GROUP A STREP IMMUNOGENIC COMPOSITIONS WITH POLYSACCHARIDE-PROTEIN CONJUGATES

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The sequence listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the sequence listing is STRO_011_04WO_ST25.txt. The text file is 321 kb, was created on Feb. 12, 2020, and is being submitted electronically via EFS-Web.

FIELD

The present application relates to the fields of microbiology and vaccine development.

BACKGROUND

Group A Streptococcus (GAS) is a preeminent human pathogen causing 700 million cases of pharyngitis (‘strep throat’) annually worldwide and increasing cases of severe invasive infections, sepsis, necrotizing fasciitis, otitis media, and toxic shock syndrome. Pharyngitis is highly prevalent in school-age children and a major source of antibiotic prescriptions worldwide; driving selective pressure for resistance throughout the human microflora. GAS is also responsible for post-infectious immune-mediated rheumatic heart disease (RHD), a leading cause of mortality in the developing world. Some 30 million people are currently affected by RHD, with over 300,000 deaths annually (60% < age 70) and 11.5 million disability-adjusted life years lost.

BRIEF SUMMARY

In some embodiments, the present disclosure provides an immunogenic composition comprising a first Group A Streptococcus (GAS) polypeptide antigen; a second GAS polypeptide antigen; and at least one polypeptide-polysaccharide conjugate, wherein the conjugate polypeptide is a third GAS polypeptide antigen or a non-GAS carrier polypeptide and comprises at least one non-natural amino acid (nnAA), and wherein the conjugate polysaccharide is a GAS polysaccharide or a variant thereof that lacks an immunodominant N-acetyl Glucosamine (GlcNAc) side chain.

In some embodiments, at least one of the GAS polypeptide antigens is a full length GAS antigen. In some embodiments, at least one of the GAS polypeptide antigens is a peptide fragment of a full length GAS antigen. In some embodiments, the first and second GAS polypeptide antigens are independently selected from C5a peptidase, streptolysin O (SLO), Sib35, and Sfb1. In some embodiments, the first and second GAS polypeptide antigens are C5a peptidase and SLO.

In some embodiments, the C5a peptidase polypeptide antigen comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, the C5a peptidase polypeptide antigen comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, the C5a peptidase polypeptide antigen comprises the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 30.

In some embodiments, the SLO polypeptide antigen comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 5 or SEQ ID NO: 7. In some embodiments, the SLO polypeptide antigen comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 7. In some embodiments, the SLO polypeptide 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. In some embodiments, 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. In some embodiments, the SLO polypeptide antigen comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 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. In some embodiments, the SLO polypeptide antigen 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.

In some embodiments, the non-GAS carrier polypeptide is ferritin. In some embodiments, the third GAS polypeptide antigen or non-GAS carrier polypeptide is selected from Streptococcus pyogenes Adhesion and Division (SpyAD) polypeptide, arginine deiminase (ADI), SEQ ID NO: 25, and Protein D. In some embodiments, the third GAS polypeptide antigen is SLO.

In some embodiments, the at least one nnAA is substituted for a lysine, a leucine, or an isoleucine in the conjugate polypeptide. In some embodiments, the at least one nnAA is substituted for a lysine, a leucine, an arginine, or an isoleucine in the conjugate polypeptide. In some embodiments, the nnAA comprises a click chemistry reactive group. In some embodiments, the 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. In some embodiments, the nnAA is pAMF.

In some embodiments, the conjugate polypeptide is a third GAS protein, which is a SpyAD polypeptide and comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 9. In some embodiments, the SpyAD polypeptide comprises a pAMF substitution at positions K64, K287, K386, and K657 of SEQ ID NO: 9. In some embodiments, the SpyAD polypeptide comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 11. In some embodiments, the conjugate polypeptide is a third GAS protein, which is a SpyAD polypeptide and comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 33. In some embodiments, the SpyAD polypeptide comprises a pAMF substitution at positions K64, K287, K386, and K657 of SEQ ID NO: 33. In some embodiments, the SpyAD polypeptide comprises the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 34.

In some embodiments, the conjugate polypeptide is a third GAS polypeptide, which is an ADI polypeptide and comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 13 or SEQ ID NO: 15. In some embodiments, the ADI polypeptide comprises a pAMF substitution at positions K15, K193, and K316 of SEQ ID NO: 13 or SEQ ID NO: 15. In some embodiments, the ADI polypeptide comprises the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 19. In some embodiments, the conjugate polypeptide is a third GAS polypeptide, which is an ADI polypeptide and comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 35 or SEQ ID NO: 36. In some embodiments, the ADI polypeptide comprises a pAMF substitution at positions K15, K193, and K316 of SEQ ID NO: 35 or SEQ ID NO: 36. In some embodiments, the ADI polypeptide comprises the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36.

In some embodiments, the conjugate polypeptide is a ferritin polypeptide and comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 21. In some embodiments, the ferritin polypeptide comprises a pAMF substitution at position I5 of SEQ ID NO: 21. In some embodiments, the ferritin polypeptide comprises the amino acid sequence of SEQ ID NO: 23.

In some embodiments, the conjugate polysaccharide comprises a polyrhamnose core. In some embodiments, the conjugate polysaccharide has an average molecular weight of about 5 kDa to about 7 kDa. In some embodiments, the conjugate polysaccharide is a tetramer, a hexamer, an octamer, or a decamer polysaccharide. In some embodiments, the conjugate polysaccharide has an average molecular weight of about 10 kDa to 40 kDa.

In some embodiments, the present disclosure provides 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 comprising a Streptococcus pyogenes Adhesion and Division (SpyAD) conjugate polypeptide and a GAS conjugate polysaccharide or a variant thereof that lacks an immunodominant N-acetyl Glucosamine (GlcNAc) side chain. In some embodiments, the SpyAD protein comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 9 and comprises a pAMF substitution at positions K64, K287, K386, and K657. In some embodiments, the SpyAD protein comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 11. In some embodiments, the SpyAD protein comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 33 and comprises a pAMF substitution at positions K64, K287, K386, and K657. In some embodiments, the SpyAD protein comprises the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 34. In some embodiments, the SpyAD protein comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 34

In some embodiments, the present disclosure provides 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 comprising an arginine deiminase (ADI) conjugate polypeptide and a GAS conjugate polysaccharide or a variant thereof that lacks an immunodominant N-acetyl Glucosamine (GlcNAc) side chain. In some embodiments, the ADI polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 13 or SEQ ID NO: 15 and comprises a pAMF substitution at positions K15, K193, and K316. In some embodiments, the ADI polypeptide comprises the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 19. In some embodiments, the ADI polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 35 or SEQ ID NO: 36 and comprises a pAMF substitution at positions K15, K193, and K316. In some embodiments, the ADI polypeptide comprises the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 38

In some embodiments, the composition comprises about 10% to about 50% of the first GAS antigen, about 10% to about 50% of the second GAS antigen, and about 10% to about 50% of the polypeptide-polysaccharide conjugate. In some embodiments, the composition further comprises one or more adjuvants selected from alum, saponin, monophosphoryl lipid A (MPL), or combinations thereof. In some embodiments, the composition comprises Alhydogel.

In some embodiments, the present disclosure provides a method of inducing a protective immune response against a Group A Streptococcus (GAS) bacterium in a subject comprising administering an immunogenic composition described herein to the subject.

In some embodiments, the subject is 18 years or older. In some embodiments, the subject is less than 18 years old. In some embodiments, the subject is between 5 years and 17 years old, between 6 months and 9 years old, or between 5 years and 9 years old.

In some embodiments, the GAS bacterium is of a serotype selected from M1, M2, M3, M4, M6, M11, M12, M22, M28, M75, and M89. In some embodiments, the GAS bacterium is a serotype selected from M1, M3, M5, M9, M12, M18, M22, M25, M28, M71, M72, and M74. In some embodiments, the GAS bacterium is a serotype selected from M1, M4, M6, M11, M12, M22, M44, M75, M77, M77, and M81. In some embodiments, the GAS bacterium is a serotype selected from M1, M2, M3, M4, M6, M9, M12, M18, M22, M75, M77, M89, and M92. In some embodiments, the GAS bacterium is a serotype selected from M1, M2, M3, M4, M5, M6, M9, M11, M12, M13, M28, M62, and M89. In some embodiments, the GAS bacterium is a serotype selected from M1, M2, M3, M4, M6, M12, M22, M28, M49, M53, M68, M77, M80, M83, M87, M89, and M92.

In some embodiments, the immunogenic composition induces an antibody response in the subject against the Group A Streptococcus (GAS) bacterium and does not induce an antibody response in the subject against human tissue. In some embodiments, the immunogenic composition induces a protective immune response against a Shigella bacterium in the subject, wherein the Shigella bacterium comprises a polysaccharide with a polyrhmanose backbone.

In some embodiments, the present disclosure provides a method of inducing a protective immune response against a Shigella bacterium in a subject comprising administering an immunogenic composition described herein to the subject. In some embodiments, the present disclosure provides a use of an immunogenic composition described herein for inducing a protective immune response against a Shigella bacterium in a subject. In some embodiments, the present disclosure provides a use of an immunogenic composition described herein in the manufacture of a medicament for inducing a protective immune response against a Shigella bacterium in a subject. In some embodiments, the subject is 18 years or older. In some embodiments, the subject is less than 18 years old. In some embodiments, the subject is between 5 years and 17 years old, between 6 months and 9 years old, or between 5 years and 9 years old.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B show the expression levels of native polypeptide antigens and non-natural amino acids containing polypeptide antigens used for making conjugates with GAS polysaccharide .

FIG. 2 shows polypeptide antigen- and polypeptide-polysaccharide conjugate-immunized sera titers compared to a pre-bleed control.

FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, and FIG. 3E show antibody titers and the IgG binding change of immune serum compared to a pre-immune serum control.

FIG. 3F shows rabbit IgG binding to SpyAD knockout GAS, confirming binding to native GAC.

FIG. 4 shows the increased killing capacity of immune serum vs. a pre-immune serum control.

FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, and FIG. 5F show the results of active immunization experiments, including antibody titers, lesion size, and bacterial burden, and percent survival.

FIG. 6A and FIG. 6B show percent survival data following passive immunization of mice subjected to an IP challenge.

FIG. 7 shows lesion size and bacterial burden data following passive immunization of mice subjected to a subdermal challenge.

FIG. 8 shows an ELISA titer analysis of immune serum against S. flexneri 2a OPS.

FIG. 9 shows the general reaction scheme for derivatization of GAC with DBCO-PEG₄-NH₂ and subsequent conjugation to pAMF-modified SpyAD.

FIG. 10 shows SEC MALS analysis of purified native GAC, purified native SpyAD[4pAMF], and purified polypeptide-polysaccharide conjugates thereof.

FIG. 11 shows a Western blot analysis assessing antigen-specific antisera for cross-reactivity to human heart lysate.

FIG. 12 is a graphical representation of fragment sequences for select antigens.

FIG. 13 shows comparative expression data of select antigens.

FIG. 14 shows expression data of select single and double SLO mutants compared to GFP.

FIG. 15 is a graphical representation of truncated SLO sequences compared to the non-truncated SLO sequence.

FIG. 16 shows expression levels (total and soluble) for truncated SLO variants.

FIG. 17 shows purity of captured peptide fragments by HisTrap column chromatography.

FIG. 18 shows purity of select expressed peptides after TEV cleavage and HisTrap purification.

FIG. 19 shows the results of purification of expressed peptides by size exclusion chromatography.

FIG. 20 shows the in vivo assessment of immunogenicity of select SLO fragments in rabbits.

FIG. 21 shows the use of serum from mock and peptide combination-immunized mice to opsonize GAS in an OPK assay.

FIG. 22A, FIG. 22B, and FIG. 22C show the results of an intradermal challenge (with M1 89155 GAS) of wild-type CD-1 mice immunized with mock or a peptide combination, including lesion size and GAS burden data.

FIG. 23 shows the results of a red blood cell hemolysis assay to assess the presence of functional antibody against SLO variants.

FIG. 24A, FIG. 24B, and FIG. 24C show the opsonophagocytic killing of GAS of different human neutrophil serotypes in the presence of immune serum from immunized rabbits, the blocking of lysis by antisera, and the oxidative burst capacity of human neutrophils exposed to GAS supernatants containing SLO.

FIG. 25A and FIG. 25B shows differential IgG binding of immune sera to additional GAS serotype surface antigens.

FIG. 26 shows expression of 10 pAMF-containing SLO polypeptide variants.

FIGS. 27A and 27B shows SDS-PAGE analysis for SLO variants during two steps of purification.

FIG. 28 shows conditions for the conjugation of SLO variants to DBCO-GAC.

FIG. 29 shows SDS-PAGE analysis of 3- and 4-pAMF variant SLO variant conjugates.

FIG. 30 shows SEC-MALS data for 3- and 4-pAMF variant SLO variant conjugates.

FIG. 31A and FIG. 31B show the antibody titers of mice post-immunization using conjugates made with select SLO(ΔC101) variants as carrier proteins, and the results of a hemolysis assay using anti-sera generated from mice vaccinated with conjugates made with those same variants. FIG. 31C shows the antibody titers of mice post-immunization using conjugates made with select SLO(ΔC101) variants, and using long polysaccharide as the coating antigen in the ELISA.

FIG. 32 shows SEC-MALS data for a conjugate of SpyAD[4pAMF] and long polysaccharide.

FIG. 33A and FIG. 33B show the expression levels of 22 SLO variants with 3-8 pAMF, and the corresponding gels for purified 5-8 pAMF containing variants.

FIG. 34 shows SDS-PAGE analysis of 5- and 6-pAMF variant SLO conjugates with long GAC.

FIG. 35 shows SEC-MALS data for 5- and 6-pAMF variant SLO conjugates with long GAC.

FIG. 36A, FIG. 36B, and FIG. 36C show NMR analysis of purified polysaccharide originating from a GAS bacterial strain expressing PS lacking GlcNAc.

FIG. 37 shows a western blot demonstrating M-protein is removed from polysaccharide when purified as described in Example 19.

DETAILED DESCRIPTION
Overview

Despite high global demand, there is no safe and efficacious commercial vaccine against GAS. Features of the pathogen pose particular challenges to vaccination, including its invariant capsule of hyaluronic acid, an immunologically inert carbohydrate ubiquitous in connective tissues. Furthermore, the immunodominant surface-anchored GAS M proteins are highly polymorphic (>200 emm types), and regions of their dimeric coiled-coil structure may provoke an autoimmune response against cardiac tissue in rheumatic heart disease (RHD). The World Health Organization, International Vaccine Institute, Wellcome Trust, Gates Foundation and others have recently convened in advisories on GAS vaccine development to address these significant scientific challenges and meet this paramount global health need.

Group A Streptococcus

Group A streptococcus (GAS) bacteria is a Gram positive, beta-hemolytic coccus in chains. It is responsible for a range of diseases in humans. These diseases include strep throat (acute pharyngitis), otitis media, and skin and soft tissue infections such impetigo and cellulitis. These can also include rare cases of invasive (serious) illnesses such as necrotizing fasciitis (flesh eating disease) and toxic shock syndrome (TSS). Several virulence factors contribute to the pathogenesis of GAS, such as M protein, hemolysins, and extracellular enzymes.

GAS vaccinology has been focused largely on the cell wall-anchored M protein. Historical studies administered crude whole M protein preparations to human volunteers, which was followed by challenge with live GAS bacteria in the pharynx. While these preparations decreased GAS colonization, an increased incidence of acute rheumatic fever (ARF) was reported for some of the immunized subjects. Two N-terminal M protein vaccine preparations have reached human clinical trials to date: a hexavalent preparation (Dale JB. 1999. Vaccine, 17:193-200; Kotloff et al, 2004. JAMA 292:709-715; Hall et al., 2004. Infect. Immun. 72:2507-2512) and a 26-valent preparation (Hu et al., 2002. Infect. Immun. 70:2171-2177; McNeil et al., 2005. Clin. Infect. Dis. 41:1114-1122). These preparations were well tolerated in adult volunteers. More recently, a similar multivalent vaccine was developed, which includes N-terminal peptides representing 30 GAS serotypes (Dale et al., 2011. Vaccine 29:8175-8178.). Sera raised against this experimental vaccine exhibited bactericidal activity against all 30 vaccine serotypes as well as 24 (of 40 tested) nonvaccine serotypes. While such preparations have been shown to have the capacity to protect against select nonvaccine serotypes, they do not protect against all globally circulating serotypes (Abdissa et al., 2006. Clin. Infect. Dis. 42:1362-1367; Ikebe et al., Working Group for Beta-Haemolytic Streptococci in Japan. 2007. 2001-2005. Epidemiol. Infect. 135:1227-1229; Nir-Paz et al., 2010. Epidemiol. Infect. 138:53-60; Steer et al., 2009. Lancet Infect. Dis. 9:611-616; O′Loughlin et al., 2007. Clin. Infect. Dis. 45:853-862). The 30 serotypes included are based on those prevalent in North America and Europe (Dale JB et al., 2011. Vaccine 29:8175-8178.), which often differ from the diverse serotypes isolated in regions of endemicity. The use of GAS proteins and Protein-GAC conjugates as vaccines has been recently reported (Di Benedetto, R. et al., 2020. J. Mol. Sci. no 22: 8558), although these use non-specific conjugation methods that can disrupt both the B/T-cell epitopes on the carrier protein and the PS backbone. Furthermore, these conjugates were also made with thenative GAC, which harbors potentially cross-reactive GlcNAc immunodominant epitope.

Thus, there is a need in the art for improved vaccines against GAS.

Combination Vaccines

In some embodiments, the present disclosure provides an immunogenic composition comprising two or more GAS polypeptide antigens and a polypeptide-polysaccharide conjugate. For clarity, in compositions described herein, each polypeptide is different. For example, in compositions comprising a first, a second, and a third polypeptide antigen or polypeptide-polysaccharide conjugate, the first, second, and third polypeptides have different sequences.

GAS Antigens

In some embodiments, the immunogenic compositions of the present disclosure comprise two or more GAS polypeptide antigens. In some embodiments, the GAS polypeptide antigens are selected from C5a peptidase, streptolysin O (SLO), streptococcal immunoglobulin-binding protein 35 (Sib35), and Fibronectin binding protein F1 (Sfb1). In some embodiments, the GAS polypeptide antigens are selected from C5a peptidase, streptolysin O (SLO), streptococcal immunoglobulin-binding protein 35 (Sib35), Fibronectin binding protein F1 (Sfb1), and Adhesion and Division polypeptide (SpyAD). UniProt Reference Identifiers for each of these GAS antigens are provided in Table 1 below.

TABLE 1

UniProt References for exemplary GAS antigens

Antigen
UniProt Ref

C5a
P15926

SLO
P0C0I3

Sib35
Q1XG74

Sfb1
Q48VN7

In some embodiments, the GAS polypeptide antigens comprise the full length polypeptide antigen sequence. In some embodiments, the GAS polypeptide antigens comprise a fragment of the full length polypeptide antigen sequence. For example, in some embodiments, the immunogenic composition comprises a C5a GAS polypeptide antigen, wherein the C5a polypeptide is a fragment of the full length C5a polypeptide. In some embodiments, the C5a fragment comprises amino acids 90-1035 of the full length protein (e.g., comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 29, or SEQ ID NO: 30). In some embodiments, the immunogenic composition comprises an SLO GAS polypeptide antigen, wherein the SLO polypeptide is a fragment of the full length SLO polypeptide. In some embodiments, the SLO fragment comprises amino acids 79-571 of the full length protein (e.g., comprises SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 31, or SEQ ID NO: 32).

In some embodiments, the GAS polypeptide antigens comprise one or more amino acid mutations in the wild-type amino acid sequence. For example, in some embodiments, the immunogenic composition comprises a C5a GAS polypeptide antigen, wherein the C5a polypeptide comprises one or more amino acid mutations. In some embodiments, the amino acid mutations are D131A and S513A (e.g., SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 30). In some embodiments, the immunogenic composition comprises an SLO GAS polypeptide antigen, wherein the SLO polypeptide comprises one or more amino acid mutations. In some embodiments, the amino acid mutation is W535A (e.g., SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 32).

Amino acid sequences of exemplary GAS antigen are provided below in Table 2. Mutated amino acids are indicated in bolded text. In some embodiments, the underlined and italicized amino acids are cleaved from the mature GAS antigens.

TABLE 2

Exemplary GAS Antigens

Antigen
Amino Acid Sequence
SEQ ID:

SLO [79-571 fragment] WT w/ His tag and TEV sequence

MHHHHHHGSGENLYFQGAPKEMPLESAEKEEKKSEDKKKSEEDHTE EINDKIYSLNYNELEVLAKNGETIENFVPKEGVKKADKFIVIERKK KNINTTPVDISIIDSVTDRTYPAALQLANKGFTENKPDAVVTKRNP QKIHIDLPGMGDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNT LPARTQYTESMVYSKSQIEAALNVNSKILDGTLGIDFKSISKGEKK VMIAAYKQIFYTVSANLPNNPADVFDKSVTFKELQRKGVSNEAPPL FVSNVAYGRTVFVKLETSSKSNDVEAAFSAALKGTDVKTNGKYSDI LENSSFTAVVLGGDAAEHNKVVTKDFDVIRNVIKDNATFSRKNPAY PISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGKINLSHQGAYVA QYEILWDEINYDDKGKEVITKRRWDNNWYSKTSPFSTVIPLGANSR NIRIMARECTGLAWEWWRKVIDERDVKLSKEINVNISGSTLSPYGS ITYK
5

SLO [79-571 fragment] W535A w/ leader

MHHHHHHGSGENLYFQGAPKEMPLESAEKEEKKSEDKKKSEEDHTE EINDKIYSLNYNELEVLAKNGETIENFVPKEGVKKADKFIVIERKK KNINTTPVDISIIDSVTDRTYPAALQLANKGFTENKPDAVVTKRNP QKIHIDLPGMGDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNT LPARTQYTESMVYSKSQIEAALNVNSKILDGTLGIDFKSISKGEKK VMIAAYKQIFYTVSANLPNNPADVFDKSVTFKELQRKGVSNEAPPL FVSNVAYGRTVFVKLETSSKSNDVEAAFSAALKGTDVKTNGKYSDI LENSSFTAVVLGGDAAEHNKVVTKDFDVIRNVIKDNATFSRKNPAY PISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGKINLSHQGAYVA QYEILWDEINYDDKGKEVITKRRWDNNWYSKTSPFSTVIPLGANSR
7

NIRIMARECTGLAAEWWRKVIDERDVKLSKEINVNISGSTLSPYGS ITYK

SLO(ΔC101) fragment W535A w/o leader, with G overhang from cleavage
GAPKEMPLESAEKEEKKSEDKKKSEEDHTEEINDKIYSLNYN ELEVLAKNGETIENFVPKEGVKKADKFIVIERKKKNINTTPV DISIIDSVTDRTYPAALQLANKGFTENKPDAVVTKRNPQKIH IDLPGMGDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNT LPARTQYTESMVYSKSQIEAALNVNSKILDGTLGIDFKSISK GEKKVMIAAYKQIFYTVSANLPNNPADVFDKSVTFKELQRKG VSNEAPPLFVSNVAYGRTVFVKLETSSKSNDVEAAFSAALKG TDVKTNGKYSDILENSSFTAWLGGDAAEHNKWTKDFDVIR NVIKDNATFSRKNPAYPISYTSVFLKNNKIAGVNNRTEYVET TSTEYTSGKINLSHQ
53

C5a [90-1035 fragment] WT w/ leader

MHHHHHHGSGENLYFQGKTADTPVTSKATIRDLNDPSQVKTLQEKA GKGAGTVVAVIDAGFDKNHEAWRLTDKTKARYQSKEDLEKAKKEHG ITYGEWVNDKVAYYHDYSKDGKTAVDQEHGTHVSGILSGNAPSETK EPYRLEGAMPEAQLLLMRVEIVNGLADYARNYAQAIRDAVNLGAKV INMSFGNAALAYANLPDETKKAFDYAKSKGVSIVTSAGNDSSFGGK TRLPLADHPDYGVVGTPAAADSTLTVASYSPDKQLTETATVKTADK QDKEMPVLSTNRFEPNKAYDYAYANRGMKEDDFKDVKGKIALIERG DIDFKDKIANAKKAGAVGVLIYDNQDKGFPIELPNVDQMPAAFISR KDGLLLKDNPKKTITFNATPKVLPTASGTKLSRFSSWGLTADGNIK PDIAAPGQDILSSVANNKYAKLSGTSMSAPLVAGIMGLLQKQYETQ YPDMTPSERLDLAKKVLMSSATALYDEDEKAYFSPRQQGAGAVDAK KASAATMYVTDKDNTSSKVHLNNVSDTFEVTVTVHNKSDKPQELYY QATVQTDKVDGKHFALAPKALYETSWQKITIPANSSKQVTVPIDAS RFSKDLLAQMKNGYFLEGFVRFKQDPTKEELMSIPYIGFRGDFGNL SALEKPIYDSKDGSSYYHEANSDAKDQLDGDGLQFYALKNNFTALT TESNPWTIIKAVKEGVENIEDIESSEITETIFAGTFAKQDDDSHYY IHRHANGKPYAAISPNGDGNRDYVQFQGTFLRNAKNLVAEVLDKEG NVVWTSEVTEQVVKNYNNDLASTLGSTRFEKTRWDGKDKDGKVVAN GTYTYRVRYTPISSGAKEQHTDFDVIVDNTTPEVATSATFSTEDRR LTLASKPKTSQPVYRERIAYTYMDEDLPTTEYISPNEDGTFTLPEE AETMEGATVPLKMSDFTYVVEDMAGNITYTPVTKLLEGHSNK
1

C5a [90-1035 fragment] D131A/S513A w/ leader

MHHHHHHGSGENLYFQGKTADTPVTSKATIRDLNDPSQVKTLQEKA GKGAGTVVAVIAAGFDKNHEAWRLTDKTKARYQSKEDLEKAKKEHG ITYGEWVNDKVAYYHDYSKDGKTAVDQEHGTHVSGILSGNAPSETK EPYRLEGAMPEAQLLLMRVEIVNGLADYARNYAQAIRDAVNLGAKV INMSFGNAALAYANLPDETKKAFDYAKSKGVSIVTSAGNDSSFGGK TRLPLADHPDYGVVGTPAAADSTLTVASYSPDKQLTETATVKTADK QDKEMPVLSTNRFEPNKAYDYAYANRGMKEDDFKDVKGKIALIERG DIDFKDKIANAKKAGAVGVLIYDNQDKGFPIELPNVDQMPAAFISR KDGLLLKDNPKKTITFNATPKVLPTASGTKLSRFSSWGLTADGNIK PDIAAPGQDILSSVANNKYAKLSGTAMSAPLVAGIMGLLQKQYETQ YPDMTPSERLDLAKKVLMSSATALYDEDEKAYFSPRQQGAGAVDAK KASAATMYVTDKDNTSSKVHLNNVSDTFEVTVTVHNKSDKPQELYY QATVQTDKVDGKHFALAPKALYETSWQKITIPANSSKQVTVPIDAS RFSKDLLAQMKNGYFLEGFVRFKQDPTKEELMSIPYIGFRGDFGNL SALEKPIYDSKDGSSYYHEANSDAKDQLDGDGLQFYALKNNFTALT TESNPWTIIKAVKEGVENIEDIESSEITETIFAGTFAKQDDDSHYY IHRHANGKPYAAISPNGDGNRDYVQFQGTFLRNAKNLVAEVLDKEG
3

NVVWTSEVTEQVVKNYNNDLASTLGSTRFEKTRWDGKDKDGKVVAN GTYTYRVRYTPISSGAKEQHTDFDVIVDNTTPEVATSATFSTEDRR LTLASKPKTSQPVYRERIAYTYMDEDLPTTEYISPNEDGTFTLPEE AETMEGATVPLKMSDFTYVVEDMAGNITYTPVTKLLEGHSNK

Residue positions in Table 2, above, and elsewhere in this disclosure, refer to the residue numbering according to the residue position in the full length sequences (e.g., as identified in Tables 1 and 3 by reference to the UniProt sequence database) for identifying the fragments and/or lysine residues modified by nnAA (e.g., pAMF replacements of certain lysine residues).

In some embodiments, the immunogenic composition comprises a C5a GAS polypeptide antigen comprising or consisting of an amino acid sequence that is at least 95%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1-4. In some embodiments, the immunogenic composition comprises a C5a GAS polypeptide antigen comprising or consisting of an amino acid sequence that is at least 95%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 29-30. In some embodiments, the immunogenic composition comprises a C5a GAS polypeptide antigen comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 1-4. In some embodiments, the immunogenic composition comprises a C5a GAS polypeptide antigen comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 29-30. In some embodiments, the immunogenic composition comprises an SLO GAS polypeptide antigen comprising or consisting of an amino acid sequence that is at least 95%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 5-8. In some embodiments, the immunogenic composition comprises an SLO GAS polypeptide antigen comprising or consisting of an amino acid sequence that is at least 95%, 95%, 97%, 98%, or 99% identical to any one 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 some embodiments, the immunogenic composition comprises an SLO GAS polypeptide antigen comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 5-8. In some embodiments, the immunogenic composition comprises an SLO GAS polypeptide antigen comprising or consisting of 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 some embodiments, the immunogenic composition comprises a SpyAD GAS polypeptide antigen comprising or consisting of an amino acid sequence that is at least 95%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 10 or 33. In some embodiments, the immunogenic composition comprises a SpyAD GAS polypeptide antigen comprising or consisting of the amino acid sequence of SEQ ID NO: 10 or 33.

Polypeptide-Polysaccharide Conjugates

In some embodiments, immunogenic compositions described herein comprise at least one polypeptide-polysaccharide conjugate. The polypeptide-polysaccharide conjugates comprise a conjugate polypeptide (also referred to as a carrier protein) and a polysaccharide conjugated to the conjugate polypeptide at one or more amino acid positions. In some embodiments, the conjugate polypeptide is selected from SpyAD, ADI, SEQ ID NO: 25, ferritin, and Protein D. UniProt Reference Identifiers for exemplary conjugate polypeptides are provided in Table 3 below.

TABLE 3

UniProt and CAS References for exemplary conjugate polypeptides

Antigen
UniProt (or CAS) Ref

SpyAD
Q9A1H3

ADI
P0C0B3

Protein D
R4R7Q5

ferritin
P02792

CRM197
(CAS: 1272033-67-6)

In some embodiments, the conjugate polypeptides comprise the full length polypeptide antigen sequence. In some embodiments, the conjugate polypeptides comprise a fragment of the full length polypeptide sequence. For example, in some embodiments, the immunogenic composition comprises a polypeptide-polysaccharide conjugate wherein the conjugate polypeptide is SpyAD and is a fragment of the full length SpyAD polypeptide (e.g., SEQ ID NO: 9 or SEQ ID NO: 10). In some embodiments, the SpyAD fragment comprises amino acids 33-849 of the full length protein (e.g., comprises SEQ ID NO: 9 or SEQ ID NO: 10). In some embodiments, the immunogenic composition comprises a polypeptide-polysaccharide conjugate wherein the conjugate polypeptide is ADI and is a fragment of the full length ADI polypeptide (e.g., comprises SEQ ID NO: 13 or SEQ ID NO: 14). In some embodiments, the immunogenic composition comprises a polypeptide-polysaccharide conjugate wherein the conjugate polypeptide is ferritin and is a fragment of the full length ferritin polypeptide (e.g., SEQ ID NO: 21 or SEQ ID NO: 22).

In some embodiments, the conjugate polypeptide comprises one or more amino acid mutations in the wild-type amino acid sequence. For example, in some embodiments, the conjugate polypeptide is SpyAD, wherein the SpyAD polypeptide comprises one or more amino acid mutations. In some embodiments, the conjugate polypeptide is ADI, wherein the ADI polypeptide comprises one or more amino acid mutations. In some embodiments, the amino acid mutation is D277A (e.g., SEQ ID NO: 15 or SEQ ID NO: 16).

In some embodiments, the conjugate polypeptide comprises one or more non-natural amino acids (nnAA). In some embodiments, the one or more nnAA comprise a click chemistry reactive group. Herein, a “click chemistry reactive group” refers to a moiety, such as an azide or an alkyne, capable of undergoing 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 instance, in International PCT Publication No. WO 2018/126229. General examples of metal-free click reactions used in biomedical applications can be found, for instance, 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 some embodiments, the conjugate polypeptide comprises one or more nnAAs, wherein each of the nnAAs are pAMF.

Amino acid sequences of exemplary conjugate polypeptides are provided in Table 4 below. Sites suitable for substitution with an nnAA are indicated in bolded and underlined text. In some embodiments, the underlined and italicized amino acids are cleaved from the mature protein during production.

