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

Home , Conformational epitope, Epitope mapping

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). PSIII PSIII-CRM197 to mild acid hydrolysis for different incubation times fragments smaller than four RUs appeared as weak inhibitors of the and obtained conjugates with decreasing sialylation levels of 100, binding of native PSIII to its specific antibodies and failed to elicit 60–70, 20–30 and <5%, as estimated by NMR analysis (SI Ap- an efficient immune response following conjugation (33, 34). Based pendix,Fig.S2andTableS1). The resulting glycoconjugates were on the above observations, it was concluded that the native PSIII characterized for their structural integrity and saccharide/protein forms a sialic acid-dependent conformational epitope that is es- content (SI Appendix,Fig.S3andTableS2). ELISA experiments sential for the elicitation and recognition of functional antibodies, using immobilized native PSIII as the antigen and soluble native or and the length dependency of this conformational epitope was as- partially desialylated PSIII-CRM197 as the competitor showed de- cribed to its localization on extended helices within a random coil creasing binding of the mAb to conjugates with increasing desia- structure (33). According to the proposed model, the side chain lylation levels (SI Appendix,Fig.S4A).Thecriticalroleofthe NeuNAc moiety would exercise a remote control over immuno- NeuNAc in elicitation of functional antibodies was confirmed by logical determinants of the PS backbone, without being directly in- immunizing female mice with three doses of the different glyco- volved in molecular interactions as part of the epitope (34). conjugates, followed by sera analysis by ELISA for quantification Structural glycobiology studies using bacterial oligosaccharides in complex with functional monoclonal antibodies (mAbs) able to protect against the target pathogen could represent the most direct methodology to gain information on PS immunological determinants at the atomic level. However, due to the high po- larity of the hydroxyl groups and flexibility of the carbohydrate structures, crystallization of carbohydrate–protein complexes is a challenging task (35). To date, only a very limited number of carbohydrate antigen–Fab complexes, in comparison with a large number of protein antigen–Fab complexes, have been resolved by X-ray crystallography (5–7, 10). As an alternative, a combination of techniques [NMR, surface plasmon resonance (SPR), ELISA, etc.] is typically used to gain insights into carbohydrate– protein interactions (36, 37). In the present study, we used saturation transfer difference–NMR (STD-NMR) in conjunction with X-ray crystallography to investigate the interaction of GBSIII oligosaccharides obtained by synthetic and depolymerization procedures with a protective antibody. Results Selection and Immunochemical Characterization of a Functional Anti- PSIII Rabbit mAb. To investigate the binding of GBS PSIII to functional antibodies at the atomic level and further decipher the role of the NeuNAc moiety, we first generated a rabbit anti-PSIII mAb capable of mediating GBS OPK. Similar to a previous report (26),rabbitsimmunizedwithnative PSIII conjugated to genetically Fig. 1. PSIII antibody specificity and functional activity toward PSIII. In- detoxified diphtheria toxin (CRM197) developed two types of anti- hibition experiments with PSIII and Pn14 of rabbit anti-PSIII polyclonal sera bodies recognizing the native PS but differing in their capacity to (pAb) and mAb in (A) ELISA, binding to immobilized PSIII conjugated to HSA, bind the PSIII desialylated core antigen. Indeed, inhibition ELISA and (B) OPK. (C) Competitive SPR of the binding between the rabbit Fab and experiments showed that binding of IgG from rabbit sera PSIII. Different length fragments were used as inhibitors. PSIII and the to immobilized PSIII unconjugated (SI Appendix,Fig.S1)or Pn14 were used as positive and negative controls, respectively.