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Synthesis, Liposomal Formulation, and Immunological Evaluation of a Minimalistic Carbohydrate-#-GalCer Vaccine Candidate Felix Broecker, Sebastian Götze, Jonathan Hudon, Dominea C. K. Rathwell, Claney L Pereira, Pierre Stallforth, Anish Chakkumkal, and Peter H Seeberger J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00312 • Publication Date (Web): 09 May 2018 Downloaded from http://pubs.acs.org on May 10, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

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Journal of Medicinal Chemistry

Synthesis, Liposomal Formulation, and Immunological Evaluation of a Minimalistic Carbohydrate-α-GalCer Vaccine Candidate

Felix Broecker,‡abc Sebastian Götze,‡ab Jonathan Hudon,‡a Dominea C. K. Rathwell,ad Claney L. Pereira,ae Pierre Stallforth,af Anish Chakkumkalag and Peter H. Seeberger*ab a

Max Planck Institute of Colloids and Interfaces, Department of Biomolecular Systems, Am Mühlenberg

1, 14424 Potsdam, Germany. b

Freie Universität Berlin, Institute of Chemistry and Biochemistry, Arnimallee 22, 14195 Berlin, Germany.

c

Present address: Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, New

York 10029, USA d

Present address: Department of Chemistry, Yonsei University, Seoul 120-749 Korea.

e

f

Present address: Vaxxilon Deutschland GmbH, 12489 Berlin, Germany.

Present address: Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll

Institute, Junior Research Group Chemistry of Microbial Communication, Beutenbergstraße 11a, 07745 Jena, Germany g

Present address: Bacterial Vaccines Discovery and Early Development, Janssen Pharmaceuticals

(Johnson & Johnson), 2333 CK Leiden, The Netherlands *Author of correspondence. E-mail: [email protected] ‡ Equal contribuHon.

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ABSTRACT Fully synthetic glycan-based vaccines hold great potential as preventive and therapeutic vaccines against infectious diseases as well as cancer. Here, we present a two-component platform based on the facile conjugation of carbohydrate antigens to α-galactosylceramide (αGalCer) to yield fully synthetic vaccine candidates. Formulation of a cancer-associated Tn antigen glycolipid model vaccine candidate into liposomes of different sizes and subsequent immunization of mice generated specific, high-affinity antibodies against the carbohydrate antigen with characteristics of T cell-dependent immunity. Liposome formulation elicited more reproducible glycan immunity than a conventional glycoconjugate vaccine bearing the same glycan antigen did. Further evaluation of the immune response revealed that the size of the liposomes influenced the glycan antibody responses towards either a cellular (Th1) or a humoral (Th2) immune phenotype. The glycolipid vaccine platform affords strong and robust anti-glycan antibody responses in vivo without the need for an external adjuvant.

INTRODUCTION Novel preventive and therapeutic vaccines are urgently needed to fight antibiotic-resistant bacteria1-3 as well as different forms of cancer4. Surface-exposed carbohydrates on pathogens and tumor cells are particularly promising antigens for vaccines against infectious diseases or cancer, respectively. Carbohydrates are usually weak, T cell independent antigens that require presention on immunogenic carrier molecules to stimulate T cell help and induce long-lasting antibody responses.5 Conventionally, isolated carbohydrate antigens are coupled to 2 ACS Paragon Plus Environment

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immunogenic protein carriers to furnish glycoconjugates. These are the constituents of licensed vaccines against various bacterial pathogens including Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae type B.6 Glycoconjugate vaccines, however, require external adjuvants to efficiently confer immunity7 and constant cooling to prevent protein degradation or aggregation8. Moreover, glycoconjugates prepared with small oligosaccharides or self-antigenic tumor-associated carbohydrate antigens (TACA) frequently fail to induce glycan-specific antibodies due to the immunodominant carrier protein.9 Oligosaccharides derived from microorganisms10 or tumor tissue11 have been shown to elicit potent anti-infective and tumor-killing antibody responses, respectively, both in model animals and humans. Yet, in many cases carbohydrate antigens cannot be accessed by isolation in sufficient purity for clinical use.12,13 In efforts to overcome these limitations, various fully synthetic vaccine candidates based on microbial or tumor-associated carbohydrate antigens have been reported. For example, three-component vaccine candidates composed of a carbohydrate antigen B cell epitope, a peptidic T cell epitope and a Toll-like receptor (TLR) agonist that serves as internal adjuvant have been shown to promote strong anti-glycan antibody responses in mice.14,15 The immunogenicity of small glycans also benefits from their multivalent presentation on peptide carriers,16,17 virus particles18,19 or self-assembling structures20. Another method to boost the immunological activity of oligosaccharides is direct conjugation with a potent immune stimulant such as monophosphoryl lipid A (MPLA)21,22 or α-galactosylceramide (α-GalCer)23, which furnishes peptide-free conjugate vaccine candidates. Synthetic vaccine candidates have the advantage of being single chemical entities that can be fully characterized with standard methods such as nuclear magnetic resonance (NMR) and high3 ACS Paragon Plus Environment

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resolution mass spectrometry (HRMS). Thus, they can be treated as a small molecule drug, enable rational structure–activity relationship (SAR) studies and may simplify the vaccine application process of regulatory agencies. On the other hand, elaborate multicomponent vaccine candidates require multistep synthetic routes and a considerable amount of resources. Therefore, a simple platform that allows for easy access to fully synthetic vaccine candidates is highly desirable. Here, we report on the in vivo efficacy of a fully synthetic saccharide-glycolipid conjugate vaccine platform that allows for the multivalent display of glycan antigens with minimal synthetic effort. The weakly immunogenic cancer-associated Tn (α-N-acetylgalactosamine linked to serine or threonine)24,25 served as model antigen that was efficiently coupled to the glycosphingolipid α-GalCer. This internal immune stimulatory moiety has recently been shown to promote strong antibody responses when co-formulated26 or covalently coupled with polysaccharides23 or peptides27,28. The liposome-formulated self-adjuvanting Tn-α-GalCer conjugate consistently induced strong anti-Tn antibody responses characterized by an immunoglobulin switch to IgG and affinity maturation in all immunized mice. Control liposomes prepared with stearoylated Tn lacking the α-GalCer moiety were also immunogenic but elicited lower antibody levels. In contrast, a semi-synthetic glycoconjugate composed of Tn antigen and the carrier protein CRM197 induced anti-glycan IgG only in a subset of immunized mice. Larger liposomes (~400 nm diameter) promoted Th1-type IgG2a antibodies, while smaller ones (~120 nm) favored the production of IgG1 associated with Th2-mediated immunity. Thus, modulating the size of liposomes allows for directing anti-glycan antibody responses towards either cellular (Th1) or humoral (Th2) immunity. Overall, this study establishes a vaccine platform based on 4 ACS Paragon Plus Environment

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fully synthetic saccharide-α-GalCer conjugates for the robust generation of high-affinity IgG even against the weakly immunogenic carbohydrate antigen Tn.

