Carbohydrates are present on the surface of bacteria making them suitable antigen candidates for vaccine development. This thesis deals with the synthesis of two carbohydrate-based components; the... Show moreCarbohydrates are present on the surface of bacteria making them suitable antigen candidates for vaccine development. This thesis deals with the synthesis of two carbohydrate-based components; the capsular polisaccharide of S. aureus type 5 and teichoic acids from staphilococci and enterococci species. The synthesis of well-defined structures can allow an in-depth characterization of antigen-antibody binding that ultimately can help the development of robust and high effective vaccine. In the case of teichoic acids for example, it has been introduced the application of a microarray technology to streamline the preferential binding of immune proteins from different sources. The binding towards teichoic acids like structures has been further explore by developing a monoclonal antibody starting from a fully sinthetic glycoconjugate. Different techniques have been used which in conclusion suggest an indirect contribution of the glycosidic appandage to the binding towards the glycerol-phosphate backbone. Show less
Carluccio, C. di; Maldonado, P.S.; Berni, F.; Haas, C.J.C. de; Temming, A.R.; Hendriks, A.; ... ; Marchetti, R. 2022
Wall teichoic acids (WTAs) are glycopolymers decorating the surface of Gram-positive bacteria and potential targets for antibody-mediated treatments against Staphylococcus aureus, including... Show moreWall teichoic acids (WTAs) are glycopolymers decorating the surface of Gram-positive bacteria and potential targets for antibody-mediated treatments against Staphylococcus aureus, including methicillin-resistant (MRSA) strains. Through a combination of glycan microarray, synthetic chemistry, crystallography, NMR, and computational studies, we unraveled the molecular and structural details of fully defined synthetic WTA fragments recognized by previously described monoclonal antibod-ies (mAbs 4461 and 4497). Our results unveiled the structural requirements for the discriminatory recognition of alpha- and beta-GlcNAc-modified WTA glycoforms by the complementarity-determining regions (CDRs) of the heavy and light chains of the mAbs. Both mAbs interacted not only with the sugar moiety but also with the phosphate groups as well as residues in the ribitol phosphate (RboP) units of the WTA backbone, highlighting their significant role in ligand specificity. Using elongated WTA fragments, containing two sugar modifications, we also demonstrated that the internal carbohydrate moiety of alpha-GlcNAc-modified WTA is preferentially accommodated in the binding pocket of mAb 4461 with respect to the terminal moiety. Our results also explained the recently documented cross-reactivity of mAb 4497 for beta-1,3/beta-1,4-GlcNAc-modified WTA, revealing that the flexibility of the RboP backbone is crucial to allow positioning of both glycans in the antibody binding pocket. Show less
Berni, F.; Kalfopoulou, E.; Cardells, A.M.G.; Carboni, F.; Es, D. van der; Romero-Saavedra, F.; ... ; Codee, J.D.C. 2021
Glycerol phosphate (GroP)-based teichoic acids (TAs) are antigenic cell-wall components found in both enterococcus and staphylococcus species. Their immunogenicity has been explored using both... Show moreGlycerol phosphate (GroP)-based teichoic acids (TAs) are antigenic cell-wall components found in both enterococcus and staphylococcus species. Their immunogenicity has been explored using both native and synthetic structures, but no details have yet been reported on the structural basis of their interaction with antibodies. This work represents the first case study in which a monoclonal antibody, generated against a synthetic TA, was developed and employed for molecular-level binding analysis using TA microarrays, ELISA, SPR-analyses, and STD-NMR spectroscopy. Our findings show that the number and the chirality of the GroP residues are crucial for interaction and that the sugar appendage contributes to the presentation of the backbone to the binding site of the antibody. Show less
Berni, F.; Kalfopoulou, E.; Gimeno Cardells, A.M.; Carboni, F.; Es, D. van der; Romero-Saavedra, F.; ... ; Codée, J.D.C. 2021
Glycerol phosphate (GroP)-based teichoic acids (TAs) are antigenic cell-wall components found in both enterococcus and staphylococcus species. Their immunogenicity has been explored using both... Show moreGlycerol phosphate (GroP)-based teichoic acids (TAs) are antigenic cell-wall components found in both enterococcus and staphylococcus species. Their immunogenicity has been explored using both native and synthetic structures, but no details have yet been reported on the structural basis of their interaction with antibodies. This work represents the first case study in which a monoclonal antibody, generated against a synthetic TA, was developed and employed for molecular-level binding analysis using TA microarrays, ELISA, SPR-analyses, and STD-NMR spectroscopy. Our findings show that the number and the chirality of the GroP residues are crucial for interaction and that the sugar appendage contributes to the presentation of the backbone to the binding site of the antibody. Show less