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1701885114 Carboni et al. Downloaded by guest on September 30, 2021 of anti-PSIII antibodies, and by OPKA to assess antibody func- Length-dependent recognition of the different fragments was tional activity. Lower levels of sialylation resulted in diminished confirmed by competitive ELISA with the selected rabbit mAb recognition of HSA-conjugated native PSIII and decreasing in vitro (SI Appendix,Fig.S8) (33). Inhibition of mAb binding increased killing of GBS bacteria (SI Appendix,Fig.S4B and C). two logs between DP1 and DP2 and then slightly increased an- other 1.5 log, changing the PS size from DP2 to DP13, and be- Selection of PSIII Glycan Fragments for Structural Studies. Antibodies came five-log higher when PSIII was used as an inhibitor (Fig. 1C elicited in mice by PSIII have been shown to bind oligosaccharide and SI Appendix,TableS4). To exclude the effect of the bivalent fragments in a length-dependent manner (33, 34, 39). Using a IgG interaction on the avidity, we performed a competitive SPR competitive ELISA, it was demonstrated that at least two un- assay where different oligosaccharide fragments (DP1–13 range) modified RUs were necessary for suboptimal binding to a GBSIII- were tested as competitors for the binding of soluble Fab fragment specific mouse mAb. Inhibition with 3–7RUPSIIIfragments to PSIII conjugated to HSA immobilized on the chip. Overall, two showed a moderate increase and became much higher above this major populations of inhibitors, DP ≥ 2andDP< 2, were dif- number of RUs (29). SPR experiments using a Fab of the same ferentiated. DP ≥ 2 oligosaccharides showed asymptotically in- mAb confirmed that the affinity (KD) was comparable between creasing affinity up to the native PSIII, with only a 2-log difference 2and6–7 RUs, it increased by threefold from 6–7 RUs to 20 RUs, between native PSIII and DP2. Concerning the single RUs, a 2-log and it remained constant beyond this point (33). difference was observed between DP2 and the synthetic DP1 RU To investigate more in detail the epitope recognized by the se- 1and2hadaβ-Glc-(1→6)–β-GlcNAc branching; conversely, a lected functional mAb, we generated a set of PSIII oligosaccharide very weak inhibition was detected for the linear structure 3, sug- fragments by partial de–N-acetylation followed by nitrosation (40). gesting that the arrangement of sugars in the RU impacts Ab Improvements in the purification process of the PSIII fragments by recognition and the ramification point is relevant for binding. using an anionic exchange HPLC in place of size exclusion HPLC, The binding affinities of the native PSIII and the DP2 fragment as described by Paoletti et al. (41), allowed for the isolation to the rabbit Fab were compared by conjugating the two PS of fragments with a degree of polymerization (DP) in the range of structures to CRM197 and immobilizing them on an SPR chip. The 2–15 with a more defined composition (SI Appendix, Fig. S5). two binding constants determined according to a 1:1 fitting model These fragments were composed of a modified RU and a variable differed by less than 10-fold (SI Appendix, Fig. S9 and Table S5) number of unmodified RUs (Fig. 2). The identity of these oligo- (33). Overall, this analysis confirmed a length-dependent affinity saccharides was established by 1H NMR, and the ratio between the of anti-PSIII GBS antibody but also indicated that DP2 contains signals of the generated hydrated aldehyde group and the H-3e the PSIII portion necessary for high-affinity antibody binding and MICROBIOLOGY (i.e., equatorial) position of NeuNAc or the H-2 position of Glc could be used for further structural analysis. was used to determine the oligosaccharide length (SI Appendix, Identification of the PSIII Antigenic Determinant by STD-NMR. To Fig. S6). The relative ratio of monosaccharide components in the map the interactions of PSIII oligosaccharides with the protective obtained oligosaccharides was ascertained by high-performance mAb, STD-NMR studies were undertaken (43). STD difference anion-exchange chromatography coupled with pulsed ampero- (STDD)-NMR spectra were derived by subtracting the STD-NMR metric detection (HPAEC-PAD) (SI Appendix,TableS3). MALDI- spectrum of the glycan in the bound state with the mAb (ligand/ TOF MS spectra in negative mode confirmed (SI Appendix,Fig.S7) protein 15–50:1 molar ratio) to the reference spectrum in the the structure of the first two peaks eluted on the analytical MonoQ unbound state. The DP3 STDD spectrum was superimposable to anionic exchange column as DP2 and DP3. Both were composed of the one obtained for the DP2–mAb complex (SI Appendix, Fig. homogeneous glycans, whereas longer oligosaccharides contained S10), highlighting that the antibody was recognizing identical re- mixtures of glycans and are hereafter indicated as average DP gions in the two fragments. Due to their higher affinity constants (avDP). A single PSIII RU was chemically synthesized by conven- and larger size, experiments carried out on longer fragments tional carbohydrate chemistry procedures (42). Given that the five (5.5 and 8 RUs) resulted in lower signal intensities that did not residues composing the RU can be arranged according to three allow unequivocal detection of saturation