1. Measurement of Bactericidal Antibody as an Indicator of Vaccine Effectiveness Wendell D. Zollinger, Ph.D. April 6, 2011

2. The importance of bactericidal antibodies in human immunity to meningococcal disease 1.Available evidence (to be discussed by others) supports the use of the hSBA as a valid serologic marker of protective immunity 2.A serologic marker of protection refers to a marker that is a measure of the biological function that is associated with immunity and can substitute for a clinical end point such as efficacy.

3. Bactericidal Assay -- Complement Mediated Lysis by Specific Antibody Assay Composition – Buffer/Diluent – Serial 2-fold dilutions of test serum – Log phase bacteria – Complement (serum or plasma without intrinsic bactericidal activity) Antibody requirements – Correct isotype: IgG3>IgG1>IgG2, IgM – High avidity Antigen requirements – Adequate surface exposure to bind antibody and fix C’ – Relatively high surface density (C’ fixation requires two IgG molecules bound close together)

4. Protective Level of Bactericidal Antibodies Work of Goldschneider, et al. was done using a titer of 1:4 with intrinsic human complement as a level that correlated with protection Some have suggested that 1:8 would be a safer level to use End point taken as the highest serum dilution giving ≥50% killing Several studies have explored the question

5. Correlation of hSBA titers with efficacy using Norwegian group B OMV Vaccine

6. Efficacy predicted on the basis of a population based marker of immunity A titer of ≥ 1:8 in the BR-SBA corresponded to the observed efficacy in UK. RC-SBA measured 1 month following vaccination with the MCC vaccine (Andrews N, et al. Clin Diagn Lab Immunol 2003;10:780-6)

7. Standardization of the SBA Principal Variables 1. Choice and management of test strains 2. Complement source 3. Assay method used

8. Assay results can vary considerably depending on the parameters of the assay The assay method used in different laboratories may vary with respect to: 1.Growth and handling of the test strain 2.Source and concentration of complement 3.Growth media and diluent composition 4.Incubation time and conditions 5.Method of determination of cell viability 6.Definition of end point titer Assay standardization is needed if lab to lab comparisons are to be meaningful

9. Assay ParameterParameter used in different Labs Initial set up of Cells CBA w/ 5% blood, Mueller-Hinton chocolate agar, GC medium w/supplement InoculumSingle colony, ~10 colonies, sweep Growth of cells for assay4-5 h on agar plates (same as set up), MH liquid medium, MH liquid medium w/ 0.25% glucose and CMP-NANA Incubation conditions37 ° C for 30 min or 60 min With shaking, gentle raking, occasional tapping DiluentHanks BSS with 0.5% or 3% or 5% BSA Dulbecco’s PBS w/ Ca & Mg Geys BSS with 1% BSA or 0.15% gelatin Cell Concentration in assay10 3 /ml to 10 5 /ml Complement10-25% Human serum, human plasma, baby rabbit SerumHeat inactivated or not Viability determinationPlate on agar by tilt plate or drizzle method, agar overlay in microtiter plate wells Range of Different Conditions Used in Different Laboratories

10. Importance of the Complement Source Used in the Bactericidal Assay 1.The studies of Goldschneider, et al. were done using human complement 2.SBA with human complement is the best serologic marker of protection as it is consistent with the in vivo biological mechanism of protection. 3.Baby rabbit complement may be more convenient and easier to standardize, but it gives significantly different results: Generally much higher titers (but differences are more complex than that) Rabbit factor H not bound by meningococcal fHbp RC appears to function with lower avidity antibodies ( human antibodies to group B polysaccharide, which are IgM and low avidity, are bactericidal with rabbit complement but not with human complement.) 4.Efforts have been made to correlate BR-SBA with hSBA but results in a population based marker of protection and is not reliable when applied to individual protection.

11. Complement Source (Cont.) 5.Donor specific differences in human complement can be averaged by creating pools from multiple donors. 6.Complement activity should be measured and verified to be within an acceptable range before use

12. Target Strain Considerations 1.Target strains vary considerably in susceptibility to killing by the same sera (differences in amount of capsule, fHbp, LOS, etc.) 2.The “same strain” present in different laboratories may not be identical due to phase variation and different handling of the strain 3.Test strains should under go thorough initial characterization and multiple samples of each strain stored as master and working cell banks. 4.Some important antigens to characterize and monitor are: capsule, LOS, Opc, factor H binding protein, and key vaccine antigens

13. Effect of LOS alpha-chain length on group B strain sensitivity to bactericidal antibody Moran, et al. Infect. Immun. 62:5290-5, 1994

14. Target Strain Considerations (cont.) 5.Use many cells to inoculate growth for use in the assay rather than a single colony 6.For group B vaccines a panel of strains is required to demonstrate the breadth of protection across group B strains. Choice of the strains for the panel may need to be vaccine specific. 7.For capsular polysaccharide vaccines only a single strain is needed for each serogroup. The sensitivity of the chosen test strain to killing by the bactericidal mechanism should be representative of the majority of case isolates.

15. Comparators No. samples with titer step of: –10123 Stationary vs. raking reaction mixture* 181841 Single colony vs. mixed population**21421111 Impact of Two Assay Parameters: hSBA titer step differences by reaction mixture motion and population type Borrow, et al. Clin. Diagn. Lab. Immunol. 12:970-976, 2005 *Raking results – Stationary results **mixed population – single colony results

16. Assay Standardization Progress (Mostly focused on Group B) International Meeting : Neisseria meningitidis group B correlates of protection and assay standardization--international meeting report Emory University, Atlanta, Georgia, United States, 16-17 March 2005. Borrow et al. Vaccine. 24:5093-107, 2006. Multi-laboratory study by Borrow, et al. (Clin Diag Lab Immunol 12:970-976, 2005). Showed good inter-laboratory reproducibility if strain, reagents, and method were controlled. Looked at the effect of different variables. New Zealand assay validation: Martin D, McCallum L, Glennie A, Ruijne N, Blatchford P, O'Hallahan J, Oster P. Validation of the serum bactericidal assay for measurement of functional antibodies against group B meningococci associated with vaccine trials. Vaccine. 2005 Mar 18;23(17-18):2218-21.

17. Interlaboratory Comparison with Assay Parameters Controlled to Different Degrees Borrow, et al. Clin. Diagn. Lab. Immunol. 12:970-976, 2005

18. Summary and Conclusions 1.Human complement-mediated bactericidal activity is the best biological marker of protection and most relevant to individual protection and assessment of vaccine immunogenicity. 2.The SBA measures functional antibodies that have been shown to be important for protection from systemic disease. 3.Standardized assay needed with important variables controlled 4.Choice and management of the target strain a critical factor in SBA 5.Good agreement can be obtained between laboratories if the assay is sufficiently standardized