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Ferretti JJ, Stevens DL, Fischetti VA, editors. Streptococcus pyogenes : Basic Biology to Clinical Manifestations [Internet]. Oklahoma City (OK): University of Oklahoma Health Sciences Center; 2016-.

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Streptococcus pyogenes : Basic Biology to Clinical Manifestations [Internet].

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Current Approaches to Group A Streptococcal Vaccine Development

, MD, , PhD, , PhD, , PhD, , BSc, MBBS, PhD, MD, DSc, , PhD, , MD, , PhD, , MD, FRCPC, , PhD, , MD, PhD, and , MB, NS, PhD, FRACP.

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Introduction and Historical Perspectives

The search for safe and effective vaccines to prevent Streptococcus pyogenes infections has been ongoing for decades. The fact that there is not a licensed vaccine is remarkable, considering that S. pyogenes is one of the most studied human bacterial pathogens. Considerable detailed information is available on the molecular pathogenesis of infection (Walker, et al., 2014), the structure and function of multiple virulence determinants, and on protective immune responses in animals (Lancefield, 1962) and humans (Wannamaker, Denny, Perry, Siegel, & Rammelkamp, Jr., 1953). The global burden of S. pyogenes disease is substantial, and excess mortality from acute rheumatic fever (ARF), rheumatic heart disease (RHD), and invasive infections is significant (see the chapter on the world disease burden of S. pyogenes). The world needs a safe, effective, and affordable S. pyogenes vaccine.

Evidence indicates that natural infection with S. pyogenes leads to a protective immunity, which could be mimicked by appropriately constructed vaccines. The peak incidence of S. pyogenes infections occurs in schoolchildren and declines in adulthood. The relative resistance of adults has been ascribed to an accumulation of protective antibodies against type-specific regions of the M protein, conserved M epitopes, or other conserved antigens that follow multiple S. pyogenes infections during childhood. Lancefield first demonstrated type-specific protective immunity in mice and subsequently showed that in humans, bactericidal M protein antibodies persisted for years after infection (Lancefield, 1959). The most direct evidence of vaccine prevention of infection was a series of studies by Fox et al. who immunized volunteers with purified M protein preparations through either the parenteral or mucosal routes, and showed the protection that followed these “challenge” infections (Fox, Pachman, Wittner, & Dorfman, 1970; Fox, Waldman, Wittner, Mauceri, & Dorfman, 1973; Polly, Waldman, High, Wittner, & Dorfman, 1975). Wannamaker and his colleagues performed a study on 131 military recruits that demonstrated that infections with homologous serotypes occurred six times more frequently in individuals without type-specific antibodies, as compared to individuals with antibodies to that particular type (Wannamaker, Denny, Perry, Siegel, & Rammelkamp, Jr., 1953). However, the same study demonstrated that type-specific serum antibodies had little effect on “transitory acquisitions” of S. pyogenes infections in the pharynx (Wannamaker, Denny, Perry, Siegel, & Rammelkamp, Jr., 1953), an observation that was confirmed in a subsequent study in families in Egypt (Guirguis, Fraser, Facklam, El Kholy, & Wannamaker, 1982). Finally, Beachey showed that serum from volunteers immunized with a purified pepsin extract of M24 contained antibodies that were bactericidal against type 24 streptococci (Beachey, Stollerman, Johnson, Ofek, & Bisno, 1979). Vaccine development has been hampered by the fact that there is not a well-defined human immune correlate of protection against S. pyogenes infection. The studies of Lancefield and Fox allude to a type-specific immunity that is mediated by antibodies against the M protein, and that bactericidal antibodies are associated with protection against symptomatic infection. However, others argue that repeated S. pyogenes infections evoke antibodies against conserved antigens (shared by most or all serotypes) that may explain the immunity acquired by adults.

Aside from the biological and technical hurdles, it has become increasingly evident that the clinical development of S. pyogenes vaccines is somehow “impeded.” There have been multiple impediments, both historical and contemporary. The major concern has been that S. pyogenes vaccine antigens may contain autoimmune epitopes that could potentially trigger ARF—one of the very diseases that vaccines are designed to prevent. Another impediment is the complexity of the epidemiology of the S. pyogenes infections, including the number of emm types, anatomic sites of infection (throat and skin), and geographic differences in the prevalence and burden of the epidemiology and diseases (Steer, Law, Matatolu, Beall, & Carapetis, 2009). A major economic impediment is the fact that 95% of all serious S. pyogenes diseases occur in low- and middle-income countries (Carapetis, Steer, Mulholland, & Weber, 2005) where the return on investment by vaccine manufacturers is predicted to be insufficient to match development costs. Additionally, there is a perception that there would be a lower demand for vaccines in high-income countries, where prevention of pharyngitis would be the major outcome, given the cost-benefit analysis.

The history of S. pyogenes vaccine development dates back more than 90 years. Clinical trials of group A streptococcal vaccines were performed as early as 1923 (Bloomfield & Felty, 1923). Children and adults have been vaccinated with everything from intravenous injections of whole, heat-killed streptococci to intramuscular injections of highly purified fragments of M proteins. The majority of the clinical trials since 1960 were performed with relatively crude preparations of M proteins or cell walls of group A streptococci (Table 1). The major problem associated with these vaccines was reactogenicity, which limited the total amount of vaccine that could be delivered, in many cases. From more recent studies, we presume that the intense inflammation associated with these crude preparations was due to contaminating antigens, which may have included known toxins, such as streptolysin S or 0, pyrogenic exotoxins (erythrogenic toxins), or unknown toxins. At least some of the reactogenicity may have been due to the presence of superantigens, which is a property associated with pyrogenic exotoxins (Kotb, 1995).

Table 1. . Summary of previously published Group A streptococcal vaccine trials in humans.

Table 1.

Summary of previously published Group A streptococcal vaccine trials in humans.

A study by Massell et al. (Massell, Michael, Amezcua, & Siner, 1968) was highly controversial because of a subsequent report that linked the vaccine to at least two (and possibly three) cases of acute rheumatic fever (Massell, Honikman, & Amezcua, 1969). The relatively crude M protein vaccine was derived from hot acid extracts of type 3 streptococci. Subjects from this trial were siblings of patients that had had documented acute rheumatic fever. The basis for their concern was the apparent increase in the "attack rate" of ARF in the 21 subjects, as compared to historical data in their clinic population. The authors admit that "final conclusions are not justified from this limited experience with only 21 vaccinated children" (Massell, Honikman, & Amezcua, 1969). Nonetheless, following the Massell publication, a US federal ban was essentially imposed on S. pyogenes vaccine testing in humans that remained in effect for over 30 years, until its reversal in 2006.

The studies by Fox (Fox, Pachman, Wittner, & Dorfman, 1970; Fox, Waldman, Wittner, Mauceri, & Dorfman, 1973; Polly, Waldman, High, Wittner, & Dorfman, 1975) and Beachey (Beachey, Stollerman, Johnson, Ofek, & Bisno, 1979) (Table 1) heralded a new age of vaccine development that employed highly purified, well-characterized M antigens. With effective methods of extracting M proteins from intact streptococci and removing potentially reactogenic contaminants, it was anticipated that multivalent vaccines could be developed in ways that paralleled those used for multivalent pneumococcal polysaccharide vaccines. Subsequent structure/function studies revealed that some M proteins contained epitopes that evoked antibodies that cross-reacted with human tissues (Cunningham, 2000). This observation resulted in a reevaluation of the use of large fragments of M proteins and led to the current approach of including only N-terminal M peptides devoid of potential autoepitopes in multivalent vaccines (Dale, 2008). Simultaneously, many investigators began to explore alternatives to M protein-based vaccines, with the goal of identifying common protective antigens that would circumvent the possibility of inducing tissue cross-reactive antibodies and also provide broad coverage against most (if not all) group A streptococci, independent of their serotype (Table 2).

Table 2. . Candidate S.

Table 2.

Candidate S. pyogenes vaccine antigens.

This chapter describes current efforts to develop safe and effective vaccines to prevent S. pyogenes infections. Although clinical development has been slow, there are a number of available approaches, based on a detailed understanding of the molecular pathogenesis of infection and protective immune responses in animals and humans. The development of M protein-based vaccines has taken full advantage of molecular techniques, rapid and reproducible emm typing methods, and modern molecular engineering that involves gene synthesis and scalable production. Common M epitopes have been engineered into a vaccine that contains a minimal B cell epitope to optimize functional antibody responses. Through genome-based reverse vaccinology, several common antigens have been identified as potential vaccine components. Although the global epidemiology of S. pyogenes infections is still not well defined, a growing amount of information is being used to inform vaccine design. As more vaccines enter clinical trials, there is a need to define common denominators in protocol design, particularly as they relate to safety assessments and efficacy.