transfer effects. different sequences, three alternative glycans were assembled with Notably, most of the 1H NMR resonances that could un- an end-terminal sugar bearing a linker for possible conjugation (Fig. equivocally be assigned to the DP2–mAb complex were related to 2). After deprotection, the defined glycans were purified on size the α-NeuNAc-(2→3)–β-GalB-(1→4) residues (Fig. 3A), indicating exclusion chromatography and characterized by NMR and MS a direct involvement of this branch in antibody binding, in addition (procedures for synthesis and characterization data are reported to the Glc and Gal residues of the backbone. In the DP2 oligo- elsewhere) (42). saccharide, it was not possible to differentiate the NAc group of NeuNAc and GlcNAc. To better discriminate the exact positions engaged in antibody interaction, the synthesized DP1 was com- plexed with the mAb and analyzed by STDD-NMR. As shown in Fig. 3 B and C, well-recognizable resonances of the branched RU 1 were recovered in the STDD spectrum with medium–high saturation intensity (>50%). The highest transfer of saturation was observed for H-5 (3.85 ppm), H-4 (3.70 ppm), H-3 (2.76 and 1.82 ppm for equatorial and axial, respectively), and H-6 (3.65 ppm) of NeuNAc (Fig. 3B), confirming that NeuNAc is the major anchoring site for mAb interaction. A similar pattern of signals related to the NeuNAc was obtained with units 3 (Fig. 3) and 2 (SI Appendix,Fig.S11). Further, the involvement of the NeuNAc N-acetyl group, not clearly distinguishable from the one of the GlcNAc residue in the DP2, was unveiled in the synthetic probes at 2.0 ppm. When the desialylated tetrasaccharide [β-Gal- → –β → –β → –α β Fig. 2. Chemical structure of GBS PSIII and glycan probes used in this study. The (1 4) -GlcNAc-(1 3) -Gal-(1 4) / -Glc] was analyzed in red highlighted GlcNAc residue is used as the reference monosaccharide to outline the presence of the mAb, no transfer of saturation was observed. the three possible sugar sequences related to PSIII RU. The 2,5-anhydri-D-mannose A high STD signal was also observed for the Glc H-2 position at residue obtained by chemical depolymerization of PSIII is indicated in blue. 3.37 ppm in the DP2 and DP1 RUs 1 and 2 (SI Appendix,Fig.S11)

Carboni et al. PNAS Early Edition | 3of6 Downloaded by guest on September 30, 2021 Fab NVS-1-19-5 shares 79% sequence identity. Excellent electron densities were observed for the Fab portions and allowed the modeling of residues Gln20–Ser245 of the heavy (H) chain and Val24–Cys238 of the light (L) chain. Additional difference densities were observed in front of the Fab complementarity determining regions (CDRs), and these were modeled with the DP2 oligosaccharide (SI Appendix,Fig.S13). Of the total 10 monosaccharide residues of DP2, 6 could be modeled with very good fitting in clear electron density maps and in both copies of the complex, whereas the terminal saccharides residues (B″,C″,D″,andE″)hadlowsigma electron densities for one of the copies of the complex only. For completeness, the full DP2 was included in the final refined model, although the terminal residues had low sigma electron density signals and subsequently high B-factors, both of which reflect uncertainty in their exact position. Importantly, the stereochemistry and geometry of final refined DP2 molecules were validated using Privateer (SI Appendix,TableS6). The coordinates of the complex were refined to final Rwork/Rfree values of 23.6%/28.2% (SI Appendix,TableS7). Structural superimposition of the two copies of the complex present in the ASU resulted in very low carbon alpha root mean square deviation (Cα rmsd) values (∼0.5 Å; the two structures are essen- tially identical), and their bound DP2 conformations were also highly similar; therefore, one copy will be used for the description of the structure. The Fab fragment binds the glycan with an extended interface (577 Å2) formed by two adjacent pockets that are located between the L and H chain of the Fab (Fig. 4 A and B and SI Appendix, Fig. S14). Most of the observed contacts with the Fab involve a single Fig. 3. GBS PSIII epitope mapping. (A) Protons interacting with the mAb identified by STD-NMR and X-ray crystallography. (B)STDDand(C) related 1H PS RU, whereas the sugar residues of the consecutive RU depart NMR spectra of the desialylated tetrasaccharide [β-Gal-(1→4)–β-GlcNAc- (1→3)–β-Gal-(1→4)–α/β-Glc in green], the linear 3 (blue) and branched 1 (red) RUs, and the DP2 fragment (black). Proton positions receiving saturation

after irradiation of the protein are indicated. ‡ indicates the CH2 signal of the Tris buffer; * refers to signals related to the protein.

