2. Evaluating Vaccine Effectiveness Using Serologic Assays
Cara R. Fiore, Ph D
Office of Vaccines Research and Review Center for Biologics Evaluation and Review
U. S. Food and Drug Administration
Vaccines Europe, Brussels
December 2011

3. Objectives Background:
Office of Vaccines Research and Review
Vaccine regulation, serological assay validation
Example: two vaccines licensed using immunogenicity data to infer effectiveness 2

4. CBER Regulation of Biologics
Biologics for human use (e.g. vaccines, blood and blood products, cell and gene therapies)
Per authority of:
Public Health Service Act, Section 351 (1944)
Federal Food, Drug and Cosmetic Act (1938)
Regulations: Title 21 of the Code of Federal Regulations (CFR) 5

5. OVRR Regulates: Vaccines for Infectious Disease Indications
Live attenuated preparations of bacteria or viruses
Inactivated or killed whole organisms
Polysaccharides (+/- protein conjugates)
Purified proteins, inactivated toxins, VLPs
DNA vaccines
Vectored vaccines 6 5

6. Development of Preventive Vaccines
Phase Phase Phase Phase 4
Safety Safety, Effectiveness
Safety, Immunogenicity
Pre IND
Initial product characterization
Preclinical Safety & Immunogenicity
Optimization of Manufacturing Process
Process Validation
Assay Development & Assay Validation (EOP2)
Final Product Specifications
Final Formulation/Dosage 7

7. Assay Validation ICH Q2 (R1) recognized standard guideline
Detection Limit
Linearity
Quantitation Limit
Range
Accuracy
Precision
Repeatability
Intermediate Precision
Specificity
Robustness
INTERNATIONAL CONFERENCE ON HARMONISATION OF TECHNICAL REQUIREMENTS FOR REGISTRATION OF PHARMACEUTICALS FOR HUMAN USE. ICH HARMONISED TRIPARTITE GUIDELINE. VALIDATION OF ANALYTICAL PROCEDURES: TEXT AND METHODOLOGY Q2(R1). Current Step 4 version. Parent Guideline dated 27 October 1994.(Complementary Guideline on Methodology dated 6 November 1996 incorporated in November 2005) 8

8. What are Clinical Serological Assays Used For?
Clinical – Immunogenicity and Effectiveness (inferred efficacy)
E.g.- Demonstration of non-inferiority of relevant immune response
Comparison to current standard of care
Comparison to sera from an efficacy trial with a clinical endpoint
Non-Interference with concomitant vaccines
Is there an acceptable correlate of protection?
CMC – Immunogenicity
Bridging manufacturing changes
New facility
Continued/additional product development
Lot consistency 9

9. Clinical Assay Considerations
Assay selection
Functional vs antibody binding
Scientific rationale and practical advantages
Assay validation should demonstrate that it is suitable for its intended purpose
Revalidation may be necessary if:
Drift in method performance
Predefined expiry
Change in procedure, equipment, or other factors 10

10. Immunological Endpoints to Infer Effectiveness
Clinical end-point efficacy studies are the Gold Standard
Why/When to use serological assay:
Clinical efficacy study not possible
Burden of disease too low
New population (age group)
(Animal Rule vaccines) 11

11. Challenges of Assay Design
Goals:
Measure meaningful immunologic biomarker
Reproducible and reliable
Reagents must be attainable
Reagents must be qualifiable
Adequate characterization, standardization and validation
Goals may be challenging for biological/ functional assays 12

12. Example 1: Meningococcal Conjugate Vaccines
Serologic Assays as a Measurement of Vaccine Effectiveness
Anti-polysaccharide IgG antibody
Serum bactericidal activity (SBA) assay
Meningococcal anti-PS antibody measured by ELISA does not always correlate with functional antibody measured by complement-mediated SBA 13

13. hSBA Meningococci Plate on MH agar Antibody Dilutions (human sera)
Complement (human)
Antibody Dilutions (human sera)
Meningococci
Incubate
Plate on MH agar + +
Survival of meningococci
Antibody titer = highest dilution that results in
killing of ≥ 50% cfu of target strain 14

14. Serum Bactericidal Activity (SBA)
Data support use of SBA as an immunological correlate for demonstrating effectiveness of meningococcal conjugate vaccines
Measures the antibody dependent complement mediated killing of the specific meningococcal strain in vitro
Critical factors - defined and adequately controlled:
Complement –
Human (h) vs. rabbit (r)
individual vs. pooled source
Target strain
Assay conditions 15

15. Factors Considered in Identifying SBA as the Primary Assay for Serologic Evaluation of Meningococcal Vaccines
Disease Pathogenesis –individuals who lack terminal complement components (complement deficiency) are at high risk of recurrent meningococcal disease
Natural History/Seroepidemiology - inverse correlation between age specific disease rates and prevalence of bactericidal activity
Historical studies of US military recruits
Antibodies that kill N. meningitidis in the presence of active complement (complement mediated bactericidal activity) were associated with protection from disease (Goldschneider et al )
Bactericidal Antibodies Play a Critical Role in Protection Against Meningococcal Disease 16

16. Highest incidence of disease occurred at the lowest bactericidal antibody prevalence
The incidence of meningococcal disease was inversely proportional to the age-specific prevalence of bactericidal antibodies to the Mn-specific strain
Adapted from Pollard et al. Vaccine 2001; 19: ; and Goldschneider I, J Exp Med 1969;129: 17

