Chemical Biology Approaches to Designing Defined Carbohydrate Vaccines

Chemistry & Biology

Review Chemical Biology Approaches to Designing Defined Carbohydrate Vaccines Chakkumkal Anish,1,3,* Benjamin Schumann,1,2,3 Claney Lebev Pereira,1 and Peter H. Seeberger1,2,* 1Department

for Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, Am Mu¨hlenberg 1, 14424 Potsdam, Germany of Chemistry and Biochemistry, Freie Universita¨t Berlin, Arnimallee 22, 14195 Berlin, Germany 3These authors contributed equally to this work *Correspondence: [email protected] (C.A.), [email protected] (P.H.S.) http://dx.doi.org/10.1016/j.chembiol.2014.01.002 2Institute

Carbohydrate antigens have shown promise as important targets for developing effective vaccines and pathogen detection strategies. Modifying purified microbial glycans through synthetic routes or completely synthesizing antigenic motifs are attractive options to advance carbohydrate vaccine development. However, limited knowledge on structure-property correlates hampers the discovery of immunoprotective carbohydrate epitopes. Recent advancements in tools for glycan modification, high-throughput screening of biological samples, and 3D structural analysis may facilitate antigen discovery process. This review focuses on advances that accelerate carbohydrate-based vaccine development and various technologies that are driving these efforts. Herein we provide a critical overview of approaches and resources available for rational design of better carbohydrate antigens. Structurally defined and fully synthetic oligosaccharides, designed based on molecular understanding of antigen-antibody interactions, offer a promising alternative for developing future carbohydrate vaccines. Carbohydrate antigens expressed by pathogens are often structurally unique, and are thus potential targets for developing vaccines and diagnostics. Vaccines based on capsular polysaccharides (CPS) against multiple pathogenic bacteria have been part of routine vaccinations for many years (Ada and Isaacs, 2003; Schumann et al., 2013; U.S. Department of Health and Human Services, 2012). These vaccines are prepared using CPS purified from bacterial cultures (European Medicines Agency, 2009; World Health Organization, 2003; Costantino et al., 2011). However, the presence of impurities that are coisolated, such as cell-wall polysaccharides, have been associated with side effects and hyporesponsiveness (Esposito et al., 2010; Goldblatt et al., 1992; Musher et al., 1990; Poolman and Borrow, 2011; Sen et al., 2005). Since isolated polysaccharides are structurally heterogeneous, multiple purification and quality control steps are required before an antigen can be formulated in a vaccine (European Medicines Agency, 2009; World Health Organization, 2000, 2003, 2005, 2006, 2009, 2012b). Efforts to improve the efficacy and safety of these vaccines are important to achieve comprehensive vaccination and eradication of the respective pathogens. Synthetic oligosaccharides based on the repeating units of CPS can be an attractive option to furnish vaccines free of contaminants that have predictable clinical outcomes (Seeberger and Werz, 2007). Designing vaccines based on synthetic oligosaccharides is not straightforward and proves to be scientifically challenging. The identification of an epitope that will eventually induce protective immunity in vivo is a major bottleneck. Innovative methodologies that can aid the design of epitopes will render this process faster and less cumbersome. Vaccines Based on Cell-Surface Glycans Glycotopes or glycan B cell epitopes are segments of antigenic polysaccharides recognized by the binding sites of membranebound immunoglobulin (Ig) molecules (B cell receptor, BCR) on

B cells. Polysaccharides are T cell-independent antigens that can induce a short-term, IgM-dependent immune response, but fail to efficiently elicit immunological memory (Mazmanian and Kasper, 2006; Mond et al., 1995; Schumann et al., 2013; Stein, 1992). Conjugation to a carrier protein converts glycans to T-cell-dependent antigens that can induce the formation of a long-lasting memory response. A T-cell-dependent immune response is accompanied by the differentiation of polysaccharide-specific B cells to plasma cells. Reinfection in the case of a pathogen or boosting in the case of a vaccine then results in proliferation of plasma cells and affinity maturation of secreted antibodies (Abbas et al., 2012). Glycoconjugate vaccines have been highly successful in preventing infectious diseases caused by Neisseria meningitidis, Haemophilus influenza, and Streptococcus pneumoniae (Garegg and Maron, 1979; Johnson et al., 2010). Despite the effectiveness of CPS-based vaccines, certain disadvantages are encountered in vaccine manufacture. One major bottleneck is the process of isolation and purification of pure capsular polysaccharides from pathogenic bacteria. Not all bacterial strains can be cultured on sufficient scale (Rappuoli et al., 2011). The production of polysaccharides requires efforts to optimize growth conditions (Cimini et al., 2010; Gonc¸alves et al., 2002; Jang et al., 2008; Jin et al., 2009). Furthermore, certain CPSs are unstable and degrade during isolation or formulation processes (Pujar et al., 2004; Sturgess et al., 1999). After isolation, the purification of polysaccharides must be monitored on multiple levels, as specified by guidelines set up by the regulatory authorities (World Health Organization, 2000, 2003, 2005, 2006, 2009, 2012b). For instance, the existence of contaminants, such as other cellular polysaccharides, proteins, and nucleic acid, must be excluded. The structural integrity of the polysaccharide is assessed by wet chemical and instrumental analyses, such as nuclear magnetic resonance (NMR) spectroscopy

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Review (Jones, 2008; Jones and Currie, 1991; Jones et al., 1991; Talaga et al., 2002). After limited depolymerization, chemical activation, and conjugation, the absence of reactive groups on the glycan fragments must be demonstrated. These and other steps of quality control must be performed frequently during glycoconjugate manufacture, raising the cost per dose of the final vaccine. Furthermore, it can be expected that vaccine formulations containing chemically derivatized polysaccharides display artificial, nonprotective epitopes that render the vaccine inefficient (Wessels et al., 1998). An important step toward the design of an effective vaccine is the elucidation of the right antigenic epitope(s) that confer(s) the production of antibodies that can protect the host from a pathogen (so-called protective epitopes). Even if information on a putative protective epitope is available, it is nearly impossible to purify the respective polysaccharide fragment to homogeneity in order to maximize vaccine efficiency. Synthetic Oligosaccharide-Based Vaccines Synthetic oligosaccharides representing the repeating units of CPS can be an attractive alternative to isolated polysaccharides to produce structurally defined vaccines free of impurities (Schumann et al., 2013). Since synthetic oligosaccharides can be easily characterized, the manufacture of vaccines based on these antigens is highly reproducible. In addition, access to molecularly defined antigenic components may result in more efficient final formulations while potentially lowering the costs for vaccine development. Recent years have seen extensive research in the field of oligosaccharide synthesis that have led to the design of highly potent antigenic targets (Berkin et al., 2002; Chong et al., 1997; Costantino et al., 2011; Kudryashov et al., 2001; Martin et al., 2013b; Morelli et al., 2011; Parameswar et al., 2009; Safari et al., 2008; Schofield et al., 2002; Verez-Bencomo et al., 2004; Zhu et al., 2009). The potency of synthetic oligosaccharidebased vaccines is exemplified by the promising protective effects of a Haemophilus influenzae type b (Hib) vaccine based on synthetic oligosaccharides that is marketed in Cuba (VerezBencomo et al., 2004). Despite the progress concerning synthetic oligosaccharidebased vaccines, designing the respective antigens remains challenging. As isolated polysaccharides are structurally complex, the corresponding glycoconjugates most likely contain the ‘‘right’’ B cell epitopes needed to confer protective immunity. Synthetic oligosaccharides, in contrast, are much smaller in size; hence, the presence of protective epitopes in such an antigen is not ensured. Identification of the right epitope is a time-consuming step. Antigen design has traditionally been an iterative process: synthetic targets are chosen based on the chemical structure of repeating units and, after conjugation to a carrier protein, evaluated in immunization experiments in animals (Robbins et al., 2009; Safari et al., 2012). If the resulting antibody response does not target the pathogen, different antigenic constructs will have to be synthesized. Since the procurement of synthetic oligosaccharides in ample quantities for mouse immunization experiments (typically 10–20 mg of the deprotected glycan) is resource intensive, this trial-and-error process in vaccine development is inefficient. Once the structure of a promising antigen is known, however, chemical synthesis can be optimized

to provide large amounts of material (Kabanova et al., 2010; Pozsgay et al., 2012). Recently, a chemoenzymatic method was devised to generate gram amounts of tumor-associated hexasaccharide antigens Globo H and SSEA-4, both of which are interesting targets in the development of antitumor vaccines (Tsai et al., 2013). Globo H is a good example of developing carbohydrate vaccines based on basic biochemical and clinical findings and further optimizing the structure to a fully synthetic epitope (Bremer et al., 1984; Gilewski et al., 2001). Glycoconjugates based on such a fully synthetic glycotope have been advanced to early stages of clinical trials (Gilewski et al., 2001). Thus, understanding the structure of a protective B cell epitope is vital before immunization experiments are performed. The currently available tools to map carbohydrate epitopes (see below) can make use of small amounts of glycans for screening purposes. Thus, the efforts of organic chemists can be shifted from providing ample amounts of few structures to generating whole libraries of structurally diverse glycans in smaller quantities. Multiple approaches have been undertaken to facilitate this process of generating diversity, such as the programmable one-pot glycosylation technique, which employs the sequential activation of different glycosylating agents based on reactivity differences to furnish oligosaccharides in one reaction vessel without intermediate purification (Lee et al., 2006; Wu and Wong, 2011). Enzymatic oligosaccharide syntheses have been developed to generate a variety of complex glycan structures (Nycholat et al., 2013; Tsai et al., 2013; Wang et al., 2013b), although this approach is still somewhat limited to the more common monosaccharides found in glycans from eukaryotes. Automated solid-phase oligosaccharide synthesis has been used to generate a plethora of synthetic oligosaccharides of different complexities. Since the process of oligosaccharide assembly is fully automated, target oligosaccharide structures can be tailored toward the needs of the experimentalist using a limited set of monosaccharide building blocks (Calin et al., 2013; Plante et al., 2001; Seeberger and Werz, 2005, 2007). It is thus conceivable that a large variety of potential antigens can be generated much faster than by using traditional solution-phase oligosaccharide assembly. Structural Attributes of Carbohydrate B Cell Epitopes B cell epitopes can be classified into two groups: sequential (continuous) epitopes and conformational (discontinuous) epitopes. A sequential B cell epitope is recognized as the primary structure of an antigen, while a conformational B cell epitope consists of residues that may be distantly separated in the primary structure and are recognized due to the close proximity within the folded 3D structure (Arnon and Van Regenmortel, 1992). The nature of an epitope is of utmost importance during antigen design: conformational epitopes are associated with a distinct secondary structure of the glycan, a feat that may only be achieved in longer sequences comprising multiple repeating units. Thus, the synthetic effort compromises the applicability of these long glycans as synthetic antigens. For instance, CPS of group B streptococci (GBS) type III adopts a helical structure above a length of five repeating units (Gonza´lez-Outeirin˜o et al., 2005; Jennings, 2012; Woods and Yongye, 2012). Polysaccharide fragments that are smaller than five repeating units fail to elicit an efficient immune response (Jennings, 2012; MacKenzie

