Contribution of cryptic epitopes in designing a group A streptococcal vaccine

Figures & data

Figure 1. Idealized schematic illustrating M-protein based vaccine targets. The amino-terminal region: 30-valent N-terminal vaccine consisting of four different multivalent fusion proteins (containing eight or nine M-protein fragments)⁴²; The B-repeat region: representing defined myosin cross-reactive epitopes²⁰; The C1-C3 repeat regions: SV1 vaccine consisting of five 14-mer amino-acid sequences (J14_i variants) combined in a single recombinant construct⁴⁶; The C2-C3 repeat regions: StreptInCor vaccine containing immunodominant T (22 amino- acids) and B-cell (25 amino-acids) epitopes (bold residues) linked by eight amino-acid residues ([] boxed residues)⁵⁸; The C3 repeat region: Minimal B-cell cryptic epitope within p145 defined as J8, bold residues are those contained within M-protein (J8_i), residues not in bold are from GCN4 protein (not from M-protein).⁹⁰

Table 1. Status of M-protein and non-M-protein-based GAS vaccines.

Name	Composition	Advantages	Disadvantages	Status	Ref.
M-protein-based vaccines
26-valent N-terminal (StreptAvax)	N-terminal subunits from 26 serotypes of GAS. Four different recombinant proteins (containing six or seven M-protein fragments linked in tandem) formulated with Alum	– Does not require carrier protein^53	– Constrained by type-specific protection	Phase II clinical trial completed: Well tolerated and immunogenic in healthy adults^55	^45^,^53^,^55
		– Ability to cross-opsonize M-types not included in vaccine^53	– Theoretical coverage of vaccine in Africa 39% and Pacific region 23.9% ^54
		– No cross-reactivity between immune sera and human heart tissue^45^,^53
30-valent N-terminal	N-terminal subunits from 30 serotypes of GAS. Four different multivalent fusion proteins (containing eight or nine M-protein fragments) formulated with Alum	– Serotypes account for 98% of all cases of pharyngitis in the U.S. and Canada and 90% of invasive diseases in the U.S. and 78% of invasive diseases in Europe^56	– Heterologous protection not reported in animal models	Pre-clinical	^43^,^56
		– Evoked bactericidal antibodies against all 30 vaccine serotypes of GAS (using serum from immunized rabbits)^43
		– Contained significant levels of bactericidal antibodies against 24 of 40 non-vaccine serotypes of GAS tested^43
StreptInCor	Based on the amino-acid sequences from the M5 protein conserved regions (C2 and C3 regions). Contains immunodominant T (22 amino- acids) and B-cell (25 amino-acids) epitopes linked by eight amino-acid residues	– Protective in BALB/c ^59, HLA class II transgenic (mice containing human HLA II alleles)^60 and SWISS mice^61	None identified yet	Pre-clinical	^59-62^,^129
		– Antibodies able to neutralize/opsonize M1, M5, M12, M22 and M87 GAS^62
		– No autoimmune pathological reactions were observed in heart or other organs^60
		– Potential to elicit B and T-memory cells^129
J8-DT/Alum (MJ8Vax)	Minimal B-cell epitope within p145 (C3-repeat region), conjugated to T-helper cell protein diphtheria toxoid (DT) and formulated with Alum	– Cryptic B-cell antigen capable of inducing protection against all GAS serotypes	– Limited efficacy against hypervirulent covR/S mutants GAS strains^65^,^66	Pilot Phase I trial completed: Well tolerated and immunogenic in healthy adults (manuscript in preparation)	^65^,^66^,^92^,^96
		– Protection against streptococcal pyoderma, bacteraemia^65 and pharyngitis (J8-Lipo-DT)^96
		– Pre-clinical data demonstrated no abnormal heart tissue pathology^91
		– Minimal epitope size enhances safety profile
J8-CRM+K4S2-CRM/Alum (MJ8CombiVax)	J8-CRM combined with a 20-mer B-cell epitope (K4S2) from SpyCEP and conjugated to CRM^66	– Act synergistically to opsonize GAS (with anti-J8 antibodies) and to block IL-8 degradation (with anti-SpyCEP antibodies)	None identified yet	In preparation for Phase I trial	^65-67
	* K4S2: A more soluble derivative of S2 (S2 with four Lysine residues)	– Protect against hypervirulent covR/S mtant GAS^65-67
