports have shown that Thl7 and Thl cell-responses are important for protection against GAS infections ( Carey, A. J. et al. Interleukin-17A contributes to the control of streptococcus pyogenes colonization and inflammation of the female genital tract. Sci. Rep. 6, 26836 (2016). Christensen, D., Mortensen, R., Rosenkrands, I., Dietrich, J. & Andersen, P. Vaccine-induced Thl7 cells are established as resident memory cells in the lung and promote local IgA responses. Mucosal. Immunol. 10, 260-270 (2017). Mortensen, R. et al. Local Thl7 / lgA immunity correlate with protection against intranasal infection with Streptococcus pyogenes. PLoS One 12, e0175707 (2017)). Flow cytometry analysis revealed that immunization with GAS EVs enhanced CD4+T-cell recruitment to the lungs as seen by an increase in the percentage (%) and total number of cells (Fig 3, uppers panels).

[0123] Moreover, a significantly greater % and total number of CD4+T-cells expressed CD44 (Fig 3, lower panels), suggesting these are activated effector and / or memory CD4+T-cells.

[0124] Activated CD4+T cells recruited to the lung are predominantly Thl7 cells as evidenced by the significant increase in IL-17 production and RORyT expression following non-specific restimulation with PMA and ionomycin (Figure 4a-b). A significant increase in I FNy production (but not T-bet expression) from CD4+T cells in the lung following GAS EVs immunization was also observed (Figure 4d-e), suggesting a small, but significant Thl response. To assess antigen-specific cytokine production, secreted IL-17A and I FNy in cell culture supernatants of lung cells stimulated ex vivo with GAS EVs were measured by ELISA. Stimulation of lung cells from GAS EVs-immunized mice induced a significant increase in the secretion of IL-17A and IFNy as compared to PBS-immunized mice (Figure 4c and 4f).

[0125] Overall, our data demonstrate that intranasal immunization with GAS EVs induces a robust Thl7 response and a significant Thl response in the lung.

[0126] Example 4: Intranasal immunization with GAS EVs induces systemic Thl7 response

[0127] In addition to eliciting a local response in the lung, intranasal immunization with GAS EVs also induced a systemic Thl7 response as evidenced by the significant increase in the % of I L-17+activated T cells in spleen (Figure 5a) and increased secretion of IL-17A by splenocytes stimulated ex vivo with GAS EVs (Figure 5c). Although there was no increase in IFNy production from CD4+T cells (Figure 5b), a significant increase in IFNy secretion by splenocytes stimulated with GAS EVs was observed (Figure 3d). These results indicate that immunization with GAS EVs can elicit the systemic activation of CD4+T cells and the induction of a Thl7 response. Example 5: Identification of immunostimulatory antigens on GAS EVs.

[0128] Examples 2-4 demonstrated that immunization with EVs elicited a humoral and cellular response in mice. Next, the inventors proceeded to identify the antigens responsible for these immunostimulatory properties of GAS EVs. Several proteins, ranging in size from ~30 to 80 kDa, were detected on GAS EVs when probed with sera from GAS EV-immunized mice (Fig 6a). The identity of these immunoreactive antigens was determined by coimmunoprecipitation coupled to mass spectrometry (Figure 6a, table to the right). Five different lipoproteins (MtsA, SPy_1228 (BMP), PrsAl, PrsA2 and SPy_0319 (MetQ)) and a transmembrane protein GlnP were identified as immunoreactive antigens. PrsAl and PrsA2 are extracellular parvulin-type peptidyl-prolyl isomerases that assist in the folding of exported proteins [3], MtsA is the substrate binding lipoprotein of the ABC transporter responsible for iron / manganese transport (Janulczyk R, Ricci S, Bjbrck L. MtsABC is important for manganese and iron transport, oxidative stress resistance, and virulence of Streptococcus pyogenes. Infect Immun. 2003 May;71(5):2656-64. doi:

[0129] 10.1128 / IAL71.5.2656-2664.2003.). SPy_1228 and GlnP have not been characterized in GAS but share sequence similarity with the substrate binding lipoprotein of ribonucleoside ABC transporters (BMP) [4] and the glutamine permease GlnP, respectively [5], SPy_0319 has not been characterized in GAS but has been classified in silico as an ABC-type transport system, periplasmic component / surface antigen based on sequence similarity.

[0130] To validate MtsA, SPy_1228 / BMP, PrsAl, PrsA2, SPy_0319 / MetQ and GlnP as immunoreactive antigens, these were expressed and purified as recombinant proteins (lacking signal peptide or membrane domains) and used to detect antigen specific IgG's by ELISA and western blot. Vaccination with GAS EVs formulated with alum, but not alum alone, significantly increased the IgG levels specific for MtsA, PrsAl, MetQ and GlnP but not BMP and PrsA2 (Figure 6b). All proteins except MetQ were detected by western blot using pooled sera from GAS EVs-immunized mice (data not shown). These results confirm that immunization with GAS EVs elicits the production of antibodies specific to six different GAS proteins: MtsA, BMP, PrsAl, PrsA2, SPy_0319 / MetQ and GlnP, highlighting their potential as novel vaccine antigens. Data also shows that GlnP is the most immunogenic of all six antigens tested (Figure 6b). The inventors further demonstrated that some of the bands observed in Figure 6a disappear in GAS strains lacking MtsA, SPy_1228, PrsAl or PrsA2 (Figure 7, left panel). Consequently, IgG binding to GAS AP lacking Aspy_1228, AmtsA and AprsA2 is slightly reduced when compared to binding to the WT strain (Fig 7, right panel).

