ABSTRACT
Methicillin-resistant Staphylococcus aureus (MRSA) is a frequent cause of difficult-to-treat, often fatal infections in humans1,2. Most humans have antibodies against S. aureus, but these are highly variable and often not protective in immunocompromised patients3. Previous vaccine development programs have not been successful4. A large percentage of human antibodies against S. aureus target wall teichoic acid (WTA), a ribitol-phosphate (RboP) surface polymer modified with N-acetylglucosamine (GlcNAc)5,6. It is currently unknown whether the immune evasion capacities of MRSA are due to variation of dominant surface epitopes such as those associated with WTA. Here we show that a considerable proportion of the prominent healthcare-associated and livestock-associated MRSA clones CC5 and CC398, respectively, contain prophages that encode an alternative WTA glycosyltransferase. This enzyme, TarP, transfers GlcNAc to a different hydroxyl group of the WTA RboP than the standard enzyme TarS7, with important consequences for immune recognition. TarP-glycosylated WTA elicits 7.5-40-fold lower levels of immunoglobulin G in mice than TarS-modified WTA. Consistent with this, human sera contained only low levels of antibodies against TarP-modified WTA. Notably, mice immunized with TarS-modified WTA were not protected against infection with tarP-expressing MRSA, indicating that TarP is crucial for the capacity of S. aureus to evade host defences. High-resolution structural analyses of TarP bound to WTA components and uridine diphosphate GlcNAc (UDP-GlcNAc) explain the mechanism of altered RboP glycosylation and form a template for targeted inhibition of TarP. Our study reveals an immune evasion strategy of S. aureus based on averting the immunogenicity of its dominant glycoantigen WTA. These results will help with the identification of invariant S. aureus vaccine antigens and may enable the development of TarP inhibitors as a new strategy for rendering MRSA susceptible to human host defences.
Subject(s)
Cell Wall/chemistry , Cell Wall/immunology , Immune Evasion , Methicillin-Resistant Staphylococcus aureus/cytology , Methicillin-Resistant Staphylococcus aureus/immunology , Pentosephosphates/immunology , Teichoic Acids/immunology , Acetylglucosamine/chemistry , Acetylglucosamine/metabolism , Adult , Animals , Bacteriophages/pathogenicity , Female , Glycosylation , Glycosyltransferases/metabolism , Humans , Male , Methicillin-Resistant Staphylococcus aureus/chemistry , Mice , Middle Aged , Models, Molecular , Pentosephosphates/chemistry , Pentosephosphates/metabolism , Teichoic Acids/chemistry , Teichoic Acids/metabolism , Uridine Diphosphate/chemistry , Uridine Diphosphate/metabolism , Young AdultABSTRACT
Interactions between glycans and glycan binding proteins are essential for numerous processes in all kingdoms of life. Glycan microarrays are an excellent tool to examine protein-glycan interactions. Here, we present a microbe-focused glycan microarray platform based on oligosaccharides obtained by chemical synthesis. Glycans were generated by combining different carbohydrate synthesis approaches including automated glycan assembly, solution-phase synthesis, and chemoenzymatic methods. The current library of more than 300 glycans is as diverse as the mammalian glycan array from the Consortium for Functional Glycomics and, due to its microbial focus, highly complementary. This glycan platform is essential for the characterization of various classes of glycan binding proteins. Applications of this glycan array platform are highlighted by the characterization of innate immune receptors and bacterial virulence factors as well as the analysis of human humoral immunity to pathogenic glycans.
