Advancing Homogeneous Antimicrobial Glycoconjugate Vaccines

May 2, 2017 - Biography. Roberto Adamo received his Ph.D. in Pharmaceutical Sciences at the University of Catania in 2004. After a postdoctoral fellow...
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Advancing Homogeneous Antimicrobial Glycoconjugate Vaccines Roberto Adamo* GSK Vaccines, Via Fiorentina 1, 53100 Siena, Italy

CONSPECTUS: Since 2004, when the first synthetic glycoconjugate vaccine against the pneumonia and meningitis causing bacterium Haemophilus influenza type b (Hib) approved for human use in Cuba was reported, 34 million doses of the synthetic vaccine have been already distributed in several countries under the commercial name of Quimi-Hib. However, despite the success of this product, no other synthetic glycoconjugate vaccine has been licensed in the following 13 years. As well as avoiding the need to handle pathogens, synthetic glycoconjugates offer clear advantages in terms of product characterization and the possibility to understand the parameters influencing immunogenicity. Nevertheless, large scale application of synthetic sugars has been perceived as challenging because of manufacturing costs and process complexity compared to natural polysaccharides. Chemoenzymatic approaches, one-pot protocols, and automated solid-phase synthesis are rendering carbohydrate production considerably more attractive for industrialization. Here we identify three areas where chemical approaches can advance this progress: (i) chemical or enzymatic methods enabling the delivery of the minimal polysaccharide portion responsible for an effective immune response; (ii) site-selective chemical or enzymatic conjugation strategies for the exploration of the conjugation point in immune responses against carbohydrate-based vaccines, and the consistent preparation of more homogeneous products; (iii) multicomponent constructs targeting receptors responsible for immune response modulation in order to control its quality and magnitude. We discuss how synthesis of bacterial oligosaccharides is useful toward understanding the polysaccharide portion responsible for immunogenicity, and for developing robust and consistent alternatives to natural heterogeneous polysaccharides. The synthesis of sugar analogues can lead to the identification of hydrolytically more stable versions of oligosaccharide antigens. The study of bacterial polysaccharide biosynthesis aids the development of in vitro hazard-free oligosaccharide production. Novel site-selective conjugation methods contribute toward deciphering the role of conjugation sites in the immunogenicity of glycoconjugates and prove to be particularly useful when glycans are conjugated to protein serving as carrier and antigen. The orthogonal incorporation of two different carbohydrate haptens enables the reduction of vaccine components. Finally, coordinated conjugation of glycans and small molecule immunopotentiators supports simplification of vaccine formulation and localization of adjuvant. Synergistic advancement of these areas, combined with competitive manufacturing processes, will contribute to a better understanding of the features guiding the immunological activity of glycoconjugates and, ultimately, to the design of improved, safer vaccines.

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INTRODUCTION In 2004, a paper appeared reporting the first synthetic glycoconjugate vaccine against the pneumonia and meningitis causing bacterium Haemophilus influenza type b (Hib) approved for human use in Cuba.1 Since then, 34 million doses of the synthetic vaccine have been distributed in several countries under the commercial name of Quimi-Hib. Hib was the first pathogen targeted by a commercial glycoconjugate product and is a good example of advances made in carbohydrate-based vaccination using microbial polysaccharides. The pioneering studies of Avery and Goebel in the 1930s2 were applied to the Hib polysaccharide by the John Robbins’ group in the 1970s3 and paved the way to the first © 2017 American Chemical Society

glycoconjugate vaccine. The covalent linkage to a carrier protein transforms carbohydrates inherently unable to provoke a T cell memory response to immunogens, triggering production of memory T cells. Upon uptake by antigen presenting cells (APCs) and intracellular digestion, the carrier protein provides peptide epitopes for the engagement of T cells by re-exposition in complexes with MHC.4 T cells induce the maturation of B cells bearing highly polysaccharide-specific IgGs. This model has been challenged recently by the finding that the peptide can anchor the covalently linked sugar to the Received: March 3, 2017 Published: May 2, 2017 1270

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Figure 1. Parameters affecting the immune-activity of glycoconjugates.

Figure 2. Synthetic bacterial glycans from C. difficile (1), C. albicans (2), S. aureus type 5 (3), and GBS type III (4).

direct this step to preselected sites. Three major areas where chemical approaches aid the engineering of modern glycoconjugates are identifiable: (i) approaches for glycan assembly, (ii) conjugation methods, and (iii) strategies for multicomponent constructs.

MHC molecule so that it can be recognized by a carbohydratespecific T cell.5 Chemical conjugation therefore assures efficacious vaccination of children with carbohydrate-based vaccines against various infectious diseases, such as Neisseria meningitidis and Streptococcus pneumoniae.4 The Cuban Hib vaccine proved that it was possible to translate a synthetic approach from the bench to a real drug product.1 As well as avoiding the handling of pathogens during polysaccharide extraction, synthetic antigens offer clear advantages such as a more defined structure, ease of characterization, lack of bacterial contaminants, higher batchto-batch reproducibility, and more robust correlation of the elicited immune response with the chemical structure. The immunogenicity of glycoconjugates is the result of the cooperation of diverse factors depending on the glycan and the protein component, summarized in Figure 1. Natural polysaccharides used for vaccine manufacturing are heterogeneous mixtures, and the impact of variables like length or substitution pattern cannot be easily established.6 Conjugation typically involves chemical manipulations of sugar residues, or the downstream terminal aldehyde, which are stochastically coupled to the protein. Consequently, for many years, it was not possible to unravel the impact of the conjugation site on immunogenicity. Recently, the attachment position is emerging as a variable to be considered in vaccine design.4 According to the classic mechanism model, the conjugation to T cell peptide epitopes could be detrimental to the activity; on the contrary it could even improve MHC presentation in the proposed mechanism. Because of their defined length and composition of glycoconjugates, the understanding of the parameters impacting their immune-activity is better achieved by means of synthetic carbohydrates. Furthermore, synthetic glycans are coupled to the protein using a linker preinstalled at the downstream end of the oligosaccharides, allowing preservation of the integrity of sugar epitopes. The number of carbohydrate moieties loaded onto the protein can be in-process monitored by mass spectrometry. Site-selective conjugation methods can now