[0131] Together, these results indicate that immunization with GAS EVs elicits the production of antibodies specific to six different GAS proteins, MtsA, SPy_1228, PrsAl, PrsA2, SPY_0319 and GlnP.

[0132] Example 6: Thl7 memory-recall after ex-vivo stimulation with GAS EVs and recombinant antigens.

[0133] Ex-vivo stimulation of lung and spleen cells was performed to investigate cellular recall responses after GAS EV vaccination. Splenocytes and lung cells from alum and GAS EV- immunized mice were stimulated ex vivo with recombinantly expressed MtsA, BMP (SPy_1228), PrsAl, PrsA2, MetQ. (SPy_0319), GlnP or MalX for 72h. Secreted IL-17 A and I FNy in cell culture supernatants were measured by ELISA. As shown in Figure 8 all antigens can induce the secretion of IL-17A by lung (Fig. 8a) and spleen (Fig. 8b) cells of mice immunized with GAS EVs but not alum alone. Besides the six immunostimulatory antigens identified in GAS EVs, MalX, a lipoprotein important for protection against pneumococcal infections (Narciso et al. Membrane particles evoke a serotype-independent cross-protection against pneumococcal infection that is dependent on the conserved lipoproteins MalX and PrsA. Proc Natl Acad Sci U S A. 2022 Jun 7;119(23):e2122386119. doi: 10.1073 / pnas.2122386119. Epub 2022 Jun 1. PMID: 35648835) was also included. Of all the tested antigens, BMP induced the highest levels of IL-17A secretion followed by SPy_0319 / MetQ. In the case of IFNy, none of the recombinant proteins induced a significant increase in secretion by lung cells, while SPy_1228 / BMP induced a significant increase in IFNy secretion by splenocytes (Figure 9a, b). These results indicate that MtsA, SPy_1228 / BMP, PrsAl, PrsA2, SPy_0319 / MetQ, GlnP and MalX are T-cell antigens that primarily induce the production and secretion of IL-17A, in addition to their humoral response.

[0134] Example 7: Vaccine candidates identified in GAS EVs are conserved across different M- types.

[0135] Ideal vaccine candidates should be conserved across different M-types and provide broad coverage across GAS strains. Additionally, we demonstrated that the same protein profile observed in GAS EVs (purified from GAS 5448 AP) can be observed in the cell lysates of different invasive GAS isolates (M-types: 1, 3, 4, 6, 12, 28, 75 and 89) (Figure 10a). Furthermore, IgG's from mice vaccinated with EVs can bind to the surface of different invasive GAS isolates as demonstrated by whole cell ELISA (Figure 10b). These results indicate that the immunostimulatory antigens identified in GAS EVs are conserved across different M-types and that the antibodies generated upon immunization with GAS 5448 AP- EVs cross react with several other M-types.

[0136] Example 8: Recombinant proteins as immunogens

[0137] All animal experiments were approved by the local ethical committee (Stockholms Norra djurfbrsbksetiska namnd). Groups ( n=5 or n=4 for control groups) of 5- to 6-week-old female C57BL / 6 mice were immunized with GAS EVs (30pg of total EV-protein) or recombinantly expressed proteins (5ug of each protein: PrsAl, PrsA2, Spy_1228, GlnP, MtsA, MalX, NIpa / MetQ) formulated with adjuvant (5ug of QS21, 2ug of 3D-MPLA and 50uL of Addavax). The control group received adjuvant alone. Mice were anesthetized by isoflurane (Abbott) inhalation and then immunized subcutaneously with lOOpL / mouse of the formulations on days 0 and 14. Fourteen days after the second immunization, serum was collected and antibody titers vs each antigen were measured by ELISA.

[0138] Nunc MaxiSorp plates (Thermo Scientific) were coated overnight at 4°C with lOOpI of recombinantly expressed antigens (lOpg / mL in PBS). Plates were blocked for 2h at room temperature with 200pl of 2.5% skim milk in PBS and then washed 3 times with PBS before the addition of mice sera (diluted 1:250 in PBS). Samples were incubated at room temperature for 2h and then washed 3 times with tPBS. Next, plates were incubated for 1.5h at room temperature with goat anti-mouse IgG-HRP diluted 1:4000 in 1% BSA in PBS. Plates were washed 3 times with PBS before developing with the TMB substrate reagent set (BD OptEIA) as specified before.

[0139] The immunization induced high titers of antigen-specific antibodies (Fig 11). Similar antibody levels can be obtained by immunization with GAS EVs in the case of MtsA, SPy_0319 / NlpA / MetQ, PrsAl, PrsA2, Spy_1228, GlnP. In the case of MalX, only recombinant proteins produced a robust response. MATERIALS AND METHODS

[0140] Bacterial strains and growth conditions

[0141] All bacterial strains used in this study are listed in Table 2. Streptococcus pyogenes was grown on tryptic soy agar (TSA) plates supplemented with 5% sheep blood at 37°C in a 5% CO2 atmosphere. Liquid cultures were grown statically at 37°C in either Todd-Hewitt broth alone (THB) or supplemented with 0.2% yeast extract (THY). Escherichia coli was grown aerobically in lysogeny broth (LB) at 37°C with constant shaking. When required, E. coli cultures were supplemented with lOOpg / mL spectinomycin or 50pg / mL kanamycin.

