Test of a novel Streptococcus pneumoniae serotype 6C type specific polyclonal antiserum (factor antiserum 6d) and characterisation of serotype 6C isolates in Denmark.

BACKGROUND: In 2007, Park et al. identified a novel serotype among Streptococcus pneumoniae serogroup 6 which they named serotype 6C. The aim of this study was to evaluate with the Neufeld test a novel S. pneumoniae serotype 6C type specific polyclonal antiserum. In addition, serotype 6C isolates found in Denmark in 2007 and 2008 as well as eight old original serotype 6A isolates were characterised. METHODS: In this study, 181 clinical Streptococcus pneumoniae isolates from Denmark 2007 and 2008 were examined; 96 isolates had previously been typed as serotype 6A and 85 as serotype 6B. In addition, eight older isolates from 1952 to 1987, earlier serotyped as 6A, were examined. Serotype 6C isolates were identified by PCR and serotyping with the Neufeld test using the novel type specific polyclonal antiserum, factor antiserum 6 d, in addition to factor antisera 6b, 6b* (absorbed free for cross-reactions to serotype 6C) and 6c. All antisera are commercially available and antiserum 6b obtained from the supplier after 1 January 2009 is antiserum 6b*. All serotype 6C isolates were further characterised using multi-locus sequence typing. RESULTS: When retesting all 96 original serotype 6A isolates by PCR and the Neufeld test, 29.6% (24 of 81) of the invasive isolates in Denmark from 2007 and 2008 were recognised as serotype 6C. In addition, three of eight old isolates originally serotyped as 6A were identified to be serotype 6C. The oldest serotype 6C isolate was from 1962. The serotype 6C isolates belonged to eleven different sequence types (ST) and nine clonal complexes (CC), ST1692 (CC395), ST386 (CC386) and ST481 (CC460) were the predominant types. CONCLUSIONS: We tested a novel polyclonal antiserum 6 d, as well as modified antiserum 6b*, provided a scheme for the serotyping of S. pneumoniae serogroup 6 using the Neufeld test and compared the serotyping method with PCR based methods. The two types of methods provided the same results. In future, it will, therefore, be possible to test also serotype 6C in accordance to the standard method for serotyping of S. pneumoniae recommended by WHO.Among all invasive isolates from Denmark 2007 and 2008, serotype 6C constituted 29.6% of the original serotype 6A isolates. The serotype 6C isolates were found to be diverse belonging to a number of different STs and CCs of which most have been observed in other countries previously. Serotype 6C is regarded as an "old" serotype being present among S. pneumoniae isolates in Denmark for at least 48 years. The genetic diversity of serotype 6C isolates and their genetic relationship to other serotypes suggested that serotype 6C strains may have arisen from several different independent recombination events involving different parental strains such as serotypes 6A, 6B, 23F and 4.

Evaluation of serotype-specific pneumococcal IgG measurement using two different polysaccharides from ATCC and SSI Diagnostica.

Streptococcus pneumoniae (pneumococcus) is a major cause of morbidity and mortality especially in infants and elderly people. Pneumococcus capsular polysaccharide has been characterised and more than 90 different serotypes have been identified. Serotype-specific antibodies against the capsular polysaccharide are produced during infection. At present, many countries follow the WHO pneumococcal ELISA IgG measurement protocol, in which polysaccharides from ATCC are used as antigens. In recent years, serotype specific polysaccharides from different producers have been tested in pneumococcal antibody assay's. In this project, purified serotype specific pneumococcal antigens from SSI Diagnostica and from ATCC were compared. In general, the data showed that both types of polysaccharide could be used as antigens. Furthermore, the effect of adsorption using different combinations of adsorption procedures was tested, showing similar results using CWPSmulti or CWPS+22F.

Purification and structure characterization of the active component in the pneumococcal 22F polysaccharide capsule used for adsorption in pneumococcal enzyme-linked immunosorbent assays.

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.