Group A streptococcal antigens and vaccine potential

Group A streptococcal antigens and vaccine potential Michael A. Kehoe

Attempts over the past seventy years to produce an effective vaccine to protect humans against group A streptococcal infections and their immunologically mediated sequelae (acute rheumatic fever and post-streptococcal glomerulonephritis) have been frustrated by two basic problems,first, the ability of the iu'ohly protective cell-surface M proteins to elicit potentially harmful host reactions and second, the existence of a large number of distinct serovars of M proteins and the fact that human immunity to group A streptococcal infections is predominantly M serovarspecific. In recent years, progress towards overcoming these problems has been greatly facilitated by an increased understanding of ti~e structural and immunological properties of protective group A streptococcal antigens, which has emerged from molecular biology studies. This article reviews these studies and discusses the potential for developing an effective group A streptococcal vaccine. Keywords:Streptococcus pyo,qenes; antigens; M proteins; opsonization

INTRODUCTION Despite the fact that highly protective antigens were first identified almost seventy years ago L2, group A streptococci continue to feature in lists of important human pathogens for which no effective vaccine is currently available 3. With the notable exception of a small number of laboratories, interest in developing a vaccine decreased in parallel with the dramatic decline in acute rheumatic fever (ARF) in developed countries'*. By the 1960s, the decline in ARF had reached the point where it was no longer considered to be an important problem. Today, there are many physicians who may not have encountered even a single case of ARF during their careers and some might argue that the development of a group A streptococcal vaccine is no longer necessary. However, in contrast to the decline of ARF in developed countries, group A streptococcal infections and their immunologically-mediated sequelae remain a serious health problem in most developing countries, where ARF is one of the major causes of heart disease. For example, in 1981 it was estimated that in India alone over six million school-aged children suffered from rheumatic heart disease 5. An effective group A streptococcal vaccine Department of Microbiology, Medical School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK

would be highly desirable in many developing countries. In addition, it would be dangerously complacent to assume that the decline in serious streptococcal diseases, such as ARF, in developed countries is irreversible. It is an oversimplification when in textbooks this decline is attributed solely to a combination of improved living conditions and penicillin prophylaxis. This is demonstrated by the fact that the dramatic decline in ARF was not accompanied by a decline in streptococcal pharyngitis, or a number of other diseases such as post-streptococcal glomerulonephritis (PSGN). Indeed, in both Europe and the United States fatalities resulting from severe group A streptococcal infections associated with the production of pyrogenic exotoxins have been increasing in recent years6-a. Similarly, beginning with an outbreak in the Salt Lake City area in 1985, ARF has been increasing in the United States, suggesting the possibility of a resurgence of this disease 9-1`*. A surprising number of these cases involved children from middle-class families in suburban or rural areas, rather than poorer families living in more crowded inner city environments. It is difficult to reconcile this with the view that the previous decline of ARF was due primarily to improved living conditions. It seems likely that a very significant factor in the decline of certain streptococcal diseases in developed countries was a change in the pattern of prevalent group A streptococcal strains, such that highly virulent strains gave way to strains that were less virulent. This is supported by a variety of observations which have been reviewed in detail elsewhere `*'14-1s. We understand almost nothing about the factors that cause a change in the pattern of prevalent group A streptococcal strains and cannot predict when the current, generally 'low virulence', pattern might be altered by the re-emergence of highly virulent strains. Indeed, there is reason to be concerned that this might be occurring already. It would also be dangerously complacent to rely indefinitely on antibiotics as our principal defensive weapon. Despite the continued sensitivity of group A streptococci to penicillin, there is no good reason to expect that this species never will acquire a fl-lactamase. On the contrary, past experience of the sudden emergence of penicillin resistance in organisms like Neisseria 9onorrhoea and Haemophilus influenza, and of the emergence of tetracycline-, chloramphenicol- and erythromycinresistant group A streptococci, suggests that it may only be a matter of time before we are faced with the problem of penicillin-resistant strains. We should take advantage of this time to determine if the remaining

0264~410X/91/11079~10 {' 1991 Butterworth-HeinemannLtd

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obstacles to designing an effective vaccine can be overcome. If feasible, an effective vaccine would have immediate applications in many developing countries and could well be required in developed countries in the future. GROUP A STREPTOCOCCAL

ANTIGENS

The very elegant studies of Rebecca Lancefield and her colleagues in the early decades of this century identified and extensively characterized many of the major surface antigens of group A streptococci, including the species-specific C carbohydrate and M, T and R proteins~,~,.~ ~,1 In the early 1970s a further surface antigen, namely lipoteichoic acid (LTA) was identified e~ and in recent years a number of laboratories have characterized several previously unidentified or poorly characterized surface antigens, including a C5 pepti-

dase 2~. lgG and IgA Fc-binding proteins 2"* 2,, a n d a fibroncctin-binding protein e7 (Figure 1). In addition. group A streptococci express a wide array of antigenic extracellular products, including the pyrogenic exotoxins, streptolysin O, streptokinase, opacity factor (a lipoproteinase which may also be loosely associated with the cell surface) and a range of degradative enzymes such as hyaluronidase, proteinases and DNAses. Although antibodies to some of these products can modulate the course of particular diseases, of the multitude of cell-surface and extracellular antigens described to date, only M proteins appear to be capable of evoking effective protection against group A streptococcal infections (see below). Therefore, this article will focus primarily on the highly protective M proteins and the reader is referred elsewhere for a recent review 2s of the properties of other antigens. The ability of M protein to elicit highly protective