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Figure 1. The Immunogenic Determinants of a Synthetic Carbohydrate Antigen Size/span of the epitope (1), terminal glycan residues (2), presence of branching points (3), side chain functional groups (4), and number of repeating units (5). L, preinstalled linker for conjugation.

and Jennings, 2003; Woods and Yongye, 2012; Zou et al., 1999). In contrast, oligosaccharides corresponding to S. pneumoniae CPS have been found to be excellent immunogens even at smaller sizes (Benaissa-Trouw et al., 2001; Jansen et al., 2001; Safari et al., 2012). Other carbohydrate-based antigens induce an efficient immune response at intermediate epitope lengths, such as CPS fragments from Hib or certain Shigella strains (Carlin et al., 1986; Chong et al., 1997; Peeters et al., 1992; Phalipon et al., 2009; Pozsgay et al., 1999; Robbins et al., 2009; Vince et al., 2003). The implications of these structural requirements are still poorly understood. In addition to glycan length, important determinants for the viability of a carbohydrate antigen comprise the stereochemistry of glycosidic linkages and the arrangement of individual glycan components within a repeating unit. In addition, the presence of branching points and different substituents like phosphates, acetates, and pyruvates may be crucial for immunogenicity, but render both design and synthesis complex (Fusco et al., 2007; McNeely et al., 1998; Szu et al., 1991; Werz and Seeberger, 2005). Critical factors determining immunogenicity of a glycan are summarized in Figure 1. Finding the Right Epitope: Approaches to Rational Oligosaccharide Antigen Design A major milestone in vaccine design is the induction of robust titer of antibodies that cross-react with the native polysaccharide and opsonize the pathogen. Thereby, it is desirable to reduce the complexity of synthetic antigens to ensure feasibility of large-scale synthesis while still maintaining the ability to raise a specific, protective immune response. Novel concepts to unveil protective epitopes include the screening of antisera on libraries of synthetic glycans, reverse engineering of neutralizing antibodies, and in silico antigen design (Figure 2). Screening of Patient Sera for Antibodies against a Synthetic Antigen Library Sera of infected or vaccinated individuals provide invaluable information on epitopes recognized by the immune system. Techniques to rapidly obtain this information have become available only recently. Anti-capsular antibodies in sera are easily detectable by conventional binding assays, such as enzyme-linked

immunosorbent assay (ELISA). The structures of the precise antigenic epitopes of CPS, however, are not uncovered by these methods. Major progress in elucidating glycotopes has been made by using glycan microarrays (de Paz and Seeberger, 2012; Feizi et al., 2003; Liu et al., 2009; Park et al., 2013; Rillahan and Paulson, 2011; Smith et al., 2010). Thereby, minute amounts of glycans of synthetic or natural origin are immobilized on surfaces via either covalent linkages or noncovalent adsorption. After incubation with antisera, specific antibody-glycan binding events can be detected by incubation with a fluorescencelabeled secondary antibody (Anish et al., 2013a; Martin et al., 2013a, 2013b). Careful design of the microarray printing pattern by inclusion of oligosaccharides with different chain lengths, frame shifts, and terminal glycan residues helps to deduce meaningful inferences on the epitope of bound antibodies. Polysaccharide-based vaccines have been highly efficient to prevent infections with a variety of pathogenic bacteria. Considerable efforts have been invested to generate synthetic variants of these and other antigens, including H. influenzae, group B Streptococcus, certain Shigella strains, and multiple serotypes of S. pneumoniae (Benaissa-Trouw et al., 2001; Berkin et al., 2002; Garegg and Norberg, 1982; Garegg et al., 1998; Hansson et al., 2001; Parameswar et al., 2009; Verez-Bencomo et al., 2004; Vince et al., 2003). Most of these antigens were prepared and tested when mapping of carbohydrate epitopes was not in experimental reach. Current endeavors by the carbohydrate research community are focused on the refinement of these and further antigens to facilitate glycoconjugate vaccine development. One recent example for rational antigen design focused on the development of vaccine candidates against Clostridium difficile. This Gram-positive bacterium is the major cause of grave hospital-acquired (nosocomial), antibiotic-induced diarrhea (Rebeaud and Bachmann, 2012). Several cell-surface glycans have been identified on C. difficile, including two phosphodiester-containing polysaccharides (PS-I and PS-II) and lipoteichoic acid (LTA or PS-III) (Ganeshapillai et al., 2008; Monteiro et al., 2013; Reid et al., 2012). The respective synthetic oligosaccharide repeating units to all of these glycans were prepared synthetically (Martin et al., 2011, 2013a, 2013b; Oberli et al., 2011). Microarray screening of both blood and stool samples of patients suffering from C. difficile infections revealed the presence of antibodies against all three structures to varying degrees (Martin et al., 2013a, 2013b). Severe disease in patients correlated with low levels of anti-PS-I IgA in stool samples, indicating a lack of protective immunity against C. difficile in these individuals. Further evaluation of the recognized epitopes was enabled by the synthesis of several PS-I oligosaccharide repeating unit substructures (Martin et al., 2013a, 2013b). Sera of reconvalescent patients exhibited a significant increase of IgG levels against the PS-I repeating unit a-Rhap-(1/3)-b-Glcp-(1/4)-[a-Rhap-(1/ 3)]-a-Glcp-(1/2)-a-Glcp, as well as a di- and a triglucoside fragment. Other substructures were recognized by serum and stool antibodies in humans with and without a history of C. difficile infection to similar degrees (Martin et al., 2013b). For further investigation of the role of PS-I in immunity against C. difficile, mice were immunized with a PS-I-CRM197 conjugate, and the antibody response was evaluated by glycan microarray analysis. Robust immune responses against a-Rhap-(1/3)-b-Glcp, a

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Figure 2. Major Steps Involved in Rational Design of Synthetic Carbohydrate Vaccines *Reprinted with permission from Oberli et al. (2010). Copyright (2010) American Chemical Society.

motif that occurs twice in the PS-I repeating unit, indicated that this disaccharide is the smallest glycotope that confers recognition of the glycan by the immune system. Consequently, a glycoconjugate harboring the respective disaccharide hapten and CRM197 as a carrier protein induced an immune response in mice that cross-reacted with the PS-I pentasaccharide repeating unit (Martin et al., 2013b). Thus, a-Rhap-(1/3)-b-Glcp is a candidate antigen to be applied in glycoconjugate vaccines against C. difficile. It will be interesting to evaluate the potency of this simplified antigen in a vaccination setting. Recent findings suggest that the phosphodiester groups present in C. difficile PS-II are crucial for eliciting an IgG response against C. difficile bacteria upon immunization with a PS-II-CRM197 conjugate (Adamo et al., 2012). Further studies will reveal whether this finding can be translated to PS-I oligosaccharides. While glycoconjugate vaccines against bacteria are marketed, recent years have seen tremendous progress in developing antigens based on unique saccharide structures on the surface of protozoan parasites, such as Leishmania, Trypanosoma, and Plasmodium. The high variability of surface proteins often hampers the manufacture of efficient protein-based vaccines against these protozoa. Therefore, glycans are of paramount interest for

vaccine research (Scherf et al., 2008; Stockdale et al., 2008). The need for synthetic oligosaccharides as antigens is of special relevance in these cases, as large-scale cultivation of protozoan parasites and isolation of the respective glycans is impossible to date (Visvesvara and Garcia, 2002). Recently, the potential of cell-surface glycans for the detection of Leishmania parasites in clinical samples has been established (Anish et al., 2013b). Leishmania are transmitted by the bite of sandflies and can cause symptoms affecting skin tissue (cutaneous leishmaniasis), but can develop into highly dangerous visceral leishmaniasis, which is frequently associated with organ failure and death (McCall et al., 2013). Development of a detection method was fuelled by the accessibility of synthetic oligosaccharides corresponding to Leishmania lipophosphoglycans (LPG) (Anish et al., 2013b; Hewitt and Seeberger, 2001; Liu et al., 2006). While the b-Galp-(1/4)-a-Manp-(1/P) repeating unit LPG backbone is a conserved motif found on the parasite surface, so-called capping oligosaccharides vary among different strains. Synthetic glycans corresponding to the capping oligosaccharides of different strains were prepared, and binding of human and canine antisera to these structures was assessed (Anish et al., 2013b). A hybrid tetrasaccharide capping structure

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Review containing the conserved the b-Galp-(1/4)-a-Manp motif was bound specifically by sera of Leishmania-infected dogs and humans. Based on glycan microarray screening, the LPG capping tetrasaccharide was selected for immunogenicity studies. Antisera raised by the respective tetrasaccharideCRM197 conjugate were used to stain heat-killed L. chagasi parasites, highlighting the relevance of synthetic glycans in developing an immune response that binds to whole pathogens. Based on synthetic oligosaccharides, the development of both a diagnostic test and a glycoconjugate vaccine against Leishmania lies within reach. Glycosylphosphatidylinositol (GPI) anchors are important glycolipids found on all eukaryotes that serve to link proteins to the plasma membrane. Protozoan parasites display a high density of structurally unique GPI anchors on the cell surface (Paulick and Bertozzi, 2008; Tsai et al., 2012). The nonvariable structures of these glycans render them ideal for use in vaccination and point-of-care diagnostic settings, especially in the case of Plasmodium falciparum, the malaria-causing parasite. A number of synthetic GPI anchors have been prepared (Azzouz et al., 2010; Hewitt et al., 2002; Kwon et al., 2005; Tamborrini et al., 2010; Tsai et al., 2012), harboring thiol linkers that can be chemoselectively coupled to microarray slides or carrier proteins. Synthetic GPI anchors include oligosaccharides consisting of three or four mannose residues (Man3-GPI and Man4-GPI, respectively), as well as smaller structures (Kwon et al., 2005). Human sera of malaria-endemic regions were assayed on microarray slides containing these oligosaccharides (Kamena et al., 2008). Robust antibody responses to longer GPI structures were observed in all serum samples. It was further revealed that the strength of this antibody response is stable throughout the wet and dry seasons in the respective areas. When sera of nonexposed humans were assayed as a control, surprisingly, an equally robust anti-GPI response to Man4-GPI was found, suggesting that the respective oligosaccharides are not Plasmodium specific. However, differences were seen in binding of sera to Man3-GPI, with samples from malaria-endemic areas exhibiting significantly increased antibody titers against these glycans. The same trend was observed in human blood samples before and after experimental Plasmodium challenge. A GPI-based malaria vaccine candidate had previously shown excellent efficacy in Plasmodium challenge studies in mice (Schofield et al., 2002). The finding that subtle differences in the GPI glycan moiety account for specificities in immune responses against protozoa paves the way for carefully designed oligosaccharide-based diagnostics and vaccines against these parasites. Infections with Trypanosoma cruzi are of serious concern in low-income regions of Latin America. T. cruzi is transmitted by triatomine bugs and is the causative for Chagas disease. Infected humans suffer from edema in the initial stages of the disease, followed by chronic symptoms such as neurodegeneration and cardiomyopathies (Chuenkova and Pereiraperrin, 2011; Nunes et al., 2013). Up to 10 million people are currently affected by Chagas disease (World Health Organization, 2012a), and treatment is hampered by resistances of the parasite toward the few available drugs (Mejia et al., 2012). T. cruzi is covered by GPI-anchored proteins that carry unique mucin-type O-glycans (Acosta-Serrano et al., 2001). Chemical synthesis of several substructures corresponding to these O-glycans was recently

