Group A Streptococcus: Challenges in multivalent M protein amino terminal based vaccine development in India and elsewhere

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1 Group A Streptococcus: Challenges in multivalent M protein amino terminal based vaccine development in India and elsewhere Meenakshi Sharma*, PhD; N K Ganguly, MD, DSc (hc) * Indian Council of Medical Research, New Delhi, India President, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, India Distinguished Biotechnology Research Professor, Dept. of Biotechnology, New Delhi, India Adviser, Sir Ganga Ram Hospital, New Delhi, India Abstract Primary prevention of rheumatic fever (RF) and rheumatic heart disease (RHD) through penicillin prophylaxis in Group A beta hemolytic streptococcus (GAS) pharyngitis cases in a population is difficult. This leaves a space for development of an anti-streptococcal vaccine which is safe, efficacious against variety of GAS strains and is affordable. However, despite widespread efforts, the only vaccine against GAS which has entered clinical trials in the last few decades is multivalent M protein s Amino (N) terminal based vaccine. As protection conferred by this multivalent vaccine is emm type specific and heterogeneity of GAS strains circulating within the same country and globally has been reported, this N terminal based vaccine will not be globally effective. Moreover, a very rapid and complete shift in the predominance of GAS serotype in a short span of time has been documented. It is possible that a lack of elicited secondary host immune response to primary infection by heterogenous GAS strains creates a scenario where probability of multiple onslaughts on heart tissue become more, resulting in development of RHD related complications in susceptible hosts. In this scenario, evolving a typing scheme which groups the more than 250 emm types of GAS into smaller number of types may reduce the number of GAS strain types to be targeted by antistreptococcal vaccine. Till the time a vaccine with broad efficacy against GAS infections is available, public health efforts in developing countries will require to be focused around secondary prevention of recurrent attacks of RF. Key Words Group A beta hemolytic streptococcus (GAS) Pharyngitis Rheumatic fever (RF) Rheumatic heart disease (RHD) M-protein M-protein s Amino (N) terminal based vaccine Introduction Streptococcus pyogenes or group A beta hemolytic streptococcus (GAS) has a remarkable ability to adapt to a variety of human tissues and produce a wide range of clinical manifestations with varying severity. The organism spreads rapidly through droplets and contact from one person to another. Through its virulence factors, GAS produces diseases ranging from noninvasive mild infections like pharyngitis, and impetigo to invasive, life threatening conditions like bacteremia, pneumonia, necrotizing fasciitis and streptococcal toxic shock syndrome (STSS). The autoimmune nonsuppurative sequelae of this bacterial pathogen include acute rheumatic fever (RF), rheumatic heart disease (RHD) and poststreptococcal glomerulonephritis (PSGN). It is not yet clear how various virulence factors interact with variety of human tissues to produce such diverse variety of diseases. It is likely that the organism is able to sense its immediate environment and accordingly modulate expression of 1 various virulence genes in different tissues. This view is supported by strain specific expression of regulatory Received: ; Revised: ; Accepted: Disclosures: This article has not received any funding and has no vested commercial interest Acknowledgements: None 276

2 Group A Streptococcus 2 networks. Ironically, this pathogen also has an ability to persist at the pharyngeal mucosa and tonsils without manifesting any active disease (often called carrier state) even following an intensive antibiotic course. In fact, recurrent GAS infections in children allow its entry into tonsillar epithelial cells, thereby escaping the human 3 immune system. These cells then act as a reservoir for recurrent infections, thereby increasing the risk of autoimmune diseases and sepsis in a subset of GAS pharyngitis patients. Carapetis et al. estimated that around 18.1 million cases of severe GAS infections (acute RF, RHD, PSGN, and invasive infections) resulting in over 500,000 deaths 4 occurred in 2005 worldwide. Of these GAS infections, RHD accounted for the major burden with about 15.6 million cases worldwide, 282,000 new cases annually and 233,000 deaths each year. These estimates included very few studies from Asia. Subsequently, it was estimated that 1.96 to 2.21 million cases of RHD occur in the 5 14-yearsage group and 10.8 to 15.9 million cases exist in all ages in 5 Asia alone. As a subset of 35 40% of RF patients convert to RHD, therefore the burden of RF is estimated to be 6 around 3 times higher. Neither the pathogenesis of the GAS caused autoimmune diseases nor is the mechanism by which GAS breaches the epidermal barrier and enters the bloodstream resulting in sepsis well understood. This lack of knowledge of the molecular and cellular basis of GAS infection sequelae has hindered the development of an efficacious and safe GAS