Method for purifying polysaccharides and their polypeptide conjugates

Abstract

The present disclosure provides methods for purifying high molecular weight bacterial polysaccharides (eg, cell wall polysaccharides) and polypeptide-polysaccharide conjugates thereof suitable for use in immunogenic compositions.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Application No. 63 / 150,516, filed February 17, 2021; U.S. Provisional Application No. 63 / 288,387, filed December 10, 2021, the disclosures of which are incorporated by reference in their entireties herein.

[0002] (Government subsidy explanation) This invention was made with U.S. Government support under Grant No. 93.360; Subaward 4500003905 awarded by the U.S. Department of Health and Human Services, Office of the Assistant Secretary for Preparedness and Response (HHS / ASPR) under the CARB-X Pass-Through Entity. The Government has certain rights in this invention.

[0003] (Description of electronically submitted text files) The sequence listing associated with this application is provided in text format in lieu of a paper copy and is incorporated herein by reference. The name of the text file containing the sequence listing is STRO_015_02WO_ST25.txt. This text file is 363 kb, was created on February 14, 2022, and was submitted via EFS-Web.

[0004] FIELD OF THE PRESENT ART The present invention relates generally to the field of bacterial polysaccharides, their purification, conjugation to polypeptides and immunogenic compositions comprising such polysaccharide-polypeptide conjugates.

[0005] (background) Bacterial cell walls and membranes are associated with many polymeric molecules, including glycoconjugates and polysaccharides (PS), which play various structural and functional roles in bacteria. In gram-negative bacteria, the outer membrane is largely composed of lipopolysaccharide (LPS). Gram-positive bacteria lack an outer membrane, but have a thick layer of peptidoglycan that contains specialized polysaccharides. Some strains of both gram-negative and gram-positive bacteria contain peptidoglycan-bound capsular polysaccharides that further aid in virulence.

[0006] Inducing immunity against many PSs, e.g., PSs of bacteria such as Neisseria meningitidis, HiB and Streptococcus pneumoniae, provides protection against disease and many effective vaccines have been developed. However, large-scale vaccine production requires a large-scale, cost-effective supply of pure, homogenous polysaccharides free of other cellular components. Several conventional methods for purifying polysaccharides rely on harsh conditions (e.g., concentrated hydrofluoric acid), which raise safety concerns and limit the scale-up of production. Furthermore, many purification methods degrade the target polysaccharide to some extent, reducing yields, or result in low molecular weight polysaccharides with limited or reduced immunogenicity. Therefore, there is a need for safer, higher-yielding, and more efficient methods for polysaccharide production.

[0007] Group A Streptococcus (GAS) is a prominent human pathogen causing 700 million cases of pharyngitis ("strep pharyngitis") annually worldwide, and increasing cases of severe invasive infections, sepsis, necrotizing fasciitis, otitis media, and toxic shock syndrome. GAS is also responsible for post-infectious immune-mediated rheumatic heart disease (RHD), a leading cause of death in developing countries. Currently, approximately 30 million people suffer from RHD, with more than 300,000 deaths annually (60% under age 70) and 11.5 million disability-adjusted life years lost. Despite high global demand, there is no safe and effective therapeutic agent. Furthermore, the immunodominant surface-anchored form of GAS M protein is highly polymorphic (more than 200 emm forms), and regions of its dimeric coiled-coil structure can trigger an autoimmune response against cardiac tissue in RHD. Thus, there is a need for immunogenic compositions that can prevent or treat GAS infections. The compositions may utilize protein-antigen conjugates, and thus there is a need for conjugates that have sufficient immunogenicity. Summary of the Invention

[0008] SUMMARY OF THEINVENTION Disclosed herein is a polypeptide-polysaccharide conjugate comprising: (a) a GAS polypeptide antigen or a non-GAS carrier polypeptide that comprises at least one unnatural amino acid (nnAA); and (b) a purified cell wall polysaccharide or peptidoglycan-bound capsular polysaccharide having a molecular weight of at least about 10 kDa to at least about 40 kDa.

[0009] Described herein is an immunogenic composition comprising a Group A Streptococcus (GAS) C5a peptidase polypeptide antigen, a GAS streptolysin O (SLO) polypeptide antigen, and a polypeptide-polysaccharide conjugate. The polypeptide-polysaccharide conjugate may comprise: (i) a Streptococcus pyogenes Adhesion and Division (SpyAD) conjugate polypeptide or a fragment thereof comprising at least one unnatural amino acid (nnAA), wherein the at least one nnAA comprises a click chemistry reactive group; and (ii) a GAS polysaccharide or a variant thereof lacking an immunodominant N-acetylglucosamine (GlcNAc) side chain. About 8 mol% to about 20 mol% of the polysaccharide repeating units of the GAS polysaccharide or a variant thereof may be derivatized with a linker. The average molecular weight of the polypeptide-polysaccharide conjugate is between about 185 kDa and about 700 kDa or between about 190 kDa and about 700 kDa.

[0010] Also disclosed herein is a method for purifying cell wall polysaccharides or peptidoglycan-bound capsular polysaccharides from bacterial cells, the method comprising: (a) hydrolyzing bacterial cells in a solution containing a base and a reducing agent to form a lysate solution containing polysaccharides; and (b) incubating the lysate solution containing polysaccharides with a mural enzyme to form a free polysaccharide solution. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 shows a flow chart outlining an exemplary polysaccharide purification process. [Diagram 2] 2A, 2B and 2C show NMR analysis of purified polysaccharide from a GAS bacterial strain expressing PS lacking GlcNAc. [Diagram 3] FIG. 3 shows a Western blot demonstrating that M protein is removed from the polysaccharide when purified as described in Example 1. [Figure 4] 4A and 4B show a flow chart outlining another exemplary polysaccharide purification method. [Diagram 5] FIG. 5 shows a scheme outlining the derivatization of GAS polysaccharide with DBCO-(PEG)-NH and subsequent conjugation to pAMF-derivatized SpyAD. [Figure 6] FIG. 6 shows SEC MALS analysis of SpyAD[4pAMF] polypeptide-polysaccharide conjugate 3.5 hours after conjugation reaction and dialysis, prepared as described in Example 1. [Figure 7] FIG. 7 shows SEC-MALS analysis of SpyAD[4pAMF] polypeptide-polysaccharide conjugate using the hydrofluoric acid-based PS purification method (Peak 1: 2070.5 kDa: Peak 2: 135,200 Da). [Figure 8] Figure 8A shows a schematic of 22 SLO(ΔC101) variants (var1-var22) and their pAMF introduction sites. Figure 8B and Figure 8C show the expression levels of 22 SLO variants containing 3-8 pAMFs and the corresponding gels of variants containing 5-8 pAMFs that were purified. Figure 8D shows a Safe Blue stained and DBCO-TAMRA labeled gel showing successful pAMF introduction into a subset of the purified variants (var1, 5, 6, 10, 11, 12, 14 and 15). Figure 8E shows a SEC-MALS analysis providing molecular weight estimates for a subset of the purified variants (var1, 5, 6, 10, 11, 12, 14 and 15). [Figure 9]Figure 9A shows SDS-PAGE analysis of 5- and 6-pAMF variant SLO conjugates with long chain GAC. Figure 9B shows SEC-MALS analysis of purified conjugates, which shows average molar mass of 97 kDa or 116 kDa for conjugates made with SLO(ΔC101) variants containing three pAMFs (introduced molar mass for GACPR: 6.2 kDa and GACPR-DBCO: 6.8 kDa), and Figure 9C shows SEC-MALS analysis of purified conjugates, which shows average molar mass of 68 kDa or 74 kDa for conjugates made with SLO(ΔC101) variants containing four pAMFs. [Figure 10] FIG. 10 shows SEC-MALS data of 5- and 6-pAMF variant SLO conjugates with long chain GAC polysaccharide. [Figure 11] Figure 11A shows antibody titers of mice after immunization with conjugates made with selected SLO(ΔC101) variants and using long-chain GAC polysaccharide as the coating antigen in an ELISA. Figure 11B shows antibody titers of another cohort of mice after immunization with conjugates made with selected SLO(ΔC101) variants and using long-chain GAC polysaccharide as the coating antigen in an ELISA. [Figure 12] FIG. 12 shows the results of an in vivo mouse immunization study with mock, SLO(ΔC101)var1-GAC conjugate or combination vaccine [SLO(ΔC101)var1+eCRM-GACPR] following challenge with GAS serotype M1. [Figure 13] FIG. 13 shows a gel comparing the conjugates CNJ-AE, CNJ-AF and CNJ-AG with native SpyAD, demonstrating that the conditions used to make CNJ-AG result in a high molecular weight conjugate. [Figure 14]FIG. 14 shows the results of an autoradiography experiment (gel and plot showing estimated expression titers) and indicates that SpyAD fragments (e.g., SEQ ID NOs:77, 78, 79 and 81) produce fewer unwanted fragments during expression, allowing for easier purification and better conjugation to activated polysaccharides. [Figure 15] FIG. 15 shows the results (antibody titers by ELISA) of an immunization study comparing SpyAD-GAC conjugates with different molecular weights and made under different conjugation conditions. [Figure 16] FIG. 16 shows the results (antibody titers by ELISA) of an immunization study comparing SpyAD-GAC conjugates with different molecular weights and made under different conjugation conditions. [Figure 17] FIG. 17 shows the results (antibody titers by ELISA) of an immunization study comparing SpyAD-GAC conjugates with different molecular weights and made under different conjugation conditions. [Figure 18] FIG. 18 shows the results (antibody titers by ELISA) of an immunization study comparing SpyAD-GAC conjugates with different molecular weights and made under different conjugation conditions. [Figure 19] FIG. 19 shows the results (antibody titers by ELISA) of an immunization study comparing SpyAD-GAC conjugates with different molecular weights and made under different conjugation conditions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Detailed Description of the Invention Described herein are methods for purifying high molecular weight polysaccharides PS (herein referred to as long chain polysaccharides) from bacterial cells. These PS have a higher average molecular weight than those isolated using methods known in the art, and therefore can be used in the production of vaccines, i.e., immunogenic compositions, etc., that are more immunogenic and / or stable. Such immunogenic compositions are described herein. In addition to increasing the size of PS, the methods described herein can result in higher yields than conventional purification methods. As a result, these methods allow for more efficient production of purified PS.

[0013] Polysaccharide purification The methods described herein are useful for purifying polysaccharides from bacterial cells. The bacterial cells can be isolated, for example, as a pellet from the culture using standard techniques known in the art. In certain embodiments, the disclosure provides methods for purifying cell wall polysaccharides or peptidoglycan-bound capsular polysaccharides from bacterial cells, for example, a pellet of bacterial cells.

[0014] "Purification," as used herein, refers to the removal or partial removal of polysaccharides from non-polysaccharide components present in the bacterial cell. Purification can mean the removal of at least about 85% of impurities and / or by-products. In certain embodiments, a "purified" polysaccharide solution or preparation contains less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, less than about 0.5%, less than about 0.4%, less than about 0.3%, less than about 0.2%, less than about 0.1%, less than about 0.09%, less than about 0.08%, less than about 0.07%, less than about 0.06%, less than about 0.05%, less than about 0.04%, less than about 0.03%, less than about 0.02%, or less than about 0.01% impurities and / or by-products.

[0015] As used herein, "cell wall polysaccharide" refers to any PS present in a bacterial cell wall, e.g., different from that present in the bacterial capsule. The cell wall polysaccharide may be bound to the cell via peptidoglycan. The cell wall polysaccharide may be derived from a gram-positive or gram-negative bacterial cell. An exemplary cell wall polysaccharide is Group A Streptococcus (GAS) PS.

[0016] "Peptidoglycan-bound capsular polysaccharides" as used herein refers to any PS from bacterial capsules that is bound to cells via peptidoglycan and is not susceptible to degradation under the conditions of the methods described herein. These include certain PSs that contain certain combinations of monosaccharides such as glucose, rhamnose, galactose, mannose, ribose, glucuronic acid, galacturonic acid and 2-acetamido-4-amino-2,4,6-trideoxygalactose (AATGal). Peptidoglycan-bound capsular polysaccharides may be derived from Gram-positive or Gram-negative bacterial cells. Peptidoglycan-bound capsular polysaccharides suitable for purification by the methods described are also characterized by little or no O-acetylation or little or no phosphodiester linkages.

[0017] In general, the method for purifying cell wall polysaccharides or peptidoglycan-linked capsular polysaccharides from bacterial cells described herein comprises hydrolyzing bacterial cells in a solution containing a base and a reducing agent; and incubating the polysaccharide-containing composition with a wall-degrading enzyme to form a free polysaccharide solution. In an embodiment, the method comprises (a) hydrolyzing bacterial cells in a solution containing a base and a reducing agent to form a polysaccharide-containing degraded solution; and (b) incubating the polysaccharide-containing degraded solution with a wall-degrading enzyme to form a free polysaccharide solution. In an embodiment, the method for producing purified cell wall polysaccharides or peptidoglycan-linked capsular polysaccharides from bacterial cells comprises (a) incubating bacterial cells with a wall-degrading enzyme to form a cleaved polysaccharide solution; and (b) incubating the cleaved polysaccharide solution with a base to form a free polysaccharide solution.

[0018] In some embodiments, the methods described herein are suitable for purifying polysaccharides that lack O-acetyl groups. The purification methods can be used for PS with N-acetylated glucose, rhamnose, mannose, ribose or galactose monomers. In some embodiments of the methods described herein, the bacterial cell is a Pseudomonas bacterial cell, a Streptococcus bacterial cell, a Staphylococcus bacterial cell, a Neisseria bacterial cell, a Haemophilus bacterial cell, a Listeria bacterial cell, an Enterococcus bacterial cell or a Clostridium bacterial cell. In certain embodiments, the bacterial cell is selected from the group consisting of Pseudomonas aeruginosa, Streptococcus viridans, Streptococcus mutans, and Streptococcus pyogenes.

[0019] In one embodiment, the bacterial cell is Streptococcus pyogenes, also known as Group A Streptococcus (GAS). GAS is a gram-positive, linear, beta-hemolytic coccus. It is responsible for a variety of diseases in humans, including streptococcal pharyngitis (acute pharyngitis), otitis media, and skin and soft tissue infections, such as impetigo or cellulitis. In rare cases, these diseases may also lead to invasive (severe) diseases, such as necrotizing fasciitis (flesh-eating disease) and toxic shock syndrome (TSS). Several virulence factors are involved in the pathogenesis of GAS, such as M protein, hemolysin, and extracellular enzymes. In certain embodiments, the Streptococcus pyogenes is selected from the following serotypes: M1, M2, M3, M4, M5, M6, M9, M11, M12, M13, M18, M22, M25, M28, M41, M43, M44, M62, M71, M72, M74, M75, M77, M80, M81, M83, M87, M89, and M92, M94, M110, or a variant of any of the foregoing. In certain embodiments of the methods described herein, the M protein (e.g., M1) is efficiently removed during purification while maintaining good yields of isolated long chain polysaccharides.

[0020] In certain embodiments, Streptococcus pyogenes bacterial cells can be genetically engineered to produce a polysaccharide or variant thereof lacking the immunodominant N-acetylglucosamine (GLcNAc) side chain (e.g., International PCT Publication No. WO 2013 / 020090, U.S. Patent No. 10,780,155, International PCT Publication No. WO 2021 / 167996, and Gao, NJ et al. Infectious Microbes and Diseases, doi: 10.1097 / IM9.000000000044).

[0021] In certain embodiments, the methods described herein may be suitable for purifying peptidoglycan-bound capsular polysaccharides from bacterial cells.

[0022] In the disclosed method, the treatment of bacterial cells with base may serve to dissociate polysaccharides from other cellular components (e.g., cell walls, etc.). Unlike known methods of PS purification that utilize acid treatment, the base used herein can avoid hydrolysis of glycosidic bonds in native polysaccharides. Specifically, the method of purifying cell wall polysaccharides or peptidoglycan-linked capsular polysaccharides from bacterial cells includes hydrolyzing bacterial cells in a solution containing a base and a reducing agent to form a disintegration solution containing the polysaccharides. Non-limiting examples of bases suitable for the disclosed method are alkali metal hydroxides (e.g., NaOH, KOH, LiOH, etc.), carbonates (e.g., Na2CO3, K2CO3, etc.), organic amines (e.g., Et3N, NH3, etc.), and alkoxides (e.g., NaOMe, NaOEt, KOtBu, etc.). In certain embodiments, the base is NaOH, KOH, or LiOH. In certain embodiments, the base is NaOH. In certain embodiments, the base is KOH. In certain embodiments, the base is LiOH.

[0023] In certain embodiments, the concentration of the base is between about 2M and about 8M. In certain embodiments, the concentration of the base is between about 2M and about 6M. In certain embodiments, the concentration of the base is between about 2M and about 4M. In certain embodiments, the concentration of the base is between about 4M and about 8M. In certain embodiments, the concentration of the base is between about 4M and about 6M. In certain embodiments, the concentration of the base is between about 6M and about 8M. In certain embodiments of the present disclosure, the solution containing the base and the reducing agent has a pH greater than about 7. In certain embodiments, the solution containing the base and the reducing agent has a pH of about 8, about pH 9, about pH 10, about pH 11, about pH 12, about pH 13, or about pH 14.

[0024] PS may be degraded under high pH conditions through a process called "elimination reaction", and a reducing agent may be added to prevent this process. Any suitable reducing agent may be selected to counteract the elimination reaction. In certain embodiments, the reducing agent is selected from sodium borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride, dithiothreitol, or β-mercaptoethanol. In certain embodiments, the reducing agent is sodium borohydride. The concentration of the reducing agent may be about 1 mM to about 500 mM. In certain embodiments, the concentration of the reducing agent is about 1 mM to about 500 mM. In certain embodiments, the concentration of the reducing agent is about 1 mM to about 400 mM, about 1 mM to about 300 mM, about 1 mM to about 200 mM, about 1 mM to about 100 mM, about 1 mM to about 50 mM, about 1 mM to about 10 mM, about 10 mM to about 100 mM, about 100 mM to about 400 mM, about 100 mM to about 300 mM, about 100 mM to about 200 mM, about 200 mM to about 400 mM, about 200 mM to about 300 mM, and about 300 mM to about 400 mM.

[0025] When hydrolyzing bacterial cells in a solution containing a base and a reducing agent to form a hydrolysis solution containing polysaccharides, the temperature can be increased to achieve higher molecular weight PS and higher yields. In some embodiments, the hydrolysis step further comprises incubating the solution between about 30°C and about 100°C. In certain embodiments, the hydrolysis step further comprises incubating the solution between the following temperatures: about 30°C to about 100°C; about 30°C to about 90°C; about 30°C to about 80°C; about 30°C to about 70°C; about 30°C to about 60°C; about 30°C to about 50°C; about 30°C to about 40°C; about 40°C to about 100°C; about 40°C to about 90°C; about 40°C to about 80°C; about 40°C to about 70°C; about 40°C to about 60°C. °C; about 40°C to about 50°C; about 50°C to about 100°C; about 50°C to about 90°C; about 50°C to about 80°C; about 50°C to about 70°C; about 50°C to about 60°C; about 60°C to about 100°C; about 60°C to about 90°C; about 60°C to about 80°C; about 60°C to about 70°C; about 70°C to about 100°C; about 70°C to about 90°C; about 70°C to about 80°C; about 80°C to about 100°C; about 80°C to about 90°C; or about 90°C to about 100°C.

[0026] The solution containing the base and the reducing agent to form a polysaccharide-containing decomposition solution may be incubated for a time sufficient for completion of the reaction. For example, in certain embodiments, the hydrolysis step further comprises incubating the solution for about 0.5 hours to about 20 hours. In certain embodiments, the hydrolysis step further comprises incubating the solution for about 0.5 hours to about 1 hour; about 1 hour to about 5 hours; about 5 hours to about 20 hours; about 5 hours to about 15 hours; about 5 hours to about 10 hours; about 10 hours to about 20 hours; about 10 hours to about 15 hours; or about 15 hours to about 20 hours. Completion of hydrolysis can be monitored, for example, by determining the yield of polysaccharide by an appropriate analytical method.

[0027] Since hydrolysis of the bacterial cells in a solution containing a base and a reducing agent to form a digestion solution containing polysaccharides results in the formation of non-PS by-products that must be removed, the method may include one or more pH adjustment steps to aid in the removal of those by-products. In one embodiment, the one or more pH adjustment steps are independently selected from: (i) increasing the pH of the digestion solution containing polysaccharides, or (ii) decreasing the pH of the digestion solution containing polysaccharides.