	^#CRM197 (CRM): Enzymatically inactive and non-toxic form of DT
SV1	Five 14-mer amino-acid sequences (J14i variants) from differing C-repeat regions combined in a single recombinant construct	– Maintains α-helical structure without the need for flanking sequence	None identified yet	Pre-clinical	^48^,^64
		– Evidence of cross-recognition between J14i variants, may provide extended M-protein coverage
		– No autoimmune responses detected in studies with the Lewis Rat model for valvulitis
		– Present in 97% of M-proteins
Self-adjuvanting J14	J14 incorporated with fibril forming peptides Q11 and lipoamino-acids (C16)	– Capable of inducing significant dendritic cell uptake and J14-specific antibody responses	None identified yet	Pre-clinical	^130
	Vaccine constructs:
	(i) J14-spacer1-Q11-spacer2-C16-C16
	(ii) J14-spacer2-C16-C16
Non-M-protein-based vaccines
GAS carbohydrate (GAS CHO)	GAS-CHO conjugated to tetanus toxoid (TT)	– GAS carbohydrate: conserved across all GAS strains^80	– Cross-reactivity between GAS carbohydrate and human tissue^131-1332	Pre-clinical	^51^,^52
		– Induction of phagocytosis promoting antibodies in rabbits^52
		– Intranasal immunization (GAS-CHO/CTB): significant reduction in GAS throat colonisation^51
GAS carbohydrate (GAC) defective for GlcNAc sidechain	Gac1 mutant bacteria (GlcNAc sidechain deficient GAC) was used to extract GlcNAc-defective GAC (ΔGAC)	– GAS carbohydrate: conserved across all GAS strains	None identified yet	Pre-clinical	^80
		– No cross-reactivity between human cardiac antigens due to removal of GlcNAc sidechain
		– Opsonisation and killing of diverse GAS serotypes
		– Passive protection in murine systemic infection model
GAS C5a peptidase (SCPA)	Recombinant, enzymatically inactive SCPA delivered intranasally (unadjuvanted)^71^,^134 or combined with MPL and formulated with Alum for subcutaneous immunization^50	– Antigenically conserved among different serotypes, may induce protection across several GAS serotypes	None identified yet	Pre-clinical	^50^,^69-71
		– Induction of high serum IgG and salivary IgA
		– Reduced the colonisation of M1, M2, M6, M11 and M49 GAS serotypes
Fibronectin binding protein (FBP54 or Sfb1)	Recombinant FBP54 (rFBP54) coupled with cholera toxin subunit B (CTB) and delivered orally and intranasally; Recombinant Sfb1 conjugated to CTB and delivered intranasally	– The fbp54 gene: highly conserved among all test strains (>98%)^72	–Intranasal immunization with Sfb1/CTB was ineffective against systemic bacterial growth and dissemination^136	Pre-clinical	^72^,^73^,^135
		– FBP54/CTB oral/intranasal immunization: antigen-specific salivary IgA and serum IgG	– GAS pyoderma predisposes individuals to severe GAS infection, despite presence of anti-Sfb1IgG titres^137
		– Subcutaneous immunization with FBP54/CFA protected mice against intraperitoneal GAS infection^135
		–Intranasal immunization with Sfb1/CTB protected mice against lethal GAS infection^73
Serine protease (SpyCEP)	Recombinant SpyCEP (rSpyCEP) administered parentally with CFA^49 or rSpyCEP combined with C-terminal peptide antigen (J8-DT) and administered subcutaneously with Alum^65	– Reduced the dissemination of GAS from local to systemic infection ^49	– Limited efficacy when administered alone^65^,^66	Pre-clinical	^50^,^66
		– Combination vaccination (J8-DT+rSpyCEP) resulted in profound protection against covR/S wild-type and mutant GAS skin challenges^65
Serum opacity factor (SOF2)	Purified recombinant SOF2 from SOF positive M-serotype 2 GAS	– Anti-rSOF2 serum able to opsonize SOF positive M2, M4 and M28 GAS types	– Anti-rSOF2 serum ineffective for SOF negative M5 GAS	Pre-clinical	^78
Streptococcal pyrogenic exotoxin A (SPE A)	SPE A purified from Staphylococcus aureus (double, triple and hexa-amino acid mutants of SPE A)	– Protection from lethal GAS infection and no symptoms of streptococcal toxic shock syndrome (STSS)^75	None identified yet	Pre-clinical	^74^,^75
		– Immunization also led to survival and no signs of illness when challenged subcutaneously with wild-type SPE A^74
