title: Purification and structure characterization of the active component in the pneumococcal 22F polysaccharide capsule used for adsorption in pneumococcal enzyme-linked immunosorbent assays.
abstract: Protection against pneumococcal disease is thought to be mediated primarily by antibodies that are opsonic [Musher DM, Chapman AJ, Goree A, Jonsson S, Briles D, Baughn RE. Natural and vaccine-related immunity to Streptococcus pneumoniae. J Infect Dis 1986;154(2):245-56]. Pneumococcal capsular polysaccharide (CPS) is immunogenic and induces type-specific protective immunity. For convenience, the protective capacity of serum antibodies is often evaluated by the measurement of antibody titers in an ELISA test. The pneumococcal capsular polysaccharide (CPS) used in ELISA contains several impurities; these include about 5% by weight of teicholic acid (CWPS) and the cholin binding protein, pneumococcal surface protein A (PspA) [Sorensen UB, Henrichsen J. C-polysaccharide in a pneumococcal vaccine. Acta Pathol Microbiol Immunol Scand C 1984;92(6):351-6; Yu J, Briles DE, Englund JA, Hollingshead SK, Glezen WP, Nahm MH. Immunogenic protein contaminants in pneumococcal vaccines. J Infect Dis 2003;187(6):1019-23]. All individuals have antibodies to CWPS possible as a result of early exposure to pneumococci, Streptocuccus mitis and Streptocuccus oralis [Bergstrom N, Jansson PE, Kilian M, Skov Sorensen UB. Structures of two cell wall-associated polysaccharides of a Streptococcus mitis biovar 1 strain. A unique teichoic acid-like polysaccharide and the group O antigen which is a C-polysaccharide in common with pneumococci. Eur J Biochem 2000;267(24):7147-57. [4]]. The concentration of the CWPS antibodies in non-immunized individuals often exceeds the concentration of the serotype-specific pneumococcal antibodies. Therefore, the pneumococcal ELISA requires an adsorption step to remove the unprotective CWPS antibodies [Konradsen HB, Sorensen UB, Henrichsen J. A modified enzyme-linked immunosorbent assay for measuring type-specific anti-pneumococcal capsular polysaccharide antibodies. J Immunol Meth 1993;164(1):13-20. [5]; Concepcion N, Frasch CE. Evaluation of previously assigned antibody concentrations in pneumococcal polysaccharide reference serum 89SF by the method of cross-standardization. Clin Diagn Lab Immunol 1998;5(2):199-204. [6]; Kayhty H, Ahman H, Ronnberg PR, Tillikainen R, Eskola J. Pneumococcal polysaccharide-meningococcal outer membrane protein complex conjugate vaccine is immunogenic in infants and children. J Infect Dis 1995;172(5):1273-8. [7]; Koskela M. Serum antibodies to pneumococcal C polysaccharide in children: response to acute pneumococcal otitis media or to vaccination. Pediatr Infect Dis J 1987;6 (6):519-26. [8]]. Recently a new pneumococcal CPS ELISA was recommended with an extra serum absorption step with 22F CPS to remove antibodies against an extra unknown common cross-reactive component. The aim of this study was to characterize the active component in the 22F capsule. A non-capsulated pneumococci was prepared from a 22F capsulated pneumococci. The cell wall polysaccharide (CWPS2) purified from this pneumococci has a better adsorption potential than 22F capsule in the pneumococci ELISA. Structure characterization of the commercial available CWPS and CWPS2 was done by nuclear magnetic resonance (NMR). The NMR results showed that commercial CWPS had one phosporylcholine per sugar repeat while the CWPS2 had two phosporylcholine per sugar repeat explaining an immunological difference between the two variants of CWPS. In addition the LicD2 gene responsible for the attachment of the second cholin in the CWPS tetra sugar repeat was inactive in the strain used for purifying the commercial CWPS but active in the strain expressing CWPS2.
authors.affiliation: Division of Microbiology and Diagnostics, Statens Serum Institut, Copenhagen, Denmark. ics@ssi.dk
authors.forename: Ian Chr
authors.initials: IC
authors.lastname: Skovsted
authors.forename: Mette B
authors.initials: MB
authors.lastname: Kerrn
authors.forename: Jacob
authors.initials: J
authors.lastname: Sonne-Hansen
authors.forename: Lis E
authors.initials: LE
authors.lastname: Sauer
authors.forename: Annie Kleis
authors.initials: AK
authors.lastname: Nielsen
authors.forename: Helle Bossen
authors.initials: HB
authors.lastname: Konradsen
authors.forename: Bent O
authors.initials: BO
authors.lastname: Petersen
authors.forename: Nils T
authors.initials: NT
authors.lastname: Nyberg
authors.forename: Jens Ø
authors.initials: JØ
authors.lastname: Duus
mesh_terms: D016477:Artifacts; D002240:Carbohydrate Sequence; D016521:Electrophoresis, Gel, Pulsed-Field; D004797:Enzyme-Linked Immunosorbent Assay; D007202:Indicators and Reagents; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010767:Phosphorylcholine / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry* / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013296:Streptococcus pneumoniae / Q000737:chemistry*
chemical_list: D007202:Indicators and Reagents; D011134:Polysaccharides; D010767:Phosphorylcholine