Subject(s)
Carrier Proteins/chemistry , Microarray Analysis/methods , Polysaccharides/chemistry , Polysaccharides/immunology , Animals , Antigens, Bacterial/chemistry , Antigens, Bacterial/immunology , CHO Cells , Cricetulus , Glycomics , Humans , Immune System , Lectins , Oligosaccharides , Polysaccharides/classification , Protein Binding , Recombinant Proteins , Species SpecificityABSTRACT
Streptococcus pneumoniae remains a deadly disease in small children and the elderly even though conjugate and polysaccharide vaccines based on isolated capsular polysaccharides (CPS) are successful. The most common serotypes that cause infection are used in vaccines around the world, but differences in geographic and demographic serotype distribution compromises protection by leading vaccines. The medicinal chemistry approach to glycoconjugate vaccine development has helped to improve the stability and immunogenicity of synthetic vaccine candidates for several serotypes leading to the induction of higher levels of specific protective antibodies. Here, we show that marketed CPS-based glycoconjugate vaccines can be improved by adding synthetic glycoconjugates representing serotypes that are not covered by existing vaccines. Combination (coformulation) of synthetic glycoconjugates with the licensed vaccines Prevnar13 (13-valent) and Synflorix (10-valent) yields improved 15- and 13-valent conjugate vaccines, respectively, in rabbits. A pentavalent semisynthetic glycoconjugate vaccine containing five serotype antigens (sPCV5) elicits antibodies with strong in vitro opsonophagocytic activity. This study illustrates that synthetic oligosaccharides can be used in coformulation with both isolated polysaccharide glycoconjugates to expand protection from existing vaccines and each other to produce precisely defined multivalent conjugated vaccines.
Subject(s)
Bacterial Vaccines/immunology , Polysaccharides/immunology , Streptococcus pneumoniae/immunology , Animals , Antibodies, Bacterial/immunology , Glycoconjugates/immunology , Pneumococcal Infections/immunology , Polysaccharides/chemical synthesis , Rabbits , Serogroup , Vaccines, Conjugate/immunology , Vaccines, Synthetic/immunologyABSTRACT
α-Galactosylceramide (α-GalCer; KRN7000) is a ligand for the glycoprotein CD1d that presents lipid antigens to natural killer T cells. Therefore, KRN7000 as well as some modified versions thereof have been widely investigated as part of novel immunotherapies. To examine the impact of structural modification, we investigated KRN7000 and C6-modified KRN7000 at the air-liquid interface using monolayer isotherms, BAM, IRRAS, GIXD, and TRXF. The amino group has no influence on the highly ordered sub-gel structures found at lateral pressures relevant for biological membranes. Neither lateral compression nor the protonation state of the amino group has a measurable effect on the lattice structure, which is defined by strong and rigid intermolecular hydrogen bonds. However, the first-order phase transition found for the C6-functionalized α-GalCer is connected with an extraordinary surface-inhibited nucleation. Our study demonstrates that KRN7000 can be functionalized at C6 without significantly changing the structural properties.
Subject(s)
Galactosylceramides/chemistry , Nitrogen/chemistry , Thermodynamics , Air , Hydrogen Bonding , Molecular Conformation , Surface PropertiesABSTRACT
Glycoconjugate vaccines based on isolated capsular polysaccharide (CPS) save millions of lives annually by preventing invasive pneumococcal disease caused by Streptococcus pneumoniae Some components of the S. pneumoniae glycoconjugate vaccine Prevnar13 that contains CPS antigens from 13 serotypes undergo modifications or degradation during isolation and conjugation, resulting in production problems and lower efficacy. We illustrate how stable, synthetic oligosaccharide analogs of labile CPS induce a specific protective immune response against native CPS using S. pneumoniae serotype 5 (ST-5), a problematic CPS component of Prevnar13. The rare aminosugar l-PneuNAc and a branched l-FucNAc present in the natural repeating unit (RU) are essential for antibody recognition and avidity. The epitope responsible for specificity differs from the part of the antigen that is stabilized by chemical modification. Glycoconjugates containing stable, monovalent synthetic oligosaccharide analogs of ST-5 CPS RU induced long-term memory and protective immune responses in rabbits superior to those elicited by the ST-5 CPS component in multivalent Prevnar13.
Subject(s)
Pneumococcal Vaccines , Streptococcus pneumoniae/immunology , Animals , Glycoconjugates , Rabbits , Serogroup , Vaccines, SyntheticABSTRACT
Streptococcus pneumoniae (SP) bacteria cause serious invasive diseases. SP bacteria are covered by a capsular polysaccharide (CPS) that is a virulence factor and the basis for SP polysaccharide and glycoconjugate vaccines. The serotype 9V is part of the currently marketed conjugate vaccine and contains an acetate modification. To better understand the importance of glycan modifications in general and acetylation in particular, defined oligosaccharide antigens are needed for serological and immunological studies. Here, we demonstrate a convergent [2 + 3] synthetic strategy to prepare the pentasaccharide repeating unit of 9V with and without an acetate group at the C-6 position of mannosamine.
