1. OPA methods in clinical vaccine trials: experience with killing and flow cytometric OPA methods Nina Ekstrom Vaccine Immunology Laboratory National Public Health Institute (KTL) Helsinki, Finland

2. OPA in clinical vaccine trials Killing type OPA (Romero-Steiner et al.1997) –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) –11-valent PncDT in Filipino infants (Puumalainen et al. 2003) –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 Pn-PD in Finnish infants (Nurkka et al. 2005) 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)

3. The Finnish Otitis Media Vaccine Trial (FinOM) Randomized, double-blind, phase 3 cohort study designed to evaluate in parallel two 7-valent Pnc conjugate vaccines (N= 2497 Finnish infants) Study vaccines ( 2, 4, 6 and 12 months of age) –PncCRM (Wyeth Vaccines)N=835 –PncOMPC (Merck & Co. Inc.)N= 831 –Hepatitis B (Merck & Co. Inc.)N=831

4. Efficacy of two PCVs against serotype-specific pneumococcal acute otitis media (AOM) compared with control vaccine Immunogenicity Quality of antibodies (avidity) Functionality of antibodies (OPA)  choice of OPA method Serological correlates of protection Aims of the FinOM Vaccine Trial

5. Comparison of OPA methods Killing type OPA (Romero-Steiner et al.1997) Radio-OPA (Vidarsson et al.1994) Flow cytometric OPA-1 (Jansen et al.1998) Flow cytometric OPA-2 (Martinez et al.1999) Sera from infants (n=10-16) immunized at 2, 4, 6 with heptavalent PncCRM and at 15 mo with PncCRM or 23-valent PncPS Pnc serotypes 6B and 19F (reference strains from CDC) IgG concentrations were determined by EIA

6. Differences in OPA protocols Killing assayRadio assayFlow assay 1Flow assay 2 Bacteria Live, untreated, grown once to log phase (1 x log) Live, radiolabelled (H 3 ), grown 1 x log Killed, labelled with FITC, grown 3 x log Killed, labelled with 5,6- carboxyfluorescein succinimidyl ester, grown 1 x log Phagocytes Fresh PMNLs HL-60 cells Bact : Phag ratio1:40010:1 4:1 Complement source Baby rabbit serumPooled serum from hypo- and agamma- globulinemic patients (6B), IgG-depleted serum from a healthy adult (19F) IgG-depleted human pooled serum Baby rabbit serum Complement concentration (%) 12.55 (6B), 12 (19F)2 12.5 Vakevainen et al. CDLI 2001

7. Relationship between killing, radio and flow-1 OPAs 6B 19F Vakevainen et al. CDLI 2001

8. Relationship between OPA and EIA 6B 19F Vakevainen et al. CDLI 2001

9. Flow-2 OPA vs. other OPA methods 6B 19F Vakevainen et al. CDLI 2001

10. Summary of comparisons Different OPAs gave comparable results –levels of OPA were different –serotype-specific differences (19F > 6B) OPA correlated with IgG concentration –Killing, radio > flow-1 Highest correlation between killing and radio OPAs Differences in sensitivity (emphasized for 19F) –Killing > Radio > Flow-2 > Flow-1

11. The Killing OPA was chosen to be used in the FinOM Trial + Most sensitive + Measured the killing of bacteria + Standardised, ”the golden standard” + Reproducibility between laboratories had been evaluated in the multilaboratory study (Romero-Steiner et al. 2003) - Laborious - Slow (max. 90 analyses/week) - Long-term repeteability ? ± Price

12. The FinOM Trial - OPA analyses N = 166 infants in Kangasala cohort –PncCRMN = 56 –PncOMPCN = 52 –Control (Hepatitis B)N = 58 Immunizations at 2, 4, 6 and 12 months of age OPA was performed for 7, 12, 13 and 24 mo samples OPA for serotypes 6B, 19F and 23F

13. Killing-OPA as described by Romero-Steiner et al. 1997 –Differentiated HL-60 cells as effector cells – S. Pneumoniae reference strains from CDC –Baby rabbit complement –Colonies counted manually IgG concentrations were determined by EIA The FinOM Trial - Killing OPA