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opsonic antibody was first suggested in 1919 by Lancefield's mouse protection experiments 1 and has been confirmed repeatedly by seven decades of subsequent research and clinical observations. It is, therefore, not surprising that most attempts to develop group A streptococcal vaccines have focused almost exclusively on this highly protective antigen. These efforts were frustrated by two basic problems. One was the ability of even highly purified M antigens to evoke potentially harmful host reactions (see later). The second was that there is a very large number of distinct serovars* of M protein (>80 identified to date); with rare exceptions only one serovar is expressed by each strain, and immunity afforded by opsonic anti-M protein antibody is predominantly M_type_specific29 31. Considerable progress in overcoming the first problem has been made in recent years, but the second problem poses greater difficulties. Therefore, before discussing the properties of M proteins and the feasibility of designing safe M-protein vaccines, it is worth considering whether the problem of M-type-specific immunity could be avoided by use of non-type-specific antigens. T Y P E - S P E C I F I C VERSUS N O N - T Y P E SPECIFIC IMMUNITY Studies on non-type-specific immunity to group A streptococcal infections have been limited and most experiments that have examined protection directly by in vivo challenge have been performed in mouse models. With the exception of an M50 strain 32, group A streptococci do not cause natural infections in mice, but many human strains can be adapted by in vivo passage to infect mice on intranasal challenge. Protection studies in mice have examined a range of different vaccine formulations (including synthetic peptides 33, recombinant antigens 3~'35 and heat-killed whole cells 36'3v) and a variety of immunization routes (subcutaneous 3v, intranasal 35-3v, and live delivery of recombinant antigens by attenuated Salmonella typhimurium or vaccinia virus34'35). All of these studies agree that systemic protection against group A streptococcal infection is very effective, but that it is due to predominantly type-specific, opsonic anti-M IgG. Therefore, any alternative to a type-specific M vaccine would need to prevent group A streptococci from successfully colonizing epithelial surfaces, prior to invasion. A number of laboratories have shown that intranasal immunization of mice or human volunteers with heat-killed whole cells can produce a significant reduction in pharyngeal colonization on subsequent challenge with the homologous M type 33'35-39. In the mouse model, immunization by this route evoked similar levels of protection against challenge with both homologous and heterologous M types, which did not correlate with the production of opsonic, systemic antibody 36'37. This was supported by more recent studies 33'35, showing that localized mucosal immunity in mice can be evoked by structurally defined, non-type-specific regions of the M proteins, which are known not to elicit opsonic antibodies ~°. It seems clear that delivery of streptococcal *In accordance with the nomenclature recommended by Bergey's Manual, the more commonly used term 'M serotype' is replaced in this article by 'M serovar'

antigens by the intranasal route can induce localized mucosal immunity in the mouse model by non-typespecific mechanisms. Has the problem of type-specific immunity finally been solved? Unfortunately, for a number of reasons, the answer is 'No'. One reason is that localized mucosal immunity would not protect against group A streptococcal invasion via the skin - a common route of streptococcal infections in humans. A second problem is that, even if a vaccine were to be targeted specifically against pharyngeal infections to prevent ARF, the non-type-specific mucosal immunity observed to date in the mouse model is limited. A significant proportion of intranasaily immunized mice still die or are effectively colonized by the challenge organism 33'35'37. Further, available evidence suggests that non-type-specific immunity is even less effective in primates than in the artificial mouse model. For understandable reasons, non-type-specific immunity has not been tested by challenging intranasally immunized human volunteers with heterologous M types, as well as the homologous organism 38'39. Such experiments, however, were performed several decades ago in monkeys, which are much more closely related to humans than mice. In 1946, Watson et al. 41 reported that intranasal inoculation of M a c a c a mulatta with any of six distinct group A streptococcal M types elicited typespecific resistance to subsequent pharyngeal colonization by the homologous M type, but pharyngeal colonization with heterologous M types was easily affected. Although studies in primates have been limited, the type-specificity of mucosal (as well as systemic) immunity observed in monkeys is consistent with 70 years of clinical observations in humans, which show that despite the production of antibodies to non-type-specific antigens during natural infections in humans, repeated infections with distinct M types are very common. At present, it is difficult to avoid the conclusion that the levels of protection in humans that could be induced by non-typespecific antigens alone are likely to be considerably lower than would be required from an effective vaccine. Thus, we return to the problems that have frustrated efforts to develop an effective vaccine, capable of evoking type-specific, opsonic anti-M antibodies. The remainder of this article will focus on the biological, structural and immunological properties of the highly protective M proteins and review the progress that has been made in overcoming at least some of these problems. M P R O T E I N S : R O L E IN V I R U L E N C E , IMMUNITY AND POTENTIALLY HARMFUL IMMUNOLOGICAL REACTIONS M protein confers group A streptococci with resistance to phagocytosis, probably due to its ability to bind host proteins such as fibrinogen and complement factor H and thereby interfere with opsonization by the alternative complement pathwayS2 ~5. In the immune host, type-specific anti-M antibodies opsonize the cell, rendering it susceptible to phagocytosis. It has been established for many decades that such antibodies provide effective and long-lasting protection against subsequent infection by the homologous M type 29''~6''~7. Earlier studies had suggested that M protein might also contribute to virulence by facilitating adhesion to epithelial surfaces, possibly by forming complexes with lipoteichoic acid that can bind specifically to fibronectin