ABSTRACT
Plesiomonas shigelloides, a pathogen responsible for frequent outbreaks of severe travelers' diarrhea, causes grave extraintestinal infections. Sepsis and meningitis due to P. shigelloides are associated with a high mortality rate as antibiotic resistance increases and vaccines are not available. Carbohydrate antigens expressed by pathogens are often structurally unique and are targets for developing vaccines and diagnostics. Here, we report a total synthesis of the highly functionalized trisaccharide repeating unit 2 from P. shigelloides serotype 51 from three monosaccharides. A judicious choice of building blocks and reaction conditions allowed for the four amino groups adorning the sugar rings to be installed with two N-acetyl (Ac) groups, rare acetamidino (Am), and d-3-hydroxybutyryl (Hb) groups. The strategy for the differentiation of amino groups in trisaccharide 2 will serve well for the syntheses of other complex glycans.
Subject(s)
Aminoglycosides/chemical synthesis , O Antigens/chemistry , Plesiomonas/chemistry , Trisaccharides/chemical synthesis , Aminoglycosides/chemistry , Carbohydrate Conformation , Trisaccharides/chemistryABSTRACT
Streptococcus pneumoniae causes life-threatening diseases including meningitis, pneumonia and sepsis. Existing glycoconjugate vaccines based on purified capsular polysaccharides are widely used and help to prevent millions of deaths every year. Herein, the total syntheses of oligosaccharides resembling portions of the S. pneumoniae serotype 7F (ST7F) capsular polysaccharide repeating unit are reported. To define minimal glycan epitopes, glycan microarrays containing the synthetic oligosaccharides were used to screen human reference serum and revealed that both side chains of the ST7F play a key role in antigen recognition. The identification of protective minimal epitopes is vital to design efficient semi- and fully-synthetic glycoconjugate vaccines.
Subject(s)
Glycoconjugates/immunology , Pneumococcal Vaccines , Polysaccharides, Bacterial/immunology , Polysaccharides/immunology , Streptococcus pneumoniae/chemistry , Epitopes/immunology , Humans , Pneumococcal Vaccines/chemistry , Pneumococcal Vaccines/immunology , Pneumococcal Vaccines/therapeutic use , Polysaccharides/chemistry , Polysaccharides, Bacterial/chemistry , SerogroupABSTRACT
Helicobacter pylori, a widespread gastric bacterial pathogen that infects 90 % of the population in developing countries, causes chronic gastritis, peptic ulcers and gastric cancer. Battling H.â pylori infection is a serious challenge due to the increased resistance to antibiotics and the lack of vaccines. The lipopolysaccharide covering the H.â pylori cell-surface outer membrane is an attractive target for the development of a glycoconjugate vaccine. Here, we report a [3+5] convergent synthesis of an outer core octasaccharide of H.â pylori employing just three orthogonally protected building blocks. A synergistic glycosylation strategy enables the creation of five pivotal 1,2-cis-α-glucosidic bonds consist of four types of linkages using just three monomers. This strategy can be expanded to many 1,2-cis-α-gluoside-containing oligosaccharides both in solution and solid phase.
Subject(s)
Anti-Bacterial Agents/therapeutic use , Helicobacter pylori/chemistry , Oligosaccharides/chemistry , Glycosylation , Helicobacter Infections/microbiology , Helicobacter Infections/pathology , Humans , Oligosaccharides/chemical synthesis , Stomach Neoplasms/etiologyABSTRACT
Invasive pneumococcal diseases (IPDs) remain the leading cause of vaccine-preventable childhood death, even though highly effective pneumococcal conjugate vaccines (PCVs) are used in national immunization programs in many developing countries. Licensed PCVs currently cover only 13 of the over 90 serotypes of Streptococcus pneumoniae (Sp), so nonvaccine serotypes are a major obstacle to the effective control of IPD. Sp serotype 2 (ST2) is such a nonvaccine serotype that is the main cause of IPD in many countries, including Nepal, Bangladesh, and Guatemala. Glycoconjugate vaccines based on synthetic oligosaccharides instead of isolated polysaccharides offer an attractive alternative to the traditional process for PCV development. To prevent the IPDs caused by ST2, we identified an effective ST2 neoglycoconjugate vaccine candidate that was identified using a medicinal chemistry approach. Glycan microarrays containing a series of synthetic glycans resembling portions of the ST2 capsular polysaccharide (CPS) repeating unit were used to screen human and rabbit sera and identify epitope hits. Synthetic hexasaccharide 2, resembling one repeating unit (RU) of ST2 CPS, emerged as a hit from the glycan array screens. Vaccination with neoglycoconjugates consisting of hexasaccharide 2 coupled to carrier protein CRM197 stimulates a T-cell-dependent B-cell response that induced CPS-specific opsonic antibodies in mice, resulting in killing of encapsulated bacteria by phagocytic activity. Subcutaneous immunization with neoglycoconjugate protected mice from transnasal challenge with the highly virulent ST2 strain NCTC 7466 by reducing the bacterial load in lung tissue and blood.
