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Pediatrics Erasmus MC Rotterdam

MOPA-7 a useful tool to monitor the 7-valent pneumococcal conjugate vaccine P.W.M. Hermans Department of Pediatrics University Medical Center St. Radboud Nijmegen, The Netherlands. Pediatrics UMC St. Radboud Nijmegen. Pediatrics Erasmus MC Rotterdam.

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Pediatrics Erasmus MC Rotterdam

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  1. MOPA-7a useful tool to monitor the 7-valent pneumococcal conjugate vaccineP.W.M. HermansDepartment of PediatricsUniversity Medical Center St. RadboudNijmegen, The Netherlands

  2. Pediatrics UMC St. Radboud Nijmegen Pediatrics Erasmus MC Rotterdam

  3. Surface-associated proteome of S. pneumoniae - SB-14 fractioning - pI 4 7 200 97 68 43 Mr (kDa) 29 18 14 Overweg, 2000

  4. Surface-associated proteome of S. pneumoniae - SB-14 fractioning - pI 4 7 200 Mass spectrometry 97 68 43 Mr (kDa) 29 18 14 Overweg, 2000

  5. Surface-associated proteome of S. pneumoniae - SB-14 fractioning - pI 4 7 200 Mass spectrometry 97 68 43 Mr (kDa) PpmA 29 18 Antigen characterization 14 Overweg, 2000

  6. FITC-labeling opsonisation phagocytosis determine percentage FITC+ PMNs Immune-protective potentials: phagocytosis assay Overweg, 2000

  7. Immune-protective potentials: phagocytosis assay α –surface protein fraction α –PpmA Overweg, 2000

  8. Effect of a pneumococcal conjugate vaccine on recurrent otitis media • Dutch OMAVAX study • R. Veenhoven, Haarlem Pediatrics, Haarlem Pediatrics, Utrecht Pediatrics, Rotterdam

  9. MOPA-7a useful tool to monitor the 7-valent pneumococcal conjugate vaccine Bogaert et al.; Vaccine; 2004; 22: 4014-4020

  10. MOPA-7: strains

  11. MOPA-7: strains . . Origin clinical R-cassette clinical natural clinical natural clinical isolate isolate selection isolate selection isolate [Antibiotic] 1.0 4.0 16 400 1.5 400 32 (mg/L)

  12. MOPA-7: materials Serum: * two-fold serial dilutions in RPMI-1640 * dilution range 2 to 128 * 30 μl per microtiter well

  13. MOPA-7: materials Serum: * two-fold serial dilutions in RPMI-1640 * dilution range 2 to 128 * 30 μl per microtiter well Bacteria: * mixture of 7 serotypes (early-log cultures) in RPMI-1640 (1x washing) * 2x105 CFU/ml for each serotype * 15 μl per microtiter well

  14. MOPA-7: materials Serum: * two-fold serial dilutions in RPMI-1640 * dilution range 2 to 128 * 30 μl per microtiter well Bacteria: * mixture of 7 serotypes (early-log cultures) in RPMI-1640 (1x washing) * 2x105 CFU/ml for each serotype * 15 μl per microtiter well HL-60: * culture in RPMI-1640 / 10% FCS / 1 % Pen-Strep * differentiation in RPMI-1640 / 0.8% DMF (7 days) * renewal of medium at day 5 * 1.4x108 cells/ml RPMI-1640 / FCS (2x washing) * 60 μl per microtiter well

  15. MOPA-7: materials Serum: * two-fold serial dilutions in RPMI-1640 * dilution range 2 to 128 * 30 μl per microtiter well Bacteria: * mixture of 7 serotypes (early-log cultures) in RPMI-1640 (1x washing) * 2x105 CFU/ml for each serotype * 15 μl per microtiter well HL-60: * culture in RPMI-1640 / 10% FCS / 1 % Pen-Strep * differentiation in RPMI-1640 / 0.8% DMF (7 days) * renewal of medium at day 5 * 1.4x108 cells/ml RPMI-1640 / FCS (2x washing) * 60 μl per microtiter well Complement: * rabbit-derived *15 μl per microtiter well