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on the surface of target cells 4s'4~. However, this view has been challenged by recent studies comparing the adhesive properties of a genetically well-defined M-negative mutant isolated by gene-targeted mutagenesis of type 6 Str~_'])tococclls pyo,qenes and the otherwise isogenic parent strain. While this review was in press, Caparon et al. s° reported that the M protein plays neither a direct nor an indirect role (e.g. by orienting lipoteichoic acid) in adhesion to buccal or tonsillar epithelial cells, but that M protein may play an important role in promoting coaggregation of streptococci and microcolony formation after initial attachment, in addition to their anti-phagocytic properties, circumstantial evidence suggests that M proteins might contribute directly to the pathogenesis of autoimmune sequelae. Structural and immunochemical studies on both pepM antigens (i.e. pepsin-generated N-terminal halves of M proteins; see following section) and intact, recombinant M proteins, have shown that at least some serovars of M proteins contain epitopes within their covalent structure that can evoke antibodies that cross-react with host antigens, such as human heart myosin 5~ ~,o, and such host-crossreactive {HCR) antibodies have been detected in patients with rheumatic fever ('~. More recently, M proteins from certain nephritogenic serovars have been shown to elicit antibodies that cross-react with kidney glomerular antigens, such as vimentin ~2 ~,s. It should be emphasized, however, that M proteins are not the only streptococcal antigens that elicit HCR antibodies sz'~ ~s and that there is no direct evidence that such antibodies are important in the pathogenesis of autoimmune sequelae. Nevertheless, the possibility that they might play a role means that any vaccine intended for human use would need to be free of HCR epitopes. Studies on pepM antigens have also suggested that M proteins might elicit potentially harmful cellular responses. Purified pepMS, pepM6 and pepM24 have been found to induce blastogenic responses in foetal cord blood lymphocytes from unimmunized human volunteers 7° and later studies indicated that pepM5, pepM6 and p e p M l 9 could stimulate lymphocytes that are cytotoxic for cultured human heart cells 7~'72 Recently, it has been reported that pepM5 is a 'superantigen '~-~'7~, i.e. a potent, non-haplotyperestricted, M H C class ll-dependent T-cell mitogen, that induces the non-specific proliferation of T cells bearing characteristic Vfi families of T-cell receptors (TCR) vs. Since each Vfl family of TCR includes T cells with a wide range of conventional antigen specificities, superantigens can elicit the non-specific proliferation of a substantial proportion of the total T-cell population and this could have serious consequences for the host. Therefore, like HCR antibody epitopes, regions of M proteins that might be responsible for these mitogenic activities would need to be omitted from a vaccine. Recent studies in our laboratory, however, have shown that neither intact recombinant M5 protein (rM5; purified from Escherichia coli expressing a cloned M5 geneVO), nor pepsin-cleaved rM5 (pep-rM5), are mitogenic for non-immune human T cells, even though pepM5 purified from streptococcal cells elicited a clear proliferative response in parallel control experiments (J.A. Goodacre, M. Pinkney, E. Holliday, J.H. Robinson and M.A. Kehoe, unpublished data). This raises the possibility that the mitogenic activities associated with pepM antigens purified from streptococci might be due to contamination with small

