Opsonization Assay: Functional Correlate of Protection

1. Opsonization Assay: Functional Correlate of Protection Dr. George M. Carlone CDC, Atlanta Elie Metchnikoff

2. OVERVIEW • Host protection • Serologic markers measured in immunogenicity trials • Opsonization – how does it work? • Opsonic assay formats • Why it’s important to measure functional activity • Correlates we observe in animals & humans

3. Host protection against pneumococcal disease is mainly mediated by phagocytosis • Phagocytosis– general process describing the engulf- ment & destruction of extracellularly-derived materials by • phagocytic cells, such as macrophages & neutrophils • Opsophagocytosis – opsonophagocytosis is a specific • mechanism by which the host protects against infection, • with the participation of serum opsonins • Opsonins – antibody and complement (e.g., IgG and C3b) • Functional antibody– leads to effective opsonization and recovery from infection

4. Serologic markers that correlate with protection in infants vaccinated with conjugate vaccine • IgG ELISA - WHO standardized and validated assay protocol (www.vaccine.UAB.edu) • Opsonophagocytic assay(OPA) - CDC standard manual • killing assay (reference method) protocol • (www.vaccine.UAB.edu) • Antibody avidity – indicator of memory maturation of antibody function & quality • Immunological memory - immune recall • (IgG, rapid, memory T & B cells) • ** Evaluated in efficacy study

5. Comparison of serologic markers that correlate with protection in infants and/or adults Laboratory Assays Conjugate Vaccine Poly. Vaccine

6. Opsonization - how does it work? antigen IgG first complement proteins complement cascade complement inserts into cell wall cell swells and bursts

7. IgG and C3b are known as opsonins and the process of attachment is called opsonization *subclasses differ in C’ deposition capacity Receptor for iC3b (high & low avidity) Receptor for IgG Phagocytic cell www.cat.cc.md.us/.../ opsonization/u1fig26n.html

8. Opsonophagocytosis of pneumococci requires bacteria, Ab, C’, and phagocytic cells With Ab and C’ Without Ab and C’ Pnc are not being engulfed Pnc are being engulfed & killed www.lsumc.edu/campus/micr/opson.htm

9. Engulfment and killing 1. attachment of bacteria / beads phagocyte 2. engulfment 4. respiratory burst stimulation of NADPH oxidase phagosome phagolysosome residual body lysosomes 3. degranulation: fusion of granules to phagosome bacterial killing and digestion www.cvm.uiuc.edu/.../ vp331/Intracell_Bacteria

10. Capsules are anti-phagocytic (interferes with attraction of phagocyte, engulfment and recognition of cell as foreign) *The thicker the capsule the more Ab required for OPA www.uic.edu/.../ slide0231.htm

11. Opsonic Assay Formats • Manual assays (viable cells) – measures killing CDC reference method (Romero-Steiner), multiplexed, antibiotic resistant (Kim, Nahm; Bogaert) radiometric (Jonsdottir), other • Flow cytometric (non-viable cells) – measures uptake single color (Martinez; Jansen) three color multiplex (Martinez) bead based target multiplexed (Martinez), other

12. Why is functional (OPA) activity important to measure for vaccine evaluation? • Opsonophagocytic activity correlates with protection, however, Ab conc. may not always corr with function • Formal efficacy trials will likely not be done • Serotypes in new vaccine formulations will not have proven efficacy • Vaccines from different manufacturers will likely be different (structurally, immunologically, etc.) • Therefore, functional activity is an essential measurement for vaccine comparison

13. What do animal models tell us about potential correlates of protection?

14. Protective activity of serotype 6B specific IgG against bacteremia after challenge with a 6B isolate Johnson, et al. 1999. JId. 180:133.

15. ` Correlation between bacteremia and OPA titer in a mouse passive protection model Serotype 1 Serotype 4 Serotype 5 Serotype 6B Serotype 18C Serotype 23 1:8 Log2 OPA Titer 75% Infant mice nonbacteremic at 48 hr (%) Johnson, et al. 1999. JId. 180:133.

16. What do human trials tell us about potential correlates of protection?

17. 11-Valent Conjugate Vaccine Response in Filipino Infants Serotype 4 Serotype 6B 18 wk old 10 mo old + EIA & OPA pre (6 wk) OPA OPA EIA Serotype 14 EIA Serotype 19F Regression- …. 18 wk old ___10 mo old OPA OPA EIA EIA Puumalainen, et al. 2003. JID. 187:1704.

18. Reverse cumulative distributions of post dose 3 ELISAAb for 7 serotypes in infants (Black, et al., North Calif. Trial) 97.9 VE = 1 - (1-.979) (1-.129) = .976 12.9 0.18 [Ab] prot ~0.2ug/ml Data from Dr. Kohberger, WHO 2003 Ignoring Ab levels in controls obtains [Ab] prot = .20 µg/ml

19. Correlation of ELISA and OPA (North CA KP infant study) Total N = 79 Jodar, et al. 2003. Vaccine R = 0.80 ( p < 0.0001) R = 0.92 ( p < 0.0001) 10000 7VPnC Control R = 0.80 ( p < 0.0001) 1000 OPA TITER 100 1:8 10 0.2ug/ml 1 0.01 0.1 1 10 ELISA Concentration (g/ml) ELISA concentration of 0.20  OPA titer of 1:8

20. SUMMARY • OPA is a correlate of protection • Animal & human OPA protection data appear to agree • ELISA & OPA are primary end-points(good correlation) • OPA formats include killing (std.) and uptake assays • Functional activity is influenced by avidity and age • Immunogenicity may be influenced by study design • and/or vaccine formulation, dose, scheduling, etc. • Formal efficacy trials will likely not be done