Immunogenicity and Diagnostic Potential of Synthetic Antigenic Cell

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Immunogenicity and Diagnostic Potential of Synthetic Antigenic Cell Surface Glycans of Leishmania Chakkumkal Anish,*,†,⊥ Christopher E. Martin,†,‡,⊥ Annette Wahlbrink,† Christian Bogdan,§ Pantelis Ntais,∥ Maria Antoniou,∥ and Peter H. Seeberger*,†,‡ †

Max-Planck Institute of Colloids and Interfaces, 14424 Potsdam, Germany Institute of Chemistry and Biochemistry, Freie Universität Berlin, Arnimallee 22, 14195 Berlin, Germany § Mikrobiologisches Institut - Klinische Mikrobiologie, Immunologie und Hygiene, Universitätsklinikum Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany ∥ Laboratory of Clinical Bacteriology Parasitology Zoonoses and Geographical Medicine, Faculty of Medicine, University of Crete, Heraklion, Greece ‡

S Supporting Information *

ABSTRACT: Detection and quantification of pathogen-derived antigenic structures is a key method for the initial diagnosis and follow-up of various infectious diseases. Complex parasitic diseases such as leishmaniasis require highly sensitive and specific tests prior to treatment with potentially toxic drugs. To investigate the diagnostic potential of cell surface glycans found on Leishmania parasites, we identified diagnostically relevant glycan epitopes and used synthetic glycan microarrays to screen sera from infected humans and dogs. On the basis of the screening results, we selected a tetrasaccharide to generate anti-glycan antibodies. The corresponding tetrasaccharidecarrier protein conjugate was immunogenic in mice, and sera obtained from immunized mice specifically detected the Leishmania parasite. These results demonstrate how synthetic glycan arrays, in combination with immunological methods, help to identify promising carbohydrate antigens for pathogen detection.

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Available confirmatory tests for VL detect the parasite by microscopy, culture, and/or polymerase chain reaction (PCR)based examination of lymph node, bone marrow, or splenic aspirates. Although the specificity is high, the sensitivity of microscopic examination varies, depending on the origin of aspirates, the ability of the laboratory workers, and the quality of the reagents.1,7 Cell culture and PCR methods are highly specific and sufficiently sensitive but require a rather complex infrastructure. The availability of parasite-specific immunological tests greatly improved rapid diagnosis of Leishmania infections. A number of such tests based on the detection of Leishmania antigen-specific antibodies in patients exist, but their performance varies depending on the antibody titers.1,8,9 Low antiLeishmania antibody titers typically occur in cases of noninvasive cutaneous leishmaniasis, can result from the initiation of treatment, and are frequently seen during Leishmania coinfection of HIV-positive patients with poor CD4+ T cell status.1,10 Antigen-based diagnostic tests avoid this variability as immunologically relevant antigenic markers are presented directly on the parasitic cell surface.1,7 Direct microscopic

eishmaniasis is a vector-borne protozoan disease caused by facultative intracellular parasites of the genus Leishmania.1 More than 20 Leishmania species are known to cause disease in humans and are endemic in many tropical and subtropical regions, home to sand fly vectors.2,3 Clinically, leishmaniasis presents in four major clinical syndromes:4 cutaneous leishmaniasis (CL), muco-cutaneous leishmaniasis, visceral leishmaniasis (VL; also known as kala-azar), and post-kalaazar dermal leishmaniasis (PKDL).1 VL is a fatal systemic disease if left untreated and is caused mainly by Leishmania donovani in East Africa and the Indian subcontinent and L. infantum or L. chagasi (the later originating from L. infantum) in Europe, North Africa, or Latin America, respectively. Two types of VL differ in their transmission characteristics: anthroponotic VL, mainly caused by L. donovani, is transmitted from humans to humans, whereas zoonotic VL, caused by L. infantum/L. chagasi, is transmitted from animals to humans.1,5,6 Dogs are the main reservoir in zoonotic VL; therefore the availability of a rapid, easy to perform, highly sensitive, and specific diagnostic test in canines is crucial for the control of zoonotic VL. Confirmatory tests are additionally required prior to clinical treatment of human patients due to the potentially high toxicity of current anti-leishmanial drugs.1,7 Diagnostic tests that can differentiate acutely diseased patients from clinically asymptomatic and non-progressive infections are highly desirable. © 2013 American Chemical Society

Received: April 2, 2013 Accepted: September 4, 2013 Published: September 4, 2013 2412

dx.doi.org/10.1021/cb400602k | ACS Chem. Biol. 2013, 8, 2412−2422

ACS Chemical Biology

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Figure 1. Synthetic LPG capping oligosaccharides 1−4. Tetrasaccharide 1 and trisaccharide 4 are found on L. donovani. Disaccharide 2 and trisaccharide 3 are found on L. chagasi (L. infantum).