[0142] Table 2 (1) Aziz, R.K., et al., Invasive M1T1 group A. Streptococcus undergoes a phase-shift in vivo to prevent proteolytic degradation of multiple virulence factors by SpeB. Mol Microbiol, 2004. 51(1): p. 123-34. (2) Chatellier, S., et al., Genetic relatedness and superantigen expression in group A. streptococcus serotype Ml isolates from patients with severe and nonsevere invasive diseases. Infect Immun, 2000. 68(6): p. 3523-34.

[0143] (3) Darenberg J, et al. . Molecular and clinical characteristics of invasive group 4 streptococcal infection in Sweden. Clin Infect Dis. 2007 Aug 15;45(4):450-8. doi: 10.1086 / 519936. Epub 2007 Jul 10. PMID: 17638193.

[0144] Isolation of extracellular vesicles

[0145] GAS 5448 AP was grown in THB to an ODeoo of 0.6. Bacteria were pellet by centrifugation at 8000 rpm for 20 minutes, the supernatant was recovered and filtered through a 0.22uM filter (Filtropur S 0.2). Samples were then concentrated 10X with a tangential flow filtration (TFF) capsule (MWCO: lOOkDa, PALL Corporation). EVs were pelleted by ultracentrifugation (30,000 rpm for 4h at 4°C) and further purified using OptiPrep (iodixanol) Density Gradient Medium (Sigma-Aldrich). Crude EVs were resuspended in 50% (v / v) Optiprep in solution A (0.25M sucrose, 6mM EDTA, 60mM HEPES-NaOH, pH 7.4) and overlaid with 35% and 5% (v / v) Optiprep in solution B (0.25M sucrose, ImM EDTA, lOmM HEPES-NaOH, pH 7.4). Separation was achieved by ultracentrifugation at 35,000 rpm for 6h. The interphase between the 5 and 35% Optiprep solutions was recovered and washed 3 times with PBS (For the first wash EVs were centrifuged at 35,000 rpm for at least 6h, 2ndand 3rdwashes were centrifuged at 30,000 rpm for 4h).

[0146] EV protein concentration was determined using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific). EVs were tested for sterility by spotting on blood agar plates and then stored at -80 until further use.

[0147] GAS EV particle size and count distribution was measured by dynamic light scattering (DLS) using a Zetasizer Ultra instrument (Malvern Panalytical). Vesicles samples were diluted 1:250 in water and equilibrated for 2 minutes at 25°C before each measurement.

[0148] Cryo EM

[0149] Sample preparation and vitrification. 10 nm gold fiducial particles (Ted Pella) were mixed with 5% BSA in a 5:1 ratio, vortexed for 1 minute and centrifuged at 17 000 x g for 15 minutes. The resulting pellet was resuspended with GAS EVs at 0.3 mg / mL in PBS. This suspension was blotted onto glow-discharged Quantifoil Cu Rl.2 / 1.3, 300-mesh EM-grids using a Vitrobot IV at room temperature and 100% humidity and plunge-frozen in liquid ethane.

[0150] Cryo-ET data collection and 3D reconstruction. Cryo-ET data was collected on a 300 kV Krios G3i microscope with a Gatan K3 detector using the Tomo-4 (Thermo Fisher) data collection software. Data was collected at a magnification of x 42,000 and a pixel-size of 2.15 A / px (binned from super-resolution). Single axis tilt-series were collected using a dose-symmetric scheme, with a 3 ° increment from -51 to +51°. Data for each tilt angle was collected as a movie stack of 10 frames and the total dose for each tilt-series was ~80 e / A2. The defocus range for the different tilt-series was between -2 and -6 pm.

[0151] 2D-preprocessing, motion correction and tilt-stack generation was done in Warp. IMOD Etomo was used for generating 3D reconstructions, including fiducial tracking, tilt-series alignment, and CTF-correction. The final tomograms were binned 3 times and reconstructed using a SI RT-like filter.

[0152] Mouse immunization

[0153] All animal experiments were approved by the local ethical committee (Stockholms Norra djurfbrsbksetiska namnd). Groups ( n=5 or n=4 for control groups) of 5- to 6-week-old male C57BL / 6 mice were immunized with GAS EVs and alum (lOpg of total EV-protein and 10 mg / mL aluminum hydroxide -Sigma-Aldrich- in PBS) or alum alone (alum, lOmg / mL in PBS). Mice were anesthetized by isoflurane (Abbott) inhalation and then immunized intranasally with 50pL / mouse of the formulations on days 0 and 14. Seven days after the second immunization, mice were euthanized and nasal washes (in PBS), blood (obtained via heart puncture), spleen and lungs were recovered under aseptic conditions.

