Prevnar 13 for adult use Pneumococcal 13-valent Conjugate Vaccine (Diphtheria CRM 197 Protein)

1. Vaccines and Related Biological Products Advisory Committee Meeting November 16, 2011 Prevnar 13 for adult usePneumococcal 13-valent Conjugate Vaccine (Diphtheria CRM197 Protein) Applicant: Wyeth Inc. Mustafa Akkoyunlu, M.D. Ph.D. Serology Reviewer FDA/CBER/OVRR/DBPAP/LBP

2. Use of Serology assays in demonstrating pneumococcal conjugate vaccine effectiveness - Efficacy studies conducted in children in different countries with pneumococcal conjugate vaccines, including Prevnar (7-valent pneumococcal vaccine) have shown that IgG antibody levels associate with protection from invasive pneumococcal disease (IPD). ≥0.35 mg/mL anti-polysaccharide IgG antibody was chosen as reference level for comparing investigational vaccines to licensed vaccine for common serotypes, and for evaluating antibody responses to new vaccine serotypes after administration of the infant series. - WHO 2009 TRS document 927, Annex 2 - Standardized ELISA. - Pneumococcal reference serum (89SF).

3. Use of serology assays in demonstrating pneumococcal conjugate vaccine effectiveness - Prevnar 13 is approved for use in children 6 weeks to 5 years old. - Efficacy of Prevnar 13 was inferred based on a non-inferiority immunogenicity study through anti-polysaccharide IgG antibody (ELISA) responses using Prevnar (7-valent pneumococcal vaccine) as a licensed comparator vaccine.

4. Opsonophagocytic assay (OPA) There have been efforts to correlate serum IgG levels measured and quantified byELISA with OPA titers. Studies conducted so far suggest that: - there may be a concordance between pediatric serum OPA titers and ELISA IgG concentrations - adult serum OPA titers do not correlate wellwith serum IgG antibody concentrations -Vaccine 25 (2007) 2518–2527 -Vaccine 26 (2008) 5521–5526

5. Demonstrating effectiveness of Prevnar 13 in adults ≥ 50 years of age - In the current submission, Wyeth Inc., is seeking an indication for Prevnar 13 for prevention of pneumococcal disease in adults (≥ 50 years old). • Use of antibody levels quantified by ELISA for non-inferiority comparisons to infer effectiveness of Prevnar 13 would likely not be valid for adults: • Pre-existing titers • Level of serum antibodies that correlate with protection in adults and the elderly have not been determined • OPA measures functional antibodies that play a role in protection against pneumococcus for vaccines directed at capsular antigens. • OPA used to infer effectiveness of Prevnar 13 in adults ≥ 50 years of age.

6. Opsonophagocytosis - The OPA used in the analysis of pneumococcal vaccines is designed to mimic the in vivo opsonophagocytosis which involves bacteria, antibodies, complement, and phagocytic cells. - It has been shown that polysaccharide bound human antibody isotypes IgG2, IgG3 and IgM activate the complement mediated opsonization through the classical pathway.

7. C4 + C2 C3 C1q Phagocytic cell C1s C1r C3b Anti-PS IgG2, IgM (C3 convertase) C4b C2a CR1/CD35 CR3/CD11b iC3b Streptococcus pneumoniae Polysaccharide Opsonophagocytosis

8. Opsonophagocytic assay (OPA) - The discovery of the importance of antibody and complement mediated opsonophagocytosis in protection against IPD led to the development of well controlled OPAs. -Vaccine 2007 25; 2518-2527. -CDLI 2005 Feb;12(2):287-95. -CVI 2006 Sep;13(9):1004-9. -CDLI 1997 July 4:415-422. - These assays use baby rabbit serum as the complement source and human promyelocytic cell line HL-60 cells as the phagocytic cells. - In all assays, the serum dilution yielding 50% killing of the initial bacterial load is defined as the OPA titer.

9. Streptococcus pneumoniae Test sera Critical Components shared by commonly used OPAs -Definition of capsule expression -Preparation and maintenance of working stocks -Bacterial count that yields target cfu after thawing of frozen bacteria - Heat inactivation (to eliminate intrinsic complement activity) - Determination of antibiotic activity

10. Baby rabbit complement (sera) HL-60 cells Critical Components of OPA -Selection of BRC lots without antibodies against S. pneumoniae (Assessment of OPA titers in heat inactivated BRC with previously qualified BRC) -Determination of lots with target complement concentration (Intrinsic opsonic OPA titers of non-heat inactivated BRC or bridging candidate lots with the existing qualified BRC lots) -The promyelocytic HL-60 cell line is differentiated into granulocytic phenotype by using N,N-dimethylformamide (DMF). -After 5 to 6 days of differentiation HL-60 cells should Have: - > 80% viability (PI/Annexin) - ≥ 55% CD35+ - ≤ 15% CD71+

11. Baby rabbit sera Serum HL-60 cells + Incubation in RT (30 min) C1q Streptococcus pneumoniae Incubation at 37oC for 45 min Plated OPA protocol

12. OPA protocol (continued…) - At the end of opsonophagocytic incubation reaction, mixtures are transferred to agar media to allow bacterial growth. - Most laboratories employ automated methods to detect and count surviving colonies. - The data gathered from colony counts are processed by software to calculate the 50% killing dilution. -

13. OPA protocol (continued…) • Each assay also contains control conditions that are used in OPA titer calculation and assay acceptance criteria determination. • - Antibiotic control: Bacterial cfu in wells containing human serum sample, bacteria, and BRC but not HL-60 cells. Helps assess the intrinsic killing ability of test serum samples. • Time 0 cfu count: Bacteria plated prior to the opsonization step (T0). Bacterial cfu in wells without BRC, human serum or HL-60 cells. • Background cfu value: Bacterial cfu in wells containing bacteria, BRC, and HL-60 cells but not human serum sample. Used to calculate 50% killing dilution. • Quality control serum (QCS): Each plate contains QCS. Measured QCS titers are compared to established QCS titers and they have to meet predetermined titration criteria.

14. 140 -------------------------------------------------------------------------- Background cfu 120 100 80 CFU -------------------------------------------------------------------------- 50% of background cfu 60 40 20 0 0 1:8 1:20 1:50 1:125 1:313 1:781 1:1953 1:4883 Serum dilution Wyeth Inc., OPA titer calculation OPA titers are calculated by interpolation between the two data points that are immediately below and above the 50% of background value.

15. Validation of Wyeth Inc., OPA Validation parameters: 1. Linearity 2. Precision a. Repeatability b. Intermediate Precision (inter-assay variability) 3. Lower Limit of Quantitation (LLOQ) 4. Limit of Detection (LOD) 5. Range

16. Summary - Although there is good correlation between ELISA IgG Abs and OPA titers in children, adult IgG levels may not accurately predict OPA activity. - OPA measures the biological activity of serum antibodies, therefore it is believed to be highly relevant in assessing vaccine efficacy. - Its experimental set up involves four critical components (bacteria, serum antibodies, complement, and phagocytic cells). - The performance of the assay and the reliability of the results requires stringent control on each of these components. - The high throughput OPA used by the applicant is sufficiently validated to show its reliability for the intended use. - The intended use is to compare the serum OPA titers induced by Prevnar 13 to those induced by Pneumovax23.