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Structure of a Protective Epitope of Group B Streptococcus Type III Capsular Polysaccharide

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Structure of a Protective Epitope of Group B Streptococcus Type III Capsular Polysaccharide Structure of a protective epitope of group B Streptococcus type III capsular polysaccharide Filippo Carbonia, Roberto Adamoa,1, Monica Fabbrinia, Riccardo De Riccoa, Vittorio Cattaneoa, Barbara Brogionia, Daniele Veggia, Vittoria Pintoa, Irene Passalacquaa, Davide Oldrinia, Rino Rappuolia,2, Enrico Malitoa, Immaculada y Ros Margarita,1, and Francesco Bertia,1,2 aGSK Vaccines, 53100 Siena, Italy Contributed by Rino Rappuoli, March 27, 2017 (sent for review February 3, 2017; reviewed by Dennis L. Kasper and Robert J. Woods) Despite substantial progress in the prevention of group B Strepto- GBS is an encapsulated Gram-positive β-hemolytic pathogen coccus (GBS) disease with the introduction of intrapartum antibiotic causing neonatal sepsis and meningitis, particularly in infants born prophylaxis, this pathogen remains a leading cause of neonatal to mothers carrying the bacteria (12). The GBS capsular PS is infection. Capsular polysaccharide conjugate vaccines have been constituted by multiple RUs (from ∼50 up to 300 per polymer) of tested in phase I/II clinical studies, showing promise for further de- four to seven monosaccharides shaped to form a backbone and one velopment. Mapping of epitopes recognized by protective anti- or two side chains. Ten serotypes presenting a unique pattern of bodies is crucial for understanding the mechanism of action of glycosidic linkages have been identified and their primary struc- vaccines and for enabling antigen design. In this study, we report β the structure of the epitope recognized by a monoclonal antibody tures elucidated (13). Three monosaccharides ( -D-glucopyranose, β β β β with opsonophagocytic activity and representative of the protective -D-Glc; -D-galactopyranose, -D-Gal; and -D-N-acetylglucosamine, response against type III GBS polysaccharide. The structure and the β-D-GlcNAc) are present in all of the described serotypes, and atomic-level interactions were determined by saturation transfer sialic acid (α-N-acetyl-neuraminic acid, NeuNAc) is always found difference (STD)-NMR and X-ray crystallography using oligosaccha- at the terminus of one chain (13). Maternal concentrations of IgG rides obtained by synthetic and depolymerization procedures. The directed to the different GBS capsular serotypes inversely corre- GBS PSIII epitope is made by six sugars. Four of them derive from late with the risk of newborn infection (14), suggesting that anti- MICROBIOLOGY two adjacent repeating units of the PSIII backbone and two of them bodies able to cross the placenta can confer serotype-specific – from the branched galactose sialic acid disaccharide contained in infant protection (15). PS conjugates of different serotypes elicit this sequence. The sialic acid residue establishes direct binding in- antibodies that mediate GBS type-specific opsonophagocytic kill- teractions with the functional antibody. The crystal structure pro- – ing (OPK) in functional assays and protection against GBS chal- vides insight into the molecular basis of antibody carbohydrate – interactions and confirms that the conformational epitope is not lenge in neonate mice (16 20). Furthermore, Ia, Ib, II, III, and V required for antigen recognition. Understanding the structural basis monovalent vaccines, as well as a trivalent combination (Ia, Ib, of immune recognition of capsular polysaccharide epitopes can aid and III), have been proven to be safe and immunogenic in in the design of novel glycoconjugate vaccines. Significance group B Streptococcus | capsular polysaccharide | antigen This article describes the characterization of the antigenic de- acterial cell surface carbohydrates are the interface of mul- terminant of the capsular polysaccharide from the clinically Btiple host interactions and have been targeted to develop relevant serotype III of group B Streptococcus (GBS). NMR and highly efficacious glycoconjugate vaccines against severe infec- X-ray crystallography have been applied to elucidate the in- tions caused by Streptococcus pneumoniae, Haemophilus influ- teraction of type III GBS oligosaccharides obtained by synthetic enzae type b, and Neisseria meningitidis (1, 2). Glycoconjugate and depolymerization procedures of the bacterial poly- vaccines against other important pathogens are under clinical or saccharide with a functional monoclonal antibody. A Fab–GBS preclinical development (2). oligosaccharide complex structure has been solved at high The mapping of polysaccharide (PS) epitopes recognized by resolution (2.7 Å). The results demonstrate the existence of a functional antibodies mediating protection from infection is sialic acid-dependent functional epitope of GBS that is fully crucial for understanding the mechanism of action of this type of contained within four consecutive sugars deriving from the vaccine. In many cases, the antigenic determinants of the im- type III GBS polysaccharide backbone and one branched di- munological properties of PS and the