TABLE 4

Exemplary Carrier Proteins

Protein
Amino Acid Sequence
SEQ ID

ADI [full length] WT w/ leader

MHHHHHHGSGENLYFQGTAQTPIHVYSEIGKLKKVLLHRPG KEIENLMPDYLERLLFDDIPFLEDAQKEHDAFAQALRDEGIEVLYL ETLAAESLVTPEIREAFIDEYLSEANIRGRATKKAIRELLMAIEDN QELIEKTMAGVQKSELPEIPASEKGLTDLVESNYPFAIDPMPNLYF TRDPFATIGTGVSLNHMFSETRNRETLYGKYIFTHHPIYGGGKVPM VYDRNETTRIEGGDELVLSKDVLAVGISQRTDAASIEKLLVNIFKQ NLGFKKVLAFEFANNRKFMHLDTVFTMVDYDKFTIHPEIEGDLRVY SVTYDNEELHIVEEKGDLAELLAANLGVEKVDLIRCGGDNLVAAGR EQWNDGSNTLTIAPGVVVVYNRNTITNAILESKGLKLIKIHGSELV RGRGGPRCMSMPFEREDI
13

ADI [full length] D277A w/ leader

MHHHHHHGSGENLYFQGTAQTPIHVYSEIGKLKKVLLHRPG KEIENLMPDYLERLLFDDIPFLEDAQKEHDAFAQALRDEGIEVLYL ETLAAESLVTPEIREAFIDEYLSEANIRGRATKKAIRELLMAIEDN QELIEKTMAGVQKSELPEIPASEKGLTDLVESNYPFAIDPMPNLYF TRDPFATIGTGVSLNHMFSETRNRETLYGKYIFTHHPIYGGGKVPM VYDRNETTRIEGGDELVLSKDVLAVGISQRTDAASIEKLLVNIFKQ NLGFKKVLAFEFANNRKFMHLATVFTMVDYDKFTIHPEIEGDLRVY SVTYDNEELHIVEEKGDLAELLAANLGVEKVDLIRCGGDNLVAAGR EQWNDGSNTLTIAPGVVVVYNRNTITNAILESKGLKLIKIHGSELV RGRGGPRCMSMPFEREDI
15

SpyAD [33-849 fragment] WT w/ leader

MHHHHHHGSGENLYFQGQVKADDRASGETKASNTHDDSLPK PETIQEAKATIDAVEKTLSQQKAELTELATALTKTTAEINHLKEQQ DNEQKALTSAQEIYTNTLASSEETLLAQGAEHQRELTATETELHNA QADQHSKETALSEQKASISAETTRAQDLVEQVKTSEQNIAKLNAMI SNPDAITKAAQTANDNTKALSSELEKAKADLENQKAKVKKQLTEEL AAQKAALAEKEAELSRLKSSAPSTQDSIVGNNTMKAPQGYPLEELK KLEASGYIGSASYNNYYKEHADQIIAKASPGNQLNQYQDIPADRNR FVDPDNLTPEVQNELAQFAAHMINSVRRQLGLPPVTVTAGSQEFAR LLSTSYKKTHGNTRPSFVYGQPGVSGHYGVGPHDKTIIEDSAGASG LIRNDDNMYENIGAFNDVHTVNGIKRGIYDSIKYMLFTDHLHGNTY GHAINFLRVDKHNPNAPVYLGFSTSNVGSLNEHFVMFPESNIANHQ RFNKTPIKAVGSTKDYAQRVGTVSDTIAAIKGKVSSLENRLSAIHQ EADIMAAQAKVSQLQGKLASTLKQSDSLNLQVRQLNDTKGSLRTEL LAAKAKQAQLEATRDQSLAKLASLKAALHQTEALAEQAAARVTALV AKKAHLQYLRDFKLNPNRLQVIRERIDNTKQDLAKTTSSLLNAQEA LAALQAKQSSLEATIATTEHQLTLLKTLANEKEYRHLDEDIATVPD LQVAPPLTGVKPLSYSKIDTTPLVQEMVKETKQLLEASARLAAENT SLVAEALVGQTSEMVASNAIVSKITSSITQPSSKTSYGSGSSTTSN LISDVDESTQR
9

hFL [full length] WT w/ leader

MHHHHHHGSGSSQIRQNYSTDVEAAVNSLVNLYLQASYTYL SLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRAL FQDIKKPAEDEWGKTPDAMKAAMALEKKLNQALLDLHALGSARTDP HLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERL TLKHD
21

eCRM-pAMF6 mutant
MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQXGIQKPKS GTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYP GLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGAS RVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAM YEYMAQACAGNRVRNSVGSSLSCINLDWDVIRDXTKTKIESLKEHG
25

PIKNKMSESPNKTVSEEKAXQYLEEFHQTALEHPELSELXTVTGTN PVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGI ADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVE SIINLFQVVHNSYNRPAYSPGHXTQPFLHDGYAVSWNTVEDSIIRT GFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNG RKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIH SNEISSDSIGVLGYQKTVDHTKVNSXLSLFFEIKS

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

In some embodiments, the conjugate polypeptide is ADI and comprises three nnAAs substituted at positions K15, K193, and K316 of SEQ ID NO: 13. In some embodiments, the conjugate polypeptide is ADI and comprises three nnAAs substituted at positions K15, K193, and K316 of SEQ ID NO: 35. In some embodiments, the three nnAAs are each pAMF. In some embodiments, the conjugate polypeptide is ADI and comprises or consists of an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 17, 18, 19, or 20. In some embodiments, the three nnAAs are each pAMF. In some embodiments, the conjugate polypeptide is SpyAD and comprises or consists of an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 26, 27, 37, or 38. In some embodiments, the conjugate polypeptide is ADI and comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 17, 18, 19, or 20. In some embodiments, the conjugate polypeptide is ADI and comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 26, 27, 37, 38.

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

In some embodiments, the conjugate polypeptide is a SLO polypeptide. In some embodiments, the conjugate polypeptide is SLO(ΔC101) and comprises five, six, seven, or eight nnAAs substituted at positions selected from selected from K98, K112, R151, K189, K272, K323, K357, K375, K407, and K464 of SEQ ID NO: 53. In some embodiments, the conjugate polypeptide is SLO(ΔC101) and comprises or consists of nnAAs substituted at positions K98, R151, K272, K357, and K407; positions K112, K189, K323, K375, and K464; positions K112, R151, K272, K357, and K407; positions K98, R151, K272, K357, K407, and K464; positions K112, R151, K189, K323, K375, and K464; positions K98, K112, K189, K323, K375, and K464; positions K112, R151, K189, K272, K357, 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 some embodiments, the five, six, seven, or eight nnAAs are each pAMF. In some embodiments, the conjugate polypeptide is a SLO polypeptide and comprises or consists of 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 some embodiments, the conjugate polypeptide is a 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.

In some embodiments, the conjugate polypeptide is ferritin and comprises one nnAA substituted at position I5 of SEQ ID NO: 21. In some embodiments, the nnAA is pAMF. In some embodiments, the conjugate polypeptide is ferritin and comprises or consists of an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 23 or SEQ ID NO: 24. In some embodiments, the conjugate polypeptide is ADI and comprises or consists of SEQ ID NO: 23 or SEQ ID NO: 24.

Polysaccharides

In some embodiments, the conjugate polysaccharide is a GAS polysaccharide, such as the group A carbohydrate (GAC). The GAC is composed of a polyrhamnose backbone with an immunodominant GlcNAc side chain and is present on the surface of strains of all GAS serotypes irrespective of M type and has been examined as a GAS vaccine candidate. The conjugate polysaccharide may be defined as the polysaccharide component of a conjugate. Affinity-purified anti-GAC antibodies successfully opsonized three tested GAS serotypes (Salvadori et al., 1995. J. Infect. Dis. 171:593-600.), and mice immunized with GAC were protected against both intraperitoneal and intranasal GAS challenge (Sabharwal et al., 2006. J. Infect. Dis. 193:129-135). However, immunological cross-reactivity between anti-GAC antibodies and host heart valve proteins (Goldstein et al., 1967. Nature 213:44-47) and cytoskeletal proteins, such as actin, keratin, myosin, and vimentin, raises important potential safety concerns regarding the use of GAC as a GAS vaccine constituent.

In some embodiments, a Streptococcus pyogenes bacterial cell may be engineered to produce a polysaccharide or a variant thereof that lacks an immunodominant N-acetyl Glucosamine (GLcNAc) side chain. Therefore, in some embodiments, the conjugate polysaccharide is a variant of the GAC that lacks the immunodominant GlcNAc side chain (See e.g., International PCT Publication No. WO 2013/020090 U.S. Pat. No. 10,780,155, and Gao, N. J. et al., Dec. 29, 2020. Infectious Microbes and Diseases, doi: 10.1097/IM9.0000000000000044). In some embodiments, the conjugate polysaccharide is a GAC comprising a polyrhamanose core. In some embodiments, the conjugate polysaccharide has an average molecular weight of about 3 kDa to about 10 kDa, about 5 kDa to about 10 kDa, about 9 kDa to about 10 kDa, about 9 kDa to about 10 kDa, about 3 kDa to about 9 kDa, about 3 kDa to about 7 kDa, about 3 kDa to about 5 kDa, about 5 kDa to about 9 kDa, or about 5 kDa to about 7 kDa. In some embodiments, the conjugate polysaccharide has an average molecular weight of about 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, or about 10 kDa. In some embodiments, the conjugate polysaccharide is a tetramer, a pentamer, a hexamer, a septamer, an octamer, a nonomer, or a decamer polysaccharide.

In some embodiments, the conjugate polysaccharide has an average molecular weight of between about 10 kDa to about 45 kDa. In some embodiments, the conjugate polysaccharide has an average molecular weight of about 10 kDa to about 15 kDa, about 15 kDa to about 20 kDa, about 20 kDa to about 25 kDa, about 25 kDa to about 30 kDa, about 30 kDa to about 35 kDa, or about about 35 kDa to about 40 kDa. In some embodiments, the conjugate polysaccharide has an average molecular weight of 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 25 kDa, or about 15 kDa to about 45 kDa. In some embodiments, the conjugate polysaccharide has an average molecular weight of about 20 kDa to about 40 kDa, about 20 kDa to about 35 kDa, about 20 kDa to about 30 kDa, or about 20 kDa to about 45 kDa. In some embodiments, the conjugate polysaccharide has an average molecular weight of about 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kD or about 45 kDa. In some embodiments, the conjugate polysaccharide has an average molecular weight of at least about 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, or about 35 kDa.

In some embodiments, the conjugate polysaccharide is purified from GAS bacterial cultures or bacterial stocks. Methods of such purification are known in the art, see e.g., WO 2010/049806, International PCT Publication No. WO 2013/020090, and van Sorge, et al., Cell Host Microbe., 2014, 15(6), 729-740. See also Example 1. In some embodiments, the conjugate polysaccharide is a synthesized polysaccharide. Methods of polysaccharide synthesis are known in the art, see e.g., Zhao, et al., Org. Chem. Front., 2019, 6, 3589-3596. See also Example 3. In some embodiments, the conjugate polysaccharides are modified with a click chemistry reactive group to facilitate conjugation to the conjugate protein. Examples of click chemistry reactive groups can be found, for instance, in International PCT Publication No. WO 2018/126229, incorporated by reference herein by reference in its entirety. For example, in some embodiments, the conjugate polysaccharides are modified with DBCO or DBCO-PEG (e.g., DBCO-PEG-NH₂). In some embodiments, the conjugate polysaccharides are modified with DBCO-(PEG)₄-NH₂.

In some embodiments, the polypeptide-polysaccharide conjugate has an average molecular mass of about 10 kDa to about 5000 kDa. In some embodiments, the polypeptide-polysaccharide conjugate has an average molecular mass of about 10 kDa to about 50 kDa. In some embodiments, the polypeptide-polysaccharide conjugate has an average molecular mass of about 10 kDa to about 100 kDa. In some embodiments, the polypeptide-polysaccharide conjugate has an average molecular mass of about 10 kDa to about 200 kDa. In some embodiments, the polypeptide-polysaccharide conjugate has an average molecular mass of about 10 kDa to about 500 kDa. In some embodiments, the polypeptide-polysaccharide conjugate has an average molecular mass of about 10 kDa to about 1000 kDa. In some embodiments, the polypeptide-polysaccharide conjugate has an average molecular mass of about 100 kDa to about 200 kDa. In some embodiments, the polypeptide-polysaccharide conjugate has an average molecular mass of about 100 kDa to about 300 kDa. In some embodiments, the polypeptide-polysaccharide conjugate has an average molecular mass of about 100 kDa to about 400 kDa. In some embodiments, the polypeptide-polysaccharide conjugate has an average molecular mass of about 100 kDa to about 500 kDa. In some embodiments, the polypeptide-polysaccharide conjugate has an average molecular mass of about 100 kDa to about 5000 kDa. In some embodiments, the polypeptide-polysaccharide conjugate has an average molecular mass of about 500 kDa to about 5000 kDa. In some embodiments, the polypeptide-polysaccharide conjugate has an average molecular mass of about 1000 kDa to about 5000 kDa. In some embodiments, the polypeptide-polysaccharide conjugate has an average molecular mass of about 2000 kDa to about 5000 kDa. In some embodiments, the polypeptide-polysaccharide conjugate has an average molecular mass of about 3000 kDa to about 5000 kDa. In some embodiments, the polypeptide-polysaccharide conjugate has an average molecular mass of about 4000 kDa to about 5000 kDa. In some embodiments, the polypeptide-polysaccharide conjugate has an average molecular mass of about 1000 kDa to about 3000 kDa. In some embodiments, the polypeptide-polysaccharide conjugate has an average molecular mass of about 1000 kDa to about 2000 kDa. In some embodiments, the polypeptide-polysaccharide conjugate has an average molecular mass of about 2000 kDa to about 3000 kDa. In some embodiments, the polypeptide-polysaccharide conjugate has an average molecular mass of about 5000 kDa or greater.

Exemplary Combination Vaccines

In some embodiments, the present disclosure provides an immunogenic composition comprising a GAS C5a peptidase polypeptide antigen; a GAS streptolysin O (SLO) polypeptide antigen; and a polypeptide-polysaccharide conjugate comprising a SpyAD conjugate polypeptide and a GAS conjugate polysaccharide or a variant thereof that lacks an immunodominant GlcNAc side chain. In some embodiments, the SLO polypeptide antigen comprises or consists of 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. In some embodiments, the SLO polypeptide antigen comprises or consists of an amino acid sequence that is at least 95% identical to SEQ ID NO: 31. In some embodiments, the SLO polypeptide antigen comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 32. In some embodiments, the SLO polypeptide antigen comprises or consists of an amino acid sequence that is at least 95% identical to SEQ ID NO: 44. In some embodiments, the SLO polypeptide antigen comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 45. In some embodiments, the SLO polypeptide antigen comprises or consists of an amino acid sequence that is at least 95% identical to SEQ ID NO: 46. In some embodiments, the SLO polypeptide antigen comprises or consists of an amino acid sequence that is at least 95% identical to SEQ ID NO: 47. In some embodiments, the SLO polypeptide antigen comprises or consists of an amino acid sequence that is at least 95% identical to SEQ ID NO: 48. In some embodiments, the SLO polypeptide antigen comprises or consists of an amino acid sequence that is at least 95% identical to SEQ ID NO: 49. In some embodiments, the SLO polypeptide antigen comprises or consists of an amino acid sequence that is at least 95% identical to SEQ ID NO: 50. In some embodiments, the SLO polypeptide antigen comprises or consists of an amino acid sequence that is at least 95% identical to SEQ ID NO: 51. In some embodiments, the SLO polypeptide antigen comprises or consists of an amino acid sequence that is at least 95% identical to SEQ ID NO: 52. In some embodiments, the SLO polypeptide antigen comprises or consists of an amino acid sequence that is at least 95% identical to SEQ ID NO: 53. In some embodiments, the SLO polypeptide antigen comprises or consists of 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 some embodiments, the present disclosure provides an immunogenic composition comprising a C5a peptidase polypeptide antigen of SEQ ID NO: 30; a SLO polypeptide antigen of SEQ ID NO: 53; and a polypeptide-polysaccharide conjugate comprising a SpyAD conjugate polypeptide of SEQ ID NO: 34 and a GAS conjugate polysaccharide. In some embodiments, the present disclosure provides an immunogenic composition comprising a C5a peptidase polypeptide antigen of SEQ ID NO: 30; a SLO polypeptide antigen of SEQ ID NO: 52; and a polypeptide-polysaccharide conjugate comprising a SpyAD conjugate polypeptide of SEQ ID NO: 34 and a GAS conjugate polysaccharide. In some embodiments, the present disclosure provides an immunogenic composition comprising a C5a peptidase polypeptide antigen of SEQ ID NO: 30; a SLO polypeptide antigen of SEQ ID NO: 32; and a polypeptide-polysaccharide conjugate comprising a SpyAD conjugate polypeptide of SEQ ID NO: 34 and a GAS conjugate polysaccharide. In some embodiments, the present disclosure provides an immunogenic composition comprising a C5a peptidase polypeptide antigen of SEQ ID NO: 29; a SLO polypeptide antigen of SEQ ID NO: 53; and a polypeptide-polysaccharide conjugate comprising a SpyAD conjugate polypeptide of SEQ ID NO: 34 and a GAS conjugate polysaccharide. In some embodiments, the present disclosure provides an immunogenic composition comprising a C5a peptidase polypeptide antigen comprising or consisting of the amino acid sequence of SEQ ID NO: 30; a SLO polypeptide antigen comprising or consisting of the amino acid sequence of SEQ ID NO: 53; and a polypeptide-polysaccharide conjugate comprising a SpyAD conjugate polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 34 and a GAS conjugate polysaccharide. In some embodiments, the present disclosure provides an immunogenic composition comprising a C5a peptidase polypeptide antigen comprising or consisting of the amino acid sequence of SEQ ID NO: 30; a SLO polypeptide antigen comprising or consisting of the amino acid sequence of SEQ ID NO: 52; and a polypeptide-polysaccharide conjugate comprising a SpyAD conjugate polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 34 and a GAS conjugate polysaccharide. In some embodiments, the present disclosure provides an immunogenic composition comprising a C5a peptidase polypeptide antigen comprising or consisting of the amino acid sequence of SEQ ID NO: 30; a SLO polypeptide antigen comprising or consisting of the amino acid sequence of SEQ ID NO: 32; and a polypeptide-polysaccharide conjugate comprising a SpyAD conjugate polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 34 and a GAS conjugate polysaccharide. In some embodiments, the present disclosure provides an immunogenic composition comprising a C5a peptidase polypeptide antigen comprising or consisting of the amino acid sequence of SEQ ID NO: 29; a SLO polypeptide antigen comprising or consisting of the amino acid sequence of SEQ ID NO: 53; and a polypeptide-polysaccharide conjugate comprising a SpyAD conjugate polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 34 and a GAS conjugate polysaccharide.

In some embodiments, the present disclosure provides an immunogenic composition comprising a C5a peptidase polypeptide antigen; a GAS SLO polypeptide antigen; and a polypeptide-polysaccharide conjugate comprising an ADI conjugate polypeptide and a GAS conjugate polysaccharide or a variant thereof that lacks an immunodominant GlcNAc side chain. In some embodiments, the ADI polypeptide comprises or consists of an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 13-16 and comprises a pAMF substitution at positions K15, K193, and K316. In some embodiments, the ADI polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 17-20. In some embodiments, the ADI polypeptide comprises or consists of an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 35-36 and comprises a pAMF substitution at positions K15, K193, and K316. In some embodiments, the ADI polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 37-38. In some embodiments, the ADI polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 17-20. In some embodiments, the ADI polypeptide comprises or consists of an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 35-36 and comprises a pAMF substitution at positions K15, K193, and K316. In some embodiments, the ADI polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 37-38.

Therapeutic Methods

In some embodiments, the present disclosure provides a method of inducing a protective immune response against a GAS bacterium in a subject comprising administering an immunogenic composition described herein to the subject. In certain embodiments are provided the use of the immunogenic compositions described herein for inducing a protective immune response against a GAS bacterium in a subject. In some embodiments, provided herein are the use of the immunogenic compositions described herein in the manufacture of a medicament for inducing a protective immune response against a GAS bacterium in a subject.

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 subjects are 18 years of age or older. In some embodiments, the human subjects are less than 18 years of age.

In some embodiments, the human subjects are between 6 months of age and 17 years of age. In some embodiments, the human subjects are between 6 months of age and 9 years of age, between 6 months of age and 8 years of age, between 6 months of age and 7 years of age, between 6 months of age and 6 years of age, between 6 months of age and 5 years of age, between 6 months of age and 4 years of age, between 6 months of age and 3 years of age, between 6 months of age and 2 years of age, or between 6 months of age and 1 year of age. In some embodiments, the human subjects are between 5 years of age and 17 years of age, between 7 years of age and 17 years of age, between 9 years of age and 17 years of age, between 11 years of age and 17 years of age, between 13 years of age and 17 years of age, or between 15 years of age and 17 years of age. In some embodiments, the human subjects are 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, or 18 years of age.

Herein, the term “protective immune response” encompasses eliciting an anti-GAS antibody response in the subject. Antibody titers generated after administration of the immunogenic compositions described herein can be determined by means known in the art, for example by ELISA assays of serum samples derived from immunized subjects (See Example 6). In some embodiments, the immunogenic compositions described herein elicit antibody responses in treated subjects, wherein the antibodies generated bind to multiple (i.e., two or more) GAS serotypes. In some embodiments, the immunogenic compositions described herein elicit antibody responses in treated subjects, wherein the antibodies generated 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 some embodiments, the immunogenic compositions described herein do not elicit antibody responses against human proteins or tissue.

In some embodiments, the immunogenic compositions described herein elicit antibody responses in treated subjects, wherein the antibodies generated 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 some embodiments, the immunogenic compositions described herein elicit antibody responses in treated subjects, wherein the antibodies generated 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.

In some embodiments, the immunogenic compositions described herein elicit antibody responses in treated subjects, wherein the antibodies generated bind to at least one GAS serotype selected from M1, M2, M3, M4, M6, M11, M12, M22, M28, M75, and M89. In some embodiments, the immunogenic compositions described herein elicit antibody responses in treated subjects, wherein the antibodies generated bind to two or more GAS serotypes selected from M1, M2, M3, M4, M6, M11, M12, M22, M28, M75, and M89.

In some embodiments, the immunogenic compositions described herein elicit antibody responses in treated subjects, wherein the antibodies generated bind to at least one GAS serotype selected from M1, M3, M5, M9, M12, M18, M22, M25, M28, M71, M72, and M74. In some embodiments, the immunogenic compositions described herein elicit antibody responses in treated subjects, wherein the antibodies generated bind to two or more GAS serotypes selected from M1, M3, M5, M9, M12, M18, M22, M25, M28, M71, M72, and M74.

In some embodiments, the immunogenic compositions described herein elicit antibody responses in treated subjects, wherein the antibodies generated bind to at least one GAS serotype selected from M1, M4, M6, M11, M12, M22, M44, M75, M77, M77, and M81. In some embodiments, the immunogenic compositions described herein elicit antibody responses in treated subjects, wherein the antibodies generated bind to two or more GAS serotypes selected from M1, M4, M6, M11, M12, M22, M44, M75, M77, M77, and M81.

In some embodiments, the immunogenic compositions described herein elicit antibody responses in treated subjects, wherein the antibodies generated bind to at least one GAS serotype selected from M1, M2, M3, M4, M6, M9, M12, M18, M22, M75, M77, M89, and M92. In some embodiments, the immunogenic compositions described herein elicit antibody responses in treated subjects, wherein the antibodies generated bind to two or more GAS serotypes selected from M1, M2, M3, M4, M6, M9, M12, M18, M22, M75, M77, M89, and M92.

In some embodiments, the immunogenic compositions described herein elicit antibody responses in treated subjects, wherein the antibodies generated bind to at least one GAS serotype selected from M1, M2, M3, M4, M5, M6, M9, M11, M12, M13, M28, M62, and M89. In some embodiments, the immunogenic compositions described herein elicit antibody responses in treated subjects, wherein the antibodies generated bind to two or more GAS serotypes selected from M1, M2, M3, M4, M5, M6, M9, M11, M12, M13, M28, M62, and M89.

In some embodiments, the immunogenic compositions described herein elicit antibody responses in treated subjects, wherein the antibodies generated 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 some embodiments, the immunogenic compositions described herein elicit antibody responses in treated subjects, wherein the antibodies generated 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.

In some embodiments, the immunogenic compositions described herein elicit antibody responses in treated subjects that bind to one or more Shigella serotypes. In some embodiments, the immunogenic compositions described herein elicit antibody responses in treated subjects that bind to one or more GAS serotypes and also bind to one or more Shigella serotypes. In some embodiments, the Shigella serotypes comprise a polysaccharide comprising a polyrhamanose backbone. Exemplary Shigella serotypes comprising such polysaccharides include S. flexneri such as S. flexneri 2A and 3A and S. flexneri 6.

FURTHER NUMBERED EMBODIMENTS

Further embodiments of the instant disclosure are provided in the numbered embodiments below:

Embodiment 1: An immunogenic composition comprising a first Group A Streptococcus (GAS) polypeptide antigen; a second GAS polypeptide antigen; and at least one polypeptide-polysaccharide conjugate, wherein the conjugate polypeptide is a third GAS polypeptide antigen or a non-GAS carrier polypeptide and comprises at least one non-natural amino acid, and wherein the conjugate polysaccharide is a GAS polysaccharide or a variant thereof that lacks an immunodominant N-acetyl Glucosamine (GlcNAc) side chain.

Embodiment 2: The immunogenic composition of Embodiment 1, wherein at least one of the GAS polypeptide antigens is a full length GAS antigen.

Embodiment 3: The immunogenic composition of Embodiment 1, wherein at least one of the GAS polypeptide antigens is a peptide fragment of a full length GAS antigen.

Embodiment 4: The immunogenic composition of any one of Embodiments 1-3, wherein the first and second GAS polypeptide antigens are independently selected from C5a peptidase, streptolysin O (SLO), Sib35, and Sfb1.

Embodiment 5: The immunogenic composition of any one of Embodiments 1-3, wherein the first and second GAS polypeptide antigens are C5a peptidase and SLO.

Embodiment 6: The immunogenic composition of Embodiment 5, wherein the C5a peptidase polypeptide antigen comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1 or SEQ ID NO: 3.

Embodiment 7: The immunogenic composition of Embodiment 5, wherein the C5a peptidase polypeptide antigen comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.

Embodiment 8: The immunogenic composition of Embodiment 5, wherein the SLO polypeptide antigen comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 5 or SEQ ID NO: 7.

Embodiment 9: The immunogenic composition of Embodiment 5, wherein the SLO polypeptide antigen comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 7.

Embodiment 10: The immunogenic composition of any one of Embodiments 1-9, wherein the non-GAS carrier polypeptide is ferritin.

Embodiment 11: The immunogenic composition of any one of Embodiments 1-10, wherein the third GAS polypeptide antigen is selected from Streptococcus pyogenes Adhesion and Division (SpyAD) polypeptide, arginine deiminase (ADI), eCRM, and Protein D.

Embodiment 12: The immunogenic composition of any one of Embodiments 1-11, wherein the at least one nnAA is substituted for a lysine, a leucine, or an isoleucine in the conjugate polypeptide.

Embodiment 13: The immunogenic composition of any one of Embodiments 1-11, wherein the nnAA comprises a click chemistry reactive group.

Embodiment 14: The immunogenic composition of Embodiment 13, wherein the nnAA is selected from 2-amino-3-(4-azidophenyl)propanoic acid(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 15: The immunogenic composition of Embodiment 13, wherein the nnAA is pAMF.

Embodiment 16: The immunogenic composition of any one of Embodiments 12-15, wherein the conjugate polypeptide is a third GAS protein, which is a SpyAD polypeptide and comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 9.

Embodiment 17: The immunogenic composition of Embodiment 16, wherein the SpyAD polypeptide comprises a pAMF substitution at positions K64, K287, K386, and K657 of SEQ ID NO: 9.

Embodiment 18: The immunogenic composition of Embodiment 17, wherein the SpyAD polypeptide comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 11.

Embodiment 19: The immunogenic composition of any one of Embodiments 12-15, wherein the conjugate polypeptide is a third GAS polypeptide, which is an ADI polypeptide and comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 13 or SEQ ID NO: 15.

Embodiment 20: The immunogenic composition of Embodiment 19, wherein the ADI polypeptide comprises a pAMF substitution at positions K15, K193, and K316 of SEQ ID NO: 13 or SEQ ID NO: 15.

Embodiment 21: The immunogenic composition of Embodiment 20, wherein the ADI polypeptide comprises the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 19.

Embodiment 22: The immunogenic composition of any one of Embodiments 12-15, wherein the conjugate polypeptide is a ferritin polypeptide and comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 21.

Embodiment 23: The immunogenic composition of Embodiment 22, wherein the ferritin polypeptide comprises a pAMF substitution at position I5 of SEQ ID NO: 21.

Embodiment 24: The immunogenic composition of Embodiment 23, wherein the ferritin polypeptide comprises the amino acid sequence of SEQ ID NO: 23.

Embodiment 25: The immunogenic composition of any one of Embodiments 1-23, wherein the conjugate polysaccharide comprises a polyrhamnose core.

Embodiment 26: The immunogenic composition of any one of Embodiments 1-23, wherein the conjugate polysaccharide has an average molecular weight of about 5 KDa to about 7 KDa.

Embodiment 27: The immunogenic composition of any one of Embodiments 1-26, wherein the conjugate polysaccharide is a tetramer, a hexamer, an octamer, or a decamer polysaccharide.

Embodiment 28: 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 comprising a Streptococcus pyogenes Adhesion and Division (SpyAD) conjugate polypeptide and a GAS conjugate polysaccharide or a variant thereof that lacks an immunodominant N-acetyl Glucosamine (GlcNAc) side chain.

Embodiment 29: The immunogenic composition of Embodiment 28, wherein the SpyAD protein comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 9 and comprises a pAMF substitution at positions K64, K287, K386, and K657.

Embodiment 30: The immunogenic composition of Embodiment 28, wherein the SpyAD protein comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 11.

Embodiment 31: 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 comprising an arginine deiminase (ADI) conjugate polypeptide and a GAS conjugate polysaccharide or a variant thereof that lacks an immunodominant N-acetyl Glucosamine (GlcNAc) side chain.

Embodiment 32: The immunogenic composition of Embodiment 31, wherein the ADI polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 13 or SEQ ID NO: 15 and comprises a pAMF substitution at positions K15, K193, and K316.

Embodiment 33: The immunogenic composition of Embodiment 31, wherein the ADI polypeptide comprises the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 19.

Embodiment 34: 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 comprising a ferritin polypeptide and a GAS conjugate polysaccharide or a variant thereof that lacks an immunodominant N-acetyl Glucosamine (GlcNAc) side chain.

Embodiment 35: The immunogenic composition of Embodiment 34, wherein the ferritin protein comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 21 and comprises a pAMF substitution at position I5.

Embodiment 35A: The immunogenic composition Embodiment 34, wherein the ferritin protein comprises the amino acid sequence of SEQ ID NO: 23.

Embodiment 36: The immunogenic composition of any one of Embodiments 1-35, wherein the composition comprises about 10% to about 50% of the first GAS antigen, about 10% to about 50% of the second GAS antigen, and about 10% to about 50% of the polypeptide-polysaccharide conjugate.

Embodiment 37: The immunogenic composition of any one of Embodiments 1-36, further comprising one or more adjuvants selected from alum, saponin, monophosphoryl lipid A (MPL), or combinations thereof.

Embodiment 38: A method of inducing a protective immune response against a Group A Streptococcus (GAS) bacterium in a subject comprising administering the immunogenic composition of any one of Embodiments 1-37 to the subject.

Embodiment 38A: Use of the immunogenic composition of any one of Embodiments 1-37 for inducing a protective immune response against a Group A Streptococcus (GAS) bacterium in a subject.

Embodiment 38B: Use of the immunogenic composition of any one of Embodiments 1-37 in the manufacture of a medicament for inducing a protective immune response against a Group A Streptococcus (GAS) bacterium in a subject.

Embodiment 39: The method of any one of Embodiments 38-38B, wherein the subject is 18 years or older.

Embodiment 40: The method of any one of Embodiments 38-38B, wherein the subject is less than 18 years old.

Embodiment 41: The method of any one of Embodiments 38-38B, wherein the subject is between 5 years and 17 years old, between 6 months and 9 years old, or between 5 years and 9 years old.

Embodiment 42: The method of any one of Embodiments 38-41, wherein the GAS bacterium is of a serotype selected from M1, M2, M3, M4, M6, M11, M12, M22, M28, M75, and M89.

Embodiment 43: The method of any one of Embodiments 38-41, wherein the GAS bacterium is a serotype selected from M1, M3, M5, M9, M12, M18, M22, M25, M28, M71, M72, and M74.

Embodiment 44: The method of any one of Embodiments 38-41, wherein the GAS bacterium is a serotype selected from M1, M4, M6, M11, M12, M22, M44, M75, M77, M77, and M81.

Embodiment 45: The method of any one of Embodiments 38-41, wherein the GAS bacterium is a serotype selected from M1, M2, M3, M4, M6, M9, M12, M18, M22, M75, M77, M89, and M92.

Embodiment 46: The method of any one of Embodiments 38-41, wherein the GAS bacterium is a serotype selected from M1, M2, M3, M4, M5, M6, M9, M11, M12, M13, M28, M62, and M89.

Embodiment 47: The method of any one of Embodiments 38-41, wherein the GAS bacterium is a serotype selected from M1, M2, M3, M4, M6, M12, M22, M28, M49, M53, M68, M77, M80, M83, M87, M89, and M92.

Embodiment 48: The method of any one of Embodiments 38-47, wherein the immunogenic composition induces an antibody response in the subject against the Group A Streptococcus (GAS) bacterium and does not induce an antibody response in the subject against human tissue.

Embodiment 49: The method of any one of Embodiments 38-48, wherein the immunogenic composition induces a protective immune response against a Shigella bacterium in the subject, wherein the Shigella bacterium comprises a polysaccharide with a polyrhmanose backbone.

Embodiment 50: A method of inducing a protective immune response against a Shigella bacterium in a subject comprising administering the immunogenic composition of any one of Embodiments 1-37 to the subject.