extensively,16,21−23 no systematic evaluation of immune responses against LPG and its substructures has been conducted.20,24 The contribution of capping oligosaccharides to the immune recognition of LPG has not been studied in detail. LPG capping oligosaccharides are vital for the parasite and its transmission as they mediate binding to the sand fly midgut.16 LPGs have a conserved inner glycan core region and a conserved repeating domain consisting of a β-Gal-(1→4)-αMan-(1→P) backbone.25,26 The distinguishing feature of LPGs responsible for the polymorphisms among Leishmania species resides in the type and number of oligosaccharide side chains that branch from the β-Gal-(1→4)-α-Man backbone repeat units and the carbohydrate residues that are attached to the backbone in the neutral cap structure.26,27 Immune recognition of LPG components to produce circulating anti-LPG antibodies prior to transformation to the amastigote stage is relatively unexplored. Though structurally different, amastigote LPG is present on the L. major surface in its flagellar pocket and binds to anti-promastigote specific monoclonal antibodies and antileishmanial rabbit and human sera.28,29 This cross reactivity is partially mapped to phosphoglycans, but the structural basis of this binding are poorly understood. The most common L. donovani caps are branched oligosaccharides consisting of βGal-(1→4)-α-Man and α-Man-(1→2)-α-Man substructures.26,27 Procyclic L. chagasi (and presumably also L. infantum) are reported to produce two types of LPG caps: the disaccharide β-Gal-(1→4)-α-Man and the trisaccharide βGlc-(1→3)-β-Gal-(1→4)-α-Man.26 Glucose has also been observed in the cap structure of the Indian strain of L. donovani.22 Developing specific immunological reagents that detect the capping oligosaccharides will lead to better structure−function analysis of LPG at the molecular level. Access to these glycans is a prerequisite to further investigate the diagnostic potential of antigenic glycans for highthroughput and point of care assay systems. Chemical synthesis can meet the need for large amounts of pure oligosaccharides. Here, we report on the creation and use of glycan arrays composed of synthetic oligosaccharides 1−4, representing part of the leishmanial LPG capping oligosaccharides from L. donovani and L. chagasi (Figure 1).26,27,30,31 To identify diagnostically relevant glycan epitopes, we first used the array to screen a positive anti-leishmanial reference serum for antiLPG capping oligosaccharide antibodies. A wider screening

examination of stain tissue samples, PCR techniques, and parasite cultures form the gold standard for confirming the diagnosis of leishmaniasis. The low sensitivity due to low parasite numbers in target organs and sometimes difficult distinction between Giemsa-stained parasites and other host cell elements in clinical specimens can be overcome by employing antibodies that bind to the parasite cell surface.11 Developing antibodies to cell surface glycans is a promising strategy for the diagnosis, follow-up, and prognosis of both parasitic and bacterial infections.12,13 Lipophosphoglycans (LPGs) are major leishmanial cell surface glycoconjugates that play an important role in disease pathophysiology and can be an attractive target for the design of novel diagnostic reagents. A single L. donovani promastigote that initiates the disease is reported to express 1.25 × 106 lipophosphoglycan molecules. LPG is a predominant promastigote antigen and is only variably expressed on amastigotes that account for the clinical manifestation of the disease and its progression.14 Leishmania major amastigotes synthesize low levels of a stagespecific LPG, and L. donovani and L. mexicana amastigotes do not express LPG at all. Amastigote LPGs are known to be structurally and antigenically distinct. LPG has been identified on the surface of L. major amastigotes by surface labeling and immunofluorescence.15,16 Amastigote LPG of L. major has no α-arabinopyranose side chain and terminates with a neutral βGal-(1→4)-α-Man capping disaccharide.17 To date, no reports on structural or antigenic variation of LPG are available for L. infantum. Although LPG is predominantly a promastigotespecific antigen, it has been demonstrated that some of the biologically active structural elements of LPG such as the repetitive phosphoglycans and the neutral cap oligosaccharides are also present on Leishmania-secreted glycoconjugates such as acid phosphatase (sAP) and the filamentous proteophosphoglycan of promastigotes. It has been shown that protein-bound phosphoglycans are also expressed in the amastigote stage, as in the case of L. mexicana, where amastigote-specific glycan modifications along with other conserved motifs such as (Manα1−2)1−5Man) or β-Gal-(1→4)-α-Man have been detected.18−20 These secreted glycans can stimulate the immune system to mount a strong antibody response that can form the basis of immune protection and diagnosis. Although the importance of leishmanial lipophosphoglycans and their role in host-parasite interactions have been studied 2413



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Figure 2. Monosaccharide building blocks 5−10 employed in the synthesis of oligosaccharides 1−3.

Scheme 1. Synthesis of Oligosaccharides 1−3 from Building Blocks 5−10a

Reagents and conditions: (A) (a) HOL, TMSOTf, DCM/Et2O, 0 °C; (b) triethylsilane, TfOH, 4 Å molecular sieves, −78 °C (11A, 57%; 11B, 59%; 12, 78%) (two steps); (c) TMSOTf, DCM, −40 to −20 °C; NEt3 (14, 89%); (d) 13A: AcCl, THF/MeOH 0 °C to rt, 86% (two steps); 13B: NaOMe, DCM/MeOH, 83% (two steps); (e) NaOMe, THF/MeOH, microwave reactor (3 W, 100 °C, 5 bar); (f) Pd/C, H2, MeOH/THF/H2O/ AcOH, 78% (two steps). (B) (a) TMSOTf, DCM/Et2O, 0 °C; (b) 15A: AcCl, THF/MeOH 0 °C to rt, 73% (two steps); 15B: NaOMe, DCM/ MeOH, 69% (two steps); (c) TMSOTf, DCM/Et2O, 0 °C, (16A, 84%; 16B, 86%); (d) 1A: NaOMe, MeOH/THF, microwave reactor (3 W, 100 °C, 5 bar); 1B: KOH, MeOH/THF/H2O; (e) 1A: H2, Pd/C, THF/MeOH/H2O/AcOH, 71% (two steps); 1B: Pd/C, H2, MeOH/THF/H2O/ AcOH; 0.25 N KOH, H2O, 54% (three steps). (C) (a) TMSOTf, DCM, −40 to −20 °C, 53%; (b) NaOMe, THF/MeOH; (c) Pd/C, H2, MeOH/ THF/H2O/AcOH, 64% (two steps). L = linker (CH2)5NBnCbz for A; (CH2)2NHCbz for B. a