Streptococcal pyrogenic exotoxin B (SPE B)	SPE B was purified from Streptococcus pyogenes A-20 (a protease producing clinical isolate)	– 100% survival in vaccinated mice following challenge with GAS A20^76	– Passive immunization with anti-SPE B IgG conferred partial protection with 50% survival in the immunized cohorot in comparison to 28% survival in the control cohort ^76	Pre-clinical	^76
		– Severity of skin lesions reduced in immunized mice^76
Streptococcal pyrogenic exotoxin C (SPE C)	Double-site Y15A/N38D and the triple-site Y15A/H35A/N38D mutants constructed from 3D-structure of SPE C	– Non-mitogenic for rabbit splenocytes and human PBMCs^77	– None identified yet	Pre-clinical	^77
		– Non-lethal in two rabbit models of STSS^77
		– Highly immunogenic and vaccination protected rabbits from challenge with wild-type SpeC^77
Streptococcal pili (T-antigen)	Vaccine comprising of a combination of recombinant pilus proteins	– Protection against mucosal infection following immunization^79	– Vast sequence diversity, protection is specific to strains that contain pilus like variants in vaccine ^56	Pre-clinical	^56^,^79
		– Cross-protection can be achieved between some T-types that share high homology, restricting number of variants required for broad coverage^56
Combo#5	Vaccine consisting of a combination of trigger factor (TF), inactivated versions of arginine deiminase (ADI), streptolysin O (SLO), Streptococcus pyogenes cell envelope proteinase (SpyCEP) and group A streptococcal C5a peptidase (SCPA) adjuvanted with Alum	– Murine serum antibodies from BALB/c and humanized plasminogen mice were able to bind to live GAS	– In a model for invasive disease, protection was compromised. M1 protein (positive control) vaccine was the only experimental vaccine that conferred protection (100% survival)	Pre-clinical	^112
		– Anti-combo#5 sera from BALB/c and humanized plasminogen mice was opsonic against pM1.200 and 5448 GAS strains respectively
		– Mice were significantly protected following skin-challenge
3-antigen combo	Vaccine consisting of Streptolysin O (SLO, aa 32–571), SPy0269 (aa 27–849), and S. pyogenes cell envelope protease (SpyCEP, aa 34–1613) tagless versions were cloned in the pET24b+ E. coli expression vector and purified	– Antigens conferred consistent cross-protection against a wide range of GAS strains in different mouse models of infection	– None identified yet	Pre-clinical	^138
		– Vaccine elicits antibodies capable of neutralizing two important virulence factors expressed by a large fraction of GAS isolates
Spy 7	Vaccine consisting of seven recombinant antigens – C5a peptidase, oligopeptide-binding protein, putative pullulanase, nucleoside-binding protein, hypothetical membrane associated protein, cell surface protein and Spy AD	– Induction of anti-streptococcal antibodies and demonstrated protective efficacy against M1 and M3 GAS strains	– Antigens have been tested in isolation as vaccine candidates, including six of those included in this study. Of these, C5a peptidase, cell surface protein, and SpyAD showed protective efficacy,^70^,^138^,^139while oligopeptide-binding protein, putative pullulanase, and hypothetical membrane associated protein were not protective^138^,^140	Pre-clinical	^141
		– Murine Spy7 antiserum demonstrated no discernible reactivity with human heart valve tissue using an ELISA based assay, suggesting an absence of cross reactive epitopes within any of the selected antigens
ADI and TF	Vaccine consisting of Arginine deiminase (ADI) and trigger factor (TF)	– These surface-exposed enzymes are expressed across multiple GAS serotypes exhibiting ≥99% amino acid sequence identity	– None identified yet	Pre-clinical	^142
		– Sera from human populations suffering repeated GAS infections and high levels of autoimmune complications do not recognize these enzymes
		– Protective efficacy demonstrated against intraperitoneal challenge with M1 GAS
		– Combination ADI and TF was observed to act synergistically, conferring significant protection against lethal subcutaneous M1 GAS challenge