Multivalent M protein-based vaccines

The surface M protein of S. pyogenes is a major virulence determinant and also a protective antigen (as discussed in the chapters on ultrastructure). Antibodies against M protein opsonize the organism and promote C3-mediated phagocytosis, which is associated with protection against infection in animals (Lancefield, 1962). These observations have served for many years as the basis for the development of M protein-based S. pyogenes vaccines. Intact M proteins not only contain protective (opsonic) epitopes, but also, contain human tissue cross-reactive epitopes in some cases (Cunningham, 2000). Because of the theoretical possibility of inducing autoantibodies, it has been challenging to separate the protective epitopes from the autoimmune epitopes so that vaccine preparations would contain only protective M protein peptides. Multiple studies from several laboratories have shown that the epitopes contained in the hypervariable, type-specific N-terminus of the M proteins evoke antibodies with the greatest bactericidal activity and that they are the least likely to cross-react with host tissues (Dale, 1999). In addition, the majority of the autoimmune epitopes of M proteins that have been identified are located in the middle of the mature M proteins and are distinct from the type-specific, protective epitopes (Dale, 1999). These observations have led some investigators to focus on the N-terminal type-specific peptides of M proteins for inclusion in multivalent vaccines (Dale, Chiang, & Lederer, 1993; Dale, Simmons, Chiang, & Chiang, 1996; Dale, 1999). Synthetic and recombinant peptides as small as 10 amino acids have been shown to protect animals against subsequent challenge infections with homologous serotypes of S. pyogenes (Dale & Chiang, 1995).

The finding that small peptides from the M proteins could evoke bactericidal antibodies that were not cross-reactive with human tissue prompted investigators to identify methods of designing and formulating vaccines that contained protective epitopes from multiple M serotypes. One approach has been to design fusion proteins that contain N-terminal M peptides linked in tandem, and the first of these was a trivalent synthetic peptide that was linked to an unrelated carrier (Beachey, Seyer, & Dale, 1987). Subsequent vaccines were produced using recombinant techniques, in which specific 5' regions of the emm genes were amplified by PCR and linked together in-frame using unique restriction sites. Vaccines containing four (Dale, Chiang, & Lederer, 1993), six (Dale, 1999), eight (Dale, Simmons, Chiang, & Chiang, 1996), twenty-six (Hu, et al., 2002), and thirty (Dale, Penfound, Chiang, & Walton, 2011) peptides from different M serotypes have been shown to evoke broadly opsonic antibodies in animals without evoking tissue cross-reactive antibodies. Clinical trials designed to assess the safety and immunogenicity of the hexavalent (Kotloff, et al., 2004) and 26-valent (McNeil, et al., 2005) vaccines have previously been performed. Both vaccines were safe, well-tolerated, and evoked bactericidal antibodies against the vaccine serotypes of S. pyogenes.

The availability of more extensive epidemiologic data from a North American pharyngitis study (Shulman, et al., 2004; Shulman, et al., 2009), the CDC's ongoing ABC surveillance in the US (O'Loughlin, et al., 2007), and the StrepEuro study of invasive S. pyogenes strains (Luca-Harari, et al., 2009) has permitted the formulation of a 30-valent vaccine (Figure 1) with greater potential efficacy (Dale, Penfound, Chiang, & Walton, 2011). The serotypes represented in the 30-valent vaccine account for 98% of all cases of pharyngitis in the US and Canada, 90% of invasive disease in the US and 78% of invasive disease in Europe. The vaccine was highly immunogenic in rabbits when delivered intramuscularly on alum. The 30-valent vaccine evoked bactericidal antibodies against all of the vaccine serotypes of S. pyogenes, which is comparable to or greater than that observed with the 26-valent vaccine (Hu, et al., 2002).

Figure 1. . Schematic diagram of the four proteins that comprise the 30-valent M protein-based S.

Figure 1.

Schematic diagram of the four proteins that comprise the 30-valent M protein-based S. pyogenes vaccine.

An unexpected observation was that the 30-valent vaccine evoked bactericidal antibodies against a number of non-vaccine serotypes of S. pyogenes. Altogether, 83 different emm types have been tested in bactericidal assays. Significant killing (>50%) was observed with 73/83 (88%) of the isolates. Of the non-vaccine types tested, 43/53 (81%) were killed, with an average killing rate of 80%. Extrapolating these results to epidemiologic studies in populations at high risk for ARF/RHD suggests a high potential coverage rate for the 30-valent vaccine. For example, the potential efficacy in preventing pharyngitis in school children in Bamako, Mali could be as high as 84% (Dale, et al., 2013) and in the Vanguard Community of Cape Town, as high as 90% (Engel, et al., 2014) when cross-opsonic antibody activity was factored into the analysis (Table 3).

Table 3. . Potential efficacy of the 30-valent vaccine considering vaccine types (VT) and non-vaccine types (NVT) opsonized by the 30-valent antisera.

Table 3.

Potential efficacy of the 30-valent vaccine considering vaccine types (VT) and non-vaccine types (NVT) opsonized by the 30-valent antisera.

The cross-opsonization of multiple non-vaccine types of S. pyogenes promoted by the 30-valent vaccine was largely unexplained, until the global M protein study group provided a collection of S. pyogenes isolates for structural analyses of M proteins. The combined results of functional antibody activity and the new cluster system (Sanderson-Smith, et al., 2014) has resulted in a revised hypothesis of cluster-specific immunity, rather than type-specific immunity (see below). These observations may form the foundation for additional computational design studies that may be able to formulate optimal M protein-based vaccines with broad efficacy throughout the world.

Molecular typing and protection against Group A Streptococcus

Traditional serotyping and emm typing of S. pyogenes

Since Lancefield’s first publication in 1919, immunity against S. pyogenes infections has been believed to be “type specific” (Dochez, Avery, & Lancefield, 1919). Subsequent pioneering work in the 1950s showed that the presence of type-specific antibodies was responsible for immunity against the homologous serotype of S. pyogenes (Denny, Jr., Perry, & Wannamaker, 1957; Wannamaker, Denny, Perry, Siegel, & Rammelkamp, Jr., 1953; Watson, Rothbard, Swift, & de Mello, 1946; Lancefield, 1959), which established the basis for “type-specific immunity.” These studies led to the development of serotyping as a method to distinguish between strains of S. pyogenes, based upon antibodies raised against heterologous strains in rabbits, also known as “M typing.” With the advent of molecular technologies and relatively easy access to sequencing facilities, time-consuming serotyping has been progressively replaced by “emm typing,” a molecular typing method based on PCR and sequencing. Molecular typing of S. pyogenes relies on sequence analysis of the emm gene, which encodes the N-terminus of the M protein. This portion of the M protein consists of a highly variable amino acid sequence that results in antigenic diversity (Whatmore, Kapur, Sullivan, Musser, & Kehoe, 1994; Beall, Facklam, & Thompson, 1996; O'Brien, et al., 2002). To date, 223 different emm types have been globally reported (McMillan, et al., 2013). When the overall architecture of the M protein is considered, all emm types fall into three main groups with distinct molecular structures that correspond to the previously described emm pattern-typing. emm patterns distinguish three distinct groupings (patterns A–C, D, and E), based on the presence and arrangement of emm and emm-like genes within the S. pyogenes genome (McMillan, et al., 2013; Bessen & Lizano, 2010). Specific emm types share the same emm pattern grouping (McMillan, et al., 2013; McGregor, et al., 2004) and the emm pattern correlates well with tissue tropism (patterns A–C for pharyngitis, pattern D for impetigo, and pattern E for both) (Bessen & Lizano, 2010). Patterns A–C and D also correspond to the previously called class I / serum opacity factor (sof) negative M proteins, whereas pattern E corresponds to the class II/sof positive (Smeesters, McMillan, & Sriprakash, 2010b). Approximately 75% of emm types belong to the pattern D and E groups (McMillan, et al., 2013). Despite their epidemiologic relevance, these emm types have not been as extensively characterized as those of the pattern A–C group.

emm-cluster typing

Standard emm-typing only considers a small region of the protein sequence to classify S. pyogenes isolates into emm types (around 15% of the complete protein) (McMillan, et al., 2013; Smeesters, McMillan, & Sriprakash, 2010b). As a consequence, emm-typing is not informative of the entire sequence, predicted conformational structure, or functional domains of the remainder of the M protein molecule. This limitation of emm-typing is relevant because M proteins are multi-functional and contain distinct domains that bind many host proteins across their entire length (Figure 2) (Smeesters, McMillan, & Sriprakash, 2010b). It is also likely that a number of subtle interactions are possible between the M protein and the immune system components, with the consequence of determining the virulence capacity of the M variants (Smeesters, McMillan, & Sriprakash, 2010b). These interactions also have the potential to interfere with the immunity induced by an S. pyogenes infection.