structural details of the saccharide present in this sequence. This finding has implica- minimal epitope targeted by specific functional antibodies are tions for the development of vaccines against GBS infection. unknown. Structural biology has been commonly practiced in the last decade for the characterization of protein antigen–antibody Author contributions: R.A., R.R., I.y.R.M., and F.B. designed research; F.C., M.F., R.D.R., V.C., B.B., D.V., V.P., I.P., D.O., and E.M. performed research; F.C., R.A., M.F., R.D.R., R.R., interactions (3). However, it has been less applied to carbohy- E.M., I.y.R.M., and F.B. analyzed data; and F.C., R.A., R.R., E.M., I.y.R.M., and F.B. wrote drate antigens, in part because of the well-known difficulty of the paper. crystallizing carbohydrates. Reviewers: D.L.K., Harvard Medical School; and R.J.W., University of Georgia. Minimal epitopes can be composed of short, defined glycans Conflict of interest statement: All authors are employees of GSK Vaccines (known as comprising 2–3 monosaccharides, as for the β-(1→2) mannans of Novartis Vaccines at the time of the study). the Candida albicans cell wall (4), Vibrio cholerae O1 (5), Shigella Freely available online through the PNAS open access option. flexneri variant Y (6), and Salmonella (7) O-antigens, or a tetra- Data deposition: The crystal structure has been deposited in the Protein Data Bank, www. saccharide, as for the repeating unit (RU) of S. pneumoniae type pdb.org (PDB ID code 5M63) (deposition ID D_1200001992). 14 PS (Pn14) (8, 9), and even six sugar residues, as in the case of 1R.A., I.y.R.M., and F.B. contributed equally to this work. S. flexneri serotype 2a O-antigen (10). In contrast, the type III PS 2To whom correspondence may be addressed. Email: [email protected] or of Streptococcus agalactiae (group B Streptococcus, GBS) has been [email protected]. proposed as a prototype of a unique length-dependent confor- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. mational epitope (11). 1073/pnas.1701885114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1701885114 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 nonpregnant and pregnant women, and elicited maternal anti- conjugated to human serum albumin (HSA) (38) (Fig. 1A) could bodies were efficiently transferred to neonates (21–23). be partially blocked by preincubation with soluble Pn14 or desia- GBS strains belonging to the serotype III (GBSIII) are epide- lylated PSIII. Pn14 also partially inhibited antibody-mediated miologically the most relevant in neonatal infections (24). Pio- GBSIII OPK (Fig. 1B), confirming functional activity of both neering immunological studies aimed at elucidating type III-specific types of NeuNAc-dependent and -independent rabbit antibodies. antigenic determinants indicated the presence of the terminal Similar results were described previously in the case of human NeuNAc residue as essential for the elicitation of protective an- responses to GBSIII (27). tibodies (25). Subsequent studies revealed that sera from rabbits An IgG1 mAb was selected by hybridoma cell line technology immunized with GBSIII bacteria and from humans vaccinated (NVS-1-19-5), and its activity was ascertained by OPKA (OPK with PSIII conjugates contained two types of anticarbohydrate titer, 1.03 μg/mL). The selected monoclonal was representative of antibodies—that is, a major population recognizing the native PS the typical response to type III PS and had the properties de- but not its incomplete core antigen derivative lacking sialic acid scribed in the literature (33, 34). Complete inhibition by native (corresponding to the S. pneumoniae PS group 14, Pn14), and a PSIII and absence of inhibition by Pn14 in ELISA and OPKA minor variable population reacting with both the native PSIII and experiments (Fig. 1 A and B) confirmed that the epitope recog- the core antigen (26, 27). Remarkably, both types of human PSIII- nized by this mAb was NeuNAc-dependent. A Fab fragment of induced antibodies were shown to mediate GBSIII OPK, whereas mAb NVS-1-19-5 was prepared by papain proteolytic cleavage, antibodies elicited by Pn14 (desialylated PSIII) did not recognize and its high-affinity binding toward the GBS PSIII was measured GBSIII bacteria and therefore did not mediate GBS OPK (27). × −8 13 by SPR (KD estimated with the Fab 4 10 M). Studies using C NMR spectroscopy highlighted ring-linkage The NeuNAc-Gal covalent link in the lateral chain of the PSIII signal displacements in the core versus the native PS, suggesting RU is the most chemically labile glycosidic bond within the PS, and that NeuNAc residues exert a specific control over the conformation complete loss of NeuNAc residues or reduction of their carboxylic of the native PS (26, 28). Molecular dynamics simulations confirmed groups results in PS structures incapable of inducing functional a more flexible and disordered structure for desialylated PSIII and antibodies (25, 26). To investigate the potential of the obtained suggested that native PSIII could form extended helical structures mAb for probing the integrity of the PSIII antigen, we subjected where each turn was made by more than four RUs (29–32).
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