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Research project (§ 26 & § 27)
Duration : 2017-11-01 - 2021-08-31

PROJECT SUMMARY A number of Abs targeting oligomannose-type glycans on the HIV envelope spike (Env) have been described in recent years that exhibit broad neutralizing activity (bnAbs). However, eliciting such nAbs by immunization has not been very successful so far. A principal problem may be the host origin of the glycans, with immune tolerance mechanisms limiting the frequency or development of B cells capable of producing Abs with specificity for mammalian oligomannose. For example, Abs elicited by glycoconjugate immunogens presenting oligomannosides are generally unable to bind oligomannose on Env and even when Env-binding Abs have been obtained, such as with recombinant yeast, they appear to bind insufficiently avid to the virus and fail to exert meaningful neutralizing activity. Here, we propose to utilize bacterially derived oligosaccharide analogs of oligomannose to overcome these challenges. We focus in this application on a fairly conserved patch of high-mannose glycans at and surrounding Asn301 and Asn332 on HIV gp120. Prototypic for Abs targeting these oligomannose-type glycans is the PGT128 family of nAbs, which are potent and broadly active, suggesting that a vaccine component able to elicit similar nAbs could offer protection at even modest serum Ab concentrations. We not long ago discovered a bacterial oligosaccharide that closely resembles the D1 arm of mammalian oligomannose and subsequently made synthetic derivatives of it with a D3 arm-like extension. One of these derivatives, in the form of a neoglycoconjugate, is bound avidly by PGT128 family members and, notably, their predicted germline predecessor. More importantly, data from a pilot immunization with the lead conjugate in transgenic animals harboring an unarranged human Ab repertoire show elicitation of oligomannose-specific Abs with HIV cross-neutralizing activity. Here, we propose to expand on these encouraging preliminary studies. Specifically, we wish to elaborate on our conjugate design to heighten Ab responses and continue to utilize transgenic animals to identify an optimal adjuvant+conjugate combination. We also will dissect antibody responses at the serum and repertoire levels to determine similarities between the elicited responses and existing nAbs. Finally, we propose to test our strategy also in macaques to assess the extent to which it may work in outbred systems. In sum, this project will investigate whether glycan mimicry can serve to readily trigger the development of cross-reactive Abs to the highly vulnerable oligomannose patch on HIV Env. If so, this work could inform strategies for targeting other glyco-epitopes on HIV-1.
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 - 2019-07-31

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

Supervised Theses and Dissertations