Research

The mammalian immune system possesses a remarkable ability to discern self from non-self, a critical function in safeguarding against infections. At the molecular level, this discrimination is facilitated by pattern recognition receptors present on eukaryotic cells, which can identify conserved non-self molecules characteristic of microorganisms. Among these molecules, lipopolysaccharide (LPS) stands out as a complex glycolipid abundantly present in Gram-negative bacterial cell wall, playing a central role in host-pathogen interaction. LPS is universally recognised by specific innate immune proteins that elicit a beneficial pro-inflammatory defense response to infection while maintaining immune homeostasis. However, bacterial pathogens possess various mechanisms to adapt their cell membranes in response to transmission between the environment, vectors, and human hosts, often altering LPS composition to modulate the host immune response. In particular, modifications to the phosphate groups of lipid A, the major immunostimulatory component of LPS, can shield bacteria from recognition by host cationic antimicrobial peptides. Yet, the impact of such modifications on LPS-specific pattern recognition receptors of the host innate immune system remains largely unexplored, particularly with regard to the recently identified cytosolic LPS-sensing proteins crucial for anti-tumor immunity. Due to the high heterogeneity of bacterial glycans and the inherent instability of modified phosphate groups, the isolation of structurally defined intact LPS fragments from bacterial sources is not feasible. Chemical synthesis, however, is a reliable method for providing molecularly defined immunomodulatory LPS motifs to study the effects of unique phosphate group modifications on the interaction with host immune receptors involved in antitumour defence. Carbohydrate chemistry, or glycochemistry, offers versatile tools for the synthesis of complex glycans, providing structurally defined, homogeneous molecules of high purity suitable for biological studies. Leveraging the glycochemistry toolbox, our project aims to develop innovative synthetic strategies for the assembly of complex phosphorylated glycans, culminating in a library of bacterial LPS motifs with phosphate group modifications reflecting those found in different bacterial species. In collaboration with international research groups in immunology and structural biology, we will investigate the immunobiological activity and interaction of our synthetic phosphorylated glycolipid-glycan library with corresponding proteins. By developing a collection of synthetic bacterial lipid A variants and LPS epitopes with uniquely modified phosphate groups, our research aims to elucidate the structural and molecular basis of their interaction with host innate immune receptors, thereby advancing our understanding of LPS-induced antibacterial defense and antitumor immunity mechanisms.

Through photosynthesis, marine algae convert gigatonnes of carbon dioxide into carbohydrates every year. In the form of algal polysaccharides, these structurally complex biomolecules determine to a large extent how much carbon is stored in the oceans. Specialised marine bacteria unlock this carbon energy by breaking down the polysaccharides through the action of carbohydrate-active enzymes (CAZymes) and releasing the carbon dioxide back into the atmosphere. However, some of the polysaccharides are not recycled quickly, but sink into the deep sea and sediments, where they can store carbon for millennia. To better understand these processes, great efforts are needed to further explore the marine carbon cycle. The same advances are also important to support emerging efforts to use algal biomass as a new sustainable resource for the bioeconomy. The enzymatic machinery responsible for the degradation of polysaccharides by marine bacteria has remained largely unexplored because of the size and heterogeneity of algal polysaccharides. Pure and defined oligosaccharides needed for systematic screenings of marine CAZymes are currently not available. Since conventional chemical synthesis is time-consuming and often not general enough, ASAP aims to obtain collections of oligosaccharides related to different classes of algal polysaccharides by using automated glycan assembly (AGA) technology. Oligosaccharides with many different sequences and sulfation patterns will be prepared from small sets of monosaccharide building blocks. Incubation of the synthetic oligosaccharides with samples containing carbohydrate-degrading activity and subsequent HPLC-MS analysis of the degradation products will provide information on: 1) the collective enzyme activities of a bacterial community in seawater and sediment samples; 2) the abilities of individual bacterial strains to degrade specific polysaccharides; 3) the substrate specificities of purified CAZymes.

The HIV-1 envelope spike (Env) bears a cluster of oligomannose-type glycans that is a target for broadly neutralizing antibodies (bnAbs). However, while nAbs to this cluster, dubbed the high-mannose patch (HMP), are known to develop in at least some HIV-infected individuals, past attempts to elicit similar antibodies by immunization have been largely unsuccessful. Most previous approaches have involved presenting clusters of natural or synthetic high-mannose glycans on the surface of carrier proteins. The difficulty in eliciting high-mannose-targeting nAbs by immunization is believed to relate, at least in part, to the ‘self’ nature of the targeted glycans. The approach that we are pursuing is based on the scientific premise that antigenic mimicry of mammalian host structures can stimulate cross-reactive antibodies if such mimics are presented in the proper ‘foreign’ milieu. Our overarching hypothesis is that, upon immunization, an antigenic mimic of mammalian oligomannose will more readily elicit antibodies that bind the HMP than native or synthetic oligomannose. In our progress report, we show that a CRM197-conjugate of our lead oligomannose mimetic is bound with high avidity by various HMP-specific bnAbs as well as their germline precursors. Furthermore, human antibody transgenic mice immunized with this neoglycoconjugate yield antibodies that bind recombinant HIV-1 SOSIP trimers, albeit only when the conjugate is formulated in the TLR4-stimulating Th1-adjuvant GLA-SE. We expect our findings to help sharpen our strategy and critically inform the pursuit of future preclinical studies. Results from this research could inform other HIV vaccine design strategies also.