RESULTS AND DISCUSSION Chemical Synthesis of Carbohydrate-lipid Conjugates. α-GalCer is a CD1d ligand and a potent stimulant of invariant natural killer T (iNKT) cells that enhances interactions with antigen presenting cells (APC) via CD40 signaling.29 This glycolipid has been extensively investigated in SAR studies as an adjuvant and as a component of synthetic vaccines.27,28,30,31 We reasoned that conjugation of α-GalCer to a defined synthetic carbohydrate antigen will furnish easily accessible but effective carbohydrate-lipid vaccine candidate conjugates to circumvent the elaborate synthetic effort required for other fully synthetic carbohydrate vaccines. Ideally, the α-GalCer conjugate should be able to induce high antibody titers even against weakly immunogenic carbohydrate antigens of synthetic origin. Hence, we selected the TACA Tn antigen as a model compound to validate the immunogenic properties of our α-GalCer based vaccine platform. It is known that the Tn antigen is only weakly immunogenic, especially in a monomeric form,32 and is usually coupled to immunogenic carrier proteins,33 peptidic T-helper cell epitopes34 or TLR ligands35 to afford strong antibody responses in mammals. In order to create a molecule to be easily conjugated with synthetic carbohydrates that have been equipped an amino linker for conjugation,36 linker-equipped α-GalCer 123 was reacted with bis(4-nitrophenyl) carbonate to yield activated α-GalCer 2 in 79% yield (Scheme 1). The 5 ACS Paragon Plus Environment

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activated carbamate represents an ideal functional group for conjugation, since carbohydrate antigens usually do not contain amino groups or other nucleophilic moieties.25,33,37,38 Thus, the subsequent attack of the amino linker equipped synthetic carbohydrate is selective. In addition, carbamate 2 can be purified by simple silica chromatography and stored at –20 °C for several days without decomposition. In the final step, synthetic Tn antigen 314 was coupled to carbamate 2 in a standard substitution reaction to form a urea linkage and glycolipid 4 in 42% yield. The resulting glycolipid represents a fully synthetic, peptide-free, homogeneous vaccine candidate. Glycolipid 5 served as control and was prepared by an amide bond forming reaction between Tn antigen 3 and stearoyl chloride in 46% yield. It was reasoned that comparing the immunogenicity of liposomes containing either 4 or 5 should reveal the effect of α-GalCer on total anti-Tn antibody levels, affinity maturation and Th-skewing. Finally, a conventional glycoconjugate 6 that consists of the carrier protein CRM197 covalently linked to 3 via di-Nsuccinimidyl adipate crosslinker chemistry was prepared to enable comparison of the immune responses.39

Liposomes Multivalently Displaying the Tn Cancer Antigen Promote Long-lasting IgG Responses In Vivo. For multivalent display of the Tn antigen, size-defined liposomes were prepared by lipid extrusion that contained either 4, 5, or equimolar amounts of 5 and 1. The liposomes contained 1,2-distearoyl-sn-glycero-3-phosphocholine

(DSPC)

and

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cholesterol

at

20.3:13.6:1

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(DSPC:cholesterol:4 or 5) or 20.3:13.6:1:1 (DSPC:cholesterol:5:1) molar ratios, respectively. The combination of DSPC and cholesterol for liposomal vaccines has been shown to induce strong antibody responses in small animals before.40 Liposomes with mean diameters (Z-average values) of about 120 and 400 nm, respectively, as determined by dynamic light scattering (DLS), were obtained using filters with pore sizes of 100 or 400 nm (Fig. S2). There was no difference in size distribution between the different antigens. Controls included micelles of 4 prepared by ultrasonication of a lipid suspension, the CRM197 glycoconjugate 6 formulated with aluminum hydroxide (Alum) adjuvant, soluble Tn 3 co-formulated with 1 by ultrasonication, and soluble 3 alone. To assess the primary antibody response to the above-mentioned antigen formulations, groups of six mice were immunized via the subcutaneous (s.c.) route three times in two-week intervals, each dose containing 1.7 μg of Tn (Fig. 1a). The serum IgG responses were followed by microarray analysis with slides presenting Tn antigen 3 and the disaccharide substructure of S. pneumoniae serotype 4 capsular polysaccharide 8 (α-GalNAc-(14)-α-Gal)41 as a control antigen (Fig. 1b). Furthermore, CRM197 was spotted on the microarray slides to detect IgG to the carrier protein in mice immunized with 6. Successful spotting of 3 and 8 was verified with the Bandeiraea (Griffonia) simplicifolia lectin-I (GS-I)42 that bound to both oligosaccharides (Fig. 1c). All mice immunized with liposomes of 4 and 5 mounted consistent serum IgG responses to Tn antigen 3 without detectable cross-reactivity to 8 (Fig. 1d). In contrast, only three of six mice immunized with Alum-adjuvanted glycoconjugate 6 elicited IgG to 3 and the magnitudes of responses were more variable. The other three mice showed weak IgG binding to 8 that shares

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the terminal α-GalNAc residue with the Tn antigen. Thus, liposomes induced more consistent and specific anti-3 IgG responses than did glycoconjugate 6. Differential IgG responses over time were quantified by microarray-inferred mean fluorescence intensity (MFI) signals of 3. A fourth immunization dose given after 21 weeks that contained 0.3 μg of Tn aimed at detecting memory responses. All mice immunized with liposomes containing 4 or 5 mounted primary anti-Tn IgG1, IgG2 and IgG3 responses (Fig. 2a). Liposomes of 4 induced detectable IgG levels, especially IgG2a and IgG3, already one week after the first immunization, whereas with 5, antibodies appeared only after the second or third injection. While T celldependent IgG responses are generally not expected before two weeks after initial immunization, it is known that murine Ig2a and IgG3, but not IgG1, can be produced without T cell help.43,44 Thus, it appeared that liposomes of 4 elicited both T cell-dependent and T cellindependent IgGs, whereas liposomes of 5 raised T cell-dependent antibodies only. Compared to 5, 120 nm liposomes of 4 elicited higher IgG3 levels. At 400 nm size, 4 elicited higher primary anti-Tn IgG responses for all three subtypes, especially IgG3. IgG of all subtypes remained detectable for 21 weeks and could then be boosted with liposomes bearing 4 or 5, indicating the presence of memory B cells. In contrast, liposomes co-formulated with 5 and 1 did not induce any detectable primary IgG response, but a weak secondary response after immunization at week 21. Micelles of 4 elicited IgGs in all immunized mice with comparable kinetics and isotype distribution to the liposomes prepared with 4, and elicited a strong secondary response. The Alum-adjuvanted glycoconjugate 6 elicited anti-Tn IgG in three of six immunized mice only and there was no detectable secondary IgG response to Tn but an increase in anti-CRM197 IgG1 and IgG2a (Fig. S3). The latter finding indicated that 6 elicited 8 ACS Paragon Plus Environment