Embodiment 51: Use of the immunogenic composition of any one of Embodiments 1-37 for inducing a protective immune response against a Shigella bacterium in a subject.

Embodiment 52: Use of the immunogenic composition of any one of Embodiments 1-37 in the manufacture of a medicament for inducing a protective immune response against a Shigella bacterium in a subject.

Embodiment 53: The method or use of any one of Embodiments 50-52, wherein the subject is 18 years or older.

Embodiment 54: The method or use of Embodiment 53, wherein the subject is less than 18 years old.

Embodiment 55: The method or use of Embodiment 54, wherein the subject is between 5 years and 17 years old, between 6 months and 9 years old, or between 5 years and 9 years old.

Embodiment II-1. An immunogenic composition comprising

a. a first Group A Streptococcus (GAS) polypeptide antigen;
b. a second GAS polypeptide antigen; and
c. at least one polypeptide-polysaccharide conjugate, wherein the conjugate polypeptide is a third GAS polypeptide antigen or a non-GAS carrier polypeptide and comprises at least one non-natural amino acid, and wherein the conjugate polysaccharide is a GAS polysaccharide or a variant thereof that lacks an immunodominant N-acetyl Glucosamine (GlcNAc) side chain.

Embodiment II-2. The immunogenic composition of embodiment II-1, wherein at least one of the GAS polypeptide antigens is a full length GAS antigen.

Embodiment II-3. The immunogenic composition of embodiment II-1, wherein at least one of the GAS polypeptide antigens is a peptide fragment of a full length GAS antigen.

Embodiment II-4. The immunogenic composition of any one of embodiments II-1 to II-3, wherein the first and second GAS polypeptide antigens are independently selected from C5a peptidase, streptolysin O (SLO), Sib35, and Sfb1.

Embodiment II-5. The immunogenic composition of any one of embodiments II-1 to II-3, wherein the first and second GAS polypeptide antigens are C5a peptidase and SLO.

Embodiment II-6. The immunogenic composition of embodiment II-5, wherein the C5a peptidase polypeptide antigen comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1 or SEQ ID NO: 3.

Embodiment II-7. The immunogenic composition of embodiment II-5, wherein the C5a peptidase polypeptide antigen comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.

Embodiment II-8. The immunogenic composition of any one of embodiments II-1 to II-6 wherein the first and second GAS polypeptide antigens are C5a peptidase and SLO, and the C5a peptidase polypeptide antigen comprises the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 30.

Embodiment II-9. The immunogenic composition of embodiment II-5, wherein the SLO polypeptide antigen comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 5 or SEQ ID NO: 7.

Embodiment II-10. The immunogenic composition of any one of embodiments II-1 to II-6 or II-9, wherein the first and second GAS polypeptide antigens are C5a peptidase and SLO, and the SLO polypeptide 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-11. The immunogenic composition of embodiment II-5, wherein the SLO polypeptide antigen comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 7.

Embodiment II-12. The immunogenic composition of any one of embodiments II-1 to II-6, wherein the first and second GAS polypeptide antigens are C5a peptidase and SLO, and 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 11-13. The immunogenic composition of any one of embodiments II-1 to II-12, wherein the non-GAS carrier polypeptide is ferritin.

Embodiment II-14. The immunogenic composition of any one of embodiments II-1 to II-13, wherein the third GAS polypeptide antigen is selected from Streptococcus pyogenes Adhesion and Division (SpyAD) polypeptide, arginine deiminase (ADI), eCRM, and Protein D.

Embodiment II-15. The immunogenic composition of any one of embodiments II-1 to II-14, wherein the at least one nnAA is substituted for a lysine, a leucine, or an isoleucine in the conjugate polypeptide.

Embodiment II-16. The immunogenic composition of any one of embodiments II-1 to II-14, wherein the nnAA comprises a click chemistry reactive group.

Embodiment II-17. The immunogenic composition of embodiment II-16, wherein the 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-18. The immunogenic composition of embodiment II-16, wherein the nnAA is pAMF.

Embodiment II-19. The immunogenic composition of any one of embodiments II-15 to II-18, wherein the conjugate polypeptide is a third GAS protein, which is a SpyAD polypeptide and comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 9.

Embodiment II-20. The immunogenic composition of embodiment II-19, wherein the SpyAD polypeptide comprises a pAMF substitution at positions K64, K287, K386, and K657 of SEQ ID NO: 9.

Embodiment II-21. The immunogenic composition of embodiment II-20, wherein the SpyAD polypeptide comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 11.

Embodiment II-22. The immunogenic composition of any one of embodiments II-15 to II-18, wherein the conjugate polypeptide is a third GAS protein, which is a SpyAD polypeptide and comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 33.

Embodiment II-23. The immunogenic composition of embodiment II-22, wherein the SpyAD polypeptide comprises a pAMF substitution at positions K64, K287, K386, and K657 of SEQ ID NO: 33.

Embodiment II-24. The immunogenic composition of embodiment II-23, wherein the SpyAD polypeptide comprises the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 34.

Embodiment II-25. The immunogenic composition of any one of embodiments II-15 to II-18, wherein the conjugate polypeptide is a third GAS polypeptide, which is an ADI polypeptide and comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 13 or SEQ ID NO: 15.

Embodiment II-26. The immunogenic composition of embodiment II-25, wherein the ADI polypeptide comprises a pAMF substitution at positions K15, K193, and K316 of SEQ ID NO: 13 or SEQ ID NO: 15.

Embodiment II-27. The immunogenic composition of embodiment II-26, wherein the ADI polypeptide comprises the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 19.

Embodiment II-28. The immunogenic composition of any one of embodiments II-15 to II-18, wherein the conjugate polypeptide is a third GAS polypeptide, which is an ADI polypeptide and comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 35 or SEQ ID NO: 36.

Embodiment II-29. The immunogenic composition of embodiment II-28, wherein the ADI polypeptide comprises a pAMF substitution at positions K15, K193, and K316 of SEQ ID NO: 35 or SEQ ID NO: 36.

Embodiment II-30. The immunogenic composition of embodiment II-29, wherein the ADI polypeptide comprises the amino acid sequence of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 37 or SEQ ID NO: 38.

Embodiment II-31. The immunogenic composition of any one of embodiments II-15 to II-18, wherein the conjugate polypeptide is a ferritin polypeptide and comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 21.

Embodiment II-32. The immunogenic composition of embodiment II-31, wherein the ferritin polypeptide comprises a pAMF substitution at position I5 of SEQ ID NO: 21.

Embodiment II-33. The immunogenic composition of embodiment II-32, wherein the ferritin polypeptide comprises the amino acid sequence of SEQ ID NO: 23.

Embodiment II-34. The immunogenic composition of any one of embodiments II-1 to II-33, wherein the conjugate polysaccharide comprises a polyrhamnose core.

Embodiment II-35. The immunogenic composition of any one of embodiments II-1 to II-33, wherein the conjugate polysaccharide has an average molecular weight of about 5 KDa to about 7 KDa.

Embodiment II-36. The immunogenic composition of any one of embodiments II-1 to II-35, wherein the conjugate polysaccharide is a tetramer, a hexamer, an octamer, or a decamer polysaccharide.

Embodiment II-37. An immunogenic composition comprising

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 a Streptococcus pyogenes Adhesion and Division (SpyAD) conjugate polypeptide and a GAS conjugate polysaccharide or a variant thereof that lacks an immunodominant N-acetyl Glucosamine (GlcNAc) side chain.

Embodiment II-38. The immunogenic composition of embodiment II-37, wherein the SpyAD protein comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 9 and comprises a pAMF substitution at positions K64, K287, K386, and K657.

Embodiment II-39. The immunogenic composition of embodiment II-37, wherein the SpyAD protein comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 11.

Embodiment II-40. The immunogenic composition of embodiment II-37, wherein the SpyAD protein comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 33 and comprises a pAMF substitution at positions K64, K287, K386, and K657.

Embodiment II-41. The immunogenic composition of embodiment II-37, wherein the SpyAD protein comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 34.

Embodiment II-42. The immunogenic composition of embodiment II-41, 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-43. The immunogenic composition of embodiment II-1 or II-37, comprising a C5a peptidase polypeptide antigen of SEQ ID NO: 30; a SLO polypeptide antigen of SEQ ID NO: 53; and a polypeptide-polysaccharide conjugate comprising a SpyAD conjugate polypeptide of SEQ ID NO: 34 and a GAS conjugate polysaccharide.

Embodiment II-44. The immunogenic composition of embodiment II-1 or II-37, comprising a C5a peptidase polypeptide antigen of SEQ ID NO: 30; a SLO polypeptide antigen of SEQ ID NO: 52; and a polypeptide-polysaccharide conjugate comprising a SpyAD conjugate polypeptide of SEQ ID NO: 34 and a GAS conjugate polysaccharide.

Embodiment II-45. The immunogenic composition of embodiment II-1 or II-37, comprising a C5a peptidase polypeptide antigen of SEQ ID NO: 30; a SLO polypeptide antigen of SEQ ID NO: 32; and a polypeptide-polysaccharide conjugate comprising a SpyAD conjugate polypeptide of SEQ ID NO: 34 and a GAS conjugate polysaccharide.

Embodiment II-46. The immunogenic composition of embodiment II-1 or II-37, comprising a C5a peptidase polypeptide antigen of SEQ ID NO: 29; a SLO polypeptide antigen of SEQ ID NO: 53; and a polypeptide-polysaccharide conjugate comprising a SpyAD conjugate polypeptide of SEQ ID NO: 34 and a GAS conjugate polysaccharide.

Embodiment II-47. An immunogenic composition comprising

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 an arginine deiminase (ADI) conjugate polypeptide and a GAS conjugate polysaccharide or a variant thereof that lacks an immunodominant N-acetyl Glucosamine (GlcNAc) side chain.

Embodiment II-48. The immunogenic composition of embodiment II-47, wherein the ADI polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 13 or SEQ ID NO: 15 and comprises a pAMF substitution at positions K15, K193, and K316.

Embodiment II-49. The immunogenic composition of embodiment II-47, wherein the ADI polypeptide comprises the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 19.

Embodiment II-50. The immunogenic composition of embodiments II-47 to II-48, wherein the ADI polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 35 or SEQ ID NO: 36 and comprises a pAMF substitution at positions K15, K193, and K316.

Embodiment II-51. The immunogenic composition of embodiments II-47 to II-48, wherein the ADI polypeptide comprises the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 38.

Embodiment II-52. An immunogenic composition comprising

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 a ferritin polypeptide and a GAS conjugate polysaccharide or a variant thereof that lacks an immunodominant N-acetyl Glucosamine (GlcNAc) side chain.

Embodiment II-53. The immunogenic composition of embodiment II-52, wherein the ferritin protein comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 21 and comprises a pAMF substitution at position I5.

Embodiment II-54. The immunogenic composition of embodiment II-52, wherein the ferritin protein comprises the amino acid sequence of SEQ ID NO: 23.

Embodiment II-55. The immunogenic composition of any one of embodiments II-1 to II-54, wherein the composition comprises about 10% to about 50% of the first GAS antigen, about 10% to about 50%of the second GAS antigen, and about 10% to about 50% of the polypeptide-polysaccharide conjugate.

Embodiment II-56. The immunogenic composition of any one of embodiments II-1 to II-55, further comprising one or more adjuvants selected from alum, saponin, monophosphoryl lipid A (MPL), or combinations thereof.

Embodiment II-57. A method of inducing a protective immune response against a Group A Streptococcus (GAS) bacterium in a subject comprising administering the immunogenic composition of any one of embodiments II-1 to II-56 to the subject.

Embodiment II-58. The method of embodiment II-57, wherein the subject is 18 years or older.

Embodiment II-59. The method of embodiment II-57, wherein the subject is less than 18 years old.

Embodiment II-60. The method of embodiment II-57, wherein the subject is between 5 years and 17 years old, between 6 months and 9 years old, or between 5 years and 9 years old.

Embodiment II-61. The method of any one of embodiments II-57 to II-60, wherein the GAS bacterium is of a serotype selected from M1, M2, M3, M4, M6, M11, M12, M22, M28, M75, and M89.

Embodiment II-62. The method of any one of embodiments II-57 to II-60, wherein the GAS bacterium is a serotype selected from M1, M3, M5, M9, M12, M18, M22, M25, M28, M71, M72, and M74.

Embodiment II-63. The method of any one of embodiments II-57 to II-60, wherein the GAS bacterium is a serotype selected from M1, M4, M6, M11, M12, M22, M44, M75, M77, M77, and M81.

Embodiment II-64. The method of any one of embodiments II-57 to II-60, wherein the GAS bacterium is a serotype selected from M1, M2, M3, M4, M6, M9, M12, M18, M22, M75, M77, M89, and M92.

Embodiment II-65. The method of any one of embodiments II-57 to II-60, wherein the GAS bacterium is a serotype selected from M1, M2, M3, M4, M5, M6, M9, M11, M12, M13, M28, M62, and M89.

Embodiment II-66. The method of any one of embodiments II-57 to II-60, wherein the GAS bacterium is a serotype selected from M1, M2, M3, M4, M6, M12, M22, M28, M49, M53, M68, M77, M80, M83, M87, M89, and M92.

Embodiment II-67. The method of any one of embodiments II-57 to II-66, wherein the immunogenic composition induces an antibody response in the subject against the Group A Streptococcus (GAS) bacterium and does not induce an antibody response in the subject against human tissue.

Embodiment II-68. The method of any one of embodiments II-57 to II-67, wherein the immunogenic composition induces a protective immune response against a Shigella bacterium in the subject, wherein the Shigella bacterium comprises a polysaccharide with a polyrhmanose backbone.

Embodiment II-69. A method of inducing a protective immune response against a Shigella bacterium in a subject comprising administering the immunogenic composition of any one of embodiments II-1 to II-56 to the subject.

Embodiment II-70. Use of the immunogenic composition of any one of embodiments II-1 to II-56 for inducing a protective immune response against a Shigella bacterium in a subj ect.

Embodiment II-71. Use of the immunogenic composition of any one of embodiments II-1 to II-56 in the manufacture of a medicament for inducing a protective immune response against a Shigella bacterium in a subject.

Embodiment II-72. The method or use of any one of embodiments II-69 to II-71, wherein the subject is 18 years or older.

Embodiment II-73. The method or use of embodiment II-72, wherein the subject is less than 18 years old.

Embodiment II-74. The method or use of embodiment II-73, wherein the subject is between 5 years and 17 years old, between 6 months and 9 years old, or between 5 years and 9 years old.

Embodiment III-1. An immunogenic composition comprising

a. a first Group A Streptococcus (GAS) polypeptide antigen;
b. a second GAS polypeptide antigen; and
c. at least one polypeptide-polysaccharide conjugate, wherein the conjugate polypeptide is a third GAS polypeptide antigen or a non-GAS carrier polypeptide and comprises at least one non-natural amino acid, and wherein the conjugate polysaccharide is a GAS polysaccharide or a variant thereof that lacks an immunodominant N-acetyl Glucosamine (GlcNAc) side chain.

Embodiment III-2. The immunogenic composition of embodiment III-1, wherein at least one of the GAS polypeptide antigens is a full length GAS antigen.

Embodiment III-3. The immunogenic composition of embodiment III-1, wherein at least one of the GAS polypeptide antigens is a peptide fragment of a full length GAS antigen.

Embodiment III-4. The immunogenic composition of any one of embodiments 111-1 to III-3, wherein the first and second GAS polypeptide antigens are independently selected from C5a peptidase, streptolysin O (SLO), Sib35, Sfb1, and SpyAD.

Embodiment III-5. The immunogenic composition of any one of embodiments 111-1 to III-3, wherein the first and second GAS polypeptide antigens are independently selected from C5a peptidase, streptolysin O (SLO), Sib35, and Sfb1.

Embodiment III-6. The immunogenic composition of any one of embodiments 111-1 to III-3, wherein the first and second GAS polypeptide antigens are C5a peptidase and SLO.

Embodiment III-7. The immunogenic composition of embodiment III-6, wherein the C5a peptidase polypeptide antigen comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1 or SEQ ID NO: 3.

Embodiment III-7A. The immunogenic composition of embodiment III-6, wherein the C5a peptidase polypeptide antigen is at least 95% identical to SEQ ID NO: 1 or SEQ ID NO: 3.

Embodiment III-8. The immunogenic composition of embodiment III-6, wherein the C5a peptidase polypeptide antigen comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.

Embodiment III-9. The immunogenic composition of any one of embodiments 111-1 to III-6 wherein the first and second GAS polypeptide antigens are C5a peptidase and SLO, and the C5a peptidase polypeptide antigen comprises the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 30.

Embodiment III-9A. The immunogenic composition of any one of embodiments III-1 to III-6 wherein the first and second GAS polypeptide antigens are C5a peptidase and SLO, and the C5a peptidase polypeptide antigen consisting of the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 30.

Embodiment III-10. The immunogenic composition of embodiment III-6, wherein the SLO polypeptide antigen comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 5 or SEQ ID NO: 7.

Embodiment III-10A. The immunogenic composition of embodiment III-6, wherein the SLO polypeptide antigen is at least 95% identical to SEQ ID NO: 5 or SEQ ID NO: 7.

Embodiment III-11. The immunogenic composition of any one of embodiments III-1 to III-7 or III-10, wherein the first and second GAS polypeptide antigens are C5a peptidase and SLO, and the SLO polypeptide 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 III-11A. The immunogenic composition of any one of embodiment III-17, III-10, III-7A or III-10A, wherein the first and second GAS polypeptide antigens are C5a peptidase and SLO, and the SLO polypeptide antigen 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 III-12. The immunogenic composition of embodiment III-6, wherein the SLO polypeptide antigen comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 7.

Embodiment III-12A. The immunogenic composition of embodiment III-6, wherein the SLO polypeptide antigen consisting of the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 7.

Embodiment III-13. The immunogenic composition of any one of embodiments III-1 to III-7, wherein the first and second GAS polypeptide antigens are C5a peptidase and SLO, and 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 III-13A. The immunogenic composition of any one of embodiments III-1 to III-7 or III-7A, wherein the first and second GAS polypeptide antigens are C5a peptidase and SLO, and the SLO polypeptide antigen consisting of 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 III-14. The immunogenic composition of any one of embodiments III-1 to III-3, wherein the first and second GAS polypeptide antigens are C5a peptidase and SpyAD.

Embodiment III-15. The immunogenic composition of embodiment III-14, 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 III-15A. The immunogenic composition of embodiment III-14, wherein the C5a peptidase polypeptide antigen is at least 95% identical to SEQ ID NO: 1 or SEQ ID NO: 3.

Embodiment III-16. The immunogenic composition of embodiment III-14, wherein the C5a peptidase polypeptide antigen comprises the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 30.

Embodiment III-16A. The immunogenic composition of embodiment III-14, wherein the C5a peptidase polypeptide antigen consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.

Embodiment III-17. The immunogenic composition of any one of embodiments III-1 to III-16, wherein the non-GAS carrier polypeptide is ferritin.

Embodiment III-18. The immunogenic composition of any one of embodiments III-1 to III-16, wherein the third GAS polypeptide antigen is selected from Streptococcus pyogenes Adhesion and Division (SpyAD) polypeptide, arginine deiminase (ADI), eCRM, and Protein D.

Embodiment III-19. The immunogenic composition of any one of embodiments III-1 to III-16, wherein the third GAS polypeptide antigen is selected from Streptococcus pyogenes Adhesion and Division (SpyAD) polypeptide, arginine deiminase (ADI), eCRM, Protein D, and SLO.

Embodiment III-20. The immunogenic composition of any one of embodiments III-1 to III-19, wherein the at least one nnAA is substituted for a lysine, a leucine, or an isoleucine in the conjugate polypeptide.

Embodiment III-21. The immunogenic composition of any one of embodiments III-1 to III-19, wherein the nnAA comprises a click chemistry reactive group.

Embodiment III-22. The immunogenic composition of embodiment III-16, wherein the 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 III-23. The immunogenic composition of embodiment III-21, wherein the nnAA is pAMF.

Embodiment III-24. The immunogenic composition of any one of embodiments III-20 to III-23, wherein the conjugate polypeptide is a third GAS protein, which is a SpyAD polypeptide and comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 9.

Embodiment III-24A. The immunogenic composition of any one of embodiments III-20 to III-23, wherein the conjugate polypeptide is a third GAS protein, which is a SpyAD polypeptide is at least 95% identical to SEQ ID NO: 9.

Embodiment III-25. The immunogenic composition of embodiment III-24, wherein the SpyAD polypeptide comprises a pAMF substitution at positions K64, K287, K386, and K657 of SEQ ID NO: 9.

Embodiment III-26. The immunogenic composition of embodiment III-25, wherein the SpyAD polypeptide comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 11.

Embodiment III-26A. The immunogenic composition of embodiment III-25, wherein the SpyAD polypeptide comprises consisting of the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 11.

Embodiment III-27. The immunogenic composition of any one of embodiments III-20 to III-23, wherein the conjugate polypeptide is a third GAS protein, which is a SpyAD polypeptide and comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 33.

Embodiment III-27A. The immunogenic composition of any one of embodiments III-20 to III-23, wherein the conjugate polypeptide is a third GAS protein, which is a SpyAD polypeptide and is at least 95% identical to SEQ ID NO: 33.

Embodiment III-28. The immunogenic composition of embodiment III-27, wherein the SpyAD polypeptide comprises a pAMF substitution at positions K64, K287, K386, and K657 of SEQ ID NO: 33.

Embodiment III-29. The immunogenic composition of embodiment III-28, wherein the SpyAD polypeptide comprises the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 34.

Embodiment III-30. The immunogenic composition of any one of embodiments III-20 to III-23, wherein the conjugate polypeptide is a third GAS polypeptide, which is an ADI polypeptide and comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 13 or SEQ ID NO: 15.

Embodiment III-30A. The immunogenic composition of any one of embodiments III-20 to III-23, wherein the conjugate polypeptide is a third GAS polypeptide, which is an ADI polypeptide and is at least 95% identical to SEQ ID NO: 13 or SEQ ID NO: 15.

Embodiment III-31. The immunogenic composition of embodiment III-30, wherein the ADI polypeptide comprises a pAMF substitution at positions K15, K193, and K316 of SEQ ID NO: 13 or SEQ ID NO: 15.

Embodiment III-32. The immunogenic composition of embodiment III-31, wherein the ADI polypeptide comprises the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 19.

Embodiment III-32A. The immunogenic composition of embodiment III-31, wherein the ADI polypeptide consisting of the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 19.

Embodiment III-33. The immunogenic composition of any one of embodiments III-20 to III-23, wherein the conjugate polypeptide is a third GAS polypeptide, which is an ADI polypeptide and comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 35 or SEQ ID NO: 36.

Embodiment III-33A. The immunogenic composition of any one of embodiments III-20 to III-23, wherein the conjugate polypeptide is a third GAS polypeptide, which is an ADI polypeptide and is at least 95% identical to SEQ ID NO: 35 or SEQ ID NO: 36.

Embodiment III-34. The immunogenic composition of embodiment III-33, wherein the ADI polypeptide comprises a pAMF substitution at positions K15, K193, and K316 of SEQ ID NO: 35 or SEQ ID NO: 36.

Embodiment III-35. The immunogenic composition of embodiment III-34, wherein the ADI polypeptide comprises the amino acid sequence of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 37 or SEQ ID NO: 38.

Embodiment III-35A. The immunogenic composition of embodiment III-34, wherein the ADI polypeptide consisting of the amino acid sequence of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 37 or SEQ ID NO: 38.

Embodiment III-36. The immunogenic composition of any one of embodiments III-20 to III-23, wherein the conjugate polypeptide is a third GAS polypeptide, which is a SLO polypeptide and 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 III-36A. The immunogenic composition of any one of embodiments III-20 to III-23, wherein the conjugate polypeptide is a third GAS polypeptide, which is a SLO polypeptide and is at least 95% identical to SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 53.

Embodiment III-37. The immunogenic composition of embodiment III-36, wherein the SLO polypeptide comprises 3 or 4 pAMF substitutions at positions selected from K98, K112, R151, K189, K272, K323, K357, K375, K407, or K464.

Embodiment III-38. The immunogenic composition of embodiment III-36 or III-37, 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 III-38A. The immunogenic composition of embodiment III-36 or III-37, wherein the SLO polypeptide consisting of 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 III-39. The immunogenic composition of any one of embodiments III-20 to III-23, wherein the conjugate polypeptide is a ferritin polypeptide and comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 21.

Embodiment III-39A. The immunogenic composition of any one of embodiments III-20 to III-23, wherein the conjugate polypeptide is a ferritin polypeptide and is at least 95% identical to SEQ ID NO: 21.

Embodiment III-40. The immunogenic composition of embodiment III-39, wherein the ferritin polypeptide comprises a pAMF substitution at position I5 of SEQ ID NO: 21.

Embodiment III-41. The immunogenic composition of embodiment III-40, wherein the ferritin polypeptide comprises the amino acid sequence of SEQ ID NO: 23.

Embodiment III-41A. The immunogenic composition of embodiment III-40, wherein the ferritin polypeptide consisting of the amino acid sequence of SEQ ID NO: 23.

Embodiment III-42. The immunogenic composition of any one of embodiments III-1 to III-41, wherein the conjugate polysaccharide comprises a polyrhamnose core.

Embodiment III-43. The immunogenic composition of any one of embodiments III-1 to III-41, wherein the conjugate polysaccharide has an average molecular weight of about 5 KDa to about 7 KDa.

Embodiment III-44. The immunogenic composition of any one of embodiments III-1 to III-43, wherein the conjugate polysaccharide is a tetramer, a hexamer, an octamer, or a decamer polysaccharide.

Embodiment III-45. An immunogenic composition comprising

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 a Streptococcus pyogenes Adhesion and Division (SpyAD) conjugate polypeptide and a GAS conjugate polysaccharide or a variant thereof that lacks an immunodominant N-acetyl Glucosamine (GlcNAc) side chain.

Embodiment III-46. The immunogenic composition of embodiment III-45, wherein the SpyAD protein comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 9 and comprises a pAMF substitution at positions K64, K287, K386, and K657.

Embodiment III-46A. The immunogenic composition of embodiment III-45, wherein the SpyAD protein is at least 95% identical to SEQ ID NO: 9 and comprises a pAMF substitution at positions K64, K287, K386, and K657.

Embodiment III-47. The immunogenic composition of embodiment III-45, wherein the SpyAD protein comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 11.

Embodiment III-47A. The immunogenic composition of embodiment III-45, wherein the SpyAD protein consisting of the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 11.

Embodiment III-48. The immunogenic composition of embodiment III-45, wherein the SpyAD protein comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 33 and comprises a pAMF substitution at positions K64, K287, K386, and K657.

Embodiment III-48A. The immunogenic composition of embodiment III-45, wherein the SpyAD protein is at least 95% identical to SEQ ID NO: 33 and comprises a pAMF substitution at positions K64, K287, K386, and K657.

Embodiment III-49. The immunogenic composition of embodiment III-45, wherein the SpyAD protein comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 34.

Embodiment III-49A. The immunogenic composition of embodiment III-45, wherein the SpyAD protein consisting of the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 34.

Embodiment III-50. The immunogenic composition of embodiment III-49, 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 III-50A. The immunogenic composition of embodiment III-49 or III-49A, wherein the SLO polypeptide antigen consisting of 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 III-51. The immunogenic composition of embodiment III-1 or III-45, comprising a C5a peptidase polypeptide antigen of SEQ ID NO: 30; a SLO polypeptide antigen of SEQ ID NO: 53; and a polypeptide-polysaccharide conjugate comprising a SpyAD conjugate polypeptide of SEQ ID NO: 34 and a GAS conjugate polysaccharide.

Embodiment III-52. The immunogenic composition of embodiment III-1 or III-45, comprising a C5a peptidase polypeptide antigen of SEQ ID NO: 30; a SLO polypeptide antigen of SEQ ID NO: 52; and a polypeptide-polysaccharide conjugate comprising a SpyAD conjugate polypeptide of SEQ ID NO: 34 and a GAS conjugate polysaccharide.

Embodiment III-53. The immunogenic composition of embodiment III-1 or III-45, comprising a C5a peptidase polypeptide antigen of SEQ ID NO: 30; a SLO polypeptide antigen of SEQ ID NO: 32; and a polypeptide-polysaccharide conjugate comprising a SpyAD conjugate polypeptide of SEQ ID NO: 34 and a GAS conjugate polysaccharide.

Embodiment III-54. The immunogenic composition of embodiment III-1 or III-45, comprising a C5a peptidase polypeptide antigen of SEQ ID NO: 29; a SLO polypeptide antigen of SEQ ID NO: 53; and a polypeptide-polysaccharide conjugate comprising a SpyAD conjugate polypeptide of SEQ ID NO: 34 and a GAS conjugate polysaccharide.

Embodiment III-55. An immunogenic composition comprising

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 an arginine deiminase (ADI) conjugate polypeptide and a GAS conjugate polysaccharide or a variant thereof that lacks an immunodominant N-acetyl Glucosamine (GlcNAc) side chain.

Embodiment III-56. The immunogenic composition of embodiment III-55, wherein the ADI polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 13 or SEQ ID NO: 15 and comprises a pAMF substitution at positions K15, K193, and K316.

Embodiment III-56A. The immunogenic composition of embodiment III-55, wherein the ADI polypeptide is at least 95% identical to SEQ ID NO: 13 or SEQ ID NO: 15 and comprises a pAMF substitution at positions K15, K193, and K316.

Embodiment III-57. The immunogenic composition of embodiment III-55, wherein the ADI polypeptide comprises the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 19.

Embodiment III-57A. The immunogenic composition of embodiment III-55, wherein the ADI polypeptide consisting of the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 19.

Embodiment III-58. The immunogenic composition of embodiments III-55 to III-56, wherein the ADI polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 35 or SEQ ID NO: 36 and comprises a pAMF substitution at positions K15, K193, and K316.

Embodiment III-58A. The immunogenic composition of any one of embodiments III-55 to III-56 or III-56A, wherein the ADI polypeptide is at least 95% identical to SEQ ID NO: 35 or SEQ ID NO: 36 and comprises a pAMF substitution at positions K15, K193, and K316.

Embodiment III-59. The immunogenic composition of embodiments III-55 to III-56, wherein the ADI polypeptide comprises the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 38.

Embodiment III-59A. The immunogenic composition of any one of embodiments III-55 to III-56 or III-56A, wherein the ADI polypeptide consisting of the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 38.

Embodiment III-60. An immunogenic composition comprising

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 a ferritin polypeptide and a GAS conjugate polysaccharide or a variant thereof that lacks an immunodominant N-acetyl Glucosamine (GlcNAc) side chain.

Embodiment III-61. The immunogenic composition of embodiment III-60, wherein the ferritin protein comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 21 and comprises a pAMF substitution at position 15.

Embodiment III-61A. The immunogenic composition of embodiment III-60, wherein the ferritin protein is at least 95% identical to SEQ ID NO: 21 and comprises a pAMF substitution at position 15.

Embodiment III-62. The immunogenic composition of embodiment III-60, wherein the ferritin protein comprises the amino acid sequence of SEQ ID NO: 23.

Embodiment III-62A. The immunogenic composition of embodiment III-60, wherein the ferritin protein consisting of the amino acid sequence of SEQ ID NO: 23.

Embodiment III-63. The immunogenic composition of any one of embodiments III-1 to III-62, wherein the composition comprises about 10% to about 50% of the first GAS antigen, about 10% to about 50%of the second GAS antigen, and about 10% to about 50% of the polypeptide-polysaccharide conjugate.

Embodiment III-64. The immunogenic composition of any one of embodiments III-1 to III-63, further comprising one or more adjuvants selected from alum, saponin, monophosphoryl lipid A (MPL), or combinations thereof.

Embodiment III-65. A method of inducing a protective immune response against a Group A Streptococcus (GAS) bacterium in a subject comprising administering the immunogenic composition of any one of embodiments III-1-56 to the subject.

Embodiment III-66. The method of embodiment III-65, wherein the subject is 18 years or older.

Embodiment III-67. The method of embodiment III-65, wherein the subject is less than 18 years old.

Embodiment III-68. The method of embodiment III-65, wherein the subject is between 5 years and 17 years old, between 6 months and 9 years old, or between 5 years and 9 years old.

Embodiment III-69. The method of any one of embodiments III-65 to III-68, wherein the GAS bacterium is of a serotype selected from M1, M2, M3, M4, M6, M11, M12, M22, M28, M75, and M89.

Embodiment III-70. The method of any one of embodiments III-65 to III-68, wherein the GAS bacterium is a serotype selected from M1, M3, M5, M9, M12, M18, M22, M25, M28, M71, M72, and M74.

Embodiment III-71. The method of any one of embodiments III-65 to III-68, wherein the GAS bacterium is a serotype selected from M1, M4, M6, M11, M12, M22, M44, M75, M77, M77, and M81.

Embodiment III-72. The method of any one of embodiments III-65 to III-68, wherein the GAS bacterium is a serotype selected from M1, M2, M3, M4, M6, M9, M12, M18, M22, M75, M77, M89, and M92.

Embodiment III-73. The method of any one of embodiments III-65 to III-68, wherein the GAS bacterium is a serotype selected from M1, M2, M3, M4, M5, M6, M9, M11, M12, M13, M28, M62, and M89.

Embodiment III-74. The method of any one of embodiments III-65 to III-68, wherein the GAS bacterium is a serotype selected from M1, M2, M3, M4, M6, M12, M22, M28, M49, M53, M68, M77, M80, M83, M87, M89, and M92.

