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Review

Contribution of cryptic epitopes in designing a group A streptococcal vaccine

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Pages 2034-2052 | Received 21 Sep 2017, Accepted 03 Apr 2018, Published online: 14 Jun 2018

ABSTRACT

A successful vaccine needs to target multiple strains of an organism. Streptococcus pyogenes is an organism that utilizes antigenic strain variation as a successful defence mechanism to circumvent the host immune response. Despite numerous efforts, there is currently no vaccine available for this organism. Here we review and discuss the significant obstacles to vaccine development, with a focus on how cryptic epitopes may provide a strategy to circumvent the obstacles of antigenic variation.

Vaccines are amongst the greatest medical achievements of modern civilisation. Today, over 70 vaccines have been licenced to prevent infection with approximately 30 different organisms.Citation1,Citation2 Vaccine development has largely focused on the concepts of live attenuated, sub-unit and whole-cell vaccine designs. Of these one-third are sub-unit vaccines that contain highly immunogenic immunodominant antigens capable of producing antibodies to a single-strain of an organism.Citation2,Citation3 However, for several infectious diseases this approach is ineffective or is associated with major disadvantages. The challenge is that many organisms are antigenically variable and due to their diversity, polyvalent vaccines have been developed. Examples include vaccines for Streptococcus pneumoniae and Human Papilloma Virus.Citation3

Streptococcus pyogenes (group A streptococcus, GAS) is an important human pathogen for which vaccines are not yet available. For this organism, antigenic diversity is extensive and this challenges even a multivalent vaccine approach. An alternative approach is to use cryptic epitopes because these are poorly immunogenic in the native organism and they are thus not under immune selection pressure.Citation3 Although they may not be recognised as a result of natural infection,Citation4 they can be highly immunogenic when presented in isolation such as a peptide or a recombinant polypeptide fragment. Furthermore, because they are conserved they may be able to induce strain-transcending immunity. Cryptic epitopes can thus be exploited in vaccine development. Despite their recognised potential there is a paucity of literature on the description and utilisation of cryptic epitopes as vaccine candidates. Here, we provide an in-depth review on the development and potential use of two separate cryptic epitopes in a vaccine to prevent infection with GAS.

GAS is a Gram-positive organism that primarily infects the upper respiratory tract (URT) and the skin.Citation5,Citation6 It is responsible for a wide array of infections ranging from superficial infections such as streptococcal pharyngitis and pyoderma to invasive necrotising fasciitis. The ‘post-streptococcal’ sequelae of rheumatic fever (RF)/rheumatic heart disease (RHD) and post-streptococcal glomerulonephritis are also of major concern. GAS infections and their sequelae are responsible for more than 500,000 deaths each year.Citation5 In 2015 there was an estimated 319 400 deaths due to RHD.Citation7

Immunopathogenesis and obstacles in GAS vaccine development

Development of auto-reactive B and T-cells

Infection with GAS can lead to acute rheumatic fever (ARF), which predominantly affects people living in resource-poor settings. Subsequent streptococcal throat infections can cause recurrent ARF. Single or repeated episodes of ARF can result in RHD.Citation8 The genetic susceptibility to RF/RHD is associated with Class II MHC molecules (HLA-DR, DQ and DP) that present peptides from extracellular pathogens to CD4+ T-cells.Citation9 These include HLA-DRB1, HLA-DRB4, HLA-DQA1 and HLA-DQB1.Citation10 The aetiology of the disease is not well understood but has been defined as an autoimmune illness (see below).Citation11 The streptococcal M-protein shares an alpha-helical coiled-coil structure and antigenic cross-reactivity with cardiac myosin. This phenomenon was first described by Kaplan,Citation12 and ZabriskieCitation13 and MeyeserianCitation12 as antibody cross-reactivity. This is a significant hindrance to GAS vaccine development where it is critical that a vaccine does not induce auto-reactive B- and T-cell responses. The evidence that an autoimmune pathogenic process might involve the M-protein was highlighted in an early study in 1969 where 21 children were vaccinated with type-3 streptococcal M-protein. Children received up to 33 injections of partially purified M-protein at doses of up to 1 mg per injection. Following vaccination, although these children developed 18 GAS infections (tonsillitis/pharyngitis), none were type-3 GAS infections. However, two of these infections were followed by RF and one by probable RF.Citation14 In other studies where subjects were immunized with three doses of M-protein there were no reported serious adverse events.Citation15,Citation16 In 1979, the US Food and Drug Administration prohibited the development of a GAS vaccine after considering the findings of the independent advisory panel “Review of Bacterial Vaccines and Bacterial Antigens”. The prohibition remained for nearly 30 years and was lifted in 2006 when subunit vaccines were being developed.Citation17

The immunopathogenesis of group A streptococcal disease has been studied and the autoimmune potential of the M-protein has been identified in a number of previous studies reviewed extensively by Cunningham.Citation18,Citation19 The B1B2/B2/B3A regions of the M-protein were found to contain myosin-cross reactive epitopes, with B2 peptide having 42% identity with cardiac myosin and B1A inducing myocardial lesions.Citation20 Therefore, the B-repeat region of the M-protein has been excluded in GAS vaccine development. Additionally, studies have also shown cross-reactivity between the C-repeat region and cardiac and skeletal myosin,Citation20-22 thus strengthening the case for the development of minimal subunit vaccines where host cross-reactive epitopes can be eliminated to reduce the risk of ARF and RHD.