afforded 11 or 12, respectively. Union of 11 and galactosylphosphate 7,33 followed by removal of the 2-O-acetate, provided disaccharide 13. Repetitive glycosylation with mannosyl-imidate 932 subsequently followed by deacetylation gave protected tetrasaccharide 16. In the case of 1A, global deprotection was achieved in two steps by saponification of the pivaloyl-ester in the presence of sodium methoxide using a microwave reactor (3 W, 5 bar, 100 °C), followed by hydrogenation for the removal of aromatic protecting groups. The deprotection sequence to give 1B was reversed by first performing a hydrogenation and then removing the pivaloyl group by treatment with an aqueous potassium hydroxide solution. Disaccharide 2 was obtained via two-step global deprotection of intermediate 13. Synthesis of trisaccharide 3 relied on the elongation of 12 with galactosyl-phosphate 8; resulting disaccharide 14 was further elongated with glucosylimidate 1034 to give fully protected trisaccharide 17. Finally, global deprotection afforded trisaccharide 3. Screening of Diagnostic Reference Dog Sera. To assess the contribution of LPG capping oligosaccharide epitopes to the immune response, we prepared a glycan array using synthetic oligosaccharides 1−3 and 4.35,36 The array was used to screen an anti-leishmanial reference serum of an infected dog

conducted on infected populations of humans and dogs further revealed high antibody level variability between infected individuals. After identifying diagnostically relevant antigens from the screens, we immunized mice with the corresponding oligosaccharide-protein conjugates and showed that the sera of immunized mice can be used for specific parasite detection. Monoclonal antibodies generated from the immunized mice may be the basis for innovative, Leishmania antigen-detection systems.

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RESULTS AND DISCUSSION Oligosaccharide Synthesis. Syntheses of three capping LPG oligosaccharide epitopes 1−3 (Figure 1), equipped with an amine linker for microarray immobilization and conjugation to carrier proteins, relied on monosaccharide building blocks 5−10 (Figure 2). Tetrasaccharides 1A equipped with a five carbon (C5) linker and 1B carrying a two carbon (C2) linker were assembled in linear fashion similarly to a procedure reported previously (Scheme 1).32 In brief, glycosylation involving mannosyl-imidates 5 or 632 with either N-protected aminopentanol or aminoethanol, followed by selective opening of the 4,6-O-benzylidene ring, 2414



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were absent in the serum (Figure 3). The positive canine reference serum contained significantly higher levels of antibodies against disaccharide 2 and trisaccharide 3 than the negative control serum. Both negative and positive sera recognized trimannose 4. Since the positive reference serum is based on infected dogs whose immune system had encountered the whole parasite, glycans 2 and 3 from L. chagasi are apparently part of antigenic structures that interact with the immune system. Consequently, detection of these oligosaccharides or antibodies in body fluids can be of diagnostic importance for zoonotic leishmaniasis caused by L. chagasi. The lack of antibodies binding to tetrasaccharide 1 was surprising since 1 contains the Gal-(1→4)-Man motif that is also conserved in 2 and 3, as well as the trimannose motif of 4. This finding may be explained by differences in oligosaccharide orientation on the parasite surface when presented to the immune system. In order to validate the hits from this first screening, we proceeded by screening sera from human patients and infected dogs, living in endemic areas. Screening of Sera from Infected Humans and Dogs. To gain further insight into immunogenic epitopes and evaluate the antibody variability in infected individuals against capping structures, we analyzed the sera of 50 infected dogs of different age, sex, and immune status from different geographical regions of Greece. Zoonotic visceral leishmaniasis (ZVL) caused by L. infantum (L. chagasi) is endemic in the Mediterranean basin. Sera from dogs with symptomatic and proven visceral leishmaniasis (i.e., positive by leishmanial confirmatory tests such as immunofluorescent-antibody test (IFAT), PCR, or parasite culture) were included as positive in the test. Twentyfive clinically asymptomatic dogs from the same endemic areas of Greece that were presumably also exposed to sand fly bites and Leishmania parasites were included as asyptomatic carriers. Twenty dogs of similar age from a non-endemic area (Berlin) formed the control group. Thirty-two experimental dogs held under specific pathogen-free conditions with presumably minimal exposure to the pathogens were included as an additional negative control group to evaluate background antibody responses. Details of the tested dogs are presented in

that was obtained as a component of a commercial immunofluorescence assay kit. The antibodies in the canine serum bound to the oligosaccharides presented on the glass surface were visualized with a corresponding anti-dog IgG DyLight 488 secondary antibody (Figure 3). IgG response

Figure 3. Glycan array screening of a reference canine serum from a commercial diagnostic kit for anti-glycan antibodies. (A) A representative well from glycan array analysis of a negative control canine serum plus the secondary anti-dog IgG DyLight 488 antibody. (B) A representative well from glycan array analysis of Leishmaniapositive canine reference serum showing reactivity of secondary antidog IgG DyLight 488 antibody. Positions of respective antigens are shown with the numbers. Fluorescent spots indicate antibody binding from the serum. Background corrected mean fluorescence intensity (MFI) of antigen-specific spots is plotted against respective antigens in the bar graph. MFI of buffer printed spots were used for background correction. MFI corresponding to 1:40 serum dilution is shown in the graph. Error bars represent mean ± SEM of eight microarray spots (****P < 0.0001, # P > 0.05).

against oligosaccharides 2, 3, and 4 were observed on the glycan array, whereas antibodies reacting to tetrasaccharide 1