Embodiment III-75. The method of any one of embodiments III-65 to III-74, wherein the immunogenic composition induces an antibody response in the subject against the Group A Streptococcus (GAS) bacterium and does not induce an antibody response in the subject against human tissue.

Embodiment III-76. The method of any one of embodiments III-65 to III-75, wherein the immunogenic composition induces a protective immune response against a Shigella bacterium in the subject, wherein the Shigella bacterium comprises a polysaccharide with a polyrhmanose backbone.

Embodiment III-77. A method of inducing a protective immune response against a Shigella bacterium in a subject comprising administering the immunogenic composition of any one of embodiments III-1-56 to the subject.

Embodiment III-78. Use of the immunogenic composition of any one of embodiments III-1 to III-54 for inducing a protective immune response against a Shigella bacterium in a subject.

Embodiment III-79. Use of the immunogenic composition of any one of embodiments III-1 to III-54 in the manufacture of a medicament for inducing a protective immune response against a Shigella bacterium in a subject.

Embodiment III-80. The method or use of any one of embodiments III-77 to III-79, wherein the subject is 18 years or older.

Embodiment III-81. The method or use of embodiment III-80, wherein the subject is less than 18 years old.

Embodiment III-82. The method or use of embodiment III-81, wherein the subject is between 5 years and 17 years old, between 6 months and 9 years old, or between 5 years and 9 years old.

Embodiment IV-1. An immunogenic composition comprising

a. a first Group A Streptococcus (GAS) polypeptide antigen;
b. a second GAS polypeptide antigen; and
c. at least one polypeptide-polysaccharide conjugate, wherein the conjugate polypeptide is a third GAS polypeptide antigen or a non-GAS carrier polypeptide and comprises at least one non-natural amino acid, and wherein the conjugate polysaccharide is a GAS polysaccharide or a variant thereof that lacks an immunodominant N-acetyl Glucosamine (GlcNAc) side chain.

Embodiment IV-2. The immunogenic composition of embodiment IV-1, wherein at least one of the GAS polypeptide antigens is a full length GAS antigen.

Embodiment IV-3. The immunogenic composition of embodiment IV-1, wherein at least one of the GAS polypeptide antigens is a peptide fragment of a full length GAS antigen.

Embodiment IV-4. The immunogenic composition of any one of embodiments IV-1 to IV-3, wherein the first and second GAS polypeptide antigens are independently selected from C5a peptidase, streptolysin O (SLO), Sib35, Sfb1, and SpyAD.

Embodiment IV-5. The immunogenic composition of any one of embodiments IV-1 to IV-3, wherein the first and second GAS polypeptide antigens are independently selected from C5a peptidase, streptolysin O (SLO), Sib35, and Sfb1.

Embodiment IV-6. The immunogenic composition of any one of embodiments IV-1 to IV-3, wherein the first and second GAS polypeptide antigens are C5a peptidase and SLO.

Embodiment IV-7. The immunogenic composition of any one of embodiments IV-1 to IV-6, 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 IV-8. The immunogenic composition of any one of embodiments IV-1 to IV-5, wherein the C5a peptidase polypeptide antigen is at least 95% identical to SEQ ID NO: 29 or SEQ ID NO: 30.

Embodiment IV-9. The immunogenic composition of embodiment IV-6, wherein the C5a peptidase polypeptide antigen comprises the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 30.

Embodiment IV-10. The immunogenic composition of any one of embodiments IV-1 to IV-6 wherein the first and second GAS polypeptide antigens are C5a peptidase and SLO, and the C5a peptidase polypeptide antigen comprises the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 30.

Embodiment IV-11. The immunogenic composition of any one of embodiments IV-1 to IV-6 wherein the first and second GAS polypeptide antigens are C5a peptidase and SLO, and the C5a peptidase polypeptide antigen consists of the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 30.

Embodiment IV-12. The immunogenic composition of any one of embodiments IV-1 to IV-11, wherein the SLO polypeptide antigen comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 31 or SEQ ID NO: 32.

Embodiment IV-13. The immunogenic composition of any one of embodiments IV-1 to IV-11, wherein the SLO polypeptide antigen is at least 95% identical to SEQ ID NO: 31 or SEQ ID NO: 32.

Embodiment IV-14. The immunogenic composition of any one of embodiments IV-1 to IV-9, wherein the first and second GAS polypeptide antigens are C5a peptidase and SLO, and the SLO polypeptide 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 IV-15. The immunogenic composition of any one of embodiments IV-1 to IV-9, wherein the first and second GAS polypeptide antigens are C5a peptidase and SLO, and the SLO polypeptide antigen 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 IV-16. The immunogenic composition of any one of embodiments IV-1 to IV-11, wherein the SLO polypeptide antigen comprises the amino acid sequence of SEQ ID NO: 31 or SEQ ID NO: 32.

Embodiment IV-17. The immunogenic composition of embodiment IV-6, wherein the SLO polypeptide antigen consists of the amino acid sequence of SEQ ID NO: 31 or SEQ ID NO: 32.

Embodiment IV-18. The immunogenic composition of any one of embodiments IV-1 to IV-9, wherein the first and second GAS polypeptide antigens are C5a peptidase and SLO, and 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 IV-19. The immunogenic composition of any one of embodiments IV-1 to IV-9, wherein the first and second GAS polypeptide antigens are C5a peptidase and SLO, and the SLO polypeptide antigen consists of 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 IV-20. The immunogenic composition of any one of embodiments IV-1 to IV-4, wherein the first and second GAS polypeptide antigens are C5a peptidase and SpyAD.

Embodiment IV-21. The immunogenic composition of any one of embodiments IV-1 to IV-6, 16-17, or 20, 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 IV-22. The immunogenic composition of embodiment IV-20 or IV-21, wherein the C5a peptidase polypeptide antigen is at least 95% identical to SEQ ID NO: 29 or SEQ ID NO: 30.

Embodiment IV-23. The immunogenic composition of any one of embodiments IV-1 to IV-6, 16-17 or 20, wherein the C5a peptidase polypeptide antigen comprises the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 30.

Embodiment IV-24. The immunogenic composition of any one of embodiments IV-1 to IV-6, 16-17, 20, or 23, wherein the C5a peptidase polypeptide antigen consists of the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 30.

Embodiment IV-25. The immunogenic composition of any one of embodiments IV-1 to IV-24, wherein the non-GAS carrier polypeptide is ferritin.

Embodiment IV-26. The immunogenic composition of any one of embodiments IV-1 to IV-24, wherein the third GAS polypeptide antigen or non-GAS polypeptide antigen is selected from Streptococcus pyogenes Adhesion and Division (SpyAD) polypeptide, arginine deiminase (ADI), SEQ ID NO: 25, and Protein D.

Embodiment IV-27. The immunogenic composition of any one of embodiments IV-1 to IV-24, wherein the third GAS polypeptide antigen or non-GAS polypeptide antigen is selected from Streptococcus pyogenes Adhesion and Division (SpyAD) polypeptide, arginine deiminase (ADI), SEQ ID NO: 25, Protein D, and SLO.

Embodiment IV-28. The immunogenic composition of any one of embodiments IV-1 to IV-27, wherein the at least one nnAA is substituted for a lysine, a leucine, an arginine, or an isoleucine in the conjugate polypeptide.

Embodiment IV-29. The immunogenic composition of any one of embodiments IV-1 to IV-27, wherein the nnAA comprises a click chemistry reactive group.

Embodiment IV-30. The immunogenic composition of any one of embodiments IV-1 to IV-29, wherein the 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 IV-31. The immunogenic composition of embodiment IV-29 or IV-30, wherein the nnAA is pAMF.

Embodiment IV-32. The immunogenic composition of any one of embodiments IV-26 to IV-31, wherein the conjugate polypeptide is a third GAS protein, which is a SpyAD polypeptide and comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 33.

Embodiment IV-33. The immunogenic composition of any one of embodiments IV-28 to IV-32, wherein the conjugate polypeptide is a third GAS protein, which is a SpyAD polypeptide and is at least 95% identical to SEQ ID NO: 33.

Embodiment IV-34. The immunogenic composition of any one of embodiments IV-26 to IV-33, wherein the SpyAD polypeptide comprises a pAMF substitution at positions K64, K287, K386, and K657 of SEQ ID NO: 33.

Embodiment IV-35. The immunogenic composition of any one of embodiments IV-26 to IV-31 or 34, wherein the SpyAD polypeptide comprises the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 34.

Embodiment IV-36. The immunogenic composition of embodiment IV-34, wherein the SpyAD polypeptide consisting of the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 34.

Embodiment IV-37. The immunogenic composition of any one of embodiments IV-26 to IV-31, wherein the conjugate polypeptide is a third GAS polypeptide, which is an ADI polypeptide and comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 35 or SEQ ID NO: 36.

Embodiment IV-38. The immunogenic composition of any one of embodiments IV-26 to IV-31, wherein the conjugate polypeptide is a third GAS polypeptide, which is an ADI polypeptide and is at least 95% identical to SEQ ID NO: 35 or SEQ ID NO: 36.

Embodiment IV-39. The immunogenic composition of any one of embodiments IV-26 to IV-31 or 37-38, wherein the ADI polypeptide comprises a pAMF substitution at positions K15, K193, and K316 of SEQ ID NO: 35 or SEQ ID NO: 36.

Embodiment IV-40. The immunogenic composition of embodiment IV-39, wherein the ADI polypeptide comprises the amino acid sequence of SEQ ID NO: 27, SEQ ID NO: 37 or SEQ ID NO: 38.

Embodiment IV-41. The immunogenic composition of any one of embodiments IV-26 to IV-31 or 39, wherein the ADI polypeptide consist of the amino acid sequence of SEQ ID NO: 27, SEQ ID NO: 37 or SEQ ID NO: 38.

Embodiment IV-42. The immunogenic composition of any one of embodiments IV-27 to IV-31, wherein the conjugate polypeptide is a third GAS polypeptide, which is a SLO polypeptide and 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 IV-43. The immunogenic composition of any one of embodiments IV-27 to IV-31, wherein the conjugate polypeptide is a third GAS polypeptide, which is a SLO polypeptide and is at least 95% identical to SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 53.

Embodiment IV-44. The immunogenic composition of embodiment IV-42 or IV-43, wherein the SLO polypeptide comprises 3 or 4 pAMF substitutions at positions selected from K98, K112, R151, K189, K272, K323, K357, K375, K407, or K464.

Embodiment IV-45. The immunogenic composition of embodiment IV-44, 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 IV-46. The immunogenic composition of embodiment IV-44, wherein the SLO polypeptide consists of 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 IV-47. The immunogenic composition of embodiment IV-42 or IV-43, 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 IV-48. The immunogenic composition of embodiment IV-47, 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 IV-49. The immunogenic composition of embodiment IV-47, wherein the SLO polypeptide consists of 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 IV-50. The immunogenic composition of any one of embodiments IV-28 to IV-31, wherein the conjugate polypeptide is a ferritin polypeptide and comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 21.

Embodiment IV-51. The immunogenic composition of any one of embodiments IV-28 to IV-31, wherein the conjugate polypeptide is a ferritin polypeptide and is at least 95% identical to SEQ ID NO: 21.

Embodiment IV-52. The immunogenic composition of embodiment IV-50 or IV-51, wherein the ferritin polypeptide comprises a pAMF substitution at position 15 of SEQ ID NO: 21.

Embodiment IV-53. The immunogenic composition of embodiment IV-52, wherein the ferritin polypeptide comprises the amino acid sequence of SEQ ID NO: 23.

Embodiment IV-54. The immunogenic composition of embodiment IV-52, wherein the ferritin polypeptide consists of the amino acid sequence of SEQ ID NO: 23.

Embodiment IV-55. The immunogenic composition of any one of embodiments IV-1 to IV-54, wherein the conjugate polysaccharide lacks the immunodominant GlcNAc side chain.

Embodiment IV-56. The immunogenic composition of any one of embodiments IV-1 to IV-56, wherein the conjugate polysaccharide has an average molecular weight of about 5 kDa to about 7 kDa.

Embodiment IV-57. The immunogenic composition of any one of embodiments IV-1 to IV-56, wherein the conjugate polysaccharide has an average molecular weight of about 10 kDa to about 45 kDa.

Embodiment IV-58. The immunogenic composition of any one of embodiments IV-1 to IV-55, wherein the conjugate polysaccharide is a tetramer, a hexamer, an octamer, or a decamer polysaccharide.

Embodiment IV-59. An immunogenic composition comprising

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 a Streptococcus pyogenes Adhesion and Division (SpyAD) conjugate polypeptide and a GAS conjugate polysaccharide or a variant thereof that lacks an immunodominant N-acetyl Glucosamine (GlcNAc) side chain.

Embodiment IV-60. The immunogenic composition of embodiment IV-59, wherein the SpyAD protein comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 33 and comprises a pAMF substitution at positions K64, K287, K386, and K657.

Embodiment IV-61. The immunogenic composition of embodiment IV-59, wherein the SpyAD protein is at least 95% identical to SEQ ID NO: 33 and comprises a pAMF substitution at positions K64, K287, K386, and K657.

Embodiment IV-62. The immunogenic composition of embodiments IV-59 to IV-61, wherein the SpyAD protein comprises the amino acid sequence of SEQ ID NO: 34.

Embodiment IV-63. The immunogenic composition of embodiments IV-59 to IV-61, wherein the SpyAD protein consists of the amino acid sequence of SEQ ID NO: 34.

Embodiment IV-64. The immunogenic composition of any one of embodiments IV-59 to IV-63, 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 IV-65. The immunogenic composition of any one of embodiments IV-59 to IV-64, wherein the SLO polypeptide antigen consists of 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 IV-66. The immunogenic composition of embodiment IV-1 or IV-59, comprising a C5a peptidase polypeptide antigen of SEQ ID NO: 30; a SLO polypeptide antigen of SEQ ID NO: 53; and a polypeptide-polysaccharide conjugate comprising a SpyAD conjugate polypeptide of SEQ ID NO: 34 and a GAS conjugate polysaccharide.

Embodiment IV-67. The immunogenic composition of embodiment IV-1 or IV-59, comprising a C5a peptidase polypeptide antigen of SEQ ID NO: 30; a SLO polypeptide antigen of SEQ ID NO: 52; and a polypeptide-polysaccharide conjugate comprising a SpyAD conjugate polypeptide of SEQ ID NO: 34 and a GAS conjugate polysaccharide.

Embodiment IV-68. The immunogenic composition of embodiment IV-1 or IV-59, comprising a C5a peptidase polypeptide antigen of SEQ ID NO: 30; a SLO polypeptide antigen of SEQ ID NO: 32; and a polypeptide-polysaccharide conjugate comprising a SpyAD conjugate polypeptide of SEQ ID NO: 34 and a GAS conjugate polysaccharide.

Embodiment IV-69. The immunogenic composition of embodiment IV-1 or IV-59, comprising a C5a peptidase polypeptide antigen of SEQ ID NO: 29; a SLO polypeptide antigen of SEQ ID NO: 53; and a polypeptide-polysaccharide conjugate comprising a SpyAD conjugate polypeptide of SEQ ID NO: 34 and a GAS conjugate polysaccharide.

Embodiment IV-70. An immunogenic composition comprising

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 an arginine deiminase (ADI) conjugate polypeptide and a GAS conjugate polysaccharide or a variant thereof that lacks an immunodominant N-acetyl Glucosamine (GlcNAc) side chain.

Embodiment IV-71. The immunogenic composition of embodiment IV-70, wherein the ADI polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 35 or SEQ ID NO: 36 and comprises a pAMF substitution at positions K15, K193, and K316.

Embodiment IV-72. The immunogenic composition of embodiment IV-70, wherein the ADI polypeptide is at least 95% identical to SEQ ID NO: 35 or SEQ ID NO: 36 and comprises a pAMF substitution at positions K15, K193, and K316.

Embodiment IV-73. The immunogenic composition of embodiment IV-72, wherein the ADI polypeptide comprises the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 38.

Embodiment IV-74. The immunogenic composition of any one of embodiment IV-72, wherein the ADI polypeptide consists of the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 38.

Embodiment IV-75. An immunogenic composition comprising

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 a ferritin polypeptide and a GAS conjugate polysaccharide or a variant thereof that lacks an immunodominant N-acetyl Glucosamine (GlcNAc) side chain.

Embodiment IV-76. The immunogenic composition of embodiment IV-75, wherein the ferritin protein comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 21 and comprises a pAMF substitution at position I5.

Embodiment IV-77. The immunogenic composition of embodiment IV-75, wherein the ferritin protein is at least 95% identical to SEQ ID NO: 21 and comprises a pAMF substitution at position I5.

Embodiment IV-78. The immunogenic composition of embodiment IV-75, wherein the ferritin protein comprises the amino acid sequence of SEQ ID NO: 23.

Embodiment IV-79. The immunogenic composition of embodiment IV-75, wherein the ferritin protein consists of the amino acid sequence of SEQ ID NO: 23.

Embodiment IV-80. The immunogenic composition of any one of embodiments IV-1 to IV-79, wherein the composition comprises about 10% to about 50% of the first GAS antigen, about 10% to about 50% of the second GAS antigen, and about 10% to about 50% of the polypeptide-polysaccharide conjugate.

Embodiment IV-81. The immunogenic composition of any one of embodiments IV-1 to IV-80, further comprising one or more adjuvants selected from alum, saponin, monophosphoryl lipid A (MPL), or combinations thereof.

Embodiment IV-82. The immunogenic composition of any one of embodiments IV-1 to IV-81, wherein the polypeptide-polysaccharide conjugate has an average molecular mass of about 10 kDa to about 5000 kDa.

Embodiment IV-83. A method of inducing a protective immune response against a Group A Streptococcus (GAS) bacterium in a subject comprising administering the immunogenic composition of any one of embodiments IV-1 to IV-82 to the subject.

Embodiment IV-84. The method of embodiment IV-83, wherein the subject is 18 years or older.

Embodiment IV-85. The method of embodiment IV-65, wherein the subject is less than 18 years old.

Embodiment IV-86. The method of embodiment IV-65, wherein the subject is between 5 years and 17 years old, between 6 months and 9 years old, or between 5 years and 9 years old.

Embodiment IV-87. The method of any one of embodiments IV-83 to IV-86, wherein the GAS bacterium is of a serotype selected from M1, M2, M3, M4, M6, M11, M12, M22, M28, M75, and M89.

Embodiment IV-88. The method of any one of embodiments IV-83 to IV-86, wherein the GAS bacterium is a serotype selected from M1, M3, M5, M9, M12, M18, M22, M25, M28, M71, M72, and M74.

Embodiment IV-89. The method of any one of embodiments IV-83 to IV-86, wherein the GAS bacterium is a serotype selected from M1, M4, M6, M11, M12, M22, M44, M75, M77, M77, and M81.

Embodiment IV-90. The method of any one of embodiments IV-83 to IV-86, wherein the GAS bacterium is a serotype selected from M1, M2, M3, M4, M6, M9, M12, M18, M22, M75, M77, M89, and M92.

Embodiment IV-91. The method of any one of embodiments IV-83 to IV-86, wherein the GAS bacterium is a serotype selected from M1, M2, M3, M4, M5, M6, M9, M11, M12, M13, M28, M62, and M89.

Embodiment IV-92. The method of any one of embodiments IV-83 to IV-86, wherein the GAS bacterium is a serotype selected from M1, M2, M3, M4, M6, M12, M22, M28, M49, M53, M68, M77, M80, M83, M87, M89, and M92.

Embodiment IV-93. The method of any one of embodiments IV-83 to IV-92, wherein the immunogenic composition induces an antibody response in the subject against the Group A Streptococcus (GAS) bacterium and does not induce an antibody response in the subject against human tissue.

Embodiment IV-94. The method of any one of embodiments IV-83 to IV-93, wherein the immunogenic composition induces a protective immune response against a Shigella bacterium in the subject, wherein the Shigella bacterium comprises a polysaccharide with a polyrhmanose backbone.

Embodiment IV-95. A method of inducing a protective immune response against a Shigella bacterium in a subject comprising administering the immunogenic composition of any one of embodiments IV-1 to IV-82 to the subject.

Embodiment IV-96. Use of the immunogenic composition of any one of embodiments IV-1 to IV-82 for inducing a protective immune response against a Shigella bacterium in a subject.

Embodiment IV-97. Use of the immunogenic composition of any one of embodiments IV-1 to IV-82 in the manufacture of a medicament for inducing a protective immune response against a Shigella bacterium in a subject.

Embodiment IV-98. Use of the immunogenic composition of any one of embodiments IV-1 to IV-82 in the manufacture of a medicament for inducing a protective immune response against a GAS bacterium in a subject.

Embodiment IV-99. Use of the immunogenic composition of any one of embodiments IV-1 to IV-82 for inducing a protective immune response against a GAS bacterium in a subject.

Embodiment IV-100. The method or use of any one of embodiments IV-93 to IV-99, wherein the subject is 18 years or older.

Embodiment IV-101. The method or use of embodiment IV-100, wherein the subject is less than 18 years old.

Embodiment IV-102. The method or use of embodiment IV-100, wherein the subject is between 5 years and 17 years old, between 6 months and 9 years old, or between 5 years and 9 years old.

INCORPORATION BY REFERENCE

All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

EXAMPLES
Example 1: Expression and Purification of GAS Polysaccharides

Experiments were performed to express and purify GAS polysaccharides from GAS bacterial cultures.

500 mL starter cultures were prepared from the appropriate recombinant GAS colony, allowing the bacteria to grow for several hours before inoculating a large 1.25 L culture for overnight growth at 37° C. without shaking. The bacteria were pelleted, washing once with PBS. 5 mL of cold, 48% aqueous hydrofluoric acid was added to the cell pellet in a 50 mL conical tube using polystyrene tips, carefully pipetting up and down using a serological pipette to disrupt the pellet. Using a small magnetic stir bar, the mixture was stirred for 48 hours at 4° C. In a fume hood, with the conical tubes on ice, 45 mL (10× volume of 48% HF) of ice cold Milli-Q® (ultrapure) water was added to the bacterial suspension in preparation for dialysis.

For dialysis, 4 L plastic beakers for dialysis were filled with deionized water. A stir bar was added to each, and the beakers were left to cool, covered, in the cold room. 1000 MWCO Regenerated Cellulose dialysis tubing was cut for each bacteria culture (~17 cm max for 4 L beakers). 3 cuts of tubing is needed for six 1.25 L cultures. Tubes were washed with DI water and rinsed with Milli-Q® (ultrapure) water before submerging in the beakers containing cold Milli-Q® (ultrapure) water. In a fume hood, the tubing was placed in the beakers before carefully being filled with the diluted bacterial suspension and sealed with a clip. The samples were dialyzed as follows: Day 1: 4° C. with stirring; Day 2: water change, 4° C., with stirring; Day 3: water change, room temperature, with stirring.

The contents of the dialysis tubing were transferred to centrifuge tubes and spun at 14,000 rpm for 20 min at 4° C. 50 mL conical tubes were rinsed with Milli-Q® (ultrapure) water 3× to remove any soluble plastic residues. Up to 30 mL of the supernatant containing polysaccharide was decanted into each tube and frozen in dry ice for 1 hour. The frozen samples were then lyophilized, and the residue was resuspended in 8 mL cold water, with gentle sonication if necessary. 1 mL of 10× proteinase K buffer (500 mM Tris HCl, 10 mM CaCl₂) and 12.5 mg of proteinase K per polysaccharide prep was added. The samples were incubated at 56° C. in a hybridization oven overnight, mixing occasionally. The reaction was terminated using 100 µL of 100 mM PMSF in ethanol (5 mM final) and was incubated for 1-2 hours at 37° C., mixing periodically. The mixture was then dialyzed in DI water with 0.05% sodium azide, with stirring, for 24 hours to remove salts. The resulting crude polysaccharide solution was frozen and lyophilized.

The crude polysaccharide was purified by size exclusion chromatography using a 100 cm x 1.5 cm Biogel P4 column pre-equilibrated with 7% ethanol, equipped with a spiral collector set for 20.0 min per sample. The crude material was re-suspended in 500 µL of 7% ethanol and centrifuged at 4000 rpm for 5 minutes to remove any insoluble particulate matter. The sample was loaded onto the column, and the preload volume and four subsequent fractions were eluted with 500 µL of 7% ethanol. The column was eluted with 7% ethanol, collecting for about 20 hours. Fractions were analyzed using the phenol-sulfuric acid method to detect the presence of polysaccharide, and samples were read at 490 nm to quantify. Fractions that tested positive in the phenol-sulfuric acid test were pooled and lyophilized to give the final purified PS product.

Example 2: Expression and Purification of pAMF-Modified Conjugate Polypeptide

Experiments were performed to express and purify pAMF-modified conjugate polypeptides from a cell free protein synthesis extract.

Conjugate polypeptides, for instance, SpyAD (SEQ ID NO: 11 or SEQ ID NO: 34), containing nnAAs (e.g., pAMF) were expressed in a cell free protein synthesis (CFPS) reaction, using an extract (XtractCF⁺) derived from E. coli engineered to produce an orthogonal tRNA for insertion of a nnAA at an amber stop codon. Sample protocols used for cloning, expression, and purification of these modified conjugate polypeptides may be found, for example, in Kapoor et al., Biochemistry, 2018, 57(5), 516-519.

As an example, controlled large scale antigen expression can be performed using a DASbox mini bioreactor system for 10 h at 25° C. with constant 650 rpm stirring, pH 7.2, dissolved oxygen 30%. After 10 h, reactions were harvested and spun down at 15,000 × g at 4° C. for 30 min, passed through a 0.45 µm filter, filtrate loaded on a 5 ml HisTrap excel column (Cytiva) equilibrated and extensively washed with Buffer A [50 mM Tris, 150 mM M NaCl, 10 mM imidazole] until absorbance returned to baseline (FIG. 17 shows sample gels from the purification of select SLO variants). Proteins were eluted using a 50% Step gradient of Buffer B [50 mM Tris, 150 mM M NaCl, 500 mM Imidazole], and elution fractions pooled, concentrated and incubated with excess purified his6-tagged TEV protease overnight while dialyzing against Buffer A (FIG. 18 shows a sample gel following the TEV cleavage of SLO peptides). The dialyzed cleavage reaction was loaded back onto a pre-equilibrated HisTrap excel 5 ml column (Cytiva) and untagged proteins collected in the flow-through (FT) fractions. Thereafter, the FT was concentrated and loaded onto a Superdex 200 26/60 size exclusion chromatography (SEC) column pre-equilibrated with Buffer S [50 mM Tris pH 8.0, 150 mM NaCl] and purity of eluted fractions assessed by SDS-PAGE gel analysis (FIG. 19 shows the purity of select SLO variants following size exclusion chromatography). Purified SpyAD[4pAMF] was incubated with excess Dibenzocyclooctyne-PEG4-tetramethylrhodamine (DBCO-TAMRA) dye for 1h at room temperature to confirm pAMF sites by SDS-PAGE gel and fluorescence readout was recorded using a Syngene G-box gel imager.

Polypeptides unmodified by nnAA were prepared using CFPS according to methods similar to the ones described above. FIG. 12 shows the modular architecture of GAS protein antigens and select immunogenic core fragments expressed in CFPS.

Single site mutagenesis studies were conducted on SpyAD (SEQ ID NO: 9), to understand how replacement of single amino acid residues with pAMF affected expression, as measured in both total protein and soluble protein. FIG. 1A shows the expression levels resulting from a lysine-scanning mutagenesis study. As shown, introduction of pAMF residues at single points in the SpyAD protein had variable effects on the total and soluble protein expression. FIG. 1B shows expression levels for antigens and antigen conjugates containing nnAAs. FIG. 13 shows greater than 95% purity of SLO variants, C5a peptidase, and SpyAD as measured by Safe Blue staining, and further shows incorporation of pAMF into SpyAD variant SpyAD[4pAMF] as confirmed by DBCO-TAMRA labeling.

Example 3: Synthesis and Purification of Rhamnose Polysaccharides Modified With Click Chemistry Reactive Groups

DBCO-modified polysaccharides were synthesized using a stepwise approach, allowing for tuning of oligomer length.

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Synthesis of Monosaccharide Intermediates 4a and 4b: Monomer 1 was mono-benzylated to furnish 2, which was further benzyl-protected to give intermediate 3a. Treatment of 3a with levulinic acid under esterification conditions provides levulinic (Lev)-ester monomer 4a. Alternatively, Fmoc-protection of 3a furnished monomer 4b.

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Synthesis of Disaccharide Donors 7a and 7b: Monosaccharide intermediates 4a/b were converted to phosphate esters 5a/b, which were subsequently treated with TMSOTf and 6a to furnish disaccharide donors 7a/b.

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Synthesis of Rhamnose Octasaccharide with a Click Chemistry Reactive Group: Azide intermediate 8 was coupled to monosaccharide intermediate 4b in the presence of NIS/TfOH to give dimer 9b, which was then subjected to three sequential deprotection/coupling reaction sequences with disaccharide donor 7b to give partially protected octamer intermediate 12b. Treatment of 12b with LiOH/methanol hydrolyzed the levulinic ester to give 13, and further treatment with H₂NNH₂/Pd(OH)₂ concomitantly reduced the pendent azide and reductively cleaved the remaining benzyl ethers to furnish 14. Coupling of 14 with DBCO-NHS gave the final octasaccharide 15.

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Polysaccharides of varying lengths, for instance the dodecasaccharide and hexasaccharide, can be prepared by altering the number of deprotection/coupling sequences with donor 7b as shown in the generic reaction scheme above.

Polysaccharides of the present disclosure can also be synthesized through solid-phase synthesis using the Fmoc-protected derivatives of the above intermediates, originating with monomer 4a.

Example 4: Linkage of Purified PS to DBCO-PEG Linker

Purified polysaccharides, for instance those produced by the methods of Example 1, can be functionalized with a DBCO-PEG linker.

Generally, to a solution of polysaccharide in water (5.5 mM final concentration after all reagents are added), borate buffer (1 M, pH 8.5) was added such that the final concentration of borate is 100 mM in the final volume. Water was then added to fill any extra reaction volume. 2.5 equivalents (with respect to the polysaccharide repeating unit) of 1-cyano-4-dimethylaminopyridinium tetra fluoroborate (CDAP; from 100 mg/mL solution in acetonitrile) was added with vigorous stirring. CDAP is stored at -20° C. and solution must be prepared immediately before use. Five minutes after the addition of CDAP (this timing is critical - any longer than 5 min results in reduced DBCO-PEG-Amine incorporation), 0.5 molar equivalents of dibenzocyclooctyne-amine linker (from DMSO stock solution, final concentration of DMSO is 5% v/v) was added. DBCO-PEG4-Amine linker is stored at -20° C. and must be prepared immediately before use. After one hour of further reaction, glycine (2 M, pH 8.35) was added 1:10 by volume to give a final concentration of 200 mM glycine to quench any unreacted cyanate esters. After 1 h of quenching, the derivatized polysaccharide was then purified via Zeba spin column. 2-3 mL of solution was added to each 10 mL Zeba column. The purified polysaccharide was analyzed on Bound/Free DBCO HPLC method to determine if residual DBCO-PEG4-Amine linker and DMAP were completely removed by column purification. The material can be further purified if necessary. The polysaccharide concentration was measured using an anthrone assay, and dibenzocyclooctyne concentration was measured using absorbance at 309 nm. These two values axcyte: were combined to give an estimate of the percentage of polysaccharide derivatized with a dibenzocyclooctyne functional group. Percent DBCO should be between 5-10% for CDAP reactions.

DBCO-PEG4 Derivatization of GAC: To a 6 mM solution of GAC in 100 mM Borate Buffer pH 8.5, three equivalents (to the polysaccharide repeating unit) of 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP; from 100 mg/mL solution in acetonitrile) were added with vigorous stirring to facilitate cyanylation at reactive hydroxyl groups. 5 minutes after addition of CDAP, 2 molar equivalents of dibenzocyclooctyne-amine linker stock in DMSO was added such that the final DMSO concentration was 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 via zeba spin desalting column and the purity of the recovered material was assessed by reverse phase. A single peak in HPLC when absorbance was monitored at 309 nm confirmed complete removal of excess DBCO linker and other reaction byproducts. Finally, the polysaccharide concentration was measured using anthrone assay (see below) and dibenzocyclooctyne concentration was measured using absorbance at 309 nm. These two values were combined to give an estimate of the percentage of polysaccharide derivatized with a dibenzocyclooctyne functional group. For conjugation, % DBCO derivatization of the GAC was kept between 5-10%.

Anthrone assay for total polysaccharide concentration: A stock of 2 mg/ml of the anthrone reagent (Sigma-Aldrich, CAS#90-44-8) was prepared in cold sulfuric acid while a 1 mM stock of polysaccharide repeating unit (PSRU) comprising 2× rhamnose was prepared in water as a standard. In triplicate wells, 100 µl of PSRU stock (serially diluted into reference standards) or the unknown samples (diluted 1:3) were plated (96-well plate) followed by addition of 200 µl / well of the anthrone reagent stock. All reactions were thoroughly mixed and sealed with a plate cover for incubation at 95° C. for 10 min. The plate was briefly placed on ice to cool to ambient temperature before absorbance is measured at 620 nm using a UV/VIS plate reader. To determine concentration of unknown samples, PSRU standard concentrations and absorbances were used to generate a least-square fit regression.