M-protein sequence variation is associated with rheumatogenic GAS strains associated with the development of ARF. Examples include M-types 5 (M5) and 6 (M6). Immunization with M6 protein was shown to induce valvulitis and myocarditis in a Lewis rat model with both CD4+ and CD8+ T-cells detected in valvular lesions.Citation23 Additionally, immunization with human cardiac myosin generated T-cells that recognized the M5 protein.Citation24 Furthermore, passive transfer of M-protein A-repeat region-specific T-cells into naïve rats produced valvulitis providing further evidence that M-protein-specific T-cells may be key mediators in valvular heart disease.Citation25 M-types 1, 3, 5, 6, 14, 18, 19, 24, 27 and 29 have been previously associated with ARF.Citation26-31 The relationship between rheumatogenic GAS strains and acute pharyngitis was evaluated in an epidemiological study in the United States. A decrease in the prevalence of ARF was associated with a significant reduction in the proportion of cases of acute streptococcal pharyngitis in children caused by rheumatogenic GAS types.Citation30 In a more recent study, it was found that GAS strains belonging to emm pattern D (skin pattern) contributed to 49% of ARF-associated GAS strains, thus also suggesting a role of skin infection in the development of ARF.Citation32

Antigenic strain variation with GAS and the need for repeat exposure to induce immune memory

A major hindrance to subunit vaccine development is the vast sequence diversity of the virulence factor, the M-protein. Strain-specific immunity is a result of the development of antibodies to the immunodominant amino-terminal epitopes on this protein.Citation27 The M-protein is encoded by the emm gene. There are over 200 distinct strains based on the serological M-types and more than 230 emm types have been identified using emm typing,Citation33,Citation34 the gold standard molecular typing method that is based on the 5′-end 150 nucleotides of the emm gene.Citation35

Early studies by Kuttner and LenertCitation36 revealed the presence of type-specific antibodies in children recovering from streptococcal pharyngitis. A follow-up study found that type-specific antibodies from adults recovering from GAS infection in the URT were able to bind to homologous heat-killed streptococci but not strains of heterologous types.Citation37 In another study, type-specific antibodies were shown to reduce the risk of homologous pharyngeal infections.Citation38 Further studies by Lancefield reported that human antisera to types 3, 6 and 13 protected mice against homologous challenge with GAS to an extent roughly proportional to the antibody concentration detected in sera.Citation39 This supported the notion that M-protein-specific antibodies, post-pharyngeal infection with GAS, persist for extended periods of time, and confer homologous strain-specific immunity.

However, there is very little knowledge on the acquisition of immunity following GAS skin infection. We used a number of epidemiologically distinct GAS strains to model the development of acquired immunity to pyoderma and demonstrated that infection leads to antibody responses to the serotype-specific determinants on the M-protein and short-lived protective immunity to homologous strains. Memory B-cells do not develop after a single infection and immunity is rapidly lost.Citation4 Similarly, sequential infections with different strains resulted in short-lived immunity only to the last strain to which the mice had been exposed and not to any previous strains. However, two sequential infections with the same strain within a short time frame did induce enduring strain-specific immunity. Along with antigenic-diversity, if the requirement for multiple consecutive exposures to each serotype of GAS to induce a memory response also occurs in humans, then this represents a further serious impediment to the development of immunity to GAS. The need for multiple infections to induce immunological memory to a given strain begs the question of whether natural infection post-vaccination will be able to boost and maintain memory. This is a critical question for all vaccine candidates. Mice exposed to multiple strains, either sequentially or simultaneously, did not develop antibodies to a conserved M-protein vaccine peptide, J8, demonstrating that this epitope is cryptic to the immune system.Citation4 However, we have recently shown that skin infection can boost J8-induced immunity and furthermore that the infection serves to broaden the nature of immunity by engaging other antigens such as SpyCEP.Citation40

GAS vaccine development

GAS vaccine development is divided into M-protein and non-M-protein-based approaches.Citation41 M-protein-based vaccines include fused recombinant peptides from the N-terminal region of the M-protein from multiple emm types of GAS (6-, 26- and 30-valent vaccines),Citation42-45 antigens from the conserved C-repeat region of the M-protein, StreptInCor (containing selected T and B-cell epitopes),Citation46 SV1 (containing five 14-mer amino-acid sequences from differing C-repeat region)Citation47 and J8/J14, a cryptic epitope-based vaccine approach (containing a single B-cell epitope from the C3 repeat region).Citation48 represents a schematic of the M-protein with the location and targets of M-protein-based vaccines in development. The non-M-protein-based vaccines include virulence factors such as SpyCEPCitation49 and C5a peptidase,Citation50 and group carbohydrates.Citation51,Citation52 A comprehensive discussion of M-protein and non-M-protein GAS vaccines is summarized in .