[0028] In certain embodiments, the method comprises lowering the pH of the degradation solution containing the polysaccharide to between about 3 and about 7. In certain embodiments, the method comprises lowering the pH of the degradation solution containing the polysaccharide to between about 3 and about 6; between about 3 and about 5; between about 3 and about 4; between about 4 and about 7; between about 4 and about 6; between about 4 and about 5; between about 5 and about 7; or between about 5 and about 6. In certain embodiments, the method comprises lowering the pH of the degradation solution containing the polysaccharide to about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, or about 7. In certain embodiments, the method comprises the following steps: (i) lowering the pH of the degradation solution containing the polysaccharide to between about 5.5 and about 7.0; (ii) lowering the pH of the degradation solution containing the polysaccharide to about 3; and (iii) increasing the pH of the degradation solution containing the polysaccharide to between about 5.5 and about 7.0. In certain embodiments, the method includes the steps of: (i) lowering the pH of the degradation solution containing the polysaccharide to about 6.5; (ii) lowering the pH of the degradation solution containing the polysaccharide to about 3; and (iii) increasing the pH of the degradation solution containing the polysaccharide to about 6.5.

[0029] In the methods disclosed herein, the degradation solution containing polysaccharides may be incubated at room temperature after one or more pH adjustment steps. "Room temperature" is defined as the ambient temperature in a laboratory environment, typically between about 20°C and about 25°C. In certain embodiments, the degradation solution containing polysaccharides is incubated at between about 4°C and about 30°C after one or more pH adjustment steps. In certain embodiments, the degradation solution containing polysaccharides is incubated after one or more pH adjustment steps at the following temperatures: about 4°C to about 30°C; about 4°C to about 25°C; about 4°C to about 20°C; about 4°C to about 15°C; about 4°C to about 10°C; about 10°C to about 30°C; about 10°C to about 25°C; about 10°C to about 20°C; about 10°C to about 15°C; about 15°C to about 30°C; about 15°C to about 25°C; about 15°C to about 20°C; about 20°C to about 30°C; about 20°C to about 25°C; or about 25°C to about 30°C.

[0030] It may be beneficial to remove solid by-products formed during the hydrolysis step. Thus, in some embodiments, the methods disclosed herein may further comprise removing solids from the decomposition solution containing polysaccharides. In some embodiments, removing solids from the decomposition solution containing polysaccharides comprises filtration, centrifugation, or a combination thereof. In some embodiments, the filtration comprises depth filtration, tangential flow filtration (TFF), sterile filtration, membrane filtration, or a combination thereof. In certain embodiments, the solids are removed from the decomposition solution containing polysaccharides by centrifugation. The various methods of removing solids may be used in combination and in any order. In some embodiments, the filtration comprises depth filtration followed by TFF.

[0031] In the disclosed method, bacterial cells or a digestion solution containing polysaccharides is treated with a wall-degrading enzyme to cleave glycosidic bonds in peptidoglycan and help dissociate PS from the amino acid backbone. The wall-degrading enzyme cleaves, for example, N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) bonds. Specifically, after hydrolysis, the enzymatic cleavage step may include incubating the digestion solution containing polysaccharides with a wall-degrading enzyme to form a free polysaccharide solution. In some embodiments, the wall-degrading enzyme is mutanolysin, lysozyme, or bacteriophage hydrolase. To further aid in purification, in some embodiments, the step of incubating the digestion solution containing polysaccharides with a wall-degrading enzyme to form a free polysaccharide solution further includes a step of incubating with a protease. In some embodiments, the protease is proteinase K. In certain embodiments, the protease is proteinase K, trypsin, chymotrypsin, endoproteinase Asp-N, endoproteinase Arg-C, endoproteinase Glu-C, endoproteinase Lys-C, pepsin, thermolysin, elastase, papain, subtilisin, clostripain, carboxypeptidase A, carboxypeptidase B, carboxypeptidase P, carboxypeptidase Y, cathepsin C, acyl amino acid releasing enzyme, or pyroglutamate.

[0032] In certain embodiments of the methods disclosed herein, the step of incubating the degradation solution containing polysaccharides with the wall-degrading enzyme to form a free polysaccharide solution further comprises the step of warming the degradation solution containing polysaccharides with the wall-degrading enzyme to between about 30° C. and about 65° C. In certain embodiments, the degradation solution containing polysaccharides together with wall-degrading enzymes is heated to between about 30°C to about 60°C; about 30°C to about 55°C; about 30°C to about 50°C; about 30°C to about 45°C; about 30°C to about 40°C; about 30°C to about 35°C; about 40°C to about 60°C; about 40°C to about 55°C; about 40°C to about 50°C; about 40°C to about 60°C; about 40°C to about 55°C; about 40°C to about 50°C; about 45°C to about 60°C; about 45°C to about 55°C; about 45°C to about 50°C; about 50°C to about 60°C; or about 55°C to about 60°C.

[0033] In one embodiment, the polysaccharide-containing degradation solution is heated together with the protease. In one embodiment, the polysaccharide-containing degradation solution is heated together with the protease to between about 45°C and about 55°C; between about 45°C and about 50°C; or between about 50°C and about 55°C.

[0034] In the case of both a degradation solution containing PS and a wall-degrading acid or both a degradation solution containing PS and a protease, the degradation solution can be heated for at least 2 hours. In certain embodiments, the degradation products are heated for the following times: about 6 hours to about 20 hours; about 6 hours to about 18 hours; about 6 hours to about 16 hours; about 6 hours to about 14 hours; about 6 hours to about 12 hours; about 6 hours to about 10 hours; about 6 hours to about 8 hours; about 8 hours to about 20 hours; about 8 hours to about 18 hours; about 8 hours to about 16 hours; about 8 hours to about 14 hours; about 8 hours to about 12 hours; about 8 hours to about 10 hours; about 10 hours to about 2 hours. 0 hours; about 10 hours to about 18 hours; about 10 hours to about 16 hours; about 10 hours to about 14 hours; about 10 hours to about 12 hours; about 12 hours to about 20 hours; about 12 hours to about 18 hours; about 12 hours to about 16 hours; about 12 hours to about 14 hours; about 14 hours to about 20 hours; about 14 hours to about 18 hours; about 14 hours to about 16 hours; about 16 hours to about 20 hours; about 16 hours to about 18 hours; or about 18 hours to about 20 hours.

[0035] To further aid in the separation of PS from by-products, the free polysaccharide solution generated during the step of incubating the polysaccharide-containing degradation solution with wall-degrading enzymes is further purified.In some embodiments, the free polysaccharide solution is further purified to reduce the concentration of nucleic acids, enzymes, host cell proteins (HCPs), or combinations thereof.In certain embodiments, the free polysaccharide solution is further purified by precipitation, anion exchange chromatography, cation exchange chromatography, hydrophobic interaction chromatography, ceramic hydroxyapatite type chromatography, or combinations thereof.

[0036] In some embodiments, the free polysaccharide solution is further purified by precipitation. This precipitation can be induced by a number of chemical or physical means. In some embodiments, the free polysaccharide solution is treated by further purification to reduce the concentration of nucleic acids, enzymes, host cell proteins (HCPs), or combinations thereof. In some embodiments, the free polysaccharide solution is treated with a detergent. In some embodiments, the detergent is cetyltrimethylammonium bromide (CTAB). In certain embodiments, the concentration of CTAB in the free polysaccharide solution is about 0.1% to about 10%. In certain embodiments, the concentration of CTAB in the free polysaccharide solution is from about 0.1% to about 0.5%; from about 0.5% to about 3%; from about 0.5% to about 2%; from about 0.5% to about 1%; from about 1% to about 10%; from about 1% to about 9%; from about 1% to about 8%; from about 1% to about 7%; from about 1% to about 6%; from about 1% to about 5%; from about 1% to about 4%; from about 1% to about 3%; from about 1% to about 2%; from about 2% to about 10%; from about 2% to about 9%; from about 2% to about 8%; from about 2% to about 7%; from about 2% to about 6%; from about 2% to about 5%; from about 2% to about 4%; from about 2% to about 3%; from about 3% to about 10%; from about 3% to about 9%. %; about 3% to about 8%; about 3% to about 7%; about 3% to about 6%; about 3% to about 5%; about 3% to about 4%; about 4% to about 10%; about 4% to about 9%; about 4% to about 8%; about 4% to about 7%; about 4% to about 6%; about 4% to about 5%; about 5% to about 10%; about 5% to about 9%; about 5% to about 8%; about 5% to about 7%; about 5% to about 6%; about 6% to about 10%; about 6% to about 9%; about 6% to about 8%; about 6% to about 7%; about 7% to about 10%; about 7% to about 9%; about 7% to about 8%; about 8% to about 10%; about 8% to about 9%; or about 9% to about 10%.

[0037] To remove excess CTAB from the solution, potassium iodide (KI) or other suitable salts can be utilized. In certain embodiments, the free polysaccharide solution is treated with potassium KI. In certain embodiments, the concentration of KI in the free polysaccharide solution is about 20 mM to about 400 mM. In certain embodiments, the concentration of KI is between about 20 mM to about 300 mM; about 20 mM to about 200 mM; about 20 mM to about 100 mM; about 20 mM to about 50 mM; about 50 mM to about 300 mM; about 50 mM to about 200 mM; about 50 mM to about 100 mM; about 100 mM to about 300 mM; about 100 mM to about 200 mM; or about 200 mM to about 300 mM.

[0038] The free polysaccharide solution resulting from the process for producing purified cell wall polysaccharides or peptidoglycan-bound capsular polysaccharides from bacterial cells can be further purified at any point by filtration, centrifugation, chromatography, or a combination of the above. In some embodiments, filtration is used. In certain embodiments, filtration comprises depth filtration, tangential flow filtration (TFF), sterile filtration, or a combination of the above. In some embodiments, chromatography comprises hydrophobic interaction chromatography (HIC), anion exchange chromatography (AEX), ceramic hydroxyapatite-based chromatography, or cation exchange chromatography (CEX).

[0039] Polypeptide-polysaccharide conjugates As mentioned above, the polymeric polysaccharides obtained by the methods described herein are suitable for the formation of polypeptide-polysaccharide conjugates for use in vaccines and the like.

[0040] The high molecular weight polysaccharides of the present disclosure may also be referred to as "long chain polysaccharides."

[0041] Thus, the polypeptide-polysaccharide conjugates disclosed herein comprise (a) a polypeptide antigen (e.g., a GAS polypeptide antigen) or a non-GAS carrier polypeptide that comprises at least one non-natural amino acid (nnAA); and (b) a purified cell wall polysaccharide or peptidoglycan-bound capsular polysaccharide having a molecular weight of at least about 10 kDa to at least about 40 kDa. As described in more detail herein, the nnAA comprises a click chemistry reactive group to facilitate conjugation between the polypeptide antigen and the PS.

[0042] In certain embodiments, the polysaccharide used in the conjugate is a purified cell wall polysaccharide or a peptidoglycan-bound capsular polysaccharide.

[0043] As mentioned above, GAS are important bacteria for human health and cause many diseases. In one embodiment, the purified cell wall polysaccharide is a GAS polysaccharide. One GAS polysaccharide, group A carbohydrate (GAC), contains a polyrhamnose backbone with immunodominant N-acetylglucosamine (GlcNAc) side chains, is present on the surface of all GAS serotype strains, regardless of M type, and has been explored as a GAS vaccine candidate. Affinity purified anti-GAC antibodies successfully opsonized the three GAS serotypes tested (Salvadori et al., 1995. J. Infect. Dis. 171:593-600.), and mice immunized with GAC were protected against intraperitoneal and intranasal GAS challenge. However, immunological cross-reactivity between anti-GAC antibodies and host cardiac valve proteins (Goldstein et al., 1967. Nature 213:44-47) and cytoskeletal proteins such as actin, keratin, myosin and vimentin raises significant safety concerns regarding the use of GAC as a GAS vaccine component. Thus, in one embodiment, the purified cell wall polysaccharide is a variant of GAC that lacks the immunodominant GlcNAc side chain (see, e.g., International PCT Publication No. WO 2013 / 020090, U.S. Patent No. 10,780,155, and Gao, NJ et al., Infectious Microbes and Diseases, 2021, 3(2), 87-100).

[0044] In certain embodiments, the purified cell wall polysaccharide or peptidoglycan-bound capsular polysaccharide has a molecular weight of from about 10 kDa to about 45 kDa; from about 10 kDa to about 40 kDa; from about 10 kDa to about 35 kDa; from about 10 kDa to about 30 kDa; from about 10 kDa to about 25 kDa; from about 10 kDa to about 20 kDa; from about 10 kDa to about 15 kDa; from 15 kDa to about 40 kDa; from about 15 kDa to about 35 kDa; from about 15 kDa to about 30 kDa. ;has an average molecular weight of about 15 kDa to about 25 kDa;about 15 kDa to about 20 kDa;20 kDa to about 40 kDa;about 20 kDa to about 35 kDa;about 20 kDa to about 30 kDa;about 20 kDa to about 25 kDa;25 kDa to about 40 kDa;about 25 kDa to about 35 kDa;about 25 kDa to about 30 kDa;about 30 kDa to about 40 kDa;about 30 kDa to about 35 kDa;or about 35 kDa to about 40 kDa. In certain embodiments, the purified cell wall polysaccharides or peptidoglycan-bound capsular polysaccharides have an average molecular weight of about 10 kDa, about 15 kDa, about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, about 40 kDa or about 45 kDa.

[0045] In some embodiments, the purified cell wall polysaccharide or peptidoglycan-bound capsular polysaccharide is modified with a click chemistry reactive group to facilitate conjugation to a conjugate protein. Examples of click chemistry reactive groups can be found, for example, in International PCT Publication No. WO2018 / 126229 and International PCT Publication No. WO2021 / 167996, each of which is incorporated herein by reference in its entirety. For example, in some embodiments, the purified cell wall polysaccharide or peptidoglycan-bound capsular polysaccharide is modified with DBCO or DBCO-PEG (e.g., DBCO-PEG-NH2). In some embodiments, the purified cell wall polysaccharide or peptidoglycan-bound capsular polysaccharide is modified with DBCO-(PEG)4-NH2.

[0046] The extent to which a purified cell wall polysaccharide or peptidoglycan-bound capsular polysaccharide is modified with click chemistry reactive groups or linkers containing click chemistry reactive groups can be defined by how many polysaccharide repeating units (PSRUs) are modified or by what percentage (e.g., mol %) are modified. Thus, in certain embodiments of the purified cell wall polysaccharide or peptidoglycan-bound capsular polysaccharide described herein, about 8 mol % to 20 mol % of the polysaccharide repeating units are derivatized with click chemistry reactive groups or linkers containing click chemistry reactive groups. In one embodiment, the proportion of PS repeating units in the polysaccharide is about 8 mol% to about 20 mol%, about 8 mol% to about 19 mol%, about 8 mol% to about 18 mol%, about 8 mol% to about 17 mol%, about 8 mol% to about 16 mol%, about 8 mol% to about 15 mol%, about 8 mol% to about 14 mol%, about 8 mol% to about 13 mol%, about 8 mol% to about 12 mol%, about 8 mol% to about 11 mol%, 8 mol% to about 10 mol%, about 8 mol% to about 9 mol%, about 9 mol% to about 20 mol%, about 9 mol% to about 19 mol%, about 9 mol% to about 18 mol%, about 9 mol% to about 17 mol%, about 9 mol% to about 16 mol%, about 9 mol% to about 15 mol%, about 9 mol% to about 14 mol%, 9 mol% to about 13 mol%, about 9 mol% to about 12 mol%, about 9 mol% to about 11 mol%, 9 mol% to about 10 mol%, about 10 mol% to about 2 0mol%, about 10mol% to about 19mol%, about 10mol% to about 18mol%, about 10mol% to about 17mol%, about 10mol% to about 16mol%, about 10mol% to about 15mol%, about 10mol% ~about 14mol%, 10mol% to about 13mol%, about 10mol% to about 12mol%, about 10mol% to about 11mol%, about 11mol% to about 20mol%, about 11mol% to about 19mol%, about 11mo l% to about 18 mol%, about 11 mol% to about 17 mol%, about 11 mol% to about 16 mol%, about 11 mol% to about 15 mol%, about 11 mol% to about 14 mol%, about 11 mol% to about 13 mol%, about 1 1 mol% to about 12 mol%, about 12 mol% to about 20 mol%, about 12 mol% to about 19 mol%, about 12 mol% to about 18 mol%, about 12 mol% to about 17 mol%, about 12 mol% to about 16 mol%,About 12 mol% to about 15 mol%, about 12 mol% to about 14 mol%, 12 mol% to about 13 mol%, about 13 mol% to about 20 mol%, about 13 mol% to about 19 mol%, about 13 mol% to about 18 mol%, about 13 mol% to about 17 mol%, about 13 mol% to about 16 mol%, about 13 mol% to about 15 mol%, about 13 mol% to about 14 mol%, about 14 mol% to about 20 mol%, about 14 mol% to about 19 mol%, about 14 mol% to about 18 mol%, about 14 mol% to about 17 mol%, about 14 mol% to about 16 mol%, about 14 mol% to about 15 mol%, about 15 mol% to about 20 mol%, about 1 5 mol% to about 19 mol%, about 15 mol% to about 18 mol%, about 15 mol% to about 17 mol%, about 15 mol% to about 16 mol%, about 16 mol% to about 20 mol%, about 16 mol% to about 19 mol%, about 16 mol% to about 18 mol%, about 16 mol% to about 17 mol%, about 17 mol% to about 20 mol%, about 17 mol% to about 19 mol%, about 17 mol% to about 18 mol%, about 18 mol% to about 20 mol%, about 18 mol% to about 19 mol%, or about 19 mol% to about 20 mol% is derivatized with a click chemistry reactive group or a linker containing a click chemistry reactive group. In certain embodiments, the polysaccharide repeating unit is a polysaccharide repeating unit of a GAS polysaccharide or a variant thereof. In certain embodiments of the purified cell wall polysaccharide or peptidoglycan-bound capsular polysaccharide described herein, about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mol % of the polysaccharide repeating units are derivatized with a click chemistry reactive group or a linker that includes a click chemistry reactive group. In certain embodiments, the GAS polysaccharide or variant thereof lacks the immunodominant N-acetylglucosamine (GlcNAc) side chain.

[0047] In general, increasing the amount (mol%) of linker introduced into the polysaccharide repeat unit of the polysaccharide can produce a polypeptide-polysaccharide conjugate with increased molecular weight. As discussed further herein, polypeptide-polysaccharide conjugates formed using polysaccharides with a higher amount of linker introduced may exhibit higher immunogenicity than those with a lower molar percentage of linker introduced. Furthermore, as discussed further herein, polysaccharides with increased amounts of linker introduced may also have a lower amount of free polysaccharide present after conjugation.

[0048] In the context of the polypeptide-polysaccharide conjugates and immunogenic compositions described herein, "free polysaccharide" refers to polysaccharides that are not conjugated to a polypeptide, polysaccharides that are derivatized with a linker and / or a click chemistry reactive group, or fragments of polysaccharides. For example, after a conjugation reaction between a derivatized polysaccharide and a polypeptide, free polysaccharides may be present in the mixture of polypeptide-polysaccharide conjugates. The concentration of polysaccharides can be measured, for example, by the method described in Example 6.

[0049] The polypeptide antigen used in the conjugates described herein can be a GAS or non-GAS polypeptide antigen. In some embodiments, the polypeptide antigen is a full-length GAS polypeptide antigen or a fragment of a full-length GAS polypeptide antigen that includes at least one non-natural amino acid (nnAA). In some embodiments, the polypeptide antigen is selected from C5a peptidase (UniProt P15926), streptolysin O (SLO, UniProt P0C0I3), streptococcal immunoglobulin binding protein 35 [Sib35, UniProt (Q1XG74)] and fibronectin binding protein F1 (Sfb1, UniProt Q48VN7) and adhesion / division polypeptide (SpyAD, UniProt Q9A1H3).

[0050] The non-GAS carrier polypeptides used in the conjugates described herein can be based on full-length non-GAS carrier polypeptides or fragments of full-length non-GAS carrier polypeptides that contain at least one non-natural amino acid. In certain embodiments, the non-GAS carrier polypeptide is selected from arginine deiminase (ADI, UniProt P0C0B3), CRM197 (1272033-67-6), ferritin (UniProt P02792) and protein D (UniProt R4R7Q5).

[0051] In some embodiments, the polypeptide antigen or non-GAS carrier protein comprises one or more amino acid mutations in the wild-type amino acid sequence. For example, in some embodiments, the polypeptide antigen is SpyAD, and the SpyAD polypeptide comprises one or more amino acid mutations. In some embodiments, the polypeptide antigen is ADI, and the ADI polypeptide comprises one or more amino acid mutations. In some embodiments, the amino acid mutation is D277A (e.g., SEQ ID NO:36).

