Bioinspired Multivalent Glycan Structures to Decipher Pathogen Immune Evasion

Background: Binding of pathogen surface specific glycans by immune cell surface lectins is the first step to activate immune defences against infection. However, some pathogens have developed strategies to exploit such recognitions to modulate immune cell responses to facilitate infection. The HIV surface is decorated with heavily glycosylated proteins with unusually large inter-glycoprotein distances, and binding of HIV by a dendritic cell (DC) surface lectin, DC-SIGN, allows the HIV to evade intracellular degradation and facilitate infection. Bacterium H. pylori also uses its surface glycans to target DC-SIGN to induce DC tolerogenic signals to establish chronic infection. However, mechanisms underlying how specific DC-SIGN-glycan interactions modulate DC immune response remain unclear. As multiple such interactions are involved and DC-SIGN has shown to form clusters on DC surface, we hypothesise that, pathogens use their specific glycan patterns to control DC-SIGN cluster formation and stability as a way to modulate DC-SIGN signaling, and hence DC immune outcomes.
Objectives/experimental: We plan to develop novel glycan-nanoparticles as multifunctional pathogen mimics to probe pathogen-DC interactions. We will prepare non-toxic gold nanoparticles (GNPs) of varied size and shape and coat them with specific oligo mannose (found on HIV) and lewis glycans (found on H. pylori). We will tune GNP surface glycan valency, inter-glycan spacing and quantify their DC-SIGN binding in solution via GNP's strong fluorescence quenching.
We will then anchor DC-SIGN onto supported lipid bilayers (SLBs) to mimic their natural presentation on cell membrane and quantify their binding by quartz crystal microbalance and spectroscopic elliposometry. We will further investigate binding induced DC-SIGN clustering in details by fluorescence microscopy and TEM, taking advantage of GNP's robust surface chemistry and high TEM contrast prpperties.
We will further stimulate DCs with glycan-GNPs and study their internalization, and modulating DC production of immune moderating signalling proteins. We will correlate the results with binding induced DC-SIGN clustering on SLBs to confirm our hypothesis.
Novelty/timeliness: This project will develop a novel bio-inspired, nano-enabled approach to elucidate pathogen immune evasion mechanisms. It will reveal the difference and correlation between multivalent bindings in solution and on 2D surfaces. The new knowledge will help develop effective immunotherapies against infectious diseases and fill the current research gap in extrapolating binding results obtained in solution using soluble fragments of receptors to their full-length native forms on cell surfaces.