[0154] Whole bacterial-cell ELISA

[0155] GAS 5448 AP or different invasive GAS isolates were grown at 37°C in THY to an ODeoo of 0.6. Bacteria were washed 2 times with PBS supplemented with EDTA-free protease inhibitor cocktail (complete, Roche) and heat inactivated at for 2.30h at 60°C. Nunc MaxiSorp plates (Thermo Scientific) were coated with lOOpI of the bacterial suspension (adjusted to an ODeoo of 0.6 in PBS + complete) and incubated overnight at 4°C. Next, plates were blocked for 2h at room temperature with 200pl of 2.5% skim milk in PBS. After blocking, plates were incubated for 2h at RT, first with lOOpL of mice sera (1:500 in PBS) and then with lOOpL of goat anti-mouse IgG-HRP diluted 1:5000 in 1% BSA in PBS. Plates were washed 3 times with PBS after each incubation step. Finally, samples were developed with the TMB substrate reagent set (BD OptEIA) for 20 minutes. The reaction was stopped wit 50pL of sulfuric acid 0.5M and the absorbance was measured at 450 and 570nm. For nasal washes, samples were not diluted and incubated overnight. Rabbit anti-mouse IgA-HRP was used at a 1:1000 dilution in 1% BSA in PBS.

[0156] Western blotting

[0157] GAS EVs were prepared as indicated above. Two micrograms of GAS EVs were mixed with IX NuPAGE LDS sample buffer (Invitrogen). For cells lysates, GAS strains were grown in THY to an ODeoo of 0.6. Cells were pelleted by centrifugation, resuspended in PBS and lysed by mechanical disruption. After centrifugation, the cleared cell lysate was recovered and mixed with IX NuPAGE LDS sample buffer (Invitrogen). Samples were boiled for 10 minutes and resolved by SDS-PAGE using 4-12% NuPAGE Bis-Tris protein gels (Invitrogen) and transferred to PVDF (polyvinylidene difluoride) membranes. Membranes were blocked overnight with 5% skim milk in tPBS (PBS containing 0.1% Tween-20). After three washes with tPBS, membranes were incubated with primary antibodies, either pooled sera from immunized mice (1:5000) or anti-GAPDH (1:20,000). HRP-conjugated antibodies (1:10,000) were used as secondary antibodies. Membranes were developed with Amersham ECL Plus Western blotting detection system (GE Healthcare Life Sciences) using ChemiDoc XRS+ (Bio-Rad Laboratories).

[0158] Coimmunoprecipitation

[0159] Protein G HP SpinTrap / Ab columns (GE Healthcare) were pre-equilibrated with PBS. Protein G Sepharose was removed from the columns and incubated with pooled sera (lOpL sera / 400pL PBS) from immunized mice (GAS EVs / alum or PBS / alum). After lh incubation at 37^C, samples were centrifuged (100 x g for 1 minute); after removing the supernatant, the pelleted Protein G Sepharose was washed 4 times with PBS. In parallel, 30pg of GAS EVs were solubilized by incubation with Pierce IP lysis buffer (thermo scientific) at 4-C for lh. Coupled Protein G Sepharose was then mixed with solubilized GAS EVs and incubated for 2h at 4^C. Finally, Protein G Sepharose was washed 6 times with PBS as described above. Two strategies were employed to elute co-immunoprecipitated proteins: i) coupled Protein G Sepharose was resuspended in IX NuPAGE LDS sample buffer (Novex) and subjected to SDS- PAGE, after 10 minutes, running was stopped, and 1 cm gel slices were cut and used for mass spectrometry, ii) proteins were eluted from Protein G Sepharose with 400pL of 0.1M glycine-HCI, pH 2.7 followed by neutralization with 40pL of IM Tris pH 8.0. Samples were concentrated using SpeedVac (Thermo Fisher) and sent for mass spectrometry.

[0160] Mass spectrometry

[0161] Coimmunoprecipitation samples. Samples were prepared using a standard (in gel or in solution) alkylation and tryptic digestion protocol. Eluted peptides were cleaned up using C18 StageTip and dried down. Resolubilized peptides were analyzed on a Q Exactive HF instrument (Thermo Scietific) using a 60 min linear gradient elution on a 50 cm long C18 column. Tandem mass spectrometry (MS / MS) was performed applying HCD (higher-energy collisional dissociation). A database search was carried out using Mascot server (v2.5.2) for peptide matches and protein searches against the 5. pyogenes ATCC 700294 (SF370, Serotype Ml) genomic database UniProtKB (Proteome ID: UP000000750).

[0162] GAS EVs samples. S. pyogenes EVs were prepared as described in previous sections. Three independent samples, consisting of 30pg of total EVs proteins were sent to mass spectrometry and processed as described above.