[0052] In some embodiments, the polypeptide antigen or non-GAS carrier protein comprises at least one non-natural amino acid (nnAA). In some embodiments, at least one nnAA is substituted for lysine, leucine, arginine, or isoleucine in the polypeptide antigen or non-GAS carrier polypeptide. In some embodiments, one or more nnAA comprises a click chemistry reactive group. Here, "click chemistry reactive group" refers to a moiety, such as an azide or an alkyne, that can undergo a click chemistry reaction with a second click chemistry reactive group. In some embodiments, one click chemistry reactive group reacts with a second click chemistry reactive group to form a substituted triazole. Examples of this type of click reaction can be found, for example, in International PCT Publication No. WO2018 / 126229. General examples of metal-free click reactions used in biomedical applications can be found, for example, in Kim, et al., Chemical Science, 2019, 10, 7835-7851. Examples of nnAAs comprising click chemistry reactive groups include 2-amino-3-(4-azidophenyl)propanoic acid (pAF), 2-amino-4-azidobutanoic acid, 2-azido-3-phenylpropionic acid, 2-amino-3-azidopropanoic acid, 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridin-2-yl)propanoic acid, 2-amino-3-(4-(azidomethyl)pyridin-2-yl)propanoic acid, 2-amino-3-(6-(azidomethyl)pyridin-3-yl)propanoic acid, and 2-amino-5-azidopentanoic acid. In an embodiment, the conjugated polypeptide comprises one or more nnAAs, each nnAA being pAMF.

[0053] The polypeptide-polysaccharide conjugates described herein may be characterized by molecular weight (e.g., average molecular weight) as defined by any reasonable characterization method (e.g., SEC-MALS). The polypeptide-polysaccharide conjugates may have, for example, a molecular weight or average molecular weight of about 185 kDa or more, or 190 kDa or more. In certain embodiments, the polypeptide-polysaccharide conjugates have a molecular weight or average molecular weight of about 185 kDa to about 700 kDa, about 185 kDa to about 600 kDa, about 185 kDa to about 500 kDa, about 185 kDa to about 400 kDa, about 185 kDa to about 300 kDa, and about 185 kDa to about 200 kDa. In certain embodiments, the polypeptide-polysaccharide conjugate is from about 185 kDa to about 700 kDa, from about 185 kDa to about 650 kDa, from about 185 kDa to about 600 kDa, from about 185 kDa to about 550 kDa, from about 185 kDa to about 500 kDa, from about 185 kDa to about 450 kDa, from about 185 kDa to about 400 kDa, from about 185 kDa to about 350 kDa, from about 185 kDa to about 300 kDa, from about 185 kDa to about 500 kDa, from about 185 kDa to about 550 kDa, from about 185 kDa to about 500 kDa, 85kDa to about 250kDa, about 185kDa to about 200kDa, about 190kDa to about 700kDa, about 190kDa to about 650kDa, about 190kDa to about 600kDa, about 190kDa to about 550kDa, about 190kDa to about 500kDa, about 190kDa to about 450kDa, about 190kDa to about 400kDa, about 190kDa to about 350kDa, about 190kDa to about 300kDa, about 190kDa~about 250kDa, about 190kDa~about 200kDa, about 200kDa~about 700kDa, about 200kDa~about 650kDa, about 200kDa~about 600kDa, about 200kDa~ Approximately 550kDa, approximately 200kDa to approximately 500kDa, approximately 200kDa to approximately 450kDa, approximately 200kDa to approximately 400kDa, approximately 200kDa to approximately 350kDa, approximately 200kDa to approximately 300kDa, Approximately 200kDa to approximately 250kDa, approximately 250kDa to approximately 700kDa, approximately 250kDa to approximately 650kDa, approximately 250kDa to approximately 600kDa, approximately 250kDa to approximately 550kDa, approximately 250kDa Approximately 500kDa, approximately 250kDa to approximately 450kDa, approximately 250kDa to approximately 400kDa, approximately 250kDa to approximately 350kDa, approximately 250kDa to approximately 300kDa, approximately 300kDa to approximately 700kDa,Approximately 300kDa to approximately 650kDa, approximately 300kDa to approximately 600kDa, approximately 300kDa to approximately 550kDa, approximately 300kDa to approximately 500kDa, approximately 300kDa to approximately Approximately 450kDa, approximately 300kDa to approximately 400kDa, approximately 300kDa to approximately 350kDa, approximately 350kDa to approximately 700kDa, approximately 350kDa to approximately 650kDa, approximately 350kDa to about 600kDa, about 350kDa to about 550kDa, about 350kDa to about 500kDa, about 350kDa to about 450kDa, about 350kDa to about 400kDa, about 400kDa to about 700kDa, about 400kDa to about 650kDa, about 400kDa to about 600kDa, about 400kDa to about 550kDa, about 00kDa to about 500kDa, about 400kDa to about 450kDa, about 450kDa to about 700kDa, about 450kDa to about 650kDa, about 450kDa to about 600kDa, about 450kDa to about 550kDa, about 450kDa to about 500kDa, about 500kDa to about 700kDa, about 500kDa to about 650kDa, about The molecular weight or average molecular weight may be between about 0 kDa and about 600 kDa, about 500 kDa and about 550 kDa, about 550 kDa and about 700 kDa, about 550 kDa and about 650 kDa, about 550 kDa and about 600 kDa, about 600 kDa and about 700 kDa, about 600 kDa and about 650 kDa and about 700 kDa. In certain embodiments, the polypeptide-polysaccharide conjugate may have a molecular weight or average molecular weight of about 185, about 190, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650 or about 700 kDa.

[0054] In some embodiments, the polypeptide-polysaccharide conjugates described herein may be characterized by their molecular weight. In some embodiments, the polypeptide-polysaccharide conjugates described herein may be characterized by the mol% of the polysaccharide repeating units of the GAS polysaccharide or variant thereof that are derivatized with a linker. In some embodiments, the polypeptide-polysaccharide conjugates described herein may be characterized by their molecular weight as well as the mol% of the polysaccharide repeating units of the GAS polysaccharide or variant thereof that are derivatized with a linker.

[0055] In certain embodiments, a polypeptide-polysaccharide conjugate described herein comprises (a) a GAS polypeptide antigen or a non-GAS carrier polypeptide that comprises at least one non-natural amino acid (nnAA), wherein the nnAA comprises a click chemistry reactive group; and (b) a purified cell wall polysaccharide or peptidoglycan-bound capsular polysaccharide having a molecular weight of at least about 10 kDa to at least about 40 kDa.

[0056] In certain embodiments, a polypeptide-polysaccharide conjugate described herein comprises: (a) a GAS polypeptide antigen or a non-GAS carrier polypeptide comprising at least one non-natural amino acid (nnAA), wherein the nnAA comprises a click chemistry reactive group; and (b) a purified cell wall polysaccharide or peptidoglycan-bound capsular polysaccharide having a molecular weight of at least about 10 kDa to at least about 40 kDa; wherein about 10 mol % to about 18 mol % of the polysaccharide repeating units of the GAS polysaccharide or variant thereof are derivatized with a linker.

[0057] Table 1 shows exemplary sequences of selected polypeptide antigens and non-GAS carrier proteins, including native, variant, truncated and nnAA-containing versions.

[0058] Table 1A shows exemplary sequences of selected polypeptide antigens containing nnAAs. [Table 1] [Table 2] [Table 3] [Table 4] [Table 5]

Table 6

Table 7

Table 8

Table 9

Table 10

Table 11

Table 12

Table 13

Table 14

Table 15

Table 16

Table 17

Table 18

Table 19

Table 20

Table 21

Table 22

Table 23

Table 24

Table 25

Table 26

Table 27

[0059] In one embodiment, the polypeptide antigen is SLO. In one embodiment, the polypeptide antigen is SLO(ΔC101) and comprises three or four nnAA substituted at positions selected from K98, K112, R151, K189, K272, K323, K357, K375, K407 and K464 of SEQ ID NO:53. In one embodiment, the polypeptide antigen is SLO(ΔC101) and comprises or consists of nnAAs substituted at positions K98, R151, K272 and K357; positions K112, K189, K323 and K375; positions R151, K272, K357 and K407; positions R151, K272, K375 and K464; positions K112, K272, K357 and K464; positions K98, K189 and K357; positions K112, K189 and K323; positions K98, R151 and K272; positions K112, K272 and K375; or positions K112, K323 and K407. In one embodiment, each of the three or four nnAAs is pAMF. The polypeptide antigen is an SLO polypeptide and comprises an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 55, 56, 57, 58, 59, 60, 61, 62, 63 or 64. In one embodiment, the conjugate polypeptide is an SLO polypeptide and comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 55, 56, 57, 58, 59, 60, 61, 62, 63 or 64.

[0060] In some embodiments, the polypeptide antigen is an SLO polypeptide. In some embodiments, the polypeptide antigen is SLO(AC101) and comprises 5, 6, 7, or 8 nnAA substituted at positions selected from K98, K112, R151, K189, K272, K323, K357, K375, K407, and K464 of SEQ ID NO:53. In one embodiment, the polypeptide antigen is SLO(ΔC101) and is at positions K98, R151, K272, K357 and K407; at positions K112, K189, K323, K375 and K464; at positions K112, R151, K272, K357 and K407; at positions K98, R151, K272, K357, K407 and K464; at positions K112, R151, K189, K323, K375 and K464; at positions K98, K112, K189, K323, K375 and K464; , K407 and K464; positions K98, R151, K189, K323, K375, K407 and K464; positions K112, K189, K272, K357, K375, K407 and K464; positions K98, K112, R151, K189, K272, K323, K357 and K375; positions K98, R151, K189, K272, K323, K357, K407 and K464; or positions K112, K189, K272, K323, K357, K375, K407 and K464. In one embodiment, 5, 6, 7 or 8 nnAAs are each pAMF. In certain embodiments, the polypeptide antigen is an SLO polypeptide and comprises an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 or 76. In certain embodiments, the polypeptide antigen is an SLO polypeptide and comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 or 76.

[0061] In one embodiment, the polypeptide antigen is SpyAD and comprises four nnAAs substituted at positions K64, K287, K396 and K657 of SEQ ID NO:9. In one embodiment, the polypeptide antigen is SpyAD and comprises four nnAAs substituted at positions K64, K287, K396 and K657 of SEQ ID NO:33. In one embodiment, the four nnAAs are each pAMF. In one embodiment, the polypeptide antigen is SpyAD and comprises an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:11 or SEQ ID NO:12. In one embodiment, the four nnAAs are each pAMF. In one embodiment, the polypeptide antigen is SpyAD and comprises an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:34. In one embodiment, the polypeptide antigen is SpyAD and comprises or consists of the amino acid sequence of SEQ ID NO:11 or SEQ ID NO:12. In one embodiment, the polypeptide antigen is SpyAD and comprises or consists of the amino acid sequence of SEQ ID NO:34.

[0062] In certain embodiments, the polypeptide antigen is SpyAD and comprises an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:80. In certain embodiments, the polypeptide antigen is SpyAD and comprises or consists of the amino acid sequence of SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:80.

[0063] In certain embodiments, the polypeptide antigen is SpyAD and comprises an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:82 or SEQ ID NO:83. In certain embodiments, the polypeptide antigen is SpyAD and comprises or consists of SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:82 or SEQ ID NO:83.

[0064] In certain embodiments, the non-GAS carrier polypeptide comprises or consists of the sequence of eCRM197. In certain embodiments, eCRM197 has the sequence of SEQ ID NO:25.

[0065] In certain embodiments, the GAS polypeptide antigen or non-GAS carrier polypeptide comprises or consists of the amino acid sequence of a polypeptide listed in Table 1. In certain embodiments, the GAS polypeptide antigen or non-GAS carrier polypeptide comprises an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs:1-76.

[0066] In certain embodiments, the GAS polypeptide antigen or non-GAS carrier polypeptide comprises or consists of the amino acid sequence of a polypeptide listed in Table 1 or 1A. In certain embodiments, the GAS polypeptide antigen or non-GAS carrier polypeptide comprises an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs:1-80.

[0067] In certain embodiments, the GAS polypeptide antigen or non-GAS carrier polypeptide comprises or consists of the amino acid sequence of a polypeptide listed in Table 1. In certain embodiments, the GAS polypeptide antigen or non-GAS carrier polypeptide comprises an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs:1-76.

[0068] In certain embodiments, the GAS polypeptide antigen or non-GAS carrier polypeptide comprises or consists of the amino acid sequence of a polypeptide listed in Table 1 A. In certain embodiments, the GAS polypeptide antigen or non-GAS carrier polypeptide comprises an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs:77-80.

[0069] immunogenic composition As mentioned above, the long chain polysaccharides obtained by the methods described herein and the polysaccharide-polypeptide conjugates made using said long chain polysaccharides are suitable for use in immunogenic compositions (e.g., vaccines for treating or preventing disease). For example, in certain embodiments, the immunogenic compositions may induce a protective immune response against GAS bacteria in a subject. In certain embodiments, provided herein is the use of the immunogenic compositions described herein in the manufacture of a medicament for inducing a protective immune response against GAS bacteria in a subject.

[0070] As used herein, the term "subject" refers to a mammal. In some embodiments, the subject is a mouse, a rat, a dog, a guinea pig, a sheep, a non-human primate or a human. In some embodiments, the subject is a human. In some embodiments, the human subject is 18 years of age or older. In some embodiments, the human subject is under 18 years of age.

[0071] In some embodiments, the human subject is between 6 months and 17 years old. In some embodiments, the human subject is between 6 months and 9 years old, between 6 months and 8 years old, between 6 months and 7 years old, between 6 months and 6 years old, between 6 months and 5 years old, between 6 months and 4 years old, between 6 months and 3 years old, between 6 months and 2 years old, or between 6 months and 1 year old. In some embodiments, the human subject is between 5 years old and under 17 years old, between 7 years old and under 17 years old, between 9 years old and under 17 years old, between 11 years old and under 17 years old, between 13 years old and under 17 years old, or between 15 years old and under 17 years old. In some embodiments, the human subject is 6 months old, 1 year old, 2 years old, 3 years old, 4 years old, 5 years old, 6 years old, 7 years old, 8 years old, 9 years old, 10 years old, 11 years old, 12 years old, 13 years old, 14 years old, 15 years old, 16 years old, 17 years old, or 18 years old.

[0072] As used herein, the term "protective immune response" includes eliciting an anti-GAS antibody response in a subject. The antibody titers generated after administration of the immunogenic compositions described herein can be determined by means known in the art, for example, ELISA assays of serum samples from immunized subjects. In some embodiments, the immunogenic compositions described herein elicit an antibody response in a treated subject, where the generated antibodies bind to multiple (i.e., two or more) GAS serotypes. In some embodiments, the immunogenic compositions described herein elicit an antibody response in a treated subject, where the generated antibodies bind to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more GAS serotypes. In certain embodiments, the immunogenic compositions described herein do not elicit an antibody response against human proteins or tissues.

[0073] In certain embodiments, the immunogenic compositions described herein elicit an antibody response in a treated subject, wherein the generated antibodies bind to at least one GAS serotype selected from M1, M2, M3, M4, M5, M6, M9, M11, M12, M13, M18, M22, M25, M28, M62, M71, M72, M74, M75, M77, M80, M81, M83, M87, M89, and M92. In certain embodiments, the immunogenic compositions described herein elicit an antibody response in a treated subject, wherein the generated antibodies bind to two or more GAS serotypes selected from M1, M2, M3, M4, M5, M6, M9, M11, M12, M13, M18, M22, M25, M28, M62, M71, M72, M74, M75, M77, M80, M81, M83, M87, M89, and M92.

[0074] In certain embodiments, the immunogenic compositions described herein elicit an antibody response in a treated subject, wherein the generated antibodies bind to at least one GAS serotype selected from M1, M2, M3, M4, M6, M11, M12, M22, M28, M75, and M89. In certain embodiments, the immunogenic compositions described herein elicit an antibody response in a treated subject, wherein the generated antibodies bind to two or more GAS serotypes selected from M1, M2, M3, M4, M6, M11, M12, M22, M28, M75, and M89.

[0075] In certain embodiments, the immunogenic compositions described herein elicit an antibody response in a treated subject, wherein the generated antibodies bind to at least one GAS serotype selected from M1, M3, M5, M9, M12, M18, M22, M25, M28, M71, M72, and M74. In certain embodiments, the immunogenic compositions described herein elicit an antibody response in a treated subject, wherein the generated antibodies bind to two or more GAS serotypes selected from M1, M3, M5, M9, M12, M18, M22, M25, M28, M71, M72, and M74.

[0076] In certain embodiments, the immunogenic compositions described herein elicit an antibody response in a treated subject, wherein the generated antibodies bind to at least one GAS serotype selected from M1, M4, M6, M11, M12, M22, M44, M75, M77, M77, and M81. In certain embodiments, the immunogenic compositions described herein elicit an antibody response in a treated subject, wherein the generated antibodies bind to two or more GAS serotypes selected from M1, M4, M6, M11, M12, M22, M44, M75, M77, M77, and M81.

[0077] In certain embodiments, the immunogenic compositions described herein elicit an antibody response in a treated subject, wherein the generated antibodies bind to at least one GAS serotype selected from M1, M2, M3, M4, M6, M9, M12, M18, M22, M75, M77, M89, and M92. In certain embodiments, the immunogenic compositions described herein elicit an antibody response in a treated subject, wherein the generated antibodies bind to two or more GAS serotypes selected from M1, M2, M3, M4, M6, M9, M12, M18, M22, M75, M77, M89, and M92.

[0078] In certain embodiments, the immunogenic compositions described herein elicit an antibody response in a treated subject, wherein the generated antibodies bind to at least one GAS serotype selected from M1, M2, M3, M4, M5, M6, M9, M11, M12, M13, M28, M62, and M89. In certain embodiments, the immunogenic compositions described herein elicit an antibody response in a treated subject, wherein the generated antibodies bind to two or more GAS serotypes selected from M1, M2, M3, M4, M5, M6, M9, M11, M12, M13, M28, M62, and M89.

[0079] In certain embodiments, the immunogenic compositions described herein elicit an antibody response in a treated subject, wherein the generated antibodies bind to at least one GAS serotype selected from M1, M2, M3, M4, M6, M12, M22, M28, M49, M53, M68, M77, M80, M83, M87, M89, and M92. In certain embodiments, the immunogenic compositions described herein elicit an antibody response in a treated subject, wherein the generated antibodies bind to two or more GAS serotypes selected from M1, M2, M3, M4, M6, M12, M22, M28, M49, M53, M68, M77, M80, M83, M87, M89, and M92.

[0080] In certain embodiments, the immunogenic compositions described herein induce an antibody response in a subject that binds to one or more Shigella serotypes. In certain embodiments, the immunogenic compositions described herein induce an antibody response in a subject that binds to one or more GAS serotypes and also binds to one or more Shigella serotypes. In certain embodiments, the Shigella serotypes comprise a polysaccharide that includes a polyrhamnose backbone. Exemplary Shigella serotypes that contain such polysaccharides include S. flexneri, e.g., S. flexneri 2A and 3A, and S. flexneri 6.

[0081] Such immunogenic compositions may generally comprise: (a) a Group A Streptococcus (GAS) C5a peptidase polypeptide antigen; (b) a GAS streptolysin O (SLO) polypeptide antigen; and (c) a polypeptide-polysaccharide conjugate.

[0082] In certain embodiments, the Group A Streptococcus (GAS) C5a peptidase polypeptide antigen may be a full-length native C5a peptidase polypeptide or a fragment thereof. In certain embodiments, the C5a peptidase polypeptide antigen or a fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:29 or SEQ ID NO:30. In certain embodiments, the C5a peptidase polypeptide antigen or a fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:30. In certain embodiments, the C5a peptidase polypeptide antigen or a fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO:30. In certain embodiments, the C5a peptidase polypeptide antigen or a fragment thereof comprises or consists of an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:29 or SEQ ID NO:30. In certain embodiments, the C5a peptidase polypeptide antigen or fragment thereof comprises an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:29 or SEQ ID NO:30. In certain embodiments, the C5a peptidase polypeptide antigen or fragment thereof has the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:29 or SEQ ID NO:30. In certain embodiments, the C5a peptidase polypeptide antigen or fragment thereof has the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:30.

[0083] Similarly, the immunogenic compositions described herein may comprise a GAS streptolysin O (SLO) polypeptide antigen, or a fragment thereof. In certain embodiments, the SLO polypeptide antigen, or a fragment thereof, comprises or consists of the following amino acid sequence: SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53. In certain embodiments, the SLO polypeptide antigen, or a fragment thereof, comprises or consists of the following amino acid sequence: SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53. In certain embodiments, the SLO antigen comprises or consists of the amino acid sequence of SEQ ID NO:53. In certain embodiments described herein, the SLO antigen or fragment thereof comprises or consists of an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 or SEQ ID NO:53. In certain embodiments described herein, the SLO antigen or fragment thereof comprises or consists of an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 or SEQ ID NO:53. In certain embodiments described herein, the SLO antigen or fragment thereof comprises or consists of an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:53.In certain embodiments, the SLO antigen or fragment thereof has the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 or SEQ ID NO:53. In certain embodiments, the SLO antigen or fragment thereof has the amino acid sequence of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 or SEQ ID NO:53. In certain embodiments, the SLO antigen or fragment thereof has the amino acid sequence of SEQ ID NO:53.