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)Citation42; The B-repeat region: representing defined myosin cross-reactive epitopesCitation20; The C1-C3 repeat regions: SV1 vaccine consisting of five 14-mer amino-acid sequences (J14i variants) combined in a single recombinant constructCitation46; 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)Citation58; The C3 repeat region: Minimal B-cell cryptic epitope within p145 defined as J8, bold residues are those contained within M-protein (J8i), residues not in bold are from GCN4 protein (not from M-protein).Citation90

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)Citation42; The B-repeat region: representing defined myosin cross-reactive epitopesCitation20; The C1-C3 repeat regions: SV1 vaccine consisting of five 14-mer amino-acid sequences (J14i variants) combined in a single recombinant constructCitation46; 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)Citation58; The C3 repeat region: Minimal B-cell cryptic epitope within p145 defined as J8, bold residues are those contained within M-protein (J8i), residues not in bold are from GCN4 protein (not from M-protein).Citation90

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

M-protein-based vaccines

To take advantage of the type-specific opsonic antibodies associated with the amino (N)-terminal region of the M-protein a multivalent M-protein vaccine was designed. The hexa-valent vaccine consisting of N-terminal subunits from 24, 5, 6, 19, 1 and 3 M-protein peptides was found to be immunogenic against all six M-protein peptides and no cross-reactivity between immune sera and human heart tissue was observed.Citation42,Citation44 However, this vaccinate candidate was constrained by type-specific protection.Citation42,Citation44 Therefore, the vaccine was advanced to a 26-valent N-terminal vaccine (StreptAvax), consisting of 26 N-terminal subunits from North American GAS isolates.Citation53 Although StreptAvax was shown to cross-opsonize non-vaccine M-types, it offered limited theoretical coverage against strains in many developing countries.Citation45,Citation53 The 26 emm types present in the vaccine accounted for only 65% of all isolates in Africa, Asia, Middle-East and Pacific region, with the theoretical coverage of the vaccine in Africa being estimated to be 39% and in the Pacific region, 23.9%.Citation54 Regardless, this is the most advanced GAS vaccine candidate with the successful completion of a Phase II clinical trial.Citation55 The vaccine has since been refined to a 30-valent vaccine consisting of 30 N-terminal subunits from North America and Europe. The serotypes included in the vaccine account for 98% of all cases of pharyngitis in the United States and Canada, 90% of invasive disease cases in the United States and 78% of invasive disease cases in Europe.Citation56 The vaccine was shown to induce antibodies in rabbits against 24 of 40 non-vaccine serotypes.Citation43,Citation57 Recently, these observations have led to the designing of M-protein-based vaccines utilizing an emm cluster-typing system in combination with computational structure-based peptide modelling. The preliminary data are promising, however, further investigations are required to confirm the feasibility of this approach.Citation58

To elicit a broader range of protection, vaccine candidates targeting the conserved C-terminal region of the M-protein have been developed. StreptInCor, comprising 55 amino-acid residues from the C2 and C3 conserved regions of the M5 protein was shown to be protective in BALB/cCitation59, HLA class II transgenic miceCitation60 and SWISS mice.Citation61 Protective efficacy was demonstrated against M1, M5, M12, M22 and M87 GAS strains.Citation62 No autoimmune pathology was observed in heart or other organsCitation60 and an epidemiological study of Brazilian GAS isolates predicted the protective coverage to be 71%.Citation62 Another C-terminal vaccine candidate in development is SV1, consisting of five 14-mer amino-acid sequences (J14i variants) from differing C-repeat regions combined in a single recombinant construct. Unlike the J8-DT vaccine candidate, SV1 maintains alpha-helical structure without the need for additional flanking sequence.Citation47,Citation63 Antibodies raised to SV1 were shown to bind to each of the 5 J14i variants which are present in 97% of M-proteins.Citation47,Citation63 The studies with the Lewis Rat model for valvulitis suggested that the vaccine is safe and will elicit antibodies that recognize a broad range of GAS serotypes.Citation63 J14 has also been combined synthetically with 7 amino-acid peptides from different emm strains and induced protective antibodies in mice to strains both represented by and not represented by the amino-terminal sequences.Citation64

An experimental vaccine J8-DT, targeting the conserved domain of M-protein and conjugated to diphtheria toxoid (focus of this review) has shown efficacy against multiple GAS strains. The efficacy of J8-DT was further improved to protect against covR/S mutant hypervirulent strains by incorporation of a SpyCEP epitope (S2) (see below). J8 conjugated to CRM 197 (enzymatically inactive non-toxic form of DT) in combination with K4S2-CRM is currently in preparation for a Phase I clinical trial.Citation65-67