[0163] Expression and purification of recombinant proteins

[0164] For protein purification, the soluble region of GlnP (codons 30-520) or the mature form (lacking the signal peptide) of MtsA (codons 22-310), Spy_1228 (BMP, (codons 22-350), PrsAl (codons 24-351), PrsA2 (codons 24-309), Spy_0319 (MetQ, codons 23-281) and MalX (codons 26-419) were cloned into pET28a, which allows the expression of C-terminally 6xHis tagged proteins. Briefly, glnP, mtsA, BMP, prsAl, prsA2, metQ and malX were amplified from gDNA using primer pairs glnP. fw / glnP.rv, mtsA.fw / mtsA.rv, BMP.fw / BMP.rv, prsAl.fw / prsAl.rv, prsA2.fw / prsA2.rv, metQ.fw / metQ.rv and malX. fw / malX.rv. The resulting PCR products were cloned into Ncol / Xhol digested pET28a using the NEBuilder HiFi assembly kit (New England biolabs) according to manufacturer's instructions. BL21 (DE3) (Invitrogen) cells containing the pET28a constructs were cultured in LB containing kanamycin at 37°C with constant shaking until an ODeoo of 0.8 was reached. Then, protein production was induced by the addition of 0.5mM IPTG (isopropyl -D-1 thiogalactopyranoside). Bacterial growth continued for 4h at 30°C, then cells were harvested by centrifugation. Cells were resuspended in lysis buffer (5mM imidazole, 150mM NaCI, 20mM Tris pH 7.5) and lysed by sonication followed by centrifugation to remove cell debris. Recombinant proteins in the cleared cell lysates were purified by affinity chromatography using Ni-sepharose (GE-healthcare). The eluted proteins were further purified by Superdex 200 size exclusion chromatography using an NGC Chromatography System (Biorad). PrsA2 was eluted in buffer containing 300mM NaCI, 5mM EDTA, 5% glycerol and 20mM Tris pH 7.5, while the other proteins were eluted in PBS. Protein concentration was determined using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific). Antigen-specific Antibody ELISA

[0165] Nunc MaxiSorp plates (Thermo Scientific) were coated overnight at 4°C with lOOpI of recombinantly expressed antigens (lOpg / mL in PBS). Plates were blocked for 2h at room temperature with 200pl of 2.5% skim milk in PBS and then washed 3 times with PBS before the addition of mice sera (diluted 1:250 in PBS). Samples were incubated at room temperature for 2h and then washed 3 times with tPBS. Next, plates were incubated for 1.5h at room temperature with goat anti-mouse IgG-HRP diluted 1:4000 in 1% BSA in PBS. Plates were washed 3 times with PBS before developing with the TMB substrate reagent set (BD OptEIA) as specified before.

[0166] Isogenic mutant generation

[0167] Bacterial strains and growth conditions

[0168] All bacterial strains used in this study are listed in Table 1. Streptococcus pyogenes [1] strains were grown on tryptic soy agar (TSA) plates supplemented with 5% sheep blood at 37°C in a 5% CO2 atmosphere. Liquid cultures were grown statically at 37°C in either Todd- Hewitt broth alone (THB) or supplemented with 0.2% yeast extract (THY). Escherichia coli was grown aerobically in lysogeny broth (LB) at 37°C with constant shaking. When required E. coli cultures, antibiotics were used at the following concentrations: lOOpg / mL spectinomycin or 50pg / mL kanamycin.

[0169] (1) Aziz, R.K., et al., Invasive M1T1 group A. Streptococcus undergoes a phase-shift in vivo to prevent proteolytic degradation of multiple virulence factors by SpeB. Mol Microbiol, 2004. 51(1): p. 123-34. (2) Chatellier, S., et al., Genetic relatedness and superantigen expression in group A streptococcus serotype Ml isolates from patients with severe and nonsevere invasive diseases. Infect Immun, 2000. 68(6): p. 3523-34.

[0170] Generation ofS. pyogenes deletion mutants.

[0171] Marker-less isogenic mutants were generated using the temperature-sensitive plasmid pLZts (Addgene #128799) following previously reported protocols (Barnett, T.C., et al., Genetic Manipulation of Group A Streptococcus-Gene Deletion by Allelic Replacement. Methods Mol Biol, 2020. 2136: p. 59-69). All deletion mutants were generated as follows: ~lkb DNA fragments up- and downstream mtsA, spy_1228, prsAl and prsA2 were amplified by PCR from GAS 5448 gDNA using the following primer pairs: mtsA.up.fw and mtsA.up.rv, mtsA.dn.fw and mtsA.dn.rv, 1228. up. fw and 1228. up. rv, 1228.dn.fw and 1228.dn.rv, prsAl.up.fw and prsAl.up.rv, prsAl.dn.fw and prsAl.dn.rv and prsA2.up.fw and prsA2.up.rv, prsA2.dn.fw and prsA2.dn.rv. pLZts was amplified by PCR with primers pLZts-S and pLZts-A and used to clone the up- and downstream flanking DNA fragments using the NEBuilder HiFi assembly kit according to manufacturer's instructions. The resulting plasmids were electroporated into GAS 5448 AP cells. Transformants were selected at 30°C on THY agar containing lOOpg / mL spectinomycin. mtsA, spy_1228, prsAl and prsA2 were deleted by allelic replacement. First, plasmid integration was promoted at 37.5°C in the presences of spectinomycin and then excised at 30°C with no antibiotic selection. Deletion strains were screened by PCR using primer pairs: seq.mtsA.fw and seq.mtsA.rv, seq.1228.fw and seq.1228.rv, seq.prsAl.fw and seq. prsAl. rv, seq.prsA2.fw and seq.prsA2.rv and confirmed by DNA sequencing. Tissue processing

[0172] Spleens and lungs were collected from mice immunized with GAS EVs / alum or PBS / alum. Lungs were diced into small pieces and digested with DNase (80 U / mL, Sigma) and liberase (0.8 U / mL, Sigma) in HBSS (Sigma) for 45 minutes at 37°C. Digestions were stopped by the addition of lung buffer (2mM EDTA, 2% FBS in PBS). Single lung and spleen-cell suspensions were prepared by homogenizing tissue through a 70 pm cell strainer. Red blood cells (RBCs) were lysed with RBS lysis buffer (BD Pharm). After washing with lung buffer, cells were resuspended in restimulation media (RPMI supplemented with 10% FBS, 1% non-essential amino acids, 1% sodium pyruvate, 1% L-glutamine, 0.1% 2-mercaptoethanol and 1% penicillin / streptomycin). Cell counts and viability were assessed by trypan blue staining.