[0084] The immunogenic compositions described herein may include a polypeptide-polysaccharide conjugate. In some embodiments, the polypeptide-polysaccharide conjugate includes: (i) a Streptococcus pyogenes adhesion / disruption (SpyAD) conjugate polypeptide or a fragment thereof, comprising at least one unnatural amino acid (nnAA), wherein the at least one nnAA comprises a click chemistry reactive group; and (ii) a GAS polysaccharide or a variant thereof lacking an immunodominant N-acetylglucosamine (GlcNAc) side chain.

[0085] As described above, the SpyAD-conjugated polypeptide or fragment thereof of the immunogenic composition comprises at least one nnAA containing a click chemistry reactive group, allowing for conjugation to a GAS polysaccharide or variant thereof. In certain embodiments, the at least one nnAA may be selected from 2-amino-3-(4-azidophenyl)propanoic acid (pAF), 2-amino-4-azidobutanoic acid, 2-azido-3-phenylpropionic acid, 2-amino-3-azidopropanoic acid, 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridin-2-yl)propanoic acid, 2-amino-3-(4-(azidomethyl)pyridin-2-yl)propanoic acid, 2-amino-3-(6-(azidomethyl)pyridin-3-yl)propanoic acid, and 2-amino-5-azidopentanoic acid. In one embodiment, at least one nnAA is pAMF.

[0086] The SpyAD conjugate polypeptide may be a native or full-length SpyAD conjugate polypeptide, or a fragment thereof. In certain embodiments, the SpyAD conjugate polypeptide, or a fragment thereof, comprises or consists of an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:33. In certain embodiments, the SpyAD conjugate polypeptide comprises or consists of an amino acid sequence that is a fragment of SEQ ID NO:33. In certain embodiments, the SpyAD conjugate polypeptide has an amino acid sequence that is a fragment of SEQ ID NO:33.

[0087] In certain embodiments, the SpyAD conjugate polypeptide, or a fragment thereof, comprises pAMF substitutions at positions K64, K287, K386 and K657 of SEQ ID NO:33 (Note: numbering herein is based on the full length native sequence). Thus, in certain embodiments, the SpyAD conjugate polypeptide, or a fragment thereof, comprises or consists of the following amino acid sequences: SEQ ID NO:34, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:82 or SEQ ID NO:83. In certain embodiments, the SpyAD conjugate polypeptide, or a fragment thereof, comprises or consists of an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:34, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:82 or SEQ ID NO:83. In certain embodiments, the SpyAD conjugate polypeptide, or a fragment thereof, comprises or consists of an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 34. In certain embodiments, the SpyAD conjugate polypeptide, or a fragment thereof, has the amino acid sequence of SEQ ID NO: 34, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 82 or SEQ ID NO: 83. In certain embodiments, the SpyAD conjugate polypeptide, or a fragment thereof, has the amino acid sequence of SEQ ID NO: 34.

[0088] In certain embodiments of the immunogenic compositions described herein, the C5a peptidase polypeptide antigen or fragment thereof comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:30; the SLO polypeptide antigen or fragment thereof comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:53; and the SpyAD conjugate polypeptide or fragment thereof comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:34. In certain embodiments, the C5a peptidase polypeptide antigen or fragment thereof comprises the amino acid sequence of SEQ ID NO:30; the SLO polypeptide antigen or fragment thereof comprises the amino acid sequence of SEQ ID NO:53; and the SpyAD conjugate polypeptide or fragment thereof comprises the amino acid sequence of SEQ ID NO:34. In certain embodiments, the C5a peptidase polypeptide antigen or fragment thereof has the amino acid sequence of SEQ ID NO:30; the SLO polypeptide antigen or fragment thereof has the amino acid sequence of SEQ ID NO:53; and the SpyAD conjugate polypeptide or fragment thereof has the amino acid sequence of SEQ ID NO:34.

[0089] In certain embodiments of any of the immunogenic compositions described herein, about 8 mol% to 20 mol% of the polysaccharide repeat units in the GAS polysaccharide or variant thereof of the polypeptide-polysaccharide conjugate are derivatized with a click chemistry reactive group or a linker comprising a click chemistry reactive group. In certain embodiments, about 8 mol% to about 20 mol%, about 8 mol% to about 19 mol%, about 8 mol% to about 18 mol%, about 8 mol% to about 17 mol%, about 8 mol% to about 16 mol%, about 8 mol% to about 15 mol%, about 8 mol% to about 14 mol%, about 8 mol% to about 13 mol%, about 8 mol% to about 12 mol%, about 8 mol% to about 11 mol%, 8 mol% to about 10 mol%, about 8 mol% to about 9 mol%, about 9 mol% to about 20 mol%, about 9 mol% to about 19 mol%. ol%, about 9 mol% to about 18 mol%, about 9 mol% to about 17 mol%, about 9 mol% to about 16 mol%, about 9 mol% to about 15 mol%, about 9 mol% to about 14 mol%, 9 mol% to about 13 mol%, about 9 mol% to about 12 mol% , about 9 mol% to about 11 mol%, 9 mol% to about 10 mol%, about 10 mol% to about 20 mol%, about 10 mol% to about 19 mol%, about 10 mol% to about 18 mol%, about 10 mol% to about 17 mol%, about 10 mol% to about 16 mol %, about 10 mol% to about 15 mol%, about 10 mol% to about 14 mol%, 10 mol% to about 13 mol%, about 10 mol% to about 12 mol%, about 10 mol% to about 11 mol%, about 11 mol% to about 20 mol%, about 11 mol% to about 1 9mol%, about 11mol% to about 18mol%, about 11mol% to about 17mol%, about 11mol% to about 16mol%, about 11mol% to about 15mol%, about 11mol% to about 14mol%, 11mol% to about 13mol%, about 11mol% ~about 12mol%, about 12mol% to about 20mol%, about 12mol% to about 19mol%, about 12mol% to about 18mol%, about 12mol% to about 17mol%, about 12mol% to about 16mol%, about 12mol% to about 15mol%, about 12 mol% to about 14 mol%, 12 mol% to about 13 mol%, about 13 mol% to about 20 mol%, about 13 mol% to about 19 mol%, about 13 mol% to about 18 mol%, about 13 mol% to about 17 mol%, about 13 mol% to about 16 mol%,About 13 mol% to about 15 mol%, about 13 mol% to about 14 mol%, about 14 mol% to about 20 mol%, about 14 mol% to about 19 mol%, about 14 mol% to about 18 mol%, about 14 mol% to about 17 mol%, about 14 mol% to about 16 mol%, about 14 mol% to about 15 mol%, about 15 mol% to about 20 mol%, about 15 mol% to about 19 mol%, about 15 mol% to about 18 mol%, about 15 mol% to about 17 mol%, about 15 mol% to about 16 mol%, about 16 mol % to about 20 mol%, about 16 mol% to about 19 mol%, about 16 mol% to about 18 mol%, about 16 mol% to about 17 mol%, about 17 mol% to about 20 mol%, about 17 mol% to about 19 mol%, about 17 mol% to about 18 mol%, about 18 mol% to about 20 mol%, about 18 mol% to about 19 mol%, or about 19 mol% to about 20 mol% of the PS repeating units of the polysaccharide are derivatized with a click chemistry reactive group or a linker containing a click chemistry reactive group. In certain embodiments, the polysaccharide repeating units are polysaccharide repeating units of a GAS polysaccharide or variants thereof. In certain embodiments of the immunogenic compositions described herein, between about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mol % of the polysaccharide repeating units in the GAS polysaccharide or variant thereof of the polypeptide-polysaccharide conjugate are derivatized with a click chemistry reactive group or a linker that includes a click chemistry reactive group. In certain embodiments, the GAS polysaccharide or variant thereof of the polypeptide-polysaccharide conjugate lacks the immunodominant N-acetylglucosamine (GlcNAc) side chain.

[0090] In one embodiment of the immunogenic composition of the invention, the linker prior to reaction with the click chemistry reactive group of the nnAA has the formula I: [ka] where X is a polysaccharide repeat unit of at least one polysaccharide and n is at least 1. In some embodiments, the polysaccharide is a GAS polysaccharide or variant thereof. In some embodiments, n is at least 1, at least 2, at least 3, at least 4, or at least 5. In some embodiments, n is 1, 2, 3, 4, or 5. When X is described here or elsewhere as being attached to a polysaccharide repeating unit of a GAS polysaccharide or variant thereof, the attachment can refer to a chemical bond to or through any suitable functional group within the polysaccharide repeating unit (e.g., reacted with an aldehyde that may result from oxidation of a vicinal diol via reductive amination). In some embodiments, X and -NH- in formula I are part of an isourea moiety.

[0091] In one embodiment, the SpyAD conjugate polypeptide has Formula II: [ka] wherein R1 is H, formyl, or at least one amino acid of a SpyAD conjugated polypeptide, or a fragment thereof; R2 is OH, or at least one amino acid of a SpyAD conjugated polypeptide, or a fragment thereof; W is CH or N; y is at least 1; n is at least 1; and X is at least one polysaccharide repeat unit of a GAS polysaccharide, or a variant thereof. In certain embodiments, R1 and R2 are both amino acids of a SpyAD conjugated polypeptide, or a fragment thereof. In certain embodiments, y is at least 1, at least 2, or at least 3. In certain embodiments, y is 1, 2, or 3. In certain embodiments, both Ws are CH. In certain embodiments, both Ws are N. In certain embodiments, one W is N and the other W is CH. In certain embodiments, n is at least 1, at least 2, at least 3, at least 4, or at least 5. In certain embodiments, n is 1, 2, 3, 4, or 5. In certain embodiments, X and -NH- of formula I are part of an isourea moiety.

[0092] In certain embodiments of the immunogenic compositions described herein, the GAS polysaccharide or variant thereof has a molecular weight of at least about 10 kDa to at least about 40 kDa. In certain embodiments, the purified cell wall polysaccharide or peptidoglycan-bound capsular polysaccharide has a molecular weight of about 10 kDa to about 40 kDa; about 10 kDa to about 35 kDa; about 10 kDa to about 30 kDa; about 10 kDa to about 25 kDa; about 10 kDa to about 20 kDa; about 10 kDa to about 15 kDa; about 15 kDa to about 40 kDa; about 15 kDa to about 35 kDa; about 15 kDa to about 30 kDa; about 15 kDa to about 15 kDa. In certain embodiments, the purified cell wall polysaccharide or peptidoglycan-bound capsular polysaccharide has an average molecular weight of about 10 kDa, about 15 kDa, about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, or about 40 kDa. In certain embodiments, the GAS polysaccharide or variant thereof lacks the immunodominant N-acetylglucosamine (GlcNAc) side chain.

[0093] In addition to having longer polysaccharides, the polypeptide-polysaccharide conjugates of the immunogenic compositions described herein may have an increased average molecular weight. In certain embodiments of any of the polypeptide-polysaccharide conjugates described herein, the average molecular weight is greater than about 185 kDa or 190 kDa. In certain embodiments, the average molecular weight is from about 185 kDa to about 700 kDa, from about 185 kDa to about 600 kDa, from about 185 kDa to about 500 kDa, from about 185 kDa to about 400 kDa, from about 185 kDa to about 300 kDa, from about 185 kDa to about 200 kDa. In certain embodiments, the polypeptide-polysaccharide conjugate has a molecular weight of about 185 kDa to about 700 kDa, about 185 kDa to about 650 kDa, about 185 kDa to about 600 kDa, about 185 kDa to about 550 kDa, about 185 kDa to about 500 kDa, about 185 kDa to about 450 kDa, about 185 kDa to about 400 kDa, about 185 kDa to about 350 kDa, about 185 kDa to about 300 kDa, about 185 kDa to about 200 kDa, or about 185 kDa to about 500 kDa. 50kDa, about 185kDa to about 200kDa, about 200kDa to about 700kDa, about 200kDa to about 650kDa, about 200kDa to about 600kDa, about 200kDa to about 550kDa, about 200k Da ~ approx. 500kDa, approx. 200kDa ~ approx. 450kDa, approx. 200kDa ~ approx. 400kDa, approx. 200kDa ~ approx. 350kDa, approx. 250kDa to about 700kDa, about 250kDa to about 650kDa, about 250kDa to about 600kDa, about 250kDa to about 550kDa, about 250kDa to about 500kDa, about 250kDa to about 450 kDa, approximately 250kDa ~ approximately 400kDa, approximately 250kDa ~ approximately 350kDa, approximately 250kDa ~ approximately 300kDa, approximately 300kDa ~ approximately 700kDa, approximately 300kDa ~ approximately 650kDa, approximately 300kDa ~ About 600kDa, about 300kDa to about 550kDa, about 300kDa to about 500kDa, about 300kDa to about 450kDa, about 300kDa to about 400kDa, about 300kDa to about 350kDa, about 350 kDa ~ about 700kDa, about 350kDa - about 650kDa, about 350kDa - about 600kDa, about 350kDa - about 550kDa, about 350kDa - about 500kDa, about 350kDa - about 450kDa,About 350 kDa to about 400 kDa, about 400 kDa to about 700 kDa, about 400 kDa to about 650 kDa, about 400 kDa to about 600 kDa, about 400 kDa to about 550 kDa, about 400 kDa to about 500 kDa, about 400 kDa to about 450 kDa, about 450 kDa to about 700 kDa, about 450 kDa to about 650 kDa, about 450 kDa to about 600 kDa, about 450 kDa to about 550 kDa, about 450 kDa to about The molecular weight or average molecular weight may be 500 kDa, about 500 kDa to about 700 kDa, about 500 kDa to about 650 kDa, about 500 kDa to about 600 kDa, about 500 kDa to about 550 kDa, about 550 kDa to about 700 kDa, about 550 kDa to about 650 kDa, about 550 kDa to about 600 kDa, about 600 kDa to about 700 kDa, about 600 kDa to about 650 kDa, or about 650 kDa to about 700 kDa. In certain embodiments, the polypeptide-polysaccharide conjugate may have a molecular weight or average molecular weight of about 185, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, or 700 kDa. In one embodiment, the GAS polysaccharide or variant thereof of the polypeptide-polysaccharide conjugate lacks the immunodominant N-acetylglucosamine (GlcNAc) side chain.

[0094] By using the long chain polysaccharides described herein, immunogenic compositions can be produced that contain reduced amounts of free GAS polysaccharides or variants thereof. For example, the immunogenic compositions may further contain less than about 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% free GAS polysaccharides or variants thereof. In certain embodiments, the immunogenic compositions further contain about 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% free GAS polysaccharides or variants thereof.

[0095] The immunogenic compositions described herein may be suitable for inducing a protective immune response against Group A Streptococcus (GAS) bacteria in a subject, characterized by administering any of the immunogenic compositions described herein. In an embodiment, the immunogenic composition may induce an antibody response in a subject against Group A Streptococcus (GAS) bacteria, but not against human tissue. In an embodiment, the present disclosure provides the use of the immunogenic compositions described herein in the manufacture of a medicament for inducing a protective immune response against GAS bacteria in a subject. It also provides the use of any of the immunogenic compositions described herein for inducing a protective immune response against GAS bacteria in a subject. In an embodiment, the subject is 18 years of age or older. In an embodiment, the subject is 18 years of age or older. In an embodiment, the subject is under 18 years of age. In an embodiment, the subject is 5 years of age or older but under 17 years of age, 6 months of age or older but under 9 years of age, or 5 years of age or older but under 9 years of age.

[0096] In some embodiments, the immunogenic compositions described herein include: (i) a Streptococcus Adhesion Disruption (SpyAD) conjugate polypeptide or fragment thereof comprising at least one unnatural amino acid (nnAA), wherein the at least one nnAA comprises a click chemistry reactive group; and (ii) a GAS polysaccharide or variant thereof lacking an immunodominant N-acetylglucosamine (GlcNAc) side chain. In some embodiments, about 8 mol% to about 20 mol% of the polysaccharide repeating units of the GAS polysaccharide or variant thereof are derivatized with a linker. Further, in some embodiments, the average molecular weight of the polypeptide-polysaccharide conjugate is between about 185 kDa and about 700 kDa. In some embodiments, the average molecular weight of the polypeptide-polysaccharide conjugate is greater than about 185 kDa. In some embodiments, the average molecular weight of the polypeptide-polysaccharide conjugate is greater than about 190 kDa.

[0097] In some embodiments, the immunogenic composition may include: (a) a Group A Streptococcus (GAS) C5a peptidase polypeptide antigen comprising or consisting of SEQ ID NO:30 or a fragment thereof; (b) a GAS streptolysin O (SLO) polypeptide antigen comprising or consisting of the amino acid sequence of SEQ ID NO:53 or a fragment thereof; and (c) (i) a Streptococcus pyogenes adhesion disruption (SpyAD) conjugate polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO:34 or a fragment thereof, and (ii) a polypeptide-polysaccharide conjugate comprising a GAS polysaccharide or variant thereof lacking an immunodominant N-acetylglucosamine (GlcNAc) side chain. In some embodiments, about 8 mol% to about 20 mol% of the polysaccharide repeating units of the GAS polysaccharide or variant thereof are derivatized with a linker. In some embodiments, the average molecular weight of the polypeptide-polysaccharide conjugate is between about 185 kDa and about 700 kDa. In certain embodiments, the average molecular weight of the polypeptide-polysaccharide conjugate is between about 200 kDa and about 700 kDa. In certain embodiments, the average molecular weight is between about 300 kDa and about 600 kDa. In certain embodiments, the average molecular weight is between about 400 kDa and about 500 kDa. In certain embodiments, the GAS polysaccharide or variant thereof has a molecular weight of at least about 10 kDa to at least about 40 kDa. In certain embodiments, the SpyAD conjugate polypeptide is a fragment of the amino acid sequence of SEQ ID NO:34, comprising or consisting of the sequence of SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:82 or SEQ ID NO:83.

[0098] In one embodiment, the immunogenic compositions described herein comprise: (a) a Group A Streptococcus (GAS) C5a peptidase polypeptide antigen; (b) a GAS streptolysin O (SLO) polypeptide antigen; and (c) a polypeptide-polysaccharide conjugate comprising: (i) a Streptococcus pyogenes adhesion / disruption (SpyAD) conjugate polypeptide or a fragment thereof comprising at least one unnatural amino acid (nnAA), wherein the at least one nnAA comprises a click chemistry reactive group; and (ii) a GAS polysaccharide or a variant thereof lacking an immunodominant N-acetylglucosamine (GlcNAc) side chain, wherein the average molecular weight of the polypeptide-polysaccharide conjugate is between about 185 kDa and about 700 kDa.

[0099] In one embodiment, the immunogenic compositions described herein comprise: (a) a Group A Streptococcus (GAS) C5a peptidase polypeptide antigen; (b) a GAS streptolysin O (SLO) polypeptide antigen; and (c) a polypeptide-polysaccharide conjugate comprising: (i) a Streptococcus pyogenes disruption of adhesion (SpyAD) conjugate polypeptide or a fragment thereof comprising at least one unnatural amino acid (nnAA), wherein the at least one nnAA comprises a click chemistry reactive group; and (ii) a GAS polysaccharide or variant thereof lacking an immunodominant N-acetylglucosamine (GlcNAc) side chain, wherein about 8 mol % to about 20 mol % of the polysaccharide repeating units of the GAS polysaccharide or variant thereof are derivatized with a linker.

[0100] In one embodiment, the immunogenic compositions described herein comprise: (a) a Group A Streptococcus (GAS) C5a peptidase polypeptide antigen; (b) a GAS streptolysin O (SLO) polypeptide antigen; and (c) a polypeptide-polysaccharide conjugate comprising: (i) a Streptococcus pyogenes adhesion / disruption (SpyAD) conjugate polypeptide or fragment thereof comprising at least one unnatural amino acid (nnAA), wherein the at least one nnAA comprises a click chemistry reactive group; and (ii) a GAS polysaccharide or variant thereof lacking an immunodominant N-acetylglucosamine (GlcNAc) side chain, wherein about 8 mol % to about 20 mol % of the polysaccharide repeating units of the GAS polysaccharide or variant thereof are derivatized with a linker, and the average molecular weight of the polypeptide-polysaccharide conjugate is between about 185 kDa and about 700 kDa.