Subject(s)
Antibodies, Bacterial/immunology , Glycoconjugates/immunology , Oligosaccharides/immunology , Pneumococcal Infections/immunology , Pneumococcal Infections/microbiology , Streptococcus pneumoniae/chemistry , Streptococcus pneumoniae/immunology , Administration, Intranasal , Animals , B-Lymphocytes/immunology , Bacterial Load , Blood/microbiology , Disease Models, Animal , Female , Glycoconjugates/chemical synthesis , Humans , Lung/microbiology , Mice , Mice, Inbred C57BL , Oligosaccharides/chemical synthesis , Phagocytosis , Pneumococcal Infections/prevention & control , Pneumococcal Vaccines/chemistry , Pneumococcal Vaccines/immunology , Polysaccharides, Bacterial/chemistry , Polysaccharides, Bacterial/immunology , Rabbits , Streptococcus pneumoniae/classification , Streptococcus pneumoniae/pathogenicity , T-Lymphocytes/immunology , Vaccination , Vaccines, Conjugate/chemistry , Vaccines, Conjugate/immunologyABSTRACT
Hospital-acquired infections are an increasingly serious health concern. Infections caused by carpabenem-resistant Klebsiella pneumoniae (CR-Kp) are especially problematic, with a 50 % average survival rate. CR-Kp are isolated from patients with ever greater frequency, 7 % within the EU but 62 % in Greece. At a time when antibiotics are becoming less effective, no vaccines to protect from this severe bacterial infection exist. Herein, we describe the convergent [3+3] synthesis of the hexasaccharide repeating unit from its capsular polysaccharide and related sequences. Immunization with the synthetic hexasaccharide 1 glycoconjugate resulted in high titers of cross-reactive antibodies against CR-Kp CPS in mice and rabbits. Whole-cell ELISA was used to establish the surface staining of CR-Kp strains. The antibodies raised were found to promote phagocytosis. Thus, this semi-synthetic glycoconjugate is a lead for the development of a vaccine against a rapidly progressing, deadly bacterium.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Vaccines/immunology , Carbapenems/pharmacology , Glycoconjugates/chemical synthesis , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/immunology , Vaccines, Synthetic/immunology , Animals , Antibodies, Bacterial/biosynthesis , Antibodies, Bacterial/immunology , Drug Resistance, Bacterial , Enzyme-Linked Immunosorbent Assay , Glycoconjugates/chemistry , Glycoconjugates/immunology , Klebsiella Infections/prevention & control , Mice , Oligosaccharides/chemistry , Phagocytosis/immunology , RabbitsABSTRACT
1,3-Dibromo-5,5-dimethylhydantoin (DBDMH), an inexpensive, non-toxic and stable reagent, is a competent activator of thioglycosides for glycosidic bond formation. Excellent yields were obtained when triflic acid (TfOH) or trimethylsilyl trifluoromethanesulfonate (TMSOTf) were employed as co-promoters in solution or automated glycan assembly on solid phase.
ABSTRACT
The Gram-positive bacterium Streptococcus pneumoniae causes severe disease globally. Vaccines that prevent S. pneumoniae infections induce antibodies against epitopes within the bacterial capsular polysaccharide (CPS). A better immunological understanding of the epitopes that protect from bacterial infection requires defined oligosaccharides obtained by total synthesis. The key to the synthesis of the S. pneumoniae serotype 12F CPS hexasaccharide repeating unit that is not contained in currently used glycoconjugate vaccines is the assembly of the trisaccharide ß-D-GalpNAc-(1â4)-[α-D-Glcp-(1â3)]-ß-D-ManpNAcA, in which the branching points are equipped with orthogonal protecting groups. A linear approach relying on the sequential assembly of monosaccharide building blocks proved superior to a convergent [3 + 3] strategy that was not successful due to steric constraints. The synthetic hexasaccharide is the starting point for further immunological investigations.