  16. MOPA-7: methods • * Mix bacterial mix and diluted serum • * Incubate for 20 min at 37 0C

  17. MOPA-7: methods • * Mix bacterial mix and diluted serum • * Incubate for 20 min at 37 0C • 2. * Add HL-60 cells and complement (ratio bacteria : HL-60 = 1 : 400) • * Incubate for 1 hour at 37 0C

  18. MOPA-7: methods • * Mix bacterial mix and diluted serum • * Incubate for 20 min at 37 0C • 2. * Add HL-60 cells and complement (ratio bacteria : HL-60 = 1 : 400) • * Incubate for 1 hour at 37 0C • * Spot 10 μl on omnitray containing blood agar / antibiotic (MOPA) • * Plate 10 μl on plate containing blood agar / antibiotic (MSOPKA) • * Incubate overnight at 37 0C (reduced O2)

  19. MOPA-7: methods • * Mix bacterial mix and diluted serum • * Incubate for 20 min at 37 0C • 2. * Add HL-60 cells and complement (ratio bacteria : HL-60 = 1 : 400) • * Incubate for 1 hour at 37 0C • * Spot 10 μl on omnitray containing blood agar / antibiotic (MOPA) • * Plate 10 μl on plate containing blood agar / antibiotic (MSOPKA) • * Incubate overnight at 37 0C (reduced O2) • * Read out: MOPA titer is the reciprocal of the highest serum dilution with 90% killing as compared with the number of bacteria in the control experiment (bacteria / HL-60 / complement) • (MSOPKA: 50% killing)

  20. Capsular IgG ELISA

  21. Bacterial dilutions Controls Vaccinees Dilution MOPA titer 1:16 16 1:32 32 1:64 64 1:128 128 1:256 256 1:512 512 1:1024 1024 1. 2. 3 4. 5. 6. 1. 2. 3. + - 1:2 1:4 1:8 1:16 MOPA-7: example 23F (Tmp-R)

  22. Bacterial dilutions Controls Vaccinees Dilution MOPA titer 1:16 16 1:32 32 1:64 64 1:128 128 1:256 256 1:512 512 1:1024 1024 1. 2. 3 4. 5. 6. 1. 2. 3. + - 1:2 1:4 1:8 1:16 MOPA-7: example 23F (Tmp-R)

  23. MOPA-7: comparison with MSOPKA

  24. MOPA-7: comparison with MSOPKA (scatter plot) Best fit line for all serotypes: logMOPA = 0.86(logMSOPKA)+0.3; R-value (Pearson): 0.94 (p<0.001)

  25. MOPA-7: In conclusion MOPA-7 allows the simultaneous (‘high’ throughput) measurement of the opsonophagocytosis activity of serum against the capsular serotypes included in the 7-valent conjugate vaccine

  26. MOPA-7: In conclusion MOPA-7 allows the simultaneous (‘high’ throughput) measurement of the opsonophagocytosis activity of serum against the capsular serotypes included in the 7-valent conjugate vaccine MOPA-7 uses limited serum volumes (30 μl)

  27. MOPA-7: In conclusion MOPA-7 allows the simultaneous (‘high’ throughput) measurement of the opsonophagocytosis activity of serum against the capsular serotypes included in the 7-valent conjugate vaccine MOPA-7 uses limited serum volumes (30 μl) Data obtained with MOPA-7 correlate well with MSOPKA

  28. MOPA-7: In conclusion MOPA-7 allows the simultaneous (‘high’ throughput) measurement of the opsonophagocytosis activity of serum against the capsular serotypes included in the 7-valent conjugate vaccine MOPA-7 uses limited serum volumes (30 μl) Data obtained with MOPA-7 correlate well with MSOPKA Inter-laboratory standardization and multi-laboratory evaluation is requested!

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