Example 5: Conjugation of Carrier Protein to DBCO-Derivatized Polysaccharides

Generally, carrier proteins ferritin (SEQ ID NO: 23), SpyAD (SEQ ID NO: 34), ADI (SEQ ID NO: 27 and SEQ ID NO: 38), SEQ ID NO: 25, and Protein D containing nnAAs (e.g., pAMF) were conjugated to the purified DBCO-derivatized polysaccharides of Example 3 and Example 4 by reacting the cyclooctyne moiety of the DBCO group with the azide moiety of the nnAA side-chain incorporated into the carrier protein. Sample protocols for the conjugation reaction between the DBCO and azide groups may be found, for example, in Zimmerman et al., Bioconjugate Chemistry, 2014, 25(2), 351-361 and Kapoor et al., Biochemistry, 2018, 57(5), 516-519.

Conjugation of pAMF-derivatized GAC to SpyAD: SpyAD[4pAMF] (SEQ ID NO: 34) was mixed with DBCO-derivatized GAC at a 1:1 ratio [0.5 mg/ml each] to facilitate conjugation via click chemistry. Post-conjugation, the reaction mixture was dialyzed against a 50 kDa cutoff membrane to remove excess unreacted free polysaccharide. The recovered conjugates were analyzed by SEC (multi-angle light scattering) MALS and the concentration was estimated using an anthrone assay. FIG. 9 is a scheme outlining the derivatization of GAC with DBCO-(PEG)₄-NH₂ and subsequent conjugation to pAMF-derivatized SpyAD. FIG. 10 shows SEC MALS analysis of purified native GAC, purified native SpyAD[4pAMF], and purified conjugates. SEC-MALS analysis of purified native GAC estimates an average molecular mass of 7.2 kDa. SEC-MALS analysis of purified native SpyAD[4pAMF] estimates an average molecular mass of 87.3 kDa. Post click chemistry SEC-MALS analysis of purified conjugates estimates an average combined molecular mass of 153.4 kDa. Inset shows the safe blue stained SDS-PAGE analysis of SpyAD[4pAMF] pre- and post-conjugation, with no unreacted protein left after conjugation.

SEC MALS-UV-RI was performed with an Agilent HPLC 1100 degasser, temperature-controlled auto-sampler (4° C.), column compartment (25° C.) and UV-VIS diode array detector (Agilent, Santa Clara, CA) in line with a DAWN-HELEOS multi-angle laser light scattering detector and Optilab T-rEX differential refractive interferometer (Wyatt Technology, Santa Barbara, CA) coupled to three TOSOH columns in series: TSKgel Guard PWXL 6.0 mm ID x 4.0 cm long, 12 µm particle; TOSOH TSKgel 6000 PWXL 7.8 mm ID x 30 cm long, 13 µm particle; and a TSKgel 3000 PWXL 7.8 mm ID x 30 cm long, 7 µm particle. A mobile phase consisting of 0.2 µm filtered 1× PBS with 5% (v/v) acetonitrile was used at a 0.5 mL/min flow rate and 50-100 µg sample was injected for analysis. Agilent Open Lab software was used to control the HPLC, and Wyatt Astra 7 software was used for data collection and molecular weight analysis.

Example 6: Antibody Responses Induced by GAS Antigens and Antigen-Polysaccharide Conjugates

Experiments were performed to assess the antibody responses produced purified GAS antigens and conjugates thereof. Briefly, rabbits were immunized intramuscularly with 50 µg of each indicated antigen or conjugate or vehicle control on days 0, 14, and 28. Animals were bled prior to immunization (pre-bleed), and on Days 7, 21, and 35 (terminal bleed) after immunizations. Serum was harvested from each blood sample and antibody titers against the immunizing antigens were assessed by ELISA.

For ELISA analysis of antibody titers, the indicated recombinant protein antigen was plated in sterile PBS at a concentration of 3 µg/well. High-binding, flat bottom 96-well plates were coated with 100 µL of prepared antigen, incubating overnight at room temperature without agitation. The plates were washed 3 times with 100 µL PBS + 0.05% Tween 20 using an Aquamax Microplate Washer, then blotted dry onto paper towels. Wells were blocked with 100 µL of Reagent Diluent 2 for 2 hours. 100 µL of PBS with 1% BSA can also be used. Plates covered with a lid and sealed can be stored in block solution at 4° C. for 3 days. Plates were then washed 3 times with 100 µL PBS + 0.05% Tween 20 as before.

Sera samples are prepared by diluting the sample into 1× Reagent Diluent 2 (typical dilution is 1:400 to 1: 10⁶). 100 µL of the prepared sera was then added to the plates, followed by 2 hours of incubation. Pooled pre-immunization rabbit serum was used as a negative control, and wells with no rabbit serum were used as a background control. Plates were washed 3 times with 100 µL PBS = 0.05% Tween 20 as before. Plates were then incubated with 100 µL of HRP-conjugated goat-anti rabbit IgG (H+L) secondary antibody (diluted 1:4000 into 1× Reagent Diluent 2) for 2 hours. Plates were then washed 3 times with 100 µL of PBS + 0.05% Tween 20 as before. TMB substrate was prepared following the manufacturer’s instructions, and equal volumes of reagent A and reagent B were added at room temperature. 100 µL of prepared TMB substrate was added per well, and the plates were incubated protected from light for no more than 30 minutes. 50 µL of 2N sulfuric acid was added per well to stop the reaction. ELISA plates are read at 450 nm and 540 nm, subtracting the values at 540 nm from the values at 450 nm to correct for background. Titers were determined based on cut off: absorbance value of background controls + 3* standard deviation of background controls.

As shown in FIG. 2, immunization with ADI (SEQ ID NO: 38), SLO (SEQ ID NO: 32), C5a (SEQ ID NO: 30), SpyCEP, Sib35, SpyAD (SEQ ID NO: 33), and SpyAD-PS conjugates (conjugates of pAMF-substituted SEQ ID NO: 34) elicited a robust antibody response as compared to pre-bleed controls. Sfb1 was also tested but generated weak antibody titers. Each individual antigen identifier in the different panels represents a different rabbit in the experiment.

Example 7: GAS-Antigen Elicited Antibody Binding to GAS Serotypes

Experiments were performed to assess the ability of GAS-antigen induced antibody responses to bind various GAS serotypes. Briefly, rabbits were immunized with 20 µg of SpyAD-PS conjugate or 50 µg of C5a peptidase on days 0, 14, and 28. Animals were bled prior to immunization (pre-immune serum), and on Days 7, 21, and 35 after immunization (immune serum). Serum was harvested from each blood sample.

All serum samples were heat inactivated at 55° C. for 30 minutes. GAS of various serotypes were grown to OD₆₀₀=0.4, washed and frozen at 2×10⁸ cfu/mL. Frozen GAS stock was thawed, 10% goat serum was added, and the mixture was incubated at room temperature for 1 hour. For the binding assay, 100 µL blocked GAS (together with goat serum) was added to 1.5 mL micro centrifuge tubes. 2 µL pre-immune or immune serum was added to each tube. The samples were mixed well and incubated at room temperature for 1 hour. The samples were spun down at 3500 G for 5 minutes washed 1 time with 500 µL PBS. Samples were then incubated with a fluorescent secondary anti-rabbit secondary antibody for. The samples were then transferred to FACS tubes in 500 µL PBS. Fluorescent signal intensity was measured by flow cytometry and calculated using FlowJo software, comparing the signal intensity of immune and pre-immune serum.

FIG. 3A shows the IgG-binding of sera post-immunization with SpyAD-conjugate (conjugate of SEQ ID NO: 34) vs. that of pre-immune sera to strains of M1 (1-21). FIG. 3B shows the IgG-binding of sera post-immunization with C5a Peptidase (SEQ ID NO: 30) vs. that of pre-immune sera to various M1 strains (1-21). The extent of IgG to SpyAD Conjugate and C5a Peptidase binding to all clinically relevant GAS strains is shown in FIG. 3C, with both showing strong binding to > 95% of clinically relevant GAS.

Additional Evaluation of GAS Vaccine Antigens for Immunogenicity: According to the protocols above, animals were immunized with 5 µg of either SLO, C5a peptidase, or SpyAD-GAC conjugate in 240 µL total volume succinate bugger adjuvanted with Adju-phos (Invivogen). The determine immunoglobulin g (IgG) titers elicited by vaccination, terminal bleed (day 35) rabbit antisera were evaluated by ELISA. For all three protein antigens (SpyAD, C5a peptidase, and SLO), the group of rabbits immunized with the recombinant protein showed significantly increased (3- to 4-log₁₀fold) antibody titers against the target antigen compared to either pooled serum from the rabbits before immunization (“pre-immune (pooled)”) or the other immunized rabbit groups (FIG. 3D, each point represents serum derived from one animal, run with 2-3 animals per group), confirming specificity of the response to each respective antigen. The anti-SpyAD protein titer generated by the SpyAD-GAC conjugate was not inferior to that of jthe SpyAD recombinant protein alone, suggesting that key B cell epitopes were not impacted by pAMF sites. Flow cytometry was used to evaluate the binding affinity of rabbit-derived IgG to the surface of eight live wild-type GAS strains of different M protein serotypes (m1-6, M12, M28). For the great preponderance of strains, the respective immunized serum yielded an increase in binding of 1000-400% over the baseline IgG binding of the pre-immune serum (FIG. 3E, numbers in the top right corner of each histogram show the mean percentage of increased signal of immunized rabbit serum over pre-immunized serum signal). For six of the eight strains, the GAS surface binding of the antisera raised against the SpyAD-GAC conjugate roughly doubled the level of IgG binding seen with antisera raised against SpyAD alone. FIG. 3F shows rabbit IgG binding to SpyAD knockout GAS, confirming binding to native GAC.

Example 8: Neutrophil Killing Assay

Experiments were performed to determine the ability of immune sera from antigen and conjugate-immunized rabbits to elicit neutrophil killing of M1, M3, and M3 strains of GAS. Rabbits were immunized with the indicated antigens according to the protocol described in Example 7. For single antigens, rabbits were immunized with 50 µg of antigen. For conjugate antigens, rabbits were immunized with 20 µg of conjugate.

Neutrophil isolation: 25 mL blood was drawn from healthy volunteers, using 21 G butterfly needles, into vacutainer heparin tubes. Neutrophils were isolated from fresh blood using Polymorphprep following the manufacturer’s protocol. All serum, including pre-immune serum, immune serum, and FBS, were heated at 65° C. for 30 minutes with occasional mixing before being used in the assay.

Neutrophil Killing Assay Protocol: Overnight cultures of GAS strains were inoculated in Todd Hewitt Broth (THB) at a ratio of 1:10. When the culture reaches OD₆₀₀=0.4, the culture was centrifuged and re-suspended in HBSS with 10% pre-immune or immune serum to 5×10⁵ colony forming units (CFU)/mL. GAS strains were kept in RPMI + serum for 30 minutes at room temperature. Prepped neutrophils were re-suspended in HBSS + 4% FBS + 4% Baby Rabbit complement (BRC) in a concentration of 5×10⁶ cells/mL. 100 µL neutrophils (5×10⁵ cells) were seeded in a 96-well flat bottom plate, with each condition run in triplicate. 100 µL of prepared bacteria (5×10⁴ cfu/well) were added to each well of neutrophils. The inoculated bacteria were plated on blood agar plate in serial dilutions to later count inoculum cfu. The 96 well plate was centrifuged at 500 G for 5 minutes and then incubated at 37° C. + 5% CO₂ for 30 minutes. Following 30 minutes of incubation, the samples were mixed well, plated on blood agar plates in serial dilutions, and incubated at 37° C. overnight. GAS colonies were counted and the % ratio of inoculum CFU versus recovered CFU is calculated as % survival. Increase in % kill compared to the pre-immune serum group was calculated by first calculating % killing (100-% survival) in both pre-immune and immune serum groups. Then, the % increase in the killing of the immune serum group was calculated compared to the % killing of the pre-immune serum group.

FIG. 4 shows the ability of serum resulting from immunization by antigens ADI 1625 (SEQ ID NO: 36), ADI conjugate mutant 1807 (conjugate of pAMF-substituted SEQ ID NO: 38), ADI conjugant mutant 1809 (conjugate of SEQ ID NO: 38), SLO 1627 (SEQ ID NO: 32), C5a peptidase 1630 (SEQ ID NO: 30), SpyAD 1642 (SEQ ID NO: 33), SpyAD-conjugate 1644, 1630+1627+1644, 1630+1627+1809, 1630+1627, and M1 to increase killing of GAS serotypes M1, M2, and M3 over pre-immune serum. The pAMF-substituted SpyAD conjugates were made using the peptide of SEQ ID NO: 12, and the ADI conjugates were made using the peptide of SEQ ID NO: 20.

Example 9: GAS Antigens and Conjugates Elicit Active Immune Responses That Increase Survival After GAS Challenge

Mice were actively immunized prior to being challenged by subdermal and IP injection.

TABLE 5

Active Immunization Experiment Timeline - Subdermal and IP Challenges

Subdermal Challenge - Steps
IP Challenge - Steps

Day 1: 1^st immunization
Day 1: 1^st immunization

Day 14: 2^nd immunization
Day 14: 2^nd immunization

Day 28: 3^rd immunization
Day 28: 3^rd immunization

Day 42: infection challenge
Day 41: shave mice

Day 49: terminate experiment
Day 42: infection challenge

Day 45: euthanize mice and collect lesions

1^st, 2^nd, and 3^rd Immunizations - Subdermal and IP Challenges: Antigen/adjuvant mixtures were prepared by combining 50 µL alum (Alhydrogel) with 10 µg antigen(s) or 5 µg of conjugate and mixing rigorously to allow antigens to adsorb onto the alum. Each antigen/adjuvant mixture was drawn into 1 mL syringes fitted with 26 ½ gauge needles. Each mouse was anesthetized with inhaled isoflurane and injected with 100 µL of the prepared vaccine into the hind leg muscle.

Preparation of Mice for Challenge - Subdermal Challenge: Mice were anesthetized with isoflurane. The backs of the mice were shaved with an electric razor, with care taken not to nick the skin. Hair depilation cream was applied to the shaved backs and was allowed to sit for a few minutes before thoroughly wiping them clean with damp paper towels. The mice were patted dry and allowed to recover from isoflurane treatment.

Preparation of Materials for Challenge - Subdermal and IP Challenges: M1 89155 strain GAS was grown to mid-logarithmic phase. The cell concentration was adjusted with sterile phosphate buffered saline, serially diluting and plating bacteria onto agar to confirm bacterial dose. For Subdermal Challenge, the targeted CFU per 10 µL per mouse was 1×10⁶, and the bacteria was drawn into 500 µL Hamilton syringes fitted with 26 ½ gauge needles. For IP Challenge on day 35, the targeted CFU per 100 µL per mouse is 1×10⁷, and the bacteria was drawn into 1 mL syringes fitted with 26 ½ gauge needles. The mice were anesthetized with inhaled isoflurane and then injected with 200 µL of M1 89155 bacteria into the peritoneal cavity. The mice were allowed to recover from the anesthetic in normal air. Survival of the mice was tracked over the course of 1 week, checking multiple times per day.

Subdermal Challenge and Lesion Collection: For subdermal challenges on day 35, the mice were anesthetized with inhaled isoflurane and then injected with 10 µL of M1 89155 bacteria into the shaved backs using a repeat dispenser for the Hamilton syringe. Lesion sizes were tracked daily over the course of 3 days by photographing isoflurane-anesthetized mice alongside a ruler. Prior to lesion collection on day 3, sterile 2 mL screw cap tubes with 1.0 mm silica beads and 1 mL PBS for each skin lesion were prepared. The weights of each tube were recorded. On day 3, when lesions were fully developed, the mice were euthanized with CO₂ and cervical dislocation. Using clean surgical instruments, each skin lesion was cut out and placed into the pre-weighed tubes. Tube weights are recorded for tissue mass calculations. The tubes were placed into a MagnaLyser bead beater, and the tubes were beat at 6000 rpm for 60 s. The tubes were then placed onto ice to cool for 60 s before repeating the beating cycle. Samples were serially diluted, and the lysate is placed onto agar to quantify bacterial burden.

Two parallel experiments were performed in which in one set of animals were bled throughout the experiment in order to test for antigen-specific antibody titers post-vaccination. In the other arm, the mice were not bled during the course of the experiment. Both set of animals were challenged similarly in the end to perform the lesion size & CFU/mg analysis. FIG. 5A shows the results of bled-arm active immunization experiments as measured by lesion size and lesion CFU/mg, demonstrating that the conjugates of the present disclosure perform better than polysaccharides alone. Similarly, FIG. 5B show the results of the no-bleed-arm active immunization experiments as measured by lesion size and lesion CFU/mg, wherein the GAC conjugates of SpyAD (conjugates of pAMF-substituted SEQ ID NO: 34) and ADI (ADImut2 conjugates use pAMF-substituted SEQ ID NO: 27, and ADImut5 conjugates use pAMF-substituted SEQ ID NO: 38) conveyed better protection than the corresponding protein alone. The best protection was conferred by C5a peptidase (SEQ ID NO: 4). FIG. 5C shows the first, second, and third antibody titers after each immunization. FIG. 5D shows survival data for mice that have undergone active immunization and were subsequently subjected to IP challenge. The only group to show some protection was the group administered a 6-antigen combination with 30 µg total protein.

Active Immunization with Antigen or Antigen Conjugate Combinations: As above, mice were actively immunized with one or more antigen or antigen conjugate prior to being challenged by IP injection.

TABLE 6

Active Immunization Experiment Timeline - Subdermal and IP Challenges

IP Challenge - Steps

Day 1: 1^st immunization

Day 14: 2^nd immunization

Day 28: 3^rd immunization

Day 42: IP challenge

Mice (n=10 per group) were immunized with one of 8 treatments: mock immunization (PBS + alum); 5 µg of SpyAD-GAC conjugate (conjugate of pAMF-substituted SEQ ID NO: 34); 5 µg of ADImut5-GAC conjugate (conjugate of pAMF-substituted SEQ ID NO: 38); 10 µg of C5a peptidase (SEQ ID NO: 30); 10 µg streptolysin O (SEQ ID NO: 32); a combination of 10 µg of C5a peptidase (SEQ ID NO: 30) and 10 µg streptolysin O (SEQ ID NO: 32); a combination of 10 µg of C5a peptidase(SEQ ID NO: 30), 10 µg streptolysin O (SEQ ID NO: 32), and 5 µg of SpyAD-GAC conjugate (conjugate of pAMF-substituted SEQ ID NO: 34); or a combination of 10 µg of C5a peptidase (SEQ ID NO: 30), 10 µg streptolysin O (SEQ ID NO: 32), and 5 µg of ADImut5-GAC conjugate (conjugate of pAMF-substituted SEQ ID NO: 38). The SpyAD conjugates were made using the peptide of SEQ ID NO: 12, and the ADImut5 conjugates were made using the peptide of SEQ ID NO: 20.

FIG. 5E shows the survival of mice actively immunized with single antigens/antigen conjugates or two antigens/antigen conjugates (combination) after IP challenge. Mice immunized with SLO along had the greatest survival, but no individual or dual antigen combination provided 100% protection six days after challenge. Notably, when mice were actively immunized with the triple combinations prior to IP challenge, 100 percent survival was observed after 6 days. These data are shown in FIG. 5F.

Example 10: GAS Antigens and Conjugates Elicit Passive Immune Responses That Increase Survival After GAS Challenge

Mice were passively immunized prior to being challenged by IP injection.

Experiments were performed to assess the protection conferred by anti-sera isolated from GAS antigen and GAS conjugate-immunized mice when transferred to mice that were subsequently challenged with GAS. Briefly, rabbits were vaccinated with 5 µg of ADI antigen (SEQ ID NO: 36), C5a antigen (SEQ ID NO: 30), Sfb1 antigen (SEQ ID NO: 28), SpyAD antigen (SEQ ID NO: 33), ADI:GAC conjugates (conjugates of pAMF-substituted SEQ ID NO: 38), or SpyAD:GAC conjugates (conjugates of pAMF-substituted SEQ ID NO: 34) at weeks 0, 3 and 6. Serum was collected from each animal on week 8.

Passive Immunization - IP Challenge: Thawed rabbit serum (approximately 200 uL per mouse), transferred on ice, was drawn into a 1 mL syringes fitted with 30 gauge needles. M1 89155 strain GAS, grown to mid-logarithmic phase, was diluted with sterile phosphate buffered saline to adjust cell concentration. The targeted CFU per 100 µL per mouse was 1×10⁷, and the bacteria was drawn into 1 mL syringes fitted with 26 ½ gauge needles. The rabbit serum was warmed in syringes to room temperature, and the mice were anesthetized with inhaled isoflurane. 200 µL of the rabbit serum was retro-orbitally transferred into each anesthetized mouse. Five minutes after serum transfer, 200 µL of the M1 81955 bacteria was injected into the peritoneal cavity, and the mice were allowed to recover from the isoflurane in normal air. Survival of the mice was tracked over the course of 1 week, checking multiple times per day.

Survival of mice that have undergone passive immunization and were subjected to subsequent GAS challenge are shown in FIG. 6A and FIG. 6B. All groups treated with antigens and antigen conjugates (pAMF conjugates) of SLO 1627, ADI-conjugate Mut5 1809, SpyAD conjugate 1644, C5a peptidase 1630, SLO+C5a peptidase, SLO+C5a+ADI-conjugate, and SLO+C5a+SpyAD-conjugate showed improved survival over the pre-immune immunized cohort.

Example 11: GAS Antigens and Conjugates Elicit Passive Immune Responses That Increase Survival After GAS Challenge

Mice were passively immunized prior to being challenged by subdermal injection.

TABLE 7

Passive Immunization Experiment Timeline - Subdermal Challenge

Subdermal Challenge - Steps

Day -1: prepare mice

Day 0: prepare materials, serum transfer, and challenge

Day 3: lesion collection

Preparation of Mice for Challenge - Subdermal Challenge: Mice were anesthetized with isoflurane. The backs of the mice were shaved with an electric razor, with care taken not to nick the skin. Hair depilation cream was applied to the shaved backs and is allowed to sit for a few minutes before thoroughly wiping them clean with damp paper towels. The mice were patted dry and allowed to recover from isoflurane treatment.

Preparation of Materials, Serum Transfer, and Challenge - Subdermal Challenge: M1 89155 strain GAS was grown to mid-logarithmic phase. The cell concentration was adjusted with sterile phosphate buffered saline, serially diluting and plating bacteria onto agar to confirm bacterial dose. For Subdermal Challenge, the targeted CFU per 10 µL per mouse was 1×10⁶, and the bacteria was drawn into 500 µL Hamilton syringes fitted with 26 ½ gauge needles. Rabbit serum was thawed and transferred on ice before being drawn into 1 mL syringes fitted with 30 gauge needles. The rabbit serum was warmed in syringes to room temperature, and the mice were anesthetized with inhaled isoflurane. 200 µL of the rabbit serum is retro-orbitally transferred into each anesthetized mouse. Five minutes after serum transfer, 10 µL of the M1 81955 bacteria was injected into the shaved backs of the mice using a repeat dispenser, and the mice were allowed to recover from the isoflurane in normal air. Lesion sizes were tracked daily over the course of 3 days by photographing isoflurane-anesthetized mice alongside a ruler.

Lesion Collection for bacterial burden analyses: Prior to lesion collection on day 3, sterile 2 mL screw cap tubes with 1.0 mm silica beads and 1 mL PBS for each skin lesion were prepared. The weights of each tube were recorded. On day 3, when lesions are fully developed, the mice were euthanized with CO₂ and cervical dislocation. Using clean surgical instruments, each skin lesion was cut out and placed into the pre-weighed tubes. Tube weights were recorded for tissue mass calculations. The tubes were placed into a MagnaLyser bead beater, and the tubes were beat at 6000 rpm for 60 s. The tubes were then placed onto ice to cool for 60 s before repeating the beating cycle. Samples are serially diluted, and the lysate was placed onto agar to quantify bacterial burden.

Lesion size and bacterial burden data of mice that have undergone passive immunization and were subjected to subdermal challenge with GAS are shown in FIG. 7. Mice (n=10 treated with antigens and antigen conjugates ADI (SEQ ID NO: 36), ADI-conjugate (SEQ ID NO: 38), SLO (SEQ ID NO: 32), C5a peptidase (SEQ ID NO: 30), sfb1 (SEQ ID NO: 28), SpyAD (SEQ ID NO: 33), SpyAD-conjugate (conjugate of pAMF-substituted SEQ ID NO: 34) exhibited reduced lesion size after 48h compared to the pre-immune cohort, and bacterial burden in most arms was significantly lower after 72 hours, with the SpyAD conjugate performing the best at reducing bacterial burden.

Example 12: Human Phase I and Phase IIA Clinical Trial Design

Initial Phase I clinical studies will be a randomized, placebo-controlled, ascending dose study in healthy adults 18-29 years of age (N=96) (Table 3). Objectives of this initial clinical study will be safety, dose response, and immunogenicity (IgG antibody). Since many individuals at this age range will have pre-existing exposure and immunity to GAS, baseline immunity will be fully evaluated to understand the impact of pre-existing immunity on vaccine responses. IgG response to each vaccine component and OPK antibody titer against a diverse panel of contemporary GAS isolates of different M serotypes will be evaluated.

TABLE 8

Phase I Clinical Trial Groups

Phase I Clinical Trial Design

Group
N
Treatment
Dose
Schedule

1A
24
GAS vaccine
Low
Day 0, 28

1B
8
Placebo

Day 0, 28

2A
24
GAS vaccine
Mid
Day 0, 28

2B
8
Placebo

Day 0, 28

3A
24
GAS vaccine
High
Day 0, 28

3B
8
Placebo

Day 0, 28

The Phase 2A clinical study will be a randomized, placebo controlled, multi-center study to evaluate the vaccine in successive cohorts of individuals from 10 to 17 years of age, followed by children 5 to 9 years old (N=96) (Table 4). The objectives of this study will be safety, immunogenicity (IgG antibody responses and opsonophagocytic activity of serum), and an evaluation of preliminary efficacy (incidence of GAS pharyngitis). Each patient will be monitored for 12 months to determine the incidence of strep pharyngitis in the treatment groups.

TABLE 9

Phase IIA Clinical Trial Groups

Phase IIA Clinical Trial Design

N
Age (yr)
Treatment
Dose
Schedule

36
10-17
GAS vaccine
Determined by PI
Day 0, 28

12
10-17
Placebo

Day 0, 28

36
5-9
GAS vaccine
Mid
Day 0, 28

12
5-9
Placebo

Day 0, 28

Example 13: GAS Conjugates Induce Generation of Anti-Sera That Binds to S. Flexneri 2a OPS in a Cross-Reactivity Experiment

Experiments were performed to assess the binding of anti-sera produced by purified GAS conjugates to S. flexneri 2a O-antigen polysaccharide (OPS).

OPS Purification: OPS was harvested directly from lipopolysaccharide (LPS) in Shigella cell biomass transformed with pSEC10-wzzB plasmid to overexpress wzzB, resulting in increased OPS chain length and conditioned growth media of fermentation (supplemented with amino acids), or shake flask (STm D65) cultures, by reducing the culture pH to 3.5-3.7 with glacial acetic acid, and incubating at 100° C. for 4 h in glass bottles submerged in a boiling water bath. Post-hydrolysis supernatants were separated from insoluble material by centrifugation at 10k x g at 4° C. for 30 minutes using a GS3 Rotor in a Sorvall RC5 refrigerated centrifuge. The supernatant fraction was brought to 1 M NaCl and filtered by tangential flow microfiltration through a 0.2 µm hollow-fiber filter at 4.5 psi transmembrane pressure (TMP), passing the full volume through, followed by flushing with an equivalent volume of 1 M NaCl. The 0.2 µm-cleared 1 M NaCl permeate was then concentrated 10-fold on a 30 kDa Hydrosart TFF membrane at 14 psi TMP and diafiltered against 35 diavolumes of 1 M NaCl, followed by 10 diavolumes of 50 mM Tris pH 7.

The retentate fraction in 20 mM Tris pH 7, 50 mM NaCl was then passed through 3 × 3 mL Sartobind NanoQ anion exchange membranes, linked in series, using an AKTA Purifier at 10 mL/min in 20 mM Tris pH 7, 50 mM NaCl. The flow-through fraction was brought to 25% (v/v) ammonium sulfate and incubated overnight at 4° C. Precipitated material was removed by centrifugation at 10k x g / 4° C. for 30 min using a GS3 rotor in a Sorvall RC5 refrigerated centrifuge followed by filtration through a 0.45 µm Stericup vacuum filter unit (Millipore, MA). Filtrates were then concentrated 10-fold by TFF with a Slice 200 TFF device using a 10 kDa Hydrosart membrane at 7.5 psi TMP, and diafiltered against 10 diavolumes of de-ionized water. TFF retentates were lyophilized and stored at -20° C. until use.

Immunization: Briefly, rabbits were immunized intramuscularly with ADI-mut2, ADI-mut5, and SpyAD conjugates according to the protocols of Example 6.

Serum IgG ELISA: Serum was harvested from each terminal blood draw, and serum IgG antibody titers against S. flexneri 2a OPS were assessed by ELISA. A working solution for the 2a OPS antigen was prepared as follows: 5.0 µg/ml of purified 2a OPS was diluted in carbonate coating buffer pH 9.6. Subsequently, Immulon 2HB “U” bottom microtiter plates (Thermo Labsystems #3655) were coated by adding 100 µl of the working solution to each well of a plate. Plates were then incubated at 37° C. for 3 h. Following this incubation, plates were washed six times with PBS-Tween (0.05%) with a two-minute soaking period between washes. Then the plates were blocked overnight at 4° C. with 1X PBS containing 10% non-fat dry milk (NFDM) at 250 µl/well. After blocking, the plates were washed again as stated above.

The test samples (rabbit sera) and pre-bleed controls were diluted in PBS-Tween 10% NFDM and were added to the plates. The specimens and controls were tested in duplicate in a series of dilutions performed on each plate. Plates were incubated for 1 h at 37° C. and then washed with PBS-Tween as described above. Next, Horseradish Peroxidase (HRP)-labeled anti-Rabbit IgG (Invitrogen #65-6120) was diluted to 1:2000 in PBS-Tween 10% non-fat dry milk (NFDM). All wells received 100 µl of the appropriate antibody solution and plates were incubated for 1 h at 37° C. Plates were again washed and 100 µl of TMB Microwell Peroxidase Substrate (SeraCare #5120-0047) was added to each well. Plates were incubated at room temperature for 15 minutes in darkness with agitation. The colorimetric reaction was stopped by adding 100 µl of 1 M phosphoric acid to all wells. Absorbance values at 450 nm were immediately measured using a Multiskan FC™ Microplate Reader.

FIG. 8 shows the result of the titer analysis. Notably, sera derived from all three GAS conjugates, ADI-mut2 (conjugates of pAMF-substituted SEQ ID NO: 27), ADI-mut5 (conjugates of pAMF-substituted SEQ ID NO: 38), and SpyAD (conjugates of pAMF-substituted SEQ ID NO: 34), exhibited up to approximately 60-fold increase in binding to S. flexneri 2a OPS at a 1:10 dilution versus pre-bleed control.

Example 14: GAS Conjugates Demonstrate Bactericidal Activity Against S. Flexneri Strains in a Serum Bactericidal Assay (SBA)

Experiments were performed, to determine the ability of immune sera from GAS conjugate-immunized rabbits to elicit neutrophil killing of S. flexneri strains. Rabbits were immunized with the indicated conjugates according to the protocol described in Example 7 prior to performing the bactericidal assay. Briefly, the serum bactericidal assay (SBA) and digital image analysis of colonies was performed according to methods previously described (Nahm, et al. mSphere, 2018, 2018 June 13;3(3), pii: e00146-18, and SBA Protocol linked to therein).

Table 9 shows the bactericidal ability of sera resulting from immunization by SpyAD conjugate 1645 (conjugate of pAMF-substituted SEQ ID NO: 34) and ADI conjugant mutants 1807, 1808, 1809, and 1810 (conjugates of pAMF-substituted SEQ ID NO: 38) against S. flexneri 2a, 3a, and 6. Values shown are SBA (serum bactericidal activity) titers, and all five conjugates demonstrated bactericidal activity versus pre-immune control. No killing was observed for S. sonnei, and titers in this arm are therefore reported as half of the lowest dilution tested. PnQC19, a human control serum, was used to normalize Shigella SBA titers as previously described (Nahm, et al. mSphere, 2018, 2018 June 13;3(3), pii: e00146-18). Titers shown for S. flexneri 6 are the average of raw titers (2 experiments). There is no assigned PnQC19 value for S. flexneri 6.

TABLE 10

Serum Bactericidal Activity of Rabbit GAS antibodies

Conjugate

S. flexneri 2a

S. flexneri 3a

S. flexneri 6

S. sonnei

pre-immune serum
nd
nd
233
nd

1645
1301
1333
980
30

1807
384
2496
553
30

1808
671
703
394
30

1809
487
1325
1401
30

1810
1654
652
532
30

PnQC19
28000
19000
6915
1100

Example 15: Immunization With SpyAD-GAC Does Not Incite Detectable Levels of Antibodies Cross-Reactive to Human Heart Epitopes

Varying amounts of normal adult human heart tissue lysate (Novus Biologicals cat# NB820-59217) with SDS-containing denaturing loading dye were separated by SDS-PAGE using 4-12% Bis-Tris gels before transfer onto a PVDF membrane using the manufacturer’s protocol on iBlot (Thermo Fisher). Finally, the blot was blocked at ambient temperature for 1 h, followed by probing with rabbit antisera generated against each of the GAS antigens (diluted 1:1000). After three 30 min washes, HRP conjugated anti-rabbit (Jackson ImmunoResearch Laboratories, Inc., Cat # 211-035-109) secondary antibody (diluted 1: 10,000) was added and chemiluminescence recorded on an Syngene G-Box F3 image scanner after incubation of the blot with the pico substrate (Thermo Fisher Scientific Cat # 34580). All blot blocking, washes, and antibody / serum dilutions were done in TBS + 5% BSA.