Table 2. List of synthetic peptides of p145.

P145:									L	R	R	D	L	D	A	S	R	E	A	K	K	Q	V	E	K	A	L	E
J1:	Q	L	E	D	K	V	K	Q	L	R	R	D	L	D	A	S	R	E	A	K	E	E	L	Q	D	K	V	K
J2:		L	E	D	K	V	K	Q	A	R	R	D	L	D	A	S	R	E	A	K	K	E	L	Q	D	K	V	K	Q
J3:			E	D	K	V	K	Q	A	E	R	D	L	D	A	S	R	E	A	K	K	Q	L	Q	D	K	V	K	Q	L
J4:				D	K	V	K	Q	A	E	D	D	L	D	A	S	R	E	A	K	K	Q	V	Q	D	K	V	K	Q	L	E
J5:					K	V	K	Q	A	E	D	K	L	D	A	S	R	E	A	K	K	Q	V	E	D	K	V	K	Q	L	E	D
J6:						V	K	Q	A	E	D	K	V	D	A	S	R	E	A	K	K	Q	V	E	K	K	V	K	Q	L	E	D	K
J7:							K	Q	A	E	D	K	V	K	A	S	R	E	A	K	K	Q	V	E	K	A	V	K	Q	L	E	D	K	V
J8:								Q	A	E	D	K	V	K	Q	S	R	E	A	K	K	Q	V	E	K	A	L	K	Q	L	E	D	K	V	Q
J9:									A	E	D	K	V	K	Q	L	R	E	A	K	K	Q	V	E	K	A	L	E	Q	L	E	D	K	V	Q	L
J14:							K	Q	A	E	D	K	V	K	A	S	R	E	A	K	K	Q	V	E	K	A	L	E	Q	L	E	D	K	V	K

Bold residues are those contained within p145.

Underlined residues represent the T-cell epitope contained within p145.

Highlighted residues represent the B-cell epitope contained within p145.

*Adapted from Hayman et al. 1997. ⁹¹

Table 3. Multiple sequence alignment of S2 variants.

S2:

N

S

D

N

I

K

E

N

Q

F

E

D

F

D

E

D

W

E

N

F

S2.1:

N

S

D

N

I

K

E

N

Q

F

G

D

F

D

E

D

W

E

N

F

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

S2 sequence alignment performed using bioinformatics program (Clustal Omega; http://www.ebi.ac.uk/Tools/msa/clustalo/). Data representing 95% homology between S2 and S2.1. An * (asterix) indicates positions which have single, fully conserved residues. Single amino-acid polymorphism represented in bold. A total of 96 BLAST hits returned with 62 hits containing 100% homology with S2 and 34 hits containing 100% homology with S2.1.

Table 4. J8-based vaccine modifications.