Figure 2. . Three representative M proteins model.

Figure 2.

Three representative M proteins model. Three representative M proteins (M5, M80 and M77) were selected as prototypes for the structural characteristics within each emm pattern group. M protein length and the size of the repeat and non-repeat regions are (more...)

A novel emm-cluster typing system has recently been proposed for S. pyogenes (Sanderson-Smith, et al., 2014). This system classifies the 223 emm types (McMillan, et al., 2013) into 48 functional emm clusters containing closely related M proteins that share structural and functional properties. emm clusters help to predict the virulence potential of any S. pyogenes isolate by ascribing M protein binding attributes to emm types that belong to the same emm cluster (Smeesters, McMillan, & Sriprakash, 2010b; Sanderson-Smith, et al., 2014). This system also correlates to the antigen content of the M protein vaccine and serves as a framework to investigate immunologic cross-protection between emm types (Sanderson-Smith, et al., 2014; Smeesters, Mardulyn, Vergison, Leplae, & Van Melderen, 2008; Smeesters, Dramaix, & Van Melderen, 2010a).

Global epidemiology and vaccine design

Strain diversity and vaccine coverage based upon emm type

Molecular epidemiology studies have shown considerable variation in emm type distribution at both the country and global regional level (Steer, Law, Matatolu, Beall, & Carapetis, 2009; Smeesters, McMillan, Sriprakash, & Georgousakis, 2009). Systematic reviews have highlighted differences in the emm type distribution of S. pyogenes, especially between high-income countries and resource-poor, predominantly tropical regions (Steer, Law, Matatolu, Beall, & Carapetis, 2009; Smeesters, McMillan, Sriprakash, & Georgousakis, 2009). While only a relatively small number of predominant emm types circulate in high-income countries, the diversity of strains associated with disease in low-income settings is much greater, which results in the possibility for low coverage of type-specific vaccines. Moreover, the profiles of the many emm types recovered from low-income countries differ considerably from one country to another, a factor that hinders vaccine development (Dey, et al., 2005; Abdissa, et al., 2006; Smeesters, et al., 2006). In high-income countries, 25 emm types accounted for 90.3% of all disease-causing isolates with the three most frequent emm types (emm1, emm12 and emm28) accounting for 40% of isolates. In contrast, 26 emm types accounted for only 61.8% of all isolates in the Pacific region with no predominant emm type. A similar distribution was observed in Africa, with 26 emm types accounting for only 62.5% of all isolates. In Asia, the Middle East, and in Latin America, obvious similarities to the emm type distribution observed in high-income countries were noted, although major differences in emm type distribution were observed between these regions and even at the country level. For example, a study in Brazil found that emm type distribution was considerably different between the slums and the wealthy suburbs from the same city of Salvador (Tartof, et al., 2010); the diversity of emm types in the suburbs had a similar profile to that of high-income countries, while the profile in the slums was more like that seen in resource-poor countries. This finding suggests that socio-economic factors have a considerable influence on the diversity of circulating S. pyogenes.

The Pacific region is characterized by a high S. pyogenes disease burden and a wide variety of circulating emm types (Steer, Law, Matatolu, Beall, & Carapetis, 2009; Smeesters, McMillan, Sriprakash, & Georgousakis, 2009). An analysis of two prospective surveillance studies of invasive S. pyogenes disease in New Caledonia in 2006 and 2012, respectively, found that 70% of strains recovered in New Caledonia in 2012 were different from those recovered six years earlier (Baroux, et al., 2014; Le Hello, et al., 2010). This observation is clearly different from those made in North America, where the overall emm type distribution has remained relatively stable over the past 10 years, with only a handful of strains responsible for the majority of infections (Shulman, et al., 2009; O'Loughlin, et al., 2007). The large diversity of strains detected in a number of Pacific countries as well as broad changes in the emm type distribution over time have implications for predicted vaccine coverage in each region, based on emm-type. Theoretical protection by the 26-valent type-specific M protein vaccine was estimated in 2009 to be 23.9% in the Pacific (Table 4). Similarly, a recent study in Hawaii showed that pharyngeal isolates of S. pyogenes represent a diverse collection of emm types, in which 50% were included in the 26-valent vaccine (Erdem, et al., 2009). In contrast, coverage of the vaccine in non-tropical, high income countries was 72.8%. Therefore, the high number of circulating emm types of S. pyogenes in S. pyogenes-endemic countries has presented a major hurdle to global vaccine development.

Table 4. . Vaccine coverage of isolates by region and disease.

Table 4.

Vaccine coverage of isolates by region and disease.

Strain diversity based on emm-cluster and the cross-protection hypothesis

The emm-cluster typing system has not yet been widely applied to global epidemiologic datasets. However, when applied to the data outlined above from New Caledonia the analysis demonstrated that emm-clusters associated with invasive infection did not vary greatly over the 6 year time course, in contrast to emm-types. Further, analysis of emm-cluster distribution in Australia, Fiji and New Caledonia combined found only a limited number of emm clusters were responsible for most of the disease burden in these three countries, while very few similarities could be found among the emm types (Baroux, et al., 2014; McDonald, Towers, Fagan, Carapetis, & Currie, 2007; Steer, et al., 2009).

While the concept of type-specific immunity against S. pyogenes is a broadly accepted paradigm, it has only been validated using a limited number of emm types; specifically those emm types that are common to high income countries, rather than those present in lower income settings. Previous studies suggest that the type-specific paradigm of immunity might not be directly applicable to the many strains of S. pyogenes currently circulating in low-incomes countries. First, preliminary analysis of the complete sequence of 51 M proteins has suggested that emm types found in low-income countries (Smeesters, et al., 2006) have complete M protein sequences that are highly related (Smeesters, Mardulyn, Vergison, Leplae, & Van Melderen, 2008; Smeesters, Dramaix, & Van Melderen, 2010a). This suggests that the immune response against the entire M protein may be similar between these emm types (Smeesters, Dramaix, & Van Melderen, 2010a; Smeesters P. R., 2014). Second, pre-clinical development of a 30-valent M protein vaccine has demonstrated in vitro cross-opsonization of emm types that are not included in the vaccine (Dale, Penfound, Chiang, & Walton, 2011; Dale, et al., 2013). Noteworthy, such emm-cluster typing appears to largely predict cross-opsonization of emm types within emm clusters, with some exceptions. As a result, the emm cluster system may serve as a framework to investigate this cross-protection phenomenon. Importantly, the emm cluster system does not contradict this concept of type specificity, because many of the “high-income emm types” occur in their own emm-cluster or with only a few other emm-types (Sanderson-Smith, et al., 2014). The concept of cross-protection across the numerous S. pyogenes emm types could lead to an M protein vaccine that could provide broad coverage in both high- and low-income settings.

Conserved region M-protein based vaccines

An alternate strategy to N-terminal M peptide vaccines that still use the extracellular domain of the M-protein has been to consider the conserved C-terminal region, and specifically, the C-repeat region. The C-repeat region is highly conserved between different S. pyogenes strains and therefore represents a possible vaccine candidate that may protect against multiple strains of S. pyogenes.

A number of research groups have explored the potential of the conserved region vaccine approach. To date, the main approaches that have been investigated include: (i) the use of the entire C-terminal region of the M6 strain as a recombinant protein (Bessen & Fischetti, 1990); (ii) the use of a 12 amino-acid minimal B-cell epitope from the C-repeat region (J8) as a synthetic peptide (Batzloff, et al., 2003); and (iii) the use of B and T cell epitopes from the C-repeat region from an M5 strain as a synthetic peptide or recombinant protein (pepVac StreptInCor vaccine) (Guilherme, et al., 2006; Guilherme, et al., 2009). Although it is believed that vaccines incorporating the conserved region will protect against all S. pyogenes strains, there are no data from human trials as yet.

Vaccine based on C-Repeat Region of the M-protein

A primary route for S. pyogenes infection in humans is through the colonization of the mucosal epithelium of the pharynx where immunoglobulin A (IgA) provides a defense mechanism against bacterial infection. By focusing on the M-protein, Bessen and Fischetti demonstrated the ability of peptides that represent the conserved region of the M-protein of an M6 S. pyogenes isolate to induce IgA antibodies that passively protect mice when peptide antisera was mixed with S. pyogenes and intranasally administered (Bessen & Fischetti, 1988b). Expanding on this concept, these peptides were conjugated to cholera toxin B subunit (CTB). Mice vaccinated with these peptide-CTB conjugates had significantly reduced pharyngeal colonization following intranasal S. pyogenes challenges, as compared to cohorts of control mice (Bessen & Fischetti, 1988a).