vaccine, which can prevent either initial colonization of the host or interrupt the crucial pathways responsible for development of GAS autoimmune and septic sequelae. However, current advances in technology have led to sequencing of 13 isolates of GAS representing serotypes 2 M1, M2, M3, M4, M5, M6, M12, M18, M28 and M49. Followed by functional genomic and proteomic approaches in GAS research, the knowledge about the molecular pathways which lead to variations in phenotypic expression in GAS strains has been enhanced. As a result, several potential GAS vaccine candidates have been identified. However, the best studied protein eliciting protection against GAS infection is the M protein and the only vaccine which has entered clinical trials in the last few decades is multivalent N-terminal based vaccine. In this review, we discuss the need for GAS vaccine, progress in development of vaccine with major emphasis on multivalent M protein amino terminal based GAS vaccine and what current public health control measures against GAS infections need to be targeted, currently, in developing countries like India. Why vaccine against GAS is required? Currently, the primary prevention of RF involves treatment of GAS infections and secondary prevention involves prevention of colonization of pharynx with GAS and recurrent attacks of RF. Both primary and secondary prevention use penicillin prophylaxis and GAS resistant to 7 penicillin has so far not been documented. Further these preventive measures have been shown to be cost effective as compared to surgical interventions requiring surgical valve management in RHD in a developing country like 8 India. However, primary prevention of RF/RHD through penicillin prophylaxis in all GAS pharyngitis cases is difficult from public health view point as described in Figure 1. Differential diagnosis of GAS from viral pharyngitis is not possible clinically and requires GAS identification through bacterial culture and other laboratory tests. This requirement of vast laboratory infrastructure and trained manpower to cover the entire country of the size of India, for identification of GAS sore throats through culture, is a formidable task, both cost and planning wise. Additionally, there are also concerns regarding development of new antimicrobial resistance following widespread usage of antibiotic for treatment of GAS sore throats. Further, penicillin treatment failure in 15 streptococcus tonsillopharyngitis has been documented. Though the exact reasons of treatment failure are not known, noncompliance is considered to be a major reason making primary prevention through penicillin prophylaxis challenging from public health perspective. Other clinical reasons for treatment failure include presence of recurrent exposures and carrier state. From mechanistic point of view, it is possible that treatment failure may be due to GAS strains evading penicillin eradication through intracellular localization, coexistence of these strains with lactamase producing normal flora in vivo, poor penetration of the antibiotic into infected vegetations or varying response to antibiotic within the bacterial vegetation. Keeping the limitation of primary prophylaxis in view, it is evident that an anti-streptococcal vaccine can be a potent tool for primary prevention of RF. However, despite widespread efforts, the only vaccine against GAS which has entered clinical trials in the last few decades is multivalent M protein s Amino (N) terminal based vaccine. The advances and limitations in this vaccine are discussed below. Multivalent M protein amino terminal based vaccine M protein is the most extensively studied GAS virulence factor. The hypervariable N terminal region of the M 277

3 Sharma M, et al 9 A. Only 26% of pharyngitis are GAS pharyngitis, with a majority being viral in origin. Differential diagnosis of GAS pharyngitis from that of other origins is difficult, therefore leading to unnecessary medication Accurate Diagnosis Requires Culture Backup/ Rapid Antigen Detection Test (RADT) Neither culture nor RADT differentiate individuals with bona fide GAS pharyngitis from GAS carriers B U T Facilitate the with holding of antibiotics from culture or RADT negative sore throat patients Important as 15% of school-age children may be asymptomatic GAS carriers Clinical findings suggestive of GAS Pharyngitis INCREASES the positive predictive value of Culture or RADT Requires laboratory infrastructure trained manpower 13,14 B. Treatment of large number of GAS pharyngitis required as only 0.3 3% convert to RF C. Not all patient s with sore throat come for clinical consultation D. Asymptomatic individual s (Carrier State) do not seek physician s advice. E. Patient s noncompliance to penicillin prophylaxis Figure 1 What makes primary prevention of RF RHD through penicillin prophylaxis difficult from public health view point? protein is followed by A, B and C repeat regions that lead 16 into D-region spanning the cell membrane. A and B repeat regions are variable between different GAS strains, whereas the C repeat region is conserved in 98% of the strains. The antiphagocytic ability of M protein is due to its ability to block deposition of C3b deposition