Non-M-protein-based vaccines

In recent years with the help of reverse vaccinology along with proteomics, whole genome sequencing, bio-informatics and microarray technology, a number of non-M-protein vaccine candidates have been identifiedCitation68 and are under pre-clinical development. Their highly-conserved nature across various serotypes and to date, no evidence of associated tissue cross-reactivity, makes them an attractive target for vaccine development.Citation56 Non-M-protein-based vaccine candidates that have been shown to play a role in immunity include C5a peptidase,Citation50,Citation69-71 streptococcal fibronectin binding protein,Citation72,Citation73 streptococcal pyrogenic exotoxins A,Citation74,Citation75 BCitation76 and C,Citation77 S. pyogenes cell envelope protease,Citation49,Citation65 serum opacity factor,Citation78 streptococcal pili,Citation79 and GAS carbohydrate.Citation51,Citation52,Citation80 However, a non-M-protein vaccine candidate has yet to progress into human clinical trials. It is believed that despite the role that non-M-protein antibodies play in GAS immunity, opsonic M-protein specific antibodies will be critical for clearing GAS infection.Citation81 A combination of M-protein and non-M-protein antigens could be exploited to improve protection which has been demonstrated with the MJ8CombiVax (J8-CRM+K4S2-CRM) vaccine.Citation67 A detailed analysis of each of these vaccine candidates is provided in .

Identifying a cryptic target for a GAS vaccine

Bessen and FischettiCitation82 demonstrated the protective potential of the conserved region of the M-protein against GAS. Mice were immunized intranasally with synthetic peptides from the highly-conserved C-repeat region of the M-protein, which had been covalently linked to cholera toxin B subunit (CTB). These peptides corresponded to antigenic epitopes shared by many emm types. It was found that intranasal immunization with the cross-reactive epitopes coupled to CTB led to significant protection against pharyngeal colonisation by GAS. In parallel, Jones and FischettiCitation83 showed that antibodies to the amino-terminal region of the M-protein, but not the conserved central region, were opsonic. Contrary to that, we demonstrated that a conserved region peptide, p145 (a 20-mer peptide from the ‘C3-repeat’ region), was able to induce opsonic antibodies in mice post-immunization.Citation84 The opsonization assay used stationary phase rather than log-phase organisms that are used in the ‘classical’ Lancefield assay. It was hypothesized that the diminished hyaluronic acid (HA) capsule associated with stationary phase GAS will allow better access of antibodies to the C-repeat region of the M-protein.Citation85 p145 peptide was identified by scanning the conserved C- region of the M-protein of GAS.Citation22,Citation84 p145-specific affinity purified human antibodies collected from a highly endemic region of Australia, were also shown to be opsonic.Citation86 These findings suggested that p145 might be a suitable vaccine candidate. However, there were concerns regarding host tissue cross reactivity. Human studies suggested that while humoral responses may initiate RF/RHD, the key mediators of heart lesions are auto reactive T-cells. By molecular mimicry these T-cells also recognize heart tissue proteins. Heart infiltrating T-cell clones isolated from RHD patients have been shown to recognize GAS M5 protein and heart tissue proteins/peptides.Citation87,Citation88 It was deemed prudent to define the minimal epitope within p145 that was immunogenic and able to induce opsonic antibodies.

The structure of the M-protein is a coiled-coil alpha helix and it was critical that the minimal epitope maintains helical folding in order to induce antibodies that recognize the native protein. To promote alpha-helical coiled-coil confirmation, small sequences (12 amino-acids in length) from p145 were flanked with a GCN4 peptide (from a DNA binding protein of yeast known to promote an alpha helical coiled-coil).Citation89 Chimeric peptides designated J1 to J9 were used to map the minimal epitope within p145 using age-stratified sera from Indigenous Australians living in a highly streptococcal endemic regionCitation86 (). Sera from over 90% of individuals in the 20+ years age group recognized peptides J1, J2, J7, and J8 but the recognition of these peptides was much less in children (approximately 20%).Citation86 The epitopes were thus cryptic in that many years of exposure were required to induce an antibody response. Additional studies revealed that human antibodies to p145 could opsonize multiple serotypes of GAS including strains that exhibited slight differences in the p145 minimal epitope sequence.Citation90 Monoclonal antibodies from mice immunized with p145 recognized J7, J8 and J9.Citation91 These three peptides induced a significant antibody response to themselves (titre >12,800), although only J8 could induce an antibody response to p145. Having noted the potential of J7, J8 and J9, an additional chimeric peptide, termed J14, was synthesized from amino-acids 7–20 of p145 (amino-acids found within J7, J8 and J9).Citation92 p145 antisera bound to J14 and antisera from mice immunized with J14 recognized J7, J8, J9 and p145. J8 and J14 did not induce p145-specific T-cell responses in mice, which was seen as a bonus in terms of the safety profile of the vaccine.Citation91 Within p145, the T-cell epitopes were mapped to J2 and J3. This corresponds to residues 3–14 located at the amino-terminal region of p145.Citation91 Thus, a minimal cryptic B-cell epitope (J8) was defined, and this did not contain a potentially deleterious T-cell epitope from GAS, yet was able to stimulate antibodies that could opsonize GAS.Citation91 Although J8 did not contain a GAS-derived T-cell epitope recognized by mice, it does nevertheless contain one or more T-cell epitopes. J8 has 12 amino-acids copying the M-protein sequence, but also contains an additional 16 non-streptococcal amino-acids (GCN4 protein) that form part of the T-cell epitope of J8.Citation89,Citation91

Table 2. List of synthetic peptides of p145.