reported (Ashmus et al., 2013). The glycans were chemically conjugated to BSA and the glycoconjugates were immobilized on 96-well plates to generate glycan arrays of a macroscopic format. Incubation with human serum samples revealed specific recognition of terminal a-Galp-(1/3)-b-Galp motifs by patient antisera. In contrast, sera from healthy control individuals did not contain antibodies that bind to these structures. Furthermore, a-Galp-b-Galp saccharides of different linkage types were not recognized by patient sera, indicating that the 1/3linked disaccharide is a specific recognition motif of T. cruzi surface glycans. Further experiments will reveal whether this glycotope is a suitable starting point for diagnostic tests and vaccination experiments. Particularly, cross-reactivity of these glycans with antibodies against different pathogens must be elucidated, preferentially in a study including more complex glycans to assess specificity. Designing Synthetic Glycan Antigens Based on Structural Information of Carbohydrates and Anti-Carbohydrate Antibodies Recent progress in synthetic carbohydrate chemistry, bioinformatics, and analysis of carbohydrate-protein interactions facilitates the 3D-structural interpretation of glycan-antibody complexes. Numerous methods are currently in practice that can aid the antigen design process. For instance, advancements in structural methods, such as X-ray crystallography, NMR, or small angle X-ray scattering (SAXS), are used to generate valuable information on the 3D structures of glycans and their interaction partners (Khan et al., 2011; Rademacher et al., 2007; Vulliez-Le Normand et al., 2008). Information on the structural requirements of glycan recognition can form the basis of designing optimized carbohydrate vaccine candidates. Structural vaccinology is a newly emerging field and has already contributed significantly to the development of modern protein-based vaccines (Dormitzer et al., 2012; Schneewind and Missiakas, 2011). Adaptation of the structural vaccinology principles to saccharides may reveal the nature of glycotopes that are recognized by the host immune system, thereby facilitating the design of more efficient vaccines. For instance, structural understanding of the epitope helps to improve biochemical characteristics of vaccine antigens and to select the combination of glycan residues that are necessary to increase the breadth of the immune response. Recently, Bundle and coworkers used structural information to design a universal carbohydrate antigen based on the antigenic determinants of A-type and M-type O antigens of Brucella (Guiard et al., 2013). Studies on the nature of antigenic determinants recognized by monoclonal antibodies facilitated the design of one single oligosaccharide that may form the basis for the diagnosis of Brucellosis. When complex multiepitope antigens like polysaccharides are conjugated to carrier proteins, the unfavorable immunodominance of few nonprotective epitopes is a key challenge in immunization (Michon et al., 1991; Michon et al., 2005). For instance, depolymerization of polysaccharides may lead to the presence of neoepitopes that may hamper immunogenicity of natural glycotopes (Csako et al., 1987; El Sabbagh et al., 1982). Structural information on the binding of epitopes may help in decoding the rules of immunodominance and provide the basis for manipulating the antigen structure to render protective epitopes immunodominant (Dormitzer et al., 2012).

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Review Certain saccharide structures harbor labile residues or modifications that render purification tedious and decrease the stability of the final vaccine formulation. The availability of validated glycan sequences can provide the basis for atomic substitutions in particularly labile positions and enhance vaccine stability. Recently, a modified trisaccharide based on the repeating unit of S. pneumoniae serotype 19F CPS (SP19F-CPS) was designed and synthesized by Lay and coworkers (Legnani et al., 2009). The SP19F-CPS consists of a trisaccharide repeating unit, (/4)-b-D-ManpNAc-(1/4)-a-D-Glcp-(1/2)-a-L-Rhap(1-OPO3-/), with a phosphodiester bridge connecting the repeating units. Based on a theoretical quantum mechanical study on the conformational behavior of a-L-rhamnose-1-phosphate analogs, it was found that a carbasugar analog of L-rhamnose is a suitable candidate to mimic the chemically labile rhamnose-1-phosphate, preserving the geometrical properties of the reference compound. Consequently, substitution of L-rhamnose for the respective carbasugar in a synthetic 19F CPS repeating unit rendered a more stable antigen with identical conformational properties as the natural trisaccharide. A competition ELISA experiment based on binding of natural CPS and anti-19F human polyclonal antiserum was employed to assess the biological activity of the analog. The designed glycan mimic showed biological activity similar to the natural repeating unit, underlining the potential of rational design in refining the structural antigenic determinants of carbohydrate antigens. Lay and coworkers recently expanded this concept to Neisseria meningitidis serotype A (MenA) CPS. Poor hydrolytic stability of MenA capsular polysaccharide, consisting of a-(1/6)-linked 2-acetamido-2-deoxy-D-mannopyranosyl phosphate repeating units, is an impediment in the development of glycoconjugates based on this antigen. Immunization of mice with hydrolysis-stable structural carbasugar analogs of MenA CPS elicited antibodies that were tested positive in a serum bactericidal assay (SBA) against MenA, indicating the potential of this strategy to generate potent antigens (Gao et al., 2013). Similar C-phosphonate MenA CPS analogs have been prepared by Oscarson and  et al., 2006). coworkers (Teodorovic Another crucial step in structural glycotope design is the identification of key substituents that mask immune responses toward a glycan. Neisseria meningitidis serotype C and Y (MenC and MenY) capsular polysaccharides are good examples for this challenge. MenC is a homopolymer of a-(2/9)-linked sialic acids that are O-acetylated at position C-8 or C-7, whereas MenY is a heteropolymer consisting of repeating disaccharides of a-(2/4)-linked sialic acids attached to the 6 position of glucose residues: a-D-Glcp-(1/4)-a-NeupNAc-(2/6). The sialic acids on the purified polysaccharide are O-acetylated to varying degrees at position C-9 or C-7 in a strain-dependent manner (Fusco et al., 2007). However, upon storage in solution after polysaccharide purification, these O-acetyl groups eventually migrate to the neighboring hydroxyl group (i.e., C-7 on MenC and C-9 on MenY) (Lemercinier and Jones, 1996). Both preclinical and clinical vaccination studies using deacetylated MenC demonstrated that the O-acetyl group masks the protective epitope in native CPS through steric hindrance or altered conformations (Fusco et al., 2007). Since modifications like acetyl groups can migrate or get lost during polysaccharide purification, controlling and defining the

degree of these modifications in vaccines based on isolated polysaccharides is challenging. This matter is of special relevance if the modifications are crucial for the induction of a protective immune response. Synthetic oligosaccharides with defined acetylation patterns can be an attractive option. Sialicacid-based synthetic vaccine candidates may accelerate this process and lead to meningococcal vaccines (Chu et al., 2011; Wang et al., 2013a). N. meningitidis serogroup W135 capsular oligosaccharides ranging in length from di- to decasaccharides have been synthesized. Sera from mice immunized with these oligosaccharide-protein conjugates showed SBA activity, indicating the immunoprotective potential of these vaccine candidates (Wang et al., 2013a). 3D-structural information on oligosaccharide antigens is becoming increasingly available. NMR spectroscopy and X-ray crystallography are the most reliable techniques to reveal glycan-antibody interactions at the molecular level. However, typical Ka values of glycan-antibody interactions are weak, in the 102–106 M 1 range, complicating the cocrystallization of both binding partners. In such instances, NMR is extremely useful to obtain insights into the solution structure and the dynamics of a ligand-antibody complex (Marcelo et al., 2012). However, due to the intrinsic complexity and flexibility of carbohydrates, a multidisciplinary strategy combining NMR parameters with molecular modeling protocols should be followed in order to generate reliable structural information (Marcelo et al., 2012). The method of choice to uncover structural requirements of glycan-antibody interactions depends on the particular problem and the required structural information. Generally, two broader approaches may be distinguished: ligand based and receptor based. In ligand-based structural glycobiology, changes in the parameters of the ligand are monitored upon passing from the free to the bound state (Marcelo et al., 2012). Thereby, NMR techniques are often preferred over other biophysical methods. NMR measurements can be employed to detect binding, to elucidate the bound conformation of the glycan, to map the bound epitope, and to provide information on molecular dynamics (Marcelo et al., 2012). Combinations of transferred nuclear Overhauser effect spectroscopy (TR-NOESY) (Ni and Zhu, 1994) and saturation transfer difference (STD) (Mayer and Meyer, 1999; Mayer and Meyer, 2001) NMR experiments have been employed extensively to study the conformations of carbohydrates bound to antibodies and to define interacting epitopes (Cle´ment et al., 2006; Herfurth et al., 2005; Maaheimo et al., 2000; Oberli et al., 2010; Rademacher et al., 2007). We revealed the structural features of the interaction between a Bacillus anthracis tetrasaccharide containing three rhamnoses and a unique terminal anthrose unit and a monoclonal antibody (MTA1-3) by STD NMR (Oberli et al., 2010). A combined approach involving glycan array screening, surface plasmon resonance (SPR), and STD NMR revealed structural insights into the molecular events of carbohydrate-antibody interactions. The use of several B. anthracis oligosaccharides demonstrated a crucial role of the anthrose residue in recognition (Oberli et al., 2010). Transferred NMR methodologies, such as TR-NOESY (Ni and Zhu, 1994) and TR-ROESY (Arepalli et al., 1995), are employed by different groups to deduce the bound conformation of