Figure 4. Distribution of anti-glycan IgG response levels against antigens 1−4 from a population of L. infantum infected domestic dogs (A) and humans (B). Bound antibody signals (MFI) of four individual carbohydrate structures, grouped by disease status and sorted by antigen. Numbers 1− 4 indicates oligosaccharide antigens. Each dot indicates a serum sample. Error bars shows mean with standard error of means (SEM). All groups were compared using one-way ANOVA with Bonnferoni post-test correction for statistical significance. Immunofluorescent-antibody test (IFAT)positive candidates are confirmed leishmaniasis patients. IFAT negative dogs are from leishmaniasis endemic areas with higher possibility of exposure to sand fly bites (Greece). Control dogs are healthy domestic dogs from a non-endemic area (Berlin, Germany). SPF dogs are healthy experimental dogs held in specific pathogen-free conditions and presumably with minimal exposure to sand fly bites. In the case of humans, IFAT negative control candidates are healthy individuals from non-endemic areas. VL and CL infected candidates are, respectively, cutaneous leishmaniasis and visceral leishmaniasis infected patients. 2415



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important to further validate these findings. The results indicate that antigen 1 and 4 motifs are recognized and antibodies against trimannose epitope are elevated in VL infection. However, anti-4 antibodies are not strictly Leishmania-specific markers since anti-mannan antibodies are also found in some patients with autoimmune diseases.37 The results from the screening of the reference serum and sera from infected dogs and human patients revealed the presence of antibodies against all four antigens. Both Gal-(1→ 4)-Man 2 and trimannose 4 appear to be immunodominant epitopes. The significant variation of antibody levels observed between individuals implicates that a diagnostic test relying exclusively on the detection of antibodies would be inaccurate. Therefore, we envisioned to develop a strategy that relies on the direct detection of the parasites using anti-capping oligosaccharide antibodies. Earlier studies using Leishmania promastigotes or LPGs as immunogens have shown the specificity of generated antibodies against the conserved Gal(1→4)-Man disaccharide portion of whole or truncated LPGs. However, some of the monoclonal antibodies that were reported did not react with the repeating unit;19,20 this observation could be attributed to the absence of capping residues and polymorphisms of isolated LPGs. It is reported that mAbs specific for the phosphorylated disaccharide repeating unit of LPG can bind the entire surface and flagellum of the Leishmania promastigote, but anti-core antibodies cannot bind to the parasite surface, presumably due to masking of the core region.38 Capping oligosaccharides expressed at the nonreducing end of the LPG can be a viable target for developing antibodies for parasite detection due to its surface exposure. Careful selection of monoclonal antibodies to species-specific capping oligosaccharides may aid studies on the parasite speciation as well as studying the immunochemical structure and surface arrangement of LPGs. To reach this goal, the synthetic glycans were used to generate polyclonal sera or monoclonal antibodies specifically detecting the parasitic cell surface glycans. For this, a single antigen capable of raising antibodies detecting a range of leishmanial glycan antigens would be ideal. The Gal-(1→4)Man substructure is common to oligosaccharides 1−3, and therefore antibodies binding to this epitope would detect all three LPG capping oligosaccharides. The trimannose epitope of 4 proved important, especially in the case of human infections. Therefore, tetrasaccharide 1 that contains both the Gal-(1→4)Man and trimannose epitopes was selected for immunogenicity studies to raise antibodies. This selection was further supported by the fact that larger oligosaccharides are more immunogenic than smaller ones and earlier reports on robust immune response observed against tetrasaccharide 1.25 Preparation of Neoglycoconjugates. Carbohydrates are T cell-independent antigens; upon immunization they usually do not induce an immunoglobulin class switch and secondary response. Covalent attachment of carbohydrate antigens to immunogenic carrier proteins produces neoglycoconjugates that induce a T cell-dependent immune response.39,40 Synthetic oligosaccharides 1A and 1B of L. donovani LPG were conjugated to the carrier protein CRM197. The diphtheria toxoid CRM197 was chosen as a carrier because it is a constituent of licensed vaccines and induces the least carrier mediated suppression of anti-hapten responses.41 A method based on the selective reaction of the primary amine with a disuccinimido adipate (DSAP) spacer was selected from a multitude of conjugation methods.42 First, the amine group of

the Supporting Information (see Supplementary Tables S5− S8). Based on mean fluorescence intensity (MFI) values of control group sera (control dogs, Figure 4), approximately 76% of sera tested positive for anti-4, 38% for anti-2, 8% for anti-3, and 42% for anti-1 antibodies, with a broad distribution of antibody levels between individuals (Figure 4). Compared to dogs from the non-endemic area the IFAT-positive infected dogs showed significantly higher levels of anti-glycan antibodies. However, within the entire group of dogs from the endemic area there were no significant differences in the antibody response between infected (IFAT-positive) and IFAT-negative (i.e., uninfected or asymptomatically infected) dogs except for antigen-1 (p = 0.0361). Receiver operating characteristic (ROC) curve analysis was used to assess the accuracy of antiglycan antibody level (MFI) to identify dogs with active leishmaniasis. For each antigen, the area under the curve (AUC) was separately calculated. Lower AUC values of 0.62, 0.53, 0.51, and 0.49 observed for antigen 1, 2, 3, and 4, respectively, indicated poor sensitivity and specificity for these antigens in predicting VL in infected versus uninfected/ asymptomatic dogs. In contrast, AUC values of 0.75, 0.69, 0.50, and 0.89 observed for 1, 2, 3, and 4, respectively, indicated higher sensitivity and specificity for predicting VL in infected versus control dogs from non-endemic area (see Supplementary Figures S16 and S17). Many (1 = 58%, 2 = 62%, 3 = 92%, 4 = 24%) infected dogs that tested positive for ZVL by PCR, culture, or IFAT showed low anti-glycan antibody levels. This may be due to an impaired humoral immune response during severe disease or may result from the anti-leishmanial therapy, which is reported to lower antibody levels. Many asymptomatic dogs from endemic areas showed significantly higher levels of anti-glycan antibodies. Frequent exposure to sand fly bites and subsequent parasite inoculation may explain this observation. Thus, although anti-glycan antibody levels have diagnostic value for predicting acute VL infection in dogs from non-endemic areas, it has limited utility in endemic areas. The high variability of antibody responses to glycan antigens may be the result of the heterogeneity of the tested dogs with respect to their genetic background their age, sex, immune status, and medication. The cross reactivity of antibodies against the Gal(1→4)-Man motif present in the LPG repeat units as well as capping oligosaccharides might also contribute to the variable and mixed response obtained. In order to investigate the response to the LPG capping glycans in human leishmanial infections, sera from five healthy controls or 23 patients with visceral leishmaniasis (following infection with L. infantum) and 6 patients with cutaneous leishmaniasis (due to various species of Leishmania parasites) were tested (see Supplementary Tables S9 and S10). Using the protocol established for canine sera, the bound antibodies were detected using antihuman IgG Alexafluor 635. Based on control group MFI values, approximately 95% of VL infected patient sera tested positive for anti-4, 48% for anti-2, 66% for anti-3, and 52% for anti-1 antibodies, with a broad distribution of antibody levels between individuals (Figure 4). Some of the sera from patients with CL also showed elevated levels of antiglycan antibodies. Especially anti-4 (33%) and anti-1 (66%) were present in many of the tested sera. Compared to canine sera analysis, relatively higher AUC values of 0.75, 0.68, 0.69, and 0.79 were observed for antigen 1, 2, 3, and 4, respectively, indicating better sensitivity and specificity for these antigens in predicting VL in infected versus non-infected humans. Testing more human samples from diverse populations would be 2416



