Biology subjects

Chao, K.

Publications and source records attributed to Chao, K..

3 recordsLinked to original sources

bioRxiv2026

Incorporation of native HCV E1E2 into a nanoparticle vaccine platform

Development of an effective HCV vaccine requires the induction of both broadly neutralizing antibodies (bnAbs) and a robust cellular response. One issue that has arisen is that HCV subunit vaccines have limited immunogenicity, thus requiring multivalent formats in order to elicit a robust anti-HCV immune response. Toward that end, nanoparticle vaccines possess the ability to facilitate a controlled multivalent presentation and trafficking to lymph nodes, where they can interact with both arms of the immune system. Here, we used a soluble, secreted form of E1E2 (sE1E2) to assemble native E1E2 into a nanoparticle platform using a post-purification coupling assembly system. Nanoparticles were assembled by purifying sE1E2 containing a C-terminal SpyTag and an mi3-SpyCatcher fusion separately and covalently coupling the components via incubation. Free sE1E2-SpyTag was removed from nanoparticle preparations via gel filtration. The sE1E2-mi3 nanoparticles are fully competent to bind conformation-dependent bnAbs, indicating retention of a native assembly in the nanoparticle format. Electron microscopy analysis showed a clear incorporation of sE1E2 on the surface of the nanoparticle. Immunogenicity of sE1E2-mi3 nanoparticles was examined relative to sE1E2 alone and membrane-bound E1E2 (mbE1E2) following inoculation of groups of CD1 mice. Assessment of the immunogenicity of the sE1E2-mi3 nanoparticles showed that the nanoparticle assembly has a similar immunogenicity profile to that of mbE1E2 after only a prime and one boost, and overall superior to sE1E2. This proof-of-principle study sets the stage for further exploration of nanoparticles and other multivalent platforms for the development of E1E2-based vaccines. ImportanceHepatitis C virus infects approximately 50 million people, and at present no effective HCV vaccine exists. Due to the high sequence variability of HCV and the resulting difficulty in developing a vaccine that elicits a broadly neutralizing response, multiple efforts are underway to enhance the immunogenicity of HCV vaccine candidates. In this study, we incorporated native soluble, secreted E1E2 (sE1E2) into a 60-mer nanoparticle via the SpyTag-SpyCatcher system and covalent isopeptide bond attachment using the purified components. These nanoparticles are antigenically intact and elicit a neutralizing antibody response at an earlier time point in the immunization regimen than the corresponding subunit vaccine. These studies show that a well-characterized sE1E2 platform compatible with multiple genotypes can be coupled to nanoparticles for use as a vaccine candidate.

microbiology↗

bioRxiv2024

Molecular mapping and functional validation of GLP-1R cholesterol binding sites in pancreatic beta cells

G protein-coupled receptors (GPCRs) are integral membrane proteins which closely interact with their plasma membrane lipid microenvironment. Cholesterol is a lipid enriched at the plasma membrane with pivotal roles in the control of membrane fluidity and maintenance of membrane microarchitecture, directly impacting on GPCR stability, dynamics and function. Cholesterol extraction from pancreatic beta cells has previously been shown to disrupt the internalisation, clustering and cAMP responses of the glucagon-like peptide-1 receptor (GLP-1R), a class B1 GPCR with key roles in the control of blood glucose levels via the potentiation of insulin secretion in beta cells and weight reduction via the modulation of brain appetite control centres. Here, we unveil the detrimental effect of a high cholesterol diet on GLP-1R-dependent glucoregulation in vivo, and the improvement in GLP-1R function that a reduction in cholesterol synthesis using simvastatin exerts in pancreatic islets. We next identify and map sites of cholesterol high occupancy and residence time on active versus inactive GLP-1Rs using coarse-grained molecular dynamics (cgMD) simulations, followed by a screen of key residues selected from these sites and detailed analyses of the effects of mutating one of these residues, Val229, to alanine on GLP-1R interactions with cholesterol, plasma membrane behaviours, clustering, trafficking and signalling in pancreatic beta cells and primary islets, unveiling an improved insulin secretion profile for the V229A mutant receptor. This study 1) highlights the role of cholesterol in regulating GLP-1R responses in vivo; 2) provides a detailed map of GLP-1R - cholesterol binding sites in model membranes; 3) validates their functional relevance in beta cells; and 4) highlights their potential as locations for the rational design of novel allosteric modulators with the capacity to fine-tune GLP-1R responses.

cell biology↗

bioRxiv2023

Structural and regulatory insights into the glideosome-associated connector from Toxoplasma gondii

The phylum of Apicomplexa groups intracellular parasites that employ substratedependent gliding motility to invade host cells, egress from the infected cells and cross biological barriers. The glideosome associated connector (GAC) is a conserved protein essential to this process. GAC facilitates the association of actin filaments with surface transmembrane adhesins and the efficient transmission of the force generated by myosin translocation of actin to the cell surface substrate. Here, we present the crystal structure of Toxoplasma gondii GAC and reveal a unique, supercoiled armadillo repeat region that adopts a closed ring conformation. Characterisation of the membrane binding interface within the C-terminal PH domain as well as an N-terminal fragment necessary for association with F-actin suggest that GAC adopts multiple conformations. A multi-conformational model for assembly of GAC within the glideosome is proposed.

biochemistry↗