Similarly, peptides corresponding to the C-repeat region of M6 were conjugated to cholera toxin B subunit (CTB) and administered to mice either orally or intranasally (Bessen & Fischetti, 1990). Cohorts of mice immunized with these conserved region peptide conjugates were significantly protected against intranasal colonization by homologous M6 or heterologous M14 streptococci, as compared to control cohorts that were only administered CTB (Bessen & Fischetti, 1990). When taken together, these data highlighted the role of conserved region peptide-specific Ig in controlling S. pyogenes colonization of the throat. Bronze et al. also highlighted the importance of a local mucosal immune response in protecting against streptococci infection (Bronze, McKinsey, Beachey, & Dale, 1988). Attenuated M24 streptococci when administered locally protected mice from subsequent intranasal infections with both homologous M24 and heterologous M6 streptococci (Bronze, McKinsey, Beachey, & Dale, 1988).

An advantage of targeting the C-repeat region as a vaccine candidate is the potential to induce host protection against all S. pyogenes strains; however, concerns about immunogenicity and the efficacy of conserved region epitopes have been raised. Jones and Fischetti investigated the potential of 19 monoclonal antibodies to opsonize M6 streptococci (Jones & Fischetti, 1988). Only one of the 19 monoclonal antibodies was capable of opsonizing the M6 strain, and this antibody was shown to target the amino-terminal region of the M-protein. Notably, the monoclonal antibodies targeting the C-repeat region, while not opsonic, were capable of fixing complement. In contrast, a peptide, SM5(164-197), towards the carboxyl terminal region of the M-protein of M5 GAS was capable of inducing antibodies that could opsonize M5, M6, M18, M19, and M49 streptococci (Sargent, Beachey, Corbett, & Dale, 1987). Antisera against this peptide, SM5(164-197), recognized sarcolemmal membranes and cardiac tissue, but not myosin (Sargent, Beachey, Corbett, & Dale, 1987). Rabbit antisera against four overlapping peptides from the C-repeat region of M6 streptococci were generated to determine if anti-C-repeat antibodies recognized myosin (Vashishtha & Fischetti, 1993). Low levels of antibodies to selected peptides in this study did bind to cleaved or denatured myosin (Vashishtha & Fischetti, 1993). This highlighted the importance of defining minimal epitopes for inclusion in an S. pyogenes vaccine.

Vaccine based on a minimal B-cell epitope

The search for S. pyogenes antigens that are conserved amongst the majority of S. pyogenes serotypes identified an epitope (referred to as P145) in the C-repeat region of the M-protein, which was recognized by individuals in a highly endemic community (Pruksakorn, Galbraith, Houghten, & Good, 1992; Pruksakorn, et al., 1994). It was found that these antibodies had opsonic potential in naturally infected individuals (Pruksakorn, Galbraith, Houghten, & Good, 1992; Brandt, et al., 1996). Interestingly, it was observed that opsonization with p145-immune sera only occurred when stationary-phase organisms were used (Brandt, et al., 1996; Hayman, Toth, Flinn, Scanlon, & Good, 2002), not log-phase organisms, as are used in the standard Lancefield assay. To minimize/eliminate any chance of tissue cross-reactivity, a minimal B-cell epitope (capable of inducing protective antibodies) from this region was defined (Relf, et al., 1996; Hayman, et al., 1997). This 12-mer epitope within P145, referred to as J8i, was a B cell epitope that did not stimulate T cells in the different mouse strains examined (Hayman, et al., 1997). As a result, J8i was poorly immunogenic. Furthermore, J8i was too small to maintain its helical structure, which is required for its antigenicity. Therefore, a technology was developed to fold J8i as a helix to result in J8, a 28-mer synthetic peptide where only the central 12 amino acids (J8i) are derived from S. pyogenes sequences, with the flanking sequences derived from a non-streptococcal peptide (Relf, et al., 1996).

The short synthetic peptide, J8, was immunologically non-responsive in some outbred genetically diverse mouse populations. To overcome this limitation, the peptide was conjugated to the diphtheria toxoid (DT) (Batzloff, et al., 2003). When the peptide-DT conjugate, “J8-DT,” was administered with CFA or with the human compatible adjuvant, alhydrogel, it was found to be highly immunogenic in inbred and outbred mice (Batzloff, et al., 2003). The induction of opsonic IgG following vaccination with J8-DT was demonstrated and the formulation was also able to significantly protect outbred mice from challenge with a S. pyogenes strain obtained from an Australian clinical isolate (Batzloff, et al., 2003). Both active and passive immunization using J8-DT induced significant protection following intraperitoneal S. pyogenes challenge (Batzloff, et al., 2003; Pandey, Batzloff, & Good, 2009; Sheel, Pandey, Good, & Batzloff, 2010). J8-DT vaccination induced vaccine-specific memory B cells (MBC) and long-lasting antibody responses, which then protected mice from systemic infection (Pandey, Wykes, Hartas, Good, & Batzloff, 2013). Most importantly, it was observed that exposure to S. pyogenes could boost the vaccine-induced antibody response and protect the immunized mice, and that the T-cell help for this boosting response could be provided by naïve T cells. Thus, even though the T-cell help for the primary response may come from the DT component of the vaccine, naïve T cells could work with the memory B cells to generate a protective response. This further highlights the uniqueness of the J8-DT vaccine, which contains a minimal GAS B cell epitope along with T-cell epitopes that do not belong to the pathogen, but to DT. Furthermore, IgG antibodies induced by J8-DT vaccination do not cross-react with human tissue (Hayman, et al., 1997). Recent data have demonstrated that J8-DT can also prevent pyoderma in an animal model that closely mimics human S. pyogenes skin infection (Pandey, 2015).

The protective potential of J8, and the closely related peptide J14, as an intranasal vaccine has also been demonstrated (Olive, Clair, Yarwood, & Good, 2002). Intranasal immunization with J14 using CTB, as well as the lipid amino terminal derivative Pam2Cys or a proteasome adjuvant, protected outbred mice from a lethal S. pyogenes challenge (Batzloff, Hartas, Zeng, Jackson, & Good, 2006; Batzloff, et al., 2005). In both cases, J14-specific mucosal IgA was generated, which resulted in reduced throat colonization following intranasal S. pyogenes challenge (Batzloff, Hartas, Zeng, Jackson, & Good, 2006; Batzloff, et al., 2005). Using a different delivery system, the J8-lipid core peptide (Toth, Danton, Flinn, & Gibbons, 1993) either with or without adjuvant was shown to induce opsonic serum IgG, which was protective against a GAS challenge (Olive, et al., 2005).

In addition to the experimental data described here, there is indirect evidence from studies of natural immunity in humans that supports the role of these epitopes in providing broad-based immunity. An earlier study in the Northern Territory of Australia found that p145 was a cryptic epitope, being poorly immunogenic as a result of natural exposure to S. pyogenes. However, after many years of S. pyogenes exposure, opsonic antibodies to p145 (from which J8 and J14 are derived), did develop, and increased with increasing age, which parallels the acquisition of immunity (Brandt, et al., 1996). J8 is hypothesized to be highly conserved because it is cryptic and as a result, it is hidden from the immune system following natural exposure, which results in the need for extensive exposure for the development of antibodies; however, a critical observation is that antibodies induced by J8 peptide immunization do recognize and opsonize S. pyogenes.

Moving forward on the basis of these immunogenicity and other safety data, J8-DT has successfully completed a human double-blinded Phase I pilot trial with no adverse events reported to date and with volunteers developing an antibody response to J8 (unpublished data).

Vaccine comprising B and T-cell epitopes

Another S. pyogenes vaccine candidate, developed by Brazilian researchers, is in progress. “StreptInCor” is based on the amino acid sequences from the M5 protein conserved region (C2 and C3 regions). To define the vaccine epitope, a large panel of approximately 900 sera and peripheral blood mononuclear cells (PBMC) were used. This enabled the identification of both B and T immunodominant epitopes, which led to the construction of StreptInCor, which is composed of 55 amino acid residues (Guilherme, et al., 2006). It has been shown that the vaccine epitope has three dimensional structural features that make it recognizable to any HLA class II (DRB1*/DRB3*/DRB4*/DRB5*), and which results in T-cell activation and differentiation into effectors and memory cells (Guilherme, et al., 2009; Guilherme, et al., 2011).

Mice vaccinated subcutaneously with this peptide using CFA as an adjuvant developed high levels of antigen-specific antibodies. Mucosal immunization with this peptide, using AFCo as a mucosal adjuvant, induced mucosal (IgA) and systemic (IgG) responses (Guilherme, et al., 2009). The protective efficacy of StreptInCor was demonstrated in various mouse strains, including BALB/C, Swiss, and HLA class II transgenic mice (Postol, et al., 2013; Guerino, et al., 2011). The vaccine did not induce cross-reactivity with cardiac proteins (Postol, et al., 2013) and no autoimmune or pathological reactions were observed in histopathological evaluations (Guerino, et al., 2011). Recent data demonstrated that anti-StreptInCor antibodies were able to opsonize several S. pyogenes strains (De Amicis, et al., 2014).