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comparison to pre-immunized serum levels (Figure 5). Isotype analysis of antigen specific antibodies in serum indicated the presence of higher IgG1 and IgG2a antibodies compared to IgG3, indicative of isotype switching (data not shown). The sera from immunized mice contained antibodies against the carrier protein as well as control conjugates C2 (Figure 5). To confirm the binding specificity, sera from immunized mice were evaluated using glycan arrays containing tetrasaccharide 1A, carrier protein CRM197, and conjugate C2. The bound antibodies were detected using anti-mouse IgG-FITC. The serum showed reactivity to carrier protein CRM197 and C1. No binding to the unconjugated 1A was observed (Figure 5B). The disparity in binding observed between the glycan array and ELISA may arise from the difference in orientation of tetrasaccharide 1A on protein and glass slides. Adipate spacer-specific antibodies elicited during immunization may contribute by cross reacting with C1 conjugate in ELISA but not with unconjugated 1A on microarrays. High levels of antiC2 antibodies were observed on microarrays and can be attributed to anti-spacer-specific antibodies (Figure 5C). Mindful of the influence of the spacer, an alternate ELISA method was adopted to evaluate the IgG response against tetrasaccharide 1A. A comparison of anti-spacer and anti-1A response indicated the presence of anti-1A IgG in the mouse sera (Figure 5D). This was also further confirmed by surface plasmon resonance by immobilizing 1A on CM5 chip and binding with post-immune sera (see Supplementary Figure S21). Though upon immunization anti-1A specific antibodies were elicited, the level was too low to detect 1A on glycan arrays. To further boost the level of anti-1A antibodies, mice were primed with CRM197-1A conjugate and subsequently boosted with CRM-tetrasaccharide 1B conjugate carrying the shorter aminoethanyl linker. Thereby, the maturation of antispacer response was limited and strong anti-tetrasaccharide 1 response was promoted. Glycan array analysis of the serum after the first week of boosting with CRM197-1B showed the presence of antibodies binding specifically to tetrasaccharide 1. As the immune response progressed, cross reactivity of antibodies to Gal(1→4)-Man 2 was also observed. Significantly higher levels of anti-2 antibodies showed that Gal-(1→4)-Man 2 is the immunodominant component of the tetrasaccharide 1A antigen

the linker moiety in tetrasaccharide 1A or 1B was reacted with one of the N-hydroxy succinimide (NHS) activated esters of DSAP to form the corresponding linker-activated oligosaccharide, and then the activated glycan was coupled with the amino groups of lysine on the CRM197 to afford the neoglycoconjugate (Scheme 2). Scheme 2. Conjugation of Oligosaccharides 1A and 1B to Carrier Protein CRM197a

a

For reagents and conditions see Methods and Supporting Information.

Conjugation was confirmed by SDS-PAGE, and the oligosaccharide/CRM197 ratio was determined by MALDITOF MS (Supporting Information). On average, eight to ten haptens were loaded on CRM197. Alternatively, tetrasaccharide 1A was attached to bovine serum albumin (BSA), yielding control neoglycoconjugate C1, which was used as a coating antigen in ELISA. Additional control glycoconjugate C2, consisting of GlcNAc conjugated to BSA was prepared; this conjugate contains the adipate spacer but no leishmanial LPG related glycan. Immunogenicity of Glycoconjugates. To test the immunogenicity of tetrasaccharide hapten 1A and raise antibodies that detect glycans on the parasite cell surface, mice were immunized with the CRM197-1A glycoconjugate. Each mouse received one priming dose with CRM197-1A conjugate (5 μg each), formulated with Complete Freund’s Adjuvant (CFA), and two boosting doses of the conjugate in Incomplete Freund’s Adjuvant (ICFA). The anti-hapten 1 antibody titers were monitored by ELISA using conjugate C1 as the coating antigen. All immunized mice showed a robust IgG response against C1 with significantly higher antibody titers in

