Glycolytic enzymes associated with the cell surface of Streptococcus pneumoniae are antigenic in humans and elicit protective immune responses in the mouse - PubMed (original) (raw)

Glycolytic enzymes associated with the cell surface of Streptococcus pneumoniae are antigenic in humans and elicit protective immune responses in the mouse

E Ling et al. Clin Exp Immunol. 2004 Nov.

Abstract

Streptococcus pneumoniae is a leading cause of otitis media, sinusitis, pneumonia, bacteraemia and meningitis worldwide. The drawbacks associated with the limited number of various capsular polysaccharides that can be included in the polysaccharide-based vaccines focuses much attention on pneumococcal proteins as vaccine candidates. We extracted an enriched cell wall fraction from S. pneumoniae WU2. Approximately 150 soluble proteins could be identified by 2D gel electrophoresis. The proteins were screened by 2D-Western blotting using sera that were obtained longitudinally from children attending day-care centres at 18, 30 and 42 months of age and sera from healthy adult volunteers. The proteins were further identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry. Seventeen proteins were antigenic in children and adults, of which 13 showed an increasing antibody response with age in all eight children analysed. Two immunogenic proteins, fructose-bisphosphate aldolase (FBA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and a control protein with known low immunogenicity, heat shock protein 70 (DnaK), were expressed in Escherichia coli, purified and used to immunize mice. Mouse antibodies elicited to the recombinant (r) FBA and rGAPDH were cross-reactive with several genetically unrelated strains of different serotypes and conferred protection to respiratory challenge with virulent pneumococci. In addition, the FBA used in this study (NP_345117) does not have a human ortholog and warrants further investigation as a candidate for a pneumococcal vaccine. In conclusion, the immunoproteomics based approach utilized in the present study appears to be a suitable tool for identification of novel S. pneumoniae vaccine candidates.

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Figures

Fig. 1

Fig. 1

2D PAGE of S. pneumoniae cell wall proteins. (a)Forty _µ_g of enriched cell wall proteins were extracted by mutanolysin treatment of S. pneumoniae, separated by 2D PAGE and stained with Coomassie brilliant blue. More than 150 proteins residing in the pI range 4–6·5 could be visualized. (b) Forty _µ_g of cytoplasmic protein extract prepared by sonication of mutanolysin treated bacteria. The increased complexity of the cytoplasmic protein mixuture is evident from the decreased spot intensity. The distribution of the major protein spots in the cytoplasmic extract (circled) was also different to that of the cell-wall extract. Extensive differences in protein concentration exemplified for two proteins (arrows)

Fig. 2

Fig. 2

Age-dependent enhancement of antibody response to S. pneumoniae surface proteins. S. pneumoniae surface proteins were separated by 2D gel electrophoresis, transferred to nitrocellulolose membrane and probed with pooled (a), 1·5, (b) 2·5 and (c) 3·5-year-old children’s and (d) adult sera. The results demonstrate enhancement of antibody response and widening of the antibody repertoire to S. pneumoniae cell wall proteins along with maturation of the host immune system. The proteins arrowed above were identified by MALDI-TOF mass spectrometry using the protein mass fingerprint technique and the Mascot search tool (Matrix Science, http://www.matrixscience.com). DnaK: heat shock protein 70; FBA: fructose–bisphosphate aldolase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Fig. 3

Fig. 3

Cloning and expression of recombinant S. pneumoniae fructose–bisphosphate aldolase and glyceraldehyde-3-phosphate dehydrogenase and DnaK-heat shock protein 70. Bacteria were transformed and the proteins as purified as described in the Methods section. (a) Following lysis of the bacteria, recombinant Hat-tagged proteins were purified on Ni-Nta columns, resolved on PAGE and stained with Coomassie brilliant blue. The representative gel demonstrates the presence of single protein bands of expected molecular weight in the eluates. (b) The recombinant FBA and GAPDH were separated on PAGE, transferred onto nitrocellulose membranes and probed with anti-HAT antibodies. (c) Total cell wall (CW) proteins of S. pneumoniae strains 9VR, 14R, 6B, 14·8, 3·8 and WU2 were separated on PAGE, transferred onto nitrocellulose membranes and probed sera from mice immunized with the respective recombinant proteins. Sera of the mice immunized with rFBA and rGAPDH recognized respective native proteins in all preparations, as judged by generation of the band of expected molecular weight. Preimmune sera were used as controls.

Fig. 4

Fig. 4

Survival of FBA or GAPDH immunized mice following S. pneumoniae challenge. Mice immunized with the respective protein were intranasally challenged with 1–2 × 108 CFU of S. pneumoniae strain WU2. Survival was monitored daily and compared to that of control sham-vaccinated mice (n = 20). (a) Mice immunization with rFBA protein (n = 21, *P < 0·05). Insert: flow cytometry of WU2 bacteria stained with mouse anti-FBA antibodies (geometric mean 9·26) in comparison to control bacteria stained with a secondary antibody only (geometric mean 2·64) (b) Mice, immunized with rGAPDH (n = 23) proteins (n = 23, *P < 0·05). Insert: flow cytometry of WU2 bacteria stained with mouse anti-GAPDH antibodies (geometric mean 10·96) in comparison to control bacteria stained with a secondary antibody only (geometric mean 2·64) (c) Mice, immunized with rDnaK (n = 20) protein (n = 20). (d) Mice immunized with rFBA (n = 10) and rGAPDH (n = 10) were intranasally challenged with 3 × 108 CFU of S. pneumoniae strain 9VR. Survival was monitored daily and compared to that of control, sham-vaccinated mice (n = 20). *P < 0·05.

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References

    1. Siber GP. Pneumococcal disease: prospects for a new generation of vaccines. Science. 1994;265:1385–7. - PubMed
    1. Zangwill KM, Vadheim CM, Vannier AM, Hemenway LS, Greenberg DP, Ward JL. Epidemiology of invasive pneumococcal disease in southern California: implications for the design and conduct of a pneumococcal conjugate vaccine efficacy trial. J Infect Dis. 1996;174:752–9. - PubMed
    1. Centers for Disease Control and Prevention. Preventing pneumococcal disease among infants and young children: recommendations of the advisory Committee on Immunization Practices (ACIP) MMWR. 2000;49:888–92. (no. RR-9). - PubMed
    1. Hutchison BG, Oxman AD, Shannon HS, Lloyd S, Altmayer CA, Thomas K. Clinical effectiveness of pneumococcal vaccine. Meta-analysis. Can Fam Physician. 1999;45:2381–93. - PMC - PubMed
    1. Wuorimaa T, Käyhty H. Current state of pneumococcal vaccines. Scand J Immunol. 2000;56:111–29. - PubMed

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