ABSTRACT
The glycosylation reaction is the key transformation in oligosaccharide synthesis, but it is still difficult to control in many cases. Stereocontrol during cis-glycosidic linkage formation relies almost exclusively on tuning the glycosylating agent or the reaction conditions. Herein, we use nucleophile-directed stereocontrol to manipulate the stereoselectivity of glycosylation reactions. Placing two fluorine atoms in close proximity to the hydroxy group of an aliphatic amino alcohol lowers the oxygen nucleophilicity and reverses the stereoselectivity of glycosylations to preferentially form the desired cis-glycosides with a broad set of substrates. This concept was applied to the design of a cis-selective linker for automated glycan assembly. Fluorination of an amino alcohol linker does not impair glycan immobilization and lectin binding as illustrated by glycan microarray experiments. These fluorinated linkers enable the facile generation of α-terminating synthetic glycans for the formation of glycoconjugates.
ABSTRACT
Vaccines against S. pneumoniae, one of the most prevalent bacterial infections causing severe disease, rely on isolated capsular polysaccharide (CPS) that are conjugated to proteins. Such isolates contain a heterogeneous oligosaccharide mixture of different chain lengths and frame shifts. Access to defined synthetic S. pneumoniae CPS structures is desirable. Known syntheses of S. pneumoniae serotype 3 CPS rely on a time-consuming and low-yielding late-stage oxidation step, or use disaccharide building blocks which limits variability. Herein, we report the first iterative automated glycan assembly (AGA) of a conjugation-ready S. pneumoniae serotype 3 CPS trisaccharide. This oligosaccharide was assembled using a novel glucuronic acid building block to circumvent the need for a late-stage oxidation. The introduction of a washing step with the activator prior to each glycosylation cycle greatly increased the yields by neutralizing any residual base from deprotection steps in the synthetic cycle. This process improvement is applicable to AGA of many other oligosaccharides.
ABSTRACT
Carbohydrate modifications are believed to strongly affect the immunogenicity of glycans. Capsular polysaccharides (CPS) from bacterial pathogens are frequently equipped with a pyruvate that can be placed across the 4,6-, 3,4-, or 2,3-positions. A trans-2,3-linked pyruvate is present on the CPS of the Gram-positive bacterium Streptococcus pneumoniae serotypeâ 4 (ST4), a pathogen responsible for pneumococcal infections. To assess the immunological importance of this modification within the CPS repeating unit, the first total synthesis of the glycan was carried out. Glycan microarrays containing a series of synthetic antigens demonstrated how antibodies raised against natural ST4â CPS specifically recognize the pyruvate within the context of the tetrasaccharide repeating unit. The pyruvate modification is a key motif for designing minimal synthetic carbohydrate vaccines for ST4.
Subject(s)
Polysaccharides, Bacterial/chemical synthesis , Pyruvic Acid/chemistry , Streptococcus pneumoniae/chemistry , Polysaccharides, Bacterial/chemistry , Polysaccharides, Bacterial/immunology , Pyruvic Acid/immunology , Serogroup , Streptococcus pneumoniae/immunologyABSTRACT
Streptococcus pneumoniae (S. pneumoniae)infections are the leading cause of child mortality globally. Currentvaccines fail to induceaprotective immune response towards a conserved part of the pathogen,resulting in newserotypescausing disease. Therefore, new vaccinestrategies are urgently needed.Described is atwo-pronged approach combiningS. pneumoniaeproteins, pneumolysin (Ply) and pneumococcal surface protein A (PspA),with aprecisely defined synthetic oligosaccharide,wherebythe carrier protein actsas a serotype-independent antigen to provideadditional protection. Proof of concept in mice and swine modelsrevealed thatthe conjugatesinhibited colonization of the nasopharynx, decreased the bacterial load and reduced disease severity in the bacteria challenge model. Immunization of piglets provided the first evidence for the immunogenicity and protective potential of synthetic glycoconjugate vaccine in a large animal model.Acombination of synthetic oligosaccharides with proteins from the target pathogen opens the path to create broadly cross-protective ("universal") pneumococcal vaccines.