on bacterial surface and binding to host fibrinogen and immunoglobulins, thereby dampening the primary host 17 defense mechanisms against this organism. The fibrillar coiled coil structure of M protein and hypervariability in its N terminal region is advantageous to the organism in providing antigenic variations and multifunctional 18 domains for evading host antibody attack. Serological cross reaction between host and bacterial proteins is also conferred due to the resemblance between the molecular structure of M protein and host proteins, such as cardiac 18,19 myosin, tropomyosin, and keratin. The antiphagocytic surface antigen, M protein, encoded by emm gene has been used in GAS serotyping scheme 20 developed by Lancefield in There are 83 GAS M serotypes and many of the GAS isolates are non-m 21 serotypable. As many of the GAS strains are non-m 21 serotypable, alternative methods of M protein serotyping have also been developed. These schemes are based on T antigen, a pilus structure associated with adherence to 278 human epithelial cells and forming biofilm, and detection 22 of streptococcal serum opacity factor (SOF). Using T serotyping scheme, 20 T serotypes have been identified. Further, reaction or non-reaction of C repeat regions of M protein with specific antibodies is used to differentiate M 23 protein into class I and class II, respectively. GAS isolates with Class I M proteins occur in four different architectural patterns ( emm patterns A, B, C, and D) of chromosomal region encoding emm and emm like genes (such as enn, mrp, fcra), whereas those with class II M protein have 24,25 predominantly pattern E. The emm patterns A, B and C are associated with pharyngeal infections, D with skin infections and E with both pharyngeal and skin 24,25 infections. Currently, emm typing is done to identify the GAS serotype by a PCR based sequencing method, which identifies the N terminal variable region of the M protein. Further, small alterations in the sequence encoding the first 50 amino acids of mature M protein can sometimes make the protein serologically different from the M serotype reference strain. CDC now ascribes a subtype to a strain, if there are any variations in the 150 bases encoding the first N terminal amino acids of M protein. More than 250 emm types and 800 subtypes have been currently described 18,27 and more types/subtypes are being added. It is likely that such a large number of emm types and subtypes is the result of adaptation of this pathogen to human host.

4 Group A Streptococcus Epidemiology of GAS types The epidemiology of GAS disease is different in different regions of the world. Though, there are strong evidences supporting the hypothesis regarding development of RF followed by GAS pharyngitis, this hypothesis is challenged in tropical countries where incidence of GAS impetigo is higher. The aboriginal communities of Australia have one of the highest prevalence rates of RHD with low incidence rates of GAS pharyngitis. In India, 28 GAS impetigo is uncommon in Northern parts but is more common (6.9 per 100 children) in the tropical climate of Southern India. A role of GAS impetigo in the pathogenesis 29,30 of RHD has been hypothesized, but proof of concept is required through well-structured research. Moreover, high degree of diversity has been observed in the stains circulating in different geographical regions. Steer et al. (2009) in their review of global distribution of GAS emm types concluded that the distribution patterns of GAS emm types was different in Africa and Pacific region as compared to other regions, particularly established market 31 economy countries. In India, although number of studies have reported a high 28,32 37 degree of heterogeneity in the GAS emm types, only one study describes the comparative distribution of emm types in throat and skin isolates collected during the same 32 period in North and South Indian pediatric population. GAS strains (n= 298) were collected from throat and skin swabs of children with and without pharyngitis/impetigo during cross sectional surveys conducted in 8026 school children (around 4000 each by two centers) in the age group of 5 14 years at Raipur Rani, Haryana in North India and Kaniyambadi block, Vellore in South India in year to Sixteen different emm types from North Indian and 59 emm types from South India were identified in this short span of 24 months indicating a high degree of heterogeneity strains circulating in the community. Interestingly, the most frequently isolated emm types 77 (29.6%), emm 81 (25.4%), emm 11 (14.1%), emm 71 and 44 (5.6% each) and emm 69 (4.2%) in North India accounted for 84.5% of all the isolates, whereas in South India emm 112 (12.3%), emm 82 (5.3%), emm 11 (4.8%), emm 105 (4.0%) and emm 108 and 110 (3.5% each) accounted for only 33.4% of all the isolates. Among the predominant emm types from Northern and Southern India, 37 only emm type 11 was common. Recently, Dhanda et al. from the North Indian compiled the epidemiological surveillance data of GAS emm types collected by them from year 1995 to The study suggested that at least 70 GAS emm types circulated in and around Chandigarh (North India) from of which 53 were known emm