Peptide	Sequence	Adjuvant/ delivery system	Application/ outcomes		Ref.
p145	LRRDLDASREAKKQVEKALE	CFA *not suitable for human use	p145/CFA: p145-spcific antibodies from mice immunized with p145/CFA were able to opsonize several serotypes of GAS		^84
J8	Highly conserved peptide from p145: QAEDKVKQSREAKKQVEKALKQLEDKVQ	Alum	J8-DT/Alum: J8 conjugated to T-helper cell carrier protein, DT, and administered subcutaneously to mice with Alum protected against streptococcal pyoderma and bacteraemia		^65
	12-mer peptide ftom p145 in bold, total 28-mer	CTB *not suitable for human use	J8-DT/CTB: Mice immunized intranasally with J8-DT/CTB had significantly increased survival following intranasal challenge with GAS and salivary IgA correlated with protection		^97
		Liposomes	J8-Lipo-DT: Mice immunized intranasally with J8-Lipo-DT induced high J8-specific salivary IgA and mice were protected following URT-challenge		^96
		LCP	LCP-J8: Mice immunized subcutaneously with LCP-J8 and LCP-J8 in CFA induced high J8-specific serum IgG and antisera from these mice was able to opsonize multiple GAS strains. Antibodies did not cross react with human heart tissue proteins.		^143^,^144
			Tetraepitopic LCP (LCP system incorporating 4 different non-host cross-reactive peptide epitopes of the GAS M-protein): Parenteral immunisation induced high antigen-specific serum IgG responses and the antisera was able to opsonize multiple GAS strains. immunized mice were also protected following systemic challenge.		^145
				LCP-8830-J8 (LCP system incorporating 2 different peptide epitopes of the GAS M-protein, 8830 and J8): Mucosal immunization induced an antigen-specific systemic IgG. Antisera was able to opsonize a homologous and heterologous GAS strain. immunized mice were protected following systemic and mucosal challenge.	^146
J14	Highly conserved peptide from p145: KQAEDKVKASREAKKQVEKALEQLEDKVK	CTB *not suitable for human use	J14-DT/CTB: Mice immunized intranasally with J14-DT/CTB had significantly increased survival following intranasal challenge with GAS and detection of salivary IgA was coincided with protection		^97
	14-mer peptide from p145 in bold, total 29-mer	Proteosomes	J14/Proteosomes: Intranasal immunization of mice induced J14-specific salivary IgA that led to decreased URT colonisation in mice post-intranasal challenge		^94
		Pam(2)Cys	P25-P2C-J14: Mice immunized intranasally with P25-P2C-J14 induced J14-specific salivary IgA that coincided with a significant reduction in throat colonisation		^98
rSpyCEP	Encompassing amino-acid residues 35–587 (GenBank No. DQ413032)	CFA *not suitable for human use	rSpyCEP/CFA: Mice immunized with rSpyCEP/CFA had reduced bacterial dissemination from local intramuscular and intranasal sites of GAS infections and anti-rSpyCEP antibodies were able to inhibit SpyCEP cleaving of IL-8		^49^,^104
		Alum	J8-DT+rSpyCEP/Alum: Combination vaccination resulted in profound protection against covR/S wild-type and mutant GAS skin challenges		^65
S2	Highly conserved peptide from rSpyCEP (AA 205–224): NSDNIKENQFEDFDEDWENF	Alum	J8-DT+S2-DT/Alum: Combination vaccination resulted in protection against pyoderma and bacteraemia following skin-infection with covR/S mutant GAS and antibodies generated to S2 could completely protect IL-8 from SpyCEP mediated proteolysis		^66
		Liposomes	J8/S2-Lipo-DT: Combination intranasal vaccination induced J8 and S2-specific salivary IgA that coincided with reduced bacterial colonisation of the URT following intranasal infection with covR/S mutant GAS		^96
K4S2 (S2 with 4 lysine residues)	A more soluble derivative of S2	Alum	J8-CRM+K4S2-CRM/Alum (MJ8CombiVax): Parenteral immunization of mice with MJ8CombiVax was immunogenic and protective against covR/S mutant skin challenge. Immunogenicity and protective efficacy was comparable to J8-DT+S2-DT vaccination.		^67
			*CRM197 (CRM): Enzymatically inactive and non-toxic form of DT

CFA: Complete Freud's adjuvant; Alum: Aluminum hydroxide; CTB: Cholera toxin B subunit; Liposomes: Liposomes composed of neutral lipids encapsulating DT and displaying lipidated peptide on surface; LCP: Lipid core peptide; Proteosomes: Bacterial outer membrane proteins; Pam(2)Cys: Lipopeptide construct containing a universal T-cell epitope and a self-adjuvanting lipid moiety.

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