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immunological memory to the carrier protein but not to the glycan antigen. The other two control groups immunized with co-formulated 3 and 1 or soluble 3 did not mount any detectable anti-Tn IgG responses. Liposomes of 4 and 5, but not those of 5 co-formulated with 1, proved to be suitable for robust induction of anti-Tn antibodies with hallmarks of T cell-dependency such as isotype switch to IgG and the formation of immunological memory. Therefore, we focused antibody analyses on these four groups of mice (liposomes of 4 or 5, each 120 or 400 nm-sized). To determine the effect of liposome size and the adjuvant effect of α-GalCer, we compared the magnitudes of primary IgG responses at week 5, one week after the third immunization, elicited by liposomes consisting of either 4 or 5 (Fig. 2b). Induction of IgG1 did not benefit from the internal adjuvant but IgG1 levels were higher for the smaller liposomes of both 4 and 5, albeit only with statistical significance in the case of 5. IgG2a responses were higher for the larger liposomes of 4 but not significantly increased when compared to liposomes of 5. In contrast, IgG3 levels were significantly higher in mice immunized with liposomes of 4 compared to 5. Liposomes with a diameter of 400 nm prepared with 4 induced the highest levels of IgG3 among all groups. Next, we compared the total primary antibody responses by using surface plasmon resonance (SPR) (Fig. 2c). Pooled serum obtained at week 5 was passed through a sensor chip functionalized with Tn antigen 3. Pre-immune serum served as control and did not show any binding signals to 3. Anti-Tn antibodies were detected in week 5 serum of all four groups. The magnitude of the binding signal expressed as response units (RUs) allowed for ranking the primary total antibody responses. Highest signals were detected for the 400 nm liposomes of 4 (~800 RUs) followed by liposomes of 5 with the same size (~400 RUs). The smaller liposomes led 9 ACS Paragon Plus Environment

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to weaker signals irrespective of the antigen (~200 RUs for both 4 and 5). The SPR studies confirmed that liposomes of 4 with a diameter of 400 nm were the most potent inducer of antiTn antibodies. This was attributed mainly to strong IgG3 responses, as indicated by the microarray studies. Furthermore, serum IgA and/or IgM might contribute to total antibody responses as well.

The Size of Liposomes Influences Th Phenotype and Affinity Maturation. Next, we sought to evaluate the quality of IgG responses elicited by the different liposomes. One factor that influences the activity of IgG is the subclass distribution. In mice, IgG1 is primarily induced by Th2-type responses, whereas Th1 favors the generation of IgG2 and IgG3.45 Therefore, we studied the IgG2a to IgG1 ratio of IgG elicited by the liposomes after the third immunization (week 5) by dividing the respective fluorescent signal intensities (Fig. 3a). We also included serum obtained from mice immunized with micelles of 4 and the Alumadjuvanted glycoconjugate 6 in this analysis. The latter adjuvant is known to primarily induce Th2-type IgG.46 Responses elicited by 400 nm liposomes showed higher IgG2a to IgG1 ratios than those induced by the smaller liposomes, but without difference between 4 and 5. Antibodies raised with micelles of 4 ranged in-between. The lowest ratio was detected for 6 that elicited primarily IgG1 likely through the action of Alum adjuvant. Finally, we investigated whether liposome size or the presence of α-GalCer influenced IgG affinity maturation. High-affinity antibodies are usually resistant to urea-mediated dissociation from the antigen. Therefore, we adapted an ELISA procedure to microarray analysis that 10 ACS Paragon Plus Environment

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includes a 10 min incubation step of bound serum IgG with 7 M urea to dissociate low-affinity antibodies.47 The ratio of measured binding signals to non-urea-treated serum samples serves as a measure of overall IgG affinity (Fig. 3b). Affinity of IgG increased from week 3 (after second immunization) to week 5 (after third immunization) in pooled serum obtained from mice immunized with all four liposome preparations, whereby larger liposomes elicited IgG of higher affinity. The α-GalCer internal adjuvant did not appear to influence affinity maturation. The size-dependent effect on affinity maturation was verified by SPR analysis of pooled week 5 serum (Fig. 3c). Binding affinity is affected by both association (ka) and dissociation (kd) rate constants, but determination of ka requires knowledge of the unknown serum antibody concentration. In contrast, kd is concentration-independent and recognized as the major parameter influencing antibody affinity.47,48 We therefore estimated the kd values by SPR through fitting the sensorgram curves during dissociation state. As expected, the larger liposomes elicited higher affinity antibodies (smaller kd values) when compared to the smaller liposomes.

CONCLUSIONS We disclose a fully synthetic vaccine platform based on readily accessible carbohydrate-αGalCer conjugates that are multivalently displayed on liposomes. Using the weakly immunogenic Tn as model antigen, the liposomes elicited strong and consistent anti-glycan IgG responses in mice with hallmarks of T cell-dependent immunity such as isotype switching to IgG1 and affinity maturation, presumably mediated by iNKT cells. While glycoconjugate6 and three-component14,15 vaccines rely on classical T cells activated by MHC-presented peptides to 11 ACS Paragon Plus Environment

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generate B cell help, iNKT cells can help B cells to mount IgG responses with affinity maturation and isotype switch in the absence of peptidic T cell epitopes26,43. Instead, the invariant T cell receptor of iNKT cells is activated by (glyco)lipids presented on the nonpolymorphic MHC class I molecule CD1d.43 Thus, B cell help was likely generated via activation of iNKT cells, because the liposomes used in this study contain only glycolipids and no peptides. The antibody responses induced by liposomes were highly specific and more reproducible than those elicited by a conventional Tn-glycoconjugate vaccine candidate with the CRM197 carrier protein. Although generating a relevant anti-tumour cell response would require the presentation of Tn antigen within mucin peptide sequences14,20,25,34, we demonstrate that our vaccine platform is suitable to generate robust IgG responses to small and weakly immunogenic carbohydrate antigens. Detailed investigation of the immune responses also revealed that the size of the liposomes influences antibody titres, affinity maturation as well as Th-skewing. Larger liposomes with a diameter of 400 nm elicited higher affinity Th1-type antibodies. The latter aspect is particularly intriguing as modifying the size of liposomes would allow for directing immune responses to the desired activity, for instance, to generate cytotoxic Th1 immunity against tumor cells or intracellular pathogens. On the other hand, smaller liposomes may be suitable to induce Th2 type immunity against extracellular pathogens. For protein antigens it has been shown before that smaller liposomes (< 155 nm) induce Th2 responses and larger ones (> 225 nm) Th1 responses in mice.49,50 This observation has been attributed to the ability of larger particles to promote secretion of the Th1-directing cytokine interleukin-12 in macrophages.49 Furthermore, larger particles (> 225 nm) are subject to phagocytosis, mainly by macrophages and perhaps dendritic cell progenitors, whereas smaller ones (< 155 nm) are 12 ACS Paragon Plus Environment

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primarily taken up via pinocytosis by other APCs such as B cells.49 Thus, the particle size determines which APC populations are activated, which in turn influences cytokine production. Large particles thereby predominantly induce Th1-associated cytokines such as interferongamma, ultimately leading to a strong induction of IgG2a.49 On the other hand, small particles mainly evoke Th2-type cytokines like interleukin-5 and interleukin-12, inducing strong IgG1 responses.49 Here, we demonstrated that this size-dependent Th skewing effect previously demonstrated for protein antigens49,50 also applies to glycan antigens. Our approach circumvents several drawbacks encountered in traditional glycoconjugate vaccine development. Organic synthesis can provide homogenous carbohydrate antigens13 that are harder and often impossible to access from natural sources since the desired microorganism or cell expressing the saccharide cannot be cultured on large scale12. The conjugation chemistry is applicable to most synthetic carbohydrates featuring an amine linker because the majority of polysaccharides or TACAs do not bear nucleophilic moieties. Thus, conjugation chemistry does not have to be developed individually for ever antigen.51 Finally, no complex quality control protocols that ensure high levels of purity of heterogeneous biological isolates and proteinglycoconjugate end products have to be established since the vaccine candidate is a defined molecular entity.52 This study opens up new venues for synthetic vaccinology as the introduced platform will facilitate SAR studies to guide rationally designed carbohydrate-based vaccines. In contrast to conventional glycoconjugate vaccines, the carbohydrate-α-GalCer conjugates do not require external adjuvants and can be easily formulated as micelles. This vaccine approach may be

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ideally suited for developing countries where the ease of preparation and administration is of crucial importance.

