Identification of B-cell Epitope Regions in Cell Surface Proteins of Streptococcus pneumoniae Serotype 19F Using Bioinformatic Tools

Pneumococcal conjugate vaccines (PCVs) were developed through chemical coupling of polysaccharide capsules of pneumococci to immunogenic carrier proteins and World Health Organization recommends inclusion of these vaccines in national immunization programs for children. However, the PCVs implementation in developing countries is prevented by the high vaccine manufacturing costs. This issue can be resolved by construction of protein based vaccines against pneumococci. We already identified three pneumococcal surface proteins including autolysin, zinc binding lipoprotein (ZBL), and plasmid stabilization protein (PSP) as appropriate protein candidates for eliciting protection against S. pneumoniae serotype 19F. The protein protective antigenicity and the absence of autoimmunity induction were used as selection criteria. However, regarding the necessity of the antibody response for protection against pneumococci, analysis of protective B-cell epitopes of these proteins is required to elucidate their usefulness in new vaccine formulations. In the present study, therefore, we aim to identify protective B-cell epitope regions of these proteins via widely used bioinformatic tools. Both of the Bepipred and BCPreds programs were used for identification of linear B-cell epitopes. The conformational B-cell epitopes were predicted using the CBTope program. The immunoprotective abilities of epitopes were evaluated using VaxiJen. We determined the linear B-cell epitope regions, which were predicted by both Bepipred and BCPreds and have common amino acids with conformational B-cell epitopes. Our results showed that all of the three studied proteins included such protective overlapped linear B-cell epitope regions. However, a truncated form of PSP had the greatest number of the protective overlapped B-cell epitope regions.Moreover, the most probable protective epitope region resides in this protein and this epitope region is completely conserved in PSPs of serotype 19F pneumococcal strains. Therefore, the truncated PSP was an appropriate candidate for development of protein based vaccines against S. pneumoniae serotype 19F. 

Strain Selection and Statistical Optimization of Culture Conditions for 19F Polysaccharide Production from Pneumococcus

Introduction:  Capsular polysaccharides of pneumococci are principle antigenic constituents of vaccines against pneumococci. Enhancing the yield of capsule production decreases costs of these vaccines and increases the vaccine coverage in developing countries. In this study therefore, we aim to optimize the capsule production from serotype 19F pneumococcus in terms of the applied pneumococcal strain and environmental culture conditions.Materials and Methods:  Thirteen serotype 19F Streptococcus pneumoniae strains were screened for the capsule production in modified Hoeprich culture medium using the stains all assay. The optimal ranges of environmental culture conditions for the selected strain were determined using single factor at a time (SFAT) strategy and utilized for the design of experiments based on the response surface methodology (RSM).Results:  S. pneumoniae 82218 showed the highest capsule production, and thus used for further studies. The maximum capsule production (1.364 mg/ml) was attained under optimal conditions (pH 7.26, 35.5 ºC, 30 rpm) predicted by the RSM derived quadratic model. The capsule production under the optimal conditions increased to 1.9 mg/ml using the buffered culture medium. Conclusion:  These results are much higher than those reported for pneumococcal capsule production in published studies [1, 2] and thus can be used to design suitable systems for the serotype 19F capsule production in the vaccine manufacturing process.

Streptococcus pneumoniae Based on B-Cell Epitopes of Pneumococcal Plasmid Stabilization Protein

Abstract Pneumococcal conjugate vaccines (PCVs) were constructed through chemical conjugation of pneumococcal capsules to immunogenic carrier proteins. The PCVs implementation in developing countries was prevented by their high manufacturing costs. This issue can be overcome by development of protein based vaccines against pneumococci. Antibody responses are necessary for protection against S. pneumoniae. The plasmid stabilization protein (PSP) was already identified as a pneumococcal surface protein able to elicit protection against S. pneumoniae serotype 19F and its protective B-cell epitope regions were determined. Whole antigens are not as potent as epitope based vaccines and every epitope in a multi epitope based vaccine can individually induce a protective immune response against the pathogen. Thus better immunoprotection can be achieved by multi epitope based vaccines. In the present study, therefore, we aim to design a multi epitope vaccine against pneumococci based on the identified B-cell epitope regions of PSP using immunoinformatic tools. These regions were joined together using the (EAAAK) 4 linker. The resulting antigen (HPBE) showed much higher immunoprotective ability compared to PSP regarding the VaxiJen scores. The codon optimization was done for HPBE using OPTIMIZER. Analysis of the mRNA secondary structure using Mfold tool revealed no stable hairpin at the 5' end and thus the antigen can be expressed appropriately. The 3D model of the antigen resulted from I-TASSER indicated the presence of alpha helix, beta sheet, turn, coil, and 3 10 helix as the protein structural elements. Analyzing physicochemical properties of the antigen using ProtParam showed that it was stable and its half life in Escherichia coli was more than 10 h. Considering the GRAVY score, HPBE possessed a hydrophilic nature and it can be expressed in the soluble form in E. coli at 79.6% probability. Our results demonstrated that HPBE is a suitable vaccine candidate, which can elicit protection against common S. pneumoniae serotypes causing invasive pneumococcal disease in children less than 5 years of age

Investigation of Appropriate Cultivation Approach for Capsular Polysaccharide Production by Streptococcus pneumoniae Serotype 19F

Abstract With the aim of determining an appropriate cultivation approach for the capsular polysaccharide production by Streptococcus pneumoniae serotype 19F, the influence of environmental and culture medium conditions on the pneumococcal culture was investigated. Using 5% CO 2 atmosphere instead of using an aeration limited environment enhanced the capsule production 3.5 fold. Buffering the cultivation medium prevented the culture pH drop to the acidic condition and increased the capsule production almost 5 fold. Utilizing casamino acid as the nitrogen source of the culture medium instead of soytone provided 1.3 fold increase in the capsule production. Glucose, sorbitol, lactose and sucrose were investigated as carbon sources of the culture medium. Regarding costs of these sugars and their effects on the capsule production, lactose was the best carbon source. Our results demonstrated that buffering the cultivation medium had the most profound effect on the serotype 19F capsule production. The capsule was produced at 1.706 mg/ml in the buffered medium. Applying this culture method allows the cost effective production of the serotype 19F pneumococcal capsule for inclusion in pneumococcal vaccines