in complex with the mAb, whereas no recovery was seen for this position in the linear RU 3, where the Glc moiety is located far from the NeuNAc (SI Appendix and Fig. 3 B and C). Previous conformational studies revealed that the orientation of the Glc backbone residue was affected by removal of sialic acid, suggesting spatial proximity of the two residues (32). The transfer of saturation to the H-2 of Glc indicated that mAb binding of this residue is determined by its intrinsic location in the sugar sequence. Owing to the better resolution of the STD-NMR spectra of the synthetic glycans, saturation transfer was more precisely quantified for some of the protons involved in binding. Experiments at increasing saturation times from 0.5 up to 5.0 s were performed to avoid possible bias in the calculation of STD effects due to different proton longitudinal relaxation times (T1) or intramolecular spin diffusion within the bound state (SI Appendix,Fig.S12). In the synthetic glycan 1, an STD relative effect of 100%, 96%, and 70% was measured for H-5, H-3eq signals of NeuNAc, and H-2 of Glc, respectively. Taken together, these results unambiguously showed that the side chain NeuNAc and Gal residues, in conjunction with the Gal and Glc backbone sugars of the RU, directly interact with the mAb. Fig. 4. Crystal structure of DP2 bound to Fab NVS-1-19-5. (A, Left) The Fab is Structural Studies of the PSIII–Fab Complex by X-Ray Crystallography. depicted with surfaces and the DP2 with sticks. Fab residues involved in di- To further elucidate the basis for the PSIII–mAb interaction at the rect binding with DP2 (the paratope) are colored in yellow, and the binding atomic level, we used X-ray crystallography to determine the 3D pockets are labeled. (A, Right) Surface electrostatic potential distribution of structure of DP2 in complex with the Fab NVS-1-19-5. Crystals of the Fab, oriented as on Left.(B) Two views (Left and Right) of the large and small pockets where the DP2 binds. (C) Details of the interactions between the complex belong to space group C2221, contain two copies of a – DP2 and Fab. Carbon atoms of the DP2 backbone are colored in yellow, and 1:1 DP2 Fab complex in the asymmetric unit (ASU), and diffract those belonging to the branches are in green, whereas carbon atoms of the to a resolution of 2.7 Å. The structure was solved by molecular Fab are colored in light and dark gray for the L and H chain, respectively. replacement, using as template input model the coordinates of a Nitrogen and oxygen atoms are colored in blue and red, respectively, and rabbit Fab [Protein Data Bank (PDB) ID code 4JO1] with which water molecules are shown as blue spheres.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1701885114 Carboni et al. Downloaded by guest on September 30, 2021 from the binding pockets, with the exception of GlcNAc-A″,which In the proposed conformational epitope structure, the PSIII is directly bound to Fab amino acid residues. The larger pocket helix structure was supposed to be stabilized by the presence of hosts the NeuNAc of the RU branch that interacts with multiple specific interactions between the side chain of the sialic acid and hydrophilic residues of the CDRs from both the H and L chains. the backbone glucosyl and galactosyl residues, influencing the Specifically, Arg56 of the L chain forms a hydrogen bond with the orientation of the side chain and stabilizing the conformation of N-acetyl group of NeuNAc-C′, and His127 interacts with its O-4 the backbone, and required a carbohydrate chain length of at least (Fig. 4C). Additional weaker interactions of NeuNAc-C′ occur four pentasaccharide RUs to form a conformational epitope through a water molecule bridge between the carboxylic group of equivalent to one present in the native PSIII (33, 34). Shorter the sugar and Asn74 of the H chain and the CH-π interaction carbohydrate chains were thought to be subjected to conforma- between Tyr52 of the L chain and the sialic NAc methyl group (Fig. tional changes so that in the absence of specific interactions the 4C). The GlcNAc-A″ residue appears involved in the binding to the overall helical structure would collapse (29). The need for a helical smaller anchoring pocket of the Fab through at least two hydrogen epitope for antibody recognition was hypothesized considering bonds formed by the L chain Arg56 and Lys74 with the NAc group that GBS PSIII showed a length-dependent binding of specific and the O-3 position of the sugar, respectively. The NAc group is monoclonal IgGs (33). Zou et al. (33) observed that KD measured also stabilized by a CH-π interaction with Tyr126 (Fig. 4C). In- by SPR for monovalent binding remained virtually constant from terestingly, the guanidine group of Arg56 is located within H-bond two to seven RUs, which demonstrated that the epitope optimi- distance from the NAc carbonyl groups of both NeuNAc-C″ and zation was occurring from two to seven RUs, whereas beyond 20 GlcNAc-A″, whose methyl groups are at the same time favorably RUs this phenomenon would overlap with the multivalent expo- oriented to make CH-π interactions with the phenyl rings of sition of stabilized epitopes. Our competitive ELISA and SPR Tyr52 of the L chain and Tyr126 of the H chain, respectively. This data indicate that the functional mAb characterized here shares arrangement allows for a peculiar symmetric network of interac- similar features to the ones used by Zou et al. (33) in that it ex- tions involving the NeuNAc of one RU and the GlcNAc of a hibits a similar length-dependent affinity. However, this length- consecutive unit (Fig. 4C). Complete de–N-acetylation of these dependent affinity could also be explained by a multivalency residues resulted in loss of Fab recognition as demonstrated by effect instead of the need for a helical conformation. competitive SPR (SI Appendix,Fig.S15). Inspection of the glycan Johnson et al. showed by STD-NMR experiments that NeuNAc structure revealed how Gal-E′, although positioned outside of the was not involved in binding to a monoclonal IgM (47), indicating two binding pockets, interacts with side chain atoms of Asn53 of the the existence of antibodies recognizing the PS backbone. Molec- L chain and of Tyr126 of the H chain. The side chain of ular dynamics simulations confirmed that sialic acid plays a role in MICROBIOLOGY – Tyr126 seems also favorably positioned to engage H-bond medi- maintaining the helical structure and led to the hypothesis that this ′ ated interactions with the O-3 position of Glc-D . Interestingly, PS conformation was pivotal for antibody binding (29). ′ GlcNAc-A that was modified into 2,5-anhydro-D-mannose during Our results demonstrate the existence of a sialic acid-dependent the depolymerization reaction appears to be far from the anchoring functional epitope of GBS that is fully contained within four pocket; hence, its chemical manipulation during depolymerization consecutive sugars deriving from the PSIII backbone and one – did not interfere with Fab binding. The DP2 Fab complex crystal branched disaccharide present in this sequence. The nature of the ′ ′ structure also revealed how NeuNAc-C and Glc-D are in close mapped epitope fully explains the well-known critical role of the contact, with a calculated distance between the O-9 atom of sialic acid and, as schematically shown in Fig. 5, does not require a NeuNAc and the O-6 of Glc of 2.8 Å (Fig. 4C). This suggests that conformational epitope (29, 33, 34). However, our study does not hydrogen bonds between these two positions might be further rule out the possibility that mAbs like those described by Johnson stabilized upon binding to the Fab. O-acetylation of the NeuNAc et al. (47) may recognize different regions of GBS PSIII. glycerol chain was noticed in PSIII from GBS clinical isolates, and Our findings have profound implications in understanding it affects inhibition of neutrophil suppression and virulence (44, 45). the protective immunity against type III GBS and in the design We observed that the mAb interacts with the O-7 of NeuNAc of conjugate vaccines and suggest that the nature of protective throughanH2O molecule, and