Carbohydrates are multifunctional, stereochemical rich natural molecules. Together with their well-known role of energy sources (glucose, glycogen, amylose) and structural components (cellulose, chitin), they serve important functions in biological processes relevant for health and disease1 such as molecular recognition, cell communication and adhesion. A dense layer of oligosaccharides (proteoglycans, glycoproteins, glycolipids and other glycoconjugates) named glycocalyx, covers the membrane of most of cells. Glycocalyx of bacteria often exhibits oligosaccharides different from those found in mammalians, which have an important role in pathogen recognition and immune system activation. For this reason, pathogenic bacteria polysaccharides are widely used for vaccines production. Moreover, the role of symbiont bacteria glycocalyx in immune system activation, development and modulation is object of increasing interest. In this thesis, the important connection between carbohydrates and immune system is discussed by two different examples. The first section of the thesis deals with the synthesis of a structural analogue of Neisseria meningitidis A (MenA) capsular polysaccharide (CPS), with the final goal of producing a stable glycoconjugate vaccine against meningococcal infections. Neisseria meningitidis is the major cause of bacterial meningitis. In particular, the serogroup A had been for a long time the leading cause of periodic meningitis epidemics in the area of Africa nicknamed “African meningitis belt”2. Despite the introduction a new vaccine named “MenAfriVac” led to a drastic decrease of MenA-related infections3, the WHO highlighted the importance to persist with a strict vaccination programme. MenAfriVac, as well as all the other vaccines targeting MenA, is obtained from the extraction and size fragmentation of the capsular polysaccharide of the bacterium. The natural CPS of MenA is made of N-acetylmannosamine repeating units linked through α-(16) phosphodiester bonds, prevalently acetylated in position 3 and partially acetylated in position 4. This structure, once isolated from the bacterium, is not stable in water due to the hydrolysis of the phosphodiester bond. Due to the instability issues, most of the licensed vaccines targeting MenA are distributed in a lyophilized form and the cold chain must be maintained during all the process of distribution and storage. In order to achieve a more stable vaccine, which could be distributed in the more convenient liquid formulation without the need of a strict temperature control, some more stable structural analogues have been developed4–8. In particular, the group of Professor Lay synthesized MenA CPS non-acetylated phosphonoester-linked oligomers up to the trimer4,5. These analogues showed good stability, however, they resulted to be poorly immunogenic. Since the acetylation in position 3 was proven to have an important role in the immunogenicity of natural MenA CPS6,9, this thesis project focused on the synthesis of the 3-O acetylated phosphonate analogues up to the trimer. The synthetic work developed for the preparation of the 3-O acetylated phosphono analogues is reported in detail and critically discussed in chapter 2.3 of the thesis. The second section of this thesis deals with the synthesis of glycolipids with the aim of elucidating the structure of new lipid A molecules extracted from Bacteroides fragilis and studying their biological potential. Bacterial lipid A modulate human’s innate immune system through their interaction with Toll like receptors (TLR), proteins expressed on dendritic cells and macrophages which play a key role in the development of the innate immunity. TLR activation by bacterial lipid A (di-glucosamines differently O- and N-acylated and phosphorylated) can cause a strong, uncontrolled infection which can even lead to septic shock10. However, the use of Monophopshoryl lipid A (MPL) as vaccine adjuvant is the proof that low-toxicity lipid A molecules can also be exploited to boost the efficiency of vaccines via a controlled immune system activation11,12. Since symbiont bacteria are well tolerated by the human immune system, the study of their influence on human’s immune system is particularly interesting. Potentially, symbiont bacteria’s lipid A could be used as a tool to modulate the human immune response. In 2019, the group of Professor Kasper, isolated novel lipid A molecules from the commensal bacterium Bacteroides fragilis13. The mixture isolated was composed of tetra- and pentaacylated di-glucosamines (acylated with 15 to 17 carbon atoms linear fatty acid chains), containing one or no phosphate groups13. However, the precise structure of the isolated compounds is still unknown. As a part of a collaboration with Professor Kasper’s group (Harvard Medical School, Boston), a small library of lipid A molecules was designed as a tool to help with B. fragilis lipid A structural elucidation. Moreover, structure-activity relationship studies will be performed on the different compounds of the library to achieve a better understanding of the biological potential of symbiont bacteria lipid A. Preliminary stages of the synthesis of a small library of lipid A molecules from B. fragilis are described in chapter 3.4 of this thesis.

SYNTHESIS OF CARBOHYDRATE ANTIGENS AND THEIR STRUCTURAL ANALOGUES FOR VACCINES DEVELOPMENT / C.m. Santi ; tutor: L. Lay ; coordinatore del corso di dottorato: E. Licandro. - : . Dipartimento di Chimica, 2021 Mar 18. ((33. ciclo, Anno Accademico 2020. [10.13130/santi-cristina-manuela_phd2021-03-18].