The immunogenicity of the J8 peptide was determined using different adjuvants.Citation48 Quackenbush (outbred) and B10.BR mice were immunized with J8 peptide and lymph node cell proliferation to the peptide was determined for each mouse. For the Quackenbush mice, lymph node cells from only 2 of the 20 mice proliferated, whereas T-cells from 7 of the 8 immunized B10.BR mice responded to the J8 peptide.Citation48

Development of a conjugate GAS vaccine

Immunological responsiveness to a vaccine is determined by T-cells being able to recognize processed fragments of an antigen (via the major histocompatibility molecule II [MHC II]). Failure of J8 to stimulate T-helper cells in an outbred population would limit its suitability as a vaccine. Therefore, J8 was conjugated to the carrier protein, diphtheria toxoid (DT), and the conjugate was used to immunize mice which were subsequently challenged via the skin or mucosal routes.Citation48

J8-DT administered subcutaneously with Alum protected against streptococcal pyoderma and bacteraemia.Citation65 In this study, a scarification method was used to mimic superficial skin infection. Vaccinated mice had significantly reduced bacterial burden in the skin in comparison to non-vaccinated mice. In addition, vaccinated mice either did not develop a systemic infection or cleared infection significantly faster compared to the non-immunized cohort.Citation65 The vaccine was shown to induce a memory response using an adoptive transfer assay. J8-DT-immunized mice were rested for 10–12 weeks and splenocytes or purified B or T-cells were then transferred to naïve immunodeficient SCID mice. Adoptive transfer of splenocytes from immunized mice or B-cells from immunized mice along with T-cells from either immunized or naïve mice resulted in the recipients being immune and showing significantly reduced bacterial burden in the skin and blood following challenge infection. At the time of challenge, the reconstituted SCID mice did not have detectable J8-specific antibodies in their serum.Citation65 These data thus demonstrated that mice could be protected even if they did not have serum antibodies at the time of challenge, providing they had memory B-cells. Presumably the memory B-cells responded quickly to the infection, producing opsonizing antibodies.

Pre-clinical data on immunogenicity and safety of J8-DT demonstrated no abnormal heart tissue pathology in a Lewis rat model for cardiac valvulitis.Citation92 In addition, a dose escalating toxicology assessment of J8-DT in rabbits demonstrated no treatment-related or toxicologically significant effects.Citation92 The vaccine has been tested in a pilot Phase I clinical trial and was shown to be immunogenic with no serious adverse events reported in the study (manuscript submitted).

J8-DT-mediated systemic protection required J8-specific IgG to mediate GAS clearance from the site of infection.Citation65,Citation66,Citation93 However, protection against URT infection may require an IgA response.Citation94,Citation95 We observed that intramuscular immunization with J8-DT/Alum resulted in high serum J8-specific IgG titres but no salivary J8-specific IgA titres. Following intranasal challenge there was minimal protection as demonstrated from estimating bacterial burden in nasal secretions, throats and Nasal Associated Lymphoid Tissue (NALT; a murine homolog to human tonsils).Citation96

We explored different approaches to induce mucosal immunity. Immunization of mice with J8-DT/CTB (cholera toxin B, CTB) (and J14-DT/CTB) led to protection following challenge via the URT route.Citation97 However, CTB is not a suitable adjuvant for human studies. We therefore explored other potential approaches to induce mucosal immunity. Immunization with J14 formulated with bacterial outer membrane proteins (J14/proteosomes) and administered intranasally to outbred mice resulted in J14-specific IgA in saliva and a decreased colonisation in mice post-challenge with GAS.Citation94 In a further study, J14 was incorporated into a lipopeptide construct to which a universal T-cell epitope and a self-adjuvanting lipid moiety, Pam(2)Cys, were attached.Citation98 This vaccine formulation (P25-P2C-J14) induced salivary J14-specific antibodies, which coincided with reduced throat colonisation post-intranasal GAS challenge.Citation99 More recently we have explored the use of liposomes composed of neutral lipids encapsulating DT and displaying lipidated J8 on their surface (J8-Lipo-DT). This liposome construct induced peptide-specific IgA and protected against intranasal GAS challenge.Citation96