Genome-based discovery of S. pyogenes vaccine candidates

Without a doubt, the completion of the genome sequence of Haemophilus influenzae in 1995 (Fleischmann, et al., 1995) opened a new era in biological sciences and their medical applications. Indeed, the possibility of simultaneously exploring each of the single genes of an organism offered completely new insights on how the synthesis of its proteome is coordinated in response to the environment and on the phylogenetic relationships both between and within different species.

The omics revolution had also a great impact in vaccinology. It became apparent that in silico data mining for bacterial secreted and surface proteins could successfully be exploited for the discovery of vaccine antigen candidates against relevant bacterial pathogens. The genes that encode potentially suitable vaccine targets could be expressed and purified by such high throughput methods as recombinant proteins in non-pathogenic hosts and tested in pre-clinical models for their immunogenicity and their ability to neutralize the original infectious agent. This genomic approach to vaccine discovery, which was termed reverse vaccinology (RV), was first applied with success to the newly available vaccine against the N. meningitidis serogroup B (Pizza, et al., 2000). In this section we will discuss how genomic based information has guided the discovery of protective antigen candidates against the group A streptococcus, one of the most elusive bacterial targets for which an effective vaccine is still not available, despite decades of intense research.

Discovery of S. pyogenes pili and their potential as vaccine candidates

The genome-based discovery of S. pyogenes pili occurred in 2005 (Mora, et al., 2005) soon after this type of structure was identified as an important vaccine target in the related species of group B streptococcus. Gram-positive pili appeared as long, flexible rods that protrude up to 3 µm from the bacterial surface (Telford, Barocchi, Margarit, Rappuoli, & Grandi, 2006). They are heteropolymeric structures that consist of a major protein subunit that constitutes the pilus backbone (BP), plus one or two minor subunits (AP1 and AP2) present at the tip and on its base respectively, all covalently assembled by a series of transpeptidase reactions catalyzed by class B and class C sortases. The sortase A that is responsible for linking proteins that bear an LPXTG motif to the peptidoglycan cell wall also anchors the pilus structure.

The observation that the genes encoding the group B streptococci pilus proteins and sortase enzymes appeared clustered together in a pathogenicity island has prompted the search for a similar island in the genomes of S. pyogenes that belong to different M types. The main candidate appeared to be the highly variable FCT genomic island, previously known for encoding fibronectin-binding proteins, collagen-binding proteins, and the T antigens. Antibodies specific to three of the proteins encoded in this region were shown to react with high molecular weight polymers on S. pyogenes extracts and pilus-like structures on the bacterial surface (Figure 3). The major S. pyogenes pilus BP protein turned out to be the T antigen that was first described by Rebecca Lancefield, the variability of which constituted the basis for the classification of the species into different T types. Knockout mutants lacking this protein, or the sortase machinery encoded in the FCT region, were deprived of pilus polymers on their surface. The relevance of pili to S. pyogenes pathogenesis was highlighted by experiments demonstrating their involvement in the development of biofilms (Manetti, et al., 2007), in mediating cell adhesion to the human epithelia (Abbot, et al., 2007) and in the formation of microcolonies that helped protect the bacteria from phagocytic killing (Becherelli, et al., 2012).

Figure 3. . Streptococcal pili visualized by electron microscopy after labeling with subunit antibodies coupled to gold particles.

Figure 3.

Streptococcal pili visualized by electron microscopy after labeling with subunit antibodies coupled to gold particles.

Notably, immunization of mice with a combination of recombinant pilus proteins was shown to confer protection against S. pyogenes challenge. However, as anticipated above, the S. pyogenes FCT region displays considerable genetic diversity, with nine different FCT variants identified to date based on their gene composition and DNA sequence. As a consequence of this variability, protection by S. pyogenes pilus proteins is specific to strains that bear the variants used for immunization.

However, sequence analysis matched with epidemiological data indicated that 90% of strains currently circulating in the US and European Union belong to 12 T types. Therefore, since immunization with the pilus backbone confers protection against S. pyogenes challenge in mice, a vaccine that included the backbone proteins of the 12 T types was predicted to cover most S. pyogenes infections (Manetti, et al., 2007). More recently, we investigated cross-protection between strains that carry pili with homologous backbone proteins. To address this question, advantage was taken of the well-established opsonophagocytic assay in group B streptococci. Different S. pyogenes pili were systematically expressed in group B streptococci, and bacterial killing mediated by antibodies to the different pilus variants was assessed. This data showed that cross-protection could potentially be achieved between some of the T types that share sufficiently high homology levels, which potentially further restricts the number of backbone proteins that are required for wide coverage (Buccato, 2015).

Regardless the number of proteins needed to achieve broad coverage, the development of an S. pyogenes pilus-based vaccine could benefit from the application of “structural vaccinology.” This approach has been successfully demonstrated in the case of group B streptococci, where a synthetic chimera that combines the protective domain of backbone proteins from different pilus variants was shown to induce functional antibodies that mediate cross-killing (Nuccitelli, et al., 2011).

Integrating genomics, proteomics and immunomics for S. pyogenes vaccine discovery

Reverse vaccinology is based on the straightforward consideration that if all annotated proteins from a given pathogen are available (for instance, by high throughput cloning and expression) and if all proteins can be screened against a robust and reliable surrogate-of-protection assay (for instance, by in vitro bactericidal assays or animal challenge models), then protective antigens will be identified. The meningococcal and group B streptococci examples demonstrate that the strategy works. However, since most of the assays available for protective antigen selection involve animal immunization, the number of antigens to be tested represents a severe bottleneck in the entire process. For this reason, more selective strategies have been applied over the last few years in order to quickly identify protective antigens. The ultimate goal, often referred to as the “Holy Grail of Vaccinology,” is to identify protective antigens by “simply” scanning the genome sequence of any given pathogen, which will let researchers avoid time-consuming “wet science” and “move straight from genome to the clinics.”

With this objective in mind, a three-technology strategy that allows narrowing the number of antigens to be tested in the animal models down to less than ten was recently described (Bensi, et al., 2012). This approach has been successfully applied to S. pyogenes, and as a result, a three-antigen vaccine candidate is currently ready to enter Phase I clinical trials. The overall approach is based on the assumption that the antigens that induce broadly protective antibody responses are those that are conserved, well expressed, and either secreted or surface-associated. To identify this specific group of antigens, the genome sequences of all S. pyogenes isolates available in the public database were first analyzed to select conserved genes that potentially encode secreted and surface-associated proteins. These genes were then expressed in E. coli and the recombinant proteins were used in two ways: to produce mouse polyclonal antibodies and to build protein arrays. Polyclonal antibodies were subsequently exploited to establish which of the corresponding surface proteins were expressed at a high level in a battery of S. pyogenes isolates by using fluorescence-activated cell sorting (FACS) analysis (Technology 1), while protein arrays were used to select immunogenic proteins by screening a panel of sera from S. pyogenes-infected human patients (Technology 2). Finally, in Technology 3, secreted and surface-exposed proteins were identified by mass spectrometry (MS) by analyzing the supernatants (secretome) and the protease-derived peptides of “shaved” bacterial cells (surfome) from different isolates. Once available, the lists of antigens identified by MS, FACS, and protein array were merged in order to establish which proteins were identified by all three technologies. These “common” proteins are those that fulfill the assumption criteria, being: 1) well expressed in a high number of strains; 2) immunogenic; and 3) are surface-exposed/secreted in multiple isolates. The three-technology strategy led to a global list of 40 antigens, with only six of these identified by all three experimental approaches. Remarkably, four of these six antigens were protective in three different mouse models (internasal, intraperitoneal and air pouch infection models) that used four S. pyogenes isolates belonging to different M types as challenge strains. The protective antigens include three particularly interesting proteins. One of them is SpyCEP, a serine protease that degrades IL-8 and other chemokines, which prevents neutrophil recruitment at the infection site (Edwards, et al., 2005). A second one is streptolysin O (SLO), a secreted toxin that kills eukaryotic cells through the formation of membrane pores. The third antigen is Spy0269, a previously uncharacterized hypothetical protein involved in bacterial cell division and probably also involved in adhesion to host cells (Gallotta, et al., 2014). The combination of SpyCEP, SLO and Spy0269 elicits robust protection in mice. Such protection is mediated by antibodies with different biological functions. They efficiently neutralize the hemolytic activity of SLO and the proteolytic activity of SpyCEP. In addition, the antibodies have bactericidal activity, as established by a whole blood bactericidal assay. Finally, the antibodies have the capacity to interfere with bacterial cell division and adhesion. Therefore, this multi-facet mechanism of protection makes this three-antigen COMBO vaccine particularly attractive for testing in human trials.