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amounts of a distinct T-cell mitogen. Alternatively, it is possible that there are conformational differences between rM5 (and pep-rMS) produced from E. toll, and pepM5 produced from S. t)yo,qenes, which may account for the difference in their mitogenic activities. In either case, it seems likely that any potential problems associated with non-specific human T-cell activation can be avoided by constructing recombinant M-protein vaccines. Intact recombinant M proteins, however, can still elicit potentially harmful HCR antibodies. For many years, the ability of M protein antigens to evoke potentially harmful host reactions was one of the major obstacles that frustrated attempts to develop safe M-protein vaccines. This particular problem can now be solved by designing defined-epitope vaccines, containing protective M-protein sequences (i.e. type-specific opsonogenic epitopes) that have been well-characterized and demonstrated to be safe. Considerable progress in identifying such sequences in a number of distinct M protein serovars has been made over the past 10 years by studying the structure and immunochemistry of M proteins. M PROTEINS: STRUCTURE AND IDENTIFICATION OF B-CELL EPITOPES Early structural and immunochemical studies on M proteins were limited by the fact that most procedures used to extract M antigens from streptococcal cells yielded highly fragmented preparations, in which contaminating antigens could not be distinguished clearly from non-type-specific M antigens. A significant advance was made in the mid 1970s, when Edwin Beachey and his colleagues in Memphis found that treatment of type 24 streptococci with pepsin released the N-terminal half of the M24 protein, which protrudes outwards from the cell surface 7v. This pepsin-released N-terminal fragment was termed 'pepM24 antigen' and could be purified to sufficient homogeneity to allow the sequence of the N-terminal 29 amino acids to be determined 78. The purified antigen elicited type-specific, protective (opsonogenic) antibodies in both animals and humans, but did not elicit HCR antibodies 69. Initially, it was hoped that pepM antigens would be safe to use in a vaccine, but with subsequent studies which showed that pepM antigens from other serovars contain HCR epitopes (see above), it soon became clear that protective epitopes would need to be defined more precisely. During the first half of the 1980s increasing amounts of primary amino acid sequence data were reported for three distinct serovars of pepM antigens, namely pepM5, pepM6 and pepM2479 8'~. Despite having distinct primary sequences, all three serovars were found to possess a common seven-residue periodicity, which was consistent with physical studies suggesting that M proteins formed a-helical coiled-coil dimers, with the C-terminal ends of the molecules closely associated with the cell surface and the N-terminal ends protruding outwards from the cell ss. The a-helical coiled-coil structure can tolerate extensive amino acid substitutions and may be important in allowing highly variable M-protein primary sequences to possess similar functional properties, such as binding host fibrinogen (see above). It has also been suggested that the similarity between this structure and a-helical coiled-coil mammalian proteins might contribute to the common occurrence of

Group A streptococcal vaccines: M.A. Kehoe

divergent families or classes of M proteins, each possessing a distinct (though related) type of conserved C-terminal sequence 9°'94. These classes appear to correspond to the long established link between the ability to express serum opacity factor (OF) and particular serovars of M protein 95. Of the M genes sequenced to date, types 5, 6, 12 and 24 are from OF-negative strains (class I) and their C-terminal sequences share very high levels of sequence homology, whereas M49 is an OF-positive serovar (class II) and has a much more divergent C-terminal sequence. Although there appear to be two different classes of C-terminal sequences, the extreme ends of both contain a hydrophobic, putative membrane anchor domain, preceded by a proline-rich cell wall-spanning domain. Similar structures have been found at the extreme C-terminal ends of a variety of streptococcal and staphylococcal surface proteins and may reflect a common mechanism for attachment to the cell surface. Another structural feature that has also been found in a variety of Gram-positive bacterial surface proteins is the occurrence of tandemly repeated sequences. A single M protein can contain several distinct types of tandem repeats, though the numbers of distinct types and their

HCR epitopes within M proteins 31. With occasional discontinuities, the a-helical coiled-coil structure is now known to extend throughout most of the length of the intact M-protein molecule, with the exception of the extreme N-terminal ~ 10-20 residues which are usually non-helical and the cell wall-spanning domains at the extreme C-terminus (see below). The partial amino acid sequences of pepM5, pepM6 and pepM24 had suggested only limited primary sequence homologies, but subsequent sequencing of cloned M-protein genes revealed that M proteins contain both highly conserved and highly variable regions. To date, the nucleotide sequences of types 5, 6, 12, 24 and 49 M genes have been reported s6-9°, as well as the sequence of the 5' half of the M1 gene 91. DNA hybridization studies with cloned M-gene probes have allowed structural comparisons to be extended to a large number of other M serovars 92'93. While structural relationships between individual serovars differ in detail, in general the C-terminal halves of M proteins are highly conserved, but there is an increasing degree of sequence variation as one progresses up towards the N-terminal ends that protrude outwards from the cell surface (Fioure 2). Recent studies suggest that there are two

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Figure 2 Relationships between sequenced M-protein genes. The boxes labelled with letters correspond to repeated sequences. Note that distinct repeated sequences are designated A, B, C in order of their appearance in an M gene and, because different genes may contain a different number of distinct repeats, in some cases a B-repeat in one corresponds to the C-repeat in another, as in M24 and M5, respectively. Sequences that are conserved between serovars are represented by thick horizontal lines and the approximate levels of sequence homologies are indicated between adjacent pairs of M genes. Note that the C-terminal half of M49 is shorter and much more divergent than (though closely related to) the corresponding regions of other sequenced M types and may represent a divergent family (see text). The conserved signal peptides represented by the thick lines at the left of the diagram are removed during M-protein export through the cytoplasmic membrane. Thus the regions immediately to the right of the solid vertical line correspond to the extreme N-terminal ends of M protein on the cell surface. Pepsin cleaves close to the continuous dashed vertical line and conveniently divides M proteins into their highly conserved C-terminal halves and highly variable N-terminal halves, but note that some sequences in the latter pepM region can be shared by a limited range of M types (e.g. the B-repeat region in M5 and M6)

