Evaluation of a Group A Streptococcus synthetic oligosaccharide as vaccine candidate
Introduction
Group A Streptococcus (GAS) infections represent a significant healthcare concern throughout the world. GAS is responsible for a broad spectrum of diseases ranging from asymptomatic colonization, uncomplicated pharyngeal and skin infections, to life-threatening invasive illnesses including sepsis, necrotizing fasciitis and toxic shock syndrome [1]. Pharyngitis may lead to delayed sequelae as rheumatic fever. In developing countries rheumatic fever remains endemic and can be followed by rheumatic heart disease, causing hundreds of thousands of deaths every year [2]. Currently, no vaccine to prevent GAS infections exists although GAS has been on the WHO priority prevention list for decades.
The diversity of GAS strains is the major challenge for the development of an anti-GAS vaccine, and therefore the identification of a common protective antigen is highly desirable. GAS bacteria contain a surface polysaccharide consisting of repeating [→3)α-l-Rhap(1→2)[β-d-GlcpNAc(1→3)]α-l-Rhap(1−]n units (Fig. 1a). These long polysaccharide chains adopt a helical conformation, where the rhamnoses form the helix core and the immunodominant N-acetylglucosamine residues are exposed on the periphery [3], [4], [5]. The GAS-polysaccharide (GAS-PS) is conserved and abundantly expressed in the vast majority of M+ GAS strain serotypes [6], [7], [8]. This feature has been used to develop the “Rapid Strep Test”, which detects the presence of the Group A Streptococcus carbohydrate antigen in the throat of infected patients.
Human anti-GAS-PS antibodies have been associated with protection against GAS throat colonization and promote bacterial phagocytosis [9], [10]. Accordingly, GAS-PS has been considered attractive for the development of an anti-GAS conjugate vaccine. Purified GAS-polysaccharide has been conjugated to tetanus toxoid carrier and elicited a protective immune response in a mouse challenge model [9]. Efforts to determine the antibody-binding epitope of GAS-PS revealed a hexamer structure of two repeating units as core antigenic determinant which is believed to be recognized by human anti-GAS humoral immune response [11]. To date it has not been established whether a minimal GAS-PS core antigen determinant can elicit an immunoprotective response.
In contrast to conventional polysaccharide isolation, chemical synthesis provides access to pure, homogeneous oligosaccharides of well-defined structure that contain single reactive groups for covalent conjugation. Access to pure synthetic oligosaccharides is of particular value as the isolated GAS-PS may potentially contain minor contaminants like M protein or hyaluronic acid capsular polysaccharide, the former associated with pathologic cross-reactive autoimmune response and the latter representing a self-antigen [12], [13], [14]. Recent progress in the chemical synthesis of oligosaccharides associated with introduction of automated systems facilitates the implementation of a synthetic vaccine approach at industrial scale, avoiding fermentation of pathogenic bacteria [15].
Here, we compared the in vivo efficacy of carbohydrate–protein conjugates containing synthetic and natural PS isolated from GAS. A set of synthetic oligosaccharides was used to study the effect of carbohydrate length and composition on immunogenicity [11], [16], [17], [18]. Conjugates with synthetic oligosaccharides and purified polysaccharide provided comparable levels of protection against bacterial challenge and promotion of serum phagocytic activity.
Section snippets
Materials
Synthetic GAS oligosaccharides 1–4, equipped with an amino-terminated two-carbon linker, were synthesized by Ancora Pharmaceuticals (Medford, USA). Final compounds were purified by size exclusion chromatography (P-4 Bio-Gel, Pharmacia XK26 × 100 cm bed, pH 3.9, H2O). Fractions with greater than 90% purity by HPLC were combined and lyophilized to give the final material as the hydrochloride salt. Each oligosaccharide was characterized by 1H NMR, MALDI-MS, elemental analysis, HPLC-ELSD (Supplemental
Design and synthesis of GAS oligosaccharides
Oligosaccharides 1–4 (Fig. 1b) were designed to evaluate the influence of antigen length and sequence on immunogenicity and immunoprotection in vivo. Based on the previously described immunodominant hexamer epitope [11], [27], two hexamers and two dodecamers were designed and synthesized with each pair differing at their non-reducing termini. An amine-terminated two-carbon linker was incorporated at the reducing end of each oligosaccharide to accommodate a variety of spacers and conjugation
Discussion
The presence of anti-GAS-PS antibodies in human sera has been evidenced since 1965 [37], [38]. Subsequently it was demonstrated that the level of human GAS-PS-specific antibodies peaks at the age of 17 in correspondence to the reduced incidence of GAS infection [39], [40], and that these antibodies might play a significant role in the protection from GAS infection [10]. Sabharwal et al. [9] showed that colonization of GAS in the human throats inversely correlated with the levels of anti-GAS-PS
Acknowledgements
A.K. is the recipient of a Novartis fellowship from the Ph.D. program in Cellular, Molecular and Industrial Biology of the University of Bologna. We want to thank the Novartis Animal Resources Center for conduction of animal studies and Claudia Magagnoli from the Technology Development Department of Novartis Vaccines and Diagnostics, Siena, Italy for her support on SEC-MALLS analysis.
References (60)
- et al.
Bivalency and epitope specificity of a high-affinity IgG3 monoclonal antibody to the Streptococcus group A carbohydrate antigen. Molecular modeling of a Fv fragment
Carbohydrate Research
(2000) - et al.