Figure 5. Evaluation of anti-tetrasaccharide 1A IgG response after immunization. Six BALB/c mice were immunized with CRM-1A conjugate for three immunization periods (2 weeks/period). (A) Comparison of groups immunized with glycoconjugates formulated in Complete Freund’s Adjuvant, conjugate without adjuvant, and unconjugated tetrasaccharide 1A. (B, C) Glycan array analysis of sera from immunized mice. (D) Comparison of IgG responses against BSA-1A conjugate (C1) and BSA-GlcNAc conjugate (C2). Fold increase in A450 nm at each dilution in comparison to pre-immunization stage is plotted as an arbitrary ELISA unit. Error bars represent mean values ± SEM. ***P ≤ 0.01 (one-way ANOVA with Tukey−Kramer multiple comparisons). 2417



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Figure 6. Evaluation of anti-1 IgG response by glycan array. Three C57BL/6J mice were primed with CRM-1A conjugate and boosted with CRM1B over 3 immunization periods (2 weeks/period). (A) A representative well from glycan array analysis of post-immune serum showing reactivity of secondary anti-mouse IgG FITC antibody at priming stage. (B, C) A representative well from glycan array analysis of post-immune serum after boosting with CRM-1B conjugate. Positions of respective antigens are shown with the numbers, GlcNAc conjugated to BSA (C2). Blue fluorescent spots indicate antibody binding from the serum. Background corrected mean fluorescence intensity (MFI) of antigen-specific spots is plotted against respective antigens in the bar graph (Day 7 boosting and Day 14 boosting in the legend indicates time point after boosting with CRM-1B conjugate with shorter aminoethanyl linker). MFI of buffer printed spots were used for background correction. MFI corresponding to 1:2000 serum dilution is shown in the graph. Error bars represent mean ± SEM of eight microarray spots (***P < 0.001).

Figure 7. CLSM images indirect immunofluorescent staining of L. chagasi by polyclonal sera against hapten 1. (A) Counterstaining of parasite DNA with DAPI. (B) Differential interference contrast microscopy (DIC). (C) FITC-specific fluorescence indicating binding of secondary antibody. (D) Overlay of layers. Top panels represent parasite cell surface labeling by antibodies from hyper-immune sera, and bottom panels show images with pre-immunization sera (back ground fluorescence). Green fluorescence of detection antibody, anti-mouse IgG FITC, in channel C indicates antibody binding from hyper-immune sera. 2418



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conserved tetrasaccharide structure, containing the Gal-(1→4)Man as well as the trimannose motifs, was selected from a primary screen, and glycoconjugates of this antigen were prepared. IgGs specific to the capping oligosaccharides were raised in the serum of the immunized mice. The immunogenicity of the spacer component in glycoconjugate was found to play an important role in driving the anti-glycan antibody responses. The mouse polyclonal sera raised against the leishmanial glycans detected the parasite with high specificity, suggesting their potential as diagnostic targets. The presence of anti-oligosaccharide antibodies in infected dogs and antiparasite antibodies in mice immunized with synthetic glycoconjugates demonstrates that the synthetic oligosaccharides could form the basis for a diagnostic test.

(Figure 6). The presence of Gal-(1→4)-Man epitope in trisaccharide 3 explains the cross reactivity of serum observed on the glycan array containing this antigen. The results indicate that tetrasaccharide 1 is sufficient to generate antibodies that can detect capping oligosaccharides 1−3 of Leishmania. The spacer immunogenicity plays a major role in driving antibody responses against the conjugated oligosaccharides. Earlier reports suggested that spacer immunogenicity mediated suppression of oligosaccharide response in glycoconjugate immunizations.43,44 Selecting an immunologically inert tether with optimal carbon length to conjugate proteins and oligosaccharides is an important design component. Detailed investigations on the contribution of spacer immunogenicity are currently underway. Immunolabeling Studies on Inactivated Parasites. Inactivated parasites were analyzed by immunofluorescence and imaged using confocal laser scanning microscopy (CLSM) to investigate the capability of antibodies produced against synthetic hapten 1 to recognize the native LPG directly on the L. chagasi surface (Figure 7). We observed specific parasite labeling indicated by localized green fluorescence of a secondary antibody (anti-mouse IgG FITC). The green fluorescence co-localized well with the blue fluorescence of counterstain DAPI used for staining parasite DNA. The green fluorescence was observed only in parasites treated with postimmune sera, and pre-immune sera-treated controls showed no signals (Figure 7). To further test the specificity of antibody binding to glycans on the parasite surface, sera were preincubated with unconjugated glycans and their conjugates, to block the binding. Though a tendency toward inhibition of binding was observed with antigens 1, 2, and their CRMconjugates, validation of results was hard due to variable immobilization and auto fluorescence levels of the parasites (see Supplementary Figures S19 and S20). The results show that specific binding of antibodies from sera to the parasite surface may provide the basis for the detection of L. chagasi in biological samples. Immunochemical investigations of clinical specimens are routine in leishmaniasis diagnosis, and the availability of immunological reagents that specifically detect the parasite will facilitate and improve the sensitivity. Incorporating synthetic surface glycans and monoclonal antibodies that recognize them into modern point of care diagnostic systems would greatly advance diagnosis of leishmaniasis. The reactivity of serum antibodies against the synthetic antigens confirmed their diagnostic importance and immunological relevance. A diagnostic test for leishmaniasis based on the detection of the LPG cap oligosaccharide antigens in their native form is conceivable. Conclusions. The immunogenicity and diagnostic potential of oligosaccharides displayed on the Leishmania cell surface were evaluated. Synthetic, structurally related oligosaccharides derived from the capping region of leishmanial lipophosphoglycans were immobilized on a glycan array to monitor the presence of anti-oligosaccharide antibodies in infected human and canine individuals. Screening with a positive canine serum revealed the importance of the Gal-(1→4)-Man motif for antibody binding. To further confirm the diagnostic potential of the oligosaccharides, a larger screening was conducted on a population of infected dogs. Significant variation in antibody levels against the glycan was observed; these results suggested the development of a diagnostic test based on the detection of the actual carbohydrate antigens rather than antibodies. A