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APPROACHES FOR GLYCAN ASSEMBLY The chemical synthesis of oligosaccharides requires a combination of efficient protective strategies7 and convergent block-wise approaches.8 Our efforts focused on obtaining a series of glycans from a variety of pathogens in order to elucidate the polysaccharide portion responsible for immunogenicity and to identify vaccine candidates (Figure 2). Clostridium difficile structures provided the information that the phosphorylated PSII hexasaccharide repeating unit 1 was capable, following conjugation to Cross Reacting Material 197 (CRM197), to mimic the conjugated polysaccharide, since they both induced comparable levels of anti-PSII antibodies.9 The synthetic approach avoids large scale fermentation of pathogen. The synthesis of a small set of glucans with β-(1 → 3) and β(1 → 6) ramifications was used to ascertain that the immune response toward conjugated Laminarin (Lam), a prototype antifungal vaccine candidate obtained from algae, was primarily directed to a linear β-(1 → 3)-glucan hexamer, suggesting that this was the minimal fragment responsible for immunogenicity.10 Conjugated hexamer 2 gave murine antibody levels comparable to the Lam conjugate. Since the Lam extracted from algae is characterized by a heterogeneous distribution of the branchings, the synthetic molecule offers a robust and process-consistent alternative. With structure 3 we verified by immune dot blotting the correctness of Staphylococcus aureus type 5 assigned structures.11 The synthesis of the counterpart amenable for conjugation was later achieved by Boons et al.12 A set of fragments from Group B Streptococcus (GBS) type III capsular polysaccharide allowed elucidation of the relevance of the Glc residue β-(1 → 6) linked to GlcNAc of 4 for antibody 1271

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Figure 3. (a) Structures of conjugated native MenA and analogues; (b) enzymatic MenA synthesis; (c) structures of MenX CPS and 4P-GlcNAc standard.

recognition,13 and mapping of the epitope recognized by protective antibodies.14 A tremendous advantage of carbohydrate synthesis is selective modification of structures to improve glycoconjugate stability. A paradigm of this approach is Neisseria meningitidis serogroup A capsular polysaccharide (MenA CPS, Figure 3). The intrinsic chemical lability of this polymer is due to the presence of phosphodiester linkages sensitive to hydrolysis. Although stability issues can be classically overcome by storage at 2−8 °C or freeze-drying, the availability of stable antigens that do not require refrigeration for vaccine distribution would be highly desirable. Lay and co-workers proposed that the replacement of either the ring oxygen or the phosphate ester of the MenA CPS repeating unit with a methylene would result in increased stability and, accordingly, carba15 or C-phosphono16 analogues of the natural polysaccharide were designed. We collaborated on this concept, engineering glycoconjugates of these carbohydrate analogues (Figure 3a). Among a set of carba-analogue mono-, di- and trimer, the latter was found to be the minimal oligomer which, when conjugated to CRM197, elicited bactericidal antibodies, although it was not comparable to a benchmark MenA conjugate.17 Human serum albumin (HSA) conjugates of C-phosphono molecules induced T cell proliferation and IL-2 release, and stimulated specific IgG antibody production in mice. However, in this class of analogues the immunogenicity of the trimer was not superior to the monomer.18 These approaches, while needing further

optimization, highlight the potential of carbohydrate chemistry for antigen stabilization. Chemical synthesis is a multistep process requiring many manipulations and chromatographic columns for purification of the intermediates; therefore, ways of accelerating this process are desirable. Along with methods like one-pot glycosylation19 or solid phase automated synthesis, 20 chemoenzymatic approaches21 offer the potential to produce glycans in a shorter amount of time. In this framework, understanding the biosynthesis of natural polysaccharide is an important prerequisite. In order to recreate in vitro the assembly of capsular polysaccharide, Gerardy-Schahn’s group isolated meningoccocal bacterial polymerases. Region A of the MenA capsule gene cluster contains the genetic information for CPS biosynthesis. The open reading frames csaA, -B, and -C were cloned and expressed to produce the UDP-N-acetyl-D-glucosamine-2-epimerase, the poly-ManNAc-1-phosphate-transferase, and the O-acetyltransferase, respectively, which cooperate in the polymer biosynthesis.22 This was established to begin with the epimerization of UDPGlcNAc to UDP-ManNAc by CsaA, followed by the CsaBmediated condensation of ManNAc-1P at position 6 of the downstream end sugar of an oligosaccharide acceptor and then a final CsaC-promoted acetylation (Figure 3b).22 By using synthetic acceptors, it was found that at least a dimer is needed to initiate polymer assembly. The use of linker equipped synthetic acceptors indicated that, in principle, oligomers ready for conjugation could be attained. CsaB was shown to be 1272

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Figure 4. Site-directed conjugation of the β-(1 → 3)-glucan hexamer.

accessibility of the structure to solvent can be predicted in silico.32 The 40 amines, deriving from 39 lysine residues and the Nterminus, are typically targeted in its conjugation. An MS-based assessment of the modified residues in meningococcal vaccines highlighted that 20 out of the 39 lysines are typically involved.33 Starting from this information we directed our attention to a method described by Barbas’ group based on the addition of 4phenyl-1,2,4-triazoline-3,5-dione (PTAD) to phenol in water.34 We reasoned that tyrosine is less accessible than lysine, thus its modification would be more regioselective. Surprisingly, when this reaction was applied to CRM197, it occurred at lysine rather than tyrosine. This outcome was due to the concurrent decomposition of PTAD in the corresponding isocyanate derivative, which was prone to reaction with amines. By using tris(hydroxymethyl)aminomethane as an isocyanate scavenger, the issue was successfully overcome. Based on this result, we designed an approach to obtain a glycoconjugate 9 with defined attachment sites (Figure 4a). The synthesis of the β-glucan hexamer antigen previously identified as a potential antiCandida vaccine was revisited, incorporating an azide linker for later Huisgen 1,3-dipolar cycloaddition (CuAAC) to tyrosine modified with an alkyne spacer. In the click chemistry-based two-step conjugation, we envisaged the advantages of orthogonality with the protein chemical groups and predetermination of the conjugation sites by ESI MS mapping. To test these concepts, CRM197 was reacted with an alkynetriazolinedione spacer, modifying Y27, 46, 358, and 380 out of a total of 18 tyrosines, as determined by MS/MS analysis of protein digests. In parallel, the sugar antigen was synthesized by a [2 + 2] convergent approach affording, in good overall yield, the deprotected hexamer, which was efficiently clicked to the four modified tyrosines in the presence of Cu(II). In spite of previous reports highlighting the need of a high glycosylation density to achieve robust immunogenicity with short oligosaccharides,35 we found that the anticarbohydrate antibodies raised in mice by the defined conjugate were at levels comparable to the heterogeneous Lam randomly conjugated to CRM 197 . This gave a first proof that conjugation of oligosaccharides with sufficient length at a limited number of sites can lead to good levels of antisugar IgGs.