[0173] Single cell suspensions were used for flow staining or ex-vivo stimulation (see below).

[0174] Flow cytometry

[0175] For intracellular cytokine analysis lung cells were stimulated with PMA (Phorbol 12- myristate 13-acetate, 30ng / mL), ionomycin (0.5pg / mL) and Brefeldin A (IX) for 3h at 37°C in a 5% CO2 atmosphere. Samples were then stained with antibodies against surface markers including CD45 (30-F11), CD4 (RM4-5), TCRb (H57-597), CD44 (IM7) in the presence of an Fc Block (Biolegend and ThermoFisher) For intracellular staining, samples were permeabilized with FOXP3 / Transcription factor staining buffer (Invitrogen) for 30 minutes before adding antibodies against IL-17 (TC11-18H10) and I FNy (XMG1.2). Samples were acquired on BD Fortessa or Sony ID700 and analyzed using FlowJo software (Tree Star). Zombie UV fixable dye (Biolegend) was used to exclude dead cells and FMOs were used as gating controls.

[0176] Ex-vivo stimulation and cytokine quantification

[0177] A total of 2xl06lung cells or splenocytes were stimulated with 0.5pg of GAS EVs, lpg of recombinantly expressed proteins or with media alone. Cells were incubated at 37°C with 5% CO2 for 72 h in restimulation media (see above). After centrifugation, culture supernatants were collected and stored at -20°C before cytokine analysis.

[0178] IL-17A and I FNy secreted into the culture supernatant was quantified using the mouse IL- 17A (homodimer) or I FNy uncoated ELISA kits (Invitrogen) following manufacturer's instructions. Data are normalized to the media stimulation controls and represented as mean +- SEM Statistical analysis

[0179] Statistical analyses were performed using GraphPad Prism 5. Results are represented as the mean ± standard error of the mean (SEM). For pairwise comparisons of samples that follow normal distribution (Shapiro-Wilk test), a two-tailed unpaired t-test was performed. Mann- Whitney test was performed when samples did not follow a normal distribution. P < 0.05, was considered significant (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant).

[0180] REFERENCES

[0181] 1. Rivera-Hernandez, T., et al., Vaccine-Induced Thl-Type Response Protects against Invasive Group A Streptococcus Infection in the Absence of Opsonizing Antibodies. mBio, 2020. 11(2).

[0182] 2. Wang, B., et al., Induction of TGF-betal and TGF-betal-dependent predominant Thl7 differentiation by group A streptococcal infection. Proc Natl Acad Sci U S A, 2010. 107(13): p. 5937-42.

[0183] 3. Wu, Z.Y., et al., Unigue virulence role of post-translocational chaperone PrsA in shaping Streptococcus pyogenes secretome. Virulence, 2021. 12(1): p. 2633-2647.

[0184] 4. Sun, X., et al., Crystal structure and metal binding properties of the lipoprotein MtsA, responsible for iron transport in Streptococcus pyogenes. Biochemistry, 2009. 48(26): p. 6184-90.

[0185] 5. Sun, X., et al., Lipoprotein MtsA of MtsABC in Streptococcus pyogenes primarily binds ferrous ion with bicarbonate as a synergistic anion. FEBS Lett, 2008. 582(9): p. 1351-4.

[0186] 6. Yousief, S.W., M. Bahey-EI-Din, and T.L Zaghloul, Immunization with the basic membrane protein (BMP) family ABC transporter elicits protection against Enterococcus faecium in a murine infection model. Microbes Infect, 2020. 22(3): p. 127- 136.

[0187] 7. Morikawa, Y., et al., Identification and functional analysis of glutamine transporter in Streptococcus mutans. J Oral Microbiol, 2020. 12(1): p. 1797320.

Claims

CLAIMS1. A composition comprising isolated Streptococcus pyogenes extracellular vesicles (EVs) and a pharmaceutically acceptable vehicle, for use in eliciting an adaptive immune response against group A streptococcus (GAS) in a subject.

2. A composition comprising isolated 5. pyogenes protein(s) MtsA, SPy_1228, PrsAl, PrsA2, GlnP, SPy_0319 and / or MalX for use in eliciting an adaptive immune response against group A streptococcus (GAS) in a subject.

3. The composition for use according to claim 1 or 2, wherein the immune response is a T-cell response, preferably a Thl7-response.

4. The composition for use according to any of the preceding claims, wherein the immune response is an IgG response, IgA response, or a combined IgG and IgA response.

5. The composition for use according to any of the preceding claims, wherein the composition further comprises an adjuvant.

6. The composition for use according to any of the preceding claims, wherein the composition further comprises an adjuvant selected from the group consisting of (i) aluminium hydroxide; (ii) aluminium phosphate; (iii) a lipid-based substance such as squalene or quillaja saponin; and (iv) a protein or other immune stimulating component from a microbe such as diphtheria (preferably CRM), cholera (preferably choleratoxoid CTB or mmCT) or Escherichia coli (preferably non-toxic heat-labile toxin such as LTK63 or dmLT).