[0101] (List of embodiments) Embodiment I-1. A method for purifying cell wall polysaccharides or peptidoglycan-bound capsular polysaccharides from bacterial cells, comprising the steps of: (a) hydrolyzing bacterial cells in a solution containing a base and a reducing agent to form a lysate solution containing polysaccharides; and (b) incubating the polysaccharide-containing degradation solution with a wall-degrading enzyme to form a free polysaccharide solution. Embodiment I-2. The method of embodiment I-1, wherein the bacterial cell is a Pseudomonas bacterial cell, a Streptococcus bacterial cell, a Staphylococcus bacterial cell, a Neisseria bacterial cell, a Haemophilus bacterial cell, a Listeria bacterial cell, an Enterococcus bacterial cell or a Clostridium bacterial cell. Embodiment I-3. The method according to embodiment I-1 or I-2, wherein the bacterial cell is selected from Pseudomonas aeruginosa, Streptococcus viridans, Streptococcus mutans or Streptococcus pyogenes, etc. Embodiment I-4. The method of embodiment I-3, wherein the Streptococcus pyogenes bacterial cell is a serotype selected from M1, M2, M3, M4, M5, M6, M9, M11, M12, M13, M18, M22, M25, M28, M62, M71, M72, M74, M75, M77, M80, M81, M83, M87, M89 or M92. Embodiment I-5. The method of embodiment I-3, wherein the Streptococcus pyogenes bacterial cell produces a polysaccharide or a variant thereof lacking an immunodominant N-acetylglucosamine (GlcNAc) side chain. Embodiment I-6. The method of embodiment I-1 for purifying peptidoglycan-bound capsular polysaccharide from bacterial cells. Embodiment I-7. The method of any one of embodiments I-1 to I-6, wherein the base in step (a) is NaOH, KOH, or LiOH. Embodiment I-8. The method of any one of embodiments I-1 to I-7, wherein the base in step (a) is NaOH. Embodiment I-9. The method of any one of embodiments I-1 to I-8, wherein the concentration of base is from about 2M to about 8M. Embodiment I-10. The method of any one of embodiments I-1 to I-9, wherein the solution containing the base and the reducing agent has a pH of about 14. Embodiment I-11. The method of any one of embodiments I-1 to I-10, wherein the reducing agent is sodium borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride, dithiothreitol or β-mercaptoethanol. Embodiment I-12. The method of any one of embodiments I-1 to I-11, wherein the reducing agent is sodium borohydride. Embodiment I-13. The method of any one of embodiments I-1 to I-12, wherein the concentration of the reducing agent is about 1 mM to 500 mM. Embodiment I-14. The method of any one of embodiments I-1 to I-13, wherein step (a) further comprises incubating the solution at between about 30°C and about 100°C. Embodiment I-15. The method of any one of embodiments I-1 to I-14, wherein step (a) further comprises incubating the solution for about 0.5 to about 20 hours. Embodiment I-16. The method of any one of embodiments I-1 to I-15, wherein step (a) further comprises one or more pH adjustment steps. Embodiment I-17. The one or more pH adjustment steps comprise the steps of: (i) increasing the pH of the degradation solution containing the polysaccharide; or (ii) reducing the pH of the degradation solution containing the polysaccharide; The method of embodiment I-16, wherein said The method of embodiment I-17, comprising lowering the pH of the degradation solution containing the polysaccharide to about 3 to 7.0. Embodiment I-19. The method of embodiment I-17 or I-18, comprising lowering the pH of the degradation solution containing the polysaccharide to about 6.5. Embodiment I-20. The method of embodiment I-17 or I-18, comprising lowering the pH of the degradation solution containing the polysaccharide to about 3 to about 4. Embodiment I-21. The method according to claim 1, further comprising the steps of: (i) lowering the pH of the degradation solution containing the polysaccharide to about 5.5 to 7.0; (ii) lowering the pH of the digestion solution containing the polysaccharide to about 3; (iii) increasing the pH of the degradation solution containing the polysaccharide to about 5.5 to 7.0; The method of embodiment I-16 or I-17, comprising: Embodiment I-22. The method of any one of embodiments I-16 to I-21, wherein the degradation solution containing the polysaccharide is incubated at about room temperature (rt) after one or more pH adjustment steps. Embodiment I-23. The method according to any one of embodiments I-17 to I-20, wherein the degradation solution containing the polysaccharide is incubated at about 4° C. to about 30° C. after one or more pH adjustment steps. Embodiment I-24. The method of any one of embodiments I-1 to I-23, further comprising removing solids from the digestion solution containing the polysaccharide. Embodiment I-25. The method of embodiment I-24, wherein the step of removing solids from the degradation solution containing the polysaccharide comprises filtration, centrifugation, or a combination thereof. Embodiment I-26. The method of embodiment I-25, wherein the filtration comprises depth filtration, tangential flow filtration (TFF), sterile filtration, or a combination thereof. Embodiment I-27. The method of embodiment I-25 or I-26, wherein the filtration comprises depth filtration followed by TFF. Embodiment I-28. The method of embodiment I-24 or I-25, wherein solids are removed from the digestion solution containing the polysaccharides by centrifugation. Embodiment I-29. The method of any one of embodiments I-1 to I-28, wherein the wall-degrading enzyme of step (b) is mutanolysin, lysozyme, or a bacteriophage hydrolase. Embodiment I-30. The method of any one of embodiments I-1 to I-29, wherein step (b) further comprises incubating with a protease. Embodiment I-31. The method of embodiment I-30, wherein the protease is proteinase K, trypsin, chymotrypsin, endoproteinase Asp-N, endoproteinase Arg-C, endoproteinase Glu-C, endoproteinase Lys-C, pepsin, thermolysin, elastase, papain, subtilisin, clostripain, carboxypeptidase A, carboxypeptidase B, carboxypeptidase P, carboxypeptidase Y, cathepsin C, acyl amino acid releasing enzyme or pyroglutamate. Embodiment I-32. The method according to any one of embodiments I-1 to I-31, wherein step (b) further comprises heating the degradation solution containing the polysaccharide together with the wall-degrading enzyme to about 30°C to about 65°C. Embodiment I-33. The method according to any one of embodiments I-30 to I-32, wherein the digestion solution containing the polysaccharide together with the protease is heated to about 45°C to about 55°C. Embodiment I-34. The method of embodiment I-32 or I-33, wherein the decomposition solution is heated for about 6 to about 20 hours. Embodiment I-35. The method of any one of embodiments I-1 to I-34, wherein the free polysaccharide solution of step (b) is further purified to reduce the concentration of nucleic acids, enzymes, host cell proteins (HCPs), or combinations thereof. Embodiment I-36. The method of any one of embodiments I-1 to I-35, wherein the free polysaccharide solution of step (b) is further purified by precipitation. Embodiment I-37. The method of any one of embodiments I-1 to I-36, wherein the free polysaccharide solution of step (b) is treated with cetyltrimethylammonium bromide (CTAB). Embodiment I-38. The method of embodiment I-37, wherein the concentration of CTAB in the free polysaccharide solution is about 0.1% to about 10%. Embodiment I-39. The method of embodiment I-37 or 38, wherein the concentration of CTAB is about 0.5% to about 3%. Embodiment I-40. The method of any one of embodiments I-37 to I-39, wherein the free polysaccharide solution of step (b) is treated with potassium iodide (KI). Embodiment I-41. The method of any one of embodiments I-32, wherein the KI concentration in the free polysaccharide solution is from about 20 mM to about 400 mM. Embodiment I-42. The method of any one of embodiments I-1 to I-41, wherein the free polysaccharide solution is further purified by filtration, centrifugation, chromatography, or a combination thereof. Embodiment I-43. The method of embodiment I-42, wherein the filtration comprises depth filtration, tangential flow filtration (TFF), sterile filtration, or a combination thereof. Embodiment I-44. The method of embodiment I-42, wherein the chromatography comprises hydrophobic interaction chromatography (HIC), anion exchange chromatography (AEX), ceramic hydroxyapatite-based chromatography or cation exchange chromatography (CEX). Embodiment I-45. (a) a GAS polypeptide antigen or a non-GAS carrier polypeptide that includes at least one non-natural amino acid (nnAA); and (b) a purified cell wall polysaccharide or peptidoglycan-bound capsular polysaccharide having a molecular weight of at least about 10 kDa to at least about 40 kDa; 2. A polypeptide-polysaccharide conjugate comprising: Embodiment I-46. The polypeptide-polysaccharide conjugate of embodiment I-45, wherein the purified cell wall polysaccharide lacks immunodominant N-acetylglucosamine (GlcNAc) side chains. Embodiment I-47. The polypeptide-polysaccharide conjugate of embodiments I-45 to I-46, wherein the polypeptide antigen is a full-length GAS polypeptide antigen or a fragment of a full-length GAS polypeptide antigen. Embodiment I-48. The polypeptide-polysaccharide conjugate of any one of embodiments I-45 to I-47, wherein at least one nnAA is substituted with lysine, leucine, isoleucine or arginine in the polypeptide antigen or non-GAS carrier polypeptide. Embodiment I-49. A polypeptide-polysaccharide conjugate according to any one of embodiments I-45 to I-48, wherein the nnAA comprises a click chemistry reactive group. Embodiment I-50. The polypeptide-polysaccharide conjugate according to any one of embodiments I-45 to I-49, wherein nnAA is selected from 2-amino-3-(4-azidophenyl)propanoic acid (pAF), 2-amino-4-azidobutanoic acid, 2-azido-3-phenylpropionic acid, 2-amino-3-azidopropanoic acid, 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridin-2-yl)propanoic acid, 2-amino-3-(4-(azidomethyl)pyridin-2-yl)propanoic acid, 2-amino-3-(6-(azidomethyl)pyridin-3-yl)propanoic acid and 2-amino-5-azidopentanoic acid. Embodiment I-51. The polypeptide-polysaccharide conjugate of any one of embodiments I-45 to I-50, wherein the nnAA is pAMF. Embodiment I-52. The polypeptide-polysaccharide conjugate of any one of embodiments I-45 to I-51, wherein the polypeptide antigen is selected from C5a peptidase, streptolysin O (SLO), SpyAD, Sib35 and Sfb1. Embodiment I-53. A polypeptide-polysaccharide conjugate according to any one of embodiments I-45 to I-52, wherein the polypeptide antigen is SLO. Embodiment I-54. The polypeptide-polysaccharide conjugate of embodiment I-53, wherein the SLO polypeptide antigen comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:31, SEQ ID NO:32 or SEQ ID NO:53. Embodiment I-55. The polypeptide-polysaccharide conjugate of embodiment I-53, wherein the SLO polypeptide antigen is at least 95% identical to SEQ ID NO:31, SEQ ID NO:32 or SEQ ID NO:53. Embodiment I-56. The polypeptide-polysaccharide conjugate of any one of embodiments I-53 to I-55, wherein the SLO polypeptide comprises three or four pAMF substitutions at positions selected from K98, K112, R151, K189, K272, K323, K357, K375, K407 or K464. Embodiment I-57. The polypeptide-polysaccharide conjugate of any one of embodiments I-53 to I-56, wherein the SLO polypeptide comprises the amino acid sequence of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63 or SEQ ID NO:64. Embodiment I-58. The polypeptide-polysaccharide conjugate of any one of embodiments I-53 to I-56, wherein the SLO polypeptide has the amino acid sequence of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63 or SEQ ID NO:64. Embodiment I-59. The polypeptide-polysaccharide conjugate of any one of embodiments I-53 to I-55, wherein the SLO polypeptide comprises 5, 6, 7 or 8 pAMF substitutions at positions selected from K98, K112, R151, K189, K272, K323, K357, K375, K407 or K464. Embodiment I-60. The polypeptide-polysaccharide conjugate of any one of embodiments I-53 to I-55 or 59, wherein the SLO polypeptide comprises the amino acid sequence of SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75 or SEQ ID NO:76. Embodiment I-61. The polypeptide-polysaccharide conjugate of any one of embodiments I-53 to I-55 or 59 to 60, wherein the SLO polypeptide has the amino acid sequence of SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75 or SEQ ID NO:76. Embodiment I-62. The polypeptide-polysaccharide conjugate of any one of embodiments I-45 to I-52, wherein the polypeptide antigen is a SpyAD polypeptide. Embodiment I-63. The polypeptide-polysaccharide conjugate of embodiment I-62, wherein the SpyAD polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:33. Embodiment I-64. A polypeptide-polysaccharide conjugate according to embodiment I-62, wherein the SpyAD polypeptide is at least 95% identical to SEQ ID NO:33. Embodiment I-65. A polypeptide-polysaccharide conjugate according to any one of embodiments I-62 to I-64, wherein the SpyAD polypeptide comprises pAMF substitutions at positions K64, K287, K386 and K657 of SEQ ID NO:33. Embodiment I-66. A polypeptide-polysaccharide conjugate according to any one of embodiments I-62 to I-65, wherein the SpyAD polypeptide comprises the amino acid sequence of SEQ ID NO:34. Embodiment I-67. A polypeptide-polysaccharide conjugate according to any one of embodiments I-62 to I-65, wherein the SpyAD polypeptide has the amino acid sequence of SEQ ID NO:34. Embodiment I-68. A polypeptide-polysaccharide conjugate according to any one of embodiments I-45 to I-45 or 48 to 51, wherein the non-GAS carrier polypeptide is selected from ADI, ferritin, protein D and eCRM197. Embodiment I-69. The polypeptide-polysaccharide conjugate of embodiment I-68, wherein the non-GAS carrier polypeptide is eCRM197. Embodiment I-70. A polypeptide-polysaccharide conjugate according to embodiment I-69, wherein eCRM197 has the sequence of SEQ ID NO:25. Embodiment I-71. A polypeptide-polysaccharide conjugate according to any one of embodiments I-45 to I-50, wherein the GAS polypeptide antigen or the non-GAS carrier polypeptide comprises or consists of the amino acid sequence of a polypeptide listed in Table 1. Embodiment II-1. (a) a GAS polypeptide antigen or a non-GAS carrier polypeptide comprising at least one non-natural amino acid (nnAA), wherein the nnAA comprises a click chemistry reactive group; and (b) a purified cell wall polysaccharide or peptidoglycan-bound capsular polysaccharide having a molecular weight of at least about 10 kDa to at least about 40 kDa; 2. A polypeptide-polysaccharide conjugate comprising: Embodiment II-2. The polypeptide-polysaccharide conjugate of embodiment II-1, wherein the purified cell wall polysaccharide lacks the immunodominant N-acetylglucosamine (GlcNAc) side chain. Embodiment II-3. A polypeptide-polysaccharide conjugate according to embodiment II-1 to II-2, wherein the GAS polypeptide antigen is a full-length GAS polypeptide antigen or a fragment of a full-length GAS polypeptide antigen. Embodiment II-4. A polypeptide-polysaccharide conjugate according to any one of embodiments II-1 to II-3, wherein the cell wall polysaccharide is a GAS polysaccharide or a variant thereof. Embodiment II-5. A polypeptide-polysaccharide conjugate according to embodiment II-4, wherein about 8 mol % to about 20 mol % of the polysaccharide repeating units of the GAS polysaccharide or variant thereof are derivatized with a linker. Embodiment II-6. A polypeptide-polysaccharide conjugate according to embodiment II-4 to II-5, wherein about 10 mol % to about 20 mol % of the polysaccharide repeat units of the GAS polysaccharide or variant thereof are derivatized with a linker. Embodiment II-7. A polypeptide-polysaccharide conjugate according to any one of embodiments II-4 to II-6, wherein about 10 mol % to about 18 mol % of the polysaccharide repeating units of the GAS polysaccharide or variant thereof are derivatized with a linker. Embodiment II-8. A polypeptide-polysaccharide conjugate according to any one of embodiments II-1 to II-6, having an average molecular weight of about 185 kDa to about 700. Embodiment II-9. A polypeptide-polysaccharide conjugate according to any one of embodiments II-1 to II-7, having an average molecular weight of about 200 kDa to about 700 kDa. Embodiment II-10. A polypeptide-polysaccharide conjugate according to any one of embodiments II-1 to II-9, wherein the average molecular weight of the polypeptide-polysaccharide conjugate is from about 300 kDa to about 600 kDa. Embodiment II-11. A polypeptide-polysaccharide conjugate according to any one of embodiments II-1 to II-10, wherein the average molecular weight of the polypeptide-polysaccharide conjugate is from about 400 kDa to about 500 kDa. Embodiment II-12. The polypeptide-polysaccharide conjugate according to any one of embodiments II-1 to II-11, wherein at least one nnAA is substituted with lysine, leucine, isoleucine or arginine in the GAS polypeptide antigen or the non-GAS carrier polypeptide. Embodiment II-13. A polypeptide-polysaccharide conjugate according to any one of embodiments II-1 to II-12, wherein at least one nnAA is selected from 2-amino-3-(4-azidophenyl)propanoic acid (pAF), 2-amino-4-azidobutanoic acid, 2-azido-3-phenylpropionic acid, 2-amino-3-azidopropanoic acid, 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridin-2-yl)propanoic acid, 2-amino-3-(4-(azidomethyl)pyridin-2-yl)propanoic acid, 2-amino-3-(6-(azidomethyl)pyridin-3-yl)propanoic acid and 2-amino-5-azidopentanoic acid. Embodiment II-14. The polypeptide-polysaccharide conjugate according to any one of embodiments II-1 to II-13, wherein the nnAA is pAMF. Embodiment II-15. The polypeptide-polysaccharide conjugate according to any one of embodiments II-1 to II-14, wherein the GAS polypeptide antigen is selected from C5a peptidase, streptolysin O (SLO), SpyAD, Sib35 and Sfb1. Embodiment II-15a. The polypeptide-polysaccharide conjugate according to any one of embodiments II-1 to II-15, wherein the GAS polypeptide antigen is SLO. Embodiment II-15b. The polypeptide-polysaccharide conjugate of embodiment II-15a, wherein the SLO polypeptide antigen comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:31, SEQ ID NO:32 or SEQ ID NO:53. Embodiment II-15c. The polypeptide-polysaccharide conjugate of embodiment II-15b, wherein the SLO polypeptide antigen is at least 95% identical to SEQ ID NO:31, SEQ ID NO:32 or SEQ ID NO:53. Embodiment II-15d. The polypeptide-polysaccharide conjugate of any one of embodiments I-15a to II-15c, wherein the SLO polypeptide comprises three or four pAMF substitutions at positions selected from K98, K112, R151, K189, K272, K323, K357, K375, K407 or K464. Embodiment II-15e. The polypeptide-polysaccharide conjugate of any one of embodiments II-15a to 15d, wherein the SLO polypeptide comprises the amino acid sequence of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63 or SEQ ID NO:64. Embodiment II-15f. The polypeptide-polysaccharide conjugate according to any one of embodiments II-15a to 15d, wherein the SLO polypeptide has the amino acid sequence of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63 or SEQ ID NO:64. Embodiment II-15g. The polypeptide-polysaccharide conjugate of any one of embodiments II-15a to 15c, wherein the SLO polypeptide comprises 5, 6, 7 or 8 pAMF substitutions selected from K98, K112, R151, K189, K272, K323, K357, K375, K407 or K464. Embodiment II-15h. The polypeptide-polysaccharide conjugate of any one of embodiments II-15a to II-15c or II-22, wherein the SLO polypeptide comprises the amino acid sequence of SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75 or SEQ ID NO:76. Embodiment II-15i. The polypeptide-polysaccharide conjugate according to any one of embodiments II-15a to 15c or II-15g to II-15f, wherein the SLO polypeptide has the amino acid sequence of SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75 or SEQ ID NO:76. Embodiment II-16. The polypeptide-polysaccharide conjugate of any one of embodiments II-1 to II-15 or II-15a to II-15i, wherein the GAS polypeptide antigen is a SpyAD polypeptide or a fragment thereof. Embodiment II-17. The polypeptide-polysaccharide conjugate of embodiment II-16, wherein the SpyAD polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:33. Embodiment II-18. The polypeptide-polysaccharide conjugate of embodiment II-16, wherein the SpyAD polypeptide is at least 95% identical to SEQ ID NO:33. Embodiment II-19. A polypeptide-polysaccharide conjugate according to any one of embodiments II-16 to II-18, wherein the SpyAD polypeptide comprises pAMF at positions K64, K287, K386 and K657 of SEQ ID NO:33. Embodiment II-20. The polypeptide-polysaccharide conjugate according to any one of embodiments II-16 to II-19, wherein the SpyAD polypeptide comprises the amino acid sequence of SEQ ID NO:34. Embodiment II-21. The polypeptide-polysaccharide conjugate according to any one of embodiments II-16 to II-19, wherein the SpyAD polypeptide has the amino acid sequence of SEQ ID NO:34. Embodiment II-22. The polypeptide-polysaccharide conjugate according to any one of embodiments II-16 to II-19, wherein the SpyAD polypeptide comprises the amino acid sequence of SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:82 or SEQ ID NO:83. Embodiment II-23. A polypeptide-polysaccharide conjugate according to any one of embodiments II-16 to II-19, wherein the SpyAD polypeptide has the amino acid sequence of SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:82 or SEQ ID NO:83. Embodiment II-24. The polypeptide-polysaccharide conjugate of any one of embodiments II-1 to II-2 or II-4 to II-14, wherein the non-GAS carrier polypeptide is selected from ADI, ferritin, protein D and eCRM197. Embodiment II-25. The polypeptide-polysaccharide conjugate of embodiment II-24, wherein the non-GAS carrier polypeptide is eCRM197. Embodiment II-26. A polypeptide-polysaccharide conjugate according to embodiment II-25, wherein eCRM197 has the sequence of SEQ ID NO:25. Embodiment II-27. A polypeptide-polysaccharide conjugate according to any one of embodiments II-1 to II-13, wherein the GAS polypeptide antigen or the non-GAS carrier polypeptide comprises or consists of the amino acid sequence of a polypeptide listed in Table 1. Embodiment II-28. A polypeptide-polysaccharide conjugate according to any one of embodiments II-1 to II-13, wherein the GAS polypeptide antigen or the non-GAS carrier polypeptide comprises or consists of the amino acid sequence of a polypeptide listed in Table 1A. Embodiment II-29. (a) Group A Streptococcus (GAS) C5a peptidase polypeptide antigen; (b) a GAS streptolysin O (SLO) polypeptide antigen; and (c) a Streptococcus pyogenes adhesion / disruption (SpyAD) conjugate polypeptide or fragment thereof comprising at least one unnatural amino acid (nnAA), wherein the at least one nnAA comprises a click chemistry reactive group; and a polypeptide-polysaccharide conjugate comprising a GAS polysaccharide or variant thereof lacking an immunodominant N-acetylglucosamine (GlcNAc) side chain; wherein about 8 mol % to about 20 mol % of the polysaccharide repeat units of said GAS polysaccharide or variant thereof are derivatized with a linker, and the average molecular weight of said polypeptide-polysaccharide conjugate is about 185 kDa to about 700 kDa. The immunogenic composition of embodiment II-29, wherein the C5a peptidase polypeptide antigen comprises the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:30. Embodiment II-31. The immunogenic composition of embodiment II-29 or II-30, wherein the C5a peptidase polypeptide antigen comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:29 or SEQ ID NO:30. Embodiment II-32. The immunogenic composition according to any one of embodiments II-29 to II-31, wherein the C5a peptidase polypeptide antigen has the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:30. Embodiment II-33. The immunogenic composition of any one of embodiments II-29 to II-32, wherein the SLO polypeptide antigen comprises the amino acid sequence of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 or SEQ ID NO:53. Embodiment II-34. An immunogenic composition described in embodiments II-29 to II-33, wherein the SLO antigen comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 or SEQ ID NO:53. Embodiment II-35. An immunogenic composition described in embodiments II-29 to II-34, wherein the SLO polypeptide antigen has the amino acid sequence of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 or SEQ ID NO:53. Embodiment II-36. The immunogenic composition of embodiments II-29 to II-35, wherein at least one nnAA is selected from 2-amino-3-(4-azidophenyl)propanoic acid (pAF), 2-amino-4-azidobutanoic acid, 2-azido-3-phenylpropionic acid, 2-amino-3-azidopropanoic acid, 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridin-2-yl)propanoic acid, 2-amino-3-(4-(azidomethyl)pyridin-2-yl)propanoic acid, 2-amino-3-(6-(azidomethyl)pyridin-3-yl)propanoic acid, and 2-amino-5-azidopentanoic acid. Embodiment II-37. The immunogenic composition of any one of embodiments II-29 to II-36, wherein at least one nnAA is pAMF. Embodiment II-38. The immunogenic composition of any one of embodiments II-29 to II-37, wherein the SpyAD conjugate polypeptide or a fragment thereof comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:33. Embodiment II-39. The immunogenic composition of any one of embodiments II-29 to II-38, wherein the SpyAD conjugate polypeptide comprises an amino acid sequence that is a fragment of SEQ ID NO:33. Embodiment II-40. The immunogenic composition of any one of embodiments II-29 to II-38, wherein the SpyAD conjugate polypeptide has an amino acid sequence that is a fragment of SEQ ID NO:33. Embodiment II-41. The immunogenic composition according to any one of embodiments II-29 to II-40, wherein the SpyAD conjugate polypeptide comprises pAMF substitutions at positions K64, K287, K386 and K657 of SEQ ID NO:33. Embodiment II-42. The immunogenic composition of any one of embodiments II-29 to II-41, wherein the SpyAD conjugate polypeptide comprises the amino acid sequence of SEQ ID NO:34, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:82 or SEQ ID NO:83. Embodiment II-43. The immunogenic composition of any one of embodiments II-29 to II-42, wherein the SpyAD conjugate polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:34, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:82 or SEQ ID NO:83. Embodiment II-44. The immunogenic composition of any one of embodiments II-29 to II-43, wherein the SpyAD conjugate polypeptide has the amino acid sequence of SEQ ID NO:34, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:82 or SEQ ID NO:83. Embodiment II-45. The immunogenic composition of any one of embodiments II-29 to II-44, wherein the C5a peptidase polypeptide antigen comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:30; the SLO polypeptide antigen comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:53; and the SpyAD conjugate polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:34. Embodiment II-46. The immunogenic composition of any one of embodiments II-29 to II-45, wherein the C5a peptidase polypeptide antigen comprises the amino acid sequence of SEQ ID NO:30; the SLO polypeptide antigen comprises the amino acid sequence of SEQ ID NO:53; and the SpyAD conjugate polypeptide comprises the amino acid sequence of SEQ ID NO:34. Embodiment II-47. The immunogenic composition of any one of embodiments II-29 to II-46, wherein the C5a peptidase polypeptide antigen has the amino acid sequence of SEQ ID NO:30; the SLO polypeptide antigen has the amino acid sequence of SEQ ID NO:53; and the SpyAD conjugate polypeptide has the amino acid sequence of SEQ ID NO:34. Embodiment II-48. An immunogenic composition according to any one of embodiments II-29 to II-47, wherein about 15 mol% to about 20 mol% of the polysaccharide repeating units of the GAS polysaccharide or its variant are derivatized with a linker. Embodiment II-49. The linker prior to reaction with the click chemistry reactive group of nnAA has the formula I: [ka] wherein X is at least one polysaccharide repeat unit of a GAS polysaccharide or variant thereof, and n is at least 1. The immunogenic composition according to any one of embodiments II-29 to II-48, comprising the structure: Embodiment II-50. The SpyAD conjugate polypeptide or fragment thereof has formula II: [ka] (In the formula, R1 is H, formyl, or at least one amino acid of a SpyAD conjugated polypeptide; R2 is OH, or at least one amino acid of a SpyAD conjugate polypeptide; W is CH or N; y is at least 1; n is at least 1; and X is at least one polysaccharide repeat unit of a GAS polysaccharide or a variant thereof. The immunogenic composition according to any one of embodiments II-29 to II-49, wherein the GAS polysaccharide is conjugated according to Embodiment II-51. An immunogenic composition according to any one of embodiments II-29 to II-50, wherein the GAS polysaccharide or its variant has a molecular weight of at least about 10 kDa to at least about 40 kDa. Embodiment II-52. The immunogenic composition according to any one of embodiments II-29 to II-51, wherein the average molecular weight of the polypeptide-polysaccharide conjugate is from about 185 kDa to about 700. Embodiment II-53. The immunogenic composition according to any one of embodiments II-29 to II-52, wherein the average molecular weight of the polypeptide-polysaccharide conjugate is from about 200 kDa to about 700. Embodiment II-54. The immunogenic composition according to any one of embodiments II-29 to II-53, wherein the average molecular weight of the polypeptide-polysaccharide conjugate is from about 300 kDa to about 700. Embodiment II-55. The immunogenic composition according to any one of embodiments II-29 to II-53, wherein the average molecular weight of the polypeptide-polysaccharide conjugate is from about 300 kDa to about 600. Embodiment II-56. The immunogenic composition of any one of embodiments II-29 to II-55, further comprising less than about 60% free GAS polysaccharide or a variant thereof. Embodiment II-57. The immunogenic composition of any one of embodiments II-29 to II-56, further comprising less than about 50% free GAS polysaccharide or a variant thereof. Embodiment II-58. The immunogenic composition of any one of embodiments II-29 to II-57, further comprising less than about 25% free GAS polysaccharide or a variant thereof. Embodiment II-59. The immunogenic composition of any one of embodiments II-29 to II-58, further comprising less than about 15% free GAS polysaccharide or a variant thereof. Embodiment II-60. The immunogenic composition of any one of embodiments II-29 to II-59, further comprising less than about 10% free GAS polysaccharide or a variant thereof. Embodiment II-61. A method for inducing a protective immune response in a subject against Group A Streptococcus (GAS) bacteria, comprising administering to the subject an immunogenic composition according to any one of embodiments II-29 to II-60. Embodiment II-62. The method of embodiment II-61, wherein the immunogenic composition induces an antibody response in the subject against Group A Streptococcus (GAS) bacteria and does not induce an antibody response in the subject against human tissue. Embodiment II-63. Use of an immunogenic composition according to any one of embodiments II-29 to II-60 in the manufacture of a medicament for inducing a protective immune response against GAS bacteria in a subject. Embodiment II-64. Use of an immunogenic composition according to any one of embodiments II-29 to II-60 for inducing a protective immune response against GAS bacteria in a subject. Embodiment II-65. A method for purifying cell wall polysaccharides or peptidoglycan-bound capsular polysaccharides from bacterial cells, comprising the steps of: (a) hydrolyzing bacterial cells in a solution containing a base and a reducing agent to form a lysate solution containing polysaccharides; and (b) incubating the degradation solution containing polysaccharides with a wall-degrading enzyme to form a free polysaccharide solution; The method includes: Embodiment II-66. The method of embodiment II-65, wherein the bacterial cell is a Pseudomonas bacterial cell, a Streptococcus bacterial cell, a Staphylococcus bacterial cell, a Neisseria bacterial cell, a Haemophilus bacterial cell, a Listeria bacterial cell, an Enterococcus bacterial cell or a Clostridium bacterial cell. Embodiment II-67. The method of embodiment II-65 or II-66, wherein the bacterial cell is selected from Pseudomonas aeruginosa, Streptococcus viridans, Streptococcus mutans or Streptococcus pyogenes. Embodiment II-68. The method of embodiment II-67, wherein the Streptococcus pyogenes bacterial cell is a serotype selected from M1, M2, M3, M4, M5, M6, M9, M11, M12, M13, M18, M22, M25, M28, M62, M71, M72, M74, M75, M77, M80, M81, M83, M87, M89 or M92. Embodiment II-69. The method of embodiment II-68, wherein the Streptococcus pyogenes bacterial cell produces a polysaccharide or a variant thereof lacking an immunodominant N-acetylglucosamine (GlcNAc) side chain. Embodiment II-70. The method of embodiment II-65 for purifying peptidoglycan-bound capsular polysaccharides from bacterial cells. Embodiment II-71. The method of any one of embodiments II-65 to II-70, wherein the base in step (a) is NaOH, KOH, or LiOH. Embodiment II-72. The method of any one of embodiments II-65 to II-71, wherein the base in step (a) is NaOH. Embodiment II-73. The method of any one of embodiments II-65 to II-72, wherein the concentration of the base is from about 2M to about 8M. Embodiment II-74. The method of any one of embodiments II-65 to II-73, wherein the solution containing the base and the reducing agent has a pH of about 14. Embodiment II-75. The method of any one of embodiments II-65 to II-72, wherein the reducing agent is sodium borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride, dithiothreitol, or β-mercaptoethanol. Embodiment II-76. The method of any one of embodiments II-65 to II-73, wherein the reducing agent is sodium borohydride. Embodiment II-77. The method of any one of embodiments II-65 to II-76, wherein the concentration of the reducing agent is about 1 mM to 500 mM. Embodiment II-78. The method of any one of embodiments II-65 to II-75, wherein step (a) further comprises incubating the solution at about 30°C to about 100°C. Embodiment II-79. The method of any one of embodiments II-65 to II-78, wherein step (a) further comprises incubating the solution for about 0.5 to about 20 hours. Embodiment II-80. The method of any one of embodiments II-65 to II-79, wherein step (a) further comprises one or more pH adjustment steps. Embodiment II-81. The one or more pH adjustment steps comprise the steps of: (i) increasing the pH of the degradation solution containing the polysaccharide; or (ii) reducing the pH of the degradation solution containing the polysaccharide; The method of embodiment II-80, wherein said Embodiment II-82. The method of embodiment II-81, comprising lowering the pH of the degradation solution containing the polysaccharide to about 3 to 7.0. Embodiment II-83. The method of embodiment II-81 or II-82, comprising lowering the pH of the degradation solution containing the polysaccharide to about 6.5. Embodiment II-84. The method according to embodiment II-81 or II-82, comprising lowering the pH of the degradation solution containing the polysaccharide to about 3 to about 4. Embodiment II-85. The method according to claim 1, further comprising the steps of: (i) lowering the pH of the degradation solution containing the polysaccharide to about 5.5 to 7.0; (ii) lowering the pH of the digestion solution containing the polysaccharide to about 3; (iii) increasing the pH of the degradation solution containing the polysaccharide to about 5.5 to 7.0; The process according to embodiment II-80 or II-81, comprising: Embodiment II-86. The method according to any one of embodiments II-80 to II-85, wherein the degradation solution containing the polysaccharide is incubated at about room temperature (rt) after one or more pH adjustment steps. Embodiment II-87. The method according to any one of embodiments II-81 to II-84, wherein the degradation solution containing the polysaccharide is incubated at about 4° C. to about 30° C. after one or more pH adjustment steps. Embodiment II-88. The method of any one of embodiments II-65 to II-87, further comprising removing solids from the digestion solution containing the polysaccharide. Embodiment II-89. The method of embodiment II-88, wherein the step of removing solids from the degradation solution containing the polysaccharide comprises filtration, centrifugation, or a combination thereof. Embodiment II-90. The method of embodiment II-89, wherein the filtration comprises depth filtration, tangential flow filtration (TFF), sterile filtration, or a combination thereof. Embodiment II-91. The method of embodiment II-89 or II-90, wherein the filtration comprises depth filtration followed by TFF. Embodiment II-92. The method of embodiment II-88 or II-89, wherein solids are removed from the polysaccharide-containing digestion solution by centrifugation. Embodiment II-93. The method of any one of embodiments II-65 to II-92, wherein the wall-degrading enzyme of step (b) is mutanolysin, lysozyme, or a bacteriophage hydrolase. Embodiment II-94. The method of any one of embodiments II-65 to II-93, wherein step (b) further comprises incubating with a protease. Embodiment II-95. The method of embodiment II-94, wherein the protease is proteinase K, trypsin, chymotrypsin, endoproteinase Asp-N, endoproteinase Arg-C, endoproteinase Glu-C, endoproteinase Lys-C, pepsin, thermolysin, elastase, papain, subtilisin, clostripain, carboxypeptidase A, carboxypeptidase B, carboxypeptidase P, carboxypeptidase Y, cathepsin C, acyl amino acid releasing enzyme or pyroglutamate. Embodiment II-96. The method according to any one of embodiments II-65 to II-95, wherein step (b) further comprises heating the degradation solution containing the polysaccharide together with the wall-degrading enzyme to about 30°C to about 65°C. Embodiment II-97. The method according to any one of embodiments II-94 to II-96, wherein the degradation solution containing polysaccharides is heated to about 45°C to about 55°C together with the protease. Embodiment II-98. The method of embodiment II-96 or II-97, wherein the decomposition solution is heated for about 6 to about 20 hours. Embodiment II-99. The method according to any one of embodiments II-65 to II-98, wherein the free polysaccharide solution of step (b) is further purified to reduce the concentration of nucleic acids, enzymes, host cell proteins (HCPs), or combinations thereof. Embodiment II-100. The method of any one of embodiments II-65 to II-99, wherein the free polysaccharide solution of step (b) is further purified by precipitation. Embodiment II-101. The method of any one of embodiments II-65 to II-100, wherein the free polysaccharide solution of step (b) is treated with cetyltrimethylammonium bromide (CTAB). Embodiment II-102. The method of embodiment II-101, wherein the concentration of CTAB in the free polysaccharide solution is about 0.1% to about 10%. Embodiment II-103. The method of embodiment II-101 or II-102, wherein the concentration of CTAB is about 0.5% to about 3%. Embodiment II-104. The method of any one of embodiments II-101 to II-103, wherein the free polysaccharide solution of step (b) is treated with potassium iodide (KI). Embodiment II-105. The method according to any one of embodiments II-104, wherein the KI concentration in the free polysaccharide solution is from about 20 mM to about 400 mM. Embodiment II-106. The method of any one of embodiments II-65 to II-105, wherein the free polysaccharide solution is further purified by filtration, centrifugation, chromatography, or a combination thereof. Embodiment II-107. The method of embodiment II-106, wherein the filtration comprises depth filtration, tangential flow filtration (TFF), sterile filtration, or a combination thereof. Embodiment II-108. The method of embodiment II-106, wherein the chromatography comprises hydrophobic interaction chromatography (HIC), anion exchange chromatography (AEX), ceramic hydroxyapatite-based chromatography, or cation exchange chromatography (CEX). Embodiment II-109. An immunogenic composition comprising: (a) a Group A Streptococcus (GAS) C5a peptidase polypeptide antigen comprising or consisting of the amino acid sequence of SEQ ID NO:30, or a fragment thereof; (b) a GAS streptolysin O (SLO) polypeptide antigen comprising or consisting of the amino acid sequence of SEQ ID NO:53 or a fragment thereof; and (c) a Streptococcus pyogenes adhesion / disruption (SpyAD) conjugate polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO:34 or a fragment thereof; and a GAS polysaccharide or variant thereof lacking an immunodominant N-acetylglucosamine (GlcNAc) side chain, wherein about 8 mol % to about 20 mol % of the polysaccharide repeat units of said GAS polysaccharide or variant thereof are derivatized with a linker. Embodiment II-110. The immunogenic composition of embodiment II-109, wherein the average molecular weight of the polypeptide-polysaccharide conjugate is between about 185 kDa and about 700 kDa. Embodiment II-111. The immunogenic composition of embodiment II-109 or II-110, wherein the average molecular weight of the polypeptide-polysaccharide conjugate is between about 200 kDa and about 700 kDa. Embodiment II-112. The immunogenic composition according to any one of embodiments II-109 or II-111, wherein the average molecular weight of the polypeptide-polysaccharide conjugate is between about 300 kDa and about 700 kDa. Embodiment II-113. A polypeptide-polysaccharide conjugate according to any one of embodiments II-109 or II-112, wherein the average molecular weight of the polypeptide-polysaccharide conjugate is between about 300 kDa and about 600 kDa. Embodiment II-114. A polypeptide-polysaccharide conjugate according to any one of embodiments II-109 or II-113, wherein the GAS polysaccharide or variant thereof has a molecular weight of at least about 10 kDa to at least about 40 kDa. Embodiment II-115. A polypeptide-polysaccharide conjugate according to any one of embodiments II-109 or II-114, wherein the SpyAD conjugate polypeptide is a fragment of the amino acid sequence of SEQ ID NO:34 and comprises or consists of the amino acid sequence of SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:82 or SEQ ID NO:83. EXAMPLES