SOF/fibronectin-binding proteins and their potential as vaccine candidates

The binding of fibronectin (Fn) is an important function for S. pyogenes, as illustrated by the fact that S. pyogenes expresses at least 11 different Fn-binding proteins (Table 5). All of these Fn-binding proteins have been found to contribute to virulence and most are multifunctional. This section will focus on evidence concerning their potential as vaccine candidates, but reviews detailing their functions and contributions to virulence are also available (Walker, et al., 2014; Yamaguchi, Terao, & Kawabata, 2013). The Fn-binding proteins of S. pyogenes can be grouped into two basic categories; those that contain a common Fn-binding repeat region and those that contain a unique Fn-binding domain. Those with a common Fn-binding domain include SOF (SfbII), SfbX, protein F1 (SfbI), protein F2 and FbaB. Figure 4 shows a generic model of these proteins. Those with a unique Fn-binding domain include FbaA, Fbp54, GAPDH, M1/M3 proteins, Scl1, and Shr.

Table 5. . Vaccine potential of Fn-binding proteins.

Table 5.

Vaccine potential of Fn-binding proteins.

Figure 4. . A schematic of S.

Figure 4.

A schematic of S. pyogenes Fn-binding proteins. Some of the Fn-binding proteins contain an upper Fn-binding domain (UFD) that immediately precedes the repeat peptide domain (Schwarz-Linek, Höök, & Potts, 2006). A single repeat (more...)

SOF/SfbII

Serum opacity factor (SOF) opacifies human serum and has multiple functions (Courtney & Pownall, 2010). SOF and SfbII (streptococcal Fn-binding protein II) were cloned by different investigators and were provided with different names, based on the functions used to select clones that express the proteins (Rakonjac, Robbins, & Fischetti, 1995; Kreikemeyer, Talay, & Chhatwal, 1995). SfbII was subsequently found to be identical to SOF (Kreikemeyer, Martin, & Chhatwal, 1999).

Similar to M proteins, SOF varies structurally in its N-terminal domain, which results in over 50 different serotypes of SOF (Courtney & Pownall, 2010). Antisera to this variable N-terminal domain have been used to serotype strains of S. pyogenes, based on the inhibition of opacification of serum. The C-terminal domain of SOF contains an Fn-binding peptide repeat that is highly conserved.

The vaccine potential of SOF was first indicated from the findings that antisera against the N-terminal domain of SOF2 (SOF2ΔFN, SOF in which DNA encoding the Fn-binding domain was deleted) opsonized not only M type 2 S. pyogenes, but also M types 4 and 28 (Courtney, Hasty, & Dale, 2003). These findings suggested that SOF may contain shared protective epitopes. A combination of antiserum to SOF and M protein resulted in greater killing of S. pyogenes in human blood than either antiserum alone, which suggests that SOF may augment the killing efficiency of M protein-based vaccines. Antibodies to SOF that were affinity-purified from human serum were found to opsonize and kill S. pyogenes, which indicates that humans can respond to SOF and produce opsonic antibodies. Notably, an affinity matrix composed of the full-length SOF provided antibodies with a greater killing efficiency than a matrix composed of SOF2ΔFN (73% vs 43% killing), a finding that suggests that the Fn-binding domain of SOF may provide an extra degree of protection. In addition, IP and SC injections protected mice against challenges with S. pyogenes. These injections were well tolerated by mice, as no overt signs of toxicity were noted (Pancholi & Chhatwal, 2003).

Gillen et al. (Gillen, et al., 2008) compared mutants of recombinant SOF75 that did not opacify serum with wild-type SOF for their ability to stimulate a protective immune response in mice. The Fn-binding domain was also deleted in these constructs. SC immunization with SOF75ΔFN provided a significant degree of protection against a challenge from M49 S. pyogenes (100% survival rate vs 38% in controls), whereas the non-opacifying mutants of SOF75ΔFN provided a slight but insignificant degree of protection (50% survival). It is not clear why the non-opacifying mutants of SOF75 failed to provide adequate protection. However, these findings did indicate that SOF from one serotype can provide protection against a heterologous serotype of S. pyogenes.

In contrast to the above findings, Schulze et al. (Schulze, Medina, & Guzmán, 2006) found that intranasal immunization of mice with SOF failed to provide protection against an intranasal challenge with S. pyogenes. Although a significant immune response was generated against SOF, it was not determined if the antibodies were opsonic. Perhaps the intranasal route is not an optimal route for SOF to generate protection against a lethal challenge.

SOF is expressed in other streptococci. S. suis express SOF that does not contain an Fn-binding domain (Baums, et al., 2006) and S. agalactiae express FbpA, which is an opacity factor with a high degree of homology to SOF from S. pyogenes (Courtney, et al., 1999). Thus, a vaccine against SOF may target other pathogenic streptococci, in addition to S. pyogenes.

Sfbx

Streptococcal Fn-binding protein x (Sfbx) is expressed by all SOF+ S. pyogenes, and its gene is found immediately downstream of sof and is cotranscribed with sof as a bicistronic message (Jeng, et al., 2003). Like SOF, Sfbx is expressed and its protein evokes an immune response during S. pyogenes infections in humans (Courtney, Nishimoto, Dale, Hasty, & Schmidt, 2004). Unlike SOF and M proteins, the N-terminal domain of SfbX does not vary (Courtney, Nishimoto, Dale, Hasty, & Schmidt, 2004). It is not known if Sfbx elicits a protective immune response, but antibodies against its Fn-binding domain should cross-react with those of SOF, because their Fn-binding repeats are almost identical. Rabbit antiserum to the N-terminal domain of Sfbx did not opsonize S. pyogenes that expresses this protein (Courtney, 2015), which suggests that this domain may not be an ideal candidate for a vaccine.

Protein F1/Sfb1

Protein F1 and SfbI (streptococcal Fn-binding protein I) are the same protein, but were discovered by different groups and were given different names (Hanski & Caparon, 1992; Talay, Valentin-Wiegand, Timmis, & Chhatwal, 1994). The N-terminal domains of SfbI from different serotypes have a 50-97% identity, whereas its Fn-binding domain is highly conserved (Towers, et al., 2003).

A number of studies have reported that IN vaccination of mice with SfbI conjugated to a variety of adjuvants provided protection against IN challenges with various serotypes of S. pyogenes (Table 5). Schulze et al. (Schulze, Medina, Talay, Towers, Chhatwal, & Guzmán, 2001) found that immunization with the Fn-binding repeats of SfbI afforded better protection than its N-terminal peptide. SfbI may also have adjuvant activity (Schulze, Medina, Chhatwal, & Guzmán, 2003; Schulze & Guzmán, 2003).

In contrast to the above studies, McArthur et al. (McArthur, et al., 2004) found that IN vaccination with Sfb1 failed to protect mice against death from an SC challenge of S. pyogenes. In addition, mouse and rabbit antisera against Sfb1 failed to opsonize S. pyogenes. The lack of protection in this study may be related to the failure to stimulate opsonic antibodies.

Protein F2, PFBP, and FbaB

To our knowledge, there has been no reported study on the stimulation of a protective immune response by protein F2, PFBP, or FbaB. Ramachandran et al. (Ramachandran, et al., 2004) proposed that PFBP (Rocha & Fischetti, 1999) and FbaB (Terao, Kawabata, Nakata, Nakagawa, & Hamada, 2002) are variants of protein F2 (Jaffe, Natanson-Yaron, Caparon, & Hanski, 1996). However, there is no significant homology between the central domains of protein F2 and FbaB, and it is unlikely that epitopes within these domains would stimulate a significant cross-protective immune response.

FbaA

FbaA is a Fn-binding protein expressed by a limited number of serotypes (Table 5). Immunization of mice with either the full-length FbA or with its Fn-binding domain provided significant protection against S. pyogenes (Terao, Okamoto, Kataoka, Hamada, & Kawabata, 2005). It is interesting to note that purified antibodies to FbaA also opsonized S. pyogenes (Terao, Okamoto, Kataoka, Hamada, & Kawabata, 2005). The highest level of opsonization was obtained with antibodies to the full length FbaA (68% killing) and intermediate levels obtained with antibodies to the N-terminal region of FbaA (57% killing) and Fn-binding repeats of FbaA (44% killing).

Fbp54

Fbp54 is an Fn-binding protein with a calculated molecular weight of 54 kDa (Courtney, Li, Dale, & Hasty, 1994), and its gene is found in all S. pyogenes strains (Kawabata, et al., 2001). The name Fbp54 is actually a misnomer and was based on the calculated mass of the cloned protein, which was thought to contain the entire gene at the time of cloning. Subsequent work (Courtney, 2015) and its sequence in S. pyogenes genomes indicated that an N-terminal fragment of 76 amino acids was missing.