Anti-J8 antibodies are not observed following a GAS infection of mice. Additionally, there is a lack of anti-J8 antibody secreting cells (ASCs) in the spleen and long lived plasma cells (LLPCs) in the bone marrow.Citation4 In contrast, following immunization with J8-DT, significant numbers of J8-specific ASCs were observed in the spleens of mice. Furthermore, following sequential infections of J8-vaccinated mice with different strains of GAS, the numbers of J8-specific ASCs increased significantly and the degree of protective immunity similarly increased. Thus, while J8 is cryptic following infection of naïve mice, J8-specific B-cells (induced by vaccination with a J8 conjugate vaccine) can nevertheless be boosted by infectionCitation4,Citation41,Citation94

J8, being highly conserved and cryptic, overcomes the barrier of antigenic variability found within circulating GAS strains. In a recent study by Sanderson-Smith et al., 2014,Citation99 J8 was found to have high sequence homology among differing emm types; 173 of the 175 emm types, collected globally, contained either the J8 or J8.1 allele.Citation99 These two J8 allelic sequences are immunologically cross-reactive. Antisera raised to both allelic sequences recognize the parent peptide (p145) equally (unpublished data). Further supporting these data is a study from Cambodia where 28% and 69% of the isolates carried the J8 or J8.1 allele respectively, thus, predicting the theoretical coverage of the vaccine to be 97%.Citation100 Likewise, in another study carried out in Lao, where among 124 GAS isolates, 34 emm types were observed: 15% and 82% of the isolates predicted to contain the J8 or J8.1 allele respectively and the theoretical coverage of the J8 vaccine was predicted to be 97%.Citation101 These studies provide encouraging data supporting the potential of cryptic epitope J8 in combating one of the major impediments to GAS vaccine development – antigenic strain variation. This is further strengthened by extensive animal studies where immunization with vaccines based on cryptic epitopes (J8-DT or J8-DT+K4S2-DT) provided protection against GAS strains from multiple emm types belonging to different clades and emm clusters.Citation65-67

Pathogenesis of covR/S mutant GAS strains

While J8-DT is a highly efficacious vaccine that protects against multiple GAS strains of various emm types, its efficacy against hyper-virulent covR/S mutant strains is compromised. The covR/S system plays an important role in regulating ∼15% of the genome of which a majority includes virulence gene expression (mostly virulence factors responsible for invasiveness of an isolate during infection).Citation102 Several virulence factor genes are upregulated as a result of covR/S mutation including S.pyogenes cell envelope proteinase (SpyCEP, cepA), streptodornase of serotype 1 (Sda1, sda1), streptolysin O (SLO, slo), streptococcal inhibitor of complement (SIC, sic) and the hyaluronic acid capsule synthesis operon (HA, hasABC).Citation103 SpyCEP, a CXC chemokine protease is a cell wall anchored serine protease that can also be released as a soluble enzyme.Citation104 SpyCEP can cleave human interleukin-8 (IL-8) and KC and MIP-2 in mice, thereby disrupting neutrophil chemotaxis to the site of infection and assisting GAS to become systemic.Citation104 Invasive blood isolates have been shown to have increased SpyCEP activity compared to non-invasive isolates.Citation105 The role of neutrophils in SpyCEP mediated pathogenesis of GAS was demonstrated utilising human microvascular endothelial cells where infection with GAS ΔcepA mutant (gene encoding SpyCEP, cepA, deleted) led to significantly higher neutrophil chemotaxis in comparison to a covR/S mutant GAS strain. In addition, it was demonstrated that covR/S mutant GAS survived neutrophil killing significantly more than ΔcepA mutant bacteria.Citation106 Furthermore, following subcutaneous skin-infection covR/S mutant GAS demonstrated increased lesion size which correlated with histopathological analysis where an impaired neutrophil recruitment to the site of infection was noted.Citation106

Hypervirulent covR/S mutant GAS have been associated with reduced colonisation capacity.Citation103 However, covR/S mutant GAS displayed enhanced ability to establish URT infection in a mouse model when compared to a ΔcepA mutant.Citation105 On the contrary, in the same study the observations were reversed when the contribution of SpyCEP to GAS adherence and invasion was examined using HEp-2 human epithelial cells. The ΔcepA mutant was found to be ∼3 fold more adherent and ∼2 fold more invasive than the covR/S mutant parent strain.Citation105 These data are supported by another study where covR/S mutant GAS had significantly decreased adherence to HEp-2 cells and HaCaT keratinocytes in comparison to wild-type GAS.Citation103 covR/S mutant GAS were found to have significantly more hyaluronic acid capsule than wild-type GAS. Hypercapsulation was associated with impaired adherence through the masking of GAS adhesins and extracellular binding proteins.Citation103