Western blot analysis of the SLO, C5a peptidase and SpyAD-GAC rabbit immune sera on normal human heart lysates separated by polyacrylamide gel electrophoresis was performed. Antisera raised against the intact GAS M1 protein using the same rabbit immunization protocol served as a control. As shown in FIG. 11, while anti-M1 antisera reacted to very high molecular weight components of the lysate, no cross-reactivity to the human heart tissue was seen for the SLO (SEQ ID NO: 8), C5a peptidase (SEQ ID NO: 4) and SpyAD-GAC (conjugate of pAMF-substituted SEQ ID NO: 12) antisera, although they recognized the respective cognate GAS protein (or protein carrier in the case of SpyAD-GAC). This analysis is consistent with (a) the lack of sequence homology of SLO, C5a peptidase or SpyAD to human proteins, and (b) knowledge that GAC is comprised solely of repeating rhamnose, a sugar absent in humans, following the genetic deletion of its GlcNAc side chain, which represented a common mammalian sugar epitope.

Example 16: Truncated SLO Peptides Were Expressed and Assessed for Their Immunogenicity in Both Rabbit and Murine Models

Truncated SLO variants were expressed, for instance, according to the above methods. FIG. 15 shows a graphical representation of 10 such variant sequences (ΔC11, ΔC21, ΔC31, ΔC41, ΔC51, ΔC61, ΔC71, ΔC81, ΔC91, and ΔC101), corresponding to SEQ ID NO: 44 to SEQ ID NO: 53 (post-TEV cleavage). FIG. 16 shows the expression of these variants, in both total and soluble protein concentration with their corresponding gels. It is worth noting the differential truncation patterns observed for each variant. Some variants, like ΔC41 and ΔC51 has several closely eluting bands around 50 kDa, whereas variants like ΔC31 or ΔC81 don’t show any discernible bands corresponding to their molecular masses.

FIG. 17 compares expression of several peptides, including truncated SLO variants after initial purification by HisTrap chromatography. Variants ΔC91 and ΔC101 expressed as single distinct bands corresponding to the correct molecular mass of the protein, along with some smaller truncation fragments. FIG. 18 and FIG. 19 compare SLO, and variants ΔC91 and ΔC101, during the steps of TEV cleavage and additional purification by HisTrap (FIG. 18) and size exclusion chromatography (FIG. 19).

Generation of immunized rabbit serum: Rabbits were intramuscularly immunized with alum + 50 µg of SLO, SLO ΔC91, or SLO ΔC101 through protocols similar to those described above. Animals were bled, and an ELISA titer against each respective antigen was performed for sera on days 0, 7, 21, and 35. FIG. 20 shows the ELISA titer for each of the three SLO variants.

Immunization with a multivalent glycoconjugate vaccine provides significant protection against systemic and intradermal GAS challenge: CD-1 mice were actively immunized with intramuscular injections of antigens at days 0, 14, and 28 to examine the contribution of enhanced bacterial clearance to the strong protective efficacy of a 3 component/4 GAS antigen formulation of SpyAD-GAC + SLO(ΔC101) + C5a peptidase. Mouse sera were collected from 9 immunized (and 10 mock immunized) animals at day 42 following the challenge, and the efficacy of these antisera tested for promoting ex vivo OPK killing of M1 GAS by freshly isolated human neutrophils (FIG. 21). When GAS were opsonized with post-immune SpyAD-GACPR290 + SLO(ΔC101) + C5a peptidase mouse antisera, ~20% of the original inoculum of M1 GAS was recovered following neutrophil exposure, compared to ~60% CFU recovery in control studies in 292 which GAS were opsonized with mock immune mouse sera (FIG. 21, P < 0.01). Also, mice immunized with SpyAD-GACPR + SLO(ΔC101) + C5a peptidase or mock (PBS and adjuvant alone) control were challenged using intradermal M1 GAS infection. The size of necrotic lesions generated by the resulting acute inflammatory response did not differ significantly between the two groups (FIGS. 22A and 22B), but the recovered bacterial CFU/gm was reduced more than twofold in the debrided tissue at the site of infection in the SpyAD-GACPR + SLO(ΔC101) + C5a peptidase immunized group (FIG. 22C, P = 0.0012).

Red blood cell hemolysis assay to assess the presence of functional antibodies against SLO variants: Antisera against SLO truncated variants were tested for their ability to block hemolytic activity of the corresponding SLO when added to red blood cells. FIG. 23 shows the results when 0.1 µg/mL of recombinant SLO was added to wells with red blood cells and antisera. Full-length SLO and SLO(ΔC101) anti-sera block cell lysis, while SLO(ΔC91) anti-sera does not block lysis.

Evaluation of vaccine antisera in GAS opsonophagocytic killing and blocking SLO activity: The ability of the rabbit antisera raised against the GAS vaccine antigens (SLO, C5a peptidase, SpyAD and SpyAD-GAC) to promote human neutrophil opsonophagocytic killing (OPK) was evaluated using GAS strains of five different M protein serotypes (M1-M5). This assay was performed with 30 min pre-opsonization with the respective heat-inactivated antisera or pre-immune sera control, then 30 min exposure to freshly isolated human neutrophils at multiplicity of infection (MOI) = 0.1 bacteria per neutrophil in the presence of 2% baby rabbit complement. All antisera induced a statistically significant increase in short-term OPK of GAS (FIG. 24A,). The assay uses a 100 to 1,000-fold greater GAS inoculum than published OPK assays employing the human promyelocytic leukemia HL-60 cell line at MOI = 0.00140 or MOI = 0.000141, to more accurately recapitulate the immunization target of supporting primary human innate immune cell function. Antisera raised against the SpyAD protein and SpyAD-GAC performed similarly. Moreover, a combination antiserum, composed of one-third of each antiserum raised against SLO, C5a peptidase, and SpyAD-GAC to maintain a consistent total serum concentration, performed similarly or better than the individual components, indicating no cross-253 interference in their OPK functions. As SLO is a secreted cytotoxin not anchored to the GAS surface, its contribution to enhanced neutrophil killing is accrued not from increased phagocytic uptake, but rather reducing SLO-mediated membrane damage and impairment of neutrophil antimicrobial functions. The anti-SLO rabbit immune serum, whether elicited by the SLO or SLO(ΔC101) antigens, significantly inhibited hemolytic activity of purified SLO against human red blood cells up to a dilution of 1 :2,048 (FIG. 24B). Furthermore, anti-SLO or anti-SLO(AC101) immune serum equally preserved neutrophil oxidative burst function (superoxide generation) against GAS supernatant (SLO)-mediated suppression (FIG. 24C).

Additional serotypes were tested in an IgG binding assay run similarly to those methods described above. Mouse sera were collected from 9 immunized (and 10 mock immunized) animals on day 42 following the three dose immunization (intramuscular injections of antigens on days 0, 14 and 28), and the efficacy of these antisera tested for IgG binding to live GAS strains via flow cytometry. Serum was collected from female CD-1 mice that were immunized with 3 doses of either mock or combination SpyAD-GACP R + SLO(ΔC101) + C5a peptidase. FIGS. : 25A and 25B show the fluorescent intensities of murine IgG from mock antisera (light histograms) or multivalent combination antisera (dark histograms) bound to GAS surface antigens of multiple serotypes as quantified via flow cytometry. Mean fluorescence intensities are summarized in column scatter plots, with each point representing individual mouse serum (circles are mock treatment mice, and square are treatment group mice). Methicillin-resistant Staphylococcus aureus USA300 was added as a control to show GAS-specificity of IgG binding.

Example 17: Truncated SLO(ΔC101) Polypeptides With 3 or 4 nnAAs Were Expressed and Conjugated

Truncated SLO(ΔC101) variants containing nnAAs were expressed, for instance, according to the above methods. The variants contain 3 or 4 pAMF residues, corresponding to SEQ ID NO: 55 to SEQ ID NO: 64. SEQ ID NO: 54 contains all of the nnAA-containing residues. FIG. 26 shows the expression levels of these 10 variants, in both total and soluble protein concentration, with their corresponding gels. Purification proceeded as described previously. In short, The CFPS was harvested, spun down and filtered before loading 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 6 ml final volume.5 ul of each step fraction was analyzed using SDS-PAGE. FIG. 27A shows the gels of the variants after initial purification by hisTRAP. The hisTRAP elution fractions were then diluted 3× in 50 mM Tris pH8 for bring the final salt concentration to 50 mM NaCl before loading onto an AEX column. Both FT and elution fractions were analyzed using the SDS-PAGE analysis to detect presence of each of the variants. FIG. 27B shows gels of the variants after the second purification step. Table 11 shows the final concentration of the expressed variants after recovery from 2-2.5 mL of solution.

TABLE 11

Final variant concentrations after purification

Protein name
Concentration (mg/ml)

SLO(ΔC101)-var1
0.8

SLO(ΔC101)-var2
1.0

SLO(ΔC101)-var3
0.6

SLO(ΔC101)-var4
1.0

SLO(ΔC101)-var5
1.2

SLO(ΔC101)-var6
2.7

SLO(ΔC101)-var7
2.3

SLO(ΔC101)-var8
1.0

SLO(ΔC101)-var9
2.2

SLO(ΔC101)-var10
1.6

The SLO (ΔC101) variants were conjugated to DBCO-derivatized GAC as described above. FIG. 28 shows specific conjugation conditions for each variant. The conjugation reactions were analyzed by SDS page, as shown in FIG. 29. The first lane of each gel shows protein pre-conjugation. The final conjugate sizes were determined by SEC-MALS, the results of which are shown in FIG. 30.

Example 18: Truncated SLO(ΔC101) Polypeptides With 3 or 4 nnAAs Were Assessed for Their Immunogenicity in Murine Models and for Their Hemolytic Titer

The 3- and 4-pAMF SLO(ΔC101) variants, and their polysaccharide conjugates, of Example 17 are assessed in murine models by methods as described above in Example 9. Mice are immunized on days 0, 7, and 14, followed by a terminal bleed on day 21 post-sacrifice.

5 µg each of the polysaccharide conjugates of pAMF-substituted SLO(ΔC101)-var1, SLO(ΔC101)-var5, SLO(ΔC101)-var6, and SLO(AC101)-var10 of Example 17 (corresponding to SEQ ID NO: 55, 59, 60, and 64) were used to immunize mice. The antibody titers against SLO(ΔC101) measured after the terminal bleed are shown in FIG. 31A. The same variants and their GAC conjugates were also evaluated in a hemolysis assay according to the protocols of Example 16. The results are shown in FIG. 31B. FIG. 31C also shows antibody titers measured after the terminal bleed, however this ELISA utilized a long polysaccharide (produced by the methods of Example 19) as the coating antigen, rather than SLO(ΔC101). Together, these data show that the conjugates of pAMF-substituted variants SLO(ΔC101)-var1, SLO(ΔC101)-var5, SLO(ΔC101)-var6, and SLO(ΔC101)-var10 produce antibodies against both the PS and peptide components, individually.

Example 19: Expression, Purification, and Conjugation of Higher Molecular Weight (Long) Polysaccharides

Experiments were performed to extract and purify GAS polysaccharides from GAS bacterial cultures.

Base Hydrolysis: A prepared GAS cell pellet was re-suspended in 50 mM NaCl solution. Using a serological pipette, 50 mL NaCl was added and vortexed until re-suspended. 10N sodium hydroxide & 1 M sodium borohydride were added to reach a final concentration of 4N NaOH and 25 mM NaBH₄. The re-suspended pellet was split between centrifuge bottles with a final target volume of 160 mL per 1L fermentation volume. When splitting the solution, constant swirling was used to ensure homogeneity. Bottles were placed on a shaker in the preheated incubator at 65° C. for 2 hours. After incubation, the hydrolysis solution was centrifuged for 30 min at 14,000 × g at 25° C. to pellet any cell debris, making sure to let the hydrolysis sample cool down to room temperature prior to centrifugation. The supernatant was collected once centrifugation stopped, in order not to disturb the pellet, and the supernatant was neutralized to pH 6.5 ± 0.3. When neutralizing, the bottle was placed on a 4° C. ice bath, using 37% HC1 to adjust pH, with 1 M NaOH used for further adjustment if necessary. The sample is then incubated at 4° C. overnight.

Filtration: After incubation, a white precipitate forms. The solution was centrifuged for 30 min at 10,000xg. A bulky white pellet is formed, containing host cell proteins (HCPs). The clear supernatant was collected. The pH was adjusted to 3.0 using 37% HCl, and the solution was incubated for 1 hour at RT. Using a Clarisolve Filter µPod -40MS, the sample was filtered. For example: at a pump flow rate of 23 mL/min, water was flushed through the filter until primed, and the valve was then opened and flushed with 120 mL volume, and equilibrated using 15 mM NaCl for a volume of 120 mL. The extract was run through the filter at the same pump flow rate of 23 mL/min, and the clear permeate coming from the filter was collected. The filter was washed with 60 mL of 15 mM NaCl, and flushed out the tubes and filter content. The filter was discarded. The sample pH was readjusted to 6.5 ± 0.3. Tangential flow filtration (TFF-10k) was then conducted to remove salts, concentrated (ultrafiltered), then buffer exchanged (diafiltered) in 10 mM NaCl.

Mutanolysin Treatment: The solution was prepared for mutanolysin treatment by adding 1 M MgC12 to reach a final conc. of 1 mM MgCl₂, and 200 mM sodium phosphate (10×) to reach a final concentration of 20 mM sodium phosphate at pH 6.8. Mutanolysin solution (5000 IU/mL) to reach 120 IU/mL. The sample was incubated at 37° C. overnight with shaking.

Proteinase-K Treatment: The sample was then treated with Proteinase-K by adding Proteinase-K solution to achieve a final concentration of 40 IU/mL (Proteinase-K at 45u/mg). The mixture was incubated at 50° C. overnight while mixing gently.

Precipitation and Filtration: To precipitate enzymes, nucleic acids and HCPs in the sample, CTAB in 20 mM sodium phosphate buffer at pH 6.8 was added, shaking for 1 hour at 30° C. The solutions used were all pre-warmed: the PS sample, 5% CTAB stock solution and 200 mM Na Phosphate pH 6.8. The PS solution was mixed (magnetic stir bar) while being heated, and the heated stir plate was set up with an internal thermometer in order to monitor temperature in the solution at all times. 200 mM sodium phosphate (pH 6.8) solution was added to the PS solution, to reach a final concentration of 20 mM sodium phosphate. 5% CTAB was added to the PS solution to reach a 1% CTAB concentration. The solution was allowed to mix for 1 hour. Using a 40MS filter, a depth filtration is conducted over the precipitating solution. For example, the system was flushed with 200 mL MilliQ H2O at a pump flow rate of 23 mL/min, and once water started to come out from the vent, it was closed so the solution is forced to come out from the outlet (priming). The system was then flushed with 75 mL of a solution of 20 mM sodium phosophate (pH 6.8) and 15 mM NaCl. The sample was filtered at 23 mL/min, and flushed with 60 mL of 20 mM sodium phosphate (pH 6.8) and15mM NaCl solution at 20 mL/min. The permeate was collected until air bubbles eluted. The filter was discarded.

The PS solution and 274 mM KI was warmed to 30° C. Enough 274 mM KI was added to the mixing PS solution to achieve a final concentration of 27.4 mM KI. The mixture was incubated at 30° C. withmixing for 1 hour. The solution was centrifuged post incubation at 30° C., 10,000xg for 30 minutes. The supernatant was collected and the pellet was discarded, be cautious as the pellet breaks up easily. The supernatant was then vacuum filtered through a 0.45 µm filter.

The sample was first concentrated by TFF-10k, followed by diafiltration with 9DVs of 350 mM NaCl and finally 2 DVs with MilliQ water. For example, the TFF system volume is around 35 mL, with pump flow set at 200 mL/min, and the diafiltration is conducted with 9 DV (50 mL) of buffer TMP: 7-8 psi

The polysaccharide solution was then purified by hydrophobic interaction chromatography using 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 in order to reach 3 M sodium chloride and 50 mM potassium phosphate (pH 6.8). The polysaccharide solution was passed over HIC resin and was operated in flow through mode. The resin is washed with the same equilibration buffer and both the flow-through and wash were collected for further processing.

A final TFF 10 kDa/ 3 kDa was conducted in order to remove high NaCl content in the PS sample by diafiltration against 9DV of 15 mM NaCl or WFI. The purified PS solution was then 0.22 um filtered.

FIG. 36A, FIG. 36B, and FIG. 36C show NMR analysis of purified polysaccharide produced by Synthetic Example 1, originating from a GAS bacterial strain expressing PS lacking GlcNAc (see PCT/US2012/049604). The NMR confirms 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. A proton NMR spectrum was obtained at 50° C. on a 400 MHz instrument, with signal averaging over 40 scans. 0.2 Hz of line broadening was applied. The purification protocol was also able to produce purified polysaccharide free of M-protein, as evidenced by the western blot of FIG. 37.

Derivitization and Conjugation of Long Polysaccharides: The higher-molecular weight (long) polysaccharides are derivatized with DBCO-PEG as previously described in the above methods (Example 4). The DBCO-derivatized PS is then conjugated to the carrier proteins described herein according to the methods of Example 4. For instance, SpyAD[4pAMF] (SEQ ID NO: 34) was conjugated to the long polysaccharide and analyzed after 3.5 hours and post-dialysis. The SEC-MALS analysis of these conjugates is shown in FIG. 32. After 3.5 hours, conjugates greater than 1 MDa were observed. Post-dialysis, the isolated conjugate size distribution showed a peak at approximately 186 kDa.

Example 20: Truncated SLO(ΔC101) Polypeptides With 5, 6, 7, or 8 nnAAs Were Expressed, Purified, and Conjugated

Truncated SLO(ΔC101) variants containing nnAAs are expressed, for instance, according to the above methods. The variants contain 5, 6, 7, or 8 pAMF residues, corresponding to 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. FIG. 33A shows the expression levels of these 12 pAMF variants (5pAMF-8pAMF), as well as for the variants of example 17, in both total and soluble protein concentration. FIG. 33B shows the corresponding gels for the expression (SEQ ID NOs: 65-76 correspond to var1-var12). Purification proceeded as described previously. In short, The CFPS was harvested, spun down and filtered before loading 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 6 ml final volume. 5 µl of each step fraction was analyzed using SDS-PAGE safe blue staining. Thereafter, the elutions were 3× diluted and loaded onto the CaptoQ column for final purification and analyzed using Safe Blue staining and DBCO TAMRA labeling. The FT fractions were concentrated and stored at -80C in 200 µl aliquots.

Conjugation of SLO(ΔC101) polypeptides with 5, 6, 7, or 8 nnAAs: The polypeptides containing 5, 6, 7, or 8 nnAAs are conjugated to GAS polysaccharides using the methods described above, including in Example 4. In this way, the 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 DBCO-derivatized GAC. The conjugation reactions were analyzed by SDS page, as shown in FIG. 34. The first lane for each variant shows the protein pre-conjugation. The final conjugate sizes were determined by SEC-MALS, the results of which are shown in FIG. 35.

Sequence identifier descriptions

Seq ID
Name/description
Amino acid sequence

1
C5a [90-1035 fragment] WT w/ leader
MHHHHHHGSGENLYFQGKTADTPVTSKATIRDLNDPSQVKTLQE KAGKGAGTVVAVIDAGFDKNHEAWRLTDKTKARYQSKEDLEKAK KEHGITYGEWVNDKVAYYHDYSKDGKTAVDQEHGTHVSGILSGN APSETKEPYRLEGAMPEAQLLLMRVEIVNGLADYARNYAQAIRD AVNLGAKVINMSFGNAALAYANLPDETKKAFDYAKSKGVSIVTS AGNDSSFGGKTRLPLADHPDYGVVGTPAAADSTLTVASYSPDKQ LTETATVKTADKQDKEMPVLSTNRFEPNKAYDYAYANRGMKEDD FKDVKGKIALIERGDIDFKDKIANAKKAGAVGVLIYDNQDKGFP IELPNVDQMPAAFISRKDGLLLKDNPKKTITFNATPKVLPTASG TKLSRFSSWGLTADGNIKPDIAAPGQDILSSVANNKYAKLSGTS MSAPLVAGIMGLLQKQYETQYPDMTPSERLDLAKKVLMSSATAL YDEDEKAYFSPRQQGAGAVDAKKASAATMYVTDKDNTSSKVHLN NVSDTFEVTVTVHNKSDKPQELYYQATVQTDKVDGKHFALAPKA LYETSWQKITIPANSSKQVTVPIDASRFSKDLLAQMKNGYFLEG FVRFKQDPTKEELMSIPYIGFRGDFGNLSALEKPIYDSKDGSSY YHEANSDAKDQLDGDGLQFYALKNNFTALTTESNPWTIIKAVKE GVENIEDIESSEITETIFAGTFAKQDDDSHYYIHRHANGKPYAA ISPNGDGNRDYVQFQGTFLRNAKNLVAEVLDKEGNVVWTSEVTE QWKNYNNDLASTLGSTRFEKTRWDGKDKDGKWANGTYTYRVR YTPISSGAKEQHTDFDVIVDNTTPEVATSATFSTEDRRLTLASK PKTSQPVYRERIAYTYMDEDLPTTEYISPNEDGTFTLPEEAETM EGATVPLKMSDFTYVVEDMAGNITYTPVTKLLEGHSNK

2
C5a [90-1035 fragment] WT w/o leader
KTADTPVTSKATIRDLNDPSQVKTLQEKAGKGAGTVVAVIDAGF DKNHEAWRLTDKTKARYQSKEDLEKAKKEHGITYGEWVNDKVAY YHDYSKDGKTAVDQEHGTHVSGILSGNAPSETKEPYRLEGAMPE AQLLLMRVEIVNGLADYARNYAQAIRDAVNLGAKVINMSFGNAA LAYANLPDETKKAFDYAKSKGVSIVTSAGNDSSFGGKTRLPLAD HPDYGVVGTPAAADSTLTVASYSPDKQLTETATVKTADKQDKEM PVLSTNRFEPNKAYDYAYANRGMKEDDFKDVKGKIALIERGDID FKDKIANAKKAGAVGVLIYDNQDKGFPIELPNVDQMPAAFISRK DGLLLKDNPKKTITFNATPKVLPTASGTKLSRFSSWGLTADGNI KPDIAAPGQDILSSVANNKYAKLSGTSMSAPLVAGIMGLLQKQY ETQYPDMTPSERLDLAKKVLMSSATALYDEDEKAYFSPRQQGAG AVDAKKASAATMYVTDKDNTSSKVHLNNVSDTFEVTVTVHNKSD KPQELYYQATVQTDKVDGKHFALAPKALYETSWQKITIPANSSK QVTVPIDASRFSKDLLAQMKNGYFLEGFVRFKQDPTKEELMSIP YIGFRGDFGNLSALEKPIYDSKDGSSYYHEANSDAKDQLDGDGL QFYALKNNFTALTTESNPWTIIKAVKEGVENIEDIESSEITETI FAGTFAKQDDDSHYYIHRHANGKPYAAISPNGDGNRDYVQFQGT FLRNAKNLVAEVLDKEGNVVWTSEVTEQVVKNYNNDLASTLGST RFEKTRWDGKDKDGKVVANGTYTYRVRYTPISSGAKEQHTDFDV IVDNTTPEVATSATFSTEDRRLTLASKPKTSQPVYRERIAYTYM DEDLPTTEYISPNEDGTFTLPEEAETMEGATVPLKMSDFTYVVE DMAGNITYTPVTKLLEGHSNK

3
C5a [90-1035 fragment] D131A/S513A w/ leader
MHHHHHHGSGENLYFQGKTADTPVTSKATIRDLNDPSQVKTLQE KAGKGAGTVVAVIAAGFDKNHEAWRLTDKTKARYQSKEDLEKAK KEHGITYGEWVNDKVAYYHDYSKDGKTAVDQEHGTHVSGILSGN APSETKEPYRLEGAMPEAQLLLMRVEIVNGLADYARNYAQAIRD AVNLGAKVINMSFGNAALAYANLPDETKKAFDYAKSKGVSIVTS AGNDSSFGGKTRLPLADHPDYGVVGTPAAADSTLTVASYSPDKQ LTETATVKTADKQDKEMPVLSTNRFEPNKAYDYAYANRGMKEDD FKDVKGKIALIERGDIDFKDKIANAKKAGAVGVLIYDNQDKGFP IELPNVDQMPAAFISRKDGLLLKDNPKKTITFNATPKVLPTASG TKLSRFSSWGLTADGNIKPDIAAPGQDILSSVANNKYAKLSGTA MSAPLVAGIMGLLQKQYETQYPDMTPSERLDLAKKVLMSSATAL YDEDEKAYFSPRQQGAGAVDAKKASAATMYVTDKDNTSSKVHLN NVSDTFEVTVTVHNKSDKPQELYYQATVQTDKVDGKHFALAPKA LYETSWQKITIPANSSKQVTVPIDASRFSKDLLAQMKNGYFLEG FVRFKQDPTKEELMSIPYIGFRGDFGNLSALEKPIYDSKDGSSY YHEANSDAKDQLDGDGLQFYALKNNFTALTTESNPWTIIKAVKE GVENIEDIESSEITETIFAGTFAKQDDDSHYYIHRHANGKPYAA ISPNGDGNRDYVQFQGTFLRNAKNLVAEVLDKEGNVVWTSEVTE QVVKNYNNDLASTLGSTRFEKTRWDGKDKDGKVVANGTYTYRVR YTPISSGAKEQHTDFDVIVDNTTPEVATSATFSTEDRRLTLASK PKTSQPVYRERIAYTYMDEDLPTTEYISPNEDGTFTLPEEAETM EGATVPLKMSDFTYVVEDMAGNITYTPVTKLLEGHSNK

4
C5a [90-1035 fragment] D131A/S513A w/o leader
KTADTPVTSKATIRDLNDPSQVKTLQEKAGKGAGTVVAVIAAGF DKNHEAWRLTDKTKARYQSKEDLEKAKKEHGITYGEWVNDKVAY YHDYSKDGKTAVDQEHGTHVSGILSGNAPSETKEPYRLEGAMPE AQLLLMRVEIVNGLADYARNYAQAIRDAVNLGAKVINMSFGNAA LAYANLPDETKKAFDYAKSKGVSIVTSAGNDSSFGGKTRLPLAD HPDYGVVGTPAAADSTLTVASYSPDKQLTETATVKTADKQDKEM PVLSTNRFEPNKAYDYAYANRGMKEDDFKDVKGKIALIERGDID FKDKIANAKKAGAVGVLIYDNQDKGFPIELPNVDQMPAAFISRK DGLLLKDNPKKTITFNATPKVLPTASGTKLSRFSSWGLTADGNI KPDIAAPGQDILSSVANNKYAKLSGTAMSAPLVAGIMGLLQKQY ETQYPDMTPSERLDLAKKVLMSSATALYDEDEKAYFSPRQQGAG AVDAKKASAATMYVTDKDNTSSKVHLNNVSDTFEVTVTVHNKSD KPQELYYQATVQTDKVDGKHFALAPKALYETSWQKITIPANSSK QVTVPIDASRFSKDLLAQMKNGYFLEGFVRFKQDPTKEELMSIP YIGFRGDFGNLSALEKPIYDSKDGSSYYHEANSDAKDQLDGDGL QFYALKNNFTALTTESNPWTIIKAVKEGVENIEDIESSEITETI FAGTFAKQDDDSHYYIHRHANGKPYAAISPNGDGNRDYVQFQGT FLRNAKNLVAEVLDKEGNVVWTSEVTEQVVKNYNNDLASTLGST RFEKTRWDGKDKDGKVVANGTYTYRVRYTPISSGAKEQHTDFDV IVDNTTPEVATSATFSTEDRRLTLASKPKTSQPVYRERIAYTYM DEDLPTTEYISPNEDGTFTLPEEAETMEGATVPLKMSDFTYVVE DMAGNITYTPVTKLLEGHSNK

5
SLO [79-571 fragment] WT w/ His tag and TEV sequence
MHHHHHHGSGENLYFQGAPKEMPLESAEKEEKKSEDKKKSEEDH TEEINDKIYSLNYNELEVLAKNGETIENFVPKEGVKKADKFIVI ERKKKNINTTPVDISIIDSVTDRTYPAALQLANKGFTENKPDAV VTKRNPQKIHIDLPGMGDKATVEVNDPTYANVSTAIDNLVNQWH DNYSGGNTLPARTQYTESMVYSKSQIEAALNVNSKILDGTLGID FKSISKGEKKVMIAAYKQIFYTVSANLPNNPADVFDKSVTFKEL QRKGVSNEAPPLFVSNVAYGRTVFVKLETSSKSNDVEAAFSAAL KGTDVKTNGKYSDILENSSFTAVVLGGDAAEHNKVVTKDFDVIR NVIKDNATFSRKNPAYPISYTSVFLKNNKIAGVNNRTEYVETTS TEYTSGKINLSHQGAYVAQYEILWDEINYDDKGKEVITKRRWDN NWYSKTSPFSTVIPLGANSRNIRIMARECTGLAWEWWRKVIDER DVKLSKEINVNISGSTLSPYGSITYK

6
SLO [79-571 fragment] WT w/o leader
APKEMPLESAEKEEKKSEDKKKSEEDHTEEINDKIYSLNYNELE VLAKNGETIENFVPKEGVKKADKFIVIERKKKNINTTPVDISII DSVTDRTYPAALQLANKGFTENKPDAVVTKRNPQKIHIDLPGMG DKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYTE SMVYSKSQIEAALNVNSKILDGTLGIDFKSISKGEKKVMIAAYK QIFYTVSANLPNNPADVFDKSVTFKELQRKGVSNEAPPLFVSNV AYGRTVFVKLETSSKSNDVEAAFSAALKGTDVKTNGKYSDILEN SSFTAVVLGGDAAEHNKVVTKDFDVIRNVIKDNATFSRKNPAYP ISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGKINLSHQGAYV AQYEILWDEINYDDKGKEVITKRRWDNNWYSKTSPFSTVIPLGA NSRNIRIMARECTGLAWEWWRKVIDERDVKLSKEINVNISGSTL SPYGSITYK

7
SLO [79-571 fragment] W535A w/ leader
MHHHHHHGSGENLYFQGAPKEMPLESAEKEEKKSEDKKKSEEDH TEEINDKIYSLNYNELEVLAKNGETIENFVPKEGVKKADKFIVI ERKKKNINTTPVDISIIDSVTDRTYPAALQLANKGFTENKPDAV VTKRNPQKIHIDLPGMGDKATVEVNDPTYANVSTAIDNLVNQWH DNYSGGNTLPARTQYTESMVYSKSQIEAALNVNSKILDGTLGID FKSISKGEKKVMIAAYKQIFYTVSANLPNNPADVFDKSVTFKEL QRKGVSNEAPPLFVSNVAYGRTVFVKLETSSKSNDVEAAFSAAL KGTDVKTNGKYSDILENSSFTAVVLGGDAAEHNKVVTKDFDVIR NVIKDNATFSRKNPAYPISYTSVFLKNNKIAGVNNRTEYVETTS TEYTSGKINLSHQGAYVAQYEILWDEINYDDKGKEVITKRRWDN NWYSKTSPFSTVIPLGANSRNIRIMARECTGLAAEWWRKVIDER DVKLSKEINVNISGSTLSPYGSITYK

8
SLO [79-571 fragment] W535A w/o leader
APKEMPLESAEKEEKKSEDKKKSEEDHTEEINDKIYSLNYNELE VLAKNGETIENFVPKEGVKKADKFIVIERKKKNINTTPVDISII DSVTDRTYPAALQLANKGFTENKPDAVVTKRNPQKIHIDLPGMG DKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYTE SMVYSKSQIEAALNVNSKILDGTLGIDFKSISKGEKKVMIAAYK QIFYTVSANLPNNPADVFDKSVTFKELQRKGVSNEAPPLFVSNV AYGRTVFVKLETSSKSNDVEAAFSAALKGTDVKTNGKYSDILEN SSFTAVVLGGDAAEHNKVVTKDFDVIRNVIKDNATFSRKNPAYP ISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGKINLSHQGAYV AQYEILWDEINYDDKGKEVITKRRWDNNWYSKTSPFSTVIPLGA NSRNIRIMARECTGLAAEWWRKVIDERDVKLSKEINVNISGSTL SPYGSITYK