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Review carbohydrates with antibodies. Bundle and coworkers combined TR-NOESY and modeling data to deduce the bound conformation of a Salmonella paratyphi oligosaccharide complexed to an antibody (Milton and Bundle, 1998). This antibody recognized the repeating unit of the Salmonella serogroup B O antigen a-D-Galp-(1/2)[a-D-Abep-(1/3)]-a-D-Manp-(1/4)a–L-Rhap-(1/3). It was shown that the monoclonal antibody (mAb) prefers one high-energy conformation of the a-DManp(1/4)-a-L-Rhap glycosidic linkage. The availability of such refined 3D-structural information of bound glycans will aid the optimization and design of immunoprotective oligosaccharide motifs. Reverse engineering of a protective monoclonal antibody (mAb C3.1) was the basis for the design of novel glycoconjugates en route to vaccines against Candida albicans (Bundle et al., 2012). The antibody was found to bind b-(1/2)-linked mannopyranose homo-oligomers. The inhibitory activity of mAb binding to the native antigen reached a maximum for a trisaccharide and then rapidly decreased with increasing oligosaccharide length (Bundle et al., 2012). This inhibition pattern is in stark contrast to the paradigm of oligosaccharide-antibody interactions, dictating that typically longer oligosaccharides are best accommodated in the antibody binding pocket (Ma¨kela¨ et al., 1984). Sequential chemical mapping by functional group replacement led to the identification of a disaccharide as a minimum sized glycotope for mAb C3.1. This disaccharide was synthesized and conjugated to a carrier protein. Synthesis of deoxygenated and methylated glycan congeners enabled the mapping of polar contacts between the antibody and the b-mannan disaccharide epitope. Protective immunity induced by immunization with rationally designed shorter oligosaccharide antigens is a great boost to the field of synthetic glycoconjugate vaccines. The scope of this work was further expanded recently by augmentation of the immunogenicity of a b-mannan trisaccharide antigen by active targeting of dendritic cells via Dectin-1, a C-type lectin receptor involved in antigen capture and processing (Lipinski et al., 2013). A shortcoming of NMR-based epitope mapping is the restriction on certain tight kinetic requirements. Such methods work optimally when the off rate is fast in the relaxation time scale of the ligand. For slow dissociation kinetics, conventional NMR methods do not provide adequate information. Consequently, STD and TR-NOESY methods are not useful for dissociation constants in the low micromolar or nanomolar ranges, (Marcelo et al., 2012) and the observation of the parameters associated with the receptor becomes the method of choice. In a receptor-based approach, methods of structural biochemistry are employed to map the antigen combining sites or complementarity determining regions (CDRs) of an antibody. However, antibodies are large macromolecules, and due to their weaker interactions, gaining detailed structural information of a carbohydrate-antibody complex represents a real challenge. NMR experiments coupled with isotope labeling are frequently employed to gain insights into antibody binding surfaces (Kato et al., 2010). X-ray crystallography would provide structural information on antigen combining sites with highest resolution. However, only few crystal structures of carbohydrate-antibody complexes have been solved thus far (Evans et al., 1995; Nagae et al., 2013; Villeneuve et al., 2000; Vulliez-Le Normand et al., 2008). Various studies have demonstrated that carbohydrate

binding sites of an anti-glycan antibody can vary significantly in the epitope size they accommodate. Cocrystal structures of carbohydrate-bound antibodies reveal the span of the bound epitope and structural details of important residues that interact with antibody CDRs. Relatively small oligosaccharides occupy the binding site in reported structures (Nagae et al., 2013; Villeneuve et al., 2000), while few reports of larger epitopes exist (Vulliez-Le Normand et al., 2008). In one special case, a(2/8)polysialic acid apparently occupies an exceptionally long binding site (Evans et al., 1995). Two key examples demonstrating the receptor based approach in carbohydrate antigen design involve Vibrio cholerae O1 antigen (Villeneuve et al., 2000) and synthetic O antigen fragments from serotype 2a Shigella flexneri (Vulliez-Le Normand et al., 2008). Vibrio cholerae serogroups differ in their lipopolysaccharide (LPS) structures. Serogroup O1 causes most cholera outbreaks worldwide and includes two major serotypes: Ogawa and Inaba. The two types differ only by a single 2-O-methyl group that is present at the nonreducing terminal perosamine unit of the Ogawa O-specific polysaccharide (O-SP) and is absent in the Inaba O-SP. The crystal structure of a murine protective antibody specific for Ogawa LPS was solved in its unliganded form and in complex with synthetic fragments of the Ogawa O-SP (Villeneuve et al., 2000). The upstream terminal O-SP monosaccharide was found to be the primary antigenic determinant and rationalized the serotype specificity of anti-Ogawa antibodies. These insights provided a basis for the development of a synthetic oligosaccharide anticholera vaccine. The repeating unit of the Shigella flexneri serotype 2a O antigen is a branched pentasaccharide with the sequence a-L-Rhap(1/2)-a-L-Rhap-(1/3)-[a-D-Glcp-(1/4)]-a-L-Rhap-(1/3)-bD-GlcNAcp (1/2). The crystal structures of two immunogenic synthetic oligosaccharides in complex with a protective Fab fragment (F22-4) have been reported (Vulliez-Le Normand et al., 2008). F22-4 binds to an epitope generated by two consecutive pentasaccharide repeat units. Six sugar residues out of a contiguous nine-residue segment were shown to form direct contacts with the CDRs. The nonreducing rhamnose and both branching glucosyl residues from the two reproductive units (RUs) were indicated as crucial components of the epitope. These results have implications for vaccine design as they suggest that a minimum of two RUs of synthetic serotype 2a oligosaccharides are required as O antigen glycotopes. Structural information of antigen binding surfaces of an antibody can form the basis for designing better antigens. Broadly neutralizing anti-HIV mAb 2G12 is a well-studied example. mAb 2G12 recognizes a conserved and unusually dense cluster of oligomannose residues (terminal a-D-Manp-(1/2)-Man residues) on gp120, an envelope protein of HIV-1. Both liganded and unliganded crystal structures of this antibody have been reported (Calarese et al., 2003), and this information has been used extensively as a template to develop strongly binding glycan ligands (Lee et al., 2004). The 2G12 antibody adopts a unique domain-swapped tertiary structure (Calarese et al., 2003). This arrangement produces an array of antibody combining sites in proximity to one another. Two conventional combining sites formed by heavy (VH) and light (VL) variable domains sandwich an unconventional site composed of two neighboring VH domain residues (Figure 3). The

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Review Figure 3. The Structure of Man9GlcNAc2 on HIV Protein gp120 Interacting with the Broadly Neutralizing mAb 2G12 Adapted from Lee et al. (2004).

crystal structure of Man9GlcNAc2 bound to Fab2G12 revealed that the conventional binding sites are occupied by the D1 arms of the Man9GlcNAc2 moieties. The terminal a-D-Man (1/2)-Man residues establish 85% of the contacts with the binding site. The nonconventional site is occupied by the D2 arms of neighboring Man9GlcNAc2 moieties. This detailed 3Dstructural information triggered the design of multivalent ligands that may form the basis of a synthetic high-mannose oligosaccharide vaccine for HIV (Enrı´quez-Navas et al., 2011; Lee et al., 2004; Wang et al., 2004, 2007). For instance, Lee et al. designed and synthesized oligomannoses by a reactivity-based, modular one-pot synthesis method that requires a minimal number of building blocks (Lee et al., 2004). Thereby, novel synthetic oligomannose glycotopes were reported that effectively inhibited the binding of 2G12 to gp120, indicating the vaccine potential of these glycans. Recently, numerous potent and broadly neutralizing antibodies such as PG 127 and PG 128 that recognize HIV glycan shield have been reported (Pejchal et al., 2011). Thus, HIV surface glycans are emerging as a major target for developing antibody based therapeutics and prevention strategies. Computational Approaches to the Rational Design of Carbohydrate Antigens Establishing structure-function correlations for carbohydrate antigenicity is a rapidly emerging area. Understanding the structural features of antigenic carbohydrates is a key step in this regard. The high-flexibility, complex shape and dynamic properties of pyranose rings and the presence of functional group modifications render structure acquisition challenging (Woods and Yongye, 2012). Computational methods can be employed to study structural determinants of affinity, specificity, and antigenicity where traditional 3D-structural methods reach their limits. In silico simulations can provide atomic level interpretations of binding events that are experimentally inaccessible. Computational methods help deducing the roles of enthalpy and entropy in driving oligosaccharide antigenicity. Such insights are crucial while selecting the right candidate for synthesis. Established structural methods, such as X-ray diffraction or NMR spectroscopy, face enormous challenges when applied to the study of large cell-surface glycans. Although NMR tech-

niques can be used for studying less homogeneous isolated polysaccharides, molecular weight limitations greatly limit the application of NMR-based methods in large antibody-polysaccharide complexes. Moreover, NMR data alone are generally insufficient to completely characterize carbohydrate conformations and need to be complemented by insight from in silico methods, such as molecular dynamics (MD) simulations (Woods and Yongye, 2012). While no method alone is adequate to characterize either the conformation of an oligosaccharide or its complex with a protein, the combination of experimental biophysical techniques with current computational methods offers an opportunity to deduce structurefunction relationships in carbohydrate-antibody interactions. A multidisciplinary approach for deciphering crucial glycan immunogenicity features was brought to bear on the polysialic acid-based Neisseria mengitidis serotype B capsular polysaccharide (MenB PSA). The immune system frequently elicits a response to long and extended glycotopes so that autoreactivity to short mammalian glycan motifs can be avoided. The two best examples for the case are constituted by CPS of group B N. meningitidis and group B Streptococcus (Jennings, 2012). Computational approaches were instrumental in defining the extended epitopes of these antigens. MenB PSA consists of chains of a-(2/8)-polysialic acid, and due to structural similarity to polysialic acids on neural cell adhesion molecules (Finne and Ma¨kela¨, 1985), MenB PSA is often considered as a self-antigen by the immune system. By adoption of aggressive immunization strategies, specific antibodies recognizing MenB PSA have been raised (Jennings, 2012). An extended helical epitope of MenB PSA was identified using a combination of inhibition studies, NMR, and conformational analysis using potential energy calculations (Jennings, 2012). MenB PSA required an unusually long motif to show efficient binding to antibodies, indicating the conformational nature of antigenic glycotopes. Conformational studies showed that, although PSA exists predominantly in the random coil form, the polysaccharide can readily adopt extended helical conformations. Detailed studies also revealed that carboxylate groups were important for maintaining the conformation and that substitution of N-acetyl with larger N-propionyl groups did not affect binding (Brisson et al., 1992). A crucial role of this large extended conformation in binding to antibodies was confirmed by X-ray analysis of an unliganded Fab fragment. Although conformational studies showed that the conformational epitope is only a minor contributor to the total number of possible epitopes formed by PSA (Brisson et al., 1992), the immune response was dominant for this less populated epitope. The reluctance of the immune system to produce antibodies associated with the more populated random

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Review coil form of PSA suggested redesigning the antigen. Based on this finding, an artificial antigen was designed carrying N-propionates instead of N-acetates (Jennings, 2012). MD simulations helped to define the conformational epitope of bacterial polysaccharides from GBS (Woods and Yongye, 2012). A complete computational analysis of GBS, in terms of its interaction with mAb 1B1, characterization of the solution conformation of the antigenic fragments, and characterization of the putative immune complex was reported (Kadirvelraj et al., 2006; Woods and Yongye, 2012). This work provided the structural and energetic explanation for the observed lack of crossreactivity between GBS and its close relative Pneumococcus type 14. Thereby, the bactericidal epitope (bound to mAb 1B1) was identified as equivalent to the solution conformational epitope (Kadirvelraj et al., 2006). Conclusions and Outlook Carbohydrate antigens are important targets for the development of vaccines and pathogen detection strategies. Vaccines based on isolated glycan structures have helped to prevent deadly infectious diseases and to approach the nearly complete eradication of certain pathogens. While it is conceivable that the majority of efficacious polysaccharide-based vaccines will still be marketed several decades from now, synthetic oligosaccharide antigens can help targeting those diseases that are difficult to tackle with purified glycans due to stability or scalability issues. Although numerous diseases fall in this category, for socioeconomic reasons, development of vaccines for such infections is generally considered nonviable. Concerted efforts of nonprofit organizations, big vaccine companies, and regulatory agencies are required to achieve this goal. Solving major technological bottlenecks may further facilitate the process. Inclusion of epitope selection steps during antigen manufacture will not only speed up the developing process, but also significantly reduce the cost for the generation of a vaccine. Development of novel protein-free synthetic carrier molecules may also be relevant, considering the carrier-mediated epitope suppression induced by carrier proteins employed in conventional vaccines. Modifying purified microbial carbohydrates through chemical synthesis or completely procuring glycan fragments through organic synthesis facilitated the antigen discovery process. An improved understanding of structural features that determine antigenicity is a prerequisite for efficient vaccine design. The identification of key epitopes that protect from a potential immune challenge or infection is a crucial milestone. Multidisciplinary approaches involving advanced oligosaccharide synthesis, glycan array screening, and biophysical and computational methods have been utilized by many groups to attain this goal. The scope of this approach has been demonstrated with numerous carbohydrate antigens. Nevertheless, fully synthetic, rationally designed carbohydrate antigen is still not available, and recent advancement in synthetic, structural, and computational methods may help to meet this goal.