SYNTHESIS OF CARBOHYDRATE ANTIGENS AND THEIR STRUCTURAL ANALOGUES FOR VACCINES DEVELOPMENT

C.M. Santi
2021

Abstract

Carbohydrates are multifunctional, stereochemical rich natural molecules. Together with their well-known role of energy sources (glucose, glycogen, amylose) and structural components (cellulose, chitin), they serve important functions in biological processes relevant for health and disease1 such as molecular recognition, cell communication and adhesion. A dense layer of oligosaccharides (proteoglycans, glycoproteins, glycolipids and other glycoconjugates) named glycocalyx, covers the membrane of most of cells. Glycocalyx of bacteria often exhibits oligosaccharides different from those found in mammalians, which have an important role in pathogen recognition and immune system activation. For this reason, pathogenic bacteria polysaccharides are widely used for vaccines production. Moreover, the role of symbiont bacteria glycocalyx in immune system activation, development and modulation is object of increasing interest. In this thesis, the important connection between carbohydrates and immune system is discussed by two different examples. The first section of the thesis deals with the synthesis of a structural analogue of Neisseria meningitidis A (MenA) capsular polysaccharide (CPS), with the final goal of producing a stable glycoconjugate vaccine against meningococcal infections. Neisseria meningitidis is the major cause of bacterial meningitis. In particular, the serogroup A had been for a long time the leading cause of periodic meningitis epidemics in the area of Africa nicknamed “African meningitis belt”2. Despite the introduction a new vaccine named “MenAfriVac” led to a drastic decrease of MenA-related infections3, the WHO highlighted the importance to persist with a strict vaccination programme. MenAfriVac, as well as all the other vaccines targeting MenA, is obtained from the extraction and size fragmentation of the capsular polysaccharide of the bacterium. The natural CPS of MenA is made of N-acetylmannosamine repeating units linked through α-(16) phosphodiester bonds, prevalently acetylated in position 3 and partially acetylated in position 4. This structure, once isolated from the bacterium, is not stable in water due to the hydrolysis of the phosphodiester bond. Due to the instability issues, most of the licensed vaccines targeting MenA are distributed in a lyophilized form and the cold chain must be maintained during all the process of distribution and storage. In order to achieve a more stable vaccine, which could be distributed in the more convenient liquid formulation without the need of a strict temperature control, some more stable structural analogues have been developed4–8. In particular, the group of Professor Lay synthesized MenA CPS non-acetylated phosphonoester-linked oligomers up to the trimer4,5. These analogues showed good stability, however, they resulted to be poorly immunogenic. Since the acetylation in position 3 was proven to have an important role in the immunogenicity of natural MenA CPS6,9, this thesis project focused on the synthesis of the 3-O acetylated phosphonate analogues up to the trimer. The synthetic work developed for the preparation of the 3-O acetylated phosphono analogues is reported in detail and critically discussed in chapter 2.3 of the thesis. The second section of this thesis deals with the synthesis of glycolipids with the aim of elucidating the structure of new lipid A molecules extracted from Bacteroides fragilis and studying their biological potential. Bacterial lipid A modulate human’s innate immune system through their interaction with Toll like receptors (TLR), proteins expressed on dendritic cells and macrophages which play a key role in the development of the innate immunity. TLR activation by bacterial lipid A (di-glucosamines differently O- and N-acylated and phosphorylated) can cause a strong, uncontrolled infection which can even lead to septic shock10. However, the use of Monophopshoryl lipid A (MPL) as vaccine adjuvant is the proof that low-toxicity lipid A molecules can also be exploited to boost the efficiency of vaccines via a controlled immune system activation11,12. Since symbiont bacteria are well tolerated by the human immune system, the study of their influence on human’s immune system is particularly interesting. Potentially, symbiont bacteria’s lipid A could be used as a tool to modulate the human immune response. In 2019, the group of Professor Kasper, isolated novel lipid A molecules from the commensal bacterium Bacteroides fragilis13. The mixture isolated was composed of tetra- and pentaacylated di-glucosamines (acylated with 15 to 17 carbon atoms linear fatty acid chains), containing one or no phosphate groups13. However, the precise structure of the isolated compounds is still unknown. As a part of a collaboration with Professor Kasper’s group (Harvard Medical School, Boston), a small library of lipid A molecules was designed as a tool to help with B. fragilis lipid A structural elucidation. Moreover, structure-activity relationship studies will be performed on the different compounds of the library to achieve a better understanding of the biological potential of symbiont bacteria lipid A. Preliminary stages of the synthesis of a small library of lipid A molecules from B. fragilis are described in chapter 3.4 of this thesis.
LAY, LUIGI
LICANDRO, EMANUELA
Carbohydrate chemistry
Settore CHIM/06 - Chimica Organica
SYNTHESIS OF CARBOHYDRATE ANTIGENS AND THEIR STRUCTURAL ANALOGUES FOR VACCINES DEVELOPMENT / C.m. Santi ; tutor: L. Lay ; coordinatore del corso di dottorato: E. Licandro. - : . Dipartimento di Chimica, 2021 Mar 18. ((33. ciclo, Anno Accademico 2020. [10.13130/santi-cristina-manuela_phd2021-03-18].
Doctoral Thesis
File in questo prodotto:
File Dimensione Formato  
phd_unimi_R11861.pdf

Open Access dal 07/03/2022

Descrizione: Tesi di dottorato - Cristina Manuela Santi
Tipologia: Tesi di dottorato completa
Dimensione 6.03 MB
Formato Adobe PDF
6.03 MB Adobe PDF Visualizza/Apri
Pubblicazioni consigliate

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/820988
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? ND
social impact