Comparative in silico analysis of two vaccine candidates for group A Streptococcus predicts that they both may have similar safety profiles
Vaccine
(2007) - et al.
Immunochemical characterization of polyclonal and monoclonal Streptococcus group A antibodies by chemically defined glycoconjugates and synthetic oligosaccharides
Carbohydrate Research
(1992) - et al.
Ultraviolet spectrophotometric determination of hexoses, pentoses, and uronic acids after their reactions with concentrated sulfuric acid
Analytical Biochemistry
(1967) - et al.
A spectrophotometric assay for soluble and immobilized N-hydroxysuccinimide esters
Analytical Biochemistry
(1982) - et al.
Correlation between the avidity of mouse-human chimeric IgG subclass monoclonal antibodies measured by solid-phase elution ELISA and biospecific interaction analysis (BIA)
Journal of Immunological Methods
(1997) - et al.
Assay of reducing sugars in the nanomole range with 2,2′-bicinchoninate
Analytical Biochemistry
(1987) - et al.
Preparative purification of soybean agglutinin by affinity chromatography and its immobilization for polysaccharide isolation
Journal of Chromatography
(2003) - et al.
Chemistry of a new investigational quadrivalent meningococcal conjugate vaccine that is immunogenic at all ages
Vaccine
(2009) - et al.
Antibody-mediated neuronal cell signaling in behavior and movement disorders
Journal of Neuroimmunology
(2006)
Immunogens related to the synthetic tetrasaccharide side chain of the Bacillus anthracis exosporium
Bioorganic & Medicinal Chemistry
The pathogenesis of streptococcal infections: from tooth decay to meningitis
Nature Reviews
Saturation transfer difference 1D-TOCSY experiments to map the topography of oligosaccharides recognized by a monoclonal antibody directed against the cell-wall polysaccharide of group A Streptococcus
Journal of the American Chemical Society
The use of streptococcal antigens to probe the mechanisms of immunity
Microbiology and Immunology
Pathogenesis of group A streptococcal infections
Clinical Microbiology Reviews
A serological differentiation of human and other groups of hemolytic Streptococci
The Journal of Experimental Medicine
Variation in the group-specific carbohydrate of group A Streptococci. I. Immunochemical studies on the carbohydrates of variant strains
The Journal of Experimental Medicine
Group A Streptococcus (GAS) carbohydrate as an immunogen for protection against GAS infection
The Journal of Infectious Diseases
Group A Streptococcus-liposome ELISA antibody titers to group A polysaccharide and opsonophagocytic capabilities of the antibodies
The Journal of Infectious Diseases
Doubly branched hexasaccharide epitope on the cell wall polysaccharide of group A Streptococci recognized by human and rabbit antisera
Infection and Immunity
The importance of the Group A Streptococcus capsule in the pathogenesis of human infections: a historical perspective
Clinical Infectious Diseases
Reactivity of rheumatic fever and scarlet fever patients’ sera with group A streptococcal M protein, cardiac myosin, and cardiac tropomyosin: a retrospective study
Infection and Immunity
Automated solid-phase synthesis of oligosaccharides
Science
Identification of the smallest structure capable of evoking opsonophagocytic antibodies against Streptococcus pneumoniae type 14
Infection and Immunity
Effect of the nonreducing end of Shigella dysenteriae type 1 O-specific oligosaccharides on their immunogenicity as conjugates in mice
Proceedings of the National Academy of Sciences of the United States of America
Determination of free amino group in proteins by trinitrobenzensulfonic acid
Analytical Biochemistry
Performance of high-throughput DNA quantification methods
BMC Biotechnology
Genetic characterization and virulence role of the RALP3/LSA locus upstream of the streptolysin S operon in invasive M1T1 Group A Streptococcus
Journal of Bacteriology
Physical chemistry of macromolecules
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2022, International Journal of Biological MacromoleculesCitation Excerpt :Symmetric active ester SIDEA is extensively employed for routine preparation of CRM197-based conjugates of synthetic glycans equipped with an amino alkyl linker. SIDEA-mediated coupling procedure provided efficient conjugation of CRM197 with to amino oligosaccharides related to the repeating unit of CPS of Clostridioides difficile cell wall glycans [132–134] (Table 1, entries 8 (PS I) and 9 (PS-II)), MenX [135] (Table 1, entry 21), S. pneumoniae serotypes 3 [136], 4 [137], 8 [138], 14 [139], 19A and 19F [140] (Table 1, entries 38, 39, 44, 50, 54, 55), S. agalactiae serotype III [141,142] (Table 1, entry 33), Streptococcus pyogenes [143] (Table 1, entry 60), phosphono- [144] and carba-analogues of MenA CPS [145]. Thus, aminoethyl glycosides of synthetic dodecasaccharide fragments of the CPS of S. pyogenes 23 and 24 (Scheme 10) were acylated with SIDEA, and the resulting active esters 25 and 26 were conjugated to CRM197 in mild conditions to give conjugates 27 and 28.
- 1
Present address: Istituto di Ricerca in Biomedicina, via Vela 6, 6500 Bellinzona, Switzerland.
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Present address: Presidio Ospedaliero Alta Val d’Elsa – USL 7, Località Campostaggia, 53036 Poggibonsi, Siena, Italy.