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Group A streptococcal vaccines. M.A. Kehoe

sizes varies between serovars (Fi.qure 2~. Recombination between copies of the same repeat can generate deletions or duplications, resulting in different strains of the same M type having different sized M proteins 9~'. Within a given set of repeats, the external (older) copies tend to be degenerate and recombination between such imperfect copies can generate new epitopes in repeated sequences 9v. This, however, does not explain the very extensive variation in important protective epitopes located in unrepeated sequences at the extreme N-terminal ends of M proteins and it is possible that extragenic recombination events involving genetic transfer between strains might contribute to antigenic variations in these unrepeated regions. This is suggested by the fact that, in addition to sequences encoding the highly conserved C-terminal halves of M proteins, sequences encoding the N-terminal signal peptides that are removed during protein export to the cell surface are also very highly conserved, even though functional constraints could allow considerable variation in these sequences. However, the mechanisms causing variation in unrepeated M-gene sequences are not understood. In parallel to structural studies, antibody epitopes in pepM antigens, and subsequently in intact M proteins, were localized by studying synthetic peptides corresponding to defined amino acid sequences. These were tested for their abilities to react in ELISAs with both polyclonal and monoclonal anti-M antibodies, to absorb type-specific, protective, and HCR antibodies from polyclonal anti-pepM sera, and to elicit such antibodies when used to immunize animals, usually after linkage to carriers such as K L H 5v 60.~,~ 6s.~8 115 Briefly, it was found that individual M proteins contain several distinct epitopes capable of eliciting opsonic antibodies. Most opsonogenic epitopes are specific for a single M type, but some anti-pepM sera were found to cross-opsonize a limited range of heterologous serovars. Some of these shared opsonogenic epitopes have been shown to elicit HCR antibodies and would need to be avoided in a vaccine, but studies with monoclonal anti-pepM5 antibodies detected others which are non-HRC. Although the M cross-opsonization was limited in serovar range, it was hoped that identification of shared, non-HCR opsonogenic epitopes would reduce the number of distinct epitopes that would be required in a multivalent M vaccine. However, the abilities of anti-pepMS, anti-pepM6 and anti-pepM19 sera to cross-opsonize heterologous M types in vitro is blocked in the presence of human fibrinogen, suggesting that the accessibility of these shared opsonogenic epitopes could be greatly restricted by fibrinogen binding to M protein in vivo ~16 This is consistent with ultrastructural studies, showing that fibrinogen binds to the N-terminal half of cell-surface M protein and may mask a considerable portion of the molecule ~7. A short N-terminal, typespecific segment of M protein remains exposed even in the presence of fibrinogen, and antibodies directed against these exposed regions are protective. The highly conserved C-terminal halves of M proteins contain epitopes that are much more widely shared between serovars than epitopes in the central regions. On the intact cell, a major part of these C-terminal sequences is masked by other cell wall components and is not accessible to antibody binding ~~8. lmmunoblotting experiments, however, have indicated that the proximal part of the conserved C repeat region of the M6 protein

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Vaccine, Vol. 9, November 1991

(Figure 2) protrudes beyond the cell wall and is accessible to antibody 11s. This contrasts with experiments employing whole-cell ELISAs, where intact types 5, 6 or 24 streptococci failed to react with antisera raised to a synthetic peptide corresponding to the most proximal C repeat sequences, even though the antisera reacted strongly with all three M antigens in immunoblots '~-~. Removal of the N-terminal half of M protein by pepsin allowed the anti-peptide antibodies to bind, suggesting that on the intact cell the corresponding epitope may normally be masked by the N-terminal half of the molecule <~. The reason for this apparent discrepancy is not clear. It may be that some C-repeat epitopes may be more accessible than others or that accessibility varies depending on the strain or density of M protein on the cell surface. One cannot, however, exclude the possibility that antibodies to some synthetic peptides may have a reduced affinity for the native molecule for conformational reasons or that immobilizing cells on filters for immunoblotting may increase the accessibility of normally masked epitopes. In either case, it has been demonstrated that antibodies to conserved C-terminal epitopes, including those that bind to whole cells in immunoblots, do not opsonize streptococci 4° and therefore these epitopes would be of no wdue in a vaccine designed to elicit effective, systemic protection. This is supported by studies on anti-M antibodies in human sera following natural streptococcal infections t ~ . Opsonic antibodies in these sera appear to be directed almost exclusively to the A repeats and upstream N-terminal unrepeated sequences (Fi,qure 2), while antibodies to the partially conserved central and highly conserved C-terminal regions of M proteins are predominantly non_opsonic 75,1 ~. The studies summarized above provide good evidence that epitopes with the greatest potential for use in an effective vaccine are most likely to be located in the N-terminal 10-20% of the M-protein molecule. Opsonogenic epitopes have been identified both in the extreme N-terminal non-helical sequences and in the closely adjacent ~-helical coiled-coil region. Selecting epitopes in the extreme N-terminal non-helical sequences could have an advantage in vaccine design, in that they might be less likely to elicit antibodies that cross-react with :z-helical coiled-coil human antigens. However, in types 1~ . 12~ and 19~° M proteins HCR epitopes have been located within 30 residues of the N-terminus, emphasizing the importance of defining and characterizing any selected epitope thoroughly. The potential for eliciting opsonic antibody to more than one serovar has been demonstrated in studies on multivalent synthetic peptides conjugated to carriers 1~1'113. For example, a trivalent synthetic peptide consisting of the N-terminal residues MS[1 10J-M6[1 11]-M24[1 12] has been shown to elicit rabbit antibodies that opsonize all three M types ~1_~.The maximum number of such epitopes that could be effectively included in a multivalent definedepitope vaccine has yet to be determined. To date, sequences of the variable N-terminal regions of the cell-surface (i.e. excluding the signal peptide that is removed during export to the cell surface) M proteins have been reported in the case of a relatively small number of distinct M serovars (types 1, 5, 6, 12, 19, 24 and 49) 6°'~' 9~. In order to facilitate vaccine design, these sequences will need to be determined for a much wider range of M types. Such studies are currently in progress