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EXPERIMENTAL SECTION

General Information. All chemicals used were reagent grade and used as supplied except where noted. All reactions were performed in oven-dried glassware under an inert atmosphere (nitrogen or argon) unless noted otherwise. Pyridine (Fisher Scientific, Karl-Fischer grade) was distilled over CaH2 prior to use. Analytical thin layer chromatography (TLC) was performed on Merck silica gel 60 F254 plates (0.25 mm). Compounds were visualized by UV irradiation or dipping the plate in a cerium sulfate ammonium molybdate (CAM) solution or sulfuric acid ethanol solution, or potassium permanganate solution. Flash column chromatography was carried out using a forced flow of the indicated solvent (Fisher Scientific, HPLC grade) on Fluka silica gel 60 (230-400 mesh, for preparative column chromatography). 1H- and

13

C-NMR were recorded either on a Varian 400 (400

MHz) or Varian 600 (600 MHz) in CDCl3, pyridine-d5 and CD3OD with chemical shifts referenced to internal standards CDCl3 (7.26 ppm 1H, 77.1 ppm

13

C), pyridine-d5 (8.74

ppm 1H, 150.4 ppm 13C) and CD3OD (3.31 ppm 1H, 49.0 ppm 13C) unless otherwise stated. Coupling constants are reported in Hertz (Hz). Splitting patterns are indicated as s, singlet; d, doublet; t, triplet; for 1H NMR data. Signals were assigned by means of 1H-1H COSY and 1H-13C HSQC spectra. ESI mass spectral analyses were performed by the MS service at the Institute of Chemistry and Biochemistry at the Free University of Berlin using a modified MAT 711 spectrometer (Varian MAT). All biologically tested compounds were highly pure as judged by 1H NMR spectroscopy. We did not assess their purity by 15 ACS Paragon Plus Environment

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qNMR, however, we here investigated vaccine candidates that act by stimulating the immune system, a mechanism of action not expected to be significantly affected by any possible minor impurities. Furthermore, the control in vivo experiments using 5 + 1 as separate compounds did not elicit any detectable immune response. This confirmed that the observed immune responses in the other groups could have only resulted from the glycolipids interacting with the immune system, and were not due to nonspecific effects.

Materials. The α-GalCer derivative 1 was prepared as reported by Cavallari et al.23 Tn antigen 3 was prepared according to procedures reported by Buskas et al.15 Disaccharide 8 was synthesized according to procedures reported by Geissner et al.41

(2S,3S,4R)-1-(6-(4-Nitrophenyl-6’-hexylcarbamate)-α-D-galactopyranosyl)-2hexacosanoylaminooctadecane-3,4-diol (activated α-GalCer, 2). α-GalCer derivative 1 (3.9 mg, 4.1 µmol) was dissolved in pyridine (0.5 mL). Bis(4nitrophenyl) carbonate (6.1 mg, 20 µmol) was added to this solution followed by triethylamine (25 µL, 0.179 mmol). The resulting yellow solution was stirred at room temperature for 12 h before it was evaporated to dryness. The crude product was purified by column chromatography (SiO2, CH2H2/MeOH 0%  20% MeOH) to yield glycolipid 2 (3.6 mg, 3.2 µmol, 79% yield) as pale yellow oil: 1H NMR (400 MHz, pyridined5) δ 9.02 (t, J = 5.6 Hz, 1H, NH of carbamate), 8.49 (d, J = 8.7 Hz, 1H, NH of amide), 8.26 (d, J = 9.2 Hz, 2H), 7.54 (d, J = 9.2 Hz, 2H), 5.56 (d, J = 3.8 Hz, 1H, Gal-1), 5.30 – 5.24 (m, 16 ACS Paragon Plus Environment

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2H), 4.73 – 4.61 (m, 2H), 4.51 (t, J = 6.0 Hz, 1H), 4.45 – 4.38 (m, 3H), 4.37 – 4.31 (m, 2H), 4.10 (dd, J = 9.8, 5.7 Hz, 1H), 4.02 (dd, J = 9.8, 6.5 Hz, 1H), 3.52 (td, J = 9.2, 2.7 Hz, 2H), 3.45 (dd, J = 13.0, 6.9 Hz, 2H), 2.46 (t, J = 7.2 Hz, 2H), 2.36 – 2.23 (m, 1H), 1.97 – 1.79 (m, 4H), 1.74 – 1.65 (m, 3H), 1.64 – 1.54 (m, 2H), 1.48 – 1.18 (m, 74H), 0.87 (t, J = 6.7 Hz, 6H, Me of lipids);

13

C NMR (101 MHz, pyridine-d5) δ 173.6 (amide), 157.8 (carbamate),

154.6, 145.2, 125.9, 123.0, 102.0 (Gal-1), 77.1, 73.0, 72.0, 71.9, 71.6, 71.3, 71.3, 70.7, 69.2, 51.8, 42.0, 37.3, 34.8, 32.6, 32.6, 30.9, 30.7, 30.6, 30.5, 30.5, 30.5, 30.4, 30.42, 30.40, 30.35, 30.28, 30.13, 30.12, 27.5, 27.0, 26.9, 26.7, 23.5, 14.8 (Me of lipids); m/z (ESI) Found: [M+Na]+, 1144.8373 C63H115N3O13 requires [M+Na]+, 1144.8323.

Tn antigen-α-GalCer conjugate (4). Activated α-GalCer 2 (9.1 mg, 8.11 µmol) was dissolved in pyridine (0.8 mL). Tn antigen 3 (4.8 mg, 11 µmol) was added to this solution. Afterwards triethylamine (2.5 µL, 18 µmol) was added to the reaction mixture that was stirred for 18 h. The reaction mixture was evaporated to dryness and redissolved in MeOH/CH2Cl2 (1 mL, 1:1). DOWEX 50WX-8 acidic resin (25 mg) was added and the slurry was stirred for 15 minutes to remove any excess amine. The filtrate was evaporated to dryness and the residue purified was purified by column chromatography (SiO2, CH2H2/MeOH 0%  20% MeOH) to yield glycolipid 4 (4.8 mg, 3.4 μmol, 42% yield) as white powder: 1H NMR (600 MHz, CDCl3/CD3OD, 1:1) δ 5.12 (d, J = 3.8 Hz, 1H, GalNAc-1), 5.09 (d, J = 3.8 Hz, 1H, Gal-1), 4.71 (d, J = 2.5 Hz, 1H, α-Thr), 4.52 – 4.43 (m, 2H, Gal-2, β-Thr), 4.41 (dd, J = 9.9, 4.2 Hz, 1H), 4.18 – 4.08 (m, 4H), 4.04 (dd, J = 10.0, 3.8 Hz, 1H, GalNAc-2), 4.02 – 3.92 (m, 4H), 3.88 17 ACS Paragon Plus Environment