Subject(s)
Pneumococcal Infections , Streptococcus pneumoniae , Animals , Antibodies, Bacterial , Bacterial Proteins , Glycoconjugates , Mice , Pneumococcal Vaccines , Serogroup , SwineABSTRACT
Extraintestinal pathogenic Escherichia coli (ExPEC) is a major health concern due to emerging antibiotic resistance. Along with O1A, O2, and O6A, E. coli O25B is a major serotype within the ExPEC group, which expresses a unique O-antigen. Clinical studies with a glycoconjugate vaccine of the above-mentioned O-types revealed O25B as the least immunogenic component, inducing relatively weak IgG titers. To evaluate the immunological properties of semisynthetic glycoconjugate vaccine candidates against E. coli O25B, we here report the chemical synthesis of an initial set of five O25B glycan antigens differing in length, from one to three repeat units, and frameshifts of the repeat unit. The oligosaccharide antigens were conjugated to the carrier protein CRM197. The resulting semisynthetic glycoconjugates induced functional IgG antibodies in mice with opsonophagocytic activity against E. coli O25B. Three of the oligosaccharide-CRM197 conjugates elicited functional IgGs in the same order of magnitude as a conventional CRM197 glycoconjugate prepared with native O25B O-antigen and therefore represent promising vaccine candidates for further investigation. Binding studies with two monoclonal antibodies (mAbs) revealed nanomolar anti-O25B IgG responses with nanomolar K D values and with varying binding epitopes. The immunogenicity and mAb binding data now allow for the rational design of additional synthetic antigens for future preclinical studies, with expected further improvements in the functional antibody responses. Moreover, acetylation of a rhamnose residue was shown to be likely dispensable for immunogenicity, as a deacylated antigen was able to elicit strong functional IgG responses. Our findings strongly support the feasibility of a semisynthetic glycoconjugate vaccine against E. coli O25B.
ABSTRACT
Streptococcus pneumoniae 19A (ST19A) and 19F (ST19F) are among the prevalent serotypes causing pneumococcal disease worldwide even after introduction of a 13-valent pneumococcal conjugate vaccine (PCV13). Synthetic glycoconjugate vaccines have defined chemical structures rather than isolated polysaccharide mixtures utilized in marketed vaccines. Ideally, a minimal number of synthetic antigens would cover as many bacterial serotypes to lower cost of goods and minimize the response to carrier proteins. To demonstrate that a chimeric oligosaccharide antigen can induce a protective immune response against multiple serotypes, we synthesized a chimeric antigen (ST19AF) that is comprised of a repeating unit of ST19A and ST19F capsular polysaccharide each. Synthetic glycan epitopes representing only ST19A, and ST19F were prepared for comparison. Semisynthetic glycoconjugates containing chimeric antigen ST19AF induced high antibody titers able to recognize native CPS from ST19A and ST19F in rabbits. The antibodies were able to kill both strains of pneumococci. Chimeric antigens are an attractive means to induce an immune response against multiple bacterial serotypes.
ABSTRACT
Vaccines based on isolated polysaccharides successfully protect humans from bacterial pathogens such as Streptococcus pneumoniae. Because polysaccharide production and isolation can be technically challenging, glycoconjugates containing synthetic antigens are an attractive alternative. Typically, the shortest possible oligosaccharide antigen is preferable as syntheses of longer structures are more difficult and time-consuming. Combining several protective epitopes or polysaccharide repeating units as blocks by bonds other than glycosidic linkages would greatly reduce the synthetic effort if the immunological response to the polysaccharide could be retained. To explore this concept, we bridged the well-understood and immunologically potent RU of S. pneumoniae serotype 14 (ST14) with an aliphatic spacer and conjugated it to the carrier protein CRM197. Mice immunized with the spacer-bridged glycan conjugates produced high levels of specific antibodies after just one or two vaccine doses, while the tetrasaccharide repeating unit alone required three doses. The antibodies recognized specifically ST14 CPS, while no significant antibody levels were raised against the spacer or unrelated CPS. Synthetic vaccines generated antibodies with opsonic activity. Mimicking polysaccharides by coupling repeating unit antigens via an aliphatic spacer may prove useful also for the development of other glycoconjugate vaccine candidates, thereby reducing the synthetic complexity while enhancing a faster immune response.