9
SpyAD [33-849 fragment] WT w/ leader
MHHHHHHGSGENLYFQGQVKADDRASGETKASNTHDDSLPKPET IQEAKATIDAVEKTLSQQKAELTELATALTKTTAEINHLKEQQD NEQKALTSAQEIYTNTLASSEETLLAQGAEHQRELTATETELHN AQADQHSKETALSEQKASISAETTRAQDLVEQVKTSEQNIAKLN AMISNPDAITKAAQTANDNTKALSSELEKAKADLENQKAKVKKQ LTEELAAQKAALAEKEAELSRLKSSAPSTQDSIVGNNTMKAPQG YPLEELKKLEASGYIGSASYNNYYKEHADQIIAKASPGNQLNQY QDIPADRNRFVDPDNLTPEVQNELAQFAAHMINSVRRQLGLPPV TVTAGSQEFARLLSTSYKKTHGNTRPSFVYGQPGVSGHYGVGPH DKTIIEDSAGASGLIRNDDNMYENIGAFNDVHTVNGIKRGIYDS IKYMLFTDHLHGNTYGHAINFLRVDKHNPNAPVYLGFSTSNVGS LNEHFVMFPESNIANHQRFNKTPIKAVGSTKDYAQRVGTVSDTI AAIKGKVSSLENRLSAIHQEADIMAAQAKVSQLQGKLASTLKQS DSLNLQVRQLNDTKGSLRTELLAAKAKQAQLEATRDQSLAKLAS LKAALHQTEALAEQAAARVTALVAKKAHLQYLRDFKLNPNRLQV IRERIDNTKQDLAKTTSSLLNAQEALAALQAKQSSLEATIATTE HQLTLLKTLANEKEYRHLDEDIATVPDLQVAPPLTGVKPLSYSK IDTTPLVQEMVKETKQLLEASARLAAENTSLVAEALVGQTSEMV ASNAIVSKITSSITQPSSKTSYGSGSSTTSNLISDVDESTQR

10
SpyAD [33-849 fragment] WT w/o leader
QVKADDRASGETKASNTHDDSLPKPETIQEAKATIDAVEKTLSQ QKAELTELATALTKTTAEINHLKEQQDNEQKALTSAQEIYTNTL ASSEETLLAQGAEHQRELTATETELHNAQADQHSKETALSEQKA SISAETTRAQDLVEQVKTSEQNIAKLNAMISNPDAITKAAQTAN DNTKALSSELEKAKADLENQKAKVKKQLTEELAAQKAALAEKEA ELSRLKSSAPSTQDSIVGNNTMKAPQGYPLEELKKLEASGYIGS ASYNNYYKEHADQIIAKASPGNQLNQYQDIPADRNRFVDPDNLT PEVQNELAQFAAHMINSVRRQLGLPPVTVTAGSQEFARLLSTSY KKTHGNTRPSFVYGQPGVSGHYGVGPHDKTIIEDSAGASGLIRN DDNMYENIGAFNDVHTVNGIKRGIYDSIKYMLFTDHLHGNTYGH AINFLRVDKHNPNAPVYLGFSTSNVGSLNEHFVMFPESNIANHQ RFNKTPIKAVGSTKDYAQRVGTVSDTIAAIKGKVSSLENRLSAI HQEADIMAAQAKVSQLQGKLASTLKQSDSLNLQVRQLNDTKGSL RTELLAAKAKQAQLEATRDQSLAKLASLKAALHQTEALAEQAAA RVTALVAKKAHLQYLRDFKLNPNRLQVIRERIDNTKQDLAKTTS SLLNAQEALAALQAKQSSLEATIATTEHQLTLLKTLANEKEYRH LDEDIATVPDLQVAPPLTGVKPLSYSKIDTTPLVQEMVKETKQL LEASARLAAENTSLVAEALVGQTSEMVASNAIVSKITSSITQPS SKTSYGSGSSTTSNLISDVDESTQR

11
SpyAD [33-849 fragment] nnAA (e.g., pAMF)mutant w/ leader X = nnAA (e.g., pAMF) positions K64/K287/K386/K 657 - numbered according to full length sequence
MHHHHHHGSGENLYFQGQVKADDRASGETKASNTHDDSLPKPET IQEAXATIDAVEKTLSQQKAELTELATALTKTTAEINHLKEQQD NEQKALTSAQEIYTNTLASSEETLLAQGAEHQRELTATETELHN AQADQHSKETALSEQKASISAETTRAQDLVEQVKTSEQNIAKLN AMISNPDAITKAAQTANDNTKALSSELEKAKADLENQKAKVKKQ LTEELAAQKAALAEKEAELSRLKSSAPSTQDSIVGNNTMKAPQG YPLEELKXLEASGYIGSASYNNYYKEHADQIIAKASPGNQLNQY QDIPADRNRFVDPDNLTPEVQNELAQFAAHMINSVRRQLGLPPV TVTAGSQEFARLLSTSYKXTHGNTRPSFVYGQPGVSGHYGVGPH DKTIIEDSAGASGLIRNDDNMYENIGAFNDVHTVNGIKRGIYDS IKYMLFTDHLHGNTYGHAINFLRVDKHNPNAPVYLGFSTSNVGS LNEHFVMFPESNIANHQRFNKTPIKAVGSTKDYAQRVGTVSDTI AAIKGKVSSLENRLSAIHQEADIMAAQAKVSQLQGKLASTLKQS DSLNLQVRQLNDTKGSLRTELLAAKAKQAQLEATRDQSLAKLAS LKAALHQTEALAEQAAARVTALVAKXAHLQYLRDFKLNPNRLQV IRERIDNTKQDLAKTTSSLLNAQEALAALQAKQSSLEATIATTE HQLTLLKTLANEKEYRHLDEDIATVPDLQVAPPLTGVKPLSYSK IDTTPLVQEMVKETKQLLEASARLAAENTSLVAEALVGQTSEMV ASNAIVSKITSSITQPSSKTSYGSGSSTTSNLISDVDESTQR

12
SpyAD [33-849 fragment] nnAA (e.g., pAMF) mutant w/o leader X = nnAA (e.g., pAMF) K64/K287/K386/K 657 - numbered according to full-length sequence
QVKADDRASGETKASNTHDDSLPKPETIQEAXATIDAVEKTLSQ QKAELTELATALTKTTAEINHLKEQQDNEQKALTSAQEIYTNTL ASSEETLLAQGAEHQRELTATETELHNAQADQHSKETALSEQKA SISAETTRAQDLVEQVKTSEQNIAKLNAMISNPDAITKAAQTAN DNTKALSSELEKAKADLENQKAKVKKQLTEELAAQKAALAEKEA ELSRLKSSAPSTQDSIVGNNTMKAPQGYPLEELKXLEASGYIGS ASYNNYYKEHADQIIAKASPGNQLNQYQDIPADRNRFVDPDNLT PEVQNELAQFAAHMINSVRRQLGLPPVTVTAGSQEFARLLSTSY KXTHGNTRPSFVYGQPGVSGHYGVGPHDKTIIEDSAGASGLIRN DDNMYENIGAFNDVHTVNGIKRGIYDSIKYMLFTDHLHGNTYGH AINFLRVDKHNPNAPVYLGFSTSNVGSLNEHFVMFPESNIANHQ RFNKTPIKAVGSTKDYAQRVGTVSDTIAAIKGKVSSLENRLSAI HQEADIMAAQAKVSQLQGKLASTLKQSDSLNLQVRQLNDTKGSL RTELLAAKAKQAQLEATRDQSLAKLASLKAALHQTEALAEQAAA RVTALVAKXAHLQYLRDFKLNPNRLQVIRERIDNTKQDLAKTTS SLLNAQEALAALQAKQSSLEATIATTEHQLTLLKTLANEKEYRH LDEDIATVPDLQVAPPLTGVKPLSYSKIDTTPLVQEMVKETKQL LEASARLAAENTSLVAEALVGQTSEMVASNAIVSKITSSITQPS SKTSYGSGSSTTSNLISDVDESTQR

13
ADI [full length] WT w/ leader
MHHHHHHGSGENLYFQGTAQTPIHVYSEIGKLKKVLLHRPGKEI ENLMPDYLERLLFDDIPFLEDAQKEHDAFAQALRDEGIEVLYLE TLAAESLVTPEIREAFIDEYLSEANIRGRATKKAIRELLMAIED NQELIEKTMAGVQKSELPEIPASEKGLTDLVESNYPFAIDPMPN LYFTRDPFATIGTGVSLNHMFSETRNRETLYGKYIFTHHPIYGG GKVPMVYDRNETTRIEGGDELVLSKDVLAVGISQRTDAASIEKL LVNIFKQNLGFKKVLAFEFANNRKFMHLDTVFTMVDYDKFTIHP EIEGDLRVYSVTYDNEELHIVEEKGDLAELLAANLGVEKVDLIR CGGDNLVAAGREQWNDGSNTLTIAPGVVVVYNRNTITNAILESK GLKLIKIHGSELVRGRGGPRCMSMPFEREDI

14
ADI [full length] WT w/o leader
TAQTPIHVYSEIGKLKKVLLHRPGKEIENLMPDYLERLLFDDIP FLEDAQKEHDAFAQALRDEGIEVLYLETLAAESLVTPEIREAFI DEYLSEANIRGRATKKAIRELLMAIEDNQELIEKTMAGVQKSEL PEIPASEKGLTDLVESNYPFAIDPMPNLYFTRDPFATIGTGVSL NHMFSETRNRETLYGKYIFTHHPIYGGGKVPMVYDRNETTRIEG GDELVLSKDVLAVGISQRTDAASIEKLLVNIFKQNLGFKKVLAF EFANNRKFMHLDTVFTMVDYDKFTIHPEIEGDLRVYSVTYDNEE LHIVEEKGDLAELLAANLGVEKVDLIRCGGDNLVAAGREQWNDG SNTLTIAPGVVVVYNRNTITNAILESKGLKLIKIHGSELVRGRG GPRCMSMPFEREDI

15
ADI [full length] D277A w/ leader
MHHHHHHGSGENLYFQGTAQTPIHVYSEIGKLKKVLLHRPGKEI ENLMPDYLERLLFDDIPFLEDAQKEHDAFAQALRDEGIEVLYLE TLAAESLVTPEIREAFIDEYLSEANIRGRATKKAIRELLMAIED NQELIEKTMAGVQKSELPEIPASEKGLTDLVESNYPFAIDPMPN LYFTRDPFATIGTGVSLNHMFSETRNRETLYGKYIFTHHPIYGG GKVPMVYDRNETTRIEGGDELVLSKDVLAVGISQRTDAASIEKL LVNIFKQNLGFKKVLAFEFANNRKFMHLATVFTMVDYDKFTIHP EIEGDLRVYSVTYDNEELHIVEEKGDLAELLAANLGVEKVDLIR CGGDNLVAAGREQWNDGSNTLTIAPGVVVVYNRNTITNAILESK GLKLIKIHGSELVRGRGGPRCMSMPFEREDI

16
ADI [full length] D277A w/o leader
TAQTPIHVYSEIGKLKKVLLHRPGKEIENLMPDYLERLLFDDIP FLEDAQKEHDAFAQALRDEGIEVLYLETLAAESLVTPEIREAFI DEYLSEANIRGRATKKAIRELLMAIEDNQELIEKTMAGVQKSEL PEIPASEKGLTDLVESNYPFAIDPMPNLYFTRDPFATIGTGVSL NHMFSETRNRETLYGKYIFTHHPIYGGGKVPMVYDRNETTRIEG GDELVLSKDVLAVGISQRTDAASIEKLLVNIFKQNLGFKKVLAF EFANNRKFMHLATVFTMVDYDKFTIHPEIEGDLRVYSVTYDNEE LHIVEEKGDLAELLAANLGVEKVDLIRCGGDNLVAAGREQWNDG SNTLTIAPGVVVVYNRNTITNAILESKGLKLIKIHGSELVRGRG GPRCMSMPFEREDI

17
ADI [full length] nnAA (e.g., pAMF) w/ leader X = nnAA (e.g., pAMF) positions -K15, K193, K316
MHHHHHHGSGENLYFQGTAQTPIHVYSEIGXLKKVLLHRPGKEI ENLMPDYLERLLFDDIPFLEDAQKEHDAFAQALRDEGIEVLYLE TLAAESLVTPEIREAFIDEYLSEANIRGRATKKAIRELLMAIED NQELIEKTMAGVQKSELPEIPASEKGLTDLVESNYPFAIDPMPN LYFTRDPFATIGTGVSLNHMFSETRNRETLYGXYIFTHHPIYGG GKVPMVYDRNETTRIEGGDELVLSKDVLAVGISQRTDAASIEKL LVNIFKQNLGFKKVLAFEFANNRKFMHLDTVFTMVDYDKFTIHP EIEGDLRVYSVTYDNEELHIVEEXGDLAELLAANLGVEKVDLIR CGGDNLVAAGREQWNDGSNTLTIAPGVVVVYNRNTITNAILESK GLKLIKIHGSELVRGRGGPRCMSMPFEREDI

18
ADI [full length] nnAA (e.g., pAMF) mutant w/o leader X = nnAA (e.g., pAMF) positions -K15, K193, K316
TAQTPIHVYSEIGXLKKVLLHRPGKEIENLMPDYLERLLFDDIP FLEDAQKEHDAFAQALRDEGIEVLYLETLAAESLVTPEIREAFI DEYLSEANIRGRATKKAIRELLMAIEDNQELIEKTMAGVQKSEL PEIPASEKGLTDLVESNYPFAIDPMPNLYFTRDPFATIGTGVSL NHMFSETRNRETLYGXYIFTHHPIYGGGKVPMVYDRNETTRIEG GDELVLSKDVLAVGISQRTDAASIEKLLVNIFKQNLGFKKVLAF EFANNRKFMHLDTVFTMVDYDKFTIHPEIEGDLRVYSVTYDNEE LHIVEEXGDLAELLAANLGVEKVDLIRCGGDNLVAAGREQWNDG SNTLTIAPGVVVVYNRNTITNAILESKGLKLIKIHGSELVRGRG GPRCMSMPFEREDI

19
ADI (mut5) [full length] D277A nnAA (e.g., pAMF) w/ leader X = nnAA (e.g., pAMF) positions -K15, K193, K316
MHHHHHHGSGENLYFQGTAQTPIHVYSEIGXLKKVLLHRPGKEI ENLMPDYLERLLFDDIPFLEDAQKEHDAFAQALRDEGIEVLYLE TLAAESLVTPEIREAFIDEYLSEANIRGRATKKAIRELLMAIED NQELIEKTMAGVQKSELPEIPASEKGLTDLVESNYPFAIDPMPN LYFTRDPFATIGTGVSLNHMFSETRNRETLYGXYIFTHHPIYGG GKVPMVYDRNETTRIEGGDELVLSKDVLAVGISQRTDAASIEKL LVNIFKQNLGFKKVLAFEFANNRKFMHLATVFTMVDYDKFTIHP EIEGDLRVYSVTYDNEELHIVEEXGDLAELLAANLGVEKVDLIR CGGDNLVAAGREQWNDGSNTLTIAPGVVVVYNRNTITNAILESK GLKLIKIHGSELVRGRGGPRCMSMPFEREDI

20
ADI (mut5) [full length] D277A nnAA (e.g., pAMF) w/o leader X = nnAA (e.g., pAMF) positions -K15, K193, K316
TAQTPIHVYSEIGXLKKVLLHRPGKEIENLMPDYLERLLFDDIP FLEDAQKEHDAFAQALRDEGIEVLYLETLAAESLVTPEIREAFI DEYLSEANIRGRATKKAIRELLMAIEDNQELIEKTMAGVQKSEL PEIPASEKGLTDLVESNYPFAIDPMPNLYFTRDPFATIGTGVSL NHMFSETRNRETLYGXYIFTHHPIYGGGKVPMVYDRNETTRIEG GDELVLSKDVLAVGISQRTDAASIEKLLVNIFKQNLGFKKVLAF EFANNRKFMHLATVFTMVDYDKFTIHPEIEGDLRVYSVTYDNEE LHIVEEXGDLAELLAANLGVEKVDLIRCGGDNLVAAGREQWNDG SNTLTIAPGVVVVYNRNTITNAILESKGLKLIKIHGSELVRGRG GPRCMSMPFEREDI

21
hFL [full length] WT w/ leader
MHHHHHHGSGSSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLG FYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALF QDIKKPAEDEWGKTPDAMKAAMALEKKLNQALLDLHALGSARTD PHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLF ERLTLKHD

22
hFL [full length] WT w/o leader
SSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVAL EGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDE WGKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETH FLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLKHD

23
hFL [full length] nnAA (e.g., pAMF) w/ leader X = nnAA (e.g., pAMF) position -I5
MHHHHHHGSGSSQXRQNYSTDVEAAVNSLVNLYLQASYTYLSLG FYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALF QDIKKPAEDEWGKTPDAMKAAMALEKKLNQALLDLHALGSARTD PHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLF ERLTLKHD

24
hFL [full length] nnAA (e.g., pAMF) w/o leader X = nnAA (e.g., pAMF) position -I5
SSQXRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVAL EGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDE WGKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETH FLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLKHD

25
eCRM-pAMF6 mutant
MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQXGIQKPKSGTQ GNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPG LTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGA SRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQ DAMYEYMAQACAGNRVRNSVGSSLSCINLDWDVIRDXTKTKIES LKEHGPIKNKMSESPNKTVSEEKAXQYLEEFHQTALEHPELSEL XTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSIL PGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELV DIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHXTQPFLHDGYA VSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPG KLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGV HANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSXL SLFFEIKS

26
ADI (mut2) [full length] D277A nnAA (e.g., pAMF) w/ leader X = nnAA (e.g., pAMF) positions -K123, K247, K316
MHHHHHHGSGENLYFQGTAQTPIHVYSEIGKLKKVLLHRPGKEI ENLMPDYLERLLFDDIPFLEDAQKEHDAFAQALRDEGIEVLYLE TLAAESLVTPEIREAFIDEYLSEANIRGRATKKAIRELLMAIED NQELIEXTMAGVQKSELPEIPASEKGLTDLVESNYPFAIDPMPN LYFTRDPFATIGTGVSLNHMFSETRNRETLYGKYIFTHHPIYGG GKVPMVYDRNETTRIEGGDELVLSKDVLAVGISQRTDAASIEXL LVNIFKQNLGFKKVLAFEFANNRKFMHLATVFTMVDYDKFTIHP EIEGDLRVYSVTYDNEELHIVEEXGDLAELLAANLGVEKVDLIR CGGDNLVAAGREQWNDGSNTLTIAPGVVVVYNRNTITNAILESK GLKLIKIHGSELVRGRGGPRCMSMPFEREDI

27
ADI (mut2) [full length] D277A nnAA (e.g., pAMF) w/o leader, with G overhang from cleavage X = nnAA (e.g., pAMF) positions -K123, K247, K316
GTAQTPIHVYSEIGKLKKVLLHRPGKEIENLMPDYLERLLFDDI PFLEDAQKEHDAFAQALRDEGIEVLYLETLAAESLVTPEIREAF IDEYLSEANIRGRATKKAIRELLMAIEDNQELIEXTMAGVQKSE LPEIPASEKGLTDLVESNYPFAIDPMPNLYFTRDPFATIGTGVS LNHMFSETRNRETLYGKYIFTHHPIYGGGKVPMVYDRNETTRIE GGDELVLSKDVLAVGISQRTDAASIEXLLVNIFKQNLGFKKVLA FEFANNRKFMHLATVFTMVDYDKFTIHPEIEGDLRVYSVTYDNE ELHIVEEXGDLAELLAANLGVEKVDLIRCGGDNLVAAGREQWND GSNTLTIAPGVVVVYNRNTITNAILESKGLKLIKIHGSELVRGR GGPRCMSMPFEREDI

28
Sfb 1 repeat region with leader
MHHHHHHGSGVETEDTKEPGVLMGGQSESVEFTKDTQTGMSGQT TPQVETEDTKEPGVLMGGQSESVEFTKDTQTGMSGQTASQVETE DTKEPGVLMGGQSESVEFTKDTQTGMSGQTTPQVETEDTKEPGV LMGGQSESVEFTKDTQTGMSGFSET

29
C5a [90-1035 fragment] WT w/o leader, with G overhang from cleavage
GKTADTPVTSKATIRDLNDPSQVKTLQEKAGKGAGTVVAVIDAG FDKNHEAWRLTDKTKARYQSKEDLEKAKKEHGITYGEWVNDKVA YYHDYSKDGKTAVDQEHGTHVSGILSGNAPSETKEPYRLEGAMP EAQLLLMRVEIVNGLADYARNYAQAIRDAVNLGAKVINMS FGNA ALAYANLPDETKKAFDYAKSKGVSIVTSAGNDSSFGGKTRLPLA DHPDYGVVGTPAAADSTLTVASYSPDKQLTETATVKTADKQDKE MPVLSTNRFEPNKAYDYAYANRGMKEDDFKDVKGKIALIERGDI DFKDKIANAKKAGAVGVLIYDNQDKGFPIELPNVDQMPAAFISR KDGLLLKDNPKKTITFNATPKVLPTASGTKLSRFSSWGLTADGN IKPDIAAPGQDILSSVANNKYAKLSGTSMSAPLVAGIMGLLQKQ YETQYPDMTPSERLDLAKKVLMSSATALYDEDEKAYFSPRQQGA GAVDAKKASAATMYVTDKDNTSSKVHLNNVSDTFEVTVTVHNKS DKPQELYYQATVQTDKVDGKHFALAPKALYETSWQKITIPANSS KQVTVPIDASRFSKDLLAQMKNGYFLEGFVRFKQDPTKEELMSI PYIGFRGDFGNLSALEKPIYDSKDGSSYYHEANSDAKDQLDGDG LQFYALKNNFTALTTESNPWTIIKAVKEGVENIEDIESSEITET IFAGTFAKQDDDSHYYIHRHANGKPYAAISPNGDGNRDYVQFQG TFLRNAKNLVAEVLDKEGNVVWTSEVTEQVVKNYNNDLASTLGS TRFEKTRWDGKDKDGKVVANGTYTYRVRYTPISSGAKEQHTDFD VIVDNTTPEVATSATFSTEDRRLTLASKPKTSQPVYRERIAYTY MDEDLPTTEYISPNEDGTFTLPEEAETMEGATVPLKMSDFTYVV EDMAGNITYTPVTKLLEGHSNK

30
C5a [90-1035 fragment] D131A/S513A w/o leader, with G overhang from cleavage
GKTADTPVTSKATIRDLNDPSQVKTLQEKAGKGAGTVVAVIAAG FDKNHEAWRLTDKTKARYQSKEDLEKAKKEHGITYGEWVNDKVA YYHDYSKDGKTAVDQEHGTHVSGILSGNAPSETKEPYRLEGAMP EAQLLLMRVEIVNGLADYARNYAQAIRDAVNLGAKVINMS FGNA ALAYANLPDETKKAFDYAKSKGVSIVTSAGNDSSFGGKTRLPLA DHPDYGVVGTPAAADSTLTVASYSPDKQLTETATVKTADKQDKE MPVLSTNRFEPNKAYDYAYANRGMKEDDFKDVKGKIALIERGDI DFKDKIANAKKAGAVGVLIYDNQDKGFPIELPNVDQMPAAFISR KDGLLLKDNPKKTITFNATPKVLPTASGTKLSRFSSWGLTADGN IKPDIAAPGQDILSSVANNKYAKLSGTAMSAPLVAGIMGLLQKQ YETQYPDMTPSERLDLAKKVLMSSATALYDEDEKAYFSPRQQGA GAVDAKKASAATMYVTDKDNTSSKVHLNNVSDTFEVTVTVHNKS DKPQELYYQATVQTDKVDGKHFALAPKALYETSWQKITIPANSS KQVTVPIDASRFSKDLLAQMKNGYFLEGFVRFKQDPTKEELMSI PYIGFRGDFGNLSALEKPIYDSKDGSSYYHEANSDAKDQLDGDG LQFYALKNNFTALTTESNPWTIIKAVKEGVENIEDIESSEITET IFAGTFAKQDDDSHYYIHRHANGKPYAAISPNGDGNRDYVQFQG TFLRNAKNLVAEVLDKEGNVVWTSEVTEQVVKNYNNDLASTLGS TRFEKTRWDGKDKDGKVVANGTYTYRVRYTPISSGAKEQHTDFD VIVDNTTPEVATSATFSTEDRRLTLASKPKTSQPVYRERIAYTY MDEDLPTTEYISPNEDGTFTLPEEAETMEGATVPLKMSDFTYVV EDMAGNITYTPVTKLLEGHSNK

31
SLO [79-571 fragment] WT w/o leader, with G overhang from cleavage
GAPKEMPLESAEKEEKKSEDKKKSEEDHTEEINDKIYSLNYNEL EVLAKNGETIENFVPKEGVKKADKFIVIERKKKNINTTPVDISI IDSVTDRTYPAALQLANKGFTENKPDAVVTKRNPQKIHIDLPGM GDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYT ESMVYSKSQIEAALNVNSKILDGTLGIDFKSISKGEKKVMIAAY KQIFYTVSANLPNNPADVFDKSVTFKELQRKGVSNEAPPLFVSN VAYGRTVFVKLETSSKSNDVEAAFSAALKGTDVKTNGKYSDILE NSSFTAVVLGGDAAEHNKVVTKDFDVIRNVIKDNATFSRKNPAY PISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGKINLSHQGAY VAQYEILWDEINYDDKGKEVITKRRWDNNWYSKTSPFSTVIPLG ANSRNIRIMARECTGLAWEWWRKVIDERDVKLSKEINVNISGST LSPYGSITYK

32
SLO [79-571 fragment] W535A w/o leader, with G overhang from cleavage
GAPKEMPLESAEKEEKKSEDKKKSEEDHTEEINDKIYSLNYNEL EVLAKNGETIENFVPKEGVKKADKFIVIERKKKNINTTPVDISI IDSVTDRTYPAALQLANKGFTENKPDAVVTKRNPQKIHIDLPGM GDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYT ESMVYSKSQIEAALNVNSKILDGTLGIDFKSISKGEKKVMIAAY KQIFYTVSANLPNNPADVFDKSVTFKELQRKGVSNEAPPLFVSN VAYGRTVFVKLETSSKSNDVEAAFSAALKGTDVKTNGKYSDILE NSSFTAVVLGGDAAEHNKVVTKDFDVIRNVIKDNATFSRKNPAY PISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGKINLSHQGAY VAQYEILWDEINYDDKGKEVITKRRWDNNWYSKTSPFSTVIPLG ANSRNIRIMARECTGLAAEWWRKVIDERDVKLSKEINVNISGST LSPYGSITYK

33
SpyAD [33-849 fragment] WT w/o leader, with G overhang from cleavage
GQVKADDRASGETKASNTHDDSLPKPETIQEAKATIDAVEKTLS QQKAELTELATALTKTTAEINHLKEQQDNEQKALTSAQEIYTNT LASSEETLLAQGAEHQRELTATETELHNAQADQHSKETALSEQK ASISAETTRAQDLVEQVKTSEQNIAKLNAMISNPDAITKAAQTA NDNTKALSSELEKAKADLENQKAKVKKQLTEELAAQKAALAEKE AELSRLKSSAPSTQDSIVGNNTMKAPQGYPLEELKKLEASGYIG SASYNNYYKEHADQIIAKASPGNQLNQYQDIPADRNRFVDPDNL TPEVQNELAQFAAHMINSVRRQLGLPPVTVTAGSQEFARLLSTS YKKTHGNTRPSFVYGQPGVSGHYGVGPHDKTIIEDSAGASGLIR NDDNMYENIGAFNDVHTVNGIKRGIYDSIKYMLFTDHLHGNTYG HAINFLRVDKHNPNAPVYLGFSTSNVGSLNEHFVMFPESNIANH QRFNKTPIKAVGSTKDYAQRVGTVSDTIAAIKGKVSSLENRLSA IHQEADIMAAQAKVSQLQGKLASTLKQSDSLNLQVRQLNDTKGS LRTELLAAKAKQAQLEATRDQSLAKLASLKAALHQTEALAEQAA ARVTALVAKKAHLQYLRDFKLNPNRLQVIRERIDNTKQDLAKTT SSLLNAQEALAALQAKQSSLEATIATTEHQLTLLKTLANEKEYR HLDEDIATVPDLQVAPPLTGVKPLSYSKIDTTPLVQEMVKETKQ LLEASARLAAENTSLVAEALVGQTSEMVASNAIVSKITSSITQP SSKTSYGSGSSTTSNLISDVDESTQR

34
SpyAD [33-849 fragment] nnAA (e.g., pAMF) w/o leader, with G overhang from cleavage X = nnAA (e.g., pAMF) positions K64/K287/K386/K 657 - numbered
GQVKADDRASGETKASNTHDDSLPKPETIQEAXATIDAVEKTLS QQKAELTELATALTKTTAEINHLKEQQDNEQKALTSAQEIYTNT LASSEETLLAQGAEHQRELTATETELHNAQADQHSKETALSEQK ASISAETTRAQDLVEQVKTSEQNIAKLNAMISNPDAITKAAQTA NDNTKALSSELEKAKADLENQKAKVKKQLTEELAAQKAALAEKE AELSRLKSSAPSTQDSIVGNNTMKAPQGYPLEELKXLEASGYIG SASYNNYYKEHADQIIAKASPGNQLNQYQDIPADRNRFVDPDNL TPEVQNELAQFAAHMINSVRRQLGLPPVTVTAGSQEFARLLSTS YKXTHGNTRPSFVYGQPGVSGHYGVGPHDKTIIEDSAGASGLIR NDDNMYENIGAFNDVHTVNGIKRGIYDSIKYMLFTDHLHGNTYG HAINFLRVDKHNPNAPVYLGFSTSNVGSLNEHFVMFPESNIANH QRFNKTPIKAVGSTKDYAQRVGTVSDTIAAIKGKVSSLENRLSA IHQEADIMAAQAKVSQLQGKLASTLKQSDSLNLQVRQLNDTKGS LRTELLAAKAKQAQLEATRDQSLAKLASLKAALHQTEALAEQAA ARVTALVAKXAHLQYLRDFKLNPNRLQVIRERIDNTKQDLAKTT SSLLNAQEALAALQAKQSSLEATIATTEHQLTLLKTLANEKEYR HLDEDIATVPDLQVAPPLTGVKPLSYSKIDTTPLVQEMVKETKQ LLEASARLAAENTSLVAEALVGQTSEMVASNAIVSKITSSITQP SSKTSYGSGSSTTSNLISDVDESTQR

35
ADI [full length] WT w/o leader, with G overhang from cleavage
GTAQTPIHVYSEIGKLKKVLLHRPGKEIENLMPDYLERLLFDDI PFLEDAQKEHDAFAQALRDEGIEVLYLETLAAESLVTPEIREAF IDEYLSEANIRGRATKKAIRELLMAIEDNQELIEKTMAGVQKSE LPEIPASEKGLTDLVESNYPFAIDPMPNLYFTRDPFATIGTGVS LNHMFSETRNRETLYGKYIFTHHPIYGGGKVPMVYDRNETTRIE GGDELVLSKDVLAVGISQRTDAASIEKLLVNIFKQNLGFKKVLA FEFANNRKFMHLDTVFTMVDYDKFTIHPEIEGDLRVYSVTYDNE ELHIVEEKGDLAELLAANLGVEKVDLIRCGGDNLVAAGREQWND GSNTLTIAPGVVVVYNRNTITNAILESKGLKLIKIHGSELVRGR GGPRCMSMPFEREDI

36
ADI [full length] D277A w/o leader, with G overhang from cleavage
GTAQTPIHVYSEIGKLKKVLLHRPGKEIENLMPDYLERLLFDDI PFLEDAQKEHDAFAQALRDEGIEVLYLETLAAESLVTPEIREAF IDEYLSEANIRGRATKKAIRELLMAIEDNQELIEKTMAGVQKSE LPEIPASEKGLTDLVESNYPFAIDPMPNLYFTRDPFATIGTGVS LNHMFSETRNRETLYGKYIFTHHPIYGGGKVPMVYDRNETTRIE GGDELVLSKDVLAVGISQRTDAASIEKLLVNIFKQNLGFKKVLA FEFANNRKFMHLATVFTMVDYDKFTIHPEIEGDLRVYSVTYDNE ELHIVEEKGDLAELLAANLGVEKVDLIRCGGDNLVAAGREQWND GSNTLTIAPGVVVVYNRNTITNAILESKGLKLIKIHGSELVRGR GGPRCMSMPFEREDI

37
ADI [full length] nnAA (e.g., pAMF) w/o leader, with G overhang from cleavage X = nnAA (e.g., pAMF) positions -K15, K193, K316
GTAQTPIHVYSEIGXLKKVLLHRPGKEIENLMPDYLERLLFDDI PFLEDAQKEHDAFAQALRDEGIEVLYLETLAAESLVTPEIREAF IDEYLSEANIRGRATKKAIRELLMAIEDNQELIEKTMAGVQKSE LPEIPASEKGLTDLVESNYPFAIDPMPNLYFTRDPFATIGTGVS LNHMFSETRNRETLYGXYIFTHHPIYGGGKVPMVYDRNETTRIE GGDELVLSKDVLAVGISQRTDAASIEKLLVNIFKQNLGFKKVLA FEFANNRKFMHLDTVFTMVDYDKFTIHPEIEGDLRVYSVTYDNE ELHIVEEXGDLAELLAANLGVEKVDLIRCGGDNLVAAGREQWND GSNTLTIAPGVVVVYNRNTITNAILESKGLKLIKIHGSELVRGR GGPRCMSMPFEREDI

38
ADI (mut5) [full length] D277A nnAA (e.g., pAMF) w/o leader, with G overhang from cleavage X = nnAA (e.g., pAMF) positions -K15, K193, K316
GTAQTPIHVYSEIGXLKKVLLHRPGKEIENLMPDYLERLLFDDI PFLEDAQKEHDAFAQALRDEGIEVLYLETLAAESLVTPEIREAF IDEYLSEANIRGRATKKAIRELLMAIEDNQELIEKTMAGVQKSE LPEIPASEKGLTDLVESNYPFAIDPMPNLYFTRDPFATIGTGVS LNHMFSETRNRETLYGXYIFTHHPIYGGGKVPMVYDRNETTRIE GGDELVLSKDVLAVGISQRTDAASIEKLLVNIFKQNLGFKKVLA FEFANNRKFMHLATVFTMVDYDKFTIHPEIEGDLRVYSVTYDNE ELHIVEEXGDLAELLAANLGVEKVDLIRCGGDNLVAAGREQWND GSNTLTIAPGVVVVYNRNTITNAILESKGLKLIKIHGSELVRGR GGPRCMSMPFEREDI