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(dd, J = 10.2, 5.3 Hz, 1H), 3.86 – 3.81 (m, 2H), 3.81 – 3.77 (m, 1H), 3.75 – 3.70 (m, 2H), 3.47 – 3.28 (m, 6H, 3x C(=O)NH-CH2), 2.44 (t, J = 7.6 Hz, 2H, HNC(=O)-CH2 of fatty acid), 2.35 (s, 3H, Me of Ac), 2.29 (s, 3H, Me of Ac), 1.89 – 1.78 (m, 6H), 1.75 – 1.69 (m, 2H) 1.64 – 1.44 (m, 79H), 1.11 (t, J = 7.0 Hz, 6H, 2x Me of lipids);

13

C NMR (151 MHz,

CDCl3/CD3OD, 1:1) δ 175.0, 173.7, 172.9, 171.4 (4x amide), 160.6 (carbamide), 100.4 (GalNAc-1), 100.2 (Gal-1), 76.7 (β-Thr), 74.8, 72.5, 72.1, 71.9, 70.9, 70.8, 70.5, 70.0, 69.7, 69.5 (GalNAc-2), 68.0, 62.2, 57.9 (α-Thr), 51.0, 50.9, 49.6, 40.5, 37.3, 37.0, 32.7, 32.5, 30.7, 30.6, 30.3, 30.2, 30.0, 29.9, 28.7, 27.1, 26.5, 26.5, 26.3, 23.2, 23.00 (2x Me of NHAc), 18.7 (Me of Thr), 14.3 (Me of lipids); m/z (ESI) Found: [M+Na]+, 1426.0300 C74H142N6O18 requires [M+Na]+, 1426.0273.

N-Acetamido-O-(2-acetamido-2-desoxy-α-D-galactopyranosyl)-L-threonine-(3-amidopropyl)stearamide (5). Tn antigen 3 (4 mg, 9.5 µmol) was dispersed in pyridine (0.7 mL) and stearoyl chloride (7.1 µL, 19 µmol, purity ≥90%) was added to this slurry. The reaction mixture was stirred for 2 h at room temperature until all solids were dissolved. LC-MS analysis showed absence of starting material and formation of the desired glycolipid. The reaction was quenched with MeOH (0.5 mL) and all solvents were evaporated to dryness. The residue was co-evaporated with toluene (3 x 2 mL) and purified using silica gel flash column chromatography (5 cm high pipette column, 10%  20% MeOH in CHCl3) to yield glycolipid 5 (3 mg, 4.4 µmol, 46%) as white solid: Rf (SiO2, CDCl3/MeOH 4:1) = 0.54; 1H NMR (400 MHz, CDCl3/CD3OD – 1:1) δ 4.84 (d, J = 3.7 Hz, 1H, GalNAc-1), 4.46 (d, J = 2.5 18 ACS Paragon Plus Environment

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Hz, 1H, α-Thr), 4.28 – 4.22 (m, 1H, β-Thr), 4.19 (dd, J = 10.9, 3.7 Hz, 1H, GalNAc-2), 3.96 (d, J = 2.5 Hz, 1H), 3.87 (t, J = 5.8 Hz, 1H), 3.80 – 3.70 (m, 3H, GalNAc-6, GalNAc-3), 3.32 – 3.28 (m, 1H, buried in solvent peak, NH-CH2-CH2-CH2-NH), 3.23 – 3.16 (m, 1H, NH-CH2CH2-CH2-NH), 3.16 – 3.03 (m, 2H, CH2, NH-CH2-CH2-CH2-NH), 2.19 – 2.14 (m, 2H, CO-CH2CH2), 2.11 (s, 3H), 2.05 (s, 3H), 1.67 – 1.53 (m, 4H, CO-CH2-CH2, NH-CH2-CH2-CH2-NH), 1.36 – 1.17 (m, 31H, 14x CH2 of lipid, Me of Thr), 0.85 (t, J = 6.9 Hz, 3H, Me of lipid); 13C NMR (151 MHz, CDCl3/CD3OD – 1:1) δ 176.1, 173.8, 173.00, 171.5 (4x amide), 100.1 (GalNAc-1), 76.6 (β-Thr), 71.4, 70.2, 69.8 (GalNAc-3), 62.4 (GalNAc-6), 58.0 (α-Thr), 50.9 (GalNAc-2), 49.9, 37.0, 36.9, 32.5, 30.22, 30.19, 30.1, 29.93, 29.89, 29.7, 26.6, 23.2, 23.0, 22.7, 18.7 (Me of Thr), 14.3 (Me of lipid); m/z (ESI) Found: [M+Na]+, 709.4724 C35H66N4O9 requires [M+Na]+, 709.4722.

Preparation and Analysis of Glycoconjugate 6. Tn antigen 3 (1.3 mg), dissolved in 120 μL anhydrous DMSO was added dropwise to a stirred solution of di-N-succinimidyl adipate spacer in 10-fold molar excess dissolved in 140 μL anhydrous DMSO with 10 μL triethylamine and reacted for 2 h at room temperature. Then, 400 μL of 100 mM sodium phosphate buffer, pH 7.4, were added and non-reacted spacer was extracted twice with 10 mL chloroform. The aqueous phase was reacted with 1 mg CRM197 (Pfénex, Inc., San Diego, CA, USA) dissolved in 1 mL 100 mM sodium phosphate buffer, pH 7.4, for 16 h at room temperature while stirring. The reaction product was desalted and concentrated with deionized water and 10 kDa centrifugal filter devices (Merck Millipore, Tullagreen, Ireland). Protein concentration of 19 ACS Paragon Plus Environment

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the resulting glycoconjugate 6 was determined with the Micro BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA) following the manufacturer’s protocol. Mass spectra

of

6

and

CRM197

were

determined

by

matrix-assisted

laser

desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis using an Autoflex Speed instrument (Bruker Daltonics, Bremen, Germany) operated in linear positive mode. Samples were spotted using the dried droplet technique employing 2’,4’dihydroxyacetophenone as matrix. For sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), proteins were dissolved in Lämmli buffer (125 mM Tris-HCl, 20% (v/v) glycerol, 4% (w/v) SDS, 5% (v/v) 2-mercaptoethanol, 0.015% (w/v) bromophenol blue, pH 6.8) and incubated at 95 °C for 5 min. Samples were run in 10% polyacrylamide gels at 20 V/cm and stained with 0.5% (w/v) Coomassie Brilliant Blue R250 in 50% (v/v) methanol and 10% (v/v) acetic acid for 30 min. The gel was destained with 50% (v/v) methanol, 10% acetic acid. PageRuler Plus Prestained Protein Ladder (Thermo Scientific, Rockford, IL, USA) was used as protein size marker (3 μL per lane).

