This research project aims to develop fluidic glycan microarrays for the quantitative profiling and characterization of pathogens, and for the screening of pathogen inhibitors and the development of vaccines. The approach targets a common mechanism at the initial stage of pathogen attack: the recognition of and attachment onto host cells via multivalent interaction between receptor proteins and glycan molecules. The tremendous variation in glycans and the complexity in multivalent interaction have necessitated the use of large-scale profiling and analysis techniques, particularly glycan microarrays. The proposed fluidic approach overcomes two major limitations of current glycan microarray technology: the lack of mobility and the difficulty in quantitatively controlling glycan density. Multivalent cell surface interactions often require mobility of the fluidic cell membrane environment and are strong functions of surface glycan density. In order to quantitatively apply the glycan microarray in profiling and characterization, one must ensure mobility and control of glycan density over a broad range.
The specific aims during phase-II are:
Aim 1 : using haemagglutinin, a predominant antigen on influenza viruses, and the dendritic cell receptor DC-SIGN, a binding receptor for mannose moieties on HIV-1 virus, as model systems and establish the roles of secondary interactions in binding affinity, avidity, and specificity. These experiments will establish the general applicability of the fluidic microarrays in profiling and characterizing complex pathogen-cell surface interactions;
Aim 2 : using several strains of E. coli with varying affinity and selectivity towards mannose as model systems and establishing that the fluidic and density gradient glycan microarray can be used to quantitatively profile the variability in binding affinity and multivalency among strains of the same species. Quantifying such variability is essential to the understanding and surveillance of how random mutations can lead to new pathogen threats, as exemplified by the recent outbreaks of avian flu and swine flu;
Aim 3 : To establish chemical procedures for the optimization of the fluidic glycan microarray, including spatial confinement of the supported lipid bilayer spots, efficient blocking of surfaces outside the spotted areas, recoverability in drying and rehydration of the microarrays, and long term stability of content glycan microarrays. These practical issues must be addressed in developing the fluidic glycan microarray as a viable product. The long-term goal of this R&D plan is to develop an effective high-throughput tool in the combat against pathogen threats.
This research project aims to develop cell-membrane mimicking microarrays of sugar molecules for the profiling and characterization of pathogens, and for the screening of vaccines and inhibitors against pathogens. )