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METHODS

General Procedure (A) for TMSOTf-Mediated Glycosylation of Mannosyl-imidates. The acceptor (1.0 equiv) and mannosyltrichloroacetimidate (1.3−3.0 equiv) were coevaporated with toluene three times and dried in vacuo. The mixture was dissolved in DCM/ Et2O (1:1 or 1:2, reaction concentration at 30−150 mm) and cooled to 0 °C, and TMSOTf (0.3 equiv) was added. After stirring at 0 °C for 30 min to 1 h, the reaction was quenched by the addition of NEt3 and concentrated under reduced pressure. Column chromatography (hexanes/EtOAc) afforded the pure product. General Procedure (B) for Selective 4,6-O-Benzylidene Opening. A mixture of 4,6-O-benzylidene mannoside (1.0 equiv) and freshly activated 4 Å molecular sieves in DCM (reaction concentration 50−100 mM) was cooled to −78 °C, and triethylsilane (3.0 equiv) was added followed by the addition of triflic acid (3.4 equiv). The reaction was stirred at −78 °C for 1−2.5 h until complete conversion of the starting material (monitored by TLC) and quenched by the addition of saturated NaHCO3. The phases were separated, and the organic phase was washed with brine, dried over MgSO4, filtered, and concentrated. Column chromatography (hexanes/EtOAc) afforded the pure product. General Procedure (C) for TMSOTf-Mediated Glycosylation of Galactosyl-phosphates. The acceptor (1.0 equiv) and galactosyldibutylphosphate (1.1 to 1.3 equiv) were coevaporated with toluene three times and dried in vacuo. The mixture was dissolved in DCM (reaction concentration 70−90 mM) and cooled to −40 °C, and TMSOTf (1.1−1.3 equiv) was added. The reaction was kept at −40 °C or warmed to −20 °C over the period of 1 h, quenched by the addition of NEt3, and concentrated under reduced pressure. Column chromatography (hexanes/EtOAc) afforded the pure product. General Procedure (D) for AcCl-Mediated Acetyl-ester Deprotection. The starting material (1.0 equiv) was dissolved in THF/MeOH (3:8, reaction concentration at 30 mM), and AcCl (10 equiv) was added dropwise at 0 °C. The reaction was warmed to RT, stirred for 24 h, and then quenched by the addition of NEt3 (10 equiv). The solvent was removed under reduced pressure, and the residue was purified by column chromatography (hexanes/EtOAc). General Procedure (E) for NaOMe-Mediated Acetyl- and Benzyl-ester Deprotection. To a solution of starting material (1.0 equiv) in DCM or THF (30 to 50 mM) was added a solution of NaOMe (1:8 v/v, 0.5 m in MeOH), and the mixture was stirred until complete conversion of the starting material (monitored by TLC). The mixture was neutralized with Amberlite IR 120 (H+) ion-exchange resin, filtered, and concentrated. Column chromatography (hexanes/ EtOAc) afforded the pure product. General Procedure (F) for Microwave-Assisted Pivaloyl- and Acetyl-ester Deprotection. To a solution of starting material (1.0 equiv) in THF (reaction concentration 7−10 mM) was added a saturated solution of NaOMe in MeOH (double the volume of THF). The mixture was heated in a microwave reactor (closed vessel, 3−5 W, 100 °C, 5 bar, ramp 10 min, hold 20−45 min), neutralized with Amberlite IR 120 (H+) ion-exchange resin, filtered, and concentrated. 2419