Preparation and Characterization of Liposomes and Micelles. Three liposome precursor solutions were prepared by mixing 0.46 μmol of either 4 (0.65 mg) or 5 (0.32 mg) or 5 (0.65 mg) and 1 (0.44 mg) with DSPC (7.39 mg, 9.35 μmol) and cholesterol (2.42 mg, 6.26 μmol) in 2.2 mL of 1:1 chlorofom:methanol. DSPC and cholesterol were purchased from Avanti Polar Lipids, Alabaster, AL, USA. The liposome precursor solutions were split evenly in 11 glass vials each. The solvent was evaporated in vacuo to a thin film on the glass and vials were stored at -20 °C under nitrogen. Each 20 ACS Paragon Plus Environment

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vial was meant to contain nine doses of liposome precursor containing 1.7 μg of the glycan antigen. The day before each immunization, the lipid film was rehydrated by adding 900 μL of sterile phosphate-buffered saline (PBS) to one glass vial and stirring at 60 °C for 30 min, yielding an opalescent suspension. The suspension was taken up in a glass syringe and extruded through a pre-heated (60 °C) lipid mini-extruder (Avanti Polar Lipids) equipped with polycarbonate membranes of 100 nm or 400 nm pore size (Whatman Nucleopore Track-Etched Membranes, Sigma-Aldrich, St. Louis, MO, USA). Each suspension was passed a minimum number of 31 times through the membrane. Liposomes were transferred to new glass vials and used for immunizations. Micelles were prepared by dissolving 4 (0.65 mg, 0.46 μmol) or 1 (0.44 mg, 0.46 μmol) in 2.2 mL of 1:1 chloroform:methanol and splitting each solution into 11 vials as described above. The lipid layer resulting from solvent evaporation was stored at -20 °C under nitrogen and rehydrated prior to immunization either in 900 μL sterile PBS (for 4) or 900 μL sterile PBS containing 17.7 μg 3 (for 1). Vials were incubated for 10 min in an ultrasonic bath to yield opaque suspensions of micelles used for immunization. For dynamic light scattering (DLS) measurements, aliquots of the liposomes or micelles were loaded into plastic UV cuvettes and analyzed in a Zetasizer μV device (Malvern Instruments Ltd., Malvern, Worcestershire, UK) using standard parameters of the accessory software.

Immunization Studies. Female, 6-8 weeks old C57BL/6 mice purchased from Charles River, Sulzfeld, Germany, were immunized subcutaneously (s.c.) with liposome or micelle suspensions, or a 21 ACS Paragon Plus Environment

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solution of 3 in PBS. For immunizations with 6, the glycoconjugate was pre-adsorbed for 24 h at 4 °C to aluminum hydroxide adjuvant (Alum Alhydrogel, Brenntag, Frederikssund, Denmark) using 1 μL Alum suspension per μg protein. Each injection contained either 1.7 μg (immunizations at weeks 0, 2 and 4) or 0.3 μg (at week 21) of glycan antigen in a total volume of 100 μL of sterile PBS. Mice were kept in individually ventilated cages (IVCs) under specific pathogen-free conditions in the Bundesamt für Risikobewertung (BfR) in Berlin, Germany. Studies were performed in strict accordance with the German regulations of the Society for Laboratory Science and the European Health Law of the Federation of Laboratory Animal Science Associations. All efforts were made to minimize suffering. This work was approved by the Landesamt für Gesundheit und Soziales (LAGeSo) of Berlin, Germany, with approval number G0104/13.

Preparation of Microarray Slides. Oligosaccharides and CRM197 were spotted at the indicated concentrations in 50 mM sodium phosphate buffer, pH 8.5 on CodeLink N-hydroxysuccinimide ester-activated glass slides (SurModics, Inc., Eden Prairie, MN, USA) using an S3 piezoelectric spotting device (Scienion, Berlin, Germany). After spotting, slides were incubated in a humidified chamber for 16 h to complete coupling reactions. Then, slides were quenched with 50 mM aminoethanol solution, pH 9, for 1 h at 50 °C and washed three times with deionized water. Slides were dried by centrifugation and stored at 4 °C until use.

Microarray Binding Assays. 22 ACS Paragon Plus Environment

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Spotted and quenched slides were blocked with 1% (w/v) bovine serum albumin (BSA) in PBS for 1 h at room temperature, washed once with PBS and dried by centrifugation. Incubation chambers with 64 wells (Grace Bio-Labs, Bend, OR, USA) were applied to the slides. Sera diluted 1:200 in PBS containing 1% (w/v) BSA and 0.01% (v/v) Tween-20 were incubated in individual wells for 1 h at room temperature. After washing three times with PBS containing 0.1% (v/v) Tween-20, slides were incubated with secondary anti-mouse IgG antibodies (Life Technologies, Carlsbad, CA, USA). Goat anti-mouse IgG1 Alexa Fluor 594 and goat anti-mouse IgG2a Alexa Fluor 647 were diluted 1:400 and goat anti-mouse IgG3 Alexa Fluor 488 was diluted 1:200 in PBS with 1% (w/v) BSA and 0.01% (v/v) Tween-20 and incubated on the microarray slides for 1 h at room temperature. After washing three times with PBS containing 0.1% (v/v) Tween-20 and rinsing once with deionized water, slides were dried by centrifugation and scanned with a GenePix 4300A microarray scanner (Molecular Devices, Sunnyvale, CA, USA). The photomultiplier tube (PMT) gain was adjusted to reveal fluorescence signals free of saturation. Likewise, spotting of oligosaccharides 3 and 8 was confirmed with lectin from Bandeiraea simplicifolia FITC conjugate (Sigma-Aldrich, St. Louis, MO, USA) that was used at a 1:100 dilution in lectin buffer (50 mM HEPES, 5 mM CaCl2, 5 mM MgCl2, pH 7.4) instead of PBS. Background-subtracted mean fluorescence intensity (MFI) values were exported to Microsoft Excel for further analysis. To determine the ratio of urea-resistant IgG, the above described procedure was modified such that the first washing step after incubation of serum samples was with 7 M urea in water for 10 min. The same serum samples analyzed in parallel were treated as described above with three washing steps 23 ACS Paragon Plus Environment

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using PBS containing 0.1% (v/v) Tween-20. The ratio of measured binding signals to nonurea-treated serum samples was calculated as a measure of overall IgG affinity, whereby goat anti-mouse IgG Alexa Fluor 647 diluted 1:400 in PBS with 1% (w/v) BSA and 0.01% (v/v) Tween-20 was used as secondary antibody.

SPR Analyses. All SPR measurements were performed using a Biacore T100 instrument (GE Healthcare, Uppsala, Sweden) and CM5 sensor chips, PBS as running buffer and a temperature of 25 °C. Tn antigen 3 was coupled to the sensor chip surface using the Amine Coupling Kit (GE Healthcare) and the standard parameters of the Immobilization procedure in the Biacore Control software provided with the instrument. Coupling conditions were as follows: 3 diluted to 2 mM in 100 mM sodium phosphate buffer, pH 7.4, a flow rate of 10 μL/min and 420 s contact time. Binding runs were performed with the standard parameters of the Kinetics function, using pooled serum at the indicated dilutions in PBS and a flow rate of 30 μL/min. The binding responses were monitored as a function of time (sensorgram) and were double-referenced with PBS injections and a blankfunctionalized flow cell. Regeneration buffer (10 mM glycine-HCl, pH 1.7) was passed through the flow cells for 30 s after each single measurement. Sensorgrams were analyzed with the accessory Biacore Evaluation software. The kd values were determined by fitting the dissociation stage of sensorgram curves with a kinetic model supplied by GE Healthcare (personal communication with Dr. Uwe Bierfreund, GE Healthcare Europe). 24 ACS Paragon Plus Environment