Fbp54 is antigenic in humans (Courtney, Dale, & Hasty, 1996) and rabbit serum against Fbp54 opsonized S. pyogenes (Kawabata, et al., 2001). Immmunization with Fbp54 by various routes in mouse models was found to evoke protection against challenges with multiple serotypes of S. pyogenes (Kawabata, et al., 2001). Fbp54 is conserved in many streptococcal species with high identity to pavA of S. pneumoniae (Holmes, et al., 2001), Sfba from group B streptococci (Mu, et al., 2014), and FbpA of S. gordonii (Christie, McNab, & Jenkinson, 2002), which suggests that vaccines that contain Fbp54 may, in theory, target other streptococci, as well as S. pyogenes.

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional, intracellular enzyme that is also found on the surface of S. pyogenes where it binds fibronectin in addition to other host proteins (Pancholi & Fischetti, 1992; Pancholi & Chhatwal, 2003; Jin, Agarwal, Agarwal, & Pancholi, 2011). Antibodies to GAPDH both opsonized S. pyogenes (Boël, Jin, & Pancholi, 2005) and protected mice from a S. pyogenes challenge (V. Pancholi, personal communication). However, there are similarities between human and bacterial GAPDH, and full autoimmune responses to GAPDH have not been thoroughly investigated (Fontán, Pancholi, Nociari, & Fischetti, 2000). In addition, GAPDH is expressed by many bacteria, and a GAPDH vaccine may target both pathogens and commensals.

Scl1

Scl1 is a collagen-like protein expressed by S. pyogenes that binds cellular Fn, but not plasma Fn (Oliver-Kozup, et al., 2013). Antibodies against Scl1 have been found in human serum and in mice infected with S. pyogenes (Hoe, Lukomska, Musser, & Lukomski, 2007). However, none of these responses have been shown to provide protection against infections. It remains to be resolved whether purified Scl1 will induce antibodies that will cross-react with human collagen and/or provide overall protection against S. pyogenes infections.

Shr

Shr (streptococcal hemoprotein receptor) is a virulence factor that, as the name implies, binds heme containing proteins and also binds extracellular matrix proteins laminin and fibronectin (Dahesh, Nizet, & Cole, 2012). Shr is highly conserved and is found in all sequenced genomes of S. pyogenes and in S. dysgalactiae subspecies equisimilis (Eichenbaum, 2012). Both IP and IN immunizations with Shr provided protection against S. pyogenes challenge infections in mice (Huang, Fisher, Nasrawi, & Eichenbaum, 2011). Furthermore, mice passively immunized with rabbit antiserum to Shr were protected against challenges with M1 and M3 S. pyogenes, which suggests that Shr may provide protection against multiple serotypes.

M1/M3 proteins

The M1 and M3 proteins bind Fn through interactions with their A and B repeat regions (Cue, Lam, & Cleary, 2001). Other serotypes of M proteins that have been tested do not bind Fn. However, the binding of Fn to M proteins has not been extensively investigated, and as a result, it is not clear if other M types may also bind Fn. The vaccine potential of both the conserved and non-conserved regions of M proteins is discussed elsewhere in this chapter.

In summary, many of the Fn-binding proteins have been found to provide protection against S. pyogenes infections in mouse models. Some of these were expressed by all types of S. pyogenes proteins (including Shr, GAPDH, and FBP54) but other, non-pathogenic bacteria may also express these proteins, and it can be debated if it would be wise to use a vaccine that targets both pathogens and commensals. Some of the Fn-binding proteins are expressed only by a limited number of serotypes (FbaA, FbaB) and may not be the best candidates to include in a multi-component vaccine. The Fn-binding domain shared by protein F1/SfbI, protein F2, SOF, and SfbX should be considered for inclusion into a multivalent vaccine, because this domain not only provided a significant degree of protection in animal models, but also had adjuvant activity. Although there is a fair degree of homology between the Fn-binding repeat domain of these proteins (Figure 3), the degree of cross-reactive immune responses to these domains has not yet been investigated. Thus, it is not known if the Fn-binding domain of one Fn-binding protein will provide cross-protection against serotypes of GAS expressing a different Fn-binding protein. Finally, the question arises whether the addition of the Fn-binding domain and/or the N-terminal domain of those Fn-binding proteins expressed by a significant number of serotypes would enhance the coverage of serotypes and protective efficacy of a multicomponent vaccine.

ScpA as a potential vaccine component

The streptococcal C5a protease (SCP) is expressed on the surface of all serotypes of S. pyogenes and most human isolates of groups B, C, and G streptococci, where it specifically destroys C5a. The enzyme also binds fibronectin and functions as a low level invasin for S. pyogenes (Courtney, Nishimoto, Dale, Hasty, & Schmidt, 2004), group B streptococci (Tenenbaum, et al., 2007), and group G streptococci (Wei, et al., 2013; Severin, et al., 2007). The genetic diversity of S. pyogenes, and the fact that other species of β hemolytic streptococci cause pharyngitis and may induce similar complications, suggest that a vaccine must include multiple, antigenically conserved proteins in order to significantly reduce the incidences of pharyngitis, skin infections, and various sequelae. The specific inhibition of complement C5a that mediates phagocyte recruitment was first discovered in S. pyogenes (Wexler, Chenoweth, & Cleary, 1985), but has since been shown to be a universal mechanism of pathogenesis. Other streptococcal species and unrelated bacteria, including staphylococci and borrelia, also interfere with C5a recruitment of phagocytes to infectious foci. Some S. pyogenes serotypes also produce proteases that destroy IL-8, another phagocyte chemoattractant (Hidalgo-Grass, et al., 2006; Sjölinder, et al., 2008). The high degree of sequence similarity of SCP from different serotypes of S. pyogenes and other β-hemolytic species, and its pivotal role in the pathogenesis of these streptococci, argue strongly for its inclusion in vaccines. Increases in SCP-specific antibodies were documented after episodes of pharyngitis in children and adults (Shet, Kaplan, Johnson, & Cleary, 2003) and human sera with high titers of anti-SCP neutralize cleavage of C5a by purified enzyme (Cleary, Matsuka, Huynh, Lam, & Olmsted, 2004). The primary goal of vaccine development is to prevent colonization of oral and vaginal mucosae and associated lymphoid tissue by all β-hemolytic streptococci that are commonly associated with human disease. Protection studies used truncated forms of recombinant SCP with mutations in the active site and employed subcutaneous, intranasal, or intravaginal rodent infection models. Intranasal and subcutaneous immunization with recombinant SCPA and adjuvants induces robust anti-SCP IgG and IgA responses that speed the clearance of streptococci from pharyngeal- and nasal-associated lymphoid tissue (NALT) after challenge with one of several serotypes tested (Cleary, Matsuka, Huynh, Lam, & Olmsted, 2004; Ji, Carlson, Kondagunta, & Cleary, 1997; Ji, McLandsborough, Kondagunta, & Cleary, 1996; Park & Cleary, 2005; Suvorov, et al., 2010). Antibody directed against SCPA inhibits cleavage of C5a and is also opsonic for both S. pyogenes and group B streptococci (Suvorov, et al., 2010). As expected, the intranasal administration of anti-SCPA prevented colonization of NALT, the mouse homologue of human tonsils (Park & Cleary, 2005).

Although GBS infections are less common, neonatal infections are associated with significant mortality. SCP is also an important virulence determinant for groups B and C streptococci, and vaccination with SCP prevented lethal infections of mice when challenged by either species (Wei, et al., 2013; Suvorov, et al., 2010; Cheng, et al., 2001; Cheng, et al., 2002). Vaccination of mice with SCP also prevented vaginal colonization of dams and protected their pups against challenge with Type III group B streptococci. Lessons learned from peptide vaccines for prevention of pertussis and other mucosal infections suggests that an efficacious vaccine will likely require multiple surface antigens. Toward this end, Severin et al. used proteomic screens to identify other surface proteins that could be combined with SCP in a multi-component vaccine-peptides that are uniformly expressed by most S. pyogenes M types and other β-hemolytic streptococcal pathogens (Severin, et al., 2007).

Group A Streptococcus: Clinical Trial Design

History of S. pyogenes vaccine trials

As described above, the slow progress toward developing a vaccine for S. pyogenes that began with clinical trials in 1923 (Steer, Batzloff, Mulholland, & Carapetis, 2009) was halted in the late 1970s, following a report in 1969 of two definite and one probable cases of rheumatic fever in recipients of a crude M protein vaccine (Massell, Honikman, & Amezcua, 1969). Although subsequent review of this study casts doubt on the role of the vaccine in the onset of rheumatic fever, in 1979, the United States Food and Drug Administration (FDA) prohibited the administration of S. pyogenes vaccines in clinical trials in the US, a prohibition that remained in place for nearly 30 years. Since the lifting of the ban in the last decade, only a few clinical trials of candidate S. pyogenes vaccines have been performed, although several vaccine candidates are approaching the clinical development stage.