Berni, F.; Wang, L.; Kalfopoulou, E.; Nguyen, D.L.; Es, D. van der; Hübner, J.; ... ; Codée, J.D.C. 2021
Lipoteichoic acids (LTAs) have been addressed as possible antigen candidates for vaccine development against several opportunistic Gram-positive pathogens. The study of structure-immunogenicity... Show moreLipoteichoic acids (LTAs) have been addressed as possible antigen candidates for vaccine development against several opportunistic Gram-positive pathogens. The study of structure-immunogenicity relationship represents a challenge due to the heterogenicity of LTA extracted from native sources. LTAs are built up from glycerol phosphate (GroP) repeating units and they can be substituted at the C-2-OH with carbohydrate appendages or d-alanine residues. The substitution pattern, but also the absolute chirality of the GroP residues can impact the interaction with chiral biomolecules including antibodies and biosynthesis enzymes. We have generated a set of diastereomeric GroP hexamers bearing a glucosyl modification at one of the residues. The chirality of the glycerol building block had an important impact on the stereoselectivity of the glycosylation reaction between the glycosyl donor and the glycerol C-2-OH acceptor. The GroP C-2-chirality also played an important role in the interaction with TA recognizing antibodies. These findings have important implications for the design and synthesis of synthetic TA fragments for diagnostic and therapeutic applications. Show less
Berni, F.; Wang, L.M.; Kalfopoulou, E.; Nguyen, D.L.; Es, D. van der; Huebner, J.; ... ; Codee, J.D.C. 2021
Lipoteichoic acids (LTAs) have been addressed as possible antigen candidates for vaccine development against several opportunistic Gram-positive pathogens. The study of structure-immunogenicity... Show moreLipoteichoic acids (LTAs) have been addressed as possible antigen candidates for vaccine development against several opportunistic Gram-positive pathogens. The study of structure-immunogenicity relationship represents a challenge due to the heterogenicity of LTA extracted from native sources. LTAs are built up from glycerol phosphate (GroP) repeating units and they can be substituted at the C-2-OH with carbohydrate appendages or d-alanine residues. The substitution pattern, but also the absolute chirality of the GroP residues can impact the interaction with chiral biomolecules including antibodies and biosynthesis enzymes. We have generated a set of diastereomeric GroP hexamers bearing a glucosyl modification at one of the residues. The chirality of the glycerol building block had an important impact on the stereoselectivity of the glycosylation reaction between the glycosyl donor and the glycerol C-2-OH acceptor. The GroP C-2-chirality also played an important role in the interaction with TA recognizing antibodies. These findings have important implications for the design and synthesis of synthetic TA fragments for diagnostic and therapeutic applications. Show less
Berni, F.; Enotarpi, J.; Voskuilen, T.; Li, S.; Marel, G.A. van der; Codée, J.D.C. 2020
Glycopolymers are found surrounding the outer layer of many bacterial species. The first uses as immunogenic component in vaccines are reported since the beginning of the XX century, but it is only... Show moreGlycopolymers are found surrounding the outer layer of many bacterial species. The first uses as immunogenic component in vaccines are reported since the beginning of the XX century, but it is only in the last decades that glycoconjugate based vaccines have been effectively applied for controlling and preventing several infectious diseases, such as H. influenzae type b (Hib), N. meningitidis, S. pneumoniae or group B Streptococcus. Methicillin resistant S. aureus (MRSA) strains has been appointed by the WHO as one of those pathogens, for which new treatments are urgently needed. Herein we present an overview of the carbohydrate-based cell wall polymers associated with different S. aureus strains and the related affords to deliver well-defined fragments through synthetic chemistry. Show less
Wang, L.; Berni, F.; Enotarpi, J.; Overkleeft, H.S.; Marel, G.A. van der; Codée, J.D.C. 2020
The stereoselective construction of 1,2-cis-glycosidic linkages is key in the assembly of biologically relevant glycans, but remains a synthetic challenge. Reagent-controlled glycosylation... Show moreThe stereoselective construction of 1,2-cis-glycosidic linkages is key in the assembly of biologically relevant glycans, but remains a synthetic challenge. Reagent-controlled glycosylation methodologies, in which external nucleophiles are employed to modulate the reactivity of the glycosylation system, have become powerful means for the construction of 1,2-cis-glycosidic linkages. Here we establish that nucleophilic additives can support the construction of α-1,2-glucans, and apply our findings in the construction of a d-alanine kojibiose functionalized glycerol phosphate teichoic acid fragment. This latter molecule can be found in the cell wall of the opportunistic Gram-positive bacterium, Enterococcus faecalis and represents a structural element that can possibly be used in the development of therapeutic vaccines and diagnostic tools. Show less
Es, D. van der; Berni, F.; Hogendorf, W.F.J.; Meeuwenoord, N.; Laverde, D.; Diepen, A. van; ... ; Codee, J.D.C. 2018