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ACKNOWLEDGEMENTS This work was funded by the Max Planck Society, the Körber Foundation (Körber Prize to PHS), the German Federal Ministry of Education and Research (grant no. 0315447 to PHS) and the German Research Council (DFG) Collaborative Research Centre SFB-TR84 (C8 to PHS). We thank Mr. Andreas Geissner and Dr. Anika Reinhardt for useful discussions and Pfénex, Inc., for kindly supplying CRM197 at a reduced price for academic institutions. Dr. Uwe Bierfreund (GE Healthcare, [email protected]) is thanked for kindly supplying the dissociation stage model used to determine kd values by SPR. We express our gratitude to Dr. Bernd Lepenies for advice and support for animal ethics.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: … Characterization of glycoconjugate 6; characterization of liposomes and micelles by DLS; microarray-inferred secondary IgG responses in mice immunized with 6; NMR spectra of compounds 2, 4, and 6 (PDF) Molecular-formula strings of the compounds (CSV)

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AUTHOR INFORMATION Corresponding Author *Tel.: 0049-331-567-9300. Fax: 0049-331-567-9302. E-mail: [email protected]. ORCID Peter Seeberger: 0000-0003-3394-8466 Author Contributions F.B. performed biological experiments. S.G., J.H., and D.C.K.R. synthesized chemical compounds. C.L.P., P.S., C.A., and P.H.S. designed the research. F.B. and S.G. wrote the manuscript with input from all authors. All authors approved the final version of the manuscript. Notes P.H.S. declares a significant financial interest in Vaxxilon, the company that commercializes carbohydrate-α-GalCer vaccine candidates. There is, however, no conflict of interest, as this is a purely scientific manuscript that was not sponsored by the company.

ABBREVIATIONS USED α-GalCer α-galactosylceramide; APC antigen presenting cell; DSPC 1,2-distearoyl-sn-glycero-3phosphocholine; HRMS high-resolution mass spectrometry; iNKT invariant natural killer T (cell); 26 ACS Paragon Plus Environment

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MFI mean fluorescence intensity; MPLA monophosphoryl lipid A; NMR nuclear magnetic resonance; RUs response units; SAR structure–activity relationship; SPR surface plasmon resonance; TACA tumor-associated carbohydrate antigen; TLR Toll-like receptor

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16. Renaudet, O.; BenMohamed L.; Dasgupta, G.; Bettahi, I.; Dumy, P. Towards a selfadjuvanting multivalent B and T cell epitope containing synthetic glycolipopeptide cancer vaccine. ChemMedChem 2008, 3, 737-741. 17. Lo-Man, R.; Bay, S.; Vichier-Guerre, S.; Dériaud, E.; Cantacuzène, D.; Leclerc, C. A fully synthetic immunogen carrying a carcinoma-associated carbohydrate for active specific immunotherapy. Cancer Res. 1999, 59, 1520-1524. 18. Miermont, A.; Barnhill, H.; Strable, E.; Lu, X.; Wall, K. A.; Wang, Q.; Finn, M. G.; Huang, X. Cowpea mosaic virus capsid: a promising carrier for the development of carbohydrate based antitumor vaccines. Chem. - Eur. J. 2008, 14, 4939−4947. 19. Liu, X.; Siegrist, S.; Amacker, M.; Zurbriggen, R.; Pluschke, G.; Seeberger, P. H. Enhancement of the immunogenicity of synthetic carbohydrates by conjugation to virosomes: a leishmaniasis vaccine candidate. ACS Chem. Biol. 2006, 1, 161-164. 20. Huang, Z. H.; Shi, L.; Ma, J. W.; Sun, Z. Y.; Cai, H.; Chen, Y. X.; Zhao, Y. F.; Li, Y.-M. A totally synthetic, self-assembling, adjuvant-free MUC1 glycopeptide vaccine for cancer therapy. J. Am. Chem. Soc. 2012, 134, 8730-8733. 21. Liao, G.; Zhou, Z.; Suryawanshi, S.; Mondal, M. A.; Guo, Z. Fully synthetic self-adjuvanting α2,9-oligosialic acid based conjugate vaccines against group C meningitis. ACS Cent. Sci. 2016, 2, 210-218. 22. Wang, Q.; Zhou, Z.; Tang, S.; Guo, Z. Carbohydrate-monophosphoryl lipid A conjugates are fully synthetic self-adjuvanting cancer vaccines eliciting robust immune responses in the mouse. ACS Chem. Biol. 2012, 7, 235-240.

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30. Zhou, X. T.; Forestier, C.; Goff, R. D.; Li, C.; Teyton, L.; Bendelac, A.; Savage, P. B. Synthesis and NKT cell stimulating properties of fluorophore- and biotin-appended 6"-amino-6"deoxy-galactosylceramides. Org. Lett. 2002, 4, 1267-1270. 31. Tashiro, T.; Nakagawa, R.; Shigeura, T.; Watarai, H.; Taniguchi, M.; Mori, K. RCAI-61 and related 6'-modified analogs of KRN7000: their synthesis and bioactivity for mouse lymphocytes to produce interferon-γ in vivo. Bioorg. Med. Chem. 2013, 21, 3066-3079. 32. Toyokuni, T.; Hakomori, S.; Singhal, A. K. Synthetic carbohydrate vaccines: synthesis and immunogenicity of Tn antigen conjugates. Bioorg. Med. Chem. 1994, 2, 1119-11132. 33. Kuduk, S. D.; Schwarz, J. B.; Chen, X.-T.; Glunz, P. W.; Sames, D.; Ragupathi, G.; Livingston, P. O.; Danishefsky, S. J. Synthetic and immunological studies on clustered modes of mucinrelated Tn and TF O-linked antigens: the preparation of a glycopeptide-based vaccine for clinical trials against prostate cancer. J. Am. Chem. Soc. 1998, 120, 12474-12485. 34. Palitzsch, B.; Gaidzik, N.; Stergiou, N.; Stahn, S.; Hartmann, S.; Gerlitzki, B.; Teusch, N.; Flemming, P.; Schmitt, E.; Kunz, H. A synthetic glycopeptide vaccine for the induction of a monoclonal antibody that differentiates between normal and tumor mammary cells and enables the diagnosis of human pancreatic cancer. Angew. Chem. Int. Ed. Engl. 2016, 55, 2894-2898. 35. Abdel-Aal, A. B.; El-Naggar, D.; Zaman, M.; Batzloff, M.; Toth, I. Design of fully synthetic, self-adjuvanting vaccine incorporating the tumor-associated carbohydrate Tn antigen and lipoamino acid-based Toll-like receptor 2 ligand. J. Med. Chem. 2012, 55, 6968-6974.

