1. Functional antibody activity as measured by opsonophagocytosis Sandra Romero-Steiner, Ph.D. Respiratory Diseases Immunology Laboratory

2. Induction of antibodies Disease Disease Vaccination Vaccination Passive immunization Passive immunization Colonization Colonization Cross reactive Cross reactive

3. Antibody Measurements Quantitative measurement Quantitative measurement (ELISA) =  g/ml (ELISA) =  g/ml Functional determinations Functional determinations How well do those Abs protect? How well do those Abs protect? Animal protection Animal protection Opsonophagocytosis Opsonophagocytosis Indicator of Memory Indicator of Memory Antibody avidity Antibody avidity B-cell stimulation (Elispot) B-cell stimulation (Elispot)

4. Opsonophagocytosis

5. OPA Players Fc  R CR1/CR3 + PMN C3b/iC3b Ab Bacteria

6. Opsonopaghocytosis Romero-Steiner et al. Romero-Steiner et al. CDLI 1997;4:415-22 CDLI 1997;4:415-22 Four components Four components Serum Serum Bacteria Bacteria Complement Complement Culturable phagocytes Culturable phagocytes Internalized Pnc are killed Internalized Pnc are killed OPA titer OPA titer

7. VIABILITY OPA Unk1 Unk2 Unk3 Unk4 QC  globulin C’ controls 1:81:161:32....1:1024 1:64.....2048

8. OPA titer determination (50% killing) Tom Taylor, PAOAW, June 2005 1/Serum Dilution Midpoint (50% Killing) Continuous Titer 50% 0% 100% Measured Killing Discontinuous Titer >50% 8 16 32 64 128 256

9. Killing OPAs Single serotype Single serotype Time consuming Time consuming Reagent demanding Reagent demanding Multivalent assays Multivalent assays 2 serotypes 2 serotypes 4 serotypes 4 serotypes 7 serotypes 7 serotypes

10. SOPA vs MOPA-4 (Burton, R. PAOAW, June 2005) R 2 =0.95 (R 2 =0.94) R 2 =0.98 R 2 =0.60 (R 2 =0.99)

11. Automation of killing OPAs

12. vOPA fOPA Bieging, K. et al. CDLI 2005; 10:1238-42 CDLI 2005; 10:1238-42 r = 0.76 to 0.97 7-valent fOPA 7-valent fOPA Bogaert, D. et al, Vaccine 2004; 22:4014-20

13. Uptake OPAs Flow cytometric methods Flow cytometric methods Uptake vs killing Uptake vs killing Killed bacteria Killed bacteria Ps-coated particles Ps-coated particles Single serotype Single serotype Multivalent assays Multivalent assays 4 serotypes 4 serotypes

14. Flow cytometric OPA (Uptake of killed bacteria or Ps-coated particles) Phagocytic cells Complement control Positive Rx OPA titer Martinez, J. et al. CDLI 1999; 6:581-6.

15. Multiplexed Flow OPA Monovalent Multivalent Martinez, J. et al. submitted to CDLI, 2005 and funded by Flow Applications r = 0.53 to 0.95 r = 0.61 to 0.91 r = 0.68 to 0.88

16. Current validation status Single serotype killing assay Single serotype killing assay Developed, standardized, evaluated and validated at the GLP level Developed, standardized, evaluated and validated at the GLP level Multivalent killing assays Multivalent killing assays Developed and standardized Developed and standardized Fluorescent killing assays Fluorescent killing assays Developed and standardized Developed and standardized Flow cytometric (uptake) assays Flow cytometric (uptake) assays Developed and standardized Developed and standardized

17. Multi-laboratory Evaluation Romero-Steiner et al. CDLI 2003; 10:1019-24 S. pneumoniae Serotype Lab 1 Lab 2 Lab 3 Lab 4 Lab 5 488 a, 100 b 79, 88100, 10096, 9696, 100 6B75, 8883, 10056, 7283, 8888, 92 9V63, 7183, 8875, 9483, 9683, 92 1463, 8375, 8381, 9492, 9288, 92 18C88, 96 56, 6988, 9288, 100 19F79, 9688, 9694, 10096, 10088, 100 23F100, 100 96, 10092, 96 a Within one dilution about the median OPA titer – 75% overall b Within two dilutions about the median OPA titer – 88% overall

18. Romero-Steiner et al. CDLI 2003;10:1019-24 Romero-Steiner et al. CDLI 2003;10:1019-24  Higher agreement in sera with low titers  Lower agreement in sera with high titers Multi-laboratory Evaluation