unable to transfer synthetic 3-O-Ac-ManNAc-1P, proving that acetylation is a postassembly modification.22 Similarly, enzymatic synthesis of N. meningitidis serogroup X CPS was developed using a recombinant version of the polymerase CsxA23 (Figure 3c). Glycoconjugates of the obtained oligomer were used to immunize mice giving results non-inferior to products prepared from the natural polymer.24 For analytical characterization of MenX and quantification25,26 of conjugated sugar, a synthetic procedure for rapid preparation of the commercially unavailable 4P-GlcNAc standard was also developed.27 This approach offers the advantage of reducing biohazards and accelerating the production of biopolymers for vaccine use, when the requisite enzyme is available.

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FROM RANDOM TO SITE SELECTIVE CONJUGATION

Classically, glycoconjugates in the market or in preclinical/ clinical development are prepared by targeting the most abundant protein functional groups (the amine of lysine, carboxyl group of aspartate/glutamate or thiol from cysteine).28 Site-selective methods would allow correlation of the immunogenicity of defined glycans not only to specific features of the sugar but also to the attachment point, fully exploiting the potential of carbohydrate synthesis.5 A seminal example of a coordinate preparation of a glycoconjugate with a single attachment point was developed by Davis’ group29 by thiol−ene addition of a synthetic oligorhamnosides from Klebsiella pneumoniae to the cysteine of the Bacillus subtilisin mutant S156C. Site-selective methods have undergone an immense development in the field of antibody modification, where produgs are linked to ensure delivery of cytotoxic molecules to targeted biological sites.30 Inspired by these approaches, we collaborated with Q.-Y. Hu to develop tyrosine ligation of carbohydrates.31 CRM197 was an attractive target since this protein is a component of commercial vaccines,4 and it differs from other carriers (Diphteriae toxin and Tetanus toxin) since it has been detoxified by mutation of Gly to Glu in position 5232 instead of formylation. The resolved crystal structure is available and 1273

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Figure 5. GBS polysaccharides and linkers used for tyrosine directed coupling to pilus proteins.

Encouraged by these results, we applied a two-step conjugation to determine the more accessible lysines of CRM197 by semiquantitative MS/MS mapping of the residues involved in the reaction with N-hydroxysuccinimidyl alkyne or azide linkers for future CuAAC with glycans. The availability of the lysine appeared to be influenced by the amino acidic contour in addition to the surface exposition.36 For instance, K244 is on the surface but forming a salt bridge with adjacent aspartic acid results in limited flexibility and low nucleophilicity. In line with computational calculations of solvent accessibility, K103, K221, K236, K242, K498, and K526 were identified as the privileged residues in conjugate 10 (Figure 4b). Guided by these findings, we synthesized a glycoconjugate targeting through CuAAC a more restricted number of lysines compared to classic random conjugation. In a follow-up immunological study the tyrosine- and lysine-directed conjugates were compared. Additionally, a third construct was prepared by a novel triazolinedione spacer enabling condensation of two sugar moieties per tyrosine (11, Figure 4b).37 This linker was designed to investigate the effect of different spatial geometries in the multivalent exposition of sugar haptens around the protein core. Anti-Lam β-glucan antibody levels induced in mice by the different constructs confirmed that four or six glycans conjugated to defined tyrosine or lysine residues, respectively, were sufficient for a robust immuneresponse. Sera from the novel type of conjugates were even more effective than anti-Lam serum itself in inhibiting the fungal adhesion to human epithelial cells. With a robust protocol for tyrosine-directed conjugation in hand, we focused on its application for connecting polysaccharides and protein antigens from different serogroups/ serotypes of one pathogen. This would expand coverage and simplify the formulation of vaccines where targeting of multiple strains is necessary to ensure adequate protection from disease. We selected as a model Group B Streptococcus, a pathogen responsible of invasive infections in pregnant women, new-

borns and elderly people. CPS conjugates of different serotypes were proven immunogenic in preclinical studies, and vaccines based on polysaccharides Ia, Ib and III, II were tested in clinical trials.38 Due to the changing epidemiology, future extension of the vaccine to CPS II and V can be foreseen. The use of alternatives to classic carriers could bypass the reduction of the anticarbohydrate immune response against glycoconjugate vaccines owing to pre-exposure to the carrier (carrier epitope suppression).39 Keeping all of these requirements in mind, we applied our protocol for tyrosine-directed conjugation of GBS carbohydrates to pathogen related proteins, with the aim of targeting a few defined amino acid residues and protecting the immune activity of the protein as antigen. Unpredictably, when coupling of the high molecular weight negatively charged GBS polysaccharides to the tyrosine residues of CRM197 modified with an azide spacer was attempted by CuAAC, scarce yields of the desired glycoconjugates were obtained.40 Conversely, condensation of short 4-pentinyl lactose was readily achieved, suggesting that the CuAAC was hampered by the reduced accessibility of the catalyst to these large molecules. We then turned our attention to the strain-promoted azide− alkyne [3 + 2] cycloaddition (SPAAC),41 which benefits from a high rate of the cycloaddition and avoids metal catalysis. The protocol was first optimized using short synthetic oligosaccharides bearing an amine linker, and then transferred to the polysaccharide (Figure 5). Despite the variety of alkyne systems for SPAAC reported in the literature, we focused on the use of the commercially available MFCO-NHS ester 12 (Figure 5). Overnight incubation of the labeled proteins at 200−300 μM azide concentration with a 2-fold excess of MFCO-glycan proved sufficient to achieve complete reaction regardless of the sugar length. Therefore, GBS CPSs were oxidized at the sialic residue to obtain an aldehyde which was then converted to amine by reductive amination with ammonium acetate and NaBH3CN. 1274

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Figure 6. (a) Disulfide rebridging with dichloroacetone (DCA); (b) mTGAase catalyzed lysine modification; (c) KDO modification.