Ministry of Education and Research, and the Ko¨rber Foundation for generous financial support. This work was supported by a Kekule´ doctoral fellowship by the Fonds der Chemischen Industrie (B.S.). REFERENCES Abbas, A.K., Lichtman, A.H., and Pillai, S. (2012). Cellular and Molecular Immunology. (Philadelphia: Elsevier Saunders). Acosta-Serrano, A., Almeida, I.C., Freitas-Junior, L.H., Yoshida, N., and Schenkman, S. (2001). The mucin-like glycoprotein super-family of Trypanosoma cruzi: structure and biological roles. Mol. Biochem. Parasitol. 114, 143–150. Ada, G., and Isaacs, D. (2003). Carbohydrate-protein conjugate vaccines. Clin. Microbiol. Infec. 9, 79–85. Adamo, R., Romano, M.R., Berti, F., Leuzzi, R., Tontini, M., Danieli, E., Cappelletti, E., Cakici, O.S., Swennen, E., Pinto, V., et al. (2012). Phosphorylation of the synthetic hexasaccharide repeating unit is essential for the induction of antibodies to Clostridium difficile PSII cell wall polysaccharide. ACS Chem. Biol. 7, 1420–1428. Anish, C., Guo, X., Wahlbrink, A., and Seeberger, P.H. (2013a). Plague detection by anti-carbohydrate antibodies. Angew. Chem. Int. Ed. Engl. 52, 9524– 9528. Anish, C., Martin, C.E., Wahlbrink, A., Bogdan, C., Ntais, P., Antoniou, M., and Seeberger, P.H. (2013b). Immunogenicity and diagnostic potential of synthetic antigenic cell surface glycans of Leishmania. ACS Chem. Biol. 8, 2412–2422. Arepalli, S.R., Glaudemans, C.P., Daves, G.D., Jr., Kovac, P., and Bax, A. (1995). Identification of protein-mediated indirect NOE effects in a disaccharide-Fab’ complex by transferred ROESY. J. Magn. Reson. B. 106, 195–198. Arnon, R., and Van Regenmortel, M.H.V. (1992). Structural basis of antigenic specificity and design of new vaccines. FASEB J. 6, 3265–3274. Ashmus, R.A., Schocker, N.S., Cordero-Mendoza, Y., Marques, A.F., Monroy, E.Y., Pardo, A., Izquierdo, L., Ga´llego, M., Gascon, J., Almeida, I.C., and Michael, K. (2013). Potential use of synthetic a-galactosyl-containing glycotopes of the parasite Trypanosoma cruzi as diagnostic antigens for Chagas disease. Org. Biomol. Chem. 11, 5579–5583. Azzouz, N., Kamena, F., and Seeberger, P.H. (2010). Synthetic glycosylphosphatidylinositol as tools for glycoparasitology research. OMICS 14, 445–454. Benaissa-Trouw, B., Lefeber, D.J., Kamerling, J.P., Vliegenthart, J.F., Kraaijeveld, K., and Snippe, H. (2001). Synthetic polysaccharide type 3-related di-, tri-, and tetrasaccharide-CRM(197) conjugates induce protection against Streptococcus pneumoniae type 3 in mice. Infect. Immun. 69, 4698–4701. Berkin, A., Coxon, B., and Pozsgay, V. (2002). Towards a synthetic glycoconjugate vaccine against Neisseria meningitidis A. Chemistry 8, 4424–4433. Bremer, E.G., Levery, S.B., Sonnino, S., Ghidoni, R., Canevari, S., Kannagi, R., and Hakomori, S.I. (1984). Characterization of a glycosphingolipid antigen defined by the monoclonal antibody MBr1 expressed in normal and neoplastic epithelial cells of human mammary gland. J. Biol. Chem. 259, 14773–14777. Brisson, J.R., Baumann, H., Imberty, A., Pe´rez, S., and Jennings, H.J. (1992). Helical epitope of the group B meningococcal alpha(2-8)-linked sialic acid polysaccharide. Biochemistry 31, 4996–5004. Bundle, D.R., Nycholat, C., Costello, C., Rennie, R., and Lipinski, T. (2012). Design of a Candida albicans disaccharide conjugate vaccine by reverse engineering a protective monoclonal antibody. ACS Chem. Biol. 7, 1754–1763. Calarese, D.A., Scanlan, C.N., Zwick, M.B., Deechongkit, S., Mimura, Y., Kunert, R., Zhu, P., Wormald, M.R., Stanfield, R.L., Roux, K.H., et al. (2003). Antibody domain exchange is an immunological solution to carbohydrate cluster recognition. Science 300, 2065–2071.

ACKNOWLEDGMENTS

Calin, O., Eller, S., and Seeberger, P.H. (2013). Automated polysaccharide synthesis: assembly of a 30mer mannoside. Angew. Chem. Int. Ed. Engl. 52, 5862–5865.

We would like to thank Bernd Lepenies for helpful discussion and critique of the manuscript. The authors thank the Max Planck Society, German Federal

Carlin, N.I.A., Wehler, T., and Lindberg, A.A. (1986). Shigella flexneri O-antigen epitopes: chemical and immunochemical analyses reveal that epitopes of type III and group 6 antigens are identical. Infect. Immun. 53, 110–115.

46 Chemistry & Biology 21, January 16, 2014 ª2014 Elsevier Ltd All rights reserved

Chemistry & Biology

Review Chong, P., Chan, N., Kandil, A., Tripet, B., James, O., Yang, Y.P., Shi, S.P., and Klein, M. (1997). A strategy for rational design of fully synthetic glycopeptide conjugate vaccines. Infect. Immun. 65, 4918–4925. Chu, K.-C., Ren, C.-T., Lu, C.-P., Hsu, C.-H., Sun, T.-H., Han, J.-L., Pal, B., Chao, T.-A., Lin, Y.-F., Wu, S.-H., et al. (2011). Efficient and stereoselective synthesis of a(2/9) oligosialic acids: from monomers to dodecamers. Angew. Chem. Int. Ed. Engl. 50, 9391–9395. Chuenkova, M.V., and Pereiraperrin, M. (2011). Neurodegeneration and neuroregeneration in Chagas disease. Adv. Parasitol. 76, 195–233. Cimini, D., Restaino, O.F., Catapano, A., De Rosa, M., and Schiraldi, C. (2010). Production of capsular polysaccharide from Escherichia coli K4 for biotechnological applications. Appl. Microbiol. Biotechnol. 85, 1779–1787. Cle´ment, M.-J., Fortune´, A., Phalipon, A., Marcel-Peyre, V., Simenel, C., Imberty, A., Delepierre, M., and Mulard, L.A. (2006). Toward a better understanding of the basis of the molecular mimicry of polysaccharide antigens by peptides: the example of Shigella flexneri 5a. J. Biol. Chem. 281, 2317– 2332. Costantino, P., Rappuoli, R., and Berti, F. (2011). The design of semi-synthetic and synthetic glycoconjugate vaccines. Expert. Opin. Drug. Discov. 6, 1045– 1066. Csako, G., Suba, E.A., Tsai, C.M., Mocca, L.F., and Elin, R.J. (1987). Dosedependent changes in the antigenicity of bacterial endotoxin exposed to ionizing radiation. J. Clin. Lab. Immunol. 24, 193–198. de Paz, J.L., and Seeberger, P.H. (2012). Recent advances and future challenges in glycan microarray technology. Methods Mol. Biol. 808, 1–12. Dormitzer, P.R., Grandi, G., and Rappuoli, R. (2012). Structural vaccinology starts to deliver. Nat. Rev. Microbiol. 10, 807–813. El Sabbagh, M., Galanos, C., Berto´k, L., Fu¨st, G., and Lu¨deritz, O. (1982). Effect of ionizing radiation on chemical and biological properties of Salmonella minnesota R595 lipopolysaccharide. Acta Microbiol. Acad. Sci. Hung. 29, 255–261. Enrı´quez-Navas, P.M., Marradi, M., Padro, D., Angulo, J., and Penade´s, S. (2011). A solution NMR study of the interactions of oligomannosides and the anti-HIV-1 2G12 antibody reveals distinct binding modes for branched ligands. Chemistry 17, 1547–1560. Esposito, S., Tansey, S., Thompson, A., Razmpour, A., Liang, J., Jones, T.R., Ferrera, G., Maida, A., Bona, G., Sabatini, C., et al. (2010). Safety and immunogenicity of a 13-valent pneumococcal conjugate vaccine compared to those of a 7-valent pneumococcal conjugate vaccine given as a three-dose series with routine vaccines in healthy infants and toddlers. Clin. Vaccine Immunol. 17, 1017–1026. European Medicines Agency (2009). Assessment Report for Synflorix. (London: European Medicines Agency). Evans, S.V., Sigurskjold, B.W., Jennings, H.J., Brisson, J.R., To, R., Tse, W.C., Altman, E., Frosch, M., Weisgerber, C., Kratzin, H.D., et al. (1995). Evidence for the extended helical nature of polysaccharide epitopes. The 2.8 A resolution structure and thermodynamics of ligand binding of an antigen binding fragment specific for alpha-(2—>8)-polysialic acid. Biochemistry 34, 6737–6744. Feizi, T., Fazio, F., Chai, W.C., and Wong, C.H. (2003). Carbohydrate microarrays - a new set of technologies at the frontiers of glycomics. Curr. Opin. Struct. Biol. 13, 637–645. Finne, J., and Ma¨kela¨, P.H. (1985). Cleavage of the polysialosyl units of brain glycoproteins by a bacteriophage endosialidase. Involvement of a long oligosaccharide segment in molecular interactions of polysialic acid. J. Biol. Chem. 260, 1265–1270. Fusco, P.C., Farley, E.K., Huang, C.H., Moore, S., and Michon, F. (2007). Protective meningococcal capsular polysaccharide epitopes and the role of O acetylation. Clin. Vaccine Immunol. 14, 577–584. Ganeshapillai, J., Vinogradov, E., Rousseau, J., Weese, J.S., and Monteiro, M.A. (2008). Clostridium difficile cell-surface polysaccharides composed of pentaglycosyl and hexaglycosyl phosphate repeating units. Carbohydr. Res. 343, 703–710. Gao, Q., Tontini, M., Brogioni, G., Nilo, A., Filippini, S., Harfouche, C., Polito, L., Romano, M.R., Costantino, P., Berti, F., et al. (2013). Immunoactivity of protein