19. Validation of killing OPA B. T. Hu et al. CDLI 2005, 12: 287-295. Specificity > 80% (> 87% CDLI, 1997) Specificity > 80% (> 87% CDLI, 1997) Heterologous Ps < 20% Heterologous Ps < 20% Intermediate Precision Intermediate Precision Overall 81% of titers within 2 dil. of median Overall 81% of titers within 2 dil. of median Linearity (9 serotypes) Linearity (9 serotypes) r = 0.982 to 1.000, slopes = -0.850 to -1.350 r = 0.982 to 1.000, slopes = -0.850 to -1.350 Accuracy (9 serotypes) Accuracy (9 serotypes) All types 100% except 14 (81%) & 23F (75%) All types 100% except 14 (81%) & 23F (75%) Robustness Robustness

20. OPA as a correlate of protection ELISA in healthy populations ELISA in healthy populations Passive protection in animals Passive protection in animals Minimum level needed for vaccine efficacy in infants Minimum level needed for vaccine efficacy in infants

22. Johnson, et al. JID 1999; 180:133 Protective OPA titer in a mouse passive protection model Type 1 Type 4 Type 5 Type 6B Type 18C Type 23 OPA = 8 75%

23. 97.9 12.9 VE = 1 - (1-.979) (1-.129) =.976 [Ab] for 97.9% protection in the target population = 0.20 µg/ml ~0.2  g/ml Reverse cumulative distributions of post dose 3 ELISA Ab for 7 serotypes in infants ELISA Ab for 7 serotypes in infants (Black, et al., North Calif. Trial) Data from Dr. Kohberger, WHO 2003

24. 0.010.1110 ELISA Concentration (  g/ml) 1 10 100 1000 10000 OPA TITER 7VPnC Control R = 0.92 ( p < 0.0001) Total N = 79 ELISA concentration of 0.20  g/ml  OPA titer of 1:8 Jodar, et al. Vaccine 2003; 21:3265-3272 Correlation of ELISA and OPA (North CA KP infant study) 1:8 0.2  g/ml

25. Functional Abs in the elderly Protective levels are unknown Protective levels are unknown Reduced function in elderly receiving PPV-23 (CID 1999;29:281-8) Reduced function in elderly receiving PPV-23 (CID 1999;29:281-8) OPA (80-89 years & >90 years) OPA (80-89 years & >90 years) Lower Ab avidity Lower Ab avidity Poor correlation with ELISA IgG (r=0.3 to 0.8) Poor correlation with ELISA IgG (r=0.3 to 0.8) Do not protect mice Do not protect mice Usinger and Lucas I&I, 1999;67:2366-70 Usinger and Lucas I&I, 1999;67:2366-70 Absorption of cross-reactive antibodies (I&A 2005; 2:1-10) Absorption of cross-reactive antibodies (I&A 2005; 2:1-10)

26. OPA in clinical vaccine trials Ekstrom, N. PAOAW, June 2005 Killing OPA (Romero-Steiner et al.1997) Killing OPA (Romero-Steiner et al.1997) Various phase 2 studies with different Pnc-conjugate vaccines in Finnish infants (Anttila et al. 1999) Various phase 2 studies with different Pnc-conjugate vaccines in Finnish infants (Anttila et al. 1999) 7-valent PncCRM and PncOMPC in Finnish infants in The Finnish Otitis Media Vaccine Trial (FinOM) 7-valent PncCRM and PncOMPC in Finnish infants in The Finnish Otitis Media Vaccine Trial (FinOM) 11-valent PncDT in Filipino infants (Puumalainen et al. 2003) 11-valent PncDT in Filipino infants (Puumalainen et al. 2003) 23-valent PS in HIV+ Ugandan adults (French et al. 2004) 23-valent PS in HIV+ Ugandan adults (French et al. 2004) 11-valent PncDT in Finnish and Israeli infants (Wuorimaa et al. 2005) 11-valent PncDT in Finnish and Israeli infants (Wuorimaa et al. 2005) 11-valent Pn-PD in Finnish infants (Nurkka et al. 2005) 11-valent Pn-PD in Finnish infants (Nurkka et al. 2005) Flow cytometric OPA (Martinez et al. 1999) Flow cytometric OPA (Martinez et al. 1999) 11-valent PncDT in Filipino infants (Lucero et al. 2004) 9-valent PncCRM in South-African infants (Mahdi et al. 2005)

27. Advantages of using OPA Laboratory correlate of protection Laboratory correlate of protection Reduce numbers of efficacy studies Reduce numbers of efficacy studies Reference method (killing sOPA) Reference method (killing sOPA) www.vaccine.uab.edu www.vaccine.uab.edu Standardized Standardized Validated Validated Culturable phagocytes Culturable phagocytes High through put High through put GLP conditions GLP conditions Data analysis Data analysis

28. Disadvantages of OPA In vitro correlate In vitro correlate Laboratory facilities Laboratory facilities Training of technical staff Training of technical staff Multiplex assays need validation Multiplex assays need validation