7. The composition for use according to any of the preceding claims, wherein the composition comprises immunogenic amount(s) of the 5. pyogenes protein(s) MtsA, SPy_1228, PrsAl, PrsA2, GlnP, SPy_0319 and / or MalX as antigen(s).

8. The composition for use according to any of the preceding claims, wherein the composition comprises EVs in an amount of at least 1 pg / ml, preferably at least 5 pg / ml, more preferably at least 10 pg / ml, even more preferably 25 pg / ml, still more preferably 50 pg / ml, yet more preferably 75 pg / ml, most preferably at least 100 pg / ml.

9. The composition for use according to claim 1 or 8, or any claim dependent thereon, wherein the EVs comprise immunogenic amount(s) of the 5. pyogenes protein(s) MtsA, SPy_1228, PrsAl, PrsA2, GlnP and / or SPy_0319.

10. The composition for use according to claim 1 or 8, or any claim dependent thereon, wherein the EVs comprise one or more of the 5. pyogenes protein(s) MtsA, SPy_1228, PrsAl, PrsA2, GlnP and / or SPy_0319 at the level of >0.001 pg / mg, preferably >0.01 pg / mg, even more preferably >0.02 pg / mg, yet more preferably >0.05 pg / mg, most preferably >0.1 pg / mg of the total protein in the EVs.

11. The composition for use according to claim 1 or 8, or any claim dependent thereon, wherein the EVs comprise an immunogenic amount of the 5. pyogenes protein MtsA, preferably at the level of >0.001 pg / mg, more preferably >0.01 pg / mg, even more preferably >0.02 pg / mg, yet more preferably >0.05 pg / mg, most preferably >0.1 pg / mg of the total protein in the EVs.

12. The composition for use according to claim 1 or 8, or any claim dependent thereon, wherein the EVs comprise an immunogenic amount of the 5. pyogenes protein SPy_1228, preferably at the level of >0.001 pg / mg, more preferably >0.01 pg / mg, even more preferably >0.02 pg / mg, yet more preferably >0.05 pg / mg, most preferably >0.1 pg / mg of the total protein in the EVs.

13. The composition for use according to claim 1 or 8, or any claim dependent thereon, wherein the EVs comprise an immunogenic amount of the 5. pyogenes protein PrsAl, preferably at the level of >0.001 pg / mg, more preferably >0.01 pg / mg, even more preferably >0.02 pg / mg, yet more preferably >0.05 pg / mg, most preferably >0.1 pg / mg of the total protein in the EVs.

14. The composition for use according to claim 1 or 8, or any claim dependent thereon, wherein the EVs comprise an immunogenic amount of the 5. pyogenes protein PrsA2, preferably at the level of >0.001 pg / mg, more preferably >0.01 pg / mg, even more preferably >0.02 pg / mg, yet more preferably >0.05 pg / mg, most preferably >0.1 pg / mg of the total protein in the EVs.

15. The composition for use according to claim 1 or 8, or any claim dependent thereon, wherein the EVs comprise an immunogenic amount of the 5. pyogenes protein GlnP, preferably at the level of >0.001 pg / mg, more preferably >0.01 pg / mg, even more preferably >0.02 pg / mg, yet more preferably >0.05 pg / mg, most preferably >0.1 pg / mg of the total protein in the EVs.

16. The composition for use according to claim 1 or 8, or any claim dependent thereon, wherein the EVs comprise an immunogenic amount of the 5. pyogenes proteinSPy_0319, preferably at the level of >0.001 pg / mg, more preferably >0.01 pg / mg, even more preferably >0.02 pg / mg, yet more preferably >0.05 pg / mg, most preferably >0.1 pg / mg of the total protein in the EVs.

17. The composition for use according to claim 1 or 8, or any claim dependent thereon, wherein the EVs comprise the 5. pyogenes protein MalX, preferably at the level of >0.001 pg / mg, more preferably >0.01 pg / mg, even more preferably >0.02 pg / mg, yet more preferably >0.05 pg / mg, most preferably >0.1 pg / mg of the total protein in the EVs.

18. The composition for use according to any of the preceding claims, wherein the composition comprises the 5. pyogenes protein(s) MtsA, SPy_1228, PrsAl, PrsA2, GlnP and / or SPy_0319 associated with EVs at a concentration of at least 0.0001 pg / ml, or in an increasing order of preference: at least 0.005 pg / ml, at least 0.001 pg / ml, at least 0.01 pg / ml, at least 0.03 pg / ml, at least 0.05 pg / ml, at least 0.08 pg / ml, at least 0.1 pg / ml, at least 0.3 pg / ml, at least 0.5 pg / ml, at least 0.8 pg / ml or at least 1 pg / ml.

19. The composition for use according to any of the preceding claims, wherein the composition comprises the 5. pyogenes protein MtsA at a concentration of at least 0.0001 pg / ml, or in an increasing order of preference: at least 0.005 pg / ml, at least 0.001 pg / ml, at least 0.01 pg / ml, at least 0.03 pg / ml, at least 0.05 pg / ml, at least 0.08 pg / ml, at least 0.1 pg / ml, at least 0.3 pg / ml, at least 0.5 pg / ml, at least 0.8 pg / ml or at least 1 pg / ml.