conjugates of carba analogues from Neisseria meningitidis a capsular polysaccharide. ACS Chem. Biol. 8, 2561–2567. Garegg, P.J., and Maron, L. (1979). Improved Synthesis of 3,4,6-Tri-O-BenzylAlpha-D-Mannopyranosides. Acta Chem. Scand. B 33, 39–41. Garegg, P.J., and Norberg, T. (1982). Syntheses of the Biological Repeating Units of Salmonella Serogroup-a, Serogroup-B and Serogroup-D1 O-Antigenic Polysaccharides. J. Chem. Soc. Perk. T 1, 2973–2982. Garegg, P.J., Oscarson, S., and Tedebark, U. (1998). Synthesis of the repeating unit of the capsular polysaccharide of Streptococcus pneumoniae type 3 as a building block suitable for formation of oligomers. J. Carbohydr. Chem. 17, 587–594. Gilewski, T., Ragupathi, G., Bhuta, S., Williams, L.J., Musselli, C., Zhang, X.F., Bencsath, K.P., Panageas, K.S., Chin, J., Hudis, C.A., et al. (2001). Immunization of metastatic breast cancer patients with a fully synthetic H conjugate: a phase I trial. Proc. Natl. Acad. Sci. USA 98, 3270–3275. Goldblatt, D., Levinsky, R.J., and Turner, M.W. (1992). Role of cell wall polysaccharide in the assessment of IgG antibodies to the capsular polysaccharides of Streptococcus pneumoniae in childhood. J. Infect. Dis. 166, 632–634. Gonc¸alves, V.M., Zangirolami, T.C., Giordano, R.L., Raw, I., Tanizaki, M.M., and Giordano, R.C. (2002). Optimization of medium and cultivation conditions for capsular polysaccharide production by Streptococcus pneumoniae serotype 23F. Appl. Microbiol. Biotechnol. 59, 713–717. Gonza´lez-Outeirin˜o, J., Kadirvelraj, R., and Woods, R.J. (2005). Structural elucidation of type III group B Streptococcus capsular polysaccharide using molecular dynamics simulations: the role of sialic acid. Carbohydr. Res. 340, 1007–1018. Guiard, J., Paszkiewicz, E., Sadowska, J., and Bundle, D.R. (2013). Design and synthesis of a universal antigen to detect brucellosis. Angew. Chem. Int. Ed. Engl. 52, 7181–7185. Hansson, J., Garegg, P.J., and Oscarson, S. (2001). Syntheses of anomerically phosphodiester-linked oligomers of the repeating units of the Haemophilus influenzae types c and f capsular polysaccharides. J. Org. Chem. 66, 6234– 6243. Herfurth, L., Ernst, B., Wagner, B., Ricklin, D., Strasser, D.S., Magnani, J.L., Benie, A.J., and Peters, T. (2005). Comparative epitope mapping with saturation transfer difference NMR of sialyl Lewis(a) compounds and derivatives bound to a monoclonal antibody. J. Med. Chem. 48, 6879–6886. Hewitt, M.C., and Seeberger, P.H. (2001). Automated solid-phase synthesis of a branched Leishmania cap tetrasaccharide. Org. Lett. 3, 3699–3702. Hewitt, M.C., Snyder, D.A., and Seeberger, P.H. (2002). Rapid synthesis of a glycosylphosphatidylinositol-based malaria vaccine using automated solidphase oligosaccharide synthesis. J. Am. Chem. Soc. 124, 13434–13436. Jang, H., Yoon, Y.K., Kim, J.A., Kim, H.S., An, S.J., Seo, J.H., Cui, C., and Carbis, R. (2008). Optimization of Vi capsular polysaccharide production during growth of Salmonella enterica serotype Typhi Ty2 in a bioreactor. J. Biotechnol. 135, 71–77. Jansen, W.T.M., Hogenboom, S., Thijssen, M.J.L., Kamerling, J.P., Vliegenthart, J.F.G., Verhoef, J., Snippe, H., and Verheul, A.F.M. (2001). Synthetic 6B di-, tri-, and tetrasaccharide-protein conjugates contain pneumococcal type 6A and 6B common and 6B-specific epitopes that elicit protective antibodies in mice. Infect. Immun. 69, 787–793. Jennings, H. (2012). The role of sialic acid in the formation of protective conformational bacterial polysaccharide epitopes. In Anticarbohydrate Antibodies, P. Kosma and S. Mu¨ller-Loennies, eds. (Vienna: Springer), pp. 55–73. Jin, S.D., Kim, Y.M., Kang, H.K., Jung, S.J., and Kim, D. (2009). Optimization of capsular polysaccharide production by Streptococcus pneumoniae type 3. J. Microbiol. Biotechnol. 19, 1374–1378. Johnson, H.L., Deloria-Knoll, M., Levine, O.S., Stoszek, S.K., Freimanis Hance, L., Reithinger, R., Muenz, L.R., and O’Brien, K.L. (2010). Systematic evaluation of serotypes causing invasive pneumococcal disease among children under five: the pneumococcal global serotype project. PLoS Med. 7, e1000348. Jones, C. (2008). The regulatory framework for glycoconjugate vaccines. In Carbohydrate-Based Vaccines, R. Roy, ed. (Washington: American Chemical Society), pp. 21–35.

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Chemistry & Biology

Review Jones, C., and Currie, F. (1991). Control of components of bacterial polysaccharide vaccines by physical methods. Biologicals 19, 41–47. Jones, C., Currie, F., and Forster, M.J. (1991). N.m.r. and conformational analysis of the capsular polysaccharide from Streptococcus pneumoniae type 4. Carbohydr. Res. 221, 95–121. Kabanova, A., Margarit, I., Berti, F., Romano, M.R., Grandi, G., Bensi, G., Chiarot, E., Proietti, D., Swennen, E., Cappelletti, E., et al. (2010). Evaluation of a Group A Streptococcus synthetic oligosaccharide as vaccine candidate. Vaccine 29, 104–114. Kadirvelraj, R., Gonzalez-Outeirin˜o, J., Foley, B.L., Beckham, M.L., Jennings, H.J., Foote, S., Ford, M.G., and Woods, R.J. (2006). Understanding the bacterial polysaccharide antigenicity of Streptococcus agalactiae versus Streptococcus pneumoniae. Proc. Natl. Acad. Sci. USA 103, 8149–8154. Kamena, F., Tamborrini, M., Liu, X.Y., Kwon, Y.U., Thompson, F., Pluschke, G., and Seeberger, P.H. (2008). Synthetic GPI array to study antitoxic malaria response. Nat. Chem. Biol. 4, 238–240. Kato, K., Yamaguchi, Y., and Arata, Y. (2010). Stable-isotope-assisted NMR approaches to glycoproteins using immunoglobulin G as a model system. Prog. Nucl. Magn. Reson. Spectrosc. 56, 346–359.

Marcelo, F., Can˜ada, F.J., and Jime´nez-Barbero, J. (2012). The interaction of saccharides with antibodies. a 3D view by using NMR. In Anticarbohydrate Antibodies, P. Kosma and S. Mu¨ller-Loennies, eds. (Vienna: Springer), pp. 385–402. Martin, C.E., Weishaupt, M.W., and Seeberger, P.H. (2011). Progress toward developing a carbohydrate-conjugate vaccine against Clostridium difficile ribotype 027: synthesis of the cell-surface polysaccharide PS-I repeating unit. Chem. Commun. (Camb.) 47, 10260–10262. Martin, C.E., Broecker, F., Eller, S., Oberli, M.A., Anish, C., Pereira, C.L., and Seeberger, P.H. (2013a). Glycan arrays containing synthetic Clostridium difficile lipoteichoic acid oligomers as tools toward a carbohydrate vaccine. Chem. Commun. (Camb.) 49, 7159–7161. Martin, C.E., Broecker, F., Oberli, M.A., Komor, J., Mattner, J., Anish, C., and Seeberger, P.H. (2013b). Immunological evaluation of a synthetic Clostridium difficile oligosaccharide conjugate vaccine candidate and identification of a minimal epitope. J. Am. Chem. Soc. 135, 9713–9722. Mayer, M., and Meyer, B. (1999). Characterization of ligand binding by saturation transfer difference NMR spectroscopy. Angew. Chem. Int. Ed. 38, 1784– 1788.

Khan, S., Rodriguez, E., Patel, R., Gor, J., Mulloy, B., and Perkins, S.J. (2011). The solution structure of heparan sulfate differs from that of heparin: implications for function. J. Biol. Chem. 286, 24842–24854.

Mayer, M., and Meyer, B. (2001). Group epitope mapping by saturation transfer difference NMR to identify segments of a ligand in direct contact with a protein receptor. J. Am. Chem. Soc. 123, 6108–6117.

Kudryashov, V., Glunz, P.W., Williams, L.J., Hintermann, S., Danishefsky, S.J., and Lloyd, K.O. (2001). Toward optimized carbohydrate-based anticancer vaccines: epitope clustering, carrier structure, and adjuvant all influence antibody responses to Lewis(y) conjugates in mice. Proc. Natl. Acad. Sci. USA 98, 3264–3269.

Mazmanian, S.K., and Kasper, D.L. (2006). The love-hate relationship between bacterial polysaccharides and the host immune system. Nat. Rev. Immunol. 6, 849–858.