detrimental to antipolysaccharide and antiprotein immuneresponses. Seeking a solution to this issue, the bifunctional spacer 15 was designed to enable installation of maleimide groups onto tyrosine.43 The spacer was tested for conjugation of GBS67 to CPSV in contrast with the previous linkers. To further expand the toolbox for lysine conjugation we turned our attention also to microbial Transglutaminases (TGase), a family of enzymes that catalyze the formation of a covalent bond between the γ-carbonyl amide group of glutamines and the primary amine of lysines. The short Cbz-Gln-Gly-NH-(PEG)3 azide linker 16 was synthesized and enzymatically condensed to K320, K340, K558, and K812 of GBS67. The polysaccharide in which NeuAc had been modified with thiol or azide groups was then coupled to the proteins by thiolmaleimide addition or click chemistry respectively, depending on the type of spacer inserted into the protein in the first step. Following immunization in mice, the combination of tyrosinedirected ligation and thiol-maleimide addition emerged as optimal to ensure production of functional antipolysaccharide and antiprotein while eliminating stimulation of antilinker antibodies. To allow precise correlation of the immunogenicity of a glycoconjugate and a certain modified residue, methods enabling a single attachment point are essential. An important step toward this was provided by the development in the Q.-Y. Hu laboratory of a method for disulfide rebridging allowing incorporation of a keto group at a defined position of CRM197.44 Of the two disulfide bridges present in the protein, the C461−C471 bond is buried inside the protein, and the C186−C201 is surface-exposed (Figure 6a). Upon treatment with TCEP, the disulfide can be opened and reacted with dichloroacetone to provide a stable stapling product incorpo-

Incorporation of the MFCO linker by reaction of the NHS derivative with the amine of CPS rendered it ready for conjugation. In tandem, GBS pili proteins were modified with the new linker for tyrosine coupling 14 where a phenyl connection between the PEG portion and the triazolinedione was removed to reduce the induction of antispacer antibodies. In the PI-1 protein GBS80, the reaction occurred at the Nterminal flexible domain of the protein and Y16, Y23, Y44, and Y135 were modified. For Pl-2a protein GBS67, Y744 resulted in a higher level of modification, whereas Y282/283, Y336/337, and Y403 were derivatized to a lower extent. After incubation of the protein and polysaccharide, SPAAC of the two partners was finally confirmed by gel electrophoresis and Western blot analysis. In a first study, the developed procedure was applied to synthesize a glycoconjugate of GBS PSII connected to the tyrosines of GBS80. The bicomponent vaccine was effective in inducing anticarbohydrate and antiprotein antibodies comparably to a control prepared by random glycan-conjugation to the lysines of the protein.42 The levels of anticarbohydrate antibodies were also similar to those induced by a PSII-CRM197 conjugate. These titers translated into effective opsonophagocytic killing of strains selectively expressing the CPS and the protein. Higher protection against strains expressing CPSII or GBS80 was achieved by this conjugate in respect to its counterpart prepared by random conjugation at lysine residues. Hence, in vivo evaluation confirmed that the immunogenicity of the tyrosine-modified protein was preserved by the CPS condensation with the added benefit of directing conjugation to predetermined sites. The major drawback in the preparation using CuAAC and SPACC was the generation of antibodies directed to the strained rings generated by the two reactions that were not 1275

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Figure 7. Orthogonal incorporation of two different ligands on CRM197.

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THE CONSTRUCTION OF MULTICOMPONENT GLYCOCONJUGATES Emerging pathogens are typically targeted by a combination of antigens, which increases the complexity of vaccine formulation. A tactic for simplifying vaccine formulation could be the creation of constructs with multiple antigens attached to the same protein molecule. For this, we developed an orthogonal conjugation strategy based on modification of the more exposed lysine residues with a maleimide linker, followed by reaction of the unoccupied lysine residues with carbohydrates bearing an N-hydroxysuccinimidyl linker, and final thiol-maleimide coupling of a second sugar hapten bearing a sulfhydryl spacer (Figure 7). Using this approach, we assembled a bivalent MenA/MenC conjugate that, at the second boost, elicited functional antibodies against the two diverse meningococcal serogroups.49 Conjugation of two different haptens can be also applied to create immunopotentiated vaccines50 where the adjuvant benefits of the multivalent exposition around the protein core for stronger receptor engagement, and codelivery with the glycoconjugate to cells responsible for uptake and processing. Initially, we showed that the synthetic β-glucan hexamer (which per se is a weak agonist of Dectin-1, a C-lectin involved in modulating the innate immune response) can be turned into a stronger binder by multivalent presentation in the form of CRM197.51 The optimal saccharide density for Dectin-1 activation was identified in vitro, demonstrating in vivo to augment the antiprotein antibody titers at a level similar to Al(OH)3, an adjuvant classically used for vaccines. This suggested that β-glucans are self-adjuvanted sugars. A similar notion was used in Bundle’s group, who incorporated Lam onto a trimannoside-TT anti Candida conjugate vaccine to enhance the antimannan response, indicating the feasibility of multicomponent antimicrobial constructs.52 Inspired by this work, we conjugated Toll Like Receptor (TLR) synthetic agonist to a MenC conjugate to take advantage of the protein internalization for the delivery of the small molecule to the receptor.53 TLRs are a family of receptors that activate innate immunity and initiate a cascade of immune responses thus affecting the magnitude and the persistence of the immune response.54 The use of TLR7 agonists as adjuvants is known; however, their proinflammatory response could produce side effects. Conjugation to the antigen could localize the adjuvant and limit undesirable events. Orthogonal conjugation based on thiolmaleimide coupling of thiolated MenC antigen to a protein