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36. Kröck, L.; Esposito, D.; Castagner, B.; Wang, C.-C.; Bindschädler, P.; Seeberger, P. H. Streamlined access to conjugation-ready glycans by automated synthesis. Chem. Sci. 2012, 3, 1617-1622. 37. Whitfield, C. Biosynthesis and assembly of capsular polysaccharides in Escherichia coli. Annu. Rev. Biochem. 2006, 75, 39-68. 38. Yother, J. Capsules of Streptococcus pneumoniae and other bacteria: paradigms for polysaccharide biosynthesis and regulation. Annu. Rev. Microbiol. 2011, 65, 563-581. 39. Martin, C. E.; Broecker, F.; Oberli, M. A.; Komor, J.; Mattner, J.; Anish, C.; Seeberger, P. H. Immunological evaluation of a synthetic Clostridium difficile oligosaccharide conjugate vaccine candidate and identification of a minimal epitope. J. Am. Chem. Soc. 2013, 135, 9713-9722. 40. Watson, D. S.; Endsley, A. N.; Huang, L. Design considerations for liposomal vaccines: influence of formulation parameters on antibody and cell-mediated immune responses to liposome associated antigens. Vaccine 2012, 30, 2256-2272. 41. Geissner, A.; Pereira, C. L.; Leddermann, M.; Anish, C.; Seeberger, P. H. Deciphering antigenic determinants of Streptococcus pneumoniae serotype 4 capsular polysaccharide using synthetic oligosaccharides. ACS Chem. Biol. 2016, 11, 335-344. 42. Wu, A. M.; Wu, J. H.; Chen, Y. Y.; Song, S. C.; Kabat, E. A. Further characterization of the combining sites of Bandeiraea (Griffonia) simplicifolia lectin-I, isolectin A(4). Glycobiology 1999, 9, 1161-1170. 43. Bai, L.; Deng, S.; Reboulet, R.; Mathew, R.; Teyton, L.; Savage, P. B.; Bendelac, A. Natural killer T (NKT)-B-cell interactions promote prolonged antibody responses and long-term 32 ACS Paragon Plus Environment

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memory to pneumococcal capsular polysaccharides. Proc. Natl. Acad. Sci. USA 2013, 110, 16097-16102. 44. Whitmire, J. K.; Slifka, M. K.; Grewal, I. S.; Flavell, R. A.; Ahmed, R. CD40 ligand-deficient mice generate a normal primary cytotoxic T-lymphocyte response but a defective humoral response to a viral infection. J. Virol. 1996, 70, 8375-8381. 45. Coffman, R. L.; Seymour, B. W.; Lebman, D. A.; Hiraki, D. D.; Christiansen, J. A.; Shrader, B.; Cherwinski, H. M.; Savelkoul, H. F.; Finkelman, F. D.; Bond, M. W.; Mosmann T. R. The role of helper T cell products in mouse B cell differentiation and isotype regulation. Immunol. Rev. 1988, 102, 5-28. 46. Brewer, J. M.; Alexander, J. Cytokines and the mechanisms of action of vaccine adjuvants. Cytokines Cell. Mol. Ther. 1997, 3, 233-246. 47. Khurana, S.; Verma, N.; Yewdell, J. W.; Hilbert, A. K.; Castellino, F.; Lattanzi, M.; Del Giudice, G.; Rappuoli, R.; Golding, H. MF59 adjuvant enhances diversity and affinity of antibodymediated immune response to pandemic influenza vaccines. Sci. Transl. Med. 2011, 3, 85ra48. 48. Reddy, S. B.; Anders, R. F.; Beeson, J. G.; Färnert, A.; Kironde, F.; Berenzon, S. K.; Wahlgren, M.; Linse, S.; Persson, K. E. High affinity antibodies to Plasmodium falciparum merozoite antigens are associated with protection from malaria. PLoS One 2012, 7, e32242. 49. Brewer, J. M.; Tetley, L.; Richmond, J.; Liew, F. Y.; Alexander, J. Lipid vesicle size determines the Th1 or Th2 response to entrapped antigen. J. Immunol. 1998, 16, 4000-4007. 50. Badiee, A.; Khamesipour, A.; Samiei, A.; Soroush, D.; Shargh, V. H.; Kheiri, M. T.; Barkhordari, F., Robert Mc Master, W.; Mahboudi, F., Jaafari, M. R. The role of liposome size 33 ACS Paragon Plus Environment

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on the type of immune response induced in BALB/c mice against leishmaniasis: rgp63 as a model antigen. Exp. Parasitol. 2012, 132, 403-409. 51. Poolman, J.; Frasch, C.; Nurkka, A.; Käyhty, H.; Biemans, R.; Schuerman, L. Impact of the conjugation method on the immunogenicity of Streptococcus pneumoniae serotype 19F polysaccharide in conjugate vaccines. Clin. Vaccine Immunol. 2011, 18, 327-336. 52. Anish, C.; Schumann, B.; Pereira, C. L.; Seeberger, P. H. Chemical biology approaches to designing defined carbohydrate vaccines. Chem. Biol. 2014, 21, 38-50.

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Scheme 1. Synthesis of carbohydrate conjugates 4, 5 and 6. Reagents and conditions: a) bis(4nitrophenyl) carbonate, trimethylamine, pyridine, 79%; b) 3, pyridine, trimethylamine, 42%; c) pyridine, stearoyl chloride, 46%; d) triethylamine, di-N-succinimidyl adipate dissolved in DMSO;

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e) 7, 100 mM sodium phosphate buffer, pH 7.4. A characterization of 6 by SDS-PAGE and MALDI-TOF MS analysis is shown in Fig. S1.

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Fig. 1. Immunogenicity of liposomes and a glycoconjugate presenting the Tn antigen. (a) Immunization regime. Groups of six mice received three injections comprising 1.7 μg of Tn antigen at the indicated time points. Serum at week 5 was subjected to microarray-assisted serum IgG1 analysis. (b) Spotting pattern of microarray slides shown in panels (c) and (d). Glycans were spotted at 0.25 and 0.5 mM (small and large circles, respectively), CRM197 at 1 μM, all in quadruplicates. (c) Representative microarray scan showing binding pattern of GS-I lectin to the spotted compounds. Blue signals represent fluorescence excited at 488 nm. (d) Exemplary microarray scans of mice immunized with the indicated antigen formulations at week 5. Each panel represents the IgG1 response of one individual mouse. Yellow signals represent fluorescence excited at 594 nm.

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Fig. 2. Serum anti-Tn antibody responses in immunized mice. (a) Microarray-inferred serum IgG expressed as mean fluorescence intensity (MFI) values are shown. Each data point represents a mean ± SEM of six mice. Statistical significance was inferred by comparing IgG levels of mice immunized with 4-containing liposomes to those immunized with 5-containing liposomes. Note that week 21 and 22 IgG levels in response to micelles of 4 represent pooled serum (n=6) instead mean of individual sera. (b) Comparison of IgG levels at week 5. Horizontal bars 38 ACS Paragon Plus Environment

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indicate mean values. (c) SPR-inferred antibody responses using pooled serum diluted 1:100. Levels of significance in panels (a) and (b) were inferred by two-sided unpaired Student’s t-Tests with *p≤0.05 and **p≤0.01.

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Fig. 3. IgG2a to IgG1 ratios and affinity maturation in immunized mice. (a) IgG2a to IgG1 ratios were calculated by dividing microarray-inferred binding signals of six mice per group except for 6 where the ratio was determined from three mice that raised anti-Tn IgG. Bars show mean ± SEM. (b) Pooled serum was used to calculate the percentage of urea-resistant IgG to Tn antigen. Bars represent mean ± SD of two experiments. (c) SPR-inferred dissociation rate constants (kd values). From top to bottom, pooled serum diluted 1:100, 1:200, 1:400, 1:800 and 1:1600 at week 5 was passed through the SPR sensor chip. Red lines indicate fitting of the dissociation stage that was used to determine the indicated kd values.

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Table of contents graphic:

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Figure 1 126x74mm (300 x 300 DPI)

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Figure 2 166x173mm (300 x 300 DPI)

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Figure 3 82x125mm (300 x 300 DPI)

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Scheme 1 181x192mm (300 x 300 DPI)

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TOC 198x48mm (300 x 300 DPI)

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