Quality Improvement of Capsular Polysaccharide in Streptococcus pneumoniae by Purification Process Optimization

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,

Possible Cross-Reactivity between SARS-CoV-2 Proteins, CRM197 and Proteins in Pneumococcal Vaccines May Protect Against Symptomatic SARS-CoV-2 Disease and Death

“…While current pneumococcal vaccines are composed primarily of capsular polysaccharides, they also contain one or both of two types of proteins. The polysaccharide component is never pure, generally containing around three percent of the cell surface proteins to which the polysaccharides are attached [ 5 , 6 , 7 ]. Proteins identified in pneumococcal vaccines include pneumococcal surface protein A (PspA) and pneumococcal surface adhesin A (PsaA) [ 8 , 9 ].…”

Cross-Reactivity Between SARS-CoV-2 Proteins and Proteins in Pneumococcal Vaccines May Protect Against Symptomatic SARS-CoV-2 Disease and Death

“…The Results of this study indicate that proteins known to contaminate pneumococcal vaccines significantly mimic SARS-CoV-2 proteins (FIGURE 1) while the CRM197 used to conjugate various polysaccharide vaccines and the proteins contained in other vaccines are statistically significantly less likely to do so (FIGURE 2). In particular, the Results point to potential cross-reactivity between SARS-CoV-2 proteins and the pneumococcal proteins PspA and PsaA, which are known to contaminate polysaccharide-based pneumococcal vaccines (WHO, 2010;Morais, et al, 2018;Lee, et al, 2020) as well as PspC, which it is reasonable to assume is another such contaminant since it derives from the same outer membrane protein complex and is highly cross-reactive with the antibodies against PspA used to demonstrate the presence of PspA in vaccines (Brooks, et al, 1999;Ogunniyi, et al, 2001).…”

“…In Prevnar-13, for example, there are 30.4 µg of capsular polysaccharides and 34.0 µg of CRM197 for a total of 64.4 micrograms of antigen per dose (FDA, 2017). Protein contaminants may make up an additional 3%, or 1.92 µg, of antigenic material according to WHO guidelines and confirmed by laboratory analysis (WHO, 2010;Morais, et al, 2018;Lee, et al, 2020). This 1.92 µg of protein is virtually identical to the 2.2 µg of each of twelve of the capsular polysaccharides present (plus 4.4 µg of serotype 6) or the 2.3 micrograms of CRM197 conjugated to each polysaccharide type (FDA, 2017) and is therefore quite sufficient to induce an immune response, especially since PspA and PspC are strongly cross-reactive.…”

“…Pneumovax-23, in contrast, has 25 µg of each capsular polysaccharide, adding up to a total of 575 µg of antigen. The three percent protein contamination allowed by WHO (WHO, 2010;Morais, et al, 2018;Lee, et al, 2020) could result in 17.25 µg of PsaA, PspA and PspC per dose, which is certainly sufficient to induce immunity. For comparison, each 0.5-mL dose of Adacel®, a diphtheriatetanus-pertussis vaccine (Sanofi Pasteur) contains 2.5 µg detoxified pertussis toxoid, 5 µg FHA, 3 µg pertactin and 5 µg FIM acellular pertussis antigens (CDC, 2020).…”

“…While current pneumococcal vaccines are composed primarily of capsular polysaccharides, they also contain one or both of two types of proteins. The polysaccharide component is never pure, generally containing around three percent of the cell surface proteins to which the polysaccharides are attached (WHO, 2010;Morais, et al, 2018;Lee, et al, 2020). Proteins identified in pneumococcal vaccines include pneumococcal surface protein A (PspA) and pneumococcal surface adhesin A (PsaA) (Yu, et al, 1999;Yu, et al, 2003).…”