rating a ketone moiety. Reaction with a bifunctional oxyamineazide linker provided access to glycoconjugates with polysaccharides modified with a strained alkyne ready for SPAAC. Having this methodology to hand, a large set of glycoconjugates were prepared; in addition to random incorporation of azide linkers at Lys and Asp/Glu, more homogeneous constructs were obtained by tyrosine-directed triazolidinone-ene reaction and controlled Lys conjugation, respectively. mTGase was also used to insert the Cbz-Gln-GlyNH-(PEG)3 (ZQC) azide linker into CRM197 (Figure 6b). Exceptional control of this reaction was achieved by tuning the pH of the reaction buffer, and selective acylation of K33 was observed at pH 8 after 18 h. By lowering the pH to 6 and increasing the reaction time to 72 h, the additional labeling of K37/39 occurred. The different modified CRM197 were clicked by SPAAC to the O-antigen of Salmonella enterica serovar typhimurium by taking advantage of the reactivity of the KDO sugar residue with a dihydrazine spacer through reductive amination and subsequent coupling of the condensed cyclopropane-cyclooctyne system. Immunological testing in mice of the different constructs led to identification of a single modification (C186−C201) giving very high functional antisaccharide antibodies, comparable to the random Lys and Asp/Glu or more homogeneous tyrosine and lysine conjugates. Contrarily, single K33 gave the poorest immune response in the series, and further addition of the second labeled lysine restored optimal biological activity. This study represented the long-sought demonstration that site-specific conjugation is useful to decipher the contribution of conjugation sites in the immunogenicity of conjugates. An emerging method for site-selective glycan conjugation is based on the genetic engineering of E. coli with the operon regulating the glycan biosynthesis in the presence of the Campilobacter jejuni glycosylstransferase PglB, which transfers a glycolipid donor to an NXST sequence. This technique, which is under development at Limmatech Biologics (formely GlycoVaxyn), allows the in vivo production of reasonably homogeneous N-glycoproteins from bacterial oligosaccharides with a 2-acetamido downstream end and with the attachment site predetermined by adequate positioning of the peptide sequence.45,46 Vaccine candidates against Shigella dysenteriae type 147 and antiextraintestinal pathogenic E. coli48 produced by this method recently entered clinical trial, emphasizing the potential of site-selective conjugation. 1276

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Accounts of Chemical Research previously coupled to the TLR7a through active ester chemistry was used to assemble the adjuvanted vaccine. The multicomponent construct elicited higher anti-MenC antibodies than coadministration of the unconjugated TLR7a molecule and the MenC-CRM197 conjugate. Despite this positive result, a limitation of this approach was found in the amount of adjuvant incorporated onto the glycoconjugate. Presentation of TLR7a phosphonate adsorbed onto Al(OH)3 resulted, in a more flexible manner, in an increased anticarbohydrate response. The adjuvant effect of the multicomponent vaccine could be tuned by identifying more potent agonists.

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ACKNOWLEDGMENTS

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REFERENCES

I am deeply grateful to internal and external collaborators: Dr. Paolo Costantino, Dr. Francesco Berti, and Dr. Maria Rosaria Romano, from GSK Vaccines, Dr. Francesca Micoli from GSK Vaccines For Global Health; Dr. Qi-Ying Hu from Novartis Institute for Biomedical Research; Prof. Luigi Lay from Milan University; Prof. Rita Gerardy-Schahn from Hannover Medical Center, and all students and associates who have contributed with passion and dedication to these projects.

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CONCLUSIONS AND PERSPECTIVES Glycoconjugates based on synthetic glycans are an attractive vaccine approach, particularly when the polysaccharide is difficult to obtain, as purification or fermentation of highly dangerous pathogens is required. However, despite the success of Quimi-Hib, no other synthetic vaccine has been licensed in the last 13 years. Large scale application of synthetic sugars has been challenged by manufacturing costs and complexity compared to natural polysaccharides. Commercial synthetic heparins and novel lectin agonists55 under clinical phase are good examples of carbohydrate production optimization through process development. Recently, methods simplifying and accelerating the preparation of glycans and corresponding glycoconjugates have been rapidly maturing. Chemoenzymatic approaches, one-pot protocols, and automated solid-phase synthesis are rendering carbohydrate production considerably more attractive for industrialization.56,57 Here we identified three areas where chemical approaches can advance this progress. Chemical or enzymatic synthesis can deliver defined polysaccharide portions responsible for an effective immune response; site-selective conjugation strategies aid the exploration of the conjugation point in the immune response against carbohydrate-based vaccines; and multicomponent constructs help to control the quality and magnitude of the immune response by targeting the vaccine to specific receptors. Synergies among these areas, accompanied by competitive manufacturing processes, will contribute to a better understanding of the parameters that govern the immunological activity of glycoconjugates, and promote the design of improved, safer vaccines.

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(1) Verez-Bencomo, V.; Fernandez-Santana, V.; Hardy, E.; Toledo, M. E.; Rodrıguez, M. C.; Heynngnezz, L.; Rodriguez, A.; Baly, A.; Herrera, L.; Izquierdo, M.; Villar, A.; Valdes, Y.; Cosme, K.; Deler, M. L.; Montane, M.; Garcia, E.; Ramos, A.; Aguilar, A.; Medina, E.; Torano, G.; Sosa, I.; Hernandez, I.; Martınez, R.; Muzachio, A.; Carmenates, A.; Costa, L.; Cardoso, F.; Campa, C.; Diaz, M.; Roy, R. A synthetic conjugate polysaccharide vaccine against Haemophilus influenzae Type b. Science 2004, 305, 522−525. (2) Avery, O. T.; Goebel, W. F. Chemo-immunological studies on conjugated carbohydrate-proteins. J. Exp. Med. 1931, 54, 437−447. (3) Schneerson, R.; Barrera, O.; Sutton, A.; Robbins, J. B. Preparation, characterization, and immunogenicity of Haemophilus influenzae type b polysaccharide-protein conjugates. J. Exp. Med. 1980, 152, 361−376. (4) Costantino, P.; Rappuoli, R.; Berti, F. The design of semisynthetic and synthetic glycoconjugate vaccines. Expert Opin. Drug Discovery 2011, 6, 1045−1067. (5) Avci, F. Y.; Li, X.; Tsuji, M.; Kasper, D. L. A mechanism for glycoconjugate vaccine activation of the adaptive immune system and its implications for vaccine design. Nat. Med. 2011, 17, 1602−1610. (6) Adamo, R.; Nilo, A.; Castagner, B.; Boutureira, O.; Berti, F.; Bernardes, G. J. L. Synthetically defined glycoprotein vaccines: Current status & future directions. Chem. Sci. 2013, 4, 2995−3008. (7) Cattaneo, V.; Oldrini, D.; Corrado, A.; Berti, F.; Adamo, R. Orthogonal cleavage of the 2-naphthylmethyl group in the presence of the p-methoxy phenyl-protected anomeric position and its use in carbohydrate synthesis. Org. Chem. Front. 2016, 3, 753−758. (8) Smoot, J. T.; Demchenko, A. V. Oligosaccharide synthesis: From conventional methods to modern expeditious strategies. Adv. Carbohydr. Chem. Biochem. 2009, 62, 161−250. (9) Adamo, R.; Romano, M. R.; Berti, F.; Leuzzi, R.; Tontini, M.; Danieli, E.; Cappelletti, E.; Cakici, O. S.; Swennen, E.; Pinto, V.; Brogioni, B.; Proietti, D.; Galeotti, C. L.; Lay, L.; Monteiro, M. A.; Scarselli, M.; Costantino, P. Phosphorylation of the synthetic hexasaccharide repeating unit is essential for the induction of antibodies to Clostridium difficile PSII cell wall polysaccharide. ACS Chem. Biol. 2012, 7, 1420−1428. (10) Adamo, R.; Tontini, M.; Brogioni, G.; Romano, M. R.; Costantini, G.; Danieli, E.; Proietti, D.; Berti, F.; Costantino, P. Synthesis of Laminarin fragments and evaluation of a β-(1,3) glucan hexasaccaride-CRM197 conjugate as vaccine candidate against Candida albicans. J. Carbohydr. Chem. 2011, 30, 249−280. (11) Danieli, E.; Proietti, D.; Brogioni, G.; Romano, M. R.; Cappelletti, E.; Tontini, M.; Berti, F.; Lay, L.; Costantino, P.; Adamo, R. Synthesis of Staphylococcus aureus type 5 capsular polysaccharide repeating unit using novel L-FucNAc and D-FucNAc synthons and immunochemical evaluation. Bioorg. Med. Chem. 2012, 20, 6403−6415. (12) Gagarinov, I. A.; Fang, T.; Liu, L.; Srivastava, A. D.; Boons, G. J. Synthesis of Staphylococcus aureus Type 5 trisaccharide repeating unit: solving the problem of lactamization. Org. Lett. 2015, 17, 928− 931. (13) Cattaneo, V.; Carboni, F.; Oldrini, D.; De Ricco, R.; Donadio, N.; Margarit y Ros, I.; Berti, F.; Adamo, R. Synthesis of Group B Streptococcus type III polysaccharide fragments for evaluation of their