14. IgG concetrations & OPA titers in The FinOM Trial

15. IgG concentration (ng/ml) required to kill 50% of Pnc (EIA:OPA ratio) Serotype- specific efficacy % against AOM ng/ml neened for 50% killing Age (mo)Vaccine 71324 6BPncCRM 84101317 PncOMPC 799*1325* 19FPncCRM 25975981 PncOMPC 375078104 23FPncCRM 59 3158 PncOMPC 5292*9665* * < 50% infants had a detectable OPA titer

16. Correlation between EIA & OPA 7 mo 13 mo 6B19F 23F

17. OPA as a correlate of protection Our results conform that Ab concentration is the primary correlate of protection, but that OPA is needed as a secondary correlate of protection: –Despite equal Ab concentrations the functionality of Ab’s may differ between serotypes –EIA:OPA ratio seems to correlate better with protection against AOM than Ab concentration at population level

18. Experience with the flow cytometric OPA method + Correlates with killing OPA + Rapid and less laborious + Unaffected by antibiotics in sera of e.g HIV+ persons + Multiplexing possible - Long-term repeteability ? - Flow cytometer needed ± Price 19F: Alexa Fluor Dye 647 23F: 5,6-carboxyfluorescein, succinimidyl ester

19. Quantitative and Qualitative Antibody Response to Pneumococcal Conjugate Vaccine Among African Human Immunodeficiency Virus-Infected and Uninfected Children Madhi SA †, Kuwanda L*, Cutland C †, Holm A ‡, Kayhty H ‡, and Klugman KP § *National Institute of Communicable Diseases/University of the Witwatersrand/Medical Research Council: Respiratory and Meningeal Pathogens Reasearch Unit, and the †Paediatric Infectious Diseases Research Unit, Wits Health Consortium, University of the Witwatersrand, Johannesburg, South Africa; the ‡National Public Health Institute, Helsinki, Finland; and the Departments of § International Health, Rollins School of Public Health and Infectious Diseases, School of Medicine, Emory University, Atlanta, GA PIDJ 2005;24:410-16 Experience with the flow cytometric OPA method – Soweto Trial

20. Soweto Trial Nested study of a larger phase 3 trial that evaluated the efficacy of a 9-valent PncCRM in 39 836 children Study vaccine or placebo were given at 6, 10 and 14 weeks of age Blood sample was taken a month after the 3 rd vaccine dose EIA and OPA analyses were performed at KTL, Finland OPA analyses were performed using the flow cytometric OPA assay (Martinez et al. 1999) –HL-60 cells as effector cells – S.pneumoniae strains from CDC –Baby rabbit complement N =HIV +HIV - PncCV 3063 Placebo 3664

21. Anti-Pnc in HIV + and HIV – children Madhi et al. PIDJ 2005

22. SerotypeHIV+PCV+HIV-PCV+p 6B% ≥878960.01 ng/ml needed for 50 % uptake 2640.0005 19F% ≥846910.00002 ng/ml needed for 50 % uptake 102890.69 23F% ≥857930.002 ng/ml needed for 50 % uptake 83410.14 IgG concentration (ng/ml) required for 50% uptake Madhi et al. PIDJ 2005

23. Conclusion Despite equal Ab concentrations PCV induced Ab’s of HIV+ and HIV- children had different functional activity Abs produced by HIV+children were dysfunctional The results suggest that although Ab concentration is the primary correlate of protection OPA is needed as a secondary correlate of protection

24. Summary – OPA methods at KTL KillingFlow OPA Multiplex Flow OPA Small sample volume +++++ High sensitivity +++++ Easy to perform ++++++ Repeteability/ Reproducibility ++ Rapidity -+++++ Antibiotics do not interfere -+++ Low cost +++++

25. Acknowledgments Vaccine Immunology Laboratory & Clinical Unit, Department of Vaccines, KTL: Merja Vakevainen Hannele Lehtonen Maijastiina Karpala Kaisa Jousimies Merja Ryynanen Nina Nikkanen Jukka Jokinen Helena Käyhty The FinOM Study Group CDC, Atlanta: Sandra Romero-Steiner Joseph Martinez George M. Carlone Eijkman-Winkler Institute for Microbiology, Utrecht University Hospital, The Netherlands: Wouter Jansen Andre Verheul Harm Snippe National University Hospital, Reykjavik, Iceland: Eirikur Saeland Ingileif Jonsdottir Paediatric Infectious Diseases Research Unit,, University of the Witwatersrand, Johannesburg, South Africa The group of Shabir Mahdi