Group A streptococcal vaccines: M.A. Kehoe

in our laboratory, using PCR primers to conserved flanking sequences to isolate and sequence the variable regions for a wide range of M-protein genes (D. Sullivan, A. Whatmore and M.A. Kehoe, unpublished results). M PROTEIN: T-CELL EPITOPES Most short synthetic peptides fail to elicit an antibody response unless they are first conjugated to larger carrier molecules, which provide T-cell epitopes necessary to activate help for the antibody-producing B cells. The structural relationships between T- and B-cell epitopes required for an effective antibody response have not been well defined, but it is clear that they must be closely linked. Unless a synthetic peptide contains a T-cell epitope, the T-cell memory evoked by carrier-peptide immunogens will be carrier-specific and may not be recalled when the host encounters the 'natural' antigen during a subsequent infection. This was demonstrated almost 20 years ago by Mitchison's elegant haptencarrier experiments, which imply that the most effective immunogens are those that elicit both B-cell and T-cell memory to the challenge antigen 12°. Thus, in principle, the most effective safe M-protein vaccine should contain defined, non-HCR, opsonogenic B-cell epitopes, linked to M-protein T-cell epitopes that have been equally well characterized and shown not to elicit potentially harmful host reactions. To date, the ability of M antigens to elicit M protein-specific T-cell memory has not been examined in any detail. Lymphocytes from rabbits immunized with peptide-carner conjugates have been reported to proliferate in vitro in response to pepM antigens 99'113. These studies were limited, in that the lymphocytes failed to respond to the synthetic peptides (i.e. the only M sequences in the immunizing antigen). For example, pepM24-responsive lymphocytes from rabbits that had been immunized with a carrier-linked 12-residue M24 peptide, failed to respond to the 12-residue peptide or even to a longer M24 peptide containing an additional 17 and 6 residues, respectively, from either side of the 12-residue sequence. At the time it seemed reasonable to assume that peptides were simply too short to stimulate T cells in vitro. This is now more difficult to accept, given recent advances in our understanding of the nature and size of T-cell epitopes and observations that peptides of < 12 residues in length, and in some cases as little as eight residues long, can effectively stimulate antigen-specific T cells in vitro 121. Although suggestive, the pepM responses of lymphocytes from rabbits immunized with M synthetic peptides are now difficult to interpret and do not provide conclusive evidence concerning the locations of Mprotein T-cell epitopes. In order to define the relationships between T- and B-cell epitopes involved in antibody responses to M proteins, T-cell responses to intact recombinant type 5 M protein are currently being studied in our laboratory. Over 80 rM5-specific, class II-restricted, CD4 ÷ T-cell clones have been isolated from a number of different hapiotypes of rM5-immunized mice and the epitopes recognized by many of these clones have been mapped by dose-response proliferation assays with overlapping M5 synthetic peptides (J. Robinson, M. Atherton and M. Kehoe, unpublished data). These studies have identified multiple T-cell epitopes located throughout most of the length of the molecule, with the exception of