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Roberto Adamo: 0000-0001-5228-6088 Notes

The author is an employee of the GSK group of companies. The author declares no competing financial interest. Biography Roberto Adamo received his Ph.D. in Pharmaceutical Sciences at the University of Catania in 2004. After a postdoctoral fellowship at the National Institutes of Health, USA, and University of Utrecht, The Netherlands, he joined Novartis Vaccines in Siena, Italy. Currently, he is Senior Scientist in GSK Vaccines R&D Siena and coordinator of the EU network Glycovax. 1277

DOI: 10.1021/acs.accounts.7b00106 Acc. Chem. Res. 2017, 50, 1270−1279

Article

Accounts of Chemical Research interactions with monoclonal antibodies. Pure Appl. Chem. 2016, DOI: 10.1515/pac-2016-0918. (14) Carboni, F.; Adamo, R.; Fabbrini, M.; De Ricco, R.; Cattaneo, V.; Brogioni, B.; Veggi, D.; Pinto, V.; Passalacqua, I.; Oldrini, D.; Rappuoli, R.; Malito, E.; Margarit, I.; Berti, F. Structure of a protective epitope of Group B Streptococcus type III capsular polysaccharide. Proc. Natl. Acad. Sci. U. S. A. 2017, 1−6. (15) Gao, Q.; Zaccaria, C.; Tontini, M.; Poletti, L.; Costantino, P.; Lay, L. Synthesis and preliminary biological evaluation of carba analogues from Neisseria meningitidis A capsular polysaccharide. Org. Biomol. Chem. 2012, 10, 6673−6681. (16) Torres-Sanchez, M. I.; Zaccaria, C.; Buzzi, B.; Miglio, G.; Lombardi, G.; Polito, L.; Russo, G.; Lay, L. Synthesis and biological evaluation of phosphono analogues of capsular polysaccharide fragments from Neisseria meningitidis A. Chem. - Eur. J. 2007, 13, 6623−6635. (17) Gao, Q.; Tontini, M.; Brogioni, G.; Nilo, A.; Filippini, S.; Harfouche, C.; Polito, L.; Romano, M. R.; Costantino, P.; Berti, F.; Adamo, R.; Lay, L. Immunoactivity of protein conjugates of carba analogues from Neisseria meningitidis a capsular polysaccharide. ACS Chem. Biol. 2013, 8, 2561−2567. (18) Fallarini, S.; Buzzi, B.; Giovarruscio, S.; Polito, L.; Brogioni, G.; Tontini, M.; Berti, F.; Adamo, R.; Lay, L.; Lombardi, G. A synthetic disaccharide analogue from Neisseria meningitidis A capsular polysaccharide stimulates immune cell responses and induces immunoglobulin G (IgG) production in mice when proteinconjugated. ACS Infect. Dis. 2015, 1, 487−496. (19) Gao, J.; Guo, Z. Chemical synthesis of the repeating unit of type V Group B Streptococcus capsular polysaccharide. Org. Lett. 2016, 18, 5552−5555. (20) Schumann, B.; Hahm, H. S.; Parameswarappa, S. G.; Reppe, K.; Wahlbrink, A.; Govindan, S.; Kaplonek, P.; Pirofski, L.-a.; Witzenrath, M.; Anish, C.; Pereira, C. L.; Seeberger, P. H. A semisynthetic Streptococcus pneumoniae serotype 8 glycoconjugate vaccine. Sci. Transl. Med. 2017, 9, eaaf5347. (21) Li, L.; Woodward, R. L.; Han, W.; Qu, J.; Song, J.; Ma, C.; Wang, P. G. Chemoenzymatic synthesis of the bacterial polysaccharide repeating unit undecaprenyl pyrophosphate and its analogs. Nat. Protoc. 2016, 11, 1280−1298. (22) Fiebig, T.; Freiberger, F.; Pinto, V.; Romano, M. R.; Black, A.; Litschko, C.; Bethe, A.; Yashunsky, D.; Adamo, R.; Nikolaev, A.; Berti, F.; Gerardy-Schahn, R. Molecular cloning and functional characterization of components of the capsule biosynthesis complex of Neisseria meningitidis serogroup A. J. Biol. Chem. 2014, 289, 19395−19407. (23) Fiebig, T.; Berti, F.; Freiberger, F.; Pinto, V.; Claus, H.; Romano, M. R.; Proietti, D.; Brogioni, B.; Stummeyer, K.; Berger, M.; Vogel, U.; Costantino, P.; Gerardy-Schahn, R. Functional expression of the capsule polymerase of Neisseria meningitidis serogroup X: A new perspective for vaccine development. Glycobiology 2014, 24, 150−158. (24) Fiebig, T.; Romano, M. R.; Oldrini, D.; Adamo, R.; Tontini, M.; Brogioni, B.; Santini, L.; Berger, M.; Costantino, P.; Berti, F.; GerardySchahn, R. An efficient cell free enzyme-based total synthesis of a meningococcal vaccine candidate. npj Vaccines 2016, 1, 16017. (25) Micoli, F.; Romano, M. R.; Tontini, M.; Cappelletti, E.; Gavini, M.; Proietti, D.; Rondini, S.; Swennen, E.; Santini, L.; Filippini, S.; Balocchi, C.; Adamo, R.; Pluschke, G.; Norheim, G.; Pollard, A.; Saul, A.; Rappuoli, R.; MacLennan, C. A.; Berti, F.; Costantino, P. Development of a glycoconjugate vaccine to prevent meningitis in Africa caused by meningococcal serogroup X. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 19077−19082. (26) Micoli, F.; Adamo, R.; Proietti, D.; Gavini, M.; Romano, M. R.; MacLennan, C. A.; Costantino, P.; Berti, F. Meningococcal X polysaccharide quantification by high-performance anion exchange chromatography using synthetic N-acetylglucosamine-4-phosphate as standard. Anal. Biochem. 2013, 442, 259−261. (27) Adamo, R.; Micoli, F.; Proietti, D.; Berti, F. Efficient synthesis of meningococcal X polysaccharide repeating unit (N-Acetylglucosamine4-phosphate) as analytical standard for polysaccharide determination. Synth. Commun. 2014, 44, 1266−1273.