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Column chromatography on silica gel (hexanes/EtOAc) afforded the pure product. General Hydrogenolysis Procedure (G). A solution of starting material (reaction concentration 4−8 mM) in a mixture of MeOH/ THF/H2O/AcOH (10/5/4/1) was purged with Ar. After that 10% Pd/C (same weight as starting material) was added, and the solution was purged with H2 for 10 min, then stirred under an H2 atmosphere for 12 h, filtered and concentrated. The crude product was dissolved in H2O, subjected to reversed phase solid phase extraction (RP SPE) (Waters Sep-Pak, C18), and lyophilized. When necessary, size exclusion chromatography on Sephadex LH-20 (MeOH) was performed. Preparation of Oligosaccharide Microarrays. Antiglycan antibody response was analyzed by antigen array made of oligosaccharides antigens, carrier protein, and spacer conjugates C1, C2, and C3. A separate array consisting of only synthetic oligosaccharide antigens 1, 2, 3, and 4 was prepared to screen the dog sera. Antigens bearing an amine linker or proteins were immobilized on CodeLink N-hydroxyl succinimide (NHS) ester activated glass slides (SurModics Inc., Eden Prairie, MN, USA) with a piezoelectric spotting device (S3; Scienion, Berlin, Germany). Sixtyfour replicate array grids were printed on each slide. Microarray slides were incubated in a humid chamber to complete reaction for 24 h, quenched with 50 mM aminoethanol solution, pH 9 for 1 h at 50 °C, washed three times with deionized water, and stored desiccated until use. (please see Supporting Information for the details of instrument parameters, printing buffers, conditions, and antigen concentrations). Antibody Binding Assays Using Synthetic Carbohydrate Antigen-Based Microarrays. A FlexWell 64 (Grace Bio-Laboratories, Bend, OR, USA) grid was applied to the slides. The resulting 64 wells were used for 64 individual experiments. The slide was blocked with 30 μL of blocking buffer (2.5% (w/v) BSA) for 1 h at RT and washed 2X with 40 μL of wash buffer (0.05% (v/v) Tween 20 in PBS). Blocked slides were incubated with serum dilutions in blocking buffer (20 μL) for 1 h at RT. Slides were washed 3X with 40 μL of washing buffer and incubated with 20 μL of secondary antibodies solution in blocking buffer [FITC conjugated goat anti-mouse IgG, Alexa Flour 594 goat anti-mouse IgM (Sigma and Invitrogen, respectively) or antidog IgG DyLight 488 (Fuller Laboratories, USA)]. Slides were washed with 4X with 40 μL of washing buffer and centrifuged to dryness before scanning with a GenePix 4300A scanner (Bucher Biotec, Basel, Switzerland) and evaluated using the GenePix Pro 7 software (Bucher Biotec). Conjugation of 1A and 1B to CRM197 Carrier Protein. Tetrasaccharide 1A or 1B (3.46 μmol) was dissolved in DMSO (100 μL) and added slowly to a solution of bifunctional linker (disuccinimido adipate, 0.33 mmol) in DMSO (100 μL). Before addition of oligosaccharide, a catalytic amount of triethylamine (10 μL) was added to the linker solution. After 1.5 h, 0.5 mL of phosphate buffer (0.1 M pH 7.4) was added to the reaction mixture, and unreacted linker was extracted with CHCl3 (15 mL). The extraction procedure was repeated three times, and the resultant aqueous layer was centrifuged (300g, 5 min) to separate traces of chloroform. The aqueous layer was separated and added to 1 mL of protein solution (CRM197, 1 mg mL‑1 in 0.1 M phosphate buffer pH 7.4). After the pH was adjusted to 7.4, the reaction was allowed to continue for 5−6 h with gentle stirring. The glycoconjugates were purified either by size exclusion chromatography (Superose 12 10/300 GL in 0.1 M phosphate buffer pH 7.4, flow rate 0.5 mL/min) or by ultrafiltration. The protein concentration was determined by micro BCA assay. Immunization Experiments in Experimental Animal Models. For initial immunogenicity studies, six female BALB/c mice (6−8 weeks old; obtained from Charles liver laboratories, Germany) per group were used. To evaluate the reproducibility of the experiments, immunogenicity was evaluated in an alternate mouse model using three 6−8 weeks old female C57BL/6J mice (obtained from Charles River Laboratories, Germany). All mice were immunized subcutaneously with 5 μg of tetrasaccharide-CRM197 glycoconjugate in Complete Freund’s adjuvant (CFA), where 5 μg refers to tetrasaccharide content. Tetrasaccharide content was estimated on

the basis of antigen loading from MALDI and carbohydrate colorimetric assay. The mice were boosted twice with 5 μg of tetrasaccharide-CRM197 glycoconjugate in Incomplete Freund’s adjuvant at 2-week intervals. After each injection, sera were collected, and serum titers (IgG) were analyzed using glycan arrays or ELISA. Immunofluorescence Studies of Antibody Binding to Parasite Surface. To study the binding of antibodies from serum to parasite surface, a glass slide with immobilized L. chagasi promastigotes (taken from the Canine Leishmaniasis IgG IFA Kit, Fuller Diagnostics, USA) was incubated with sera in different dilutions at 4 °C. After incubation for 1 h, the supernatant was aspirated, and the slide was washed three times with PBS with 0.1% Tween. The bound antibodies were probed with a secondary anti-mouse IgG FITC by incubation of the slide with 1:200 dilution of secondary antibody (Santa Cruz Biotechnology, USA) for 1 h. After incubation the slide was washed four times with PBS with 0.1% Tween. The slide was finally washed and imaged using confocal laser scanning microscopy (Imager M.2, Carl Zeiss). A series of images were collected. Parasites treated with a polyclonal normal mouse serum were used as the negative control to adjust background fluorescence.

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ASSOCIATED CONTENT

* Supporting Information S

Experimental procedures including the synthesis of all compounds, conjugation to carrier protein, NMR experiments, data for microarray analysis, and protocols for in vitro and in vivo experiments. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions ⊥

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Max-Planck Society and the Körber Foundation for generous financial support. C.A. acknowledges support from the German Federal Ministry of Education and Research (Grant 0315447). C.B. acknowledges support from the Interdisciplinary Center for Clinical Research (University of Erlangen, project A49) and the Bavarian State Ministry of the Environment and Public Health within the research consortium “Vector-borne infectious diseases in climate change investigations (VICCI)” coordinated by the ‘Bavarian Health and Food Safety Authority’, No. 08/18. We acknowledge Pfenex Inc. (reagent proteins) for providing CRM197 at a discounted price. The contributions of F. Matsumura and D. Grünstein for synthetic glycans and B. P. Monnanda for microscopy studies are gratefully acknowledged. We acknowledge the advice of B. Lepenies on institutional animal ethics policy. We thank C. L. Pereira and J. Hudon for careful review of the manuscript. We acknowledge A. Geissner and F. Bröcker for glycan array printing. We gratefully acknowledge R. K. Straubinger, Institut für Infektionsmedizin und Zoonosen, Veterinärwissenschaftliches Department, Ludwig-Maximilians-Universität München, Germany for providing sera from experimental dogs housed under specific pathogen-free conditions. We thank G. S. Himmelstjerna, J. Demeler, J. Krücken, and S. Miltsch, Institut of Parasitology and Tropical Veterinary Medicine Robert-von2420



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Ostertag-Str. 7-13, 14163 Berlin, Germany for providing sera from control dogs from non-endemic area (Berlin).

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ABBREVIATIONS CRM197, cross reactive material 197; BSA, bovine serum albumin; DSAP, disuccinimoido adipate

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REFERENCES

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Immunogenicity and Diagnostic Potential of Synthetic Antigenic Cell

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