20. The composition for use according to any of the preceding claims, wherein the composition comprises the 5. pyogenes protein SPy_1228 at a concentration of at least 0.0001 pg / ml, or in an increasing order of preference: at least 0.005 pg / ml, at least 0.001 pg / ml, at least 0.01 pg / ml, at least 0.03 pg / ml, at least 0.05 pg / ml, at least 0.08 pg / ml, at least 0.1 pg / ml, at least 0.3 pg / ml, at least 0.5 pg / ml, at least 0.8 pg / ml or at least 1 pg / ml.

21. The composition for use according to any of the preceding claims, wherein the composition comprises the 5. pyogenes protein PrsAl at a concentration of at least 0.0001 pg / ml, or in an increasing order of preference: at least 0.005 pg / ml, at least 0.001 pg / ml, at least 0.01 pg / ml, at least 0.03 pg / ml, at least 0.05 pg / ml, at least0.08 pg / ml, at least 0.1 pg / ml, at least 0.3 pg / ml, at least 0.5 pg / ml, at least 0.8 pg / ml or at least 1 pg / ml22. The composition for use according to any of the preceding claims, wherein the composition comprises the 5. pyogenes protein PrsA2 at a concentration of at least 0.0001 pg / ml, or in an increasing order of preference: at least 0.005 pg / ml, at least 0.001 pg / ml, at least 0.01 pg / ml, at least 0.03 pg / ml, at least 0.05 pg / ml, at least 0.08 pg / ml, at least 0.1 pg / ml, at least 0.3 pg / ml, at least 0.5 pg / ml, at least 0.8 pg / ml or at least 1 pg / ml.

23. The composition for use according to any of the preceding claims, wherein the composition comprises the 5. pyogenes protein GlnP at a concentration of at least 0.0001 pg / ml, or in an increasing order of preference: at least 0.005 pg / ml, at least 0.001 pg / ml, at least 0.01 pg / ml, at least 0.03 pg / ml, at least 0.05 pg / ml, at least 0.08 pg / ml, at least 0.1 pg / ml, at least 0.3 pg / ml, at least 0.5 pg / ml, at least 0.8 pg / ml or at least 1 pg / ml.

24. The composition for use according to any of the preceding claims, wherein the composition comprises the 5. pyogenes protein SPy_0319 at a concentration of at least 0.0001 pg / ml, or in an increasing order of preference: at least 0.005 pg / ml, at least 0.001 pg / ml, at least 0.01 pg / ml, at least 0.03 pg / ml, at least 0.05 pg / ml, at least 0.08 pg / ml, at least 0.1 pg / ml, at least 0.3 pg / ml, at least 0.5 pg / ml, at least 0.8 pg / ml or at least 1 pg / ml.

25. The composition for use according to any of the preceding claims, wherein the composition comprises the 5. pyogenes protein MalX at a concentration of at least 0.0001 pg / ml, or in an increasing order of preference: at least 0.005 pg / ml, at least 0.001 pg / ml, at least 0.01 pg / ml, at least 0.03 pg / ml, at least 0.05 pg / ml, at least 0.08 pg / ml, at least 0.1 pg / ml, at least 0.3 pg / ml, at least 0.5 pg / ml, at least 0.8 pg / ml or at least 1 pg / ml.

26. The composition for use according to any of the preceding claims, wherein the composition comprises each of the 5. pyogenes protein(s) MtsA, SPy_1228, PrsAl, PrsA2, GlnP and SPy_0319 at a concentration of at least 0.0001 pg / ml, or in an increasing order of preference: at least 0.005 pg / ml, at least 0.001 pg / ml, at least 0.01 pg / ml, at least 0.03 pg / ml, at least 0.05 pg / ml, at least 0.08 pg / ml, at least 0.1 pg / ml, at least 0.3 pg / ml, at least 0.5 pg / ml, at least 0.8 pg / ml or at least 1 pg / ml.

27. The composition for use according to any of the preceding claims, wherein the composition is for use by injection (including intramuscular, intracutaneous, subcutaneous, intravenous), buccal administration, oral administration or intranasal administration.

28. The composition for use according to any of the preceding claims, wherein the composition further comprises one or more group A streptococcus antigen(s) in addition to MtsA, SPy_1228, PrsAl, PrsA2, GlnP, MalX and / or SPy_0319.

29. The composition for use according to any of the preceding claims, wherein the composition is essentially free of Streptococcus pyogenes whole cells.

30. The composition for use according to any of the preceding claims, wherein the composition is essentially free of Streptococcus pyogenes whole cell lysate.

31. The composition for use according to any of the preceding claims, wherein the composition is an immunogenic composition.

32. The composition for use according to any of the preceding claims, wherein the composition is a vaccine.

33. The composition for use according to any of the preceding claims, for use in a method for inducing protective immunity in a subject against a GAS infection.

34. The composition for use according to any of the preceding claims, for use in a method for inducing protective immunity in a subject against colonization by GAS.

35. The composition for use according to any of the preceding claims, for use in a method for inducing protective immunity in a subject against a GAS infection selected from streptococcal pharyngitis, scarlet fever, impetigo, streptococcal meningitis, streptococcal sinusitis, necrotizing fasciitis, cellulitis, streptococcal toxic shock syndrome, rheumatic fever, and post-streptococcal glomerulonephritis.