residues 1-20 corresponding to the extreme N-terminus of cell-surface M protein (i.e. minus the signal sequence). No region is strongly immunodominant, though a peptide corresponding to residues 20-35 is recognized by a larger than average number of clones. Thus, M protein appears to differ from most other antigens in which T-cell epitopes have been mapped systematically, in that it contains a larger number of T-cell epitopes of approximately similar 'strength', rather than a smaller number of epitopes displaying a clear hierarchy of immunodominance. The reasons for this are not understood, but it is interesting to note that the M5 protein is the first extensively s-helical coiled-coil antigen to have been subjected to detailed T-cell epitope mapping. It is possible that the a-helical coiled-coil structure has a tendency to include T-cell epitopes. This would be consistent with Bersofsky's T-cell epitope motifs 122, even though the predictive value of these motifs may be limited when applied to more globular proteins123. DEFINED-EPITOPE M-PROTEIN VACCINES: DESIGN AND DELIVERY The observation that M5 contains multiple T-cell epitopes, located in both the highly variable N-terminal and highly conserved C-terminal halves of the molecule, indicates that many of these epitopes will be shared with a wide range of other M types. The problems of accessibility or masking that may apply to antibody recognition of epitopes in the conserved C-terminal sequences are less likely to apply to T-cell epitopes, which are frequently 'buried' in internal regions of an antigen and exposed during processing by antigen-presenting cells. Given our current undersanding of antigen presentation to T cells, it is reasonable to hope that T-cell epitopes in the conserved C-terminal halves of M proteins can evoke T-cell help for the type-specific N-terminal B-cell epitopes. If so, then a single set of conserved C-terminal T-cell epitopes could prove to be the ideal carrier for multivalent M-protein vaccines, containing many distinct, type-specific, B-cell epitopes. It must be emphasized, however, that the relative locations of specific T- and B-cell epitopes in an antigen could, in principle, affect the ability of a specific B cell to process the antigen in a manner that allows it to present a particular T-cell epitope effectively to activated T cells. Thus, the ability of conserved M-protein T-cell epitopes to evoke effective help for N-terminal specific B cells will need to be tested by experiment. In addition, any potential 'T-cell epitope' carrier would need to be well characterized and demonstrated not to elicit potentially harmful host reactions. Experiments in small animal models should establish the basic principles for the design of effective defined-epitope vaccines, but these principles will need to be tested in primates and eventually in humans before an effective defined-epitope vaccine could be produced. Defined M-protein vaccines based on synthetic peptides would be expensive to produce and impose a limitation on host delivery systems. A potentially more useful approach is to construct recombinant vaccines, by linking DNA sequences encoding defined B-cell epitopes to sequences encoding 'carrier' T-cell epitopes, such that the resulting recombinant antigens could be produced in a bacterial expression system. This also offers the

Vaccine, Vol. 9, November 1991 803

Group A streptococcal vaccines. M.A. K e h o e

potential for delivering antigens by live oral delivery systems, such as attenuated Salmon,ella strains. We have already demonstrated that oral immunization of mice with aroA Salmom'lla l y p h i m m ' i m ~ expressing recombinant type 5 M protein evokes very effective, type-specific protection against intranasal challenge by Streptococcus pyogenes "s4 and we are currently exploring the design of defined-epitope recombinant M antigens. The major limitation is likely to be on the number of distinct type-specific B-cell epitopes that could be effectively included in such constructs. One could speculate that, with a better understanding of the structural basis for type-specific antibody-epitope recognition, it might eventually be possible to design novel 'mimetopes' that would reduce the number of distinct epitopes required in a vaccine. Although there is no evidence to support this highly speculative suggestion, it has been demonstrated that the sequential substitution of residues in an epitope can produce 'mimetopes' with a greatly improved affinity for monoclonal antibodies evoked by the parent epitope le4. Our sequencing studies on the type-specific regions of a wide range of M serovars have already identifed 'clusters' of structurally related sequences (D. Sullivan, A. Whatmore and M. Kehoe, unpublished data) and it would be interesting to explore the feasibility of representing particular clusters or related type-specific epitopes with a novel mimiotope. If possible, this might also reduce the potential problem of new serovars emerging in response to vaccination. Unless such a novel solution to the problem of type-specific immunity is found, one may be limited to designing vaccines targeted on a limited range of important M types, associated with either a particular disease or geographical location. Studies in recent years have solved a number of previously intractable problems and suggest that it would be feasible to design effective vaccines to protect against a limited range of M types. It may, however, be some time before an effective vaccine that could protect against a wide range of group A streptococcal M types will be produced, or even demonstrated to be feasible. I1 is clear that further studies are required to determine if the currently limited efficacy of non-type-specific immunogens could be improved and to explore possible solutions to limitations associated with designing multivalent, type-specific epitope vaccines. Even though all of the problems have not been solved, studies aimed at developing group A streptococcal vaccines have, over the decades, made significant contributions to our basic understanding of pathogenesis and immunity, and should continue to do so.