[0102] Synthesis Example 1: Purification of high molecular weight (long chain) polysaccharides Experiments were performed to extract and purify GAS polysaccharides from GAS bacterial cultures. Figure 1 shows a simplified flow chart of the sample purification described herein.

[0103] Base hydrolysis: The prepared GAS cell pellet was resuspended in 50 mM NaCl solution. 50 mL of NaCl was added using a serological pipette and vortexed until resuspended. 10 N sodium hydroxide and 1 M sodium borohydride were added to a final concentration of 4 N NaOH and 25 mM NaBH4. The resuspended pellet was dispensed into centrifuge vessels with a final target volume of 160 mL per 1 L of fermentation volume. Constant swirling was used to ensure homogeneity when dispensing the solution. The bottles were placed on a shaker for 2 hours in a 65°C pre-incubator. After incubation, the hydrolysis solution was centrifuged at 14,000xg at 25°C for 30 minutes to pellet the cell debris, making sure to cool the hydrolysis sample to room temperature before centrifugation. After centrifugation stopped, the supernatant was collected without disrupting the pellet and the supernatant was neutralized to pH 6.5 ± 0.3. Upon neutralization, the bottles were placed in an ice bath at 4° C. and the pH was adjusted with 37% HCl and further adjusted with 1 M NaOH if necessary. The samples were then incubated overnight at 4° C.

[0104] Filtration: After incubation, a white precipitate forms. The solution was centrifuged at 10,000xg for 30 min. A large white pellet containing the host cell proteins (HCPs) forms. The clear supernatant was collected. The pH was adjusted to 3.0 with 37% HCl and the solution was incubated at RT for 1 h. The sample was filtered using a Clarisolve Filter μPod -40MS. For example, flush the filter with water at a pump flow rate of 23 mL / min until ready, open the valve to flush a volume of 120 mL, and equilibrate with 15 mM NaCl at a volume of 120 mL. The extract was flushed through the filter at the same pump flow rate of 23 mL / min and the clear filtrate coming out of the filter was collected. The filter was washed with 60 mL of 15 mM NaCl to flush the tubing and the filter contents. The filter was discarded. The sample pH was readjusted to 6.5 ± 0.3. This was followed by tangential flow filtration (TFF-10k) to remove salts, concentration (ultrafiltration), and then buffer exchange with 10 mM NaCl (diafiltration).

[0105] Mutanolysin treatment: A solution for mutanolysin treatment was prepared by adding 1M MgCl2 to a final concentration of 1 mM MgCl2 and 200 mM sodium phosphate (pH 6.8) (10x) to a final concentration of 20 mM sodium phosphate. Mutanolysin solution (5000 IU / mL) was adjusted to 120 IU / mL. Samples were incubated overnight at 37°C with shaking.