General design issues

As with any vaccine, potential S. pyogenes vaccines will need to first undergo phase 1 clinical trials to demonstrate their initial safety in humans. Dose-ranging studies will explore the optimal dose of the vaccine antigens, based on findings from pre-clinical animal studies. The number of doses and dose intervals will also be determined, which are typically based on both animal studies and clinical trials of vaccines with similar antigen compositions. For example, a purified or recombinant protein-based vaccine may initially be studied using a three-dose schedule, similar to that used with hepatitis B vaccines (two closely spaced injections and a third injection after a longer interval). Inclusion of an adjuvant will require demonstration of improved immunogenicity compared to the antigen alone. Inclusion of a control (either placebo or a licensed, non- S. pyogenes vaccine) will permit blinding of the study and provide an unbiased assessment of adverse events. While smaller phase 1 studies often use a placebo, larger phase 2 studies and studies in children may benefit from use of a licensed, non-S. pyogenes vaccine as the comparator vaccine so that all participants can derive a potential benefit from participation in the study. Although the target age for an S. pyogenes vaccine is likely to be pre-school-aged children, prior to the peak of S. pyogenes pharyngitis and rheumatic fever, clinical trials of S. pyogenes vaccine candidates will, out of necessity, first be performed in adults. Although regulatory agencies frequently require vaccines targeting infants to first be studied in older children, it is not clear whether subsequent studies of an S. pyogenes vaccine could be immediately undertaken in pre-school-aged children after phase 1 and early phase 2 studies in adults. An argument could be made that following experience with vaccination of approximately 100 adults with a candidate S. pyogenes vaccine, a phase 2 study in 3–5 year olds could be initiated.

Immunogenicity outcomes

Immunogenicity outcomes will be an important component of any clinical trial of an S. pyogenes vaccine. Whatever the antigen employed, demonstrating induction of a specific immune response to the antigen will be required, using validated immunological assays. For M protein vaccine candidates, assessment of antibody response against each component M type will be required; for the 30-valent S. pyogenes vaccine currently under development, this will require 30 assays for each participant at each time point. In contrast, single-antigen S. pyogenes vaccine candidates, such as the J8 vaccine from the conserved C-repeat section of the M protein, require only a single serological assay (Batzloff, et al., 2003). Vaccine candidates, such as the StrepInCor vaccine that uses B and T cell epitopes from the C-repeat section, will likely require assays that measure T cell response in addition to the antibody responses (Guilherme, et al., 2006).

The lack of a definitive correlate of protection for S. pyogenes vaccines has led to the development of a number of functional assays to measure the biological activity of the antibodies elicited. The clinical trials undertaken with the hexavalent and 26-valent M protein-based S. pyogenes vaccines measured antibody responses by both enzyme immunoassay and by a functional opsonophagocytosis assay (Kotloff, et al., 2004; McNeil, et al., 2005). Functional assays are tedious to perform and are not easily adaptable for high throughput methodologies. While functional assays may be required in the early development of the 30-valent M protein S. pyogenes vaccine candidate, limiting such assays in larger, later-phase studies may be necessary for reasons of feasibility. For example, 30 opsonophagocyosis assays for each participant at every blood collection would amount to tens of thousands of assays in a large phase 2 or 3 study. It is not yet known whether the development and validation of functional assays will be required for S. pyogenes vaccine candidates that use non-M protein antigens. The identification of a correlate of protection using an easily performed, high-throughput assay would greatly alleviate these issues. Alternately, performing functional assays on a subset of participants (and with a subset of M proteins in the case of a multivalent M protein vaccine) might be an acceptable option.

Safety outcomes

Because of the history of safety concerns, adverse event monitoring in clinical trials of S. pyogenes vaccines will be subjected to even greater scrutiny than in other vaccine clinical trials. Routine adverse event monitoring will be required, including injection site reactions (such as erythema, swelling, tenderness) and systemic adverse events (such as fever, headache, fatigue, anorexia). In addition to these common adverse events and reporting of all serious adverse events, surveillance for adverse events of special interest to S. pyogenes vaccines, including carditis and arthritis, will be required. In early phase trials, monitoring will require regular interval physical examinations, baseline and routine serum chemistry and hematology, and assays to measure complement (C3) and inflammatory markers (C reactive proteins). In the clinical trials of the 26-valent M protein vaccine, baseline and follow-up electrocardiograms and echocardiograms were performed, as well as baseline and follow up assays for tissue cross-reactive antibodies (heart, kidney, cartilage and brain) (Kotloff, et al., 2004; McNeil, et al., 2005). As with the functional serological assays, tissue cross-reactive antibodies are not routinely available, and these tests are burdensome to perform. Further discussions with regulatory authorities and scientific experts will be needed to determine the stage in the clinical development process at which these assays will no longer be required. The use of echocardiograms as a screening tool is problematic, as use during clinical trials of the 26-valent M protein vaccine demonstrated a wide range of normal variation of non-pathological findings in normal, healthy individuals. The standardized, reproducible interpretation of echocardiograms in healthy adults also proved to be a challenge; these challenges may be even greater in healthy pre-school-aged children. The necessity for echocardiograms and electrocardiograms through all phases of the clinical vaccine development program will also be an important topic for discussion with regulatory authorities.

Efficacy studies

In the absence of a definitive immunological correlate of protection, phase 3 studies of candidate S. pyogenes vaccines will require efficacy outcomes. Ideally, these efficacy outcomes will be easily measured and confirmed, and will be sufficiently common so that study sample sizes will be manageable (Steer, Dale, & Carapetis, 2013). Pharyngitis is the most readily measured efficacy outcome, because its clinical symptoms are readily observed, and laboratory confirmation through rapid antigen testing and culture is easily performed and is readily available, inexpensive, and reproducible. However, the prevalence of asymptomatic streptococcal carriage, and the fact that pharyngitis is most common in school-aged children, yet the target population for vaccination is the pre-school-aged child, are important factors that must be considered, as they may impact the assessment of efficacy when pharyngitis is the outcome of interest. The latter issue could be addressed by focusing enrollment of older pre-school-aged children, prolonging the duration of follow-ups, and marginally increasing the sample size. Impetigo is another common manifestation of S. pyogenes infection that could be used as an efficacy outcome, but it is unclear whether vaccination will protect against skin infection and its presence is less commonly confirmed by laboratory testing. However, the J8 vaccine, when administered, subcutaneously does protect mice from pyoderma that is due to multiple strains of S. pyogenes (Pandey, et al., 2015).

Acute rheumatic fever and invasive streptococcal infections are the most severe forms of S. pyogenes infection and would be important targets for prevention by a candidate S. pyogenes vaccine. However, ARF and invasive infections are uncommon, even in the developing world, where the incidence of these complications far exceeds rates observed in industrialized countries. A phase 3 efficacy study with prevention of rheumatic fever as the outcome would be problematic as it would require follow-up for participants to identify cases of acute rheumatic fever. In the context of this follow-up, should episodes of pharyngitis be identified, treatment with antibiotics would be required and cases of rheumatic fever would be avoided. As a result, the ethical issues involved in the design of such a study would be challenging. The efficacy of S. pyogenes vaccines against ARF may best be assessed in post-licensure phase 4 studies.

Development of an S. pyogenes human challenge model might provide an opportunity to identify correlates of protection that could be used in the design of a phase 3 S. pyogenes vaccine field trial. Challenge studies with S. pyogenes were used frequently in the 1960s and 1970s, prior to the FDA’s hold on S. pyogenes vaccine clinical trials (Polly, Waldman, High, Wittner, & Dorfman, 1975; D'Alessandri, et al., 1978). These studies could only be undertaken in adults and would be used to inform the design of the pivotal phase 3 study in children, rather than to directly support the licensure of a candidate vaccine.

Conclusion

The overall goal of S. pyogenes vaccine development is to introduce vaccines that will significantly impact the global burden of disease. As summarized in this chapter, there are a number of candidate vaccine antigens that have been proven to be efficacious in animal models of S. pyogenes challenge infections. The impediments to bringing an effective and affordable vaccine to market have proven to be significant—but are not insurmountable. The greatest challenge will in developing vaccines that address the global need and that are effective in preventing the infections that may trigger ARF and RHD, as well as serious invasive infections. This may require a systematic experimental approach to develop combination vaccines that contain multiple protective antigens to achieve the desired level of protective immunity. The successful global deployment of safe and affordable vaccines could have a significant, positive impact on the morbidity and mortality that is attributable to S. pyogenes infections.

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