SpyCEP is highly conserved between GAS isolates.Citation104,Citation107 Initial studies by Rodriguez-Ortega et al., 2006,Citation68 using a whole genome proteomic bioinformatic approach identified SpyCEP (Spy0416) as a potential vaccine candidate that led to partial protection following intranasal infection with M23 GAS. In another study, SpyCEP immunization led to reduced dissemination of GAS to the blood and spleen following challenge.Citation49 Similarly, intranasal immunization with rSpyCEP significantly reduced covR/S mutant GAS dissemination from URT to blood liver or spleen.Citation49 Furthermore, SpyCEP vaccination has been shown to reduce the intensity of intranasal infection with bioluminescent GAS (covR/S wild-type).Citation108 However, bacterial counts in nasal tissues on day-4 post-infection were not significantly different between vaccinated and control groups, indicating that SpyCEP alone was unlikely to be a viable vaccine candidate.Citation108

Development of a combination vaccine to broaden the scope of J8-DT

The data on the mechanism of J8-DT-mediated protection highlighted a critical role of neutrophils.Citation65 Following skin challenge with covR/S wild-type GAS, vaccinated neutrophil-depleted mice suffered significantly higher bacterial burdens in skin and blood when compared to vaccinated neutrophil-sufficient mice.Citation65 These data suggested that J8-DT may have compromised efficacy against strains of GAS that have a mutation in the covR/S regulon, preventing neutrophil ingress to the site of infection and hampering phagocytosis. This was supported by histological examination that demonstrated a lack of neutrophils at the site of infection.Citation65 To protect neutrophil-attracting CXC chemokines from degradation, antibodies were generated using a truncated recombinant SpyCEP fragment (rSpyCEP: amino-acid residues 35–587)Citation49 combined with J8-DT. Vaccination with this combination vaccine (J8-DT+ rSpyCEP) led to significant protection against pyoderma and bacteraemia.Citation65 In-vitro studies showed that anti-SpyCEP antibodies protected IL-8 from degradation mediated by supernatants from covR/S mutant GAS strains.Citation106 These data demonstrated that J8-DT and rSpyCEP act synergistically to opsonize GAS (with anti-J8 antibodies) and to block IL-8 degradation (with anti-SpyCEP antibodies). The combination vaccine resulted in profound protection against covR/S wild-type and mutant GAS skin challenges.

The combination J8-DT+rSpyCEP is promising; however, rSpyCEP is a large protein, which may have the ability to induce an unwanted autoimmune response. Although rSpyCEP has been previously used as a vaccine candidate with no known side effectsCitation49; to eliminate any potential risks that may impede future vaccine progress, epitope mapping of rSpyCEP was undertaken. Peptide S2 (AA 205–224) was recognized by antisera from rSpyCEP-immunized mice. Antibodies generated to S2 could completely protect IL-8 from SpyCEP-mediated proteolysis.Citation66 We also demonstrated that human plasma samples with a confirmed antibody response to GAS could only partially protect IL-8 from degradation, suggesting that native SpyCEP may be cryptic or subdominant conferring a survival advantage to the organism.Citation67 Like J8, S2 is highly conserved with 95% homology found between the vaccine candidate S2 and S2.1 (), further suggesting that it is not under immune pressure. Both rSpyCEP- and S2-antisera also protected the related mouse chemokine, MIP-2, against degradation. Subsequently, mice vaccinated with the combination vaccine (J8-DT+S2-DT) and challenged via the skin route with stationary or log phase covR/S mutant organisms had significantly reduced bacterial burden in skin and blood when compared to PBS controls.Citation66 Furthermore, histological examination revealed that immunized mice had a large influx of neutrophils to the site of infection. Mucosal immunity was also assessed in the context of J8 and S2 mediated protection. J8 and S2 expressed on the surface of liposomes (J8/S2-Lipo-DT) and administered to mice intranasally elicited J8- and S2-specific IgA titres that were comparable to the titres induced by the individual vaccine constructs (J8-Lipo-DT and S2-Lipo-DT respectively).Citation96 Following intranasal-challenge with 5448AP GAS (a covR/S mutant), immunized mice had significantly reduced bacterial colonisation in comparison to PBS controls in throat swabs and NALT.Citation96 Recently a more soluble derivative of S2 (S2 with four Lysine residues; K4S2) in combination with J8-DT has demonstrated comparable efficacy.Citation67 A comprehensive summary of cryptic/B-cell epitopes utilized in vaccines designed by our group is presented in .

Table 3. Multiple sequence alignment of S2 variants.

Table 4. J8-based vaccine modifications.

Animal models in GAS vaccine development

GAS is a human-specific pathogen; consequently, use of an animal model to study vaccine efficacy and immunopathogenesis of the organism poses several challenges. GAS isolated from humans rarely show natural virulence for mice and serial passaging is required to increase the virulence of the organism. Additionally, lack of responsiveness to GAS superantigens further limits the utility of animal models to assess vaccine efficacy in the context of humans; colonization is often difficult to achieve and true pharyngitis does not occur.Citation109 A potential way forward would be to develop a human GAS pharyngeal challenge model and efforts to implement this strategy are currently underway.Citation110