types with 33 subtypes, 12 sequence-types and five novel MNT strains. The seven most prevalent emm types in this region were emm 81 (15.7%), 11 (7.9%), 112 (7.6%), 77 (7.4%), 44 (4.4%), 49 (3.7%) and 15 (3.4%) and constituted 50% of the isolates. Importantly, as shown in Table 1, the predominant GAS emm types varied from year to year in this small geographical region. The findings from India are similar to a community based study by Kaplan et al. where a very rapid and complete shift in the predominance of GAS serotype from M1 (92% of all isolated strains) to M6 (84% of all isolated strains) was 38 observed in a short span of time. Also, an individual can become infected with more than one GAS type during his 39 lifetime, thus the organism has a dynamic epidemiology. The epidemiological data also suggests that the development of RF and RHD, following GAS pharyngitis, may be result of reinfections with different GAS emm types in susceptible individual. We suggest that a lack of elicited secondary host immune response to primary infection by heterogenous GAS strains creates a scenario where probability of multiple onslaughts on heart tissue become more, resulting in development of RHD related complications in susceptible hosts. The resemblance of epitopes of M protein with several of cardiac proteins further makes the pathogenesis of this disease complex. Our hypothesis is supported by the fact that: 1. Animals immunized with M protein or its fragments elicit an immune response which is specific for the homologous strains only; GAS infection in humans also induces, type specific antibody response providing protection against reinfections by the same 18,19 serotype 2. GAS emm types keep on varying even in small regions studied till date in very short intervals of time providing greater chances of reinfection by GAS strain 37,38 different from the previous one 3. Difficulties in development of animal model which may be because of requirement of onslaught of different GAS emm types in a susceptible host by providing multiple GAS protein epitopes for development of RHD lesions; prospective population based studies will be required to test this hypothesis It is thus evident that the presence of wide variety of GAS emm types, regional distribution patterns and replacement of GAS emm types within short time span will pose several challenges in developing a GAS vaccine with proven efficacy against different strains. 279

5 Sharma M, et al Study area Peri urban slums near Chandigarh Chandigarh hospital and villages of district Panchkula, Haryana Raipur Rani, Panchkula, Haryana Slums, rural and urban areas of Chandigarh Rural areas of Haryana Rural areas of Punjab Table 1: Yearly variations in the most prevalent emm type isolated from 38 areas in and around Chandigarh, North India Year Number of isolates (number of different emm types) emm types - emm 49 and (33) emm (17) 40 (23) 139 (27) - emm 77 and emm 81 emm 49 emm 81 and emm 112 emm 57 Progress and challenges in designing amino (N) terminal of M protein based vaccine The host immune response against N terminal hypervariable region of M protein is elicited in a type 40 specific manner, making it a potential vaccine candidate. A recombinant multivalent vaccine containing N terminal peptides of M protein initially from four different GAS serotypes ( emm type 24, 5, 6 and 19), followed by six and subsequently 26 different GAS serotypes was developed by Dales group. The selection of GAS serotypes in this multivalent vaccine was based on: 1. The most common emm types isolated from pediatric cases of GAS pharyngitis in USA between the fall of 2000 and spring of emm types that were isolated most frequently from cases of invasive diseases between The ones which were currently ( emm 18 and 5) or historically ( emm 19 and 24) considered to be 43 associated with Rf An amino terminal peptide fragment of Spa, an antigen expressed by several serotypes, was also incorporated into the multivalent vaccine. The inclusion of amino acid sequence of M protein and Spa antigen was based on the absence of matching of more than five contiguous amino acids in the amino terminal of protein with any human protein. This 26 valent vaccine elicited a strong immunological response against the 25 of the 26 GAS serotypes as well as against emm subtypes following 43,44 intramuscular immunization in rabbits. Meanwhile, intranasal immunization of mice with hexavalent GAS M protein based vaccine also induced protective antibody response. The safety concerns for 26 valent vaccine delivered intramuscularly were further discerned by the inability of antisera to cross react with human tissues. Successful Phase I and Phase II clinical trials of this 46 multivalent vaccine were carried out in This is the most advanced vaccine against GAS infections. The major drawback for M protein N terminal based vaccine is the heterogenic distribution patterns of GAS emm types in different geographical regions as discussed 31 previously. A comparison of frequency distribution of GAS emm types (from all types of GAS diseases) represented in Dale s multivalent vaccine in six different global regions is shown in Figure Established market economy countries Pacific Island/ Australia Middle East Latin America Asia Africa emm type Figure 2 Comparison of frequency distribution of GAS emm types (from all types of GAS diseases) which are also present in multivalent vaccine from six different global regions (data from Steer et al CDC website) As is clear, the multivalent vaccine will be of limited use in Asia and the Middle East and of poor coverage in Africa