(28) Hu, Q.-Y.; Berti, F.; Adamo, R. Towards the next generation of biomedicines by site-selective conjugation. Chem. Soc. Rev. 2016, 45, 1691−1719. (29) Grayson, E. J.; Bernardes, G. J.; Chalker, J. M.; Boutureira, O.; Koeppe, J. R.; Davis, B. G. A coordinated synthesis and conjugation strategy for the preparation of homogeneous glycoconjugate vaccine candidates. Angew. Chem., Int. Ed. 2011, 50, 4127−4132. (30) Chari, R. V. J.; Miller, M. L.; Widdison, W. C. Antibody−drug conjugates: An emerging concept in cancer therapy. Angew. Chem., Int. Ed. 2014, 53, 3796−3827. (31) Hu, Q.-Y.; Allan, M.; Adamo, R.; Quinn, D.; Zhai, H.; Wu, G.; Clark, K.; Zhou, J.; Ortiz, S.; Wang, B.; Danieli, E.; Crotti, S.; Tontini, M.; Brogioni, G.; Berti, F. Synthesis of a well-defined glycoconjugate vaccine by a tyrosine-selective conjugation strategy. Chem. Sci. 2013, 4, 3827−3832. (32) Malito, E.; Bursulaya, B.; Chen, C.; Surdo, P. L.; Picchianti, M.; Balducci, E.; Biancucci, M.; Brock, A.; Berti, F.; Bottomley, M. J.; Nissum, M.; Costantino, P.; Rappuoli, R.; Spraggon, G. Structural basis for lack of toxicity of the diphtheria toxin mutant CRM197. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 5229−5234. (33) Bardotti, A.; Averani, G.; Berti, F.; Berti, S.; Carinci, V.; D’Ascenzi, S.; Fabbri, B.; Giannini, S.; Giannozzi, A.; Magagnoli, C.; Proietti, D.; Norelli, F.; Rappuoli, R.; Ricci, S.; Costantino, P. Physicochemical characterisation of glycoconjugate vaccines for prevention of meningococcal diseases. Vaccine 2008, 26, 2284−2296. (34) Ban, H.; Nagano, M.; Gavrilyuk, J.; Hakamata, W.; Inokuma, T.; Barbas, C. F. I. Facile and stabile linkages through tyrosine: Bioconjugation strategies with the tyrosine-click reaction. Bioconjugate Chem. 2013, 24, 520−532. (35) Pozsgay, V.; Chu, C.; Pannell, L.; Wolfe, J.; Robbins, J. B.; Schneerson, R. 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. U. S. A. 1999, 96, 5194−5197. (36) Crotti, S.; Zhai, H.; Zhou, J.; Allan, M.; Proietti, D.; Pansegrau, W.; Hu, Q.-Y.; Berti, F.; Adamo, R. Defined conjugation of glycans to the lysines of CRM197 guided by their reactivity mapping. ChemBioChem 2014, 15, 836−843. (37) Adamo, R.; Hu, Q.-Y.; Torosantucci, A.; Crotti, S.; Brogioni, G.; Allan, M.; Chiani, P.; Bromuro, C.; Quinn, D.; Tontini, M.; Berti, B. Deciphering the structure−immunogenicity relationship of antiCandida glycoconjugate vaccines. Chem. Sci. 2014, 5, 4302−4311. (38) Nuccitelli, A.; Rinaudo, C. D.; Maione, D. Group B Streptococcus vaccine: state of the art. Ther. Adv. Vaccines 2015, 3, 76−90. (39) Dagan, R.; Poolman, J.; Siegrist, C. A. Glycoconjugate vaccines and immune interference: A review. Vaccine 2010, 28, 5513−5523. (40) Nilo, A.; Allan, M.; Brogioni, B.; Proietti, D.; Cattaneo, V.; Crotti, S.; Sokup, S.; Zhai, H.; Margarit, I.; Berti, F.; Hu, Q.; Adamo, R. Tyrosine-directed conjugation of large glycans to proteins via copperfree click chemistry. Bioconjugate Chem. 2014, 25, 2105−2111. (41) Baskin, J. M.; Prescher, J. A.; Laughlin, S. T.; Agard, N. J.; Chang, P. V.; Miller, I. A.; Lo, A.; Codelli, J. A.; Bertozzi, C. R. Copper-free click chemistry for dynamic in vivo imaging. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 16793−16797. (42) Nilo, A.; Morelli, L.; Passalacqua, I.; Brogioni, B.; Allan, M.; Carboni, F.; Pezzicoli, A.; Maione, D.; Fabbrini, M.; Romano, M. R.; Hu, Q.-Y.; Margarit, I.; Berti, F.; Adamo, R. Anti-Group B Streptococcus carbohydrate-based vaccines using pilus proteins as carriers: Comparing lysine and tyrosine-directed conjugation. ACS Chem. Biol. 2015, 10, 1737−1746. (43) Nilo, A.; Passalacqua, I.; Fabbrini, M.; Allan, M.; Usera, A.; Carboni, F.; Brogioni, B.; Pezzicoli, A.; Cobb, J.; Romano, M. R.; Margarit, I.; Hu, Q.-Y.; Berti, F.; Adamo, R. Exploring the effect of conjugation site and chemistry on the immunogenicity of an antiGroup B Streptococcus glycoconjugate vaccine based on GBS67 pilus protein and type V polysaccharide. Bioconjugate Chem. 2015, 26, 1839−1849. 1278

DOI: 10.1021/acs.accounts.7b00106 Acc. Chem. Res. 2017, 50, 1270−1279

Article

Accounts of Chemical Research

(57) Seeberger, P. H. The logic of automated glycan assembly. Acc. Chem. Res. 2015, 48, 1450−1463.