dimensionally, this space could also epitopes should be investigated in all PS vaccines, especially host an acetyl group. This could explain the data from the literature those where the helical structure has been proposed (29). reporting that O-acetylation does not interfere with protection (46). Carbohydrates present a higher level of structural complexity In summary, our structure clearly showed that the side chain compared with other classes on natural biopolymers such as NeuNAc and the backbone Gal and Glc of the first RU, as well as polypeptides or polynucleotides, because of the presence of the backbone GlcNAc of the consecutive RU, interact with mul- α-orβ-glycosidic bonds and the occurrence of connections tiple hydrophilic residues of the CDRs from both the H and the L at different positions of the ring that create ramifications. chain, whereas the NeuNAc glycerol moiety is involved in internal interactions rather than in direct binding to the Fab. Discussion Conjugate vaccines have been one of the major developments of the last 40 y. The nature of the repetitive protective epitopes present in the PS has been difficult to define, mostly because of the difficulty in obtaining crystals from PSs. In many instances, it has been speculated that the protective epitopes would be formed from distant residues that would become close in a helical structure assumed by the PS (28, 29). In this study, we determined the molecular structure of a protective epitope of GBS PSIII. Based on NMR simulation studies indicating the formation of several potential helical conformations (29, 32), where sialylated side chains are arranged on the exterior surface of the helix, the protective epitope dependent on a conformational structure has been proposed for GBS PSIII. In Fig. 5. Schematic representation of GBS PSIII–Fab interaction according to marked contrast, the data reported in this paper show that the (A) the helical model previously proposed (29–34) and (B) the model that protective epitope is linear and overlaps two RUs of the PS. fully explains the well-known critical role of NeuNAc.

Carboni et al. PNAS Early Edition | 5of6 Downloaded by guest on September 30, 2021 These unique features may result in spatial motifs relevant for anti- Materials and Methods body recognition and specificity even in the presence of a limited SI Appendix, Materials and Methods feature additional information to that number of sugar residues, as it has been demonstrated here for GBS provided here. PSIII. In this manner, the hypothesized need for the human immune The clone producing the rabbit mAb anti-PSIII NVS-1-19-5 was obtained using system to recognize GBS PS by means of length-dependent helical hybridoma technology by EPITOMICS Inc., and the relative Fab was prepared by structures, thus avoiding recognition of self-antigens containing sialic using a “Fab Preparation Kit” (Pierce). The GBS PSIII fragments were prepared acid, would need to be reconsidered (34). The existence of helical by deamination (40), and different chain-length oligosaccharides were sepa- conformational epitopes, which would imply the necessity of using a rated by anionic exchange chromatography. NMR experiments were carried long PS antigen, has been considered a challenge when applying out on a Bruker 500 MHz NMR instrument, and the X-ray diffraction data were these technologies to certain pathogens (2). The elucidation and collected at the European Synchrotron Radiation Facility (ESRF). understanding of the structural base for the immune recognition of ACKNOWLEDGMENTS. We thank Matthew James Bottomley of the GSK group carbohydrate epitopes reported here paves the way for designing of companies for useful discussions and critical reading of the manuscript. This modern glycoconjugate vaccines with short oligosaccharides obtained study was sponsored by Novartis Vaccines, now part of the GSK group of by synthetic, chemoenzymatic, or bioengineering methods (48). companies, which was involved in all stages of the study conduct and analysis.

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