6 Group A Streptococcus 31 and Pacific region. Region specific N terminal based multivalent vaccine are also not feasible as the differences between GAS emm types are not restricted to six geographical areas. The diversity of GAS emm typing data from India indicates that a multivalent vaccine based on N terminal region of M protein of prevalent emm types will provide poor coverage in North and South of India. Further, the cumulative data from North India from a period 37 between 1995 to 2007 indicates that even a region specific M protein N terminal based vaccine is not going to be useful as the prevalent GAS emm types keep on varying even in a very small region over a period of time. The quest for M protein N terminal based vaccine is however yet not over. Recently, a new 30 valent vaccine, containing N terminal M protein peptides from GAS serotypes prevalent in North America and Europe, has raised hopes as it was observed to have higher efficacy against different GAS serotypes than could be predicted by the type specific M peptides present in it. The antisera against this 30 valent were found to be effective not only against 30 vaccine GAS serotypes in rabbit model but also 47 against 24 of 40 tested non-vaccine serotypes. This study raises the possibility of developing an N terminal of M protein based vaccine with a broad efficacy against a wide range of GAS emm types. Though this task may appear to be herculean task at present, it may be possible to achieve this target through development of a typing system which groups the GAS M types to smaller number of types. Falugi et al. in 2008 proposed a tee gene sequence typing, a tool to substitute serological T typing system in a manner similar to which emm gene typing has been used for substituting 48 serological M typing scheme. Using this tool, it was established that the 12 predominant tee types in Eurpoe and USA could provide protection against 90% of the circulating GAS strains. Development of such typing procedures, which can club the emm types into smaller number of groups, hold a promise for development of multivalent GAS vaccine with broader efficacy. Efforts would be required to undertake concerted research efforts to reduce the time for developing a system of clustering different emm types and then testing a model multivalent N terminal of M protein based vaccine to be efficacious against the emm types. A public health approach to prevent and control RF RHD in absence of vaccine in developing countries Secondary prophylaxis involving administration of benzathine penicillin G injections every 2 3 weeks for years together in patients with history of RF to prevent recurrent episodes, through RF RHD registries, is cost effective and practical in developing countries like India. Such a RF RHD registry based program will require a continuous supply of benzathine penicillin in all Indian States. The shortage of benzathine penicillin in the market, policies in states like Tamil Nadu where use of injectable penicillin is prohibited and fear of allergic reaction to benzathine penicillin contribute to inadequate management of RF/RHD patients. To achieve greater compliance to benzathine penicillin, it is imperative to discern the fear of allergic reactions through systemic advocacy among all stakeholders of the fact that allergic 10 reaction to penicillin is rare in children and occurs only in a small percentage of individuals. In fact, allergic reactions can be circumvented by obtaining careful history regarding allergic reaction to penicillin and administration of the injection by medical practitioner only. Conclusion In conclusion, prevention and control of RF RHD in developing countries like India will require development of vaccine with proven efficacy against GAS strains circulating in the country. The developing countries will be required to enhance the research potential in this area so that a vaccine as per the local needs could be developed. Meanwhile, prevention and control methods through primary prophylaxis for treatment of GAS pharyngitis and secondary prophylaxis for RF prevention are available. 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8 Group A Streptococcus 46. Steer AC, Batzloff MR, Mulholland K, Carapetis JR. Group A streptococcal vaccines: facts versus fantasy. Curr Opin Infect Dis. 2009;22(6): Dale JB, Penfound TA, Chiang EY, Walton WJ. New 30-valent M protein-based vaccine evokes cross-opsonic antibodies against non-vaccine serotypes of group A streptococci. Vaccine. 2011;29(46): Falugi F, Zingaretti C, Pinto V, Mariani M, Amodeo L, Manetti AG, et al. Sequence variation in group A Streptococcus pili and association of pilus backbone types with lancefield T serotypes. J Infect Dis. 2008;198 (12): Address for correspondence Dr. Meenakshi Sharma: smeenakshi@hotmail.com 283