S. pneumoniae is a human pathogen primarily that is disseminated through airborne particles. It is commonly inhibited through the use of vaccines. The pivotal immunogenic component of this pathogen is its CPS. The CPS of S. pneumoniae forms a mucous or gelatinous layer that envelops the bacterial cell wall, acting as a shield against host immune responses. This crucial structure is made up of a complex polymer. The constituent oligosaccharides are chemically linked to the cell wall through covalent bonds. The CPS enhances the pathogen's defense mechanisms that make it evade detection by the host's immune system. Different serotypes of pneumococcal strains exhibit varying levels of virulence and invasiveness, which depend on the chemical composition and amount of CPS they produce. These variations determine the bacteria's endurance within the circulation and their potential to cause invasive diseases. Some capsules are polar and hydrophilic which disrupts the interactions between the bacteria and phagocytes.
Fig.1 Schematic cross-section of S. pneumoniae cell wall. (Brooks, et al., 2018)
CD BioGlyco provides an alcohol-free method for the production of S. pneumoniae polysaccharides. This process uses cationic detergents, specifically hexadecyl 1-trimethylammonium bromide, to remove protein and nucleic acid contaminants. It is noteworthy that some polysaccharides show resistance to precipitation even at extremely low detergent concentrations, whereas other serotypes are co-precipitated along with impurities. However, we are able to effectively resuspend the latter in a buffer that prevents the resolubilization of contaminants. Most pneumococcal polysaccharide types tend to precipitate when hexadecyl-trimethylammonium bromide (1-4%) is used, except for types 7F, 14, and 33F. Our single-step precipitation procedure successfully removes C-polysaccharide and other contaminants and simplifies the subsequent purification process. This approach involves using activated carbon filters and then performing hydroxyapatite chromatography as a subsequent step. Through this streamlined approach, we generate high-purity polysaccharides. We utilize the additional step, anion exchange chromatography, to obtain equally pure material for serotypes 7F, 14, and 33F, which do not precipitate with the detergent.
Fig.2 The process of pneumococcal CPS conjugation. (CD BioGlyco)
We utilize copper-catalyzed azide-alkyne cycloaddition (CuAAC) which is a groundbreaking technique within click chemistry for the development of our pneumococcal CPS conjugate vaccine. The carrier protein we chose is Tetanus Toxoid (TT). By adding a long spacer arm in our pneumococcal CPS conjugate vaccine design, our conjugate reduces steric hindrance between CPS and TT. Finally, we purify and analyze the conjugates by 1H NMR spectra.
Fig.3 Pneumococcal CPS conjugate with long spacer arm. (CD BioGlyco)
CD BioGlyco specializes in the field of Carbohydrate-based Vaccine Development, where we have made significant breakthroughs. At the heart of our success is the innovative Glyco Vaccine Development Platform, which helps our clients resolve problems. If you would like more in-depth information about our services, please do not hesitate to contact us .
Reference:
