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Research project (§ 26 & § 27)
Duration : 2016-06-01 - 2019-05-31

Recognition of the endotoxic portion of the Gram-negative bacterial lipopolysaccharide (LPS), a glycophospholipid Lipid A, by the transmembrane protein complex Toll-like receptor 4 (TLR4)-myeloid differentiation factor 2 (MD-2) and by the intracellular serine protease Caspase-4/11 initiates activation of the pro-inflammatory signaling cascade and is essential for the control of infectious diseases. Activation of TLR4 was also shown to bridge the innate and adaptive immunity, which highlights stimulation of TLR4 complex by the non-pyrogenic ligands as a useful approach for development of novel vaccine adjuvants. Though, LPS-induced TLR4 signaling may result in the development of a dysregulated innate immune response leading to variety of inflammatory conditions. To explore the molecular basis for Lipid A – induced Caspase-4/11 (non-canonical TLR4-independent inflammasome activation) and TLR4 activation, chemical synthesis and immuno-functional studies of innovative conformationally confined Lipid A mimetics is intended. Lipid A is composed of a 1,4′ -bisphosphorylated βGlcN(1→6)GlcN polar head group which carries a variable number of long-chain (R)-3-hydroxyacyl- and (R)-3-acyloxyacyl residues in symmetric or asymmetric distribution. Synthesis of Lipid A mimetics wherein the flexible three-bond β(1→6) connection is exchanged for a rigid two-bond β,β-(1↔1) glycosidic linkage will provide potentially agonistic Caspase-4/11 and TLR4 ligands. Restricting conformational flexibility of Lipid A by fixing the molecular shape of its carbohydrate backbone in a predefined conformation attained by a rigid β,β-(1↔1)-linked disaccharide scaffold would allow to attain a specific topology of the functional groups of the ligands (phosphates and long-chain (R)-3-acyloxyacyl residues) in the ligand-protein complexes. Evaluation of the tetriary structure of the ligand in respect to its immuno-stimulating activity will ensure a reliable correlation of structure-activity relationships.
Research project (§ 26 & § 27)
Duration : 2016-03-01 - 2019-02-28

Many Gram-negative bacteria have various efficient defense mechanisms against the immune system of their respective host at their disposal. The outer membrane of the bacterial cell wall exerts a protective function and is characterized by the presence of numerous negatively charged substituents such as sugar acids and sugar phosphates. The barrier function of the cell membrane, however, is counteracted by positively charged proteins (cationic antimicrobial peptides) provided by the innate immune system. Conversely, by decorating their membrane with positively charged aminosugars such as aminoarabinose, bacteria become resistant. These modifications are relevant in plant-pathogenic Burkholderia, being used in agricultural applications and which have meanwhile become major human pathogens of increasing clinical importance. Colonization of the lung by B. cepacia strains leads to dysfunction of the respiratory tract and to the lethal “cepacia syndrome”. In addition, Burkholderia strains are notoriously multiresistant against many common antibiotics. The enzymes involved in the transfer of aminoarabinose onto the bacterial lipopolysaccharide have only been incompletely characterized. This is also the case for other enzymes of the biosynthetic pathway, which generate the activated form of aminoarabinose as sugar-phosphate lipids. Since isolation of the substrates from native sources only generates tiny amounts, the project aims to prepare the native substrates, inhibitors as well as fluorescence-labeled derivatives by chemical synthesis. The central synthetic steps will first be studied using simplified lipids and then transferred to the synthesis of the complex, long-chain lipids (hepta- and undecaprenol containing 35 and 55 carbon atoms). In addition, carbon-connected derivatives (C-glycosyl phosphonates, monofluoro-C-phosphonates) are planned, which could inhibit the transfer reaction, thereby restoring efficacy of antimicrobial peptides. The synthetic substrates should also help to clarify, if one or two different aminoarabinosyl transferases are present. The synthetic compounds will be tested in collaboration with Miguel Valvano (Queens University Belfast, UK), who is a leading expert in the microbiology and genetics of Burkholderia. Appropriate inhibitors and substrates will also be used in binding studies (NMR spectroscopy, crystallography). In summary, the project should contribute substantially to the understanding of transfer mechanisms of phospholipid-activated carbohydrates onto bacterial acceptor substrates with far-reaching implications for future therapies of infections caused by multiresistant Burkholderia and other Gram-negative pathogens
Research project (§ 26 & § 27)
Duration : 2014-08-01 - 2018-03-31

Combating the worldwide pandemic caused by the human immunodeficiency virus (HIV-1) is still a formidable challenge despite the progress made in antiviral drug design. The development of broadly cross-protective and neutralizing antibodies is counteracted by various immune evasion strategies of the virus. Still, the tight glycan clusters at the viral cell envelope spikes being present in the oligomannosidic glycoprotein gp120 may be exploited as „Achilles heel“ target for antibody recognition. A limited number of gp120 directed monoclonal antibodies has been elaborated with highly unique epitope specificities but due to the close similarity of these epitopes with mammalian glycoprotein glycans the binding of gp120-specific antibodies is usually characterized by a low immunogenicity and low affinity. Recently, a gp120-related oligomannosyl glycan has been found connected to the core unit of the Gram-negative lipopolysaccharide of the soil bacterium Rhizobium radiobacter. In preliminary experiments these glycan structures cross-reacted with HIV-specific antibodies with modest affinity. Structural data of a gp120 ligand and the bacterial glycan in the binding sites of two monoclonal anti HIV antibodies indicated that the bacterial scaffold might provide additional binding interactions with mannosyl residues. Moreover, since the bacterial sugars are inherently recognized as foreign by the immune system, an enhanced immune response against these antigens and reduced autoreactivity may be expected. Two major hypotheses may thus be put forward: Do novel, modified HIV-related bacterial glycans mimic the viral gp120 epitopes? Second, will these glycans be more efficiently recognized by anti-HIV monoclonal antibodies and could elicit neutralizing antibodies? The project will focus on the chemical synthesis of oligosaccharides up to the undecasaccharide level as modified and chain-extended mimics of gp120 glycan epitopes by combining the structurally conserved bacterial lipopolysaccharide component with two oligomannosyl chains. As controls, the mannosyl units will also be provided without the bacterial scaffold. The ligands will be prepared as spacer derivatives to be conjugated biotin and to protein carriers for testing of monoclonal anti-HIV antibodies. In addition large glycan clusters will be assembled, which should lead to a substantial increase in binding affinities due to the multivalent presentation of ligands. NMR spectroscopic studies will be performed to get insight into the detailed recognitions motifs of HIV-specific antibodies with the ligands. Immunochemical studies will be jointly performed with Dr. Kunert at the Department of Biotechnology (BOKU) and with Dr. Pantophlet at Simon Fraser University in Canada. Provided the binding studies are successful, future studies will include immunization with the neoglycoconjugates in order to elicit cross-reactive and neutralizing antibodies. The outcome of the project should thus substantially contribute to novel approaches in anti-HIV vaccine development and/or supplement combination therapies and will have significant impact on anti-AIDS immunogen design strategies.

Supervised Theses and Dissertations