(44) Stefanetti, G.; Hu, Q.-Y.; Usera, A.; Robinson, Z.; Allan, M.; Singh, A.; Imase, H.; Cobb, J.; Zhai, H.; Quinn, D.; Lei, M.; Saul, A.; Adamo, R.; MacLennan, C. A.; Micoli, F. Sugar-protein connectivity impacts on the immunogenicity of site-selective Salmonella O-antigen glycoconjugate vaccines. Angew. Chem., Int. Ed. 2015, 54, 13198− 131203. (45) Kowarik, M.; Numao, S.; Feldman, M. F.; Schulz, B. L.; Callewaert, N.; Kiermaier, E.; Catrein, I.; Aebi, M. N-Linked glycosylation of folded proteins by the bacterial oligosaccharyltransferase. Science 2006, 314, 1148−1150. (46) Feldman, M. F.; Wacker, M.; Hernandez, M.; Hitchen, P. G.; Marolda, C. L.; Kowarik, M.; Morris, H. R.; Dell, A.; Valvano, M. A.; Aebi, M. Engineering N-linked protein glycosylation with diverse Oantigen lipopolysaccharide structures in Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 3016−3021. (47) Hatz, C. F.; Bally, B.; Rohrer, S.; Steffen, R.; Kramme, S.; Siegrist, C. A.; Wacker, M.; Alaimo, C.; Fonck, V. G. Safety and immunogenicity of a candidate bioconjugate vaccine against Shigella dysenteriae type 1 administered to healthy adults: A single blind, partially randomized Phase I study. Vaccine 2015, 33, 4594−4601. (48) Huttner, A.; Hatz, C.; van den Dobbelsteen, G.; Abbanat, D.; Hornacek, A.; Frölich, R.; Dreyer, A. M.; Martin, P.; Davies, T.; Fae, K.; van den Nieuwenhof, I.; Thoelen, S.; de Vallière, S.; Kuhn, A.; Bernasconi, E.; Viereck, V.; Kavvadias, T.; Kling, K.; Ryu, G.; Hülder, T.; Gröger, S.; Scheiner, D.; Alaimo, C.; Harbarth, S.; Poolman, J.; Fonck, V. G. Safety, immunogenicity, and preliminary clinical efficacy of a vaccine against extraintestinal pathogenic Escherichia coli in women with a history of recurrent urinary tract infection: a randomised, single-blind, placebo-controlled phase 1b trial. Lancet Infect. Dis. 2017, 17, 528−537. (49) Adamo, R.; Nilo, A.; Harfouche, C.; Brogioni, B.; Pecetta, S.; Brogioni, G.; Balducci, E.; Pinto, V.; Filippini, S.; Mori, E.; Tontini, M.; Romano, M. R.; Costantino, P.; Berti, F. Investigating the immunodominance of carbohydrate antigens in a bivalent unimolecular glycoconjugate vaccine against serogroup A and C meningococcal disease. Glycoconjugate J. 2014, 31, 637−647. (50) Bongat, A. F. G.; Saksena, R.; Adamo, R.; Fujimoto, Y.; Shiokawa, Z.; Peterson, D. C.; Fukase, K.; Vann, W. F.; Kovác,̌ P. Multimeric bivalent immunogens from recombinant tetanus toxin HC fragment, synthetic hexasaccharides, and a glycopeptide adjuvant. Glycoconjugate J. 2010, 27, 69−77. (51) Donadei, A.; Gallorini, S.; Berti, F.; O’Hagan, D. T.; Adamo, R.; Baudner, B. C. Rational design of adjuvant for skin delivery: Conjugation of synthetic β-Glucan dectin-1 agonist to protein antigen. Mol. Pharmaceutics 2015, 12, 1662−1672. (52) Lipinski, T.; Fitieh, A.; St. Pierre, J.; Ostergaard, H. L.; Bundle, D. R.; Toure, N. Enhanced immunogenicity of a tricomponent mannan Tetanus Toxoid conjugate vaccine targeted to dendritic cells via Dectin-1 by incorporating β-glucan. J. Immunol. 2013, 190, 4116− 4128. (53) Donadei, A.; Balocchi, C.; Mancini, F.; Proietti, D.; Gallorini, S.; O’Hagan, D. T.; D’Oro, U.; Berti, F.; Baudner, B. C.; Adamo, R. The adjuvant effect of TLR7 agonist conjugated to a meningococcal serogroup C glycoconjugate vaccine. Eur. J. Pharm. Biopharm. 2016, 107, 110−119. (54) Kasturi, S. P.; Skountzou, I.; Albrecht, R.; Koutsonanos, D.; Hua, T.; Nakaya, H. I.; Ravindran, R.; Stewart, S.; Alam, M.; Kwissa, M.; Villinger, F.; Murthy, N.; Steel, J.; Jacob, J.; Hogan, R. J.; GarcíaSastre, A.; Compans, R.; Pulendran, B. Programming the magnitude and persistence of antibody responses with innate immunity. Nature 2011, 470, 543−547. (55) Egger, J.; Weckerle, C.; Cutting, B.; Schwardt, O.; Rabbani, S.; Lemme, K.; Ernst, B. Nanomolar E-selectin antagonists with prolonged half-lives by a fragment-based approach. J. Am. Chem. Soc. 2013, 135, 9820−9828. (56) Wang, L. X.; Amin, M. N. Chemical and chemoenzymatic synthesis of glycoproteins for deciphering functions. Chem. Biol. 2014, 21, 51−66. 1279

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