[0106] Proteinase-K treatment: The samples were then treated with proteinase-K by adding proteinase-K solution to a final concentration of 40 IU / mL (proteinase-K 45u / mg). The mixture was incubated at 50°C overnight with gentle mixing.

[0107] Precipitation and filtration: To precipitate enzymes, nucleic acids and HCPs in the samples, CTAB / 20 mM sodium phosphate buffer (pH 6.8) was added and shaken at 30°C for 1 hour. All solutions used were pre-warmed: PS samples, 5% CTAB stock solution, 200 mM sodium phosphate (pH 6.8). The PS solution was heated and stirred (magnetic stir bar) and the heated stir plate was equipped with a thermometer so that the temperature of the solution could be constantly monitored. 200 mM sodium phosphate (pH 6.8) solution was added to the PS solution to give a final concentration of 20 mM sodium phosphate. 5% CTAB was added to the PS solution to give a CTAB concentration of 1%. The solutions were mixed for 1 hour. The precipitated solutions were depth filtered using a 40MS filter. For example, the system was flushed with 200 mL of MilliQ H20 at a pump flow rate of 23 mL / min, the sample was filtered at 23 mL / min, and flushed with 60 mL of a solution of 20 mM sodium phosphate (pH 6.8) and 15 mM NaCl at 20 mL / min. The filtrate was collected until the air bubbles were eluted. The filter was discarded.

[0108] The PS solution and 274 mM KI were warmed to 30°C. A sufficient amount of 274 mM KI was added to the mixed PS solution to give a final concentration of 27.4 mM KI. The mixed solution was incubated at 30°C for 1 hour. After incubation, the solution was centrifuged at 10,000 x g for 30 minutes at 30°C. The supernatant was collected and the pellet was discarded. Note that the pellet is easily friable. The supernatant was then vacuum filtered through a 0.45 μm filter.

[0109] The sample was first concentrated with TFF-10k, then diafiltered with 9 DV of 350 mM NaCl and finally with 2 DV of MilliQ water. For example, the volume of the TFF system is about 35 mL, the pump flow rate is set to 200 mL / min, and diafiltration is performed with 9 DV (50 mL) of buffer TMP: 7-8 psi.

[0110] The polysaccharide solution was then purified by hydrophobic interaction chromatography using a HiPrep® Butyl Fast Flow 16 / 10 pre-equilibrated with 3 M sodium chloride and 50 mM sodium phosphate, pH 6.8. Sodium chloride and phosphate buffer were added to the polysaccharide solution to give 3 M sodium chloride and 50 mM potassium phosphate, pH 6.8. The polysaccharide solution was passed through the HIC resin and operated in flow-through mode. The resin was washed with the same equilibration buffer, and both the flow-through and the wash solution were collected for further processing.

[0111] A final TFF 10 kDa / 3 kDa was performed to remove high NaCl concentrations in the PS samples by diafiltration with 9 DV of 15 mM NaCl or WFI. The purified PS solution was filtered through a 0.22 μm filter.

[0112] Figures 2A, 2B and 2C show NMR analysis of purified polysaccharide prepared according to synthesis example 1 from a GAS bacterial strain expressing PS lacking GlcNAc (see PCT / US2012 / 049604). NMR confirmed the presence of polyrhamnose and the absence of GlcNAc. For sample preparation, the purified polysaccharide solution was lyophilized and exchanged into D2O with TSP added as an internal standard. Proton NMR spectra were obtained on a 400 MHz instrument at 50°C averaging the signal over 40 scans. A line broadening of 0.2 Hz was applied. This purification protocol also allowed the yield of purified polysaccharide free of M-protein, as demonstrated by the Western blot in Figure 3. Figures 4A and 4B show a flow chart outlining another exemplary polysaccharide purification method.

[0113] Synthesis Example 2: General Protocol for Derivatization of Polymeric Polysaccharides Purified polysaccharides, such as those prepared by the method of Example 1, can be functionalized with a DBCO-PEG linker.

[0114] Typically, borate buffer (1M, pH 8.5) was added to an aqueous solution of polysaccharide (final concentration 5.5 mM after all reagents were added) such that the final concentration of borate was 100 mM in the final volume. Additional water was then added to fill up the reaction volume. 2.5 equivalents (relative to the repeating unit of the polysaccharide) of 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP; of a 100 mg / mL solution in acetonitrile) were added with vigorous stirring. CDAP is stored at -20°C, so the solution should be prepared immediately before use. Five minutes after the addition of CDAP (this timing is important, as more than 5 minutes reduces the incorporation of DBCO-PEG-amine), 0.5 molar equivalents of dibenzocyclooctyne-amine linker (from a DMSO stock solution, final concentration of DMSO was 5% v / v) were added. DBCO-PEG4-amine linker is stored at -20°C, so it should be prepared immediately before use. After an additional hour of reaction, glycine (2M, pH 8.35) was added 1:10 by volume to a final concentration of 200 mM to quench any unreacted cyanate esters. After 1 hour of quenching, the derivatized polysaccharides were purified on Zeba spin columns. 2-3 mL of the solution was added to each 10 mL Zeba column. The purified polysaccharides were analyzed by the Bound / Free DBCO HPLC method to determine whether the residual DBCO-PEG4-amine linker and DMAP were completely removed by the column purification. If necessary, the material can be further purified. The polysaccharide concentration was measured using the anthrone assay, and the concentration of dibenzocyclooctyne was measured using absorbance at 309 nm. The two values ​​were combined to estimate the percentage of polysaccharide derivatized with dibenzocyclooctyne functional groups. The percentage of DBCO should be 5-10% in the CDAP reaction.

[0115] DBCO-PEG4 derivatization of GAS polysaccharide: To a 6 mM solution of GAS polysaccharide in 100 mM borate buffer (pH 8.5), 3 equivalents (relative to polysaccharide repeat unit) of 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP; from a 100 mg / mL solution in acetonitrile) were added with vigorous stirring to promote cyanylation at the reactive hydroxyl groups. Five minutes after CDAP addition, 2 molar equivalents of dibenzocyclooctylamine linker stock / DMSO were added to a final DMSO concentration of 5% (v / v). After DBCO derivatization, 200 mM glycine was added to the reaction to quench unreacted cyanate esters. The DBCO-derivatized polysaccharide was purified on a zeba spin desalting column, and the purity of the recovered material was assessed by reversed phase. A single peak was observed by HPLC monitoring absorbance at 309 nm, confirming that the excess DBCO linker and other reaction by-products had been completely removed. Finally, polysaccharide concentration was measured using the anthrone assay (see below), and dibenzocyclooctyne concentration was measured using absorbance at 309 nm. These two values ​​were combined to estimate the percentage of polysaccharide derivatized with dibenzocyclooctyne functional groups. For conjugation, the DBCO derivatization rate of GAS polysaccharide was kept between 5 and 10%.

[0116] Anthrone assay for total polysaccharide concentration: A 2 mg / ml stock of Anthrone reagent (Sigma-Aldrich, CAS#90-44-8) was prepared in cold sulfuric acid, and a 1 mM stock of 2x rhamnose-containing polysaccharide repeating unit (PSRU) was prepared in water as a standard. Triplicate wells were plated (96-well plate) with 100 μl of PSRU stock (serial dilution to reference standard) or unknown samples (diluted 1:3), followed by the addition of 200 μl / well of Anthrone reagent stock. All reactions were mixed thoroughly, sealed with a plate cover and incubated at 95°C for 10 min. Plates were placed on ice and cooled to room temperature before measuring absorbance at 620 nm using a UV / VIS plate reader. To determine the concentration of unknown samples, a least squares regression analysis was performed using the PSRU standard concentrations and absorbance.

[0117] Synthesis Example 3: Expression and purification of pAMF modified conjugate polypeptides This experiment was performed to express and purify pAMF modified conjugate polypeptides from cell-free protein synthesis extracts. Polypeptides containing nnAAs (e.g., pAMF) are expressed in cell-free protein synthesis (CFPS) reactions using extracts (XtractCF+) from Escherichia coli engineered to produce orthogonal tRNAs for inserting nnAAs at amber stop codons. Sample protocols used for cloning, expression and purification of these engineered conjugated polypeptides can be found, for example, in Kapoor et al., Biochemistry, 2018, 57(5), 516-519.

[0118] Synthetic Example 4: General Protocol for Conjugation of High Molecular Weight Polysaccharides to Polypeptide Antigens or Non-GAS Carrier Polypeptides to Form Polypeptide-Polysaccharide Conjugates Generally, a polypeptide antigen or non-GAS carrier polypeptide is conjugated to the purified DBCO-derivatized polysaccharide of Example 2 by reacting the cyclooctyne group of the DBCO group with an azide group of an nnAA side chain incorporated into the polypeptide antigen or non-GAS carrier polypeptide. Sample protocols for conjugation reactions between DBCO and azide groups can be found, for example, in Zimmerman et al., Bioconjugate Chemistry, 2014, 25(2), 351-361 and Kapoor et al., Biochemistry, 018, 57(5), 516-519.

[0119] Conjugation of pAMF-derivatized GAS polysaccharide with SpyAD: SpyAD[4pAMF] (SEQ ID NO:34) was mixed 1:1 with DBCO-derivatized GAS polysaccharide [0.5 mg / ml each] to facilitate conjugation by click chemistry. After conjugation, the reaction mixture was dialyzed against a 50 kDa cutoff membrane to remove excess unreacted free polysaccharide. The recovered conjugate was analyzed by SEC (multi-angle light scattering) MALS and the concentration was estimated using anthrone assay. Figure 5 shows a scheme outlining the derivatization of GAS polysaccharide with DBCO-(PEG)4-NH2 followed by conjugation to pAMF-derivatized SpyAD. Figure 6 shows SEC MALS analysis of SpyAD[4pAMF] (SEQ ID NO:34) polypeptide-polysaccharide conjugate after 3.5 hours of conjugation reaction and after dialysis. After 3.5 hours, conjugates of over 1 MDa were observed. After dialysis, the size distribution of the isolated conjugates showed a peak at approximately 186 kDa. Figure 7 shows SEC-MALS analysis of polypeptide-polysaccharide conjugates using the same SpyAD polypeptide as in this example, but utilizing hydrofluoric acid-based PS purification (see, e.g., van Sorge, NM et al. 2014, Cell Host Microbe. 15(6): 729-740). The molecular weight peak was 153 kDa when using traditionally purified PS, compared to ∼186 kDa for the purified polysaccharide reported here.

[0120] SEC MALS-UV-RI was performed using an Agilent HPLC 1100 degasser, temperature-controlled autosampler (4 °C), column compartment (25 °C) and UV-VIS diode array detector (Agilent, Santa Clara, CA) coupled to a DAWN-HELEOS multi-angle laser light scattering detector and an Optilab T-rEX differential refractometer (Wyatt Technology, Santa Barbara, CA) connected in series to three TOSOH columns: TSKgel Guard PWXL 6.0 mm i.d. × 4.0 cm length, 12 μm particle size; TOSOH TSKgel 6000 PWXL 7.8 mm i.d. × 30 cm length, 13 μm particle size; and TSKgel 3000 PWXL 7.8 mm i.d. × 30 cm length, 7 μm particle size. Samples (50–100 μg) were injected and analyzed using a mobile phase consisting of 1xPBS containing 5% (v / v) acetonitrile filtered through 0.2 μm at a flow rate of 0.5 mL / min. The HPLC was controlled using Agilent Open Lab software, and data collection and molecular weight analysis were performed using Wyatt Astra 7 software.

[0121] Synthesis Example 5: Expression, purification and conjugation of truncated SLO(ΔC101) polypeptides with 3, 4, 5, 6, 7 or 8 nnAAs Truncated SLO(ΔC101) variants containing nnAA are expressed, for example, according to the methods described above (see, for example, Synthesis Example 3). The variants contain 3, 4, 5, 6, 7 or 8 pAMF residues and correspond to SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75 and SEQ ID NO:76. Figure 8A is a schematic diagram of the 22 SLO(ΔC101) variants (var1 to var22) and their pAMF introduction sites. Figure 8B shows the expression levels of these 22 pAMF variants (3pAMF to 8pAMF) in both total and soluble protein concentrations. Figure 8C shows the gel corresponding to expression (SEQ ID NOs: 65-76 correspond to var11-var22). 8D shows the gel revealing successful pAMF introduction in a subset of purified variants as confirmed by DBCO-TAMRA labeling (var1, 5, 6, 10, 11, 12, 14 and 15). Briefly, for purification, CFPS was collected, spun down, filtered and then loaded onto a pre-equilibrated hisTRAP excel 1 ml column at 0.75 ml / min and eluted in a single step of 250 mM imidazole in a final volume of 6 ml. Fractions (5 μl) from each step were analyzed by SDS-PAGE safe blue staining. The eluate was then diluted 3-fold and loaded onto a CaptoQ column for final purification and analyzed by safe blue staining and DBCO TAMRA labeling. The FT fraction was concentrated and 200 μl aliquots were stored at -80 °C. The molecular weights of a subset of the purified variants were estimated by SEC-MALS analysis (Figure 8E), which showed that the 5-pAMF(var11) and 6-pAMF(var14) variants exhibited highly oligomeric states in solution.

[0122] Conjugation of SLO(ΔC101) polypeptides containing 5, 6, 7 or 8 nnAAs: Polypeptides containing 5, 6, 7 or 8 nnAAs are conjugated to GAS polysaccharides using the methods described above, including synthesis example 4. In this manner, pAMF-containing SLO(ΔC101) variants of SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:68 and SEQ ID NO:69 were conjugated to long-chain DBCO-derivatized GAC (synthesis example 1). The conjugation reactions were analyzed by SDS-PAGE, as shown in FIG. 9A. The first lane of each variant shows the protein before conjugation. The final conjugate size was determined by SEC-MALS, and the results are shown in FIG. 10.

[0123] Biophysical characterization and immunogenicity evaluation of conjugates made using 3- and 5-pAMF SLO variants as carrier proteins To create conjugates with purified SLO(ΔC101) variants, three pAMF (var1 and var5) and four pAMF (var6 and var10)-containing SLO(ΔC101) variants were conjugated with a DBCO-derivatized polyrhamnose-rich core (i.e., GACPR)-defining species of membrane-anchored GAS carbohydrate (GACPR). PR Each protein was used in a copper-free click chemistry reaction with DBCO-GAC PR The conjugates were mixed with 0.1% EDTA for 4 h at room temperature under stirring. The reaction products were then collected and dialyzed against buffer to remove excess free PS. The purified conjugates were then subjected to SEC-MALS analysis, which showed that the average molar mass of the conjugates made with the SLO(ΔC101) variant containing three pAMFs was 97 or 116 kDa (Figure 9B), and that of the conjugates made with the SLO(ΔC101) variant containing four pAMFs was 68 or 74 kDa (Figure 9C). Finally, SDS-PAGE analysis of the purified conjugates was followed by SafeBlue TM Staining confirmed that the carrier protein was completely consumed for the generation of each conjugate after the click chemistry reaction, as demonstrated by the results (Figures 9B-9C).

[0124] Synthesis Example 6: Optimization of Linker Introduction into Polysaccharide Repeating Units As described above, a purified polysaccharide (e.g., a GAS polysaccharide), such as one prepared by the method of Example 1, can be derivatized with a linker (e.g., DBCO-PEG). The mol% of polysaccharide repeating units (PSRU) of a GAS polysaccharide that are derivatized with DBCO-PEG4-amine can be defined in several relevant ways (e.g., % incorporation of DBCO-PEG4-amine measured in moles of linker per mole of PSRU; mol% of PSRU that are derivatized with linker).

[0125] To an aqueous solution containing a group A carbohydrate (GAC) polysaccharide lacking the immunodominant N-acetylglucosamine side chain, sodium borate (pH 8.8), and dimethyl sulfoxide, 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP, 0.5–4 molar equivalents relative to the GAC polysaccharide repeating unit) in acetonitrile was added. After 5 min, a solution of DBCO-PEG4-amine (0.5–2 molar equivalents) in dimethyl sulfoxide was added. The final concentrations of the reaction components were as follows: GAC, 4 mg / mL; sodium borate, 0.1 M; dimethyl sulfoxide, 10% v / v. After 1 h, glycine was added to a final concentration of 0.2 M. After 2 h, the reaction mixture was purified by gel filtration chromatography or tangential flow filtration using saline and water as diafiltration buffers. Activated GAC (APS, also called activated polysaccharide) was analyzed for polysaccharide concentration and DBCO-PEG4-amine incorporation rate.

[0126] By varying the reaction conditions, the above protocol was used to produce GAS polysaccharides with 6-15% (mol % relative to PSRU) loaded with DBCO-PEG4-amine. Table 2 shows examples that led to the noted loading percentages. [Table 28]

[0127] Synthesis Example 7: Optimization of conjugation conditions for long chain polysaccharides and SpyAD The conjugation efficiency of the long-chain polysaccharides described herein depends on the mol % amount of the linker introduced (see, for example, Synthesis Example 6), the reaction concentration of the polypeptide, and the amount of activated polysaccharide relative to the polypeptide.

[0128] Sample conjugation protocol: SpyAD-4pAMF (SEQ ID NO:34) was added to an aqueous solution containing activated polysaccharide (see Synthesis Example 6) and phosphate buffered saline. The final concentrations of the reaction components were as follows: activated PS with DBCO linker, 0.075-1.35 mg / mL; SpyAD-4pAMF, 0.5-3.7 mg / mL. After 16-20 hours, sodium azide (4 equivalents relative to the repeating unit of the polysaccharide) was added. After 2 hours, the reaction mixture was purified by dialysis or tangential flow filtration using phosphate buffered saline as the diafiltration buffer. The conjugates were filtered through a 0.22 micron rated filter (Pall KM2EKVS) and analyzed for polysaccharide concentration, protein concentration, % free sugar and molecular weight. Examples of conjugates made are shown in Table 3. [Table 29]

[0129] As discussed further in the Biological Examples below, among the conjugates made with the long chain polysaccharides of the present disclosure, the high molecular weight SpyAD-GAC conjugates (e.g., made by the method of Synthetic Example 7) yielded stronger potency against SpyAD and GAC than the low molecular weight conjugates. Several factors are important in the production of high molecular weight conjugates. First, the % incorporation of DBCO-PEG4-amine into the activated polysaccharide (e.g., Synthetic Example 4) affects the size of the conjugate. In the range of 6-15%, higher DBCO incorporation rates (mol%) generally yield larger conjugates. Second, higher mass ratios of GAC:SpyAD generally yield smaller conjugates, while lower mass ratios yield unconjugated SpyAD polypeptides. A mass ratio of 0.10-0.15 generally limits the amount of unconjugated SpyAD and activated polysaccharide and yields larger conjugates. Finally, at a given GAC:SpyAD ratio, higher concentrations of both reaction products generally yield larger conjugates.

[0130] The % DBCO-PEG4-amine incorporated into the activated polysaccharide was reported as the molar ratio (total DBCO-PEG4-amine)*(% bound DBCO-PEG4-amine) / (PSRU concentration). These values ​​were obtained as follows: for total DBCO-PEG4-amine, the absorbance of the APS sample was measured at 307 nm. The total DBCO-PEG4-amine concentration in the sample was determined using the extinction coefficient determined for DBCO-PEG4-amine. The % bound DBCO-PEG4-amine was measured by HPLC analysis. Samples were injected onto a Sepax Zenix-C SEC-300 column and eluted with a mobile phase containing 50 mM potassium chloride, 15% v / v acetonitrile and 0.1% v / v trifluoroacetic acid. The % bound DBCO-PEG4-amine was determined as the peak area ratio of (APS) / (APS+free DBCO-PEG4-amine) at 310 nm.

[0131] Polysaccharide concentration was measured using the anthrone assay as described above. Protein concentration of the samples was measured using the Pierce Modified Lowry assay kit according to the manufacturer's protocol. To measure free sugars, a sodium deoxycholate solution (1% w / v in water) was prepared and the pH was adjusted to 6.8 with hydrochloric acid. To the solution of the conjugate, 0.1 volume of the deoxycholate stock solution and 0.05 volume of HCl (1M) were added. The sample was spun, the supernatant was collected, and the procedure was repeated once more. The polysaccharide content of the supernatant was measured by the anthrone assay (as described above), and the free sugar content of the conjugate was determined as the ratio of the polysaccharide concentration of the deoxycholate supernatant to that of the conjugate. The molecular weight of the conjugate was determined by SEC-MALS as described above.

[0132] As an illustration, SpyAD conjugates CNJ-AE, CNJ-AF and CNJ-AG were made by varying the parameters mentioned above. Notably, the average molecular weight of the conjugate CNJ-AG (determined by SEC-MALS) was approximately 469 kDa, compared to approximately 170 kDa for both CNJ-AE and CNJ-AF (see FIG. 13). This may be due to the higher degree of DBCO incorporation in the activated polysaccharide (15% vs. 6%), the higher concentration of SpyAD polypeptide in the reaction mixture, and the lower input ratio of activated polysaccharide to polypeptide. In addition, CNJ-AG was made with a lower amount of free polysaccharide, which simplified the purification procedure and made the active pharmaceutical ingredient more stable for use in the immunogenic compositions described herein. This also means that the production is more efficient, cost-effective, and reagent-saving.

