Abstract
Streptococcus pneumoniae is the causative agent of many diseases, most notably pneumonia. Most of the currently used vaccines to protect against this pathogen employ pneumococcal capsular polysaccharides (CPSs) as antigens, but purifying CPS of sufficient quality has been challenging. A purification process for CPS comprising conventional methods such as ultrafiltration, CTAB precipitation, and chromatography was previously established; however, this method resulted in high cell wall polysaccharide (CWPS) contamination, especially for serotype 5. Thus, a better purification method that yields CPS of a higher quality is needed for vaccine development. In this study, we significantly reduced CWPS contamination in serotype 5 CPS by improving the ultrafiltration and CTAB precipitation steps. Moreover, by applying an acid precipitation process to further remove other impurities, serotype 5 CPS was obtained with a lower impurity such as decreased nucleic acid contamination. This improved method was also successfully applied to 14 other serotypes (1, 3, 4, 6A, 6B, 7F, 9V, 11A, 14, 18C, 19A, 19F, 22F, and 23F). To assess the immunogenicity of the CPS from the 15 serotypes, two sets of 15-valent pneumococcal conjugate vaccines were prepared using the previous purification method and the improved method developed here; these vaccines were administered to a rabbit model. Enzyme-linked immunosorbent assay and opsonophagocytic assay demonstrated higher immunogenicity of the conjugate vaccine prepared using CPS produced by the improved purification process.
Highlights
Streptococcus pneumoniae, which was first isolated by Louis Pasteur and George Sternberg independently in 1880 (Grabenstein and Klugman, 2012), is a gram-positive bacterium and major cause of pneumonia
The molecular weights of cell wall polysaccharide (CWPS) were measured by Gel Permeation Chromatography (GPC) to determine if CWPS can be separated from capsular polysaccharides (CPSs) based on size differences (Figure 2)
We developed our own pneumococcal CPS purification method by combining several traditional separation techniques such as ultrafiltration, CTAB precipitation, and chromatography; concerns about CWPS contamination remained, as high CWPS concentrations in CPS were observed by Nuclear Magnetic Resonance (NMR) spectroscopy
Summary
Streptococcus pneumoniae, which was first isolated by Louis Pasteur and George Sternberg independently in 1880 (Grabenstein and Klugman, 2012), is a gram-positive bacterium and major cause of pneumonia. Because pneumococcal CPS is a major factor of pathogenicity and involved in the antigen-specific immune response against S. pneumoniae, it has been a primary target for the development of pneumococcal disease vaccines by pharmaceutical companies worldwide (Melegaro et al, 2006). We developed an improved process for CPS purification from the fermentation broth of S. pneumoniae that reduces CWPS as well as other contaminants such as nucleic acids and proteins. Higher-quality CPS and CPS with high CWPS content were conjugated to the carrier protein CRM197 to overcome the limited effectiveness of polysaccharide vaccines since polysaccharide alone cannot elicit T-cell dependent immune responses (Beuvery et al, 1982; Lesinski and Westerink, 2001). Animal studies were conducted to compare the immunogenicity between the conjugate vaccines produced by the previous and new methods
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