Despite these limitations, mouse models provide a complex multi-factorial immune system that cannot be recapitulated in an in-vitro environment. The recent emergence of humanized mice is a pivotal step in the advancement of translational vaccine research. Humanized mice expressing human MHC recognize GAS superantigensCitation111 and therefore can be utilized to assess vaccine efficacy against clinical GAS isolates that rarely show natural virulence in mice. Humanized plasminogen mice can be used to model GAS invasive disease in humans. Since GAS streptokinase has a higher affinity for human plasminogen than mouse plasminogen, these mice can mimic the activation of human plasminogen by streptokinase which is vital for systemic dissemination.Citation112 Another alternative would be to use non-human primate (NHP) models that are biologically closer to humans. Streptococcal pharyngitis has been previously assessed in NHPs.Citation113,Citation114 In addition, different experimental vaccine candidates inducing significantly different level of protection in two different mouse models;Citation112 suggests that progression to human clinical trials requires standardisation of animal models for the advancement of GAS vaccine development.Citation112 Overall, a combination of various readouts (in-vivo protection studies in mouse and in-vitro opsonophagocytic assays) may provide valuable insight into the mechanistic aspects as well as protective efficacy of vaccines in humans.

Many pre-clinical studies in GAS vaccine development rely on hypothesis-driven research in mice. Recently, the translation of mouse data into humans has been questioned. A recent study claimed that genomic responses in mouse models correlate poorly with the human condition.Citation115 A subsequent report reevaluated the same gene expression dataset in a more rigorous and less biased manner and reported the exact opposite findings.Citation116 To combat the caveats associated with in-vivo research, rigorous standards need to be implemented when undertaking mouse studies. Proper use of controls, sufficient statistical power to determine cohort sizes and attention to data interpretation will improve the translational impact of these experiments.Citation117 Additionally, discounting the practicality and the utility of mouse-based research may compromise future scientific discoveries.Citation118 Thus, an ongoing discussion on mouse models in all disease states is necessary to advance translational research in a more efficient and effective way.

Other cryptic vaccines in preclinical development

The implementation of cryptic epitopes as vaccine candidates is not unique to GAS vaccine development and has been employed in other fields as well. Plasmodium spp. parasites evade immunity through switching antigen expression and/or by expressing antigens that exist in multiple allelic forms. However, some important antigens/epitopes are cryptic and such as not under immune pressure. The circumsporozoite protein (CSP) protein is found on the surface of sporozoites (introduced into the blood stream following a mosquito bite). The amino-terminal region of the CSP is responsible for liver invasion by sporozoites.Citation119 A cryptic, 21 amino-acid epitope, from the amino-terminal region of the CSP protein, was identified that induced antibodies capable of blocking liver cell invasion.Citation120 However, in the native state the epitope was not immunogenic, protecting the parasite's ability to invade hepatocytes.Citation120

Bacillus anthracis is the causative agent of anthrax in animals and humans. Anthrax toxin is composed of a protective antigen (PA), a cell binding protein, and two enzyme components. PA-based vaccination has shown protective efficacy following anthrax challenge.Citation121-123 The licensed Bioanthrax/AVA vaccine, composed predominantly of PA, requires multiple injections and yearly boosts to maintain immunity. It has also demonstrated a high degree of reactogenicity.Citation124-126 PA-specific neutralising antibody repertoire has been shown to be limited to a few dominant specificities thus leaving the vaccine vulnerable to B.anthracis strains resistant to PA-specific humoral immunity.Citation127,Citation128 A protective cryptic antigen within PA was identified that could elicit humoral immunity and potent neutralisation of lethal toxin in-vitro.Citation128 Immunization with full-length PA did not induce antibodies specific for the epitope.Citation128

Conclusion

Vaccine development strategies have primarily focused on dominant epitopes; however, immunodominance can be a hindrance to the progression of a vaccine due to its common association with antigenic polymorphism. Therefore, a focus should be placed on defining cryptic epitopes that induce protective immune responses to a vast array of antigenically variable organisms. While cryptic epitopes are not recognized, or recognized poorly, as a result of natural infection,Citation4 they can induce antibodies that may recognize the organism and induce strain-transcending immunity.Citation65,Citation66

The cryptic epitope, J8, is minimal and this enhances its safety profile, and S2 contains only 20 amino-acids. They work synergistically to induce strain-transcending immunity that prevents infection with virulent streptococci. This strategy of identifying non-dominant/cryptic epitopes has been successfully applied to a few organisms that readily evade immunity and enable the design of highly immunogenic and effective vaccines.

Acknowledgments

We thank Emma Langshaw for critically reviewing the manuscript and Ainslie Calcutt for her assistance with analysis of the S2 data sequence.

Additional information

Funding

This work was supported by grants from the National Heart Foundation of Australia (APP1044023), a National Health and Medical Research Council (NHMRC) (Australia Program grant (APP1037304) and a NHMRC project grant (APP1083548). We also acknowledge funding from the National Foundation of Medical Research and Innovation (NFMRI, Australia), and the Australian Tropical Medicine Commercialisation grant. An APA and a GLYPRS Scholarship awarded to VO and a NHMRC Fellowship grant to MFG.

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