[0133] Synthetic Example 8: Expression of SpyAD Fragments SpyAD polypeptide fragments of SEQ ID NO:34 (SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, and SEQ ID NO:81) were expressed according to the methods of Synthesis Example 3. Each fragment was expressed using a construct encoding a polypeptide having a 2x6-histag leader sequence (e.g., SEQ ID NO:80), with SEQ ID NOs:77, 78, 79, and 81 corresponding to the sequence after purification and cleavage of the leader tag.

[0134] Fragments of SpyAD may be useful for improving expression, purification and / or immunogenicity compared to the native or full-length SpyAD sequence. To monitor production of a desired polypeptide fragment, 14 C-leucine (GE Life Sciences, Piscataway, NJ) was added to the CFPS reaction to synthesize SEQ ID NOs: 77, 78, 79 and 81 and incorporated into the translated polypeptide. After shaking at 23°C for 10 hours and spinning at 4,500 rpm for 10 minutes, the supernatant was collected. Total and soluble protein expression titers were 14 The total number of nucleoside residues was estimated by C count. A total of 4 μl of the supernatant was loaded onto a non-reducing 4-12% SDS-PAGE gel. After dehydration of the protein gel at 80° C. for 2 hours, native SpyAD (SEQ ID NO: 33), SpyAD(4pAMF) (SEQ ID NO: 34) and fragments (SEQ ID NO: 77, 78, 79 and 81) were analyzed by autoradiography using a Storm 820 PhosphoImager.

[0135] Figure 14 shows the results of autoradiography for the expression of native SpyAD, SpyAD(4pAMF) and the four fragments, as well as the expression titers of total and soluble proteins in each sample. It is noteworthy that the expression mixture of native SpyAD and SpyAD(4pAMF) has six contaminant fragment bands between 50 and 75 kDa, whereas the SpyAD fragments of SEQ ID NOs: 77-79 and 81 show fewer bands in the same region. The improved protein expression level in the CFPS reaction results in a higher recovery rate. This simplifies the purification procedure of the polypeptide and allows for higher purity material to be obtained by conjugation to derivatized polysaccharides.

[0136] Biological Example 1: Active Immunization of Mice with Polysaccharides or Polysaccharide Conjugates Mice are actively immunized prior to challenge by subcutaneous and IP injection. [Table 30]

[0137] 1st, 2nd and 3rd immunizations - subcutaneous and IP challenge: Antigen / adjuvant mixtures are prepared by adding 10 μg of antigen or 5 μg of conjugate to alum (Alhydrogel) (50 μL) and mixing thoroughly to allow the antigen to adsorb to the alum. Each antigen / adjuvant mixture is placed into a 1 mL syringe fitted with a 26 1 / 2 gauge needle. Each mouse is anesthetized by isoflurane inhalation and 100 μL of the prepared vaccine is injected into the hind leg muscle.

[0138] Preparation of mice for challenge - Subcutaneous challenge: Mice were anesthetized with isoflurane. Shave the back of the mouse with an electric razor, taking care not to damage the skin. Apply depilatory cream to the shaved back and leave it for a few minutes before wiping thoroughly with a damp paper towel. Allow the mouse to dry and recover from the isoflurane treatment.

[0139] Preparation of materials for challenge - Subcutaneous and IP challenge: Grow GAS cultures to mid-log phase. Adjust cell concentration in sterile phosphate-buffered saline and plate serial dilutions on agar to confirm bacterial load. For subcutaneous challenge, the target CFU in 10 μL / mouse is 1x10 6 and bacteria were collected in a 500 μL Hamilton syringe with a 26 1 / 2 gauge needle. For IP challenge on day 35, the target CFU in 100 μL / mouse was 1x10 7 and bacteria were collected with a 1 mL syringe fitted with a 26 1 / 2 gauge needle. Mice were anesthetized with isoflurane inhalation and 200 μL of M1 89155 bacteria were injected intraperitoneally. Mice were allowed to recover from anesthesia in normal air. Mice were followed for survival over a week, with checks multiple times per day.

[0140] Subcutaneous challenge and lesion collection: For subcutaneous challenge on day 35, mice are anesthetized by isoflurane inhalation and 10 μL of GAS bacteria are injected into the shaved back using a repeat dispenser for Hamilton syringes. Lesion size is tracked daily for 3 days by photographing mice under isoflurane anesthesia alongside a ruler. Prior to lesion collection on day 3, a 2 mL sterile screw-cap tube containing 1.0 mm silica beads and 1 mL of PBS was prepared for each skin lesion. The weight of each tube was recorded. On day 3, when the lesions were fully developed, mice were euthanized with CO2 and cervical dislocation. Each skin lesion was excised using clean surgical instruments and placed into a pre-weighed tube. The weight of the tube was recorded for calculation of tissue volume. The tube was placed in a MagnaLyser bead beater and spun at 6000 rpm for 60 seconds. Samples were serially diluted and the lysate was placed on agar plates to quantify bacterial load.

[0141] Two parallel studies were performed: in one group, mice were bled throughout the experiment to test antigen-specific antibody titers after vaccination, and in the other group, mice were not bled throughout the experiment, and both groups of animals were challenged identically at the end to perform lesion size and CFU / mg analysis.

[0142] Biological Example 2: Evaluating the immunogenicity of polypeptide antigens or non-GAS carrier polypeptides, and their polypeptide-polysaccharide conjugates in a mouse model Polypeptide antigens of synthetic Examples 2 and 3 or non-GAS carrier polypeptides, and their polysaccharide conjugates, were evaluated in a mouse model by the methods described above in Biological Example 1. Mice were immunized on days 0, 7 and 14, followed by terminal bleeding after sacrifice on day 21.

[0143] Mice are immunized with 5 μg of each polysaccharide conjugate. Antibody titers against the polypeptide and polysaccharide were measured after the final bleed. Long-chain polysaccharides prepared according to the method of Example 1 can be used as coating antigens in the ELISA analysis of these immunogenicity experiments. Figure 11A shows the ELISA after 5 μg of each of the polysaccharide conjugates of pAMF-substituted SLO(ΔC101)-var1, SLO(ΔC101)-var5, SLO(ΔC101)-var6 and SLO(ΔC101)-var10 (corresponding to SEQ ID NOs: 55, 59, 60 and 64) were used to immunize mice. Although these polypeptide-polysaccharide conjugates used short-chain polysaccharides, the first plot of Figure 11A shows the antibody titers measured after the final bleed when the ELISA utilized long-chain polysaccharides (prepared by the method of Example 1) as the coating antigen. The second plot of Figure 11A shows the antibody titers in the ELISA utilizing the polypeptide coating antigen.

[0144] The above experiment was repeated in a different cohort of mice, and as shown in Figure 11B, antibody titers against SLO (>10 6) were recorded when antisera from the protein alone or conjugate groups were analyzed. This analysis confirmed that the careful selection of pAMF-introduced residues for site-selective conjugation did not destroy important immunogenic epitopes of SLO as a carrier protein. Finally, GAC PR Strong antibody responses (>10 6 ) was recorded ( FIG. 11B ). Overall, this indicates that CFPS provides an effective platform for expressing and purifying pathogen-specific protein antigens, including nnAAs, which can be successfully utilized to create immunogenic conjugates.

[0145] Biological Example 3: Stability of Polysaccharide and Polypeptide-Polysaccharide Conjugates Experiments were performed to measure the stability of the polysaccharide and its conjugates of Example 1. The polysaccharide or polypeptide-polysaccharide conjugates are kept for at least 6 months at -20°C, 5°C and 25°C. Samples are taken after 6 months, 12 months and 24 months and each sample is analyzed for pH and molecular weight to determine stability.

[0146] Biological Example 4: Comparison of SLO variant conjugates and combined administration of SLO variant conjugates and eCRM197-polysaccharide conjugates in an M1 GAS challenge Mice (N = 10 per group) were treated with mock, SLO(ΔC101)var1-GAC PR Conjugate or combination vaccine [SLO(ΔC101)var1 + eCRM-GAC PR Wild-type female CD-1 mice (Charles River) were immunized three times every 14 days starting at 5 weeks of age. Intramuscular immunizations were performed in a total volume of 100 μl / mouse per dose with 50 μl Alhydrogel; 2% aluminum hydroxide adjuvant (Invivogen) prepared according to the manufacturer's instructions. 14 days after the final immunization, mice were immunized with 1x10 7CFU M1 89155GAS was injected ip and survival was followed. Kaplan-Meier survival curve statistics were calculated using the log-rank Mantel-Cox test. As shown in Figure 12, unlike the combined vaccine group, SLO(ΔC101)var1-GAC PR Immunization with GAC alone resulted in a significantly higher protection rate (p=0.012) compared to the mock group. This indicates that, unlike the conventional method of preparing conjugates using unrelated carrier proteins, GAC PR We show that site-specific conjugation of CFPS-generated GAS-specific protein antigens confers superior protection in vivo.

[0147] Biological Example 5: Evaluation of the immunogenicity of SpyAD-PS conjugates using an immunogenic composition in NZW rabbits Test materials (shown in Table 5) were prepared containing varying amounts of SpyAD-PS conjugate in combination with equal amounts of the polypeptide antigens C5a (SEQ ID NO:30) and SLO (SEQ ID NO:50). Conjugates CNJ-AE, CNJ-AF and CNJ-AG (see Synthesis Examples 6 and 7) were tested. [Table 31]

[0148] All test substances were in a buffer containing 5 mM sodium succinate (pH 5.8), 150 mM sodium chloride and 0.02% polysorbate 80. Each dose contained 31.2 μg aluminum phosphate. Conjugates were administered intramuscularly to female New Zealand White rabbits at 0.25 mL / side (0.125 mL / both limbs). Dosing occurred on days 0, 21, 42, with blood drawn on days -1, 14, 35 and 56. Ten rabbits were dosed per arm. Titers against each antigen were measured by ELISA using SLO, C5A, SpyAD, SpyAD-GAC and eCRM-GAC as coating antigens).

[0149] For each ELISA experiment, the coating solution containing the antigen of interest was diluted (100 μL in total) with sterile filtered PBS (pH 7.4 ± 0.2) and added to each well at 0.5 μg / mL. The plate was placed in a sealed container and kept at 2°C to 8°C for 16 to 24 hours. The plate was then washed three times with plate washing buffer (350 μL / well). The plate was then blocked by adding 200 μL of PBS + 3% bovine serum albumin (BSA) and incubated at room temperature for 60 to 65 minutes. After washing the plate three times with plate washing buffer, five-fold serial dilutions of serum samples (50 μL) from animals administered the test substances were prepared starting from a 1:10 concentration and added to the designated wells. Controls (sample diluent, 1XPBS + 3% BSA; a negative control of commercially available normal serum [Jackson ImmunoResearch]; and a positive control serially diluted at 3- or 5-fold dilutions) were also added to the designated wells. Serum samples of test substances in duplicate and controls in single plates were preferably placed on a single plate and incubated at 35°C-39°C for 60-65 minutes. Plates were washed six times with plate wash buffer, peroxidase-conjugated donkey anti-rabbit IgG (H+L) was added, plates were incubated at room temperature for 60-65 minutes, and then washed six times again. ABTS substrate was added and plates were incubated at room temperature for 30 minutes. Plates were read at 415 nm and 570 nm.

[0150] The results of each ELISA shown in Figures 15, 16, 17, 18 and 19 (C5a, SLO, SpyAD, SpyAD-GAC and eCRM-GAC coating antigens, respectively) show that the high molecular weight SpyAD-GAC conjugate (CNJ-AG) induces stronger titers against both SpyAD and GAC than the low molecular weight conjugates (CNJ-AE and CNJ-AF). In some cases, the high molecular weight conjugate induces 3-30 times higher titers than the low molecular weight conjugate (CNJ-AE) at the same dose on day 35 (e.g., 0.062 μg and 0.25 μg; group 6 vs. group 2).

[0151] The stability of the conjugates is measured in various buffers to assess changes in mass recovery, conjugate molecular weight, and particle formation upon storage at 25° C. or multiple freeze-thaw cycles, including: (i) 20 mM potassium phosphate pH 7.4, 10% sorbitol; (ii) 20 mM potassium phosphate pH 7.4, 10% sorbitol, 10 mM sodium chloride; (iii) 20 mM potassium phosphate pH 7.4, 10% sorbitol, 10 mM sodium chloride, 0.02% polysorbate 80; and (iv) 20 mM tris(hydroxymethyl)aminomethane pH 8.0, 10% sorbitol, 50 mM sodium chloride.

[0152] In vivo immunogenicity studies will also be performed, as described above, with conjugates of different molecular weights, at one or two doses, before and after being subjected to accelerated stability conditions (e.g., temperatures higher than those likely to be used in the manufactured vaccine product) or freeze / thaw cycles. For example, conjugates will be below 250 kDa, between 250-400 kDa, and / or above 400 kDa. These experiments will further evaluate the effect of conjugation conditions that affect yield and immunogenicity, evaluate what changes in immunogenicity occur when the conjugates described herein are subjected to freeze-thaw cycles, and evaluate both of these variables at one or multiple dose levels.

[0153] Biological Example 6: Human Phase I and Phase IIA Clinical Trial Design The initial Phase I clinical trial will be a randomized, placebo-controlled, ascending dose study in healthy adults aged 18-29 years (N=96) (Table 3). The objectives of this initial clinical trial will be safety, dose-responsiveness, and immunogenicity (IgG antibodies). As many in this age group have pre-existing exposure and immunity to GAS, baseline immunity will be adequately assessed to understand the impact of pre-existing immunity on vaccine response. IgG responses to each vaccine component and OPK antibody titers to a diverse panel of contemporary GAS isolates with different M serotypes will be evaluated. [Table 32]

[0154] The Phase 2A clinical trial is a randomized, placebo-controlled, multicenter study evaluating the vaccine in consecutive cohorts of children aged 10 to 17 years, then 5 to 9 years (N=96) (Table 4). The objectives of the study are to evaluate safety, immunogenicity (serum IgG antibody responses and opsonophagocytic activity) and preliminary efficacy (incidence of GAS pharyngitis). Each patient was monitored for 12 months to determine the incidence of streptococcal pharyngitis in the treatment groups. [Table 33]

Claims

1. (a) a Group A Streptococcus (GAS) polypeptide antigen or a non-GAS carrier polypeptide that includes at least one unnatural amino acid (nnAA); and (b) a purified cell wall polysaccharide or peptidoglycan-bound capsular polysaccharide having a molecular weight of at least about 10 kDa to at least about 40 kDa; 2. A polypeptide-polysaccharide conjugate comprising:

2. 2. The polypeptide-polysaccharide conjugate of claim 1, wherein the purified cell wall polysaccharide lacks immunodominant N-acetylglucosamine (GlcNAc) side chains.

3. 3. The polypeptide-polysaccharide conjugate of claim 1 or 2, wherein the cell wall polysaccharide is a GAS polysaccharide or a variant thereof, or a Group A carbohydrate (GAC).

4. 4. The polypeptide-polysaccharide conjugate of claim 3, wherein about 8 mol % to about 20 mol %, about 10 mol % to about 20 mol %, or about 10 mol % to about 18 mol % of the polysaccharide repeating units of the GAS polysaccharide or variant thereof are derivatized with a linker.

5. 5. The polypeptide-polysaccharide conjugate of any one of claims 1 to 4, wherein at least one nnAA is substituted with lysine, leucine, isoleucine or arginine in the GAS polypeptide antigen or non-GAS carrier polypeptide.

6. A polypeptide-polysaccharide conjugate described in any one of claims 1 to 5, wherein a purified cell wall polysaccharide or peptidoglycan-bound capsular polysaccharide is bound to a triazole group formed by a click chemistry reaction with an nnAA, the nnAA being pAMF.

7. The polypeptide-polysaccharide conjugate of any one of claims 1 to 6, wherein the GAS polypeptide antigen is selected from the group consisting of C5a peptidase, streptolysin O (SLO), SpyAD, Sib35 and Sfb1.

8. The polypeptide-polysaccharide conjugate of any one of claims 1 to 7, wherein the GAS polypeptide antigen is a SpyAD polypeptide or a fragment thereof.

9. 9. The polypeptide-polysaccharide conjugate of claim 8, wherein the SpyAD polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:33 or an amino acid sequence that is a fragment of SEQ ID NO:

33.

10. The polypeptide-polysaccharide conjugate of claim 8, wherein the SpyAD polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:

83.

11. 10. The polypeptide-polysaccharide conjugate of claim 8 or 9, wherein the SpyAD polypeptide comprises nnAA substitutions at positions K64, K287, K386 and K657 with reference to SEQ ID NO:

33.

12. The polypeptide-polysaccharide conjugate according to any one of claims 8 to 11, wherein the SpyAD polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 78 or a sequence which is at least 95% identical thereto.

13. (a) Group A Streptococcus (GAS) C5a peptidase polypeptide antigen; (b) a GAS streptolysin O (SLO) polypeptide antigen; and (c) a polypeptide-polysaccharide conjugate comprising a Streptococcus pyogenes adhesion / disruption (SpyAD) conjugate polypeptide or a fragment thereof comprising at least one unnatural amino acid (nnAA), said at least one nnAA comprising a click chemistry reactive group, and a GAS polysaccharide or a variant thereof lacking an immunodominant N-acetylglucosamine (GlcNAc) side chain; 20. An immunogenic composition comprising:

14. The immunogenic composition of claim 13, wherein the C5a peptidase polypeptide antigen comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:29 or SEQ ID NO:30, or an amino acid sequence that is at least 95% identical thereto.

15. The immunogenic composition of claim 13 or 14, wherein the SLO polypeptide antigen comprises an amino acid sequence as set forth in SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 or SEQ ID NO:53, or a sequence at least 95% identical thereto.

16. An immunogenic composition described in any one of claims 13 to 15, wherein the conjugated polysaccharide is bound to a triazole group formed by a click chemistry reaction with an nnAA, and the nnAA is pAMF.

17. The immunogenic composition of any one of claims 13 to 16, wherein the SpyAD conjugate polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 33 or an amino acid sequence that is a fragment of SEQ ID NO:

33.

18. The immunogenic composition of any one of claims 13 to 17, wherein the SpyAD conjugate polypeptide comprises pAMF substitutions at positions K64, K287, K386 and K657 of SEQ ID NO:

33.

19. The immunogenic composition of any one of claims 13 to 18, wherein the SpyAD conjugate polypeptide comprises an amino acid sequence as set forth in SEQ ID NO:34, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:82 or SEQ ID NO:83 or an amino acid sequence that is at least 95% identical thereto.

20. 20. The immunogenic composition of any one of claims 13 to 19, wherein the C5a peptidase polypeptide antigen comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:29, or SEQ ID NO:30; the SLO polypeptide antigen comprises an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53; and the SpyAD conjugate polypeptide comprises an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO:34, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:82, or SEQ ID NO:

83.

21. The immunogenic composition of any one of claims 13 to 20, wherein about 8 mol % to about 20 mol % of the polysaccharide repeating units of the GAS polysaccharide or variant thereof are derivatized with a linker.

22. The linker before reacting with the click chemistry reactive group of the nnAA has the formula I: 【Chemistry 1】 where X is at least one polysaccharide repeat unit of a GAS polysaccharide or a fragment thereof and n is at least 1. The immunogenic composition according to any one of claims 13 to 21, comprising the structure:

23. The SpyAD conjugate polypeptide or fragment thereof has the formula II: 【Chemistry 2】 (In the formula, R 1 is H, formyl, or at least one amino acid of a SpyAD conjugate polypeptide; R 2 is OH or at least one amino acid of the SpyAD conjugate polypeptide; W is CH or N; y is at least 1; n is at least 1; and X is at least one polysaccharide repeat unit of a GAS polysaccharide or a variant thereof.

23. The immunogenic composition according to any one of claims 13 to 22, wherein the GAS polysaccharide is conjugated according to

24. The immunogenic composition according to any one of claims 13 to 23, wherein the GAS polysaccharide or variant thereof has a molecular weight of at least about 10 kDa to at least about 40 kDa.

25. 25. The immunogenic composition of any one of claims 13 to 24, further comprising less than about 60% to less than about 10% free GAS polysaccharide or a variant thereof.

26. A pharmaceutical composition for inducing a protective immune response against GAS bacteria in a subject, the pharmaceutical composition comprising an immunogenic composition according to any one of claims 13 to 25.

27. 1. A method for purifying cell wall polysaccharides or peptidoglycan-bound capsular polysaccharides from bacterial cells, the method comprising the steps of: (a) hydrolyzing bacterial cells in a solution containing a base and a reducing agent to form a lysate solution containing polysaccharides; and (b) incubating the polysaccharide-containing degradation solution with a wall-degrading enzyme to form a free polysaccharide solution.

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