Kwon, Y.U., Soucy, R.L., Snyder, D.A., and Seeberger, P.H. (2005). Assembly of a series of malarial glycosylphosphatidylinositol anchor oligosaccharides. Chemistry 11, 2493–2504. Lee, H.-K., Scanlan, C.N., Huang, C.-Y., Chang, A.Y., Calarese, D.A., Dwek, R.A., Rudd, P.M., Burton, D.R., Wilson, I.A., and Wong, C.-H. (2004). Reactivity-based one-pot synthesis of oligomannoses: defining antigens recognized by 2G12, a broadly neutralizing anti-HIV-1 antibody. Angew. Chem. Int. Ed. Engl. 43, 1000–1003. Lee, J.C., Greenberg, W.A., and Wong, C.H. (2006). Programmable reactivitybased one-pot oligosaccharide synthesis. Nat. Protoc. 1, 3143–3152. Legnani, L., Ronchi, S., Fallarini, S., Lombardi, G., Campo, F., Panza, L., Lay, L., Poletti, L., Toma, L., Ronchetti, F., and Compostella, F. (2009). Synthesis, molecular dynamics simulations, and biology of a carba-analogue of the trisaccharide repeating unit of Streptococcus pneumoniae 19F capsular polysaccharide. Org. Biomol. Chem. 7, 4428–4436. Lemercinier, X., and Jones, C. (1996). Full 1H NMR assignment and detailed O-acetylation patterns of capsular polysaccharides from Neisseria meningitidis used in vaccine production. Carbohydr. Res. 296, 83–96. Lipinski, T., Fitieh, A., St Pierre, J., Ostergaard, H.L., Bundle, D.R., and Touret, N. (2013). Enhanced immunogenicity of a tricomponent mannan tetanus toxoid conjugate vaccine targeted to dendritic cells via Dectin-1 by incorporating beta-glucan. J. Immunol. 190, 4116–4128. Liu, X., Siegrist, S., Amacker, M., Zurbriggen, R., Pluschke, G., and Seeberger, P.H. (2006). Enhancement of the immunogenicity of synthetic carbohydrates by conjugation to virosomes: a leishmaniasis vaccine candidate. ACS Chem. Biol. 1, 161–164. Liu, Y., Palma, A.S., and Feizi, T. (2009). Carbohydrate microarrays: key developments in glycobiology. Biol. Chem. 390, 647–656. Maaheimo, H., Kosma, P., Brade, L., Brade, H., and Peters, T. (2000). Mapping the binding of synthetic disaccharides representing epitopes of chlamydial lipopolysaccharide to antibodies with NMR. Biochemistry 39, 12778–12788.

McCall, L.I., Zhang, W.W., and Matlashewski, G. (2013). Determinants for the development of visceral leishmaniasis disease. PLoS Pathog. 9, e1003053. McNeely, T.B., Staub, J.M., Rusk, C.M., Blum, M.J., and Donnelly, J.J. (1998). Antibody responses to capsular polysaccharide backbone and O-acetate side groups of Streptococcus pneumoniae type 9V in humans and rhesus macaques. Infect. Immun. 66, 3705–3710. Mejia, A.M., Hall, B.S., Taylor, M.C., Go´mez-Palacio, A., Wilkinson, S.R., Triana-Cha´vez, O., and Kelly, J.M. (2012). Benznidazole-resistance in Trypanosoma cruzi is a readily acquired trait that can arise independently in a single population. J. Infect. Dis. 206, 220–228. Michon, F., Chalifour, R., Feldman, R., Wessels, M., Kasper, D.L., Gamian, A., Pozsgay, V., and Jennings, H.J. (1991). The alpha-L-(1——2)-trirhamnopyranoside epitope on the group-specific polysaccharide of group B streptococci. Infect. Immun. 59, 1690–1696. Michon, F., Moore, S.L., Kim, J., Blake, M.S., Auzanneau, F.I., Johnston, B.D., Johnson, M.A., and Pinto, B.M. (2005). Doubly branched hexasaccharide epitope on the cell wall polysaccharide of group A streptococci recognized by human and rabbit antisera. Infect. Immun. 73, 6383–6389. Milton, M.J., and Bundle, D.R. (1998). Observation of the anti conformation of a glycosidic linkage in an antibody-bound oligosaccharide. J. Am. Chem. Soc. 120, 10547–10548. Mond, J.J., Lees, A., and Snapper, C.M. (1995). T cell-independent antigens type 2. Annu. Rev. Immunol. 13, 655–692. Monteiro, M.A., Ma, Z., Bertolo, L., Jiao, Y., Arroyo, L., Hodgins, D., Mallozzi, M., Vedantam, G., Sagermann, M., Sundsmo, J., and Chow, H. (2013). Carbohydrate-based Clostridium difficile vaccines. Expert Rev. Vaccines 12, 421–431. Morelli, L., Poletti, L., and Lay, L. (2011). Carbohydrates and immunology: synthetic oligosaccharide antigens for vaccine formulation. Eur. J. Org. Chem. 2011, 5723–5777.

MacKenzie, C.R., and Jennings, H.J. (2003). Characterization of polysaccharide conformational epitopes by surface plasmon resonance. Methods Enzymol. 363, 340–354.

Musher, D.M., Luchi, M.J., Watson, D.A., Hamilton, R., and Baughn, R.E. (1990). Pneumococcal polysaccharide vaccine in young adults and older bronchitics: determination of IgG responses by ELISA and the effect of adsorption of serum with non-type-specific cell wall polysaccharide. J. Infect. Dis. 161, 728–735.

Ma¨kela¨, O., Pe´terfy, F., Outschoorn, I.G., Richter, A.W., and Seppa¨la¨, I. (1984). Immunogenic properties of alpha (1——6) dextran, its protein conjugates, and conjugates of its breakdown products in mice. Scand. J. Immunol. 19, 541–550.

Nagae, M., Ikeda, A., Hane, M., Hanashima, S., Kitajima, K., Sato, C., and Yamaguchi, Y. (2013). Crystal structure of anti-polysialic acid antibody single chain Fv fragment complexed with octasialic acid: insight into the binding preference for polysialic acid. J. Biol. Chem. 288, 33784–33796.

48 Chemistry & Biology 21, January 16, 2014 ª2014 Elsevier Ltd All rights reserved

Chemistry & Biology

Review Ni, F., and Zhu, Y. (1994). Accounting for ligand-protein interactions in the relaxation-matrix analysis of transferred nuclear Overhauser effects. J. Magn. Reson. B. 103, 180–184. Nunes, M.C., Dones, W., Morillo, C.A., Encina, J.J., and Ribeiro, A.L.; Council on Chagas Disease of the Interamerican Society of Cardiology (2013). Chagas disease: an overview of clinical and epidemiological aspects. J. Am. Coll. Cardiol. 62, 767–776. Nycholat, C.M., Peng, W., McBride, R., Antonopoulos, A., de Vries, R.P., Polonskaya, Z., Finn, M.G., Dell, A., Haslam, S.M., and Paulson, J.C. (2013). Synthesis of biologically active N- and O-linked glycans with multisialylated poly-N-acetyllactosamine extensions using P. damsela a2-6 sialyltransferase. J. Am. Chem. Soc. 135, 18280–18283. Oberli, M.A., Tamborrini, M., Tsai, Y.H., Werz, D.B., Horlacher, T., Adibekian, A., Gauss, D., Mo¨ller, H.M., Pluschke, G., and Seeberger, P.H. (2010). Molecular analysis of carbohydrate-antibody interactions: case study using a Bacillus anthracis tetrasaccharide. J. Am. Chem. Soc. 132, 10239–10241. Oberli, M.A., Hecht, M.L., Bindscha¨dler, P., Adibekian, A., Adam, T., and Seeberger, P.H. (2011). A possible oligosaccharide-conjugate vaccine candidate for Clostridium difficile is antigenic and immunogenic. Chem. Biol. 18, 580–588. Parameswar, A.R., Park, I.H., Saksena, R., Kova´c, P., Nahm, M.H., and Demchenko, A.V. (2009). Synthesis, conjugation, and immunological evaluation of the serogroup 6 pneumococcal oligosaccharides. ChemBioChem 10, 2893– 2899. Park, S., Gildersleeve, J.C., Blixt, O., and Shin, I. (2013). Carbohydrate microarrays. Chem. Soc. Rev. 42, 4310–4326. Paulick, M.G., and Bertozzi, C.R. (2008). The glycosylphosphatidylinositol anchor: a complex membrane-anchoring structure for proteins. Biochemistry 47, 6991–7000. Peeters, C.C., Evenberg, D., Hoogerhout, P., Ka¨yhty, H., Saarinen, L., van Boeckel, C.A., van der Marel, G.A., van Boom, J.H., and Poolman, J.T. (1992). Synthetic trimer and tetramer of 3-beta-D-ribose-(1-1)-D-ribitol-5phosphate conjugated to protein induce antibody responses to Haemophilus influenzae type b capsular polysaccharide in mice and monkeys. Infect. Immun. 60, 1826–1833. Pejchal, R., Doores, K.J., Walker, L.M., Khayat, R., Huang, P.S., Wang, S.K., Stanfield, R.L., Julien, J.P., Ramos, A., Crispin, M., et al. (2011). A potent and broad neutralizing antibody recognizes and penetrates the HIV glycan shield. Science 334, 1097–1103.

Rappuoli, R., Black, S., and Lambert, P.H. (2011). Vaccine discovery and translation of new vaccine technology. Lancet 378, 360–368. Rebeaud, F., and Bachmann, M.F. (2012). Immunization strategies for Clostridium difficile infections. Expert Rev. Vaccines 11, 469–479. Reid, C.W., Vinogradov, E., Li, J., Jarrell, H.C., Logan, S.M., and Brisson, J.R. (2012). Structural characterization of surface glycans from Clostridium difficile. Carbohydr. Res. 354, 65–73. Rillahan, C.D., and Paulson, J.C. (2011). Glycan microarrays for decoding the glycome. Annu. Rev. Biochem. 80, 797–823. Robbins, J.B., Kubler-Kielb, J., Vinogradov, E., Mocca, C., Pozsgay, V., Shiloach, J., and Schneerson, R. (2009). Synthesis, characterization, and immunogenicity in mice of Shigella sonnei O-specific oligosaccharide-core-protein conjugates. Proc. Natl. Acad. Sci. USA 106, 7974–7978. Safari, D., Dekker, H.A.T., Joosten, J.A.F., Michalik, D., de Souza, A.C., Adamo, R., Lahmann, M., Sundgren, A., Oscarson, S., Kamerling, J.P., and Snippe, H. (2008). Identification of the smallest structure capable of evoking opsonophagocytic antibodies against Streptococcus pneumoniae type 14. Infect. Immun. 76, 4615–4623. Safari, D., Rijkers, G., and Snippe, H. (2012). The future of synthetic carbohydrate vaccines: immunological studies on Streptococcus pneumoniae type 14. In The Complex World of Polysaccharides, D.N. Karunaratne, ed. (Rijeka: InTech), pp. 617–634. Scherf, A., Lopez-Rubio, J.J., and Riviere, L. (2008). Antigenic variation in Plasmodium falciparum. Annu. Rev. Microbiol. 62, 445–470. Schneewind, O., and Missiakas, D. (2011). Structural vaccinology to thwart antigenic variation in microbial pathogens. Proc. Natl. Acad. Sci. USA 108, 10029–10030. Schofield, L., Hewitt, M.C., Evans, K., Siomos, M.A., and Seeberger, P.H. (2002). Synthetic GPI as a candidate anti-toxic vaccine in a model of malaria. Nature 418, 785–789. Schumann, B., Anish, C., Pereira, C.L., and Seeberger, P.H. (2013). Chapter 3 carbohydrate vaccines. In Biotherapeutics: Recent Developments using Chemical and Molecular Biology, L.H. Jones and A.J. McKnight, eds. (London: The Royal Society of Chemistry), pp. 68–104. Seeberger, P.H., and Werz, D.B. (2005). Automated synthesis of oligosaccharides as a basis for drug discovery. Nat. Rev. Drug Discov. 4, 751–763. Seeberger, P.H., and Werz, D.B. (2007). Synthesis and medical applications of oligosaccharides. Nature 446, 1046–1051.