39
hFL [full length] WT w/ leader
MHHHHHHGSGSSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLG FYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALF QDIKKPAEDEWGKTPDAMKAAMALEKKLNQALLDLHALGSARTD PHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLF ERLTLKHD

40
hFL [full length] WT w/o leader
SSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVAL EGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDE WGKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETH FLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLKHD

41
hFL [full length] nnAA (e.g., pAMF) w/ leader X = nnAA (e.g., pAMF) position -I5
MHHHHHHGSGSSQXRQNYSTDVEAAVNSLVNLYLQASYTYLSLG FYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALF QDIKKPAEDEWGKTPDAMKAAMALEKKLNQALLDLHALGSARTD PHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLF ERLTLKHD

42
hFL [full length] nnAA (e.g., pAMF) w/o leader X = nnAA (e.g., pAMF) position -I5
SSQXRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVAL EGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDE WGKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETH FLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLKHD

43
eCRM-pAMF6 mutant
GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQXGIQKPKSGTQG NYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGL TKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGAS RVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQD AMYEYMAQACAGNRVRNSVGSSLSCINLDWDVIRDXTKTKIESL KEHGPIKNKMSESPNKTVSEEKAXQYLEEFHQTALEHPELSELX TVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILP GIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVD IGFAAYNFVESIINLFQVVHNSYNRPAYSPGHXTQPFLHDGYAV SWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGK LDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVH ANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSXLS LFFEIKS

44
SLO(ΔC11) W535A w/o leader, with G overhang from cleavage
GAPKEMPLESAEKEEKKSEDKKKSEEDHTEEINDKIYSLNYNEL EVLAKNGETIENFVPKEGVKKADKFIVIERKKKNINTTPVDISI IDSVTDRTYPAALQLANKGFTENKPDAVVTKRNPQKIHIDLPGM GDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYT ESMVYSKSQIEAALNVNSKILDGTLGIDFKSISKGEKKVMIAAY KQIFYTVSANLPNNPADVFDKSVTFKELQRKGVSNEAPPLFVSN VAYGRTVFVKLETSSKSNDVEAAFSAALKGTDVKTNGKYSDILE NSSFTAVVLGGDAAEHNKVVTKDFDVIRNVIKDNATFSRKNPAY PISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGKINLSHQGAY VAQYEILWDEINYDDKGKEVITKRRWDNNWYSKTSPFSTVIPLG ANSRNIRIMARECTGLAAEWWRKVIDERDVKLSKEINVNISGS

45
SLO(ΔC21) fragment W535A w/o leader, with G overhang from cleavage
GAPKEMPLESAEKEEKKSEDKKKSEEDHTEEINDKIYSLNYNEL EVLAKNGETIENFVPKEGVKKADKFIVIERKKKNINTTPVDISI IDSVTDRTYPAALQLANKGFTENKPDAVVTKRNPQKIHIDLPGM GDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYT ESMVYSKSQIEAALNVNSKILDGTLGIDFKSISKGEKKVMIAAY KQIFYTVSANLPNNPADVFDKSVTFKELQRKGVSNEAPPLFVSN VAYGRTVFVKLETSSKSNDVEAAFSAALKGTDVKTNGKYSDILE NSSFTAVVLGGDAAEHNKVVTKDFDVIRNVIKDNATFSRKNPAY PISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGKINLSHQGAY VAQYEILWDEINYDDKGKEVITKRRWDNNWYSKTSPFSTVIPLG ANSRNIRIMARECTGLAAEWWRKVIDERDVKLS

46
SLO(ΔC31) fragment W535A w/o leader, with G overhang from cleavage
GAPKEMPLESAEKEEKKSEDKKKSEEDHTEEINDKIYSLNYNEL EVLAKNGETIENFVPKEGVKKADKFIVIERKKKNINTTPVDISI IDSVTDRTYPAALQLANKGFTENKPDAVVTKRNPQKIHIDLPGM GDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYT ESMVYSKSQIEAALNVNSKILDGTLGIDFKSISKGEKKVMIAAY KQIFYTVSANLPNNPADVFDKSVTFKELQRKGVSNEAPPLFVSN VAYGRTVFVKLETSSKSNDVEAAFSAALKGTDVKTNGKYSDILE NSSFTAVVLGGDAAEHNKVVTKDFDVIRNVIKDNATFSRKNPAY PISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGKINLSHQGAY VAQYEILWDEINYDDKGKEVITKRRWDNNWYSKTSPFSTVIPLG ANSRNIRIMARECTGLAAEWWRK

47
SLO(ΔC41) fragment W535A w/o leader, with G overhang from cleavage
GAPKEMPLESAEKEEKKSEDKKKSEEDHTEEINDKIYSLNYNEL EVLAKNGETIENFVPKEGVKKADKFIVIERKKKNINTTPVDISI IDSVTDRTYPAALQLANKGFTENKPDAVVTKRNPQKIHIDLPGM GDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYT ESMVYSKSQIEAALNVNSKILDGTLGIDFKSISKGEKKVMIAAY KQIFYTVSANLPNNPADVFDKSVTFKELQRKGVSNEAPPLFVSN VAYGRTVFVKLETSSKSNDVEAAFSAALKGTDVKTNGKYSDILE NSSFTAVVLGGDAAEHNKVVTKDFDVIRNVIKDNATFSRKNPAY PISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGKINLSHQGAY VAQYEILWDEINYDDKGKEVITKRRWDNNWYSKTSPFSTVIPLG ANSRNIRIMAREC

48
SLO(ΔC51) fragment W535A w/o leader, with G overhang from cleavage
GAPKEMPLESAEKEEKKSEDKKKSEEDHTEEINDKIYSLNYNEL EVLAKNGETIENFVPKEGVKKADKFIVIERKKKNINTTPVDISI IDSVTDRTYPAALQLANKGFTENKPDAVVTKRNPQKIHIDLPGM GDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYT ESMVYSKSQIEAALNVNSKILDGTLGIDFKSISKGEKKVMIAAY KQIFYTVSANLPNNPADVFDKSVTFKELQRKGVSNEAPPLFVSN VAYGRTVFVKLETSSKSNDVEAAFSAALKGTDVKTNGKYSDILE NSSFTAVVLGGDAAEHNKVVTKDFDVIRNVIKDNATFSRKNPAY PISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGKINLSHQGAY VAQYEILWDEINYDDKGKEVITKRRWDNNWYSKTSPFSTVIPLG ANS

49
SLO(ΔC61) fragment W535A w/o leader, with G overhang from cleavage
GAPKEMPLESAEKEEKKSEDKKKSEEDHTEEINDKIYSLNYNEL EVLAKNGETIENFVPKEGVKKADKFIVIERKKKNINTTPVDISI IDSVTDRTYPAALQLANKGFTENKPDAVVTKRNPQKIHIDLPGM GDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYT ESMVYSKSQIEAALNVNSKILDGTLGIDFKSISKGEKKVMIAAY KQIFYTVSANLPNNPADVFDKSVTFKELQRKGVSNEAPPLFVSN VAYGRTVFVKLETSSKSNDVEAAFSAALKGTDVKTNGKYSDILE NSSFTAVVLGGDAAEHNKVVTKDFDVIRNVIKDNATFSRKNPAY PISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGKINLSHQGAY VAQYEILWDEINYDDKGKEVITKRRWDNNWYSKTSPF

50
SLO(ΔC71) fragment W535A w/o leader, with G overhang from cleavage
GAPKEMPLESAEKEEKKSEDKKKSEEDHTEEINDKIYSLNYNEL EVLAKNGETIENFVPKEGVKKADKFIVIERKKKNINTTPVDISI IDSVTDRTYPAALQLANKGFTENKPDAVVTKRNPQKIHIDLPGM GDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYT ESMVYSKSQIEAALNVNSKILDGTLGIDFKSISKGEKKVMIAAY KQIFYTVSANLPNNPADVFDKSVTFKELQRKGVSNEAPPLFVSN VAYGRTVFVKLETSSKSNDVEAAFSAALKGTDVKTNGKYSDILE NSSFTAVVLGGDAAEHNKVVTKDFDVIRNVIKDNATFSRKNPAY PISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGKINLSHQGAY VAQYEILWDEINYDDKGKEVITKRRWD

51
SLO(ΔC81) fragment W535A w/o leader, with G overhang from cleavage
GAPKEMPLESAEKEEKKSEDKKKSEEDHTEEINDKIYSLNYNEL EVLAKNGETIENFVPKEGVKKADKFIVIERKKKNINTTPVDISI IDSVTDRTYPAALQLANKGFTENKPDAVVTKRNPQKIHIDLPGM GDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYT ESMVYSKSQIEAALNVNSKILDGTLGIDFKSISKGEKKVMIAAY KQIFYTVSANLPNNPADVFDKSVTFKELQRKGVSNEAPPLFVSN VAYGRTVFVKLETSSKSNDVEAAFSAALKGTDVKTNGKYSDILE NSSFTAVVLGGDAAEHNKVVTKDFDVIRNVIKDNATFSRKNPAY PISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGKINLSHQGAY VAQYEILWDEINYDDKG

52
SLO(AC91) fragment W535A w/o leader, with G overhang from cleavage
GAPKEMPLESAEKEEKKSEDKKKSEEDHTEEINDKIYSLNYNEL EVLAKNGETIENFVPKEGVKKADKFIVIERKKKNINTTPVDISI IDSVTDRTYPAALQLANKGFTENKPDAVVTKRNPQKIHIDLPGM GDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYT ESMVYSKSQIEAALNVNSKILDGTLGIDFKSISKGEKKVMIAAY KQIFYTVSANLPNNPADVFDKSVTFKELQRKGVSNEAPPLFVSN VAYGRTVFVKLETSSKSNDVEAAFSAALKGTDVKTNGKYSDILE NSSFTAVVLGGDAAEHNKVVTKDFDVIRNVIKDNATFSRKNPAY PISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGKINLSHQGAY VAQYEIL

53
SLO(ΔC101) fragment W535A w/o leader, with G overhang from cleavage
GAPKEMPLESAEKEEKKSEDKKKSEEDHTEEINDKIYSLNYNEL EVLAKNGETIENFVPKEGVKKADKFIVIERKKKNINTTPVDISI IDSVTDRTYPAALQLANKGFTENKPDAVVTKRNPQKIHIDLPGM GDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYT ESMVYSKSQIEAALNVNSKILDGTLGIDFKSISKGEKKVMIAAY KQIFYTVSANLPNNPADVFDKSVTFKELQRKGVSNEAPPLFVSN VAYGRTVFVKLETSSKSNDVEAAFSAALKGTDVKTNGKYSDILE NSSFTAVVLGGDAAEHNKVVTKDFDVIRNVIKDNATFSRKNPAY PISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGKINLSHQ

54
SLO(ΔC101) fragment W535A w/o leader, with G overhang from cleavage X = nnAA (e.g., pAMF) or native amino acid (e.g.:K98, K112, R151, K189, K272, K323, K357, K375, K407, K464; or K or R.)
GAPKEMPLESAEKEEKKSEDXKKSEEDHTEEINDXIYSLNYNEL EVLAKNGETIENFVPKEGVKKADKFIVIEXKKKNINTTPVDISI IDSVTDRTYPAALQLANKGFTENXPDAVVTKRNPQKIHIDLPGM GDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYT ESMVYSKSQIEAALNVNSXILDGTLGIDFKSISKGEKKVMIAAY KQIFYTVSANLPNNPADVFDKSVTFXELQRKGVSNEAPPLFVSN VAYGRTVFVKLETSSXSNDVEAAFSAALKGTDVXTNGKYSDILE NSSFTAVVLGGDAAEHNKVVTXDFDVIRNVIKDNATFSRKNPAY PISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGXINLSHQ

55
SLO(ΔC101) fragment W535A -nnAA (e.g., pAMF) 1 - w/o leader, with G overhang from cleavage (420995) X = nnAA (e.g., pAMF) positions -K98, R151, K272, K357
GAPKEMPLESAEKEEKKSEDXKKSEEDHTEEINDKIYSLNYNEL EVLAKNGETIENFVPKEGVKKADKFIVIEXKKKNINTTPVDISI IDSVTDRTYPAALQLANKGFTENKPDAVVTKRNPQKIHIDLPGM GDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYT ESMVYSKSQIEAALNVNSXILDGTLGIDFKSISKGEKKVMIAAY KQIFYTVSANLPNNPADVFDKSVTFKELQRKGVSNEAPPLFVSN VAYGRTVFVKLETSSXSNDVEAAFSAALKGTDVKTNGKYSDILE NSSFTAVVLGGDAAEHNKVVTKDFDVIRNVIKDNATFSRKNPAY PISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGKINLSHQ

56
SLO(ΔC101) fragment W535A -nnAA (e.g., pAMF) 2 - w/o leader, with G overhang from cleavage (420996) X = nnAA (e.g., pAMF) positions -K112, K189, K323, K375
GAPKEMPLESAEKEEKKSEDKKKSEEDHTEEINDXIYSLNYNEL EVLAKNGETIENFVPKEGVKKADKFIVIERKKKNINTTPVDISI IDSVTDRTYPAALQLANKGFTENXPDAVVTKRNPQKIHIDLPGM GDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYT ESMVYSKSQIEAALNVNSKILDGTLGIDFKSISKGEKKVMIAAY KQIFYTVSANLPNNPADVFDKSVTFXELQRKGVSNEAPPLFVSN VAYGRTVFVKLETSSKSNDVEAAFSAALKGTDVXTNGKYSDILE NSSFTAVVLGGDAAEHNKVVTKDFDVIRNVIKDNATFSRKNPAY PISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGKINLSHQ

57
SLO(ΔC101) fragment W535A -nnAA (e.g., pAMF) 3 - w/o leader, with G overhang from cleavage (420997) X = nnAA (e.g., pAMF) positions -R151, K272, K357, K407
GAPKEMPLESAEKEEKKSEDKKKSEEDHTEEINDKIYSLNYNEL EVLAKNGETIENFVPKEGVKKADKFIVIEXKKKNINTTPVDISI IDSVTDRTYPAALQLANKGFTENKPDAVVTKRNPQKIHIDLPGM GDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYT ESMVYSKSQIEAALNVNSXILDGTLGIDFKSISKGEKKVMIAAY KQIFYTVSANLPNNPADVFDKSVTFKELQRKGVSNEAPPLFVSN VAYGRTVFVKLETSSXSNDVEAAFSAALKGTDVKTNGKYSDILE NSSFTAVVLGGDAAEHNKVVTXDFDVIRNVIKDNATFSRKNPAY PISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGKINLSHQ

58
SLO(ΔC101) fragment W535A -nnAA (e.g., pAMF) 4 - w/o leader, with G overhang from cleavage (420998) X = nnAA (e.g., pAMF) positions -R151, K272, K375, K464
GAPKEMPLESAEKEEKKSEDKKKSEEDHTEEINDKIYSLNYNEL EVLAKNGETIENFVPKEGVKKADKFIVIEXKKKNINTTPVDISI IDSVTDRTYPAALQLANKGFTENKPDAVVTKRNPQKIHIDLPGM GDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYT ESMVYSKSQIEAALNVNSXILDGTLGIDFKSISKGEKKVMIAAY KQIFYTVSANLPNNPADVFDKSVTFKELQRKGVSNEAPPLFVSN VAYGRTVFVKLETSSKSNDVEAAFSAALKGTDVXTNGKYSDILE NSSFTAVVLGGDAAEHNKVVTKDFDVIRNVIKDNATFSRKNPAY PISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGXINLSHQ

59
SLO(ΔC101) fragment W535A -nnAA (e.g., pAMF)5 - w/o leader, with G overhang from cleavage (420999) X = nnAA (e.g., pAMF) positions -K112, K272, K357, K464
GAPKEMPLESAEKEEKKSEDKKKSEEDHTEEINDXIYSLNYNEL EVLAKNGETIENFVPKEGVKKADKFIVIERKKKNINTTPVDISI IDSVTDRTYPAALQLANKGFTENKPDAVVTKRNPQKIHIDLPGM GDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYT ESMVYSKSQIEAALNVNSXILDGTLGIDFKSISKGEKKVMIAAY KQIFYTVSANLPNNPADVFDKSVTFKELQRKGVSNEAPPLFVSN VAYGRTVFVKLETSSXSNDVEAAFSAALKGTDVKTNGKYSDILE NSSFTAVVLGGDAAEHNKVVTKDFDVIRNVIKDNATFSRKNPAY PISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGXINLSHQ

60
SLO(ΔC101) fragment W535A -nnAA (e.g., pAMF) 6 - w/o leader, with G overhang from cleavage (421000) X = nnAA (e.g., pAMF) positions -K98, K189, K357
GAPKEMPLESAEKEEKKSEDXKKSEEDHTEEINDKIYSLNYNEL EVLAKNGETIENFVPKEGVKKADKFIVIERKKKNINTTPVDISI IDSVTDRTYPAALQLANKGFTENXPDAVVTKRNPQKIHIDLPGM GDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYT ESMVYSKSQIEAALNVNSKILDGTLGIDFKSISKGEKKVMIAAY KQIFYTVSANLPNNPADVFDKSVTFKELQRKGVSNEAPPLFVSN VAYGRTVFVKLETSSXSNDVEAAFSAALKGTDVKTNGKYSDILE NSSFTAVVLGGDAAEHNKVVTKDFDVIRNVIKDNATFSRKNPAY PISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGKINLSHQ

61
SLO(ΔC101) fragment W535A -nnAA (e.g., pAMF) 7 - w/o leader, with G overhang from cleavage (421001) X = nnAA (e.g., pAMF) positions -K112, K189, K323
GAPKEMPLESAEKEEKKSEDXKKSEEDHTEEINDXIYSLNYNEL EVLAKNGETIENFVPKEGVKKADKFIVIERKKKNINTTPVDISI IDSVTDRTYPAALQLANKGFTENXPDAVVTKRNPQKIHIDLPGM GDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYT ESMVYSKSQIEAALNVNSKILDGTLGIDFKSISKGEKKVMIAAY KQIFYTVSANLPNNPADVFDKSVTFXELQRKGVSNEAPPLFVSN VAYGRTVFVKLETSSKSNDVEAAFSAALKGTDVKTNGKYSDILE NSSFTAVVLGGDAAEHNKVVTKDFDVIRNVIKDNATFSRKNPAY PISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGKINLSHQ

62
SLO(ΔC101) fragment W535A -nnAA (e.g., pAMF) 8 - w/o leader, with G overhang from cleavage (421002) X = nnAA (e.g., pAMF) positions -K98, R151, K272
GAPKEMPLESAEKEEKKSEDXKKSEEDHTEEINDKIYSLNYNEL EVLAKNGETIENFVPKEGVKKADKFIVIEXKKKNINTTPVDISI IDSVTDRTYPAALQLANKGFTENKPDAVVTKRNPQKIHIDLPGM GDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYT ESMVYSKSQIEAALNVNSXILDGTLGIDFKSISKGEKKVMIAAY KQIFYTVSANLPNNPADVFDKSVTFKELQRKGVSNEAPPLFVSN VAYGRTVFVKLETSSKSNDVEAAFSAALKGTDVKTNGKYSDILE NSSFTAVVLGGDAAEHNKVVTKDFDVIRNVIKDNATFSRKNPAY PISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGKINLSHQ

63
SLO(ΔC101) fragment W535A -nnAA (e.g., pAMF) 9 - w/o leader, with G overhang from cleavage (421003) X = nnAA (e.g., pAMF) positions -K112, K272, K375
GAPKEMPLESAEKEEKKSEDKKKSEEDHTEEINDXIYSLNYNEL EVLAKNGETIENFVPKEGVKKADKFIVIERKKKNINTTPVDISI IDSVTDRTYPAALQLANKGFTENKPDAVVTKRNPQKIHIDLPGM GDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYT ESMVYSKSQIEAALNVNSXILDGTLGIDFKSISKGEKKVMIAAY KQIFYTVSANLPNNPADVFDKSVTFKELQRKGVSNEAPPLFVSN VAYGRTVFVKLETSSKSNDVEAAFSAALKGTDVXTNGKYSDILE NSSFTAVVLGGDAAEHNKVVTKDFDVIRNVIKDNATFSRKNPAY PISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGKINLSHQ

64
SLO(ΔC101) fragment W535A -nnAA (e.g., pAMF) 10 - w/o leader, with G overhang from cleavage (421004) X = nnAA (e.g., pAMF) positions -K112, K323, K407
GAPKEMPLESAEKEEKKSEDKKKSEEDHTEEINDXIYSLNYNEL EVLAKNGETIENFVPKEGVKKADKFIVIERKKKNINTTPVDISI IDSVTDRTYPAALQLANKGFTENKPDAVVTKRNPQKIHIDLPGM GDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYT ESMVYSKSQIEAALNVNSKILDGTLGIDFKSISKGEKKVMIAAY KQIFYTVSANLPNNPADVFDKSVTFXELQRKGVSNEAPPLFVSN VAYGRTVFVKLETSSKSNDVEAAFSAALKGTDVKTNGKYSDILE NSSFTAVVLGGDAAEHNKVVTXDFDVIRNVIKDNATFSRKNPAY PISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGKINLSHQ

65
SLO(ΔC101) fragment W535A -nnAA (e.g., pAMF) 10 - w/o leader, with G overhang from cleavage (426507) X = nnAA (e.g., pAMF) positions -K98, R151, K272, K357, K407
GAPKEMPLESAEKEEKKSEDXKKSEEDHTEEINDKIYSLNYNEL EVLAKNGETIENFVPKEGVKKADKFIVIEXKKKNINTTPVDISI IDSVTDRTYPAALQLANKGFTENKPDAVVTKRNPQKIHIDLPGM GDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYT ESMVYSKSQIEAALNVNSXILDGTLGIDFKSISKGEKKVMIAAY KQIFYTVSANLPNNPADVFDKSVTFKELQRKGVSNEAPPLFVSN VAYGRTVFVKLETSSXSNDVEAAFSAALKGTDVKTNGKYSDILE NSSFTAVVLGGDAAEHNKVVTXDFDVIRNVIKDNATFSRKNPAY PISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGKINLSHQ

66
SLO(ΔC101) fragment W535A -nnAA (e.g., pAMF) 10 - w/o leader, with G overhang from cleavage (426508) X = nnAA (e.g., pAMF) positions -K112, K189, K323, K375, K464
GAPKEMPLESAEKEEKKSEDKKKSEEDHTEEINDXIYSLNYNEL EVLAKNGETIENFVPKEGVKKADKFIVIERKKKNINTTPVDISI IDSVTDRTYPAALQLANKGFTENXPDAVVTKRNPQKIHIDLPGM GDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYT ESMVYSKSQIEAALNVNSKILDGTLGIDFKSISKGEKKVMIAAY KQIFYTVSANLPNNPADVFDKSVTFXELQRKGVSNEAPPLFVSN VAYGRTVFVKLETSSKSNDVEAAFSAALKGTDVXTNGKYSDILE NSSFTAVVLGGDAAEHNKVVTKDFDVIRNVIKDNATFSRKNPAY PISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGXINLSHQ

67
SLO(ΔC101) fragment W535A -nnAA (e.g., pAMF) 10 - w/o leader, with G overhang from cleavage (426509) X = nnAA (e.g., pAMF) positions -K112, R151, K272, K357, K407
GAPKEMPLESAEKEEKKSEDKKKSEEDHTEEINDXIYSLNYNEL EVLAKNGETIENFVPKEGVKKADKFIVIEXKKKNINTTPVDISI IDSVTDRTYPAALQLANKGFTENKPDAVVTKRNPQKIHIDLPGM GDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYT ESMVYSKSQIEAALNVNSXILDGTLGIDFKSISKGEKKVMIAAY KQIFYTVSANLPNNPADVFDKSVTFKELQRKGVSNEAPPLFVSN VAYGRTVFVKLETSSXSNDVEAAFSAALKGTDVKTNGKYSDILE NSSFTAVVLGGDAAEHNKVVTXDFDVIRNVIKDNATFSRKNPAY PISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGKINLSHQ

68
SLO(ΔC101) fragment W535A -nnAA (e.g., pAMF) 10 - w/o leader, with G overhang from cleavage (426510) X = nnAA (e.g., pAMF) positions -K98, R151, K272, K357, K407, K464
GAPKEMPLESAEKEEKKSEDXKKSEEDHTEEINDKIYSLNYNEL EVLAKNGETIENFVPKEGVKKADKFIVIEXKKKNINTTPVDISI IDSVTDRTYPAALQLANKGFTENKPDAVVTKRNPQKIHIDLPGM GDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYT ESMVYSKSQIEAALNVNSXILDGTLGIDFKSISKGEKKVMIAAY KQIFYTVSANLPNNPADVFDKSVTFKELQRKGVSNEAPPLFVSN VAYGRTVFVKLETSSXSNDVEAAFSAALKGTDVKTNGKYSDILE NSSFTAVVLGGDAAEHNKVVTXDFDVIRNVIKDNATFSRKNPAY PISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGXINLSHQ

69
SLO(ΔC101) fragment W535A -nnAA (e.g., pAMF) 10 - w/o leader, with G overhang from cleavage (426511) X = nnAA (e.g., pAMF) positions -K112, R151, K189, K323, K375, K464
GAPKEMPLESAEKEEKKSEDKKKSEEDHTEEINDXIYSLNYNEL EVLAKNGETIENFVPKEGVKKADKFIVIEXKKKNINTTPVDISI IDSVTDRTYPAALQLANKGFTENXPDAVVTKRNPQKIHIDLPGM GDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYT ESMVYSKSQIEAALNVNSKILDGTLGIDFKSISKGEKKVMIAAY KQIFYTVSANLPNNPADVFDKSVTFXELQRKGVSNEAPPLFVSN VAYGRTVFVKLETSSKSNDVEAAFSAALKGTDVXTNGKYSDILE NSSFTAVVLGGDAAEHNKVVTKDFDVIRNVIKDNATFSRKNPAY PISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGXINLSHQ

70
SLO(ΔC101) fragment W535A -nnAA (e.g., pAMF) 10 - w/o leader, with G overhang from cleavage (426512) X = nnAA (e.g., pAMF) positions -K98, K112, K189, K323, K375, K464
GAPKEMPLESAEKEEKKSEDXKKSEEDHTEEINDXIYSLNYNEL EVLAKNGETIENFVPKEGVKKADKFIVIERKKKNINTTPVDISI IDSVTDRTYPAALQLANKGFTENXPDAVVTKRNPQKIHIDLPGM GDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYT ESMVYSKSQIEAALNVNSKILDGTLGIDFKSISKGEKKVMIAAY KQIFYTVSANLPNNPADVFDKSVTFXELQRKGVSNEAPPLFVSN VAYGRTVFVKLETSSKSNDVEAAFSAALKGTDVXTNGKYSDILE NSSFTAVVLGGDAAEHNKVVTKDFDVIRNVIKDNATFSRKNPAY PISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGXINLSHQ

71
SLO(ΔC101) fragment W535A -nnAA (e.g., pAMF) 10 - w/o leader, with G overhang from cleavage (426513) X = nnAA (e.g., pAMF) positions -K112, R151, K189, K272, K357, K407, K464
GAPKEMPLESAEKEEKKSEDKKKSEEDHTEEINDXIYSLNYNEL EVLAKNGETIENFVPKEGVKKADKFIVIEXKKKNINTTPVDISI IDSVTDRTYPAALQLANKGFTENXPDAVVTKRNPQKIHIDLPGM GDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYT ESMVYSKSQIEAALNVNSXILDGTLGIDFKSISKGEKKVMIAAY KQIFYTVSANLPNNPADVFDKSVTFKELQRKGVSNEAPPLFVSN VAYGRTVFVKLETSSXSNDVEAAFSAALKGTDVKTNGKYSDILE NSSFTAVVLGGDAAEHNKVVTXDFDVIRNVIKDNATFSRKNPAY PISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGXINLSHQ

72
SLO(ΔC101) fragment W535A -nnAA (e.g., pAMF) 10 - w/o leader, with G overhang from cleavage (426514) X = nnAA (e.g., pAMF) positions -K98, R151, K189, K323, K375, K407, K464
GAPKEMPLESAEKEEKKSEDXKKSEEDHTEEINDKIYSLNYNEL EVLAKNGETIENFVPKEGVKKADKFIVIEXKKKNINTTPVDISI IDSVTDRTYPAALQLANKGFTENXPDAVVTKRNPQKIHIDLPGM GDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYT ESMVYSKSQIEAALNVNSKILDGTLGIDFKSISKGEKKVMIAAY KQIFYTVSANLPNNPADVFDKSVTFXELQRKGVSNEAPPLFVSN VAYGRTVFVKLETSSKSNDVEAAFSAALKGTDVXTNGKYSDILE NSSFTAVVLGGDAAEHNKVVTXDFDVIRNVIKDNATFSRKNPAY PISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGXINLSHQ

73
SLO(ΔC101) fragment W535A -nnAA (e.g., pAMF) 10 - w/o leader, with G overhang from cleavage (426515) X = nnAA (e.g., pAMF) positions -K112, K189, K272, K357, K375, K407, K464
GAPKEMPLESAEKEEKKSEDKKKSEEDHTEEINDXIYSLNYNEL EVLAKNGETIENFVPKEGVKKADKFIVIERKKKNINTTPVDISI IDSVTDRTYPAALQLANKGFTENXPDAVVTKRNPQKIHIDLPGM GDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYT ESMVYSKSQIEAALNVNSXILDGTLGIDFKSISKGEKKVMIAAY KQIFYTVSANLPNNPADVFDKSVTFKELQRKGVSNEAPPLFVSN VAYGRTVFVKLETSSXSNDVEAAFSAALKGTDVXTNGKYSDILE NSSFTAVVLGGDAAEHNKVVTXDFDVIRNVIKDNATFSRKNPAY PISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGXINLSHQ

74
SLO(ΔC101) fragment W535A -nnAA (e.g., pAMF) 10 - w/o leader, with G overhang from cleavage (426516) X = nnAA (e.g., pAMF) positions -K98, K112, R151, K189, K272, K323, K357, K375,
GAPKEMPLESAEKEEKKSEDXKKSEEDHTEEINDXIYSLNYNEL EVLAKNGETIENFVPKEGVKKADKFIVIEXKKKNINTTPVDISI IDSVTDRTYPAALQLANKGFTENXPDAVVTKRNPQKIHIDLPGM GDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYT ESMVYSKSQIEAALNVNSXILDGTLGIDFKSISKGEKKVMIAAY KQIFYTVSANLPNNPADVFDKSVTFXELQRKGVSNEAPPLFVSN VAYGRTVFVKLETSSXSNDVEAAFSAALKGTDVXTNGKYSDILE NSSFTAVVLGGDAAEHNKVVTKDFDVIRNVIKDNATFSRKNPAY PISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGKINLSHQ

75
SLO(ΔC101) fragment W535A -nnAA (e.g., pAMF) 10 - w/o leader, with G overhang from cleavage (426510) X = nnAA (e.g., pAMF) positions -K98, R151, K189, K272, K323, K357, K407, K464
GAPKEMPLESAEKEEKKSEDXKKSEEDHTEEINDKIYSLNYNEL EVLAKNGETIENFVPKEGVKKADKFIVIEXKKKNINTTPVDISI IDSVTDRTYPAALQLANKGFTENXPDAVVTKRNPQKIHIDLPGM GDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYT ESMVYSKSQIEAALNVNSXILDGTLGIDFKSISKGEKKVMIAAY KQIFYTVSANLPNNPADVFDKSVTFXELQRKGVSNEAPPLFVSN VAYGRTVFVKLETSSXSNDVEAAFSAALKGTDVKTNGKYSDILE NSSFTAVVLGGDAAEHNKVVTXDFDVIRNVIKDNATFSRKNPAY PISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGXINLSHQ

76
SLO(ΔC101) fragment W535A -nnAA (e.g., pAMF) 10 - w/o leader, with G overhang from cleavage (426515) X = nnAA (e.g., pAMF) positions -K112, K189, K272, K323, K357, K375, K407, K464
GAPKEMPLESAEKEEKKSEDKKKSEEDHTEEINDXIYSLNYNEL EVLAKNGETIENFVPKEGVKKADKFIVIERKKKNINTTPVDISI IDSVTDRTYPAALQLANKGFTENXPDAVVTKRNPQKIHIDLPGM GDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYT ESMVYSKSQIEAALNVNSXILDGTLGIDFKSISKGEKKVMIAAY KQIFYTVSANLPNNPADVFDKSVTFXELQRKGVSNEAPPLFVSN VAYGRTVFVKLETSSXSNDVEAAFSAALKGTDVXTNGKYSDILE NSSFTAVVLGGDAAEHNKVVTXDFDVIRNVIKDNATFSRKNPAY PISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGXINLSHQ

Number	Date	Country
63123293	Dec 2020	US
63090069	Oct 2020	US
63013924	Apr 2020	US
62977886	Feb 2020	US

	Number	Date	Country
Parent	PCT/US2021/018402	Feb 2021	WO
Child	17820554		US

GROUP A STREP IMMUNOGENIC COMPOSITIONS WITH POLYSACCHARIDE-PROTEIN CONJUGATES

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