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Cunningham, M.W., McCormack, J.M., Fenderson, P.G., Ho, M.K., Beachey, E.H. and Dale, J.B. Human and murine antibodies cross-reactive with streptococcal M protein and myosin recognize the sequence GIn-Lys-Ser-Lys-GIn in M protein. J. Immunol. 1989, 14,3, 2677-2683 Bronze, M.S., Beachey, E.H. and Dale, J.B. Protective and heart cross-reactive epitopes located within the NH 2 terminus of type 19 streptococcal M protein. J. Exp. Med. 1988, 167, 1849-1859 Bisno, A.L., Berrios, X., Quesney, F., Monroe, D.M., Dale, J.B. and Beachey, E.H. Type-specific antibodies to structurally defined fragments of streptococcal M proteins in patients with acute rheumatic fever. Infect. Immun. 1982, :3~, 573-579 Goroncy-Bermes, P., Dale, J.B., Beachey, E.H. and Opferkuch, W. Monoclonal antibody to human renal glomeruli cross-reacts with streptococcal M protein. Infect. Immun. 1987, ~5, 2416-2419 Kraus, W. and Beachey, E.H. Renal autoimmune epitope of group A streptococci specified by M protein tetrapeptide Ile-Arg-Leu-Arg. Proc. Natl Acad. Sci. USA 1988, I~, 4516-4520 Kraus, W., Seyer, J.M. and Beachey, E.H. Vimentin-cross-reactive epitope of type 12 streptococcal M protein. Infect. Immun. 1989, 57, 2457~461 Kraus, W., Ohyama, K., Snyder, S. and Beachey, E.H. Autoimmune sequence of streptococcal M protein shared with the intermediate filament protein, vimentin. J. Exp. Med. 1989, 169, 481-492 Zabriskie, J.B. and Freimer, E.H An immunological relationship between the group A streptococcus and mammalian muscle. J. Exp. Med. 1966, 124, 661478 Van de Rijn, I., Zabriskie, J.B. and McCarty, M. Group A streptococcal antigens cross-reactive with myocardium. Purification of heart-reactive antibody and isolation and characterization of the streptococcal antigen. J. Exp. Med. 1977, 146, 579-599 Barnett, L.A. and Cunningham, M.W. A new heart cross-reactive antigen in Streptococcus pyogenes is not M protein. J. Infect. Dis. 1990, 162, 876-882 Beachey, E.H., Stollerman, G.H., Johnson, R.H., Ofek, I. and Bisno, A.L. Human immune response to immunization with a structurally defined polypeptide fragment of streptococcal M protein. J. Exp. Med. 1979, 156, 862-877 Dale, J.B., Simpson, A.W., Ofek, I. and Beachey, E.H. Blastogenic responses of human lymphocytes to structurally defined polypeptide fragments of streptococcal M protein. J. Immunol. 1981, 126, 14991505 Dale, J.B. and Beachey, E.H. Human cytotoxic T lymphocytes evoked by group A streptococcal M proteins. J. Exp. Med. 1987, 166, 1825-1835 Kotb, M., Courtney, H.S., Dale, J.B. and Beachey, E.H. Cellular and biochemical responses of human T lymphocytes stimulated with streptococcal M proteins. J. Immunol. 1989, 142, 986-970 Tomai, M., Kotb, M., Majumdar, G. and Beachey, E.H. Superantigenic properties of streptococcal M protein. J. Exp. Med. 1990, 172, 359-362 Kotb, M., Majumbdar, G., Tomai, M. and Beachey, E.H. Accessory cell-independent stimulation of human T cells by streptococcal M protein superantigen. J. Immunol. 1990, 145, 1332-1336 Marrack, P. and Kappler, J.W. The staphylococcal enterotoxins and their relatives. Science 1990, 246, 705-711 Kehoe, M.A., Poirier, T.P., Beachey, E.H. and Timmis, K.N. Cloning and genetic analysis of serotype 5 M protein determinant of group A streptococci: evidence for multiple copies of the M5 determinant in the Streptococcus pyogenes genome. Infect. Immune. 1985, 48, 190-197 Beachey, E.H., Campbell, G.L. and Ofek, I. Peptic digestion of streptococcal M protein. II. Extraction of M antigen from group A streptococci with pepsin. Infect. Immun. 1974, 9, 891~896 Beachey, E.H., Stollerman, G.H., Chiang, E.Y., Chiang, T.M., Seyer, J.M. and Kang, A.H. Purification and properties of M protein extracted from group A streptococci with pepsin: covalent structure of the amino terminal region of type 24 M antigen. J. Exp. Med. 1977, 145, 1459-1483 Beachey, E.H., Seyer, J.M and Kang, A.H. Repeating covalent structure of streptococcal M protein. Proc. Natl Acad. Sci. USA 1978, 75, 3163-3167 Beachey, E.H., Seyer, J.M. and Kang, A.H. Studies of the primary structure of streptococcal M protein antigens. In: Streptococcal Diseases and the Immune Response (Eds Read, S.E. and Zabriskie, J.B.). Academic Press, New York, 1980, pp 149-160 Seyer, J.M., Kang, A.H. and Beachey, E.H. Primary structural similarities between types 5 and 24 M proteins of Streptococcus pyogenes. Biochem. Biophys. Res. Commun. 1980, 9~), 546-553 Manjula, B.N. and Fischetti, V.A. Studies on group A streptococcal M proteins: purification of type 5 M protein and comparison of its

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