17. US Licensed Meningococcal Vaccines
Menomune 1981 (>2 yo). Quadrivalent PS vaccine, licensed based on efficacy data for A and C only. Not enough disease in W-135 and Y. W-135 and Y were based on 4 fold rise of SBA in 90% of vaccinees.
Menactra - Quadravalent (A, C, Y, W-135) PS conjugate
2005: 11-55yo
4-fold rise in rSBA non-inferiority to Menomune.
2007: 2-10 yo
% ≥ 1:8 with hSBA non-inferiority to Menomune
2011, 9-23mo
% ≥ 1:8 hSBA
Menveo - Quadravalent (A, C, Y, W-135) PS conjugate
2010: yo
% ≥ 1:8 with hSBA non-inferiority to Menactra.
2011: 2-10 yo
% ≥ 1:8 with hSBA non-inferiority to Menactra
ACIP recommends MCV4 for years of age, as well as others 2-55 years of age at increased risk of meningococcal disease. 18

18. Vaccines and Related Biological Products Advisory Committee Meeting
VRBPAC 1999
Vaccines and Related Biological Products Advisory Committee Meeting
VRBPAC agreed that new meningococcal vaccines could be evaluated using immunologic assays
Serum bactericidal activity was an appropriate parameter to evaluate immunogenicity of a new vaccine in age groups for which the current meningococcal vaccine is licensed for use
FDA implementation: SBA has been used to evaluate the immunogenicity of new meningococcal vaccines in comparison to currently licensed vaccines . 19

19. VRBPAC 2011
VRBPAC was asked to “comment on the use of hSBA as an immune measure to infer effectiveness of meningococcal conjugate vaccine for children younger than two years old.”
The Advisory Committee agreed that data supported the role of functional antibody in protection from meningococcal disease and that vaccine effectiveness can be inferred from serum bactericidal activity measurements in children less than 2 years of age.  20

20. Example 2: Pneumococcal Conjugate Vaccines
Serologic Assays as a Measurement of Vaccine Effectiveness
ELISA – Serotype specific IgG
Infants
IgG antibody levels are associated with protection from invasive pneumococcal disease
Good correlation between IgG and pediatric serum OPA titers
Older children and adults
Poor correlation with OPA for many serotypes
Not considered to be an appropriate endpoint in adults.
Opsonophagocytic Antibody (OPA) Assay
OPA measures functional antibodies that play a role in protection against pneumococcus for vaccines directed at capsular antigens 21

21. OPA Methodology
The OPA assay measures the ability of functional antibody to bind and opsonize the target bacteria in the presence of a complement source, engulfment by phagocytic human cell line (HL-60 cells.)
Polysaccharide bound human antibodies activate the complement mediated opsonization through the classical pathyway.
4 components
Human Sera + pneumococcus + complement + HL-60 cells
OPA titer = reciprocal of the lowest serum dilution that results in complement-dependent killing of 50% of the bacteria in vitro. 22

22. OPA Phagocytic Human Cell Source Pneumococci Plate on agar Incubate
Complement (human)
Antibody Dilutions (human sera)
Pneumococci +
Incubate
Plate on agar
Phagocytic Human Cell Source
Survival of pneumococci
Antibody titer = highest dilution that results in
killing of ≥ 50% cfu of target strain 23

23. US Licensed Pneumococcal Vaccines
Pneumovax 23 (1983) Multivalent (23) polysaccharide vaccine.
50 years of age or older and persons aged ≥2 years who are at increased risk for pneumococcal disease.
Efficacy of PS vaccines evaluated in several clinical trials
Prevnar - 7 valent polysaccharide conjugate vaccine
2000: Immunization of infants 2, 4, 6 and months of age to prevent invasive pneumococcal disease.
2002: Immunization of infants and toddlers against otitis media caused by vaccine serotypes .
Clinical endpoint efficacy trail
VRBPAC 2001 – advised that for new pneumococcal vaccines effectiveness could be inferred from non-inferiority studies using ELISA 24

24. Prevnar 13 2010: 13 valent polysaccharide conjugate vaccine.
Licensed in 6 weeks through 5 years of age.
Prevention of invasive disease caused by S.pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F.
Prevention of otitis media caused by S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F.
The efficacy was inferred from comparisons to Prevnar 7 using IgG (ELISA) to measured the production of vaccine type (VT) functional antibody.
Under Review in adults ≥ 50 years of age
IgG does not correlate with functional antibody for older children and adults. Therefore, IgG measurement was not considered to be an appropriate endpoint in these age groups.
VRBPAC 2005 – emphasized the need for clinical endpoint studies while acknowledging challenges, accelerate approval reasonable path
OPA - used as the “surrogate endpoint that is reasonably likely… to predict clinical benefit” of Prevnar 13 in adults
VRBPAC November 2011 25

25. Challenges Practical challenges: Clinical assay challenges:
Availability of patient sera: age group, study size, number of different assays
Clinical assay challenges:
Background titers
Functional quality, and robustness of immune responses
Interpretation of results
Study design issues
need for blinding and controls vs. pairing sera pre and post
require dilution of post immune sera 26

26. Summary: Demonstration of Effectiveness
Gold Standard: Clinical endpoint efficacy study
Immunogenicity Assessment: Serologic endpoint:
Non inferiority: Licensure on the basis of a comparison to licensed product
In populations where there is no comparator vaccine, no direct comparison is possible
Accepted correlate of protection
E.g., Menactra® licensed for use in children 9 month to 23 months of age based on the proportion of subjects with hSBA titer ≥ 1:8 27

27. Using serologic endpoints to infer vaccine efficacy:
Take Home Message
Using serologic endpoints to infer vaccine efficacy:
Ideally, there is a well established correlate of protection
Ideally, we understand the immunologic basis for the correlate of protection
Need to develop an assay that can be well validated and conducted to provide accurate results reliably 28

28. Thanks! Margaret Bash, MD, MPH Elizabeth Sutkowski, Ph D
Wellington Sun, MD
Nicolette deVore, Ph D 29