Phalipon, A., Tanguy, M., Grandjean, C., Guerreiro, C., Be´lot, F., Cohen, D., Sansonetti, P.J., and Mulard, L.A. (2009). A synthetic carbohydrate-protein conjugate vaccine candidate against Shigella flexneri 2a infection. J. Immunol. 182, 2241–2247.

Sen, G., Khan, A.Q., Chen, Q., and Snapper, C.M. (2005). In vivo humoral immune responses to isolated pneumococcal polysaccharides are dependent on the presence of associated TLR ligands. J. Immunol. 175, 3084–3091.

Plante, O.J., Palmacci, E.R., and Seeberger, P.H. (2001). Automated solidphase synthesis of oligosaccharides. Science 291, 1523–1527.

Smith, D.F., Song, X.Z., and Cummings, R.D. (2010). Use of glycan microarrays to explore specificity of glycan-binding proteins. Methods Enzymol. 480, 417–444.

Poolman, J., and Borrow, R. (2011). Hyporesponsiveness and its clinical implications after vaccination with polysaccharide or glycoconjugate vaccines. Expert Rev. Vaccines 10, 307–322.

Stein, K.E. (1992). Thymus-independent and thymus-dependent responses to polysaccharide antigens. J. Infect. Dis. 165 (Suppl 1 ), S49–S52.

Pozsgay, V., Chu, C.Y., Pannell, L., Wolfe, J., Robbins, J.B., and Schneerson, R. (1999). Protein conjugates of synthetic saccharides elicit higher levels of serum IgG lipopolysaccharide antibodies in mice than do those of the O-specific polysaccharide from Shigella dysenteriae type 1. Proc. Natl. Acad. Sci. USA 96, 5194–5197. Pozsgay, V., Kubler-Kielb, J., Coxon, B., Santacroce, P., Robbins, J.B., and Schneerson, R. (2012). Synthetic oligosaccharides as tools to demonstrate cross-reactivity between polysaccharide antigens. J. Org. Chem. 77, 5922– 5941. Pujar, N.S., Huang, N.F., Daniels, C.L., Dieter, L., Gayton, M.G., and Lee, A.L. (2004). Base hydrolysis of phosphodiester bonds in pneumococcal polysaccharides. Biopolymers 75, 71–84. Rademacher, C., Shoemaker, G.K., Kim, H.S., Zheng, R.B., Taha, H., Liu, C., Nacario, R.C., Schriemer, D.C., Klassen, J.S., Peters, T., and Lowary, T.L. (2007). Ligand specificity of CS-35, a monoclonal antibody that recognizes mycobacterial lipoarabinomannan: a model system for oligofuranosideprotein recognition. J. Am. Chem. Soc. 129, 10489–10502.

Stockdale, C., Swiderski, M.R., Barry, J.D., and McCulloch, R. (2008). Antigenic variation in Trypanosoma brucei: joining the DOTs. PLoS Biol. 6, e185. Sturgess, A.W., Rush, K., Charbonneau, R.J., Lee, J.I., West, D.J., Sitrin, R.D., and Hennessy, J.P., Jr. (1999). Haemophilus influenzae type b conjugate vaccine stability: catalytic depolymerization of PRP in the presence of aluminum hydroxide. Vaccine 17, 1169–1178. Szu, S.C., Li, X.R., Stone, A.L., and Robbins, J.B. (1991). Relation between structure and immunologic properties of the Vi capsular polysaccharide. Infect. Immun. 59, 4555–4561. Talaga, P., Vialle, S., and Moreau, M. (2002). Development of a high-performance anion-exchange chromatography with pulsed-amperometric detection based quantification assay for pneumococcal polysaccharides and conjugates. Vaccine 20, 2474–2484. Tamborrini, M., Liu, X., Mugasa, J.P., Kwon, Y.U., Kamena, F., Seeberger, P.H., and Pluschke, G. (2010). Synthetic glycosylphosphatidylinositol microarray reveals differential antibody levels and fine specificities in children with mild and severe malaria. Bioorg. Med. Chem. 18, 3747–3752.

Chemistry & Biology 21, January 16, 2014 ª2014 Elsevier Ltd All rights reserved 49

Chemistry & Biology

Review , P., Sla¨ttega˚rd, R., and Oscarson, S. (2006). Synthesis of stable Teodorovic C-phosphonate analogues of Neisseria meningitidis group A capsular polysaccharide structures using modified Mitsunobu reaction conditions. Org. Biomol. Chem. 4, 4485–4490.

Wang, Z., Chinoy, Z.S., Ambre, S.G., Peng, W., McBride, R., de Vries, R.P., Glushka, J., Paulson, J.C., and Boons, G.J. (2013b). A general strategy for the chemoenzymatic synthesis of asymmetrically branched N-glycans. Science 341, 379–383.

Tsai, Y.H., Liu, X., and Seeberger, P.H. (2012). Chemical biology of glycosylphosphatidylinositol anchors. Angew. Chem. Int. Ed. Engl. 51, 11438–11456.

Werz, D.B., and Seeberger, P.H. (2005). Total synthesis of antigen bacillus anthracis tetrasaccharide–creation of an anthrax vaccine candidate. Angew. Chem. Int. Ed. 44, 6315–6318.

Tsai, T.I., Lee, H.Y., Chang, S.H., Wang, C.H., Tu, Y.C., Lin, Y.C., Hwang, D.R., Wu, C.Y., and Wong, C.H. (2013). Effective sugar nucleotide regeneration for the large-scale enzymatic synthesis of Globo H and SSEA4. J. Am. Chem. Soc. 135, 14831–14839. U.S. Department of Health and Human Services (2012). Recommended Immunizations for Children. (Washington: U.S. Department of Health and Human Services). Verez-Bencomo, V., Ferna´ndez-Santana, V., Hardy, E., Toledo, M.E., Rodrı´guez, M.C., Heynngnezz, L., Rodriguez, A., Baly, A., Herrera, L., Izquierdo, M., et al. (2004). A synthetic conjugate polysaccharide vaccine against Haemophilus influenzae type b. Science 305, 522–525. Villeneuve, S., Souchon, H., Riottot, M.-M., Mazie´, J.-C., Lei, P., Glaudemans, C.P.J., Kova´c, P., Fournier, J.-M., and Alzari, P.M. (2000). Crystal structure of an anti-carbohydrate antibody directed against Vibrio cholerae O1 in complex with antigen: molecular basis for serotype specificity. Proc. Natl. Acad. Sci. USA 97, 8433–8438. Vince, P., Bruce, C., Cornelis, P.J.G., Rachel, S., and John, B.R. (2003). Towards an oligosaccharide-based glycoconjugate vaccine against shigella dysenteriae type 1. ChemInform. Published online July 9, 2003. http://dx. doi.org/10.1002/chin.200329260. Visvesvara, G.S., and Garcia, L.S. (2002). Culture of protozoan parasites. Clin. Microbiol. Rev. 15, 327–328. Vulliez-Le Normand, B., Saul, F.A., Phalipon, A., Be´lot, F., Guerreiro, C., Mulard, L.A., and Bentley, G.A. (2008). Structures of synthetic O-antigen fragments from serotype 2a Shigella flexneri in complex with a protective monoclonal antibody. Proc. Natl. Acad. Sci. USA 105, 9976–9981.

Wessels, M.R., Paoletti, L.C., Guttormsen, H.K., Michon, F., D’Ambra, A.J., and Kasper, D.L. (1998). Structural properties of group B streptococcal type III polysaccharide conjugate vaccines that influence immunogenicity and efficacy. Infect. Immun. 66, 2186–2192. Woods, R., and Yongye, A. (2012). Computational techniques applied to defining carbohydrate antigenicity. In Anticarbohydrate Antibodies, P. Kosma and S. Mu¨ller-Loennies, eds. (Vienna: Springer), pp. 361–383. World Health Organization (2000). Recommendations for the production and control of Haemophilus influenzae type b conjugate vaccines. World Health Organ. Tech. Rep. Ser. 897, 27–60. World Health Organization (2003). Recommendations for the Production & Control of Pneumococcal Conjugate Vaccines. (Geneva: World Health Organization). World Health Organization (2005). Recommendations for diphtheria, tetanus, pertussis and combined vaccines (Amendments 2003). World Health Organ. Tech. Rep. Ser. 927, 138–147. World Health Organization (2006). Recommendations to Assure the Quality, Safety and Efficacy of Group A Meningococcal Conjugate Vaccines. (Geneva: World Health Organization). World Health Organization (2009). Recommendations to Assure the Quality, Safety and Efficacy of Pneumococcal Conjugate Vaccines. (Geneva: World Health Organization). World Health Organization (2012a). Chagas disease (American trypanosomiasis) – fact sheet (revised in August 2012). Wkly. Epidemiol. Rec. 87, 519–522.

Wang, L.X., Ni, J., Singh, S., and Li, H. (2004). Binding of high-mannose-type oligosaccharides and synthetic oligomannose clusters to human antibody 2G12: implications for HIV-1 vaccine design. Chem. Biol. 11, 127–134.

World Health Organization (2012b). Recommendations to Assure the Quality, Safety and Efficacy of DT-based Combined Vaccines. (Geneva: World Health Organization).

Wang, J., Li, H., Zou, G., and Wang, L.X. (2007). Novel template-assembled oligosaccharide clusters as epitope mimics for HIV-neutralizing antibody 2G12. Design, synthesis, and antibody binding study. Org. Biomol. Chem. 5, 1529–1540.

Wu, C.Y., and Wong, C.H. (2011). Programmable one-pot glycosylation. Top. Curr. Chem. 301, 223–252.

Wang, C.H., Li, S.T., Lin, T.L., Cheng, Y.Y., Sun, T.H., Wang, J.T., Cheng, T.J.R., Mong, K.K.T., Wong, C.H., and Wu, C.Y. (2013a). Synthesis of Neisseria meningitidis serogroup W135 capsular oligosaccharides for immunogenicity comparison and vaccine development. Angew. Chem. Int. Ed. Engl. 52, 9157–9161.

Zhu, J., Warren, J.D., and Danishefsky, S.J. (2009). Synthetic carbohydratebased anticancer vaccines: the Memorial Sloan-Kettering experience. Expert Rev. Vaccines 8, 1399–1413. Zou, W., Mackenzie, R., The´rien, L., Hirama, T., Yang, Q., Gidney, M.A., and Jennings, H.J. (1999). Conformational epitope of the type III group B Streptococcus capsular polysaccharide. J. Immunol. 163, 820–825.

50 Chemistry & Biology 21, January 16, 2014 ª2014 Elsevier Ltd All rights reserved